Biology subjects

Srivastava, S.

Publications and source records attributed to Srivastava, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Spatially resolved niche and tumor microenvironmental alterations in gastric cancer peritoneal metastases

Peritoneal metastases (PM) in gastric cancer (GC) portend a poor prognosis, yet our understanding of tumor microenvironmental (TME) characteristics associated with GCPM remain limited. Here, we analyzed intrinsic genomic alterations and transcriptomic programs predictive of GCPM in a prospective cohort of 248 patients, identifying CDH1, PIGR, and ELF3 mutations as predictors. By inspecting the spatial dynamics of the TME, we find that tumor compartment infiltration of pro-tumorigenic cell types such as inflammatory cancer-associated fibroblasts (CAFs) predict peritoneal recurrence. Next, in a cross-sectional study of 205 samples from 55 patients, distinct pathways and immune compositions in GCPM relative to liver metastases highlight the TMEs significance in transcoelomic metastases. Notably, several putative therapeutic targets exhibited distinct expression patterns between PTs and PMs. We also observed increased immune infiltration in GCPMs treated with systemic immunotherapy and intraperitoneal chemotherapy. Our findings highlight transcriptomic variations and niche reprogramming in the GCPM peritoneal environment, revealing roles of myeloid dendritic cells, effector memory CD8+ T cells, and CAFs in metastatic progression. Statement of significanceComprehensive molecular profiling of gastric cancer primary and peritoneal tumors unveils crucial insights into the distinct molecular and immune landscape of peritoneal metastases. Identifying predictive markers and therapeutic targets emphasizes the significance of tumor microenvironment alterations in guiding future therapies for gastric cancer peritoneal metastasis.

cancer biology↗

bioRxiv2024

Spiking GABAergic OPC tumor cells prolong survival in IDH1 mutant glioma

Prior studies have described the complex interplay that exists between glioma cells and neurons, however, the electrophysiological properties endogenous to tumor cells remain obscure. To address this, we employed Patch-sequencing on human glioma specimens and found that one third of patched cells in IDH mutant (IDHmut) tumors demonstrate properties of both neurons and glia by firing single, short action potentials. To define these hybrid cells (HCs) and discern if they are tumor in origin, we developed a computational tool, Single Cell Rule Association Mining (SCRAM), to annotate each cell individually. SCRAM revealed that HCs represent tumor and non-tumor cells that feature GABAergic neuron and oligodendrocyte precursor cell signatures. These studies are the first to characterize the combined electrophysiological and molecular properties of human glioma cells and describe a new cell type in human glioma with unique electrophysiological and transcriptomic properties that are likely also present in the non-tumor mammalian brain.

cancer biology↗

bioRxiv2024

Steroidal glycoalkaloid biosynthesis and fungal tolerance are regulated by ELONGATED HYPOCOTYL 5, SlHY5, in tomato

Tomato (Solanum lycopersicum L.) is one of the highest consumable fruit crops, rich in nutrients, and has been an important target for enhancing the accumulation of various metabolites. Tomato also contains cholesterol-derived molecules, steroidal glycoalkaloids (SGAs), which contribute to pathogen defence but are toxic to humans and considered anti-nutritional compounds. Previous studies suggest the role of various transcription factors in SGA biosynthesis; however, the role of light and associated regulatory factors has not been studied in tomatoes. Here, we demonstrated that SGA biosynthesis is regulated by light through the ELONGATED HYPOCOTYL 5 homolog, SlHY5, by binding to light-responsive G-boxes present in the promoters of the structural and regulatory genes. Our analysis suggests that SlHY5 could complement the Arabidopsis thaliana and Nicotiana tabacum, hy5 mutants at molecular, morphological, and biochemical levels. We report the development of CRISPR/Cas9-based knockout mutant plants of tomato, slhy5CR, and show down-regulation of the SGA and phenylpropanoid pathway genes leading to a significant reduction in SGA (-tomatine and dehydrotomatine) and flavonol contents, whereas SlHY5 overexpression (SlHY5OX) plants show opposite effect. An enhanced SGA and flavonol levels in SlHY5OX lines provided tolerance against Alternaria solani fungus, while SlHY5CR was susceptible to the pathogen. This study advances our understanding of the HY5-dependent light-regulated biosynthesis of SGAs and flavonoids and their role in biotic stress in tomatoes. One Sentence SummaryLight-associated transcription factor, ELONGATED HYPOCOTYL 5, regulates biosynthesis of anti-nutrient molecules, steroidal glycoalkaloids, and fungal tolerance in tomato

