Biology subjects

Gupta, T.

Publications and source records attributed to Gupta, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Immune determinants of leuprolide-associated cardiovascular risk in prostate cancer

Leuprolide, a widely used androgen-deprivation therapy (ADT) for prostate cancer (PC), has been associated with increased cardiovascular events (CVE), although the underlying mechanisms remain unclear. Through integrated spatial transcriptomic analyses of murine heart and immune-stromal profiling in patient blood, we identified immune programs linked to leuprolide-associated CVE risk. Spatial analyses revealed the distinct organization of lymphoid (T+B cells) aggregates and inflammatory macrophages in the plaque following leuprolide exposure that accompanied by ventricular hypertrophy, relative to normal vasculature. Parallel profiling of circulatory soluble and cellular components in patients demonstrated CD8+T cell enriched pattern in ~44% of patients, including memory and type 1 cytotoxic subsets that linked to treatment-induced CVE, such as ventricular dysfunctions, but no contribution of adipocytokines. These translational findings highlight the stratification of leuprolide-associated CVE risk in PC patients based on circulating T cell profiling and could guide the risk mitigation with alternative ADT agents.

immunology↗

bioRxiv2026

Deep shifts in Evolutionary Rate Trajectories of Ancient Bacterial Genes

Reconstruction of ancestral gene repertoires from extant genomes captures only the genes that survived; those lost from the record are invisible. Among the genes that did persist, selective pressures change not only across lineages but across time. Here, we resolve evolutionary rate trajectories across 528 genes in the Last Bacterial Common Ancestor (LBCA). We hypothesized that LBCA genes would show distinct evolutionary rate trajectories across bacterial history and tested this by resolving normalized branch lengths across five calibrated taxonomic intervals (phylum, class, order, family, and genus), spanning approximately 2.1 billion years of bacterial diversification. The distribution of rate trajectories is continuous, but four clusters capture the major patterns: Decelerating, Class-Peaking, Constant, and Accelerating. The Decelerating and Class-Peaking clusters are composed predominantly of Genetic Information Processing genes, whereas the Constant and Accelerating clusters are enriched for Metabolic genes. Specifically, the Decelerating cluster is enriched for core informational machinery, including translation initiation factors and components of the expressome, the molecular complex physically coupling transcription and translation, suggesting that transcription-translation interfaces locked in early in bacterial history. Cofactor-dependence and cofactor biosynthesis are decoupled: biosynthetic pathways producing metallocofactors such as heme, molybdopterin, and cobalamin, concentrated in the Constant and Accelerating clusters but proportionally more proteins use inorganic cofactors in the Decelerating and Class-Peaking clusters. This offset coincides with the shift in metal bioavailability associated with the Great Oxidation Event and reveals genomic fingerprints of the co-evolution of bacterial metabolism with planetary geochemistry.

evolutionary biology↗

bioRxiv2026

Exosome-Derived Proteomic Signatures Highlight Pathogenic Mechanisms in Moyamoya Disease

BackgroundThe etiology and molecular mechanisms of Moyamoya disease (MMD) remain unclear. Exosomes, as carriers of bioactive molecules, may reflect disease-specific alterations and serve as potential biomarkers. This study aimed to investigate disease mechanisms using proteomic profiling of serum-derived exosomes (SDEs) in MMD. Materials and MethodsPeripheral blood each from 15 MMD patients and 15 healthy-controls were used to isolate SDEs via ultracentrifugation. Proteins from pooled SDEs were extracted, digested, and analyzed by LC-MS/MS. Differentially expressed proteins were examined using MetaboAnalyst, DAVID, Enrichr, STRING, and Cytoscape. Key targets were validated at transcript and protein levels using RT-qPCR and ELISA in independent cohorts. ResultsA total of 2,554 proteins were identified, with 213 showing differential expression (118 upregulated, 95 downregulated; p [≤] 0.05). Functional and pathway analyses revealed enrichment in angiogenesis, cytoskeletal remodeling, and endothelial signaling. PRKG2 and MYC were upregulated, while RHOA was downregulated, highlighting their involvement in focal adhesion and PI3K-AKT pathways. Validation confirmed these findings. ConclusionDysregulated proteins were linked to RHOA-ROCK and PI3K-Akt signaling, suggesting their role in driving VSMC phenotypic switching, contributing to vascular occlusion. These findings indicate that altered exosomal-proteins may participate in maladaptive vascular remodeling, although the initial trigger for VSMC transition remains unknown.

