@article {9074, title = {Approaches for Identifying LncRNA-Associated Proteins for Therapeutic Targets and Cancer Biomarker Discovery [Biologics Characterization Facility, BCF at C-CAMP]}, journal = {Targets}, volume = {3}, year = {2025}, abstract = {

Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression and cellular signaling in cancer. Their functions are primarily mediated through interactions with specific protein partners that modulate chromatin structure, epigenetic remodeling, transcription, and signal transduction. In this review, we explore reports and strategies for the proteomic characterization of lncRNA-associated proteins, particularly emphasizing high-throughput liquid chromatography{\textendash}mass spectrometry (LC-MS)-based techniques. Affinity-based methods such as RNA pull-down, ChIRP MS, RAP-MS, BioID-MS, and SILAC-MS enable sensitive and specific mapping of lncRNA and protein complexes. These approaches reveal cancer-specific proteomic signatures, post-translational modifications, and mechanistic insights into tumor biology. The use of label-free quantification, bituminization, and crosslinking strategies further enhances the resolution of dynamic RNA{\textendash}protein networks. Validation tools following bioinformatic analyses, such as Western blotting, immunohistochemistry, immunofluorescence, and ELISA, are used to prioritize and confirm findings. Candidate biomarkers from hepatocellular carcinoma to colorectal and prostate cancers, profiling lncRNA-associated proteins, hold promise for identifying clinically actionable biomarkers and therapeutic targets. This review highlights the translational relevance of lncRNA protein studies and advocates for their broader adoption in oncological research. In LC-MS workflows, proteins bound to lncRNAs are enzymatically digested into peptides, separated via nano-LC, and analyzed using high-resolution tandem MS. Label-free or isotope-labeled methods quantify differential enrichment, followed by bioinformatics-driven pathway annotation.

}, keywords = {cancer biomarkers, LC{\textendash}mass spectrometry, long non-coding RNAs (LncRNAs), protein interactions, Proteomics, therapeutic targets}, issn = {2813-3137}, doi = {10.3390/targets3030027}, url = {https://www.mdpi.com/2813-3137/3/3/27}, author = {Hussain, Mohammad Shabir and Vij, Puneet and Kotnala, Sudhir and Ahmad, Shadab and Chauhan, Subhash C. and Tripathi, Manish K.} } @article {8993, title = {Compositional characterisation of colostrum and proline rich polypeptides of indigenous cattle (Bos indicus family) and Holstein Friesian (Bos taurus) and their crossbred Karan Fries cattle [Mass Spectrometry Facility]}, journal = {International Dairy Journal}, year = {2025}, pages = {106264}, abstract = {

Proline-rich polypeptides (PRPs) are polypeptides characterized by a high proportion of hydrophobic amino acids (\~{} 45\%) and over 20\% proline, with molecular weights ranging from 0.5 to 17 kDa. Colostrum is the only source of PRPs; therefore, excess colostrum could be utilized for PRPs production. These PRPs mostly originate from caseins, such as β-casein and α-casein. The role of PRPs in preventing pathological processes in the central nervous system is well established and many formulations included PRPs or colostrum in their medications. Present study envisages composition of colostrum and quantify PRPs content across select indigenous breeds with exotic breed (Holstein Friesian, HF) and one cross breed of HF and Tharparkar (Karan Fries, KF), with time period after parturition. Furthermore, PRPs were characterized using SDS-PAGE, HPLC, amino acid composition, and LC-ESI-MS/MS to understand molecular weight distribution and sequential changes. The HF and Sahiwal breed consistently exhibited the higher PRPs yields at 12, 24 and 36 h postpartum. HF (114.10{\textpm}19.41 mg/L of colostrum) and Sahiwal (102.20{\textpm}2.35 mg/L of colostrum) showed the highest PRPs concentrations at 12 h, followed by a decline, highlighting colostrum collected within 24 h of parturition as the optimal PRPs source. Indigenous breeds such as Sahiwal (24.50\%), Gir (21.91\%), and Tharparkar (22.30\%) has shown higher concentrations of proline compared to the crossbred KF (17.49\%) and the exotic HF (18.25\%).

}, keywords = {Amino acid profile, Colostrinin, Colostrum, Composition, Immunonoglobulins, Proline-rich polypeptides}, issn = {0958-6946}, doi = {https://doi.org/10.1016/j.idairyj.2025.106264}, url = {https://www.sciencedirect.com/science/article/pii/S0958694625000834}, author = {Mamta Kumari and Usha Devi and T.M. Amrutha and Sandeep N and M.H. Sathish Kumar and Suman Kapila and Abdul S. Hussain and C.T. Manoj Kumar} } @article {9056, title = {Cryo-EM structures of Mycobacterium tuberculosis imidazole glycerol phosphate dehydratase in the apo state and in the presence of small molecules [National Cryo-EM Facility, BLiSC]}, journal = {Acta Crystallogr F Struct Biol Commun}, year = {2025}, month = {2025 Jul 01}, abstract = {

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

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

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

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

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

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

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

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

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

}, keywords = {Bacterial Proteins, Mycobacterium tuberculosis, Ribosomal Proteins, Ribosomes, RNA, Ribosomal, 16S}, issn = {2041-1723}, doi = {10.1038/s41467-024-44879-y}, author = {Kumar, Niraj and Sharma, Shivani and Kaushal, Prem S} } @article {8897, title = {Embryonic spatiotemporal expression pattern of Folded gastrulation suggests roles in multiple morphogenetic events and regulation by AbdA [Transgenic Fly (Drosophila) Facility]}, journal = {G3 (Bethesda)}, volume = {14}, year = {2024}, month = {2024 May 07}, abstract = {

In Drosophila, the signaling pathway activated by the ligand Folded gastrulation (Fog) is among the few known G protein-coupled receptor (GPCR) pathways to regulate cell shape change with a well-characterized role in gastrulation. However, an understanding of the spectrum of morphogenetic events regulated by Fog signaling is still lacking. Here, we present an analysis of the expression pattern and regulation of fog using a genome-engineered Fog::sfGFP line. We show that Fog expression is widespread and in tissues previously not associated with the signaling pathway including germ cells, trachea, and amnioserosa. In the central nervous system (CNS), we find that the ligand is expressed in multiple types of glia indicating a prominent role in the development of these cells. Consistent with this, we have identified 3 intronic enhancers whose expression in the CNS overlaps with Fog::sfGFP. Further, we show that enhancer-1, (fogintenh-1) located proximal to the coding exon is responsive to AbdA. Supporting this, we find that fog expression is downregulated in abdA mutants. Together, our study highlights the broad scope of Fog-GPCR signaling during embryogenesis and identifies Hox gene AbdA as a novel regulator of fog expression.

}, keywords = {Animals, Central Nervous System, Drosophila melanogaster, Drosophila Proteins, Embryo, Nonmammalian, Enhancer Elements, Genetic, Gastrulation, Gene Expression Regulation, Developmental, Morphogenesis, Signal Transduction, Transcription Factors}, issn = {2160-1836}, doi = {10.1093/g3journal/jkae032}, author = {Katagade, Vrushali and Kandroo, Manisha and Ratnaparkhi, Anuradha} } @article {8887, title = {Inhibition of dipeptidyl peptidase-IV by hydrolysates of beta-lactoglobulin isolated from Gir cow milk [Mass Spectrometry - Proteomics]}, journal = {Food Chemistry Advances}, year = {2024}, pages = {100842}, abstract = {

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

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

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

}, url = {https://www.agilent.com/cs/library/applications/an-liraglutide-impurities-characterization-5994-7727en-agilent.pdf}, author = {Shadab Ahmad and Nirpendra Singh and Ashish Pargaonkar and Dheeraj Vig and Mike Knierman} } @article {8813, title = {A novel acidic pH-dependent metacaspase governs defense-response against pathogens in tomato [Mass Spectrometry - Proteomics Facility]}, journal = {Plant Physiology and Biochemistry}, volume = {213}, year = {2024}, pages = {108850}, abstract = {

The importance of metacaspases in programmed cell death and tissue differentiation is known, but their significance in disease stress response, particularly in a crop plant, remained enigmatic. We show the tomato metacaspase expression landscape undergoes differential reprogramming during biotrophic and necrotrophic modes of pathogenesis; also, the metacaspase activity dynamics correlate with the disease progression. These stresses have contrasting effects on the expression pattern of SlMC8, a Type II metacaspase, indicating that SlMC8 is crucial for stress response. In accordance, selected biotic stress-related transcription factors repress SlMC8 promoter activity. Interestingly, SlMC8 exhibits maximum proteolysis at an acidic pH range of 5{\textendash}6. Molecular dynamics simulation identified the low pH-driven protonation event of Glu246 as critical to stabilize the interaction of SlMC8 with its substrate. Mutagenesis of Glu246 to charge-neutral glutamine suppressed SlMC8{\textquoteright}s proteolytic activity, corroborating the importance of the amino acid in SlMC8 activation. The glutamic acid residue is found in an equivalent position in metacaspases having acidic pH dependence. SlMC8 overexpression leads to heightened ROS levels, cell death, and tolerance to PstDC3000, and SlMC8 repression reversed the phenomena. However, the overexpression of SlMC8 increases tomato susceptibility to necrotrophic Alternaria solani. We propose that SlMC8 activation due to concurrent changes in cellular pH during infection contributes to the basal resistance of the plant by promoting cell death at the site of infection, and the low pH dependence acts as a guard against unwarranted cell death. Our study confirms the essentiality of a low pH-driven Type II metacaspase in tomato biotic stress-response regulation.