molecular biology↗

bioRxiv2024

Proteomic analysis of gut in Labeo rohita reveals ECM as Key Player in host's Response to Aeromonas hydrophila Infection

In the aquaculture sector, one of the challenges include disease outbreaks such as bacterial infections, particularly from Aeromonas hydrophila (Ah), impacting both wild and farmed fish. In this study, we conducted a proteomic analysis of the gut tissue in Labeo rohita following Ah infection to elucidate the protein alterations and its implications for immune response. Our findings reveal significant dysregulation in extracellular matrix (ECM) associated proteins during Ah infection, with increased abundance of elastin and Collagen alpha-3(VI) contributing to matrix rigidity. Pathway and enrichment analysis of differentially expressed proteins (DEPs) highlights the involvement of ECM-related pathways, including Focal adhesions, Integrin cell surface interactions, and actin cytoskeleton organization. Focal adhesions, crucial for connecting intracellular actin bundles to the ECM, play a pivotal role in immune response during infections. Increased abundance of integrin alpha 1, integrin beta 1, and Tetraspanin suggests their involvement in the hosts response to Ah infection. Proteins associated with actin cytoskeleton reorganization, such as myosin, tropomyosin, and phosphoglucomutase, exhibit increased abundance, influencing changes in cell behavior. Additionally, upregulated proteins like LTBP1 and Fibrillin-2 contribute to TGF-{beta} signaling and focal adhesion, indicating their role in immune regulation. The study also identifies elevated levels of laminin, galectin 3, and tenascin-C, which interact with integrins and other ECM components, influencing immune cell migration and function. These proteins, along with decorin and lumican, act as immunomodulators, coordinating pro- and anti-inflammatory responses. ECM fragments released during pathogen invasion serve as "danger signals," initiating pathogen clearance and tissue repair through Toll-like receptor signaling. IMPORTANCEThe study underscores the critical role of the extracellular matrix (ECM) and its associated proteins in the immune response of aquatic organisms during bacterial infections like Aeromonas hydrophila (Ah). Understanding the intricate interplay between ECM alterations and immune response pathways provides crucial insights for developing effective disease control strategies in aquaculture. By identifying key proteins and pathways involved in host defense mechanisms, this research lays the groundwork for targeted interventions to mitigate the impact of bacterial infections on fish health and aquaculture production.

microbiology↗

bioRxiv2024

Spatial interactions modulate tumor growth and immune infiltration

Direct observation of immune cell trafficking patterns and tumor-immune interactions is unlikely in human tumors with currently available technology, but computational simulations based on clinical data can provide insight to test hypotheses. It is hypothesized that patterns of collagen formation evolve as a mechanism of immune escape, but the exact nature of the interaction between immune cells and collagen is poorly understood. Spatial data quantifying the degree of collagen fiber alignment in squamous cell carcinomas indicates that late stage disease is associated with highly aligned fibers. Here, we introduce a computational modeling framework (called Lenia) to discriminate between two hypotheses: immune cell migration that moves 1) parallel or 2) perpendicular to collagen fiber orientation. The modeling recapitulates immune-ECM interactions where collagen patterns provide immune protection, leading to an emergent inverse relationship between disease stage and immune coverage. We also illustrate the capabilities of Lenia to model the evolution of tumor progression and immune predation. Lenia provides a flexible framework for considering a spectrum of local (cell-scale) to global (tumor-scale) dynamics by defining a kernel cell-cell interaction function that governs tumor growth dynamics under immune predation with immune cell migration. Mathematical modeling provides important mechanistic insights into cell interactions. Short-range interaction kernels provide a mechanism for tumor cell survival under conditions with strong Allee effects, while asymmetric tumor-immune interaction kernels lead to poor immune response. Thus, the length scale of tumor-immune interactions drives tumor growth and infiltration.

