@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 {9059, 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 {8824, title = {Macrophage-conditioned medium enhances tunneling nanotube formation in breast cancer cells via PKC, Src, NF-κB, and p38 MAPK signaling [Next Gen Genomics Facility]}, journal = {Cell Signal}, year = {2024}, month = {2024 Jun 25}, pages = {111274}, abstract = {

Tumor-associated macrophages (TAMs) secrete cytokines, chemokines, and growth factors in the tumor microenvironment (TME) to support cancer progression. Higher TAM infiltration in the breast TME is associated with a poor prognosis. Previous studies have demonstrated the role of macrophages in stimulating long-range intercellular bridges referred to as tunneling nanotubes (TNTs) in cancer cells. Intercellular communication between cancer cells via TNTs promotes cancer growth, invasion, metastasis, and therapy resistance. Given the important role of TNTs and macrophages in cancer, the role of macrophage-induced TNTs in chemotherapy drug doxorubicin resistance is not known. Furthermore, the mechanism of macrophage-mediated TNT formation is elusive. In this study, it is shown that the macrophage-conditioned medium (MΦCM) partially mimicked inflammatory TME, induced an EMT phenotype, and increased migration in MCF-7 breast cancer cells. Additionally, secreted proteins in MΦCM induced TNT formation in MCF-7 cells, which led to increased resistance to doxorubicin. Transcriptomic analysis of MΦCM-treated MCF-7 cells showed enrichment of the NF-κB and focal adhesion pathways, as well as upregulation of genes involved in EMT, extracellular remodeling, and actin cytoskeleton reorganization. Interestingly, inhibitors of PKC, Src, NF-κB, and p38 decreased macrophage-induced TNT formation in MCF-7 cells. These results reveal the novel role of PKC and Src in inducing TNT formation in cancer cells and suggest that inhibition of PKC and Src activity may likely contribute to reduced macrophage-breast cancer cell interaction and the potential therapeutic strategy of cancer.

}, issn = {1873-3913}, doi = {10.1016/j.cellsig.2024.111274}, author = {Melwani, Pooja Kamal and Balla, Murali Mohan Sagar and Bhamani, Aman and Nandha, Shivani R and Checker, Rahul and Pandey, Badri Narain} } @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 {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 {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 {8062, title = {CRISPR/Cas9 and FLP-FRT mediated regulatory dissection of the BX-C of Drosophila melanogaster [Transgenic Fly Facility]}, journal = {Chromosome Res}, volume = {31}, year = {2023}, month = {2023 Jan 31}, pages = {7}, abstract = {

The homeotic genes or Hox define the anterior-posterior (AP) body axis formation in bilaterians and are often present on the chromosome in an order collinear to their function across the AP axis. However, there are many cases wherein the Hox are not collinear, but their expression pattern is conserved across the AP axis. The expression pattern of Hox is attributed to the cis-regulatory modules (CRMs) consisting of enhancers, initiators, or repressor elements that regulate the genes in a segment-specific manner. In the Drosophila melanogaster Hox complex, the bithorax complex (BX-C) and even the CRMs are organized in an order that is collinear to their function in the thoracic and abdominal segments. In the present study, the regulatorily inert regions were targeted using CRISPR/Cas9 to generate a series of transgenic lines with the insertion of FRT sequences. These FRT lines are repurposed to shuffle the CRMs associated with Abd-B to generate modular deletion, duplication, or inversion of multiple CRMs. The rearrangements yielded entirely novel phenotypes in the fly suggesting the requirement of such complex manipulations to address the significance of higher order arrangement of the CRMs. The functional map and the transgenic flies generated in this study are important resources to decipher the collective ability of multiple regulatory elements in the eukaryotic genome to function as complex modules.

}, keywords = {Animals, CRISPR-Cas Systems, Drosophila melanogaster, Drosophila Proteins, Gene Expression Regulation, Developmental, Homeodomain Proteins, Regulatory Sequences, Nucleic Acid}, issn = {1573-6849}, doi = {10.1007/s10577-023-09716-w}, author = {Hajirnis, Nikhil and Pandey, Shubhanshu and Mishra, Rakesh K} } @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 {8097, title = {Identification of key amino acid residues in OqxB mediated efflux of fluoroquinolones using site-directed mutagenesis [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Res Microbiol}, year = {2023}, month = {2023 Feb 02}, pages = {104039}, abstract = {

OqxB belongs to the RND (Resistance-Nodulation-Division) efflux pump family, recognized widely as a major contributor towards enhancing antimicrobial resistance. It is known to be predominantly present in all Klebsiella spp. and is attributed for its role in increasing resistance against an array of antibiotics like nitrofurantoin, quinolones, β-lactams and colistin. However, the presence of oqxB encoding this efflux pump is not limited only to Klebsiella spp., but is also found to occur via horizontal gene transfer in other bacterial genera like Escherichia coli, Enterobacter cloacae and Salmonella spp. Recently, we reported the crystal structure of OqxB and its structure-function relationship required for the efflux of fluoroquinolones. Extending these findings further, we characterized the structural architecture of this efflux pump along with identifying some critical amino acids at the substrate binding domain of OqxB. Based on our in silico modelling studies, both, hydrophobic residues (F180, L280, L621, F626) and polar residues (R48, E50, E184, R157, R774) were found to be located at this site. The present work reports the importance of these key amino acid residues and the crucial ion-pair interactions at the substrate-binding pocket, thereby establishing their role in OqxB mediated efflux and the resultant resistance development against fluoroquinolones.

}, issn = {1769-7123}, doi = {10.1016/j.resmic.2023.104039}, author = {Bhowmik, Purnendu and Bharatham, Nagakumar and Murakami, Satoshi and Ramachandran, Vasanthi and Datta, Santanu} } @article {8588, title = {A new species of Bufoides Pillai and Yazdani 1973 (Amphibia: Bufonidae) from Mizoram (India) and the delimitation of the distribution range of Bufoides meghalayanus (Yazdani \& Chanda 1971) to the Khasi hills, Meghalaya (India) [Next Gen Genomics Facility]}, journal = { Biodiversitas Journal of Biological Diversity}, volume = {Vol. 24 No. 9}, year = {2023}, month = {10/2023}, chapter = {4617}, abstract = {

Naveen RS, Tapley B, Chandramouli SR, Jervis PA, Babu S, Meetei AB, Karunakaran PV. 2023. A new species of\ Bufoides\ Pillai and Yazdani 1973 (Amphibia: Bufonidae) from Mizoram\ (India) and the delimitation of the distribution range of\ Bufoides meghalayanus\ (Yazdani \& Chanda 1971) to the Khasi hills, Meghalaya\ (India). Biodiversitas 24: 4617-4627.\ The Oriental toad genus\ Bufoides\ currently comprises two species:\ Bufoides meghalayanus\ and\ B. kempi. Populations of\ Bufoides\ from Mizoram were previously considered to be conspecific with\ Bufoides meghalayanus,\ although it has been hypothesized that these populations could represent an undescribed species. An uncorrected p-distance at the 16S rDNA gene between the Mizoram population and each of the two congeneric species was 2.74-3.0\% and 3.5\% for\ B. meghalayanus\ and\ B. kempi\ respectively. We describe the population from Dampa Tiger Reserve, Mizoram, as new based on molecular from two specimens and morphological data from two adult males and one adult female. We confirm that\ B. meghalayanus\ is endemic to the Khasi Hills in Meghalaya and it does not occur in Mizoram. The new species from Mizoram differs from congeneric species by differences in interdigital webbing, coloration, skin tuberculation and the presence of ovoid, tuberculated and depressed parotoid glands. Like other\ Bufoides\ species, it is a microhabitat specialist and utilizes streamside rock crevices as refugia, which might make it vulnerable to changes in habitat. The new species is currently only known to occur in Dampa Tiger Reserve and it is probably range-restricted and likely meets the International Union for Conservation of Nature{\textquoteright}s criteria for being assessed as Critically Endangered.

}, keywords = {Amphibian, conservation, cryptic diversity, Indo-Burma region, systematics, taxonomy}, issn = {1412-033X}, doi = {0.13057/biodiv/d240901}, url = {https://smujo.id/biodiv/article/view/14616}, author = {R.S. NAVEEN and BENJAMIN TAPLEY and S.R. CHANDRAMOULI and PHILLIP A. JERVIS and S. BABU and A.B. MEETEI and P.V. KARUNAKARAN} } @article {4869, title = {Dual control of dopamine in Drosophila myeloid-like progenitor cell proliferation and regulation of lymph gland growth. [Central Imaging \& Flow Cytometry Facility]}, journal = {EMBO Rep}, year = {2022}, month = {2022 Apr 27}, pages = {e52951}, type = {Journal Article}, abstract = {

In Drosophila, definitive haematopoiesis takes place in a specialized organ termed "lymph gland". It harbours multi-potent stem-like blood progenitor cells whose development controls overall growth of this haematopoietic tissue and formation of mature blood cells. With respect to its development, neurotransmitters have emerged as potent regulators of blood-progenitor cell development and function. In this study, we extend our understanding of neurotransmitters and show that progenitors are self-sufficient with regard to synthesizing dopamine, a well-established neurotransmitter. These cells also have modules for dopamine sensing through the receptor and transporter. We found that modulating expression of these components in progenitor cells affected lymph gland growth, which suggested growth-promoting function of dopamine in blood-progenitor cells. Cell-cycle analysis of developing lymph glands revealed an unexpected requirement for intracellular dopamine in moderating the progression of early progenitor cells from S to G2 phase of the cell cycle, while activation of dopamine receptor signalling later in development regulated their progression from G2 and entry into mitosis. The dual capacity in which dopamine operated, first intracellularly to coordinate S/G2 transition and later extracellularly in G2/M transition, was critical for the growth of the lymph gland. Overall, the data presented highlight a novel non-canonical use of dopamine in the myeloid system that reveals an uncharacterized function of intracellular dopamine in cell-cycle phasing with outcomes on haematopoietic growth and immunity as well.

