Biology subjects

Pandey, S.

Publications and source records attributed to Pandey, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Role Of Pcdh15 In The Development Of Intrinsic Polarity Of Inner Ear Hair Cells

In vertebrates, auditory information is transduced in the cochlea by mechanosensory hair cells (HC) through an eccentrically organised structure known as the hair bundle. This consists of a true cilium, known as the kinocilium, and modified microvilli, known as stereocilia. The hair bundle has a distinct structure with stereocilia organised in graded rows, with the longest abutting the kinocilia. The hair bundles of all HC are aligned to the tissue axis and are planar polarised. Important in the development and physiology of HC are protein bridges consisting of cadherin-23 and protocadherin-15. These link the tips of stereocilia, where they play a role in mechanotransduction, and between the kinocilia and the stereocilia, where they are involved in development. Both cadherin-23 and protocadherin-15 mutations result in defects in planar polarity; however, the mechanism through which this defect arises is unclear. Using a novel mutant for the Pcdh15-CD2 isoform, we show that while the initial deflection of the kinocilium occurs, its peripheral migration to register with Gi3 is perturbed. Pcdh15-CD2 genetically interacts with Gpsm2, perturbing vestibular function. We find that the earliest expression of Pcdh15-CD2 is at the base of the kinocilia, and the defects in morphogenesis occur before the formation of kinocilial links. By re-introducing functional Pcdh15-CD2, we show that polarity can be restored, suggesting that Pcdh15-CD2 couples kinocilial and stereocilial development through a mechanism independent of kinocilial links.

developmental biology↗

bioRxiv2024

Insights into the conformational dynamics of the cytoplasmic domain of metal-sensing sensor histidine kinase ZraS

ZraS is a metal sensor integral to ZraPSR, a two-component signaling system found in enterobacters. It belongs to a family of bifunctional sensor histidine kinases (SHKs) and is speculated to sense zinc-induced stress on the bacterial envelope. Information on the structure-function relationship of sensor kinases is elusive due to the lack of full-length structures, intrinsically dynamic behavior, and difficulty trapping them in active state conformations. While the kinase domains of a few sensor histidine kinases are well-characterized, they exhibit significant functional diversity attributed to their modular multi-domain arrangement in the cytoplasmic region, combined with other signal transducing elements such as simple helices, HAMP and PAS domains. We report the crystal structure of the entire cytoplasmic region of Escherichia coli ZraS (EcZraS-CD) resolved at resolution 2.49 [A], comprising of a unique helical linker and the kinase domain. In the asymmetric unit, four molecules of ZraS assemble as homodimers trapped as two ligand-bound occluded conformers. Our analysis using these conformers show that modulation of the dimer bundle through segmental helical bending, sliding, and rotation leads to reorganization of the dimerization interface during kinase activation. Further, our analysis reveals the significance of aromatic amino acid interactions and loop residues at the dimer base in regulating the directionality of rotation during autophosphorylation. We also performed an invitro coupled assay to determine ATPase activity. Overall, our findings provide structure-based mechanistic insights into the process of autophosphorylation in trans-acting sensor histidine kinases.

biochemistry↗

bioRxiv2024

MAPK/ERK signaling in gliomas modulates interferon responses, T cell recruitment, enhances tumor-microglia crosstalk, and drives immune checkpoint blockade efficacy.

