Biology subjects

Mehta, N.

Publications and source records attributed to Mehta, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Dual Pathways of Extracellular ATP Action in Cancer Cells: Purinergic Signaling Driven Senescence and Macropinocytic ATP Internalization

BackgroundOpportunistic nutrient uptake is a hallmark of cancer metabolism. Cancer cells upregulate macropinocytosis to acquire extracellular nutrients to support growth and stress adaptation. We previously showed that extracellular ATP (eATP) is internalized by macropinocytosis and promotes multiple cancer phenotypes. Here, we tested whether eATP uptake is prevalent across cancers and whether eATP also induces senescence through purinergic receptor (PR) signaling. MethodsIntracellular ATP (iATP) levels were measured following eATP exposure across multiple cancer cell lines. eATP internalization was visualized in vitro and in vivo using a non-hydrolyzable fluorescent ATP analog together with high-molecular-weight dextran as a macropinocytosis marker. Senescence was quantified using three SA-{beta}-galactosidase assays and flow cytometry. Pharmacologic inhibitors of macropinocytosis and purinergic receptors were used to define pathway dependence. Combination treatments with the glucose transporter inhibitor DRB18 and the senolytic navitoclax were evaluated for antiproliferative effects. ResultseATP produced dose- and time-dependent increases in iATP across diverse cancer cell types. Imaging demonstrated widespread macropinocytic internalization of ATP in vitro and in tumor xenografts. eATP induced senescence in NSCLC cells, confirmed by multiple {beta}-gal assays and flow cytometry. PR inhibition significantly reduced senescence, whereas macropinocytosis inhibition had minimal effect on senescence induction. ConclusionseATP acts through dual pathways in cancer cells: macropinocytic internalization that elevates iATP and PR signaling that drives senescence. Targeting metabolic uptake together with senolytic therapy may offer a novel anticancer strategy.

cancer biology↗

bioRxiv2025

Intracellular amorphous calcium carbonate biomineralization in methanotrophic gammaproteobacteria was acquired by horizontal gene transfer from cyanobacteria

Some bacteria genetically control the biomineralization of intracellular amorphous calcium carbonates (iACC) with potential implications for microbial physiology, evolution, bioremediation and biogeochemical cycling. Until now, this capacity has been documented in Cyanobacteria, the giant gammaproteobacterium Achromatium and a few magnetotactic Pseudomonadota and Nitrospirota. Here, we report the discovery of iACC biomineralization in members of the Methylococcaceae, a family of aerobic methanotrophic Gammaproteobacteria. A homolog of the ccyA gene, previously considered a diagnostic marker for iACC formation in Cyanobacteria, was identified in several Methylococcaceae genomes, based on a search of the conserved C-terminal (GlyZip)3 domain of the encoded calcyanin protein, with a sequence coverage higher than 60% and an E-value lower than 1e-20. Moreover, two cultivated strains, Methylococcus geothermalis and Methylococcus mesophilus, whose genomes contained the ccyA gene, were consistently shown to form iACC granules. The ccyA genes of Methylococcaceae and Microcystis share higher sequence similarity (47%) than with other Cyanobacteria (around 30%) within their common (GlyZip)3 domain, suggesting horizontal gene transfer (HGT) from an ancestral Microcystis-like cyanobacterium to Methylococcaceae. This finding extends the known taxonomic distribution of ccyA and suggests that the capability to biomineralize iACC was acquired by HGT, possibly in environments such as those close to the oxyclines of lakes, where Cyanobacteria and Methylococcaceae commonly co-exist. The discovery of iACC in methane-oxidizing Methylococcaceae highlights a previously unrecognized coupling between calcium carbonate biomineralization and methane cycling in aquatic environments, suggesting that iACC formation may play an overlooked role in microbial carbon storage and local geochemical regulation.

microbiology↗

bioRxiv2025

Alternative splicing of synaptotagmin 7 regulates oligomerization and short-term synaptic plasticity