}, keywords = {cell cycle, dopamine signalling, dopamine synthesis, haematopoietic progenitors, proliferation}, issn = {1469-3178}, doi = {10.15252/embr.202152951}, url = {embopress.org/doi/abs/10.15252/embr.202152951}, author = {Kapoor, Ankita and Padmavathi, Achalla and Madhwal, Sukanya and Mukherjee, Tina} } @article {8883, title = {Effect of Altitude on Volatile Organic and Phenolic Compounds of Artemisia brevifolia Wall ex Dc. From the Western Himalayas [Mass Spectrometry Facility]}, journal = {Frontiers in Ecology and Evolution}, volume = {10}, year = {2022}, abstract = {

Adaptation to changing environmental conditions is a driver of plant diversification. Elevational gradients offer a unique opportunity for investigating adaptation to a range of climatic conditions. The use of specialized metabolites as volatile and phenolic compounds is a major adaptation in plants, affecting their reproductive success and survival by attracting pollinators and protecting themselves from herbivores and other stressors. The wormseed Artemisia brevifolia can be found across multiple elevations in the Western Himalayas, a region that is considered a biodiversity hotspot and is highly impacted by climate change. This study aims at understanding the volatile and phenolic compounds produced by A. brevifolia in the high elevation cold deserts of the Western Himalayas with the view to understanding the survival strategies employed by plants under harsh conditions. Across four sampling sites with different elevations, polydimethylsiloxane (PDMS) sampling and subsequent GCMS analyses showed that the total number of volatile compounds in the plant headspace increased with elevation and that this trend was largely driven by an increase in compounds with low volatility, which might improve the plant{\textquoteright}s resilience to abiotic stress. HPLC analyses showed no effect of elevation on the total number of phenolic compounds detected in both young and mature leaves. However, the concentration of the majority of phenolic compounds decreased with elevation. As the production of phenolic defense compounds is a costly trait, plants at higher elevations might face a trade-off between energy expenditure and protecting themselves from herbivores. This study can therefore help us understand how plants adjust secondary metabolite production to cope with harsh environments and reveal the climate adaptability of such species in highly threatened regions of our planet such as the Himalayas.

}, issn = {2296-701X}, doi = {10.3389/fevo.2022.864728}, url = {https://www.frontiersin.org/journals/ecology-and-evolution/articles/10.3389/fevo.2022.864728}, author = {Nataraj, Nandita and Hussain, Manzoor and Ibrahim, Mohd and Hausmann, Alexander E. and Rao, Srinivas and Kaur, Satwinderjeet and Khazir, Jabeena and Mir, Bilal Ahmad and Olsson, Shannon B.} } @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 {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 {6781, title = {Techno-commercial application of microencapsulated basil oil and thyme oil and their antibacterial activity against aquatic pathogens [Electron Microscopy Facility]}, journal = {International Journal of Fisheries and Aquatic Studies}, volume = {10}, year = {2022}, month = {10/2022}, chapter = {101-106}, 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 = {2394-0506}, doi = {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 Ganesh Muguli and Sameer Pai and Babu UV} } @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 {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 {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 {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 {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 {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 {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 {1917, title = {Novel non intrusive continuous use ZeBox technology to trap and kill airborne microbes [Biomoneta Research Pvt. Ltd., a C-CAMP Startup / Electron Microscopy Facility]}, journal = {Scientific Reports}, volume = {11}, year = {2021}, month = {11/2021}, pages = {Article number: 22779 }, abstract = {

Preventing nosocomial infection is a major unmet need of our times. Existing air decontamination technologies suffer from demerits such as toxicity of exposure, species specificity, noxious gas emission, environment-dependent performance and high power consumption. Here, we present a novel technology called {\textquotedblleft}ZeBox{\textquotedblright} that transcends the conventional limitations and achieves high microbicidal efficiency. In ZeBox, a non-ionizing electric field extracts naturally charged microbes from flowing air and deposits them on engineered microbicidal surfaces. The surface{\textquoteright}s three dimensional topography traps the microbes long enough for them to be inactivated. The electric field and chemical surfaces synergistically achieve rapid inactivation of a broad spectrum of microbes. ZeBox achieved near complete kill of airborne microbes in challenge tests (5{\textendash}9 log reduction) and\ \>90\%\ efficiency in a fully functional stem cell research facility in the presence of humans. Thus, ZeBox fulfills the dire need for a real-time, continuous, safe, trap-and-kill air decontamination technology.

}, doi = {doi.org/10.1038/s41598-021-02184-4}, author = {Kruttika S. Phadke and Deepak G. Madival and Janani Venkataraman and Debosmita Kundu and K. S. Ramanujan and Nisha Holla and Jaywant Arakeri and Gaurav Tomar and Santanu Datta and Arindam Ghatak} } @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 {1743, title = {Xeno-Free Human Wharton{\textquoteright}s Jelly Mesenchymal Stromal Cells Maintain Their Characteristic Properties after Long-Term Cryopreservation [OCT Therapies \& Research, a C-CAMP Startup] }, journal = {Cell Journal (Yakhteh)}, volume = {23}, year = {2021}, month = {07/2021}, chapter = {145}, abstract = {

Objective: The past decade has witnessed a rapid growth in harnessing the potential of adult stem cells for regenerative medicine. An investigational new drug (IND) or a regenerative medicine advanced therapy (RMAT) product must fulfil many requirements, such as stability studies, after cryopreservation. Such studies are important to ascertain the utility of off-the-shelf allogeneic cells for clinical applications. The present work describes a complete characterisation of xenofree human Wharton{\textquoteright}s Jelly mesenchymal stromal cells (hWJ-MSCs) before and up to 28 months post-cryopreservation.

Materials and Methods: In this experimental study, culture methods that involved plasma derived human serum and recombinant trypsin were used to develop clinical grade cells. Complete cell characterisation involved flow cytometry studies for viability, positive and negative markers, colony forming unit (CFU) potential, population doubling time (PDT), soft agar assay to evaluate in vitro tumourigenicity, karyotype analysis and differentiation studies which were performed before and at 6, 12, 18 and 28 months post-cryopreservation.

Results: Our data showed consistency in the flow cytometry, CFU assay, PDT, soft agar assay, karyotyping and differentiation studies.

Conclusion: Using our protocols for extended xeno-free culture and cryopreservation of hWJ-MSCs, we could establish the shelf life of the cell-based product for up to 28 months.

}, keywords = {Mesenchymal Stromal Cells, Stability, Umbilical Cord, Wharton{\textquoteright}s Jelly}, doi = {10.22074/cellj.2021.7131}, author = {Mathen, Caroline and D{\textquoteright}souza, Wilfrid} } @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 {1248, title = {ELYS Regulates Dorsal Dynamics during Development [Transgenic Fly Facility]}, journal = {J Biol Chem}, year = {2020}, month = {2020 Jan 15}, abstract = {

Embryonic large molecule derived from yolk sac (ELYS) is a constituent protein of nuclear pores. It initiates assembly of nuclear pore complexes (NPCs) into functional nuclear pores toward the end of mitosis. Using cellular, molecular and genetic tools, including fluorescence and electron microscopy, quantitative PCR and RNAi mediated depletion, here; we report that ELYS ortholog (Elys) plays critical roles during development. Elys localized to the nuclear rim in interphase cells, but during mitosis, it was absent from kinetochores and enveloped chromatin. We observed that RNAi-mediated depletion leads to aberrant development and, at the cellular level, to defects in the nuclear pore and nuclear lamina assembly. Further genetic analyses indicated that depletion re-activates the Dorsal (NF-κB) pathway during late larval stages. Re-activated Dorsal caused untimely expression of the Dorsal target genes in the post-embryonic stages. We also demonstrate that activated Dorsal triggers apoptosis during later developmental stages by up-regulating the pro-apoptotic genes and The apoptosis induced by Reaper and Hid was probably the underlying cause for developmental abnormalities observed upon depletion. Moreover, we noted that Elys has conserved structural features, but contains a non-canonical AT-hook like motif through which it strongly binds to DNA. Together, our results uncover a novel epistatic interaction that regulates Dorsal dynamics by Elys during development.

}, issn = {1083-351X}, doi = {10.1074/jbc.RA119.009451}, author = {Mehta, Saurabh Jayesh Kumar and Kumar, Vimlesh and Mishra, Ram Kumar} } @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 {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 {1422, title = {Structural Characterization of an Exopolysaccharide Isolated from Enterococcus faecalis, and Study on its Antioxidant Activity, and Cytotoxicity Against HeLa Cells [Mass Spectrometry - Glycomics]}, journal = {Curr Microbiol}, year = {2020}, month = {2020 Jul 28}, abstract = {

An exopolysaccharide (EPS-I) having the molecular weight ~ 2.6 {\texttimes} 10\ Da, was isolated from a Zinc resistant strain of Enterococcus faecalis from costal area. The exopolysaccharide consists of D-mannose, D-glucose, and L-fucose in molar ratio of 9:4:1. The monosaccharide units in the EPS-1 were determined through chemical (total acid hydrolysis and methylation analysis) and spectroscopic (FTIR and H NMR experiment) analysis. The mannose-rich EPS-1 showed total antioxidant activity (1\ mg\ mL of EPS-I as functional as approximately to 500 {\textpm} 5.2\ {\textmu}M of ascorbic acid) and Fe metal ion chelation activity (EC = 405.6\ {\textmu}g\ mL) and hydroxyl radical scavenging activity (EC = 219.5\ {\textmu}g\ mL). The in vitro cytotoxicity experiment of EPS-I against cervical carcinoma cell line, HeLa cells showed strong cytotoxic effect (LC = 267.3\ {\textmu}g\ mL) and at that concentration, it found almost nontoxic against normal healthy cells (HEK-293).

