@article {9056, title = {Cryo-EM structures of Mycobacterium tuberculosis imidazole glycerol phosphate dehydratase in the apo state and in the presence of small molecules [National Cryo-EM Facility, BLiSC]}, journal = {Acta Crystallogr F Struct Biol Commun}, year = {2025}, month = {2025 Jul 01}, abstract = {

Unlike humans, Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, has a de novo histidine-biosynthesis pathway. The enzyme imidazole glycerol phosphate dehydratase (IGPD), which catalyses the conversion of imidazole glycerol phosphate to imidazole acetol phosphate, has been studied extensively from various organisms and has become a major target for the development of antibacterial, antiweed and antifungal small molecules. In our previous studies, we have shown that in crystals IGPD forms a 24-mer oligomeric state in which the monomers are arranged in 432 symmetry. In order to gain insights into the oligomeric state of Mtb IGPD in solution, we determined cryogenic sample electron microscopy (cryo-EM) structures of apo IGPD at 2.2 and 3.1 {\r A} resolution. In addition, we also determined the cryo-EM structure of IGPD in the presence of 3-amino-1,2,4-triazole (ATZ) to 2.8 {\r A} resolution. The results of this work, which corroborate those from the crystallographic studies, indicate that IGPD forms a homo-oligomeric structure in solution comprising of 24 subunits. ATZ binds in the active-site pocket of the enzyme, which is located at the interface of three monomers and tethers 24 ATZ molecules. The results of this study suggest that cryo-EM, in addition to being a rapidly evolving and complementary imaging technology for elucidating 3D structures of biological macromolecules, can be useful in pinpointing the mode of binding small molecules of low mass (here \~{}85 Da) and mapping protein-ligand interactions, which could assist in the design of accurate (high-potency) inhibitors.

}, issn = {2053-230X}, doi = {10.1107/S2053230X25004595}, author = {Raina, Rahul and Kar, Deepsikha and Singla, Mohini and Tiwari, Satish and Kumari, Swati and Aneja, Sonanjali and Kumar, Varun and Banerjee, Soumya and Goyal, Shivika and Pal, Ravi Kant and Vinothkumar, Kutti R and Biswal, Bichitra} } @inbook {9067, title = {The Diagnostic Success Story: C-CAMP COVID-19 Innovations Deployment Accelerator (C-CIDA)}, booktitle = {At War with the Single Strand: How Indian Science Fought the COVID-19 Pandemic}, year = {2025}, pages = {53{\textendash}56}, publisher = {Springer Nature Singapore}, organization = {Springer Nature Singapore}, address = {Singapore}, abstract = {

Novel coronavirus COVID-19 emerged as one of the gravest public healthcare challenges in recent history. Even before the first lockdown started, we at C-CAMP had already felt the need to identify and work with such indigenously developed and indigenously produced COVID-19 innovations for rapid deployment. One of the first such innovations we fast-tracked in early March was a rapid diagnostics kit that has since received regulatory nods and was deployed in the field. As the virus marched on with no sign of reprieve, we recognized the importance of being more organized and focused in our efforts. Through the last decade or so of India{\textquoteright}s biotech boom, India{\textquoteright}s bio start-ups have been putting forward Made-in-India globally competitive solutions. Now was the time to capitalize on these technologies and align them with unmet needs for rapid deployment at an unprecedented scale.

}, isbn = {978-981-96-8122-8}, doi = {10.1007/978-981-96-8122-8_5}, url = {https://doi.org/10.1007/978-981-96-8122-8_5}, author = {Saiyed, Taslimarif and Chandavarkar, Nandita and Chakraborty, Debarshini and Janardan, Neelanjana and Alam, Mohammad Atif and Patel, Paresh and Gupta, Preksha and Bhardwaj, Vishal and Wala, Bhavisha and Joshi, Niranjan and Mukherjee, Titash and Vuppala, Kiran and Kiran, Amritanjali and Das, Deboleena Raha and Jain, Neeraj and Kumar, Manoj and Chandran, Subhashini and Rao, Madhusudhana and Joshi, Madhav and Kakkar, Abhishek and Khirani, Sekhar}, editor = {Swarup, Renu and Padmanaban, Govindarajan and Arora, Narendra Kumar} } @article {9060, title = {Identifying bellwether sewershed sites for sustainable disease surveillance in Bengaluru, India: a longitudinal study [Next Gen Genomics Facility (INT)]}, journal = {The Lancet Regional Health - Southeast Asia}, volume = {39}, year = {2025}, pages = {100619}, abstract = {

Summary Background Throughout the COVID-19 pandemic, wastewater surveillance emerged as an important tool as an important tool by providing data that are more representative of the population than case reporting, which is often biased towards individuals with health-seeking behaviour or access to healthcare. With changing phases of the pandemic, decreased testing, and varying viral shedding rates, it is crucial to have a robust, sustainable, and flexible wastewater surveillance system that can serve as an independent signal of disease outbreaks. We aimed to identify {\textquoteleft}bellwether{\textquoteright} sewershed sites for sustainable disease surveillance in Bengaluru, India. Methods We conducted this longitudinal study from December 2021 to January 2024 at 26 centralised sewershed sites in Bengaluru city (\~{}11 million inhabitants). We quantified weekly SARS-CoV-2 RNA concentrations to track infection dynamics and identify {\textquoteleft}bellwether{\textquoteright} sewershed sites. This was achieved by integrating established metrics for wastewater analysis, calculating sample-to-sample percentage rate of change, and applying algorithms to differentiate signal from noise, thereby validating factors contributing to the precision and reliability of outbreak predictions. Findings Using 2873 wastewater samples, we applied a modified algorithm (COVID-SURGE algorithm) to identify {\textquoteleft}bellwether{\textquoteright} sewershed sites using longitudinal wastewater data on SARS-CoV-2 from 26 sewershed sites in Bengaluru. We utilised an Excel-based calculator (COVID-SURGE calculator) for user-entered wastewater data that differentiates signal from noise (underlying variability) based on the algorithm, with adjustments made to the input format of viral data and a specified limit of detection (LOD) value from the reverse transcriptase-quantitative PCR kit. We identified 11 {\textquoteleft}bellwether{\textquoteright} sites: four with large catchment sizes (KC Valley 1, KC Valley 2, Rajacanal, Doddabelee); four with medium sizes (Agaram, Nagasandra, KR Puram, Yelahanka); and three with small sizes (Chikkabegur, Chikkabanavara, Lalbagh). These were the best performers and can serve as a useful subset of sewage treatment plants for an early warning system at the city level. Interpretation Using wastewater metrics helps in selecting permanent sewershed sites and identifying sub-sites that can be scaled up during peak outbreak periods to detect disease hotspots, or scaled down during lean periods, especially when clinical data are unavailable. In a post-pandemic world, particularly in low-resource settings, focusing on the best-performing sewershed sites will ensure high-quality data that captures valid signals amid the noise from wastewater, conserves resources, and optimises public health actions beyond SARS-CoV-2. Funding This work has been supported by funding from the Rockefeller Foundation (grant 2021 HTH018) to National Centre for Biological Sciences (TIFR) and the Indian Council of Medical Research grant to (FI) Tata Institute for Genetics and Society and Tata Trusts.

}, keywords = {Bengaluru, COVID-19, COVID-19 pandemic, COVID-SURGE, India, Low-resource settings, Pandemic, SARS-CoV-2, Variants, Wastewater surveillance}, issn = {2772-3682}, doi = {https://doi.org/10.1016/j.lansea.2025.100619}, url = {https://www.sciencedirect.com/science/article/pii/S2772368225000903}, author = {Rebecca Fern Daniel and Subash K. Kannan and Namrta Daroch and Sutharsan Ganesan and Farhina Mozaffer and Vishwanath Srikantaiah and Lingadahalli Subrahmanya Shashidhara and Rakesh Mishra and Farah Ishtiaq} } @article {8961, title = {Integrated omics analysis of rice gene expression profiles and microRNAs identifies crucial target genes for nitrogen use efficiency [Next Gen Genomics Facility]}, journal = {Plant Physiology Reports}, year = {2025}, month = {Feb}, abstract = {

Nitrogen use efficiency is a complex trait in rice involving nitrogen uptake, utilization, and assimilation. The coding mRNAs and non-coding regulatory RNAs have combinatorial involvement in molecular mechanisms underlying nutrient use efficiency. In this study, we generated the microRNA expression profiles of panicle tissues in N-efficient (Basmati370, Thurur bhog) and non-efficient genotypes (Kola Joha3, IC463254) at the booting stage using microRNA sequencing and microarray expression profiles of root and shoot tissues at the seedling stage in N-efficient (Varadhan) and non-efficient (Prasanna) genotypes under hydroponic condition with N (+) and without N supplementation N (-) using Affymetrix rice microarray chip. To understand holistically the differential expression of mRNAs and microRNAs regulating NUE, we integrated the newly generated microRNA sequencing and microarray datasets with earlier generated mRNA gene expression profile data using bioinformatics tools. We present a consolidated set of genes operating tissue and stage-wise in rice for NUE. The microRNAs viz. miR2118o, miR1442, miR5149 were up-regulated and miR164, miR2867, miR171i were down-regulated in N efficient genotypes under low N conditions. Interestingly, we have identified the consistent differential expression of known and novel microRNAs in N efficient genotypes viz. miR1859 (down-regulated), miR1441, miR3979-5p, miR5149 (up-regulated) under low nitrogen conditions. The GARP-MYB/SANT domain, early nodulin genes were exclusively present in the shoot of Varadhan; genes related to polycomb complex, enhancer of zeste, were downregulated under N0 condition. Under low N conditions, brassinosteroid insensitive receptor kinase-1, IAA31, DUF581, DUF617, DUF803, NIN, nodulin, oligopeptide transporter, OsPOP1, calmodulin kinases were upregulated across roots and shoots. The genes upregulated in efficient genotypes expressed in shoots as well as in panicle tissues and regulated by downregulation of their miRNAs are targets for further studies. Remarkably, the downregulation of specific metabolic pathways was observed as an adaptive strategy to cope with nitrogen limitation, highlighting the complexity of NUE regulation in rice. These findings elucidate the intricate molecular networks governing NUE in rice and underscore the potential of specific microRNAs and genes as targets for precision breeding strategies aimed at enhancing nitrogen utilization efficiency in rice cultivation. The integrative transcriptomic approach presented here provides a foundation for further functional genomic studies, facilitating the development of novel breeding strategies to optimize NUE in rice.

}, issn = {2662-2548}, doi = {10.1007/s40502-024-00846-9}, url = {https://doi.org/10.1007/s40502-024-00846-9}, author = {Barbadikar, Kalyani M. and Talapanti, Krishna Kant and Bej, Sonali and Karre, Suman and Kothandaraman, Harish and Malathi, S. and Puskur, Raghuveer Rao and Mangrauthia, Satendra Kumar and Desiraju, Subrahmanyam and Voleti, S. R. and Sureshrao Sakhare, Akshay and Anantha, M. S. and Sundaram, R. M. and Chirravuri, Neeraja N.} } @article {8674, title = {Cryo-EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH [National Cryo-EM Facility, BLiSC]}, journal = {Nat Commun}, volume = {15}, year = {2024}, month = {2024 Jan 20}, pages = {638}, abstract = {

Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome. Mycobacterium tuberculosis encounters hypoxia (low oxygen) as a major stress in the host macrophages, and upregulates the expression of RafH protein, which is crucial for its survival. The RafH, a dual domain HPF, an orthologue of bacterial long HPF (HPF), hibernates ribosome in 70S monosome form, whereas in other bacteria, the HPF induces 70S ribosome dimerization and hibernates its ribosome in 100S disome form. Here, we report the cryo- EM structure of M. smegmatis, a close homolog of M. tuberculosis, 70S ribosome in complex with the RafH factor at an overall 2.8 {\r A} resolution. The N- terminus domain (NTD) of RafH binds to the decoding center, similarly to HPF NTD. In contrast, the C- terminus domain (CTD) of RafH, which is larger than the HPF CTD, binds to a distinct site at the platform binding center of the ribosomal small subunit. The two domain-connecting linker regions, which remain mostly disordered in earlier reported HPF structures, interact mainly with the anti-Shine Dalgarno sequence of the 16S rRNA.

}, keywords = {Bacterial Proteins, Mycobacterium tuberculosis, Ribosomal Proteins, Ribosomes, RNA, Ribosomal, 16S}, issn = {2041-1723}, doi = {10.1038/s41467-024-44879-y}, author = {Kumar, Niraj and Sharma, Shivani and Kaushal, Prem S} } @article {8832, title = {Cryo-EM structures reveal the molecular mechanism of HflX-mediated erythromycin resistance in mycobacteria [National Cryo-EM Facility, BLiSC]}, journal = {Structure}, year = {2024}, month = {2024 Jul 08}, abstract = {

Mycobacterial HflX confers resistance against macrolide antibiotics. However, the exact molecular mechanism is poorly understood. To gain further insights, we determined the cryo-EM structures of M.\ smegmatis (Msm) HflX-50S subunit and 50S subunit-erythromycin (ERY) complexes at a global resolution of approximately 3\ {\r A}. A conserved nucleotide A2286 at the gate of nascent peptide exit tunnel (NPET) adopts a swayed conformation in HflX-50S complex and interacts with a loop within the linker helical (LH) domain of MsmHflX that contains an additional 9 residues insertion. Interestingly, the swaying of this nucleotide, which is usually found in the non-swayed conformation, is induced by erythromycin binding. Furthermore, we observed that erythromycin decreases HflX{\textquoteright}s ribosome-dependent GTP hydrolysis, resulting in its enhanced binding and anti-association activity on the 50S subunit. Our findings reveal how mycobacterial HflX senses the presence of macrolides at the peptide tunnel entrance and confers antibiotic resistance in mycobacteria.

}, issn = {1878-4186}, doi = {10.1016/j.str.2024.06.016}, author = {Srinivasan, Krishnamoorthi and Banerjee, Aneek and Sengupta, Jayati} } @article {8675, title = {Generation and characterization of retinal pigment epithelium from patient iPSC line to model oculocutaneous albinism (OCA)1A disease [EyeStem Research Pvt. Ltd. - a C-CAMP Startup and Discovery to Innovation Accelerator, C-CAMP]}, journal = {J. Biosciences }, volume = {49}, year = {2024}, month = {01/2024}, abstract = {

Oculocutaneous albinism (OCA) is characterized by reduced melanin biosynthesis affecting the retina, thus impairing visual function. The disease pathology of OCA is poorly understood at the cellular level due to unavailability of suitable biological model systems. This study aimed to develop a disease-specific\ in vitro\ model for OCA type 1A, the most severe form caused by\ TYR\ (tyrosinase) gene mutations, using retinal pigment epithelium (RPE) differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs). A comparative study between healthy and OCA1A RPE cells revealed that while healthy RPE cells exhibited timely onest of pigmentation during differentiation, OCA1A RPE cells failed to pigment even after an extended culture period. This observation was validated by ultrastructural studies using electron microscopy, hinting at melanosome-specific defects. Immunocytochemistry demonstrated abnormal expression patterns of melanogenesis-specific protein markers in OCA1A RPE cells, indicating reduced or absence of melanin synthesis. Next, a quantitative assay was\ performed to\ confirm the\ absence of melanin production in\ OCA1A RPE cells. Tyrosinase assay showed no activity in OCA1A compared with healthy RPE, suggesting non-functionality of\ TYR, further corroborated by western blot analysis showing complete absence of the protein. Gene expression by RNA sequencing of healthy and OCA1A RPE cells uncovered differential gene expression associated with lens development, visual perception, transmembrane transporter activity, and key signaling pathways. This disease-in-a-dish model of OCA1A provides an excellent platform to understand disease mechanism, identify potential therapeutic targets, and facilitate gene therapy or gene correction.

}, doi = {/doi.org/10.1007/s12038-023-00406-7}, url = {https://link.springer.com/article/10.1007/s12038-023-00406-7}, author = {Janavi Subramani and Niharika Patlolla and Rajani Battu and Taslimarif Saiyed and Rajarshi Pal} } @article {8887, title = {Inhibition of dipeptidyl peptidase-IV by hydrolysates of beta-lactoglobulin isolated from Gir cow milk [Mass Spectrometry - Proteomics]}, journal = {Food Chemistry Advances}, year = {2024}, pages = {100842}, abstract = {

The current study attempts to assess the dipeptidyl peptidase {\textendash} IV (DPP-IV) inhibitory potential of β-lactoglobulin (β-Lg) isolated from milk of Gir cow. Whey was separated from skim milk by precipitation of casein followed by ultrafiltration (UF) to remove lactose and salts as permeate. β-Lg was separated from the UF retentate using salting out technique. Its purity was confirmed by SDS-PAGE and RP-HPLC. Hydrolysates of β-Lg were prepared using three different enzymes viz., pepsin, trypsin and Flavourzyme at different enzyme to substrate (E:S) ratios (1:25, 1:50, 1:100) and treated for 2-12 h durations. Pepsin-treated hydrolysate showed maximum DPP-IV inhibition of 77.62 {\textpm} 0.98 \% (IC50 =3.78 mg/mL). Hydrolysate was passed serially through UF filters of 10 and 3 kDa. The permeate of 3 kDa, which exhibited 52.10 {\textpm} 0.72 \% DPP-IV inhibition was fractionated through RP-HPLC and time based fractions were collected. All 41 fractions were again analysed for DPP-IV inhibition activity and 3 fractions which showed maximum DPP-IV inhibition activity were chosen for LC-MS/MS analysis. One novel peptide was identified through LC-MS/MS, which showed adequate DPP-IV inhibition (IC50 = 6.64 mmolL-1).

}, keywords = {Antidiabetic peptides, Degree of hydrolysis, DPP-IV inhibition, IC value, β-lactoglobulin}, issn = {2772-753X}, doi = {https://doi.org/10.1016/j.focha.2024.100842}, url = {https://www.sciencedirect.com/science/article/pii/S2772753X24002375}, author = {Ashok Kumar and M H Sathish Kumar and C S Rajani and Latha Sabikhi and Harshita Sonarthi} } @article {8863, title = {LC/MS Based Characterization Workflow of GLP-1 Therapeutic Peptide Liraglutide and Its Impurities [Agilent Technologies - C-CAMP Collaboration]}, year = {2024}, abstract = {

This application note demonstrates the power of the Agilent 1290 Infinity II liquid chromatograph (LC) coupled with the Agilent 6545XT AdvanceBio liquid chromatography/quadrupole time-of-flight mass spectrometer (LC/Q-TOF MS) for identification and characterization of therapeutic peptides. This application is exemplified for the GLP-1 agonist liraglutide and its impurities. The therapeutic peptide workflow within Agilent MassHunter BioConfirm software allows for accurate mass-based identification, sequence conformation, and identification of impurities, with extra or missing amino acid impurities that can arise during solid phase synthesis. These impurities may pose a potential safety risk and eventually may impact the efficacy of the product.

}, url = {https://www.agilent.com/cs/library/applications/an-liraglutide-impurities-characterization-5994-7727en-agilent.pdf}, author = {Shadab Ahmad and Nirpendra Singh and Ashish Pargaonkar and Dheeraj Vig and Mike Knierman} } @article {8708, title = {Multiplexed fluorescence and scatter detection with single cell resolution using on-chip fiber optics for droplet microfluidic applications [Discovery to Innovation Accelerator, C-CAMP]}, journal = {Microsyst Nanoeng}, volume = {10}, year = {2024}, month = {2024}, pages = {35}, abstract = {

Droplet microfluidics has emerged as a critical component of several high-throughput single-cell analysis techniques in biomedical research and diagnostics. Despite significant progress in the development of individual assays, multiparametric optical sensing of droplets and their encapsulated contents has been challenging. The current approaches, most commonly involving microscopy-based high-speed imaging of droplets, are technically complex and require expensive instrumentation, limiting their widespread adoption. To address these limitations, we developed the OptiDrop platform; this platform is a novel optofluidic setup that leverages the principles of flow cytometry. Our platform enables on-chip detection of the scatter and multiple fluorescence signals from the microfluidic droplets and their contents using optical fibers. The highly customizable on-chip optical fiber-based signal detection system enables simplified, miniaturized, low-cost, multiparametric sensing of optical signals with high sensitivity and single-cell resolution within each droplet. To demonstrate the ability of the OptiDrop platform, we conducted a differential expression analysis of the major histocompatibility complex (MHC) protein in response to IFN stimulation. Our results showed the platform{\textquoteright}s ability to sensitively detect cell surface biomarkers using fluorescently labeled antibodies. Thus, the OptiDrop platform combines the versatility of flow cytometry with the power of droplet microfluidics to provide wide-ranging, scalable optical sensing solutions for research and diagnostics.

}, issn = {2055-7434}, doi = {10.1038/s41378-024-00665-w}, author = {Gupta, Preksha and Mohan, Ambili and Mishra, Apurv and Nair, Atindra and Chowdhury, Neeladri and Balekai, Dhanush and Rai, Kavyashree and Prabhakar, Anil and Saiyed, Taslimarif} } @article {8740, title = {Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB. [Mass Spectrometry - Proteomics Facility]}, journal = {Cell Commun Signal}, volume = {22}, year = {2024}, month = {2024 Apr 23}, pages = {237}, abstract = {

BACKGROUND: A water-soluble ingredient of mature leaves of the tropical mahogany {\textquoteright}Neem{\textquoteright} (Azadirachta indica), was identified as glycoprotein, thus being named as {\textquoteright}Neem Leaf Glycoprotein{\textquoteright} (NLGP). This non-toxic leaf-component regressed cancerous murine tumors (melanoma, carcinoma, sarcoma) recurrently in different experimental circumstances by boosting prime antitumor immune attributes. Such antitumor immunomodulation, aid cytotoxic T cell (T)-based annihilation of tumor cells. This study focused on identifying and characterizing the signaling gateway that initiate this systemic immunomodulation. In search of this gateway, antigen-presenting cells (APCs) were explored, which activate and induce the cytotoxic thrust in T cells.

METHODS: Six glycoprotein-binding C-type lectins found on APCs, namely, MBR, Dectin-1, Dectin-2, DC-SIGN, DEC205 and DNGR-1 were screened on bone marrow-derived dendritic cells from C57BL/6\ J mice. Fluorescence microscopy, RT-PCR, flow cytometry and ELISA revealed Dectin-1 as the NLGP-binding receptor, followed by verifications through RNAi. Following detection of β-Glucans in NLGP, their interactions with Dectin-1 were explored in silico. Roles of second messengers and transcription factors in the downstream signal were studied by co-immunoprecipitation, western blotting, and chromatin-immunoprecipitation. Intracellularization of FITC-coupled NLGP was observed by processing confocal micrographs of DCs.

RESULTS: Considering extents of hindrance in NLGP-driven transcription rates of the cytokines IL-10 and IL-12p35 by receptor-neutralization, Dectin-1 receptors on dendritic cells were found to bind NLGP through the ligand{\textquoteright}s peripheral β-Glucan chains. The resulting signal phosphorylates PKCδ, forming a trimolecular complex of CARD9, Bcl10 and MALT1, which in turn activates the canonical NFκB-pathway of transcription-regulation. Consequently, the NFκB-heterodimer p65:p50 enhances Il12a transcription and the p50:p50 homodimer represses Il10 transcription, bringing about a cytokine-based systemic-bias towards type-1 immune environment. Further, NLGP gets engulfed within dendritic cells, possibly through endocytic activities of Dectin-1.

CONCLUSION: NLGP{\textquoteright}s binding to Dectin-1 receptors on murine dendritic cells, followed by the intracellular signal, lead to NFκB-mediated contrasting regulation of cytokine-transcriptions, initiating a pro-inflammatory immunopolarization, which amplifies further by the responding immune cells including T cells, alongside their enhanced cytotoxicity. These insights into the initiation of mammalian systemic immunomodulation by NLGP at cellular and molecular levels, may help uncovering its mode of action as a novel immunomodulator against human cancers, following clinical trials.

}, keywords = {Animals, Azadirachta, CARD Signaling Adaptor Proteins, Dendritic Cells, Lectins, C-Type, Mice, Mice, Inbred C57BL, NF-kappa B, Plant Leaves, Protein Binding, Signal Transduction}, issn = {1478-811X}, doi = {10.1186/s12964-024-01576-z}, author = {Ganguly, Nilanjan and Das, Tapasi and Bhuniya, Avishek and Guha, Ipsita and Chakravarti, Mohona and Dhar, Sukanya and Sarkar, Anirban and Bera, Saurav and Dhar, Jesmita and Dasgupta, Shayani and Saha, Akata and Ghosh, Tithi and Das, Juhina and Sk, Ugir Hossain and Banerjee, Saptak and Laskar, Subrata and Bose, Anamika and Baral, Rathindranath} } @article {8739, title = {Proteomic and metabolomic insights into seed germination of Ferula assa-foetida [Mass Spectrometry - Proteomics Facility]}, journal = {J Proteomics}, volume = {300}, year = {2024}, month = {2024 May 30}, pages = {105176}, abstract = {

Cold stratification is known to affect the speed of seed germination; however, its regulation at the molecular level in Ferula assa-foetida remains ambiguous. Here, we used cold stratification (4\ {\textdegree}C in the dark) to induce germination in F. assa-foetida and adopted a proteomic and metabolomic approach to understand the molecular mechanism of germination. Compared to the control, we identified 209 non-redundant proteins and 96 metabolites in germinated F. assa-foetida seed. Results highlight the common and unique regulatory mechanisms like signaling cascade, reactivation of energy metabolism, activation of ROS scavenging system, DNA repair, gene expression cascade, cytoskeleton, and cell wall modulation in F. assa-foetida germination. A protein-protein interaction network identifies 18 hub protein species central to the interactome and could be a key player in F. assa-foetida germination. Further, the predominant metabolic pathways like glucosinolate biosynthesis, arginine and proline metabolism, cysteine and methionine metabolism, aminoacyl-tRNA biosynthesis, and carotenoid biosynthesis in germinating seed may indicate the regulation of carbon and nitrogen metabolism is prime essential to maintain the physiology of germinating seedlings. The findings of this study provide a better understanding of cold stratification-induced seed germination, which might be utilized for genetic modification and traditional breeding of Ferula assa-foetida. SIGNIFICANCE: Seed germination is the fundamental checkpoint for plant growth and development, which has ecological significance. Ferula assa-foetida L., commonly known as "asafoetida," is a medicinal and food crop with huge therapeutic potential. To date, our understanding of F. assa-foetida seed germination is rudimentary. Therefore, studying the molecular mechanism that governs dormancy decay and the onset of germination in F. assa-foetida is essential for understanding the basic principle of seed germination, which could offer to improve genetic modification and traditional breeding.

}, keywords = {Ferula, Gene Expression Regulation, Plant, Germination, Metabolomics, Plant Proteins, Protein Interaction Maps, Proteome, Proteomics, Seeds}, issn = {1876-7737}, doi = {10.1016/j.jprot.2024.105176}, author = {Punia, Ashwani and Kumari, Manglesh and Chouhan, Monika and Saini, Vishal and Joshi, Robin and Kumar, Ashok and Kumar, Rajiv} } @article {8882, title = {Role of Mating Receptivity Peptides in Helicoverpa armigera: Molecular and Evolutionary Perspectives [Mass Spectrometry - Proteomics Facility]}, journal = {Journal of Advances in Biology \& Biotechnology}, volume = {27 }, year = {2024}, month = {08/2024}, chapter = {1427}, abstract = {

Previous studies have implicated the role of peptides/proteins in male accessory glands (MAGs), responsible for the induction of behavioral and reproductive changes in\ Helicoverpa armigera\ females. Our and several other studies have seen the MAG showed post-mating behaviors such as accelerated unreceptivity to re-mating and other post-mating traits. We have reported several peptides responsible for reduced mating receptivity, including a putative 4.9 kDa, a novel peptide with various functions in female post-copulatory reproductive behavior (PMR). In this study, we report a detailed analysis of the proteins from a biochemical, physiological, molecular and evolutionary perspective. 5 major classes of multigene family proteins were detected after protein database searches. All proteins were found to bind nucleic acids, possess signal peptides, and undergo posttranslational modification by seminal fluid proteins (SFPs). The Porphobilinogen synthetase protein (PBGS) was unique to the lepidopteran order. Our molecular studies support the observation that the molecular weight of MAG proteins varies significantly across species, suggesting the existence of different target sites or faster evolution of SFP. MAG proteins not only ensure male reproductive fitness by constantly evolving under postcopulatory sexual selection (PCSS), but also maintain species-specific barriers to fertilization thus potentially contribute to the formation of new species and mediate reproductive conflicts between the sexes.

}, doi = {doi.org/10.9734/jabb/2024/v27i81266}, url = {https://journaljabb.com/index.php/JABB/article/view/1266$\#$:~:text=MAG\%20proteins\%20not\%20only\%20ensure,reproductive\%20conflicts\%20between\%20the\%20sexes.}, author = {Rama Thyloor and Kiran Kumar Halagur Bhogegowda and Saraswathi Saraswathi and Doddamane Manjulakumari} } @article {8098, title = {Analysis of smart biomaterial containing umbilical cord blood serum protein conjugated with P-(NIPAAM) using spectroscopy [Bio-incubation Services]}, journal = {Materials Today: Proceedings}, year = {2023}, abstract = {

Human Umbilical Cord Blood Serum (HUCBS) is a complex and evolving collection of proteins that promote fetal development. In the realm of regenerative medicine, the important proteins found in HUCBS are of great interest. The smart biomaterial generated from HUCBS is described in this paper. To characterize this novel biomaterial, human umbilical cord blood was obtained in sterile vacutainers from mothers and left to clot for 24\ h at 37 {\textdegree}C. The supernatant serum was collected, centrifuged and lyophilized. The lyophilized HUCBS was homogenized with smart polymer. This sample was subjected to physico-chemical characterization using Attenuated Total Reflectance-Fourier-Transform Infrared (ATR-FTIR) Spectroscopy and Nuclear Magnetic Resonance (NMR). The quantification of protein-polymer conjugate using ATR-FTIR revealed peaks ranging between 3264 and 531\ cm-1 and that of NMR showed wide resonances in the region 0{\textendash}5\ ppm. ATR-FTIR and NMR investigations were used to determine the structural stability of protein molecules in protein-polymer complex which helps in understanding the possible clinical effectiveness of the smart biomaterial in drug delivery.

}, keywords = {ATR-FTIR, H NMR, HUCBS, P-NIPAAM, Protein-polymer conjugate}, issn = {2214-7853}, doi = {https://doi.org/10.1016/j.matpr.2023.01.285}, url = {https://www.sciencedirect.com/science/article/pii/S2214785323003759}, author = {Manasa Biligowda Latha and Ashmitha Kishan Shetty and Rajamanickam Deveswaran and Ashish Jagannath Rai and Serene Joy and Hadonahalli Munegowda Shashanka and Siddique Sha Muhammad Hussain and Suraksha Shetty} } @article {8553, title = {Characterizing nucleotide binding site domain (NBD) of ZzR1 resistance gene from Zingiber zerumbet: in silico ligand docking and optimizing heterologous expression [Bio-incubation Services]}, journal = {Archives of Phytopathology and Plant Protection }, year = {2023}, month = {08/2023}, abstract = {

The Nucleotide-binding site domain (NBD) of plant resistance (R) genes plays a vital role during plant defense signaling. The functional significance of CC-NBS-LRR (Coiled coil-NBS-Leucine Rich Repeat) class of R gene designated\ ZzR1, characterized from\ Zingiber zerumbet\ in earlier studies, was determined by molecular modeling and docking studies. Docked complex showed the ligand GTP interacts with amino acid residues in the cleft made by GLPL and P-loop motifs of the NBD. Heterologous expression of ZzR1 NBS protein was optimized using expression vectors, pEcoli-Nterm-6xHN and pET Directional TOPO and transformed in five\ Escherichia coli\ strains namely DH5α, TOP10, BL21DE3, BL21DE3 star and BL21plysS cells. The NBS protein of 36 kDa molecular size was expressed in\ E. coli\ BL21DE3 strain using pET TOPO vector. Optimum induction was detected at 30 {\textdegree}C using isopropyl-1-thio-β-D-galactopyranoside (IPTG) (1 mM). The present study provides valuable information on ligand interactions and heterologous expression of ZzR1 NBD protein.

}, doi = {https://doi.org/10.1080/03235408.2023.2251789}, author = {Aswati Ravindranathan Nair and Sumna Sasidharan} } @article {8562, title = {Comparative stability study and aggregate analysis of Bevacizumab marketed formulations using advanced analytical techniques [Biologics / Biopharmaceutical Characterization Facility]}, journal = {Heliyon}, volume = {9}, year = {2023}, pages = {e19478}, abstract = {

Bevacizumab (Bvz) is the most preferred recombinant humanized monoclonal antibody in biosimilar development due to its prominence as a standard treatment in the oncology space. Therapeutic monoclonal antibodies are typically more complex and unlikely to produce a replica. As a result, regulatory agencies allow approval of biosimilars that differ structurally and functionally from their reference product, but these differences should not have any clinical significance. To identify these significant discrepancies, it is essential to perform a thorough characterization of critical product attributes both in real-time and after storage until the product{\textquoteright}s expiration. In the present study, two Bvz biosimilar brands (Bio-1 and Bio-2) marketed in India were evaluated and compared with the reference product Avastin{\textregistered} to assess their degree of similarity. A comprehensive physicochemical characterization of biosimilars and reference product was performed using orthogonal techniques including LC-ESI-QTOF, MALDI-TOF, FTIR-ATR, iCIEF, rCE, nrCE, UV280, and RP-HPLC. Furthermore, Bvz formulations under study were subjected to various stress conditions of thermal (elevated temperature 50\ {\textpm}\ 2\ {\textdegree}C), chemical (acidic pH 3.0\ {\textpm}\ 0.2, neutral pH 7.0\ {\textpm}\ 0.2, and basic pH 10.0\ {\textpm}\ 0.2), and mechanical (agitation 200\ rpm) for comparative stability evaluation. Any alteration in the secondary structure of the native protein was detected and quantified using far-UV circular dichroism (CD), indicating an average of 15\% and 11\% loss in native antiparallel β-sheet conformation respectively in Bio-1 and Bio-2 upon exposure to elevated temperature and high pH. Additionally, covalent or non-covalent aggregates formed as a function of elevated temperature and agitation were quantified using SEC-MALS.

}, keywords = {Aggregate analysis, Bevacizumab, Biosimilars, Intact mass analysis, stress study}, issn = {2405-8440}, doi = {https://doi.org/10.1016/j.heliyon.2023.e19478}, url = {https://www.sciencedirect.com/science/article/pii/S2405844023066860}, author = {Arpit Arunkumar Bana and Nithin Sajeev and Sabyasachi Halder and Haidar Abbas Masi and Shikha Patel and Priti Mehta} } @article {8462, title = {A curated list of targeted optimized promiscuous ketoreductases (TOP-K). [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Biochem J}, volume = {480}, year = {2023}, month = {2023 Jul 12}, pages = {975-997}, abstract = {

Enzymes are either specific or promiscuous catalysts in nature. The latter is portrayed by protein families like CYP450Es, Aldo-ketoreductases and short/medium-chain dehydrogenases which participate in detoxification or secondary metabolite production. However, enzymes are evolutionarily {\textquoteright}blind{\textquoteright} to an ever-increasing synthetic substrate library. Industries and laboratories have circumvented this by high-throughput screening or site-specific engineering to synthesize the product of interest. However, this paradigm entails cost and time-intensive one-enzyme, one-substrate catalysis model. One of the superfamilies regularly used for chiral alcohol synthesis are short-chain dehydrogenases/reductases (SDRs). Our objective is to determine a superset of promiscuous SDRs that can catalyze multiple ketones. They are typically classified into shorter {\textquoteright}Classical{\textquoteright} and longer {\textquoteright}Extended{\textquoteright} type ketoreductases. However, current analysis of modelled SDRs reveals a length-independent conserved N-terminus Rossmann-fold and a variable substrate-binding C-terminus substrate-binding region for both categories. The latter is recognized to influence the enzyme{\textquoteright}s flexibility and substrate promiscuity and we hypothesize these properties are directly linked with each other. We tested this by catalyzing ketone intermediates with the essential and specific enzyme: FabG_E, as well as non-essential SDRs such as UcpA and IdnO. The experimental results confirmed this biochemical-biophysical association, making it an interesting filter for ascertaining promiscuous enzymes. Hence, we created a dataset of physicochemical properties derived from the protein sequences and employed machine learning algorithms to examine potential candidates. This resulted in 24 targeted optimized ketoreductases (TOP-K) from 81 014 members. The experimental validation of select TOP-Ks demonstrated the correlation between the C-terminal lid-loop structure, enzyme flexibility and turnover rate on pro-pharmaceutical substrates.

}, keywords = {ketoreductases, machine learning, medium-chain dehydrogenases, short-chain dehydrogenases}, issn = {1470-8728}, doi = {10.1042/BCJ20230051}, url = {https://pubmed.ncbi.nlm.nih.gov/37335080/}, author = {Shanbhag, Anirudh P and Rajagopal, Sreenath and Ghatak, Arindam and Katagihallimath, Nainesh and Subramanian, Ramaswamy and Datta, Santanu} } @article {8508, title = {Defining a research agenda for environmental wastewater surveillance of pathogens.}, journal = {Nat Med}, year = {2023}, month = {2023 Aug 03}, issn = {1546-170X}, doi = {10.1038/s41591-023-02457-7}, author = {Shaw, Alexander G and Troman, Catherine and Akello, Joyce Odeke and O{\textquoteright}Reilly, Kathleen M and Gauld, Jillian and Grow, Stephanie and Grassly, Nicholas and Steele, Duncan and Blazes, David and Kumar, Supriya} } @article {8544, title = {Distinct evolution of type I glutamine synthetase in Plasmodium and its species-specific requirement [Mass Spectrometry Facility - Metabolomics]}, journal = {Nat Commun}, volume = {14}, year = {2023}, month = {2023 Jul 14}, pages = {4216}, abstract = {

Malaria parasite lacks canonical pathways for amino acid biosynthesis and depends primarily on hemoglobin degradation and extracellular resources for amino acids. Interestingly, a putative gene for glutamine synthetase (GS) is retained despite glutamine being an abundant amino acid in human and mosquito hosts. Here we show Plasmodium GS has evolved as a unique type I enzyme with distinct structural and regulatory properties to adapt to the asexual niche. Methionine sulfoximine (MSO) and phosphinothricin (PPT) inhibit parasite GS activity. GS is localized to the parasite cytosol and abundantly expressed in all the life cycle stages. Parasite GS displays species-specific requirement in Plasmodium falciparum (Pf) having asparagine-rich proteome. Targeting PfGS affects asparagine levels and inhibits protein synthesis through eIF2α phosphorylation leading to parasite death. Exposure of artemisinin-resistant Pf parasites to MSO and PPT inhibits the emergence of viable parasites upon artemisinin treatment.

