Biology subjects

Raj, N.

Publications and source records attributed to Raj, N..

16 recordsLinked to original sources

bioRxiv2026

Neutrophils are critical for placental and fetal infection with the human pathogen Listeria monocytogenes

Fetal listeriosis, caused by Listeria monocytogenes (Lm), represents a severe infectious disease. During pregnancy, the unique intrauterine environment permits Lm to breach the maternal-fetal barrier by infiltrating the placental trophoblast layer and ultimately leading to significant fetal morbidity and mortality. However, the exact pathways remain incompletely understood. In this study, we sought to elucidate the molecular mechanisms underlying the early stages of placental and fetal Lm infection. Our results reveal that neutrophils serve as a survival niche for Lm within the vasculature, facilitating placental and fetal infection in the mouse in vivo. This finding was further substantiated by in vitro studies using TLR2-stimulated trophoblast cells and Lm-infected neutrophils. Additionally, tissue-clearing analysis of Lm-infected placentas demonstrated neutrophil colocalization with Lm is rare within placental tissue. Collectively, our findings identify maternal neutrophils as an essential viability niche for Lm enabling their entry into trophoblast cells, thereby promoting placental and fetal infection. One Sentence SummaryMaternal circulating neutrophils act as survival niche for Listeria monocytogenes and facilitate placental/fetal infection with Listeria during pregnancy.

immunology↗

bioRxiv2026

THERM-D Uncovers Distinct Neural Mechanisms Separating Morning and Evening Body Temperature Rhythms in Drosophila

Animal body temperature rises throughout the day and peaks in the evening, a pattern conserved across diurnal endotherms and ectotherms. However, the mechanisms driving the robust body temperature rhythms (BTR) remain largely unclear. Here, we developed a machine learning-based platform, temperature homeostasis evaluation of rhythmicity in model Drosophila (THERM-D), enabling continuous, high-throughput analyses of BTR. Using THERM-D, we identified robust BTR patterns reflecting flies morning and evening behaviors and revealed the function of CRYPTOCHROME (CRY)-negative clock neurons. About half of all clock neurons lack CRY, yet their function was unclear. Newly developed Gal4 drivers targeting CRY-negative neurons demonstrated that these neurons control the morning temperature rise without affecting evening BTR. The data suggests that separate clock circuits regulate morning and evening BTR. Thus, THERM-D elucidated the role of CRY-negative clock neurons, which are specialized for BTR regulation and distinct from the circuits controlling sleep-wake cycles.

neuroscience↗

bioRxiv2026

Genomic signatures of asymmetric selection on membrane and cytoplasmic proteins.

Protein evolution is influenced by functional, structural, and ecological factors. Here, we propose that subcellular localization adds a critical but less understood dimension to evolutionary constraints. Specifically, we hypothesize that membrane proteins evolve under distinct pressures compared to cytoplasmic proteins, owing to the (a) association between co-translational folding and membrane integration and (b) roles at the interface with dynamic environments. Drawing from comparative genomics data of over 1,000 strains each of Escherichia coli and Saccharomyces cerevisiae, we present evidence suggesting that membrane proteins exhibit increased non-synonymous variation and reduced synonymous variation relative to cytoplasmic proteins. These patterns are stronger in E. coli, as compared to S. cerevisiae. Our data reveal systematic trends consistent with our hypothesis. We argue that recognizing localization-dependent selection could refine our understanding of evolutionary dynamics, inform molecular evolution models, and guide future synthetic and evolutionary studies.

evolutionary biology↗

bioRxiv2025

Facultative Cheating and Hybrid Vigor Resolves Cooperator-Cheater Conflict in a Yeast Public Goods System.

The persistence of cooperation in the face of cheating is a central paradox in evolutionary biology. Microbial public goods systems employ diverse solutions to this dilemma, yet most studies assume fixed strategies wherein genotypes function strictly as cooperators or cheaters. Here, using the GAL/MEL regulon of Saccharomyces cerevisiae, we uncover a dynamic resolution to this conflict through facultative strategy switching. When haploid cheater-cooperator strains were co-evolved in melibiose, we observed the repeated emergence of same-mating-type diploid hybrids. These hybrids arise early in evolution and ultimately spread in the population. The hybrids exploit the public good produced by cooperator strains when present, acting as facultative cheaters. However, following cooperator extinction, hybrids switch to a cooperative phenotype. This dynamic role transition enables the hybrid to persist across shifting ecological contexts. Our findings reveal a novel, context-dependent mechanism of cooperation maintenance, whereby facultative cheating and genotype plasticity resolve the tension between individual fitness and collective benefit. This work expands the conceptual framework of social evolution by demonstrating that phenotypic flexibility, facilitated through hybridization, can stabilize cooperation even in fully exploitable public goods systems.

