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Kashyap, R.

Publications and source records attributed to Kashyap, R..

10 recordsLinked to original sources

Spatial Decoupling of the Endodermal Functions Establishes the Innermost Cortex as the Gravity Sensor in Rice Stems

Gravitropism is a remarkable example of coordinated growth, enabling plants to perceive gravity and generate asymmetric cell expansion that restores organ orientation. In Arabidopsis, gravity sensing in shoots occurs within starch-rich endodermis cells. In rice, however, the stem must accommodate gravitropic bending while preserving integrity of tissues surrounding emerging crown roots, raising the possibility that gravity sensing relies on distinct structural and physiological adaptations. Here, using high-resolution imaging together with auxin-response analyses, we identify a specialized cell layer at the inner boundary of the rice stem cortex, operationally defined by its position adjacent to the lignified endodermis and associated with gravitropic responses. This layer is characterized by circumferentially elongated cells, starch sedimentation, a confined auxin-response domain, and expression of gravity-sensing markers. Following gravistimulation, starch granules realign and the auxin response domain expands asymmetrically within this layer, coinciding with differential growth during stem bending. Our results further indicate that this response depends on polar auxin transport and conserved components of the gravitropic regulatory network. Together, these findings support a model in which gravity sensing and signaling in rice stems are spatially separated from the lignified endodermal barrier. Such tissue organization may represent a developmental adaptation that allows the stem to reconcile barrier integrity with growth flexibility.

plant biology↗

A Phosphorylation Switch Modulates Configurational Codes in the Oncofetal IGF2BP RNA Binding Paralogs

The insulin like growth factor 2 mRNA binding proteins (IGF2BP1-3) are oncofetal RNA regulators that control translation, stability, and localization of several transcripts, yet display paralog specific functions despite high structural similarity. Each paralog contains six RNA binding domains (two RRMs and four KH domains) linked by intrinsically disordered segments. mTORC2 phosphorylates IGF2BP1 and IGF2BP3 at a single conserved serine within the disordered linker between the RRM2 and KH1 domains, a modification required for proper regulation of mRNA translational fate. Pairing site specific phosphoserine incorporation with structural and biophysical interrogations, we show that this phosphorylation acts as a configurational switch that reorganizes long range arrangements of RNA binding domains and linkers without altering the secondary structure, and with only modest effects on RNA binding affinity. Critically, pSer driven rearrangements occur both in the RNA free state and upon RNA engagement, and the resulting architectures differ markedly between IGF2BP1 and IGF2BP3 despite >70% sequence identity. These paralog specific, phosphorylation dependent configurational landscapes likely underlie differences in mRNA recognition modes and functional outcomes. Our work identifies a post translational mechanism that tunes IGF2BP paralog dynamics across free and RNA bound states to program target mRNA selection, processing, and translational fate.

biochemistry↗

Cryo-EM structure of a methanogen nitrogenase-PII protein supercomplex

Nitrogenases are metalloenzymes that catalyze the reduction of atmospheric dinitrogen to ammonia, sustaining the global nitrogen cycle. While bacterial nitrogenase has been extensively characterized, the architecture and regulation of archaeal nitrogenases remain unknown despite longstanding evidence of nitrogen fixation in methanogens. Here we report a 3.2 [A] cryo-electron microscopy structure of a native nitrogenase-PII protein supercomplex from Methanosarcina acetivorans. The structure reveals an unprecedented assembly of three NifDK heterotetramers bridged by six NifI1,2 heterotrimeric PII complexes, which sterically block NifH association and lock the enzyme in an inactive state. The PII complexes display asymmetric binding of ADP and 2-oxoglutarate, coupling nitrogenase inhibition directly to cellular energy and nitrogen status. Addition of 2-oxoglutarate and ATP releases the NifI complexes, stimulating a threefold increase in NifDK activity in vitro. This higher-order architecture uncovers a previously unrecognized regulatory strategy in methanogens, in which PII proteins drive nitrogenase oligomerization to control activity. The discovery that nitrogenase activity may be modulated through direct assembly into higher-order structures opens new avenues for exploring nitrogenase evolution, regulation, and biotechnological applications. One sentence summaryDiscovery of a nitrogenase-PII protein supercomplex in methanogens, uncovering a metabolite-gated assembly mechanism for nitrogenase inhibition.

