@article {6592, title = {Cryo-EM research in India.}, journal = {Structure}, volume = {32}, year = {2024}, month = {2024 Feb 01}, pages = {113-119}, abstract = {

To celebrate the 50th anniversary of Cell Press and the Cell special issue focusing on structural biology, we want to highlight the rapid progress of cryo-EM related research in India in this collection of Voices. We have asked structural biologists to introduce their research and the national cryo-EM facilities throughout the country.

}, keywords = {Cryoelectron Microscopy, India}, issn = {1878-4186}, doi = {10.1016/j.str.2024.01.002}, author = {Shukla, Arun K and Banerjee, Manidipa and Singh, Appu K and Penmatsa, Aravind and Dutta, Somnath and Anand, Ruchi and Sirajuddin, Minhajuddin and Srivastava, Shanti Swaroop} } @article {2466, title = {The CCR5 Gene Edited CD34+CD90+ Hematopoietic Stem Cell Population Serves as an Optimal Graft Source for HIV Gene Therapy}, journal = {Front. Immunol}, year = {2022}, abstract = {

Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90\% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.

}, doi = {https://doi.org/10.3389/fimmu.2022.792684}, author = {Karthik V. Karuppusamy and John Paul Demosthenes and Vigneshwaran Venkatesan and Abisha Crystal Christopher and Prathibha Babu and Manojkumar K. Azhagiri and Annlin Jacob and Veena Vadhini Ramalingam and Sumathi Rangaraj and Mohankumar Kumarasamypet Murugesan and Srujan Marepally and George Varghese and Alok Srivastava and Rajesh Kannangai and Saravanabhavan Thangavel} } @article {3340, title = {CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications.}, journal = {J Vis Exp}, year = {2022}, month = {2022 08 09}, abstract = {

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

}, keywords = {Animals, CRISPR-Cas Systems, Gene Editing, Genetic Therapy, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Mice}, issn = {1940-087X}, doi = {10.3791/64064}, author = {Venkatesan, Vigneshwaran and Christopher, Abisha Crystal and Karuppusamy, Karthik V and Babu, Prathibha and Alagiri, Manoj Kumar K and Thangavel, Saravanabhavan} } @article {2272, title = {Chromatin remodelling complexes in cerebral cortex development and neurodevelopmental disorders.}, journal = {Neurochem Int}, volume = {147}, year = {2021}, month = {2021 Jul}, pages = {105055}, abstract = {

The diverse number of neurons in the cerebral cortex are generated during development by neural stem cells lining the ventricle, and they continue maturing postnatally. Dynamic chromatin regulation in these neural stem cells is a fundamental determinant of the emerging property of the functional neural network, and the chromatin remodellers are critical determinants of this process. Chromatin remodellers participate in several steps of this process from proliferation, differentiation, migration leading to complex network formation which forms the basis of higher-order functions of cognition and behaviour. Here we review the role of these ATP-dependent chromatin remodellers in cortical development in health and disease and highlight several key mouse mutants of the subunits of the complexes which have revealed how the remodelling mechanisms control the cortical stem cell chromatin landscape for expression of stage-specific transcripts. Consistent with their role in cortical development, several putative risk variants in the subunits of the remodelling complexes have been identified as the underlying causes of several neurodevelopmental disorders. A basic understanding of the detailed molecular mechanism of their action is key to understating how mutations in the same networks lead to disease pathologies and perhaps pave the way for therapeutic development for these complex multifactorial disorders.

}, issn = {1872-9754}, doi = {10.1016/j.neuint.2021.105055}, author = {D{\textquoteright}Souza, Leora and Channakkar, Asha S and Muralidharan, Bhavana} } @article {2207, title = {Comprehensive annotation and characterization of planarian tRNA and tRNA-derived fragments (tRFs).}, journal = {RNA}, year = {2021}, month = {2021 Jan 14}, abstract = {

tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological process such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration suggesting a possible role for these small RNAs in the regulation of regeneration. In order to characterise planarian tRFs, we first annotated 457 tRNAs in S.mediterranea combining two tRNA prediction algorithms. Annotation of tRNAs facilitated the identification of three main species of tRFs in planarians - the shorter tRF-5s and itRFs, and the abundantly expressed 5{\textquoteright}-tsRNAs. Spatial profiling of tRFs in sequential transverse sections of planarians revealed diverse expression patterns of these small RNAs, including those that are enriched in the head and pharyngeal regions. Expression analysis of these tRF species revealed dynamic expression of these small RNAs over the course of regeneration suggesting an important role in planarian anterior and posterior regeneration. Finally, we show that 5{\textquoteright}-tsRNA in planaria interact with all three SMEDWI proteins and an involvement of Ago1 in the processing of itRFs. In summary, our findings implicate a novel role for tRFs in planarian regeneration, highlighting their importance in regulating complex systemic processes. Our study adds to the catalogue of post-transcriptional regulatory systems in planarian, providing valuable insights on the biogenesis and the function of tRFs in neoblasts and planarian regeneration.