BackgroundGlioblastoma (GB) remains a formidable challenge in neuro-oncology, with immune checkpoint blockade (ICB) showing limited efficacy in unselected patients. We previously recently established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GB. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain poorly understood. MethodWe conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T cell responses and performed survival studies to validate the most relevant genes. Additionally, paired single cell RNA- sequencing (scRNA-seq) with p-ERK staining, spatial transcriptomics on GB samples, and ex-vivo slice culture of a BRAFV600E mutant GB tumor treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. ResultsCRISPR/Cas9 screens identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to CD8+ T cells, and anti-PD-1 across all kinases. Experimentally-induced ERK phosphorylation in gliomas enhanced survival with ICB treatment, led to durable anti-tumoral immunity upon re-challenge and memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells correlated with increased interferon responses, antigen presentation and T cell infiltration in GB. Moreover, spatial transcriptomics and scRNA-seq analysis revealed the modulation of interferon responses by the MAPK/ERK pathway in BRAFV600E human GB cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GB tissue. Notably, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia in slice cultures from BRAFV600E mutant GB. ConclusionOur data indicate that the MAPK/ERK pathway is a critical regulator of GB cell susceptibility to anti-tumoral immunity, modulating interferon responses, and antigen-presentation in glioma cells, as well as tumor cell interaction with microglia. These findings not only elucidate the mechanistic underpinnings of immunotherapy resistance in GB but also highlight the MAPK/ERK pathway as a promising target for enhancing immunotherapeutic efficacy in this challenging malignancy.

cancer biology↗

bioRxiv2024

Mouse olfactory system acts as anemo-detector and -discriminator

Airflow detection while smelling is a fundamental requirement for olfaction, yet the mechanisms underlying such multimodal processing in the olfactory system remain unknown. We report here that mice can learn to accurately discriminate airflow with parallel processing of both mechanical and chemical stimuli revealed by modulated sniffing and refined calcium signaling in the olfactory bulb inhibitory network. Genetic perturbation of AMPAR function and optogenetic control bidirectionally shifted airflow discrimination learning pace, with contrasting phenotypes observed for odor learning, engagement of inhibitory circuits, and setting the optimal inhibition level for stimulus refinement. Multimodal odor-airflow stimuli at subthreshold levels enhanced learning, demonstrating that mechanical stimuli heighten olfactory perception. Our results, thus explain the multimodality of olfaction, and reveal an unexplored dimensionality of odor perception.

neuroscience↗

bioRxiv2024

Therapeutically targeting the classical complement pathway with antisense oligonucleotides in Alzheimer's disease

The complement classical pathway (CP) is a key mediator of synapse loss and neurodegeneration in mouse models of Alzheimers (AD) and other neurodegenerative diseases. We analyzed human brain proteomics and found consistent elevations of all CP proteins, but not other complement pathways, in AD patient brains. We performed human genetics analysis that identified a rare variant in the C1S gene within the Finnish population that is associated with AD and we found that a common AD-associated C1S variant correlates with increased C1S protein levels. A targeted assay detected elevated C1S activation in AD patient CSF. Given this specific implication of the CP in AD, we next evaluated the therapeutic approach of targeting the CP in the brain using antisense oligonucleotides (ASOs). To identify promising CP targets for knockdown using ASOs we first tested for rescue of synapse loss in an AD mouse model using heterozygous and homozygous complement knockout mice and examined the relative brain expression levels of different CP genes. Based on these experiments we prioritized C1r, C1s and C4 as promising targets for therapeutic knockdown using ASOs. We then screened for ASOs for each target, evaluating in vitro and in vivo knockdown and toxicity, and identified optimal ASOs targeting C1r, C1s and C4. Experiments with AD model mice demonstrated significant rescue of synapse loss following treatment with C1r, C1s or C4 ASOs. Overall, our findings provide proof of concept for using nucleic acid-based medicine to target the CP in AD and demonstrate the translational potential of this approach.

