Biology subjects

Singh, D.

Publications and source records attributed to Singh, D..

At least 127 records · Page 7Linked to original sources

bioRxiv2020

Effects of individual base-pairs on in vivo target search and destruction kinetics of small RNA

Base-pairing interactions mediate intermolecular target recognition in many biological systems and applications, including DNA repair, CRISPR, microRNA, small RNA (sRNA) and antisense oligo therapies. Even a single base-pair mismatch can cause a substantial difference in biological activity but presently we do not yet know how the target search kinetics in vivo are influenced by single nucleotide level changes. Here, we used high-throughput sequencing to identify functionally relevant single point mutants of the bacterial sRNA, SgrS, and quantitative super-resolution microscopy to probe the mutational impact on the regulation of its primary target, ptsG mRNA. Our super-resolution imaging and analysis platform allowed us to further dissect mutational effects on SgrS lifetimes, and even subtle changes in the in vivo rates of target association, kon, and dissociation, koff. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decreased kon and increased koff, providing an in vivo demonstration that Hfq directly facilitates sRNA-mRNA annealing. Single base-pair mismatches in the annealing region reduced kon by 24-31% and increased koff by 14-25%, extending the time it takes to find and destroy the target mRNA by about a third, depending on whether an AU or GC base-pair is disrupted. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq also buffers base-pair disruptions.

molecular biology↗

bioRxiv2020

SARS-CoV2 genome analysis of Indian isolates and molecular modelling of D614G mutated spike protein with TMPRSS2 depicted its enhanced interaction and virus infectivity

COVID-19 that emerged as a global pandemic is caused by SARS-CoV-2 virus. The virus genome analysis during disease spread reveals about its evolution and transmission. We did whole genome sequencing of 225 clinical strains from the state of Odisha in eastern India using ARTIC protocol-based amplicon sequencing. Phylogenetic analysis identified the presence of all five reported clades 19A, 19B, 20A, 20B and 20C in the population. The analyses revealed two major routes for the introduction of the disease in India i.e. Europe and South-east Asia followed by local transmission. Interestingly, 19B clade was found to be much more prevalent in our sequenced genomes (17%) as compared to other genomes reported so far from India. The haplogroup analysis for clades showed evolution of 19A and 19B in parallel whereas the 20B and 20C appeared to evolve from 20A. Majority of the 19A and 19B clades were present in cases that migrated from Gujarat state in India suggesting it to be one of the major initial points of disease transmission in India during month of March and April. We found that with the time 20A and 20B clades evolved drastically that originated from central Europe. At the same time, it has been observed that 20A and 20B clades depicted selection of four common mutations i.e. 241 C>T (5UTR), P323L in RdRP, F942F in NSP3 and D614G in the spike protein. We found an increase in the concordance of G614 mutation evolution with the viral load in clinical samples as evident from decreased Ct value of spike and Orf1ab gene in qPCR. Molecular modelling and docking analysis identified that D614G mutation enhanced interaction of spike with TMPRSS2 protease, which could impact the shedding of S1 domain and infectivity of the virus in host cells.

genomics↗

bioRxiv2020

Cell-type and cytosine context-specific evolution of DNA methylation in the human brain

Cell-type specific epigenetic modifications are critical for brain development and neuropsychiatric diseases. Here we elucidate evolutionary origins of neuron- and oligodendrocyte-specific DNA methylation in human prefrontal cortex, and demonstrate dynamic and distinctive changes of CG and CH methylation. We show that the human brain has experienced pronounced reduction of CG methylation during evolution, which significantly contributed to cell-type specific active regulatory regions. On the other hand, a substantial increase of CH methylation occurred during human brain evolution, associated with fine-tuning expression in development and neuronal subtypes. The majority of differential CG methylation between neurons and oligodendrocytes originated before the divergence of hominoids and catarrhine monkeys, and carries strong signal for genetic risk for schizophrenia. Remarkably, a substantial portion of differential CG methylation between neurons and oligodendrocytes emerged in the human lineage and harbors additional genetic risk for schizophrenia, implicating epigenetic evolution of human cortex in increased vulnerability to neuropsychiatric diseases.

genomics↗

bioRxiv2020

Artificial light at night amplifies seasonal relapse of haemosporidian parasites in a widespread songbird