}, issn = {1469-9001}, doi = {10.1261/rna.077701.120}, author = {Lakshmanan, Vairavan and T N, Sujith and Bansal, Dhiru and Padubidri, Shivaprasad V and Palakodeti, Dasaradhi and Krishna, Srikar} } @article {2293, title = {Contribution of NMDA Receptors to Synaptic Function in Rat Hippocampal Interneurons.}, journal = {eNeuro}, volume = {8}, year = {2021}, month = {2021 Jul-Aug}, abstract = {

The ability of neurons to produce behaviorally relevant activity in the absence of pathology relies on the fine balance of synaptic inhibition to excitation. In the hippocampal CA1 microcircuit, this balance is maintained by a diverse population of inhibitory interneurons that receive largely similar glutamatergic afferents as their target pyramidal cells, with EPSCs generated by both AMPA receptors (AMPARs) and NMDA receptors (NMDARs). In this study, we take advantage of a recently generated GluN2A-null rat model to assess the contribution of GluN2A subunits to glutamatergic synaptic currents in three subclasses of interneuron found in the CA1 region of the hippocampus. For both parvalbumin-positive and somatostatin-positive interneurons, the GluN2A subunit is expressed at glutamatergic synapses and contributes to the EPSC. In contrast, in cholecystokinin (CCK)-positive interneurons, the contribution of GluN2A to the EPSC is negligible. Furthermore, synaptic potentiation at glutamatergic synapses on CCK-positive interneurons does not require the activation of GluN2A-containing NMDARs but does rely on the activation of NMDARs containing GluN2B and GluN2D subunits.

}, issn = {2373-2822}, doi = {10.1523/ENEURO.0552-20.2021}, author = {Booker, Sam A and Sumera, Anna and Kind, Peter C and Wyllie, David J A} } @article {2326, title = {Correction of amygdalar dysfunction in a rat model of fragile X syndrome.}, journal = {Cell Rep}, volume = {37}, year = {2021}, month = {2021 Oct 12}, pages = {109805}, abstract = {

Fragile X syndrome (FXS), a commonly inherited form of autism and intellectual disability, is associated with emotional symptoms that implicate dysfunction of the amygdala. However, current understanding of the pathogenesis of the disease is based primarily on studies in the hippocampus and neocortex, where FXS defects have been corrected by inhibiting group I metabotropic glutamate receptors (mGluRs). Here, we observe that activation, rather than inhibition, of mGluRs in the basolateral amygdala reverses impairments in a rat model of FXS. FXS rats exhibit deficient recall of auditory conditioned fear, which is accompanied by a range of in\ vitro and in\ vivo deficits in synaptic transmission and plasticity. We find presynaptic mGluR5 in the amygdala, activation of which reverses deficient synaptic transmission and plasticity, thereby restoring normal fear learning in FXS rats. This highlights the importance of modifying the prevailing mGluR-based framework for therapeutic strategies to include circuit-specific differences in FXS pathophysiology.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.109805}, author = {Fernandes, Giselle and Mishra, Pradeep K and Nawaz, Mohammad Sarfaraz and Donlin-Asp, Paul G and Rahman, Mohammed Mostafizur and Hazra, Anupam and Kedia, Sonal and Kayenaat, Aiman and Songara, Dheeraj and Wyllie, David J A and Schuman, Erin M and Kind, Peter C and Chattarji, Sumantra} } @article {2292, title = {Cross-diagnostic evaluation of minor physical anomalies in psychiatric disorders.}, journal = {J Psychiatr Res}, volume = {142}, year = {2021}, month = {2021 Jul 20}, pages = {54-62}, abstract = {

BACKGROUND: Minor physical anomalies (MPA) are markers of impaired neurodevelopment during the prenatal stage. Assessing MPA across psychiatric disorders may help understand their shared nature. In addition, MPA in family members would indicate a shared liability and endophenotype potential. We examined familial aggregation of MPA and their role as transdiagnostic and disorder-specific markers of 5 major psychiatric/neuropsychiatric conditions (schizophrenia, bipolar disorder, substance dependence, obsessive-compulsive disorder, and Alzheimer{\textquoteright}s dementia).

METHODS: Modified Waldrop{\textquoteright}s MPA scale was applied on 1321 individuals from 439 transdiagnostic multiplex families and 125 healthy population controls (HC). Stage of fetal development (morphogenetic/phenogenetic)- and anatomical location (craniofacial/peripheral)-based sub-scores were calculated. Familiality and endophenotypic potential of MPA were analyzed with serial negative binomial mixed-effect regression. Cross-diagnostic differences and the effect of family history density (FHD) of each diagnosis on MPA were assessed. Mixed-effects Cox models estimated the influence of MPA on age-at-onset of illness (AAO).