neuroscience↗

bioRxiv2024

EGFR does not directly interact with cortical actin: A SRRF'n'TIRF Study

The epidermal growth factor receptor (EGFR) governs pivotal signaling pathways in cell proliferation and survival, with mutations implicated in numerous cancers. The organization of EGFR on the plasma membrane (PM) is influenced by the lipids and the cortical actin (CA) cytoskeleton. Despite the presence of a putative actin-binding domain (ABD) spanning 13 residues, a direct interaction between EGFR and CA has not been definitively established. While disrupting the cytoskeleton can impact EGFR behavior, suggesting a connection, the influence of the static actin cytoskeleton has been found to be indirect. Here, we investigate the potential interaction between EGFR and CA, as well as the extent to which CA regulates EGFRs distribution on the PM using SRRFnTIRF, a spatiotemporal super-resolution microscopy technique that provides sub-100 nm resolution and ms-scale dynamics from the same dataset. To label CA, we constructed PMT-mEGFP-F-tractin, which combines an inner leaflet targeting domain PMT, fluorescent probe mEGFP, and the actin-binding protein F-tractin. In addition to EGFR-mEGFP, we included two control constructs: a) an ABD deletion mutant, EGFR{Delta}ABD-mEGFP serving as a negative control, and b) EGFR-mApple-F-tractin, where F-tractin is fused to the C-terminus of EGFR-mApple, serving as the positive control. We find that EGFR-mEGFP and EGFR{Delta}ABD-mEGFP show similar membrane dynamics, implying that EGFR-mEGFP dynamics and organization are independent of CA. EGFR dynamics show CA dependence when F-tractin is anchored to the cytoplasmic tail. Together, our results demonstrate that EGFR does not directly interact with the CA in its resting and activated state. SIGNIFICANCESRRFnTIRF is a spatiotemporal super-resolution microscopy technique that allows for the investigation of plasma membrane-cytoskeleton interactions. We investigate how cortical actin (CA) influences the dynamic behavior and structural organization of EGFR, employing specific probe targeting CA structure and dynamics. Our results suggest that EGFR, whether in its resting or activated state, does not directly bind to or interact with the CA. Any influence of CA on EGFR is indirect through membrane modulating activities of CA.

biophysics↗

bioRxiv2024

Early changes in microRNA expression in Arabidopsis plants infected with the fungal pathogen Fusarium graminearum

Plants respond to biotic stressors by modulating various processes in an attempt to limit the attack by a pathogen or herbivore. Triggering these different defense processes requires orchestration of a network of proteins and RNA molecules that includes microRNAs (miRNAs). These short RNA molecules (20-22 nucleotides) have been shown to be important players in the early responses of plants to stresses because they can rapidly regulate the expression levels of a network of downstream genes. The ascomycete Fusarium graminearum is an important fungal pathogen that causes significant losses in cereal crops worldwide. Using the well-characterized Fusarium-Arabidopsis pathosystem, we investigated how plants change expression of their miRNAs globally during the early stages of infection by F. graminearum. In addition to miRNAs that have been previously implicated in stress responses, we have also identified evolutionarily young miRNAs whose levels change significantly in response to fungal infection. Some of these young miRNAs have homologs present in cereals. Thus, manipulating expression of these miRNAs may provide a unique path toward development of plants with increased resistance to fungal pathogens.