}, keywords = {Amino Acids, Animals, Artemisinins, Asparagine, Glutamate-Ammonia Ligase, Glutamine, Humans, Parasites, Plasmodium falciparum}, issn = {2041-1723}, doi = {10.1038/s41467-023-39670-4}, author = {Ghosh, Sourav and Kundu, Rajib and Chandana, Manjunatha and Das, Rahul and Anand, Aditya and Beura, Subhashree and Bobde, Ruchir Chandrakant and Jain, Vishal and Prabhu, Sowmya Ramakant and Behera, Prativa Kumari and Mohanty, Akshaya Kumar and Chakrapani, Mahabala and Satyamoorthy, Kapaettu and Suryawanshi, Amol Ratnakar and Dixit, Anshuman and Padmanaban, Govindarajan and Nagaraj, Viswanathan Arun} } @article {8318, title = {High-quality single amplicon sequencing method for illumina MiSeq platform using pool of {\textquoteright}N{\textquoteright} (0-10) spacer-linked target specific primers without PhiX spike-in [Next Gen Genomics Facility (INT)]}, journal = {BMC Genomics}, volume = {24}, year = {2023}, month = {2023 Mar 23}, pages = {141}, abstract = {

BACKGROUND: Illumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms, we developed a high throughput single amplicon sequencing method by introducing {\textquoteright}N{\textquoteright} (0-10) spacers in target gene amplification primers that are pooled for simple handling.

RESULT: We evaluated the efficiency of {\textquoteright}N{\textquoteright} (0-10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of {\textquoteright}N{\textquoteright} (0-10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software,"MetReTrim", to trim the {\textquoteright}N{\textquoteright} (0-10) spacers from the raw reads ( https://github.com/Mohak91/MetReTrim ). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS{\texttrademark} microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We observed no difference between the communities represented by our method and standard illumina V3-V4 primer method.

CONCLUSION: This method eliminates the need for PhiX spike-in for single amplicon sequencing on illumina MiSeq platform. This allows for sequencing of more number of samples in a run and a reduction in the overall cost. Given that Illumina sequencing works on SBS chemistry irrespective of the platform (such as HiSeq, MiSeq, NextSeq, NovaSeq, etc.) we propose that this strategy of using {\textquoteright}N{\textquoteright} (0-10) spacer-linked primer design can be adopted for generating high-quality single locus amplicon sequencing in a high throughput manner across the illumina platform subject to further validation.

}, keywords = {Bacteria, Gene Library, High-Throughput Nucleotide Sequencing, Microbiota, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {1471-2164}, doi = {10.1186/s12864-023-09233-4}, author = {Naik, Tejali and Sharda, Mohak and C P, Lakshminarayanan and Virbhadra, Kumar and Pandit, Awadhesh} } @article {8472, title = {Marinobacterium lacunae sp. nov. isolated from estuarine sediment [Next Gen Genomics Facility]}, journal = {Arch Microbiol}, volume = {205}, year = {2023}, month = {2023 Jul 22}, pages = {294}, abstract = {

A novel motile bacterium was isolated from a sediment sample collected in Kochi backwaters, Kerala, India. This bacterium is Gram negative, rod shaped, 1.0-1.5\ {\textmu}m wide, and 2.0-3.0\ {\textmu}m long. It was designated as strain AK27. Colonies were grown on marine agar displayed circular, off-white, shiny, moist, translucent, flat, margin entire, 1-2\ mm in diameter. The major fatty acids identified in this strain were C ω7c, C, and summed in feature 3. The composition of polar lipids in the strain AK27 included phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unidentified amino lipid, two unidentified aminophospholipids, two unidentified phospholipids, and six unidentified lipids. The genomic DNA of strain AK27 exhibited a G+C content of 56.4\ mol\%. Based on the analysis of 16S rRNA gene sequence, strain AK27 showed sequence similarity to M. ramblicola D7 and M. zhoushanense WM3 as 98.99\% and 98.58\%, respectively. Compared to other type strains of the Marinobacterium genus, strain AK27 exhibited sequence similarities ranging from 91.7\% to 96.4\%. When compared to Marinobacterium zhoushanense WM3 and Marinobacterium ramblicola D7, strain AK27 exhibited average nucleotide identity values of 80.25\% and 79.97\%, and dDDH values of 22.9\% and 22.6\%, respectively. The genome size of the strain AK27 was 4.55\ Mb, with 4,229 coding sequences. Based on the observed phenotypic and chemotaxonomic features, and the results of phylogenetic and phylogenomic analysis, this study proposes the classification of strain AK27 as a novel species within the genus Marinobacterium. The proposed name for this novel species is Marinobacterium lacunae sp. nov.

}, keywords = {Agar, Alteromonadaceae, Cardiolipins, Phylogeny, RNA, Ribosomal, 16S}, issn = {1432-072X}, doi = {10.1007/s00203-023-03627-4}, author = {Tanuku, Srinivas Naga Radha and Pinnaka, Anil Kumar and Behera, Swarnaprava and Singh, Aditya and Pydi, Sudharani and Vasudeva, Gunjan and Vaidya, Bhumika and Sharma, Gunjan and Ganta, Sampath Kumar and Garbhapu, Naveen Sagar} } @article {8556, title = {Purification and characterization of an asialofetuin specific lectin from the rhizome of Xanthosoma violaceum Schott [Mass Spectrometry - Proteomics Facility]}, journal = {Protein Expr Purif}, year = {2023}, month = {2023 Aug 29}, pages = {106357}, abstract = {

Lectins are proteins or glycoproteins that bind specifically and reversibly to the carbohydrate or glycoconjugates. A new lectin is purified from the rhizome of Xanthosoma violaceum Schott. by successive steps of ammonium sulfate fractionation and affinity chromatography with asialofetuin as ligand. The purified lectin was found to be a homotetramer of approximately 49 kDa with a subunit molecular weight of 12 kDa linked by non-covalent bonds. Characterization of the lectin shows that the hemagglutination activity is inhibited by asialofetuin and d-galacturonic acid. Hemagglutination activity is shown only in rabbit RBC but not in the human RBC of all blood groups. It is a metal ion-independent glycoprotein of 1.87\% carbohydrate content, stable upto 40 {\textdegree}C and pH from 5.5 to 9. The lectin shows its optimum hemagglutination activity at 0 {\textdegree}C-40 {\textdegree}C and pH 6 to 8.5. From LC-MS/MS analysis it is confirmed that the purified lectin was not purified and characterized earlier.

}, issn = {1096-0279}, doi = {10.1016/j.pep.2023.106357}, author = {Devi, Oinam Sangita and Singh, Senjam Sunil and Rana, K and Singh, Sorokhaibam Jibankumar and Singh, Wayenbam Sobhachandra} } @article {8319, title = {Significance of Plasmodium berghei Amino Acid Transporter 1 in Food Vacuole Functionality and Its Association with Cerebral Pathogenesis [Mass Spectrometry Facility - Metabolomics]}, journal = {Microbiol Spectr}, year = {2023}, month = {2023 Mar 28}, pages = {e0494322}, abstract = {

The food vacuole plays a central role in the blood stage of parasite development by digesting host hemoglobin acquired from red blood cells and detoxifying the host heme released during hemoglobin digestion into hemozoin. Blood-stage parasites undergo periodic schizont bursts, releasing food vacuoles containing hemozoin. Clinical studies in malaria-infected patients and animal studies have shown the association of hemozoin with disease pathogenesis and abnormal host immune responses in malaria. Here, we perform a detailed characterization of putative Plasmodium berghei amino acid transporter 1 localized in the food vacuole to understand its significance in the malaria parasite. We show that the targeted deletion of amino acid transporter 1 in Plasmodium berghei leads to a swollen food vacuole phenotype with the accumulation of host hemoglobin-derived peptides. Plasmodium berghei amino acid transporter 1-knockout parasites produce less hemozoin, and the hemozoin crystals display a thin morphology compared with wild-type parasites. The knockout parasites show reduced sensitivity to chloroquine and amodiaquine by showing recrudescence. More importantly, mice infected with the knockout parasites are protected from cerebral malaria and display reduced neuronal inflammation and cerebral complications. Genetic complementation of the knockout parasites restores the food vacuole morphology with hemozoin levels similar to that of wild-type parasites, causing cerebral malaria in the infected mice. The knockout parasites also show a significant delay in male gametocyte exflagellation. Our findings highlight the significance of amino acid transporter 1 in food vacuole functionality and its association with malaria pathogenesis and gametocyte development. Food vacuoles of the malaria parasite are involved in the degradation of red blood cell hemoglobin. The amino acids derived from hemoglobin degradation support parasite growth, and the heme released is detoxified into hemozoin. Antimalarials such as quinolines target hemozoin formation in the food vacuole. Food vacuole transporters transport hemoglobin-derived amino acids and peptides from the food vacuole to the parasite cytosol. Such transporters are also associated with drug resistance. Here, we show that the deletion of amino acid transporter 1 in Plasmodium berghei leads to swollen food vacuoles with the accumulation of hemoglobin-derived peptides. The transporter-deleted parasites generate less hemozoin with thin crystal morphology and show reduced sensitivity to quinolines. Mice infected with transporter-deleted parasites are protected from cerebral malaria. There is also a delay in male gametocyte exflagellation, affecting transmission. Our findings uncover the functional significance of amino acid transporter 1 in the life cycle of the malaria parasite.

}, issn = {2165-0497}, doi = {10.1128/spectrum.04943-22}, author = {Anand, Aditya and Chandana, Manjunatha and Ghosh, Sourav and Das, Rahul and Singh, Nalini and Vaishalli, Pradeep Mini and Gantasala, Nagavara Prasad and Padmanaban, Govindarajan and Nagaraj, Viswanathan Arun} } @article {8587, title = {Systematic identification of CAZymes and transcription factors in the hypercellulolytic fungus Penicillium funiculosum NCIM1228 involved in lignocellulosic biomass degradation [Next Gen Genomics Facility]}, journal = {Biotechnol Biofuels Bioprod}, volume = {16}, year = {2023}, month = {2023 Oct 04}, pages = {150}, abstract = {

BACKGROUND: Penicillium funiculosum NCIM1228 is a filamentous fungus that was identified in our laboratory to have high cellulolytic activity. Analysis of its secretome suggested that it responds to different carbon substrates by secreting\ specific enzymes capable of digesting those substrates. This phenomenon indicated the presence of a regulatory system guiding the expression of these hydrolyzing enzymes. Since transcription factors (TFs) are the key players in regulating the expression of enzymes, this study aimed first to identify the complete repertoire of Carbohydrate Active Enzymes (CAZymes) and TFs coded in its genome. The regulation of CAZymes was then analysed by studying the expression pattern of these CAZymes and TFs in different carbon substrates-Avicel (cellulosic substrate), wheat bran (WB; hemicellulosic substrate), Avicel + wheat bran, pre-treated wheat straw (a potential substrate for lignocellulosic ethanol), and glucose (control).

RESULTS: The P. funiculosum NCIM1228 genome was sequenced, and 10,739 genes were identified in its genome. These genes included a total of 298 CAZymes and 451 TF coding genes. A distinct expression pattern of the CAZymes was observed in different carbon substrates tested. Core cellulose hydrolyzing enzymes were highly expressed in the presence of Avicel, while pre-treated wheat straw and Avicel + wheat bran induced a mixture of CAZymes because of their heterogeneous nature. Wheat bran mainly induced hemicellulases, and the least number of CAZymes were expressed in glucose. TFs also exhibited distinct expression patterns in each of the carbon substrates. Though most of these TFs have not been functionally characterized before, homologs of NosA, Fcr1, and ATF21, which have been known to be involved in fruiting body development, protein secretion and\ stress response, were identified.

CONCLUSIONS: Overall, the P. funiculosum NCIM1228 genome was sequenced, and the CAZymes and TFs present in its genome were annotated. The expression of the CAZymes and TFs in response to various polymeric sugars present in the lignocellulosic biomass was identified. This work thus provides a comprehensive mapping of transcription factors (TFs) involved in regulating the production of biomass hydrolyzing enzymes.

}, issn = {2731-3654}, doi = {10.1186/s13068-023-02399-9}, author = {Pasari, Nandita and Gupta, Mayank and Sinha, Tulika and Ogunmolu, Funso Emmanuel and Yazdani, Syed Shams} } @article {8359, title = {Understanding the mode of action of AgroGain{\textregistered}, a biostimulant derived from the red seaweed Kappaphycus alvarezii in the stimulation of cotyledon expansion and growth of Cucumis sativa (cucumber) [Sea6Energy Pvt. Ltd, a C-CAMP Start-up]}, journal = {Front. Plant Sci.}, volume = {14}, year = {2023}, month = {06/2023}, abstract = {

Seaweed-based biostimulants are sustainable agriculture inputs that are known to have a multitude of beneficial effects on plant growth and productivity. This study demonstrates that Agrogain{\textregistered}\ (Product code: LBS6), a\ Kappaphycus alvarezii-derived biostimulant induced the expansion of cucumber cotyledons. Seven days treatment of LBS6-supplementation showed a 29.2\% increase in area of expanded cotyledons, as compared to the control. LBS6-treated cotyledons also showed higher amylase activity, suggesting starch to sucrose conversion was used efficiently as an energy source during expansion. To understand the mechanisms of LBS6-induced expansion, real time gene expression analysis was carried out. This revealed that LBS6-treated cotyledons differentially modulated the expression of genes involved in cell division, cell number, cell expansion and cell size. LBS6 treatment also differentially regulated the expression of those genes involved in auxin and cytokinin metabolism. Further, foliar application of LBS6 on cucumber plants being grown under hydroponic conditions showed improved plant growth as compared to the control. The total leaf area of LBS6-sprayed plants increased by 19.1\%, as compared to control. LBS6-sprayed plants efficiently regulated photosynthetic quenching by reducing loss\ via\ non-photochemical and non-regulatory quenching. LBS6 applications also modulated changes in the steady-state photosynthetic parameters of the cucumber leaves. It was demonstrated that LBS6 treatment modulated the electron and proton transport related pathways which help plants to efficiently utilize the photosynthetic radiation for optimal growth. These results provide clear evidence that bioactive compounds present in LBS6 improved the growth of cucumber plants by regulating the physiological as well as developmental pathways.

}, doi = {https://doi.org/10.3389/fpls.2023.1136563}, author = {Pushp Sheel Shukla and Nagarajan Nivetha and Sri Sailaja Nori and Debayan Bose and Sawan Kumar and Sachin Khandelwal and Alan Critchley and Shrikumar Suryanarayan} } @article {2916, title = {Characterization of ACE inhibitory and antioxidant peptides in yak and cow milk hard chhurpi cheese of the Sikkim Himalayan region [Mass Spectrometry Proteomics Facility]}, journal = {Food Chemistry: X}, volume = {13}, year = {2022}, month = {03/2022}, pages = {100231}, type = {Journal Article}, abstract = {

In this study, simulated in vitro GI digestion of the Himalayan hard chhurpi cheese resulted in the increase of hydrolyzed protein content, antioxidant and ACE-inhibitory activities. LC-MS/MS-based peptidomics revealed a total of 1473 peptides in the samples originating from different milk proteins, including α-S1-casein, α-S2-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin, out of which 60 peptides have been reported for different functional properties. A total of 101 peptides were predicted to be antihypertensive using the bioactivity prediction web servers, AHTpin and mAHTPred. In silico molecular docking studies predicted 20 antihypertensive peptides, exhibiting non-bond interactions between hard chhurpi peptides and ACE catalytic residues. A peptide, SLVYPFPGPI, identified in GI digested cow hard chhurpi and undigested, and GI digested samples of yak hard chhurpi, showed a stronger binding affinity towards ACE. Identifying antioxidant and ACE inhibitory peptides in hard cheese products adds value to them as functional foods of the Himalayan region.

}, keywords = {Antihypertensive, Bioactive peptides, Hard chhurpi, Molecular docking, Proteomics, Sikkim Himalaya}, doi = {https://doi.org/10.1016/j.fochx.2022.100231}, url = {https://www.sciencedirect.com/science/article/pii/S2590157522000293}, author = {Abedin, Md Minhajul and Chourasia, Rounak and Phukon, Loreni Chiring and Singh, Sudhir P and Rai, Amit Kumar} } @article {3706, title = {Effectiveness of a novel, non-intrusive, continuous-use air decontamination technology to reduce microbial contamination in clinical settings: a multi-centric study [Biomoneta Research Pvt. Ltd., a C-CAMP Startup]}, journal = {J Hosp Infect}, volume = {123}, year = {2022}, month = {2022 Feb 16}, pages = {15-22}, abstract = {

BACKGROUND: Despite rigorous disinfection and fumigation, healthcare-associated infection (HAI) remains a significant concern in healthcare settings. We have developed a novel airborne-microbicidal technology {\textquoteright}ZeBox{\textquoteright} which clears \>99.999\% of airborne microbial load under controlled laboratory conditions.

AIM: To evaluate the clinical performance of ZeBox in reducing airborne and surface microbial load.

METHODS: The study was conducted in single-bed and multi-bed intensive care units (ICUs) of two hospitals. Airborne and surface microbial loads were sampled pre and post deployment of ZeBox at pre-determined sites. Statistical significance of the reduction was determined using the Mann-Whitney U-test.

FINDINGS: ZeBox brought statistically significant reduction of both airborne and surface bacterial and fungal load. In both hospital ICUs, airborne and surface bacterial load decreased by 90\% and 75\% on average respectively, providing a low bioburden zone of 10-15 feet diameter around the unit. The reduced microbial level was maintained during ZeBox{\textquoteright}s operation over several weeks. Most clinical bacterial isolates recovered from one of the hospitals were antibiotic resistant, highlighting ZeBox{\textquoteright}s ability to eliminate antimicrobial-resistant bacteria among others.

CONCLUSION: ZeBox significantly reduces airborne and surface microbial burden in clinical settings. It thereby serves an unmet need for reducing the incidence of HAI.

}, issn = {1532-2939}, doi = {10.1016/j.jhin.2022.02.002}, author = {Nagaraj, S and Chandrasingh, S and Jose, S and Sofia, B and Sampath, S and Krishna, B and Menon, I and Kundu, D and Parekh, S and Madival, D and Nandi, V and Ghatak, A} } @article {3630, title = {Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences [C-CAMP Bioincubation Facility]}, journal = {Molecular Therapy - Cell Press}, year = {2022}, month = {2022 Jan 07}, type = {Journal Article}, abstract = {

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in\ India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T\ cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune\ response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.

}, keywords = {antibody-dependent enhancement, consensus sequence, dengue, dengue surveillance, DNA vaccine, EDIII domain, NS1 protein}, issn = {1525-0024}, doi = {10.1016/j.ymthe.2022.01.013}, url = {https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(22)00013-2$\#$secsectitle0165}, author = {Sankaradoss, Arun and Jagtap, Suraj and Nazir, Junaid and Moula, Shefta E and Modak, Ayan and Fialho, Joshuah and Iyer, Meenakshi and Shastri, Jayanthi S and Dias, Mary and Gadepalli, Ravisekhar and Aggarwal, Alisha and Vedpathak, Manoj and Agrawal, Sachee and Pandit, Awadhesh and Nisheetha, Amul and Kumar, Anuj and Bordoloi, Mahasweta and Shafi, Mohamed and Shelar, Bhagyashree and Balachandra, Swathi S and Damodar, Tina and Masika, Moses Muia and Mwaura, Patrick and Anzala, Omu and Muthumani, Kar and Sowdhamini, Ramanathan and Medigeshi, Guruprasad R and Roy, Rahul and Pattabiraman, Chitra and Krishna, Sudhir and Sreekumar, Easwaran} } @article {3480, title = {Production and characterization of bioactive peptides from rice beans using Bacillus subtilis [Mass Spectrometry - Proteomics Facility]}, journal = {Bioresource Technology}, year = {2022}, month = {01/2022}, pages = {126932}, type = {Journal Article}, abstract = {

A bioprocess was developed for production of bioactive peptides on microbial fermentation of rice beans using proteolytic Bacillus subtilis strains. The peptides produced were identified by LC-MS/MS analysis, revealing the presence of many unique peptide sequences to individual hydrolysates. On functional properties prediction, antihypertensive peptides (3.90\%) were found to be higher in comparison to other bioactive peptides. Among different strains, B. subtilis KN2B fermented hydrolysate exhibited highest angiotensin converting enzyme (ACE)-inhibitory activity (45.73\%). Furthermore, 19 selected peptides, including the common and unique peptides were examined for their affinity towards the binding cavity of ACE using molecular docking. The results showed a common peptide PFPIPFPIPIPLP, and another IPFPPIPFLPPI unique to B. subtilis KN2B fermented hydrolysate exhibited promising binding at the ACE binding site with substantial free binding energy. The process developed can be used for the production of bioactive peptides from rice bean for application in nutraceutical industries.

}, keywords = {ACE-inhibitory peptide, Bacillus subtilis, Fermented legume, Molecular docking, Rice bean}, doi = {https://doi.org/10.1016/j.biortech.2022.126932}, url = {https://www.sciencedirect.com/science/article/abs/pii/S0960852422002619}, author = {Padhi, Srichandan and Chourasia, Rounak and Kumari, Megha and Singh, Sudhir P and Rai, Amit Kumar} } @article {3918, title = {Profilin is involved in G1 to S phase progression and mitotic spindle orientation during Leishmania donovani cell division cycle [Mass Spectrometry - Proteomics Facility]}, journal = {PLoS One}, volume = {17}, year = {2022}, month = {2022}, pages = {e0265692}, abstract = {

Profilin is a multi-ligand binding protein, which is a key regulator of actin dynamics and involved in regulating several cellular functions. It is present in all eukaryotes, including trypanosomatids such as Leishmania. However, not much is known about its functions in these organisms. Our earlier studies have shown that Leishmania parasites express a single homologue of profilin (LdPfn) that binds actin, phosphoinositides and poly- L- proline motives, and depletion of its intracellular pool to 50\%of normal levels affects the cell growth and intracellular trafficking. Here, we show, employing affinity pull-down and mass spectroscopy, that LdPfn interacted with a large number of proteins, including those involved in mRNA processing and protein translation initiation, such as eIF4A1. Further, we reveal, using mRNA Seq analysis, that depletion of LdPfn in Leishmania cells (LdPfn+/-) resulted in significantly reduced expression of genes which encode proteins involved in cell cycle regulation, mRNA translation initiation, nucleosides and amino acids transport. In addition, we show that in LdPfn+/- cells, cellular levels of eIF4A1 protein were significantly decreased, and during their cell division cycle, G1-to-S phase progression was delayed and orientation of mitotic spindle altered. These changes were, however, reversed to normal by episomal expression of GFP-LdPfn in LdPfn+/- cells. Taken together, our results indicate that profilin is involved in regulation of G1-to-S phase progression and mitotic spindle orientation in Leishmania cell cycle, perhaps through its interaction with elF4A1 protein.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0265692}, author = {Ambaru, Bindu and Gangadharan, Ganesh Muthu and Subramanya, Hosahalli S and Gupta, Chhitar M} } @article {4451, title = {SARS-CoV-2 infection of human-induced pluripotent stem cells-derived lung lineage cells evokes inflammatory and chemosensory responses by targeting mitochondrial pathways [Eyestem Research Pvt. Ltd., a C-CAMP Startup]}, journal = {J Cell Physiol}, year = {2022}, month = {2022 Apr 23}, abstract = {

The COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2\ (SARS-CoV-2) primarily affects the lung, particularly the proximal airway and distal alveolar cells. NKX2.1+ primordial lung progenitors of the foregut (anterior) endoderm are the developmental precursors to all adult lung epithelial lineages and are postulated to play an important role in viral tropism. Here, we show that SARS-CoV-2 readily infected and replicated in human-induced pluripotent stem cell-derived proximal airway cells, distal alveolar cells, and lung progenitors. In addition to the upregulation of antiviral defense and immune responses, transcriptomics data uncovered a robust epithelial cell-specific response, including perturbation of metabolic processes and disruption in the alveolar maturation program. We also identified spatiotemporal dysregulation of mitochondrial heme oxygenase 1 (HMOX1), which is associated with defense against antioxidant-induced lung injury. Cytokines, such as TNF-α, INF-γ, IL-6, and IL-13, were upregulated in infected cells sparking mitochondrial ROS production and change in electron transport chain complexes. Increased mitochondrial ROS then activated additional proinflammatory cytokines leading to an aberrant cell cycle resulting in apoptosis. Notably, we are the first to report a chemosensory response resulting from SARS-CoV-2 infection similar to that seen in COVID-19 patients. Some of our key findings were validated using COVID-19-affected postmortem lung tissue sections. These results suggest that our in vitro system could serve as a suitable model to investigate the pathogenetic mechanisms of SARS-CoV-2 infection and to discover and test therapeutic drugs against COVID-19 or its consequences.

}, issn = {1097-4652}, doi = {10.1002/jcp.30755}, author = {Surendran, Harshini and Kumar, Saurabh and Narasimhaiah, Swathi and Ananthamurthy, Anuradha and Varghese, P S and D{\textquoteright}Souza, George A and Medigeshi, Guruprasad and Pal, Rajarshi} } @article {7317, title = {SPAD-1, a serine proteinase associated disintegrin from Russell{\textquoteright}s viper venom disrupts adhesion of MCF7 human breast cancer cells. [Mass Spectrometry - Proteomics]}, journal = {Toxicon}, volume = {221}, year = {2022}, month = {2022 Nov 21}, pages = {106979}, type = {Journal Article}, abstract = {

Serine Proteinase Associated Disintegrin-1 (SPAD-1) is a low molecular mass (26\ kDa) positively charged protein purified from Russell{\textquoteright}s viper venom (RVV) possessing cytotoxic activity on MCF7, human breast cancer cells. Primary sequence analysis of the protein confirms that it is a novel Snake Venom Serine Proteinase (SVSP) and a member of the trypsin family. SPAD-1 contains a conserved triad of Histidine (H), Aspartic acid(D) and Serine(S) residues at its active site for proteinase activity and also an adjacent histidine-glycine-aspartic acid (HGD) disintegrin-like motif. The serine proteinase and disintegrin parts are functionally active and independent. SPAD-1 showed proteolytic digestion of fibrinogen and fibronectin, but laminin digestion was below the detectable limit. Proteolytically inactivated SPAD-1 inhibited collagen and ADP-induced platelet aggregation. This study proposes considering Serine Proteinase Associated Disintegrin (SPAD) as a new group of snake venom proteins. Members of this group contain a serine proteinase catalytic triad and a disintegrin-like motif. SPAD-1 caused visible morphological changes in MCF7 cells, including a reduction of the cell-to-cell attachments, rounding of cell shape and death, in vitro. SPAD-1 also showed a dose-dependent significant decrease in the invasive potency of breast cancer cells. Confocal microscopic analysis revealed the breakage of nuclei of the SPAD-1-treated cells. SPAD-1 also increased cell detachment from the poly L-lysine-coated, laminin-coated and fibronectin-coated culture plate matrices, confirming the disintegrin activity. This study concludes that SPAD-1 may be a good candidate for anti-tumour drug design in the future.

}, keywords = {Cytotoxic, RGD-Like disintegrin motifs, Russell{\textquoteright}s viper Venom toxin, Snake venom serine proteinases}, issn = {1879-3150}, doi = {10.1016/j.toxicon.2022.106979}, url = {https://www.sciencedirect.com/science/article/abs/pii/S0041010122003300}, author = {Bhattacharya, Navodipa and Kolvekar, Nivedita and Mondal, Sukanta and Sarkar, Angshuman and Chakrabarty, Dibakar} } @article {7318, title = {Techno-commercial application of microencapsulated basil oil and thyme oil and their antibacterial activity against aquatic pathogens [EM Facility]}, journal = {International Journal of Fisheries and Aquatic Studies 2022}, volume = {10(5)}, year = {2022}, month = {23/06/2022}, pages = {101-106}, type = {Journal Article}, chapter = {101}, abstract = {

A complete study was conducted through manufacturing of Microencapsulation of Basil Oil Powder (MEBOP) and Microencapsulation of Thyme Oil Powder (METOP) to evaluate the anti-bacterial activity against aquatic pathogens. An oil-in-water emulsion were prepared using gum with dextrin and the resultant matrix was spray dried with the average yield of 45\% w/w of MEBOP and 50\% w/w of METOP. Total oil content in the encapsulated powder in both were found to be 24.39\% w/w of MEBOP 25.80\% w/w of METOP respectively. Scanning Electron Microscopic (SEM) analysis was also done to confirm the encapsulation of oil and study possible structure of matrix. Microencapsulated powders were further subjected for antibacterial activity against aquaculture pathogens like Vibrio harvey, Vibrio parahaemolyticus, Bacillus cereus, Aeromonas sp, E. coli and Salmonella sp. Results were compared against the respective oils at different concentrations. Phyto- constituents from Basil oil and Thyme oil like Thymol, Linalool, and Methyl chavicol were quantified using HPLC (High-Performance Liquid Chromatography) and GC (Gas Chromatography) which may be responsible for this activity. Furthermore, stress studies were conducted at 60 {\textdegree}C to understand the stability of Encapsulated oil powders to this and its commercial usage in formulations.

}, keywords = {anti-bacterial, aqua gut health, Microencapsulated oil powder, stress study}, issn = {E-ISSN: 2347-5129}, doi = {https://doi.org/10.22271/fish.2022.v10.i5b.2730}, url = {https://www.fisheriesjournal.com/archives/2022/vol10issue5/PartB/10-4-44-468.pdf}, author = {Sampath, M and Muguli, Ganesh and Pai, Sameer and Babu, UV} } @article {2733, title = {Transcriptome analysis at mid-stage seed development in litchi with contrasting seed size [Next Gen Genomics Facility]}, journal = {3 Biotech }, volume = {12}, year = {2022}, month = {01/2022}, abstract = {

Litchi is a sub-tropical fruit crop with genotypes that bear fruits with variable seed size. Small seed size is a desirable trait\ in litchi, as it improves consumers{\textquoteright} preference and facilitates fruit processing. Seed specific transcriptome analysis was performed in two litchi genotypes with contrasting seed size to identify the genes associated with seed development. The transcriptomic sequence data from seeds at mid-development stages (16{\textendash}28\ days after anthesis) were de-novo assembled into 1,39,608 Trinity transcripts. Out of these, 6325 transcripts expressed differentially between the two contrasting genotypes. Several putative genes for salicylic acid, jasmonic acid and brassinosteriod pathways were down-regulated in seeds of the small-seeded litchi. The putative regulators of seed maturation and seed storage were down-regulated in the small-seeded genotype. Embryogenesis, cell\ expansion, seed size and stress related Trinity transcripts exhibited differential expression. Further studies on gene characterization will reveal the early regulators of seed size in litchi.

}, keywords = {Embryogenesis, Litchi, Seed size, Small seed, Transcriptome}, doi = {doi.org/10.1007/s13205-021-03098-8}, url = {https://link.springer.com/article/10.1007/s13205-021-03098-8$\#$citeas}, author = {Ashish K. Pathak and Sudhir P. Singh and Ritika Sharma and Vishal Nath and Rakesh Tuli} } @article {7323, title = {Ultrashort Peptide-Based Hydrogel for the Healing of Critical Bone Defects in Rabbits [C-CAMP BIG Grantee/Startup]}, journal = {ACS Appl Mater Interfaces}, year = {2022}, month = {2022 Nov 19}, abstract = {

The use of hydrogels as scaffolds for three-dimensional (3D) cell growth is an active area of research in tissue engineering. Herein, we report the self-assembly of an ultrashort peptide, a tetrapeptide, Asp-Leu-IIe-IIe, the shortest peptide sequence from a highly fibrillogenic protein TDP-43, into the hydrogel. The hydrogel was mechanically strong and highly stable, with storage modulus values in MPa ranges. The hydrogel supported the proliferation and successful differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) in its matrix as assessed by cell viability, calcium deposition, alkaline phosphatase (ALP) activity, and the expression of osteogenic marker gene studies. To check whether the hydrogel supports 3D growth and regeneration in conditions, a rabbit critical bone defect model was used. Micro-computed tomography (CT) and X-ray analysis demonstrated the formation of mineralized neobone in the defect areas, with significantly higher bone mineralization and relative bone densities in animals treated with the peptide hydrogel compared to nontreated and matrigel treatment groups. The ultrashort peptide-based hydrogel developed in this work holds great potential for its further development as tissue regeneration and/or engineering scaffolds.

}, issn = {1944-8252}, doi = {10.1021/acsami.2c18733}, author = {Yadav, Nitin and Kumar, Utkarsh and Roopmani, Purandhi and Krishnan, Uma Maheswari and Sethuraman, Swaminathan and Chauhan, Meenakshi K and Chauhan, Virander S} } @article {2598, title = {Validated In Silico Model for Biofilm Formation in Escherichia coli [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {ACS Synthetic Biology}, year = {2022}, month = {01/2022}, abstract = {

Using\ Escherichia coli\ as the representative biofilm former, we report here the development of an in silico model built by simulating events that transform a free-living bacterial entity into self-encased multicellular biofilms. Published literature on \~{}300 genes associated with pathways involved in biofilm formation was curated, static maps were created, and suitably interconnected with their respective metabolites using ordinary differential equations. Precise interplay of genetic networks that regulate the transitory switching of bacterial growth pattern in response to environmental changes and the resultant multicomponent synthesis of the extracellular matrix were appropriately represented. Subsequently, the in silico model was analyzed by simulating time-dependent changes in the concentration of components by using the R and python environment. The model was validated by simulating and verifying the impact of key gene knockouts (KOs) and systematic knockdowns on biofilm formation, thus ensuring the outcomes were comparable with the reported literature. Similarly, specific gene KOs in laboratory and pathogenic\ E. coli\ were constructed and assessed. MiaA, YdeO, and YgiV were found to be crucial in biofilm development. Furthermore, qRT-PCR confirmed the elevation of expression in biofilm-forming clinical isolates. Findings reported in this study offer opportunities for identifying biofilm inhibitors with applications in multiple industries. The application of this model can be extended to the health care sector specifically to develop novel adjunct therapies that prevent biofilms in medical implants and reduce emergence of biofilm-associated resistant polymicrobial-chronic infections. The in silico framework reported here is open source and accessible for further enhancements.

}, doi = {10.1021/acssynbio.1c00445}, url = {https://doi.org/10.1021/acssynbio.1c00445}, author = {Bhowmik, Purnendu and Rajagopal, Sreenath and Hmar, Rothangamawi Victoria and Singh, Purnima and Saxena, Pragya and Amar, Prakruthi and Thomas, Teby and Ravishankar, Rajani and Nagaraj, Savitha and Katagihallimath, Nainesh and Sarangapani, Ramanujan Kadambi and Ramachandran, Vasanthi and Datta, Santanu} } @article {1863, title = {Antimicrobial and antibiofilm activity of GNP-Tannic Acid-Ag nanocomposite and their epoxy-based coatings [Log9 Materials, a C-CAMP startup]}, journal = {Progress in Organic Coatings}, volume = {159}, year = {2021}, month = {07/2021}, pages = {106421}, type = {Journal Article}, abstract = {

In order to deal with the challenge of biofilms and its associated infections, it is important to develop some efficient methodology which prevents the formation of biofilms over the surfaces. Herein, we have successfully developed an antibacterial and antibiofilm composite using non-toxic and hydrophobic nanomaterial, graphene nanoplatelets, via facile two step methodology including functionalization and subsequent hydrothermal treatment. The as-prepared composites were characterized using FE-SEM, FTIR and Raman spectroscopy to obtain morphological, compositional and structural details. In addition, the antibacterial efficiency of these composites was investigated against antibiotic resistant S. aureus and E. coli. Afterwards, graphene composite based epoxy coatings were prepared on glass substrate and tested for their antibiofilm efficiency against methicillin resistant S. aureus strain. The abundant presence of hydroxyl groups over the surface due to tannic acid, hydrophobicity of graphene and epoxy and remarkable antibacterial efficiency of tannic acid (TA), silver (Ag) and graphene synergistically enhance the antibiofilm efficiency of such coatings. This work presents a new strategy to develop a multifunctional coating and subsequently lessen the risk of biofilm assisted infections.

}, keywords = {Antibiofilm, Antimicrobial, Composite, Cytotoxicity, Graphene nanoplatelets}, doi = {https://doi.org/10.1016/j.porgcoat.2021.106421}, url = {https://www.sciencedirect.com/science/article/pii/S0300944021002927$\#$}, author = {Singhal, Akshay V and Malwal, Deepika and Thiyagarajan, Shankar and Lahiri, Indranil} } @article {1858, title = {Azaindole Based Potentiator of Antibiotics against Gram-Negative Bacteria [C-CAMP Startup Bugworks]}, journal = {ACS Infectious Diseases}, year = {2021}, month = {10/2021}, type = {Journal Article}, abstract = {
We discovered azaindole-based compounds with weak innate activity that exhibit substantial potentiation of antibacterial activities of different antibiotics, viz., rifampicin, erythromycin, solithromycin, and novobiocin in Gram-negative bacteria. In the presence of the azaindole derivatives, these antibiotics exhibited submicromolar minimum inhibitory concentrations (MICs) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The fold improvements in MIC of these antibiotics that were otherwise weak or inactive on their own against these bacteria were also observed against drug-resistant clinical isolates. Our studies indicate that this selective potentiation is probably through destabilization of the outer membrane{\textquoteright}s integrity, known to be regulated by the lipopolysaccharides (LPS). Thus, the azaindole based compounds described here open opportunities for those antibiotics that are otherwise ineffective due to LPS mediated entry barriers in Gram-negative bacteria.
}, keywords = {Azaindole, Bacterial Permeability, Lipopolysachhardies (LPS), Polymyxin, Synergy}, doi = {10.1021/acsinfecdis.1c00171}, url = {https://pubs.acs.org/doi/abs/10.1021/acsinfecdis.1c00171$\#$}, author = {Sharma, Sreevalli and Rao, Ranga and Reeve, Stephanie M and Phelps, Gregory A and Bharatham, Nagakumar and Katagihallimath, Nainesh and Ramachandran, Vasanthi and Raveendran, Savitha and Sarma, Maitrayee and Nath, Anubha} } @article {1679, title = {Chemical intervention for enhancing growth and reducing grain arsenic accumulation in rice [Mass Spectrometry - Metabolomics Facility]}, journal = {Environ Pollut}, volume = {276}, year = {2021}, month = {2021 Feb 13}, pages = {116719}, abstract = {

Arsenic (As) is a ubiquitous environmental carcinogen that enters the human food chain mainly through rice grains. In the present study, we evaluated the potential of thiourea (TU; non-physiological reactive oxygen species scavenger) in mitigating the negative effects of arsenic (As) stress in indica rice variety IR64, with the overall aim to reduce grain As accumulation. At seedling stage, As\ +\ TU treatment induced the formation of more numerous and longer crown roots compared with As alone. The As accumulation in main root, crown root, lower leaf and upper leaf was significantly reduced to 0.1-, 0.14-, 0.16-, 0.14-fold, respectively in As\ +\ TU treated seedlings compared with those of As alone. This reduced As accumulation was also coincided with light-dependent suppression in the expression levels of aquaporins and photosynthesis-related genes in As\ +\ TU treated roots. In addition, the foliar-supplemented TU under As-stress maintained reducing redox conditions which decreased the rate of As accumulation in flag leaves and, eventually grain As by 0.53-fold compared with those of As treatment. The agronomic feasibility of TU was validated under naturally As contaminated sites of Nadia (West Bengal, India). The tiller numbers and crop productivity (kg seed/ha) of TU-sprayed plants were increased by 1.5- and 1.18-fold, respectively; while, grain As accumulation was reduced by 0.36-fold compared with those of water-sprayed control. Thus, this study established TU application as a sustainable solution for cultivating rice in As-contaminated field conditions.

