Publications by authors named "Abhyudai Singh"

71 Publications

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast.

PLoS Genet 2021 Apr 7;17(4):e1009520. Epub 2021 Apr 7.

Instituto de Biotecnología y Biomedicina (Biotecmed), Universitat de València, Burjassot, Spain.

The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.
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http://dx.doi.org/10.1371/journal.pgen.1009520DOI Listing
April 2021

Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level.

Nat Commun 2021 03 23;12(1):1836. Epub 2021 Mar 23.

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

To prevent damage to the host or its commensal microbiota, epithelial tissues must match the intensity of the immune response to the severity of a biological threat. Toll-like receptors allow epithelial cells to identify microbe associated molecular patterns. However, the mechanisms that mitigate biological noise in single cells to ensure quantitatively appropriate responses remain unclear. Here we address this question using single cell and single molecule approaches in mammary epithelial cells and primary organoids. We find that epithelial tissues respond to bacterial microbe associated molecular patterns by activating a subset of cells in an all-or-nothing (i.e. digital) manner. The maximum fraction of responsive cells is regulated by a bimodal epigenetic switch that licenses the TLR2 promoter for transcription across multiple generations. This mechanism confers a flexible memory of inflammatory events as well as unique spatio-temporal control of epithelial tissue-level immune responses. We propose that epigenetic licensing in individual cells allows for long-term, quantitative fine-tuning of population-level responses.
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http://dx.doi.org/10.1038/s41467-021-22070-xDOI Listing
March 2021

Cell size distribution of lineage data: analytic results and parameter inference.

iScience 2021 Mar 24;24(3):102220. Epub 2021 Feb 24.

School of Biological Sciences, University of Edinburgh, EH9 3JH, UK.

Recent advances in single-cell technologies have enabled time-resolved measurements of the cell size over several cell cycles. These data encode information on how cells correct size aberrations so that they do not grow abnormally large or small. Here, we formulate a piecewise deterministic Markov model describing the evolution of the cell size over many generations, for all three cell size homeostasis strategies (timer, sizer, and adder). The model is solved to obtain an analytical expression for the non-Gaussian cell size distribution in a cell lineage; the theory is used to understand how the shape of the distribution is influenced by the parameters controlling the dynamics of the cell cycle and by the choice of cell tracking protocol. The theoretical cell size distribution is found to provide an excellent match to the experimental cell size distribution of lineage data collected under various growth conditions.
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http://dx.doi.org/10.1016/j.isci.2021.102220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961097PMC
March 2021

Glycinergic Transmission in the Presence and Absence of Functional GlyT2: Lessons From the Auditory Brainstem.

Front Synaptic Neurosci 2020 9;12:560008. Epub 2021 Feb 9.

Animal Physiology Group, Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany.

Synaptic transmission is controlled by re-uptake systems that reduce transmitter concentrations in the synaptic cleft and recycle the transmitter into presynaptic terminals. The re-uptake systems are thought to ensure cytosolic concentrations in the terminals that are sufficient for reloading empty synaptic vesicles (SVs). Genetic deletion of glycine transporter 2 (GlyT2) results in severely disrupted inhibitory neurotransmission and ultimately to death. Here we investigated the role of GlyT2 at inhibitory glycinergic synapses in the mammalian auditory brainstem. These synapses are tuned for resilience, reliability, and precision, even during sustained high-frequency stimulation when endocytosis and refilling of SVs probably contribute substantially to efficient replenishment of the readily releasable pool (). Such robust synapses are formed between MNTB and LSO neurons (medial nucleus of the trapezoid body, lateral superior olive). By means of patch-clamp recordings, we assessed the synaptic performance in controls, in GlyT2 knockout mice (KOs), and upon acute pharmacological GlyT2 blockade. Via computational modeling, we calculated the reoccupation rate of empty release sites and replenishment kinetics during 60-s challenge and 60-s recovery periods. Control MNTB-LSO inputs maintained high fidelity neurotransmission at 50 Hz for 60 s and recovered very efficiently from synaptic depression. During 'marathon-experiments' (30,600 stimuli in 20 min), replenishment accumulated to 1,260-fold. In contrast, KO inputs featured severe impairments. For example, the input number was reduced to ~1 (vs. ~4 in controls), implying massive functional degeneration of the MNTB-LSO microcircuit and a role of GlyT2 during synapse maturation. Surprisingly, neurotransmission did not collapse completely in KOs as inputs still replenished their small 80-fold upon 50 Hz | 60 s challenge. However, they totally failed to do so for extended periods. Upon acute pharmacological GlyT2 inactivation, synaptic performance remained robust, in stark contrast to KOs. replenishment was 865-fold in marathon-experiments, only ~1/3 lower than in controls. Collectively, our empirical and modeling results demonstrate that GlyT2 re-uptake activity is not the dominant factor in the SV recycling pathway that imparts indefatigability to MNTB-LSO synapses. We postulate that additional glycine sources, possibly the antiporter Asc-1, contribute to replenishment at these high-fidelity brainstem synapses.
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http://dx.doi.org/10.3389/fnsyn.2020.560008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900164PMC
February 2021

