Biology subjects

Jain, N.

Publications and source records attributed to Jain, N..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Insights into the Evolution of Ancient Shark and Ray Sex Chromosomes

While sex-determining mechanisms have been extensively characterized in many vertebrates, they have not been explored in chondrichthyan fishes until relatively recently. In the present study, we used high-quality whole genome reference assemblies to examine the putative sex chromosomes of 14 elasmobranch species spanning nine orders. We describe four newly assembled reference genomes belonging to the white shark Carcharodon carcharias, the Atlantic stingray Hypanus sabinus, the smalltooth sawfish Pristis pectinata, and the zebra shark Stegostoma tigrinum. We conducted sex chromosome identification and verification using short-read sequence data collected for multiple individuals for three of the species. This revealed putative pseudoautosomal regions (PARs) and, in one instance, a candidate sex chromosome reassignment. A synteny analysis revealed an ancient and shared origin of the chromosomes within elasmobranchs considerably older than any previously proposed scenario, and a potential candidate gene involved in sex determination shared across all examined species. The synteny analysis also revealed a historical fusion and the formation of neo-Y chromosomes between two myliobatiform species. Our results show that there has been strong conservation and homology of the X chromosomes among elasmobranchs in spite of their varied features and different evolutionary histories.

evolutionary biology↗

bioRxiv2025

Prenatal maternal infection promotes maternal microchimeric cells to alter infection risk in male offspring

Vertically transferred maternal cells or maternal microchimeric cells (MMCs) engraft the fetus and persist in offspring for long periods of time. How altered maternal immune states arising from infection affect MMCs and their function in offspring is poorly understood. Here, we show that pregnancy-associated transient maternal infection alters MMCs to differentially regulate immunity in offspring. In male offspring of dams previously infected with Yersinia pseudotuberculosis, MMCs confer a pro-inflammatory type 17 T effector phenotype that leads to enhanced protective immunity to an unrelated Salmonella infection. Thus, acquired maternal cells imprinted by microbial exposure during pregnancy exert an antigen agnostic and sex-differential effect on offspring immunity, and may potentially be targeted to deliver immune benefits to infants in the vulnerable early life period.

immunology↗

bioRxiv2024

Heterologous Expression of the carbon monoxide dehydrogenase Gene from Clostridium sp. to Enhance Acetic Acid and Alcohol Production from CO2

This study evaluates the performance of carbon monoxide dehydrogenase (codh) embedded strains in bench-scale microbial electrochemical systems (MES) for CO2 reduction to biofuels and biochemicals. CO2 fermentation efficiency was evaluated by comparing the wild-type Clostridium acetobutylicum (Wild), a negative control E. coli strain lacking the codh gene (NC-BL21), and engineered E. coli strain (Eng) alone and with IPTG induction (Eng+IPTG). Four electrochemical systems were used viz. Wild+E, NC-BL21+E, Eng+E, and Eng+IPTG+E, with a poised potential of 0.6 V applied to the working electrode. CO2 and bicarbonate were supplemented to a total inorganic carbon (IC) concentration of 40 g/L, with a retention time of 60 h. The engineered strains demonstrated enhanced metabolic performance compared to the wild-type and negative control strains, yielding a maximum formic acid concentration of 2.1 g/L and acetic acid concentration of 7.8 g/L under the Eng+IPTG condition. Ethanol yield was highest at 3.9 g/L under the Eng+IPTG+E condition, substantially exceeding the 2.4 g/L acetic acid yield observed in the wild-type strain. The engineered strains showed superior cumulative yields (0.4075 g/g), improved codh charge flux stability (60 vs. 5 for Wild), and upregulated expression of genes in the Wood-Ljungdahl pathway. Bioelectrochemical performance analysis demonstrated elevated reductive catalytic currents, enhanced CO2 reduction, and optimal charge transfer kinetics. This study highlights the effectiveness of genetic and process engineering, particularly codh overexpression and IPTG induction, in optimizing microbial electrosynthesis for biofuel and biochemical production from C1 gases.

