Biology subjects

Jain, K.

Publications and source records attributed to Jain, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Pulsatile basal gene expression as a fitness determinant in bacteria

Active regulation of gene expression, orchestrated by complex interactions of activators and repressors at promoters, controls the fate of organisms. In contrast, basal expression at uninduced promoters is considered to be a dynamically inert mode of non-functional "promoter leakiness", merely a byproduct of transcriptional regulation. Here, we investigate the basal expression mode of the mar operon, the main regulator of intrinsic multiple antibiotic resistance in Escherichia coli, and link its dynamic properties to the non-canonical, yet highly conserved start codon of marR across Enterobacteriaceae. Real-time single-cell measurements across tens of generations, reveal that basal expression consists of rare stochastic gene expression pulses, which maximize variability in wildtype and, surprisingly, transiently accelerate cellular elongation rates. Competition experiments show that basal expression confers fitness advantages to wildtype across several transitions between exponential and stationary growth by shortening lag times. The dynamically rich basal expression of the mar operon, has likely been evolutionarily maintained for its role in growth homeostasis of Enterobacteria within the gut environment, thereby allowing other ancillary gene regulatory roles to evolve, e.g. control of costly-to-induce multi-drug efflux pumps. Understanding the complex selection forces governing genetic systems involved in intrinsic multi-drug resistance is crucial for effective public health measures.

systems biology↗

bioRxiv2024

Genetic diversity during selective sweeps in non-recombining populations

Selective sweeps, resulting from the spread of beneficial, neutral, or deleterious mutations through a population, shape patterns of genetic variation at linked neutral sites. While many theoretical, computational, and statistical advances have been made in understanding the genomic signatures of selective sweeps in recombining populations, relatively less is understood in populations with little/no recombination, and arbitrary dominance and inbreeding. Using diffusion theory, we obtain the full expression for the expected site frequency spectrum (SFS) at linked neutral sites immediately post and during the fixation of moderately or strongly beneficial mutations. When a single hard sweep occurs, the SFS decays as 1/x for low derived allele frequencies (x), similar to the neutral SFS at equilibrium, whereas at higher derived allele frequencies, it follows a 1/x2 power law as also seen in a rapidly expanding neutral population. We show that these power laws are universal in the sense that they are independent of the dominance and inbreeding coefficients, and also characterize the SFS during the sweep. Additionally, we find that the derived allele frequency where the SFS shifts from the 1/x to 1/x2 power law is inversely proportional to the selection strength; thus under strong selection, the SFS follows the 1/x2 dependence for most allele frequencies. When clonal interference is pervasive, the SFS immediately post-fixation becomes U-shaped and can be approximated by the equilibrium SFS of selected sites. Our results will be important in developing statistical methods to infer the timing and strength of recent selective sweeps in asexual populations, genomic regions that lack recombination, and clonally propagating tumor populations.

genetics↗

bioRxiv2024

Cell-free chromatin particles damage genomic DNA of healthy cells via an ROS-independent mechanism

Several hundred billion cells die daily in the human body, releasing cell-free chromatin particles (cfChPs) in the process, which then enter the blood circulation and get taken up by healthy cells. We have previously reported that, these internalized cfChPs damage genomic DNA as well cause physical damage to mitochondria, resulting in increased mitochondrial ROS production. In the current study, we evaluated the potential damaging effects of the cfChP-induced increase in ROS production on genomic DNA. NIH3T3 mouse fibroblast cells were treated with cfChPs isolated from the sera of healthy individuals (H-cfChPs) or patients with cancer (C-cfChPs) in the presence or absence of the ROS scavenger Mito-TEMPO. The pre-incubation of cfChP-treated cells with Mito-TEMPO abolished ROS production, but did not prevent genomic DNA damage induced by H-cfChPs and C-cfChPs. Our results suggest that cfChP-induced genomic DNA damage occurs in an ROS-independent manner. These findings align with emerging evidence suggesting that mitochondrial ROS may not be a direct cause of genomic DNA damage and suggest that DNA damage attributed to ROS may in-fact be induced by cfChPs. This possibility opens up new therapeutic approaches involving deactivation of cfChPs to retard ageing and other degenerative conditions traditionally attributed to oxidative stress.

cell biology↗

bioRxiv2024

Spectrotemporal profiling of ultrasonic vocalizations during neonatal opioid withdrawal reveals a kappa opioid receptor component in female mice

