Biology subjects

Jain, V.

Publications and source records attributed to Jain, V..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Inhibition of UBE2N in regulatory T-cells boosts immunity against cancer

Regulatory T (Treg) cells prevent autoimmunity and facilitate cancer immune evasion. Depletion of Tregs is a promising cancer therapy, but risks of autoimmune reactions hamper its clinical translation. Here, we demonstrate that temporally induced deletion of Ube2n in Tregs (Ube2nTreg-KO) of adult mice results in a robust expansion and activation of cytotoxic CD8+ T-cells in response to cancer cell challenges, producing a long-lasting survival benefit without autoimmune complications. The anti-tumor effect persists following adoptive T-cell transfer to T-cell-deficient Rag1-knockout mice. Single-cell transcriptomic analysis revealed that UBE2N deletion shifted immunosuppressive Tregs to effector-like T-cells. This shift is characterized by the downregulation of c-Myc target genes, resembling that observed in tumor-infiltrating Tregs of melanoma patients. Further analyses confirm that UBE2N maintains c-Myc protein stability via suppression of K48-Ubiquitin-mediated proteasomal degradation. Taken together, our studies uncover a hitherto unexplored and potentially druggable UBE2N/c-Myc signaling axis to eradicate Treg-enabled cancer immune escape.

cancer biology↗

bioRxiv2024

DeepFocus: A Transnasal Approach for Optimized Deep Brain Stimulation of Reward Circuit Nodes

ObjectiveTranscranial electrical stimulation (TES) is an effective technique to modulate brain activity and treat diseases. However, TES is primarily used to stimulate superficial brain regions and is unable to reach deeper targets. The spread of injected currents in the head is affected by volume conduction and the additional spreading of currents as they move through head layers with different conductivities, as is discussed in [1]. In this paper, we introduce DeepFocus, a technique aimed at stimulating deep brain structures in the brains "reward circuit" (e.g. the orbitofrontal cortex, Brodmann area 25, amygdala, etc.). ApproachTo accomplish this, DeepFocus utilizes transnasal electrode placement (under the cribriform plate and within the sphenoid sinus) in addition to electrodes placed on the scalp, and optimizes current injection patterns across these electrodes. To quantify the benefit of DeepFocus, we develop the DeepROAST simulation and optimization platform. DeepROAST simulates the effect of complex skull-base bones geometries on the electric fields generated by DeepFocus configurations using realistic head models. It also uses optimization methods to search for focal and efficient current injection patterns, which we use in our simulation and cadaver studies. Main ResultsIn simulations, optimized DeepFocus patterns created larger and more focal fields in several regions of interest than scalp-only electrodes. In cadaver studies, DeepFocus patterns created large fields at the medial orbitofrontal cortex (OFC) with magnitudes comparable to stimulation studies, and, in conjunction with established cortical stimulation thresholds, suggest that the field intensity is sufficient to create neural response, e.g. at the OFC. SignificanceThis minimally invasive stimulation technique can enable more efficient and less risky targeting of deep brain structures to treat multiple neural conditions.

neuroscience↗

bioRxiv2024

Gli2 Facilitates Tumor Immune Evasion and Immunotherapeutic Resistance by Coordinating Wnt Ligand and Prostaglandin Signaling

Therapeutic resistance to immune checkpoint blockade has been commonly linked to the process of mesenchymal transformation (MT) and remains a prevalent obstacle across many cancer types. An improved mechanistic understanding for MT-mediated immune evasion promises to lead to more effective combination therapeutic regimens. Herein, we identify the Hedgehog transcription factor, Gli2, as a key node of tumor-mediated immune evasion and immunotherapy resistance during MT. Mechanistic studies reveal that Gli2 generates an immunotolerant tumor microenvironment through the upregulation of Wnt ligand production and increased prostaglandin synthesis. This pathway drives the recruitment, viability, and function of granulocytic myeloid-derived suppressor cells (PMN-MDSCs) while also impairing type I conventional dendritic cell, CD8+ T cell, and NK cell functionality. Pharmacologic EP2/EP4 prostaglandin receptor inhibition and Wnt ligand inhibition each reverses a subset of these effects, while preventing primary and adaptive resistance to anti-PD-1 immunotherapy, respectively. A transcriptional Gli2 signature correlates with resistance to anti-PD-1 immunotherapy in stage IV melanoma patients, providing a translational roadmap to direct combination immunotherapeutics in the clinic. SIGNIFICANCEGli2-induced EMT promotes immune evasion and immunotherapeutic resistance via coordinated prostaglandin and Wnt signaling.