RESULTS: MPA were found to be heritable in families with psychiatric disorders, with a familiality of 0.52. MPA were higher in psychotic disorders after controlling for effects of sex and intrafamilial correlation. Morphogenetic variant MPA was noted to be lower in dementia in comparison to HC. FHD of schizophrenia and bipolar disorder predicted higher, and that of dementia and substance dependence predicted lower MPA. MPA brought forward the AAO [HR:1.07 (1.03-1.11)], and this was more apparent in psychotic disorders.

CONCLUSION: MPA are transmissible in families, are specifically related to the risk of developing psychoses, and predict an earlier age at onset. Neurodevelopmentally informed classification of MPA has the potential to enhance the etiopathogenic and translational understanding of psychiatric disorders.

}, issn = {1879-1379}, doi = {10.1016/j.jpsychires.2021.07.028}, author = {Sreeraj, Vanteemar S and Puzhakkal, Joan C and Holla, Bharath and Nadella, Ravi Kumar and Sheth, Sweta and Balachander, Srinivas and Ithal, Dhruva and Ali, Furkhan and Viswanath, Biju and Muralidharan, Kesavan and Venkatasubramanian, Ganesan and John, John P and Benegal, Vivek and Murthy, Pratima and Varghese, Mathew and Reddy, Yc Janardhan and Jain, Sanjeev} } @article {2241, title = {Cycles, sources, and sinks: Conceptualizing how phosphate balance modulates carbon flux using yeast metabolic networks.}, journal = {Elife}, volume = {10}, year = {2021}, month = {2021 Feb 05}, abstract = {

Phosphates are ubiquitous molecules that enable critical intracellular biochemical reactions. Therefore, cells have elaborate responses to phosphate limitation. Our understanding of long-term transcriptional responses to phosphate limitation is extensive. Contrastingly, a systems-level perspective presenting unifying biochemical concepts to interpret how phosphate balance is critically coupled to (and controls) metabolic information flow is missing. To conceptualize such processes, utilizing yeast metabolic networks we categorize phosphates utilized in metabolism into cycles, sources and sinks. Through this, we identify metabolic reactions leading to putative phosphate sources or sinks. With this conceptualization, we illustrate how mass action driven flux towards sources and sinks enable cells to manage phosphate availability during transient/immediate phosphate limitations. We thereby identify how intracellular phosphate availability will predictably alter specific nodes in carbon metabolism, and determine signature cellular metabolic states. Finally, we identify a need to understand intracellular phosphate pools, in order to address mechanisms of phosphate regulation and restoration.

}, issn = {2050-084X}, doi = {10.7554/eLife.63341}, author = {Gupta, Ritu and Laxman, Sunil} } @article {2217, title = {Comparison of CryoEM and X-ray structures of dimethylformamidase.}, journal = {Prog Biophys Mol Biol}, year = {2020}, month = {2020 Jul 28}, abstract = {

Dimethylformamidase (DMFase) catalyzes the hydrolysis of dimethylformamide, an industrial solvent, introduced into the environment by humans. Recently, we determined the structures of dimethylformamidase by electron cryo microscopy and X-ray crystallography revealing a tetrameric enzyme with a mononuclear iron at the active site. DMFase from Paracoccus sp. isolated from a waste water treatment plant around the city of Kanpur in India shows maximal activity at 54\ {\textdegree}C and is halotolerant. The structures determined by both techniques are mostly identical and the largest difference is in a loop near the active site. This loop could play a role in co-operativity between the monomers. A number of non-protein densities are observed in the EM map, which are modelled as water molecules. Comparison of the structures determined by the two methods reveals conserved water molecules that could play a structural role. The higher stability, unusual active site and negligible activity at low temperature makes this a very good model to study enzyme mechanism by cryoEM.

}, issn = {1873-1732}, doi = {10.1016/j.pbiomolbio.2020.06.008}, author = {Vinothkumar, Kutti R and Arya, Chetan Kumar and Ramanathan, Gurunath and Subramanian, Ramaswamy} } @article {2065, title = {Convolvulus pluricaulis extract can modulate synaptic plasticity in rat brain hippocampus.}, journal = {Neuroreport}, volume = {31}, year = {2020}, month = {2020 May 22}, pages = {597-604}, abstract = {