}, issn = {1873-6424}, doi = {10.1016/j.envpol.2021.116719}, author = {Srivastava, Ashish Kumar and Pandey, Manish and Ghate, Tejashree and Kumar, Vikash and Upadhyay, Munish Kumar and Majumdar, Arnab and Sanjukta, Abhay Kumar and Agrawal, Ashish Kumar and Bose, Sutapa and Srivastava, Sudhakar and Suprasanna, Penna} } @article {1865, title = {Chrysin modulates protein kinase IKK$\varepsilon$/TBK1, insulin sensitivity and hepatic fatty infiltration in diet-induced obese mice [Mass Spectrometry - Lipidomics]}, journal = {Drug Development Research}, year = {2021}, month = {08/2021}, type = {Journal Article}, abstract = {

Nuclear factor kappa B cells (NF-κB) activation causes induction of the noncanonical IκB kinases (I-kappa-B kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) in liver and fat after high fat diet which followed activating of cascade of counter-inflammation that conserves energy storage. Chrysin (5,7-dihydroxyflavone), a natural flavonoid, present in many plants, honey and propolis, used conventionally to treat numerous ailments. The present study was aimed to identify the protective role of chrysin on the glucose lowering and insulin sensitivity in diet induced obese (DIO) mice by regulating IKKε/TBK1. Chrysin administered therapeutically (60, 100, 200 mg/kg body weight) and preventive mode (200 mg/kg body weight) for 4 and 10 weeks respectively to DIO mice. At last fasting blood glucose, oral glucose tolerance test, serum lipid profile, as well as the expression level of IKKε/TBK1 and triglyceride in the liver tissue were assessed. DIO mice showed impaired glucose tolerance, reduced weight gain, elevated hepatic IKKε/TBK1 expression, fatty acid infiltration triglyceride and increased in plasma insulin and glucose. Chrysin in both therapeutic and preventive mode normalized the altered levels of the same. Overall chrysin improves glycemic control and insulin sensitivity through regulating expression of IKKε/TBK1 in liver of DIO mice.

}, keywords = {chrysin, DIO, high fat diet, IKKε/TBK1, insulin.}, doi = {https://doi.org/10.1002/ddr.21859}, url = {https://onlinelibrary.wiley.com/doi/10.1002/ddr.21859}, author = {Amir Siddiqui, Mohammad and Akhtar, Juber and Uddin, Shahab and Chandrashekharan, Sidda Madappa and Ahmad, Mohammad and Khan, Mohammad Irfan and Khalid, Mohammad} } @article {1699, title = {Derivation of Induced Pluripotent Stem Cell (iPSC) Lines from Patient-Specific Peripheral Blood Mononuclear Cells (PBMC) Using Episomal Vectors [Eyestem Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Methods Mol Biol}, year = {2021}, month = {2021 Mar 27}, abstract = {

Inherited retinal diseases (IRDs) are a diverse group of rare eye disorders, resulting in vision loss or blindness. The underlying reason is mutation in one or more than 250 different genes associated with the development and normal physiology of retina largely comprising of rod/cone photoreceptors and retinal pigment epithelium. Interestingly, the sub retinal region of an eye has been shown to be immune privileged, broadening the scope of cell-replacement therapies for patients suffering from retinal degeneration. Several groups around the globe, including ours, have demonstrated safety and efficacy in preclinical studies by employing various approaches of retinal cell therapy. This had largely been possible with the advent of induced pluripotent stem cells (iPSC)-reprogrammed from adult somatic cells, that serves as a starting material for generating retinal cells de novo. Here, we describe a detailed procedure for reprogramming peripheral blood mononuclear cells (PBMC) into iPSC using episomal vectors without any physical disruption in the host genome. The lines thus created were tested for sterility, cytogenetic stability, identity, absence of episomal plasmids and further authenticated for pluripotency and tri-lineage differentiation capacity by embryoid body formation and immunocytochemistry. We believe that this feeder-cell free, animal-product free and gene-insertion free protocol would help people to develop and bank patient-specific cell lines for autologous cell therapies for incurable rare diseases.

}, issn = {1940-6029}, doi = {10.1007/7651_2021_385}, author = {Konala, Vijay Bhaskar Reddy and Nandakumar, Swapna and Surendran, Harshini and Pal, Rajarshi} } @article {1717, title = { Differential proteins associated with plasma membrane in X- and/or Y-chromosome bearing spermatozoa in indicus cattle [Mass Spectrmetry - Propteomics Facility]}, year = {2021}, month = {04/2021}, abstract = {

The differential proteins associated with plasma membrane of spermatozoa are less known, identification of which shall help overcome limitations of currently used methods of sperm sexing, considered as a high priority for livestock sector of many countries. This study has reported plasma membrane proteomics of unsorted spermatozoa and differential expression of plasma membrane-associated proteins between X- and Y-chromosome bearing spermatozoa of indicus cattle (Bos indicus). Isolation of plasma membrane fraction using percoll gradient, relatively a rapid method, from bovine spermatozoa has been reported to enrich isolation of plasma membrane proteins. Significant enrichment for plasma membrane-associated proteins was observed in plasma membrane fraction (p \< .05) as compared to the total cell lysate using LC-MS/MS. Furthermore, these experiments were conducted in flow cytometry sorted, sexed-semen samples. Thirteen proteins were identified as differentially abundant between X- and Y-sorted spermatozoa. Among these, two proteins were downregulated in Y-sorted spermatozoa compared to the X-sorted spermatozoa (p \< .05), while four and seven proteins could be noted in X- and Y-sorted spermatozoa, respectively. Proteins that are presumed to support sperm capacitation and sperm migration velocity were found to be abundant in Y-sorted spermatozoa while those associated with structural molecule activity were identified as abundant in X-sorted spermatozoa in the present study. Our study provides better insight into the plasma membrane proteomics of spermatozoa of indicus cattle and furnishes data that might aid in design and development of alternate and open technology for sex-sorting of semen.

https://pubmed.ncbi.nlm.nih.gov/33829570/

}, keywords = {X/Y-sorted spermatozoa; plasma membrane; pre-determination of sex; proteome; sperm sexing; unsorted spermatozoa.}, doi = {10.1111/rda.13936}, author = {Rongala Laxmivandana and Chhaya Patole and Tilak Raj Sharma and Kewal Krishan Sharma and Soumen Naskar} } @article {1867, title = {Downregulation of CRX, a Group 3-specific oncogenic transcription factor, inhibits TGF-β activin signaling in medulloblastoma cells [Genomics Facility]}, journal = {Biochemical and Biophysical Research Communications}, volume = {568}, year = {2021}, month = {06/2021}, pages = {76{\textendash}82}, type = {Journal Article}, chapter = {76}, abstract = {

Medulloblastoma, the most common malignant brain tumor in children, consists of four molecular subgroups WNT, SHH, Group 3, and Group 4. Group 3 has the worst survival rate among the four subgroups and is characterized by the expression of retina-specific genes. CRX, the master regulator of the photoreceptor differentiation, is aberrantly expressed in Group 3 medulloblastomas. CRX expression increased the proliferation, anchorage-independent growth, invasion potential, and tumorigenicity of medulloblastoma cells indicating the oncogenic role of CRX in medulloblastoma pathogenesis. CRX knockdown resulted in the downregulation of expression of several retina-specific genes like IMPG2, PDC, RCVRN. and Group 3 specific genes like GABRA5, MYC, PROM1. Thus, CRX plays a major role not only in the expression of retina-specific genes but also in defining Group 3 identity. Increased expression of several pro-apoptotic genes upon CRX knockdown suggests that CRX could protect Group 3 medulloblastoma cells from cell death. Several negative regulators of the TGF-β signaling pathway like SMAD7, PMEPA1, KLF2 were upregulated upon the CRX knockdown. Western blot analysis showed a decrease in the levels of (Phospho)-SMAD2, total levels of SMAD2, SMAD4, and an increase in the levels of SMAD7 indicating inhibition of the TGF-β signaling pathway upon CRX knockdown. Copy number variations in several genes involved in the TGF-β signaling pathway occur in a subset of Group 3 tumors. Autocrine TGF-β/activin signaling has recently been reported to be active in a subset of Group 3 medulloblastomas. CRX knockdown resulting in the inhibition of the TGF-β/activin signaling pathway demonstrates an interaction between the two Group 3 specific oncogenic pathways and suggests simultaneous targeting of both CRX and TGF-β signaling as a possible therapeutic strategy.

}, keywords = {CRX, Group 3, Medulloblastoma, TGF-β/activin signaling}, issn = {0006-291X}, doi = {https://doi.org/10.1016/j.bbrc.2021.06.064}, url = {https://www.sciencedirect.com/science/article/abs/pii/S0006291X21009888}, author = {Masurkar, Shalaka Arun and Deogharkar, Akash and Bharambe, Harish Shrikrishna and Shirsat, Neelam Vishwanath} } @article {1760, title = {Effect of nanoparticle exposure in a living system: probed by quantification of Fetuin-B in plasma proteome and kidney tissue imaging using MALDI imaging mass spectrometry in a rat model [Mass Spectrometry - Proteomics Facility]}, journal = {Journal of Nanoparticle Research }, volume = {23}, year = {2021}, type = {Article number: 125}, abstract = {

Nanoparticles have gained importance in various biomedical field viz cosmetics, diagnostics, therapeutics, and food additives. Due to small size, NPs penetrate tissues and cells, thereby interacting with biomolecules such as proteins and nucleic acids. This raises the major concern to investigate the effect of NPs in a living system. Previously, we reported downregulation of Fetuin-B in rat plasma, with the increasing dose level of NP-Fe4(P2O7)3, in rats treated with NP-Fe4(P2O7)3 as a food-fortificant. In the present study, we validated the dose response of Fetuin-B in rats exposed to NP-Fe4(P2O7)3, NP-ZnO, and NP-SiO2. Fetuin-B showed similar dose responses in all the groups irrespective of their physicochemical properties. Previously, we hypothesized that the immune response was triggered upon exposure to NP-Fe4(P2O7)3 in vivo, which led to the differential regulation of Fetuin-B. To evaluate the hypothesis, immunological response was assessed in rats by estimating the pro-inflammatory cytokines in plasma. We observed Interleukin-1α and Interleukin-1β increased with the increasing dose levels of the three NPs. The impact of NPs on the proteomic profile in the kidney tissues of rats exposed to three NPs, showed differential spatial distribution of α-Enolase across the medulla and the cortex region with respect to the control group, suggesting an overexpression of α-Enolase. Overexpression of α-Enolase might be an indicative of cellular apoptosis in response to cytotoxicity caused due to NP perforation across the cellular membrane. In the present study, we propose that Fetuin-B might be considered a plasma biomarker to detect nanotoxicity at an early stage in a living system.

}, doi = {://doi.org/10.1007/s11051-021-05251-z}, url = {https://link.springer.com/article/10.1007/s13204-021-01914-4}, author = {Bindu Y. Srinivasu and Arun Arumugaperumal and Amrita Mitra and Monita Muralidharan and Rajdeep Das and Amit Kumar Mandal} } @article {1761, title = {Elucidation of the liver proteome in response to an antioxidant intake in rabbits [Mass Spectrometry - Proteomics Facility]}, journal = {Egyptian Liver Journal }, volume = {11}, year = {2021}, month = {06/2021}, abstract = {

Background

Antioxidant intakes are one of the most cherished dietary approaches for the management of oxidative stress-induced liver damages. These antioxidants exist as the bioactive compounds present in plants and other natural sources functioning in varieties of ways from acting as direct scavengers of the free radicals to acting as the modifiers of genes and proteins expressions.\ Chlorella vulgaris\ is one of such antioxidants; it is a unicellular microalga and a rich source of polyphenols which has been reported for its capacity of reducing oxidative stress by upregulation of antioxidant genes. However, there are scarce reports on its effect on antioxidant protein expressions and functions in the liver. This situation necessitates untargeted proteomic profiling of the liver due to the antioxidant intakes as carried out in this present study. Sixteen laboratory weaner rabbits of 8 weeks old with initial average bodyweight of 1060 {\textpm} 29.42 g were randomly divided into two groups (n\ = 8 per group); the first group served as control while the second served as the treatment group were used for this study.

Results

After a period of 120 days daily consumption of 500 mg of\ Chlorella vulgaris\ biomass per kg bodyweight of the rabbit models, the animals were sacrificed and their livers were harvested followed by protein extraction for the untargeted proteomic profiling using LC-MS/Orbitrap Fusion Tribrid{\texttrademark} peptides quantifier and sequencer. Also, there was an assessment of the oxidative stress biomarkers in the liver and serum of the rabbits. Five-hundred and forty-four (544) proteins were identified out of which 204 were unique to the control, 198 were unique to the treatment group, while 142 were common to both groups of the rabbits. Antioxidant proteins commonly found in both groups were upregulated in the treatment group and were significantly associated with oxidative stress-protective activities. There was a reduction in oxidative stress biomarkers of the supplemented group as indicated by the assessment of the liver malondialdehyde concentrations (p\ \< 0.05), total antioxidant capacities (p\ \< 0.05), and antioxidant enzyme activities (p\ \< 0.05). Similarly, these biomarkers were significantly reduced in the serum of the supplemented rabbits (p\ \< 0.05).

Conclusion

The study concluded that\ Chlorella vulgaris\ is an antioxidant agent that could be suitable for reducing liver oxidative stress damage and it is a potential drug candidate for protecting the liver against oxidative stress damages as revealed in the rabbit models.

}, doi = {https://doi.org/10.1186/s43066-021-00118-3}, url = {https://link.springer.com/article/10.1186/s43066-021-00118-3$\#$Ack1}, author = {Akeem Babatunde Sikiru and Arunachalam Arangasamy and Stephen Sunday Acheneje Egena and Sejian Veerasamy and Ippala Janardhan Reddy and Bhatta Raghavendra} } @article {1691, title = {Geometry encoded functional programming of tumor homing peptides for targeted drug delivery [Image Analysis Support]}, journal = {J Control Release}, year = {2021}, month = {2021 Mar 12}, abstract = {

Poly-peptide molecules have shown promising applications in drug delivery and tumor targeting. A series of tumor homing peptides were designed by exhaustively sampling low energy geometrical basins of amino acids at specific sites of a peptide molecule to induce a conformational lock. This peptide library was pruned to a limited set of eight molecules, employing electrostatic interactions, docking, and molecular dynamics simulations. These designed and optimized peptides were synthesized and tested on various cell lines, including breast cancer (MDA-MB-231), cervical cancer (HeLa), osteosarcoma (U2-OS), and non-cancerous mammary epithelial cells (MCF-10A) using confocal microscopy and flow cytometry. Peptides show differential uptake in cancerous MDA-MB-231, HeLa, U2-OS, and non-cancerous MCF-10A cells. Confocal imaging verified their ability to penetrate even in 3D tumorospheres of MDA-MB-231 cells. Further, experiments of mitochondrial membrane potential depolarization and Caspase-3 activation confirmed that their cytotoxic effects are by apoptosis. Homing ability of the designed peptides in in vivo system and fluorescence imaging with clinical samples of human origin have further confirmed that the in vitro studies are qualitatively identical and quantitatively comparable in their ability to selectively recognize tumor cells. Overall, we present a roadmap for the functional programming of peptide-based homing and penetrating molecules that can perform selective tumor targeting.

}, issn = {1873-4995}, doi = {10.1016/j.jconrel.2021.03.010}, author = {Goyal, Ruchika and Jerath, Gaurav and Akhil, R and Chandrasekharan, Aneesh and Puppala, Eswara Rao and Ponneganti, Srikanth and Sarma, Anupam and Naidu, V G M and Santhoshkumar, T R and Ramakrishnan, Vibin} } @article {1860, title = {Glycomic and glycotranscriptomic profiling of mucin-type O-glycans in planarian Schmidtea mediterranea [Mass Spectrometry - Glycomics]}, journal = {Glycobiology}, year = {2021}, month = {09/2021}, type = {Journal Article}, abstract = {

O-Glycans on cell surfaces play important roles in cell-cell, cell-matrix, and receptor-ligand interaction. Therefore, glycan-based interactions are important for tissue regeneration and homeostasis. Free-living flatworm Schmidtea mediterranea, because of its robust regenerative potential, is of great interest in the field of stem cell biology and tissue regeneration. Nevertheless, information on the composition and structure of O-glycans in planaria is unknown. Using mass spectrometry and in silico approaches, we characterized the glycome and the related transcriptome of mucin-type O-glycans of planarian S. mediterranea. Mucin-type O-glycans were composed of multiple isomeric, methylated, and unusually extended mono- and di-substituted O-GalNAc structures. Extensions made of hexoses and 3-O methyl hexoses were the glycoforms observed. From glycotranscriptomic analysis, sixty genes belonging to five distinct enzyme classes were identified to be involved in mucin-type O-glycan biosynthesis. These genes shared homology with those in other invertebrate systems. While a majority of the genes involved in mucin-type O-glycan biosynthesis was highly expressed during organogenesis and in differentiated cells, a few select genes in each enzyme class were specifically enriched during early embryogenesis. Our results indicate a unique temporal and spatial role for mucin-type O-glycans during embryogenesis and organogenesis and in adulthood. In summary, this is the first report on O-glycans in planaria. This study expands the structural and biosynthetic possibilities in cellular glycosylation in the invertebrate glycome and provides a framework towards understanding the biological role of mucin-type O-glycans in tissue regeneration using planarians.

}, keywords = {Flatworm, GC-MS, Invertebrates, MALDI-TOF mass spectrometry, ScRNAseq}, doi = {https://doi.org/10.1093/glycob/cwab097}, url = {https://academic.oup.com/glycob/advance-article-abstract/doi/10.1093/glycob/cwab097/6361079}, author = {Subramanian, Sabarinath Peruvemba and Lakshmanan, Vairavan and Palakodeti, Dasaradhi and Subramanian, Ramaswamy} } @article {1724, title = {Identification of novel vaccine candidates in the whole-cell Aeromonas hydrophila biofilm vaccine through reverse vaccinology approach [Mass Spectrometry - Proteomics Facility]}, journal = {Fish \& Shellfish Immunology}, volume = {114}, year = {2021}, pages = {132-141}, abstract = {

Biofilm vaccine has been recognised as one of the successful strategy to reduce the Aeromonas hydrophila infection in fish. But, the vaccine contains the protective and non-protective proteins, which may lead to show altered heterologous adaptive immunity response. Moreover, cross protection and effectiveness of previously developed biofilm vaccine was not tested against different geographical A. hydrophila isolates. Therefore, in the present study, whole-cell A. hydrophila biofilm vaccine was evaluated in rohu, vaccinated group showed increased antibody titer and protection against the different geographical A. hydrophila isolates namely KAH1 and AAH2 with 78.9\% and 84.2\% relative percentage survival, respectively. In addition, by using the immune sera of biofilm vaccinated group, a total of six protective proteins were detected using western blot assay. Further, the same proteins were identified by nano LC-MS/MS method, a total of fourteen candidate proteins showing the immunogenic property including highly expressed OMP{\textquoteright}s tolC, bamA, lamb, AH4AK4_2542, AHGSH82_029580 were identified as potential vaccine candidates. The STRING analysis revealed that, top candidate proteins identified may potentially interact with other intracellular proteins; involved in ribosomal and (tricarboxylic acid) TCA pathway. Importantly, all the selected vaccine candidate proteins contain the B-cell epitope region. Finally, the present study concludes that, whole-cell A. hydrophila biofilm vaccine able to protect the fish against the different geographical A. hydrophila isolates. Further, through reverse vaccinology approach, a total of fourteen proteins were identified as potential vaccine candidates against A. hydrophila pathogen.

}, keywords = {Biofilm vaccine, isolates, Protection, Protective protein, Proteomics, Reverse vaccinology}, issn = {1050-4648}, doi = {https://doi.org/10.1016/j.fsi.2021.04.019}, url = {https://www.sciencedirect.com/science/article/pii/S1050464821001091}, author = {Basmeet Kaur and B.T. Naveen Kumar and Anuj Tyagi and Shanthanagouda Admane Holeyappa and Niraj Kumar Singh} } @article {1859, title = {In Vitro Anticancer Activity of Imperata cylindrica Root{\textquoteright}s Extract toward Human Cervical Cancer and Identification of Potential Bioactive Compounds [Mass Spectrometry - Metabolomics Facility]}, journal = {BioMed research international}, volume = {2021}, year = {2021}, month = {10/2021}, type = {Journal Article}, abstract = {

Imperata cylindrica is traditionally used to cure several diseases including cancer, wounds, and hypertension. The present study was designed to investigate the anticancer activity of the methanolic root extract of I. cylindrica (IC-MeOH). The water-soluble tetrazolium-1 and colony formation assays were used to check the proliferation ability of the cells. Cell apoptosis and cell cycle were measured by flow cytometry-based fluorescence-activated cell sorting. The ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis was used for the metabolites profiling of IC-MeOH. Based on high-mass accuracy, spectral data, and previous reports, tentative compound identifications were assigned. Our findings revealed that IC-MeOH inhibited the proliferation of HeLa and CaSki cells. The plant extract was also found to induce a concentration- and time-dependent apoptosis and cell cycle arrest in the G0/G1 phase (IC50 value) in CaSki cell line. Analysis of IC-MeOH permitted the identification of 10 compounds already reported for their anticancer activity, epicatechin, curcumin, (-)-yatein, caffeic acid, myricetin, jatrorrhizine, harmaline, cinnamaldehyde, dobutamine, and syringin. In conclusion, IC-MeOH is a rich source of cytotoxic metabolites that inhibits human cervical cancer proliferation via apoptosis and cell cycle arrest.

}, doi = {https://doi.org/10.1155/2021/4259777}, url = {https://www.hindawi.com/journals/bmri/2021/4259777/$\#$materials-and-methods}, author = {Nayim, Paul and Sudhir, Krishna and Mbaveng, Armelle T and Kuete, Victor and Sanjukta, Mukherjee} } @article {1854, title = {Interventional Strategies for Parkinson Disease: Can Neural Precursor Cells Forge a Path Ahead? [Eyestem Research, a C-CAMP startup]}, journal = {ACS Chemical Neuroscience}, year = {2021}, month = {10/2021}, type = {Journal Article}, abstract = {

Neural precursor cells (NPCs), derived from pluripotent stem cells (PSCs), with their unique ability to generate multiple neuronal and glial cell types are extremely useful for understanding biological mechanisms in normal and diseased states. However, generation of specific neuronal subtypes (mature) from NPCs in large numbers adequate for cell therapy is challenging due to lack of a thorough understanding of the cues that govern their differentiation. Interestingly, neural stem cells (NSCs) themselves are in consideration for therapy given their potency to form different neural cell types, release of trophic factors, and immunomodulatory effects that confer neuroprotection. With the recent COVID-19 outbreak and its accompanying neurological indications, the immunomodulatory role of NSCs may gain additional significance in the prevention of disease progression in vulnerable populations. In this regard, small-molecule mediated NPC generation from PSCs via NSC formation has become an important strategy that ensures consistency and robustness of the process. The development of the mammalian brain occurs along the rostro-caudal axis, and the establishment of anterior identity is an early event. Wnt signaling, along with fibroblast growth factor and retinoic acid, acts as a caudalization signal. Further, the increasing amount of epigenetic data available from human fetal brain development has enhanced both our understanding of and ability to experimentally manipulate these developmental regulatory programs in vitro. However, the impact on homing and engraftment after transplantation and subsequently on therapeutic efficacy of NPCs based on their derivation strategy is not yet clear. Another formidable challenge in cell replacement therapy for neurodegenerative disorders is the mode of delivery. In this Perspective, we discuss these core ideas with insights from our preliminary studies exploring the role of PSC-derived NPCs in rat models of MPTP-induced Parkinson{\textquoteright}s disease following intranasal injections.

}, keywords = {cell transplantation, induced pluripotent stem cells, neural precursor cell, neural stem cell., Parkinson disease}, doi = {https://doi.org/10.1021/acschemneuro.1c00525}, url = {https://pubs.acs.org/doi/abs/10.1021/acschemneuro.1c00525}, author = {Nandakumar, Swapna and Shahani, Pradnya and Datta, Indrani and Pal, Rajarshi} } @article {1678, title = {Methanol Skin Mucus Extract of Mrigal (Cirrhinus mrigala) Fish Peptide Targeting Viral Particles of Infectious Pancreatic Necrosis Virus (IPNV) and Infectious Salmon Anemia Virus (ISAV): an in silico Approach [Mass Spectrometry Facility]}, journal = {International Journal of Peptide Research and Therapeutics}, volume = {71}, year = {2021}, month = {02/2021}, type = {Research Article}, abstract = {

The teleost fish skin mucus acts as an important physical and biological barrier that prevents fish from the surrounding environment. Many studies reported the presence of various immunological molecules in fish skin mucus that involve in protection against invading microbes. In the present study, the skin mucus proteins of freshwater fish\ Cirrhinus mrigala\ (mrigal) were extracted using organic solvent (methanol) and further analyzed by liquid chromatography-tandem mass spectrometry (LC{\textendash}MS/MS) to identify proteins by database retrieval. LC{\textendash}MS/MS analysis revealed the presence of diverse proteins in the methanol skin mucus extract. The identified proteins were classified into biological process, cellular process and molecular functions by Gene Ontology (GO) enrichment analysis. A peptide was selected, modelled and compared with other antimicrobial peptides sequences through phylogenetic analysis and showed that the modelled peptide shared high similarity with Arminin-1 of Cnidaria animals. We also investigated the potentiality of the modelled peptide against Infectious Pancreatic Necrosis Virus sub viral particle and Infectious Salmon Anemia Virus hemagglutinin-esterase protein through protein-peptide docking using ClusPro. The docking results confirmed that the modelled peptide has good interactions with viral particles. Therefore, these results suggest that the modelled peptide molecule from\ C. mrigala\ methanol skin mucus extract can be further studied that aid in the development of novel peptide candidate for the control of aquaculture viral diseases.

}, doi = {https://doi.org/10.1007/s10989-021-10179-y}, author = {Arun Sridhar and Dinesh Babu Manikandan and Sathish Kumar Marimuthu and Manikandan Murugesan and Thirumurugan Ramasamy} } @article {1862, title = {Mito-targeted antioxidant prevents cardiovascular remodelling in spontaneously hypertensive rat by modulation of energy metabolism [Mass Spectrometry - Proteomics Facility]}, journal = {Clinical and Experimental Pharmacology and Physiology}, year = {2021}, month = {08/2021}, type = {Journal Article}, abstract = {

Hypertension induced left ventricular hypertrophy (LVH) augments the risk of cardiovascular anomalies. Mitochondrial alterations result in oxidative stress, accompanied by decrease in fatty acid oxidation, leading to the activation of the hypertrophic program. Targeted antioxidants are expected to reduce mitochondrial reactive oxygen species more effectively than general antioxidants. This study was designed to assess whether the mito-targeted antioxidant, Mito-Tempol (Mito-TEMP) is more effective than the general oxidant, Tempol (TEMP) in reduction of hypertension and hypertrophy and prevention of shift in cardiac energy metabolism. Spontaneously hypertensive rats were administered either TEMP (20 mg/kg/day) or Mito-TEMP (2 mg/kg/day) intraperitoneally for 30 days. Post treatment, animals were subjected to 2D-echocardiography. Myocardial lysates were subjected to RPLC {\textendash} LTQ-Orbitrap-MS analysis. Mid-ventricular sections were probed for markers of energy metabolism and fibrosis. The beneficial effect on cardiovascular structure and function was significantly higher for Mito-TEMP. Increase in mitochondrial antioxidants and stimulation of fatty acid metabolism; with significant improvement in cardiovascular function was apparent in spontaneously hypertensive rats (SHR) treated with Mito-TEMP. The study indicates that Mito-TEMP is superior to its non- targeted isoform in preventing hypertension induced LVH, and the beneficial effects on heart are possibly mediated by reversal of metabolic remodelling.

}, keywords = {cardiac hypertrophy, cardiac metabolism, LC-MS, mitochondria targeted antioxidant, proteomic analysis, spontaneously hypertensive rat}, doi = {https://doi.org/10.1111/1440-1681.13585}, url = {https://onlinelibrary.wiley.com/doi/10.1111/1440-1681.13585}, author = {Potnuri, Ajay Godwin and Purushothaman, Sreeja and Saheera, Sherin and Nair, Renuka R} } @article {1638, title = {A Novel N4-Like Bacteriophage Isolated from a Wastewater Source in South India with Activity against Several Multidrug-Resistant Clinical Pseudomonas aeruginosa Isolates [Next Gen Genomics \& National Electron Cryo-Microscopy Facilities]}, journal = {mSphere}, volume = {6}, year = {2021}, month = {2021 Jan 13}, abstract = {

Multidrug-resistant community-acquired infections caused by the opportunistic human pathogen are increasingly reported in India and other locations globally. Since this organism is ubiquitous in the environment, samples such as sewage and wastewater are rich reservoirs of bacteriophages. In this study, we report the isolation and characterization of a novel N4-like lytic bacteriophage, vB_Pae_AM.P2 (AM.P2), from wastewater in Kerala, India. AM.P2 is a double-stranded DNA podovirus that efficiently lyses the model strain, PAO1, at a multiplicity of infection as low as 0.1 phage per bacterium and resistance frequency of 6.59 {\texttimes} 10 Synergy in bactericidal activity was observed between AM.P2 and subinhibitory concentrations of the antibiotic ciprofloxacin. Genome sequencing of AM.P2 revealed features similar to those of the N4-like phages LUZ7 and KPP21. As judged by two independent assay methods, spot tests and growth inhibition, AM.P2 successfully inhibited the growth of almost 30\% of strains from a contemporary collection of multidrug-resistant clinical isolates from South India. Thus, AM.P2 may represent an intriguing candidate for inclusion in bacteriophage cocktails developed for various applications, including water decontamination and clinical bacteriophage therapy. In India, multidrug resistance determinants are much more abundant in community-associated bacterial pathogens due to the improper treatment of domestic and industrial effluents. In particular, a high bacterial load of the opportunistic pathogen in sewage and water bodies in India is well documented. The isolation and characterization of bacteriophages that could target emerging strains, representing possible epicenters for community-acquired infections, could serve as a useful alternative tool for various applications, such as phage therapy and environmental treatment. Continuing to supplement the repertoire of broad-spectrum bacteriophages is an essential tool in confronting this problem.

}, issn = {2379-5042}, doi = {10.1128/mSphere.01215-20}, author = {Menon, Nitasha D and Kumar, Megha S and Satheesh Babu, T G and Bose, Sucharita and Vijayakumar, Gayathri and Baswe, Manasi and Chatterjee, Meghna and D{\textquoteright}Silva, Jovita Rowena and Shetty, Kavya and Haripriyan, Jayalekshmi and Kumar, Anil and Nair, Samitha and Somanath, Priyanka and Nair, Bipin G and Nizet, Victor and Kumar, Geetha B} } @article {1864, title = {Organic mineral supplementation on differential protein profile of Osmanabadi bucks (Capra hircus) [Mass Spectrometry - Proteomics Facility]}, journal = {Reproductive Biology}, volume = {21}, year = {2021}, month = {07/2021}, pages = {100533}, type = {Journal Article}, abstract = {

The present study aimed to determine the differential protein profile of seminal plasma proteins of bucks supplemented with trace minerals. Forty bucks of uniform size and body weight were assigned as ten groups (n = 4). The control group (T1) was fed with the control diet (concentration mixture and roughages) whereas the remaining groups were supplemented the control diet with Zn20 mg (T2), Zn40 mg (T3), Zn60 mg (T4), Cu12.5 mg (T5), Cu25 mg (T6), Cu37.5 mg (T7), Zn20 mg + Cu12.5 mg (T8), Zn40 mg + Cu25 mg (T9), and Zn60 mg + Cu37.5 mg (T10) for eight months. Seminal plasma proteins from each group were subjected to two-dimensional electrophoresis and fifteen differential proteins were selected based on differential expression, subjected to identification using Nano-LC{\textendash}MS/MS (LTQ-Qrbitrap-MS). The identified proteins were Triacylglycerol lipase, EGF like repeats and discoidin domains 3, Lipocalin, Iodothyronine deiodinase, Transcription factor AP2-delta, 60S ribosomal protein L13, IST1 factor associated with ESCRT-III, Lysozyme, Uncharacterized protein (BRI3-binding protein), Uncharacterized protein, Histone deacetylase 11, General transcription factor IIF subunit 2, Nudix hydrolase 6, Protein kinase cAMP-activated catalytic subunit beta and Elongin C. The organic Cu supplemented group is the better than the organic Zn and organic Zn + Cu supplemented groups.

}, keywords = {Bucks, Differential protein, Fecundity, Organic mineral, Seminal plasma}, doi = {https://doi.org/10.1016/j.repbio.2021.100533}, url = {https://www.sciencedirect.com/science/article/abs/pii/S1642431X21000541$\#$}, author = {Sekar, Backialakshmi and Arangasamy, Arunachalam and Naidu, Sharanya Jeevendra and Reddy, Ippala Janardhan and Bhatta, Raghavendra} } @article {1716, title = {ROS Dependent Antifungal and Anticancer Modulations of Piper colubrinum Osmotin [Bio-Incubation Core Equipment/Protein Expression Facility]}, journal = {Molecules}, volume = {26}, year = {2021}, abstract = {

Osmotin, a plant defense protein, has functional similarity to adiponectin, an insulin sensitizingsensitising hormone secreted by adipocytes. We speculated that Piper colubrinum Osmotin (PcOSM) could have functional roles in obesity-related cancers, especially breast cancer. Immunofluorescence assays, flow cytometry, cell cycle analysis and a senescence assay were employed to delineate the activity in MDAMB231 breast cancer cell line. PcOSM pre-treated P. nigrum leaves showed significant reduction in disease symptoms correlated with high ROS production. In silico analysis predicted that PcOSM has higher binding efficiency with adiponectin receptor compared to adiponectin. PcOSM was effectively taken up by MDAMB231 cancer cells which resulted in marked increase in intracellular ROS levels leading to senescence and cell cycle arrest in G2/M stage. This study provides evidence on the ROS mediated direct inhibitory activity of the plant derived osmotin protein on the phytopathogen Phytophthora capsici, and the additional functional roles of this plant defense protein on cancer cells through inducing ROS associated senescence. The strong leads produced from this study could be pursued further to obtain more insights into the therapeutic potential of osmotin in human cancers.

}, issn = {1420-3049}, doi = {10.3390/molecules26082239}, url = {https://www.mdpi.com/1420-3049/26/8/2239}, author = {Geetha, Rajeswari Gopal and Krishnankutty Nair Chandrika, Sivakumar and Saraswathy, Gayathri G. and Nair Sivakumari, Asha and Sakuntala, Manjula} } @article {1861, title = {Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae [Bugworks, a C-CAMP startup]}, journal = {Nature communications}, volume = {12}, year = {2021}, month = {09/2021}, pages = {1{\textendash}12}, type = {Journal Article}, abstract = {

OqxB is an RND (Resistance-Nodulation-Division) efflux pump that has emerged as a factor contributing to the antibiotic resistance in Klebsiella pneumoniae. OqxB underwent horizontal gene transfer and is now seen in other Gram-negative bacterial pathogens including Escherichia coli, Enterobacter cloacae and Salmonella spp., further disseminating multi-drug resistance. In this study, we describe crystal structure of OqxB with n-dodecyl-β-D-maltoside (DDM) molecules bound in its substrate-binding pocket, at 1.85 {\r A} resolution. We utilize this structure in computational studies to predict the key amino acids contributing to the efflux of fluoroquinolones by OqxB, distinct from analogous residues in related transporters AcrB and MexB. Finally, our complementation assays with mutated OqxB and minimum inhibitory concentration (MIC) experiments with clinical isolates of E. coli provide further evidence that the predicted structural features are indeed involved in ciprofloxacin efflux.

}, doi = {https://doi.org/10.1038/s41467-021-25679-0}, url = {https://www.nature.com/articles/s41467-021-25679-0}, author = {Bharatham, Nagakumar and Bhowmik, Purnendu and Aoki, Maho and Okada, Ui and Sharma, Sreevalli and Yamashita, Eiki and Shanbhag, Anirudh P and Rajagopal, Sreenath and Thomas, Teby and Sarma, Maitrayee and others} } @article {1639, title = {Transplantation of retinal pigment epithelium and photoreceptors generated concomitantly via small molecule-mediated differentiation rescues visual function in rodent models of retinal degeneration [Eyestem Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Stem Cell Res Ther}, volume = {12}, year = {2021}, month = {2021 Jan 19}, pages = {70}, abstract = {

BACKGROUND: Age-related macular degeneration (AMD) is a result of degeneration/damage of the retinal pigment epithelium (RPE) while retinitis pigmentosa (RP), an inherited early-onset disease, results from premature loss of photoreceptors. A promising therapeutic approach for both is the replacement of lost/damaged cells with human induced pluripotent stem cell (hiPSC)-derived retinal cells.

METHODS: The aim of this study was to investigate the in vivo functionality of RPE and photoreceptor progenitor (PRP) cells derived from a clinical-grade hiPSC line through a unified protocol. De novo-generated RPE and PRP were characterized extensively to validate their identity, purity, and potency.

RESULTS: RPE expressed tight junction proteins, showed pigmentation and ciliation, and secreted polarization-related factors vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PRP expressed neural retina proteins and cone and rod markers, and responded to KCl-induced polarization. Transcriptomic analysis demonstrated an increase in the expression of mature retinal tissue-specific genes coupled with concomitant downregulation of genes from undesired lineages. RPE transplantation rescued visual function in RCS rats shown via optokinetic tracking and photoreceptor rescue. PRP transplantation improved light perception in NOD.SCID-rd1 mice, and positive electroretinography signals indicated functional photoreceptor activity in the host{\textquoteright}s outer nuclear layer. Graft survival and integration were confirmed using immunohistochemistry, and no animals showed teratoma formation or any kind of ectopic growth in the eye.

CONCLUSIONS: To our knowledge, this is the first demonstration of a unified, scalable, and GMP-adaptable protocol indicating strong animal efficacy and safety data with hiPSC-derived RPE and PRP cells. These findings provide robust proof-of-principle results for IND-enabling studies to test these potential regenerative cell therapies in patients.

}, issn = {1757-6512}, doi = {10.1186/s13287-021-02134-x}, author = {Surendran, Harshini and Nandakumar, Swapna and Reddy K, Vijay Bhaskar and Stoddard, Jonathan and Mohan K, Varsha and Upadhyay, Pramod K and McGill, Trevor J and Pal, Rajarshi} } @article {1677, title = {The truth about scats and dogs: Next-generation sequencing and spatial capture{\textendash}recapture models offer opportunities for conservation monitoring of an endangered social canid [Next Gen Genomics Facility (INT)].}, journal = {Biological Conservation}, volume = {256}, year = {2021}, pages = {109028}, abstract = {

Obtaining accurate population counts of endangered species is central to conservation biology, with implications for gaining ecological insights, informing management strategies, and judicial use of conservation funds. Despite decades of progress in methodological developments in the realm of population ecology, reliable density estimates are unavailable for many species of conservation concern. The dhole (Asiatic wild dog Cuon alpinus) is one such endangered large carnivore found in the tropical forests of south and southeast Asia. Here, we (i) develop next-generation sequencing resources to identify individual dholes from genetic samples, (ii) apply these methods to identify individuals in the wild, from scat (fecal) samples collected through systematic field surveys and (iii) generate reliable estimates of dhole densities in Wayanad Wildlife Sanctuary (Western Ghats, India) using Spatial Capture{\textendash}Recapture {\textquoteleft}SCR{\textquoteright} models. We estimate dhole densities to be 12{\textendash}14.2 individuals/100 sq. km based on a set of SCR models, with \ 50 individuals within Wayanad{\textquoteright}s administrative boundary. Our study presents a methodological improvement in generating population estimates of an important apex predator while also offering ecologically informative insights on a species in dire need of science-based management efforts. Replicating this study across connected reserves and over time can serve as a unified framework for understanding population dynamics, population structures, landscape connectivity and metapopulation-level conservation requirements. We propose that the approach presented here may be adopted as an economically and logistically feasible protocol for conservation monitoring of dholes and other ecologically important species plagued by similar issues of data-deficiency, and insufficient funding and resources.