cancer biology↗

bioRxiv2024

Light-microscopy based dense connectomic reconstruction of mammalian brain tissue

The information-processing capability of the brains cellular network depends on the physical wiring pattern between neurons and their molecular and functional characteristics. Mapping neurons and resolving their individual synaptic connections can be achieved by volumetric imaging at nanoscale resolution with dense cellular labeling. Light microscopy is uniquely positioned to visualize specific molecules but dense, synapse-level circuit reconstruction by light microscopy has been out of reach due to limitations in resolution, contrast, and volumetric imaging capability. Here we developed light-microscopy based connectomics (LICONN). We integrated specifically engineered hydrogel embedding and expansion with comprehensive deep-learning based segmentation and analysis of connectivity, thus directly incorporating molecular information in synapse-level brain tissue reconstructions. LICONN will allow synapse-level brain tissue phenotyping in biological experiments in a readily adoptable manner. One-Sentence SummaryHydrogel expansion enables molecularly informed reconstruction of brain tissue at synaptic resolution with light microscopy.

neuroscience↗

bioRxiv2023

UBE2N is essential for maintenance of skin homeostasis and suppression of inflammation

UBE2N, a Lys63-ubiquitin conjugating enzyme, plays critical roles in embryogenesis and immune system development and function. However, its roles in adult epithelial tissue homeostasis and pathogenesis are unclear. We generated conditional mouse models that deleted Ube2n in skin cells in a temporally and spatially controlled manner. We found that Ube2n- knockout (KO) in the adult skin keratinocytes induced a range of inflammatory skin defects characteristic of psoriatic and actinic keratosis. These included eczematous inflammation, epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration, as well as signs of edema and blistering. Single cell transcriptomic analyses and RT-qPCR showed that Ube2n KO keratinocytes expressed elevated myeloid cell chemo-attractants such as Cxcl1 and Cxcl2 and decreased the homeostatic T lymphocyte chemo-attractant, Ccl27a. Consistently, the infiltrating immune cells of Ube2n-KO skin were predominantly myeloid-derived cells including neutrophils and M1-like macrophages that were highly inflammatory, as indicated by expression of Il1{beta} and Il24. Pharmacological blockade of the IL-1 receptor associated kinases (IRAK1/4) alleviated eczema, epidermal and dermal thickening, and immune infiltration of the Ube2n mutant skin. Together, these findings highlight a key role of keratinocyte-UBE2N in maintenance of epidermal homeostasis and skin immunity and identify IRAK1/4 as potential therapeutic target for inflammatory skin disorders.

pathology↗

bioRxiv2023

Determining the effects of temperature on the evolution of bacterial tRNA pools

The genetic code consists of 61 codon coding for 20 amino acids. These codons are recognized by transfer RNAs (tRNA) that bind to specific codons during protein synthesis. Most organisms utilize less than all 61 possible anticodons due to base pair wobble: the ability to have a mismatch with a codon at its third nucleotide. Previous studies observed a correlation between the tRNA pool of bacteria and the temperature of their respective environments. However, it is unclear if these patterns represent biological adaptations to maintain the efficiency and accuracy of protein synthesis in different environments. A mechanistic mathematical model of mRNA translation is used to quantify the expected elongation rates and error rate for each codon based on an organisms tRNA pool. A comparative analysis across a range of bacteria that accounts for covariance due to shared ancestry is performed to quantify the impact of environmental temperature on the evolution of the tRNA pool. We find that thermophiles generally have more anticodons represented in their tRNA pool than mesophiles or psychrophiles. Based on our model, this increased diversity is expected to lead to increased missense errors. The implications of this for protein evolution in thermophiles are discussed. SignificanceProtein synthesis is a vital biological process; however, our understanding of the impact of environmental factors, such as temperature, on the evolution of the molecular mechanisms involved in protein synthesis is limited. In this study, we investigated the impact of environmental temperature on the evolution of the tRNA pool. Our analyses revealed that heat-loving bacteria (thermophiles) generally have more anticodons represented in their tRNA pool. Based on a simple model of ribosome elongation, this observed increase in tRNA diversity in thermophiles is expected to also increase the frequency of translation errors. We speculate that the increased diversity of the tRNA pool could be due to the decreased efficiency of wobble base pairing at higher temperatures, necessitating more tRNA with exact codon-anticodon pairings. Our findings provide key insights into the role of the environment in shaping the tRNA pool.