biochemistry↗

Cryo-EM captures the coordination of long-range allostery and asymmetric electron transfer by a bi-copper cluster in the nitrogenase-like DPOR complex

Enzymes that catalyze long-range electron transfer reactions are often structurally evolved to possess two symmetrical halves. The functional advantages and mechanistic principles for such architecture remain a mystery. Using Cryo-EM we capture snapshots of the nitrogenase-like Dark-operative Protochlorophyllide Oxidoreductase (DPOR) enzyme during substrate recognition and turnover. The structures reveal that asymmetry is enforced upon substrate binding and leads to an allosteric inhibition of protein-protein interactions and electron transfer in one half. Residues that form a conduit for electron transfer are aligned in one half while misaligned in the other. An ATP-turnover coupled switch is triggered once electron transfer is accomplished in one half and relayed through a bi-copper cluster at the oligomeric interface, leading to activation of enzymatic events in the other. The findings provide a mechanistic blueprint for regulation of asymmetric long-range electron transfer. One-Sentence SummaryA bi-copper cluster coordinates electron transfer for substrate reduction in the nitrogenase-like DPOR enzyme and the structures reveal how allostery and asymmetry are enacted over 100[A] and utilized for sequential electron transfer.

biochemistry↗

Insights into assembly of constitutive filament-forming proteins of novel fold through domain dissection and EGFP insertion-based screen: A case study of Spiroplasma Fibril

Spiroplasma is a cell-wall less, helical bacterium possessing a cytoskeletal ribbon consisting Fibril and 5 MreB paralogs. In absence of any information regarding the filament interface of Fibril, a cytoskeleton protein of novel fold, multiple short constructs of Fibril were designed to identify minimal domains required for polymerization. Purification trials of these short constructs resulted in insoluble protein products. In a parallel approach, we incorporated EGFP at the loop regions aimed to hinder polymerization and obtain non-polymerising constructs. We were able to identify probable interfilament/intrafilament interface residues based on the fluorescence screen. We show that EGFP insertions at many of the positions are tolerated as Fibril filaments were readily observed for these constructs in transmission electron microscopy. Both the approaches suggest that Fibril requires N and C-terminal domains for its polymerization. Earlier studies have shown that FibrilWT interacts with MreB5. Our Fibril-EGFP constructs and MreB5 showed interaction similar to the wild-type. Co-sedimentation assay and visualization of Fibril-EGFP proteins proves that the fluorescent constructs are folded and functional and possess structural and biochemical properties similar to the wild type protein.

microbiology↗

Astroglia proliferate upon biogenesis of tunneling nanotubes and clearance of α-synuclein toxicities