cancer biology↗

bioRxiv2023

Development and clinical validation of molecular subgrouping in medulloblastoma by targeted methylation sequencing

BackgroundThe WHO classification of CNS tumors confers promising prognostic value to the molecular classification of medulloblastoma (MB). Next-generation sequencing (NGS) has been the primary method employed for molecular classification through transcriptomic, genomic, or methylation profiling. However, due to cost and infrastructural needs, particularly in developing countries, we propose a relatively simple, rapid, and economical Sanger sequencing-based targeted methylation sequencing method for MB classification and prognostication. MethodsEleven epigenetic targets were amplified using optimized primers and bisulfite-converted DNA for Sanger sequencing. Chromas software was used for low-quality data trimming and NCBIs Needleman Wunsch alignment tool was used for sequence alignment to reference. The developed method was applied to tissues from twelve cases of medulloblastoma. ResultsSuccessful interpretation of methylation status in ten out of eleven targets was achieved which was sufficient for classification according to the latest WHO classification of Medulloblastoma tumors. Twelve medulloblastoma cases were classified into WNT (n=2), Group 3 (n=5), and Group 4 (n=5). ConclusionThe developed Sanger sequencing method is a cost-effective, in-house solution that can be used for molecular subgrouping of medulloblastoma. It offers an alternative to NGS, can be done on a case-to-case basis, and does not require high-end infrastructure, sample pooling, or extensive bioinformatics knowledge. Impact statementMolecular classification is imperative for determining the prognosis of medulloblastoma and is recommended by WHO. However, NGS proves to be an expensive option in developing countries. This study has come up with an affordable targeted methylation Sanger sequencing method requiring minimal bioinformatic skills, by utilizing epigenetic targets, for prognostication and risk stratification in medulloblastoma patients. The molecular subgroups of all recruited cases were successfully determined according to WHO classification which is crucial information that, when combined with clinical findings, will enable the clinicians to determine effective treatment strategies.

cancer biology↗

bioRxiv2023

Targeting endothelial FOXO1 protects diabetic β-cells and improves wound healing

The forkhead box O1 (FOXO1) transcription factor plays critical roles in regulating not only metabolic activity but also angiogenesis in the vascular endothelium1-4. Our previous studies show that epsin endocytic adaptors can regulate both angiogenesis and lymphangiogenesis5-7. Endothelial cells (ECs) lining the inside of blood vessels are continuously exposed to circulating insulin and insulin-like growth factors (IGFs). Emerging evidences suggest that ECs can affect {beta}-cell function8-11. Excessive IGF2, especially elevated local IGF2 levels in islets, may represent a risk factor for developing diabetes12-15; however, the underlying molecular mechanisms by which aberrant angiogenesis and endothelium-derived factors regulate pancreatic {beta}-cell function in diabetes remain unclear. Here, we report that the pancreas of diabetic patients as well as the pancreas, skin, and plasma of streptozotocin/high fat diet (STZ/HFD)-induced diabetic mice and db/db mice contains excess IGF2, which can lead to {beta}-cell dysfunction and apoptosis. Single-cell transcriptomics combined with mass spectrometry analysis reveal that endothelial-specific knockout of FOXO1 increases circulating soluble and cell-membrane or intracellular expression levels of IGF type 2 receptor (IGF2R) and CCCTC-binding factor (CTCF), while decreasing IGF2 levels in diabetes. Both IGFR215-17 and CTCF18-21 can reduce IGF2 levels and may ameliorate {beta}-cell decline associated with excess IGF2 in diabetes. Furthermore, depletion of FOXO1, epsins, or knockdown of ULK1 inhibits autophagy formation in ECs, preventing degradation of vascular endothelial growth factor receptor 2 (VEGFR2) to promote angiogenesis and improve wound healing in diabetes. Our findings reveal that endothelial FOXO1 regulates epsin-dependent angiogenesis and affects {beta}-cell function and fate through CTCF and IGF2-IGF2R, providing a potential strategy for ameliorating diabetes and accelerating cutaneous wound healing.

