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Kumar, J.

Publications and source records attributed to Kumar, J..

At least 37 records · Page 2Linked to original sources
bioRxiv2024

P66Shc Mediates SUMO2-induced Endothelial Dysfunction

BackgroundSumoylation is a post-translational modification that can regulate different physiological functions. Increased sumoylation, specifically conjugation of SUMO2/3 (small ubiquitin-like modifier 2/3), is detrimental to vascular health. However, the molecular mechanism mediating this effect is poorly understood. MethodsWe used cell-based assays and mass spectrometry to show that p66Shc is a direct target of SUMO2 and SUMO2 regulates p66Shc function via lysine-81 modification. To determine the effects of SUMO2-p66ShcK81 on vascular function, we generated p66ShcK81R knockin mice and crossbred to LDLr-/- mice to induce hyperlipidemia. Next, to determine p66ShcK81-SUMO2ylation-induced changes in endothelial cell signaling, we performed mass spectrometry followed by Ingenuity Pathway Analysis. ResultsOur data reveal that p66Shc mediates the effects of SUMO2 on endothelial cells. Mass spectrometry identified that SUMO2 modified lysine-81 in the unique collagen homology-2 domain of p66Shc. SUMO2ylation of p66Shc increased phosphorylation at serine-36, causing it to translocate to the mitochondria, a step critical for oxidative function of p66Shc. Notably, sumoylation-deficient p66Shc (p66ShcK81R) was resistant to SUMO2-induced p66ShcS36 phosphorylation and mitochondrial translocation. P66ShcK81R knockin mice were resistant to endothelial dysfunction induced by SUMO2ylation and hyperlipidemia. Ingenuity Pathway Analysis revealed multiple signaling pathways regulated by p66ShcK81-SUMO2ylation in endothelial cells, highlighting JAK-STAT as a major pathway affected by SUMO2-p66ShcK81. ConclusionsCollectively, our work reveals SUMO2-p66Shc signaling as a fundamental regulator of vascular endothelial function. We discovered that p66ShcK81 is an upstream modification regulating p66Shc signaling and mediates hyperlipidemia-induced endothelial dysfunction and oxidative stress.

cell biology↗
bioRxiv2023

Analytical Method Development for Evaluation of Pharmacokinetics and Tissue Distribution of Thiourea-Based Antivirals through nLC/MS-MS

Thiourea-based antivirals are organosulfur chemical compounds. Due to their various medicinal uses, including antiviral, antioxidant, and anticancer properties, they are becoming more popular. In this study, pharmacokinetics, Metabolism, bioavailability, and distribution of thiourea derivatives to organs of rats. Thiourea derivatives, namely DSA-00, DSA-02, and DSA-09, exhibit characteristics similar to those of conventional drugs when evaluated in terms of pharmacokinetics, drug-likeness, and medicinal chemistry through in-silico analysis. Then a simple and sensitive analytical method was developed for quantified DSA-00, DSA-02, and DSA-09, using prednisolone as an internal standard (IS) and nano liquid chromatography tandem mass spectrometry (nLC-MS/MS). The plasma and tissue samples were preprocessing with acetonitrile before chromatographic separation by C18 column with isocratic elution using a mobile phase of water-methanol (30:70, v/v) at a flow rate of 0.6 mL/min. A triplequadrupole tandem mass spectrometer in multiple-reaction monitoring (MRM) scanning via an electrospray ionization (ESI) source functioning in negative and positive mode detection. For DSA-00, DSA-02, DSA-09 and, internal standard the optimized mass transition ion-pairs (m/z) for quantification were 297.2, 311.2, 309.09, and 361.19 respectively. Each analytic run required only 15 minutes between injections. The calibration curve for thiourea derivatives showed good linear regression coefficient (R2 >.99) over a range of 1.00-10000 pg/mL except DSA-09. The lower limit of quantification (LLOQ) was 1.00 ng/mL. The intra- and inter-day precisions were no more than 10.8%, and relative errors (RE) ranged from 0.5 to 5.98%. The validated method was effectively used to explore the pharmacokinetics of orally administrative thiourea derivatives in rats.

