@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 {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 {8957, title = {Lactobacillus fermentum SNR1: Probiotic efficiency and characterization of the antimicrobial peptide against dental caries and foodborne pathogens [Mass Spectrometry - Proteomics]}, journal = {The Microbe}, volume = {5}, year = {2024}, pages = {100190}, abstract = {

In this study, fermented rice gruel, a traditional food in South Telangana, India, was used as a source for the isolation of biologically significant probiotics. A total of 56 morphologically diverse strains were isolated and screened for their ability to inhibit dental biofilms as well as foodborne and aquaculture bacterial pathogens. Among these, isolate SNR1 was selected for further characterization due to its potential antimicrobial activity against bacterial pathogens such as Streptococcus mutans, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus and Salmonella typhi. The results of 16S rDNA sequencing showed that isolate SNR1 was closely related to Lactobacillus fermentum strains from different sources. The bacteriocin peptide with potential activity was extracted and purified from the cell-free supernatant using ammonium sulphate precipitation, gel permeation chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and was named peptide SNR1. Tricine SDS-PAGE followed by MALDI-TOF-MS analysis revealed that the purified peptide had a molecular mass of 4.33 kDa, with the N-terminal sequence identified as MPTTSHFHLSPQGK. The purified peptide SNR1 showed good stability with various physiological enzymes, temperatures and pH conditions.

}, keywords = {Anti-biofilm activity, Anti-microbial activity, Dental caries, SNR1}, issn = {2950-1946}, doi = {https://doi.org/10.1016/j.microb.2024.100190}, url = {https://www.sciencedirect.com/science/article/pii/S2950194624001572}, author = {Ayyanna Repally and Esakkiraj Palanichamy and Natwar Jha and Arul Venkatesan and Ankaiah Dasari} } @article {8740, title = {Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB. [Mass Spectrometry - Proteomics Facility]}, journal = {Cell Commun Signal}, volume = {22}, year = {2024}, month = {2024 Apr 23}, pages = {237}, abstract = {

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

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

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

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

}, keywords = {Animals, Azadirachta, CARD Signaling Adaptor Proteins, Dendritic Cells, Lectins, C-Type, Mice, Mice, Inbred C57BL, NF-kappa B, Plant Leaves, Protein Binding, Signal Transduction}, issn = {1478-811X}, doi = {10.1186/s12964-024-01576-z}, author = {Ganguly, Nilanjan and Das, Tapasi and Bhuniya, Avishek and Guha, Ipsita and Chakravarti, Mohona and Dhar, Sukanya and Sarkar, Anirban and Bera, Saurav and Dhar, Jesmita and Dasgupta, Shayani and Saha, Akata and Ghosh, Tithi and Das, Juhina and Sk, Ugir Hossain and Banerjee, Saptak and Laskar, Subrata and Bose, Anamika and Baral, Rathindranath} } @article {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 {8098, title = {Analysis of smart biomaterial containing umbilical cord blood serum protein conjugated with P-(NIPAAM) using spectroscopy [Bio-incubation Services]}, journal = {Materials Today: Proceedings}, year = {2023}, abstract = {

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

}, keywords = {ATR-FTIR, H NMR, HUCBS, P-NIPAAM, Protein-polymer conjugate}, issn = {2214-7853}, doi = {https://doi.org/10.1016/j.matpr.2023.01.285}, url = {https://www.sciencedirect.com/science/article/pii/S2214785323003759}, author = {Manasa Biligowda Latha and Ashmitha Kishan Shetty and Rajamanickam Deveswaran and Ashish Jagannath Rai and Serene Joy and Hadonahalli Munegowda Shashanka and Siddique Sha Muhammad Hussain and Suraksha Shetty} } @article {8467, title = {The conundrum of bacteria-specific antibiotics [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {J Antimicrob Chemother}, volume = {78}, year = {2023}, month = {2023 Jun 01}, pages = {1354-1358}, abstract = {