Insights on the Control of Yeast Single-Cell Growth Variability by Members of the Trehalose Phosphate Synthase (TPS) Complex.

Front Cell Dev Biol 2021 28;9:607628. Epub 2021 Jan 28.

TBI, Université de Toulouse, CNRS, INRAE INSA, Toulouse, France.

Single-cell variability of growth is a biological phenomenon that has attracted growing interest in recent years. Important progress has been made in the knowledge of the origin of cell-to-cell heterogeneity of growth, especially in microbial cells. To better understand the origins of such heterogeneity at the single-cell level, we developed a new methodological pipeline that coupled cytometry-based cell sorting with automatized microscopy and image analysis to score the growth rate of thousands of single cells. This allowed investigating the influence of the initial amount of proteins of interest on the subsequent growth of the microcolony. As a preliminary step to validate this experimental setup, we referred to previous findings in yeast where the expression level of Tsl1, a member of the Trehalose Phosphate Synthase (TPS) complex, negatively correlated with cell division rate. We unfortunately could not find any influence of the initial expression level on the growth rate of the microcolonies. We also analyzed the effect of the natural variations of trehalose-6-phosphate synthase () expression on cell-to-cell growth heterogeneity, but we did not find any correlation. However, due to the already known altered growth of the Δ mutants, we tested this strain at the single-cell level on a permissive carbon source. This mutant showed an outstanding lack of reproducibility of growth rate distributions as compared to the wild-type strain, with variable proportions of non-growing cells between cultivations and more heterogeneous microcolonies in terms of individual growth rates. Interestingly, this variable behavior at the single-cell level was reminiscent to the high variability that is also stochastically suffered at the population level when cultivating this Δ strain, even when using controlled bioreactors.
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http://dx.doi.org/10.3389/fcell.2021.607628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876269PMC
January 2021

Subcellular localization of the J-protein Sis1 regulates the heat shock response.

J Cell Biol 2021 Jan;220(1)

Whitehead Institute for Biomedical Research, Cambridge, MA.

Cells exposed to heat shock induce a conserved gene expression program, the heat shock response (HSR), encoding protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1, a cofactor for the chaperone Hsp70, controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm, where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the endoplasmic reticulum. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.
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http://dx.doi.org/10.1083/jcb.202005165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7748816PMC
January 2021

Microbial metabolic noise.

Wiley Interdiscip Rev Syst Biol Med 2020 Nov 23:e1512. Epub 2020 Nov 23.

Department of Electrical and Computer Engineering, University of Delaware, Newark, Delaware, USA.

From the time a cell was first placed under the microscope, it became apparent that identifying two clonal cells that "look" identical is extremely challenging. Since then, cell-to-cell differences in shape, size, and protein content have been carefully examined, informing us of the ultimate limits that hinder two cells from occupying an identical phenotypic state. Here, we present recent experimental and computational evidence that similar limits emerge also in cellular metabolism. These limits pertain to stochastic metabolic dynamics and, thus, cell-to-cell metabolic variability, including the resulting adapting benefits. We review these phenomena with a focus on microbial metabolism and conclude with a brief outlook on the potential relationship between metabolic noise and adaptive evolution. This article is categorized under: Metabolic Diseases > Computational Models Metabolic Diseases > Biomedical Engineering.
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http://dx.doi.org/10.1002/wsbm.1512DOI Listing
November 2020

Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration.