}, keywords = {Cell death, host-pathogen interaction, Metacaspase, pH sensitivity, Tomato}, issn = {0981-9428}, doi = {https://doi.org/10.1016/j.plaphy.2024.108850}, url = {https://www.sciencedirect.com/science/article/pii/S0981942824005187}, author = {Shrabani Basak and Debarati Paul and Rohit Das and Shubhra Ghosh Dastidar and Pallob Kundu} } @article {8739, title = {Proteomic and metabolomic insights into seed germination of Ferula assa-foetida [Mass Spectrometry - Proteomics Facility]}, journal = {J Proteomics}, volume = {300}, year = {2024}, month = {2024 May 30}, pages = {105176}, abstract = {

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

}, keywords = {Ferula, Gene Expression Regulation, Plant, Germination, Metabolomics, Plant Proteins, Protein Interaction Maps, Proteome, Proteomics, Seeds}, issn = {1876-7737}, doi = {10.1016/j.jprot.2024.105176}, author = {Punia, Ashwani and Kumari, Manglesh and Chouhan, Monika and Saini, Vishal and Joshi, Robin and Kumar, Ashok and Kumar, Rajiv} } @article {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 {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 {8508, title = {Defining a research agenda for environmental wastewater surveillance of pathogens.}, journal = {Nat Med}, year = {2023}, month = {2023 Aug 03}, issn = {1546-170X}, doi = {10.1038/s41591-023-02457-7}, author = {Shaw, Alexander G and Troman, Catherine and Akello, Joyce Odeke and O{\textquoteright}Reilly, Kathleen M and Gauld, Jillian and Grow, Stephanie and Grassly, Nicholas and Steele, Duncan and Blazes, David and Kumar, Supriya} } @article {8544, title = {Distinct evolution of type I glutamine synthetase in Plasmodium and its species-specific requirement [Mass Spectrometry Facility - Metabolomics]}, journal = {Nat Commun}, volume = {14}, year = {2023}, month = {2023 Jul 14}, pages = {4216}, abstract = {

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

}, keywords = {Amino Acids, Animals, Artemisinins, Asparagine, Glutamate-Ammonia Ligase, Glutamine, Humans, Parasites, Plasmodium falciparum}, issn = {2041-1723}, doi = {10.1038/s41467-023-39670-4}, author = {Ghosh, Sourav and Kundu, Rajib and Chandana, Manjunatha and Das, Rahul and Anand, Aditya and Beura, Subhashree and Bobde, Ruchir Chandrakant and Jain, Vishal and Prabhu, Sowmya Ramakant and Behera, Prativa Kumari and Mohanty, Akshaya Kumar and Chakrapani, Mahabala and Satyamoorthy, Kapaettu and Suryawanshi, Amol Ratnakar and Dixit, Anshuman and Padmanaban, Govindarajan and Nagaraj, Viswanathan Arun} } @article {8441, title = {Gene flow drives genomic diversity in Asian Pikas distributed along the core and range-edge habitats in the Himalayas [Next Gen Genomics Facility (INT)]}, journal = {Ecol Evol.}, volume = {13}, year = {2023}, month = {05/2023}, chapter = {e10129}, abstract = {

Studying the genetic variation among different species distributed across their core and range-edge habitats can provide valuable insights into how genetic variation changes across the species{\textquoteright} distribution range. This information can be important for understanding local adaptation, as well as for conservation and management efforts. In this study, we have carried out genomic characterization of six species of Asian Pikas distributed along their core and range-edge habitats in the Himalayas. We utilized a population genomics approach using ~28,000 genome-wide SNP markers obtained from restriction-site associated DNA sequencing. We identified low nucleotide diversity and high inbreeding coefficients in all six species across their core and range-edge habitats. We also identified evidence of gene flow among genetically diverse species. Our results provide evidence of reduced genetic diversity in Asian pikas distributed across the Himalayas and the neighboring regions and indicate that recurrent gene flow is possibly a key mechanism for maintaining genetic diversity and adaptive potential in these pikas. However, full-scale genomics studies that utilize whole-genome sequencing approaches will be needed to quantify the direction and timing of gene flow and functional changes associated with introgressed regions in the genome. Our results represent an important step toward understanding the patterns and consequences of gene flow in species, sampled at the least studied, yet climatically vulnerable part of their habitat that can be further used to inform conservation strategies that promote connectivity and gene flow between populations.

}, keywords = {gene flow, genetic diversity, Himalayas, pika}, doi = {10.1002/ece3.10129}, url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10208896/}, author = {Nishma Dahal and Melia G. Romine and Sunita Khatiwara and Uma Ramakrishnan and Sangeet Lamichhaney} } @article {8886, title = {Grain Characteristics, Moisture, and Specific Peptides Produced by Contribute to False Smut Disease in Rice ( L.) [Mass Spectrometry - Proteomics Facility]}, journal = {Biomolecules}, volume = {13}, year = {2023}, month = {2023 Apr 12}, abstract = {

The fungus the causative agent of false smut in rice ( L.), is responsible for one of the severe grain diseases that lead to significant losses worldwide. In this research, microscopic and proteomic analyses were performed by comparing infected and non-infected grains of the susceptible and resistant rice varieties to provide insights into the molecular and ultrastructural factors involved in false smut formation. Prominent differentially expressed peptide bands and spots were detected due to false smut formation as revealed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) SDS-PAGE profiles and were identified using liquid chromatography-mass spectrometry (LC-MS/MS). The proteins identified from the resistant grains were involved in diverse biological processes such as cell redox homeostasis, energy, stress tolerance, enzymatic activities, and metabolic pathways. It was found that produces diverse degrading enzymes such as β-1, 3-endoglucanase, subtilisin-like protease, putative nuclease S1, transaldolase, putative palmitoyl-protein thioesterase, adenosine kinase, and DNase 1 that could discretely alter the host morphophysiology resulting in false smut. The fungus also produced superoxide dismutase, small secreted proteins, and peroxidases during the smut formation. This study revealed that the dimension of rice grain spikes, their elemental composition, moisture content, and the specific peptides produced by the grains and the fungi play a vital role in the formation of false smut.

}, keywords = {Chromatography, Liquid, Edible Grain, Oryza, Peptides, Proteomics, Tandem Mass Spectrometry}, issn = {2218-273X}, doi = {10.3390/biom13040669}, author = {Jose, Robinson C and Kanchal, Thangjam and Louis, Bengyella and Talukdar, Narayan C and Chowdhury, Devasish} } @article {8416, title = {Grain Characteristics, Moisture, and Specific Peptides Produced by Ustilaginoidea virens Contribute to False Smut Disease in Rice (Oryza\ sativa L.) [Mass Spectrometry - Proteomics Facility]}, journal = {Biomolecules}, volume = {13}, year = {2023}, abstract = {

The fungus Ustilaginoidea virens, the causative agent of false smut in rice (Oryza sativa L.), is responsible for one of the severe grain diseases that lead to significant losses worldwide. In this research, microscopic and proteomic analyses were performed by comparing U. virens infected and non-infected grains of the susceptible and resistant rice varieties to provide insights into the molecular and ultrastructural factors involved in false smut formation. Prominent differentially expressed peptide bands and spots were detected due to false smut formation as revealed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) SDS-PAGE profiles and were identified using liquid chromatography-mass spectrometry (LC-MS/MS). The proteins identified from the resistant grains were involved in diverse biological processes such as cell redox homeostasis, energy, stress tolerance, enzymatic activities, and metabolic pathways. It was found that U. virens produces diverse degrading enzymes such as β-1, 3-endoglucanase, subtilisin-like protease, putative nuclease S1, transaldolase, putative palmitoyl-protein thioesterase, adenosine kinase, and DNase 1 that could discretely alter the host morphophysiology resulting in false smut. The fungus also produced superoxide dismutase, small secreted proteins, and peroxidases during the smut formation. This study revealed that the dimension of rice grain spikes, their elemental composition, moisture content, and the specific peptides produced by the grains and the fungi U. virens play a vital role in the formation of false smut.