Urban habitats can shape interactions between hosts and parasites by altering not only exposure rates but also within-host processes. Artificial light at night is common in urban environments, and chronic exposure can impair host immunity in ways that may increase infection. However, studies of causal links between this stressor, immunity, and infection dynamics are rare, particularly in migratory animals. Here, we experimentally tested how artificial light at night affects cellular immunity and haemosporidian parasite intensity across the annual cycle of migrant and resident subspecies of the dark-eyed junco (Junco hyemalis). We monitored an experimental group exposed to light at night and a control group under natural light/dark cycles as they passed through short days simulating early spring to longer days simulating the breeding season, followed by fall migration. Using generalized additive models, we show that artificial light at night increased inflammation, and leukocyte counts were greatest in early spring and fall. At the start of the experiment, few birds had active infections based on microscopy, but PCR revealed many birds had chronic infections. Artificial light at night increased parasitemia across the annual cycle, with strong peaks in spring and fall that were largely absent in control birds. As birds were kept in indoor aviaries to prevent vector exposure, this increased parasitemia indicates relapse of chronic infection during costly life history stages (i.e., reproduction). Although the immunological and parasitological time series were in phase for control birds, cross-correlation analyses also revealed artificial light at night desynchronized leukocyte profiles and parasitemia, which could suggest a general exaggerated inflammatory response. Our study shows how a common anthropogenic influence can shape within-host processes to affect infection dynamics.

ecology↗

bioRxiv2020

Exposure to artificial light at night accelerates but does not override latitude-dependent seasonal reproductive response in dark-eyed juncos

In the modern era of industrialization, illuminated nights have become a common defining feature of human-occupied environments, particularly cities. Artificial light at night (ALAN) imposes several known negative impacts on the neuroendocrine system, metabolism, and seasonal reproduction of species living in the wild. However, we know little about the impact of ALAN on populations of birds that either live year-round in the same location or move to different latitudes across seasons. To test whether ALAN has differing impact on reproductive timing of the bird populations that winter in sympatry but breed at different latitudes, we monitored sedentary and migratory male dark-eyed juncos that were or were not exposed to low intensity (~2.5 {+/-} 0.5 lux) ALAN. All groups were held in common conditions and day length was gradually increased to mimic natural day length changes (NDL). We assessed seasonal reproductive response from initiation to termination of the breeding cycle. As expected based on earlier research, the sedentary birds exhibited earlier gonadal recrudescence and terminated breeding later than the migratory birds. In addition, resident and migrant birds exposed to ALAN initiated gonadal recrudescence earlier and terminated reproduction sooner as compared to their conspecifics experiencing NDL. Importantly, the difference in the reproductive timing of sedentary and migratory populations was maintained even when exposed to ALAN. This variation in the seasonal reproductive timing may likely have a genetic ground or early developmental effects imposed due to different latitude of origin. This study reveals first that latitude-dependent variation in reproductive timing is maintained despite exposure to ALAN, and second that ALAN accelerated reproductive development across both migrants and residents. The results corroborating relationship between latitude, population, and ALAN impact on seasonal reproductive timing, may provide a potential mechanism to test the fitness of a population and its range expansion to exploit urban environment.

ecology↗

bioRxiv2020

Cdk1-dependent destabilization of long astral microtubules is required for spindle orientation

The precise execution of mitotic spindle orientation in response to cell shape cues is important for tissue organization and development. The presence of astral microtubules extending from the centrosome towards the cell cortex is essential for this process, but little is understood about the contribution of astral microtubule dynamics to spindle positioning, or how astral microtubule dynamics are regulated spatiotemporally. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells transition from interphase to mitosis, but how Cdk1 activity specifically modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules to ensure spindle reorientation in response to cell shape. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus-ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus-ends in mitosis. This decreases the catastrophe frequency of astral microtubules, and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules must thus not only be present, but also dynamic to allow the spindle to reorient in response to cell shape, a state achieved by selective destabilization of long astral microtubules via Cdk1.

cell biology↗

bioRxiv2020

Effect of dioxins in milk on 3D-cultured primary buffalo granulosa cells, a pilot study for a prospective RT-LAMP colour reaction for dioxin toxicity