plant biology↗

bioRxiv2024

Nutrient Sensing Receptor GPRC6A Regulates mTORC1 Signaling and Tau Biology

Tauopathies, including Alzheimers disease (AD), comprise microtubule-associated protein tau aggregates that cause neuronal cell death and clinical cognitive decline. Reducing overall tau abundance remains a central strategy for therapeutics; however, no disease-modifying treatment exists to date. One principal pathway for balancing cellular proteostasis includes the mechanistic target of rapamycin complex 1 (mTORC1) signaling. Recently, arginine emerged as one of the primary amino acids to activate mTORC1 through several intracellular arginine sensors and an extracellular arginine receptor, namely the G protein-coupled receptor (GPCR) family C, group 6, member A (GPRC6A). Human AD brains were previously reported with elevated mTORC1 signaling; however, it is unclear whether arginine sensing and signaling to mTORC1 plays a role in tauopathies. Herein, we examined arginine sensing associated with mTORC1 signaling in the human AD and animal models of tauopathy. We found that human AD brains maintained elevated levels of arginine sensors with potential uncoupling of arginine sensing pathways. Furthermore, we observed increased GPRC6A and arginine in the brain, accompanied by increased mTORC1 signaling and decreased autophagy in a mouse model of tauopathy (Tau PS19). We also discovered that both supplementing arginine and overexpressing GPRC6A in cell culture models could independently activate mTORC1 and promote tau accumulation. In addition, we found that suppressing GPRC6A signaling by either genetic reduction or pharmacological antagonism reduced tau accumulation, phosphorylation, and oligomerization. Overall, these findings uncover the crucial role of arginine sensing pathways in deregulating mTORC1 signaling in tauopathies and identify GPRC6A as a promising target for future therapeutics in tauopathies and other proteinopathies. Significance StatementTauopathies, including Alzheimers disease (AD), accumulate pathogenic tau protein inclusions that potentially contribute to the hyperactive mechanistic target of rapamycin complex 1 (mTORC1) signaling and eventually cause neuronal cell death. Here, we presented novel findings that AD and animal models of tauopathy maintained increased expression of arginine sensors and uncoupling of arginine sensing associated with mTORC1 signaling. We investigated the role of a putative extracellular arginine and basic L-amino acid sensing G protein-coupled receptor (GPCR) family C, group 6, member A (GPRC6A) in activating mTORC1 and accelerating pathogenic tau phenotypes in several cell models. Additionally, we showed that genetic repression or antagonism of GPRC6A signaling provides a novel therapeutic target for tauopathies and other proteinopathies.

neuroscience↗

bioRxiv2024

How motility drives the glassy dynamics in confluent epithelial monolayers?

As wounds heal, embryos develop, cancer spreads, or asthma progresses, the cellular monolayer undergoes glass transition between solid-like jammed and fluid-like flowing states. During some of these processes, the cells undergo an epithelial-to-mesenchymal transition (EMT): they acquire in-plane polarity and become motile. Thus, how motility drives the glassy dynamics in epithelial systems is critical for the EMT process. However, no analytical framework that is indispensable for deeper insights exists. Here, we develop such a theory inspired by a well-known glass theory. One crucial result of this work is that the confluency affects the effective persistence time-scale of active force, described by its rotational diffusivity, [Formula]. [Formula] differs from the bare rotational diffusivity, Dr, of the motile force due to cell shape dynamics, which acts to rectify the force dynamics: [Formula] is equal to Dr when Dr is small and saturates when Dr is large. We test the theoretical prediction of [Formula] and how it affects the relaxation dynamics in our simulations of active Vertex model. This novel effect of [Formula] is crucial to understanding the new and previously published simulation data of active glassy dynamics in epithelial monolayers.

biophysics↗

bioRxiv2024

Macrophage Polarizations in the Placenta and Lung are Associated with Bronchopulmonary Dysplasia

The intricate interplay between macrophage polarization and placenta vascular dysfunction has garnered increasing attention in the context of placental inflammatory diseases. This study delves into the complex relationship between macrophage polarization within the placenta and its potential impact on the development of vascular dysfunction and inflammatory conditions. The placenta, a crucial organ in fetal development, relies on a finely tuned balance of immune responses for proper functioning. Disruptions in this delicate equilibrium can lead to pathological conditions, including inflammatory diseases affecting the fetus and newborn infant. We explored the interconnectedness between placental macrophage polarization and its relevance to lung macrophages, particularly in the context of early life lung development. Bronchopulmonary dysplasia (BPD), the most common chronic lung disease of prematurity, has been associated with abnormal immune responses, and understanding the role of macrophages in this context is pivotal. The investigation aims to shed light on how alterations in placental macrophage polarization may contribute to lung macrophage behavior and, consequently, influence the development of BPD. By unraveling the intricate mechanisms linking macrophage polarization, placental dysfunction and BPD, this research seeks to provide insights that could pave the way for targeted therapeutic interventions. The findings may offer novel perspectives on preventing and managing placental and lung-related pathologies, ultimately contributing to improved maternal and neonatal health outcomes.