}, issn = {2218-273X}, doi = {10.3390/biom13040669}, url = {https://www.mdpi.com/2218-273X/13/4/669}, author = {Jose, Robinson C. and Kanchal, Thangjam and Louis, Bengyella and Talukdar, Narayan C. and Chowdhury, Devasish} } @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 {8359, title = {Understanding the mode of action of AgroGain{\textregistered}, a biostimulant derived from the red seaweed Kappaphycus alvarezii in the stimulation of cotyledon expansion and growth of Cucumis sativa (cucumber) [Sea6Energy Pvt. Ltd, a C-CAMP Start-up]}, journal = {Front. Plant Sci.}, volume = {14}, year = {2023}, month = {06/2023}, abstract = {

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

}, doi = {https://doi.org/10.3389/fpls.2023.1136563}, author = {Pushp Sheel Shukla and Nagarajan Nivetha and Sri Sailaja Nori and Debayan Bose and Sawan Kumar and Sachin Khandelwal and Alan Critchley and Shrikumar Suryanarayan} } @article {5253, title = {Analysis of water soluble fractions of crude oil by gas chromatography: Mass spectroscopy [Mass Spectrometry - Metabolomics Facility]}, journal = {The Pharma Innovation Journal}, volume = {SP-11(4)}, year = {2022}, month = {06/2022}, chapter = {1119}, abstract = {

Crude oil is the major source of energy in the modern society to meet the global energy demand by which exploitation of crude oil and its transportation increasing rapidly leading to frequent catastrophic oil spills. When oil spill occurs or when oil is discharged into aquatic environment the components of the crude oil present in it are mostly volatile, evaporates into the environment and the fraction of the oil soluble in water (i.e., WSF) is available to the organisms directly which is the main determinant of crude oil toxicity to aquatic organisms. Although this fraction is present only in relatively low concentrations, it is this fraction which is in most intimate contact with fish and other pelagic organisms with carcinogenic and mutagenic potential. So, the aim of the study was to determine different hydrocarbons dissolved in water soluble fraction (WSF) of crude oil qualitatively and quantitatively. The determination of different hydrocarbons present in water soluble fraction of crude oil can be used as reference point for many studies in future for determining the toxicity of water soluble fraction of crude oil to fishes.

}, keywords = {crude oil, gas chromatography, Water soluble fraction}, url = {https://www.thepharmajournal.com/archives/2022/vol11issue4S/PartP/S-11-4-135-968.pdf}, author = {Rishika, V. and Lakshmipathi, M.T. and Sampath Kumar, B.} } @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 {3704, title = {Genome-wide SNP markers from fecal samples reveal anthropogenic impacts on connectivity: case of a small carnivore in the central Indian landscape [Next Gen Genomics Facility]}, journal = {Animal Conservation}, year = {2022}, month = {03/2022}, abstract = {

Maintaining gene flow among fragmented habitat patches is critical for the long-term persistence of wild species. Landscape genetics tools are often used to understand the impact of landscape features on gene flow among fragmented populations. The ability to detect the relationship between gene flow and landscape depends on the power of the genetic tools used, which increases with the number of genotyped loci. Next-generation sequencing (NGS) based methods allow genotyping of a high number of loci but are challenging to implement for non-invasive samples, which are commonly used in conservation genetics research. Here we assess the impact of landscape heterogeneity on jungle cat (Felis chaus) movement using genome-wide single nucleotide polymorphism (SNP) markers obtained from fecal samples, using a methylation-based DNA (MBD) enrichment method. We successfully genotyped 20 jungle cat individuals at 2246 SNP loci and compared our results to a previous study that used microsatellite markers and 93 individuals. Our results demonstrate the efficiency and robustness of the MBD enrichment approach with fecal samples in generating genome-wide data for endangered and cryptic species of conservation concern. Our landscape analyses revealed that roads and human-dominated land-use negatively impact jungle cat movement in central India. We explicitly quantified the uncertainty in our analyses and concluded that several thousand SNPs from fewer individuals provide more power than tens of microsatellites from more individuals, in quantifying the effects of landscape on gene flow. Our results provide insight into the impacts of anthropogenic habitat modification on an often-ignored small carnivore species. Insights on connectivity for such species can help policymakers and wildlife managers move beyond connectivity contingent on charismatic species to devise holistic landscape-level management plans for multiple carnivores.

}, doi = {https://doi.org/10.1111/acv.12770}, author = {Tyagi, A. and Khan, A. and Thatte, P. and Ramakrishnan, U.} } @article {7616, title = {Identification and molecular characterization of drug targets of methicillin resistant Staphylococcus aureus [Mass Spectrometry - Proteomics]}, journal = {Journal of Applied and Natural Science}, volume = {14}, year = {2022}, month = {11/2022}, chapter = {1152}, abstract = {

Antimicrobial resistance is a major world health concern and drug-resistant Staphylococcus aureus is a serious threat. Due to the emergence of multidrug-resistant bacterial strains, there is an urgent need to develop novel drug targets to meet the challenge of multidrug-resistant organisms. The main objective of the current study was to determine molecular targets against S. aureus using by computational approach. S. aureus was cultured in brain heart infusion broth medium and MRSA (Methicillin resistant S. aureus) protein was extracted acetone-sodium dodecyl sulfate method. The cell lysate was treated with various antibiotics and proteinase K stable proteins were analyzed. The molecular weight of Geninthiocin-targeted protein of interest in S. aureus ranged from 46 to 50 kDa. A prominent protein band in SDS-PAGE indicated that the protein corresponding 50 kDa was resistant against proteinase K. The SDS-PAGE separated sample was excised and trypsinated, and the peptides were characterized using Nano Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) analysis. Spectrum with clusters of molecular peptides and peptide fragments ranging from 110.0716 to 1002.7093 mass/charge ratio (m/z) were displayed against intensity or relative abundance in the excised gel band. The spectral data from nano LC-MS/MS was subjected to mascot search in the NCBIprot database (taxonomy-bacteria (eubacteria), resulting in seven bacterial proteins. Geninthiocin target proteins were determined against MRSA. To conclude, antibiotic target proteins were identified using a machine learning approach and these targets may have a lot of applications in developing a novel lead molecule against drug-resistant bacteria.\ 

}, keywords = {Bacteria, Drug resistance, Drug target, Geninthiocin, Virulence}, url = {https://journals.ansfoundation.org/index.php/jans/article/view/3693}, author = {Subha Lakshmi and Gopalakrishnan Nair and Santha Kumari and Samuel Gnana and Prakash Vincent} } @article {3630, title = {Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences [C-CAMP Bioincubation Facility]}, journal = {Molecular Therapy - Cell Press}, year = {2022}, month = {2022 Jan 07}, type = {Journal Article}, abstract = {

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

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

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

}, keywords = {ACE-inhibitory peptide, Bacillus subtilis, Fermented legume, Molecular docking, Rice bean}, doi = {https://doi.org/10.1016/j.biortech.2022.126932}, url = {https://www.sciencedirect.com/science/article/abs/pii/S0960852422002619}, author = {Padhi, Srichandan and Chourasia, Rounak and Kumari, Megha and Singh, Sudhir P and Rai, Amit Kumar} } @article {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 {7323, title = {Ultrashort Peptide-Based Hydrogel for the Healing of Critical Bone Defects in Rabbits [C-CAMP BIG Grantee/Startup]}, journal = {ACS Appl Mater Interfaces}, year = {2022}, month = {2022 Nov 19}, abstract = {

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

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

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

}, doi = {10.1021/acssynbio.1c00445}, url = {https://doi.org/10.1021/acssynbio.1c00445}, author = {Bhowmik, Purnendu and Rajagopal, Sreenath and Hmar, Rothangamawi Victoria and Singh, Purnima and Saxena, Pragya and Amar, Prakruthi and Thomas, Teby and Ravishankar, Rajani and Nagaraj, Savitha and Katagihallimath, Nainesh and Sarangapani, Ramanujan Kadambi and Ramachandran, Vasanthi and Datta, Santanu} } @article {1858, title = {Azaindole Based Potentiator of Antibiotics against Gram-Negative Bacteria [C-CAMP Startup Bugworks]}, journal = {ACS Infectious Diseases}, year = {2021}, month = {10/2021}, type = {Journal Article}, abstract = {
We discovered azaindole-based compounds with weak innate activity that exhibit substantial potentiation of antibacterial activities of different antibiotics, viz., rifampicin, erythromycin, solithromycin, and novobiocin in Gram-negative bacteria. In the presence of the azaindole derivatives, these antibiotics exhibited submicromolar minimum inhibitory concentrations (MICs) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The fold improvements in MIC of these antibiotics that were otherwise weak or inactive on their own against these bacteria were also observed against drug-resistant clinical isolates. Our studies indicate that this selective potentiation is probably through destabilization of the outer membrane{\textquoteright}s integrity, known to be regulated by the lipopolysaccharides (LPS). Thus, the azaindole based compounds described here open opportunities for those antibiotics that are otherwise ineffective due to LPS mediated entry barriers in Gram-negative bacteria.
}, keywords = {Azaindole, Bacterial Permeability, Lipopolysachhardies (LPS), Polymyxin, Synergy}, doi = {10.1021/acsinfecdis.1c00171}, url = {https://pubs.acs.org/doi/abs/10.1021/acsinfecdis.1c00171$\#$}, author = {Sharma, Sreevalli and Rao, Ranga and Reeve, Stephanie M and Phelps, Gregory A and Bharatham, Nagakumar and Katagihallimath, Nainesh and Ramachandran, Vasanthi and Raveendran, Savitha and Sarma, Maitrayee and Nath, Anubha} } @article {1679, title = {Chemical intervention for enhancing growth and reducing grain arsenic accumulation in rice [Mass Spectrometry - Metabolomics Facility]}, journal = {Environ Pollut}, volume = {276}, year = {2021}, month = {2021 Feb 13}, pages = {116719}, abstract = {