Astrocytic cells are a subtype of glial cells that engulf pathogenic aggregates derived from degenerative neurons to facilitate its degradation. Here, we show that exposure to -SYN protofibrils caused a transient increase in biogenesis of tunneling nanotubes (TNTs) in primary astrocytes and astrocyte-origin cancer cell-lines (U-87 MG, U251). Biogenesis of nascent TNTs corresponds to -SYN protofibril-induced organelle toxicities, increased reactive oxygen species (ROS), and oxidative stress-induced premature cellular senescence. These TNTs mediate cell-to-cell transfer of -SYN protofibrils, toxic lysosomes and mitochondria. Biogenesis of TNTs precedes clearance of -SYN-induced organelle toxicities, cellular ROS levels and reversal of cellular senescence. Consequences of cellular clearance results in enhanced cell proliferation. Further, we have shown -SYN-induced senescence promotes transient localization of focal adhesion kinase (FAK) in the nucleus. FAK mediated regulation of Rho-associated kinases may have a role in the biogenesis of TNTs, successively proliferation. Our study emphasizes that TNT biogenesis may have a potential role in the clearance of -SYN toxicities and reversal of stress-induced cellular senescence, consequences of which cause enhanced proliferation in the post-recovered astroglia cells. HighlightsO_LI-SYN protofibrils treated astroglia cells proliferate upon transient biogenesis of TNTs. C_LIO_LITransient TNT biogenesis precedes clearance of -SYN toxicities and reversal of senescence. C_LIO_LIStress-induced senescence results in nuclear localization of FAK and ROCK mediated TNT biogenesis. C_LIO_LIThe rescued cells enhance proliferation through ROCK mediated ERK1/2 and NF{kappa}B signalling cascades. C_LI Synopsis O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/554645v1_figa1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@9dc5bcorg.highwire.dtl.DTLVardef@20e924org.highwire.dtl.DTLVardef@aa7920org.highwire.dtl.DTLVardef@1f6274b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO -SYN protofibrils-induced biogenesis of tunneling nanotubes (TNTs) aids to enhance cellular clearance of toxic burdens as a cellular survival strategy. -SYN protofibrils treated toxic senescence cells regulate FAK mediated modulation of ROCK signalling cascades to promote TNT biogenesis and rescue the cellular toxicities. The rescued cells eventually enhance cell proliferation. C_FIG

cell biology↗

Single session of rTMS enhances brain metastability and intrinsic ignition

BackgroundEmerging evidence support the view that brain stimulation might improve essential tremor (ET) by altering brain networks and facilitating plasticity. Yet, we are still missing a mechanistic explanation of the whole brain dynamics underlying these plasticity defining changes. MethodIn this study, we explored the effect of low-frequency repetitive transcranial magnetic stimulation (rTMS) over left primary motor cortex (L-M1) on functional connectivity dynamics (FCD) in patients with ET. Resting-state fMRI (RsfMRI) was acquired before and after a single session of rTMS in 30 patients with ET and compared with RsfMRI of 20 age and gender matched healthy controls (HCs). We have measured the effect of brain stimulation using network topological re-organization through whole brain integration and segregation, brain stability and capacity of neural propagation through metastability and intrinsic ignition. ResultsPatients with ET had altered FCD measures compared to controls. After a single session rTMS, the brain connectivity measures approached normality and patients with ET revealed significantly higher integration, lower segregation with higher metastability and increased intrinsic ignition. ConclusionBrain metastability and intrinsic ignition measures could be valuable tools in appreciating mechanisms of brain stimulation in ET and other neurological diseases.

neuroscience↗

-SATA: A MATLAB based toolbox to estimate Current Density generated by Transcranial Direct Current Stimulation in an Individual Brain

BackgroundTranscranial Direct Current Stimulation (tDCS) is a technique where a weak current is passed through the electrodes placed on the scalp. The distribution of the electric current induced in the brain due to tDCS is provided by simulation toolbox like Realistic-volumetric-Approach-based-Simulator-for-Transcranial-electric-stimulation (ROAST). However, the procedure to estimate the total current density induced at the target and the intermediary region of the cortex is complex. The Systematic-Approach-for-tDCS-Analysis (SATA) was developed to overcome this problem. However, SATA is limited to standardized headspace only. Here we develop individual-SATA (-SATA) to extend it to individual head. MethodT1-weighted images of 15 subjects were taken from two Magnetic Resonance Imaging (MRI) scanners of different strengths. Across the subjects, the montages were simulated in ROAST. -SATA converts the ROAST output to Talairach space. The x, y and z coordinates of the anterior commissure (AC), posterior commissure (PC), and Mid-Sagittal (MS) points are necessary for the conversion. AC and PC are detected using the acpcdetect toolbox. We developed a method to determine the MS in the image and cross-verified its location manually using BrainSight(R). ResultDetermination of points with -SATA is fast and accurate. The -SATA provided estimates of the current-density induced across an individuals cortical lobes and gyri as tested on images from two different scanners. ConclusionResearchers can use -SATA for customizing tDCS-montages. With -SATA it is also easier to compute the inter-individual variation in current-density across the target and intermediary regions of the brain. The software is publicly available.