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

}, keywords = {Anti-Bacterial Agents, Bacteria, Bacterial Infections, Drug Resistance, Bacterial, Dysbiosis, Humans}, issn = {1460-2091}, doi = {10.1093/jac/dkad130}, author = {Datta, Santanu} } @article {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 {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 {8097, title = {Identification of key amino acid residues in OqxB mediated efflux of fluoroquinolones using site-directed mutagenesis [Bugworks Research Pvt. Ltd., a C-CAMP Startup]}, journal = {Res Microbiol}, year = {2023}, month = {2023 Feb 02}, pages = {104039}, abstract = {

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

}, issn = {1769-7123}, doi = {10.1016/j.resmic.2023.104039}, author = {Bhowmik, Purnendu and Bharatham, Nagakumar and Murakami, Satoshi and Ramachandran, Vasanthi and Datta, Santanu} } @article {8556, title = {Purification and characterization of an asialofetuin specific lectin from the rhizome of Xanthosoma violaceum Schott [Mass Spectrometry - Proteomics Facility]}, journal = {Protein Expr Purif}, year = {2023}, month = {2023 Aug 29}, pages = {106357}, abstract = {

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

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

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

}, issn = {2165-0497}, doi = {10.1128/spectrum.04943-22}, author = {Anand, Aditya and Chandana, Manjunatha and Ghosh, Sourav and Das, Rahul and Singh, Nalini and Vaishalli, Pradeep Mini and Gantasala, Nagavara Prasad and Padmanaban, Govindarajan and Nagaraj, Viswanathan Arun} } @article {3630, title = {Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences [C-CAMP Bioincubation Facility]}, journal = {Molecular Therapy - Cell Press}, year = {2022}, month = {2022 Jan 07}, type = {Journal Article}, abstract = {

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

}, keywords = {antibody-dependent enhancement, consensus sequence, dengue, dengue surveillance, DNA vaccine, EDIII domain, NS1 protein}, issn = {1525-0024}, doi = {10.1016/j.ymthe.2022.01.013}, url = {https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(22)00013-2$\#$secsectitle0165}, author = {Sankaradoss, Arun and Jagtap, Suraj and Nazir, Junaid and Moula, Shefta E and Modak, Ayan and Fialho, Joshuah and Iyer, Meenakshi and Shastri, Jayanthi S and Dias, Mary and Gadepalli, Ravisekhar and Aggarwal, Alisha and Vedpathak, Manoj and Agrawal, Sachee and Pandit, Awadhesh and Nisheetha, Amul and Kumar, Anuj and Bordoloi, Mahasweta and Shafi, Mohamed and Shelar, Bhagyashree and Balachandra, Swathi S and Damodar, Tina and Masika, Moses Muia and Mwaura, Patrick and Anzala, Omu and Muthumani, Kar and Sowdhamini, Ramanathan and Medigeshi, Guruprasad R and Roy, Rahul and Pattabiraman, Chitra and Krishna, Sudhir and Sreekumar, Easwaran} } @article {4451, title = {SARS-CoV-2 infection of human-induced pluripotent stem cells-derived lung lineage cells evokes inflammatory and chemosensory responses by targeting mitochondrial pathways [Eyestem Research Pvt. Ltd., a C-CAMP Startup]}, journal = {J Cell Physiol}, year = {2022}, month = {2022 Apr 23}, abstract = {

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

}, issn = {1097-4652}, doi = {10.1002/jcp.30755}, author = {Surendran, Harshini and Kumar, Saurabh and Narasimhaiah, Swathi and Ananthamurthy, Anuradha and Varghese, P S and D{\textquoteright}Souza, George A and Medigeshi, Guruprasad and Pal, Rajarshi} } @article {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 {1867, title = {Downregulation of CRX, a Group 3-specific oncogenic transcription factor, inhibits TGF-β activin signaling in medulloblastoma cells [Genomics Facility]}, journal = {Biochemical and Biophysical Research Communications}, volume = {568}, year = {2021}, month = {06/2021}, pages = {76{\textendash}82}, type = {Journal Article}, chapter = {76}, abstract = {