evolutionary biology↗

bioRxiv2023

Probing Molecular Diversity and Ultrastructure of Brain Cells with Fluorescent Aptamers

Detergent-free immunolabeling has been proven feasible for correlated light and electron microscopy, but its application is restricted by the availability of suitable affinity reagents. Here we introduce CAptVE, a method using slow off-rate modified aptamers for cell fluorescence labeling on ultrastructurally reconstructable electron micrographs. CAptVE provides labeling for a wide range of biomarkers, offering a pathway to integrate molecular analysis into recent approaches to delineate neural circuits via connectomics.

neuroscience↗

bioRxiv2023

Multiplexed Volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

neuroscience↗

bioRxiv2023

Somatic small-variant calling methods in Illumina DRAGEN™ Secondary Analysis

We present the DRAGEN somatic pipeline for calling small somatic variants from tumor samples, with or without paired normal samples. The DRAGEN somatic variant caller offers 1) a flexible architecture that can be used on a wide array of somatic use cases; 2) built-in noise models enabling robustness against various sources of noise artifacts (mapping, genome context, or sample specific); 3) performance of joint analysis of tumor and normal samples in the case of a tumor-normal workflow yielding improved accuracy; 4) benefits from FPGA acceleration for efficient run time. We demonstrate the speed and accuracy of the DRAGEN tumor-normal pipeline across a range of whole genome sequencing (WGS) datasets and compare against third party tools such as Mutect2/GATK4 [1] and Strelka2 [2]. DRAGEN secondary analysis outperforms all other tools with its ability to complete a 110x/40x T/N whole-genome analysis in less than two hours. It offers exceptional accuracy, with higher sensitivity and precision than third party tools. We also show that the DRAGEN T/N workflow supports analysis of liquid and late-stage solid tumors by tolerating tumor-in-normal (TiN) contamination.

bioinformatics↗

bioRxiv2023

Automated microarray for single-cell sorting and collection of lymphocytes following HIV reactivation

A promising strategy to cure HIV infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T-lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of various LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5 %, 11.2 %, and 12.1 % percentage of cells respectively, reactivated similar to that observed in other experimental systems. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T-lymphocytes that were either fast or slow reactivators were sorted, and single-cell RNA-sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found. These results advance our conceptual understanding of HIV reactivation dynamics at the single-cell level toward a cure for HIV.

bioengineering↗

bioRxiv2023

Sex-biased expression is associated with chromatin state in D. melanogaster and D. simulans

We propose a new model for the association of chromatin state and sex-bias in expression. We hypothesize enrichment of open chromatin in the sex where we see expression bias (OS) and closed chromatin in the opposite sex (CO). In this study of D. melanogaster and D. simulans head tissue, sex-bias in expression is associated with H3K4me3 (open mark) in males for male-biased genes and in females for female-biased genes in both species. Sex-bias in expression is also largely conserved in direction and magnitude between the two species on the X and autosomes. In male-biased orthologs, the sex-bias ratio is more divergent between species if both species have H3K27me2me3 marks in females compared to when either or neither species has H3K27me2me3 in females. H3K27me2me3 marks in females are associated with male-bias in expression on the autosomes in both species, but on the X only in D. melanogaster. In female-biased orthologs the relationship between the species for the sex-bias ratio is similar regardless of the H3K27me2me3 marks in males. Female-biased orthologs are more similar in the ratio of sex-bias than male-biased orthologs and there is an excess of male-bias in expression in orthologs that gain/lose sex-bias. There is an excess of male-bias in sex-limited expression in both species suggesting excess male-bias is due to rapid evolution between the species. The X chromosome has an enrichment in male-limited H3K4me3 in both species and an enrichment of sex-bias in expression compared to the autosomes.