neuroscience↗

Maximizing Dissimilarity in Resting State detects Heterogeneous Subtypes in Healthy population associated with High Substance-Use and Problems in Antisocial Personality

Patterns in resting-state fMRI (rs-fMRI) are widely used to characterize the trait effects of brain function. In this aspect, multiple rs-fMRI scans from single subjects can provide interesting clues about the rs-fMRI patterns, though scan-to-scan variability pose challenges. Therefore, rs-fMRIs are either concatenated or the functional connectivity is averaged. This leads to loss of information. Here, we use an alternative way to extract the rs-fMRI features that are common across all the scans by applying Common-and-Orthogonal-Basis-Extraction (COBE) technique. To address this, we employed rs-fMRI of 788 subjects from the human connectome project and estimated the common-COBE-component of each subject from the four rs-fMRI runs. Since the common-COBE-component are specific to a subject, the pattern was used to classify the subjects based on the similarity/dissimilarity of the features. The subset of subjects (n=107) with maximal-COBE-Dissimilarity (MCD) was extracted and the remaining subjects (n = 681) formed the COBE-similarity (CS) group. The distribution of weights of the common-COBE-component for the two groups across rs-fMRI networks and subcortical regions was evaluated. We found the weights in the default mode network to be lower in the MCD compared to the CS. We compared the scores of 69 behavioral measures and found 6 behaviors related to the use of marijuana, illicit drugs, alcohol, and tobacco; and including a measure of antisocial personality to differentiate the two groups. Gender differences were also significant. Altogether findings suggested that subtypes exist even in healthy control population and comparison studies (Case vs Control) need to be mindful of it.

neuroscience↗

Individual-Specific fMRI-Subspaces Improve Functional Connectivity Prediction of Behavior

There is significant interest in using resting-state functional connectivity (RSFC) to predict human behavior. Good behavioral prediction should in theory require RSFC to be sufficiently distinct across participants; if RSFC were the same across participants, then behavioral prediction would obviously be poor. Therefore, we hypothesize that removing common resting-state functional magnetic resonance imaging (rs-fMRI) signals that are shared across participants would improve behavioral prediction. Here, we considered 803 participants from the human connectome project (HCP) with four rs-fMRI runs. We applied the common and orthogonal basis extraction (COBE) technique to decompose each HCP run into two subspaces: a common (group-level) subspace shared across all participants and a subject-specific subspace. We found that the first common COBE component of the first HCP run was localized to the visual cortex and was unique to the run. On the other hand, the second common COBE component of the first HCP run and the first common COBE component of the remaining HCP runs were highly similar and localized to regions within the default network, including the posterior cingulate cortex and precuneus. Overall, this suggests the presence of run-specific (state-specific) effects that were shared across participants. By removing the first and second common COBE components from the first HCP run, and the first common COBE component from the remaining HCP runs, the resulting RSFC improves behavioral prediction by an average of 11.7% across 58 behavioral measures spanning cognition, emotion and personality.\n\nHighlightsO_LIWe decomposed rs-fMRI signals into common subspace & individual-specific subspace\nC_LIO_LICommon subspace is shared across all Human Connectome Project (HCP) participants\nC_LIO_LICommon subspaces are different across runs, suggesting state-specific effects\nC_LIO_LIIndividual-specific subspaces are unique to individuals\nC_LIO_LIRemoval of common subspace signals improve behavioral prediction by 11.7%\nC_LI

neuroscience↗