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

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

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

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

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

}, issn = {1940-6029}, doi = {10.1007/7651_2021_385}, author = {Konala, Vijay Bhaskar Reddy and Nandakumar, Swapna and Surendran, Harshini and Pal, Rajarshi} } @article {1724, title = {Identification of novel vaccine candidates in the whole-cell Aeromonas hydrophila biofilm vaccine through reverse vaccinology approach [Mass Spectrometry - Proteomics Facility]}, journal = {Fish \& Shellfish Immunology}, volume = {114}, year = {2021}, pages = {132-141}, abstract = {

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

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

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

}, doi = {https://doi.org/10.1155/2021/4259777}, url = {https://www.hindawi.com/journals/bmri/2021/4259777/$\#$materials-and-methods}, author = {Nayim, Paul and Sudhir, Krishna and Mbaveng, Armelle T and Kuete, Victor and Sanjukta, Mukherjee} } @article {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 {1848, title = {SUMOylation of Arginyl tRNA Synthetase Modulates the Drosophila Innate Immune Response [Transgenic Fly Facility]}, journal = {Frontiers in Cell and Developmental Biology }, year = {2021}, month = {09/2021}, abstract = {

SUMO conjugation of a substrate protein can modify its activity, localization, interaction or function. A large number of SUMO targets in cells have been identified by Proteomics, but biological roles for SUMO conjugation for most targets remains elusive. The multi-aminoacyl tRNA synthetase complex (MARS) is a sensor and regulator of immune signaling. The proteins of this 1.2 MDa complex are targets of SUMO conjugation, in response to infection. Arginyl tRNA Synthetase (RRS), a member of the sub-complex II of MARS, is one such SUMO conjugation target. The sites for SUMO conjugation are Lys 147 and 383. Replacement of these residues by Arg (RRSK147R,K383R), creates a SUMO conjugation resistant variant (RRSSCR). Transgenic Drosophila lines for RRSWT and RRSSCR were generated by expressing these variants in a RRS loss of function (lof) animal, using the UAS-Gal4 system. The RRS-lof line was itself generated using CRISPR/Cas9 genome editing. Expression of both RRSWT and RRSSCR rescue the RRS-lof lethality. Adult animals expressing RRSWT and RRSSCR are compared and contrasted for their response to bacterial infection by gram positive M. luteus and gram negative Ecc15. We find that RRSSCR, when compared to RRSWT\ shows modulation of the transcriptional response, as measured by quantitative 3' mRNA sequencing. Our study uncovers a possible non-canonical role for SUMOylation of RRS, a member of the MARS complex, in host-defense.

}, keywords = {ArgRS, Cas9, CRISPR, MARS complex, NFkB, signaling}, doi = {https://doi.org/10.3389/fcell.2021.695630}, url = {https://www.frontiersin.org/articles/10.3389/fcell.2021.695630/full$\#$h9}, author = {Prajna Nayak and Aarti Kejriwal and Girish S. Ratnaparkhi} } @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 {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 {1323, title = {Dataset for the combined transcriptome assembly of M. oleifera and functional annotation [Next Gen Genomics Facility (INT)]}, journal = {Data in Brief}, year = {2020}, pages = {105416}, abstract = {

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

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

BACKGROUND

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

RESULTS

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

CONCLUSION

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

}, doi = {https://doi.org/10.1002/jsfa.10708}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/jsfa.10708}, author = {Paul, Shyam Sundar and Venkata, Hemanth Giri Rao Vantharam and Raju, MVLN and Rama Rao, SV and Nori, Sri Sailaja and Suryanarayan, Shrikumar and Kumar, Vikas and Perveen, Zeba and Srinivas Prasad, Cadaba} } @article {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 {1342, title = {Metagenomics analysis reveals features unique to Indian distal gut microbiota [Next Gen Genomics Facility]}, journal = {PLoS One}, volume = {15}, year = {2020}, month = {2020}, pages = {e0231197}, abstract = {

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

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0231197}, author = {Kaur, Kamaldeep and Khatri, Indu and Akhtar, Akil and Subramanian, Srikrishna and Ramya, T N C} } @article {1288, title = {Microbial Diversity and Metabolite Profiles of Palm Wine Produced From Three Different Palm Tree Species in C{\^o}te d{\textquoteright}Ivoire [Mass Spectrometry - Metabolomics Facility]}, journal = {Sci Rep}, volume = {10}, year = {2020}, month = {2020 Feb 03}, pages = {1715}, abstract = {

Palm wine, the most commonly consumed traditional alcoholic beverage in Western Africa, harbours a complex microbiota and metabolites, which plays a crucial role in the overall quality and value of the product. In the present study, a combined metagenomic and metabolomic approach was applied to describe the microbial community structure and metabolites profile of fermented saps from three palm species (Elaeis guineensis, Raphia hookeri, Borassus aethiopum) in C{\^o}te d{\textquoteright}Ivoire. Lactobacillaceae (47\%), Leuconostocaceae (16\%) and Acetobacteriaceae (28\%) were the most abundant bacteria and Saccharomyces cerevisiae (87\%) the predominant yeasts in these beverages. The microbial community structure of Raphia wine was distinctly different from the others. Multivariate analysis based on the metabolites profile clearly separated the three palm wine types. The main differentiating metabolites were putatively identified as gevotroline hydrochloride, sesartemin and methylisocitrate in Elaeis wine; derivative of homoserine, mitoxantrone in Raphia wine; pyrimidine nucleotide sugars (UDP-D-galacturonate) and myo-Inositol derivatives in Borassus wine. The enriched presence of gevotroline (an antipsychotic agent) and mitoxantrone (an anticancer drug) in palm wine supports its therapeutic potential. This work provides a valuable insight into the microbiology and biochemistry of palm wines and a rationale for selecting functional microorganisms for potential biotechnology applications.

}, issn = {2045-2322}, doi = {10.1038/s41598-020-58587-2}, author = {Djeni, Theodore N and Kouame, Karen H and Ake, Francine D M and Amoikon, Laurent S T and Dje, Marcellin K and Jeyaram, Kumaraswamy} } @article {1627, title = {RNA-binding proteins La and HuR cooperatively modulate translation repression of PDCD4 mRNA [Mass Spectrometry Facility - Proteomics]}, journal = {J Biol Chem .}, volume = {doi: 10.1074/jbc.RA120.014894. Online ahead of print.}, year = {2020}, month = {12/2020}, abstract = {

Post-transcriptional regulation of gene expression plays a critical role in controlling the inflammatory response. An uncontrolled inflammatory response results in chronic inflammation, often leading to tumorigenesis. Programmed cell death 4 (PDCD4) is a pro-inflammatory tumor-suppressor gene which helps to prevent the transition from chronic inflammation to cancer. PDCD4 mRNA translation is regulated by an interplay between the oncogenic microRNA miR-21 and the RNA-binding protein (RBP) HuR in response to LPS stimulation, but the role of other regulatory factors remain unknown. Here we report that the RBP Lupus antigen (La) interacts with the 3{\textquoteright}UTR of PDCD4 mRNA and prevents miR-21-mediated translation repression. While LPS causes nuclear-cytoplasmic translocation of HuR, it enhances cellular La expression. Remarkably, La and HuR were found to bind cooperatively to the PDCD4 mRNA and mitigate miR-21-mediated translation repression. The cooperative action of La and HuR reduced cell proliferation and enhanced apoptosis, reversing the pro-oncogenic function of miR-21. Together, these observations demonstrate a cooperative interplay between two RBPs, triggered differentially by the same stimulus, which exerts a synergistic effect on PDCD4 expression and thereby helps maintain a balance between inflammation and tumorigenesis.