evolutionary biology↗

bioRxiv2022

Ganglioglioma deep transcriptomics reveals primitive neuroectoderm neural precursor-like population

Gangliogliomas are brain tumors composed of neuron-like and macroglia-like components that occur in children and young adults. Gangliogliomas are often characterized by a rare population of immature astrocyte-appearing cells expressing CD34, a marker expressed in the neuroectoderm (neural precursor cells) during embryogenesis. New insights are needed to refine tumor classification and to identify therapeutic approaches. We evaluated five gangliogliomas with single nucleus RNA-seq, cellular indexing of transcriptomes and epitopes by sequencing, and/or spatially-resolved RNA-seq. We uncovered a population of CD34+ neoplastic cells with mixed neuroectodermal, immature astrocyte, and neuronal markers. Gene regulatory network interrogation in these neuroectoderm-like cells revealed control of transcriptional programming by TCF7L2/MEIS1-PAX6 and SOX2, similar to that found during neuroectodermal/neural development. Developmental trajectory analyses place neuroectoderm-like tumor cells as precursor cells that give rise to neuron-like and macroglia-like neoplastic cells. Spatially-resolved transcriptomics revealed a neuroectoderm-like tumor cell niche with relative lack of vascular and immune cells. We used these high resolution results to deconvolute clinically-annotated transcriptomic data, confirming that CD34+ cell-associated gene programs associate with gangliogliomas compared to other glial brain tumors. Together, these deep transcriptomic approaches characterized a ganglioglioma cellular hierarchy - confirming CD34+ neuroectoderm-like tumor precursor cells, controlling transcription programs, cell signaling, and associated immune cell states. These findings may guide tumor classification, diagnosis, prognostication, and therapeutic investigations.

cancer biology↗

bioRxiv2022

Population History and Genome Wide Association Studies of Birth Weight in a Native High Altitude Ladakhi Population

Pathological low birth weight due to fetal growth restriction (FGR)is an important predictor of adverse obstetric and neonatal outcomes. It is more common amongst native lowlanders when gestating in the hypoxic environment of high altitude, whilst populations who have resided at high altitude for many generations are relatively protected. Genetic study of pregnant populations at high altitude permits exploration of the role of hypoxic in FGR pathogenesis, and perhaps of FGR pathogenesis more broadly. We studied the umbilical cord blood DNA of 316 neonates born to pregnant women managed at the Sonam Norboo Memorial Hospital, Ladakh (altitude 3540m) between February 2017-January 2019. Principal component, admixture and genome wide association studies (GWAS) were applied to dense single nucleotide polymorphism (SNP) genetic data, to explore ancestry and genetic predictors of low birth weight. Our findings support Tibetan ancestry in the Ladakhi population, with subsequent admixture with neighboring Indo-Aryan populations. Fetal growth protection was evident in Ladakhi neonates. Although no variants achieved genome wide significance, we observed nominal association of seven variants across genes (ZBTB38, ZFP36L2, HMGA2, CDKAL1, PLCG1) previously associated with birthweight.

genetics↗

bioRxiv2022

Multi-Layered Maps of Neuropil with Segmentation-Guided Contrastive Learning

Maps of the nervous system that identify individual cells along with their type, subcellular components, and connectivity have the potential to reveal fundamental organizational principles of neural circuits. Volumetric nanometer-resolution imaging of brain tissue provides the raw data needed to build such maps, but inferring all the relevant cellular and subcellular annotation layers is challenging. Here, we present Segmentation-Guided Contrastive Learning of Representations ("SegCLR"), a self-supervised machine learning technique that produces highly informative representations of cells directly from 3d electron microscope imagery and segmentations. When applied to volumes of human and mouse cerebral cortex, SegCLR enabled the classification of cellular subcompartments (axon, dendrite, soma, astrocytic process) with 4,000-fold less labeled data compared to fully supervised approaches. Surprisingly, SegCLR also enabled inference of cell types (neurons, glia, and subtypes of each) from fragments with lengths as small as 10 micrometers, a task that can be difficult for humans to perform and whose feasibility greatly enhances the utility of imaging portions of brains in which many neuron fragments terminate at a volume boundary. These predictions were further augmented via Gaussian process uncertainty estimation to enable analyses restricted to high confidence subsets of the data. Finally, SegCLR enabled detailed exploration of layer-5 pyramidal cell subtypes and automated large-scale statistical analysis of upstream and downstream synaptic partners in mouse visual cortex.