}, keywords = {Carnivores, Conservation monitoring, Genetic markers, Non-invasive surveys, Population estimation, Single nucleotide polymorphisms, Tropics}, issn = {0006-3207}, doi = {https://doi.org/10.1016/j.biocon.2021.109028}, url = {https://www.sciencedirect.com/science/article/pii/S000632072100080X}, author = {Arjun Srivathsa and Ryan G. Rodrigues and Kok Ben Toh and Arun Zachariah and Ryan W. Taylor and Madan K. Oli and Uma Ramakrishnan} } @article {1295, title = {Cone snail analogs of the pituitary hormones oxytocin/vasopressin and their carrier protein neurophysin. Proteomic and transcriptomic identification of conopressins and conophysins [Next Gen Genomics Facility (INT)]}, journal = {Biochim Biophys Acta Proteins Proteom}, year = {2020}, month = {2020 Feb 10}, pages = {140391}, abstract = {

Transcriptomic analysis of cone snail venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (* indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing.

}, issn = {1878-1454}, doi = {10.1016/j.bbapap.2020.140391}, author = {Kumar, Sanjeev and Vijayasarathy, M and Venkatesha, M A and Sunita, P and Balaram, P} } @article {1323, title = {Dataset for the combined transcriptome assembly of M. oleifera and functional annotation [Next Gen Genomics Facility (INT)]}, journal = {Data in Brief}, year = {2020}, pages = {105416}, abstract = {

In this paper, we present the data acquired during transcriptome analysis of the plant Moringa oleifera [1] from five different tissues (root, stem, leaf, flower and seed) by RNA sequencing. A total of 271 million reads were assembled with an N50 of 2094bp. The combined transcriptome was assessed for transcript abundance across five tissues. The protein coding genes identified from the transcripts were annotated and used for orthology analysis. Further, enzymes involved in the biosynthesis of select medicinally important secondary metabolites, vitamins and ion transporters were identified and their expression levels across tissues were examined. The data generated by RNA sequencing has been deposited to NCBI public repository under the accession number PRJNA394193 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA394193).

}, keywords = {Annotation, Enrichment analysis, Gene Expression, Metabolic pathway, Orthology, Transcriptome}, issn = {2352-3409}, doi = {https://doi.org/10.1016/j.dib.2020.105416}, url = {http://www.sciencedirect.com/science/article/pii/S2352340920303103}, author = {K. Mohamed Shafi and Adwait G. Joshi and Iyer Meenakshi and Shaik Naseer Pasha and K. Harini and Jarjapu Mahita and Radha Sivarajan Sajeevan and Snehal D. Karpe and Pritha Ghosh and Sathyanarayanan Nitish and A. Gandhimathi and Oommen K. Mathew and Subramanian Hari Prasanna and Manoharan Malini and Eshita Mutt and Mahantesha Naika and Nithin Ravooru and Rajas M. Rao and Prashant N. Shingate and Anshul Sukhwal and Margaret S. Sunitha and Atul K. Upadhyay and Rithvik S. Vinekar and Ramanathan Sowdhamini} } @article {1447, title = {Dietary supplementation of extracts of red sea weed (Kappaphycus alvarezii) improves growth, intestinal morphology, expression of intestinal genes and immune responses in broiler chickens [Sea6Energy Pvt. Ltd, a C-CAMP Startup]}, journal = {Journal of the Science of Food and Agriculture}, year = {2020}, month = {08, 2020}, type = {Research Article}, abstract = {

BACKGROUND

Effects of supplementation of dried alkaline (referred to as MVP1) and aqueous (referred to as PBD1) extracts of\ K. alvarezii\ , were evaluated in broiler (Vencobb 400) chickens (1{\textendash}35 d post-hatch). In experiment I, each of the seven diets (basal diet with three levels (0.5, 1.5 or 5.0 g kg-1\ diet) of MVP1 or PBD1 and a negative control) was fed to twelve pen replicates containing five birds in each. In experiment II, each of three diets (a negative control, and PBD1 at two levels (1.0 or 1.5 g kg-1\ diet)) was fed to sixteen pen replicates of five chicks in each.

RESULTS

Concentrations of total phenolics, phycobillins and free radical scavenging activity were higher (P\<0.01) whereas carrageenan was lower in PBD1 than in MVP1. In the experiment I, PBD1 at 1.5 g kg-1\ diet improved (P\<0.05) body weight (7.11\% higher). In the experiment II, both the treatments improved (P\<0.01) BW (9.18\% and 8.47\%, respectively) as compared to control. The group fed with PBD1@ 1.0 g kg-1\ had higher (P\<0.05) HI titre, expression of intestinal claudin 2, TLR2A, NOD1, avian beta defensin 4, interleukin 2 and 6 genes than control. Treatments did not influence feed efficiency or levels of most of the antioxidant enzymes. Villus width and crypt depth were significantly higher in the group fed with 1.5 g kg-1\ of PBD1.

CONCLUSION

Supplementing dried aqueous extract of\ Kappaphycus alvarezii\ at 1 g kg-1\ diet may be an effective strategy to increase growth and immunity in broiler chicken.

}, doi = {https://doi.org/10.1002/jsfa.10708}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/jsfa.10708}, author = {Paul, Shyam Sundar and Venkata, Hemanth Giri Rao Vantharam and Raju, MVLN and Rama Rao, SV and Nori, Sri Sailaja and Suryanarayan, Shrikumar and Kumar, Vikas and Perveen, Zeba and Srinivas Prasad, Cadaba} } @article {1530, title = {Elucidating the processes and pathways enriched in buffalo sperm proteome in regulating semen quality [Mass Spectrometry Facility - Proteomics]}, journal = {Cell Tissue Res}, year = {2020}, month = {2020 Nov 05}, abstract = {

Sperm carries a reservoir of proteins regulating the molecular functions to attain functional competence. Semen samples collected from buffalo bulls were assessed for sperm functional attributes (n\ =\ 11) and proteome profiling (n\ =\ 6). Sperm proteins were extracted and profiled by employing LC-MS/MS. Overall, the buffalo sperm contained 1365 proteins, of which 458 were common between the groups. The unique proteins were 477 and 430 in good and poor quality semen, respectively. In the whole proteome of buffalo sperm, sexual reproduction with phosphatidylethanolamine-binding protein1 (PEBP1), fetuin-B (FETUB) and acrosin (ACR) was the most enriched (p\ =\ 8.44E-19) biological process, also with thermogenesis (p\ =\ 0.003), oocyte meiosis (p\ =\ 0.007) and vascular smooth muscle contraction (p\ =\ 0.009) apart from metabolic pathways. In good quality semen, mesenchyme migration (p\ =\ 1.24E-07) and morphogenesis (p\ =\ 0.001) were abundant biological processes. In good quality semen, the fluid shear stress (p\ =\ 0.01) and, in poor quality semen, valine, leucine and isoleucine degradation (p\ =\ 3.8E-05) pathways were enriched. In good quality semen, 7 proteins were significantly (p\ \<\ 0.05) upregulated and 33 proteins were significantly (p\ \<\ 0.05) downregulated. On validating the abundantly expressed sperm proteins, serine protease inhibitor Kazal-type 2-like (SPINK2; 2.17-fold) and neddylin (NEDD8; 1.13-fold) were upregulated and YBX2 was downregulated (0.41-fold) in good quality semen as compared with poor quality semen (1-fold). The present findings revealed the importance of sperm proteins in oocyte maturation, fertilization process and early embryonic development. The variations in the proteomic composition can be used as potential markers for the selection of breeding bulls.

}, issn = {1432-0878}, doi = {10.1007/s00441-020-03303-9}, author = {Binsila, Bala Krishnan and Archana, Santhanahalli Siddalingappa and Ramya, Laxman and Swathi, Divakar and Selvaraju, Sellappan and Gowda, N K Shivakumar and Pal, Din Taran and Rafay, Abu and Bhatta, Raghavendra} } @article {1464, title = {Human induced pluripotent stem cell-derived lung epithelial system for SARS-CoV-2 infection modeling and its potential in drug repurposing [Eyestem Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Stem Cells Dev}, year = {2020}, month = {2020 Aug 31}, abstract = {

The lung is the most vulnerable target for the SARS-CoV-2 infection and respiratory failure causing Acute Respiratory Distress Syndrome (ARDS) is its foremost outcome. However, the current primary in vitro models in use for SARS-CoV-2 display apparent limitations for modeling such complex human respiratory disease. While patient cells can directly model the effects of a drug, their availability and capacity for expansion are limited compared to the transformed/immortalized cells or the tumor-derived cell lines. What{\textquoteright}s more, the latter may harbor genetic and metabolic abnormalities in the course of their derivation, making them unsuitable for drug screening. Therefore, it is important to create physiologically relevant human-cell models that can replicate the pathophysiology of SARS-CoV-2, thus, facilitating drug testing. Here, we show preliminary data on how human induced pluripotent stem cells (hiPSC)-based lung epithelial cell system could emerge as a relevant and sensitive platform for modelling SARS-CoV-2 infection and drug screening.

}, issn = {1557-8534}, doi = {10.1089/scd.2020.0152}, author = {Surendran, Harshini and Nandakumar, Swapna and Pal, Rajarshi} } @article {1465, title = {A knowledge-driven protocol for prediction of proteins of interest with an emphasis on biosynthetic pathways [Next Gen Genomics Facility (INT)]}, journal = {MethodsX}, year = {2020}, pages = {101053}, abstract = {

This protocol describes a stepwise process to identify proteins of interest from a query proteome derived from NGS data. We implemented this protocol on Moringa oleifera transcriptome to identify proteins involved in secondary metabolite and vitamin biosynthesis and ion transport. This knowledge-driven protocol identifies proteins using an integrated approach involving sensitive sequence search and evolutionary relationships. We make use of functionally important residues (FIR) specific for the query protein family identified through its homologous sequences and literature. We screen protein hits based on the clustering with true homologues through phylogenetic tree reconstruction complemented with the FIR mapping. The protocol was validated for the protein hits through qRT-PCR and transcriptome quantification. Our protocol demonstrated a higher specificity as compared to other methods, particularly in distinguishing cross-family hits. This protocol was effective in transcriptome data analysis of M. oleifera as described in Pasha et. al.{\textbullet}Knowledge-driven protocol to identify secondary metabolite synthesizing protein in a highly specific manner.{\textbullet}Use of functionally important residues for screening of true hits.{\textbullet}Beneficial for metabolite pathway reconstruction in any (species, metagenomics) NGS data.

}, keywords = {functionally important residue, homology, multiple sequence alignment, Pathway, phylogenetic analysis}, issn = {2215-0161}, doi = {https://doi.org/10.1016/j.mex.2020.101053}, url = {http://www.sciencedirect.com/science/article/pii/S2215016120302739}, author = {Adwait G. Joshi and K. Harini and Iyer Meenakshi and K. Mohamed Shafi and Shaik Naseer Pasha and Jarjapu Mahita and Radha Sivarajan Sajeevan and Snehal D. Karpe and Pritha Ghosh and Sathyanarayanan Nitish and A. Gandhimathi and Oommen K. Mathew and Subramanian Hari Prasanna and Manoharan Malini and Eshita Mutt and Mahantesha Naika and Nithin Ravooru and Rajas M. Rao and Prashant N. Shingate and Anshul Sukhwal and Margaret S. Sunitha and Atul K. Upadhyay and Rithvik S. Vinekar and Ramanathan Sowdhamini} } @article {1637, title = {Metabolic control of cellular immune-competency by odors in Drosophila [Mass Spectrometry - Metabolomics Facility (INT)]}, journal = {Elife}, volume = {9}, year = {2020}, month = {2020 Dec 29}, abstract = {

Studies in different animal model systems have revealed the impact of odors on immune cells; however, any understanding on why and how odors control cellular immunity remained unclear. We find that employ an olfactory-immune cross-talk to tune a specific cell type, the lamellocytes, from hematopoietic-progenitor cells. We show that neuronally released GABA derived upon olfactory stimulation is utilized by blood-progenitor cells as a metabolite and through its catabolism, these cells stabilize Sima/HIFα protein. Sima capacitates blood-progenitor cells with the ability to initiate lamellocyte differentiation. This systemic axis becomes relevant for larvae dwelling in wasp-infested environments where chances of infection are high. By co-opting the olfactory route, the preconditioned animals elevate their systemic GABA levels leading to the upregulation of blood-progenitor cell Sima expression. This elevates their immune-potential and primes them to respond rapidly when infected with parasitic wasps. The present work highlights the importance of the olfaction in immunity and shows how odor detection during animal development is utilized to establish a long-range axis in the control of blood-progenitor competency and immune-priming.

}, issn = {2050-084X}, doi = {10.7554/eLife.60376}, author = {Madhwal, Sukanya and Shin, Mingyu and Kapoor, Ankita and Goyal, Manisha and Joshi, Manish K and Ur Rehman, Pirzada Mujeeb and Gor, Kavan and Shim, Jiwon and Mukherjee, Tina} } @article {1342, title = {Metagenomics analysis reveals features unique to Indian distal gut microbiota [Next Gen Genomics Facility]}, journal = {PLoS One}, volume = {15}, year = {2020}, month = {2020}, pages = {e0231197}, abstract = {

Various factors including diet, age, geography, culture and socio-economic status have a role in determining the composition of the human gut microbiota. The human gut microbial composition is known to be altered in disease conditions. Considering the important role of the gut microbiome in maintaining homeostasis and overall health, it is important to understand the microbial diversity and the functional metagenome of the healthy gut. Here, we characterized the microbiota of 31 fecal samples from healthy individuals of Indian ethnic tribes from Ladakh, Jaisalmer and Khargone by shotgun metagenomic sequencing. Sequence analysis revealed that Bifidobacterium and Prevotella were the key microbes contributing to the differences among Jaisalmer, Khargone and Ladakh samples at the genus level. Our correlation network study identified carbohydrate-active enzymes and carbohydrate binding proteins that are associated with specific genera in the different Indian geographical regions studied. Network analysis of carbohydrate-active enzymes and genus abundance revealed that the presence of different carbohydrate-active enzymes is driven by differential abundance of genera. The correlation networks were different in the different geographical regions, and these interactions suggest the role of less abundant genera in shaping the gut environment. We compared our data with samples from different countries and found significant differences in taxonomic composition and abundance of carbohydrate-active enzymes in the gut microbiota as compared to the other countries.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0231197}, author = {Kaur, Kamaldeep and Khatri, Indu and Akhtar, Akil and Subramanian, Srikrishna and Ramya, T N C} } @article {1480, title = {A microfluidic flow analyzer with integrated lensed optical fibers [Discovery to Innovation Accelerator]}, journal = {Biomicrofluidics}, volume = {14}, year = {2020}, pages = {054104}, doi = {10.1063/5.0013250}, url = {https://doi.org/10.1063/5.0013250}, author = {Mohan,A. and Gupta,P. and Nair,A. P. and Prabhakar,A. and Saiyed,T.} } @article {1459, title = {Sequential activation of Notch and~Grainyhead~gives apoptotic competence to Abdominal-B expressing larval neuroblasts in Drosophila Central nervous system [Transgenic Fly Facility]}, journal = {PLoS Genet}, volume = {16}, year = {2020}, month = {2020 Aug 31}, pages = {e1008976}, abstract = {

Neural circuitry for mating and reproduction resides within the terminal segments of central nervous system (CNS) which express Hox paralogous group 9-13 (in vertebrates) or Abdominal-B (Abd-B) in Drosophila. Terminal neuroblasts (NBs) in A8-A10 segments of Drosophila larval CNS are subdivided into two groups based on expression of transcription factor Doublesex (Dsx). While the sex specific fate of Dsx-positive NBs is well investigated, the fate of Dsx-negative NBs is not known so far. Our studies with Dsx-negative NBs suggests that these cells, like their abdominal counterparts (in A3-A7 segments) use Hox, Grainyhead (Grh) and Notch to undergo cell death during larval development. This cell death also happens by transcriptionally activating RHG family of apoptotic genes through a common apoptotic enhancer in early to mid L3 stages. However, unlike abdominal NBs (in A3-A7 segments) which use resident Hox gene Abdominal-A (Abd-A) as an apoptosis trigger, Dsx-negative NBs (in A8-A10 segments) keep the levels of resident Hox gene Abd-B constant. These cells instead utilize increasing levels of the temporal transcription factor Grh and a rise in Notch activity to gain apoptotic competence. Biochemical and in vivo analysis suggest that Abdominal-A and Grh binding motifs in the common apoptotic enhancer also function as Abdominal-B and Grh binding motifs and maintains the enhancer activity in A8-A10 NBs. Finally, the deletion of this enhancer by the CRISPR-Cas9 method blocks the apoptosis of Dsx-negative NBs. These results highlight the fact that Hox dependent NB apoptosis in abdominal and terminal regions utilizes common molecular players (Hox, Grh and Notch), but seems to have evolved different molecular strategies to pattern CNS.

}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1008976}, author = {Bakshi, Asif and Sipani, Rashmi and Ghosh, Neha and Joshi, Rohit} } @article {1343, title = {Tracing the origin of olive ridley turtles entangled in ghost nets in the Maldives: A phylogeographic assessment of populations at risk [Next Gen Genomics Facility]}, journal = {Biological Conservation}, volume = {245}, year = {2020}, pages = {108499}, abstract = {

Abandoned, lost or discarded fishing nets, (ghost nets) represent a major threat to marine vertebrates. However, thorough assessments of their impact on threatened species are largely missing. In the Maldives, olive ridley sea turtles (Lepidochelys olivacea) are frequently caught in ghost nets however the archipelago does not support a significant nesting population. Our aim in this study was to determine the origin of olive ridleys entangled in ghost nets found in the Maldives and evaluate potential impacts on respective source populations. Based on a citizen science and conservation program, we recorded 132 olive ridley turtles entangled in ghost nets in just one year. Genetic analyses (mtDNA) of entangled individuals and of potential source populations revealed that most captured olive ridleys originated from Sri Lanka and eastern India. Oman could be excluded as source population, even during the prevalence of the south west monsoon. Based on our results and already available published literature, we were able to estimate that the recorded ghost net entanglements accounted for a relatively small amount (0.48\%) of the eastern Indian population. However, the entangled turtles accounted for a much larger percentage (41\%) of the Sri Lankan populations. However, it should be noted that our estimates of population-level mortality are linked to substantial uncertainty due to the lack of reliable information on population dynamics. Consequently, any precautionary protection measures applied should be complemented with improved quantification of turtle recruitment and life-stage specific mortalities.

}, keywords = {Citizen science, Entanglement, Ghost nets, mtDNA, Phylogenetics, Plastics}, issn = {0006-3207}, doi = {https://doi.org/10.1016/j.biocon.2020.108499}, url = {http://www.sciencedirect.com/science/article/pii/S0006320719313874}, author = {Martin Stelfox and Alfred Burian and Kartik Shanker and Alan F. Rees and Claire Jean and Ma{\"\i}a S. Willson and Nashwa Ahmed Manik and Michael Sweet} } @article {1524, title = {Tracking the time-dependent and tissue-specific processes of arsenic accumulation and stress responses in rice (Oryza sativa L.) [Mass Spectrometry - Metabolomics Facility]}, journal = {Journal of Hazardous Materials}, year = {2020}, pages = {124307}, abstract = {

The present study analysed time (0.5h to 24h) and tissue [roots, old leaves (OL) and young leaves (YL)] dependent nature of arsenic (As) accumulation and ensuing responses in two contrasting varieties of rice (Oryza sativa L.); Pooja (tolerant) and CO-50 (moderately sensitive). Arsenic accumulation was 5.4-, 4.7- and 7.3-fold higher at 24h in roots, OL and YL, respectively of var. CO-50 than that in var. Pooja. Arsenic accumulation in YL depicted a delayed accumulation; at 2h onwards in var. Pooja (0.23{\textmu}gg-1 dw) while at 1h onwards in var. CO50 (0.26{\textmu}gg-1 dw). The responses of oxidative stress parameters, antioxidant enzymes, metabolites and ions were also found to be tissue- and time-dependent and depicted differential pattern in the two varieties. Among hormone, salicylic acid and abscisic acid showed variable response in var. Pooja and var. CO-50. Metabolite analysis depicted an involvement of various metabolites in As stress responses of two varieties. In conclusion, an early sensing of the As stress, proper coordination of hormones, biochemical responses, ionic and metabolic profiles allowed var. Pooja to resist As stress and reduce As accumulation more effectively as compared to that of var. CO-50.

}, keywords = {Arsenic, Hormones, Metabolites, ROS, Signalling, Stress}, issn = {0304-3894}, doi = {https://doi.org/10.1016/j.jhazmat.2020.124307}, url = {http://www.sciencedirect.com/science/article/pii/S0304389420322974}, author = {Poonam Yadav and Sudhakar Srivastva and Tanmayi Patil and Rishiraj Raghuvanshi and Ashish K. Srivastava and Penna Suprasanna} } @article {1145, title = {Aspartic protease from Aspergillus niger: Molecular characterization and interaction with pepstatin A [Mass Spectrometry Facility - Proteomics].}, journal = {Int J Biol Macromol}, year = {2019}, month = {2019 Jul 30}, abstract = {

In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 {\textpm} 0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 {\textdegree}C respectively. The enzyme was stable for 60 min at 50 {\textdegree}C. The purified enzyme had specific activity of 40,000 {\textpm} 1800 U/mg. The enzyme had 85\% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a K value of 0.045 μM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in β-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin \> defatted soya flour \> gluten \> gelatin \> skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. This aspartic protease was found to be an excellent candidate for genetic manipulation for biotechnological application in food and feed industries, due to its high catalytic turn over number and thermostability.

}, issn = {1879-0003}, doi = {10.1016/j.ijbiomac.2019.07.133}, author = {Purushothaman, Kavya and Bhat, Sagar Krishna and Singh, Sridevi Annapurna and Marathe, Gopal Kedihithlu and Appu Rao, Appu Rao G} } @article {1140, title = {Cell spread area and traction forces determine myosin-II-based cortex thickness regulation. [Central Imaging and Flow Cytometry Facility]}, journal = {Biochim Biophys Acta Mol Cell Res}, year = {2019}, month = {2019 Jul 23}, abstract = {

Actomyosin network under the plasma membrane of cells forms a cortical layer that regulates cellular deformations during different processes. What regulates the cortex? Characterized by its thickness, it is believed to be regulated by actin dynamics, filament-length regulators and myosin motor proteins. However, its regulation by cellular morphology (e.g. cell spread area) or mechanical microenvironment (e.g. substrate stiffness) has remained largely unexplored. In this study, super- and high-resolution imaging of actin in CHO cells demonstrates that at high spread areas (\>450 μm), the cortex is thinner, better separated as layers, and sensitive to deactivation of myosin II motors or reduction of substrate stiffness (and traction forces). In less spread cells (\<400 μm) such perturbations do not elicit a response. Myosin IIA{\textquoteright}s mechanosensing is limited here due to its lowered actin-bound fraction and higher turnover rate. Cofilin, in line with its competitive inhibitory role, is found to be overexpressed in these cells. To establish the causal relation, we initiate a spread area drop by de-adhesion and find enhanced actin dynamics and fragmentation along with oscillations and increase in thickness. This is more correlated to the reduction of traction forces than the endocytosis-based reduction in cell volume. Cortex thickness control by spread area is also found be true during differentiation of THP-1 monocytes to macrophages. Thus, we propose that spread area regulates cortex and its thickness by traction-based mechanosensing of myosin II.

}, issn = {1879-2596}, doi = {10.1016/j.bbamcr.2019.07.011}, author = {Kumar, Rinku and Saha, Sajjita and Sinha, Bidisha} } @article {1161, title = {Chronic Pressure Overload Results in Deficiency of Mitochondrial Membrane Transporter ABCB7 Which Contributes to Iron Overload, Mitochondrial Dysfunction, Metabolic Shift and Worsens Cardiac Function [Mass Spectrometry - Metabolomics Facility].}, journal = {Sci Rep}, volume = {9}, year = {2019}, month = {2019 Sep 11}, pages = {13170}, abstract = {

We examined the hitherto unexplored role of mitochondrial transporters and iron metabolism in advancing metabolic and mitochondrial dysfunction in the heart during long term pressure overload. We also investigated the link between mitochondrial dysfunction and fluctuation in mitochondrial transporters associated with pressure overload cardiac hypertrophy. Left ventricular hypertrophy (LVH) was induced in 3-month-old male Wistar rats by constriction of the aorta using titanium clips. After sacrifice at the end of 6 and 15 months after constriction, tissues from the left ventricle (LV) from all animals were collected for histology, biochemical studies, proteomic and metabolic profiling, and gene and protein expression studies. LV tissues from rats with LVH had a significant decrease in the expression of ABCB7 and mitochondrial oxidative phosphorylation (mt-OXPHOS) enzymes, an increased level of lipid metabolites, decrease in the level of intermediate metabolites of pentose phosphate pathway and elevated levels of cytoplasmic and mitochondrial iron, reactive oxygen species (ROS) and autophagy-related proteins. Knockdown of ABCB7 in H9C2 cells and stimulation with angiotensin II resulted in increased ROS levels, ferritin, and transferrin receptor expression and iron overload in both mitochondria and cytoplasm. A decrease in mRNA and protein levels of mt-OXPHOS specific enzymes, mt-dynamics and autophagy clearance and activation of IGF-1 signaling were also seen in these cells. ABCB7 overexpression rescued all these changes. ABCB7 was found to interact with mitochondrial complexes IV and V. We conclude that in chronic pressure overload, ABCB7 deficiency results in iron overload and mitochondrial dysfunction, contributing to heart failure.

}, issn = {2045-2322}, doi = {10.1038/s41598-019-49666-0}, author = {Kumar, Vikas and A, Aneesh Kumar and Sanawar, Rahul and Jaleel, Abdul and Santhosh Kumar, T R and Kartha, C C} } @article {1194, title = {Deciphering the in vivo redox behavior of human peroxiredoxins I and II by expressing in budding yeast [Mass Spectrometry - Proteomics].}, journal = {Free Radic Biol Med}, volume = {145}, year = {2019}, month = {2019 Sep 30}, pages = {321-329}, abstract = {

Peroxiredoxins (Prxs), scavenge cellular peroxides by forming recyclable disulfides but under high oxidative stress, hyperoxidation of their active-site Cys residue results in loss of their peroxidase activity. Saccharomyces cerevisiae deficient in human Prx (hPrx) orthologue TSA1 show growth defects under oxidative stress. They can be complemented with hPRXI but not by hPRXII, but it is not clear how the disulfide and hyperoxidation states of the hPrx vary in yeast under oxidative stress. To understand this, we used oxidative-stress sensitive tsa1tsa2Δ yeast strain to express hPRXI or hPRXII. We found that hPrxI in yeast exists as a mixture of disulfide-linked dimer and reduced monomer but becomes hyperoxidized upon elevated oxidative stress as analyzed under denaturing conditions (SDS-PAGE). In contrast, hPrxII was present predominantly as the disulfide in unstressed cells and readily converted to its hyperoxidized, peroxidase-inactive form even with mild oxidative stress. Interestingly, we found that plant extracts containing polyphenol antioxidants provided further protection against the growth defects of the tsa1tsa2Δ strain expressing hPrx and preserved the peroxidase-active forms of the Prxs. The extracts also helped to protect against hyperoxidation of hPrxs in HeLa cells. Based on these findings we can conclude that resistance to oxidative stress of yeast cells expressing individual hPrxs requires the hPrx to be maintained in a redox state that permits redox cycling and peroxidase activity. Peroxidase activity decreases as the hPrx becomes hyperoxidized and the limited protection by hPrxII compared with hPrxI can be explained by its greater sensitivity to hyperoxidation.

}, issn = {1873-4596}, doi = {10.1016/j.freeradbiomed.2019.09.034}, author = {Kumar, Rakesh and Mohammad, Ashu and Saini, Reena V and Chahal, Anterpreet and Wong, Chi-Ming and Sharma, Deepak and Kaur, Sukhvir and Kumar, Vikas and Winterbourn, Christine C and Saini, Adesh K} } @article {893, title = {Deep sequencing identified potential miRNAs involved in defence response, stress and plant growth characteristics of wild genotypes of cardamom [Next Gen Genomics Facility]}, journal = {Plant Biol (Stuttg)}, volume = {21}, year = {2019}, month = {2019 Jan}, pages = {3-14}, abstract = {

Cardamom has long been used as a food flavouring agent and in ayurvedic medicines for mouth ulcers, digestive problems and even depression. Extensive occurrence of pests and diseases adversely affect its cultivation and result in substantial reductions in total production and productivity. Numerous studies revealed the significant role of miRNAs in plant biotic stress responses. In the current study, miRNA profiling of cultivar and wild cardamom genotypes was performed using an Ion Proton sequencer. We identified 161 potential miRNAs representing 42 families, including monocot/tissue-specific and 14 novel miRNAs in both genotypes. Significant differences in miRNA family abundance between the libraries were observed in read frequencies. A total of 19 miRNAs (from known miRNAs) displayed a twofold difference in expression between wild and cultivar genotypes. We found 1168 unique potential targets for 40 known miRNA families in wild and 1025 potential targets for 42 known miRNA families in cultivar genotypes. The differential expression analysis revealed that most miRNAs identified were highly expressed in cultivars and, furthermore, lower expression of miR169 and higher expression of miR529 in wild cardamom proved evidence that wild genotypes have stronger drought stress tolerance and floral development than cultivars. Potential targets predicted for the newly identified miRNAs from the miRNA libraries of wild and cultivar cardamom genotypes involved in metabolic and developmental processes and in response to various stimuli. qRT-PCR confirmed miRNAs were differentially expressed between wild and cultivar genotypes. Furthermore, four target genes were validated experimentally to confirm miRNA-mRNA target pairing using RNA ligase-mediated 5{\textquoteright} Rapid Amplification of cDNA Ends (5{\textquoteright}RLM-RACE) PCR.

}, issn = {1438-8677}, doi = {10.1111/plb.12888}, author = {Nadiya, F and Anjali, N and Thomas, J and Gangaprasad, A and Sabu, K K} } @article {1061, title = {Differentiating Human Induced Pluripotent Stem Cells (iPSCs) Into Lung Epithelial Cells. [Eyestem, a C-CAMP Startup]}, journal = {Curr Protoc Stem Cell Biol}, year = {2019}, month = {2019 Apr 18}, pages = {e86}, abstract = {

Human induced pluripotent\ stem cells\ (hiPSCs) not only offer great opportunities for the study of human development but also have tremendous potential for future clinical\ cell-based therapies.\ The protocol outlined here is used to differentiate hiPSCs into lung epithelial cell types through a process that faithfully recapitulates the stepwise events observed in vivo. From pluripotency, cells are differentiated to a definitive endoderm fate, followed by progression into anteriorized foregut endoderm that has the ability to give rise to both proximal and distal epithelial cells. Furthermore, this methodology allows for the study of lung dysfunction and disease modeling using patient-derived cells, as well as high-throughput pharmacological screening and eventually personalized therapies. Recently we were able to reproduce this protocol using the working cell bank of an hiPSC line made under current Good Manufacturing Practice (cGMP) conditions, a necessary step for the future clinical application of these\ cells. {\textcopyright} 2019 by John Wiley \& Sons, Inc.

}, issn = {1938-8969}, doi = {10.1002/cpsc.86}, author = {Surendran, Harshini and Rajamoorthy, Mohanapriya and Pal, Rajarshi} } @article {1138, title = {Discovery of a deeply divergent new lineage of vine snake (Colubridae: Ahaetuliinae: Proahaetulla gen. nov.) from the southern Western Ghats of Peninsular India with a revised key for Ahaetuliinae [Next Gen Genomics Facility].}, journal = {PLoS One}, volume = {14}, year = {2019}, month = {2019}, pages = {e0218851}, abstract = {

The Western Ghats are well known as a biodiversity hotspot, but the full extent of its snake diversity is yet to be uncovered. Here, we describe a new genus and species of vine snake Proahaetulla antiqua gen. et sp. nov., from the Agasthyamalai hills in the southern Western Ghats. It was found to be a member of the Ahaetuliinae clade, which currently comprises the arboreal snake genera Ahaetulla, Dryophiops, Dendrelaphis and Chrysopelea, distributed in South and Southeast Asia. Proahaetulla shows a sister relationship with all currently known taxa belonging to the genus Ahaetulla, and shares ancestry with Dryophiops. In addition to its phylogenetic position and significant genetic divergence, this new taxon is also different in morphology from members of Ahaetuliinae in a combination of characters, having 12-13 partially serrated keels on the dorsal scale rows, 20 maxillary teeth and 3 postocular scales. Divergence dating reveals that the new genus is ancient, dating back to the Mid-Oligocene, and is one of the oldest persisting monotypic lineages of snakes in the Western Ghats. This discovery adds to the growing list of ancient lineages endemic to the Agasthyamalai hills and underscores the biogeographic significance of this isolated massif in the southern Western Ghats.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0218851}, author = {Mallik, Ashok Kumar and Achyuthan, N Srikanthan and Ganesh, Sumaithangi R and Pal, Saunak P and Vijayakumar, S P and Shanker, Kartik} } @article {986, title = {Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway [Discovery to Innovation Accelerator]}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 Jan 09}, pages = {89}, abstract = {

The importance of gut microbiota in human health and pathophysiology is undisputable. Despite the abundance of metagenomics data, the functional dynamics of gut microbiota in human health and disease remain elusive. Urolithin A (UroA), a major microbial metabolite derived from polyphenolics of berries and pomegranate fruits displays anti-inflammatory, anti-oxidative, and anti-ageing activities. Here, we show that UroA and its potent synthetic analogue (UAS03) significantly enhance gut barrier function and inhibit unwarranted inflammation. We demonstrate that UroA and UAS03 exert their barrier functions through activation of aryl hydrocarbon receptor (AhR)- nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways to upregulate epithelial tight junction proteins. Importantly, treatment with these compounds attenuated colitis in pre-clinical models by remedying barrier dysfunction in addition to anti-inflammatory activities. Cumulatively, the results highlight how microbial metabolites provide two-pronged beneficial activities at gut epithelium by enhancing barrier functions and reducing inflammation to protect from colonic diseases.

}, issn = {2041-1723}, doi = {10.1038/s41467-018-07859-7}, author = {Singh, Rajbir and Chandrashekharappa, Sandeep and Bodduluri, Sobha R and Baby, Becca V and Hegde, Bindu and Kotla, Niranjan G and Hiwale, Ankita A and Saiyed, Taslimarif and Patel, Paresh and Vijay-Kumar, Matam and Langille, Morgan G I and Douglas, Gavin M and Cheng, Xi and Rouchka, Eric C and Waigel, Sabine J and Dryden, Gerald W and Alatassi, Houda and Zhang, Huang-Ge and Haribabu, Bodduluri and Vemula, Praveen K and Jala, Venkatakrishna R} } @article {1115, title = {Hematite Nanoparticles: synthesis, characterization and aquatic ecotoxicity effects [Central Imaging and Flow Cytometry Facility]}, journal = {Research Journal of Biotechnology}, volume = {14}, year = {2019}, chapter = {21}, abstract = {

Iron oxide nanoparticles have been investigated recently for their useful applications in numerous biomedical areas, in environmental remediation and in different industrial applications. In any case, additional risks have been identified with the release of nanoparticles into the environment. In the present study the toxicity of hematite nanoparticles to marine algae, Chlorella vulgaris was studied with focus on oxidative stress and cytotoxicity analysis. The synthesized hematite nanoparticles are in the range of 26-50 nm. Result showed that Chlorella vulgaris growth reduced with increasing concentrations. The nanoparticles induced oxidative stress was the main toxic mechanism. The nanoparticles and Chlorella vulgaris cell physical interaction also contributed to the nanotoxicity. Field Emission Scanning Electron Microscope shows the morphological changes and cell damage. In 24h, treatment mortality was 20 {\textendash} 70 \% and LC50 value for 24h was 393.60 mg/L. Toxicity study on copepod showed mortality increased from 20- 100\% for 48hr and LC50 value for 44h was 221.34 mg/L.

}, author = {Suman Thodhal Yoganandham and Radhika Rajasree Santha Ravindranath* and Gayathri Sathyamoorthy and Remya Rajan Renuka and Aranganathan Lakshminarayanan} } @article {1151, title = {Isolation and Characterization of Conotoxin Protein from Conus inscriptus and Its Potential Anticancer Activity Against Cervical Cancer (HeLa-HPV 16 Associated) Cell Lines [Mass Spectrometry - Proteomics/Glycomics]}, journal = {International Journal of Peptide Research and Therapeutics}, year = {2019}, month = {Aug}, abstract = {

Marine snails are abundant sources of biologically important conopeptides with their potential applications in drug development. The present study aimed to identify the potential conopeptides from the venom duct of Conus inscriptus. After extraction conopeptides were characterized by liquid chromatography{\textendash}mass spectrometric analysis showing totally 29 protein sequences with disulfide linkages of different molecular mass distributed in the range of 387{\textendash}1536\ m/z and the peptides showing mostly to T-superfamily of conotoxins with 78\ kDa heat shock proteins. The venom peptides showed six different molecular weight bands (37, 51, 60, 70, 80 and 90\ kDa) above 30\ kDa after in-gel enzymatic digestion. Furthermore, the conopeptides exhibit potential cytotoxic activity against HeLa-HPV 16 associated, Vero (normal) cell lines and brine shrimp. Fourier transform infra-red spectroscopy analysis confirms the structural and functional groups. The observed results suggests the venom peptides from C. inscriptus as a potential anticancer agent.