cell biology↗

bioRxiv2023

AAO2 impairment improves aldehyde detoxification by AAO3 in Arabidopsis leaves exposed to UVC or Rose Bengal.

Among the three active aldehyde oxidases in Arabidopsis thaliana leaves (AAO1-3), AAO3, which catalyzes the oxidation of abscisic-aldehyde to abscisic-acid, was shown recently to function as a reactive aldehyde detoxifier. Notably, aao2KO mutants exhibited less senescence symptoms and lower aldehyde accumulation, such as acrolein, benzaldehyde, and HNE than in wild-type leaves exposed to UV-C or Rose-Bengal. The effect of the absence of AAO2 expression on aldehyde detoxification by AAO3 and/or AAO1 was studied by comparing the response of wild-type plants to the response of aao1Single mutant, aao2KO mutants and single mutants of aao3Ss. Notably, aao3Ss exhibited similar aldehyde accumulation and chlorophyll content to aao2KO treated with UV-C or Rose-Bengal. In contrast, wild-type and aao1S exhibited higher aldehyde accumulation that resulted in lower remaining chlorophyll than in aao2KO leaves, indicating that the absence of active AAO2 enhanced AAO3 detoxification activity in aao2KO mutants. In support of this notion, employing abscisic-aldehyde as a specific substrate marker for AAO3 activity revealed enhanced AAO3 activity in aao2KO and aao3Ss leaves compared to wild-type treated with UV-C or Rose Bengal. The similar abscisic acid level accumulated in leaves of unstressed or stressed genotypes indicates that aldehyde detoxification by AAO3 is the cause for better stress resistance in aao2KO mutants. Employing the sulfuration process (known to activate aldehyde oxidases) in wild-type, aao2KO, and molybdenum-cofactor sulfurase (aba3-1) mutant plants revealed that the active AAO2 in WT employs sulfuration processes essential for AAO3 activity level, resulting in the lower AAO3 activity in WT than AAO3 activity in aao2KO.

plant biology↗

bioRxiv2023

Neuronal Population Statistics, Computations, and Mechanisms of a Feedforward Convolutional Neural Network that Learns to Covertly Attend

