Biology subjects

Pande, V.

Publications and source records attributed to Pande, V..

12 recordsLinked to original sources

bioRxiv2026

A lipid cue drives the subcellular localization of a self-inserting bacterial transmembrane protein

The correct subcellular localization of proteins is a critical step underlying myriad biological processes, but the cues that drive the specific localization of integral membrane proteins in bacteria remain largely undeciphered. During sporulation in Bacillus subtilis, a rod-shaped outer "mother cell" constructs an internal spherical "forespore" cell that eventually matures into the spore. The integral membrane protein ShfA is made in the mother cell cytosol and localizes to the surface of the forespore. Here, we report that, despite being a multi-pass transmembrane protein, ShfA spontaneously inserts into the lipid bilayer via its N-terminal "YabQ" domain without the apparent need for a pre-localized insertase. ShfA preferentially inserts into cell division septa in multiple bacterial species, indicating that a widely conserved septal cue drives ShfA localization. Structural modeling suggested that the YabQ domain harbors a specific intramembrane groove that can bind the universal lipid carrier undecaprenyl phosphate (UndP), and UndP depletion in vivo disrupted proper ShfA localization. We propose that ShfA localizes to sites of active cell wall synthesis by binding to UndP and/or molecules like lipid I and lipid II that contain UndP and speculate that the function of ShfA is to stabilize these precursors of cell wall biogenesis from the harsh cytosolic nanoenvironment that surrounds the forespore during sporulation.

microbiology↗

bioRxiv2025

Unbiased Quantification of Persistent Postural and Motor Deficits Following Spinal Cord Injury in Mice

Spinal cord injury (SCI) causes multifaceted postural and motor impairments that are challenging to quantify. Conventional behavioral tests, such as the Basso mouse scale (BMS), rely on qualitative observations, which do not detect subtle yet significant functional deficits. To perform unbiased and quantitative evaluation of motor function and posture after SCI, we employed high-speed video tracking with machine learning-based whole body pose estimation and weight-bearing analyses using the Blackbox system in freely moving mice. We found enduring alterations in posture induced by SCI not captured by conventional metrics. Postural deficits included reduced hindpaw spacing with concomitant increased forepaw spacing, narrowed femur width, and altered hind paw angles, which remained evident beyond 42 days post-injury (dpi). Furthermore, sustained deficits in locomotor activity were identified as decreased distance traveled, decreased exploratory behavior, and disrupted fore-to-hindpaw speed ratio. Additionally, we analyzed behavioral motifs using Keypoint MoSeq software and found frequency changes in unique motor syllables correlating with forward acceleration and turning after SCI. Interestingly, while motor deficits persisted, sensory deficits, such as thermal and mechanical sensitivity, returned to baseline levels by 21 days after injury in C57Bl/6J mice, with no subsequent hypersensitivity. Lastly, we developed a web-based application to assist in visualization and analyses of Blackbox-based kinematic data. Altogether, our study identifies distinct postural and motor deficits pre- and post-SCI using accurate, unbiased, and quantitative behavioral assessments. By tracking the unique features of motor recovery trajectories, researchers can more accurately assess the effectiveness of SCI therapeutic strategies.

neuroscience↗

bioRxiv2024

Genotyping of paired clinical isolates using PvCSP, PvMSP3α PvMSP3βand exploring STRs to differentiate between relapse and reinfection in P. vivax

The challenge of eliminating Vivax malaria is due to the relapses caused by hypnozoites. Despite several attempts to identify molecular markers to differentiate between relapse and new infection, a reliable marker has not yet been established. To address this issue, a genomic study was conducted on paired samples of patients who had experienced Plasmodium vivax infection twice. Genotyping was performed on paired samples from 10 vivax malaria patients using five molecular markers to distinguish between relapse and new infection. Our findings indicate that one sample represented a second episode that was a relapse of the first, while another sample had three episodes, two of which were relapse episodes. We combined our clinical records with molecular inferences to identify each pair as a relapse. This particular study provides a momentary view of forthcoming research endeavors that could be undertaken to distinguish between a relapse and a novel infection. Given the notable genetic variability of Plasmodium vivax, it is crucial to harness various markers to discern between relapse and new infection. The findings of this study are poised to be of immense utility for upcoming marker research in the aforesaid aspect.

genomics↗

bioRxiv2023

Evolution of tandem repeats in putative CSP to enhance its function: A recent and exclusive event in Plasmodium vivax in India