pharmacology and toxicology↗
bioRxiv2023

Safety Assessment and Evaluation of Novel Thiourea Derivatives as Antivirals

DSA-00 and its two derivatives (DSA-02 & DSA-09) are new thiourea derivatives that exhibit strong antiviral activity against hepatitis B virus comparable to Entecavir. Here these compounds were evaluated for in-vitro cytotoxicity and genotoxicity, and in vivo acute toxicity for their potential therapeutic use. The cytotoxicity of thiourea derivatives was assessed in HepG2 and HepG2.2.15 cells by MTT assay whereas their genotoxicity was measured by Ames II test. The acute toxicity study was carried out in the Sprague-Dawley rats by observing the following parameters: mortality, clinical symptoms, hematological parameters, urine and changes in animal body & organs weight, gross necropsy and histopathology. DSA-00, DSA-02, and DSA-09 were non-cytotoxic even at the 320M concentration with respective CC50 values of 329.6, 323.5, and 349.7 M. The Ames II test revealed that these molecules were non-mutagenic at a [~]1M concentration. The acute toxicity studies revealed LD50 values belong from the moderate range of toxicity of DSA-00, DSA-02 and DSA-09. Importantly, there were no abnormal change in body weight and organs weight. Moreover, no abnormal clinical signs such as hematological parameter, urinalysis, gross necropsy and or histopathological in any of the animals after receiving oral doses of thiourea derivatives. Thiourea derivatives DSA-00, DSA-02, and DSA-09 appear to have a moderate range of acute toxicity at dosages used and thus, appear to be generally safe. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/546447v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@8c8abaorg.highwire.dtl.DTLVardef@e7bcb1org.highwire.dtl.DTLVardef@15446dorg.highwire.dtl.DTLVardef@3f5d7b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Illustration of the study design and the findings for each aspect of the study: in-vitro cytotoxicity (1), genotoxicity by the Ames test (2), and oral toxicity in rats (3). C_FIG

pharmacology and toxicology↗
bioRxiv2023

Functional Implications of the Exon 9 Splice Insert in GluK1 Kainate Receptors

Kainate receptors are key modulators of synaptic transmission and plasticity in the central nervous system. Different kainate receptor isoforms with distinct spatiotemporal expressions have been identified in the brain. The GluK1-1 splice variant receptors, which are abundant in the adult brain, have an extra fifteen amino acids inserted in the amino-terminal domain (ATD) of the receptor resulting from alternative splicing of exon 9. However, the functional implications of this post-transcriptional modification are not yet clear. We employed a multi-pronged approach using cryogenic electron microscopy, electrophysiology, and other biophysical and biochemical tools to understand the structural and functional impact of this splice insert in the extracellular domain of GluK1 receptors. Our study reveals that the splice insert alters the key gating properties of GluK1 receptors and their modulation by the cognate auxiliary Neuropilin and tolloid-like (Neto) proteins 1 and 2. Mutational analysis identified the role of crucial splice residues that influence receptor properties and their modulation. Furthermore, the cryoEM structure of the variant shows that the presence of exon 9 in GluK1 does not affect the receptor architecture or domain arrangement in the desensitized state. Our study thus provides the first detailed structural and functional characterization of GluK1-1a receptors, highlighting the role of the splice insert in modulating receptor properties and their modulation.

neuroscience↗
bioRxiv2023

Efficient protein tagging and cis-regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants

Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient NHEJ-mediated targeted insertion method was recently developed by combining CRISPR-Cas9 with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet this approach often led to imprecise insertions with no control over the insertion direction. In this study, we first quantitatively compared the impact of the chemical protection on efficiency of targeted insertion. With the observation that CRISPR-SpCas9 could frequently induce staggered cleavages with 5' 1-nucleotide overhangs, we then evaluated the impact of the donor end structures on the direction and preciseness of targeted insertions. Our study demonstrated that the chemically protected dsODNs with 5' 1-nt overhangs could significantly improve the precision and direction control of target insertions in all tested CRIPSR targeting sites. Lastly, we applied this method to endogenous gene tagging in Setaria viridis, and cis-regulatory element engineering for disease resistance in rice. Two distinct TAL effector binding elements were directionally inserted into the promoter region of a recessive rice bacterial blight resistance gene at up to 24.4% efficiency. The resulting rice lines with heritable insertions exhibited strong resistance to the infection of Xanthomonas oryzae pv. oryzae pathogen in an inducible and strain-specific manner.

plant biology↗
bioRxiv2023

Automatic Abdominal Multi Organ Segmentationusing Residual UNet

Automated segmentation of abdominal organs plays an important role in supporting computer-assisted diagnosis, radiotherapy, biomarker extraction, surgery navigation, and treatment planning. Segmenting multiple abdominal organs using a single algorithm would improve model development efficiency and accelerate model deployment into clinical workflows. To achieve broadly generalized performance, we trained a residual UNet using 500 CT/MRI scans collected from multi-center, multi-vendor, multi-phase, multi-disease patients, each with voxel-level annotation of 15 abdominal organs. Using the model trained on multimodality (CT/MRI), we achieved an average dice of 0.8990 in the held-out test dataset with only CT scans (N=100). An average dice of 0.8948 was achieved in the held-out test dataset with both CT and MRI scans (N=120. Our results demonstrate broad generalization of the model.

bioengineering↗
bioRxiv2023

Automatic Whole Body FDG PET/CT Lesion Segmentation using Residual UNet and Adaptive Ensemble

Multimodal Positron Emission Tomography/Computed Tomography (PET/CT) plays a key role in the diagnosis, staging, restaging, treatment response assessment, and radiotherapy planning of malignant tumors. The complementary nature of high-resolution anatomic CT and high sensitivity/specificity molecular PET imaging provides accurate assessment of disease status [14] In oncology, 18-fluorodeoxyglucose (FDG) PET/CT is the most widely used method to identify and analyze metabolically active tumors. In particular, FDG uptake allows for more accurate detection of both nodal and distant forms of metastatic disease. Accurate quantification and staging of tumors is the most important prognostic factor for predicting the survival of patients and for designing personalized patient management plans. [8,3] Analyzing PET/CT quantitatively by experienced medical imaging experts/radiologists is timeconsuming and error-prone. Automated quantitative analysis by deep learning algorithms to segment tumor lesions will enable accurate feature extraction, tumor staging, radiotherapy planning, and treatment response assessment. The AutoPET Challenge 2022 provided an opensource platform to develop and benchmark deep learning models for automated PET lesion segmentation by providing large open-source wholebody FDG-PET/CT data. Using the multimodal PET/CT data from 900 subjects with 1014 studies provided by the AutoPET MICCAI 2022 Challenge, we applied fivefold cross-validation on residual UNETs to automatically segment lesions. We then utilized the output from adaptive ensemble highly contributive models as the final segmentation. Our method achieved a 10th ranking with a dice score of 0.5541 in the heldout test dataset (N=150 studies).

bioengineering↗
bioRxiv2022

An Extensible Vector Toolkit and Parts Library for Advanced Engineering of Plant Genomes

Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

plant biology↗
bioRxiv2021

Quantitative prediction of variant effects on alternative splicing using endogenous pre-messenger RNA structure probing