bioengineering↗

bioRxiv2024

Programming the elongation of mammalian cell aggregates with synthetic gene circuits

A key goal of synthetic morphogenesis is the identification and implementation of methods to control morphogenesis. One line of research is the use of synthetic genetic circuits guiding the self-organization of cell ensembles. This approach has led to several recent successes, including control of cellular rearrangements in 3D via control of cell-cell adhesion by user-designed artificial genetic circuits. However, the methods employed to reach such achievements can still be optimized along three lines: identification of circuits happens by hand, 3D structures are spherical, and effectors are limited to cell-cell adhesion. Here we show the identification, in a computational framework, of genetic circuits for volumetric axial elongation via control of proliferation, tissue fluidity, and cell-cell signaling. We then seek to implement this design in mammalian cell aggregates in vitro. We start by identifying effectors to control tissue growth and fluidity in vitro. We then combine these new modules to construct complete circuits that control cell behaviors of interest in space and time, resulting in measurable tissue deformation along an axis that depends on the engineered signaling modules. Finally, we contextualize in vitro and in silico implementations within a unified morphospace to suggest further elaboration of this initial family of circuits towards more robust programmed axial elongation. These results and integrated in vitro/in silico pipeline demonstrate a promising method for designing, screening, and implementing synthetic genetic circuits of morphogenesis, opening the way to the programming of various user-defined tissue shapes.

synthetic biology↗

bioRxiv2024

Genetic requirements of Mycobacterium tuberculosis for survival under pathogen-specific immunity

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), remains a persistent global health challenge due to the lack of an effective vaccine. The only licensed TB vaccine, Bacille Calmette-Guerin (BCG), is a live attenuated strain of Mycobacterium bovis, that protects young children from severe disease but fails to provide protection through adulthood. It is unclear why BCG-induced immunity provides incomplete protection despite inducing a robust CD4+ T cell response, a known critical correlate of protection against Mtb. We set out to interrogate mycobacterial determinants of vaccine escape using a functional genomics approach, TnSeq, to define mycobacterial genes required for survival in mice vaccinated with BCG, the live attenuated Mtb vaccine, {Delta}LprG, and in mice with Mtb immunity conferred by prior infection. We find that critical virulence genes associated with acute infection and exponential growth are less essential in hosts with adaptive immunity, including genes encoding the Esx-1 and Mce1 systems. Genetic requirements for Mtb growth in vaccinated and previously Mtb-infected hosts mirror the genetic requirements reported for bacteria grown under in vitro conditions that reflect aspects of the adaptive immune response as well as in mouse genetic backgrounds where bacterial growth is restricted. Across distinct immunization conditions, differences in genetic requirements between live attenuated vaccines and vaccination routes are observed, suggesting that different immunization strategies impose distinct bacterial stressors. Collectively, these data support the idea that Mtb requires genes that enable stress adaptation and growth arrest upon encountering the restrictive host environment induced by the adaptive immune response. We demonstrate that TnSeq can be used to understand the bacterial genetic requirements for survival in vaccinated hosts across pre-clinical live attenuated vaccines and therefore may be applied to other vaccine modalities. Understanding how Mtb survives in the setting of vaccine-induced immunity could uncover bacterial vulnerabilities to inform the development and prioritization of new vaccines or adjuvant therapies.

microbiology↗

bioRxiv2024

Sub-chronic and acute toxicity of 6PPD-quinone to early-life stage rainbow trout (Oncorhynchus mykiss)