Dioxins are highly toxic environmental persistent organic pollutants. In several countries, their presence was also reported in cow and human milk samples in the range of 0.023-26.46 and 0.88-19.0 pg/gm of fat, respectively. The detection of dioxns in food samples has been relied on several expensive technologies, which do not represent their toxic effects on consumers. However, mammalian cell based bioassays have such potential to detect the toxins, while representing their toxic effects. Therefore, we tried a three-dimensionally (3D) cultured buffalo granulosa cell based RT-LAMP colour reaction for detecting the presence of added model dioxin, 2,3,7,8-tetrachlorodibenzodioxin (TCDD), in commercial milk. The 3D spheroids on the fifth day of culture were treated with different concentrations of TCDD (i.e. 0.02- 20 pg/ml) directly as well as indirectly through milk fat. After 24hrs of treatment, gene expression studies were performed on certain granulosa cell-specific (CYP19A1, ER-beta, FSHR and LHR) and selective TCDD-responsive (CYP1A1, CYP1B1 and AHR) genes to identify the potential dioxin responsive gene for further RT-LAMP reaction. As the AHR expression in 3D cultured buffalo granulosa cells appears to be a potential gene marker for sensing the added TCDD in the milk, a colour based RT-LAMP reaction was successfully attempted for its expression. However, future studies are needed to develop a dose-responsive colour reaction by considering the treatment time less than 24 hrs.

pharmacology and toxicology↗

bioRxiv2020

TDP-43 and HSP70 phase separate into anisotropic, intranuclear liquid spherical annuli

The RNA binding protein TDP-43 naturally phase separates within cell nuclei and forms cytoplasmic aggregates in age-related neurodegenerative diseases. Here we show that acetylation-mediated inhibition of TDP-43 binding to RNA produces co-de-mixing of acetylated and unmodified TDP-43 into symmetrical, intranuclear spherical annuli whose shells and cores have liquid properties. Shells are anisotropic, like liquid crystals. Consistent with our modelling predictions that annulus formation is driven by components with strong self-interactions but weak interaction with TDP-43, the major components of annuli cores are identified to be HSP70 family proteins, whose chaperone activity is required to maintain liquidity of the core. Proteasome inhibition, mimicking reduction in proteasome activity during aging, induces TDP-43-containing annuli in neurons in rodents. Thus, we identify that TDP-43 phase separation is regulated by acetylation, proteolysis, and ATPase-dependent chaperone activity of HSP70. One Sentence SummaryAcetylation of TDP-43 drives its phase separation into spherical annuli that form a liquid-inside-a-liquid-inside-a-liquid.

cell biology↗

bioRxiv2020

Detection of HER2 expression and its structural alterations in gastric cancer tissues infected with cagA+ H. pylori

BackgroundHelicobacter pylori (HP) cagA is the causing agent for development of gastric cancer (GC). H. pylori also involves to trigger the EGFR (epidermal growth factor receptor) expression in gastric cancer cells. However, the prognostic relation of cagA with HER2 status in GC was not well understood. ObjectiveThe main aim of this study was to investigate the link of HER2 expression with CagA+ H. pylori in GC tissues. Materials and MethodsThe study was performed on 85 GC tissues of GC patients. The specific primers of 16S rDNA and cagA for PCR amplification were used. For investigation of HER2 status in GC tissues, immunohistochemistry and PCR amplification were performed. In silico study was performed for the investigation of interactive potential of HER2 with CagA protein. ResultsPCR amplified the 54 (63.52 %) of 85 GC tissues for HP that showed 34 (62.96 %) cagA+ HP. Immunohistochemistry of tissues revealed 57 (67.05 %) diffuse and 28 (32.94 %) intestinal type cancer. Of 85 cases, 21 GC tissues scored (2 + or 3 +) for positive HER2 expression and score (0 or 1 +) of 64 (75.29 %) showed negative. Of 21 HER2 + GC tissue, 15 biopsies had cagA+ HP and 2 were negative. PCR amplified single amplicon in 17 (20 %) CagA+ tissues and 3 (5.55 %) in CagA - HP. The molecular interactions of CagA was also showed its efficiency for HER2 expression. ConclusionThe study concluded that CagA+ HP may induce HER2 overexpression in GC tissues.

cancer biology↗

bioRxiv2020

Emergent properties of the HNF4α-PPARγ may drive consequent phenotypic plasticity in NAFLD

Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common form of chronic liver disease in adults and children. It is characterized by excessive accumulation of lipids in the hepatocytes of patients without any excess alcohol intake. With a global presence of 24% and limited therapeutic options, the disease burden of NAFLD is increasing. Thus, it becomes imperative to attempt to understand the dynamics of disease progression at a systems-level. Here, we decode the emergent dynamics of underlying gene regulatory networks that have been identified to drive the initiation and progression of NAFLD. We have developed a mathematical model to elucidate the dynamics of the HNF4-PPAR{gamma} gene regulatory network. Our simulations reveal that this network can enable multiple co-existing phenotypes under certain biological conditions: an adipocyte, a hepatocyte, and a "hybrid" adipocyte-like state of the hepatocyte. These phenotypes may also switch among each other, thus enabling phenotypic plasticity and consequently leading to simultaneous deregulation of the levels of molecules that maintain a hepatic identity and/or facilitate a partial or complete acquisition of adipocytic traits. These predicted trends are supported by the analysis of clinical data, further substantiating the putative role of phenotypic plasticity in driving NAFLD. Our results unravel how the emergent dynamics of underlying regulatory networks can promote phenotypic plasticity, thereby propelling the clinically observed changes in gene expression often associated with NAFLD.

systems biology↗

bioRxiv2020

Ancient BED-domain-containing immune receptor from wild barley confers widely effective resistance to leaf rust

Leaf rust, caused by Puccinia hordei is a devastating fungal disease affecting barley (Hordeum vulgare subsp. vulgare) production globally. Race-specific resistance (R) genes have been deployed widely; however, their durability is often compromised due to the rapid emergence of virulent P. hordei races, prompting the search for new sources of broad-spectrum resistance. Here we report on the cloning of Rph15, a broadly effective resistance gene derived from the wild progenitor Hordeum vulgare subsp. spontaneum. We demonstrate using introgression mapping, mutation and complementation that Rph15 encodes a coiled-coil nucleotide-binding leucine-rich repeat (NLR) protein with an integrated Zinc-finger BED (ZF-BED) domain. The allelic variation at the Rph15 locus was assessed using barley exome capture data that traced its origin to the western region of the Fertile Crescent bordering Jordan and Israel. To unravel the genetic relationship of two other leaf rust resistance genes (Rph14 and Rph16) mapped at similar locus on chromosome 2H, we re-sequenced the Rph15 gene from the near-isogenic line for Rph15 (Bowman+Rph15) and the two donor accessions of Rph14 (PI 584760) and Rph16, (PI 405292, Hs 680). Both whole genome and Sanger sequencing confirmed that Hs 680 carried Rph15, while Rph14 in PI 584760 was an independent locus. A perfect diagnostic KASP marker was developed and validated to permit efficient introduction of Rph15 into cultivated barley.

plant biology↗

bioRxiv2020

CD4+ follicular helper-like T cells are key players in anti-tumor immunity

To determine the nature of CD4+ T cells that provide help for generating robust anti-tumor CD8+ cytotoxic T cell (CTL) responses, we profiled the transcriptomes of patient-matched CD4+ and CD8+ T cells present in the tumor micro-environment (TME) and analyzed them jointly using integrated weighted gene correlation network analysis. We found the follicular helper T cell (TFH) program in CD4+ T cells was strongly associated with proliferation and tissue-residency in CD8+ CTLs. Single-cell analysis demonstrated the presence of TFH-like cells and features linked to cytotoxic function and their provision of CD8+ T cell help. Tumor-infiltrating TFH-like cells expressed PD-1 and were enriched in tumors following checkpoint blockade, suggesting that they may respond to anti-PD-1 therapy. Adoptive transfer or induction of TFH cells in mouse models resulted in augmented CD8+ CTL responses and impairment of tumor growth, indicating an important role of TFH-like CD4+ T cells in anti-tumor immunity.

immunology↗

bioRxiv2019

Structural features recapitulate collective dynamics of inhibitory networks

Inhibitory interneurons are ubiquitous through-out the central nervous system (CNS) and play an important role in organizing the excitatory neuronal populations into spatiotemporal patterns. These spatiotemporal patterns are believed to play a vital role in encoding sensory information. The olfactory system is a wellknown example where odor information is encoded in temporally evolving activity of the principal neurons and inhibitory interneurons play an important role in generating these patterns. In this work we study how inhibitory interactions generate such patterns in the con-text of odor encoding by simulating random biophysical models of mitral cells. Using the Newman community clustering algorithm we identify synchronously firing groups of neurons that switch in their activity. Our study presents a new method of inferring the dynamics of inhibitory networks from their structure.

neuroscience↗

bioRxiv2019

Breeding at higher latitude as measured by stable isotope is associated with higher photoperiod threshold and delayed reproductive development in a songbird