Covert visual attention allows the brain to select different regions of the visual world without eye movements. Predictive cues of a target location orient covert attention and improve perceptual performance. In most computational models, researchers explicitly incorporate an attentional mechanism that alters processing at the attended location (gain, noise reduction, divisive normalization, biased competition, Bayesian priors). Here, we assess the emergent neuronal mechanisms of Convolutional Neural Networks (CNNs) that exhibit behavioral signatures of covert attention, despite lacking a built-in attention mechanism. We use neuroscience-inspired approaches to analyze 1.8M units of CNNs trained on the cueing paradigm. Consistent with neurophysiology, we show early layers with retinotopic neurons separately tuned to the target or cue, and later layers with neurons with joint tuning and increased cue influence on target responses. CNN computational stages mirror a Bayesian ideal observer (BIO), but with more gradual transitions. The cue influences the target sensitivity through four mechanisms. A BIO-like cue-weighted location summation, and three mechanisms absent in the BIO: an opponency across locations, a summation/opponency location combination, and interaction with the thresholding Rectified Linear Unit (ReLU). Re-analyses of mices superior colliculus neuronal activity during a cueing task show CNN-predicted but previously unreported cue-inhibitory, location- summation, and location-opponent cells in addition to the commonly reported cue (attention) excitatory cells. The novel single-unit CNN analysis approach establishes a likely system-wide characterization mediating covert attention and a framework to identify new neuron types and emergent computational mechanisms contributing to perceptual behavior. Significance StatementCues predictive of a target location orient covert attention and improve perceptual performance. Studies have focused on how attention influences neural activity, but how cues activate attention and how entire neuronal populations result in behavioral signatures of attention is not understood. Using neuroscience-inspired approaches to characterize properties of 1.8M CNN neurons, we predict new neuron types and emergent mechanisms. A re-analysis of mices superior colliculus activity shows new neuron types predicted by the CNN. The findings might explain how organisms, from primates and crows to insects, show behavioral signatures of covert attention using a variety of cue-target-location neuronal properties. The new CNN single-neuron analysis can guide neurophysiological studies to identify potential new neuron types and their computational contributions to perception.

neuroscience↗

bioRxiv2023

BrainProt(™) 3.0: Understanding Human Brain Diseases using comprehensively curated & Integrated OMICS datasets

BrainProt 3.0 is an integrative and simplified omics-based knowledge base of the human brain and its associated diseases. The current version of BrainProt includes six domains, which provide simplified, robust, and comprehensive data visualization to understand the human brain and its diseases/disorders based on proteomics, transcriptomics, public data curation, and integration strategies. Firstly, the HBDA (Human Brain Disease Atlas), index and navigator of BrainProt provides a resource table for 56 brain diseases. Secondly, Brain Disease Marker Curator (BDMC) and Brain Disease Drug Finder (BDDF) include a total of 20,202 diseases associated genes, more than 1,30,000 Chemical Target interactions, and around 2,145 Clinical Trial Information for more than 50 Brain Diseases. Thirdly, Brain Disease Transcriptome Map (BDTM) and Brain Disease Proteome Map (BDPM) integrate multi-omics data for 11 and 6 alarming brain diseases respectively. Currently, these two domains feature an expressional profile of 52 datasets, information of 1,868 samples, 3,657 DEPs, and 6,256 DEGs. Lastly, BrainProt also modifies and integrates the proteome and phosphoproteome data of the Inter-hemispheric Brain Proteome Map (IBPM). Overall, BrainProt is the first knowledgebase that connects different omics level information of brain diseases and provides a powerful scoring-based ranking platform to select and identify brain disease-associated markers, along with exploration of clinical trials, and drugs/chemical compounds to accelerate the identification of new disease markers and novel therapeutic strategies. The objectives of BrainProt are to support and follow the footsteps of the HBPP (Human Brain Proteome Project) by integrating different datasets to unravel the complexity of Human Brain and its associated diseases.

neuroscience↗

bioRxiv2023

Identification of potential molecular mimicry in pathogen-host interactions

Pathogens have evolved sophisticated strategies to manipulate host signaling pathways, including the phenomenon of molecular mimicry, where pathogen-derived biomolecules imitate host biomolecules. In this study, we used a sequence-based bioinformatics pipeline to identify potential molecular mimicry candidates between humans and 32 pathogenic species--bacteria, fungi, protozoa, and helminths--of global health importance. We observed considerable variation in the number of mimicry candidates across pathogenic species, with pathogenic bacteria exhibiting fewer candidates compared to fungi and protozoans. Further analysis revealed that the candidate mimicry regions were enriched in solvent-accessible regions, highlighting their potential functional relevance. We identified a total of 1878 mimicked regions in 1439 human proteins, and clustering analysis indicated diverse target proteins across pathogen species. The human proteins containing mimicked regions revealed significant associations between these proteins and various biological processes, with an emphasis on host extracellular matrix organization and cytoskeletal processes. However, immune-related proteins were underrepresented as targets of mimicry. Our findings provide insights into the broad range of host-pathogen interactions mediated by molecular mimicry and highlight potential targets for further investigation. This comprehensive analysis contributes to our understanding of the complex mechanisms employed by pathogens to subvert host defenses and promotes the development of novel therapeutic strategies.