}, issn = {1573-3904}, doi = {10.1007/s10989-019-09907-2}, url = {https://doi.org/10.1007/s10989-019-09907-2}, author = {Kumari, Anjali and Ameri, Shijin and Ravikrishna, Palavancha and Dhayalan, Arul and Kamala-Kannan, S. and Selvankumar, T. and Govarthanan, M.} } @article {1014, title = {Loss of Hepatic Oscillatory Fed microRNAs Abrogates Refed Transition and Causes Liver Dysfunctions [Next Gen Genomics Facility].}, journal = {Cell Rep}, volume = {26}, year = {2019}, month = {2019 Feb 19}, pages = {2212-2226.e7}, abstract = {

Inability to mediate fed-fast transitions in the liver is known to cause metabolic dysfunctions and diseases. Intuitively, a failure to inhibit futile translation of state-specific transcripts during fed-fast cycles would abrogate dynamic physiological transitions. Here, we have discovered hepatic fed microRNAs that target fasting-induced genes and are essential for a refed transition. Our findings highlight the role of these fed microRNAs in orchestrating system-level control over liver physiology and whole-body energetics. By targeting SIRT1, PGC1α, and their downstream genes, fed microRNAs regulate metabolic and mitochondrial pathways. MicroRNA expression, processing, and RISC loading oscillate during these cycles and possibly constitute an anticipatory mechanism. Fed-microRNA oscillations are deregulated during aging. Scavenging of hepatic fed microRNAs causes uncontrolled gluconeogenesis and failure in\ the catabolic-to-anabolic switching upon feeding, which are hallmarks of metabolic diseases. Besides identifying mechanisms that enable efficient physiological toggling, our study highlights fed microRNAs as candidate therapeutic targets.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2019.01.087}, author = {Maniyadath, Babukrishna and Chattopadhyay, Tandrika and Verma, Srikant and Kumari, Sujata and Kulkarni, Prineeta and Banerjee, Kushal and Lazarus, Asmitha and Kokane, Saurabh S and Shetty, Trupti and Anamika, Krishanpal and Kolthur-Seetharam, Ullas} } @article {1160, title = {Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway [National Cryo-Electron Microscopy Facility (INT)]}, journal = {Nature Communications}, volume = {10}, year = {2019}, month = {09, 2019}, type = {Article}, chapter = {4127}, abstract = {Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.}, keywords = {Bacterial structural biology, Cryoelectron microscopy, Multienzyme complexes}, author = {Nitish Sathyanarayanan and Giuseppe Cannone and Lokesh Gakhar and Nainesh Katagihallimath and Ramanathan Sowdhamini and Subramanian Ramaswamy and Kutti R. Vinothkumar} } @article {1011, title = {Molecular mechanism of interactions between Chrysin and I-kappa-B kinase epsilon (IKKe)/Tank Binding Kinase-1(TBK1): Cell based assay and insilico molecular docking studies [High Throughput Screening Facility].}, journal = {J Biomol Struct Dyn}, year = {2019}, month = {2019 Feb 15}, pages = {1-9}, abstract = {

Chrysin, a bioactive flavonoid, was investigated for its potential to inhibit the activity of I-kappa-B kinase epsilon (IKKe) / Tank Binding Kinase-1(TBK1) an enzyme responsible for production of pro inflammatory cytokine and suppression of energy expenditure genes, finally causing insulin resistance and obesity. Expressions of majority of polyinosinic-polycytidylic acid (poly IC) mediated genes are mediated through Toll/interleukin-1 receptor domainߝcontaining adapter-inducing interferon (TRIF) dependent signalling pathway. To check the therapeutic potential of chrysin its effect was examined on Toll/interleukin-1 receptor domainߝcontaining adapter-inducing interferon (TRIF) dependent pathway. Chrysin showed significant inhibition of I-kappa-B kinase epsilon (IKKe) / Tank Binding Kinase-1(TBK1) enzyme activity by kinase assay. Chrysin suppressed the I-kappa-B kinase epsilon expression induced by polyinosinic-polycytidylic acid resulting into decrease expression of target genes interferon gamma-induced protein 10 (IP10), monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) in macrophage-like cell line. Chrysin also showed increase in oxygen consumption in osteosarcoma cell line hence alleviating energy expenditure and thermogenesis. Moreover, insilico analysis shows that chrysin interact weakly with I-kappa-B kinase epsilon (IKKe) but displayed good interaction with Tank Binding Kinase-1 (TBK1). Overall these results suggested that I-kappa-B kinase epsilon (IKKe) / Tank Binding Kinase-1(TBK1) may be a novel target for chrysin that possesses anti-inflammatory and insulin sensitivity effects at I-kappa-B kinase epsilon (IKKe) / Tank Binding Kinase-1(TBK1) binding sites.

}, issn = {1538-0254}, doi = {10.1080/07391102.2019.1581086}, author = {Siddiqui, Amir M and Akhtar, Juber and M S, Shahab Uddin and Khan, Mohammad Irfan and Khalid, Mohammad} } @article {1136, title = {Mon1 constitutes a novel node in the brain-gonad axis that is essential for female germline maturation. [Transgenic Fly Facility]}, journal = {Development}, volume = {146}, year = {2019}, month = {2019 Jul 10}, abstract = {

Monensin-sensitive 1 (Mon1) is an endocytic regulator that participates in the conversion of Rab5-positive early endosomes to Rab7-positive late endosomes. In , loss of leads to sterility as the mutant females have extremely small ovaries with complete absence of late stage egg chambers - a phenotype reminiscent of mutations in the insulin pathway genes. Here, we show that expression of many insulin-like peptides (ILPs) is reduced in mutants and feeding adults an insulin-rich diet can rescue the ovarian defects. Surprisingly, however, functions in the tyramine/octopaminergic neurons (OPNs) and not in the ovaries or the insulin-producing cells (IPCs). Consistently, knockdown of in only the OPNs is sufficient to mimic the ovarian phenotype, while expression of the gene in the OPNs alone can {\textquoteright}rescue{\textquoteright} the mutant defect. Last, we have identified and as critical targets of This study thus identifies as a novel molecular player in the brain-gonad axis and underscores the significance of inter-organ systemic communication during development.

}, issn = {1477-9129}, doi = {10.1242/dev.166504}, author = {Dhiman, Neena and Shweta, Kumari and Tendulkar, Shweta and Deshpande, Girish and Ratnaparkhi, Girish S and Ratnaparkhi, Anuradha} } @book {1188, title = {The Neem Genome [Next Gen Genomics Facility]}, year = {2019}, publisher = {Springer Nature Switzerland AG}, organization = {Springer Nature Switzerland AG}, abstract = { }, keywords = {C-CAMP Genomics Facility, Illumina HiSeq 1000, Next Generation Sequencing, paired-end (PE) (2 {\texttimes} 100 nts) sequencing chemistry}, author = {Gowda, Malali and Sheetal, Ambardar and Kole, Chittaranjan} } @conference {1311, title = {Overcoming Barriers in Commercializing Bio-Tech Innovations in India: A Case of Center for Cellular and Molecular Platforms}, booktitle = {2019 Portland International Conference on Management of Engineering and Technology (PICMET)}, year = {2019}, month = {Aug}, abstract = {

Building capabilities to successfully commercialize biotech research into products or solutions, is paramount but challenging to develop, especially in developing ecosystems or countries. Once accomplished, they can turn-around the socioeconomic condition in such countries and make them self-reliant, at least in the healthcare sector. It may also encourage the incumbent research community to transform their research findings into scientific, entrepreneurial or commercial ventures. However, building such {\textquoteleft}innovation ecosystems{\textquoteright} in developing countries, requires scientific, financial and infrastructural support to overcome their existing barriers. In India, one such government-funded non-profit organization which is trying to overcome the existing barriers and building an ecosystem to encourage biotech innovation and entrepreneurship, is the Centre for Cellular and Molecular Platforms (C-CAMP). Since its inception, it has been able to support more than 90 biotech start-ups in funding, mentorship and incubation. In this paper, we start our discussion by understanding some of the major challenges faced by contemporary biotech organizations in India while commercializing their innovations. Subsequently, we attempt to understand how C-CAMP was conceptualized to overcome some of these barriers and how it evolved over the years, to become a nodal agency for inspiring biotech innovations and entrepreneurship. This case study highlights some of the best practices followed by C-CAMP in managing biotech innovation and commercialization. Top Management Teams in biotech-based academia, industry, government or venture capital funding agencies from any country may find these barriers and best practices worth studying and analyzing.

}, keywords = {biotech innovation, biotech organizations, biotech start-ups funding, biotechnology, Centre for Cellular and Molecular Platforms, commercial ventures, commercialization, entrepreneurial ventures, entrepreneurship, financial management, financial support, government, government-funded nonprofit organization, health care, healthcare sector, incumbent research community, India, infrastructural support, innovation management, mentorship, organisational aspects, product research, scientific support, scientific ventures, socio-economic effects, socioeconomic condition, top management teams, venture capital, venture capital funding agencies}, doi = {10.23919/PICMET.2019.8893754}, author = {G. D. Tikas and T. Saiyed and A. Katte} } @article {1201, title = {Overexpression of native Musa-miR397 enhances plant biomass without compromising abiotic stress tolerance in banana [Mass Spectrometry - Metabolomics Facility].}, journal = {Sci Rep}, volume = {9}, year = {2019}, month = {2019 Nov 11}, pages = {16434}, abstract = {

Plant micro RNAs (miRNAs) control growth, development and stress tolerance but are comparatively unexplored in banana, whose cultivation is threatened by abiotic stress and nutrient deficiencies. In this study, a native Musa-miR397 precursor harboring 11 copper-responsive GTAC motifs in its promoter element was identified from banana genome. Musa-miR397 was significantly upregulated (8-10) fold in banana roots and leaves under copper deficiency, correlating with expression of root copper deficiency marker genes such as Musa-COPT and Musa-FRO2. Correspondingly, target laccases were significantly downregulated (\>-2 fold), indicating miRNA-mediated silencing for Cu salvaging. No significant expression changes in the miR397-laccase module were observed under iron stress. Musa-miR397 was also significantly upregulated (\>2 fold) under ABA, MV and heat treatments but downregulated under NaCl stress, indicating universal stress-responsiveness. Further, Musa-miR397 overexpression in banana significantly increased plant growth by 2-3 fold compared with wild-type but did not compromise tolerance towards Cu deficiency and NaCl stress. RNA-seq of transgenic and wild type plants revealed modulation in expression of 71 genes related to diverse aspects of growth and development, collectively promoting enhanced biomass. Summing up, our results not only portray Musa-miR397 as a candidate for enhancing plant biomass but also highlight it at the crossroads of growth-defense trade-offs.

}, issn = {2045-2322}, doi = {10.1038/s41598-019-52858-3}, author = {Patel, Prashanti and Yadav, Karuna and Srivastava, Ashish Kumar and Suprasanna, Penna and Ganapathi, Thumballi Ramabhatta} } @article {1178, title = {Serotonin is essential for eye regeneration in planaria Schmidtea~mediterranea [Mass Spectrometry (Metabolomics) and Central Imaging \& Flow Cytometry Facilities (INT)]}, journal = {FEBS Lett}, year = {2019}, month = {2019 Sep 17}, abstract = {

Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC-MS)-based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidtea\ mediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a noncanonical enzyme, phenylalanine hydroxylase. Knockdown of Smed-pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescues the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.

}, issn = {1873-3468}, doi = {10.1002/1873-3468.13607}, author = {Sarkar, Arunabha and Mukundan, Namita and Sowndarya, Sai and Dubey, Vinay Kumar and Babu, Rosana and Lakshmanan, Vairavan and Rangiah, Kannan and Panicker, Mitradas M and Palakodeti, Dasaradhi and Subramanian, Sabarinath Peruvemba and Subramanian, Ramaswamy} } @article {1016, title = {Serum biomarkers identification by iTRAQ and verification by MRM: S100A8/S100A9 levels predict tumor-stroma involvement and prognosis in Glioblastoma [Mass Spectrometry - Metabolomics Facility].}, journal = {Sci Rep}, volume = {9}, year = {2019}, month = {2019 Feb 26}, pages = {2749}, abstract = {

Despite advances in biology and treatment modalities, the prognosis of glioblastoma (GBM) remains poor. Serum reflects disease macroenvironment and thus provides a less invasive means to diagnose and monitor a diseased condition. By employing 4-plex iTRAQ methodology, we identified 40 proteins with differential abundance in GBM sera. The high abundance of serum S100A8/S100A9 was verified by multiple reaction monitoring (MRM). ELISA and MRM-based quantitation showed a significant positive correlation. Further, an integrated investigation using stromal, tumor purity and cell type scores demonstrated an enrichment of myeloid cell lineage in the GBM tumor microenvironment. Transcript levels of S100A8/S100A9 were found to be independent poor prognostic indicators in GBM. Medium levels of pre-operative and three-month post-operative follow-up serum S100A8 levels predicted poor prognosis in GBM patients who lived beyond median survival. In vitro experiments showed that recombinant S100A8/S100A9 proteins promoted integrin signalling dependent glioma cell migration and invasion up to a threshold level of concentrations. Thus, we have discovered GBM serum marker by iTRAQ and verified by MRM. We also demonstrate interplay between tumor micro and macroenvironment and identified S100A8 as a potential marker with diagnostic and prognostic value in GBM.

}, issn = {2045-2322}, doi = {10.1038/s41598-019-39067-8}, author = {Arora, Anjali and Patil, Vikas and Kundu, Paramita and Kondaiah, Paturu and Hegde, A S and Arivazhagan, A and Santosh, Vani and Pal, Debnath and Somasundaram, Kumaravel} } @article {1231, title = {A sleep-inducing gene, nemuri, links sleep and immune function in Drosophila [Transgenic Fly Facility]}, journal = {Science}, volume = {363}, year = {2019}, month = {2019 02 01}, pages = {509-515}, abstract = {

Sleep remains a major mystery of biology. In particular, little is known about the mechanisms that account for the drive to sleep. In an unbiased screen of more than 12,000 lines, we identified a single gene, , that induces sleep. The NEMURI protein is an antimicrobial peptide that can be secreted ectopically to drive prolonged sleep (with resistance to arousal) and to promote survival after infection. Loss of increased arousability during daily sleep and attenuated the acute increase in sleep induced by sleep deprivation or bacterial infection. Conditions that increase sleep drive induced expression of in a small number of fly brain neurons and targeted it to the sleep-promoting, dorsal fan-shaped body. We propose that NEMURI is a bona fide sleep homeostasis factor that is particularly important under conditions of high sleep need; because these conditions include sickness, our findings provide a link between sleep and immune function.

}, keywords = {Animals, Antimicrobial Cationic Peptides, Arousal, Bacterial Infections, Brain, Drosophila melanogaster, Drosophila Proteins, Female, Gain of Function Mutation, Gene Knockout Techniques, Homeostasis, Immune System, Male, Neurons, Sleep}, issn = {1095-9203}, doi = {10.1126/science.aat1650}, author = {Toda, Hirofumi and Williams, Julie A and Gulledge, Michael and Sehgal, Amita} } @article {1202, title = {Small molecule modulator of aggrephagy regulates neuroinflammation to curb pathogenesis of neurodegeneration [Discovery to Innovation Accelerator (INT)].}, journal = {EBioMedicine}, year = {2019}, month = {2019 Nov 11}, abstract = {

BACKGROUND: Plethora of efforts fails to yield a single drug to reverse the pathogenesis of Parkinson{\textquoteright}s disease (PD) and related α-synucleopathies.

METHODS: Using chemical biology, we identified a small molecule inhibitor of c-abl kinase, PD180970 that could potentially clear the toxic protein aggregates. Genetic, molecular, cell biological and immunological assays were performed to understand the mechanism of action. In vivo preclinical disease model of PD was used to assess its neuroprotection efficacy.

FINDINGS: In this report, we show the ability of a small molecule inhibitor of tyrosine kinases, PD180970, to induce autophagy (cell lines and mice midbrain) in an mTOR-independent manner and ameliorate the α-synuclein mediated toxicity. PD180970 also exerts anti-neuroinflammatory potential by inhibiting the release of proinflammatory cytokines such as IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1) through reduction of TLR-4 (toll like receptor-4) mediated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation. In vivo studies show that PD180970 is neuroprotective by degrading the toxic protein oligomers through induction of autophagy and subsiding the microglial activation.

INTERPRETATION: These protective mechanisms ensure the negation of Parkinson{\textquoteright}s disease related motor impairments. FUND: This work was supported by Wellcome Trust/DBT India Alliance Intermediate Fellowship (500159-Z-09-Z), DST-SERB grant (EMR/2015/001946), DBT (BT/INF/22/SP27679/2018) and JNCASR intramural funds to RM, and SERB, DST (SR/SO/HS/0121/2012) to PAA, and DST-SERB (SB/YS/LS-215/2013) to JPC and BIRAC funding to ETA C-CAMP.

}, issn = {2352-3964}, doi = {10.1016/j.ebiom.2019.10.036}, author = {Sn, Suresh and Pandurangi, Janhavi and Murumalla, Ravi and Dj, Vidyadhara and Garimella, Lakshmi and Acharya, Achyuth and Rai, Shashank and Paul, Abhik and Yarreiphang, Haorei and Pillai, Malini S and Giridharan, Mridhula and Clement, James P and Alladi, Phalguni Anand and Saiyed, Taslimarif and Manjithaya, Ravi} } @article {1135, title = {Spoiled for Choice: Diverse Endocytic Pathways Function at the Cell Surface [BLiSC - NCBS, inStem, C-CAMP]}, journal = {Annu Rev Cell Dev Biol}, year = {2019}, month = {2019 Jul 05}, abstract = {

Endocytosis has long been identified as a key cellular process involved in bringing in nutrients, in clearing cellular debris in tissue, in the regulation of signaling, and in maintaining cell membrane compositional homeostasis. While clathrin-mediated endocytosis has been most extensively studied, a number of clathrin-independent endocytic pathways are continuing to be delineated. Here we provide a current survey of the different types of endocytic pathways available at the cell surface and discuss a new classification and plausible molecular mechanisms for some of the less characterized pathways. Along with an evolutionary perspective of the origins of some of these pathways, we provide an appreciation of the distinct roles that these pathways play in various aspects of cellular physiology, including the control of signaling and membrane tension. Expected final online publication date for the Volume 35 is October 7, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

}, issn = {1530-8995}, doi = {10.1146/annurev-cellbio-100617-062710}, author = {Thottacherry, Joseph Jose and Sathe, Mugdha and Prabhakara, Chaitra and Mayor, Satyajit} } @article {1228, title = {Transcriptome analysis reveals plasticity in gene regulation due to environmental cues in Primula sikkimensis, a high altitude plant species [Next Gen Genomics Facility (INT)].}, journal = {BMC Genomics}, volume = {20}, year = {2019}, month = {2019 Dec 17}, pages = {989}, abstract = {

BACKGROUND: Studying plasticity in gene expression in natural systems is crucial, for predicting and managing the effects of climate change on plant species. To understand the contribution of gene expression level variations to abiotic stress compensation in a Himalaya plant (Primula sikkimensis), we carried out a transplant experiment within (Ambient), and beyond (Below Ambient and Above Ambient) the altitudinal range limit of species. We sequenced nine transcriptomes (three each from each altitudinal range condition) using Illumina sequencing technology. We compared the fitness variation of transplants among three transplant conditions.

RESULTS: A large number of significantly differentially expressed genes (DEGs) between below ambient versus ambient (109) and above ambient versus ambient (85) were identified. Transcripts involved in plant growth and development were mostly up-regulated in below ambient conditions. Transcripts involved in signalling, defence, and membrane transport were mostly up-regulated in above ambient condition. Pathway analysis revealed that most of the genes involved in metabolic processes, secondary metabolism, and flavonoid biosynthesis were differentially expressed in below ambient conditions, whereas most of the genes involved in photosynthesis and plant hormone signalling were differentially expressed in above ambient conditions. In addition, we observed higher reproductive fitness in transplant individuals at below ambient condition compared to above ambient conditions; contrary to what we expect from the cold adaptive P. sikkimensis plants.

CONCLUSIONS: We reveal P. sikkimensis{\textquoteright}s capacity for rapid adaptation to climate change through transcriptome variation, which may facilitate the phenotypic plasticity observed in morphological and life history traits. The genes and pathways identified provide a genetic resource for understanding the temperature stress (both the hot and cold stress) tolerance mechanism of P. sikkimensis in their natural environment.

}, issn = {1471-2164}, doi = {10.1186/s12864-019-6354-1}, author = {Gurung, Priya Darshini and Upadhyay, Atul Kumar and Bhardwaj, Pardeep Kumar and Sowdhamini, Ramanathan and Ramakrishnan, Uma} } @article {1084, title = {The transcriptome enables the identification of candidate genes behind medicinal value of Drumstick tree (Moringa oleifera) [Next Gen Genomics Facility (INT)].}, journal = {Genomics}, year = {2019}, month = {2019 Apr 29}, abstract = {

Moringa oleifera is a plant well-known for its nutrition value, drought resistance and medicinal properties. cDNA libraries from five different tissues (leaf, root, stem, seed and flower) of M. oleifera cultivar Bhagya were generated and sequenced. We developed a bioinformatics pipeline to assemble transcriptome, along with the previously published M. oleifera genome, to predict 17,148 gene models. Few candidate genes related to biosynthesis of secondary metabolites, vitamins and ion transporters were identified. Expressions were further confirmed by real-time quantitative PCR experiments for few promising leads. Quantitative estimation of metabolites, as well as elemental analysis, was also carried out to support our observations. Enzymes in the biosynthesis of vitamins and metabolites like quercetin and kaempferol are highly expressed in leaves, flowers and seeds. The expression of iron transporters and calcium storage proteins were observed in root and leaves. In general, leaves retain the highest amount of small molecules of interest.

}, issn = {1089-8646}, doi = {10.1016/j.ygeno.2019.04.014}, author = {Pasha, Shaik Naseer and Shafi, K Mohamed and Joshi, Adwait G and Meenakshi, Iyer and Harini, K and Mahita, Jarjapu and Sajeevan, Radha Sivarajan and Karpe, Snehal D and Ghosh, Pritha and Nitish, Sathyanarayanan and Gandhimathi, A and Mathew, Oommen K and Prasanna, Subramanian Hari and Malini, Manoharan and Mutt, Eshita and Naika, Mahantesha and Ravooru, Nithin and Rao, Rajas M and Shingate, Prashant N and Sukhwal, Anshul and Sunitha, Margaret S and Upadhyay, Atul K and Vinekar, Rithvik S and Sowdhamini, Ramanathan} } @article {1020, title = {Transcriptomics analysis of propiconazole-treated Cochliobolus sativus reveals new putative azole targets in the plant pathogen [Next Gen Genomics Facility].}, journal = {Funct Integr Genomics}, year = {2019}, month = {2019 Mar 06}, abstract = {

Cochliobolus sativus (anamorph: Bipolaris sorokiniana) is a filamentous fungus from the class Dothideomycetes. It is a pathogen of cereals including wheat and barley, and causes foliar spot blotch, root rot, black point on grains, head blight, leaf blight, and seedling blight diseases. Annual yields of these economically important cereals are severely reduced due to this pathogen attack. Evolution of fungicide resistant pathogen strains, availability of a limited number of potent antifungal compounds, and their efficacy are the acute issues in field management of phytopathogenic fungi. Propiconazole is a widely used azole fungicide to control the disease in fields. The known targets of azoles are the demethylase enzymes involved in ergosterol biosynthesis. Nonetheless, azoles have multiple modes of action, some of which have not been explored yet. Identifying the off-target effects of fungicides by dissecting gene expression profiles in response to them can provide insights into their modes of action and possible mechanisms of fungicide resistance. Moreover it can also reveal additional targets for development of new fungicides. Hence, we analyzed the global gene expression profile of C. sativus on exposure to sub-lethal doses of propiconazole in a time series. The gene expression patterns were confirmed using quantitative reverse transcriptase PCR (qRT-PCR). This study revealed overexpression of target genes from the sterol biosynthesis pathway supporting the reported mode of resistance against azoles. In addition, some new potential targets have also been identified, which could be explored to develop new fungicides and plant protection strategies.

}, issn = {1438-7948}, doi = {10.1007/s10142-019-00660-9}, author = {Somani, Deepika and Adhav, Ragini and Prashant, Ramya and Kadoo, Narendra Y} } @article {690, title = {Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. [Protein Technology (INT)]}, year = {2018}, author = {Sneha Bairy and Lakshmi Narayanan Gopalan and Thanuja Gangi Setty and Sathya Srinivasachari and Lavanyaa Manjunath and Jay Prakash Kumar and Sai R Guntupalli and Sucharita Bose and Vinod Nayak and Swagatha Ghosh and Nitish Sathyanarayanan and Rhawnie Caing-Carlsson and Weixiao Yuan Wahlgren and Rosmarie Friemann and S. Ramaswamy and Muniasamy Neerathilingam} } @article {993, title = {Complete assembly of a dengue virus type 3 genome from a recent genotype III clade by metagenomic sequencing of serum.[Next Gen Genomics Facility (INT)]}, journal = {Wellcome Open Res}, volume = {3}, year = {2018}, month = {2018}, pages = {44}, abstract = {

Mosquito-borne flaviviruses, such as dengue and Japanese encephalitis virus (JEV), cause life-threatening diseases, particularly in the tropics. Here we performed unbiased metagenomic sequencing of RNA extracted from the serum of four patients and the plasma of one patient, all hospitalized at a tertiary care centre in South India with severe or prolonged febrile illness, together with the serum from one healthy control, in 2014. We identified and assembled a complete dengue virus type 3 sequence from a case of severe dengue fever. We also identified a small number of JEV sequences in the serum of two adults with febrile illness, including one with severe dengue. Phylogenetic analysis revealed that the dengue sequence belonged to genotype III. It has an estimated divergence time of 13.86 years from the most highly related Indian strains. In total, 11 amino acid substitutions were predicted for this strain in the antigenic envelope protein, when compared to the parent strain used for development of the first commercial dengue vaccine.\  We demonstrate that both genome assembly and detection of a low number of viral sequences are possible through the unbiased sequencing of clinical material. These methods may help ascertain causal agents for febrile illnesses with no known cause.

}, issn = {2398-502X}, doi = {10.12688/wellcomeopenres.14438.2}, author = {Dias, Mary and Pattabiraman, Chitra and Siddappa, Shilpa and Gowda, Malali and Shet, Anita and Smith, Derek and Muehlemann, Barbara and Tamma, Krishnapriya and Solomon, Tom and Jones, Terry and Krishna, Sudhir} } @article {771, title = {Enhanced delignification of lignocellulosic substrates by Pichia GS115 expressed recombinant laccase. [Mass Spectrometry - Proteomics]}, journal = {J Gen Appl Microbiol}, year = {2018}, month = {2018 Apr 25}, abstract = {

Utilization of energy-rich crop residues by ruminants is restricted by the presence of lignin, which is recalcitrant to digestion. Application of lignin degrading enzymes on the lignocellulosic biomass exposes the cellulose for easy digestion by ruminants. Laccases have been found to be considerably effective in improving the digestibility by way of delignification. However, laccase yields from natural hosts are not sufficient for industrial scale applications, which restricts their use. A viable option would be to express the laccase gene in compatible hosts to achieve higher production yields. A codon-optimized synthetic variant of Schizophyllum commune laccase gene was cloned into a pPIC9K vector and expressed in P. pastoris GS115 (his4) under the control of an alcohol oxidase promoter. Colonies were screened for G418 resistance and the methanol utilization phenotype was established. The transformant yielded a laccase activity of 344 U{\textperiodcentered}mL after 5 days of growth at 30{\textdegree}C (0.019 g{\textperiodcentered}mL wet cell weight). The laccase protein produced by the recombinant Pichia clone was detected as two bands with apparent molecular weights of 55 kDa and 70 kDa on SDS-PAGE. Activity staining on native PAGE confirmed the presence of bioactive laccase. Treatment of five common crop residues with recombinant laccase recorded a lignin loss ranging between 1.64\% in sorghum stover, to 4.83\% in finger millet, with an enhancement in digestibility ranging between 8.71\% in maize straw to 24.61\% in finger millet straw. Treatment with recombinant laccase was effective in enhancing the digestibility of lignocellulosic biomass for ruminant feeding through delignification. To date, a number of hosts have been adventured to produce laccase in large quantities, but, to our knowledge, there are no reports of the expression of laccase protein from Schizophyllum commune in Pichia pastoris, and also on the treatment of crop residues using recombinant laccase for ruminant feeding.

}, issn = {1349-8037}, doi = {10.2323/jgam.2017.11.006}, author = {Kumar, Vidya Pradeep and Kolte, Atul P and Dhali, Arindam and Naik, Chandrashekar and Sridhar, Manpal} } @article {686, title = {Exploiting a water network to achieve enthalpy-driven, bromodomain-selective BET inhibitors}, journal = {Bioorganic \& Medicinal Chemistry}, volume = {26}, year = {2018}, pages = {25 - 36}, issn = {0968-0896}, doi = {https://doi.org/10.1016/j.bmc.2017.10.042}, url = {http://www.sciencedirect.com/science/article/pii/S0968089617315948}, author = {William R. Shadrick and Peter J. Slavish and Sergio C. Chai and Brett Waddell and Michele Connelly and Jonathan A. Low and Cynthia Tallant and Brandon M. Young and Nagakumar Bharatham and Stefan Knapp and Vincent A. Boyd and Marie Morfouace and Martine F. Roussel and Taosheng Chen and Richard E. Lee and R. Kiplin Guy and Anang A. Shelat and Philip M. Potter} } @article {888, title = {Gene expression is implicated in the ability of pikas to occupy Himalayan elevational gradient. [Next Gen Genomics Facility (INT)]}, journal = {PLoS One}, volume = {13}, year = {2018}, month = {2018}, pages = {e0207936}, abstract = {

Species are shifting their ranges due to climate change, many moving to cooler and higher locations. However, with elevation increase comes oxygen decline, potentially limiting a species{\textquoteright} ability to track its environment depending on what mechanisms it has available to compensate for hypoxic stress. Pikas (Family Ochotonidae), cold-specialist small mammal species, are already undergoing elevational range shifts. We collected RNA samples from one population of Ochotona roylei in the western Himalaya at three sites- 3,600, 4,000, and 5,000 meters-and found no evidence of significant population genetic structure nor positive selection among sites. However, out of over 10,000 expressed transcripts, 26 were significantly upregulated at the 5,000 m site and were significantly enriched for pathways consistent with physiological compensation for limited oxygen. These results suggest that differences in gene expression may play a key role in enabling hypoxia tolerance on this local scale, indicating elevational flexibility that may facilitate successful range shifts in response to climate change.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0207936}, author = {Solari, Katherine A and Ramakrishnan, Uma and Hadly, Elizabeth A} } @article {1062, title = {Generation of Transplantable Retinal Pigmented Epithelial (RPE) Cells for Treatment of Age-Related Macular Degeneration (AMD). [Eyestem, a C-CAMP Startup]}, journal = {Methods Mol Biol}, year = {2018}, month = {2018 Jun 13}, abstract = {

Age-related macular degeneration (AMD) is the foremost cause of blindness in people over the age of 60 worldwide. Clinically, this disease starts with distortion in central vision eventually leading to legal blindness. Vision loss has a significant impact on quality of life and incurs a substantial cost to the economy. Furthermore, AMD is a complex and progressive neurodegenerative disorder that triggers visual impairment due to the loss of retinal pigmented epithelium (RPE) and the light-sensitive photoreceptors that they support, protect and provide nutrition. Currently, there is no curative treatment for the most common form of this disease, i.e., dry AMD. A novel approach to treat AMD involves the transplantation of RPE cells derived from human induced pluripotent stem cells (iPSCs) in the outer retina. These iPSC-derived RPE cells not only show characteristics similar to native RPE but also could replace as well as regenerate damaged pathologic RPE and produce supportive growth factors and cytokines. Several clinical trials are being conducted taking advantage of a variety of cell- and tissue engineering-based approaches. Here, we present a simple, cost effective, and scalable cell-culture model for generation of purified RPE thus providing the foundation for developing an allogeneic cell therapy for AMD.

}, issn = {1940-6029}, doi = {10.1007/7651_2018_140}, author = {Surendran, Harshini and Rathod, Reena J and Pal, Rajarshi} } @article {775, title = {Genome-wide differential expression profiling in wild and cultivar genotypes of cardamom reveals regulation of key pathways in plant growth and development [Next Gen Genomics Facility]}, journal = {Agri Gene}, volume = {8}, year = {2018}, pages = {18 - 27}, keywords = {Cardamom, Differential expression, Down regulation, Essential oil biosynthesis, Transcriptome, Up regulation}, issn = {2352-2151}, doi = {https://doi.org/10.1016/j.aggene.2018.03.002}, url = {http://www.sciencedirect.com/science/article/pii/S2352215118300060}, author = {F Nadiya and N Anjali and Jinu Thomas and AGangaprasad and K K Sabu} } @article {1013, title = {Highly Sensitive LC-MS/MS-ESI method Development for the Determination of 5,7-dihydroxyflavone in Mouse Plasma and Pharmacokinetic Study in Lean and Diet Induced Obese Mice [High Throughput Screening Facility].}, journal = {Der Pharmacia Lettre}, volume = {10}, year = {2018}, type = {Research Article}, chapter = {1-12}, abstract = {

Diet-induced obese (DIO) mice have been commonly used extensively as an animal model of obesity and diabetes in the efficacy assessment for new drug candidates. Physiological and biochemical alterations are reported in DIO mice due to modulations of drug-metabolizing enzymes and high calorie intake. Limited studies have been reported regarding the effect of obesity/diabetes on pharmacokinetics (PK) in animals. A simple, specific and sensitive rapid LC-ESI-MS/MS method has been developed and validated for the quantification of chrysin (5,7-dihydroxyflavone) using tolbutamide as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a simple protein precipitation. Chromatographic separation of 5,7-dihydroxyflavone and IS was performed on Atlantis C-18 column using an isocratic mobile phase comprising 0.2\% formic acid in water and acetonitrile (20:80, v/v) at a flow rate of 0.9 mL/min. Elution of 5,7-dihydroxyflavone and IS occurred at ~2.49 and 2.34 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 4.50-4500 ng/mL. This novel method has been applied to a pharmacokinetic study in lean and DIO mice.

}, author = {Amir Siddiqui and . Badruddeen and Juber Akhtar and Shahab Uddin MS and Chandrashekaran S and Mohammad Irfan Khan and Mohammad Khalid} } @article {769, title = {Identification and characterization of a calcium dependent bacillopeptidase from Bacillus subtilis CFR5 with novel kunitz trypsin inhibitor degradation activity. [Mass Spectrometry Facility - Proteomics]}, journal = {Food Res Int}, volume = {103}, year = {2018}, month = {2018 Jan}, pages = {263-272}, abstract = {

The cereals and pulses are considered to be an important component in the food chain due to their proteinaceous nature, but the presence of anti-nutritional factors (KTI) decreases their nutrient absorption rate. Kunitz trypsin inhibitors (KTI) reduce the bioavailability of trypsin and are the primary cause for the existence of various metabolic disorders. To overcome the inhibitory effect of KTI, a KTI degrading protein (BPC) was identified and characterized from Bacillus subtilis CFR5. BPC possesses 60\% identity with bacillopeptidase of B. subtilis 168. BPC cleaves at DFVLD and DFFNNY sites of KTI which results in the formation of three inactive KTI fragments. Subsequently, BPC was cloned in pHY300PLK and recombinant protein was used for the biochemical characterization, sequence alignment and mutational studies. The optimal temperature and pH of the BPC was 40{\textdegree}C and 8.0, respectively. BPC is a calcium dependent metalloprotease and its activity was significantly increased by 41.2-fold in the presence of 2.5mM Ca. BPC also showed moderate thermostability with the half-life of 4h at 55{\textdegree}C. Site directed mutagenesis studies in recombinant BPC revealed that mutation of Tyr with Phe, Tyr with Phe, and Pro to Arg affects the catalytic activity without affecting the conformation of BPC. Hence, Tyr, Tyr and Pro were identified as the unique residues responsible for KTI cleavage. Thus, this study leads to the identification of a novel KTI degrading protease from B. subtilis CFR5 which cleaves and deactivates the kunitz trypsin inhibitor.

}, issn = {1873-7145}, doi = {10.1016/j.foodres.2017.10.049}, author = {Sharmila, Giliyaru Ramachandra and Halami, Prakash Motiram and Venkateswaran, Gonvindarajalu} } @article {890, title = {Identification and characterization of drought responsive microRNAs and their target genes in cardamom (Elettaria cardamomum Maton) [Next Gen Genomics Facility]}, journal = {Plant Growth Regulation}, year = {2018}, month = {Dec}, abstract = {

Plant miRNAs are found to be present throughout the genome and they regulate gene expression either by cleaving mRNA or inhibiting the translational process at the post transcriptional level. Drought is one of the major limiting factors that negatively affect productivity of plants. Cardamom cultivation is having good production potential but the plants are vulnerable to biotic and abiotic stress factors. To date, nothing is known about the regulatory roles of miRNAs in response to drought stress in cardamom. Ion Torrent sequencing of two small RNA libraries prepared from control (C) and treated (T) plants raised under well irrigated and drought stressed treatments respectively created 3,938,342 and 4,083,181 primary reads. A total of 150 conserved and 20 novel microRNAs were identified from both the control and treated libraries. Discovery of 17 differentially expressed miRNAs under drought stress suggests that these miRNAs might have involved in various biological processes to improve plant tolerance to water stress. Several target genes for drought stress regulating miRNAs were identified including miR156l and miR169c which cleave the target mRNA involved in response to water deprivation. miR530b and miR156a target mRNAs which respond to water deprivation and inhibit the translational process. The expression patterns of some of the miRNAs and their targets were validated by qRT-PCR. This study is the first report of drought responsive miRNAs and their targets in cardamom. The outcome of this research could provide insights into the miRNA based regulatory mechanisms in response to drought stress in monocot plants.

}, issn = {1573-5087}, doi = {10.1007/s10725-018-0462-9}, url = {https://doi.org/10.1007/s10725-018-0462-9}, author = {Anjali, N. and Nadiya, F. and Thomas, Jinu and Sabu, K. K.} } @article {765, title = {Identification of multiple isomeric core chitobiose-modified high-mannose and paucimannose -glycans in the planarian. [Mass Spectrometry Facility - Glycomics (INT)]}, journal = {J Biol Chem}, volume = {293}, year = {2018}, month = {2018 May 04}, pages = {6707-6720}, abstract = {

Cell surface-associated glycans mediate many cellular processes, including adhesion, migration, signaling, and extracellular matrix organization. The galactosylation of core fucose (GalFuc epitope) in paucimannose and complex-type -glycans is characteristic of protostome organisms, including flatworms (planarians). Although uninvestigated, the structures of these glycans may play a role in planarian regeneration. Whole-organism MALDI-MS analysis of -linked oligosaccharides from the planarian revealed the presence of multiple isomeric high-mannose and paucimannose structures with unusual mono-, di-, and polygalactosylated ( = 3-5) core fucose structures; the latter structures have not been reported in other systems. Di- and trigalactosylated core fucoses were the most dominant glycomers. -Glycans showed extensive, yet selective, methylation patterns, ranging from non-methylated to polymethylated glycoforms. Although the majority of glycoforms were polymethylated, a small fraction also consisted of non-methylated glycans. Remarkably, monogalactosylated core fucose remained unmethylated, whereas its polygalactosylated forms were methylated, indicating structurally selective methylation. Using database searches, we identified two potential homologs of the Galβ1-4Fuc-synthesizing enzyme from nematodes (GALT-1) that were expressed in the prepharyngeal, pharyngeal, and mesenchymal regions in The presence of two GALT-1 homologs suggests different requirements for mono- and polygalactosylation of core fucose for the formation of multiple isomers. Furthermore, we observed variations in core fucose glycosylation patterns in different planarian strains, suggesting evolutionary adaptation in fucose glycosylation. The various core chitobiose modifications and methylations create \>60 different glycoforms in These results contribute greatly to our understanding of -glycan biosynthesis and suggest the presence of a GlcNAc-independent biosynthetic pathway in

}, issn = {1083-351X}, doi = {10.1074/jbc.RA117.000782}, author = {Subramanian, Sabarinath Peruvemba and Babu, Ponnusamy and Palakodeti, Dasaradhi and Subramanian, Ramaswamy} } @article {766, title = {Integrated transcriptomic and proteomic analyses~suggest the participation of endogenous protease inhibitors in the regulation of protease gene expression in [Next Gen Genomics Facility]}, journal = {Mol Cell Proteomics}, year = {2018}, month = {2018 Apr 16}, abstract = {

Insects adapt to plant protease inhibitors (PIs) present in their diet by differentially regulating multiple digestive proteases. However, mechanisms regulating protease gene expression in insects are largely enigmatic. Ingestion of multi-domain recombinant Capsicum annuum protease inhibitor-7 (CanPI-7) arrests growth and development of Helicoverpa armigera (Lepidoptera: Noctuidae). Using de novo RNA sequencing and proteomic analysis, we examined the response of H. armigera larvae fed on recombinant CanPI-7 at different time intervals. Here, we present evidence supporting a dynamic transition in H. armigera protease expression upon CanPI-7 feeding with general down-regulation of protease genes at early time points (0.5 to 6 h) and significant up-regulation of specific trypsin, chymotrypsin and aminopeptidase genes at later time points (12 to 48 h). Further, co-expression of H. armigera endogenous PIs with several digestive protease genes were apparent. In addition to the differential expression of endogenous H. armigera PIs, we also observed a distinct novel isoform of endogenous PI in CanPI-7 fed H. armigera larvae. Based on present and earlier studies, we propose potential mechanism of protease regulation in H. armigera and subsequent adaptation strategy to cope with anti-nutritional components of plants.

