@article {8674, title = {Cryo-EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH [National Cryo-EM Facility, BLiSC]}, journal = {Nat Commun}, volume = {15}, year = {2024}, month = {2024 Jan 20}, pages = {638}, abstract = {

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

}, keywords = {Bacterial Proteins, Mycobacterium tuberculosis, Ribosomal Proteins, Ribosomes, RNA, Ribosomal, 16S}, issn = {2041-1723}, doi = {10.1038/s41467-024-44879-y}, author = {Kumar, Niraj and Sharma, Shivani and Kaushal, Prem S} } @article {8465, title = {Characterization and techno-functional properties of Tenebrio molitor larvae protein concentrate [Mass Spectrometry - Proteomics Facility]}, journal = {Food Bioscience}, volume = {54}, year = {2023}, month = {07/2023}, abstract = {

Global population growth and an alarming rate of global warming conditions have driven the need for protein from alternative non-conventional sources, namely, insect proteins, through a sustainable approach. The protein concentrate of\ Tenebrio molitor\ larvae was examined for its suitability and applicability as a\ food\ ingredient. Protein was extracted through the acid-alkali method with \~{}79\% of extraction efficiency. The protein-bound polyphenols were extracted and quantified, and the anti-oxidant potential of protein-bound polyphenols and protein concentrate was assessed. The colour parameters of protein concentrate showed brighter colour (L*, 57.3) than the whole insect powder (L*, 39.0). The surface\ hydrophobicity\ of protein concentrate increased to 650, and an increase in the total and free sulfhydryl content (15.39 and 9.158\ μmol/g, respectively) was also observed compared to the whole insect powder. Significantly higher protein solubility was observed at pH 2 (72.3\%) and pH 11 (66.4\%), as compared to near the pI (6.9\% at pH 5 and 30.15\% at pH 4). Several prominent proteins, including 86 and 56\ kDa early-staged encapsulation-inducing proteins, cuticle proteins, and\ structural proteins\ were identified in protein concentrate. The techno-functional properties revealed that mealworm protein concentrate was superior to whole insect powder and comparable to\ whey protein isolate. These findings confirmed the excellent functional properties of mealworm protein concentrate, which can be further explored as a\ novel food ingredient.

}, doi = {https://doi.org/10.1016/j.fbio.2023.102882}, url = {https://www.sciencedirect.com/science/article/abs/pii/S2212429223005333}, author = {Siddaraju Anusha and Pradeep Singh Negi} } @article {8553, title = {Characterizing nucleotide binding site domain (NBD) of ZzR1 resistance gene from Zingiber zerumbet: in silico ligand docking and optimizing heterologous expression [Bio-incubation Services]}, journal = {Archives of Phytopathology and Plant Protection }, year = {2023}, month = {08/2023}, abstract = {

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

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

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

}, keywords = {Aggregate analysis, Bevacizumab, Biosimilars, Intact mass analysis, stress study}, issn = {2405-8440}, doi = {https://doi.org/10.1016/j.heliyon.2023.e19478}, url = {https://www.sciencedirect.com/science/article/pii/S2405844023066860}, author = {Arpit Arunkumar Bana and Nithin Sajeev and Sabyasachi Halder and Haidar Abbas Masi and Shikha Patel and Priti Mehta} } @article {8125, title = {Conformationally restricted, dipeptide-based, self-assembled nanoparticles for efficient vancomycin delivery. [C-CAMP BIG Grantee/Startup]}, journal = {Nanomedicine (Lond)}, year = {2023}, month = {2023 Jan 16}, type = {Journal Article}, abstract = {

Emergence of vancomycin (Van) resistance, and usage of its higher dose and short half-life are posing a serious concern. Slow and sustained release of Van using a nanodelivery system may overcome these problems. Arginine-α,β-dehydrophenylalanine (RΔF) was synthesized using solution-phase synthesis which self-assembled into nanospheres. Van was entrapped in the nanoparticles (NPs). and efficacy of Van-RΔF was determined using broth microdilution and the mouse thigh infection model, respectively. Van-RΔF NPs efficiently inhibited bacterial growth (), while Van alone showed limited growth inhibition in . Intravenous administration of Van-RΔF in mice with bacterial thigh infection showed enhanced efficacy (double) compared with Van alone, which indicates its high potential for further development.