RNA Biol 2020 Dec 1:1-14. Epub 2020 Dec 1.

Instituto de Biomedicina de Sevilla, Universidad de Sevilla-CSIC-Hospital Universitario V. Del Rocío, Seville, Spain.

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in . We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5' exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3' was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.
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http://dx.doi.org/10.1080/15476286.2020.1845504DOI Listing
December 2020

Robust Filtering and Noise Suppression in Intragenic miRNA-Mediated Host Regulation.

iScience 2020 Oct 21;23(10):101595. Epub 2020 Sep 21.

Bioengineering Department, University of Texas at Dallas, Richardson, TX 75080, USA.

MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate gene expression post-transcriptionally by binding to target messenger RNAs (mRNAs). Many human miRNAs are intragenic, located within introns of protein-coding sequence (host). Intriguingly, a percentage of intragenic miRNAs downregulate the host transcript forming an incoherent feedforward motif topology. Here, we study intragenic miRNA-mediated host gene regulation using a synthetic gene circuit stably integrated within a safe-harbor locus of human cells. When the intragenic miRNA is directed to inhibit the host transcript, we observe a reduction in reporter expression accompanied by output filtering and noise reduction. Specifically, the system operates as a filter with respect to promoter strength, with the threshold being robust to promoter strength and measurement time. Additionally, the intragenic miRNA regulation reduces expression noise compared to splicing-alone architecture. Our results provide a new insight into miRNA-mediated gene expression, with direct implications to gene therapy and synthetic biology applications.
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http://dx.doi.org/10.1016/j.isci.2020.101595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554026PMC
October 2020

Modeling homeostasis mechanisms that set the target cell size.

Sci Rep 2020 08 18;10(1):13963. Epub 2020 Aug 18.

Department of Biomedical Engineering, University of Delaware, Newark, Delaware, USA.

How organisms maintain cell size homeostasis is a long-standing problem that remains unresolved, especially in multicellular organisms. Recent experiments in diverse animal cell types demonstrate that within a cell population, cellular proliferation is low for small and large cells, but high at intermediate sizes. Here we use mathematical models to explore size-control strategies that drive such a non-monotonic profile resulting in the proliferation capacity being maximized at a target cell size. Our analysis reveals that most models of size control yield proliferation capacities that vary monotonically with cell size, and non-monotonicity requires two key mechanisms: (1) the growth rate decreases with increasing size for excessively large cells; and (2) cell division occurs as per the Adder model (division is triggered upon adding a fixed size from birth), or a Sizer-Adder combination. Consistent with theory, Jurkat T cell growth rates increase with size for small cells, but decrease with size for large cells. In summary, our models show that regulation of both growth and cell-division timing is necessary for size control in animal cells, and this joint mechanism leads to a target cell size where cellular proliferation capacity is maximized.
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http://dx.doi.org/10.1038/s41598-020-70923-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434900PMC
August 2020

Memory Sequencing Reveals Heritable Single-Cell Gene Expression Programs Associated with Distinct Cellular Behaviors.

Cell 2020 08 30;182(4):947-959.e17. Epub 2020 Jul 30.

Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, Philadelphia, PA, USA; Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Non-genetic factors can cause individual cells to fluctuate substantially in gene expression levels over time. It remains unclear whether these fluctuations can persist for much longer than the time of one cell division. Current methods for measuring gene expression in single cells mostly rely on single time point measurements, making the duration of gene expression fluctuations or cellular memory difficult to measure. Here, we combined Luria and Delbrück's fluctuation analysis with population-based RNA sequencing (MemorySeq) for identifying genes transcriptome-wide whose fluctuations persist for several divisions. MemorySeq revealed multiple gene modules that expressed together in rare cells within otherwise homogeneous clonal populations. These rare cell subpopulations were associated with biologically distinct behaviors like proliferation in the face of anti-cancer therapeutics. The identification of non-genetic, multigenerational fluctuations can reveal new forms of biological memory in single cells and suggests that non-genetic heritability of cellular state may be a quantitative property.
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http://dx.doi.org/10.1016/j.cell.2020.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496637PMC
August 2020

Global redistribution and local migration in semi-discrete host-parasitoid population dynamic models.