}, issn = {1535-9484}, doi = {10.1074/mcp.RA117.000533}, author = {Lomate, Purushottam R and Dewangan, Veena and Mahajan, Neha and Kumar, Yashwant and Kulkarni, Abhijeet and Wang, Li and Saxsena, Smita and Gupta, Vidya S and Giri, Ashok P} } @article {846, title = {Isolation and characterization of two lytic bacteriophages against Staphylococcus aureus from India: newer therapeutic agents against Bovine mastitis. [Electron Microscopy Facility]}, journal = {Vet Res Commun}, year = {2018}, month = {2018 Sep 15}, abstract = {

Bovine mastitis causes severe economic losses to dairy farmers. Staphylococcus aureus, is one of the most important pathogen implicated in etiology of clinical and subclinical mastitis in bovines. In view of increasing antimicrobial resistance alternatives to antibiotic therapy are much needed. The present decade has witnessed a renewed interest in phage based therapeutics and diagnostics. The present study, describes isolation and characterization of two lytic phages SAJK-IND and MSP against Staphylococcus aureus having a potential to be used in therapy against mastitis. SAJK-IND and MSP phages belonged to Myoviridae and Podoviridae families, respectively. TEM imaging of the two phages revealed an iscosahedral head. MSP phage has a short non contractile tail. SAJK-IND and MSP have a burst size of 44 {\textpm} 3 and 25 {\textpm} 5 PFU/ infected cell, respectively. SAJK-IND and MSP phages revealed ̴ 12 and ̴16 proteins, respectively on SDS-PAGE analysis. The lytic activity of the phages was specific for Staphylococcus aureus. SAJK-IND revealed 100\% lytic activity against several strains of Staphylococcus aureus isolated from mastitis milk samples whereas, MSP had only 40\% lytic activity. SAJK-IND phage genome was sequenced, assembled and deposited in Genbank under accession no MG010123.

}, issn = {1573-7446}, doi = {10.1007/s11259-018-9736-y}, author = {Ganaie, M Y and Qureshi, S and Kashoo, Z and Wani, S A and Hussain, M I and Kumar, R and Maqbool, R and Sikander, P and Banday, M S and Malla, W A and Mondal, P and Khan, R I N} } @article {1015, title = {Mechanochemical feedback control of dynamin independent endocytosis modulates membrane tension in adherent cells. [Microfluidics and Microfabrication Facility (INT)]}, journal = {Nat Commun}, volume = {9}, year = {2018}, month = {2018 10 11}, pages = {4217}, abstract = {

Plasma membrane tension regulates many key cellular processes. It is modulated by, and can modulate, membrane trafficking. However, the cellular pathway(s) involved in this interplay is poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon a sudden reduction of tension. Moreover, inhibition (activation) of the CG pathway results in lower (higher) membrane tension. However, alteration in membrane tension does not directly modulate CG endocytosis. This requires vinculin, a mechano-transducer recruited to focal adhesion in adherent cells. Vinculin acts by controlling the levels of a key regulator of the CG pathway, GBF1, at the plasma membrane. Thus, the CG pathway directly regulates membrane tension and is in turn controlled via a mechano-chemical feedback inhibition, potentially leading to homeostatic regulation of membrane tension in adherent cells.

}, keywords = {Animals, Biomechanical Phenomena, Cell Adhesion, Cell Membrane, Dynamins, Endocytosis, Feedback, Physiological, Mechanotransduction, Cellular, Mice, Signal Transduction, Temperature, Vinculin}, issn = {2041-1723}, doi = {10.1038/s41467-018-06738-5}, author = {Thottacherry, Joseph Jose and Kosmalska, Anita Joanna and Kumar, Amit and Vishen, Amit Singh and Elosegui-Artola, Alberto and Pradhan, Susav and Sharma, Sumit and Singh, Parvinder P and Guadamillas, Marta C and Chaudhary, Natasha and Vishwakarma, Ram and Trepat, Xavier and Del Pozo, Miguel A and Parton, Robert G and Rao, Madan and Pullarkat, Pramod and Roca-Cusachs, Pere and Mayor, Satyajit} } @article {869, title = {Methionine coordinates a hierarchically organized anabolic program enabling proliferation. [Mass Spectrometry - Lipidomics \& Metabolomics and Next Gen Sequencing Facilities (INT)]}, journal = {Mol Biol Cell}, year = {2018}, month = {2018 Oct 24}, pages = {mbcE18080515}, abstract = {

Methionine availability during overall amino acid limitation metabolically reprograms cells to support proliferation, the underlying basis for which remains unclear. Here, we construct the organization of this methionine mediated anabolic program, using yeast. Combining comparative transcriptome analysis, biochemical and metabolic flux based approaches, we discover that methionine rewires overall metabolic outputs by increasing the activity of a key regulatory node. This comprises of: the pentose phosphate pathway (PPP) coupled with reductive biosynthesis, the glutamate dehydrogenase (GDH) dependent synthesis of glutamate/glutamine, and pyridoxal-5-phosphate (PLP) dependent transamination capacity. This PPP-GDH-PLP node provides the required cofactors and/or substrates for subsequent rate-limiting reactions in the synthesis of amino acids, and therefore nucleotides. These rate-limiting steps in amino acid biosynthesis are also induced in a methionine-dependent manner. This thereby results in a biochemical cascade establishing a hierarchically organized anabolic program. For this methionine mediated anabolic program to be sustained, cells co-opt a "starvation stress response" regulator, Gcn4p. Collectively, our data suggest a hierarchical metabolic framework explaining how methionine mediates an anabolic switch.

}, issn = {1939-4586}, doi = {10.1091/mbc.E18-08-0515}, author = {Walvekar, Adhish S and Srinivasan, Rajalakshmi and Gupta, Ritu and Laxman, Sunil} } @article {1012, title = {A Naturally Occurring Flavone (Chrysin): Chemistry, Occurrence, Pharmacokinetic, Toxicity, Molecular Targets and Medicinal Properties [High Throughput Screening Facility].}, journal = {Journal of Biologically Active Products from Nature}, volume = {8}, year = {2018}, pages = {208-227}, doi = {10.1080/22311866.2018.1498750}, url = {https://doi.org/10.1080/22311866.2018.1498750}, author = {Amir Siddiqui and . Badruddeen and Juber Akhtar and Shahab Uddin M.S. and Mohammad Irfan Khan and Mohammad Khalid and Mohammad Ahmad} } @article {764, title = {Nitrothiophene carboxamides, a novel narrow spectrum antibacterial series: Mechanism of action and Efficacy [Bugworks Res. Pvt. Ltd., a C-CAMP Startup]}, journal = {Sci Rep}, volume = {8}, year = {2018}, month = {2018 May 08}, pages = {7263}, abstract = {

The mechanism of efflux is a tour-de-force in the bacterial armoury that has thwarted the development of novel antibiotics. We report the discovery of a novel chemical series with potent antibacterial properties that was engineered to overcome efflux liability. Compounds liable to efflux specifically via the Resistance Nodulation and cell Division (RND) pump, AcrAB-TolC were chosen for a hit to lead progression. Using structure-based design, the compounds were optimised to lose their binding to the efflux pump, thereby making them potent on wild-type bacteria. We discovered these compounds to be pro-drugs that require activation in E. coli by specific bacterial nitroreductases NfsA and NfsB. Hit to lead chemistry led to the generation of compounds that were potent on wild-type and multi-drug resistant clinical isolates of E. coli, Shigella spp., and Salmonella spp. These compounds are bactericidal and efficacious in a mouse thigh infection model.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-25407-7}, author = {Hameed P, Shahul and Bharatham, Nagakumar and Katagihallimath, Nainesh and Sharma, Sreevalli and Nandishaiah, Radha and Shanbhag, Anirudh P and Thomas, Teby and Narjari, Riya and Sarma, Maitrayee and Bhowmik, Purnendu and Amar, Prakruthi and Ravishankar, Rajani and Jayaraman, Ramesh and Muthan, Kubendran and Subbiah, Ramesh and Ramachandran, Vasanthi and Balasubramanian, V and Datta, Santanu} } @article {1018, title = {Pharmacokinetics of colistin in patients with multidrug-resistant Gram-negative infections: A pilot study [Mass Spectrometry - Metabolomics Facility].}, journal = {Indian J Med Res}, volume = {147}, year = {2018}, month = {2018 04}, pages = {407-412}, abstract = {

Background \& objectives: There is little information concerning intravenously (i.v.) administered colistin in patients with multidrug-resistant (MDR) Gram-negative infections. Thus, this pilot prospective study was undertaken to characterize efficacy and pharmacokinetics of colistin in patients with MDR Gram-negative infections.

Methods: Nine patients with age \>12 yr and MDR Gram-negative infections were included, of whom six were given colistin at the doses of 2 MU, while three patients were given 1 MU i.v. dose every 8 h. Blood samples were collected at different time intervals. Determination of colistin concentration was done by a ultra-high-performance liquid chromatography/mass spectrometry/selected reaction monitoring assay.

Results: The area under the plasma concentration-versus-time curve over eight hours (AUC) for colistin after the 1 dose ranged from 3.3 to 16.4 mg{\texttimes}h/l (median, 4.59). After the 5 dose, AUCfor colistin ranged from 4.4 to 15.8 mg{\texttimes}h/l (median, 6.0). With minimal inhibitory concentration (MIC) value of 0.125 mg/l, AUC/MIC ranged from 26.7 to 131.4 (median, 36.7) and 35.5 to 126.0 (median, 48.0) after the 1 and the 5 doses of 2 MU every 8 h, respectively.

Interpretation \& conclusions: As there is a paucity of information on AUC/MIC for colistin, it may not be possible to conclude whether AUC/MIC values in our patients were adequate. There is a microbiological clearance of organism, which goes in favour of the dosing schedule being adequate. Further studies need to be done to understand the pharmacokinetics of colistin in patients with infections.

}, keywords = {Anti-Bacterial Agents, Colistin, Drug Resistance, Multiple, Bacterial, Gram-Negative Bacterial Infections, Humans, Pilot Projects, Prospective Studies}, issn = {0971-5916}, doi = {10.4103/ijmr.IJMR_1464_16}, author = {Gautam, Vikas and Shafiq, Nusrat and Mouton, Johan W and Malhotra, Sameer and Kaur, Satinder and Ray, Pallab} } @article {865, title = {Prevention of pesticide-induced neuronal dysfunction and mortality with nucleophilic poly-Oxime topical gel. [BIG Grant Supported Entrepreneur]}, journal = {Sci Adv}, volume = {4}, year = {2018}, month = {2018 Oct}, pages = {eaau1780}, abstract = {

Organophosphate-based pesticides inhibit acetylcholinesterase (AChE), which plays a pivotal role in neuromuscular function. While spraying in the field, farmworkers get exposed to pesticides through the dermal route. Internalized pesticide inhibits AChE, which leads to neurotoxicity, cardiotoxicity, cognitive dysfunction, loss of endurance, and death in severe cases. Here, we present a nucleophilic pyridine-2-aldoxime-functionalized chitosan-based topical gel (-Oxime gel) that rapidly deactivates organophosphates, methyl parathion (MPT), on the skin of rats, which leads to reduced AChE inhibition in the blood and tissues. Testing the robustness of -Oxime gel, we report reduction in AChE inhibition following repeated dermal administration of MPT in the presence of -Oxime gel. Furthermore, -Oxime gel prevented MPT-induced neuromuscular dysfunction, loss of endurance, and locomotor coordination. We observe a 100\% survival in rats following topical MPT administration in the presence of -Oxime gel. This prophylactic gel may therefore help farmworkers by limiting pesticide-induced toxicity and mortality.

}, issn = {2375-2548}, doi = {10.1126/sciadv.aau1780}, author = {Thorat, Ketan and Pandey, Subhashini and Chandrashekharappa, Sandeep and Vavilthota, Nikitha and Hiwale, Ankita A and Shah, Purna and Sreekumar, Sneha and Upadhyay, Shubhangi and Phuntsok, Tenzin and Mahato, Manohar and Mudnakudu-Nagaraju, Kiran K and Sunnapu, Omprakash and Vemula, Praveen K} } @article {824, title = {A proteomic approach of biomarker candidate discovery for alcoholic liver cirrhosis [Mass Spectrometry - Proteomics Facility].}, journal = {J Circ Biomark}, volume = {7}, year = {2018}, month = {2018 Jan-Dec}, pages = {1849454418788417}, abstract = {

Alcoholic liver disease (ALD) progresses from steatosis to alcoholic hepatitis to fibrosis and cirrhosis. Liver biopsy is considered as the gold standard method for diagnosis of liver cirrhosis and provides useful information about damaging process which is an invasive procedure with complications. Existing biomarkers in clinical practice have narrow applicability due to lack of specificity and lack of sensitivity. The objective of this article is to identify proteomic biomarker candidates for alcoholic liver cirrhosis by differential expression analysis between alcoholic liver cirrhotic and healthy subjects. Blood samples were collected from 20 subjects (10 alcoholic liver cirrhosis and 10 healthy) from R. L. Jalapa Hospital and Research Centre, Kolar, Karnataka, India. Differential protein analysis was carried out by two-dimensional electrophoresis after albumin depletion, followed by liquid chromatography-mass spectrometry. The image analysis found 46 spots in cirrhotic gel and 69 spots in healthy gel, of which 14 spots were identified with significant altered expression levels. Based on the protein score and clinical significance, among 14 spots, a total of 28 protein biomarker candidates were identified: 13 with increased expression and 15 with decreased expression were categorized in alcoholic liver cirrhosis compared to healthy subjects. Protein biomarker candidates identified by "-omics" approach based on differential expression between alcoholic liver cirrhotic subjects and healthy subjects may give better insights for diagnosis of ALD. Prioritization of candidates identified is a prerequisite for validation regimen. Biomarker candidates require verification that demonstrates the differential expression will remain detectable by assay to be used for validation.

}, issn = {1849-4544}, doi = {10.1177/1849454418788417}, author = {Nallagangula, Krishna Sumanth and Lakshmaiah, V and Muninarayana, C and Deepa, K V and Shashidhar, K N} } @article {814, title = {Recovery of Five Complete Influenza A(H1N1)pdm09 Genome Sequences from the 2015 Influenza Outbreak in India by Metagenomic Sequencing. [Next Gen Genomics Facility (INT)]}, journal = {Genome Announc}, volume = {6}, year = {2018}, month = {2018 Jun 28}, abstract = {

Five complete (H1N1)pdm09 viral sequences were recovered from hospitalized individuals during the 2015 influenza outbreak by metagenomic sequencing. Four of the genomes are from oropharyngeal swabs, and one is from an isolate. All five sequences belong to an emerging 6B clade. Studying them further is critical for outbreak preparedness.

}, issn = {2169-8287}, doi = {10.1128/genomeA.00511-18}, author = {Dash, Paban Kumar and Pattabiraman, Chitra and Tandel, Kundan and Sharma, Shashi and Kumar, Jyoti S and Siddappa, Shilpa and Gowda, Malali and Krishna, Sudhir and Parida, Manmohan} } @article {767, title = {Regulation of Global Transcription in by Rsd and 6S RNA. [Next Gen Genomics Facility (INT)]}, journal = {G3 (Bethesda)}, year = {2018}, month = {2018 Apr 23}, abstract = {

In , the sigma factor σ directs RNA polymerase to transcribe growth-related genes, while σ directs transcription of stress response genes during stationary phase. Two molecules hypothesized to regulate RNA polymerase are the protein Rsd, which binds to σ, and the non-coding 6S RNA which binds to the RNA polymerase-σ holoenzyme. Despite multiple studies, the functions of Rsd and 6S RNA remain controversial. Here we use RNA-Seq in five phases of growth to elucidate their function on a genome-wide scale. We show for the first time that Rsd and 6S RNA facilitate σ activity throughout bacterial growth, while 6S RNA also regulates widely different genes depending upon growth phase. We discover novel interactions between 6S RNA and Rsd and show widespread expression changes in a strain lacking both regulators. Finally, we present a mathematical model of transcription which highlights the crosstalk between Rsd and 6S RNA as a crucial factor in controlling sigma factor competition and global gene expression.

}, issn = {2160-1836}, doi = {10.1534/g3.118.200265}, author = {Lal, Avantika and Krishna, Sandeep and Seshasayee, Aswin Sai Narain} } @article {742, title = {The role of ZA channel water-mediated interactions in the design of bromodomain-selective BET inhibitors.}, journal = {J Mol Graph Model}, volume = {81}, year = {2018}, month = {2018 Mar 22}, pages = {197-210}, abstract = {

The Bromodomain and Extra-Terminal domain (BET) family of proteins are involved in the regulation of gene transcription, and their dysregulation is implicated in several diseases including cancer. BET proteins contain two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine residues and appear to have distinct biological roles. We compared several published co-crystal structures and found five positions near the substrate binding pocket that vary between BET bromodomains. One position located in the ZA loop has unique properties. In BRD2-4, this residue is glutamine in BD1 and lysine in BD2; in BRDT, this residue is arginine in BD1 and asparagine in BD2. Using molecular modeling, we identified differences in the water-mediated network at this position between bromodomains. Molecular dynamics simulations helped rationalize the observed bromodomain selectivity for exemplar BET inhibitors and a congeneric series of tetrahydroquinolines (THQ) that differed by a single heteroatom near the ZA channel. The 2-furan SJ830599, the most BD2-selective THQ analog, did not disrupt the water-mediated networks in either domain, but was electrostatically-repulsed by the specific arrangement of the W5 water dipole in BD1. Our work underscores the value of exploring water-mediated interactions to study ligand binding, and highlights the difficulty of optimizing polar interactions due to high desolvation penalties. Finally, we suggest further modifications to THQ-based BET inhibitors that would increase BD2-selectivity in BRD2-4, while minimizing affinity for one or both bromodomains of BRDT.

}, issn = {1873-4243}, doi = {10.1016/j.jmgm.2018.03.005}, author = {Bharatham, Nagakumar and Slavish, Peter J and Young, Brandon M and Shelat, Anang A} } @article {889, title = {A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation [Bugworks Res. Pvt. Ltd., a C-CAMP Startup]}, journal = {Microb Cell Fact}, volume = {17}, year = {2018}, month = {2018 Dec 03}, pages = {192}, abstract = {

INTRODUCTION: Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E.\ coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure\ to predict product chirality.

RESULTS: Six enzymes originating from Sulfolobus\ sulfotaricus, Zygosaccharomyces\ rouxii, Hansenula\ polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC\ 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole-cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E.\ coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z.\ rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565\ (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95\% (S)-specific alcohol with a chemical yield of 23-79\% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85-99\%.

CONCLUSIONS: Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces\ rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole-cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield.

}, issn = {1475-2859}, doi = {10.1186/s12934-018-1036-2}, author = {Haq, Saiful F and Shanbhag, Anirudh P and Karthikeyan, Subbulakshmi and Hassan, Imran and Thanukrishnan, Kannan and Ashok, Abhishek and Sukumaran, Sunilkumar and Ramaswamy, S and Bharatham, Nagakumar and Datta, Santanu and Samant, Shalaka and Katagihallimath, Nainesh} } @article {683, title = {Combinatorial action of Grainyhead, Extradenticle and Notch in regulating Hox mediated apoptosis in Drosophila larval CNS.}, journal = {PLoS Genet}, volume = {13}, year = {2017}, month = {2017 Oct}, pages = {e1007043}, abstract = {

Hox mediated neuroblast apoptosis is a prevalent way to pattern larval central nervous system (CNS) by different Hox genes, but the mechanism of this apoptosis is not understood. Our studies with Abdominal-A (Abd-A) mediated larval neuroblast (pNB) apoptosis suggests that AbdA, its cofactor Extradenticle (Exd), a helix-loop-helix transcription factor Grainyhead (Grh), and Notch signaling transcriptionally contribute to expression of RHG family of apoptotic genes. We find that Grh, AbdA, and Exd function together at multiple motifs on the apoptotic enhancer. In vivo mutagenesis of these motifs suggest that they are important for the maintenance of the activity of the enhancer rather than its initiation. We also find that Exd function is independent of its known partner homothorax in this apoptosis. We extend some of our findings to Deformed expressing region of sub-esophageal ganglia where pNBs undergo a similar Hox dependent apoptosis. We propose a mechanism where common players like Exd-Grh-Notch work with different Hox genes through region specific enhancers to pattern respective segments of larval central nervous system.

}, keywords = {Amino Acid Sequence, Animals, Apoptosis, Central Nervous System, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Enhancer Elements, Genetic, Female, Gene Expression Regulation, Developmental, Genes, Homeobox, Homeodomain Proteins, Larva, Male, Nuclear Proteins, Receptors, Notch, Transcription Factors}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1007043}, author = {Khandelwal, Risha and Sipani, Rashmi and Govinda Rajan, Sriivatsan and Kumar, Raviranjan and Joshi, Rohit} } @article {8903, title = {Comparative sperm protein profiling in bulls differing in fertility and identification of phosphatidylethanolamine-binding protein 4, a potential fertility marker [Mass Spectrometry - Proteomics]}, journal = {Andrology}, volume = {5}, year = {2017}, month = {2017 Sep}, pages = {1032-1051}, abstract = {

This study aimed to identify sperm proteomic signatures regulating sperm functions and fertility by: (i) comparing the sperm electrophoretic protein profiles and identifying the differentially abundant proteins among breeding bulls differing in fertility status and (ii) elucidating the possible role of one of the identified novel proteins, PEBP4 on sperm function and fertility. The grouping of bulls as fertile (n\ =\ 6) and low fertile (n\ =\ 6) was performed based on bull fertility index and infertile (n\ =\ 6) based on semen rejection rate (\>33\%). The sperm motility, fructolysis index, acrosomal reaction, intracellular calcium levels, and seminal plasma fructose and calcium levels were studied among fertility groups. The differentially expressed sperm proteins observed in single- and two-dimensional gel electrophoresis (2DE) were identified using Nano-LC-MS/MS. In the fertile bulls, the expression levels of calmodulin (CALM1), spermadhesinZ13 (SPADH2), and phosphatidylethanolamine-binding protein 4 (PEBP4) were significantly (p\ \<\ 0.05) higher than in other fertility groups. In bovine, expression of PEBP4 a novel seminal protein was not observed in spermatozoa of infertile bulls. When the bulls were grouped based on the presence (n\ =\ 8) or absence (n\ =\ 10) of PEBP4 protein in spermatozoa, a positive significant (p\ \<\ 0.05) association of this protein with the percentage of motile, type-A spermatozoa, and sperm fructose uptake was observed. Further, PEBP4 was localized in elongated spermatids, Leydig cells, excurrent duct system, and principal piece of spermatozoa. These findings suggest a crucial role for the PEBP4 protein in spermiogenesis, epididymal sperm maturation, and sperm motility. This first study in bovine indicates the positive association of PEBP4 in regulating sperm maturation, functions, and fertility and could be a potential marker for predicting semen quality and fertility.

}, keywords = {Acrosome Reaction, Animals, Biomarkers, Calcium Signaling, Cattle, Cattle Diseases, Epididymis, Fertility, Fructose, Infertility, Male, Male, Phosphatidylethanolamine Binding Protein, Proteome, Semen, Sperm Maturation, Sperm Motility, Spermatozoa}, issn = {2047-2927}, doi = {10.1111/andr.12404}, author = {Somashekar, L and Selvaraju, S and Parthipan, S and Patil, S K and Binsila, B K and Venkataswamy, M M and Karthik Bhat, S and Ravindra, J P} } @article {892, title = {Data on identification of conserved and novel miRNAs in Elettaria cardamomum [Next Gen Genomics Facility]}, journal = {Data Brief}, volume = {14}, year = {2017}, month = {2017 Oct}, pages = {789-792}, abstract = {

(L.) Maton, or small cardamom referred as {\textquoteright}queen of spices{\textquoteright}, is a perennial herbaceous rhizomatous monocot of the family Zingiberaceae. Cardamom seeds and fruits are the economically significant parts and effectively used as a traditional medicine, food additive and flavoring agent. In the present study, using Ion Proton next generation sequencing technology we performed the small RNA sequencing, conserved and novel miRNA predictions of a wild and five cultivar genotypes of cardamom. Small RNA sequencing generated a total of 5,451,328 and 2,756,250 raw reads for wild and cultivar cardamom respectively. The raw data was submitted to SRA database of NCBI under the accession numbers and SRX2273863 (wild) and SRX2273862 (cultivars). The raw reads were quality filtered and predicted conserved and novel miRNAs for wild and cultivar cardamom. The predicted miRNAs, miRNA-targets and functional annotations might provide valuable insights into differences between wild progenitor and cultivated cardamom.

}, issn = {2352-3409}, doi = {10.1016/j.dib.2017.08.037}, author = {Nadiya, F and Anjali, N and Thomas, Jinu and Gangaprasad, A and Sabu, K K} } @article {524, title = {Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii [Bugworks Res. Pvt. Ltd., a C-CAMP Startup]}, journal = {PLoS One}, volume = {12}, year = {2017}, month = {2017}, pages = {e0170202}, abstract = {

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0170202}, author = {Ghatak, Arindam and Bharatham, Nagakumar and Shanbhag, Anirudh P and Datta, Santanu and Venkatraman, Janani} } @article {689, title = {Discovery of MicroRNAs in Cardamom ( Elettaria cardamomum Maton) under Drought Stress}, journal = {Dataset Papers in Science}, volume = {2017}, year = {2017}, month = {07}, pages = {1-4}, author = {Narayanannair, Anjali and Nadiya, F and Thomas, Jinu and Sabu, Kallevettankuzhy} } @article {8888, title = {Genome-wide changes in microRNA expression during short and prolonged heat stress and recovery in contrasting rice cultivars [Next Gen Genomics Facility]}, journal = {J Exp Bot}, volume = {68}, year = {2017}, month = {2017 Apr 01}, pages = {2399-2412}, abstract = {

MicroRNAs (miRNAs) are known to regulate expression of genes under stress. We report here the deep sequencing of small RNAs expressed during control, short and prolonged heat stress and recovery. Genome-wide identification of miRNAs in tolerant (Nagina 22) and susceptible (Vandana) rice cultivars was performed in 16 samples representing root and shoot of 13-day-old seedlings. The expression profile of miRNAs was analysed in 36 pairwise combinations to identify the genotype-, treatment- and tissue-dependent expression of miRNAs. Small-RNA sequencing of 16 libraries yielded ~271 million high-quality raw sequences; 162 miRNA families were identified. The highly expressed miRNAs in rice tissues were miR166, miR168, miR1425, miR529, mR162, miR1876, and miR1862. Expression of osa-miR1436, osa-miR5076, osa-miR5161, and osa-miR6253 was observed only in stressed tissue of both genotypes indicating their general role in heat stress response. Expression of osa-miR1439, osa-miR1848, osa-miR2096, osa-miR2106, osa-miR2875, osa-miR3981, osa-miR5079, osa-miR5151, osa-miR5484, osa-miR5792, and osa-miR5812 was observed only in Nagina 22 during high temperature, suggesting a specific role of these miRNAs in heat stress tolerance. This study provides details of the repertoire of miRNAs expressed in root and shoot of heat susceptible and tolerant rice genotypes under heat stress and recovery.

}, keywords = {Gene Expression Regulation, Plant, Genome, Plant, High-Throughput Nucleotide Sequencing, Hot Temperature, MicroRNAs, Oryza, RNA, Plant, Seedlings, Sequence Analysis, RNA, Time Factors}, issn = {1460-2431}, doi = {10.1093/jxb/erx111}, author = {Mangrauthia, Satendra K and Bhogireddy, Sailaja and Agarwal, Surekha and Prasanth, Vishnu V and Voleti, S R and Neelamraju, Sarla and Subrahmanyam, Desiraju} } @article {471, title = {Gut microbial degradation of organophosphate insecticides-induces glucose intolerance via gluconeogenesis. [Next Generation Genomics facility]}, journal = {Genome Biol}, volume = {18}, year = {2017}, month = {2017 Jan 24}, pages = {8}, abstract = {

BACKGROUND: Organophosphates are the most frequently and largely applied insecticide in the world due to their biodegradable nature. Gut microbes were shown to degrade organophosphates and cause intestinal dysfunction. The diabetogenic nature of organophosphates was recently reported but the underlying molecular mechanism is unclear. We aimed to understand the role of gut microbiota in organophosphate-induced hyperglycemia and to unravel the molecular mechanism behind this process.

RESULTS: Here we demonstrate a high prevalence of diabetes among people directly exposed to organophosphates in rural India (n = 3080). Correlation and linear regression analysis reveal a strong association between plasma organophosphate residues and HbA1c but no association with acetylcholine esterase was noticed. Chronic treatment of mice with organophosphate for 180\ days confirms the induction of glucose intolerance with no significant change in acetylcholine esterase. Further fecal transplantation and culture transplantation experiments confirm the involvement of gut microbiota in organophosphate-induced glucose intolerance. Intestinal metatranscriptomic and host metabolomic analyses reveal that gut microbial organophosphate degradation produces short chain fatty acids like acetic acid, which induces gluconeogenesis and thereby accounts for glucose intolerance. Plasma organophosphate residues are positively correlated with fecal esterase activity and acetate level of human diabetes.

CONCLUSION: Collectively, our results implicate gluconeogenesis as the key mechanism behind organophosphate-induced hyperglycemia, mediated by the organophosphate-degrading potential of gut microbiota. This study reveals the gut microbiome-mediated diabetogenic nature of organophosphates and hence that the usage of these insecticides should be reconsidered.

}, issn = {1474-760X}, doi = {10.1186/s13059-016-1134-6}, author = {Velmurugan, Ganesan and Ramprasath, Tharmarajan and Swaminathan, Krishnan and Mithieux, Gilles and Rajendhran, Jeyaprakash and Dhivakar, Mani and Parthasarathy, Ayothi and Babu, D D Venkatesh and Thumburaj, Leishman John and Freddy, Allen J and Dinakaran, Vasudevan and Puhari, Shanavas Syed Mohamed and Rekha, Balakrishnan and Christy, Yacob Jenifer and Anusha, Sivakumar and Divya, Ganesan and Suganya, Kannan and Meganathan, Boominathan and Kalyanaraman, Narayanan and Vasudevan, Varadaraj and Kamaraj, Raju and Karthik, Maruthan and Jeyakumar, Balakrishnan and Abhishek, Albert and Paul, Eldho and Pushpanathan, Muthuirulan and Rajmohan, Rajamani Koushick and Velayutham, Kumaravel and Lyon, Alexander R and Ramasamy, Subbiah} } @article {1019, title = {Insulin Plant (Costus pictus) Extract Restores Thyroid Hormone Levels in Experimental Hypothyroidism [Mass Spectrometry - Metabolomics Facility].}, journal = {Pharmacognosy Res}, volume = {9}, year = {2017}, month = {2017 Jan-Mar}, pages = {51-59}, abstract = {

BACKGROUND: The aim of the present study was to investigate the preventive effect of leaf extract in experimental hypothyroidism.

MATERIALS AND METHODS: Forty male Wistar rats were randomly divided into four groups with ten rats in each group: Control (C), hypothyroid (H), control+extract (C+E), and hypothyroid+extract (H+E). Rats in C group did not receive any intervention throughout the experimental period. The rats in the C+E and H+E groups received pretreatment with leaf extract for 4 weeks. Subsequently, for the next 6 weeks, rats in the H group received 0.05\% propylthiouracil in drinking water while C+E group received leaf extract and H+E group received propyl thiouracil and leaf extract.

RESULTS: Hypothyroid group rats exhibited dramatic increase in thyroid-stimulating hormone (TSH) levels with concomitant depletion in the levels of thyroid hormones. Treatment with the extract resulted in remarkable improvement in thyroid profile. Extract produced 10.59-fold increase in plasma free T3, 8.65-fold increase in free T4, and 3.59-fold decrease in TSH levels in H+E group in comparison with H group. Treatment with the extract ameliorated hypercholesterolemia, decreased levels of plasma C-reactive protein and tumor necrosis factor alpha, suppressed tissue oxidative stress and prevented hepatic and renal damage caused due to thyroid hormone depletion in the H+E group. Pentacyclic triterpenes alpha and beta amyrins were identified and quantified in the extract.

CONCLUSIONS: This is the first study to reveal that extract has therapeutic potential to restore thyroid hormone levels and prevent the biochemical complications due to thyroid hormone insufficiency in the animal model of experimental hypothyroidism.

SUMMARY: The preventive effect of leaf extract in experimental hypothyroidism was evaluated in the present study.Hypothyroidism was induced in the experimental animals by giving 0.05\% propylthiouracil in drinking water.Hypothyroid rats exhibited dramatic increase in thyroid-stimulating hormone (TSH) levels with concomitant depletion in the levels of thyroid hormones.Treatment with leaf extract in hypothyroid rats significantly improved the thyroid profile. It also ameliorated hypercholesterolemia, decreased the levels of plasma inflammatory markers, suppressed tissue oxidative stress and prevented hepatic and renal damage caused due to thyroid hormone depletion.The possible active principles alpha and beta amyrins were identified and quantified in the extract through LC-MS. APCI: Atmospheric pressure chemical ionization; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; C group: Control group; C+E group: Control+extract group; : ; CRP: C-reactive protein; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FRAP: Ferric reducing antioxidant power; HDL: High-density lipoprotein; H group: Hypothyroid group; H+E group: Hypothyroid+extract group; LDL: Low-density lipoprotein; LC-MS: Liquid chromatography-mass spectrometry; MDA: Malondialdehyde; PTU: 6-Propyl-2-thiouracil; SRM: Single reaction monitoring; TSH: Thyroid-stimulating hormone; TPTZ: 2,4,6-tri-(2-pyridyl)-5-triazine; TBA: 2-Thiobarbituric acid; TG: Triglyceride; TNFα: Tumor necrosis factor alpha; TAS: Total antioxidant status.

}, issn = {0976-4836}, doi = {10.4103/0974-8490.199766}, author = {Ashwini, S and Bobby, Zachariah and Sridhar, M G and Cleetus, C C} } @article {737, title = {Lipid metabolic perturbation is an early-onset phenotype in adultmutants: amodel for lysosomal storage disorders. [Mass Spectrometry - Lipidomics]}, journal = {Mol Biol Cell}, volume = {28}, year = {2017}, month = {2017 Dec 15}, pages = {3728-3740}, abstract = {

Intracellular accumulation of lipids and swollen dysfunctional lysosomes are linked to several neurodegenerative diseases, including lysosomal storage disorders (LSD). Detailed characterization of lipid metabolic changes in relation to the onset and progression of neurodegeneration is currently missing. We systematically analyzed lipid perturbations inmutants, amodel of LSD-like neurodegeneration. Our results highlight an imbalance in brain ceramide and sphingosine in the early stages of neurodegeneration, preceding the accumulation of endomembranous structures, manifestation of altered behavior, and buildup of lipofuscin. Manipulating levels ofand altering these lipids inmutants allowed us to conclude that ceramide homeostasis is the driving force in disease progression and is integral tofunction in the adult nervous system. We identified 29 novel physical interaction partners of Spin and focused on the lipid carrier protein, Lipophorin (Lpp). A subset of Lpp and Spin colocalize in the brain and within organs specialized for lipid metabolism (fat bodies and oenocytes). Reduced Lpp protein was observed inmutant tissues. Finally, increased levels of lipid metabolites produced by oenocytes inmutants allude to a functional interaction between Spin and Lpp, underscoring the systemic nature of lipid perturbation in LSD.

}, keywords = {Animals, Carrier Proteins, Disease Models, Animal, Drosophila, Drosophila Proteins, Lipid Metabolism, Lipids, Lipoproteins, Lysosomal Storage Diseases, Lysosomes, Membrane Proteins, Mutation, Nervous System, Neurodegenerative Diseases, Phenotype}, issn = {1939-4586}, doi = {10.1091/mbc.E16-09-0674}, author = {Hebbar, Sarita and Khandelwal, Avinash and Jayashree, R and Hindle, Samantha J and Chiang, Yin Ning and Yew, Joanne Y and Sweeney, Sean T and Schwudke, Dominik} } @article {532, title = {Targeted, Site-specific quantitation of N- and O-glycopeptides using (18)O-labeling and product ion based mass spectrometry.}, journal = {Glycoconj J}, volume = {34}, year = {2017}, month = {2017 Feb}, pages = {95-105}, abstract = {

The site-specific quantitation of N- and O-glycosylation is vital to understanding the function(s) of different glycans expressed at a given site of a protein under physiological and disease conditions. Most commonly used precursor ion intensity based quantification method is less accurate and other labeled methods are expensive and require enrichment of glycopeptides. Here, we used glycopeptide product (y and Y0) ions and (18)O-labeling of C-terminal carboxyl group as a strategy to obtain quantitative information about fold-change and relative abundance of most of the glycoforms attached to the glycopeptides. As a proof of concept, the accuracy and robustness of this targeted, relative quantification LC-MS method was demonstrated using Rituximab. Furthermore, the N-glycopeptide quantification results were compared with a biosimilar of Rituximab and validated with quantitative data obtained from 2-AB-UHPLC-FL method. We further demonstrated the intensity fold-change and relative abundance of 46 unique N- and O-glycopeptides and aglycopeptides from innovator and biosimilar samples of Etanercept using both the normal-MS and product ion based quantitation. The results showed a very similar site-specific expression of N- and O-glycopeptides between the samples but with subtle differences. Interestingly, we have also been able to quantify macro-heterogeneity of all N- and O-glycopetides of Etanercept. In addition to applications in biotherapeutics, the developed method can also be used for site-specific quantitation of N- and O-glycopeptides and aglycopeptides of glycoproteins with known glycosylation pattern.