}, issn = {1432-0991}, doi = {10.1007/s00284-020-02130-z}, author = {Choudhuri, Indranil and Khanra, Kalyani and Pariya, Prasenjit and Maity, Gajendra Nath and Mondal, Soumitra and Pati, Bikas Ranjan and Bhattacharyya, Nandan} } @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 {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 {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 {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 {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 {1193, title = {Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration [Mass Spectrometry - Proteomics Facility]}, journal = {Mol Pharm}, year = {2019}, month = {2019 Oct 22}, abstract = {

Recombinant adeno-associated virus (AAV)-based gene therapy has been promising, but several host-related transduction or immune challenges remain. For this mode of therapy to be widely applicable, it is crucial to develop high transduction and permeating vectors that infect the target at significantly low doses. Because glycosylation of capsid proteins is known to be rate limiting in the life cycle of many viruses, we reasoned that perturbation of glycosylation sites in AAV2 capsid will enhance gene delivery. In our first set experiments, pharmacological modulation of the glycosylation status in host cells, modestly decreased (1-fold) AAV2 packaging efficacy while it improved their gene expression (\~{}74\%) in vitro. We then generated 24 mutant AAV2 vectors modified to potentially create or disrupt a glycosylation site in its capsid. Three of them demonstrated a 1.3-2.5-fold increase in transgene expression in multiple cell lines (HeLa, Huh7, and ARPE-19). Hepatic gene transfer of these vectors in hemophilia B mice, resulted in a 2-fold increase in human coagulation factor (F)IX levels, while its T/B-cell immunogenic response was unaltered. Subsequently, intravitreal gene transfer of glycosylation site-modified vectors in C57BL6/J mice demonstrated an increase in green fluorescence protein expression (\~{}2- to 4-fold) and enhanced permeation across retina. Subretinal administration of these modified vectors containing RPE65 gene further rescued the photoreceptor response in a murine model of Leber congenital amarousis. Our studies highlight the translational potential of glycosylation site-modified AAV2 vectors for hepatic and ocular gene therapy applications.

}, issn = {1543-8392}, doi = {10.1021/acs.molpharmaceut.9b00959}, author = {Mary, Bertin and Maurya, Shubham and Kumar, Mohit and Bammidi, Sridhar and Kumar, Vikas and Jayandharan, Giridhara R} } @article {1025, title = {Myosin heavy chain mutations that cause Freeman-Sheldon syndrome lead to muscle structural and functional defects in Drosophila. [Transgenic Fly Facility]}, journal = {Dev Biol}, volume = {449}, year = {2019}, month = {2019 May 15}, chapter = {90}, abstract = {

Missense mutations in the MYH3 gene encoding myosin heavy chain-embryonic (MyHC-embryonic) have been reported to cause two skeletal muscle contracture syndromes, Freeman Sheldon Syndrome (FSS) and Sheldon Hall Syndrome (SHS). Two residues in MyHC-embryonic that are most frequently mutated, leading to FSS, R672 and T178, are evolutionarily conserved across myosin heavy chains in vertebrates and Drosophila. We generated transgenic Drosophila expressing myosin heavy chain (Mhc) transgenes with the FSS mutations and characterized the effect of their expression on Drosophila muscle structure and function. Our results indicate that expressing these mutant Mhc transgenes lead to structural abnormalities in the muscle, which increase in severity with age and muscle use. We find that flies expressing the FSS mutant Mhc transgenes in the muscle exhibit shortening of the inter-Z disc distance of sarcomeres, reduction in the Z-disc width, aberrant deposition of Z-disc proteins, and muscle fiber splitting. The ATPase activity of the three FSS mutant MHC proteins are reduced compared to wild type MHC, with the most severe reduction observed in the T178I mutation. Structurally, the FSS mutations occur close to the ATP binding pocket, disrupting the ATPase activity of the protein. Functionally, expression of the FSS mutant Mhc transgenes in muscle lead to significantly reduced climbing capability in adult flies. Thus, our findings indicate that the FSS contracture syndrome mutations lead to muscle structural defects and functional deficits in Drosophila, possibly mediated by the reduced ATPase activity of the mutant MHC proteins.

}, issn = {1095-564X}, doi = {10.1016/j.ydbio.2019.02.017}, author = {Das, Shreyasi and Kumar, Pankaj and Verma, Aakanksha and Maiti, Tushar K and Mathew, Sam J} } @article {1232, title = {Phosphoproteomic analysis reveals Akt isoform-specific regulation of cytoskeleton proteins in human temporal lobe epilepsy with hippocampal sclerosis [Mass Spectrometry - Proteomics]}, journal = {Neurochem Int}, year = {2019}, month = {2019 Dec 26}, pages = {104654}, abstract = {

Akt is one of the most important downstream effectors of phosphatidylinositol 3-kinase/mTOR pathway. Hyper activation and expression of this pathway are shown in a variety of neurological disorders including human temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). Nevertheless, the expression and activation profiles of the Akt isoforms, Akt1, Akt2, and Akt3 and their functional roles in human TLE-HS have not been studied. We examined the protein expression and activation (phosphorylation) patterns of Akt and its isoforms in human hippocampal tissue from TLE and non-TLE patients. A phosphoproteomic approach followed by interactome analysis of each Akt isoform was used to understand protein-protein interactions and their role in TLE-HS pathology. Our results demonstrated activation of the Akt/mTOR pathway as well as activation of Akt downstream substrates like GSK3β, mTOR, and S6 in TLE-HS samples. Akt1 isoform levels were significantly increased in the TLE-HS samples as compared to the non-TLE samples. Most importantly, different isoforms were activated in different TLE-HS samples, Akt2 was activated in three samples, Akt2 and Akt1 were simultaneously activated in one sample and Akt3 was activated in two samples. Our phosphoproteomic screen across six TLE-HS samples identified 183 proteins phosphorylated by Akt isoforms, 29 of these proteins belong to cytoskeletal modification. Also, we were able to identify proteins of several other classes involved in glycolysis, neuronal development, protein folding and excitatory amino acid transport functions as Akt substrates. Taken together, our data offer clues to understand the role of Akt and its isoforms in underlying the pathology of TLE-HS and further, modulation of Akt/mTOR pathway using Akt isoforms specific inhibitors may offer a new therapeutic window for treatment of human TLE-HS.

}, issn = {1872-9754}, doi = {10.1016/j.neuint.2019.104654}, author = {Valmiki, Rajesh Ramanna and Venkatesalu, Subhashini and Chacko, Ari George and Prabhu, Krishna and Thomas, Maya Mary and Mathew, Vivek and Yoganathan, Sangeetha and Muthusamy, Karthik and Chacko, Geeta and Vanjare, Harshad Arvind and Krothapalli, Srinivasa Babu} } @article {1139, title = {Post-translational modifications in capsid proteins of recombinant adeno-associated virus (AAV) 1-rh10 serotypes [Mass Spectrometry Facility - Proteomics \& Glycomics].}, journal = {FEBS J}, year = {2019}, month = {2019 Jul 22}, abstract = {

Post-translational modifications in viral capsids are known to fine-tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post-translational modifications during intra-cellular maturation of its capsid proteins. Data on such post-translational modifications in the capsid of recombinant adeno-associated virus serotypes (AAV1-rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post-translational modifications in AAV1-rh10 capsid protein. Our analysis revealed a total of 52 post-translational modifications in AAV2-AAVrh10 capsids, including ubiquitination (17\%), glycosylation (36\%), phosphorylation (21\%), SUMOylation (13\%) and acetylation (11\%). While AAV1 had no detectable post-translational modification, at least four AAV serotypes had \>7 post-translational modifications in their capsid protein. About 82\% of these post-translational modifications are novel. A limited validation of AAV2 capsids by MALDI-TOF and western blot analysis demonstrated minimal glycosylation and ubiquitination of AAV2 capsids. To further validate this, we disrupted a glycosylation site identified in AAV2 capsid (AAV2-N253Q), which severely compromised its packaging efficiency (~100-fold vs AAV2 wildtype vectors). In order to confirm other post-translational modifications detected such as SUMOylation, mutagenesis of a SUMOylation site(K258Q) in AAV2 was performed. This mutant vector demonstrated reduced levels of SUMO-1/2/3 proteins and negligible transduction, two weeks after ocular gene transfer. Our study underscores the heterogeneity of post-translational modifications in AAV vectors. The data presented here, should facilitate further studies to understand the biological relevance of post-translational modifications in AAV life cycle and the development of novel bioengineered AAV vectors for gene therapy applications. This article is protected by copyright. All rights reserved.

}, issn = {1742-4658}, doi = {10.1111/febs.15013}, author = {Mary, Bertin and Maurya, Shubham and Arumugam, Sathyathithan and Kumar, Vikas and Jayandharan, Giridhara R} } @article {1079, title = {ROS Inhibits Cell Growth by Regulating 4EBP and S6K, Independent of TOR, during Development [Transgenic Fly Facility].}, journal = {Dev Cell}, volume = {49}, year = {2019}, month = {2019 May 06}, pages = {473-489.e9}, abstract = {

Reactive oxygen species (ROS), despite having damaging roles, serve as signaling molecules regulating diverse biological and physiological processes. Employing in\ vivo genetic studies in Drosophila, we show that besides causing G1-S arrest by activation of Dacapo, ROS can simultaneously inhibit cell growth by regulating the expression of 4EBP and S6K. This is achieved by triggering a signaling cascade that includes Ask1, JNK, and FOXO independent of the Tsc-TOR growth regulatory pathway. Qualitative and quantitative differences in the types of ROS molecules generated dictate whether cells undergo G1-S arrest only or experience blocks in both cell proliferation and growth. Importantly, during normal development, this signaling cascade is triggered by ecdysone in late larval fat body cells to restrict their growth prior to pupation by antagonizing insulin signaling. The present work reveals an unexpected role of ROS in systemic control of growth in response to steroid hormone signaling to establish organismal size.

}, issn = {1878-1551}, doi = {10.1016/j.devcel.2019.04.008}, author = {Toshniwal, Ashish G and Gupta, Sakshi and Mandal, Lolitika and Mandal, Sudip} } @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 {1008, title = {Shotgun proteomics provides an insight into pathogenesis related proteins using anamorphic stage of the biotroph, Erysiphe pisi pathogen of garden pea [Mass Spectrometry Facility - Proteomics]}, journal = {Microbiological Research}, year = {2019}, abstract = {

E. pisi is an ascomycete member causing powdery mildew disease of garden pea. It is a biotrophic pathogen requiring a living host for its survival. Our understanding of molecular mechanisms underlying its pathogenesis is limited. The identification of proteins expressed in the pathogen is required to gain an insight into the functional mechanisms of an obligate biotrophic fungal pathogen. In this study, the proteome of the anamorphic stage of E. pisi pathogen has been elucidated through the nano LC-MS/MS approach. A total of 328 distinct proteins were detected from Erysiphe isolates infecting the susceptible pea cultivar, Arkel. The proteome is available via ProteomeXchange with identifier PXD010238. The functional classification of protein accessions based on Gene Ontology revealed proteins related to signal transduction, secondary metabolite formation and stress which might be involved in virulence and pathogenesis. The functional validation carried through differential expression of genes encoding G-protein beta subunit, a Cyclophilin (Peptidyl prolyl cis-transisomerase) and ABC transporter in a time course study confirmed their putative role in pathogenesis between resistant and susceptible genotypes, JI2480 and Arkel. The garden pea-powdery mildew pathosystem is largely unexplored, therefore, the identified proteome provides a first-hand information and will form a basis to analyse mechanisms involving pathogen survival, pathogenesis and virulence.