systems biology↗

bioRxiv2026

Postnatal Reprogramming Shapes Human Intestinal Epithelial Immune Competency

At birth, the intestine must rapidly adapt to enable nutritional function and immune microbial tolerance. Here, integrating single-cell multi-omics and spatial transcriptomics we define the circuits underpinning this process. We identify asynchronous developmental trajectories with postnatal epithelial reprogramming characterised by coordinated changes in metabolism, junctional structure and innate defence. At birth epithelial stem cells demonstrate dynamic enhancer remodelling, with accessibility often preceding transcription. Fetal stemness elements remain accessible despite reduced transcription across epithelial lineages, retaining plasticity potential. Post-natal epithelia experience sequential homing of myeloid cells followed by innate T cells with peri-epithelial B cells localising later in infancy. Using developmentally staged organoids, we show that epithelial responses to inflammatory stimuli are age-dependent and constrained in early life. We identify BHLHE40 as an early-life regulator that attenuates the impact of interferon- and NF-{kappa}B-driven signalling. Altogether we define the events driving epithelial licensing and barrier adaptation at birth and through infancy.

cell biology↗

bioRxiv2025

Basal Ganglia Dopamine Availability and Immune Activation Interact and Relate to Anhedonia Severity among Youth with Depression

Anhedonia emerges in adolescence, has putative substrates in neural reward and dopamine systems, and is a hallmark of poor course in depression. Psychoneuroimmunology models suggest altered immune and reward systems may jointly underlie its development. When dopamine availability (rather than function) is considered, immune activation may be understood as a moderator amplifying how lower dopamine levels translate into motivational deficits. This cross-sectional study analyzed data from 55 youth with current depression (Mage=21.4 years; Female=75%). Plasma was assayed for relative quantification of 92 immune proteins, followed by dimensionality reduction via principal component (PC) analysis, forming five PC scores. Dopamine availability was indexed by basal ganglia tissue iron, quantified by MRI R2, a measure detectable in youth. Anhedonia was assessed using the Snaith-Hamilton Pleasure Scale (SHAPS), thought to capture overall anhedonia, and Positive Valence Systems Scale (PVSS), which provides overall, anticipatory, and consummatory anhedonia scores. PC1 scores--whose loadings indicated broadly distributed immune protein elevations consistent with general immune activation--moderated the relationship between basal ganglia dopamine availability and anhedonia: At higher PC1 levels, lower dopamine availability was linked to greater overall anhedonia (as measured by SHAPS) and anticipatory anhedonia, but not to consummatory anhedonia. Lower PC2 scores were associated with greater PVSS overall and consummatory anhedonia. Analyses examining individual basal ganglia regions suggest potential differences in dopaminergic-immune interactions across these regions, with effects most consistently observed in the putamen. Findings highlight nuanced neuroimmune pathways underlying anhedonia components in youth and support inflammation-based models of depression emphasizing dopaminergic-immune interplay.

neuroscience↗

bioRxiv2025

Chromosome-associated spot formation by human cytomegalovirus immediate early 1 (IE1) protein

HCMV is associated with severe disease in immunocompromised patients and is a cause of congenital disease. Already more than 30 years ago, IE1 was described to bind to mitotic chromosomes, however the implications of this finding have yet to be determined. HCMV can infect a wide range of cell types and was also detected in different tumours; however, the majority of molecular studies relating to this virus are performed in fibroblasts or monocytic cells. We examined the localization of HCMV IE1 across multiple cell types, including tumour cell lines, to better understand how the protein behaves in these cellular contexts. IE1 is one of the first HCMV genes expressed after lytic infection of the cell and it was observed to distribute evenly over the chromosomes in so-called "chromosome painting" pattern. In leukemia and glioblastoma cells we detected a novel mitotic localization pattern of IE1 - in chromosome-associated spots (CAS), in addition to "painting the chromosomes", as reported before. We demonstrate that the IE1 core domain mediates CAS localization and that the distribution pattern of IE1 on mitotic chromosomes is influenced by both the cell type being infected and the level of IE1 expression. The novel CAS localization of IE1 suggests a new, possibly cell-type specific function of this protein, distinct from its role in transcriptional regulation.

microbiology↗

bioRxiv2025

Peptide-functionalized fluorescent polymeric nanoparticles: polysarcosine length defines stealth properties