The molecular hitchhiking model proposes that linked non-coding regions also undergo fixation, while fixing a beneficial allele in a population. This concept can be applied to identify loci with functional and evolutionary significance. Putative circumsporozoite protein (CSP) in Plasmodium vivax (PvpuCSP) identified following the molecular hitchhiking model, holds evolutionary significance. We investigated the extent of genetic polymorphism in PvpuCSP and the role of natural selection which shapes the genetic composition and maintains the diversity in P. vivax isolates from India. Sequencing the putative CSP of P. vivax (PvpuCSP) in 71 isolates revealed a well-conserved N- and C-terminal, constituting around 80% of the gene. PCR amplification and sequencing validated extensive diversity in the repeat region, ranging from 1.8 to 2.2 kb towards the C-terminal, identifying 37 different alleles from 71 samples. The recent and exclusive accumulation of repeats in puCSP within P. vivax highlights its highly variable length polymorphism, making it a potential marker for estimating diversity and infection complexity. Episodic diversifying selection in the PvpuCSP repeat region, evidenced by statistically significant p-values and likelihood ratios, enhances amino acid diversity at various phylogenetic levels, facilitating adaptation for accommodating different substrates for degradation.

evolutionary biology↗

bioRxiv2021

Filament dynamics driven by ATP hydrolysis modulates membrane binding of the bacterial actin MreB

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. While the eukaryotic actin utilizes ATP hydrolysis to drive filament treadmilling, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we report mechanistic insights into the interplay between nucleotide-binding, ATP hydrolysis and membrane-binding of Spiroplasma citri MreB5 (ScMreB5). Antiparallel double protofilament assembly of ScMreB5WT with ATP, ADP or AMPPNP and an ATPase deficient mutant ScMreB5E134A demonstrate that the filaments assemble independent of ATP hydrolysis. However, capture of the filament dynamics revealed that efficient filament formation, bundling through lateral interactions and filament disassembly are affected in ScMreB5E134A. Hence, the catalytic glutamate (Glu134 in ScMreB5) plays a dual role - it functions as a switch by sensing the ATP-bound state for filament assembly, and by assisting hydrolysis for triggering disassembly. Glu134 mutation also exhibits an allosteric effect on membrane binding, as observed from the reduced liposome binding compared to that of the wild type. Thus, ATP hydrolysis can modulate filament length and bundling, and consequently the orientation of MreB filaments on the cell membrane depending on the curvature. Binding of ScMreB5 with liposomes is mediated by surface charge-based interactions, demonstrating paralog and organism specific features for MreB function. We conclude that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis has been repurposed for modulating curvature-dependent organization of filaments on the membrane.

biochemistry↗

bioRxiv2021

Intragenomic sequence variations in the second internal transcribed spacer (ITS2) ribosomal DNA of the malaria vector Anopheles stephensi

Second Internal Transcribed Spacer (ITS2) ribosomal DNA (rDNA) sequence is a widely used molecular marker for species-identification or -delimitation due to observed concerted evolution which is believed to homogenize rDNA copies in an interbreeding population. However, intra-specific differences in ITS2 of Anopheles stephensi have been reported. This study reports the presence of intragenomic sequence variation in the ITS2-rDNA of An. stephensi and hypothesizes that observed intra-specific differences in this species may have resulted due to ambiguous DNA sequence-chromatogram resulting from intragenomic heterogeneity. Anopheles stephensi collected from different parts of India were sequenced for complete ITS2 and the variable region of 28S-rDNA (d1-d3 domains). Intragenomic variations were found in ITS2 region of all An. stephensi sequenced, but no such variation was observed in d1 to d3 domains of 28S-rDNA. Cloning and sequencing of ITS2 through the d3 domain of the 28S region of rDNA from representative samples from northern, central, and southern India confirmed the presence of intragenomic variation in ITS2 due to transitions at three loci and two bp indel in a di-nucleotide microsatellite locus. Multiple haplotypes were observed in ITS2 raised from such variations. Due to the absence of detectable intragenomic sequence variation in the d1 to d3 domain of 28S rDNA of An. stephensi, this region can serve as an ideal reference sequence for taxonomic and phylogenetic studies. The presence of intragenomic variation in rDNA should be carefully examined before using this as a molecular marker for species delimitation or phylogenetic analyses.

genomics↗

bioRxiv2020

Clustered γ-Protocadherins Regulate Cortical Interneuron Programmed Cell Death.