Synaptic plasticity is crucial for learning and memory. The presynaptic calcium sensor synaptotagmin 7 (syt7) regulates aspects of short-term plasticity (STP), but the underlying mechanisms remain unclear. Here, we show that alternative splicing of the syt7 juxtamembrane linker acts as a molecular switch at both biochemical and functional levels. The and {beta} variants undergo liquid-liquid phase separation to form condensates, while the {gamma} variant forms aggregates. Using iGluSnFR imaging, we found that when expressed at equal levels, these three isoforms also diverge regarding their abilities to regulate two key aspects of STP: paired-pulse facilitation and synaptic depression. STED microscopy showed that all three isoforms form active zone-associated clusters that colocalize with syt1, while MINFLUX super-resolution microscopy resolved syt7 clusters within the active zone, well-positioned to directly control synaptic vesicle dynamics. Thus, alternative splicing might fine-tune STP by differentially impacting syt7 oligomerization.

neuroscience↗

bioRxiv2025

A comparison of long-read single-cell transcriptomic approaches

Long-read sequencing enables the incorporation of isoform-level expression into single-cell transcriptomic studies, offering detail beyond those accessible with short-read methods. Although insightful, these approaches have typically been costly and yielded limited data for each individual cell. Recent advances in library preparation approaches and sequencing throughput have brought long-read single-cell studies closer to the mainstream. Here, we present a comparative analysis of commercial approaches for single-cell long-read sequencing. We have performed parallel analyses of the same cDNA material, generated using the 10X genomics platform, on Illumina short-read, and PacBio and Oxford Nanopore long-read platforms. We also demonstrate the impact of CRISPR-based depletion of libraries, to remove highly expressed transcripts, prior to long-read sequencing in these experiments. By analysing single-source cDNA libraries in parallel, we enable a direct comparison of each platform, evaluating standard metrics alongside concordance in clustering and cell type identification. While each approach generates usable gene and isoform expression data, we identify limitations common across platforms, primarily linked to cDNA synthesis inefficiencies and read filtering strategies. Our work demonstrates the increasing utility of single-cell long-read sequencing for isoform-resolved analyses, such as direct immunoglobulin chain reconstruction without additional amplification, and the detection of alternative splicing patterns across immune cell subtypes in CD45, a key gene for immune cell activation and differentiation. Our benchmarking of current platform options provides a foundation for researchers looking to adopt single-cell long-read sequencing into their transcriptomic studies, providing a framework for its integration into diverse biological questions.

cell biology↗

bioRxiv2024

MORC-1 is a key component of the C. elegans CSR-1 germline gene licensing mechanism

The Argonaute CSR-1 is essential for germline development in C. elegans. Mutation of csr-1 downregulates thousands of germline-expressed genes, leading to the model that the CSR-1-mediated small RNA pathway promotes, or "licenses," gene expression by an unknown mechanism. CSR-1 also silences a limited number of genes through its canonical endonucleolytic "slicer" activity. We show that the GHKL-type ATPase MORC-1, a CSR-1 slicing target, over-accumulates at CSR-1 "licensed" target genes in csr-1(-), which correlates with ectopic gain of H3K9me3, H3K36me3 loss, and gene downregulation. Loss of morc-1 rescues csr-1(-) defects, while overexpressing MORC-1 in the germline of wild-type worms is sufficient to cause sterility and downregulate CSR-1 targets. These results show that MORC-1 overexpression in csr-1(-) is a primary driver of the CSR-1-mediated gene licensing mechanism. One-Sentence SummaryMORC-1 acts downstream of CSR-1 to regulate germline chromatin states and is a key component of the gene licensing mechanism.

molecular biology↗

bioRxiv2024

Olfactory basis for essential amino acid perception during foraging in Caenorhabditis elegans