evolutionary biology↗

bioRxiv2025

Daily light exposure habits of youth with migraine: A prospective pilot study

BackgroundEighty percent of youth with migraine report photophobia. It is unknown if photophobia leads to light avoidant behavior, and if such behaviors worsen light sensitivity and disrupt sleep. Recently developed wearable, continuous light loggers allow us to address these open questions. We conducted a pilot study to determine the feasibility of measuring light exposure using wearable light loggers in youth with migraine. MethodsYouth 10 - 21 years old with a headache-specialist confirmed ICHD-3 diagnosis of migraine were recruited from CHOP headache clinics. Each participant recorded 7 consecutive days of light logging data from the ActLumus device worn as a pendant around the neck paired with a text-based daily migraine symptom diary during a typical school week between November and March 2024. Validated questionnaires were used to capture any headache and bad headache frequency, headache-related disability, visual sensitivity, fear-of-pain, and sleep disturbance and impairment. Percent time spent within recommended light exposure levels was calculated for the day, 3 hours prior to bedtime, and night. Power analysis was calculated to determine sample size needed for group comparison of baseline characteristics across light intensity and light timing metrics to aid in the design of larger studies. ResultsTwenty youth with a median age 17 years [IQR 16, 19], 70% of whom were female completed 7 days of continuous light logger recording and daily headache diary. Data completion rates were high with 136/140 (97.1%) useable days of light logger data, and 100% compliance on the daily headache diary. Participant feedback on the study was positive; 85% would recommend the study to others. On average, participants received recommended light exposure during only 14.5% +/- SD 7.0 of daylight hours. By contrast, participants were more consistently below the recommended maximum light levels 3 hours prior to bed (77.5% +/- 21.6 of the time), and at night (99.1% +/- 2.9 of the time). Youth with chronic migraine (i.e., at least 15 headache days and 8 bad headache days per month) had daily light exposure patterns that were phase shifted 60 minutes later as compared to participants with non-chronic migraine. Power analyses suggest that future tests for differences in light exposure between migraine-characteristic groups (e.g., differing by headache frequency, severity, or disability) will require sample sizes on the order of 50 to 150 to reach 80% power with an alpha of 0.05. ConclusionMeasuring daily light exposure is feasible in pediatric populations with photophobia and reveals intriguing trends in youth with migraine that warrant further study.

neuroscience↗

bioRxiv2024

Variations and predictability of epistasis on an intragenic fitness landscape.

How epistasis hinders or facilitates movement on fitness landscapes has been a longstanding question in evolutionary biology. High-throughput experiments have revealed that, despite their idiosyncratic nature, epistatic interactions can exhibit reproducible global statistical patterns. Recently, Papkou et al. constructed a fitness landscape for a 9-base pair region of the folA gene in Escherichia coli, which encodes dihydrofolate reductase (DHFR), and showed that this landscape is both rugged and highly navigable. Here, we analyze this landscape to address two questions: How does the nature of epistasis between two mutations change with genetic background? and How predictable is epistasis within a gene? We find that epistasis is "fluid": higher-order interactions cause the relationship between two mutations to shift strongly across genetic backgrounds. Mutations fall into two distinct categories: a small subset exhibit strong global epistasis, while the majority do not. Nonetheless, we find that the distribution of fitness effects (DFE) of a genotype is highly predictable from its fitness. These findings provide a gene-level perspective on how epistasis operates, revealing both its unpredictability and its statistical regularities, and offer a framework for predicting mutational effects from high-dimensional fitness landscapes. Significance Statement.The effect of a mutation on fitness depends on the genome in which it occurs, a phenomenon known as epistasis. Epistasis makes evolution difficult to predict, but recent work has uncovered statistical regularities in how it manifests. Using a fitness landscape of [~]260,000 variants of an E. coli gene, we show that higher-order interactions make pair-wise epistasis "fluid": the relationship between two mutations changes with genetic background. We also find that epistasis is "binary" - a small subset of mutations exhibits strong global statistical patterns, while most do not. These findings reveal new principles of how epistasis shapes protein evolution and, ultimately, organismal adaptation.