Stealth properties of nanoparticles are essential for their proper functionalization in biological systems. To address limitations of polyethylene glycol (PEG), commonly used for this purpose, we explore the potential of polysarcosine (PSar) as stealth shell in peptide-functionalized dye-loaded polymeric NPs. To this end, polymeric NPs loaded with rhodamine dye with bulky hydrophobic counterion and bearing azide groups at their surface were grafted with PSar of different lengths ranging from 5 to 19 sarcosine units using strain-promoted cycloaddition. The obtained peptide-functionalized NPs showed remarkable colloidal stability in physiological media. The length of PSar showed a profound effect on stealth properties of NPs. The increase in the length of grafted PSar lead to decrease in the negative surface charge to nearly neutral values and decreased protein adsorption according to fluorescence correlation spectroscopy. The NPs with 19mer PSar showed minimal interactions with live cells and glass surfaces in a complex biological medium, in contrast to its shorter PSar analogues. These stealth NPs bearing HaloTag ligand enabled specific targeting of proteins at the cell surface. The obtained results show that a relatively short PSar peptide can be used for achieving stealth properties in polymeric NPs, allowing specific protein targeting with minimized non-specific interactions. The obtained PSar-functionalized polymeric NPs appear as a powerful platform for the fabrication of the next generation of nanomaterials for bioimaging and biosensing applications.

pharmacology and toxicology↗

bioRxiv2025

Turning blood to brain cells: a plasma mediated reprograming model

The lack of effective stem cell protocols for generating personalized neurovascular niches poses a critical challenge in precision medicine. While iPSC-based methods are explored, their clinical use is hindered by high costs, long timelines, and cancer risks. Recent advancements in plasma-driven differentiation, using circulating monocytes, offer a promising solution as they can be reprogrammed into neuron-like, endothelial-like, and hematopoietic cells without genetic manipulation, by inducing growth factors mediated transdifferentiation. Vasculature is integral to neurodevelopment, with early blood supply transitioning from the perineural to intrinsic vascular plexus, driven by neuro-hematovascular signaling. The choroid plexus selectively transports proteins and growth factors from blood to CSF, supporting neural proliferation and differentiation. Building on these insights, we leveraged the innate reprograming potential of blood-derived cells to generate neuro-hematovascular niches using a novel PITTRep methodology, devoid of transgene and growth factor mediated transdifferentiation opening new avenues for regenerative and investigative neurovascular studies.

cell biology↗

bioRxiv2024

Mutations in the microexon splicing regulator srrm4 have minor phenotypic effects on zebrafish neural development

Achieving a diversity of neuronal cell types and circuits during brain development requires alternative splicing of developmentally regulated mRNA transcripts. Microexons are a type of alternatively spliced exon that are 3-27 nucleotides in length and are predominantly expressed in neuronal tissues. A key regulator of microexon splicing is the RNA-binding protein Serine/arginine repetitive matrix 4 (Srrm4). Srrm4 is a highly conserved, vertebrate splicing factor that is part of an ancient family of splicing proteins. To better understand the function of Srrm4 during brain development, we examined neural expression of zebrafish srrm4 from days 1-5 of development using fluorescence in situ hybridization. We found that srrm4 has a dynamically changing expression pattern, with expression in diverse cell types and stages during development. We then used CRISPR-based mutagenesis to generate zebrafish srrm4 mutants. Unlike previously described morphant phenotypes, srrm4 mutants did not show overt morphological defects. Moreover, sequencing of wild-type and mutant transcriptomes revealed only minor changes in splicing. srrm4 thus appears to have a limited role in zebrafish neural development.