RationaleOpioid use during pregnancy can lead to negative infant health outcomes, including neonatal opioid withdrawal syndrome (NOWS). NOWS comprises gastrointestinal, autonomic nervous system, and neurological dysfunction that manifest during spontaneous withdrawal. Variability in NOWS severity necessitates a more individualized treatment approach. Ultrasonic vocalizations (USVs) in neonatal mice are emitted in isolation as a stress response and are increased during opioid withdrawal, thus modeling a negative affective state that can be utilized to test new treatments. ObjectivesWe sought to identify the behavioral and USV profile, brainstem transcriptomic adaptations, and role of kappa opioid receptors in USVs during neonatal opioid withdrawal. MethodsWe employed a third trimester-approximate opioid exposure model, where neonatal inbred FVB/NJ pups were injected twice-daily with morphine (10mg/kg, s.c.) or saline (0.9%, 20 ul/g, s.c.) from postnatal day(P) 1 to P14. This protocol induces reduced weight gain, hypothermia, thermal hyperalgesia, and increased USVs during spontaneous morphine withdrawal. ResultsOn P14, there were increased USV emissions and altered USV syllables during withdrawal, including an increase in Complex 3 syllables in FVB/NJ females (but not males). Brainstem bulk mRNA sequencing revealed an upregulation of the kappa opioid receptor (Oprk1), which contributes to withdrawal-induced dysphoria. The kappa opioid receptor (KOR) antagonist, nor-BNI (30 mg/kg, s.c.), significantly reduced USVs in FVB/NJ females, but not males during spontaneous morphine withdrawal. Furthermore, the KOR agonist, U50,488h (0.625 mg/kg, s.c.), was sufficient to increase USVs on P10 (both sexes) and P14 (females only) in FVB/NJ mice. ConclusionsWe identified an elevated USV syllable, Complex 3, and a female-specific recruitment of the dynorphin/KOR system in increased USVs associated with neonatal opioid withdrawal severity.

neuroscience↗

bioRxiv2024

H3K18 & H3K23 acetylation directs establishment of MLL-mediated H3K4 methylation

In an unmodified state, positively charged histone N-terminal tails engage nucleosomal DNA in a manner which restricts access to not only the underlying DNA, but also key tail residues subject to binding and/or modification. Charge-neutralizing modifications, such as histone acetylation, serve to disrupt this DNA-tail interaction, facilitating access to such residues. We previously showed that a polyacetylation-mediated chromatin "switch" governs the read-write capability of H3K4me3 by the MLL1 methyltransferase complex. Here, we discern the relative contributions of site-specific acetylation states along the H3 tail and extend our interrogation to other chromatin modifiers. We show that the contributions of H3 tail acetylation to H3K4 methylation by MLL1 are highly variable, with H3K18 and H3K23 acetylation exhibiting robust stimulatory effects, and that this extends to the related H3K4 methyltransferase complex, MLL4. We show that H3K4me1 and H3K4me3 are found preferentially co-enriched with H3 N-terminal tail proteoforms bearing dual H3K18 and H3K23 acetylation (H3{K18acK23ac}). We further show that this effect is specific to H3K4 methylation, while methyltransferases targeting other H3 tail residues (H3K9, H3K27, & H3K36), a methyltransferase targeting the nucleosome core (H3K79), and a kinase targeting a residue directly adjacent to H3K4 (H3T3) are insensitive to tail acetylation. Together, these findings indicate a unique and robust stimulation of H3K4 methylation by H3K18 and H3K23 acetylation and provide key insight into why H3K4 methylation is often associated with histone acetylation in the context of active gene expression.

biochemistry↗

bioRxiv2024

Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignalling in focal adhesions

Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity (increasing the signal-to-noise ratio) and robustness (signalling above a threshold rather than activation by a single receptor) of the signal compared to signalling by single receptors. Compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, if nanoclusters function as signalling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at sub-diffraction limited resolution of [~]100nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in immobile population of integrin {beta}3 organised as nanoclusters, which (ii) in turn serve to organise nanoclusters of associated key adhesome proteins-vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signalling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct dynamics dependent on function. (iv) long-lived nanoclusters function as signalling hubs-wherein phosphorylated FAK and paxillin formed stable nanoclusters in close proximity to immobile integrin nanoclusters which are disassembled in response to inactivation signal by phosphatase PTPN12 (v) signalling takes place in response to an external signal such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. Taken together, these results demonstrate that signalling downstream of transmembrane receptors is organised as hubs of signalling proteins (FAK, paxillin, vinculin) seeded by nanoclusters of the transmembrane receptor (integrin).