}, keywords = {antibiotic delivery, nanostructures, self-assembly, sustained release, ultrashort peptides}, issn = {1748-6963}, doi = {10.2217/nnm-2022-0144}, url = {https://www.futuremedicine.com/doi/abs/10.2217/nnm-2022-0144}, author = {Yadav, Nitin and Kumar, Utkarsh and Chauhan, Virander Singh} } @article {8467, title = {The conundrum of bacteria-specific antibiotics [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {J Antimicrob Chemother}, volume = {78}, year = {2023}, month = {2023 Jun 01}, pages = {1354-1358}, abstract = {

There is a continual debate on the pros and cons of broad-spectrum versus pathogen-specific antibiotics. The unmet need for a solution for antimicrobial resistance (AMR) has put this argument into sharper focus. A shortage of clinically differentiated antibiotics in late-stage clinical development coupled with the global unmet need in the face of the AMR onslaught has exacerbated the treatment options of drug-resistant bacterial infections. An added dimension to this problem is the current understanding of dysbiosis caused by antibiotics, often leading to negative fallout in immunocompromised patients. We attempt to deconstruct the nuances of this debate from an antibiotics discovery and a clinical standpoint.

}, keywords = {Anti-Bacterial Agents, Bacteria, Bacterial Infections, Drug Resistance, Bacterial, Dysbiosis, Humans}, issn = {1460-2091}, doi = {10.1093/jac/dkad130}, author = {Datta, Santanu} } @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 {8466, title = {Cryo-EM structure of GABA transporter 1 reveals substrate recognition and transport mechanism [National Cryo-Electron Microscopy Facility]}, journal = {Nat Struct Mol Biol}, year = {2023}, month = {2023 Jul 03}, abstract = {

The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 {\r A}. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.

}, issn = {1545-9985}, doi = {10.1038/s41594-023-01011-w}, url = {https://www.nature.com/articles/s41594-023-01011-w}, author = {Nayak, Smruti Ranjan and Joseph, Deepthi and H{\"o}fner, Georg and Dakua, Archishman and Athreya, Arunabh and Wanner, Klaus T and Kanner, Baruch I and Penmatsa, Aravind} } @article {8462, title = {A curated list of targeted optimized promiscuous ketoreductases (TOP-K). [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Biochem J}, volume = {480}, year = {2023}, month = {2023 Jul 12}, pages = {975-997}, abstract = {

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

}, keywords = {ketoreductases, machine learning, medium-chain dehydrogenases, short-chain dehydrogenases}, issn = {1470-8728}, doi = {10.1042/BCJ20230051}, url = {https://pubmed.ncbi.nlm.nih.gov/37335080/}, author = {Shanbhag, Anirudh P and Rajagopal, Sreenath and Ghatak, Arindam and Katagihallimath, Nainesh and Subramanian, Ramaswamy and Datta, Santanu} } @article {2916, title = {Characterization of ACE inhibitory and antioxidant peptides in yak and cow milk hard chhurpi cheese of the Sikkim Himalayan region [Mass Spectrometry Proteomics Facility]}, journal = {Food Chemistry: X}, volume = {13}, year = {2022}, month = {03/2022}, pages = {100231}, type = {Journal Article}, abstract = {

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

}, keywords = {Antihypertensive, Bioactive peptides, Hard chhurpi, Molecular docking, Proteomics, Sikkim Himalaya}, doi = {https://doi.org/10.1016/j.fochx.2022.100231}, url = {https://www.sciencedirect.com/science/article/pii/S2590157522000293}, author = {Abedin, Md Minhajul and Chourasia, Rounak and Phukon, Loreni Chiring and Singh, Sudhir P and Rai, Amit Kumar} } @article {1679, title = {Chemical intervention for enhancing growth and reducing grain arsenic accumulation in rice [Mass Spectrometry - Metabolomics Facility]}, journal = {Environ Pollut}, volume = {276}, year = {2021}, month = {2021 Feb 13}, pages = {116719}, abstract = {

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

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

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

}, keywords = {chrysin, DIO, high fat diet, IKKε/TBK1, insulin.}, doi = {https://doi.org/10.1002/ddr.21859}, url = {https://onlinelibrary.wiley.com/doi/10.1002/ddr.21859}, author = {Amir Siddiqui, Mohammad and Akhtar, Juber and Uddin, Shahab and Chandrashekharan, Sidda Madappa and Ahmad, Mohammad and Khan, Mohammad Irfan and Khalid, Mohammad} } @article {1759, title = {Conformationally constrained dipeptide-based hydrogel as a platform for 3D cell growth and tissue engineering applications [Dr. Nitin Yadav, a BIG Funding Grantee/Start-up]}, journal = {Applied Nanoscience }, volume = {34}, year = {2021}, month = {06/2021}, abstract = {