}, keywords = {garden pea, nano-LC-MS/MS, powdery mildew, Proteome, shotgun proteomics}, issn = {0944-5013}, doi = {https://doi.org/10.1016/j.micres.2019.02.006}, url = {http://www.sciencedirect.com/science/article/pii/S0944501318308826}, author = {Malathi Bheri and Sheetal M. Bhosle and Ragiba Makandar} } @article {8898, title = {Shotgun proteomics provides an insight into pathogenesis-related proteins using anamorphic stage of the biotroph, Erysiphe pisi pathogen of garden pea [Mass Spectrometry - Proteomics Facility]}, journal = {Microbiol Res}, volume = {222}, year = {2019}, month = {2019 May}, pages = {25-34}, abstract = {

E. pisi is an ascomycete member causing powdery mildew disease of garden pea. It is a biotrophic pathogen, requiring a living host for its survival. Our understanding of molecular mechanisms underlying its pathogenesis is limited. The identification of proteins expressed in the pathogen is required to gain an insight into the functional mechanisms of an obligate biotrophic fungal pathogen. In this study, the proteome of the anamorphic stage of E. pisi pathogen has been elucidated through the nano LC-MS/MS approach. A total of 328 distinct proteins were detected from Erysiphe isolates infecting the susceptible pea cultivar, Arkel. The proteome is available via ProteomeXchange with identifier PXD010238. The functional classification of protein accessions based on Gene Ontology revealed proteins related to signal transduction, secondary metabolite formation and stress which might be involved in virulence and pathogenesis. The functional validation carried through differential expression of genes encoding G-protein beta subunit, a Cyclophilin (Peptidyl prolyl cis-transisomerase) and ABC transporter in a time course study confirmed their putative role in pathogenesis between resistant and susceptible genotypes, JI2480 and Arkel. The garden pea-powdery mildew pathosystem is largely unexplored, therefore, the identified proteome provides a first-hand information and will form a basis to analyze mechanisms involving pathogen survival, pathogenesis and virulence.

}, keywords = {Ascomycota, Chromatography, Liquid, Disease Resistance, Fungal Proteins, Gardens, Gene Ontology, Genes, Plant, Genotype, Host-Pathogen Interactions, Pisum sativum, Plant Diseases, Plant Leaves, Proteome, Proteomics, Tandem Mass Spectrometry}, issn = {1618-0623}, doi = {10.1016/j.micres.2019.02.006}, author = {Bheri, Malathi and M Bhosle, Sheetal and Makandar, Ragiba} } @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 {1175, title = {Stromal cells downregulate miR-23a-5p to activate protective autophagy in acute myeloid leukemia [Next Gen Genomics Facility (INT)].}, journal = {Cell Death Dis}, volume = {10}, year = {2019}, month = {2019 Sep 30}, pages = {736}, abstract = {

Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

}, issn = {2041-4889}, doi = {10.1038/s41419-019-1964-8}, author = {Ganesan, Saravanan and Palani, Hamenth Kumar and Lakshmanan, Vairavan and Balasundaram, Nithya and Alex, Ansu Abu and David, Sachin and Venkatraman, Arvind and Korula, Anu and George, Biju and Balasubramanian, Poonkuzhali and Palakodeti, Dasaradhi and Vyas, Neha and Mathews, Vikram} } @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 {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 {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 {708, title = {First report of the characterization of a snake venom apyrase (Ruviapyrase) from Indian Russell{\textquoteright}s viper (Daboia russelii) venom. [Mass Spectromety Facility - Proteomics]}, journal = {Int J Biol Macromol}, volume = {111}, year = {2018}, month = {2018 Jan 08}, pages = {639-648}, abstract = {

A novel apyrase from Russell{\textquoteright}s viper venom (RVV) was purified and characterized, and it was named Ruviapyrase (Russell{\textquoteright}s viper apyrase). It is a high molecular weight (79.4 kDa) monomeric glycoprotein that contains 2.4\% neutral sugars and 58.4\% N-linked oligosaccharides and strongly binds to Concanavalin A. The LC-MS/MS analysis did not identify any protein in NCBI protein database, nevertheless some de novo sequences of Ruviapyrase showed putative conserved domain of apyrase superfamily. Ruviapyrase hydrolysed adenosine triphosphate (ATP) to a significantly greater extent (p \< .05) as compared to adenosine diphosphate (ADP); however, it was devoid of 5{\textquoteright}-nucleotidase and phosphodiesterase activities. The Km and Vmax values for Ruviapyrase towards ATP were 2.54 μM and 615 μM of Pi released min-1, respectively with a turnover number (Kcat) of 24,600 min-1. Spectrofluorometric analysis demonstrated interaction of Ruviapyrase with ATP and ADP at Kd values of 0.92 nM and 1.25 nM, respectively. Ruviapyrase did not show cytotoxicity against breast cancer (MCF-7) cells and haemolytic activity, it exhibited marginal anticoagulant and strong antiplatelet activity, and dose-dependently reversed the ADP-induced platelet aggregation. The catalytic activity and platelet deaggregation property of Ruviapyrase was significantly inhibited by EDTA, DTT and IAA, and neutralized by commercial monovalent and polyvalent antivenom.

}, issn = {1879-0003}, doi = {10.1016/j.ijbiomac.2018.01.038}, author = {Kalita, Bhargab and Patra, Aparup and Jahan, Shagufta and Mukherjee, Ashis K} } @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 {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 {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 {891, title = {RagC phosphorylation autoregulates mTOR complex 1. [Drosophila Facility]}, journal = {EMBO J}, year = {2018}, month = {2018 Dec 14}, abstract = {

The mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) controls cell growth, proliferation, and metabolism in response to diverse stimuli. Two major parallel pathways are implicated in mTORC1 regulation including a growth factor-responsive pathway mediated via TSC2/Rheb and an amino acid-responsive pathway mediated via the Rag GTPases. Here, we identify and characterize three highly conserved growth factor-responsive phosphorylation sites on RagC, a component of the Rag heterodimer, implicating cross talk between amino acid and growth factor-mediated regulation of mTORC1. We find that RagC phosphorylation is associated with destabilization of mTORC1 and is essential for both growth factor and amino acid-induced mTORC1 activation. Functionally, RagC phosphorylation suppresses starvation-induced autophagy, and genetic studies in reveal that RagC phosphorylation plays an essential role in regulation of cell growth. Finally, we identify mTORC1 as the upstream kinase of RagC on S21. Our data highlight the importance of RagC phosphorylation in its function and identify a previously unappreciated auto-regulatory mechanism of mTORC1 activity.

}, issn = {1460-2075}, doi = {10.15252/embj.201899548}, author = {Yang, Guang and Humphrey, Sean J and Murashige, Danielle S and Francis, Deanne and Wang, Qiao-Ping and Cooke, Kristen C and Neely, Greg and James, David E} } @article {868, title = {A versatile LC-MS/MS approach for comprehensive, quantitative analysis of central metabolic pathways. [Mass Spectrometry - Lipidomics \& Metabolomics (INT)]}, journal = {Wellcome Open Res}, volume = {3}, year = {2018}, month = {2018}, pages = {122}, abstract = {

Liquid chromatography-mass spectrometry (LC-MS/MS) based approaches are widely used for the identification and quantitation of specific metabolites, and are a preferred approach towards analyzing cellular metabolism. Most methods developed come with specific requirements such as unique columns, ion-pairing reagents and pH conditions, and typically allow measurements in a specific pathway alone. Here, we present a single column-based set of methods for simultaneous coverage of multiple pathways, primarily focusing on central carbon, amino acid, and nucleotide metabolism. We further demonstrate the use of this method for quantitative, stable isotope-based metabolic flux experiments, expanding its use beyond steady-state level measurements of metabolites. The expected kinetics of label accumulation pertinent to the pathway under study are presented with some examples. The methods discussed here are broadly applicable, minimize the need for multiple chromatographic resolution methods, and highlight how simple labeling experiments can be valuable in facilitating a comprehensive understanding of the metabolic state of cells.

}, issn = {2398-502X}, doi = {10.12688/wellcomeopenres.14832.1}, author = {Walvekar, Adhish and Rashida, Zeenat and Maddali, Hemanth and Laxman, Sunil} } @article {588, title = {A comparative intracellular proteomic profiling of Pseudomonas aeruginosa strain ASP-53 grown on pyrene or glucose as sole source of carbon and identification of some key enzymes of pyrene biodegradation pathway.}, journal = {J Proteomics}, volume = {167}, year = {2017}, month = {2017 Sep 07}, pages = {25-35}, abstract = {

Pseudomonas aeruginosa strain ASP-53, isolated from a petroleum oil-contaminated soil sample, was found to be an efficient degrader of pyrene. PCR amplification of selected hydrocarbon catabolic genes (alkB gene, which encodes for monooxygenase, and the C12O, C23O, and PAH-RHDα genes encoding for the dioxygenase enzyme) from the genomic DNA of P. aeruginosa strain ASP-53 suggested its hydrocarbon degradation potential. The GC-MS analysis demonstrated 30.1\% pyrene degradation by P. aeruginosa strain ASP-53 after 144h of incubation at pH6.5, 37{\textdegree}C. Expressions of 115 and 196 intracellular proteins were unambiguously identified and quantitated by shotgun proteomics analysis when the isolate was grown in medium containing pyrene and glucose, respectively. The pyrene-induced uniquely expressed and up-regulated proteins in P. aeruginosa strain ASP-53 in addition to substrate (pyrene) metabolism are also likely to be associated with different cellular functions for example-related to protein folding (molecular chaperone), stress response, metabolism of carbohydrate, proteins and amino acids, and fatty acids; transport of metabolites, energy generation such as ATP synthesis, electron transport and nitrate assimilation, and other oxidation-reduction reactions. Proteomic analyses identified some important enzymes involved in pyrene degradation by P. aeruginosa ASP-53 which shows that this bacterium follows the salicylate pathway of pyrene degradation.