Acquisition of essential nutrients through diet is crucial for the survival of animals. Dietary odors might enable animals to forage for nutrient-rich diets. We asked if Caenorhabditis elegans, a bacterivorous nematode, uses olfactory cues to forage for essential amino acid-rich (EAA) diets. Using the native microbiome of C. elegans, we show that worms rely on olfaction to select leucine (EAA)-enriched bacteria. Using gas chromatography, we find that leucine-enriched bacteria produce isoamyl alcohol (IAA) odor in the highest abundance. Prior adaptation of worms to IAA diminishes the diet preference of worms. Several wild isolates of C. elegans display robust responses to IAA emphasizing its ecological relevance. We find that foraging for a leucine-enriched diet is mediated via the AWC olfactory neurons. Finally, we identify SNIF-1 G protein-coupled receptor in AWC neurons as a receptor for IAA and a mediator of dietary decisions in worms. Our study identifies a receptor-ligand module underpinning foraging behavior in C. elegans.

animal behavior and cognition↗

bioRxiv2024

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR (FMC63) as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion and memory development of CD19 CAR-T cells in both syngeneic and humanized mouse models of B-ALL/Lymphoma, and enhanced control of disease progression. This approach thus enables vaccine boosting to be applied to any clinically relevant CAR-T cell product.

bioengineering↗

bioRxiv2024

Anchor-Enhanced Bead Design for Reduced Oligonucleotide Synthesis Errors in Single-cell sequencing

Single-cell transcriptomics, reliant on the incorporation of barcodes and unique molecular identifiers (UMIs) into captured polyA+ mRNA, faces a significant challenge due to synthesis errors in oligonucleotide capture sequences. These inaccuracies, which are especially problematic in long-read sequencing, impair the precise identification of sequences and result in inaccuracies in UMI deduplication. To mitigate this issue, we have modified the oligonucleotide capture design, which integrates an interposed anchor between the barcode and UMI, and a V base anchor adjacent to the polyA capture region. This configuration is devised to ensure compatibility with both short and long-read sequencing technologies, facilitating improved UMI recovery and enhanced feature detection, thereby improving the efficacy of droplet-based sequencing methods.

molecular biology↗

bioRxiv2024

A systems-based approach to uterine fibroids identifies differential splicing associated with abnormal uterine bleeding

Uterine fibroids (UFs), benign tumours prevalent in up to 80% of women of reproductive age, are associated with significant morbidity, including abnormal uterine bleeding, pain and infertility. Despite identification of key genomic alterations in MED12 and HMGA2, the pathogenic mechanisms underlying UFs and heavy menstrual bleeding (HMB) remain poorly understood. To correlate systematically genetic, transcriptional and proteomic phenotypes, our study involved an integrative analysis of fibroid, myometrium and endometrium tissues from 137 patients, utilising genome-wide SNP arrays, targeted sequencing, RNA sequencing and proteomics. Our findings reveal 39.7% of UFs possess MED12 mutations, alongside novel variants in genes such as COL4A5 and COL4A6. Multi-omics factor analysis of integrated protein and mRNA highlighted differential regulation related to extracellular matrix remodelling, proteolysis and homeostasis in fibroid versus myometrium tissues, and distinct gene sets associated with RNA splicing in the endometrium of patients with HMB, particularly in MED12-mutated fibroids. Our study proposes a model, which is supported by in vivo evidence, where altered signalling of MED12-mutated fibroids influences RNA transcript isoform expression in endometrium, potentially leading to abnormal uterine bleeding. This integrative approach unravels complex molecular pathways in UF pathogenesis and HMB, offering novel insights for targeted therapeutic development.

systems biology↗

bioRxiv2023

The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation

Synaptotagmin (syt) 1, a Ca2+ sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca2+ via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca2+-sensitivity of syt1. Moreover, Ca2+ and anionic phospholipids facilitate the observed phase separation, and increases in [Ca2+]i promote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca2+ binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS.

neuroscience↗

bioRxiv2023

A developmental gradient reveals biosynthetic pathways to eukaryotic toxins in monocot geophytes