Many organisms time reproduction to photoperiod, a constant from year to year. Predicting how anthropogenic change will influence future timing demands greater knowledge of the current role of photoperiod. We held two closely related bird populations in a common environment. One population is resident; the other winters in sympatry with the resident population but migrates north prior to reproducing. We increased photoperiod gradually and measured preparation for migration and reproduction, using feather stable isotopes to estimate breeding latitude. We predicted population differences in the minimum stimulatory day length to elicit a response (CPP, critical photoperiod) and co-variation between CPP and distance migrated. We found clear population differences in CPP and greater CPP in longer distance migrants. We conclude that current geographic variation in reproductive timing has a genetic or early developmental basis and recommend that future research focus on how anthropogenic changes will interact with CPP to adjust timing of reproduction and migration.

ecology↗

bioRxiv2019

Bivariate analysis of barley scald resistance with relative maturity reveals a new major QTL on chromosome 3H

The disease scald of barley is caused by the pathogen Rhynchosporium commune and can cause up to 30-40% yield loss in susceptible varieties. In this study, the Australian barley cultivar Yerong was demonstrated to have resistance that differed from Turk (Rrs1) based on seedling tests with 11 R. commune isolates. A doubled haploid population with 177 lines derived from a cross between Yerong and Franklin was used to identify quantitative trait loci (QTL) for scald resistance. Scald resistance against four pathogen isolates was assessed at the seedling growth stage in a glasshouse experiment and at the adult growth stage in field experiments with natural infection over three consecutive years. A QTL on chromosome 3H was identified with large effect, consistent with a major gene conferring scald resistance at the seedling stage. Under field conditions, scald percentage was negatively correlated with early relative maturity. A bivariate analysis was used to model scald percentage and relative maturity together, residuals from the regression of scald percentage on relative maturity were used as our phenotype for QTL analysis. This analysis identified one major QTL on chromosome 3H, which mapped to the same position as the QTL identified for scald resistance at seedling stage. The identified QTL on 3H is proposed to be different from the Rrs1 on the basis of seedling resistance against different R. commune isolates and physical map position. The analysis also identified an additional novel QTL on chromosome 7H. This study increases the current understanding of scald resistance and identifies genetic material possessing QTLs useful for the marker-assisted selection of scald resistance in barley breeding programs.

genetics↗

bioRxiv2019

Proteome-wide characterization and biomarker identification of intracranial aneurysm

The scientific basis of intracranial aneurysm (IA) formation, its rupture and further development of cerebral vasospasm remains incompletely understood. Deciphering the molecular mechanisms underlying these events will lead to identification of early detection biomarkers and in turn, improved treatment outcomes. Aberrant protein expression may drive structural alterations of vasculature found in IA. To unravel these aberrations, we performed untargeted, global, quantitative proteomic analysis of aneurysm tissue and serum from patients with IA. Samples were derived from patients of three clinical sub groups- 1) unruptured aneurysm 2) ruptured aneurysm without vasospasm 3) ruptured aneurysm who developed vasospasm. A total of 937 and 294 proteins were identified in aneurysm tissue and serum samples respectively. Several proteins that are known to maintain the structural integrity of vasculature were found to be dysregulated. ORM1, a glycoprotein, was significantly upregulated in both the aneurysm tissue and serum samples of unruptured IA patients. We employed a larger cohort of patients and validated ORM1 as a potential biomarker for screening of unruptured aneurysm using ELISA. Samples from ruptured aneurysm with vasospasm showed significant upregulation of MMP9 as compared to ruptured aneurysm without vasospasm. Using a cohort of ruptured aneurysm patients with and without vasospasm, we validated MMP9 as a potential biomarker for vasospasm. This study reveals pathophysiology underlying different clinical sub groups of IA and also suggests potential biomarkers.

neuroscience↗

bioRxiv2019

Improving interpretability of deep learning models: splicing codes as a case study

Despite the success and fast adaptation of deep learning models in a wide range of fields, lack of interpretability remains an issue, especially in biomedical domains. A recent promising method to address this limitation is Integrated Gradients (IG), which identifies features associated with a prediction by traversing a linear path from a baseline to a sample. We extend IG with nonlinear paths, embedding in latent space, alternative baselines, and a framework to identify important features which make it suitable for interpretation of deep models for genomics.

bioinformatics↗

bioRxiv2019

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Cas9 has made a wide range of genome engineering applications possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity but at the cost of the on-target activity. DNA unwinding is the primary checkpoint before cleavage by Cas9 and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in Cas9. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two guanines added were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5 end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

biophysics↗