}, author = {Kumar, Ravi and Poria, Dipak Kumar and Ray, Partho Sarothi} } @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 {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 {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 {986, title = {Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway [Discovery to Innovation Accelerator]}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 Jan 09}, pages = {89}, abstract = {

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

}, issn = {2041-1723}, doi = {10.1038/s41467-018-07859-7}, author = {Singh, Rajbir and Chandrashekharappa, Sandeep and Bodduluri, Sobha R and Baby, Becca V and Hegde, Bindu and Kotla, Niranjan G and Hiwale, Ankita A and Saiyed, Taslimarif and Patel, Paresh and Vijay-Kumar, Matam and Langille, Morgan G I and Douglas, Gavin M and Cheng, Xi and Rouchka, Eric C and Waigel, Sabine J and Dryden, Gerald W and Alatassi, Houda and Zhang, Huang-Ge and Haribabu, Bodduluri and Vemula, Praveen K and Jala, Venkatakrishna R} } @article {1458, title = {The Hox gene uses Doublesex as a cofactor to promote neuroblast apoptosis in the central nervous system [Transgenic Fly Facility]}, journal = {Development}, volume = {146}, year = {2019}, month = {2019 08 22}, abstract = {

Highly conserved DM domain-containing transcription factors (Doublesex/MAB-3/DMRT1) are responsible for generating sexually dimorphic features. In the central nervous system, a set of Doublesex (Dsx)-expressing neuroblasts undergo apoptosis in females whereas their male counterparts proliferate and give rise to serotonergic neurons crucial for adult mating behaviour. Our study demonstrates that the female-specific isoform of Dsx collaborates with Hox gene () to bring about this apoptosis. Biochemical results suggest that proteins AbdB and Dsx interact through their highly conserved homeodomain and DM domain, respectively. This interaction is translated into a cooperative binding of the two proteins on the apoptotic enhancer in the case of females but not in the case of males, resulting in female-specific activation of apoptotic genes. The capacity of AbdB to use the sex-specific isoform of Dsx as a cofactor underlines the possibility that these two classes of protein are capable of cooperating in selection and regulation of target genes in a tissue- and sex-specific manner. We propose that this interaction could be a common theme in generating sexual dimorphism in different tissues across different species.

}, keywords = {Animals, Apoptosis, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Female, Gene Expression Regulation, Developmental, Genes, Homeobox, Homeodomain Proteins, Male, Neural Stem Cells, Protein Isoforms, Sex Characteristics}, issn = {1477-9129}, doi = {10.1242/dev.175158}, author = {Ghosh, Neha and Bakshi, Asif and Khandelwal, Risha and Rajan, Sriivatsan Govinda and Joshi, Rohit} } @article {1151, title = {Isolation and Characterization of Conotoxin Protein from Conus inscriptus and Its Potential Anticancer Activity Against Cervical Cancer (HeLa-HPV 16 Associated) Cell Lines [Mass Spectrometry - Proteomics/Glycomics]}, journal = {International Journal of Peptide Research and Therapeutics}, year = {2019}, month = {Aug}, abstract = {

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

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

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

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

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

}, issn = {1538-0254}, doi = {10.1080/07391102.2019.1581086}, author = {Siddiqui, Amir M and Akhtar, Juber and M S, Shahab Uddin and Khan, Mohammad Irfan and Khalid, Mohammad} } @article {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} } @book {1188, title = {The Neem Genome [Next Gen Genomics Facility]}, year = {2019}, publisher = {Springer Nature Switzerland AG}, organization = {Springer Nature Switzerland AG}, abstract = { }, keywords = {C-CAMP Genomics Facility, Illumina HiSeq 1000, Next Generation Sequencing, paired-end (PE) (2 {\texttimes} 100 nts) sequencing chemistry}, author = {Gowda, Malali and Sheetal, Ambardar and Kole, Chittaranjan} } @conference {1311, title = {Overcoming Barriers in Commercializing Bio-Tech Innovations in India: A Case of Center for Cellular and Molecular Platforms}, booktitle = {2019 Portland International Conference on Management of Engineering and Technology (PICMET)}, year = {2019}, month = {Aug}, abstract = {

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

}, keywords = {biotech innovation, biotech organizations, biotech start-ups funding, biotechnology, Centre for Cellular and Molecular Platforms, commercial ventures, commercialization, entrepreneurial ventures, entrepreneurship, financial management, financial support, government, government-funded nonprofit organization, health care, healthcare sector, incumbent research community, India, infrastructural support, innovation management, mentorship, organisational aspects, product research, scientific support, scientific ventures, socio-economic effects, socioeconomic condition, top management teams, venture capital, venture capital funding agencies}, doi = {10.23919/PICMET.2019.8893754}, author = {G. D. Tikas and T. Saiyed and A. Katte} } @article {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 {1016, title = {Serum biomarkers identification by iTRAQ and verification by MRM: S100A8/S100A9 levels predict tumor-stroma involvement and prognosis in Glioblastoma [Mass Spectrometry - Metabolomics Facility].}, journal = {Sci Rep}, volume = {9}, year = {2019}, month = {2019 Feb 26}, pages = {2749}, abstract = {

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

}, issn = {2045-2322}, doi = {10.1038/s41598-019-39067-8}, author = {Arora, Anjali and Patil, Vikas and Kundu, Paramita and Kondaiah, Paturu and Hegde, A S and Arivazhagan, A and Santosh, Vani and Pal, Debnath and Somasundaram, Kumaravel} } @article {1007, title = {SOD1 activity threshold and TOR signalling modulate VAP(P58S) aggregation via reactive oxygen species-induced proteasomal degradation in a model of amyotrophic lateral sclerosis. [High Throughput Screening Facility]}, journal = {Dis Model Mech}, volume = {12}, year = {2019}, month = {2019 Feb 07}, abstract = {

Familial amyotrophic lateral sclerosis (ALS) is an incurable, late-onset motor neuron disease, linked strongly to various causative genetic loci. codes for a missense mutation, P56S, in VAMP-associated protein B (VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase our understanding of the disease and lead to potential therapeutics. We developed a quantitative high-throughput S2R+ cell-based kinetic assay coupled with fluorescent microscopy to score for genes involved in the modulation of aggregates of the fly orthologue, VAP(P58S), fused with GFP. A targeted RNA interference screen against 900 genes identified 150 hits that modify aggregation, including the ALS loci and (also known as ), as well as genes belonging to the mTOR pathway. Further, a system to measure the extent of VAP(P58S) aggregation in the larval brain was developed in order to validate the hits from the cell-based screen. In the larval brain, we find that reduction of SOD1 levels or decreased mTOR signalling reduces aggregation, presumably by increasing the levels of cellular reactive oxygen species (ROS). The mechanism of aggregate clearance is, primarily, proteasomal degradation, which appears to be triggered by an increase in ROS. We have thus uncovered an interesting interplay between SOD1, ROS and mTOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead to better understanding of the initiation and progression of ALS.This article has an associated First Person interview with the first author of the paper.

}, issn = {1754-8411}, doi = {10.1242/dmm.033803}, author = {Chaplot, Kriti and Pimpale, Lokesh and Ramalingam, Balaji and Deivasigamani, Senthilkumar and Kamat, Siddhesh S and Ratnaparkhi, Girish S} } @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 {1159, title = {Structural and Functional Insights into GluK3-kainate Receptor Desensitization and Recovery [National Cryo-Electron Microscopy Facility]}, journal = {Sci Rep}, volume = {9}, year = {2019}, month = {2019 Jul 16}, pages = {10254}, abstract = {

GluK3-kainate receptors are atypical members of the iGluR family that reside at both the pre- and postsynapse and play a vital role in the regulation of synaptic transmission. For a better understanding of structural changes that underlie receptor functions, GluK3 receptors were trapped in desensitized and resting/closed states and structures analyzed using single particle cryo-electron microscopy. While the desensitized GluK3 has domain organization as seen earlier for another kainate receptor-GluK2, antagonist bound GluK3 trapped a resting state with only two LBD domains in dimeric arrangement necessary for receptor activation. Using structures as a guide, we show that the N-linked glycans at the interface of GluK3 ATD and LBD likely mediate inter-domain interactions and attune receptor-gating properties. The mutational analysis also identified putative N-glycan interacting residues. Our results provide a molecular framework for understanding gating properties unique to GluK3 and exploring the role of N-linked glycosylation in their modulation.