microbiology↗

bioRxiv2023

Investigating miRNA signatures in metastatic breast cancer: An in silico analysis.

miRNAs are small non-coding RNAs that regulate most cellular processes. Tumorigenesis disrupts the normal balance in the cell, which leads to changes in the cell cycle, cell signalling, activation of growth factors, miRNA deregulation, etc. Thus, the variations in miRNA expression between normal and tumor stages can be used to predict, diagnose, detect and identify different cancer stages, thereby suggesting the potential use of the miRNAs as biomarkers or potential therapeutic targets for cancer. In this study we aim to identify differentially expressed miRNAs which could serve as potential biomarkers or therapeutic targets for breast cancer. Microarray-based expression analysis was performed on tissue samples from patients with early and locally advanced breast cancer and nCounter analysis was performed to detect differentially expressed miRNA. Additionally, functional enrichment analysis, miRNA linked gene prediction, and in silico analysis for miRNA expression in cancer databases were carried out. This analysis revealed increased expression of Hsa-miR-199a and b. Further these miRs had increased expression levels across various cancer. Importantly, genes such as CALR, SSR2, and YWHAZ predicted as miRs target were highly expressed in breast cancer. However, these genes lack therapeutic drugs targeting them. Overall, our study identified previously unexplored miRNAs and their gene targets, which could be exploited as potential targets for future therapeutic interventions.

cancer biology↗

bioRxiv2023

Genomic and transcriptomic analysis of camptothecin producing novel fungal endophyte - Alternaria burnsii NCIM 1409

Camptothecin is an important anticancer alkaloid produced by particular plant species. No suitable synthetic route has been established for camptothecin production yet, imposing a stress on plant-based production systems. Endophytes associated with these camptothecin-producing plants have been reported to also produce camptothecin and other high-value phytochemicals. A previous study identified a fungal endophyte Alternaria burnsii NCIM 1409, isolated from Nothapodytes nimmoniana, to be a sustainable producer of camptothecin. Our study provides key insights on camptothecin biosynthesis in this recently discovered endophyte. The whole genome sequence of Alternaria burnsii NCIM 1409 was assembled and screened for biosynthetic gene clusters. Comparative studies with related fungi supported the identification of candidate genes involved in camptothecin synthesis and also helped to understand some aspects of the endophytes defense against the toxic effects of camptothecin. No evidence for horizontal gene transfer of the camptothecin biosynthetic genes from the host plant to the endophyte was detected suggesting an independent evolution of the camptothecin biosynthesis in this fungus.

genomics↗

bioRxiv2023

Transcriptomic Evaluation of Stress Vulnerability Network using Single Cell RNA-Seq in mouse Prefrontal Cortex

