Biology subjects

Trivedi, V.

Publications and source records attributed to Trivedi, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Dehydrozingerone mitigates energy deficits and cognitive impairments induced by cranial irradiation

Radiotherapy is widely used in the management of brain tumors; however, it is often associated with delayed adverse effects, including cognitive decline and depression-like behavior. These effects are thought to arise, in part, from suppressed hippocampal neurogenesis, altered neuronal architecture, and microglial dysfunction. Despite this, the precise mechanisms underlying irradiation-induced cognitive deficits, as well as effective therapeutic interventions, remain poorly understood. In the present study, six-month-old male mice were subjected to a single 9 Gy dose of cranial irradiation, followed by behavioral assessments several weeks post-exposure. We observed that cranial irradiation significantly impaired hippocampal-, prefrontal cortex-, and cortical-dependent memory functions. Notably, treatment with dehydrozingerone (DH), a curcumin analog (50 mg/kg, oral administration for two weeks), markedly prevented these cognitive deficits. At the molecular level, irradiation disrupted the activity of key enzymes involved in the tricarboxylic acid (TCA) cycle and the glutamate-glutamine/GABA cycle, both of which were restored following DH treatment. Furthermore, irradiation induced dysregulation of genes and proteins associated with glycolysis (Atp2b1, mt-Nd2, mt-Atp6), mitochondrial energetics (mt-Atp8, mt-Cytb), glucose transport (Slc4a5), insulin resistance (Etnppl), lipid metabolism (Pla2g3, Plin4), and inflammation (Ighg2c), all of which were significantly normalized by DH. Importantly, DH also prevented irradiation-induced loss of cell-type-specific glucose transporter expression, including GLUT3 in neurons and GLUT5 in microglia. In conclusion, our findings suggest that DH is a promising therapeutic candidate for mitigating irradiation-induced energy deficits and cognitive impairments, likely through modulation of metabolic and mitochondrial pathways.

neuroscience↗

bioRxiv2025

Dysregulated Microglial Synaptic Engulfment in Diffuse Midline Glioma

Diffuse midline glioma (DMG) is a near-universally lethal form of pediatric high-grade glioma, driven by neuronal activity-regulated paracrine signaling and synaptic integration of malignant cells into neural circuits. In turn, DMG increases neuronal excitability, augmenting neuron-to-glioma signaling. In the healthy brain, microglia, the resident immune cells of the central nervous system (CNS), regulate neuronal excitability and synaptic connectivity. However, the role of microglia in promoting tumor-associated hyperexcitable neural networks in glioma remains unknown. Here, we investigate the activity-regulated engulfment of neuronal synapses by microglia in both healthy and glioma-bearing mice, and further explore how glioma cells alter microglia-mediated circuit refinement, contributing to pathogenic neuronal hyperexcitability. Microglia-mediated circuit refinement in the glioma microenvironment was characterized through synaptic engulfment analysis of both excitatory and inhibitory synapses by microglia in healthy mice and patient-derived DMG xenograft models, paired with optogenetic stimulation in the neocortex. We found that glutamatergic neuronal activity in the healthy brain increased excitatory synaptic engulfment by microglia in a previously unappreciated negative feedback mechanism that may guard against hyperexcitability. In contrast, this activity-regulated increase in excitatory synaptic engulfment was abrogated in DMG-infiltrated brains. Instead, inhibitory synaptic engulfment was significantly increased in DMG in response to glutamatergic neuronal activity. Together, these dysregulated synaptic engulfment mechanisms may create imbalance in the excitatory to inhibitory (E:I) synapse ratio predicted to increase neuronal excitability. Complementary single-nuclei sequencing studies revealed concordant tumor-specific, activity-regulated changes in microglia-neuron signaling showing reduced expression of excitatory synaptic refinement gene programs in microglia, potentially mediating the aberrant synaptic engulfment observed in DMG. These findings reveal novel cancer-neuron-immune interactions in DMG and provide an opportunity to potentially modulate tumor-associated neuronal hyperexcitability by targeting aberrant microglial synaptic engulfment. HIGHLIGHTSO_LIExcitatory synaptic engulfment by microglia increases in a glutamatergic neuronal activity-dependent manner in the healthy brain. C_LIO_LIIn the glioma-infiltrated brain, this feedback regulatory mechanism is inverted, with loss of activity-regulated excitatory synaptic engulfment and aberrant increased inhibitory synaptic engulfment. C_LIO_LITranscriptomic changes in glioma-associated microglia contribute to differential excitatory and inhibitory synaptic engulfment, with reduced excitatory synaptic refinement gene programs in tumor-associated microglia. C_LIO_LIActivity-regulated soluble factors drive this imbalanced excitatory-inhibitory postsynaptic engulfment by glioma-associated microglia. C_LI