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

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

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

}, doi = {://doi.org/10.1007/s11051-021-05251-z}, url = {https://link.springer.com/article/10.1007/s13204-021-01914-4}, author = {Bindu Y. Srinivasu and Arun Arumugaperumal and Amrita Mitra and Monita Muralidharan and Rajdeep Das and Amit Kumar Mandal} } @article {1854, title = {Interventional Strategies for Parkinson Disease: Can Neural Precursor Cells Forge a Path Ahead? [Eyestem Research, a C-CAMP startup]}, journal = {ACS Chemical Neuroscience}, year = {2021}, month = {10/2021}, type = {Journal Article}, abstract = {

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

}, keywords = {cell transplantation, induced pluripotent stem cells, neural precursor cell, neural stem cell., Parkinson disease}, doi = {https://doi.org/10.1021/acschemneuro.1c00525}, url = {https://pubs.acs.org/doi/abs/10.1021/acschemneuro.1c00525}, author = {Nandakumar, Swapna and Shahani, Pradnya and Datta, Indrani and Pal, Rajarshi} } @article {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 {1743, title = {Xeno-Free Human Wharton{\textquoteright}s Jelly Mesenchymal Stromal Cells Maintain Their Characteristic Properties after Long-Term Cryopreservation [OCT Therapies \& Research, a C-CAMP Startup] }, journal = {Cell Journal (Yakhteh)}, volume = {23}, year = {2021}, month = {07/2021}, chapter = {145}, abstract = {

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

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

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

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

}, keywords = {Mesenchymal Stromal Cells, Stability, Umbilical Cord, Wharton{\textquoteright}s Jelly}, doi = {10.22074/cellj.2021.7131}, author = {Mathen, Caroline and D{\textquoteright}souza, Wilfrid} } @article {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 {1554, title = {Pitchers of Nepenthes khasiana express several digestive-enzyme encoding genes, harbor mostly fungi and probably evolved through changes in the expression of leaf polarity genes [Next Gen Genomics Facility]}, journal = {BMC Plant Biol}, volume = {20}, year = {2020}, month = {2020 Nov 17}, pages = {524}, abstract = {

BACKGROUND: A structural phenomenon seen in certain lineages of angiosperms that has captivated many scholars including Charles Darwin is the evolution of plant carnivory. Evidently, these structural features collectively termed carnivorous syndrome, evolved to aid nutritional acquisition from attracted, captured and digested prey. We now understand why plant carnivory evolved but how carnivorous plants acquired these attributes remains a mystery. In an attempt to understand the evolution of Nepenthes pitcher and to shed more light on its role in prey digestion, we analyzed the transcriptome data of the highly specialized Nepenthes khasiana leaf comprising the leaf base lamina, tendril and the different parts/zones of the pitcher tube viz. digestive zone, waxy zone and lid.

RESULTS: In total, we generated around 262 million high-quality Illumina reads. Reads were pooled, normalized and de novo assembled to generate a reference transcriptome of about 412,224 transcripts. We then estimated transcript abundance along the N. khasiana leaf by mapping individual reads from each part/zone to the reference transcriptome. Correlation-based hierarchical clustering analysis of 27,208 commonly expressed genes indicated functional relationship and similar cellular processes underlying the development of the leaf base and the pitcher, thereby implying that the Nepenthes pitcher is indeed a modified leaf. From a list of 2386 differentially expressed genes (DEGs), we identified transcripts encoding key enzymes involved in prey digestion and protection against pathogen attack, some of which are expressed at high levels in the digestive zone. Interestingly, many of these enzyme-encoding genes are also expressed in the unopened N. khasiana pitcher. Transcripts showing homology to both bacteria and fungi were also detected; and in the digestive zone, fungi are more predominant as compared to bacteria. Taking cues from histology and scanning electron microscopy (SEM) photomicrographs, we found altered expressions of key regulatory genes involved in leaf development. Of particular interest, the expression of class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPIII) and ARGONAUTE (AGO) genes were upregulated in the tendril.

CONCLUSIONS: Our findings suggest that N. khasiana pitchers employ a wide range of enzymes for prey digestion and plant defense, harbor microbes and probably evolved through altered expression of leaf polarity genes.