systems biology↗

bioRxiv2024

C1QA is an invariant biomarker for tissue macrophages

Macrophages play a pivotal role in immune responses, particularly in the context of combating microbial threats within tissues. The identification of reliable biomarkers associated with macrophage function is essential for understanding their diverse roles in host defense. This study investigates the potential of C1QA as an invariant biomarker for tissue macrophages, focusing on its correlation with the anti-microbial pathway. C1QA, a component of the complement system, has been previously implicated in various immune functions. Our research delves into the specific association of C1QA with tissue-resident macrophages and its implications in the context of anti-microbial responses. Through comprehensive systems biology and Boolean analysis of gene expression, we aim to establish C1QA as a consistent and reliable marker for identifying tissue macrophages. Furthermore, we explore the functional significance of C1QA in the anti-microbial pathway. This research seeks to provide valuable insights into the molecular mechanisms underlying the anti-microbial functions of tissue macrophages, with C1QA emerging as a potential key player in this intricate regulatory network. Understanding the relationship between C1QA, tissue macrophages, and the anti-microbial pathway could pave the way for the development of targeted therapeutic strategies aimed at enhancing the hosts ability to combat infections. Ultimately, our findings contribute to the expanding knowledge of macrophage biology and may have implications for the diagnosis and treatment of infectious diseases. One Sentence SummaryC1QA is a fundamental biomarker of tissue macrophages

systems biology↗

bioRxiv2023

Targeted inCITE-Seq Analysis Identifies the Loss of Nuclear TDP-43 in Endothelium as a Mediator of Blood Brain Barrier Signaling Pathway Dysfunction in Neurodegeneration

AbstractDespite the importance of the endothelium in the regulation of the blood brain barrier (BBB) in aging and neurodegenerative disease, difficulties in extracting endothelial cell (EC) nuclei have limited analysis of these cells. In addition, nearly all Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Degeneration (FTD), and a large portion of Alzheimers Disease (AD) exhibit neuronal TDP-43 aggregation, leading to loss of nuclear function, but whether TDP-43 is similarly altered in human BBB ECs is unknown. Here we utilize a novel technique for the enrichment of endothelial and microglial nuclei from human cortical brain tissues, combined with inCITE-seq, to analyze nuclear proteins and RNA transcripts in a large cohort of healthy and diseased donors. Our findings reveal a unique transcriptional signature in nearly half of the capillary endothelial cells across neurodegenerative states, characterized by reduced levels of nuclear {beta}-Catenin and canonical downstream genes, and an increase in TNF/NF-kB target genes. We demonstrate that this does not correlate with increased nuclear p65/NF-kB, but rather a specific loss of nuclear TDP-43 in these disease associated ECs. Comparative analysis in animal models with targeted disruption of TDP-43 shows that this is sufficient to drive these transcriptional alterations. This work reveals that TDP-43 is a critical governor of the transcriptional output from nuclear p65/NF-kB, which has paradoxical roles in barrier maintenance and also barrier compromising inflammatory responses, and suggests that disease specific loss in ECs contributes to BBB defects observed in the progression of AD, ALS and FTD.

neuroscience↗

bioRxiv2023

Homologs of SD6 and ICE2 from rice may be involved in regulation of ABA in woody perennial buds

The availability of ABA, a central component in the regulation of the dormancy cycle in grapevine buds, is controlled by coordinated and opposite regulation of NCED and ABA8OX expression, as shown during natural dormancy release and following treatment with Hydrogen Cyanamide (HC). This implies the existence of a shared regulatory entity, which serves as an upstream switch. A molecular switch for integrated and opposite regulation of NCED and ABA8OX was recently described in rice, involving a pair of bHLH transcription factors (OsSD6-OsICE2) that directly regulate ABA8OX3 expression and indirectly regulate NCED2 expression, by direct regulation of the expression of the NCED repressor OsbHLH048. Here, we tested whether expression of the Vitis homologs of the rice SD6 and ICE2 are regulated by dormancy release stimuli, and whether the direction of regulation agrees with that of ABA8OX. Treatment with two independent stimuli of bud break (HC and hypoxia), as well as natural dormancy release, resulted in upregulation of OsSD6 homologs and down regulation of OsICE2 homologs, in agreement with the rice model. In unexpected contrast, the homolog of OsbHLH048 was down-regulated. Our results suggest a grapevine model in which (1) the homologs of OsSD6 and OsICE2 act as direct activators and repressors of ABA8OX3 expression, as for rice, (2) they have opposed effects on the expression of an OsbHLH048 homolog, which serves as direct activator of NCED expression, as for Arabidopsis, and (3) together they act as a switch that allows removal of ABA repression, followed by meristem reactivation and bud break. HIGHLIGHTA molecular switch for integrated and opposite regulation of NCED and ABA8OX in rice seeds, operated by three bHLH transcription factors, is conserved in grapevine buds and regulates dormancy release