neuroscience↗

bioRxiv2025

Species-specific basal fluidization shapes early forebrain development

The human forebrain exhibits an expanded surface area compared with other great apes. Despite progress in dissecting species-specific gene expression programs and cell lineages, the tissue-level physical mechanisms underlying human-specific neurodevelopment remain unclear. Here, by quantifying tissue dynamics and mechanics using oil microdroplets in cerebral organoids from humans, gorillas, chimpanzees, and mice, we uncover a transient tissue fluidization occurring exclusively in the basal region of human neuroepithelia. Enhanced droplet motility and frequent cell rearrangements reveal this basal fluidization, which gradually diminishes at the onset of neurogenesis in humans. By contrast, such basal fluidization is not detected in non-human neuroepithelia examined here. Mechanistically, basal fluidization is driven by basal nuclear fluctuations and facilitated by expanded intercellular spaces. Increasing N-cadherin expression in human neuroepithelia to gorilla levels suppresses both nuclear fluctuations and basal fluidization. Integrating simulations of neuroepithelial dynamics with experimental data, we further find that nuclear fluctuations underlying basal fluidization promote the insertion of newly divided nuclei into the basal side to support tangential surface expansion, a hallmark of human forebrain development.

developmental biology↗

bioRxiv2025

ETS-guided iPSC-endothelial models recapitulate malaria pathogenesis

The sequestration of the malaria parasite Plasmodium falciparum in the microvasculature is a major driver of severe malaria, but its pathogenic mechanisms still remain unknown. Advancements in induced pluripotent stem cell (iPSC) technologies offer unique opportunities to study parasite interactions with blood vessels in a well-defined host environment. However, endothelial iPSC-differentiation methods often result in cells with mixed epithelial identity. Here, we have generated an iPSC line with inducible and simultaneous expression of ETS transcription factors (ETV2, FLI1, ERG), which resulted in improved endothelial cell identity and strong barrier function. These cells display a high affinity to infected red blood cells. Exposure to parasite products caused significant endothelial metabolic changes and splicing alterations. Furthermore, it disrupted the iPSC-endothelial barrier, as a consequence of transcriptional downregulation of key barrier processes, and alteration of severe malaria biomarkers. Our novel iPSC-based approach represents a new in vitro platform to study the pathogenesis of vascular infections.

microbiology↗

bioRxiv2025

Axial patterning of gastruloids via diverse cell type compositions

The formation of the germ layers and antero-posterior (AP) axial patterning are interlinked milestones of embryogenesis. Gastruloids, in vitro models from aggregated embryonic stem cells (ESCs), permit the study and deconstruction of these events. Gastruloids are successfully generated from ESCs of variable pluripotent states, but it remains unknown how the initial conditions influence cell type composition and to what degree resulting variations in spatial patterning can converge onto an elongated body axis. To address this, we comparatively study aggregates from varying proportions of primed and naive ESCs. Despite differences in AP symmetry-breaking dynamics and distinct trajectories toward either anterior mesodermal or neuro-ectodermal fates, all conditions produce elongated gastruloids. Furthermore, timed modulation of Activin/Nodal signaling in aggregates equalizes AP polarization modes and mixing of initial ESC pluripotent states generates gastruloids with enhanced tissue type diversity. This work therefore uncovers a previously unappreciated developmental flexibility underlying mammalian AP axial patterning.

developmental biology↗

bioRxiv2024

Cell-cell communication controls the timing of gastruloid symmetry-breaking

How cell fate decisions coordinate with tissue-scale morphogenesis remains a major challenge in developmental biology. Gastruloids, three-dimensional aggregates of pluripotent stem cells that self-organise and break symmetry via polarised Brachyury/T expression, provide an ideal system to address this question. By generating gastruloids with defined initial proportions of T-expressing cells we show that fate decisions occur collectively, with cell-fate proportions influencing the transition rates. Mechanical measurements reveal differences in surface tension between T-positive and T-negative tissues, consistent with radial cell sorting. Finally, incorporating fate dynamics and mechanics into a computational model recapitulates the sequential symmetry-breaking events observed in vitro. Our findings identify a mechanochemical mechanism underlying axis formation, and demonstrate how multicellular systems can robustly self-organise without external signalling cues.

biophysics↗

bioRxiv2024

Optimized genome-wide CRISPR screening enables rapid engineering of growth-based phenotypes in Yarrowia lipolytica