Conventional two-dimensional culture has greatly assisted tissue engineering research but the absence of tissue-like architectures and availability of unidirectional cell growth limited its use in biomedical applications. Hydrogels, particularly peptide-based, due to their solid-like porous structures, high water content, and high biocompatibility offer tissue-like three-dimensional microenvironments for cell growth and help to mimic in vivo tissue-like conditions in in vitro. Here, we described spontaneous self-assembly of a conformationally constrained dipeptide, Leucine-α,β-dehydrophenylalanine (Leu-ΔPhe), into a strong hydrogel. Leu-ΔPhe hydrogel showed high mechanical strength in mega-pascal with self-healing properties. The hydrogel supported culture of three different mammalian cells including HEK293T, HeLa, and HepG2 in its matrix. Results of cell viability, confocal microscopy and protein expression for HEK293T cells cultured in the hydrogel have indicated that the cells were healthy and functional. In addition, Leu-ΔPhe hydrogel was stable against proteases which makes it a good candidate for use in tissue engineering applications.

}, doi = {https://doi.org/10.1007/s13204-021-01914-4}, url = {https://link.springer.com/article/10.1007/s13204-021-01914-4}, author = {Yadav, N. and Chauhan, M.K. and Chauhan, V.S.} } @article {1442, title = {Caspar SUMOylation regulates lifespan [Transgenic Fly Facility]}, journal = {MicroPubl Biol}, volume = {2020}, year = {2020}, month = {2020 Aug 02}, issn = {2578-9430}, doi = {10.17912/micropub.biology.000288}, author = {Kaduskar, Bhagyashree and Trivedi, Deepti and Ratnaparkhi, Girish S} } @article {1295, title = {Cone snail analogs of the pituitary hormones oxytocin/vasopressin and their carrier protein neurophysin. Proteomic and transcriptomic identification of conopressins and conophysins [Next Gen Genomics Facility (INT)]}, journal = {Biochim Biophys Acta Proteins Proteom}, year = {2020}, month = {2020 Feb 10}, pages = {140391}, abstract = {

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

}, issn = {1878-1454}, doi = {10.1016/j.bbapap.2020.140391}, author = {Kumar, Sanjeev and Vijayasarathy, M and Venkatesha, M A and Sunita, P and Balaram, P} } @article {1140, title = {Cell spread area and traction forces determine myosin-II-based cortex thickness regulation. [Central Imaging and Flow Cytometry Facility]}, journal = {Biochim Biophys Acta Mol Cell Res}, year = {2019}, month = {2019 Jul 23}, abstract = {

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

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

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

}, issn = {2045-2322}, doi = {10.1038/s41598-019-49666-0}, author = {Kumar, Vikas and A, Aneesh Kumar and Sanawar, Rahul and Jaleel, Abdul and Santhosh Kumar, T R and Kartha, C C} } @article {1017, title = {Comparative analysis of the gut microbiota in centenarians and young adults shows a common signature across genotypically non-related populations [Mass Spectrometry - Metabolomics Facility].}, journal = {Mech Ageing Dev}, volume = {179}, year = {2019}, month = {2019 Feb 06}, pages = {23-35}, abstract = {

Gut microbiota is among the factors that may be involved in healthy aging. Broader and geographically spread studies on gut microbiota of centenarians can help in identifying a common signature of longevity. We identified an endogamous Indian population with high centenarian prevalence. Here, we compared the gut microbiota composition and fecal metabolites of a centenarians group (\~{}100 years) with young people (25-45 years) of the region with the high centenarian prevalence and the nearby region of low centenarian prevalence to decipher microbial-related longevity signatures. Also, we compared our results with publicly available datasets of similar groups including 125 centenarians from three countries (Italy, Japan, China). Our comparative analysis resulted in higher biodiversity within Ruminococcaceae in centenarians, with respect to younger adults, irrespective of their nationality. We observed bacterial signatures that are common among extremely old people of different nationality. Comparative metabolites profiling identified the fecal metabolic signature of extreme aging in the Indian study population. Our analysis of the co-occurrence network and bimodal distribution of several taxa suggested the establishment of a pervasive change in the gut ecology during extreme aging. Our study might pave the way to develop gut microbiota based biomarkers for healthy aging.