Splicing is a highly regulated process that depends on numerous factors. It is particularly challenging to quantitatively predict how a mutation will affect precursor messenger RNA (mRNA) structure and the subsequent functional consequences. Here we use a novel Mutational Profiling (-MaP) methodology to obtain highly reproducible endogenous precursor and mature mRNA structural probing data in vivo. We use these data to estimate Boltzmann suboptimal ensembles, and predict the structural consequences of mutations on precursor mRNA structure. Together with a structural analysis of recent cryo-EM spliceosome structures at different stages of the splicing cycle, we determined that the footprint of the Bact complex on precursor mRNA is best able to predict splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene. However, structure alone only achieves 74% accuracy. We therefore developed a {beta}-regression weighting framework that incorporates splice site strength, structure and exonic/intronic splicing regulatory elements which together achieves 90% accuracy for 47 known and six newly discovered splice-altering variants. This combined experimental/computational framework represents a path forward for accurate prediction of splicing related disease-causing variants.

genomics↗
bioRxiv2021

Knockdown of a novel ATPase domain of capsid protein inhibits genome packaging in potato leaf roll virus

Potato leaf roll virus (PLRV) uses powerful molecular machines to package its genome into a viral capsid employing ATP as fuel. Although, recent bioinformatics and structural studies have revealed detailed mechanism of DNA packaging, little is known about the mechanochemistry of genome packaging in small plant viruses such as PLRV. We have identified a novel P-loop-containing ATPase domain with two Walker A-like motifs, two arginine fingers, and two sensor motifs distributed throughout the polypeptide chain of PLRV capsid protein (CP). The composition and arrangement of the ATP binding and hydrolysis domain of PLRV CP is unique and rarely reported. The discovery of the system sheds new light on the mechanism of viral genome packaging, regulation of viral assembly process, and evolution of plant viruses. Here, we used the RNAi approach to suppress CP gene expression, which in turn prevented PLRV genome packaging and assembly in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with siRNA constructs against the ATPase domain of CP exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants. Moreover, our findings provide a robust approach to generate PLRV-resistant potato plants, which can be further extended to other species. Finally, we propose a new mechanism of genome packaging and assembly in plant viruses.

molecular biology↗
bioRxiv2021

Knowledge production and translation of science between Academia and Industry: assessing the impact of R&D in India

India is at the 3rd position worldwide in terms of publication of scientific literature. However, in terms of productivity, it has been consistently failing to transform the research knowledge into industrial output. This study compares India with the leading countries to understand its lacuna in terms of R&D policy and outputs. Although scientific publications are regarded as the output of basic research, patent applications serve as a better indicator of the applied research. This paper assesses the important determinants for patent filings of a nation. It also focuses on the role of academia and industry collaboration in R&D and the productivity of a nation. We found that the higher the GERD (total Gross Domestic Expenditure of R&D) and the R&D personnel in a nation, the higher the patent filings of the nation. Moreover, we show that academia-industrial collaboration plays a key role in transforming basic research into real-world applications, as we illustrate the governments role in making necessary policies to make the collaboration successful. This paper highlights the significance of investing in R&D to improve the productivity of a nation, as also the need to design policies to strengthen the applied research environment by fostering solution-centric collaborations between academia and industry.

scientific communication and education↗
bioRxiv2021

Non-coding enhancer RNA regulates NPAS4 in the mPFC to control chronic stress-induced anhedonia-like behavior

Chronic stress can produce reward system deficits (i.e. anhedonia) and other common symptoms associated with depressive disorders, as well as neural circuit hypofunction in the medial prefrontal cortex (mPFC). However, the molecular mechanisms by which chronic stress promotes depressive-like behavior and hypofrontality remain unclear. We show here that the neuronal activity-regulated transcription factor, NPAS4, in the mPFC is regulated by chronic social defeat stress (CSDS), and its required in this brain region for CSDS-induced changes in sucrose preference and natural reward motivation. Interestingly, NPAS4 is not required for CSDS-induced social avoidance or anxiety-like behavior. We also find that mPFC NPAS4 is required for CSDS-induced reduction of pyramidal cell dendritic spine density, revealing a relationship between mPFC dendritic spine changes and anhedonia-like behavior, but not social avoidance behavior. Finally, transcriptomic analysis from the mPFC revealed that NPAS4 influences expression of numerous genes linked to glutamatergic synapses and ribosomal function, as well as many dysregulated genes observed in common neuropsychiatric disorders, including depression. Together our findings reveal an essential role for the activity-regulated transcription factor, NPAS4, in chronic stress-induced mPFC hypofunction and anhedonia.