Numerous eukaryotic toxins that accumulate in geophytic plants are valuable in the clinic, yet their biosynthetic pathways have remained elusive. A lead example is the >150 Amaryllidaceae alkaloids (AmAs) including galantamine, an FDA-approved treatment for Alzheimers disease. We show that while AmAs accumulate to high levels in many tissues in daffodils, biosynthesis is localized to nascent, growing tissue at the base of leaves. A similar trend is found for the production of steroidal alkaloids (e.g. cyclopamine) in corn lily. This model of active biosynthesis enabled elucidation of a complete set of biosynthetic genes for the production of AmAs. Taken together, our work sheds light on the developmental and enzymatic logic of diverse alkaloid biosynthesis in daffodil. More broadly, it suggests a paradigm for biosynthesis regulation in monocot geophytes where plants are protected from herbivory through active charging of newly formed cells with eukaryotic toxins that persist as aboveground tissue develops.

biochemistry↗

bioRxiv2023

Correcting PCR amplification errors in unique molecular identifiers to generate absolute numbers of sequencing molecules.

Unique Molecular Identifiers (UMIs) are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are the main source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing UMIs using homotrimeric nucleotide blocks provides an error correcting solution, that allows absolute counting of sequenced molecules.

genomics↗

bioRxiv2023

Pupil dilation and microsaccades provide complementary insights into the dynamics of arousal and instantaneous attention during effortful listening

Listening in noisy environments requires effort - the active engagement of attention and other cognitive abilities, as well as increased arousal. The ability to separately quantify the contribution of these components is key to understanding the dynamics of effort and how it may change across listening situations and in certain populations. We concurrently measured two types of ocular data in young participants (both sexes) - Pupil dilation (PD) (thought to index arousal aspects of effort) and Microsaccades (MS) (hypothesized to reflect automatic visual exploratory sampling) whilst listeners were performing a speech-in-noise task under high- (HL) and low-(LL) perceptual load conditions. Sentences were manipulated so that the behaviourally relevant information (keywords) appeared at the end (Exp1) or beginning (Exp2) of the sentence, resulting in different temporal demands on focused attention. In line with previous reports, PD effects were associated with increased dilation under load. We observed a sustained difference between HL and LL conditions, consistent with increased phasic and tonic arousal. Importantly we show that MS rate was also modulated by perceptual load, manifested as a reduced MS rate in HL relative to LL. Critically, in contrast to the sustained difference seen for PD, MS effects were localised in time, specifically during periods when demands on auditory attention were greatest. These results demonstrate that auditory selective attention interfaces with the mechanisms controlling MS-generation, establishing MS as an informative measure, complementary to PD, with which to quantify the temporal dynamics of auditory attentional processing under effortful listening conditions. Significance StatementListening effort, reflecting the "cognitive bandwidth" deployed to effectively process sound in adverse environments, contributes critically to listening success. Understanding listening effort and the processes involved in its allocation is a major challenge in auditory neuroscience. Here we demonstrate that the microsaccade rate can be used to index a specific sub-component of listening effort - the allocation of instantaneous auditory attention - that is distinct from the modulation of arousal indexed by pupil dilation (currently the dominant measure of listening effot). These results reveal the push-pull process through which auditory attention interfaces with the (visual) attention network that controls microsaccades, establishing microsaccades as a powerful tool for measuring auditory attention and its deficits.

neuroscience↗

bioRxiv2023

Spatially aware deep learning reveals tumor heterogeneity patterns that encode distinct kidney cancer states

Background Abstract Background Results Discussion Methods Data Availability Code Availability Author Contributions Competing Interests Extended Data References Renal cell carcinoma (RCC) is among the 10 most common cancers worldwide and is comprised of several histological subtypes1. The clear cell histological subtype (ccRCC) is the most common form of RCC and accounts for the vast majority (75-80%) of metastatic cases1. In addition to highly recurrent mutations in hypoxia (VHL) and chromatin regulator genes (e.g. PBRM1, BAP1, SETD2), ccRCC exhibits extensive genomic intratumoral heterogeneity (ITH)2, which wa ...

cancer biology↗

bioRxiv2022

Description of Gloeomargarita ahousahtiae sp. nov., a thermophilic member of the order Gloeomargaritales with intracellular carbonate inclusions