SIGNIFICANCE: This study is the first report on proteomic analysis of pyrene biodegradation pathway by Pseudomonas aeruginosa, isolated from a petroleum-oil contaminated soil sample. The pathway displays partial similarity with deduced pyrene degradation mechanisms of Mycobacterium vanbaalenii PYR-1. The GC-MS analysis as well as PCR amplification of hydrocarbon catabolic genes substantiated the potency of the bacterium under study to effectively degrade high molecular weight, toxic PAH such as pyrene for its filed scale bioremediation experiments. The proteomics approach (LC-MS/MS analysis) identified the differentially regulated intracellular proteins of the isolate P. aeruginosa ASP-53 when grown in pyrene medium. This study identified some important pyrene biodegradation enzymes in Pseudomonas aeruginosa ASP-53 and highlights that the bacterium follows salicylate pathway for pyrene degradation.

}, issn = {1876-7737}, doi = {10.1016/j.jprot.2017.07.020}, author = {Mukherjee, Ashis K and Bhagowati, Pabitra and Biswa, Bhim Bahadur and Chanda, Abhishek and Kalita, Bhargab} } @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 {645, title = {Performance of a docking/molecular dynamics protocol for virtual screening of nutlin-class inhibitors of Mdmx.}, journal = {J Mol Graph Model}, volume = {74}, year = {2017}, month = {2017 Jun}, pages = {54-60}, abstract = {

A virtual screening protocol involving docking and molecular dynamics has been tested against the results of fluorescence polarization assays testing the potency of a series of compounds of the nutlin class for inhibition of the interaction between p53 and Mdmx, an interaction identified as a driver of certain cancers. The protocol uses a standard docking method (AutoDock) with a cutoff based on the AutoDock score (ADscore), followed by molecular dynamics simulation with a cutoff based on root-mean-square-deviation (RMSD) from the docked pose. An analysis of the experimental and computational results shows modest performance of ADscore alone, but dramatically improved performance when RMSD is also used.

}, issn = {1873-4243}, doi = {10.1016/j.jmgm.2017.02.014}, author = {Bharatham, Nagakumar and Finch, Kristin E and Min, Jaeki and Mayasundari, Anand and Dyer, Michael A and Guy, R Kiplin and Bashford, Donald} } @article {707, title = {Proteomics and antivenomics of Echis carinatus carinatus venom: Correlation with pharmacological properties and pathophysiology of envenomation.}, journal = {Sci Rep}, volume = {7}, year = {2017}, month = {2017 Dec 07}, pages = {17119}, abstract = {

The proteome composition of Echis carinatus carinatus venom (ECV) from India was studied for the first time by tandem mass spectrometry analysis. A total of 90, 47, and 22 distinct enzymatic and non-enzymatic proteins belonging to 15, 10, and 6 snake venom protein families were identified in ECV by searching the ESI-LC-MS/MS data against non-redundant protein databases of Viperidae (taxid 8689), Echis (taxid 8699) and Echis carinatus (taxid 40353), respectively. However, analysis of MS/MS data against the Transcriptome Shotgun Assembly sequences (87 entries) of conger E. coloratus identified only 14 proteins in ECV. Snake venom metalloproteases and snaclecs, the most abundant enzymatic and non-enzymatic proteins, respectively in ECV account for defibrinogenation and the strong in vitro pro-coagulant activity. Further, glutaminyl cyclase, aspartic protease, aminopeptidase, phospholipase B, vascular endothelial growth factor, and nerve growth factor were reported for the first time in ECV. The proteome composition of ECV was well correlated with its biochemical and pharmacological properties and clinical manifestations observed in Echis envenomed patients. Neutralization of enzymes and pharmacological properties of ECV, and immuno-cross-reactivity studies unequivocally point to the poor recognition of \<20 kDa ECV proteins, such as PLA2, subunits of snaclec, and disintegrin by commercial polyvalent antivenom.

}, issn = {2045-2322}, doi = {10.1038/s41598-017-17227-y}, author = {Patra, Aparup and Kalita, Bhargab and Chanda, Abhishek and Mukherjee, Ashis K} } @article {510, title = {Sirtuin 1 regulates cardiac electrical activity by deacetylating the cardiac sodium channel.}, journal = {Nat Med}, year = {2017}, month = {2017 Feb 13}, abstract = {

The voltage-gated cardiac Na(+) channel (Nav1.5), encoded by the SCN5A gene, conducts the inward depolarizing cardiac Na(+) current (INa) and is vital for normal cardiac electrical activity. Inherited loss-of-function mutations in SCN5A lead to defects in the generation and conduction of the cardiac electrical impulse and are associated with various arrhythmia phenotypes. Here we show that sirtuin 1 deacetylase (Sirt1) deacetylates Nav1.5 at lysine 1479 (K1479) and stimulates INa via lysine-deacetylation-mediated trafficking of Nav1.5 to the plasma membrane. Cardiac Sirt1 deficiency in mice induces hyperacetylation of K1479 in Nav1.5, decreases expression of Nav1.5 on the cardiomyocyte membrane, reduces INa and leads to cardiac conduction abnormalities and premature death owing to arrhythmia. The arrhythmic phenotype of cardiac-Sirt1-deficient mice recapitulated human cardiac arrhythmias resulting from loss of function of Nav1.5. Increased Sirt1 activity or expression results in decreased lysine acetylation of Nav1.5, which promotes the trafficking of Nav1.5 to the plasma membrane and stimulation of INa. As compared to wild-type Nav1.5, Nav1.5 with K1479 mutated to a nonacetylatable residue increases peak INa and is not regulated by Sirt1, whereas Nav1.5 with K1479 mutated to mimic acetylation decreases INa. Nav1.5 is hyperacetylated on K1479 in the hearts of patients with cardiomyopathy and clinical conduction disease. Thus, Sirt1, by deacetylating Nav1.5, plays an essential part in the regulation of INa and cardiac electrical activity.

}, issn = {1546-170X}, doi = {10.1038/nm.4284}, author = {Vikram, Ajit and Lewarchik, Christopher M and Yoon, Jin-Young and Naqvi, Asma and Kumar, Santosh and Morgan, Gina M and Jacobs, Julia S and Li, Qiuxia and Kim, Young-Rae and Kassan, Modar and Liu, Jing and Gabani, Mohanad and Kumar, Ajay and Mehdi, Haider and Zhu, Xiaodong and Guan, Xiaoqun and Kutschke, William and Zhang, Xiaoming and Boudreau, Ryan L and Dai, Shengchuan and Matasic, Daniel S and Jung, Saet-Byel and Margulies, Kenneth B and Kumar, Vikas* and Bachschmid, Markus M and London, Barry and Irani, Kaikobad} } @article {505, title = {Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase). [Protein Technology Core]}, journal = {PLoS One}, volume = {11}, year = {2016}, month = {2016}, pages = {e0150153}, abstract = {

Intrinsically disordered proteins (IDPs) play a major role in various cellular functions ranging from transcription to cell migration. Mutations/modifications in such IDPs are shown to be associated with various diseases. Current strategies to study the mode of action and regulatory mechanisms of disordered proteins at the structural level are time consuming and challenging. Therefore, using simple and swift strategies for identifying functionally important regions in unstructured segments and understanding their underlying mechanisms is critical for many applications. Here we propose a simple strategy that employs dissection of human paxillin (residues 1-313) that comprises intrinsically disordered regions, followed by its interaction study using FAT (Focal adhesion targeting domain of focal adhesion kinase) as its binding partner to retrace structural behavior. Our findings show that the paxillin interaction with FAT exhibits a masking and unmasking effect by a putative intra-molecular regulatory region. This phenomenon suggests how cancer associated mutations in paxillin affect its interactions with Focal Adhesion Kinase (FAK). The strategy could be used to decipher the mode of regulations and identify functionally relevant constructs for other studies.

}, keywords = {Focal Adhesion Protein-Tyrosine Kinases, Focal Adhesions, Humans, Intrinsically Disordered Proteins, Models, Molecular, Paxillin, Peptide Fragments, Protein Binding, Protein Structure, Tertiary}, issn = {1932-6203}, doi = {10.1371/journal.pone.0150153}, author = {Neerathilingam, Muniasamy and Bairy, Sneha G and Mysore, Sumukh} } @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 {454, title = {Mechanism of apoptosis induction in human breast cancer MCF-7 cell by Ruviprase, a small peptide from Daboia russelii russelii venom.[Mass Spectrometry]}, journal = {Chem Biol Interact}, volume = {258}, year = {2016}, month = {2016 Oct 25}, pages = {297-304}, abstract = {

Ruviprase, a 4.4~kDa peptide isolated from Daboia russelii russelii venom demonstrated antiproliferative activity against EMT6/AR1, U-87MG, HeLa and MCF-7 cancer cells with an IC50 value of 23.0, 8.8, 5.8 and 4.0~μg ml(-1), respectively. However, it was nontoxic to non-cancerous human embryonic kidney cell and human peripheral blood lymphocytes. Flow-cytometric analysis confirmed the apoptosis induction in MCF-7 cells by Ruviprase where it induced DNA condensation but did not cause mitotic blockage or chromosomal aberration in treated-cells. Immunofluorescence microscopic analysis indicated Ruviprase induced apoptosis in MCF-7 cells through p53 and p21-mediated pathways. Ruviprase generated reactive oxygen species (ROS), altered the mitochondrial transmembrane potential, and significantly decreased the cellular glutathione (GSH) content of MCF-7 cells. Immunoblotting and quantitative real-time PCR (qRT-PCR) analyses suggested that Ruviprase down-regulated the expression of anti-apoptotic protein Bcl-2, increased cleavage of poly (ADP-ribose) polymerase (PARP) protein, and up-regulated the expression of pro-apoptotic protein Bax, as well as executer protein caspase-7 to induced apoptosis in MCF-7 cells via intrinsic pathway. This is the first report on the characterization of the anticancer potential of a small, non-toxic and anticoagulant peptide purified from Russell{\textquoteright}s viper venom.