neuroscience↗
bioRxiv2020

Swi6/HP1 binding to RNA-DNA hybrids initiates heterochromatin assembly at the centromeric dg-dh repeats in Fission Yeast

Canonically, heterochromatin formation in fission yeast and metazoans is initiated by di/trimethylation of histone H3 at lysine 9 position by the histone methyltransferase Suv39H1/Clr4, followed by binding of Swi6/HP1 to H3-K9-me2/me3 via its chromodomain. Subsequent self-association of Swi6/HP1 on adjacent nucleosomes or a cooperative interaction between Clr4 and Swi6/HP1 leads to folded heterochromatin structure. HP1 binding to RNA is shown to facilitate its localization at and assembly of heterochromatin in metazoans. Likewise, recruitment of Swi6/HP1 to centromere depends on the RNAi pathway in fission yeast; paradoxically, Swi6/HP1 is also thought to play a role in RNA turnover. Here we provide evidence in support of RNAi-independent recruitment of Swi6. We show that, apart from the low affinity binding to RNAs through its hinge domain, as already reported, Swi6/HP1 displays a hierarchy of increasing binding affinity through its chromodomain to the siRNAs corresponding to specific dg-dh repeats and even stronger binding to the cognate siRNA-DNA hybrids than to the siRNA precursors or general RNAs. Our results support a mechanism of recruitment of Swi6, which is dependent on its specific and high affinity binding to siRNA-DNA hybrid at the dg-dh repeats. This binding, which is independent of, albeit augmented by binding to H3-K9-Me2, leads to heterochromatin formation and silencing. We suggest that the net role of Swi6 in RNA physiology may be regulated by a balance between abundance and affinity of Swi6 towards heterochromatic and euchromatic RNAs and siRNAs.

genetics↗
bioRxiv2020

Phylogenomic analysis of SARS-CoV-2 genomes from western India reveals unique linked mutations

India has become the third worst-hit nation by the COVID-19 pandemic caused by the SARS-CoV-2 virus. Here, we investigated the molecular, phylogenomic, and evolutionary dynamics of SARS-CoV-2 in western India, the most affected region of the country. A total of 90 genomes were sequenced. Four nucleotide variants, namely C241T, C3037T, C14408T (Pro4715Leu), and A23403G (Asp614Gly), located at 5UTR, Orf1a, Orf1b, and Spike protein regions of the genome, respectively, were predominant and ubiquitous (90%). Phylogenetic analysis of the genomes revealed four distinct clusters, formed owing to different variants. The major cluster (cluster 4) is distinguished by mutations C313T, C5700A, G28881A are unique patterns and observed in 45% of samples. We thus report a newly emerging pattern of linked mutations. The predominance of these linked mutations suggests that they are likely a part of the viral fitness landscape. A novel and distinct pattern of mutations in the viral strains of each of the districts was observed. The Satara district viral strains showed mutations primarily at the 3' end of the genome, while Nashik district viral strains displayed mutations at the 5' end of the genome. Characterization of Pune strains showed that a novel variant has overtaken the other strains. Examination of the frequency of three mutations i.e., C313T, C5700A, G28881A in symptomatic versus asymptomatic patients indicated an increased occurrence in symptomatic cases, which is more prominent in females. The age-wise specific pattern of mutation is observed. Mutations C18877T, G20326A, G24794T, G25563T, G26152T, and C26735T are found in more than 30% study samples in the age group of 10-25. Intriguingly, these mutations are not detected in the higher age range 61-80. These findings portray the prevalence of unique linked mutations in SARS-CoV-2 in western India and their prevalence in symptomatic patients. ImportanceElucidation of the SARS-CoV-2 mutational landscape within a specific geographical location, and its relationship with age and symptoms, is essential to understand its local transmission dynamics and control. Here we present the first comprehensive study on genome and mutation pattern analysis of SARS-CoV-2 from the western part of India, the worst affected region by the pandemic. Our analysis revealed three unique linked mutations, which are prevalent in most of the sequences studied. These may serve as a molecular marker to track the spread of this viral variant to different places.