The memory-boosting property of Indian traditional herb, Convolvulus pluricaulis, has been documented in literature; however, its effect on synaptic plasticity has not yet been reported. Two important forms of synaptic plasticity known to be involved in the processes of memory formation are long-term potentiation (LTP) and long-term depression (LTD). In the present study, the effect of C. pluricaulis plant extract on LTP and LTD were evaluated. The adult male Wistar rats were fed orally with 250, 500 and 1000 mg/kg of this extract for 4 weeks and the effect was determined on LTP and LTD in the Schaffer collaterals of the hippocampal cornu ammonis region CA1. We found that the 500 mg/kg dose of the extract could significantly enhance LTP compared to the vehicle treated ones. Moreover, the same dose could also reduce LTD while used in a separate set of animals. Also, a fresh group of animals treated with the effective dose (500 mg/kg) of plant extract were examined for memory retention in two behavioral platforms namely, contextual fear conditioning (CFC) and novel object recognition test (NORT). Increased fear response to the conditioned stimulus and enhanced recognition of objects were observed in CFC and NORT, respectively, both indicating strengthening of memory. Following up, ex-vivo electrophysiology experiments were performed with the active single molecule scopoletin, present in C. pluricaulis extract and similar patterns in synaptic plasticity changes were obtained. These findings suggest that prolonged treatment of C. pluricaulis extract, at a specific dose in healthy animals, can augment memory functions by modulating hippocampal plasticity.

}, issn = {1473-558X}, doi = {10.1097/WNR.0000000000001446}, author = {Das, Rishi and Sengupta, Tathagata and Roy, Shubhrajit and Chattarji, Sumantra and Ray, Jharna} } @article {2110, title = {Cortical neurons derived from human pluripotent stem cells lacking FMRP display altered spontaneous firing patterns.}, journal = {Mol Autism}, volume = {11}, year = {2020}, month = {2020 Jun 19}, pages = {52}, abstract = {

BACKGROUND: Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP).

METHODS: Electrophysiological whole-cell voltage- and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced.

RESULTS: Neurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca-activated K currents and the persistent Na current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines.

CONCLUSIONS: Pharmacological manipulations can alter the action potential burst profiles in both control and FMRP-null human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered.

}, issn = {2040-2392}, doi = {10.1186/s13229-020-00351-4}, author = {Das Sharma, Shreya and Pal, Rakhi and Reddy, Bharath Kumar and Selvaraj, Bhuvaneish T and Raj, Nisha and Samaga, Krishna Kumar and Srinivasan, Durga J and Ornelas, Loren and Sareen, Dhruv and Livesey, Matthew R and Bassell, Gary J and Svendsen, Clive N and Kind, Peter C and Chandran, Siddharthan and Chattarji, Sumantra and Wyllie, David J A} } @article {2112, title = {Corticosterone after acute stress prevents the delayed effects on the amygdala.}, journal = {Neuropsychopharmacology}, year = {2020}, month = {2020 Jul 06}, abstract = {

Even a single 2-hour episode of immobilization stress is known to trigger anxiety-like behavior and increase spine-density in the basolateral amygdala (BLA) of rats 10 days later. This delayed build-up of morphological and behavioral effects offers a stress-free time window of intervention after acute stress, which we used to test a protective role for glucocorticoids against stress. We observed that post-stress corticosterone, given 1 day after acute stress in drinking water, reversed enhanced anxiety-like behavior 10 days later. Quantification of spine-density on Golgi-stained BLA principal neurons showed that the same intervention also prevented the increase in spine numbers in the amygdala, at the same delayed time-point. Further, stress elevated serum corticosterone levels in rats that received vehicle in the drinking water. However, when stress was followed 24 h later by corticosterone in the drinking water, the surge in corticosterone was prevented. Together, these observations suggest that corticosterone, delivered through drinking water even 24 h after acute stress, is capable of reversing the delayed enhancing effects on BLA synaptic connectivity and anxiety-like behavior. Strikingly, although the immobilization-induced surge in corticosterone by itself has delayed detrimental effects on amygdalar structure and function, there exists a window of opportunity even after stress to mitigate its impact with a second surge of exogenously administered corticosterone. This provides a framework in the amygdala for analyzing how the initial physiological and endocrine processes triggered by traumatic stress eventually give rise to debilitating emotional symptoms, as well as the protective effects of glucocorticoids against their development.

}, issn = {1740-634X}, doi = {10.1038/s41386-020-0758-0}, author = {Chakraborty, Prabahan and Datta, Siddhartha and McEwen, Bruce S and Chattarji, Sumantra} } @article {1734, title = {Characterization of new variant human ES line VH9 hESC (INSTEMe001-a): a tool for human stem cell and cancer research.}, journal = {Stem Cell Res}, volume = {37}, year = {2019}, month = {2019 May}, pages = {101444}, abstract = {

Human pluripotent stem cells (hPSCs) acquire changes at the genomic level upon proliferation and differentiation (Peterson and Loring, 2014). Studies from International Stem Cell Initiative and independent laboratories identified a copy number variant (CNV) in hES cell lines displaying a normal karyotype, which provided a selective advantage to hES cells in culture. In our laboratory we have identified variant H9-hESC (derived from H9-hESC) with normal karyotype, pluripotency expression, differentiation profile but with altered traits of high cell survival and low E-CADHERIN expression.