A unicellular cyanobacterium, strain VI4D9, was isolated from thermophilic microbial mats thriving in a hot spring of the Ahousaht territory of Vancouver Island, Canada. The cells were elongated rods (5.1 m in length and 1.2 m in width on average). Their UV-visible absorption spectra revealed that they contain chlorophyll a, phycocyanin, and carotenoids. Transmission electron microscopy showed the presence of thylakoids concentrated on one side of the cells. The strain grew within a temperature range of 37-50{degrees}C, with an optimum at 45{degrees}C. Its genome had a size of 3,049,282 bp and a DNA G+C content of 51.8 mol%. The cells contained numerous intracellular spherical granules easily visible under scanning electron microscopy. Energy-dispersive x-ray spectroscopy revealed that these granules were made of Ca-, Ba- and Sr-containing carbonates. A phylogenetic 16S rRNA gene tree robustly placed this strain as sister to several environmental sequences and the described species Gloeomargarita lithophora, also characterized by the possession of intracellular carbonate inclusions. We consider strain VI4D9 to represent a new Gloeomargarita species based on its marked phenotypic differences with G. lithophora, notably, its thermophilic nature and different thylakoid organization. We propose the name Gloeomargarita ahousahtiae sp. nov. for this newly isolated thermophilic cyanobacterium. The type strain is VI4D9 (Culture Collection of Algae and Protozoa strain 1472/1; Laboratorio de Algas Continentales Mexico strain LAC 140). G. ahousahtiae is the second species described within the recently discovered order Gloeomargaritales.

microbiology↗

bioRxiv2022

Syntaxin 3-SPI 2 dependent cross-talk facilitates the division of Salmonella containing vacuole (SCV)

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in the vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. A report showed that Salmonella containing vacuole (SCV) harbors recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis is unclear. Upon knockdown of STX3, we have observed a reduction in bacterial proliferation and is restored upon the overexpression of STX3. Post infected live-cell imaging of cells showed STX3 localises to the SCV membranes and thus might help in fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found this interaction abrogated when we infected with SPI-2 encoded T3SS apparatus mutant (STM {Delta}ssaV) but not with SPI-1 encoded T3SS (STM{Delta} invC). These observations were also consistent in mice model of Salmonella infection. Together, these results shed a light on the effector molecules secreted through SPI-2 encoded by T3SS possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and maintenance the principle single bacterium per vacuole. SynopsisSalmonella Typhimurium infection in murine macrophage leads to upregulation of host Syntaxin 3 both at transcript and protein levels at late stage of infection. Syntaxin 3 cross-talk with Salmonella containing vacuoles (SCVs) is essential for establishment of replicative niche in host macrophages. The cross-talk between STX3 and SCVs is Salmonella pathogenicity island 2 (SPI-2) dependent and is consistent in mice model of Salmonella Typhimurium infection.

microbiology↗

bioRxiv2022

Synaptotagmin 7 outperforms synaptotagmin 1 to open and stabilize nascent fusion pores via robust membrane penetration

Synaptotagmin-1 and synaptotagmin-7 are two prominent Ca2+ sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding Ca2+, both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examined the biophysical properties of these two synaptotagmin isoforms and compared their interactions with phospholipid membranes. We discovered that synaptotagmin-1*membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a "super-penetrator". We exploited these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to Ca2+.

neuroscience↗

bioRxiv2022

The canonical Wnt signaling pathway is not a promising therapeutic target for PNS regeneration enhancement

PNS injury initiates transcriptional changes in Schwann cells, satellite glial cells and PNS neurons that facilitate regeneration. The signaling pathways that control these transcriptional changes are not fully understood. The canonical Wnt signaling pathway is active during early stages of PNS development, where it controls radial axonal sorting and the onset of PNS myelination. Upon PNS injury, the Wnt signaling pathway is re-activated, suggesting that Wnt signaling plays an important role in PNS regeneration. To explore the potential of the Wnt pathway as a therapeutic target for enhancement of PNS recovery, we used a combination of genetic and pharmacological approaches to either activate or inhibit the Wnt signaling pathway during PNS recovery. We found that manipulating the Wnt signaling pathway does not alter PNS regeneration. Our data suggests that the Wnt signaling pathway is not a strong therapeutic target for the enhancement of PNS regeneration.

neuroscience↗