}, issn = {2045-2322}, doi = {10.1038/s41598-019-46770-z}, author = {Kumari, Jyoti and Vinnakota, Rajesh and Kumar, Janesh} } @article {1084, title = {The transcriptome enables the identification of candidate genes behind medicinal value of Drumstick tree (Moringa oleifera) [Next Gen Genomics Facility (INT)].}, journal = {Genomics}, year = {2019}, month = {2019 Apr 29}, abstract = {

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

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

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

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

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

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

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

}, issn = {1349-8037}, doi = {10.2323/jgam.2017.11.006}, author = {Kumar, Vidya Pradeep and Kolte, Atul P and Dhali, Arindam and Naik, Chandrashekar and Sridhar, Manpal} } @article {686, title = {Exploiting a water network to achieve enthalpy-driven, bromodomain-selective BET inhibitors}, journal = {Bioorganic \& Medicinal Chemistry}, volume = {26}, year = {2018}, pages = {25 - 36}, issn = {0968-0896}, doi = {https://doi.org/10.1016/j.bmc.2017.10.042}, url = {http://www.sciencedirect.com/science/article/pii/S0968089617315948}, author = {William R. Shadrick and Peter J. Slavish and Sergio C. Chai and Brett Waddell and Michele Connelly and Jonathan A. Low and Cynthia Tallant and Brandon M. Young and Nagakumar Bharatham and Stefan Knapp and Vincent A. Boyd and Marie Morfouace and Martine F. Roussel and Taosheng Chen and Richard E. Lee and R. Kiplin Guy and Anang A. Shelat and Philip M. Potter} } @article {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 {814, title = {Recovery of Five Complete Influenza A(H1N1)pdm09 Genome Sequences from the 2015 Influenza Outbreak in India by Metagenomic Sequencing. [Next Gen Genomics Facility (INT)]}, journal = {Genome Announc}, volume = {6}, year = {2018}, month = {2018 Jun 28}, abstract = {

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

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

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

}, issn = {2160-1836}, doi = {10.1534/g3.118.200265}, author = {Lal, Avantika and Krishna, Sandeep and Seshasayee, Aswin Sai Narain} } @article {685, title = {S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils. [Mass Spectrometry Facility - Proteomics]}, journal = {Biochim Biophys Acta}, volume = {1865}, year = {2018}, month = {2018 Feb}, pages = {444-454}, abstract = {

Post-translational modifications (PTMs) induced conformational changes of proteins can cause their activation or inactivation. Neutrophils clear pathogen through phagocytosis and oxidative burst generation, while participate in inflammation through sustained and uncontrolled generation of ROS. In activated PMNs, cytosolic NOX-2 subunit p47phox following phosphorylation interacts with p67phox, p40phox and along with Rac2 translocate to the membrane. Phosphorylation of p47phox subunit occurs in both short spurts as well as sustained ROS generation, suggesting towards the unidentified molecular mechanism(s) driving these two diverse outcomes by various stimuli. The present study demonstrates that in PMA or NO treated neutrophils a subunit of NOX2, p47phox gets glutathionylated to sustain ROS generation along with a decrease in catalase, Grx-1 activity and change in GSH/GSSG ratio. Surprisingly, fMLP treated cells neither showed sustained ROS production nor glutathionylation of p47phox. S-Glutathionylation was always secondary to phosphorylation of p47phox and inhibition of glutathionylation did not alter phosphorylation but specifically impaired sustained ROS production. Interestingly, forced S-glutathionylation of p47phox converted the fMLP induced ROS generation into sustained release of ROS. We then identified the glutathionylation susceptible cysteine residues of p47phox by LC-MS/MS with IAM switch mapping. Site-directed mutagenesis of cysteine residues further mitigated p47phox S-glutathionylation. Thus, we demonstrate that p47phox S-glutathionylation plays an essential key role in the sustained ROS generation by human neutrophils.

}, issn = {0006-3002}, doi = {10.1016/j.bbamcr.2017.11.014}, author = {Nagarkoti, Sheela and Dubey, Megha and Awasthi, Deepika and Kumar, Vikas and Chandra, Tulika and Kumar, Sachin and Dikshit, Madhu} } @article {889, title = {A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation [Bugworks Res. Pvt. Ltd., a C-CAMP Startup]}, journal = {Microb Cell Fact}, volume = {17}, year = {2018}, month = {2018 Dec 03}, pages = {192}, abstract = {

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

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

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

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

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

}, keywords = {Amino Acid Sequence, Animals, Apoptosis, Central Nervous System, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Enhancer Elements, Genetic, Female, Gene Expression Regulation, Developmental, Genes, Homeobox, Homeodomain Proteins, Larva, Male, Nuclear Proteins, Receptors, Notch, Transcription Factors}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1007043}, author = {Khandelwal, Risha and Sipani, Rashmi and Govinda Rajan, Sriivatsan and Kumar, Raviranjan and Joshi, Rohit} } @article {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 {627, title = {Developmentally regulated higher-order chromatin interactions orchestrate B cell fate commitment.}, journal = {Nucleic Acids Res}, year = {2017}, month = {2017 Aug 17}, abstract = {

Genome organization in 3D nuclear-space is important for regulation of gene expression. However, the alterations of chromatin architecture that impinge on the B cell-fate choice of multi-potent progenitors are still unclear. By integrating in situ Hi-C analyses with epigenetic landscapes and genome-wide expression profiles, we tracked the changes in genome architecture as the cells transit from a progenitor to a committed state. We identified the genomic loci that undergo developmental switch between A and B compartments during B-cell fate determination. Furthermore, although, topologically associating domains (TADs) are stable, a significant number of TADs display structural alterations that are associated with changes in cis-regulatory interaction landscape. Finally, we demonstrate the potential roles for Ebf1 and its downstream factor, Pax5, in chromatin reorganization and transcription regulation. Collectively, our studies provide a general paradigm of the dynamic relationship between chromatin reorganization and lineage-specific gene expression pattern that dictates cell-fate determination.

}, issn = {1362-4962}, doi = {10.1093/nar/gkx722}, author = {Boya, Ravi and Yadavalli, Anurupa Devi and Nikhat, Sameena and Kurukuti, Sreenivasulu and Palakodeti, Dasaradhi and Pongubala, Jagan M R} } @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 {737, title = {Lipid metabolic perturbation is an early-onset phenotype in adultmutants: amodel for lysosomal storage disorders. [Mass Spectrometry - Lipidomics]}, journal = {Mol Biol Cell}, volume = {28}, year = {2017}, month = {2017 Dec 15}, pages = {3728-3740}, abstract = {

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

}, keywords = {Animals, Carrier Proteins, Disease Models, Animal, Drosophila, Drosophila Proteins, Lipid Metabolism, Lipids, Lipoproteins, Lysosomal Storage Diseases, Lysosomes, Membrane Proteins, Mutation, Nervous System, Neurodegenerative Diseases, Phenotype}, issn = {1939-4586}, doi = {10.1091/mbc.E16-09-0674}, author = {Hebbar, Sarita and Khandelwal, Avinash and Jayashree, R and Hindle, Samantha J and Chiang, Yin Ning and Yew, Joanne Y and Sweeney, Sean T and Schwudke, Dominik} } @article {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 {511, title = {Sirtuin1-regulated lysine acetylation of p66Shc governs diabetes-induced vascular oxidative stress and endothelial dysfunction.}, journal = {Proc Natl Acad Sci U S A}, year = {2017}, month = {2017 Jan 30}, abstract = {

The 66-kDa Src homology 2 domain-containing protein (p66Shc) is a master regulator of reactive oxygen species (ROS). It is expressed in many tissues where it contributes to organ dysfunction by promoting oxidative stress. In the vasculature, p66Shc-induced ROS engenders endothelial dysfunction. Here we show that p66Shc is a direct target of the Sirtuin1 lysine deacetylase (Sirt1), and Sirt1-regulated acetylation of p66Shc governs its capacity to induce ROS. Using diabetes as an oxidative stimulus, we demonstrate that p66Shc is acetylated under high glucose conditions and is deacetylated by Sirt1 on lysine 81. High glucose-stimulated lysine acetylation of p66Shc facilitates its phosphorylation on serine 36 and translocation to the mitochondria, where it promotes hydrogen peroxide production. Endothelium-specific transgenic and global knockin mice expressing p66Shc that is not acetylatable on lysine 81 are protected from diabetic oxidative stress and vascular endothelial dysfunction. These findings show that p66Shc is a target of Sirt1, uncover a unique Sirt1-regulated lysine acetylation-dependent mechanism that governs the oxidative function of p66Shc, and demonstrate the importance of p66Shc lysine acetylation in vascular oxidative stress and diabetic vascular pathophysiology.