}, issn = {1471-2229}, doi = {10.1186/s12870-020-02663-2}, author = {Dkhar, Jeremy and Bhaskar, Yogendra Kumar and Lynn, Andrew and Pareek, Ashwani} } @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 {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 {1136, title = {Mon1 constitutes a novel node in the brain-gonad axis that is essential for female germline maturation. [Transgenic Fly Facility]}, journal = {Development}, volume = {146}, year = {2019}, month = {2019 Jul 10}, abstract = {

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

}, issn = {1477-9129}, doi = {10.1242/dev.166504}, author = {Dhiman, Neena and Shweta, Kumari and Tendulkar, Shweta and Deshpande, Girish and Ratnaparkhi, Girish S and Ratnaparkhi, Anuradha} } @article {1025, title = {Myosin heavy chain mutations that cause Freeman-Sheldon syndrome lead to muscle structural and functional defects in Drosophila. [Transgenic Fly Facility]}, journal = {Dev Biol}, volume = {449}, year = {2019}, month = {2019 May 15}, chapter = {90}, abstract = {

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

}, issn = {1095-564X}, doi = {10.1016/j.ydbio.2019.02.017}, author = {Das, Shreyasi and Kumar, Pankaj and Verma, Aakanksha and Maiti, Tushar K and Mathew, Sam J} } @article {1178, title = {Serotonin is essential for eye regeneration in planaria Schmidtea~mediterranea [Mass Spectrometry (Metabolomics) and Central Imaging \& Flow Cytometry Facilities (INT)]}, journal = {FEBS Lett}, year = {2019}, month = {2019 Sep 17}, abstract = {

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

}, issn = {1873-3468}, doi = {10.1002/1873-3468.13607}, author = {Sarkar, Arunabha and Mukundan, Namita and Sowndarya, Sai and Dubey, Vinay Kumar and Babu, Rosana and Lakshmanan, Vairavan and Rangiah, Kannan and Panicker, Mitradas M and Palakodeti, Dasaradhi and Subramanian, Sabarinath Peruvemba and Subramanian, Ramaswamy} } @article {1202, title = {Small molecule modulator of aggrephagy regulates neuroinflammation to curb pathogenesis of neurodegeneration [Discovery to Innovation Accelerator (INT)].}, journal = {EBioMedicine}, year = {2019}, month = {2019 Nov 11}, abstract = {

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

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

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

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

}, issn = {2352-3964}, doi = {10.1016/j.ebiom.2019.10.036}, author = {Sn, Suresh and Pandurangi, Janhavi and Murumalla, Ravi and Dj, Vidyadhara and Garimella, Lakshmi and Acharya, Achyuth and Rai, Shashank and Paul, Abhik and Yarreiphang, Haorei and Pillai, Malini S and Giridharan, Mridhula and Clement, James P and Alladi, Phalguni Anand and Saiyed, Taslimarif and Manjithaya, Ravi} } @article {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 {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 {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 {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 {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 {824, title = {A proteomic approach of biomarker candidate discovery for alcoholic liver cirrhosis [Mass Spectrometry - Proteomics Facility].}, journal = {J Circ Biomark}, volume = {7}, year = {2018}, month = {2018 Jan-Dec}, pages = {1849454418788417}, abstract = {

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

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

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

}, issn = {2169-8287}, doi = {10.1128/genomeA.00511-18}, author = {Dash, Paban Kumar and Pattabiraman, Chitra and Tandel, Kundan and Sharma, Shashi and Kumar, Jyoti S and Siddappa, Shilpa and Gowda, Malali and Krishna, Sudhir and Parida, Manmohan} } @article {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 {768, title = {Species-specific and differential expression of BSP-5 and other BSP variants in normozoospermic and asthenozoospermic buffalo (Bubalus bubalis) and cattle (Bos taurus) seminal plasma. [Mass Spectrometry Facility - Proteomics]}, journal = {Theriogenology}, volume = {106}, year = {2018}, month = {2018 Jan 15}, pages = {279-286}, abstract = {