N-(1,3-Dimethylbutyl)-N-phenyl-p-phenylenediamine-quinone (6PPD-Q) is a derivative of rubber tires which leaches into surface waters when tire particles are swept into roadway runoff. 6PPD-Q has been identified as a potential cause of urban runoff mortality syndrome in coho salmon, and subsequent research has determined a wide species variation in toxicity among fishes. While adult rainbow trout are known to be sensitive, there is limited research on their early-life stages. Given that early-life stages of fish are often more sensitive than adults, the aim of these studies was to assess the acute and sub-chronic toxicity of 6PPD-Q in early-life stage rainbow trout. Rainbow trout alevins were exposed from hatch until 28 days post-hatch (dph) to time-weighted average 6PPD-Q concentrations ranging from 0.06-2.35 g/L. From these studies, a 28-day median lethal dose (LC50) of 0.56 g/L was derived. Morphological deformities were observed during the exposure period, including pooling of blood in the caudal fin. A follow-up acute study with exogenously feeding rainbow trout fry revealed a 96-hour LC50 of 0.47 g/L. These studies indicate that early-life stage rainbow trout are more sensitive than previously studied sub-adults, with exogenously feeding fry being the most sensitive life stage studied so far, and that sub-chronic exposure to 6PPD-Q can result in developmental abnormalities. This research highlights the importance of utilizing early-life stage studies to determine the most sensitive benchmark concentrations and their value in determining sub-lethal effects.

pharmacology and toxicology↗

bioRxiv2024

Cyanobacteria from marine oxygen deficient zones encode both form I and form II rubiscos

Cyanobacteria are highly abundant in the marine photic zone and primary drivers of the conversion of inorganic carbon to biomass. To date, all studied Cyanobacterial lineages encode carbon fixation machinery hinged upon form I rubisco enzymes within a CO2-concentrating carboxysome. Here, we report that the AMZ IB lineage of Prochlorococcus from global oxygen deficient zones (ODZs) harbor both form I and form II rubisco enzymes, the latter of which are typically non-carboxysomal and possess biochemical properties tuned towards low oxygen environments. Our analyses reveal that these cyanobacterial form II enzymes are functional in vitro and were likely acquired via lateral gene transfer from proteobacteria. Global metagenomic read recruitment demonstrates that Prochlorococcus with form II rubisco are essentially restricted to ODZs in the Eastern Tropical Pacific, suggesting that acquisition may confer an advantage specifically under low-O2 conditions. Populations of AMZ IB Prochlorococcus express both forms of rubisco in situ, with the highest form II rubisco expression at depths where both oxygen and light are particularly low, possibly as a mechanism to increase the efficiency of photoautotrophy under energy limitation. Our findings expand the diversity of carbon fixation configurations in the microbial world and may have implications for the overall capacity of ODZs to sequester carbon.

microbiology↗

bioRxiv2024

AP-1 Mediates Cellular Adaptation and Memory Formation During Therapy Resistance

Cellular responses to environmental stimuli are typically thought to be governed by genetically encoded programs. We demonstrate that melanoma cells can form and maintain cellular memories during the acquisition of therapy resistance that exhibit characteristics of cellular learning and are dependent on the transcription factor AP-1. We show that cells exposed to a low dose of therapy adapt to become resistant to a high dose, demonstrating that resistance was not purely selective. The application of therapy itself results in the encoding of transient gene expression into cellular memory and that this encoding occurs for both transiently induced and probabilistically arising expression. Chromatin accessibility showed concomitant persistence. A two-color AP-1 reporter system showed that these memories are encoded in cis, constituting an example of activating cis epigenetics. Our findings establish the formation and maintenance of cellular memories as a critical aspect of gene regulation during the development of therapy resistance.

systems biology↗

bioRxiv2024

A mouse model of TB-associated lung fibrosis reveals persistent inflammatory macrophage populations during treatment

Post-TB lung disease (PTLD) causes a significant burden of global disease. Fibrosis is a central component of many clinical features of PTLD. To date, we have a limited understanding of the mechanisms of TB-associated fibrosis and how these mechanisms are similar to or dissimilar from other fibrotic lung pathologies. We have adapted a mouse model of TB infection to facilitate the mechanistic study of TB-associated lung fibrosis. We find that the morphologies of fibrosis that develop in the mouse model are similar to the morphologies of fibrosis observed in human tissue samples. Using Second Harmonic Generation (SHG) microscopy, we are able to quantify a major component of fibrosis, fibrillar collagen, over time and with treatment. Inflammatory macrophage subpopulations persist during treatment; matrix remodeling enzymes and inflammatory gene signatures remain elevated. Our mouse model suggests that there is a therapeutic window during which adjunctive therapies could change matrix remodeling or inflammatory drivers of tissue pathology to improve functional outcomes after treatment for TB infection.