}, issn = {1876-7753}, doi = {10.1016/j.scr.2019.101444}, author = {Arasala, Radhika Rao and Jayaram, Manjunath and Chattai, Jagamohan and Kumarasamy, Thangaraj and Sambasivan, Ramkumar and Rampalli, Shravanti} } @article {1605, title = {Chemical fuel-driven living and transient supramolecular polymerization.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 01 25}, pages = {450}, abstract = {

Temporal control over self-assembly process is a desirable trait in the quest towards adaptable and controllable materials. The ability to devise synthetic ways to control the growth, as well as decay of materials has long been a property which only the biological systems could perform seamlessly. A common synthetic strategy which works on the biological principles such as chemical fuel-driven control over temporal self-assembly profile has not been completely realized synthetically. Here we show, we filled this dearth by showing that a chemical fuel driven self-assembling system can not only be grown in a controlled manner, but it can also result in precise control over the assembly and disassembly kinetics. Herein, we elaborate strategies which clearly show that once a chemical fuel driven self-assembly is established it can be made receptive to multiple molecular cues such that the inherent growth and decay characteristics are programmed into the ensemble.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-08308-9}, author = {Jain, Ankit and Dhiman, Shikha and Dhayani, Ashish and Vemula, Praveen K and George, Subi J} } @article {1737, title = {Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation.}, journal = {Sci Rep}, volume = {9}, year = {2019}, month = {2019 Jun 05}, pages = {8302}, abstract = {

Muscle differentiation is controlled by adhesion and growth factor-dependent signalling through common effectors that regulate muscle-specific transcriptional programs. Here we report that mDiaphanous1, an effector of adhesion-dependent RhoA-signalling, negatively regulates myogenesis at the level of Myogenin expression. In myotubes, over-expression of mDia1ΔN3, a RhoA-independent mutant, suppresses Myogenin promoter activity and expression. We investigated mDia1-interacting proteins that may counteract mDia1 to permit Myogenin expression and timely differentiation. Using yeast two-hybrid and mass-spectrometric analysis, we report that mDia1 has a stage-specific interactome, including Prohibitin2, MyoD, Akt2, and β-Catenin, along with a number of proteosomal and mitochondrial components. Of these interacting partners, Prohibitin2 colocalises with mDia1 in cytoplasmic punctae in myotubes. We mapped the interacting domains of mDia1 and Phb2, and used interacting (mDia1ΔN3/Phb2 FL or mDia1ΔN3/Phb2-Carboxy) and non-interacting pairs (mDia1H + P/Phb2 FL or mDia1ΔN3/Phb2-Amino) to dissect the functional consequences of this partnership on Myogenin promoter activity. Co-expression of full-length as well as mDia1-interacting domains of Prohibitin2 reverse the anti-myogenic effects of mDia1ΔN3, while non-interacting regions do not. Our results suggest that Prohibitin2 sequesters mDia1, dampens its anti-myogenic activity and fine-tunes RhoA-mDia1 signalling to promote differentiation. Overall, we report that mDia1 is multi-functional signalling effector whose anti-myogenic activity is modulated by a differentiation-dependent interactome.\ The data have been deposited to the ProteomeXchange with identifier PXD012257.

}, issn = {2045-2322}, doi = {10.1038/s41598-019-44749-4}, author = {Saleh, Amena and Subramaniam, Gunasekaran and Raychaudhuri, Swasti and Dhawan, Jyotsna} } @article {1209, title = {Cell density overrides the effect of substrate stiffness on human mesenchymal stem cells{\textquoteright} morphology and proliferation.}, journal = {Biomater Sci}, volume = {6}, year = {2018}, month = {2018 May 01}, pages = {1109-1119}, abstract = {

The effect of substrate stiffness on the cellular morphology, proliferation, and differentiation of human mesenchymal stem cells (hMSCs) has been extensively researched and well established. However, the majority of these studies are done with a low seeding density where cell to cell interactions do not play a significant role. While these conditions permit an analysis of cell-substratum interactions at the single cell level, such a model system fails to capture a critical aspect of the cellular micro-environment in vivo, i.e. the cell-cell interaction via matrix deformation (i.e., strain). To address this question, we seeded hMSCs on soft poly-acrylamide (PAA) gels, at a seeding density that permits cells to be mechanically interacting via the underlying substrate. We found that as the intercellular distance decreases with the increasing seeding density, cellular sensitivity towards the substrate rigidity becomes significantly diminished. With the increasing seeding density, the cell spread area increased on a soft substrate (500 Pa) but reduced on an even slightly stiffer substrate (2 kPa) as well as on glass making them indistinguishable at a high seeding density. Not only in terms of cell spread area but also at a high seeding density, cells formed mature focal adhesions and prominent stress fibres on a soft substrate similar to that of the cells being cultured on a stiff substrate. The decreased intercellular distance also influenced the proliferation rate of the cells: higher seeding density on the soft substrate showed cell cycle progression similar to that of the cells on glass substrates. In summary, this paper demonstrates how the effect of substrate rigidity on the cell morphology and fate is a function of inter-cellular distance when seeded on a soft substrate. Our AFM data suggest that such changes happen due to local strain stiffening of the soft PAA gel, an effect that has been rarely reported in the literature so far.