Math Biosci 2020 09 29;327:108409. Epub 2020 Jun 29.

Department of Mathematics, Kutztown University, Kutztown, PA 19530, United States of America. Electronic address:

Host-parasitoid population dynamics is often probed using a semi-discrete/hybrid modeling framework. Here, the update functions in the discrete-time model connecting year-to-year changes in the population densities are obtained by solving ordinary differential equations that mechanistically describe interactions when hosts become vulnerable to parasitoid attacks. We use this semi-discrete formalism to study two key spatial effects: local movement (migration) of parasitoids between patches during the vulnerable period; and yearly redistribution of populations across patches outside the vulnerable period. Our results show that in the absence of any redistribution, constant density-independent migration and parasitoid attack rates are unable to stabilize an otherwise unstable host-parasitoid population dynamics. Interestingly, inclusion of host redistribution (but not parasitoid redistribution) before the start of the vulnerable period can lead to stable coexistence of both species. Next, we consider a Type-III functional response (parasitoid attack rate increases with host density), where the absence of any spatial effects leads to a neutrally stable host-parasitoid equilibrium. As before, density-independent parasitoid migration by itself is again insufficient to stabilize the population dynamics and host redistribution provides a stabilizing influence. Finally, we show that a Type-III functional response combined with density-dependent parasitoid migration leads to stable coexistence, even in the absence of population redistributions. In summary, we have systematically characterized parameter regimes leading to stable/unstable population dynamics with different forms of spatial heterogeneity coupled to the parasitoid's functional response using mechanistically formulated semi-discrete models.
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http://dx.doi.org/10.1016/j.mbs.2020.108409DOI Listing
September 2020

Mixture distributions in a stochastic gene expression model with delayed feedback: a WKB approximation approach.

J Math Biol 2020 07 24;81(1):343-367. Epub 2020 Jun 24.

University of Delaware, Newark, DE, USA.

Noise in gene expression can be substantively affected by the presence of production delay. Here we consider a mathematical model with bursty production of protein, a one-step production delay (the passage of which activates the protein), and feedback in the frequency of bursts. We specifically focus on examining the steady-state behaviour of the model in the slow-activation (i.e. large-delay) regime. Using a formal asymptotic approach, we derive an autonomous ordinary differential equation for the inactive protein that applies in the slow-activation regime. If the differential equation is monostable, the steady-state distribution of the inactive (active) protein is approximated by a single Gaussian (Poisson) mode located at the globally stable fixed point of the differential equation. If the differential equation is bistable (due to cooperative positive feedback), the steady-state distribution of the inactive (active) protein is approximated by a mixture of Gaussian (Poisson) modes located at the stable fixed points; the weights of the modes are determined from a WKB approximation to the stationary distribution. The asymptotic results are compared to numerical solutions of the chemical master equation.
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http://dx.doi.org/10.1007/s00285-020-01512-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363733PMC
July 2020

Optimum Threshold Minimizes Noise in Timing of Intracellular Events.

iScience 2020 Jun 21;23(6):101186. Epub 2020 May 21.

Biology Department, Queens College of The City University of New York, Queens, NY, USA; The Graduate Center of The City University of New York, New York City, NY, USA. Electronic address:

How the noisy expression of regulatory proteins affects timing of intracellular events is an intriguing fundamental problem that influences diverse cellular processes. Here we use the bacteriophage λ to study event timing in individual cells where cell lysis is the result of expression and accumulation of a single protein (holin) in the Escherichia coli cell membrane up to a critical threshold level. Site-directed mutagenesis of the holin gene generated phage variants that vary in their lysis times from 30 to 190 min. Observation of the lysis times of single cells reveals an intriguing finding-the noise in lysis timing first decreases with increasing lysis time to reach a minimum and then sharply increases at longer lysis times. A mathematical model with stochastic expression of holin together with dilution from cell growth was sufficient to explain the non-monotonic noise profile and identify holin accumulation thresholds that generate precision in lysis timing.
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http://dx.doi.org/10.1016/j.isci.2020.101186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276437PMC
June 2020

Enhancement of gene expression noise from transcription factor binding to genomic decoy sites.