developmental biology↗

bioRxiv2024

Building neurovascular tissue from autologous blood for modeling brain-activity

There are no faithful individualized stem cell-based bioengineered neuro-vascularized models that can recapitulate the physiological hemodynamic phenomenon of neuro-vascular coupling (NVC)-the principal behind BOLD (blood oxygen level-dependent) signal in functional neuroimaging, thereby dissuading the research in exploring the brain activity-based investigative studies in neurological/neurosensory diseases. This encouraged us to establish a preclinical optoacoustic (Hb/dHb hemoglobin/deoxyhemoglobin) imaging-competent in vitro neuro-vascularized model by employing a novel cellular reprograming PITTRep (Plasma Induced Transcriptomics/ epi-Transcriptomics Reprograming) approach. The current reprograming approach is based on coaxing autologous blood components to ecto-mesodermal lineage intermediates that can subsequently self-pattern into neurovascular tissue by harnessing the hemorheological properties of RBCs. The nature of blood flow is non-Newtonian and is a function of RBC concentration /haematocrit when they flow through the regions of low shear rates as seen in cerebral microcirculation. The current reprograming approach is a modification of our previous cellular reprograming approach that employed a Newtonian plasma fluid. The autologous blood-derived neurovascular tissue is free from exogenous genetic modification, external growth factors, and induced pluripotent stem cell (iPSC) derivation. This model uniquely integrates functional vasculature and neurogenesis. The current reprogramming approach resulted (in part) serendipitously while testing a potential (yet completely unexplored) hypothesis of haemodynamic reprograming by leveraging the fluid mechanic feature of blood erythrocytes as seen in thrombus formation during cerebral ischemic stroke, that is characterized by physiologically intriguing yet clinically meaningful neurological recovery (neuroplasticity) during an early time window. The current study attempted to induce "a post stroke-like model" of adult neurogenesis with functional synaptogenesis by instructing autologous blood components into thrombus formation through incorporation of erythrocytes in varying concentrations. We tried to instruct adult neurogenesis and neuroplasticity (a relatively non-resilient phenomenon under in vitro conditions) by co-induction of a neuro-vascular niche (NVN). These NVNs are marked by dendrites, synapses, astrogliosis, microglia activation, and growth factor signaling, thus phenocopying molecular and cellular aspects of post-stroke recovery window. The induction of neuro-vascularized niches and functional neuro-vascular coupling (NVC) was characterized by confocal microscopy, scanning electron microscopy, proteomic profiling, and Hb/dHb spectra based optoacoustic imaging. The blood thrombus formation was checked by rotational thromboelastometry (ROTEM), and switching of adult-to-embryonic hemoglobin was confirmed by routine hemoglobin typing. We also attempted to establish patient-specific neuro-vascularized niches from autologous blood of sensorineural hearing loss (SNHL) patients. The individualized neovascularised tissues are intended to be employed for investigating deregulated synaptic plasticity/ long term potentiation underlying poor auditory comprehension outcomes in school going kids suffering from SNHL that greatly compromises their academic performance and socio-behavioural-cognitive development. The attendant multiomics of patient-specific NVNs may have potential implications in developing stem-cell based therapies for neurosensory and cerebrovascular diseases.

neuroscience↗

bioRxiv2024

Prefrontal dopamine circuits are required for active avoidance learning but not fear learning

To survive, animals must rapidly learn to avoid predictable threats. Such learning depends on detecting reliable cue-outcome relationships that efficiently drive behavioral adaptations. The medial prefrontal cortex (mPFC) integrates learned information about the environment to guide adaptive behaviors1-7 and is critical for threat avoidance8-15. However, as most studies have focused on well-learned threat avoidance strategies, the specific inputs that signal avoidability and drive rapid avoidance learning remain poorly understood. Dopamine (DA) inputs from the ventral tegmental area (VTA) potently modulate prefrontal function and are preferentially engaged by aversive stimuli16-21. Previous studies implicated VTA and prefrontal DA in aversive learning22-25, but their findings have been constrained by limited spatiotemporal resolution. Here, we used high-resolution tools to dissect the role of the VTA-mPFC DA circuit in rapid avoidance learning. Optogenetic suppression of VTA DA terminals in mPFC selectively impaired learning of a cued avoidance response, without affecting cue-shock association learning, reactive escape behaviors, or expression of previously learned avoidance. Using a fluorescent DA sensor, we observe rapid, event-locked DA activity that emerged transiently during the initiation of learning. Increased DA encoded aversive outcomes and their predictive cues, while decreased DA encoded their omission and predicted how quickly mice learned to avoid. In yoked mice lacking control over shock omission, these dynamics were largely absent. Together, these findings demonstrate that the VTA-mPFC DA circuit is necessary for rapid acquisition of proactive avoidance behaviors and reveal transient event-related DA signals that underlie this form of learning.

neuroscience↗

bioRxiv2023

Critical Behavioral Traits Foster Peer Engagement in Online Mental Health Communities