Binder of sperm-5 (BSP-5) is one of the fertility-associated proteins of cattle seminal plasma. Binding of sperm to the oviductal epithelium is mediated by BSP group of proteins. However, it is not clear, whether this protein is also involved in sperm motility. In the present study, attempts were made to characterize BSP-5 protein in both normozoospermic (NS) and asthenozoospermic (AS) Murrah buffalo (n~=~18; Bubalus bubalis), Holstein Friesian (n~=~8, Bos taurus) and Jersey cattle (n~=~8; Bos taurus) bull seminal plasma and also study its expression pattern in these species. 1-D Western blot demonstrated three major BSP-5 immunoreactive protein bands (24.2~kDa, 20.5~kDa, and 12.3~kDa) in buffalo seminal plasma. Of these, the intensities of 24.2 and 20.5~kDa protein bands reduced significantly (P~<=~0.05) in seminal plasma of AS group compared to that of NS group. On the contrary, the expression of 12.3~kDa protein band did not vary significantly between the groups. In Holstein Friesian seminal plasma, at least six BSP-5 immunoreactive protein bands (25.1, 23.6, 19.5, 13.8, 13.1 and 12.3~kDa) could be detected. Of these, the intensities of 23.6, 13.8/13.1 and 12.3~kDa protein bands decreased (P~=~0.058, 0.111, 0.053) in AS group bulls compared to NS bulls. Holstein Friesian bull seminal plasma demonstrated a BSP-5 immunoreactive duplex protein band of 13.8/13.1~kDa, which was not evident in buffalo seminal plasma. In 2-D Western blot, a train of five BSP-5 immunoreactive duplex protein spots (Mr 21.0-27.6~kDa, pI of \~{}3.9-5.1) was detected. Mass spectrometry of one of the representative duplex spot confirmed that these were BSP-5 and BSP-3 proteins, respectively. Indirect immunofluorescence studies showed that BSP-5 is primarily localized to the mid-piece/mitochondrial region of buffalo spermatozoa. To conclude, the findings of the present study could establish the significance and association of BSP-5 proteins in sperm motility and how their level differ in semen from two different clinical groups of buffalo bull (NS vs. AS). Further, the study also demonstrated that the expression pattern of BSP-5 and other BSP variants in seminal plasma of bulls is species-specific.

}, issn = {1879-3231}, doi = {10.1016/j.theriogenology.2017.10.014}, author = {Divyashree, B C and Roy, Sudhir C} } @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 {524, title = {Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii [Bugworks Res. Pvt. Ltd., a C-CAMP Startup]}, journal = {PLoS One}, volume = {12}, year = {2017}, month = {2017}, pages = {e0170202}, abstract = {

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

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0170202}, author = {Ghatak, Arindam and Bharatham, Nagakumar and Shanbhag, Anirudh P and Datta, Santanu and Venkatraman, Janani} } @article {471, title = {Gut microbial degradation of organophosphate insecticides-induces glucose intolerance via gluconeogenesis. [Next Generation Genomics facility]}, journal = {Genome Biol}, volume = {18}, year = {2017}, month = {2017 Jan 24}, pages = {8}, abstract = {

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

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

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

}, issn = {1474-760X}, doi = {10.1186/s13059-016-1134-6}, author = {Velmurugan, Ganesan and Ramprasath, Tharmarajan and Swaminathan, Krishnan and Mithieux, Gilles and Rajendhran, Jeyaprakash and Dhivakar, Mani and Parthasarathy, Ayothi and Babu, D D Venkatesh and Thumburaj, Leishman John and Freddy, Allen J and Dinakaran, Vasudevan and Puhari, Shanavas Syed Mohamed and Rekha, Balakrishnan and Christy, Yacob Jenifer and Anusha, Sivakumar and Divya, Ganesan and Suganya, Kannan and Meganathan, Boominathan and Kalyanaraman, Narayanan and Vasudevan, Varadaraj and Kamaraj, Raju and Karthik, Maruthan and Jeyakumar, Balakrishnan and Abhishek, Albert and Paul, Eldho and Pushpanathan, Muthuirulan and Rajmohan, Rajamani Koushick and Velayutham, Kumaravel and Lyon, Alexander R and Ramasamy, Subbiah} } @article {645, title = {Performance of a docking/molecular dynamics protocol for virtual screening of nutlin-class inhibitors of Mdmx.}, journal = {J Mol Graph Model}, volume = {74}, year = {2017}, month = {2017 Jun}, pages = {54-60}, abstract = {