Sci Rep 2020 06 4;10(1):9126. Epub 2020 Jun 4.

Department of Electrical and Computer Engineering, University of Delaware, Newark, DE, 19716, USA.

The genome contains several high-affinity non-functional binding sites for transcription factors (TFs) creating a hidden and unexplored layer of gene regulation. We investigate the role of such "decoy sites" in controlling noise (random fluctuations) in the level of a TF that is synthesized in stochastic bursts. Prior studies have assumed that decoy-bound TFs are protected from degradation, and in this case decoys function to buffer noise. Relaxing this assumption to consider arbitrary degradation rates for both bound/unbound TF states, we find rich noise behaviors. For low-affinity decoys, noise in the level of unbound TF always monotonically decreases to the Poisson limit with increasing decoy numbers. In contrast, for high-affinity decoys, noise levels first increase with increasing decoy numbers, before decreasing back to the Poisson limit. Interestingly, while protection of bound TFs from degradation slows the time-scale of fluctuations in the unbound TF levels, the decay of bound TFs leads to faster fluctuations and smaller noise propagation to downstream target proteins. In summary, our analysis reveals stochastic dynamics emerging from nonspecific binding of TFs and highlights the dual role of decoys as attenuators or amplifiers of gene expression noise depending on their binding affinity and stability of the bound TF.
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http://dx.doi.org/10.1038/s41598-020-65750-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272470PMC
June 2020

Gene Networks with Transcriptional Bursting Recapitulate Rare Transient Coordinated High Expression States in Cancer.

Cell Syst 2020 04;10(4):363-378.e12

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Genetics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA. Electronic address:

Non-genetic transcriptional variability is a potential mechanism for therapy resistance in melanoma. Specifically, rare subpopulations of cells occupy a transient pre-resistant state characterized by coordinated high expression of several genes and survive therapy. How might these rare states arise and disappear within the population? It is unclear whether the canonical models of probabilistic transcriptional pulsing can explain this behavior, or if it requires special, hitherto unidentified mechanisms. We show that a minimal model of transcriptional bursting and gene interactions can give rise to rare coordinated high expression states. These states occur more frequently in networks with low connectivity and depend on three parameters. While entry into these states is initiated by a long transcriptional burst that also triggers entry of other genes, the exit occurs through independent inactivation of individual genes. Together, we demonstrate that established principles of gene regulation are sufficient to describe this behavior and argue for its more general existence. A record of this paper's transparent peer review process is included in the Supplemental Information.
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http://dx.doi.org/10.1016/j.cels.2020.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293108PMC
April 2020

Network inference in systems biology: recent developments, challenges, and applications.

Curr Opin Biotechnol 2020 06 9;63:89-98. Epub 2020 Jan 9.

Electrical and Computer Engineering, University of Delaware, Newark, Delaware 19716, USA. Electronic address:

One of the most interesting, difficult, and potentially useful topics in computational biology is the inference of gene regulatory networks (GRNs) from expression data. Although researchers have been working on this topic for more than a decade and much progress has been made, it remains an unsolved problem and even the most sophisticated inference algorithms are far from perfect. In this paper, we review the latest developments in network inference, including state-of-the-art algorithms like PIDC, Phixer, and more. We also discuss unsolved computational challenges, including the optimal combination of algorithms, integration of multiple data sources, and pseudo-temporal ordering of static expression data. Lastly, we discuss some exciting applications of network inference in cancer research, and provide a list of useful software tools for researchers hoping to conduct their own network inference analyses.
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http://dx.doi.org/10.1016/j.copbio.2019.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308210PMC
June 2020

Efficient computation of stochastic cell-size transient dynamics.

BMC Bioinformatics 2019 Dec 27;20(Suppl 23):647. Epub 2019 Dec 27.

Physics department, Universidad de los Andes, Bogotá, South America, Colombia.

Background: How small, fast-growing bacteria ensure tight cell-size distributions remains elusive. High-throughput measurement techniques have propelled efforts to build modeling tools that help to shed light on the relationships between cell size, growth and cycle progression. Most proposed models describe cell division as a discrete map between size at birth and size at division with stochastic fluctuations assumed. However, such models underestimate the role of cell size transient dynamics by excluding them.