}, issn = {1091-6490}, doi = {10.1073/pnas.1614112114}, author = {Kumar, Santosh and Kim, Young-Rae and Vikram, Ajit and Naqvi, Asma and Li, Qiuxia and Kassan, Modar and Kumar, Vikas* and Bachschmid, Markus M and Jacobs, Julia S and Kumar, Ajay and Irani, Kaikobad} } @article {8889, title = {Transcriptional transitions in Alphonso mango (Mangifera indica L.) during fruit development and ripening explain its distinct aroma and shelf life characteristics [Next Gen Genomics Facility]}, journal = {Sci Rep}, volume = {7}, year = {2017}, month = {2017 Aug 18}, pages = {8711}, abstract = {

Alphonso is known as the "King of mangos" due to its unique flavor, attractive color, low fiber pulp and long shelf life. We analyzed the transcriptome of Alphonso mango through Illumina sequencing from seven stages of fruit development and ripening as well as flower. Total transcriptome data from these stages ranged between 65 and 143 Mb. Importantly, 20,755 unique transcripts were annotated and 4,611 were assigned enzyme commission numbers, which encoded 142 biological pathways. These included ethylene and flavor related secondary metabolite biosynthesis pathways, as well as those involved in metabolism of starch, sucrose, amino acids and fatty acids. Differential regulation (p-value <= 0.05) of thousands of transcripts was evident in various stages of fruit development and ripening. Novel transcripts for biosynthesis of mono-terpenes, sesqui-terpenes, di-terpenes, lactones and furanones involved in flavor formation were identified. Large number of transcripts encoding cell wall modifying enzymes was found to be steady in their expression, while few were differentially regulated through these stages. Novel 79 transcripts of inhibitors of cell wall modifying enzymes were simultaneously detected throughout Alphonso fruit development and ripening, suggesting controlled activity of these enzymes involved in fruit softening.

}, keywords = {Cell Wall, Enzyme Inhibitors, Flowers, Fruit, Gene Expression Profiling, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant, Gene Ontology, Genes, Plant, Glycoside Hydrolases, Mangifera, Odorants, Phylogeny, Reproducibility of Results, RNA, Messenger, Transcription, Genetic, Transcriptome}, issn = {2045-2322}, doi = {10.1038/s41598-017-08499-5}, author = {Deshpande, Ashish B and Anamika, Krishanpal and Jha, Vineet and Chidley, Hemangi G and Oak, Pranjali S and Kadoo, Narendra Y and Pujari, Keshav H and Giri, Ashok P and Gupta, Vidya S} } @article {780, title = {Exploring Packaged Microvesicle Proteome Composition of Chinese Hamster Ovary Secretome [Mass Spectrometry - Proteomics]}, journal = {Journal of Bioprocessing \& Biotechniques}, volume = {6}, year = {2016}, pages = {1-11}, keywords = {

CHO, Microvesicles, Proteome, Recombinant protein production

, Secretome}, issn = {2155-9821}, doi = {10.4172/2155-9821.1000274}, url = {https://www.omicsonline.org/open-access/exploring-packaged-microvesicle-proteome-composition-of-chinesehamster-ovary-secretome-2155-9821-1000274.php?aid=71599}, author = {Niraj Kumar and Dixat Gopal Gupta and Srikant Kumar and et. al.} } @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 {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 {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 {473, title = {A dPIP5K dependent pool of phosphatidylinositol 4,5 bisphosphate (PIP2) is required for G-protein coupled signal transduction in Drosophila photoreceptors.[Drosophila facility]}, journal = {PLoS Genet}, volume = {11}, year = {2015}, month = {2015 Jan}, pages = {e1004948}, abstract = {

Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. We identify dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction. Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

}, keywords = {Animals, Cell Membrane, Cytoskeleton, Drosophila, Drosophila melanogaster, Light Signal Transduction, Membrane Proteins, Ocular Physiological Phenomena, Phosphatidylinositol 4,5-Diphosphate, Phosphoinositide Phospholipase C, Phosphotransferases (Alcohol Group Acceptor), Photoreceptor Cells, Retina, Signal Transduction}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1004948}, author = {Chakrabarti, Purbani and Kolay, Sourav and Yadav, Shweta and Kumari, Kamalesh and Nair, Amit and Trivedi, Deepti and Raghu, Padinjat} } @article {8884, title = {Genome Sequence, Comparative Analysis, and Evolutionary Insights into Chitinases of Entomopathogenic Fungus Hirsutella thompsonii [Next Gen Genomics Facility]}, journal = {Genome Biology and Evolution}, volume = {7}, year = {2015}, month = {02}, pages = {916-930}, abstract = {

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

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

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

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

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

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

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

}, keywords = {Actins, Adult, Amino Acid Sequence, Animals, Case-Control Studies, Cell Polarity, Chemotaxis, Diabetes Mellitus, Female, Glutathione, HEK293 Cells, Humans, Male, Mice, Inbred C57BL, Mice, Obese, Microfilament Proteins, Middle Aged, Molecular Sequence Data, Neutrophils, Nitric Oxide, Oxidative Stress, Protein Binding, Protein Processing, Post-Translational, Young Adult}, issn = {1873-4596}, doi = {10.1016/j.freeradbiomed.2015.04.008}, author = {Dubey, Megha and Singh, Abhishek K and Awasthi, Deepika and Nagarkoti, Sheela and Kumar, Sachin and Ali, Wahid and Chandra, Tulika and Kumar, Vikas and Barthwal, Manoj K and Jagavelu, Kumaravelu and S{\'a}nchez-G{\'o}mez, Francisco J and Lamas, Santiago and Dikshit, Madhu} } @article {533, title = {A rapid, nonradioactive assay for measuring heparan sulfate C-5 epimerase activity using hydrogen/deuterium exchange-mass spectrometry.}, journal = {Methods Mol Biol}, volume = {1229}, year = {2015}, month = {2015}, pages = {209-19}, abstract = {

Heparin and heparan sulfate (HS) glycosaminoglycans have important roles in anticoagulation, human development, and human diseases. HS C5-epimerase, which catalyzes the epimerization of GlcA to IdoA, is a crucial enzyme involved in the biosynthesis of heparin-related biomolecules. Here, we describe a detailed method for measuring the total activity of HS C5-epimerase that involves the following steps: H/D exchange upon epimerization of the substrate with HS C5-epimerase, low-pH nitrous acid treatment of the substrate, the separation of low-pH nitrous acid-cleaved disaccharides using HPLC, and mass spectrometry analysis. This nonradioactive method is rapid and sensitive and, importantly, allows us to study the reversible nature of HS C5-epimerase.

}, keywords = {Animals, Biocatalysis, Carbohydrate Epimerases, Chromatography, Ion Exchange, Chromatography, Liquid, Deuterium Exchange Measurement, Disaccharides, Enzyme Assays, Glucuronic Acid, Heparitin Sulfate, Humans, Iduronic Acid, Mass Spectrometry, Sf9 Cells}, issn = {1940-6029}, doi = {10.1007/978-1-4939-1714-3_19}, author = {Babu, Ponnusamy and Victor, Xylophone V and Raman, Karthik and Kuberan, Balagurunathan} } @article {684, title = {Role of Homothorax in region specific regulation of Deformed in embryonic neuroblasts.}, journal = {Mech Dev}, volume = {138 Pt 2}, year = {2015}, month = {2015 Nov}, pages = {190-7}, abstract = {

The expression and regulation of Hox genes in developing central nervous system (CNS) lack important details like specific cell types where Hox genes are expressed and the transcriptional regulatory players involved in these cells. In this study we have investigated the expression and regulation of Drosophila Hox gene Deformed (Dfd) in specific cell types of embryonic CNS. Using Dfd neural autoregulatory enhancer we find that Dfd autoregulates itself in cells of mandibular neuromere. We have also investigated the role of a Hox cofactor Homothorax (Hth) for its role in regulating Dfd expression in CNS. We find that Hth exhibits a region specific role in controlling the expression of Dfd, but has no direct role in mandibular Dfd neural autoregulatory circuit. Our results also suggest that homeodomain of Hth is not required for regulating Dfd expression in embryonic CNS.

}, keywords = {Animals, Central Nervous System, Drosophila, Drosophila Proteins, Enhancer Elements, Genetic, Gene Expression Regulation, Developmental, Genes, Homeobox, Homeodomain Proteins, Neural Stem Cells, Organogenesis}, issn = {1872-6356}, doi = {10.1016/j.mod.2015.09.003}, author = {Kumar, Raviranjan and Chotaliya, Maheshvari and Vuppala, Sruthakeerthi and Auradkar, Ankush and Palasamudrum, Kalyani and Joshi, Rohit} } @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 {8356, title = {Lunatimonas lonarensis gen. nov., sp. nov., a haloalkaline bacterium of the family Cyclobacteriaceae with nitrate reducing activity [Next Gen Genomics Facility]}, journal = {Syst Appl Microbiol}, volume = {37}, year = {2014}, month = {2014 Feb}, pages = {10-6}, abstract = {

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

}, keywords = {Bacterial Typing Techniques, Bacteroidetes, Cluster Analysis, DNA, Bacterial, DNA, Ribosomal, Fatty Acids, Fresh Water, Genome, Bacterial, Geologic Sediments, India, Molecular Sequence Data, Nitrates, Oxidation-Reduction, Phospholipids, Phylogeny, Quinones, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {1618-0984}, doi = {10.1016/j.syapm.2013.10.003}, author = {Srinivas, T N R and Aditya, S and Bhumika, V and Kumar, P Anil} } @article {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 {493, title = {Deep sequencing reveals unique small RNA repertoire that is regulated during head regeneration in Hydra magnipapillata. [Next Generation Genomics facility]}, journal = {Nucleic Acids Res}, volume = {41}, year = {2013}, month = {2013 Jan 07}, pages = {599-616}, abstract = {