microbiology↗

bioRxiv2024

Clonal evolution of hematopoietic stem cells after cancer chemotherapy

Normal hematopoietic stem and progenitor cells (HSPCs) inherently accumulate somatic mutations and lose clonal diversity with age, processes implicated in the development of myeloid malignancies1. The impact of exogenous stressors, such as cancer chemotherapies, on the genomic integrity and clonal dynamics of normal HSPCs is not well defined. We conducted whole-genome sequencing on 1,032 single-cell-derived HSPC colonies from 10 patients with multiple myeloma (MM), who had undergone various chemotherapy regimens. Our findings reveal that melphalan treatment distinctly increases mutational burden with a unique mutation signature, whereas other MM chemotherapies do not significantly affect the normal mutation rate of HSPCs. Among these therapy-induced mutations were several oncogenic drivers such as TET2 and PPM1D. Phylogenetic analysis showed a clonal architecture in post-treatment HSPCs characterized by extensive convergent evolution of mutations in genes such as TP53 and PPM1D. Consequently, the clonal diversity and structure of post-treatment HSPCs mirror those observed in normal elderly individuals, suggesting an accelerated clonal aging due to chemotherapy. Furthermore, analysis of matched therapy-related myeloid neoplasm (t-MN) samples, which occurred 1-8 years later, enabled us to trace the clonal origin of t-MNs to a single HSPC clone among a group of clones with competing malignant potential, indicating the critical role of secondary mutations in dictating clonal dominance and malignant transformation. Our findings suggest that cancer chemotherapy promotes an oligoclonal architecture with multiple HSPC clones possessing competing leukemic potentials, setting the stage for the selective emergence of a singular clone that evolves into t-MNs after acquiring secondary mutations. These results underscore the importance of further systematic research to elucidate the long-term hematological consequences of cancer chemotherapy.

cancer biology↗

bioRxiv2024

Complete list of canonical post-transcriptional modifications in the Bacillus subtilis ribosome and their link to RbgA driven large subunit assembly

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. B. subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis. While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after E. coli and T. thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

microbiology↗

bioRxiv2024

Acute and sub-chronic toxicity of 6PPD-quinone to early-life stage lake trout (Salvelinus namaycush)

N-(1,3-Dimethylbutyl)-N-phenyl-p-phenylenediamine-quinone (6PPD-q) is a rubber-tire derivative which leaches into surface waters from roadway runoff, from tire particles and has been identified as a possible driver of urban runoff mortality syndrome in coho salmon. Sensitivity to this toxicant is highly variable across fish species and life stages. With environmental concentrations meeting or exceeding toxicity thresholds in sensitive fishes, the potential for ecologically relevant effects is significant. There is currently no data regarding the sensitivity of lake trout (Salvelinus namaycush) to 6PPD-q. As early-life stages of fishes are typically more sensitive than adults, the goal of these studies was to evaluate the acute and sub-chronic toxicity of 6PPD-q to early-life stage lake trout. Alevins exposed from hatch until 45 days post hatch (dph) to time-weighted average 6PPD-q concentrations ranging from 0.22-13.5 g/L exhibited a 45-day median lethal dose (LC50) of 0.39 g/L. Deformities throughout growth were observed, with a unique pooling of blood observed in the caudal fin and eye. A subsequent acute study with exogenously feeding lake trout fry determined a 96-hr LC50 of 0.50 g/L. From these studies we can conclude that lake trout alevins and exogenously feeding fry are sensitive to 6PPD-q, which underscores the relevance of this chemical to inland freshwater ecosystems.

physiology↗

bioRxiv2024

Piscis: a novel loss estimator of the F1 score enables accurate spot detection in fluorescence microscopy images via deep learning