Results: We propose an efficient approach for estimation of cell size transient dynamics. Our technique approximates the transient size distribution and statistical moment dynamics of exponential growing cells following an adder strategy with arbitrary precision.

Conclusions: We approximate, up to arbitrary precision, the distribution of division times and size across time for the adder strategy in rod-shaped bacteria cells. Our approach is able to compute statistical moments like mean size and its variance from such distributions efficiently, showing close match with numerical simulations. Additionally, we observed that these distributions have periodic properties. Our approach further might shed light on the mechanisms behind gene product homeostasis.
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http://dx.doi.org/10.1186/s12859-019-3213-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933677PMC
December 2019

Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modelling.

PLoS Pathog 2019 11 18;15(11):e1008164. Epub 2019 Nov 18.

Department of Infectious Diseases, Faculty of Medicine, Imperial College London, London, United Kingdom.

The human T cell leukemia virus HTLV-1 establishes a persistent infection in vivo in which the viral sense-strand transcription is usually silent at a given time in each cell. However, cellular stress responses trigger the reactivation of HTLV-1, enabling the virus to transmit to a new host cell. Using single-molecule RNA FISH, we measured the kinetics of the HTLV-1 transcriptional reactivation in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1+ individuals. The abundance of the HTLV-1 sense and antisense transcripts was quantified hourly during incubation of the HTLV-1-infected PBMCs ex vivo. We found that, in each cell, the sense-strand transcription occurs in two distinct phases: the initial low-rate transcription is followed by a phase of rapid transcription. The onset of transcription peaked between 1 and 3 hours after the start of in vitro incubation. The variance in the transcription intensity was similar in polyclonal HTLV-1+ PBMCs (with tens of thousands of distinct provirus insertion sites), and in samples with a single dominant HTLV-1+ clone. A stochastic simulation model was developed to estimate the parameters of HTLV-1 proviral transcription kinetics. In PBMCs from a leukemic subject with one dominant T-cell clone, the model indicated that the average duration of HTLV-1 sense-strand activation by Tax (i.e. the rapid transcription) was less than one hour. HTLV-1 antisense transcription was stable during reactivation of the sense-strand. The antisense transcript HBZ was produced at an average rate of ~0.1 molecules per hour per HTLV-1+ cell; however, between 20% and 70% of HTLV-1-infected cells were HBZ-negative at a given time, the percentage depending on the individual subject. HTLV-1-infected cells are exposed to a range of stresses when they are drawn from the host, which initiate the viral reactivation. We conclude that whereas antisense-strand transcription is stable throughout the stress response, the HTLV-1 sense-strand reactivation is highly heterogeneous and occurs in short, self-terminating bursts.
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http://dx.doi.org/10.1371/journal.ppat.1008164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886867PMC
November 2019

p53 pulse modulation differentially regulates target gene promoters to regulate cell fate decisions.

Mol Syst Biol 2019 09;15(9):e8685

Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

The p53 tumor suppressor regulates distinct responses to cellular stresses. Although different stresses generate different p53 dynamics, the mechanisms by which cells decode p53 dynamics to differentially regulate target genes are not well understood. Here, we determined in individual cells how canonical p53 target gene promoters vary in responsiveness to features of p53 dynamics. Employing a chemical perturbation approach, we independently modulated p53 pulse amplitude, duration, or frequency, and we then monitored p53 levels and target promoter activation in individual cells. We identified distinct signal processing features-thresholding in response to amplitude modulation, a refractory period in response to duration modulation, and dynamic filtering in response to frequency modulation. We then showed that the signal processing features not only affect p53 target promoter activation, they also affect p53 regulation and downstream cellular functions. Our study shows how different promoters can differentially decode features of p53 dynamics to generate distinct responses, providing insight into how perturbing p53 dynamics can be used to generate distinct cell fates.
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http://dx.doi.org/10.15252/msb.20188685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761572PMC
September 2019

Synaptic vesicle fusion is modulated through feedback inhibition by dopamine auto-receptors.

Synapse 2020 01 23;74(1):e22131. Epub 2019 Sep 23.

Department of Biological Sciences, Delaware State University, Dover, Delaware.