}, issn = {2047-4849}, doi = {10.1039/c7bm00853h}, author = {Venugopal, Balu and Mogha, Pankaj and Dhawan, Jyotsna and Majumder, Abhijit} } @article {1217, title = {Chemically diverse small molecule fluorescent chemosensors for copper ion}, journal = {Coordination Chemistry Reviews}, volume = {357}, year = {2018}, pages = {50 - 104}, keywords = {Chemosensors, Fluorescence, Live cell imaging, Small molecules, Turn-off, Turn-on}, issn = {0010-8545}, doi = {https://doi.org/10.1016/j.ccr.2017.11.020}, url = {http://www.sciencedirect.com/science/article/pii/S0010854517306537}, author = {Gandhi Sivaraman and Murugan Iniya and Thangaraj Anand and Niranjan G. Kotla and Omprakash Sunnapu and Subramanian Singaravadivel and Akash Gulyani and Duraisamy Chellappa} } @article {1597, title = {Crystal structures and kinetics of N-acetylneuraminate lyase from Fusobacterium nucleatum.}, journal = {Acta Crystallogr F Struct Biol Commun}, volume = {74}, year = {2018}, month = {2018 Nov 01}, pages = {725-732}, abstract = {

N-Acetyl-D-neuraminic acid lyase (NanA) catalyzes the breakdown of sialic acid (Neu5Ac) to N-acetyl-D-mannosamine (ManNAc) and pyruvate. NanA plays a key role in Neu5Ac catabolism in many pathogenic and bacterial commensals where sialic acid is available as a carbon and nitrogen source. Several pathogens or commensals decorate their surfaces with sialic acids as a strategy to escape host innate immunity. Catabolism of sialic acid is key to a range of host-pathogen interactions. In this study, atomic resolution structures of NanA from Fusobacterium nucleatum (FnNanA) in ligand-free and ligand-bound forms are reported at 2.32 and 1.76 {\r A} resolution, respectively. F. nucleatum is a Gram-negative pathogen that causes gingival and periodontal diseases in human hosts. Like other bacterial N-acetylneuraminate lyases, FnNanA also shares the triosephosphate isomerase (TIM)-barrel fold. As observed in other homologous enzymes, FnNanA forms a tetramer. In order to characterize the structure-function relationship, the steady-state kinetic parameters of the enzyme are also reported.

}, keywords = {Bacterial Proteins, Crystallography, X-Ray, Fusobacterium nucleatum, Hydrogen Bonding, Models, Molecular, N-Acetylneuraminic Acid, Oxo-Acid-Lyases, Protein Conformation, Protein Folding, Pyruvic Acid, Schiff Bases, Sequence Alignment, Tyrosine}, issn = {2053-230X}, doi = {10.1107/S2053230X18012992}, author = {Kumar, Jay Prakash and Rao, Harshvardhan and Nayak, Vinod and Ramaswamy, S} } @article {1207, title = {Cycling to Meet Fate: Connecting Pluripotency to the Cell Cycle}, journal = {Frontiers in Cell and Developmental Biology}, volume = {6}, year = {2018}, pages = {57}, abstract = {

Pluripotent stem cells are characterised by their high proliferative rates, their ability to self-renew and their potential to differentiate to all the three germ layers. This rapid proliferation is brought about by a highly modified cell cycle that allows the cells to quickly shuttle from DNA synthesis to cell division, by reducing the time spent in the intervening gap phases. Many key regulators that define the somatic cell cycle are either absent or exhibit altered behaviour, allowing the pluripotent cell to bypass cell cycle checkpoints typical of somatic cells. Experimental analysis of this modified stem cell cycle has been challenging due to the strong link between rapid proliferation and pluripotency, since perturbations to the cell cycle or pluripotency factors result in differentiation. Despite these hurdles, our understanding of this unique cell cycle has greatly improved over the past decade, in part because of the availability of new technologies that permit the analysis of single cells in heterogeneous populations. This review aims to highlight some of the recent discoveries in this area with a special emphasis on different states of pluripotency. We also discuss the highly interlinked network that connects pluripotency factors and key cell cycle genes and review evidence for how this interdependency may promote the rapid cell cycle. This issue gains translational importance since disruptions in stem cell proliferation and differentiation can impact disorders at opposite ends of a spectrum, from cancer to degenerative disease.