Online Mental Health Communities (OMHCs), such as Reddit, have witnessed a surge in popularity as go-to platforms for seeking information and support in managing mental health needs. Platforms like Reddit offer immediate interactions with peers, granting users a vital space for seeking mental health assistance. However, the largely unregulated nature of these platforms introduces intricate challenges for both users and society at large. This study explores the factors that drive peer engagement within counseling threads, aiming to enhance our understanding of this critical phenomenon. We introduce BeCOPE, a novel behavior encoded Peer counseling dataset comprising over 10,118 posts and 58,279 comments sourced from 21 mental health-specific subreddits. The dataset is annotated using three major fine-grained behavior labels: (a) intent, (b) criticism, and (c) readability, along with the emotion labels. Our analysis indicates the prominence of "self-criticism" as the most prevalent form of criticism expressed by help-seekers, accounting for a significant 43% of interactions. Intriguingly, we observe that individuals who explicitly express their need for help are 18.01% more likely to receive assistance compared to those who present "surveys" or engage in "rants." Furthermore, we highlight the pivotal role of well-articulated problem descriptions, showing that superior readability effectively doubles the likelihood of receiving the sought-after support. Our study emphasizes the essential role of OMHCs in offering personalized guidance and unveils behavior-driven engagement patterns.

neuroscience↗

bioRxiv2023

Maternal regulation of the vertebrate egg-to-embryo transition

Egg activation and early embryonic development are complex and highly regulated processes that involve a series of coordinated cellular and molecular events after fertilization. While significant progress has been made in unraveling the mechanisms underlying these processes, there remains a need to comprehensively understand the precise molecular pathways and regulatory factors involved. We characterized four recessive maternal-effect mutants identified from a zebrafish forward genetic screen that function during the egg-to-embryo transition. We found that these genes encompass distinct aspects of egg activation, including cortical granule biology, cytoplasmic segregation, and the suppression of microtubule organizing center (MTOC) assembly and ectopic aster-like microtubule formation. These genes are essential to the development of the early embryo and the establishment of the basic body plan. Notably, we discovered a novel gene that we named krang, which is highly conserved across metazoans. Maternal Krang was found to be associated with the function of cortical granules during egg activation. This collection of mutants represents valuable tools to understand the genetic architecture underlying phenotypic traits shaping the egg-to-embryo transition. Furthermore, these results highlight the evolutionary conservation of maternal functions in diverse species. By deepening our understanding of these findings, we will improve our knowledge of reproductive traits and potentially develop new diagnostic tools to address human reproductive disorders. Author SummaryDuring egg activation, the egg undergoes a series of biochemical and cellular modifications that activate its dormant metabolism, preparing it to initiate embryonic development. Egg activation initiates the completion of meiosis, and the newly formed zygote undergoes a series of additional changes, including the cell cycle establishment. However, our knowledge of the molecular mechanisms controlling these processes remain limited. We identified four recessive maternal-effect mutants in zebrafish that exhibit a range of developmental alterations during egg activation and early embryogenesis. We found that these maternally acting mutant genes are associated with defects in cortical granule exocytosis, yolk-cytoplasm segregation, microtubule nucleation and dynamics that are critical for the egg-to-embryo transition. Our results suggest that the proper regulation of these processes is essential for successful egg development and embryogenesis. We characterized the Krang factor, which regulates aspects of egg activation likely by modulating the secretory pathway. Further studies on this gene may provide new insights into the molecular mechanisms underlying oocyte development and egg quality acquisition. Overall, our collection of maternal-effect mutants sheds light on the proper regulation of key molecular and cellular events for successful egg development, with important implications for reproductive medicine and assisted reproductive technologies.