evolutionary biology↗

bioRxiv2024

Membrane tension regulation is required for wound repair

Disruptions of the eukaryotic plasma membrane due to chemical and mechanical challenges are frequent and detrimental, and thus need to be repaired to maintain proper cell function and avoid cell death. However, the cellular mechanisms involved in wound resealing and restoration of homeostasis are diverse and contended. Here, we show that clathrin-mediated endocytosis is induced at later stages of plasma membrane wound repair following the actual resealing of the wound. This compensatory endocytosis occurs near the wound, predominantly at sites of previous early endosome exocytosis which is required in the initial stage of membrane resealing, suggesting a spatio-temporal co-ordination of exo- and endocytosis during wound repair. Using cytoskeletal alterations and modulation of membrane tension and membrane area, we identify membrane tension as a major regulator of the wounding-associated exo- and endocytic events that mediate efficient wound repair. Thus, membrane tension changes are a universal trigger for plasma membrane wound repair modulating the exocytosis of early endosomes required for resealing and subsequent clathrin-mediated endocytosis acting at later stages to restore cell homeostasis and function.

cell biology↗

bioRxiv2024

Empirical evidence of resource dependent evolution of payoff matrices in Saccharomyces cerevisiae populations.

In evolutionary game theory, a relative comparison of the cost and benefit associated with obtaining a resource, called payoff, is used as an indicator of fitness of an organism. Such payoff matrices are used to understand complex inter-species and intra-species interactions like cooperation, mutualism, and altruism. In the absence of any empirical data, the evolution of these payoff matrices has been investigated theoretically by tweaking well-established game theory models. In this paper, we present empirical evidence of three types of resource-dependent changes in the payoff matrices of evolving Saccharomyces cerevisiae populations. We show that depending on the carbon source and participating genotypes, the payoff matrix could either (a) evolve quantitatively yet maintain a cheater-cooperator game, (b) change qualitatively such that the cheater-cooperator game collapses, or (c) change qualitatively to result in the birth of a cheater-cooperator game. Our results highlight the need to consider the dynamic nature of payoff matrices while making even short-term predictions about population interactions and dynamics.

evolutionary biology↗

bioRxiv2023

Transcriptomic analysis identifies synapse-enriched lncRNAs required for glutamatergic synapse development and fear memory consolidation.

Regulatory functions of lncRNAs in neurons have been majorly limited to the nucleus. The identity of synaptic lncRNAs and their functional roles associated with synapse development and plasticity are poorly understood. Transcriptomic analysis from synaptoneurosomes identified 94 synapse-enriched lncRNAs from the mouse hippocampus. We find Pvt1 to be a specific regulator of excitatory, not inhibitory, synapse development. Synapse-specific loss of Pvt1 regulates synaptic activity and the downregulation of mRNAs pertinent to synapse development. We also report a synapse-centric role for an uncharacterized lncRNA; 2410006H16Rik (SynLAMP); in translation and memory formation. SynLAMP demonstrates enhanced localization to the synapse following fear conditioning and regulates dendritic translation by sequestering the translation repressor FUS; acting as a molecular decoy. Synapse-specific knockdown of SynLAMP inhibits the localised, activity-dependent translation of CamK2a, a FUS target. SynLAMP RNAi occludes fear-memory formation. Comprehensively, our study highlights that de novo activity of lncRNAs are involved in diverse synaptic functions.

neuroscience↗

bioRxiv2023

Maintenance of cooperation in a yeast population in a public-good driven system.

The phenomenon of cooperation is prevalent at all levels of life. In microbial communities, some groups of cells exhibit cooperative behaviour by producing costly extracellular resources that are freely available to others. These resources are referred to as public goods. Saccharomyces cerevisiae secretes invertase (public good) in the periplasm to hydrolyse sucrose into glucose and fructose, which are further imported by the cells. After hydrolysis of sucrose, the cells retain only 1% of the monosaccharides, while 99% diffuse into the environment and can be utilised by all neighbouring cells. The non-producers of invertase (cheaters) exploit the invertase-producing cells (cooperators) by utilising the monosaccharides and paying nothing for the latter. In this work, we investigate the evolutionary dynamics of this cheater-cooperator system. If cheaters are selected for their higher fitness, the population will collapse. For cooperators to survive cheating and thrive in nature, they should have evolved some survival strategies. To understand the adaptation of cooperators in sucrose, we performed a coevolution experiment in sucrose. Our results show that cooperators increase in fitness as the experiment progresses. This phenomenon was not observed in environments which involved a non-public good system. Genome sequencing reveals the molecular basis for the cooperator adaptating is because of increased privatization of the public-good released carbon resource.