}, issn = {1872-7786}, doi = {10.1016/j.cbi.2016.09.004}, author = {Thakur, Rupamoni and Kini, Sudarshan and Kurkalang, Sillarine and Banerjee, Atanu and Chatterjee, Purba and Chanda, Abhishek and Chatterjee, Anupam and Panda, Dulal and Mukherjee, Ashis K} } @article {457, title = {A proteomic analysis of Pakistan Daboia russelii russelii venom and assessment of potency of Indian polyvalent and monovalent antivenom.[Mass Spectrometry]}, journal = {J Proteomics}, volume = {144}, year = {2016}, month = {2016 Jul 20}, pages = {73-86}, abstract = {

UNLABELLED: To address the dearth of knowledge on the biochemical composition of Pakistan Russell{\textquoteright}s Viper (Daboia russelii russelii) venom (RVV), the venom proteome has been analyzed and several biochemical and pharmacological properties of the venom were investigated. SDS-PAGE (reduced) analysis indicated that proteins/peptides in the molecular mass range of ~56.0-105.0kDa, 31.6-51.0kDa, 15.6-30.0kDa, 9.0-14.2kDa and 5.6-7.2kDa contribute approximately 9.8\%, 12.1\%, 13.4\%, 34.1\% and 30.5\%, respectively of Pakistan RVV. Proteomics analysis of gel-filtration peaks of RVV resulted in identification of 75 proteins/peptides which belong to 14 distinct snake venom protein families. Phospholipases A2 (32.8\%), Kunitz type serine protease inhibitors (28.4\%), and snake venom metalloproteases (21.8\%) comprised the majority of Pakistan RVV proteins, while 11 additional families accounted for 6.5-0.2\%. Occurrence of aminotransferase, endo-β-glycosidase, and disintegrins is reported for the first time in RVV. Several of RVV proteins/peptides share significant sequence homology across Viperidae subfamilies. Pakistan RVV was well recognized by both the polyvalent (PAV) and monovalent (MAV) antivenom manufactured in India; nonetheless, immunological cross-reactivity determined by ELISA and neutralization of pro-coagulant/anticoagulant activity of RVV and its fractions by MAV surpassed that of PAV.

BIOLOGICAL SIGNIFICANCE: The study establishes the proteome profile of the Pakistan RVV, thereby indicating the presence of diverse proteins and peptides that play a significant role in the pathophysiology of RVV bite. Further, the proteomic findings will contribute to understand the variation in venom composition owing to different geographical location and identification of pharmacologically important proteins in Pakistan RVV.

}, issn = {1876-7737}, doi = {10.1016/j.jprot.2016.06.001}, author = {Mukherjee, Ashis K and Kalita, Bhargab and Mackessy, Stephen P} } @article {458, title = {Proteomics and Metabolomics Analyses to Elucidate the Desulfurization Pathway of Chelatococcus sp.[Mass Spectrometry]}, journal = {PLoS One}, volume = {11}, year = {2016}, month = {2016}, pages = {e0153547}, abstract = {

Desulfurization of dibenzothiophene (DBT) and alkylated DBT derivatives present in transport fuel through specific cleavage of carbon-sulfur (C-S) bonds by a newly isolated bacterium Chelatococcus sp. is reported for the first time. Gas chromatography-mass spectrometry (GC-MS) analysis of the products of DBT degradation by Chelatococcus sp. showed the transient formation of 2-hydroxybiphenyl (2-HBP) which was subsequently converted to 2-methoxybiphenyl (2-MBP) by methylation at the hydroxyl group of 2-HBP. The relative ratio of 2-HBP and 2-MBP formed after 96 h of bacterial growth was determined at 4:1 suggesting partial conversion of 2-HBP or rapid degradation of 2-MBP. Nevertheless, the enzyme involved in this conversion process remains to be identified. This production of 2-MBP rather than 2-HBP from DBT desulfurization has a significant metabolic advantage for enhancing the growth and sulfur utilization from DBT by Chelatococcus sp. and it also reduces the environmental pollution by 2-HBP. Furthermore, desulfurization of DBT derivatives such as 4-M-DBT and 4, 6-DM-DBT by Chelatococcus sp. resulted in formation of 2-hydroxy-3-methyl-biphenyl and 2-hydroxy -3, 3/- dimethyl-biphenyl, respectively as end product. The GC and X-ray fluorescence studies revealed that Chelatococcus sp. after 24 h of treatment at 37{\textdegree}C reduced the total sulfur content of diesel fuel by 12\% by per gram resting cells, without compromising the quality of fuel. The LC-MS/MS analysis of tryptic digested intracellular proteins of Chelatococcus sp. when grown in DBT demonstrated the biosynthesis of 4S pathway desulfurizing enzymes viz. monoxygenases (DszC, DszA), desulfinase (DszB), and an NADH-dependent flavin reductase (DszD). Besides, several other intracellular proteins of Chelatococcus sp. having diverse biological functions were also identified by LC-MS/MS analysis. Many of these enzymes are directly involved with desulfurization process whereas the other enzymes/proteins support growth of bacteria at an expense of DBT. These combined results suggest that Chelatococcus sp. prefers sulfur-specific extended 4S pathway for deep-desulphurization which may have an advantage for its intended future application as a promising biodesulfurizing agent.

}, keywords = {Air Pollutants, Bacterial Proteins, Beijerinckiaceae, Gas Chromatography-Mass Spectrometry, Gasoline, Metabolomics, Phylogeny, Proteomics, Signal Transduction, Sulfur}, issn = {1932-6203}, doi = {10.1371/journal.pone.0153547}, author = {Bordoloi, Naba K and Bhagowati, Pabitra and Chaudhuri, Mihir K and Mukherjee, Ashis K} } @article {456, title = {Structural and functional characterization of complex formation between two Kunitz-type serine protease inhibitors from Russell{\textquoteright}s Viper venom.[Mass Spectrometry]}, journal = {Biochimie}, volume = {128-129}, year = {2016}, month = {2016 Sep-Oct}, pages = {138-47}, abstract = {

Snake venom Kunitz-type serine protease inhibitors (KSPIs) exhibit various biological functions including anticoagulant activity. This study elucidates the occurrence and subunit stoichiometry of a putative complex formed between two KSPIs (Rusvikunin and Rusvikunin-II) purified from the native Rusvikunin complex of Pakistan Russell{\textquoteright}s Viper (Daboia russelii russelii) venom (RVV). The protein components of the Rusvikunin complex were identified by LC-MS/MS analysis. The non-covalent interaction between two major components of the complex (Rusvikunin and Rusvikunin-II) at 1:2 stoichiometric ratio to form a stable complex was demonstrated by biophysical techniques such as spectrofluorometric, classical gel-filtration, equilibrium gel-filtration, circular dichroism (CD), dynamic light scattering (DLS), RP-HPLC and SDS-PAGE analyses. CD measurement showed that interaction between Rusvikunin and Rusvikunin-II did not change their overall secondary structure; however, the protein complex exhibited enhanced hydrodynamic diameter and anticoagulant activity as compared to the individual components of the complex. This study may lay the foundation for understanding the basis of protein complexes in snake venoms and their role in pathophysiology of snakebite.

}, issn = {1638-6183}, doi = {10.1016/j.biochi.2016.08.005}, author = {Mukherjee, Ashis K and Dutta, Sumita and Kalita, Bhargab and Jha, Deepak K and Deb, Pritam and Mackessy, Stephen P} } @article {455, title = {Unraveling the Proteome Composition and Immuno-profiling of Western India Russell{\textquoteright}s Viper Venom for In-Depth Understanding of Its Pharmacological Properties, Clinical Manifestations, and Effective Antivenom Treatment.[Mass Spectrometry]}, journal = {J Proteome Res}, year = {2016}, month = {2016 Dec 12}, abstract = {

The proteome composition of western India (WI) Russell{\textquoteright}s viper venom (RVV) was correlated with pharmacological properties and pathological manifestations of RV envenomation. Proteins in the 5-19 and 100-110 kDa mass ranges were the most predominate (\~{}35.1\%) and least abundant (\~{}3.4\%) components, respectively, of WI RVV. Non-reduced SDS-PAGE indicated the occurrence of multiple subunits, non-covalent oligomers, self-aggregation, and/or interactions among the RVV proteins. A total of 55 proteins belonging to 13 distinct snake venom families were unambiguously identified by ESI-LC-MS/MS analysis. Phospholipase A2 (32.5\%) and Kunitz-type serine protease inhibitors (12.5\%) represented the most abundant enzymatic and non-enzymatic proteins, respectively. However, ATPase, ADPase, and hyaluronidase, detected by enzyme assays, were not identified by proteomic analysis owing to limitations in protein database deposition. Several biochemical and pharmacological properties of WI RVV were also investigated. Neurological symptoms exhibited by some RV-bite patients in WI may be correlated to the presence of neurotoxic phospholipase A2 enzymes and Kunitz-type serine protease inhibitor complex in this venom. Monovalent antivenom was found to be better than polyvalent antivenom in immuno-recognition and neutralization of the tested pharmacological properties and enzyme activities of WI RVV; nevertheless, both antivenoms demonstrated poor cross-reactivity and neutralization of pharmacological activities shown by low-molecular-mass proteins (<18 kDa) of this venom.

}, issn = {1535-3907}, doi = {10.1021/acs.jproteome.6b00693}, author = {Kalita, Bhargab and Patra, Aparup and Mukherjee, Ashis K} } @article {515, title = {Biochemical and pharmacological characterization of a toxic fraction and its cytotoxin-like component isolated from Russell{\textquoteright}s viper (Daboia russelii russelii) venom. (Mass Spectrometry)}, journal = {Comp Biochem Physiol C Toxicol Pharmacol}, volume = {168}, year = {2015}, month = {2015 Feb}, pages = {55-65}, abstract = {

The pathophysiological significance of a toxic fraction (GF-VI DEAE-II) isolated from Russell{\textquoteright}s viper venom (RVV) is characterized. GF-VI DEAE-II represents 1.6\% of the total RVV protein and it comprises of a 27.6kDa minor component (RP-I) (0.04\%, w/w) and a major 6.6kDa non-enzymatic peptide (1.11\%, w/w), named Rusvitoxin. The LC-MS/MS analysis of RP-I showed its identity to snake venom serine proteases, whereas Rusvitoxin demonstrated its close identity with snake venom three finger toxins, cytotoxins and cardiotoxins particularly from Naja sp. GF-VI DEAE-II was found to be non-cytotoxic to the tested mammalian cancer cells and non-hemolytic; nevertheless, it demonstrated α-fibrin(ogen)ase activity and in vivo toxicity in BALB/c mice with an LD50 (i.p.) of 2.3mg/kg. GF-VI DEAE-II induced lethargy and hind-leg paralysis in mice within 10min of i.p. injection. GF-VI DEAE-II induced hyperfibrinogenomia, and significantly altered (p\<0.05) the plasma levels of factor X, pro- and anti-inflammatory cytokines viz. TNF-α, IL-6 and IL-10 in treated mice. Histological observations of tissues and biochemical properties of serum from GF-VI DEAE-II-treated mice suggested multiple organ dysfunctions. Conversely, Rusvitoxin at a dose of 5mg/kg did not induce toxicity in BALB/c mice. At 1:15 (antigen: antivenom, w/w) ratio, commercially polyvalent and monovalent antivenoms neutralized more than 80\% of the fibrinolytic and anticoagulant activities of GF-VI DEAE-II. The present study suggests the significant role of GF-VI DEAE-II in RVV-induced pathogenesis in victim/prey.