CRISPR-Cas9 functional genomic screens uncover gene targets linked to various phenotypes for metabolic engineering with remarkable efficiency. However, these genome-wide screens face a number of design challenges, including variable guide RNA activity, ensuring sufficient genome coverage, and maintaining high transformation efficiencies to ensure full library representation. These challenges are prevalent in non-conventional yeast, many of which exhibit traits that are well suited to metabolic engineering and bioprocessing. To address these hurdles in the oleaginous yeast Yarrowia lipolytica, we designed a compact, high-activity genome-wide sgRNA library. The library was designed using DeepGuide, a sgRNA activity prediction algorithm, and a large dataset of [~]50,000 sgRNAs with known activity. Three guides per gene enables redundant targeting of 98.8% of genes in the genome in a library of 23,900 sgRNAs. We deployed the optimized library to uncover genes essential to the tolerance of acetate, a promising alternative carbon source, and various hydrocarbons present in many waste streams. Our screens yielded several gene knockouts that improve acetate tolerance on their own and as double knockouts in media containing acetate as the sole carbon source. Analysis of the hydrocarbon screens revealed genes related to fatty acid and alkane metabolism in Y. lipolytica. The optimized CRISPR gRNA library and its successful use in Y. lipolytica led to the discovery of alternative carbon source-related genes and provides a workflow for creating high-activity, compact genome-wide libraries for strain engineering. HighlightsO_LIDesigned a compact, high activity CRISPR sgRNA knockout library for Yarrowia lipolytica. C_LIO_LIDeveloped an efficient pipeline for discovering genes involved in alternative carbon-source utilization. C_LIO_LIIdentified single and double gene knockouts that improve growth on acetate. C_LIO_LIIdentified genes with improved fitness and essentiality for hydrocarbon growth. C_LI

synthetic biology↗

bioRxiv2024

Functional genomic screening in Komagataella phaffii enabled by high-activity CRISPR-Cas9 library

CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast Komagataella phaffii is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in K. phaffii, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guides targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of K. phaffii essential genes, many of which relate to protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in K. phaffii, applied here to gene essentiality classification, and promises to enable other genetic screens. HighlightsO_LIDesigned and validated a high activity genome-wide CRISPR-Cas9 library for K. phaffii C_LIO_LIDisabling NHEJ DNA repair enables the generation of genome-wide guide activity profiles C_LIO_LIActivity-corrected fitness screens identify a high confidence set of essential genes in K. phaffii C_LIO_LIProtein production, secretion, and glycosylation pathways are essential in K. phaffii but not in other yeasts C_LI

systems biology↗

bioRxiv2023

Metabolic control of germ layer proportions through regulation of Nodal and Wnt signalling

During embryonic development, cells exit pluripotency to give rise to the three germ layers. Metabolic pathways influence cell fate decisions by modulating the epigenetic, transcriptional, and signalling states of cells. However, the interplay between metabolism and the major signalling pathways that drive the emergence of ectoderm, mesoderm, and endoderm remains poorly understood. Here, we demonstrate an instructive role of glycolytic activity in activating signalling pathways involved in mesoderm and endoderm induction. Using an in vitro model system for mouse gastrulation, we observed that inhibiting glycolysis prevents the upregulation of primitive streak markers, resulting in a significant increase in ectodermal cell fates at the expense of mesodermal and endodermal lineages. We demonstrate that this relationship is dose-dependent, enabling metabolic control of germ layer proportions through exogenous glucose levels. Mechanistically, we found that glycolysis inhibition leads to the downregulation of Wnt, Nodal, and Fgf signalling. Notably, this metabolic phenotype was rescued by Nodal or Wnt signalling agonists in the absence of glycolytic activity, suggesting that glycolytic activity acts upstream of both signalling pathways. Our work underscores the dependence of specific signalling pathways on metabolic conditions and provides mechanistic insight into the nutritional regulation of cell fate decision making.

developmental biology↗

bioRxiv2023

Population genomics-guided engineering of phenazine biosynthesis in Pseudomonas chlororaphis