evolutionary biology↗

bioRxiv2023

Cross-species transcriptomic analysis identifies mitochondrial dysregulation as a functional consequence of the schizophrenia-associated 3q29 deletion

Recent advances in the genetics of schizophrenia (SCZ) have identified rare variants that confer high disease risk, including a 1.6 Mb deletion at chromosome 3q29 with a staggeringly large effect size (O.R. > 40). Understanding the impact of the 3q29 deletion (3q29Del) on the developing CNS may therefore lead to insights about the pathobiology of schizophrenia. To gain clues about the molecular and cellular perturbations caused by the 3q29 deletion, we interrogated transcriptomic effects in two experimental model systems with complementary advantages: isogenic human forebrain cortical organoids and isocortex from the 3q29Del mouse model. We first created isogenic lines by engineering the full 3q29Del into an induced pluripotent stem cell line from a neurotypical individual. We profiled transcriptomes from isogenic cortical organoids that were aged for 2 months and 12 months, as well as day p7 perinatal mouse isocortex, all at single cell resolution. Differential expression analysis by genotype in each cell-type cluster revealed that more than half of the differentially expressed genes identified in mouse cortex were also differentially expressed in human cortical organoids, and strong correlations were observed in mouse-human differential gene expression across most major cell-types. We systematically filtered differentially expressed genes to identify changes occurring in both model systems. Pathway analysis on this filtered gene set implicated dysregulation of mitochondrial function and energy metabolism, although the direction of the effect was dependent on developmental timepoint. Transcriptomic changes were validated at the protein level by analysis of oxidative phosphorylation protein complexes in mouse brain tissue. Assays of mitochondrial function in human heterologous cells further confirmed robust mitochondrial dysregulation in 3q29Del cells, and these effects are partially recapitulated by ablation of the 3q29Del gene PAK2. Taken together these data indicate that metabolic disruption is associated with 3q29Del and is conserved across species. These results converge with data from other rare SCZ-associated variants as well as idiopathic schizophrenia, suggesting that mitochondrial dysfunction may be a significant but overlooked contributing factor to the development of psychotic disorders. This cross-species scRNA-seq analysis of the SCZ-associated 3q29 deletion reveals that this copy number variant may produce early and persistent changes in cellular metabolism that are relevant to human neurodevelopment.

genomics↗

bioRxiv2022

Metabolic Engineering Of Lactococcus Lactis For The Production Of Heparosan

Heparosan is a precursor molecule for the widely used anticoagulant heparin, which also has other uses such as certain drug delivery applications and as a scaffold for tissue engineering in biomaterials. Traditionally, pathogenic bacteria such as E.Coli have been used as a host to produce heparosan as an alternative to animal and chemoenzymatic synthesis. Using GRAS status organisms like Lactococcus Lactis as the host for production of heparosan provides a safe alternative as well as being a well-established organism for genetic manipulation and reengineering. In this study, a functional heparosan synthesis pathway was successfully expressed in Lactococcus Lactis by the expression of E.coli K5 genes KfiA and KfiC, along with the overexpression of ugd, glmu and pgma genes present natively in the host organism. The genes were activated using the tightly controlled NICE expression system. The genes were cloned into plasmid p8148 and transformed into two strains, Lactococcus Lactis NZ9000 and Lactococcus Lactis NZ9020, totaling six different recombinant strains were created using these two hosts and various combinations of the heterologous genes. The recombinant Lactococcus Lactis SH6 strain, expressing the genes ugd-KfiA-KfiC-pgma yielded a maximum concentration of 754 mg/l in batch bioreactor experiments and the titer was increased to 1263 mg/l in fed-batch fermentation. NMR imaging successfully determined that the structure of the product derived from Lactococcus Lactis was indeed similar to E.coli heparosan. The molecular weight of heparosan varied from 10-20 KDa, indicating its potential use for chemoenzymatic heparin biosynthesis.

bioengineering↗

bioRxiv2022

Early endosomes act as local exocytosis hubs to repair endothelial membrane damage