developmental biology↗

bioRxiv2023

Nucleo-cytoplasmic environment modulates spatio-temporal p53 phase separation

Phase separation of various transcription factors and nucleic acids into biomolecular condensates is known to play an essential role in the regulation of gene expression. Here, we show that p53, a tumor suppressor and transcription factor, phase separates and forms biomolecular condensates in the nucleus of cancer cells as well as when overexpressed in the various cell lines. Although the nuclear condensates of wild-type (WT) p53 maintain their liquid state and are able to bind DNA, cancer-associated mutations not only promote misfolding but also partially rigidify the p53 condensates, which are unable to bind the DNA. Irrespective of WT or mutant form, the cytoplasmic partitioning of p53 with time also results in biomolecular condensate formation, which eventually undergoes rigidification. In vitro, WT p53 core domain (p53C) forms biomolecular condensates, which rigidify with time and the process is further promoted by cancer-associated mutations. Both RNA and non-specific DNA promote LLPS of p53C, but specific DNA promotes the dissolution of p53C condensates. The result suggests that the cellular microenvironment regulates p53 LLPS, material property and its functions.

biophysics↗

bioRxiv2023

Red cabbage juice-mediated gut microbiota modulation improves intestinal epithelial homeostasis and ameliorates colitis

Gut microbiota plays a crucial role in inflammatory bowel disease (IBD) and has therapeutic benefits. Thus, targeting the gut microbiota is a promising therapeutic approach for IBD treatment. We recently found that red cabbage juice (RCJ) ameliorates dextran sulfate sodium (DSS)-induced colitis in mice. However, the underlying mechanisms remain unknown. The current study investigated the modulation of gut microbiota in response to treatment with RCJ to ameliorate the DSS colitis. The initial results demonstrated that mice treated with DSS + RCJ showed increased body weight and decreased diarrhea and blood in feces compared to the DSS alone group. RCJ ameliorated colitis by regulating the intestinal barrier function by reducing the number of apoptotic cells, improving colonic protective mucin, and increasing tight junction protein in RCJ + DSS groups compared to the DSS group. Short-gun metagenomic analysis revealed significant enrichment of short-chain fatty acid (SCFAs)-producing bacteria (Butyrivibrio, Ruminococcaceae, Acetatifactor muris, Rosburia Sp. CAG:303, Dorea Sp. 5-2) increased PPAR-(C) activation, leading to repression of the nuclear factor {kappa}B (NF{kappa}B) signaling pathway, thus decreasing the production of crucial inflammatory cytokines and chemokines in the RCJ + DSS groups compared to the DSS group. Pathway abundance analysis showed an increased abundance of the SCFA pathway, reduced histidine degradation (Bacteroides sartorii, and Bacteroides caecimuris), and LCFA production in the RCJ+DSS treated group, suggesting the promotion of good colonic health. Furthermore, increased T-reg (FOXP3+) cells in the colon were due to SCFAs produced by the gut microbiota, which was corroborated by an increase in IL-10, a vital anti-inflammatory cytokine. Thus, our study provides the first evidence that RCJ ameliorates colonic inflammation by modulating the gut microbiota.