Single-molecule RNA fluorescence in situ hybridization (RNA FISH)-based spatial transcriptomics methods have enabled the accurate quantification of gene expression at single-cell resolution by visualizing transcripts as diffraction-limited spots. While these methods generally scale to large samples, image analysis remains challenging, often requiring manual parameter tuning. We present Piscis, a fully automatic deep learning algorithm for spot detection trained using a novel loss function, the SmoothF1 loss, that approximates the F1 score to directly penalize false positives and false negatives but remains differentiable and hence usable for training by deep learning approaches. Piscis was trained and tested on a diverse dataset composed of 358 manually annotated experimental RNA FISH images representing multiple cell types and 240 additional synthetic images. Piscis outperforms other state-of-the-art spot detection methods, enabling accurate, high-throughput analysis of RNA FISH-derived imaging data without the need for manual parameter tuning.

bioinformatics↗

bioRxiv2023

Neutrophil-derived IL-17 limits protective host responses and promotes tuberculosis pathogenesis

The protective correlates of Mycobacterium tuberculosis (Mtb) infection-elicited host immune responses are incompletely understood. Here, we report pro-pathogenic crosstalk involving Ly6G+granulocytes (Ly6G+Gra), IL-17 and COX2. We show that in the lungs of Mtb-infected wildtype mice, either BCG-vaccinated or not, most intracellular bacilli are Ly6G+Gra-resident four weeks post-infection onwards. In the genetically susceptible IFN{psi} -/- mice, excessive Ly6G+Gra infiltration correlates with severe bacteraemia. Neutralizing IL-17 (anti-IL17mAb) and COX2 inhibition by celecoxib reverse Ly6G+Gra infiltration, associated pathology and death in IFN{psi} -/- mice. Surprisingly, Ly6G+Gra also serves as the major source of IL-17 in the lungs of Mtb-infected WT or IFN{psi} -/- mice. The IL-17-COX2-Ly6G+Gra interplay also operates in WT mice. Inhibiting ROR{psi}t, the key transcription factor for IL-17 production or COX2, reduces the bacterial burden in Ly6G+Gra, leading to reduced bacterial burden and pathology in the lungs of WT mice. In the Mtb-infected WT mice, COX2 inhibition abrogates IL-17 levels in the lung homogenates and significantly enhances BCGs protective efficacy, mainly by targeting the Ly6G+Gra-resident Mtb pool, a phenotype also observed when IL-17 is blocked by ROR{psi}t inhibitor. Furthermore, in pulmonary TB patients, high neutrophil count and IL-17 correlated with adverse treatment outcomes. Together, our results suggest that IL-17 and PGE2 are the negative correlates of protection, and we propose targeting the pro-pathogenic IL-17-COX2-Ly6G+Gra axis for TB prevention and therapy.

immunology↗

bioRxiv2023

Vps18 contributes to phagosome membrane integrity in Mycobacterium tuberculosis-infected macrophages.

Mycobacterium tuberculosis (Mtb) has evolved to be exquisitely adapted to survive within host macrophages. The capacity to damage the phagosomal membrane has emerged as central to Mtb virulence. While Mtb factors driving membrane damage have been described, host factors that repair that damage to contain the pathogen remain largely unknown. We used a genome-wide CRISPR screen to identify novel host factors required to repair Mtb-damaged phagosomal membranes. Vacuolar protein sorting-associated protein 18 (Vps18), a member of the HOPS and CORVET trafficking complexes, was among the top hits. Vps18 colocalized with Mtb in macrophages beginning shortly after infection, and Vps18-knockout macrophages demonstrated increased damage of Mtb-containing phagosomes without impaired autophagy. Mtb grew more robustly in Vps18-knockout cells, and the first-line anti-tuberculosis antibiotic pyrazinamide was less effective. Our results identify Vps18 as required for phagosomal membrane integrity in Mtb-infected cells and suggest that modulating phagosome integrity may hold promise for improving the efficacy of antibiotic treatment for TB.

microbiology↗

bioRxiv2023

Toxicokinetic Characterization of the Inter-Species Differences in 6PPD-Quinone Toxicity Across Seven Fish Species: Metabolite Identification and Semi-Quantification