}, issn = {1573-4986}, doi = {10.1007/s10719-016-9733-8}, author = {Srikanth, Jandhyam and Agalyadevi, Rathinasamy and Babu, Ponnusamy} } @article {688, title = {Transcriptome profiling of Elettaria cardamomum (L.) Maton (small cardamom).}, journal = {Genom Data}, volume = {11}, year = {2017}, month = {2017 Mar}, pages = {102-103}, abstract = {

Elettaria cardamomum (L.) Maton, known as {\textquoteright}queen of spices, is a perennial herbaceous monocot of the family Zingiberaceae, native to southern India. Cardamom is an economically valuable spice crop and used widely in culinary and medicinal purposes. In the present study, using Ion Proton RNA sequencing technology, we performed transcriptome sequencing and de novo transcriptome assembly of a wild and five cultivar genotypes of cardamom. RNA-seq generated a total of 22,811,983 (92 base) and 24,889,197 (75 base) raw reads accounting for approximately 8.21GB and 7.65GB of sequence data for wild and cultivar genotypes of cardamom respectively. The raw data were submitted to SRA database of NCBI under the accession numbers SRX1141272 (wild) and SRX1141276 (cultivars). The raw reads were quality filtered and assembled using MIRA assembler resulted with 112,208 and 264,161contigs having N50 value 616 and 664 for wild and cultivar cardamom respectively. The assembled unigenes were functionally annotated using several databases including PlantCyc for pathway annotation. This work represents the first report on cardamom transcriptome sequencing. In order to generate a comprehensive reference transcriptome, we further assembled the raw reads of wild and cultivar genotypes which might enrich the plant transcriptome database and trigger advanced research in cardamom genomics.

}, issn = {2213-5960}, doi = {10.1016/j.gdata.2016.12.013}, author = {Nadiya, F and Anjali, N and Thomas, Jinu and Gangaprasad, A and Sabu, K K} } @article {8890, title = {Transcriptomic resources for the medicinal legume Mucuna pruriens: de novo transcriptome assembly, annotation, identification and validation of EST-SSR markers [Next Gen Genomics Facility]}, journal = {BMC Genomics}, volume = {18}, year = {2017}, month = {2017 May 25}, pages = {409}, abstract = {

BACKGROUND: The medicinal legume Mucuna pruriens (L.) DC. has attracted attention worldwide as a source of the anti-Parkinson{\textquoteright}s drug L-Dopa. It is also a popular green manure cover crop that offers many agronomic benefits including high protein content, nitrogen fixation and soil nutrients. The plant currently lacks genomic resources and there is limited knowledge on gene expression, metabolic pathways, and genetics of secondary metabolite production. Here, we present transcriptomic resources for M. pruriens, including a de novo transcriptome assembly and annotation, as well as differential transcript expression analyses between root, leaf, and pod tissues. We also develop microsatellite markers and analyze genetic diversity and population structure within a set of Indian germplasm accessions.

RESULTS: One-hundred ninety-one million two hundred thirty-three thousand two hundred forty-two\ bp cleaned reads were assembled into 67,561 transcripts with mean length of 626\ bp and N50 of 987\ bp. Assembled sequences were annotated using BLASTX against public databases with over 80\% of transcripts annotated. We identified 7,493 simple sequence repeat (SSR) motifs, including 787 polymorphic repeats between the parents of a mapping population. 134 SSRs from expressed sequenced tags (ESTs) were screened against 23 M. pruriens accessions from India, with 52 EST-SSRs retained after quality control. Population structure analysis using a Bayesian framework implemented in fastSTRUCTURE showed nearly similar groupings as with distance-based (neighbor-joining) and principal component analyses, with most of the accessions clustering per geographical origins. Pair-wise comparison of transcript expression in leaves, roots and pods identified 4,387 differentially expressed transcripts with the highest number occurring between roots and leaves. Differentially expressed transcripts were enriched with transcription factors and transcripts annotated as belonging to secondary metabolite pathways.

CONCLUSIONS: The M. pruriens transcriptomic resources generated in this study provide foundational resources for gene discovery and development of molecular markers. Polymorphic SSRs identified can be used for genetic diversity, marker-trait analyses, and development of functional markers for crop improvement. The results of differential expression studies can be used to investigate genes involved in L-Dopa synthesis and other key metabolic pathways in M. pruriens.

}, keywords = {Data Mining, Expressed Sequence Tags, Gene Expression Profiling, Microsatellite Repeats, Molecular Sequence Annotation, Mucuna, Polymorphism, Genetic, RNA, Messenger, Transcription Factors}, issn = {1471-2164}, doi = {10.1186/s12864-017-3780-9}, author = {Sathyanarayana, N and Pittala, Ranjith Kumar and Tripathi, Pankaj Kumar and Chopra, Ratan and Singh, Heikham Russiachand and Belamkar, Vikas and Bhardwaj, Pardeep Kumar and Doyle, Jeff J and Egan, Ashley N} } @article {8885, title = {Comparative Metagenomics Reveal Phylum Level Temporal and Spatial Changes in Mycobiome of Belowground Parts of Crocus sativus [Next Gen Genomics Facility]}, journal = {PLoS One}, volume = {11}, year = {2016}, month = {2016}, pages = {e0163300}, abstract = {

Plant-fungal associations have been explored by routine cultivation based approaches and cultivation based approaches cannot catalogue more than 5\% of fungal diversity associated with any niche. In the present study, an attempt has been made to catalogue fungal diversity associated with belowground parts i.e. rhizosphere and cormosphere, of Crocus sativus (an economically important herb) during two growth stages, using cultivation independent ITS gene targeted approach, taking bulk soil as reference. The 454 pyrosequencing sequence data analysis suggests that the fungal diversity was niche and growth stage specific. Fungi diversity, in the present case, was not only different between the two organs (roots and corm) but the dominance pattern varies between the cormosphere during two growth stages. Zygomycota was dominant fungal phylum in the rhizosphere whereas Basidiomycota was dominant in cormosphere during flowering stage. However in cormosphere though Basidiomycota was dominant phylum during flowering stage but Zygomycota was dominant during dormant stage. Interestingly, in cormosphere, the phyla which was dominant at dormant stage was rare at flowering stage and vice-versa (Basidiomycota: Flowering = 93.2\% Dormant = 0.05\% and Zygomycota: Flowering = 0.8\% Dormant = 99.7\%). At genus level, Rhizopus was dominant in dormant stage but was rare in flowering stage (Rhizopus: Dormant = 99.7\% Flowering = 0.55\%). This dynamics is not followed by the bulk soil fungi which was dominated by Ascomycota during both stages under study. The genus Fusarium, whose species F. oxysporum causes corm rot in C. sativus, was present during both stages with slightly higher abundance in roots. Interestingly, the abundance of Rhizopus varied a great deal in two stages in cormosphere but the abundance of Fusarium was comparable in two growth stages (Bulk soil Flowering = 0.05\%, Rhizosphere Flowering = 1.4\%, Cormosphere Flowering = 0.06\%, Bulk soil Dormant = 2.47\% and cormosphere dormant = 0.05\%). This is the first report on the fungal diversity associated with the root of Crocus sativus and first report on the fungi associated with corm of any plant with the temporal and spatial variation in the fungal community structure.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0163300}, author = {Ambardar, Sheetal and Singh, Heikham Russiachand and Gowda, Malali and Vakhlu, Jyoti} } @article {706, title = {Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes.}, journal = {Cell}, volume = {164}, year = {2016}, month = {2016 Feb 11}, pages = {722-34}, abstract = {

Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes.

}, keywords = {Animals, Biological Transport, Cell Line, Dictyostelium, Dyneins, Glycosphingolipids, Leishmania donovani, Lysosomes, Membrane Microdomains, Mice, Phagosomes}, issn = {1097-4172}, doi = {10.1016/j.cell.2015.12.054}, author = {Rai, Ashim and Pathak, Divya and Thakur, Shreyasi and Singh, Shampa and Dubey, Alok Kumar and Mallik, Roop} } @article {509, title = {Structure of a heterogeneous, glycosylated, lipid-bound, in vivo-grown protein crystal at atomic resolution from the viviparous cockroach Diploptera punctata. [Mass spectrometry - Glycomics]}, journal = {IUCrJ}, volume = {3}, year = {2016}, month = {2016 Jul 01}, pages = {282-93}, abstract = {

Macromolecular crystals for X-ray diffraction studies are typically grown in vitro from pure and homogeneous samples; however, there are examples of protein crystals that have been identified in vivo. Recent developments in micro-crystallography techniques and the advent of X-ray free-electron lasers have allowed the determination of several protein structures from crystals grown in cellulo. Here, an atomic resolution (1.2 {\r A}) crystal structure is reported of heterogeneous milk proteins grown inside a living organism in their functional niche. These in vivo-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach, Diploptera punctata. The milk proteins crystallized in space group P1, and a structure was determined by anomalous dispersion from the native S atoms. The data revealed glycosylated proteins that adopt a lipocalin fold, bind lipids and organize to form a tightly packed crystalline lattice. A single crystal is estimated to contain more than three times the energy of an equivalent mass of dairy milk. This unique storage form of nourishment for developing embryos allows access to a constant supply of complete nutrients. Notably, the crystalline cockroach-milk proteins are highly heterogeneous with respect to amino-acid sequence, glycosylation and bound fatty-acid composition. These data present a unique example of protein heterogeneity within a single in vivo-grown crystal of a natural protein in its native environment at atomic resolution.

}, doi = {10.1107/S2052252516008903}, author = {Banerjee, Sanchari and Coussens, Nathan P and Gallat, Fran{\c c}ois-Xavier and Sathyanarayanan, Nitish and Srikanth, Jandhyam and Yagi, Koichiro J and Gray, James S S and Tobe, Stephen S and Stay, Barbara and Chavas, Leonard M G and Ramaswamy, Subramanian} } @article {499, title = {Comprehensive analyses of genomes, transcriptomes and metabolites of neem tree. [Mass spectrometry - Metabolomics]}, journal = {PeerJ}, volume = {3}, year = {2015}, month = {2015}, pages = {e1066}, abstract = {

Neem (Azadirachta indica A. Juss) is one of the most versatile tropical evergreen tree species known in India since the Vedic period (1500 BC-600 BC). Neem tree is a rich source of limonoids, having a wide spectrum of activity against insect pests and microbial pathogens. Complex tetranortriterpenoids such as azadirachtin, salanin and nimbin are the major active principles isolated from neem seed. Absolutely nothing is known about the biochemical pathways of these metabolites in neem tree. To identify genes and pathways in neem, we sequenced neem genomes and transcriptomes using next generation sequencing technologies. Assembly of Illumina and 454 sequencing reads resulted in 267 Mb, which accounts for 70\% of estimated size of neem genome. We predicted 44,495 genes in the neem genome, of which 32,278 genes were expressed in neem tissues. Neem genome consists about 32.5\% (87 Mb) of repetitive DNA elements. Neem tree is phylogenetically related to citrus, Citrus sinensis. Comparative analysis anchored 62\% (161 Mb) of assembled neem genomic contigs onto citrus chromomes. Ultrahigh performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was used to quantify azadirachtin, nimbin, and salanin from neem tissues. Weighted Correlation Network Analysis (WCGNA) of expressed genes and metabolites resulted in identification of possible candidate genes involved in azadirachtin biosynthesis pathway. This study provides genomic, transcriptomic and quantity of top three neem metabolites resource, which will accelerate basic research in neem to understand biochemical pathways.

}, doi = {10.7717/peerj.1066}, author = {Kuravadi, Nagesh A and Yenagi, Vijay and Rangiah, Kannan and Mahesh, H B and Rajamani, Anantharamanan and Shirke, Meghana D and Russiachand, Heikham and Loganathan, Ramya Malarini and Shankara Lingu, Chandana and Siddappa, Shilpa and Ramamurthy, Aishwarya and Sathyanarayana, B N and Gowda, Malali} } @article {538, title = {Genome analysis of rice-blast fungus Magnaporthe oryzae field isolates from southern India.}, journal = {Genom Data}, volume = {5}, year = {2015}, month = {2015 Sep}, pages = {284-91}, abstract = {

The Indian subcontinent is the center of origin and diversity for rice (Oryza sativa L.). The O. sativa ssp. indica is a major food crop grown in India, which occupies the first and second position in area and production, respectively. Blast disease caused by Magnaporthe oryzae is a major constraint to rice production. Here, we report the analysis of genome architecture and sequence variation of two field isolates, B157 and MG01, of the blast fungus from southern India. The 40\ Mb genome of B157 and 43\ Mb genome of MG01 contained 11,344 and 11,733 predicted genes, respectively. Genomic comparisons unveiled a large set of SNPs and several isolate specific genes in the Indian blast isolates. Avr genes were analyzed in several sequenced Magnaporthe strains; this analysis revealed the presence of Avr-Pizt and Avr-Ace1 genes in all the sequenced isolates. Availability of whole genomes of field isolates from India will contribute to global efforts to understand genetic diversity of M. oryzae population and to track the emergence of virulent pathotypes.

}, doi = {10.1016/j.gdata.2015.06.018}, author = {Gowda, Malali and Shirke, Meghana D and Mahesh, H B and Chandarana, Pinal and Rajamani, Anantharamanan and Chattoo, Bharat B} } @article {8884, title = {Genome Sequence, Comparative Analysis, and Evolutionary Insights into Chitinases of Entomopathogenic Fungus Hirsutella thompsonii [Next Gen Genomics Facility]}, journal = {Genome Biology and Evolution}, volume = {7}, year = {2015}, month = {02}, pages = {916-930}, abstract = {

{Hirsutella thompsonii (Ht) is a fungal pathogen of acarines and the primary cause of epizootics among mites. The draft genomes of two isolates of Ht (MTCC 3556: Ht3, 34.6 Mb and MTCC 6686: Ht6, 34.7 Mb) are presented and compared with the genomes of Beauveria bassiana (Bb) ARSEF 2860 and Ophiocordyceps sinensis (Os) CO18. Comparative analysis of carbohydrate active enzymes, pathogen{\textendash}host interaction genes, metabolism-associated genes, and genes involved in biosynthesis of secondary metabolites in the four genomes was carried out. Reduction in gene family sizes in Ht3 and Os as compared with Ht6 and Bb is observed. Analysis of the mating type genes in Ht reveals the presence of MAT idiomorphs which is suggestive of cryptic sexual traits in Ht. We further identify and classify putative chitinases that may function as virulence factors in fungal entomopathogens due to their role in degradation of arthropod cuticle.}

}, issn = {1759-6653}, doi = {10.1093/gbe/evv037}, url = {https://doi.org/10.1093/gbe/evv037}, author = {Agrawal, Yamini and Khatri, Indu and Subramanian, Srikrishna and Shenoy, Belle Damodara} } @article {498, title = {Genome sequencing of herb Tulsi (Ocimum tenuiflorum) unravels key genes behind its strong medicinal properties.[Mass spectrometry - Metabolomics]}, journal = {BMC Plant Biol}, volume = {15}, year = {2015}, month = {2015 Aug 28}, pages = {212}, abstract = {

BACKGROUND: Krishna Tulsi, a member of Lamiaceae family, is a herb well known for its spiritual, religious and medicinal importance in India. The common name of this plant is {\textquoteright}Tulsi{\textquoteright} (or {\textquoteright}Tulasi{\textquoteright} or {\textquoteright}Thulasi{\textquoteright}) and is considered sacred by Hindus. We present the draft genome of Ocimum tenuiflurum L (subtype Krishna Tulsi) in this report. The paired-end and mate-pair sequence libraries were generated for the whole genome sequenced with the Illumina Hiseq 1000, resulting in an assembled genome of 374\ Mb, with a genome coverage of 61\ \% (612\ Mb estimated genome size). We have also studied transcriptomes (RNA-Seq) of two subtypes of O. tenuiflorum, Krishna and Rama Tulsi and report the relative expression of genes in both the varieties.

RESULTS: The pathways leading to the production of medicinally-important specialized metabolites have been studied in detail, in relation to similar pathways in Arabidopsis thaliana and other plants. Expression levels of anthocyanin biosynthesis-related genes in leaf samples of Krishna Tulsi were observed to be relatively high, explaining the purple colouration of Krishna Tulsi leaves. The expression of six important genes identified from genome data were validated by performing q-RT-PCR in different tissues of five different species, which shows the high extent of urosolic acid-producing genes in young leaves of the Rama subtype. In addition, the presence of eugenol and ursolic acid, implied as potential drugs in the cure of many diseases including cancer was confirmed using mass spectrometry.

CONCLUSIONS: The availability of the whole genome of O.tenuiflorum and our sequence analysis suggests that small amino acid changes at the functional sites of genes involved in metabolite synthesis pathways confer special medicinal properties to this herb.

}, keywords = {Gene Expression Regulation, Plant, Genome, Plant, India, Ocimum, Plant Leaves, Plants, Medicinal}, issn = {1471-2229}, doi = {10.1186/s12870-015-0562-x}, author = {Upadhyay, Atul K and Chacko, Anita R and Gandhimathi, A and Ghosh, Pritha and Harini, K and Joseph, Agnel P and Joshi, Adwait G and Karpe, Snehal D and Kaushik, Swati and Kuravadi, Nagesh and Lingu, Chandana S and Mahita, J and Malarini, Ramya and Malhotra, Sony and Malini, Manoharan and Mathew, Oommen K and Mutt, Eshita and Naika, Mahantesha and Nitish, Sathyanarayanan and Pasha, Shaik Naseer and Raghavender, Upadhyayula S and Rajamani, Anantharamanan and Shilpa, S and Shingate, Prashant N and Singh, Heikham Russiachand and Sukhwal, Anshul and Sunitha, Margaret S and Sumathi, Manojkumar and Ramaswamy, S and Gowda, Malali and Sowdhamini, Ramanathan} } @article {513, title = {L-Plastin S-glutathionylation promotes reduced binding to β-actin and affects neutrophil functions. (Mass Spectrometry)}, journal = {Free Radic Biol Med}, volume = {86}, year = {2015}, month = {2015 Sep}, pages = {1-15}, abstract = {

Posttranslational modifications (PTMs) of cytoskeleton proteins due to oxidative stress associated with several pathological conditions often lead to alterations in cell function. The current study evaluates the effect of nitric oxide (DETA-NO)-induced oxidative stress-related S-glutathionylation of cytoskeleton proteins in human PMNs. By using in vitro and genetic approaches, we showed that S-glutathionylation of L-plastin (LPL) and β-actin promotes reduced chemotaxis, polarization, bactericidal activity, and phagocytosis. We identified Cys-206, Cys-283, and Cys-460as S-thiolated residues in the β-actin-binding domain of LPL, where cys-460 had the maximum score. Site-directed mutagenesis of LPL Cys-460 further confirmed the role in the redox regulation of LPL. S-Thiolation diminished binding as well as the bundling activity of LPL. The presence of S-thiolated LPL was detected in neutrophils from both diabetic patients and db/db mice with impaired PMN functions. Thus, enhanced nitroxidative stress may results in LPL S-glutathionylation leading to impaired chemotaxis, polarization, and bactericidal activity of human PMNs, providing a mechanistic basis for their impaired functions in diabetes mellitus.

}, keywords = {Actins, Adult, Amino Acid Sequence, Animals, Case-Control Studies, Cell Polarity, Chemotaxis, Diabetes Mellitus, Female, Glutathione, HEK293 Cells, Humans, Male, Mice, Inbred C57BL, Mice, Obese, Microfilament Proteins, Middle Aged, Molecular Sequence Data, Neutrophils, Nitric Oxide, Oxidative Stress, Protein Binding, Protein Processing, Post-Translational, Young Adult}, issn = {1873-4596}, doi = {10.1016/j.freeradbiomed.2015.04.008}, author = {Dubey, Megha and Singh, Abhishek K and Awasthi, Deepika and Nagarkoti, Sheela and Kumar, Sachin and Ali, Wahid and Chandra, Tulika and Kumar, Vikas and Barthwal, Manoj K and Jagavelu, Kumaravelu and S{\'a}nchez-G{\'o}mez, Francisco J and Lamas, Santiago and Dikshit, Madhu} } @article {500, title = {Nickel azamacrocyclic complex activated persulphate based oxidative degradation of methyl orange: recovery and reuse of complex using adsorbents [Mass spectrometry - Metabolomics]}, journal = {RSC Adv.}, volume = {5}, year = {2015}, pages = {31716-31724}, abstract = {

Adsorbents are useful for the removal of metal complex based catalysts from the reaction medium. Moreover{,} effective catalysts may be recycled with the use of adsorbents. These facts inspired us to investigate the use of adsorbents for the recovery and reuse of a metal complex that could activate persulphate to effectively degrade an organic pollutant in water. Herein{,} we report the nickel complex (C1) activated persulphate based degradation of methyl orange (MO) in water and the removal of C1 using activated carbon (AC) and Amberlite (Am) as adsorbents. C1 adsorbed onto AC (C1-AC) was reused in the solid form to activate persulphate and degrade MO without leaching C1 into water. Additionally{,} solid C1-Am recovered from the degraded MO solution was ion exchanged using sodium chloride to obtain C1{,} which was reused for MO degradation. The study demonstrates the application of adsorbents such as AC and Am for the adsorptive recovery and reuse of a metal complex based persulphate activator.

}, doi = {10.1039/C5RA03350K}, url = {http://dx.doi.org/10.1039/C5RA03350K}, author = {Subramanian, Gokulakrishnan and Nalawade, Pranav and Hinder, Steven J. and Pillai, Suresh C. and Prakash, Halan} } @article {506, title = {Simple Detection Methods for Antinutritive Factor β-ODAP Present in Lathyrus sativus L. by High Pressure Liquid Chromatography and Thin Layer Chromatography. [Protein Technology Core]}, journal = {PLoS One}, volume = {10}, year = {2015}, month = {2015}, pages = {e0140649}, abstract = {

Lathyrus sativus L. (Grass pea) is the source for cheap and nutritious food choice in drought and famine susceptible zones in greater part of North India and Africa. The non-protein amino acid β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) has been known for decades for its potent neurotoxic effect, causing irreversible neurodegenerative disease "neurolathyrism", present in both seed and leaf of Lathyrus sativus L. and other species in varying proportions. It is crucial to establish a rapid as well as reliable detection methodology for β-ODAP content in various Lathyrus plants. Currently available HPLC based methods involve multi-step derivatization of the sample. To overcome this, we have developed β-ODAP analysis method by HPLC without any prior derivatization. This method is statistically significant in the range of 2 to 100μg/ml and exhibited linear response with r2 \> 0.99. Limit of detection and quantitation of the later method was determined to be 5.56 μg/ml and 16.86 μg/ml, respectively. In addition to this, a TLC based method has also been developed. The limit of detection of β-ODAP is 0.6μg and for its substrate, L-1,2-diaminopropionic acid is 5μg. Both HPLC and TLC methods were validated by conducting in-vitro bioconversion test to detect the presence of biocatalyst in plant extract. This method is economical, rapid and simple.

}, keywords = {Africa, Amino Acids, Diamino, Chromatography, High Pressure Liquid, Chromatography, Thin Layer, India, Lathyrus, Neurotoxins, Plant Extracts, Plant Leaves, Seeds}, issn = {1932-6203}, doi = {10.1371/journal.pone.0140649}, author = {Ghosh, Bidisha and Mitra, Joy and Chakraborty, Saikat and Bhattacharyya, Jagannath and Chakraborty, Anirban and Sen, Soumitra Kumar and Neerathilingam, Muniasamy} } @article {507, title = {Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro. [Protein Technology Core]}, journal = {PLoS One}, volume = {10}, year = {2015}, month = {2015}, pages = {e0123428}, abstract = {

Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.

}, keywords = {Actins, Animals, Avian Proteins, Chickens, Cytoskeletal Proteins, In Vitro Techniques, Microfilament Proteins, Models, Molecular, Protein Interaction Domains and Motifs, Recombinant Fusion Proteins}, issn = {1932-6203}, doi = {10.1371/journal.pone.0123428}, author = {Shrivastava, Rohini and K{\"o}ster, Darius and Kalme, Sheetal and Mayor, Satyajit and Neerathilingam, Muniasamy} } @article {536, title = {Does reversible cysteine oxidation link the Western diet to cardiac dysfunction?}, journal = {FASEB J}, volume = {28}, year = {2014}, month = {2014 May}, pages = {1975-87}, abstract = {

Using a novel cysteine thiol labeling strategy coupled with mass spectrometric analysis, we identified and quantified the changes in global reversible cysteine oxidation of proteins in the left ventricle of hearts from mice with metabolic syndrome-associated diastolic dysfunction. This phenotype was induced by feeding a high-fat, high-sucrose, type-2 diabetogenic diet to C57BL/6J mice for 8 mo. The extent of reversible thiol oxidation in relationship to the total available (free and reducible) level of each cysteine could be confidently determined for 173 proteins, of which 98 contained cysteines differentially modified >=1.5-fold by the diet. Our findings suggest that the metabolic syndrome leads to potentially deleterious changes in the oxidative modification of metabolically active proteins. These alterations may adversely regulate energy substrate flux through glycolysis, β-oxidation, citric acid (TCA) cycle, and oxidative phosphorylation (oxphos), thereby contributing to maladaptive tissue remodeling that is associated with, and possibly contributing to, diastolic left ventricular dysfunction.

}, keywords = {Animals, Chromatography, Liquid, Citric Acid Cycle, Cysteine, Diet, Fatty Acids, Glycolysis, Heart Diseases, Male, Mice, Mice, Inbred C57BL, Myocardial Contraction, Myocardium, Obesity, Oxidative Phosphorylation, Oxygen, Phenotype, Protein Processing, Post-Translational, Proteomics, Reactive Nitrogen Species, Reactive Oxygen Species, Sulfhydryl Compounds, Tandem Mass Spectrometry}, issn = {1530-6860}, doi = {10.1096/fj.13-233445}, author = {Behring, Jessica B and Kumar, Vikas and Whelan, Stephen A and Chauhan, Pratibha and Siwik, Deborah A and Costello, Catherine E and Colucci, Wilson S and Cohen, Richard A and McComb, Mark E and Bachschmid, Markus M} } @article {475, title = {Genetic transformation of structural and functional circuitry rewires the Drosophila brain. [Drosophila Facility]}, journal = {Elife}, volume = {3}, year = {2014}, month = {2014 Dec 29}, abstract = {

Acquisition of distinct neuronal identities during development is critical for the assembly of diverse functional neural circuits in the brain. In both vertebrates and invertebrates, intrinsic determinants are thought to act in neural progenitors to specify their identity and the identity of their neuronal progeny. However, the extent to which individual factors can contribute to this is poorly understood. We investigate the role of orthodenticle in the specification of an identified neuroblast (neuronal progenitor) lineage in the Drosophila brain. Loss of orthodenticle from this neuroblast affects molecular properties, neuroanatomical features, and functional inputs of progeny neurons, such that an entire central complex lineage transforms into a functional olfactory projection neuron lineage. This ability to change functional macrocircuitry of the brain through changes in gene expression in a single neuroblast reveals a surprising capacity for novel circuit formation in the brain and provides a paradigm for large-scale evolutionary modification of circuitry.

}, keywords = {Animals, Brain, Cell Lineage, Drosophila, Morphogenesis, Neurons}, issn = {2050-084X}, doi = {10.7554/eLife.04407}, author = {Sen, Sonia and Cao, Deshou and Choudhary, Ramveer and Biagini, Silvia and Wang, Jing W and Reichert, Heinrich and VijayRaghavan, K} } @article {8356, title = {Lunatimonas lonarensis gen. nov., sp. nov., a haloalkaline bacterium of the family Cyclobacteriaceae with nitrate reducing activity [Next Gen Genomics Facility]}, journal = {Syst Appl Microbiol}, volume = {37}, year = {2014}, month = {2014 Feb}, pages = {10-6}, abstract = {

Novel pinkish-orange pigmented, Gram-negative staining, half-moon shaped, non-motile, strictly aerobic strains designated AK24(T) and AK26 were isolated from water and sediment samples of Lonar Lake, Buldhana district, Maharahstra, India. Both strains were positive for oxidase, catalase and β-galactosidase activities. The predominant fatty acids were iso-C15:0 (41.5\%), anteiso-C15:0 (9.7\%), iso-C17:0 3OH (9.6\%), iso-C17:1 ω9c (10.2\%) and C16:1 ω7c/C16:1 ω6c/iso-C15:0 2OH (summed feature 3) (14.4\%). The strains contained MK-7 as the major respiratory quinone, and phosphatidylethanolamine and five unidentified lipids as the polar lipids. Blast analysis of the 16S rRNA gene sequence of strain AK24(T) showed that it was closely related to Aquiflexum balticum, with a pair-wise sequence similarity of 91.6\%, as well as to Fontibacter ferrireducens, Belliella baltica and Indibacter alkaliphilus (91.3, 91.2 and 91.2\% pair-wise sequence similarity, respectively), but it only had between 88.6 and 91.0\% pair-wise sequence similarity to the rest of the family members. The MALDI-TOF assay reported no significant similarities for AK24(T) and AK26, since they potentially represented a new species. A MALDI MSP dendrogram showed close similarity between the two strains, but they maintained a distance from their phylogenetic neighbors. The genome of AK24(T) showed the presence of heavy metal tolerance genes, including the genes providing resistance to arsenic, cadmium, cobalt and zinc. A cluster of heat shock resistance genes was also found in the genome. Two lantibiotic producing genes, LanR and LasB, were also found in the genome of AK24(T). Strains AK24(T) and AK26 were very closely related to each other with 99.5\% pair-wise sequence similarity. Phylogenetic analysis indicated that the strains were members of the family Cyclobacteriaceae and they clustered with the genus Mariniradius, as well as with the genera Aquiflexum, Cecembia, Fontibacter, Indibacter, and Shivajiella. DNA-DNA hybridization between strains AK24(T) and AK26 showed a relatedness of 82\% and their rep-PCR banding patterns were very similar. Based on data from the current polyphasic study, it is proposed that the isolates be placed in a new genus and species with the name Lunatimonas lonarensis gen. nov., sp. nov. The type strain of Lunatimonas lonarensis is AK24(T) (=JCM 18822(T)=MTCC 11627(T)).

}, keywords = {Bacterial Typing Techniques, Bacteroidetes, Cluster Analysis, DNA, Bacterial, DNA, Ribosomal, Fatty Acids, Fresh Water, Genome, Bacterial, Geologic Sediments, India, Molecular Sequence Data, Nitrates, Oxidation-Reduction, Phospholipids, Phylogeny, Quinones, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {1618-0984}, doi = {10.1016/j.syapm.2013.10.003}, author = {Srinivas, T N R and Aditya, S and Bhumika, V and Kumar, P Anil} } @article {535, title = {Unique, polyfucosylated glycan-receptor interactions are essential for regeneration of Hydra magnipapillata.}, journal = {ACS Chem Biol}, volume = {9}, year = {2014}, month = {2014 Jan 17}, pages = {147-55}, abstract = {

Cell-cell communications, cell-matrix interactions, and cell migrations play a major role in regeneration. However, little is known about the molecular players involved in these critical events, especially cell surface molecules. Here, we demonstrate the role of specific glycan-receptor interactions in the regenerative process using Hydra magnipapillata as a model system. Global characterization of the N- and O-glycans expressed by H. magnipapillata using ultrasensitive mass spectrometry revealed mainly polyfucosylated LacdiNAc antennary structures. Affinity purification showed that a putative C-type lectin (accession number Q6SIX6) is a likely endogenous receptor for the novel polyfucosylated glycans. Disruption of glycan-receptor interactions led to complete shutdown of the regeneration machinery in live Hydra. A time-dependent, lack-of-regeneration phenotype observed upon incubation with exogenous fuco-lectins suggests the involvement of a polyfucose receptor-mediated signaling mechanism during regeneration. Thus, for the first time, the results presented here provide direct evidence for the role of polyfucosylated glycan-receptor interactions in the regeneration of H. magnipapillata.

}, keywords = {Animals, Carbohydrate Sequence, Hydra, Lectins, C-Type, Molecular Sequence Data, Polysaccharides, Regeneration}, issn = {1554-8937}, doi = {10.1021/cb400486t}, author = {Sahadevan, Sonu and Antonopoulos, Aristotelis and Haslam, Stuart M and Dell, Anne and Ramaswamy, Subramanian and Babu, Ponnusamy} } @article {521, title = {Draft Genome Sequence of Acinetobacter baumannii Strain MSP4-16. (Next Generation Genomics)}, journal = {Genome Announc}, volume = {1}, year = {2013}, month = {2013 Apr 04}, pages = {e0013713}, abstract = {

We report the 4.0-Mb draft genome sequence of Acinetobacter baumannii strain MSP4-16, isolated from a mangrove soil sample from Parangipettai (11{\textdegree}30{\textquoteright}N, 79{\textdegree}47{\textquoteright}E), Tamil Nadu, India. The draft genome sequence of strain MSP4-16 consists of 3,944,542\ bp, with a G+C content of 39\%, 5,387 protein coding genes, and 69 RNAs.

}, doi = {10.1128/genomeA.00137-13}, author = {Singh, Nitin Kumar and Kumar, Shailesh and Raghava, Gajendra Pal Singh and Mayilraj, Shanmugam} } @article {523, title = {Draft Genome Sequence of Streptomyces gancidicus Strain BKS 13-15. (Next Generation Genomics)}, journal = {Genome Announc}, volume = {1}, year = {2013}, month = {2013 Apr 18}, pages = {e0015013}, abstract = {

We report the 7.3-Mbp genome sequence of Streptomyces gancidicus strain BKS 13-15, isolated from mangrove sediment samples collected from the Bhitar Kanika Mangrove Reserve Forest, Odissha, India. The draft genome of strain Streptomyces gancidicus strain BKS 13-15 consists of 7,300,479\ bp with 72.6\% G+C content, 6,631 protein-coding genes, and 71 RNAs.

}, doi = {10.1128/genomeA.00150-13}, author = {Kumar, Shailesh and Kaur, Navjot and Singh, Nitin Kumar and Raghava, Gajendra Pal Singh and Mayilraj, Shanmugam} } @article {477, title = {A genetic RNAi screen for IP$_{3}$/Ca{\texttwosuperior}$^{+}$ coupled GPCRs in Drosophila identifies the PdfR as a regulator of insect flight. [Drosophila facility]}, journal = {PLoS Genet}, volume = {9}, year = {2013}, month = {2013}, pages = {e1003849}, abstract = {

Insect flight is regulated by various sensory inputs and neuromodulatory circuits which function in synchrony to control and fine-tune the final behavioral outcome. The cellular and molecular bases of flight neuromodulatory circuits are not well defined. In Drosophila melanogaster, it is known that neuronal IP3 receptor mediated Ca{\texttwosuperior}$^{+}$ signaling and store-operated Ca{\texttwosuperior}$^{+}$ entry (SOCE) are required for air-puff stimulated adult flight. However, G-protein coupled receptors (GPCRs) that activate intracellular Ca{\texttwosuperior}$^{+}$ signaling in the context of flight are unknown in Drosophila. We performed a genetic RNAi screen to identify GPCRs that regulate flight by activating the IPIP$_{3}$ receptor. Among the 108 GPCRs screened, we discovered 5 IPIP$_{3}$/Ca{\texttwosuperior}$^{+}$ linked GPCRs that are necessary for maintenance of air-puff stimulated flight. Analysis of their temporal requirement established that while some GPCRs are required only during flight circuit development, others are required both in pupal development as well as during adult flight. Interestingly, our study identified the Pigment Dispersing Factor Receptor (PdfR) as a regulator of flight circuit development and as a modulator of acute flight. From the analysis of PdfR expressing neurons relevant for flight and its well-defined roles in other behavioral paradigms, we propose that PdfR signaling functions systemically to integrate multiple sensory inputs and modulate downstream motor behavior.

}, keywords = {Adult, Animals, Calcium Signaling, Drosophila melanogaster, Drosophila Proteins, Flight, Animal, Humans, Inositol 1,4,5-Trisphosphate Receptors, Neurons, Receptors, G-Protein-Coupled, RNA Interference, Signal Transduction}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1003849}, author = {Agrawal, Tarjani and Sadaf, Sufia and Hasan, Gaiti} } @article {488, title = {Genome sequencing unveils a novel sea enterotoxin-carrying PVL phage in Staphylococcus aureus ST772 from India. [Next Generation Genomics facility]}, journal = {PLoS One}, volume = {8}, year = {2013}, month = {2013}, pages = {e60013}, abstract = {

Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772 suggests that widespread mobility of the sea enterotoxin might be a selective force behind its {\textquoteright}transfer{\textquoteright} to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside the genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.

}, keywords = {Bacterial Toxins, Base Sequence, Enterotoxins, Exotoxins, Genome, Bacterial, Hemolysin Proteins, Humans, India, Leukocidins, Molecular Sequence Data, Prophages, RNA, Messenger, Sequence Analysis, DNA, Sphingomyelin Phosphodiesterase, Staphylococcus aureus}, issn = {1932-6203}, doi = {10.1371/journal.pone.0060013}, author = {Prabhakara, Sushma and Khedkar, Supriya and Shambat, Srikanth Mairpady and Srinivasan, Rajalakshmi and Basu, Atanu and Norrby-Teglund, Anna and Seshasayee, Aswin Sai Narain and Arakere, Gayathri} } @article {484, title = {Genomic analysis reveals epistatic silencing of "expensive" genes in Escherichia coli K-12. [Next Generation Genomics facility]}, journal = {Mol Biosyst}, volume = {9}, year = {2013}, month = {2013 Aug}, pages = {2021-33}, abstract = {

A barrier for horizontal gene transfer is high gene expression, which is metabolically expensive. Silencing of horizontally-acquired genes in the bacterium Escherichia coli is caused by the global transcriptional repressor H-NS. The activity of H-NS is enhanced or diminished by other proteins including its homologue StpA, and Hha and YdgT. The interconnections of H-NS with these regulators and their role in silencing gene expression in E. coli are not well understood on a genomic scale. In this study, we use transcriptome sequencing to show that there is a bi-layered gene silencing system - involving the homologous H-NS and StpA - operating on horizontally-acquired genes among others. We show that H-NS-repressed genes belong to two types, termed "epistatic" and "unilateral". In the absence of H-NS, the expression of "epistatically controlled genes" is repressed by StpA, whereas that of "unilaterally controlled genes" is not. Epistatic genes show a higher tendency to be non-essential and recently acquired, when compared to unilateral genes. Epistatic genes reach much higher expression levels than unilateral genes in the absence of the silencing system. Finally, epistatic genes contain more high affinity H-NS binding motifs than unilateral genes. Therefore, both the DNA binding sites of H-NS as well as the function of StpA as a backup system might be selected for silencing highly transcribable genes.