The emergence of next-generation sequencing (NGS) technologies has made it possible to not only sequence entire genomes, but also identify metabolic engineering targets across the pangenome of a microbial population. This study leverages NGS data as well as existing molecular biology and bioinformatics tools to identify and validate genomic signatures for improving phenazine biosynthesis in Pseudomonas chlororaphis. We sequenced a diverse collection of 34 Pseudomonas isolates using short- and long-read sequencing techniques and assembled whole genomes using the NGS reads. In addition, we assayed three industrially relevant phenotypes (phenazine production, biofilm formation, and growth temperature) for these isolates in two different media conditions. We then provided the whole genomes and phenazine production data to a unitig-based microbial genome-wide association study (mGWAS) tool to identify novel genomic signatures responsible for phenazine production in P. chlororaphis. Post-processing of the mGWAS analysis results yielded 330 significant hits influencing the biosynthesis of one or more phenazine compounds. Based on a quantitative metric (called the phenotype score), we elucidated the most influential hits for phenazine production and experimentally validated them in vivo in the most optimal phenazine producing strain. Two genes significantly increased phenazine-1-carboxamide (PCN) production: a histidine transporter (ProY_1), and a putative carboxypeptidase (PS__04251). A putative MarR-family transcriptional regulator decreased PCN titer when overexpressed in a high PCN producing isolate. Overall, this work seeks to demonstrate the utility of a population genomics approach as an effective strategy in enabling identification of targets for metabolic engineering of bioproduction hosts.

systems biology↗

bioRxiv2022

A stem cell zoo uncovers intracellular scaling of developmental tempo across mammals

Differential speeds in biochemical reactions have been proposed to be responsible for the differences in developmental tempo between mice and humans. However, the underlying mechanism controlling the species-specific kinetics remains to be determined. Using in vitro differentiation of pluripotent stem cells, we recapitulated the segmentation clocks of diverse mammalian species varying in body weight and taxa: marmoset, rabbit, cattle and rhinoceros. Together with the mouse and human, the segmentation clock periods of the six species did not scale with the animal body weight, but were rather grouped according to phylogeny. The biochemical kinetics of the core clock gene HES7 displayed clear scaling with the species-specific segmentation clock period. However, the cellular metabolic rates did not show an evident correlation. Instead, genes involving biochemical reactions showed an expression pattern that scales with the segmentation clock period. Altogether, our stem cell zoo uncovered general scaling laws governing species-specific developmental tempo.*

developmental biology↗

bioRxiv2022

acCRISPR: An activity-correction method for improving the accuracy of CRISPR screens

High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of novel and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes, thus determining the fitness effect of disrupted genes. This is accomplished by calculating an optimization metric that quantifies the tradeoff between guide activity and library coverage, which is maximized to accurately classify genes essential to screening conditions. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in gain-and loss-of-function screens under high salt and low pH conditions to identify known and novel genes that were related to stress tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.

synthetic biology↗

bioRxiv2022

Single-cell Bayesian deconvolution

Individual cells exhibit substantial heterogeneity in protein abundance and activity, which is frequently reflected in broad distributions of fluorescently labeled reporters. Since all cellular components are intrinsically fluorescent to some extent, the observed distributions contain background noise that masks the natural heterogeneity of cellular populations. This limits our ability to characterize cell-fate decision processes that are key for development, immune response, tissue homeostasis, and many other biological functions. It is therefore important to separate the contributions from signal and noise in single-cell measurements. Addressing this issue rigorously requires deconvolving the noise distribution from the signal, but approaches in that direction are still limited. Here we present a non-parametric Bayesian formalism that performs such a deconvolution efficiently on multidimensional measurements, in a way that allows us to estimate confidence intervals. We use the approach to study the expression of the mesodermal transcription factor Brachyury in mouse embryonic stem cells undergoing differentiation.

developmental biology↗

bioRxiv2021

Flow Cytometric Evaluation of Yeast-Bacterial Cell-Cell Interactions

Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting (FACS) to enrich for a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports characterization of a therapeutically relevant nanobody-antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic E. coli. Overall, our findings indicate that the yeast-bacteria format supports efficient evaluation of ligand-target interactions. With further development, this format may facilitate systematic characterization and high throughput discovery of bacterial surface-binding molecules.

synthetic biology↗

bioRxiv2021

CD36: Hemin interaction axis to control immune responses and cytokine secretion from macrophages involving Lyn kinase.

The intensity and duration of TNF- production are mutually correlated with the level of CD36 expression level. The macrophages exposed to hemin exhibits modulation of non-opsonic phagocytosis of aged RBCs and ability to kill bacteria. Immuno-fluorescence study indicates translocation and sequestration of CD36 within the intracellular storage in the hemin treated macrophages. It in-tern dysregulate the global cytokine secretion from macrophages. CD36 has suitable hemin biophoric environment involving R292, D372 and Q382 to bind and the mutation in biophore residues (R292A, D372A or Q382A) significantly reduced the affinity. Ectopic expression of CD36 in MG63 cells showed several folds increment in cytokines TNF-, MCP-1, RANTES and CCL1 in response to hemin stimulation but no significant amount of cytokines released with mutants (R292A, D372A or Q382A), highlights the relevance of CD36-hemin interaction for immune-dysfunction. Hemin is driving down-stream signalling involving CD36 and subsequent recruitment of adaptor proteins to the cytosolic domain of CD36. Immuno-precipitation of membrane bound CD36 and detection of adaptor proteins indicate change in level of Lyn proteins with CD36 fractions after hemin stimulation to macrophages. The Lyn targeted siRNA restored the phagocytic activity, reduced the secretion of pro-inflammatory cytokine levels clearly suggests the Src family protein Lyn is crucial for CD36-hemin mediated immune dysregulation and cytokine secretion. In summary, hemin-CD36-Lyn cytokine signalling axis could be a contribution factor to severe malaria pathology and prognosis.