}, keywords = {Animals, Anticoagulants, Antivenins, Cytotoxins, Fibrinolytic Agents, Interleukin-10, Interleukin-6, Mice, Mice, Inbred BALB C, Mollusk Venoms, Russell{\textquoteright}s Viper, Tumor Necrosis Factor-alpha, Viper Venoms}, issn = {1532-0456}, doi = {10.1016/j.cbpc.2014.12.001}, author = {Thakur, Rupamoni and Chattopadhyay, Pronobesh and Mukherjee, Ashis K} } @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 {514, title = {Elucidation of procoagulant mechanism and pathophysiological significance of a new prothrombin activating metalloprotease purified from Daboia russelii russelii venom. (Mass Spectrometry)}, journal = {Toxicon}, volume = {100}, year = {2015}, month = {2015 Jun 15}, pages = {1-12}, abstract = {

The procoagulant proteases present in Russell{\textquoteright}s Viper venom (RVV) are responsible for promoting consumption coagulopathy in victims. In this study, a procoagulant metalloprotease (Rusviprotease) possessing prothrombin activating and α-fibrinogenase properties has been purified and characterized from RVV. Rusviprotease is a 26.8\ kDa glycoprotein which also exists in other multimeric forms. The peptide mass fingerprinting and secondary structure analyses of Rusviprotease revealed its similarity with snake venom prothrombin activators and metalloproteases. Similar to group A prothrombin activators, Rusviprotease cleaved prothrombin independent of any co-factor requirement generating meizothrombin which is further cleaved to form thrombin. The Km and Vmax values of Rusviprotease towards prothrombin were determined to be 1.73\ μM, and 153.5\ nM thrombin generated/min/μmoles of Rusviprotease, respectively. The Km and Vmax values of Rusviprotease towards fibrinogen were calculated to be 3.14\ μM and 78.7\ nmol/min, respectively. Spectrofluorometric study provided the evidence of interaction between Rusviprotease and factor Xa with a Kd value of\ 6.64\ nM. This interaction augmented the prothrombin activating property of the factor Xa-prothrombinase-Rusviprotease complex by 2.5 fold. Intravenous injection of Rusviprotease to BALB/c mice (0.1\ mg/kg) resulted in in\ vivo defibrinogenation rendering the blood incoagulable. In conclusion, Rusviprotease is the first example of a prothrombin activator with fibrinogenolytic property purified from Daboia russelii russelii venom.

}, keywords = {Animals, Blood Coagulation, Blood Platelets, Chemical Fractionation, Coagulants, Erythrocytes, Goats, Metalloproteases, Mice, Inbred BALB C, Plasma, Russell{\textquoteright}s Viper, Toxicity Tests, Acute, Viper Venoms}, issn = {1879-3150}, doi = {10.1016/j.toxicon.2015.03.019}, author = {Thakur, Rupamoni and Chattopadhyay, Pronobesh and Ghosh, Siddharth S and Mukherjee, Ashis K} } @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 {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 {520, title = {Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods. (Mass spectrometry - Proteomics)}, journal = {PLoS One}, volume = {10}, year = {2015}, month = {2015}, pages = {e0145686}, abstract = {

BACKGROUND: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described.

AIM: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC).

METHODS AND RESULTS: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4{\textdegree}C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4{\textdegree}C, or UC performed at 37{\textdegree}C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin.

CONCLUSION: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

}, keywords = {Animals, Chromatography, Gel, Exosomes, Male, Plasma, Rats, Wistar, Ultracentrifugation}, issn = {1932-6203}, doi = {10.1371/journal.pone.0145686}, author = {Baranyai, Tam{\'a}s and Herczeg, Kata and On{\'o}di, Zs{\'o}fia and Voszka, Istv{\'a}n and M{\'o}dos, K{\'a}roly and Marton, Nikolett and Nagy, Gy{\"o}rgy and M{\"a}ger, Imre and Wood, Matthew J and El Andaloussi, Samir and P{\'a}link{\'a}s, Zolt{\'a}n and Kumar, Vikas and Nagy, P{\'e}ter and Kittel, {\'A}gnes and Buz{\'a}s, Edit Ir{\'e}n and Ferdinandy, P{\'e}ter and Giricz, Zolt{\'a}n} } @article {472, title = {RDGBα, a PtdIns-PtdOH transfer protein, regulates G-protein-coupled PtdIns(4,5)P2 signalling during Drosophila phototransduction.[Drosophila Facility]}, journal = {J Cell Sci}, volume = {128}, year = {2015}, month = {2015 Sep 01}, pages = {3330-44}, abstract = {

Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in the levels of several plasma membrane lipids including phosphatidylinositol (PtdIns), phosphatidic acid (PtdOH) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. It is widely believed that exchange of lipids between the plasma membrane and endoplasmic reticulum (ER) is required to restore lipid homeostasis during PLC signalling, yet the mechanism remains unresolved. RDGBα (hereafter RDGB) is a multi-domain protein with a PtdIns transfer protein (PITP) domain (RDGB-PITPd). We find that, in vitro, the RDGB-PITPd binds and transfers both PtdOH and PtdIns. In Drosophila photoreceptors, which experience high rates of PLC activity, RDGB function is essential for phototransduction. We show that binding of PtdIns to RDGB-PITPd is essential for normal phototransduction; however, this property is insufficient to explain the in vivo function because another Drosophila PITP (encoded by vib) that also binds PtdIns cannot rescue the phenotypes of RDGB deletion. In RDGB mutants, PtdIns(4,5)P2 resynthesis at the plasma membrane following PLC activation is delayed and PtdOH levels elevate. Thus RDGB couples the turnover of both PtdIns and PtdOH, key lipid intermediates during G-protein-coupled PtdIns(4,5)P2 turnover.

}, keywords = {Animals, Drosophila melanogaster, Drosophila Proteins, Eye Proteins, Light Signal Transduction, Membrane Proteins, Phosphatidic Acids, Phosphatidylinositol 4,5-Diphosphate, Type C Phospholipases}, issn = {1477-9137}, doi = {10.1242/jcs.173476}, author = {Yadav, Shweta and Garner, Kathryn and Georgiev, Plamen and Li, Michelle and Gomez-Espinosa, Evelyn and Panda, Aniruddha and Mathre, Swarna and Okkenhaug, Hanneke and Cockcroft, Shamshad and Raghu, Padinjat} } @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 {516, title = {A new C-type lectin (RVsnaclec) purified from venom of Daboia russelii russelii shows anticoagulant activity via inhibition of FXa and concentration-dependent differential response to platelets in a Ca{\texttwosuperior}$^{+}$-independent manner. (Mass spectrometry)}, journal = {Thromb Res}, volume = {134}, year = {2014}, month = {2014 Nov}, pages = {1150-6}, abstract = {

This is the first report on the characterization of a snaclec (RVsnaclec) purified from Daboia russelii russelii venom. The RVsnaclec is a heterodimer of two subunits, α (15.1 kDa) and β (9 kDa). These subunits are covalently linked to form multimeric (αβ)$_{2}$ and (αβ)$_{4}$ structures. Peptide mass fingerprinting analysis of RVsnaclec via LC-MS/MS demonstrated its similarity to snaclecs purified from other viperid snake venoms. Two tryptic peptide sequences of RVsnaclec revealed the putative conserved domains of C-type lectin (CTL). RVsnaclec dose-dependently increased the Ca-clotting time and prothrombin time of platelet-poor plasma (PPP); however, it did not affect the partial thromboplastin time (APTT) or thrombin time of PPP. The in vitro and in vivo anticoagulant activity of RVsnaclec is correlated to its binding and subsequent uncompetitive inhibition of FXa (Ki = 0.52 μmole) in a Ca(2+)-independent manner; however, supplementation with 0.25 mM Ca(2+) enhanced the Xa binding potency of RVsnaclec. Monovalent or polyvalent antivenom failed to neutralize its anticoagulant potency, and RVsnaclec did not inhibit trypsin, chymotrypsin, thrombin or plasmin. RVsnaclec was devoid of hemolytic activity or cytotoxicity against several human cancer cell lines, demonstrated concentration-dependent aggregation and deaggregation of human platelets, and inhibited the ADP-induced aggregation of platelet. RVsnaclec (5.0 mg/kg body weight) was non-lethal to mice and showed no adverse pharmacological effects, suggesting that it has potential as a lead compound for future therapeutic applications in cardiovascular disorders.

}, keywords = {Animals, Anticoagulants, Blood Coagulation, Blood Platelets, Calcium, Factor Xa, Factor Xa Inhibitors, Goats, Humans, Lectins, C-Type, Mice, Platelet Aggregation, Russell{\textquoteright}s Viper, Viper Venoms}, issn = {1879-2472}, doi = {10.1016/j.thromres.2014.09.009}, author = {Mukherjee, Ashis K and Dutta, Sumita and Mackessy, Stephen P} } @article {508, title = {Soni-removal of nucleic acids from inclusion bodies. [Protein Technology Core]}, journal = {Biochem Biophys Res Commun}, volume = {448}, year = {2014}, month = {2014 May 23}, pages = {45-9}, abstract = {

Inclusion bodies (IBs) are commonly formed in Escherichia coli due to over expression of recombinant proteins in non-native state. Isolation, denaturation and refolding of these IBs is generally performed to obtain functional protein. However, during this process IBs tend to form non-specific interactions with sheared nucleic acids from the genome, thus getting carried over into downstream processes. This may hinder the refolding of IBs into their native state. To circumvent this, we demonstrate a methodology termed soni-removal which involves disruption of nucleic acid-inclusion body interaction using sonication; followed by solvent based separation. As opposed to conventional techniques that use enzymes and column-based separations, soni-removal is a cost effective alternative for complete elimination of buried and/or strongly bound short nucleic acid contaminants from IBs.