Cortical function critically depends on inhibitory/excitatory balance. GABAergic cortical inhibitory interneurons (cINs) are born in the ventral forebrain. After completing their migration into cortex, their final numbers are adjusted-during a period of postnatal development - by programmed cell death (PCD). The mechanisms that regulate cIN elimination remain controversial. Here we show that genes in the protocadherin (Pcdh)-{gamma} gene cluster, but not in the Pcdh- or Pcdh-{beta} clusters, are required for survival of cINs through a BAX-dependent mechanism. Surprisingly, the physiological and morphological properties of Pcdh-{gamma} deficient and wild type cINs during PCD were indistinguishable. Co-transplantation of wild type and Pcdh-{gamma} deficient interneuron precursor cells demonstrate that: 1) the number of mutant cINs eliminated was much higher than that of wild type cells, but the proportion of mutant or WT cells undergoing cell death was not affected by their density; 2) the presence of mutant cINs increases cell death among wild-type counterparts, and 3) cIN survival is dependent on the expression of Pcdh-{gamma} C3, C4, and C5. We conclude that Pcdh-{gamma}, and specifically {gamma}C3, {gamma}C4, and {gamma}C5, play a critical role in regulating cIN survival during the endogenous period of PCD. SignificanceGABAergic cortical inhibitory interneurons (cINs) in the cerebral cortex originate from the ventral embryonic forebrain. After a long migration, they come together with local excitatory neurons to form cortical circuits. These circuits are responsible for higher brain functions, and the improper balance of excitation/inhibition in the cortex can result in mental diseases. Therefore, an understanding of how the final number of cINs is determined is both biologically and, likely, therapeutically significant. Here we show that cell surface homophilic binding proteins belonging to the clustered protocadherin gene family, specifically three isoforms in the Pcdh-{gamma} cluster, play a key role in the regulation cIN programmed cell death. Co-transplantation of mutant and wild-type cINs shows that Pcdh-{gamma} genes have cell-autonomous and non-cell autonomous roles in the regulation of cIN cell death. This work will help identify the molecular mechanisms and cell-cell interactions that determine how the proper ratio of excitatory to inhibitory neurons is determined in the cerebral cortex.

neuroscience↗

bioRxiv2019

REP-X: An Evolution-guided Strategy for the Rational Design of Cysteine-less Protein Variants

Site-specific labeling of proteins is often a prerequisite for biophysical and biochemical characterization. Chemical modification of a unique cysteine residue is among the most facile methods for site-specific labeling of proteins. However, many proteins have multiple reactive cysteines, which must be mutated to other residues to enable labeling of unique positions. This trial-and-error process often results in cysteine-free proteins with reduced activity or stability. Herein we describe a general methodology to rationally engineer cysteine-less proteins. Briefly, natural variation across orthologues is exploited to identify suitable cysteine replacements compatible with protein activity and stability. As a proof-of-concept, we recount the successful engineering of a cysteine-less mutant of the group II chaperonin from methanogenic archaeon Methanococcus maripaludis. A webapp, REP-X (Replacement at Endogenous Positions from eXtant sequences), which enables users to design their own cysteine-less protein variants, will make this rational approach widely available.

biochemistry↗

bioRxiv2019

Genetic changes of P. vivax tempers host tissue-specific responses in Anopheles stephensi

In our preceding study (Sharma et al., 2019; BioRxiv) we showed that in the gut lumen Plasmodium vivax follows a unique strategy of immuno-suppression by disabling gut flora proliferation. Here, we further demonstrate that post gut invasion, a shrewd molecular relationship with individual tissues such as midgut, hemocyte, salivary glands, and strategic changes in the genetic makeup of P. vivax favors its survival in the mosquito host. A transient suppression of metabolic machinery by early oocysts, and increased immunity against late oocysts suggested a unique mechanism of gut homeostasis restoration and Plasmodium population regulation. Though a hyper immune response of hemocyte was a key to remove free circulating sporozoites, but a strong suppression of salivary metabolic activities, may favor successful survival of invaded sporozoites. Finally, genetic alteration of P. vivax ensures evasion of mosquito responses. Conclusively, our system-wide RNAseq analysis provides first genetic evidences of direct mosquito-Plasmodium interaction and establishes a functional correlation.\n\nAuthor SummaryMalaria transmission dynamics is heavily influenced by mosquito -parasite interaction. When passing through tissue specific barriers, Plasmodium have to compromise by losing its own population, but genetic relation is unknown. To win the developmental race Plasmodium need to overcome two important immuno-physiological barriers. First one accounts an indirect 24-30hr long pre-invasive gut-microbe-parasite interaction in the gut lumen. And second one follows a direct post gut invasive 14-18 days interaction with midgut, hemocyte and salivary glands. During pre-invasive phase of interaction, we showed Plasmodium vivax follows immuno-suppression strategy by restricting microbial growth in the gut lumen. Here, we demonstrate that switch of parasite from one stage to another stage within mosquito vector is accompanied by genetic changes of parasite. Our data suggests genetic makeup change enables the parasite to manipulate the metabolism of mosquito tissues. This strategy not only clear off multifaceted mosquitos tissue specific immune responses, but also favors Plasmodium own survival and transmission. Comprehending this tissue specific interaction between host and parasite at molecular level could provide new tool to intervene the plasmodium life cycle within vector.