cell biology↗

bioRxiv2023

Intrinsic self-organization of integrin nanoclusters within focal adhesions is required for cellular mechanotransduction

Upon interaction with the extracellular matrix, the integrin receptors form nanoclusters as a first biochemical response to ligand binding. Here, we uncover a critical biodesign principle where these nanoclusters are spatially self-organized, facilitating effective mechanotransduction. Mouse Embryonic Fibroblasts (MEFs) with integrin {beta}3 nanoclusters organized themselves with an intercluster distance of [~]550 nm on uniformly coated fibronectin substrates, leading to larger focal adhesions. We determined that this spatial organization was driven by cell-intrinsic factors since there was no pre-existing pattern on the substrates. Altering this spatial organization using cyclo-RGD functionalized Titanium nanodiscs (of 100 nm, corroborating to the integrin nanocluster size) spaced at intervals of 300 nm (almost half), 600 nm (normal) or 1000 nm (almost double) resulted in abrogation in mechanotransduction, indicating that a new parameter i.e., an optimal intercluster distance is necessary for downstream function. Overexpression of -actinin, which induces a kink in the integrin tail, disrupted the establishment of the optimal intercluster distance, while simultaneous co-overexpression of talin head with -actinin rescued it, indicating a concentration-dependent competition, and that cytoplasmic activation of integrin by talin head is required for the optimal intercluster organization. Additionally, talin head-mediated recruitment of FHOD1 that facilitates local actin polymerization at nanoclusters, and actomyosin contractility were also crucial for establishing the optimal intercluster distance and a robust mechanotransduction response. These findings demonstrate that cell-intrinsic machinery plays a vital role in organizing integrin receptor nanoclusters within focal adhesions, encoding essential information for downstream mechanotransduction signalling.

cell biology↗

bioRxiv2023

Nature and location of modifier alleles determine the resolution of intralocus sexual conflict

When selection favours different levels of expression for a shared trait in males and females, the population is said to be in Intralocus Sexual Conflict (IaSC). In a landmark study, Connallon and Clark (2010) showed that modifiers (alleles at a different locus that alter the expression of the trait under IaSC in a sex-specific manner), effectively resolve IaSC. Their model assumed that modifier alleles are "perfect"; i.e., they fully rescue the harmful effects of the deleterious allele while leaving the positive effects of the beneficial allele unaffected. In the present study, we relaxed this assumption to include more realistic "imperfect" modifier alleles. Imperfect modifiers are those that reduce the expressions of both detrimental allele and beneficial allele to different extents in a given sex. We also ask whether autosomes or X chromosomes are more conducive to resolution of IaSC. Our results suggest that the resolution of IaSC may not always be guaranteed when the modifier allele is "imperfect." Interestingly, IaSC gets resolved more easily on autosomes only when modifier alleles are "perfect" or near "perfect" in concordance with the assumption made by Connallon and Clark (2010). However, in general, X chromosomes are more conducive to the resolution of IaSC.

evolutionary biology↗

bioRxiv2023

Polygenic adaptation dynamics in large, finite populations

Although many phenotypic traits are determined by a large number of genetic variants, the behavior of allele frequencies in a polygenic trait is not completely understood. The problem is especially challenging when the quantitative trait of interest is under epistatic selection as the allele frequency at a locus is affected by those at other loci. Here, we consider a panmictic, diploid finite population evolving under stabilizing selection and symmetric mutations when the population is in linkage equilibrium. In the stationary state, using a diffusion theory, we calculate the marginal distribution of allele frequency, and find parameter regimes where fitness epistasis can not be ignored for an accurate description of the frequency distribution. For such parameters, the mean deviation in the phenotypic optimum and genic variance are, however, found to be well captured even when epistatic interactions are neglected. Thus, while the presence of epistasis may not be evident in phenotypic quantities, it can strongly affect the allele frequency distribution. We also find that the allele frequency distribution at a locus is unimodal if its effect size is below a threshold effect and bimodal otherwise; these results are the stochastic analog of the deterministic ones where the stable allele frequency becomes bistable when the effect size exceeds a threshold. Our analytical results are verified against Monte Carlo simulations and numerical integration of a Langevin equation.