}, issn = {2296-634X}, doi = {10.3389/fcell.2018.00057}, url = {https://www.frontiersin.org/article/10.3389/fcell.2018.00057}, author = {Zaveri, Lamuk and Dhawan, Jyotsna} } @article {1158, title = {Co-expression of Tbx6 and Sox2 identifies a novel transient neuromesoderm progenitor cell state.}, journal = {Development}, volume = {144}, year = {2017}, month = {2017 12 15}, pages = {4522-4529}, abstract = {

Elongation of the body axis is a key aspect of body plan development. Bipotential neuromesoderm progenitors (NMPs) ensure axial growth of embryos by contributing both to the spinal cord and mesoderm. The current model for the mechanism controlling NMP deployment invokes Tbx6, a T-box factor, to drive mesoderm differentiation of NMPs. Here, we identify a new population of Tbx6 cells in a subdomain of the NMP niche in mouse embryos. Based on co-expression of a progenitor marker, Sox2, we identify this population as representing a transient cell state in the mesoderm-fated NMP lineage. Genetic lineage tracing confirms the presence of the NMP cell state. Furthermore, we report a novel aspect of the documented mutant phenotype, namely an increase from two to four ectopic neural tubes, corresponding to the switch in NMP niche, thus highlighting the importance of function in NMP fate decision. This study emphasizes the function of Tbx6 as a bistable switch that turns mesoderm fate {\textquoteright}on{\textquoteright} and progenitor state {\textquoteright}off{\textquoteright}, and thus has implications for the molecular mechanism driving NMP fate choice.

}, keywords = {Animals, Body Patterning, Cell Differentiation, Cell Lineage, Embryonic Stem Cells, Gene Expression Regulation, Developmental, Mesoderm, Mice, Mice, Transgenic, Neural Tube, SOXB1 Transcription Factors, Spinal Cord, Transcription Factors}, issn = {1477-9129}, doi = {10.1242/dev.153262}, author = {Javali, Alok and Misra, Aritra and Leonavicius, Karolis and Acharyya, Debalina and Vyas, Bhakti and Sambasivan, Ramkumar} } @article {1157, title = {Conceptualizing Eukaryotic Metabolic Sensing and Signaling.}, journal = {J Indian Inst Sci}, volume = {97}, year = {2017}, month = {2017 Mar}, pages = {59-77}, abstract = {

For almost all cells, nutrient availability, from glucose to amino acids, dictates their growth or developmental programs. This nutrient availability is closely coupled to the overall intracellular metabolic state of the cell. Therefore, cells have evolved diverse, robust and versatile modules to sense intracellular metabolic states, activate signaling outputs and regulate outcomes to these states. Yet, signaling and metabolism have been viewed as important but separate. This short review attempts to position aspects of intracellular signaling from a metabolic perspective, highlighting how conserved, core principles of metabolic sensing and signaling can emerge from an understanding of metabolic regulation. I briefly explain the nature of metabolic sensors, using the example of the AMP activated protein kinase (AMPK) as an "energy sensing" hub. Subsequently, I explore how specific central metabolites, particularly acetyl-CoA, but also -adenosyl methionine and SAICAR, can act as signaling molecules. I extensively illustrate the nature of a metabolic signaling hub using the specific example of the Target of Rapamycin Complex 1 (TORC1), and amino acid sensing. A highlight is the emergence of the lysosome/vacuole as a metabolic and signaling hub. Finally, the need to expand our understanding of the intracellular dynamics (in concentration and localization) of several metabolites, and their signaling hubs is emphasized.

}, issn = {0970-4140}, doi = {10.1007/s41745-016-0013-1}, author = {Laxman, Sunil} } @article {1185, title = {Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum.}, journal = {Acta Crystallogr F Struct Biol Commun}, volume = {73}, year = {2017}, month = {2017 Jun 01}, pages = {356-362}, abstract = {

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5{\textquoteright}-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 {\r A} resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

}, keywords = {Adenosine Triphosphate, Amino Acid Sequence, Bacterial Proteins, Binding Sites, Cloning, Molecular, Crystallography, X-Ray, Escherichia coli, Fusobacterium nucleatum, Gene Expression, Genetic Vectors, Hexosamines, Models, Molecular, Phosphotransferases (Alcohol Group Acceptor), Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Multimerization, Recombinant Proteins, Sequence Alignment, Sequence Homology, Amino Acid, Substrate Specificity}, issn = {2053-230X}, doi = {10.1107/S2053230X17007439}, author = {Caing-Carlsson, Rhawnie and Goyal, Parveen and Sharma, Amit and Ghosh, Swagatha and Setty, Thanuja Gangi and North, Rachel A and Friemann, Rosmarie and Ramaswamy, S} } @article {1210, title = {C-State: an interactive web app for simultaneous multi-gene visualization and comparative epigenetic pattern search.}, journal = {BMC Bioinformatics}, volume = {18}, year = {2017}, month = {2017 Sep 13}, pages = {392}, abstract = {

BACKGROUND: Comparative epigenomic analysis across multiple genes presents a bottleneck for bench biologists working with NGS data. Despite the development of standardized peak analysis algorithms, the identification of novel epigenetic patterns and their visualization across gene subsets remains a challenge.