genomics↗

bioRxiv2018

CFTR misfolds during native-centric simulations due to entropic penalties of native state formation

Cystic fibrosis (CF) is a common genetic disorder that affects approximately 70,000 people worldwide. It is caused by mutation-induced defects in synthesis, folding, processing, or function of the Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR), a chloride-selective ion channel required for the proper functioning of secretory epithelia in tissues such as the lung, pancreas, and skin. The most common cause of CF is the single-residue deletion of F508 (F508del), a mutation present in one or both alleles in 90% of patients that induces severe folding defects and results in greatly reduced expression of the protein. Despite its medical importance, high-resolution mechanistic information about CFTR folding is lacking. In this study, we used molecular dynamics simulation with a native-centric force field to examine the folding and assembly of both full-length CFTR and the isolated first nucleotide-binding domain (NBD1). We observed that the protein was capable of substantial misfolding on both the intradomain and interdomain scale due to entropically favorable kinetic traps that exist on CFTRs folding free energy surface. These results suggest that even wild type CFTR, in the absence of any disease-related mutations, has suboptimal folding efficiency. We speculate that such entropically-driven misfolding also occurs in disease-prone mutants such as F508del and contributes to the proteins poor in vivo activity.

molecular biology↗

bioRxiv2017

Towards simple kinetic models of functional dynamics for a kinase subfamily

Kinases are ubiquitous enzymes involved in the regulation of critical cellular pathways and have been implicated in several cancers. Consequently, the kinetics and thermodynamics of prototypical kinases are of interest and have been the subject of numerous experimental studies. In-silico modeling of the conformational ensembles of these enzymes, on the other hand, is lacking due to inherent computational limitations. Recent algorithmic advances combined with homology modeling and parallel simulations allow us to address this computational sampling bottleneck. Here, we present the results of molecular dynamics (MD) studies for seven Src family kinase (SFK) members Fyn, Lyn, Lck, Hck, Fgr, Yes, and Blk. We present a sequence invariant extension to Markov state models (MSMs), which allows us to quantitatively compare the structural ensembles of the seven kinases. Our findings indicate that in the absence of their regulatory partners, SFK members have similar in-silico dynamics with active state populations ranging from 4-40% and activation timescales in the hundreds of microseconds. Furthermore, we observe several potentially druggable intermediate states, including a pocket next to the ATP binding site that could be potentially targeted via a small molecule inhibitors. These results establish the utility of MSMs for studying protein families.

biophysics↗

bioRxiv2017

Transferable tICA-Metadynamics: Efficient sampling of protein mutants by transferring information from the wild type’s Markov state model

We recently showed that the time-structure based independent component analysis method from Markov state model literature provided a set of variationally optimal slow collective variables for Metadynamics (tICA-Metadynamics). In this paper, we extend the methodology towards efficient sampling of related mutants by borrowing ideas from transfer learning methods in machine learning. Our method explicitly assumes that a similar set of slow modes and metastable states are found in both the wild type (base line) and its mutants. Under this assumption, we describe a few simple techniques using sequence mapping for transferring the slow modes and structural information contained in the wild type simulation to a mutant model for performing enhanced sampling. The resulting simulations can then be reweighted onto the full-phase space using Multi-state Bennett Acceptance Ratio, allowing for thermodynamic comparison against the wild type. We first benchmark our methodology by re-capturing alanine dipeptide dynamics across a range of different atomistic force fields, including the polarizable Amoeba force field, after learning a set of slow modes using Amber ff99sb-ILDN. We next extend the method by including structural data from the wild type simulation and apply the technique to recapturing the affects of the GTT mutation on the FIP35 WW domain.

biophysics↗