immunology↗

bioRxiv2021

Dynamics of anteroposterior axis establishment in a mammalian embryo-like system

1.In the mammalian embryo, specification of the anteroposterior (AP) axis demarcates one of the first steps of body plan formation. While this process requires interactions with extra-embryonic tissues in the native embryo, minimal in vitro systems from embryonic stem cells (ESCs) undergo initial AP polarization in the absence of any localized, external cues. This self-organizing potential of stem cells remains not well understood. Here, we study such an initial symmetry breaking event in gastruloids, an established in vitro model for mammalian body plan formation, using the mesodermal marker gene Brachyury or T (Bra/T) to denote the onset of AP axis specification and concomitant germ layer formation. Through aggregate fusion experiments and manipulation of initial culture conditions as well as key developmental signalling pathways, we probe the dynamics of Bra/T polarization. We further conduct single-cell (sc) RNA sequencing of gastruloids at early stages to identify incipient molecular signatures of germ layer commitment and differences between Bra/T+ and Bra/T- populations during as well as after symmetry breaking. Moreover, we transcriptionally compare early development of gastruloids to the mouse embryo and conclude that gastruloids reproducibly undergo AP axis and germ layer specification in a parallel, but distinct manner: While their primed pluripotent cell populations adopt a more mesenchymal state in lieu of an epithelial epiblast-like transcriptome, the emerging mesendodermal lineages in vitro are nevertheless similar to their in vivo equivalents. Altogether, this study provides a comprehensive analysis of self-organized body plan establishment in a minimal in vitro system of early mammalian patterning and highlights the regulative capacity of mESCs, thereby shedding light on underlying principles of axial polarity formation.

developmental biology↗

bioRxiv2020

linus: Conveniently explore, share and present large-scale biological trajectory data from a web browser.

In biology, we are often confronted with information-rich, large-scale trajectory data, but exploring and communicating patterns in such data is often a cumbersome task. Ideally, the data should be wrapped with an interactive visualisation in one concise package that makes it straightforward to create and test hypotheses collaboratively. To address these challenges, we have developed a tool, linus, which makes the process of exploring and sharing 3D trajectories as easy as browsing a website. We provide a python script that reads trajectory data and enriches them with additional features, such as edge bundling or custom axes and generates an interactive web-based visualisation that can be shared offline and online. The goal of linus is to facilitate the collaborative discovery of patterns in complex trajectory data.

bioinformatics↗

bioRxiv2019

Self-organised symmetry breaking in zebrafish reveals feedback from morphogenesis to pattern formation

A fundamental question in developmental biology is how the early embryo breaks initial symmetry to establish the spatial coordinate system later important for the organisation of the embryonic body plan. In zebrafish, this is thought to depend on the inheritance of maternal mRNAs [1-3], cortical rotation to generate a dorsal pole of beta-catenin activity [4-8] and the release of Nodal signals from the yolk syncytial layer (YSL) [9-12]. Recent work aggregating mouse embryonic stem cells has shown that symmetry breaking can occur in the absence of extra-embryonic tissue [19,20]. To test whether this is also true in zebrafish, we separated embryonic cells from the yolk and allowed them to develop as aggregates. These aggregates break symmetry autonomously to form elongated structures with an anterior-posterior pattern. Extensive cell mixing shows that any pre-existing asymmetry is lost prior to the breaking morphological symmetry, revealing that the maternal pre-pattern is not strictly required for early embryo patterning. Following early signalling events after isolation of embryonic cells reveals that a pole of Nodal activity precedes and is required for elongation. The blocking of PCP-dependent convergence and extension movements disrupts the establishment of opposing poles of BMP and Wnt/TCF activity and the patterning of anterior-posterior neural tissue. These results lead us to suggest that convergence and extension plays a causal role in the establishment of morphogen gradients and pattern formation during zebrafish gastrulation.

developmental biology↗