RESULTS: We developed a fast and interactive web app, C-State (Chromatin-State), to query and plot chromatin landscapes across multiple loci and cell types. C-State has an interactive, JavaScript-based graphical user interface and runs locally in modern web browsers that are pre-installed on all computers, thus eliminating the need for cumbersome data transfer, pre-processing and prior programming knowledge.

CONCLUSIONS: C-State is unique in its ability to extract and analyze multi-gene epigenetic information. It allows for powerful GUI-based pattern searching and visualization. We include a case study to demonstrate its potential for identifying user-defined epigenetic trends in context of gene expression profiles.

}, keywords = {Algorithms, Embryonic Stem Cells, Epigenomics, Genes, Genomics, HeLa Cells, Humans, Internet, K562 Cells, Promoter Regions, Genetic, Software, Transcription, Genetic, Web Browser}, issn = {1471-2105}, doi = {10.1186/s12859-017-1786-6}, author = {Sowpati, Divya Tej and Srivastava, Surabhi and Dhawan, Jyotsna and Mishra, Rakesh K} } @article {1160, title = {Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian .}, journal = {Development}, volume = {144}, year = {2017}, month = {2017 09 01}, pages = {3066-3079}, abstract = {

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including , are translationally regulated by SMED-PABPC2 Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration.

}, keywords = {Animals, Cell Lineage, Cell Proliferation, Cytoplasm, Epidermis, Epithelium, Extracellular Matrix, Gene Knockdown Techniques, Homeostasis, Models, Biological, Planarians, Poly(A)-Binding Protein I, Regeneration, RNA, Messenger, Wound Healing}, issn = {1477-9129}, doi = {10.1242/dev.152942}, author = {Bansal, Dhiru and Kulkarni, Jahnavi and Nadahalli, Kavana and Lakshmanan, Vairavan and Krishna, Srikar and Sasidharan, Vidyanand and Geo, Jini and Dilipkumar, Shilpa and Pasricha, Renu and Gulyani, Akash and Raghavan, Srikala and Palakodeti, Dasaradhi} } @article {398, title = {Conserved hippocampal cellular pathophysiology but distinct behavioural deficits in a new rat model of FXS.}, journal = {Hum Mol Genet}, volume = {24}, year = {2015}, month = {2015 Nov 1}, pages = {5977-84}, abstract = {

Recent advances in techniques for manipulating genomes have allowed the generation of transgenic animals other than mice. These new models enable cross-mammalian comparison of neurological disease from core cellular pathophysiology to circuit and behavioural endophenotypes. Moreover they will enable us to directly test whether common cellular dysfunction or behavioural outcomes of a genetic mutation are more conserved across species. Using a new rat model of Fragile X Syndrome, we report that Fmr1 knockout (KO) rats exhibit elevated basal protein synthesis and an increase in mGluR-dependent long-term depression in CA1 of the hippocampus that is independent of new protein synthesis. These defects in plasticity are accompanied by an increase in dendritic spine density selectively in apical dendrites and subtle changes in dendritic spine morphology of CA1 pyramidal neurons. Behaviourally, Fmr1 KO rats show deficits in hippocampal-dependent, but not hippocampal-independent, forms of associative recognition memory indicating that the loss of fragile X mental retardation protein (FMRP) causes defects in episodic-like memory. In contrast to previous reports from mice, Fmr1 KO rats show no deficits in spatial reference memory reversal learning. One-trial spatial learning in a delayed matching to place water maze task was also not affected by the loss of FMRP in rats. This is the first evidence for conservation across mammalian species of cellular and physiological hippocampal phenotypes associated with the loss of FMRP. Furthermore, while key cellular phenotypes are conserved they manifest in distinct behavioural dysfunction. Finally, our data reveal novel information about the selective role of FMRP in hippocampus-dependent associative memory.

}, issn = {1460-2083}, doi = {10.1093/hmg/ddv299}, author = {Till, Sally M and Asiminas, Antonis and Jackson, Adam D and Katsanevaki, Danai and Barnes, Stephanie A and Osterweil, Emily K and Bear, Mark F and Chattarji, Sumantra and Wood, Emma R and Wyllie, David J A and Kind, Peter C} }