}, issn = {1872-6216}, doi = {10.1016/j.mad.2019.02.001}, author = {Tuikhar, Ngangyola and Keisam, Santosh and Labala, Rajendra Kumar and Ramakrishnan, Padma and Arunkumar, Moirangthem Cha and Ahmed, Giasuddin and Biagi, Elena and Jeyaram, Kumaraswamy} } @article {870, title = {Cellular and proteomic events associated with localized formation of smut-gall during Zizania latifolia-Ustilago esculenta interaction. [Mass Spectrometry - Proteomics Facility]}, journal = {Microb Pathog}, year = {2018}, month = {2018 Oct 24}, abstract = {

The perennial wild rice Zizania latifolia is confined in the swampy habitat and wetland of the Indo-Burma biodiversity hotspot of India and infection by the biotrophic fungus Ustilago esculenta is hallmarked by swellings that develop to form localized smut-gall at the topmost internodal region. The cellular and proteomic events involved in the non-systemic colonization of Z. latifolia by U. esculenta leading to smut-gall formation is poorly understood. Proteins were extracted from the smut-gall region at the topmost internodal region below the apical meristematic tissue from the infected and uninfected parts of Z. latifolia. By combining transmission electron microscopy (TEM) and fluorescent microscopy (FM), we showed that U. esculenta hyphal morphological transitions and movement occurred both intercellularly and intracellularly while sporulation occurred intracellularly in selective cells. Following proteome profiling using two dimensional SDS-PAGE at different phenotypic phases of smut-gall development and U. esculenta infection, differentially expressed proteins bands and their relative abundance were detected and subjected to liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Importantly, the fungus explores at least 7 metabolic pathways and 5 major biological processes to subdue the host defense and thrive successfully on Z. latifolia. The fungus U. esculenta produces proteases and energy acquisition proteins that enhances it{\textquoteright}s defensive and survival mode in the host. The identified differentially regulated proteins shed-light into why inflorescence is being replaced by bulbous smut-gall at late stages of the disease, as well as the development of resistance in some Z. latifolia plants against U. esculenta infection.

}, issn = {1096-1208}, doi = {10.1016/j.micpath.2018.10.028}, author = {Jose, Robinson C and Bengyella, Louis and Handique, Pratap J and Talukdar, Narayan C} } @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 {683, title = {Combinatorial action of Grainyhead, Extradenticle and Notch in regulating Hox mediated apoptosis in Drosophila larval CNS.}, journal = {PLoS Genet}, volume = {13}, year = {2017}, month = {2017 Oct}, pages = {e1007043}, abstract = {

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

}, keywords = {Amino Acid Sequence, Animals, Apoptosis, Central Nervous System, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Enhancer Elements, Genetic, Female, Gene Expression Regulation, Developmental, Genes, Homeobox, Homeodomain Proteins, Larva, Male, Nuclear Proteins, Receptors, Notch, Transcription Factors}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1007043}, author = {Khandelwal, Risha and Sipani, Rashmi and Govinda Rajan, Sriivatsan and Kumar, Raviranjan and Joshi, Rohit} } @article {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 {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 {503, title = {Comprehensive analysis of neurotransmitters from regenerating planarian extract using an ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring method.}, journal = {Rapid Commun Mass Spectrom}, volume = {27}, year = {2013}, month = {2013 Nov 15}, pages = {2439-52}, abstract = {

RATIONALE: Absolute quantification of neurotransmitters (NTs) from biological systems is imperative to track how changes in concentration of active neurochemicals may affect biological behavior. A sensitive method for the absolute quantification of multiple NTs in a single method is highly needed.

METHODS: A stable-isotope dilution ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC/MS/SRM) assay has been developed for a sensitive and quantitative assessment of NTs in planaria. We used this method for the simultaneous quantification of 16 NTs. All analytes showed a linear relationship between concentrations (0.78-50 ng/mL), regression coefficients higher than 0.97, accuracy (91-109\%) and low coefficients of variation (CVs). The inter-day CVs for the lowest quality controls (1.56 ng/mL) were in the range between 2-11\%.

RESULTS: The levels of most of the NTs were similar in both sexual and asexual planarians except for glutamic acid, which was about two-fold higher in asexual compared to sexual planarians. We identified high levels of serotonin and failed to detect tryptamine suggesting that the pathway essential for the conversion of tryptophan into tryptamine is absent in planarians. Interestingly, we also found high levels of dopamine and L-DOPA in regenerating planarians suggesting their possible role in regeneration.

CONCLUSIONS: For the first time, we developed novel methodology based on UHPLC/MS/SRM and quantified 16 NTs with high sensitivity and specificity from sexual and asexual strains of planarian Schmidtea mediterranea. This method will also have great application in quantifying various NTs with great precision in different model systems.

}, keywords = {Animals, Chromatography, High Pressure Liquid, Glutamic Acid, Limit of Detection, Neurotransmitter Agents, Planarians, Reproduction, Reproduction, Asexual, Serotonin, Spectrometry, Mass, Electrospray Ionization, Tryptamines}, issn = {1097-0231}, doi = {10.1002/rcm.6706}, author = {Rangiah, Kannan and Palakodeti, Dasaradhi} }