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

}, issn = {1873-4243}, doi = {10.1016/j.jmgm.2017.02.014}, author = {Bharatham, Nagakumar and Finch, Kristin E and Min, Jaeki and Mayasundari, Anand and Dyer, Michael A and Guy, R Kiplin and Bashford, Donald} } @article {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 {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 {8890, title = {Transcriptomic resources for the medicinal legume Mucuna pruriens: de novo transcriptome assembly, annotation, identification and validation of EST-SSR markers [Next Gen Genomics Facility]}, journal = {BMC Genomics}, volume = {18}, year = {2017}, month = {2017 May 25}, pages = {409}, abstract = {

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

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

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

}, keywords = {Data Mining, Expressed Sequence Tags, Gene Expression Profiling, Microsatellite Repeats, Molecular Sequence Annotation, Mucuna, Polymorphism, Genetic, RNA, Messenger, Transcription Factors}, issn = {1471-2164}, doi = {10.1186/s12864-017-3780-9}, author = {Sathyanarayana, N and Pittala, Ranjith Kumar and Tripathi, Pankaj Kumar and Chopra, Ratan and Singh, Heikham Russiachand and Belamkar, Vikas and Bhardwaj, Pardeep Kumar and Doyle, Jeff J and Egan, Ashley N} } @article {706, title = {Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes.}, journal = {Cell}, volume = {164}, year = {2016}, month = {2016 Feb 11}, pages = {722-34}, abstract = {

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

}, keywords = {Animals, Biological Transport, Cell Line, Dictyostelium, Dyneins, Glycosphingolipids, Leishmania donovani, Lysosomes, Membrane Microdomains, Mice, Phagosomes}, issn = {1097-4172}, doi = {10.1016/j.cell.2015.12.054}, author = {Rai, Ashim and Pathak, Divya and Thakur, Shreyasi and Singh, Shampa and Dubey, Alok Kumar and Mallik, Roop} } @article {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 {476, title = {The E3 ligase ube3a is required for learning in Drosophila melanogaster. [Drosophila facility]}, journal = {Biochem Biophys Res Commun}, volume = {462}, year = {2015}, month = {2015 Jun 19}, pages = {71-7}, abstract = {

Angelman syndrome and autism are neurodevelopmental disorders linked to mutations and duplications of an E3 ligase called ube3a respectively. Since cognitive deficits and learning disabilities are hallmark symptoms of both these disorders, we investigated a role for dube3a in the learning ability of flies using the aversive phototaxis suppression assay. We show that down and up-regulation of dube3a are both detrimental to learning in larvae and adults. Using conditional gene expression we found that dube3a is required for normal brain development and during adulthood. Furthermore, we suggest that dube3a could be interacting with other learning and memory genes such as derailed. Along with firmly establishing dube3a as a gene that is required for learning, our work also opens avenues for further understanding the role played by this gene in brain development and behavior.

}, keywords = {Animals, Animals, Genetically Modified, Brain, Drosophila melanogaster, Drosophila Proteins, Gene Expression Regulation, Developmental, Immunoblotting, Larva, Learning, Memory, Motor Activity, Mushroom Bodies, Mutation, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Ubiquitin-Protein Ligases}, issn = {1090-2104}, doi = {10.1016/j.bbrc.2015.04.110}, author = {Chakraborty, Moumita and Paul, Blesson K and Nayak, Tanmoyita and Das, Aniruddha and Jana, Nihar R and Bhutani, Supriya} } @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 {516, title = {A new C-type lectin (RVsnaclec) purified from venom of Daboia russelii russelii shows anticoagulant activity via inhibition of FXa and concentration-dependent differential response to platelets in a Ca{\texttwosuperior}$^{+}$-independent manner. (Mass spectrometry)}, journal = {Thromb Res}, volume = {134}, year = {2014}, month = {2014 Nov}, pages = {1150-6}, abstract = {