neuroscience↗

bioRxiv2022

The RNA helicase DDX39B activates FOXP3 RNA splicing to control T regulatory cell fate

Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor- gene (IL7R) in MS-risk. Based on evolutionary and functional arguments we postulated that DDX39B enhances immune tolerance decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified ForkheadBox P3 (FOXP3), which codes for the master transcriptional factor in CD4+/CD25+ T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized subclass of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.

molecular biology↗

bioRxiv2021

Denoising-based Image Compression for Connectomics

Connectomic reconstruction of neural circuits relies on nanometer resolution microscopy which produces on the order of a petabyte of imagery for each cubic millimeter of brain tissue. The cost of storing such data is a significant barrier to broadening the use of connectomic approaches and scaling to even larger volumes. We present an image compression approach that uses machine learning-based denoising and standard image codecs to compress raw electron microscopy imagery of neuropil up to 17-fold with negligible loss of 3d reconstruction and synaptic detection accuracy.

neuroscience↗

bioRxiv2021

A connectomic study of a petascale fragment of human cerebral cortex

We acquired a rapidly preserved human surgical sample from the temporal lobe of the cerebral cortex. We stained a 1 mm3 volume with heavy metals, embedded it in resin, cut more than 5000 slices at [~]30 nm and imaged these sections using a high-speed multibeam scanning electron microscope. We used computational methods to render the three-dimensional structure containing 57,216 cells, hundreds of millions of neurites and 133.7 million synaptic connections. The 1.4 petabyte electron microscopy volume, the segmented cells, cell parts, blood vessels, myelin, inhibitory and excitatory synapses, and 104 manually proofread cells are available to peruse online. Many interesting and unusual features were evident in this dataset. Glia outnumbered neurons 2:1 and oligodendrocytes were the most common cell type in the volume. Excitatory spiny neurons comprised 69% of the neuronal population, and excitatory synapses also were in the majority (76%). The synaptic drive onto spiny neurons was biased more strongly toward excitation (70%) than was the case for inhibitory interneurons (48%). Despite incompleteness of the automated segmentation caused by split and merge errors, we could automatically generate (and then validate) connections between most of the excitatory and inhibitory neuron types both within and between layers. In studying these neurons we found that deep layer excitatory cell types can be classified into new subsets, based on structural and connectivity differences, and that chandelier interneurons not only innervate excitatory neuron initial segments as previously described, but also each others initial segments. Furthermore, among the thousands of weak connections established on each neuron, there exist rarer highly powerful axonal inputs that establish multi-synaptic contacts (up to [~]20 synapses) with target neurons. Our analysis indicates that these strong inputs are specific, and allow small numbers of axons to have an outsized role in the activity of some of their postsynaptic partners.

neuroscience↗

bioRxiv2021

Gain control by sparse, ultra-slow glycinergic synapses

SO_SCPLOWUMMARYC_SCPLOWRetinal ON starburst amacrine cells (SACs) play a critical role in computing stimulus direction, partly in service of image stabilization by optokinetic nystagmus. ON SAC responses are sculpted by rich GABAergic innervation, mostly from neighbouring SACs. Surprisingly, however, we find that glycinergic narrow field amacrine cells (NACs) serve as their dominant source of inhibition during sustained activity. Although NAC inputs constitute only [~]5% of inhibitory synapses to ON SACs, their distinct input patterns enable them to drive glycine inhibition during the both light increments and decrements. NAC-to-ON-SAC inhibition appears to be mediated by ultra-slow non-canonical glycine receptors containing the 4 subunit, which effectively summate during repetitive stimulation. Glycinergic inhibition strongly decreases the output gain of the SACs, ensuring that their direction-selective output is maintained over their operating range. These results reveal an unexpected role for glycinergic pathways and receptor kinetics in modulating direction selectivity in the retina.

neuroscience↗