Increased vulnerability to stress is a major risk factor for the manifestation of several mood disorders, including major depressive disorder (MDD). Despite the status of MDD as a significant donor to global disability, the complex integration of genetic and environmental factors that contribute to the behavioral display of such disorders has made a thorough understanding of related etiology elusive. Recent developments suggest that a brain-wide network approach is needed, taking into account the complex interplay of cell types spanning multiple brain regions. Single cell RNA-sequencing technologies can provide transcriptomic profiling at the single-cell level across heterogenous samples. Furthermore, we have previously used local field potential oscillations and machine learning to identify an electrical brain network that is indicative of a predisposed vulnerability state. Thus, this study combined single cell RNA-sequencing (scRNA-Seq) with electrical brain network measures of the stress-vulnerable state, providing a unique opportunity to access the relationship between stress network activity and transcriptomic changes within individual cell types. We found especially high numbers of differentially expressed genes between animals with high and low stress vulnerability brain network activity in astrocytes and glutamatergic neurons but we estimated that vulnerability network activity depends most on GABAergic neurons. High vulnerability network activity included upregulation of microglia and mitochondrial and metabolic pathways, while lower vulnerability involved synaptic regulation. Genes that were differentially regulated with vulnerability network activity significantly overlapped with genes identified as having significant SNPs by human GWAS for depression. Taken together, these data provide the gene expression architecture of a previously uncharacterized stress vulnerability brain state, enabling new understanding and intervention of predisposition to stress susceptibility. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/540705v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1ebe036org.highwire.dtl.DTLVardef@cd2131org.highwire.dtl.DTLVardef@13e3519org.highwire.dtl.DTLVardef@1100e62_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

A novel biomechanical indicator for impaired ankle dorsiflexion function during walking in individuals with chronic stroke

Ankle dorsiflexion function during swing phase of the gait cycle contributes to foot clearance and plays an important role in walking ability post-stroke. Commonly used biomechanical measures such as foot clearance and ankle joint excursion have limited ability to accurately evaluate dorsiflexor function in stroke gait. We retrospectively evaluated ankle angular velocity and ankle angular acceleration as direct measures for swing phase dorsiflexor function in post-stroke gait of 61 chronic stroke survivors. Our linear regression models revealed that peak ankle angular velocity (AAVP), peak ankle angular acceleration (AAAP), peak dorsiflexion angle (DFAP) and peak foot clearance (FCLP) during swing had a significant relationship (p < 0.05) with impaired dorsiflexion function. AAAP and DFAP accounted for the most variance of dorsiflexion function. Additionally, AAVP, AAAP, FCLP during swing, correlated significantly with all clinical outcome measures of walking ability. DFAP during swing had a positive correlation only with FMA-LE. Post-hoc Williams t-tests, used to compare the magnitude of difference between two non-independent correlations, revealed that the correlation between all clinical measures and DFAP were significantly weaker than with AAVP and AAAP. We also found that correlation between FMA-LE and FCLP was weaker than with AAVP and AAAP. We found an excellent test-retest reliability for both AAVP (ICC = 0.968) and AAAP (ICC = 0.947). These results suggest that DFAP may only be associated with non-task specific isolated dorsiflexion movement, but not during walking. FCLP is associated with dorsiflexion function and walking ability measures but not as strongly as AAVP and AAAP possibly because FCLP is influenced by contribution from hip and knee joint movements during walking. Therefore, we believe that AAVP and AAAP both can be used as reliable measures of impaired dorsiflexion function in post-stroke gait.

bioengineering↗

bioRxiv2023

Exploration of T-cell immune responses by expression of a dominant-negative SHP1 and SHP2

SHP1 and SHP2 are SH2 domain-containing proteins which have inhibitory phosphatase activity when recruited to phosphorylated ITIMs and ITSMs on inhibitory immune receptors. Consequently, SHP1 and SHP2 are key proteins in the transmission of inhibitory signals within T cells, constituting an important point of convergence for diverse inhibitory receptors. Therefore, SHP1 and SHP2 inhibition may represent a strategy for preventing immunosuppression of T cells mediated by cancers hence improving immunotherapies directed against these malignancies. Both SHP1 and SHP2 contain dual SH2 domains responsible for localization to the endodomain of inhibitory receptors and a protein tyrosine phosphatase domain which dephosphorylates and thus inhibits key mediators of T cell activation. We explored the interaction of the isolated SH2 domains of SHP1 and SHP2 to inhibitory motifs from PD1 and identified strong binding of both SH2 domains from SHP2 and more moderate binding in the case of SHP1. We next explored whether these a truncated form of SHP1/2 comprising only of SH2 domains (dSHP1/2) could act in a dominant negative fashion by preventing docking of the wild type proteins. When co-expressed with CARs we found that dSHP2 but not dSHP1 could alleviate immunosuppression mediated by PD1. We next explored the capacity of dSHP2 to bind with other inhibitory receptors and observed several potential interactions. In vivo we observed that the expression of PDL1 on tumor cells impaired the ability of CAR T cells to mediate tumor rejection and this effect was partially reversed by the co-expression of dSHP2 albeit at the cost of reduced CAR T cell proliferation. Modulation of SHP1 and SHP2 activity in engineered T cells through the expression of these truncated variants may enhance T cell activity and hence efficacy in the context of cancer immunotherapy.