}, keywords = {Binding Sites, DNA-Binding Proteins, Epistasis, Genetic, Escherichia coli K12, Escherichia coli Proteins, Fimbriae Proteins, Gene Expression Regulation, Bacterial, Gene Silencing, Gene Transfer, Horizontal, Genome, Bacterial, Molecular Chaperones, Protein Binding, Repressor Proteins, Sequence Analysis, DNA, Transcription, Genetic, Transcriptome}, issn = {1742-2051}, doi = {10.1039/c3mb70035f}, author = {Srinivasan, Rajalakshmi and Chandraprakash, Deepti and Krishnamurthi, Revathy and Singh, Parul and Scolari, Vittore F and Krishna, Sandeep and Seshasayee, Aswin Sai Narain} } @article {1010, title = {Light driven ultrafast electron transfer in oxidative redding of Green Fluorescent Proteins. [Protein Technology Facility]}, journal = {Sci Rep}, volume = {3}, year = {2013}, month = {2013}, pages = {1580}, abstract = {

Fluorescent proteins undergoing green to red (G/R) photoconversion have proved to be potential tools for investigating dynamic processes in living cells and for photo-localization nanoscopy. However, the photochemical reaction during light induced G/R photoconversion of fluorescent proteins remains unclear. Here we report the direct observation of ultrafast time-resolved electron transfer (ET) during the photoexcitation of the fluorescent proteins EGFP and mEos2 in presence of electron acceptor, p-benzoquinone (BQ). Our results show that in the excited state, the neutral EGFP chromophore accepts electrons from an anionic electron donor, Glu222, and G/R photoconversion is facilitated by ET to nearby electron acceptors. By contrast, mEos2 fails to produce the red emitting state in the presence of BQ; ET depletes the excited state configuration en route to the red-emitting fluorophore. These results show that ultrafast ET plays a pivotal role in multiple photoconversion mechanisms and provide a method to modulate the G/R photoconversion process.

}, keywords = {Benzoquinones, Electron Transport, Green Fluorescent Proteins, Light, Oxidation-Reduction}, issn = {2045-2322}, doi = {10.1038/srep01580}, author = {Saha, Ranajay and Verma, Pramod Kumar and Rakshit, Surajit and Saha, Suvrajit and Mayor, Satyajit and Pal, Samir Kumar} } @article {1009, title = {Tender coconut water an economical growth medium for the production of recombinant proteins in Escherichia coli. [Protein Technology Facility]}, journal = {BMC Biotechnol}, volume = {13}, year = {2013}, month = {2013 Sep 02}, pages = {70}, abstract = {

BACKGROUND: Escherichia coli is most widely used prokaryotic expression system for the production of recombinant proteins. Several strategies have been employed for expressing recombinant proteins in E.coli. This includes the development of novel host systems, expression vectors and cost effective media. In this study, we exploit tender coconut water (TCW) as a natural and cheaper growth medium for E.coli and Pichia pastoris.

RESULT: E.coli and P.pastoris were cultivated in TCW and the growth rate was monitored by measuring optical density at 600 nm (OD(600nm)), where 1.55 for E.coli and 8.7 for P.pastoris was obtained after 12 and 60 hours, respectively. However, variation in growth rate was observed among TCW when collected from different localities (0.15-2.5 at OD(600nm)), which is attributed to the varying chemical profile among samples. In this regard, we attempted the supplementation of TCW with different carbon and nitrogen sources to attain consistency in growth rate. Here, supplementation of TCW with 25 mM ammonium sulphate (TCW-S) was noted efficient for the normalization of inconsistency, which further increased the biomass of E.coli by 2 to 10 folds, and 1.5 to 2 fold in P.pastoris. These results indicate that nitrogen source is the major limiting factor for growth. This was supported by total nitrogen and carbon estimation where, nitrogen varies from 20 to 60 mg/100 ml while carbohydrates showed no considerable variation (2.32 to 3.96 g/100 ml). In this study, we also employed TCW as an expression media for recombinant proteins by demonstrating successful expression of maltose binding protein (MBP), MBP-TEV protease fusion and a photo switchable fluorescent protein (mEos2) using TCW and the expression level was found to be equivalent to Luria Broth (LB).

CONCLUSION: This study highlights the possible application of TCW-S as a media for cultivation of a variety of microorganisms and recombinant protein expression.

}, keywords = {Ammonium Sulfate, Biomass, Carbohydrates, Carbon, Cocos, Culture Media, Escherichia coli, Gene Expression, Nitrogen, Pichia, Recombinant Proteins}, issn = {1472-6750}, doi = {10.1186/1472-6750-13-70}, author = {Sekar, Narendrakumar and Veetil, Soumya Kariyadan and Neerathilingam, Muniasamy} } @article {713, title = {Active remodeling of cortical actin regulates spatiotemporal organization of cell surface molecules.}, journal = {Cell}, volume = {149}, year = {2012}, month = {2012 Jun 08}, pages = {1353-67}, abstract = {

Many lipid-tethered proteins and glycolipids exist as monomers and nanoclusters on the surface of living cells. The spatial distribution and dynamics of formation and breakup of nanoclusters does not reflect thermal and chemical equilibrium and is controlled by active remodeling of the underlying cortical actin. We propose a model for nanoclustering based on active hydrodynamics, wherein cell surface molecules bound to dynamic actin are actively driven to form transient clusters. This consistently explains all of our experimental observations. Using FCS and TIRF microscopy, we provide evidence for the existence of short, dynamic, polymerizing actin filaments at the cortex, a key assumption of the theoretical framework. Our theory predicts that lipid-anchored proteins that interact with dynamic actin must exhibit anomalous concentration fluctuations, and a cell membrane protein capable of binding directly to actin can form nanoclusters. These we confirm experimentally, providing an active mechanism for molecular organization and its spatiotemporal regulation on the plasma membrane.

}, keywords = {Actins, Animals, Cell Line, Tumor, Cell Membrane, CHO Cells, Cricetinae, Cytoskeleton, Humans, Membrane Proteins, Models, Biological, Spectrometry, Fluorescence}, issn = {1097-4172}, doi = {10.1016/j.cell.2012.05.008}, author = {Gowrishankar, Kripa and Ghosh, Subhasri and Saha, Suvrajit and C, Rumamol and Mayor, Satyajit and Rao, Madan} } @article {720, title = {Antiproliferative property of n-hexane and chloroform extracts of Anisomeles malabarica (L). R. Br. in HPV16-positive human cervical cancer cells.}, journal = {J Pharmacol Pharmacother}, volume = {3}, year = {2012}, month = {2012 Jan}, pages = {26-34}, abstract = {

OBJECTIVES: To find the efficacy of serial extracts of Anisomeles malabarica in inhibiting proliferation of and inducing apoptosis in human cervical cancer cells, SiHa and ME 180, that are HPV 16-positive.

MATERIALS AND METHODS: The whole plant was extracted in n-hexane, chloroform, ethyl acetate, n-butanol, methanol, and water. The cells were treated with the extracts at increasing concentrations to find the IC(50), adopting MTT ([3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay. Acridine orange (AO) and ethidium bromide (EB) and Hoechst 33258 staining were adopted to assess the mode of cell death, Annexin V-Cy3 staining to evaluate one of the early apoptotic features, JC-1 staining to assess the mitochondrial membrane depolarization, comet assay for DNA fragmentation, and cell cycle analysis for the distribution of cells after treatment.

RESULTS: n-Hexane and chloroform extracts were cytotoxic to the cervical cancer cells in dose- and duration-dependent manner. The cells that responded to the treatments revealed typical apoptotic features. Early features of apoptosis, phosphatidyl serine translocation and loss of mitochondrial trans-membrane potential, were observed in the treated cells, and comet assay revealed DNA damage. In the FACS analysis, the cells accumulated in the sub-G0/G1 phase of the cell cycle, except in n-hexane- and chloroform extract-treated SiHa cells at 24 h, which showed arrest in S- and G2/M phases.

CONCLUSIONS: n-Hexane and chloroform extracts of A. malabarica inhibit proliferation of and induce death in HPV16-positive cervical cancer cells, mostly by apoptosis and to some extent by necrosis.

}, issn = {0976-5018}, doi = {10.4103/0976-500X.92500}, author = {Preethy, Christo Paul and Padmapriya, Ramamoorthy and Periasamy, Vaiyapuri Subbarayan and Riyasdeen, Anvarbatcha and Srinag, Suresh and Krishnamurthy, Hanumanthappa and Alshatwi, Ali Abdullah and Akbarsha, Mohammad Abdulkader} } @article {718, title = {Development of the structural core and of conformational heterogeneity during the conversion of oligomers of the mouse prion protein to worm-like amyloid fibrils.}, journal = {J Mol Biol}, volume = {423}, year = {2012}, month = {2012 Oct 19}, pages = {217-31}, abstract = {

Understanding how structure develops during the course of amyloid fibril formation by the prion protein is important for understanding prion diseases. Determining how conformational heterogeneity manifests itself in the fibrillar and pre-fibrillar amyloid aggregates is critical for understanding prion strain phenotypes. In this study, the formation of worm-like amyloid fibrils by the mouse prion protein has been characterized structurally by hydrogen-deuterium exchange coupled to mass spectrometry. The structural cores of these fibrils and of the oligomer on the direct pathway of amyloid fibril formation have been defined, showing how structure develops during fibril formation. The structural core of the oligomer not on the direct pathway has also been defined, allowing the delineation of the structural features that make this off-pathway oligomer incompetent to directly form fibrils. Sequence segments that exhibit multiple local conformations in the three amyloid aggregates have been identified, and the development of structural heterogeneity during fibril formation has been characterized. It is shown that conformational heterogeneity is not restricted to only the C-terminal domain region, which forms the structural core of the aggregates; it manifests itself in the N-terminal domain of the protein as well. Importantly, all three amyloid aggregates are shown to be capable of disrupting lipid membrane structure, pointing to a mechanism by which they may be toxic.

}, keywords = {Amino Acid Sequence, Amyloid, Animals, Deuterium Exchange Measurement, Kinetics, Mice, Models, Molecular, Molecular Sequence Data, Prion Proteins, Prions, Protein Conformation, Protein Folding}, issn = {1089-8638}, doi = {10.1016/j.jmb.2012.06.040}, author = {Singh, Jogender and Sabareesan, A T and Mathew, M K and Udgaonkar, Jayant B} } @article {717, title = {Developmental heterogeneity in DNA packaging patterns influences T-cell activation and transmigration.}, journal = {PLoS One}, volume = {7}, year = {2012}, month = {2012}, pages = {e43718}, abstract = {

Cellular differentiation programs are accompanied by large-scale changes in nuclear organization and gene expression. In this context, accompanying transitions in chromatin assembly that facilitates changes in gene expression and cell behavior in a developmental system are poorly understood. Here, we address this gap and map structural changes in chromatin organization during murine T-cell development, to describe an unusual heterogeneity in chromatin organization and associated functional correlates in T-cell lineage. Confocal imaging of DNA assembly in cells isolated from bone marrow, thymus and spleen reveal the emergence of heterogeneous patterns in DNA organization in mature T-cells following their exit from the thymus. The central DNA pattern dominated in immature precursor cells in the thymus whereas both central and peripheral DNA patterns were observed in na{\"\i}ve and memory cells in circulation. Na{\"\i}ve T-cells with central DNA patterns exhibited higher mechanical pliability in response to compressive loads in vitro and transmigration assays in vivo, and demonstrated accelerated expression of activation-induced marker CD69. T-cell activation was characterized by marked redistribution of DNA assembly to a central DNA pattern and increased nuclear size. Notably, heterogeneity in DNA patterns recovered in cells induced into quiescence in culture, suggesting an internal regulatory mechanism for chromatin reorganization. Taken together, our results uncover an important component of plasticity in nuclear organization, reflected in chromatin assembly, during T-cell development, differentiation and transmigration.

}, keywords = {Animals, Antigens, CD, Antigens, Differentiation, T-Lymphocyte, Bone Marrow Cells, Cell Lineage, Cell Movement, Cell Nucleus, Chromatin, DNA, Hematopoietic Stem Cells, Lectins, C-Type, Lymphocyte Activation, Mice, Microscopy, Confocal, Models, Biological, Models, Statistical, Sequence Analysis, DNA, Spleen, T-Lymphocytes}, issn = {1932-6203}, doi = {10.1371/journal.pone.0043718}, author = {Gupta, Soumya and Marcel, Nimi and Talwar, Shefali and Garg, Megha and R, Indulaxmi and Perumalsamy, Lakshmi R and Sarin, Apurva and Shivashankar, G V} } @article {714, title = {Distinct spatial and molecular features of notch pathway assembly in regulatory T cells.}, journal = {Sci Signal}, volume = {5}, year = {2012}, month = {2012 Jul 24}, pages = {ra53}, abstract = {

Variations in the spatial localization of signaling components and crosstalk among signaling cascades are mechanisms through which diversity in signaling networks is generated. The receptor Notch provides an example of regulation by spatial localization: In the canonical Notch signaling pathway, Notch is cleaved to produce the Notch intracellular domain (NICD, also known as NIC), which translocates to the nucleus to regulate gene expression. We describe a T cell receptor-dependent, non-nuclear distribution and function of the processed receptor Notch, which was associated with the improved survival of regulatory T cells (T(regs)) in vitro and in vivo and was compromised by T cell-specific deletion of Notch1. Unlike a nuclear-restricted mutant of NICD, mutant NICD that underwent nuclear export or was targeted to the plasma membrane protected Notch1(-/-) T(regs) from apoptosis induced by nutrient deprivation and oxidative stress. Notch signaling integrated with phosphatidylinositol 3-kinase signaling and mammalian target of rapamycin complex 2 (mTORC2) for this cell survival function. Biochemical and imaging approaches revealed a membrane-proximal complex containing NICD and the mTORC2 component Rictor, and this complex was stabilized by specific interactions with the Notch ligand Delta-like-1 and mediated the survival of T(regs). Together, our evidence for the spatial control of Notch and the crosstalk of Notch signaling with other pathways reveals coupling between the localization of Notch and diverse intracellular signaling pathways.

}, keywords = {Animals, Apoptosis, Blotting, Western, Carrier Proteins, Cell Line, Cell Membrane, Cell Survival, Flow Cytometry, Gene Knockout Techniques, Humans, Immunoprecipitation, Intercellular Signaling Peptides and Proteins, Mice, Mice, Inbred C57BL, Microscopy, Fluorescence, Phosphatidylinositol 3-Kinase, Rapamycin-Insensitive Companion of mTOR Protein, Receptor Cross-Talk, Receptor, Notch1, Signal Transduction, T-Lymphocytes, Regulatory, Trans-Activators, Transcription Factors}, issn = {1937-9145}, doi = {10.1126/scisignal.2002859}, author = {Perumalsamy, Lakshmi R and Marcel, Nimi and Kulkarni, Sneha and Radtke, Freddy and Sarin, Apurva} } @article {491, title = {Draft genome sequence of Rhodovulum sp. strain PH10, a phototrophic alphaproteobacterium isolated from a soil sample of mangrove of Namkhana, India. [Next Generation Genomics facility]}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Nov}, pages = {6363}, abstract = {

We report the 4.8-Mb draft genome of Rhodovulum sp. strain PH10, a phototrophic bacterium belonging to class Alphaproteobacteria, isolated from a soil sample collected from the mangrove forest of Namkhana in India. This genome is the first from the genus Rhodovulum and will lead to a better understanding of the genes/pathways involved in activities like phototrophic growth and nitrogen fixation in this group of bacteria.

}, keywords = {Genome, Bacterial, India, Molecular Sequence Data, Rhodovulum, Soil Microbiology, Wetlands}, issn = {1098-5530}, doi = {10.1128/JB.01695-12}, author = {Khatri, Indu and Korpole, Suresh and Subramanian, Srikrishna and Pinnaka, Anil Kumar} } @article {495, title = {Draft genome sequence of Staphylococcus aureus 118 (ST772), a major disease clone from India. [Next Generation Genomics facility]}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Jul}, pages = {3727-8}, abstract = {

We report the draft genome sequence of an ST772 Staphylococcus aureus disease isolate carrying staphylococcal cassette chromosome mec (SCCmec) type V from a pyomyositis patient. Our de novo short read assembly is \~{}2.8 Mb and encodes a unique Panton-Valentine leukocidin (PVL) phage with structural genes similar to those of ϕ7247PVL and novel lysogenic genes at the N termini.

}, keywords = {Cloning, Molecular, Genome, Bacterial, India, Molecular Sequence Data, Pyomyositis, Staphylococcal Infections, Staphylococcus aureus}, issn = {1098-5530}, doi = {10.1128/JB.00480-12}, author = {Prabhakara, Sushma and Khedkar, Supriya and Loganathan, Ramya Malarini and Chandana, S and Gowda, Malali and Arakere, Gayathri and Seshasayee, Aswin Sai Narain} } @article {494, title = {Draft genome sequence of Staphylococcus aureus ST672, an emerging disease clone from India. [Next Generation Genomics facility]}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Dec}, pages = {6946-7}, abstract = {

We report the draft genome sequence of methicillin-resistant Staphylococcus aureus (MRSA) strain ST672, an emerging disease clone in India, from a septicemia patient. The genome size is about 2.82 Mb with 2,485 open reading frames (ORFs). The staphylococcal cassette chromosome mec (SCCmec) element (type V) and immune evasion cluster appear to be different from those of strain ST772 on preliminary examination.

}, keywords = {Bacteremia, Bacterial Proteins, Bacterial Typing Techniques, Base Sequence, DNA, Bacterial, Genome, Bacterial, Humans, Methicillin-Resistant Staphylococcus aureus, Molecular Sequence Data, Open Reading Frames, Penicillin-Binding Proteins, Sequence Analysis, DNA, Staphylococcal Infections}, issn = {1098-5530}, doi = {10.1128/JB.01868-12}, author = {Khedkar, Supriya and Prabhakara, Sushma and Loganathan, Ramya Malarini and S, Chandana and Gowda, Malali and Arakere, Gayathri and Seshasayee, Aswin Sai Narain} } @article {492, title = {Draft genome sequence of the nitrophenol-degrading actinomycete Rhodococcus imtechensis RKJ300. [Next Generation Genomics facility]}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Jul}, pages = {3543}, abstract = {

We report the 8.231-Mb genome sequence of Rhodococcus imtechensis RKJ300, isolated from pesticide-contaminated soil in Punjab, India. The genome sequence of the strain RKJ300 will be helpful in exploring the molecular pathways involved in the degradation of nitrophenols.

}, keywords = {Biodegradation, Environmental, Genome, Bacterial, India, Molecular Sequence Data, Nitrophenols, Pesticides, Rhodococcus, Sequence Analysis, DNA, Soil Microbiology, Soil Pollutants}, issn = {1098-5530}, doi = {10.1128/JB.00532-12}, author = {Vikram, Surendra and Kumar, Shailesh and Subramanian, Srikrishna and Raghava, Gajendra Pal Singh} } @article {715, title = {Dynamic imaging of homo-FRET in live cells by fluorescence anisotropy microscopy.}, journal = {Methods Enzymol}, volume = {505}, year = {2012}, month = {2012}, pages = {291-327}, abstract = {

Multiple lipid and protein components of the plasma membrane of a living cell are organized, both compositionally and functionally, at different spatial and temporal scales. For instance, Rab protein domains in membranes the clathrin-coated pit, or the immunological synapse are exquisite examples of functional compartmentalization in cell membranes. These assemblies consist in part of nanoscale complexes of lipids and proteins and are necessary to facilitate some specific sorting and signaling functions. It is evident that cellular functions require a regulated spatiotemporal organization of components at the nanoscale, often comprising of countable number of molecular species. Here, we describe multiple homo-FRET-based imaging methods that provide information about nanoscale interactions between fluorescently tagged molecules in live cells, at optically resolved spatial resolution.

}, keywords = {Animals, Cell Membrane, Cell Tracking, Drosophila, Fluorescence Polarization, Fluorescence Resonance Energy Transfer, Green Fluorescent Proteins, Image Processing, Computer-Assisted, Lipid Metabolism, Microscopy, Confocal, Microscopy, Fluorescence}, issn = {1557-7988}, doi = {10.1016/B978-0-12-388448-0.00024-3}, author = {Ghosh, Subhasri and Saha, Suvrajit and Goswami, Debanjan and Bilgrami, Sameera and Mayor, Satyajit} } @article {719, title = {Functional tumor infiltrating TH1 and TH2 effectors in large early-stage cervical cancer are suppressed by regulatory T cells.}, journal = {Int J Gynecol Cancer}, volume = {22}, year = {2012}, month = {2012 Sep}, pages = {1130-7}, abstract = {

OBJECTIVE: Analysis of tumor-infiltrating lymphocytes (TILs) is one of the cornerstones for the understanding of immune responses prevailing in the tumor microenvironment. We studied TILs from squamous cell carcinoma of the cervix ex vivo without proliferating them in vitro before analysis.

METHODS: Whereas TILs were magnetic activated cell separation enriched and flow sorted into CD4 CD25 (regulatory T cells [Tregs]), CD4 CD25 (effector T cells [Teffs]) were directly purified by flow cytometry, and both these subsets were characterized phenotypically and functionally. Tissue sections were probed for interleukin 4 (IL-4) and interferon γ.

RESULTS: Effector T cells constitutively expressed both interferon γ and IL-4 prototypical cytokines of TH1 and TH2, respectively, and were able to proliferate and secrete higher quantities of both cytokines in response to anti-CD3/anti-CD28 and autologous tumor lysates. Only 53\% of cervical cancer Tregs were FOXP3, elaborated transforming growth factor β1, and IL-10 and were able to inhibit both T helper subsets.

CONCLUSIONS: Intratumoral Teffs represented functionally active subsets of both TH1 and TH2 that were not anergic but were suppressed by multiple Treg subsets, which comprised FOXP3 + Tregs and Tregs secreting transforming growth factor β1 and IL-10. These results imply that the microenvironment of cervical carcinomas harbored both TH1 and TH2 subsets of CD4 Teffs that were functionally active but were perhaps unable to perform because of the overpowering effect of Tregs.

}, keywords = {Carcinoma, Squamous Cell, Cytokines, Female, Flow Cytometry, Humans, Immune Tolerance, Interferon-gamma, Lymphocyte Activation, Lymphocytes, Tumor-Infiltrating, Neoplasm Staging, T-Lymphocytes, Regulatory, Th1 Cells, Th2 Cells, Uterine Cervical Neoplasms}, issn = {1525-1438}, doi = {10.1097/IGC.0b013e318262aa53}, author = {Adurthi, Sreenivas and Mukherjee, Geetashree and Krishnamurthy, H and Sudhir, Krishna and Bafna, Uttamchand D and Umadevi, Kswamy and Jayshree, Rudrapatna Subramanyam} } @article {490, title = {Genome sequence of the halotolerant bacterium Imtechella halotolerans K1T. [Next Generation Genomics facility]}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Jul}, pages = {3731}, abstract = {

We report the 3.087-Mb genome sequence of Imtechella halotolerans K1(T), isolated from an estuarine water sample collected from Kochi, Kerala, India. Strain K1 was recently reported as a novel genus of the family Flavobacteriaceae.

}, keywords = {Gene Expression Regulation, Bacterial, Genome, Bacterial, Gram-Negative Aerobic Bacteria, Molecular Sequence Data}, issn = {1098-5530}, doi = {10.1128/JB.00506-12}, author = {Kumar, Shailesh and Vikram, Surendra and Subramanian, Srikrishna and Raghava, Gajendra Pal Singh and Pinnaka, Anil Kumar} } @article {489, title = {Genome sequence of the marine bacterium Marinilabilia salmonicolor JCM 21150T. [Next Generation Genomics facility]}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Jul}, pages = {3746}, abstract = {

We report the 4.98-Mb genome sequence of Marinilabilia salmonicolor JCM 21150(T), which was isolated from marine mud in the year 1961. The draft genome of strain Marinilabilia salmonicolor JCM 21150(T) contains 4,982,627 bp with a G+C content of 41.92\% and 4,227 protein coding genes, 52 tRNAs, and 3 rRNAs.

}, keywords = {Bacteria, Gene Expression Regulation, Bacterial, Genome, Bacterial, Molecular Sequence Data}, issn = {1098-5530}, doi = {10.1128/JB.00649-12}, author = {Kumar, Shailesh and Subramanian, Srikrishna and Raghava, Gajendra Pal Singh and Pinnaka, Anil Kumar} } @article {723, title = {Germ cell abnormalities in streptozotocin induced diabetic mice do not correlate with blood glucose level.}, journal = {J Assist Reprod Genet}, volume = {29}, year = {2012}, month = {2012 Dec}, pages = {1405-13}, abstract = {

PURPOSE: To assess the effect of streptozotocin induced hyperglycemia on germ cell integrity, DNA ploidy and methylation status for a period of two spermatogenesis cycles in adult male Swiss albino mice.

METHODS: Streptozotocin injected mice were monitored for hyperglycemia at a regular interval for a period of 36 and 72\ days. The DNA integrity in epididymal spermatozoa was determined by the comet assay. Flow cytometric analysis was done in germ cells to assess the DNA ploidy. The global methylation analysis in germ cells was done by 5-methyl cytosine immunostaining.

RESULTS: Streptozotocin administration successfully resulted in hyperglycemic response which significantly affected serum testosterone level, sperm DNA integrity and DNA ploidy at the end of 36\ days. However, no changes were observed in either epididymal sperm concentration or germ cell methylation status. In contrast, at the end of 76\ days, although serum testosterone level, sperm DNA integrity and DNA ploidy status were unperturbed significantly in hyperglycemic group, the epididymal sperm concentration and methylation status of preleptotene/zygotene cells were significantly altered. Importantly, an attempt to find out the association between the blood glucose levels and the abnormalities in hyperglycemic group failed to demonstrate any correlation.

CONCLUSIONS: The germ cell abnormalities observed in hyperglycemic group could be interpreted as a primary effect of streptozotocin and not due to hyperglycemia. Our results call for further evaluation of streptozotocin before its application to study the hyperglycemic responses on male germ cells.

}, keywords = {Animals, Blood Glucose, Diabetes Mellitus, Experimental, DNA Methylation, Epididymis, Germ Cells, Humans, Hyperglycemia, Male, Mice, Ploidies, Sperm Count, Spermatogenesis, Spermatozoa, Streptozocin, Testosterone}, issn = {1573-7330}, doi = {10.1007/s10815-012-9873-0}, author = {Bose, Rohini and Adiga, Satish K and D{\textquoteright}Souza, Fiona and Salian, Sujith R and Uppangala, Shubhashree and Kalthur, Guruprasad and Jain, Navya and Radhakrishnan, Raghu A and Bhat, Nalini and Krishnamurthy, Hanumantappa and Kumar, Pratap} } @article {712, title = {An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression.}, journal = {Cell}, volume = {151}, year = {2012}, month = {2012 Dec 21}, pages = {1474-87}, abstract = {

DNA Ligase IV is responsible for sealing of double-strand breaks (DSBs) during nonhomologous end-joining (NHEJ). Inhibiting Ligase IV could result in amassing of DSBs, thereby serving as a strategy toward treatment of cancer. Here, we identify a molecule, SCR7 that inhibits joining of DSBs in cell-free repair system. SCR7 blocks Ligase IV-mediated joining by interfering with its DNA binding but not that of T4 DNA Ligase or Ligase I. SCR7 inhibits NHEJ in a Ligase IV-dependent manner within cells,\ and activates the intrinsic apoptotic pathway. More importantly, SCR7 impedes tumor progression in mouse models and when coadministered with DSB-inducing therapeutic modalities enhances their sensitivity significantly. This inhibitor to target NHEJ offers a strategy toward the treatment of cancer and improvement of existing regimens.

}, keywords = {Amino Acid Sequence, Animals, Cell Line, Tumor, Disease Models, Animal, DNA Breaks, Double-Stranded, DNA End-Joining Repair, DNA Ligase ATP, DNA Ligases, Drug Design, Drug Resistance, Neoplasm, Humans, Lymphocytes, Lymphoma, Male, Mice, Mice, Inbred BALB C, Mice, Nude, Models, Molecular, Molecular Sequence Data, Neoplasms, Protein Structure, Tertiary, Pyrimidines, Radiation Tolerance, Rats, Schiff Bases, Sequence Alignment}, issn = {1097-4172}, doi = {10.1016/j.cell.2012.11.054}, author = {Srivastava, Mrinal and Nambiar, Mridula and Sharma, Sheetal and Karki, Subhas S and Goldsmith, G and Hegde, Mahesh and Kumar, Sujeet and Pandey, Monica and Singh, Ram K and Ray, Pritha and Natarajan, Renuka and Kelkar, Madhura and De, Abhijit and Choudhary, Bibha and Raghavan, Sathees C} } @article {479, title = {Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse. [Drosophila facility]}, journal = {Traffic}, volume = {13}, year = {2012}, month = {2012 Jul}, pages = {979-91}, abstract = {

Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin-based movement of large protein-aggregates aids this process. Choline acetyltransferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates toward the synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. Here, we show that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution.

}, keywords = {Animals, Animals, Genetically Modified, Axonal Transport, Carrier Proteins, Choline O-Acetyltransferase, Cholinergic Neurons, Drosophila, Drosophila Proteins, Fluorescence Recovery After Photobleaching, Kinesin, Larva, Microtubule-Associated Proteins, Protein Interaction Domains and Motifs, Synapses}, issn = {1600-0854}, doi = {10.1111/j.1600-0854.2012.01361.x}, author = {Sadananda, Aparna and Hamid, Runa and Doodhi, Harinath and Ghosal, Debnath and Girotra, Mukul and Jana, Swadhin Chandra and Ray, Krishanu} } @article {724, title = {Neutrophil extracellular traps contain mitochondrial as well as nuclear DNA and exhibit inflammatory potential.}, journal = {Cytometry A}, volume = {81}, year = {2012}, month = {2012 Mar}, pages = {238-47}, abstract = {

Neutrophils expel extracellular traps (NETs) to entrap and exterminate the invaded micro-organisms. Acute/chronic inflammatory disorders are often observed with aberrantly enhanced NETs formation and high nitric oxide (NO) availability. Recent study from this laboratory demonstrated release of NETs from human neutrophils following treatment with SNP or SNAP. This study is an extension of our previous finding to explore the extracellular bacterial killing, source of DNA in the expelled NETs, their ability to induce proinflammatory cytokines release from platelets/THP-1 cells, and assessment of NO-mediated free radical formation by using a consistent NO donor, DETA-NONOate. NO-mediated NETs exhibited extracellular bacterial killing as determined by colony forming units. NO-mediated NETs formation was due to the activation of NADPH oxidase and myeloperoxidase. NO- or PMA-mediated NETs were positive for both nuclear and mitochondrial DNA as well as proteolytic enzymes. Incubation of NETs with human platelets enhanced the release of IL-1β and IL-8, while with THP-1 cells, release of IL-1β, IL-8, and TNFα was observed. This study demonstrates that NO by augmenting enzymatic free radical generation release NETs to promote extracellular bacterial killing. These NETs were made up of mitochondrial and nuclear DNA and potentiated release of proinflammatory cytokines.

}, keywords = {Adult, Blood Platelets, DNA, DNA, Mitochondrial, Free Radicals, Humans, Inflammation, Interleukin-1beta, Interleukin-8, Mitochondria, NADPH Oxidases, Neutrophil Activation, Neutrophils, Nitric Oxide, Peroxidase, Tumor Necrosis Factor-alpha}, issn = {1552-4930}, doi = {10.1002/cyto.a.21178}, author = {Keshari, Ravi S and Jyoti, Anupam and Kumar, Sachin and Dubey, Megha and Verma, Anupam and Srinag, Bangalore S and Krishnamurthy, Hanumanthappa and Barthwal, Manoj K and Dikshit, Madhu} } @article {722, title = {Poor sperm quality and advancing age are associated with increased sperm DNA damage in infertile men.}, journal = {Andrologia}, volume = {44 Suppl 1}, year = {2012}, month = {2012 May}, pages = {642-9}, abstract = {

With increasing evidence for faulty paternal contribution to reproduction, there has been a steady increase in studies highlighting an association between sperm DNA damage, failed/delayed fertilisation and aberrant embryo development. Owing to prevailing ambiguity, the aims of the study were to analyse the genetic integrity of the male gamete and then to understand its association with age, standard semen parameters, lifestyle and occupational factors. The study included 504 subjects, attending university infertility clinic for fertility evaluation and treatment. Semen characteristics were analysed by standard criteria; terminal deoxynucelotidyl transferase-mediated nick end-labelling assay was employed for DNA damage assessment. The average incidence of sperm DNA damage in patients with normozoospermic semen parameters was \<10\%. Patients with oligozoospermia, severe oligozoospermia, oligoasthenoteratospermia, asthenoteratozoospermia and necrozoospermia had significantly higher level of sperm DNA damage (P \< 0.001). Patients above 40 years of age had significantly high levels of DNA damage (P \< 0.001) compared with their counterparts. Patients with varicocele and a history of alcohol consumption had higher incidence of spermatozoa with DNA damage (P \< 0.01). Poor sperm characteristics in the ejaculate are associated with increased sperm DNA damage. Age-related increase in sperm DNA damage and association of the same with varicocele and alcohol consumption are also demonstrated.

}, keywords = {Aging, DNA Damage, Humans, Infertility, Male, Life Style, Male, Occupations, Spermatozoa}, issn = {1439-0272}, doi = {10.1111/j.1439-0272.2011.01243.x}, author = {Varshini, J and Srinag, B S and Kalthur, G and Krishnamurthy, H and Kumar, P and Rao, S B-S and Adiga, S K} } @article {721, title = {Synaptic activity in serotonergic neurons is required for air-puff stimulated flight in Drosophila melanogaster.}, journal = {PLoS One}, volume = {7}, year = {2012}, month = {2012}, pages = {e46405}, abstract = {

BACKGROUND: Flight is an integral component of many complex behavioral patterns in insects. The giant fiber circuit has been well studied in several insects including Drosophila. However, components of the insect flight circuit that respond to an air-puff stimulus and comprise the flight central pattern generator are poorly defined. Aminergic neurons have been implicated in locust, moth and Drosophila flight. Here we have investigated the requirement of neuronal activity in serotonergic neurons, during development and in adults, on air-puff induced flight in Drosophila.

METHODOLOGY/PRINCIPAL FINDINGS: To target serotonergic neurons specifically, a Drosophila strain that contains regulatory regions from the TRH (Tryptophan Hydroxylase) gene linked to the yeast transcription factor GAL4 was used. By blocking synaptic transmission from serotonergic neurons with a tetanus toxin transgene or by hyperpolarisation with Kir2.1, close to 50\% adults became flightless. Temporal expression of a temperature sensitive Dynamin mutant transgene (Shi(ts)) suggests that synaptic function in serotonergic neurons is required both during development and in adults. Depletion of IP(3)R in serotonergic neurons via RNAi did not affect flight. Interestingly, at all stages a partial requirement for synaptic activity in serotonergic neurons was observed. The status of serotonergic neurons was investigated in the central nervous system of larvae and adults expressing tetanus toxin. A small but significant reduction was observed in serotonergic cell number in adult second thoracic segments from flightless tetanus toxin expressing animals.

CONCLUSIONS: These studies show that loss of synaptic activity in serotonergic neurons causes a flight deficit. The temporal focus of the flight deficit is during pupal development and in adults. The cause of the flight deficit is likely to be loss of neurons and reduced synaptic function. Based on the partial phenotypes, serotonergic neurons appear to be modulatory, rather than an intrinsic part of the flight circuit.

}, keywords = {Animals, Cell Count, Central Nervous System, DNA-Binding Proteins, Drosophila melanogaster, Drosophila Proteins, Dynamins, Flight, Animal, Gene Expression Regulation, Developmental, Larva, Potassium Channels, Inwardly Rectifying, Pupa, Saccharomyces cerevisiae Proteins, Serotonergic Neurons, Synaptic Transmission, Tetanus Toxin, Transcription Factors, Transgenes, Tryptophan Hydroxylase}, issn = {1932-6203}, doi = {10.1371/journal.pone.0046405}, author = {Sadaf, Sufia and Birman, Serge and Hasan, Gaiti} } @article {482, title = {A protein complex network of Drosophila melanogaster. [Drosophila facility]}, journal = {Cell}, volume = {147}, year = {2011}, month = {2011 Oct 28}, pages = {690-703}, abstract = {

Determining the composition of protein complexes is an essential step toward understanding the cell as an integrated system. Using coaffinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly 5,000 individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a statistical framework designed to define individual protein-protein interactions, led to the generation of a Drosophila protein interaction map (DPiM) encompassing 556 protein complexes. The high quality of the DPiM and its usefulness as a paradigm for metazoan proteomes are apparent from the recovery of many known complexes, significant enrichment for shared functional attributes, and validation in human cells. The DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. The DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution.

}, keywords = {Animals, Drosophila melanogaster, Drosophila Proteins, Proteasome Endopeptidase Complex, Protein Interaction Mapping, Proteomics, SNARE Proteins}, issn = {1097-4172}, doi = {10.1016/j.cell.2011.08.047}, author = {Guruharsha, K G and Rual, Jean-Fran{\c c}ois and Zhai, Bo and Mintseris, Julian and Vaidya, Pujita and Vaidya, Namita and Beekman, Chapman and Wong, Christina and Rhee, David Y and Cenaj, Odise and McKillip, Emily and Shah, Saumini and Stapleton, Mark and Wan, Kenneth H and Yu, Charles and Parsa, Bayan and Carlson, Joseph W and Chen, Xiao and Kapadia, Bhaveen and VijayRaghavan, K and Gygi, Steven P and Celniker, Susan E and Obar, Robert A and Artavanis-Tsakonas, Spyros} } @article {483, title = {Inositol 1,4,5-trisphosphate receptor and dSTIM function in Drosophila insulin-producing neurons regulates systemic intracellular calcium homeostasis and flight. [Drosophila facility]}, journal = {J Neurosci}, volume = {30}, year = {2010}, month = {2010 Jan 27}, pages = {1301-13}, abstract = {

Calcium (Ca(2+)) signaling is known to regulate the development, maintenance and modulation of activity in neuronal circuits that underlie organismal behavior. In Drosophila, intracellular Ca(2+) signaling by the inositol 1,4,5-trisphosphate receptor and the store-operated channel (dOrai) regulates the formation and function of neuronal circuits that control flight. Here, we show that restoring InsP(3)R activity in insulin-producing neurons of flightless InsP(3)R mutants (itpr) during pupal development can rescue systemic flight ability. Expression of the store operated Ca(2+) entry (SOCE) regulator dSTIM in insulin-producing neurons also suppresses compromised flight ability of InsP(3)R mutants suggesting that SOCE can compensate for impaired InsP(3)R function. Despite restricted expression of wild-type InsP(3)R and dSTIM in insulin-producing neurons, a global restoration of SOCE and store Ca(2+) is observed in primary neuronal cultures from the itpr mutant. These results suggest that restoring InsP(3)R-mediated Ca(2+) release and SOCE in a limited subset of neuromodulatory cells can influence systemic behaviors such as flight by regulating intracellular Ca(2+) homeostasis in a large population of neurons through a non-cell-autonomous mechanism.

}, keywords = {Animals, Calcium, Calcium Signaling, Cell Membrane, Cells, Cultured, Central Nervous System, Drosophila, Drosophila Proteins, Flight, Animal, Homeostasis, Inositol 1,4,5-Trisphosphate Receptors, Insulin, Intracellular Fluid, Membrane Proteins, Mutation, Neural Pathways, Neurons, Pupa, Stromal Interaction Molecule 1}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.3668-09.2010}, author = {Agrawal, Neha and Venkiteswaran, Gayatri and Sadaf, Sufia and Padmanabhan, Nisha and Banerjee, Santanu and Hasan, Gaiti} }