N-(1,3-Dimethylbutyl)-N-Phenyl-P-Phenylenediamine-Quinone (6PPD-Q) is a recently identified contaminant that originates from the oxidation of the tire anti-degradant 6PPD. 6PPD-Q is acutely toxic to select salmonids at environmentally relevant concentrations, while other fish species display tolerance to concentrations surpassing those measured in the environment. The reasons for these marked differences in sensitivity are presently unknown. The objective of this research was to explore potential toxicokinetic drivers of species sensitivity by characterizing biliary metabolites of 6PPD-Q in sensitive and tolerant fishes. For the first time, we identified an O-glucuronide metabolite of 6PPD-Q using high-resolution mass spectrometry. The semi-quantified levels of this metabolite in tolerant species or life stages, including white sturgeon (Acipenser transmontanus), chinook salmon (Oncorhynchus tshawytscha), westslope cutthroat trout (Oncorhynchus clarkia lewisi) and non-fry life stages of Atlantic salmon (Salmo salar), were greater than those in sensitive species, including coho salmon (Oncorhynchus kisutch), brook trout (Salvelinus fontinalis), and rainbow trout (Oncorhynchus mykiss), suggesting that tolerant species might more effectively detoxify 6PPD-Q. Thus, we hypothesize that differences in species sensitivity are a result of differences in basal expression of biotransformation enzyme across various fish species. Moreover, the semi-quantification of 6PPD-Q metabolites in bile extracted from wild-caught fish might be a useful biomarker of exposure to 6PPD-Q, thereby being invaluable to environmental monitoring and risk assessment.

pharmacology and toxicology↗

bioRxiv2023

Single cell susceptibility to SARS-CoV-2 infection is driven by variable cell states

The ability of a virus to infect a cell type is at least in part determined by the presence of host factors required for the viral life cycle. However, even within cell types that express known factors needed for infection, not every cell is equally susceptible, suggesting that our knowledge of the full spectrum of factors that promote infection is incomplete. Profiling the most susceptible subsets of cells within a population may reveal additional factors that promote infection. However, because viral infection dramatically alters the state of the cell, new approaches are needed to reveal the state of these cells prior to infection with virus. Here, we used single-cell clone tracing to retrospectively identify and characterize lung epithelial cells that are highly susceptible to infection with SARS-CoV-2. The transcriptional state of these highly susceptible cells includes markers of retinoic acid signaling and epithelial differentiation. Loss of candidate factors identified by our approach revealed that many of these factors play roles in viral entry. Moreover, a subset of these factors exert control over the infectable cell state itself, regulating the expression of key factors associated with viral infection and entry. Analysis of patient samples revealed the heterogeneous expression of these factors across both cells and patients in vivo. Further, the expression of these factors is upregulated in particular inflammatory pathologies. Altogether, our results show that the variable expression of intrinsic cell states is a major determinant of whether a cell can be infected by SARS-CoV-2.

bioengineering↗

bioRxiv2023

Scalable, accessible, and reproducible reference genome assembly and evaluation in Galaxy

Improvements in genome sequencing and assembly are enabling high-quality reference genomes for all species. However, the assembly process is still laborious, computationally and technically demanding, lacks standards for reproducibility, and is not readily scalable. Here we present the latest Vertebrate Genomes Project assembly pipeline and demonstrate that it delivers high-quality reference genomes at scale across a set of vertebrate species arising over the last [~]500 million years. The pipeline is versatile and combines PacBio HiFi long-reads and Hi-C-based haplotype phasing in a new graph-based paradigm. Standardized quality control is performed automatically to troubleshoot assembly issues and assess biological complexities. We make the pipeline freely accessible through Galaxy, accommodating researchers even without local computational resources and enhanced reproducibility by democratizing the training and assembly process. We demonstrate the flexibility and reliability of the pipeline by assembling reference genomes for 51 vertebrate species from major taxonomic groups (fish, amphibians, reptiles, birds, and mammals).

bioinformatics↗