immunology↗

bioRxiv2023

Enhancing CAR T cell therapy using Fab-Based Constitutively Heterodimeric Cytokine Receptors

Adoptive T cell therapy aims to achieve lasting tumour clearance, requiring enhanced engraftment and survival of the immune cells. Cytokines are paramount modulators of T cell survival and proliferation. Cytokine receptors signal via ligand-induced dimerization, and this principle has been hijacked utilising non-native dimerization domains. A major limitation of current technologies resides in the absence of a module that recapitulates the natural cytokine receptor heterodimeric pairing. To circumvent this, we created a new engineered cytokine receptor able to constitutively recreate receptor-heterodimer utilising the heterodimerization domain derived from the IgG1 antibody (dFab_CCR). We found that the signal delivered by the dFab_CCR-IL2 proficiently mimics the cytokine receptor heterodimerization, with transcriptomic signatures similar to that obtained by the activation of IL2 native receptor. Importantly, we found that this dimerization structure is agnostic, efficiently activating signaling through four cytokine receptor families. Using a combination of in vivo and in vitro screening approaches, we characterized a library of 18 dFab_CCRs co-expressed with a clinically relevant solid tumor-specific GD2 CAR. Based on this characterization we suggest that the co-expression of either the common {beta}-chain GMCSF or the IL18 dFab_CCRs is optimal to improve CAR T cell expansion, engraftment, and efficacy. Our results demonstrate how the Fab dimerization is efficient and versatile in recapitulating a cytokine receptor heterodimerization signal. This module could be applied for the enhancement of adoptive T cell therapies, and therapies based on other immune cell types. Furthermore, these results provide a choice of cytokine signal to incorporate with adoptive T cells therapies.

immunology↗

bioRxiv2023

Spatiotemporal Genomic Profiling of Intestinal Metaplasia Reveals Clonal Dynamics of Gastric Cancer Progression

Intestinal metaplasia (IM) is a pre-malignant condition of the gastric mucosa associated with increased gastric cancer (GC) risk. We analyzed 1256 gastric samples (1152 IMs) from 692 subjects through a prospective 10-year study. We identified 26 IM driver genes in diverse pathways including chromatin regulation (ARID1A) and intestinal homeostasis (SOX9), largely occurring as small clonal events. Analysis of clonal dynamics between and within subjects, and also longitudinally across time, revealed that IM clones are likely transient but increase in size upon progression to dysplasia, with eventual transmission of somatic events to paired GCs. Single-cell and spatial profiling highlighted changes in tissue ecology and lineage heterogeneity in IM, including an intestinal stem-cell dominant cellular compartment linked to early malignancy. Expanded transcriptome profiling revealed expression-based molecular subtypes of IM, including a body-resident "pseudoantralized" subtype associated with incomplete histology, antral/intestinal cell types, ARID1A mutations, inflammation, and microbial communities normally associated with the healthy oral tract. We demonstrate that combined clinical- genomic models outperform clinical-only models in predicting IMs likely to progress. Our results raise opportunities for GC precision prevention and interception by highlighting strategies for accurately identifying IM patients at high GC risk and a role for microbial dysbiosis in IM progression.

cancer biology↗