pathology↗

bioRxiv2023

Modeling of the Network Mediated by IL-36 Involved inPsoriasis

Pathogenesis of inflammatory, chronic and common skin disease Psoriasis involves immune cells, skin cells (keratinocytes) and cytokines secreted by them. Hyperproliferation and abnormal differentiation of keratinocytes are believed to be a hallmark of it. Roles of several cytokines such as TNF, IL-15, IL-17 and IL-23 in Psoriasis have been explored through mathematical/computational models as well as experimentally. However, the role of pro-inflammatory cytokine IL-36 is still elusive, especially in the case of General Pustular Psoriasis, a prevalent type of Psoriasis. To explore the role of that here, we construct a network embodying indirect cell-cell interactions of a few immune and skin cells mediated by IL-36 based on the existing knowledge. Further, we develop a mathematical model for the network and study the steady-state behaviour of that. Our results demonstrate that an increase in the level of IL-36 could lead to the hyper-proliferation of keratinocytes and, thus, Psoriasis. In addition, the analysis suggests that the plaque formation and progression of Psoriasis could occur via a gradual or switch-like increase in the population of keratinocytes. The switch-like increase would be due to the bistable behaviour of the network and could be used as a novel treatment strategy, as proposed and demonstrated earlier.

systems biology↗

bioRxiv2023

MORPHOGENIC VERSUS MITOGENIC ROLES OF SHH ARE SEGREGATED ON DISTINCT EXOSOMES REGULATED BY CELLULAR RAB7 LEVELS

The secreted signaling molecule, Sonic hedgehog (Shh) is involved in patterning and growth of various embryonic tissues across species. This lipid-anchored protein is secreted on extracellular vesicles to activate signaling. Shh has two functions during spinal cord development, acting as a morphogen to pattern the ventral neural tube and a mitogen to maintain neural progenitors. Here, we find that these activities are segregated on to two distinct pools of exosomes. Shh secreted on a classical exosomal pool (Shh-P150) is able to activate ventral threshold targets in the neural tube. In contrast, the mitogenic activity, is elicited by a lighter exosomal pool (Shh-P450). We further show that cellular environment plays a major role in regulating the biogenesis of P150 versus P450 by modulating Rab7 levels. We find that active Rab7 is necessary for ventral neural tube patterning through the regulation of Shh-P150 secretion. The cellular mechanisms involved in packaging and secretion of Shh with different partners on P150 or P450 pools may be more general, enabling the separation of functional activities of signaling molecules during development and disease.

developmental biology↗

bioRxiv2023

Enhanced protective efficacy of a novel, thermostable, RBD-S2 fusion immunogen against SARS-CoV-2 and its variants

With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly protective immune responses. However, the contribution of the S2 subunit in improving the overall efficacy of vaccines remains unclear. Therefore, we designed, characterized, and evaluated the immunogenicity and protective potential of a stabilized SARS-CoV-2 Receptor Binding Domain (RBD) fused to a stabilized S2. Designed immunogens were expressed as soluble proteins with approximately fivefold higher purified yield than the Spike ectodomain and formulated along with Squalene-in-water emulsion (SWE) adjuvant. S2 immunization failed to elicit a neutralizing immune response but significantly reduced lung viral titers in mice challenged with the heterologous Beta variant. In hamsters, SWE-formulated RS2 showed enhanced immunogenicity and efficacy relative to corresponding RBD and Spike formulations. Despite being based on the ancestral Wuhan strain of SARS-CoV-2, RS2 exhibited broad neutralization, including against Omicron variants (BA.1, BA.5 and BF.7), as well as the clade 1a WIV-1 and SARS-CoV-1 strains. RS2 sera also showed enhanced competition with both S2 directed and RBD Class 4 directed broadly neutralizing antibodies, relative to RBD and Spike elicited sera. When lyophilized, RS2 retained antigenicity and immunogenicity even after incubation at 37 {degrees}C for a month. The data collectively suggest that the RS2 immunogen is a promising modality to combat SARS-CoV-2 variants.

biophysics↗