genomics↗
bioRxiv2020

Composite modeling of leaf shape across shoots discriminates Vitis species better than individual leaves

Premise of studyLeaf morphology is dynamic, continuously deforming during leaf expansion and among leaves within a shoot. We measured leaf morphology from over 200 vines over four years, and modeled changes in leaf shape along the shoot to determine if a composite "shape of shapes" can better capture variation and predict species identity compared to individual leaves. MethodsUsing homologous universal landmarks found in grapevine leaves, we modeled various morphological features as a polynomial function of leaf node. The resulting functions are used to reconstruct modeled leaf shapes across shoots, generating composite leaves that comprehensively capture the spectrum of possible leaf morphologies. ResultsWe found that composite leaves are better predictors of species identity than individual leaves from the same plant. We were able to use composite leaves to predict species identity of previously unassigned vines, which were verified with genotyping. DiscussionObservations of individual leaf shape fail to capture the true diversity between species. Composite leaf shape--an assemblage of modeled leaf snapshots across the shoot--is a better representation of the dynamic and essential shapes of leaves, as well as serving as a better predictor of species identity than individual leaves.

plant biology↗
bioRxiv2020

CASSPER: A Semantic Segmentation based Particle Picking Algorithm for Single Particle Cryo-Electron Microscopy

Single-particle cryo-electron microscopy has emerged as the method of choice for structure determination of proteins and protein complexes. However, particle identification and selection which is a prerequisite for achieving high-resolution still poses a major bottleneck for automating the steps of structure determination. Here, we present a generalised deep learning tool, CASSPER, for the automated detection and isolation of protein particles in transmission microscope images. This deep learning tool uses Semantic Segmentation and a collection of visually prepared training samples to capture the differences in the transmission intensities of protein, ice, carbon and other impurities found in the micrograph. CASSPER is the first method to do pixel level classification and completely eliminates the need of manual particle picking. Integration of Contrast Limited Adaptive Histogram Equalization (CLAHE) in CASSPER enables high-fidelity particle detection even in micrographs with variable ice thickness and contrast. In addition, our generalized model for cross molecule picking works with high efficiency on unseen datasets and can potentially pick particles on-the-fly, thereby, enabling automation of data processing.

biophysics↗
bioRxiv2020

The Architecture of GluD2 Ionotropic Delta Glutamate Receptor Elucidated by cryo-EM

GluD2 receptors belong to the orphan delta receptor family of glutamate receptor ion channels. These receptors play key roles in synaptogenesis and synaptic plasticity and are associated with multiple neuronal disorders like schizophrenia, autism spectrum disorder, cerebellar ataxia, intellectual disability, paraplegia, retinal dystrophy, etc. Despite the importance of these receptors in CNS, insights into full-length GluD2 receptor structure is missing till-date. Here we report cryo-electron microscopy structure of the rat GluD2 receptor in the presence of calcium ions and the ligand 7-chlorokynurenic acid, elucidating its 3D architecture. The structure reveals a non-swapped architecture at the extracellular aminoterminal (ATD), and ligand-binding domain (LBD) interface similar to that observed in GluD1; however, the organization and arrangement of the ATD and LBD domains are unique. While our results demonstrate that non-swapped architecture is conserved in the delta receptor family, they also highlight the differences that exist between the two member receptors; GluD1 and GluD2.

biochemistry↗