developmental biology↗

bioRxiv2023

Maternal regulation of the vertebrate oocyte-to-egg transition

Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the oocyte and egg to acquire developmental competence and ensure the production of viable embryos. The oocyte-to-egg transition consists of the regulation of multiple molecular processes both cytoplasmic and nuclear acting in the late oocyte during a process called oocyte maturation. However, the molecular nature and functional importance of factors acting at this stage remain poorly understood. Here, we present a collection of 5 recessive maternal-effect mutants identified in a zebrafish forward genetic screen that reveal unique molecular insights into the mechanisms controlling the vertebrate oviparous oocyte-to-egg transition. We identified critical cytoplasmic regulators of yolk globule formation and maturation that are essential for egg development and embryogenesis. Specifically, the maternal-effect genes, over easy, poached, p33bjta, and black caviar control yolk globule sizing and/or protein cleavage during oogenesis, likely through endolysosomal organization independent of nuclear oocyte maturation. Furthermore, we cloned one of the mutant genes, identifying a subunit of the Adaptor Protein complex 5, which regulates intracellular trafficking, and yolk vesicle formation. Together, these mutant genes represent novel genetic entry points to decipher the molecular mechanisms functioning in the oocyte-to-egg transition, fertility, and human disease. Additionally, our genetic screen provides valuable functional tools for exploring the evolutionary fates of maternal factors and their contribution to developmental strategies for reproductive success in metazoans. Author SummaryThe oocyte-to-egg transition consists of the coordinated regulation of multiple molecular processes acting in the late oocyte. This transcriptionally silent period requires the precisely timed function of maternally-supplied gene products during oogenesis. However, knowledge of their molecular nature and in vivo function remains incomplete. The mutants reported here provide access to maternal factors regulating the processes that prepare an oocyte for reproductive competence and embryogenesis. We have identified essential regulators of yolk granule formation and protein processing. Specifically, we found that the highly conserved maternal Ap5m1 protein regulates yolk granule maturation, which generate essential nutrients and immunity for growth and development in oviparous animals. The mutants presented here represent attractive genetic models to investigate the molecular and cell biological mechanisms that control the oocyte-to-egg transition, as well as reveal a collection of genetic factors indispensable for reproduction and survival. Importantly, knowledge of their genetic underpinnings and biological importance in reproduction will also pave the way to investigate the evolution of maternal genes during vertebrate development.

developmental biology↗

bioRxiv2023

Age-associated elevated inflammation and immune pathways in the mice heart are linked to Adiponectin-Adipor1-RelA signaling

Inflammatory gene profiles using RNA seq analysis were studied by measuring pro-inflammatory cytokines and chemokines levels. qRT-PCR and Western blot analysis were used to validate the expression profile of these inflammatory mediators. Using flow cytometry analysis, CD11b+ monocytes and CD64+ Ly6C were quantified in the young and old hearts. The inflammatory response, Adipor1 and Adipor2 gene expression, RelA nuclear translocation and the effects of adiponectin in LPS-stimulated or Adipor1 silenced H9C2 cells were studied. Gene ontology analysis using differentially expressed genes revealed an enrichment of immune response pathways in the old mice hearts when compared to young mice hearts. Western blot analysis confirmed the down regulation of several anti-inflammatory proteins and the upregulation of pro-inflammatory proteins including CD68, NF-kB1 and Rel-A, in the old mice hearts. Flow cytometry suggested an infiltration of CD11b+monocytes and CD64+ Ly6C-high macrophages in the old mice hearts compared to younger hearts confirming an increased inflammation in the older hearts. Mechanistically, to understand if the Adiponectin-Adipor1-NFkB axis regulates inflammation in the aging heart, Adipor1 and Adipor2 genes were silenced in H9c2 cardiomyocytes. Immune response genes were elevated in the Adipor1 silenced H9c2 cells but not in Adipor2 silenced cells. Pretreatment with Adiponectin (APN) attenuated the Adipor1 silenced or lipopolysaccharides (LPS)-stimulated expression of inflammatory genes in H9c2 cardiomyocytes. APN also attenuated the nuclear translocation of RelA and induction of immune response genes in Adipor1 silenced or LPS-challenged H9c2 cardiomyocytes. APN-AdipoR1-RelA signaling might be a novel therapeutic target for the treatment of inflamed elderly hearts.

immunology↗

bioRxiv2022

First report of classical knockdown resistance (kdr) mutation, L1014F, in human head louse Pediculus humanus capitis (Phthiraptera: Anoplura)

There are at least three known knockdown resistance (kdr) mutations reported globally in the human head louse Pediculus humanus capitis (Phthiraptera: Anoplura) that are associated with reduced sensitivity to pyrethroids. However, prevalence of kdr mutation is not known in the Indian subcontinent. To identify kdr mutations in the Indian population, the genomic region of the voltage-gated sodium channel gene encompassing IIS1-2 linker to IIS6 segments was PCR-amplified and sequenced from P. humanus capitis samples collected from different geographic localities of India. DNA sequencing revealed the presence of four kdr mutations, i.e., M827I, T929I, L932F and L1014F. The presence of a classical kdr mutation, L1014F, a most widely reported mutation across insect-taxa associated with kdr-trait, is being reported for the first time in P. humanus capitis.