The plasma membrane of a cell is subject to stresses causing ruptures that must be repaired immediately to preserve membrane integrity and ensure cell survival. Yet, the spatio-temporal membrane dynamics at the wound site and the source of membrane required for wound repair are poorly understood. Here, we show that early endosomes, previously only known to function in the uptake of extracellular material and its endocytic transport, are involved in plasma membrane repair in human endothelial cells. Using live-cell imaging and correlative light and electron microscopy, we demonstrate that membrane injury triggers a previously unknown exocytosis of early endosomes that is induced by Ca2+ entering through the wound. This exocytosis is restricted to the vicinity of the wound site and mediated by the endosomal SNARE VAMP2, which is crucial for efficient membrane repair. Thus, the here identified Ca2+-evoked and localized exocytosis of early endosomes supplies the membrane material required for rapid resealing of a damaged plasma membrane, thereby providing the first line of defense against damage in mechanically challenged endothelial cells.

cell biology↗

bioRxiv2021

The Mettl3 epitranscriptomic writer amplifies p53 stress responses

The p53 transcription factor, encoded by the most frequently mutated gene in human cancer, plays a critical role in tissue homeostasis in response to stress signals. The mechanisms through which p53 promotes downstream tumor suppressive gene expression programs remain, however, only superficially understood. Here, we used tandem affinity purification and mass spectrometry to reveal new components of the p53 response. This approach uncovered Mettl3, a component of the m6A RNA methyltransferase complex (MTC), as a p53-interacting protein. Analysis of Mettl3- deficient cells revealed that Mettl3 promotes p53 protein stabilization and target gene expression in response to DNA damage. Mettl3 acts in part by competing with the p53 negative regulator, Mdm2, for binding to the p53 transactivation domains to promote methyltransferase-independent stabilization of p53. In addition, Mettl3 relies on its catalytic activity to augment p53 responses, with p53 recruiting Mettl3 to p53 target genes to co-transcriptionally direct m6A modification of p53 pathway transcripts to enhance their expression. Mettl3 also promotes p53 activity downstream of oncogenic signals in vivo, in both allograft and autochthonous lung adenocarcinoma models, suggesting cooperative action of p53 and Mettl3 in tumor suppression. Accordingly, we found in diverse human cancers that mutations in MTC components perturb expression of p53 target genes and that MTC mutations are mutually exclusive with TP53 mutations, suggesting that the MTC enhances the p53 transcriptional program in human cancer. Together, these studies reveal a fundamental role for Mettl3 in amplifying p53 signaling through protein stabilization and epitranscriptome regulation.

cancer biology↗

bioRxiv2020

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes, such as limb and craniofacial defects as well as bone marrow failure. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining comprehensive mouse genetics and in vivo ribosome profiling, we observe limb patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos and uncover corresponding selective translational changes of transcripts controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are reconciled by the unexpected identification that p53 functions as a master regulator of protein synthesis through transcriptional activation of 4E-BP1. 4E-BP1, a key regulator of translation, in turn, facilitates selective changes in the translatome downstream of p53 and thereby explains, at least in part, how RP haploinsufficiency elicits specificity to gene expression. These results provide an integrative model to explain how in vivo tissue-specific phenotypes emerge from a mutation in a ribosome component.

developmental biology↗

bioRxiv2019

FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex sequences

The sorting of RNA molecules to distinct subcellular locations facilitates the activity of spatially restricted processes through local protein synthesis. This process affects thousands of transcripts yet precisely how these RNAs are trafficked to their destinations remains generally unclear. Here we have analyzed subcellular transcriptomes of FMRP-null mouse neuronal cells to identify transcripts that depend on FMRP for efficient transport to neurites. We found that these FMRP RNA localization targets contain a large enrichment of G-quadruplex sequences, particularly in their 3' UTRs, suggesting that FMRP recognizes these sequences to promote the localization of transcripts that contain them. Fractionation of neurons derived from human Fragile X Syndrome patients revealed a high degree of conservation in the identity of FMRP localization targets between human and mouse as well as an enrichment of G-quadruplex sequences in human FMRP RNA localization targets. Using high-throughput RNA/protein interaction assays and single-molecule RNA FISH, we identified the RGG domain of FMRP as important for both interaction with G-quadruplex RNA sequences and the neuronal transport of G-quadruplex-containing transcripts. Finally, we used ribosome footprinting to identify translational regulatory targets of FMRP. The translational regulatory targets were not enriched for G-quadruplex sequences and were largely distinct from the RNA localization targets of FMRP, indicating that the two functions can be biochemically separated and are mediated through different target recognition mechanisms. These results establish a molecular mechanism underlying FMRP-mediated neuronal RNA localization and provide a framework for the elucidation of similar mechanisms governed by other RNA-binding proteins.

molecular biology↗