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

}, keywords = {Animals, Anticoagulants, Blood Coagulation, Blood Platelets, Calcium, Factor Xa, Factor Xa Inhibitors, Goats, Humans, Lectins, C-Type, Mice, Platelet Aggregation, Russell{\textquoteright}s Viper, Viper Venoms}, issn = {1879-2472}, doi = {10.1016/j.thromres.2014.09.009}, author = {Mukherjee, Ashis K and Dutta, Sumita and Mackessy, Stephen P} } @article {535, title = {Unique, polyfucosylated glycan-receptor interactions are essential for regeneration of Hydra magnipapillata.}, journal = {ACS Chem Biol}, volume = {9}, year = {2014}, month = {2014 Jan 17}, pages = {147-55}, abstract = {

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

}, keywords = {Animals, Carbohydrate Sequence, Hydra, Lectins, C-Type, Molecular Sequence Data, Polysaccharides, Regeneration}, issn = {1554-8937}, doi = {10.1021/cb400486t}, author = {Sahadevan, Sonu and Antonopoulos, Aristotelis and Haslam, Stuart M and Dell, Anne and Ramaswamy, Subramanian and Babu, Ponnusamy} } @article {518, title = {Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties. (Mass spectrometry - Proteomics)}, journal = {Sci Rep}, volume = {4}, year = {2014}, month = {2014 Dec 08}, pages = {7357}, abstract = {

Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3β and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation.

}, keywords = {Animals, Chick Embryo, Exosomes, Extracellular Space, Hedgehog Proteins, HEK293 Cells, Humans, MicroRNAs, Models, Biological, Protein Transport, Signal Transduction, Vertebrates}, issn = {2045-2322}, doi = {10.1038/srep07357}, author = {Vyas, Neha and Walvekar, Ankita and Tate, Dhananjay and Lakshmanan, Vairavan and Bansal, Dhiru and Lo Cicero, Alessandra and Raposo, Graca and Palakodeti, Dasaradhi and Dhawan, Jyotsna} } @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 {481, title = {Dysregulation of core components of SCF complex in poly-glutamine disorders. [Drosophila facility]}, journal = {Cell Death Dis}, volume = {3}, year = {2012}, month = {2012 Nov 22}, pages = {e428}, abstract = {

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

}, keywords = {Animals, Cullin Proteins, Drosophila, Female, Humans, Huntington Disease, Machado-Joseph Disease, Male, Mice, Mice, Transgenic, Peptides, SKP Cullin F-Box Protein Ligases}, issn = {2041-4889}, doi = {10.1038/cddis.2012.166}, author = {Bhutani, S and Das, A and Maheshwari, M and Lakhotia, S C and Jana, N R} } @article {723, title = {Germ cell abnormalities in streptozotocin induced diabetic mice do not correlate with blood glucose level.}, journal = {J Assist Reprod Genet}, volume = {29}, year = {2012}, month = {2012 Dec}, pages = {1405-13}, abstract = {

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

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

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

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

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

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

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

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

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

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

}, keywords = {Adult, Blood Platelets, DNA, DNA, Mitochondrial, Free Radicals, Humans, Inflammation, Interleukin-1beta, Interleukin-8, Mitochondria, NADPH Oxidases, Neutrophil Activation, Neutrophils, Nitric Oxide, Peroxidase, Tumor Necrosis Factor-alpha}, issn = {1552-4930}, doi = {10.1002/cyto.a.21178}, author = {Keshari, Ravi S and Jyoti, Anupam and Kumar, Sachin and Dubey, Megha and Verma, Anupam and Srinag, Bangalore S and Krishnamurthy, Hanumanthappa and Barthwal, Manoj K and Dikshit, Madhu} }