}, keywords = {Antigens, CD44, Cell Fractionation, Dengue Virus, Inclusion Bodies, Nucleic Acids, Protein Denaturation, Protein Folding, Recombinant Proteins, Solubility, Sonication, Viral Envelope Proteins}, issn = {1090-2104}, doi = {10.1016/j.bbrc.2014.04.049}, author = {Neerathilingam, Muniasamy and Mysore, Sumukh and Gandham, Sai Hari A} } @article {517, title = {Two acidic, anticoagulant PLA2 isoenzymes purified from the venom of monocled cobra Naja kaouthia exhibit different potency to inhibit thrombin and factor Xa via phospholipids independent, non-enzymatic mechanism. (Mass Spectrometry - Proteomics)}, journal = {PLoS One}, volume = {9}, year = {2014}, month = {2014}, pages = {e101334}, abstract = {

BACKGROUND: The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC 3.1.1.4) is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA2 isoenzymes, Nk-PLA2α (13463.91 Da) and Nk-PLA2β (13282.38 Da) purified from the venom of N. kaouthia.

PRINCIPAL FINDINGS: By LC-MS/MS analysis, these PLA2s showed highest similarity (23.5\% sequence coverage) with PLA2 III isolated from monocled cobra venom. The catalytic activity of Nk-PLA2β exceeds that of Nk-PLA2α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA2 isoenzymes. The anticoagulant potency of Nk-PLA2α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA2β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA2s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA2α and Nk-PLA2β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA2 isoenzymes inhibit their "pharmacological target(s)" by uncompetitive mechanism without the requirement of phospholipids/Ca(2+). The anticoagulant potency of Nk-PLA2β which is higher than that of Nk-PLA2α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA2 isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA2β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation.

CONCLUSION/SIGNIFICANCE: In order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA2α and Nk-PLA2β for the treatment and/or prevention of cardiovascular disorders are proposed.

}, keywords = {Anticoagulants, Blood Platelets, Chemical Fractionation, Chromatography, Liquid, Cobra Venoms, Factor Xa, Factor Xa Inhibitors, Isoenzymes, Kinetics, Phospholipases A2, Phospholipids, Sequence Analysis, Protein, Tandem Mass Spectrometry, Thrombin}, issn = {1932-6203}, doi = {10.1371/journal.pone.0101334}, author = {Mukherjee, Ashis K and Kalita, Bhargab and Thakur, Rupamoni} } @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 {486, title = {Draft Genome Sequence of Amycolatopsis decaplanina Strain DSM 44594T. [Next Generation Genomics facility]}, journal = {Genome Announc}, volume = {1}, year = {2013}, month = {2013 Apr 04}, pages = {e0013813}, abstract = {

We report the 8.5-Mb genome sequence of Amycolatopsis decaplanina strain DSM 44594(T), isolated from a soil sample from India. The draft genome of strain DSM 44594(T) consists of 8,533,276\ bp with a 68.6\% G+C content, 7,899 protein-coding genes, and 57 RNAs.

}, doi = {10.1128/genomeA.00138-13}, author = {Kaur, Navjot and Kumar, Shailesh and Bala, Monu and Raghava, Gajendra Pal Singh and Mayilraj, Shanmugam} } @article {522, title = {Draft Genome Sequence of Rhodococcus qingshengii Strain BKS 20-40. (Next Generation Genomics)}, journal = {Genome Announc}, volume = {1}, year = {2013}, month = {2013 Mar 28}, pages = {e0012813}, abstract = {

We report the 5.8-Mb genome sequence of Rhodococcus qingshengii strain BKS 20-40, isolated from a palm tree rhizosphere soil sample from Bhitarkanika National Park, Odisha, India. The strain is capable of degrading cholesterol moiety. The draft genome of strain BKS 20-40 consists of 6,601,618\ bp, with 62.4\% G+C content.

}, doi = {10.1128/genomeA.00128-13}, author = {Bala, Monu and Kumar, Shailesh and Raghava, Gajendra Pal Singh and Mayilraj, Shanmugam} } @article {487, title = {Draft Genome Sequence of Rhodococcus ruber Strain BKS 20-38. [Next Generation Genomics facility]}, journal = {Genome Announc}, volume = {1}, year = {2013}, month = {2013 Apr 04}, pages = {e0013913}, abstract = {

We report the 6.1-Mb genome sequence of Rhodococcus ruber strain BKS 20-38, isolated from the palm tree rhizosphere soil of Bhitarkanika National Park, Odhisha, India. The draft genome sequence of strain BKS 20-38 consists of 6,126,900\ bp, with a G+C content of 69.72\%, 5,716 protein-coding genes, and 49 RNAs.

}, doi = {10.1128/genomeA.00139-13}, author = {Bala, Monu and Kumar, Shailesh and Raghava, Gajendra Pal Singh and Mayilraj, Shanmugam} } @article {485, title = {Draft Genome Sequence of Rhodococcus triatomae Strain BKS 15-14. [Next Generation Genomics facility]}, journal = {Genome Announc}, volume = {1}, year = {2013}, month = {2013 Mar 28}, pages = {e0012913}, abstract = {

We report the 5.8-Mb genome sequence of Rhodococcus triatomae BKS 15-14, isolated from an ant hill soil sample, collected from Bhitarkanika Mangrove Reserve Forest, Odisha, India. The draft genome of strain BKS 15-14 consists of 5,824,349 bp, with a G+C content of 69\%, 5,387 protein-coding genes, and 57 RNAs.

}, doi = {10.1128/genomeA.00129-13}, author = {Kumar, Shailesh and Bala, Monu 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 {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 {519, title = {Unexpected histone H3 tail-clipping activity of glutamate dehydrogenase. (Mass spectrometry - Proteomics)}, journal = {J Biol Chem}, volume = {288}, year = {2013}, month = {2013 Jun 28}, pages = {18743-57}, abstract = {

Clipping of histone tails has been reported in several organisms. However, the significance and regulation of histone tail clipping largely remains unclear. According to recent discoveries H3 clipping has been found to be involved in regulation of gene expression and chromatin dynamics. Earlier we had provided evidence of tissue-specific proteolytic processing of histone H3 in White Leghorn chicken liver nuclei. In this study we identify a novel activity of glutamate dehydrogenase (GDH) as a histone H3-specific protease in chicken liver tissue. This protease activity is regulated by divalent ions and thiol-disulfide conversion in vitro. GDH specifically clips H3 in its free as well as chromatin-bound form. Furthermore, we have found an inhibitor that inhibits the H3-clipping activity of GDH. Like previously reported proteases, GDH too may have the potential to regulate/modulate post-translational modifications of histone H3 by removing the N-terminal residues of the histone. In short, our findings identify an unexpected proteolytic activity of GDH specific to histone H3 that is regulated by redox state, ionic concentrations, and a cellular inhibitor in vitro.

}, keywords = {Amino Acid Sequence, Animals, Binding Sites, Brain, Chickens, Chromatin, Cysteine Proteases, Disulfides, Epigenesis, Genetic, Gene Expression Regulation, Enzymologic, Glutamate Dehydrogenase, Histones, Hydrogen-Ion Concentration, Liver, Mass Spectrometry, Mice, Molecular Sequence Data, Rats, Recombinant Proteins, Salts, Sulfhydryl Compounds, Temperature}, issn = {1083-351X}, doi = {10.1074/jbc.M113.462531}, author = {Mandal, Papita and Verma, Naveen and Chauhan, Sakshi and Tomar, Raghuvir S} } @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 {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 {480, title = {Drosophila protein interaction map (DPiM): a paradigm for metazoan protein complex interactions. [Drosophila facility]}, journal = {Fly (Austin)}, volume = {6}, year = {2012}, month = {2012 Oct-Dec}, pages = {246-53}, abstract = {

Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein-especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex "map" provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map.

}, keywords = {Animals, Cell Line, Computational Biology, Drosophila melanogaster, Drosophila Proteins, Models, Biological, Protein Interaction Mapping, Protein Interaction Maps}, issn = {1933-6942}, doi = {10.4161/fly.22108}, author = {Guruharsha, K G and Obar, Robert A and Mintseris, Julian and Aishwarya, K and Krishnan, R T and VijayRaghavan, K and Artavanis-Tsakonas, Spyros} } @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 {481, title = {Dysregulation of core components of SCF complex in poly-glutamine disorders. [Drosophila facility]}, journal = {Cell Death Dis}, volume = {3}, year = {2012}, month = {2012 Nov 22}, pages = {e428}, abstract = {

Poly-glutamine (polyQ) diseases are neurodegenerative disorders characterised by expanded CAG repeats in the causative genes whose proteins form inclusion bodies. Various E3 ubiquitin ligases are implicated in neurodegenerative disorders. We report that dysfunction of the SCF (Skp1-Cul1-F-box protein) complex, one of the most well-characterised ubiquitin ligases, is associated with pathology in polyQ diseases like Huntington{\textquoteright}s disease (HD) and Machado-Joseph disease (MJD). We found that Cullin1 (Cul1) and Skp1, core components of the SCF complex, are reduced in HD mice brain. A reduction in Cul1 levels was also observed in cellular HD model and fly models of both HD and MJD. We show that Cul1 is able to genetically modify mutant huntingtin aggregates because its silencing results in increased aggregate load in cultured cells. Moreover, we demonstrate that silencing dCul1 and dSkp1 in Drosophila results in increased aggregate load and enhanced polyQ-induced toxicity. Our results imply that reduced levels of SCF complex might contribute to polyQ disease pathology.

}, keywords = {Animals, Cullin Proteins, Drosophila, Female, Humans, Huntington Disease, Machado-Joseph Disease, Male, Mice, Mice, Transgenic, Peptides, SKP Cullin F-Box Protein Ligases}, issn = {2041-4889}, doi = {10.1038/cddis.2012.166}, author = {Bhutani, S and Das, A and Maheshwari, M and Lakhotia, S C and Jana, N R} } @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 {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} }