Small non-coding RNAs such as miRNAs, piRNAs and endo-siRNAs fine-tune gene expression through post-transcriptional regulation, modulating important processes in development, differentiation, homeostasis and regeneration. Using deep sequencing, we have profiled small non-coding RNAs in Hydra magnipapillata and investigated changes in small RNA expression pattern during head regeneration. Our results reveal a unique repertoire of small RNAs in hydra. We have identified 126 miRNA loci; 123 of these miRNAs are unique to hydra. Less than 50\% are conserved across two different strains of Hydra vulgaris tested in this study, indicating a highly diverse nature of hydra miRNAs in contrast to bilaterian miRNAs. We also identified siRNAs derived from precursors with perfect stem-loop structure and that arise from inverted repeats. piRNAs were the most abundant small RNAs in hydra, mapping to transposable elements, the annotated transcriptome and unique non-coding regions on the genome. piRNAs that map to transposable elements and the annotated transcriptome display a ping-pong signature. Further, we have identified several miRNAs and piRNAs whose expression is regulated during hydra head regeneration. Our study defines different classes of small RNAs in this cnidarian model system, which may play a role in orchestrating gene expression essential for hydra regeneration.

}, keywords = {Animals, Gene Expression Regulation, Head, High-Throughput Nucleotide Sequencing, Hydra, MicroRNAs, Regeneration, RNA, Small Interfering, RNA, Small Untranslated, Sequence Analysis, RNA, Transcriptome}, issn = {1362-4962}, doi = {10.1093/nar/gks1020}, author = {Krishna, Srikar and Nair, Aparna and Cheedipudi, Sirisha and Poduval, Deepak and Dhawan, Jyotsna and Palakodeti, Dasaradhi and Ghanekar, Yashoda} } @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 {488, title = {Genome sequencing unveils a novel sea enterotoxin-carrying PVL phage in Staphylococcus aureus ST772 from India. [Next Generation Genomics facility]}, journal = {PLoS One}, volume = {8}, year = {2013}, month = {2013}, pages = {e60013}, abstract = {

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

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

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

}, keywords = {Binding Sites, DNA-Binding Proteins, Epistasis, Genetic, Escherichia coli K12, Escherichia coli Proteins, Fimbriae Proteins, Gene Expression Regulation, Bacterial, Gene Silencing, Gene Transfer, Horizontal, Genome, Bacterial, Molecular Chaperones, Protein Binding, Repressor Proteins, Sequence Analysis, DNA, Transcription, Genetic, Transcriptome}, issn = {1742-2051}, doi = {10.1039/c3mb70035f}, author = {Srinivasan, Rajalakshmi and Chandraprakash, Deepti and Krishnamurthi, Revathy and Singh, Parul and Scolari, Vittore F and Krishna, Sandeep and Seshasayee, Aswin Sai Narain} } @article {537, title = {Redox proteomics of thiol proteins in mouse heart during ischemia/reperfusion using ICAT reagents and mass spectrometry.}, journal = {Free Radic Biol Med}, volume = {58}, year = {2013}, month = {2013 May}, pages = {109-17}, abstract = {

There is strong evidence for the involvement of reactive oxygen species in ischemia/reperfusion injury. Although oxidation of individual thiol proteins has been reported, more extensive redox proteomics of hearts subjected to ischemia/reperfusion has not been performed. We have carried out an exploratory study using mass spectrometry with isotope-coded affinity tags (ICAT) aimed at identifying reversible oxidative changes to protein thiols in Langendorff perfused isolated mouse hearts subjected to 20 min ischemia with or without aerobic reperfusion for 5 or 30 min. Reduced thiols were blocked by adding N-ethylmaleimide during protein extraction, then reversibly oxidized thiols in extracts of control perfused and treated hearts were reduced and labeled with the light and heavy ICAT reagents, respectively. Protein extracts were mixed in equal amounts and relative proportions of the isotope-labeled peaks were used to quantify oxidative changes between the control and the treated groups. Approximately 300 peptides with ICAT signatures were reliably identified in each sample, with 181 peptides from 118 proteins common to all treatments. A proportion showed elevated ICAT ratios, consistent with reversible thiol oxidation. This was most evident after early reperfusion, with apparent reversal after longer reperfusion. In comparison, there was gradual accumulation of protein carbonyls and loss of GSH with longer reperfusion. Many of the thiol changes were in mitochondrial proteins, including components of electron transport complexes and enzymes involved in lipid metabolism. The results are consistent with mitochondria being a major site of oxidant generation during early cardiac reperfusion and mitochondrial thiol proteins being targets for oxidation.

}, keywords = {Animals, Isotope Labeling, Mass Spectrometry, Mice, Mitochondrial Proteins, Myocardium, Oxidation-Reduction, Proteomics, Reactive Oxygen Species, Reperfusion Injury, Sulfhydryl Compounds}, issn = {1873-4596}, doi = {10.1016/j.freeradbiomed.2013.01.021}, author = {Kumar, Vikas and Kleffmann, Torsten and Hampton, Mark B and Cannell, Mark B and Winterbourn, Christine C} } @article {720, title = {Antiproliferative property of n-hexane and chloroform extracts of Anisomeles malabarica (L). R. Br. in HPV16-positive human cervical cancer cells.}, journal = {J Pharmacol Pharmacother}, volume = {3}, year = {2012}, month = {2012 Jan}, pages = {26-34}, abstract = {

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

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

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

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

}, issn = {0976-5018}, doi = {10.4103/0976-500X.92500}, author = {Preethy, Christo Paul and Padmapriya, Ramamoorthy and Periasamy, Vaiyapuri Subbarayan and Riyasdeen, Anvarbatcha and Srinag, Suresh and Krishnamurthy, Hanumanthappa and Alshatwi, Ali Abdullah and Akbarsha, Mohammad Abdulkader} } @article {714, title = {Distinct spatial and molecular features of notch pathway assembly in regulatory T cells.}, journal = {Sci Signal}, volume = {5}, year = {2012}, month = {2012 Jul 24}, pages = {ra53}, abstract = {

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

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

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

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

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

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

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

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

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

}, keywords = {Biodegradation, Environmental, Genome, Bacterial, India, Molecular Sequence Data, Nitrophenols, Pesticides, Rhodococcus, Sequence Analysis, DNA, Soil Microbiology, Soil Pollutants}, issn = {1098-5530}, doi = {10.1128/JB.00532-12}, author = {Vikram, Surendra and Kumar, Shailesh and Subramanian, Srikrishna and Raghava, Gajendra Pal Singh} } @article {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 {719, title = {Functional tumor infiltrating TH1 and TH2 effectors in large early-stage cervical cancer are suppressed by regulatory T cells.}, journal = {Int J Gynecol Cancer}, volume = {22}, year = {2012}, month = {2012 Sep}, pages = {1130-7}, abstract = {

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

}, keywords = {Amino Acid Sequence, Animals, Cell Line, Tumor, Disease Models, Animal, DNA Breaks, Double-Stranded, DNA End-Joining Repair, DNA Ligase ATP, DNA Ligases, Drug Design, Drug Resistance, Neoplasm, Humans, Lymphocytes, Lymphoma, Male, Mice, Mice, Inbred BALB C, Mice, Nude, Models, Molecular, Molecular Sequence Data, Neoplasms, Protein Structure, Tertiary, Pyrimidines, Radiation Tolerance, Rats, Schiff Bases, Sequence Alignment}, issn = {1097-4172}, doi = {10.1016/j.cell.2012.11.054}, author = {Srivastava, Mrinal and Nambiar, Mridula and Sharma, Sheetal and Karki, Subhas S and Goldsmith, G and Hegde, Mahesh and Kumar, Sujeet and Pandey, Monica and Singh, Ram K and Ray, Pritha and Natarajan, Renuka and Kelkar, Madhura and De, Abhijit and Choudhary, Bibha and Raghavan, Sathees C} } @article {724, title = {Neutrophil extracellular traps contain mitochondrial as well as nuclear DNA and exhibit inflammatory potential.}, journal = {Cytometry A}, volume = {81}, year = {2012}, month = {2012 Mar}, pages = {238-47}, abstract = {

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

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

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

}, keywords = {Aging, DNA Damage, Humans, Infertility, Male, Life Style, Male, Occupations, Spermatozoa}, issn = {1439-0272}, doi = {10.1111/j.1439-0272.2011.01243.x}, author = {Varshini, J and Srinag, B S and Kalthur, G and Krishnamurthy, H and Kumar, P and Rao, S B-S and Adiga, S K} } @article {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} }