molecular biology↗

bioRxiv2022

Contributions and synaptic basis of diverse cortical neuron responses to task performance

Neuronal responses during behavior are diverse, ranging from highly reliable classical responses to irregular or seemingly-random non-classically responsive firing. While a continuum of response properties is frequently observed across neural systems, little is known about the synaptic origins and contributions of diverse response profiles to network function, perception, and behavior. Here we use a task-performing, spiking recurrent neural network model incorporating spike-timing-dependent plasticity that captures heterogeneous responses measured from auditory cortex of behaving rodents. Classically responsive and non-classically responsive model units contributed to task performance via output and recurrent connections, respectively. Excitatory and inhibitory plasticity independently shaped spiking responses and task performance. Local patterns of synaptic inputs predicted spiking response properties of network units as well as the responses of auditory cortical neurons from in vivo whole-cell recordings during behavior. Thus a diversity of neural response profiles emerges from synaptic plasticity rules with distinctly important functions for network performance.

neuroscience↗

bioRxiv2021

Conformational flexibility is a key determinant of the lytic activity of the pore forming protein, Cytolysin A

Bacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes forming nanopores, that cause cell lysis to promote a wide range of bacterial infections. Cytolysin A (ClyA), an alpha()-PFT, is known to undergo one of the largest conformational changes during its transition from a water soluble monomeric form to the membrane embedded dodecameric nanopore assembly. Despite extensive work on the structure and assembly of ClyA, a complete molecular picture of the interplay between the protein segments and membrane lipids driving this transformation remains elusive. In this study, we combine experiments and all-atom molecular dynamics (MD) simulations of ClyA and its mutants to unravel the role of the two key membrane interacting motifs, namely, the {beta}-tongue and N-terminus helix, in facilitating this critical transition. Erythrocyte turbidity and vesicle leakage assays reveal a loss of activity for {beta}-tongue mutant (Y178F), and delayed kinetics for the N-terminus mutants (Y27A and Y27F). All atom, thermal unfolding molecular dynamics simulations of the monomer carried out at 310, 350 and 400 K reveal a distinct reduction in the flexibility in both the {beta}-tongue and N-terminal regions of the mutants when compared with the wild type. This decreased loss of conformational flexbility correlates positively with the reduced lytic and leakage activity observed in experiments, indicating that the tendency to lose secondary structure in the {beta}-tongue region is an important step in the conformational transition bistability of the ClyA protein. Simulations with the membrane inserted oligomeric arcs representing the pore state reveal a greater destabilization tendency among the {beta}-tongue mutant as inferred from secondary structure and N-terminal positioning. Our combined experimental and simulation study, reveals that conformational flexibility is indispensable for the outward movement of the {beta}-tongue and the tendency to induce disorder in the {beta}-tongue is an important step in the transition to the membrane mediated helix-turn-helix motif integral to ClyA pore formation. This observed loss of secondary structure is akin to the structural transitions observed in intrinsically disordered proteins (IDPs) to support protein function. Our finding suggest that inherent flexibility in the protein could play a wider and hitherto unrecognized role in the membrane mediated conformational transitions of PFTs in general. Author summaryBacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes to form bilayer spanning nanopores. Unregulated pore formation disrupts ionic balance that compromises the permeability of the cell, leading to cell death and infection. PFTs display remarkable structural plasticity that allow these proteins to interconvert between the two physiochemically distinct water soluble and membrane bound forms. However the molecular mechanism for this robust interconversion is poorly understood. For example, upon membrane binding, cytolysin A (ClyA), an -PFT, shows one of the largest conformational changes in protein structure among the PFT family of proteins. In order to understand this transtition we characterized several point mutations in ClyA using experiments and molecular dynamics (MD) simulations to understand the role of two essential ClyA motifs ({beta} tongue and N-terminus) implicated in the conformational changes responsible for oligomerization and conferring stability to the pore state. Our study reveals that the innate conformational flexibility of the {beta} tongue results in a disordered intermediate state that facilitate the complete transition to the pore state. This tendency to disorder is compromised to varying degrees in ClyA mutants, correlating with the loss of lytic activity. Our results suggest that the finely tuned conformational flexibility in the membrane motifs of ClyA are critical to its function, revealing a broader paradigm that could be at play during membrane associated secondary structure transitions of proteins in general.

biophysics↗