evolutionary biology↗

bioRxiv2022

An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability

In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g., K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has broader extension. Here we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation chromatin switch on the H3 tail that modulates read-write accessibility in nucleosomes and resolve the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.

biochemistry↗

bioRxiv2022

Joint effect of changing selection and demography on the site frequency spectrum

The site frequency spectrum (SFS) is an important statistic that summarizes the molecular variation in a population, and used to estimate population-genetic parameters and detect natural selection. While the equilibrium SFS in a constant environment is quite well studied, recent research has focused on nonequilibrium SFS to elucidate the role of demography when selection is constant in time and of fluctuating selection in a population of constant size. However, the joint effect of time-dependent selection and population size has not been investigated so far. Here, we study the SFS in a randomly mating, diploid population in which both the population size and selection coefficient vary periodically with time using a diffusion theory approach, and derive simple analytical expressions for the time-averaged SFS in slowly and rapidly changing environments. We show that for strong selection and in slowly changing environments, the time-averaged SFS differs significantly from the equilibrium SFS when the population experiences both positive and negative cycles of the selection coefficient. The deviation depends on the time spent by the population in the deleterious part of the selection cycle and the phase difference between the selection coefficient and population size. In particular, we find that the time-averaged SFS in slowly to moderately fast varying, on-average neutral environment has the same qualitative shape as the equilibrium SFS for positively selected mutant but differs quantitatively from it which can be captured by an effective population size.

evolutionary biology↗

bioRxiv2021

The Escherichia coli serS gene promoter region overlaps with the rarA gene

Deletion of the entire gene encoding the RarA protein of Escherichia coli results in a growth defect and additional deficiencies that were initially ascribed to a lack of RarA function. Further work revealed that most of the effects reflected the presence of sequences in the rarA gene that affect expression of the downstream gene, serS. The serS gene encodes the seryl aminoacyl-tRNA synthetase. Decreases in the expression of serS can trigger the stringent response. The sequences that affect serS expression are located in the last 15 nucleotides of the rarA gene.

genetics↗

bioRxiv2021

Effect of plant tubulin kinetic diversification on microtubule lengths

Microtubules (MTs) are dynamic polymers vital for cellular physiology. Bulk tubulin polymerization is nucleation dependent, while individual filaments exhibit dynamic instability driven by GTP hydrolysis rates. Although MTs assembled from well-studied animal brain tubulins have very comparable nucleation and GTP-hydrolysis rates, the kinetic rates of evolutionarily more distant species could diverge. Here we focus on a plant tubulin, the legume Vigna sp. (mung bean) to test the effect of kinetic diversification on MT polymerization. We activity purify tubulin from seedlings and find MT filaments are fewer and shorter than animal brain tubulin. We find mung tubulin polymerization kinetics is nucleation dependent with a high rate of GTP hydrolysis and a critical concentration lower than previously reported for tubulins. A computational model of the kinetics based on the relative influence of rates of nucleation and hydrolysis demonstrates increased rates of hydrolysis can affect MT filament numbers and their lengths, as compared to increasing nucleation rates. Our approach provides a framework to compare the effect of evolutionary diversification of MT nucleation and elongation.

biophysics↗

bioRxiv2021

Time to fixation in changing environments

Although a large number of experimental and theoretical studies have been carried out in a constant environment, as natural environments vary in time, it is important to ask if and how these results are affected by a changing environment. Here, we study the properties of the conditional fixation time of a mutant in a finite, randomly mating diploid population which is evolving in a periodically changing environment. In a static environment, as the conditional mean fixation time of a co-dominant beneficial mutant is equal to that of a deleterious mutant with the same magnitude of selection coefficient, similar patterns for beneficial and deleterious sweeps may result. We find that this symmetry breaks even when the environment is changing slowly. Furthermore, for intermediate dominance, the conditional mean fixation time of a beneficial mutant in a slowly changing environment depends weakly on the dominance coefficient and is close to the corresponding results in the static environment; however, the fixation time for a deleterious mutant under moderate selection with a slowly varying selection coefficient differs substantially from that in the constant environment when the mutant is recessive. Our results thus suggest that the variability patterns and levels for beneficial sweeps are mildly affected by temporally varying environment but changing environment is likely to strongly impact those due to recessive deleterious sweeps.

evolutionary biology↗

bioRxiv2020

PRAK-03202: A triple antigen VLP vaccine candidate against SARS CoV-2

The rapid development of safe and effective vaccines against SARS CoV-2 is the need of the hour for the coronavirus outbreak. Here, we have developed PRAK-03202, the worlds first triple antigen VLP vaccine candidate in a highly characterized S. cerevisiae-based D-CryptTM platform, which induced SARS CoV-2 specific neutralizing antibodies in BALB/c mice. Immunizations using three different doses of PRAK-03202 induces antigen specific (Spike, envelope and membrane proteins) humoral response and neutralizing potential. PBMCs from convalescent patients, when exposed to PRAK-03202, showed lymphocyte proliferation and elevated IFN-{gamma} levels suggestive of conservation of epitopes and induction of T helper 1 (Th1)-biased cellular immune responses. These data support the clinical development and testing of PRAK-03202 for use in humans.

immunology↗

bioRxiv2020

A novel humanized mouse model to study mucosal HIV-1 transmission and prevention

Humanized mice have been critical for HIV-1 research, but are inefficient at mucosal HIV-1 transmission. We present a fetal tissue-independent model called CD34T+ with enhanced human leukocyte levels in the blood and improved T cell homing to the gut-associated lymphoid tissue. CD34T+ mice are highly permissive to intra-rectal HIV-1 infection, which can be prevented by infusion of broadly neutralizing antibodies. Therefore, CD34T+ mice provide a novel platform for mucosal HIV-1 transmission and prevention studies.

immunology↗

bioRxiv2020

The impact of dominance on adaptation in changing environments

Natural environments are seldom static and therefore it is important to ask how a population adapts in a changing environment. We consider a finite, diploid population evolving in a periodically changing environment and study how the fixation probability of a rare mutant depends on its dominance coefficient and the rate of environmental change. We find that in slowly changing environments, the effect of dominance is the same as in the static environment, that is, if a mutant is beneficial (deleterious) when it appears, it is more (less) likely to fix if it is dominant. But in fast changing environments, the effect of dominance can be different from that in the static environment and is determined by the mutants fitness at the time of appearance as well as that in the time-averaged environment. We find that in a rapidly varying environment which is neutral on average, an initially beneficial (deleterious) mutant that arises while selection is decreasing (increasing) has a fixation probability lower (higher) than that for a neutral mutant as a result of which the recessive (dominant) mutant is favored. If the environment is beneficial (deleterious) on average but the mutant is deleterious (beneficial) when it appears in the population, the dominant (recessive) mutant is favored in a fast changing environment. We also find that when recurrent mutations occur, dominance does not have a strong influence on evolutionary dynamics.

evolutionary biology↗

bioRxiv2020

Rhizobium desertarenae sp. nov., isolated from the Saline Desert Soil from the Rann of Kachchh, India.

A novel bacterial strain designated ADMK78T was isolated from the saline desert soil. The cells were rod-shaped, Gram-negative, and non-motile. The strain ADMK78T grows best at 28{degrees}C and pH 7.0 and can tolerate up to 2% (w/v) NaCl. Based on 16S rRNA gene phylogeny, the strain ADMK78T belongs to the genus Rhizobium, with the highest similarity to Rhizobium wuzhouense W44T (98.7%) and Rhizobium ipomoeae shin9-1T (97.9%). Core-genes based phylogenetic analysis revealed that the strain ADMK78T forms a distinct branch in between Rhizobium ipomoeae shin9-1T and Rhizobium selenitireducens BAA-1503T. The average nucleotide identity of ADMK78T was less than 82%, to members of the family Rhizobiaceae. The genomic DNA G+C content of strain ADMK78T is 58.6 mol%. The major fatty acids of strain ADMK78T were C18:0 and C18:1 {omega}7c. The strain ADMK78T showed differences in physiological, phenotypic, and protein profiles estimated by MALDI-TOF MS to its closest relatives. Based on the phenotypic, chemotaxonomic properties, and phylogenetic analyses, the strain ADMK78T could be distinguished from the recognized species of the genus Rhizobium. It is suggested to represent a novel species of this genus, for which the name Rhizobium desertarenae sp. nov. is proposed. The type strain is ADMK78T (=MCC 3400T; KACC 21383T; JCM 33657T).

microbiology↗