Mechanisms of synaptic vesicular fusion and neurotransmitter clearance are highly controlled processes whose finely-tuned regulation is critical for neural function. This modulation has been suggested to involve pre-synaptic auto-receptors; however, their underlying mechanisms of action remain unclear. Previous studies with the well-defined C. elegans nervous system have used functional imaging to implicate acid sensing ion channels (ASIC-1) to describe synaptic vesicle fusion dynamics within its eight dopaminergic neurons. Implementing a similar imaging approach with a pH-sensitive fluorescent reporter and fluorescence resonance after photobleaching (FRAP), we analyzed dynamic imaging data collected from individual synaptic termini in live animals. We present evidence that constitutive fusion of neurotransmitter vesicles on dopaminergic synaptic termini is modulated through DOP-2 auto-receptors via a negative feedback loop. Integrating our previous results showing the role of ASIC-1 in a positive feedback loop, we also put forth an updated model for synaptic vesicle fusion in which, along with DAT-1 and ASIC-1, the dopamine auto-receptor DOP-2 lies at a modulatory hub at dopaminergic synapses. Our findings are of potential broader significance as similar mechanisms are likely to be used by auto-receptors for other small molecule neurotransmitters across species.
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http://dx.doi.org/10.1002/syn.22131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336876PMC
January 2020

MicroRNA Based Feedforward Control of Intrinsic Gene Expression Noise.

IEEE/ACM Trans Comput Biol Bioinform 2021 Jan-Feb;18(1):272-282. Epub 2021 Feb 3.

Intrinsic noise, which arises in gene expression at low copy numbers, can be controlled by diverse regulatory motifs, including feedforward loops. Here, we study an example of a feedforward control system based on the interaction between an mRNA molecule and an antagonistic microRNA molecule encoded by the same gene, aiming to quantify the variability (or noise) in molecular copy numbers. Using linear noise approximation, we show that the mRNA noise is sub-Poissonian in case of non-bursty transcription, and exhibits a nonmonotonic response both to the species natural lifetime ratio and to the strength of the antagonistic interaction. Additionally, we use the Chemical Reaction Network Theory to prove that the mRNA copy number distribution is Poissonian in the absence of spontaneous mRNA decay channel. In case of transcriptional bursts, we show that feedforward control can attenuate the super-Poissonian gene-expression noise that is due to bursting, and that the effect is more considerable at the protein than at the mRNA level. Our results indicate that the strong coupling between mRNA and microRNA in the sense of burst stoichiometry and also of timing of production events renders the microRNA based feedforward motif an effective mechanism for the control of gene expression noise.
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http://dx.doi.org/10.1109/TCBB.2019.2938502DOI Listing
February 2021

Considerable differences between auditory medulla, auditory midbrain, and hippocampal synapses during sustained high-frequency stimulation: Exceptional vesicle replenishment restricted to sound localization circuit.

Hear Res 2019 09 16;381:107771. Epub 2019 Jul 16.

Animal Physiology Group, Department of Biology, University of Kaiserslautern, D-67663, Kaiserslautern, Germany. Electronic address:

Reliable synaptic transmission is essential for interneuronal communication. Synaptic inputs to auditory brainstem neurons, particularly those involved in sound localization, are characterized by resilience during sustained activity and temporal precision in the sub-millisecond range. Both features are obtained by synchronous release of a high number of synaptic vesicles following a single action potential. Here, we compare transmission behavior of three heterogeneous types of inputs in the auditory midbrain and medulla. The first terminate in the central inferior colliculus (ICc) and are glutamatergic (activated from the lateral lemniscus, LL). The medullary inputs terminate in the lateral superior olive (LSO) and are glutamatergic (from the cochlear nuclear complex, CN) or glycinergic (from the medial nucleus of the trapezoid body, MNTB). LSO neurons are the first to integrate binaural information and compute interaural level differences, whereas ICc neurons receive information from almost all auditory brainstem nuclei and construct an initial auditory image used for reflexive behavior. We hypothesized that CN-LSO and MNTB-LSO inputs are more resilient to synaptic fatigue during sustained stimulation than LL-ICc inputs. To test the hypothesis, we performed whole-cell patch-clamp recordings in acute brainstem slices of juvenile mice. We investigated the synaptic performance during prolonged periods of high-frequency stimulation (60 s, up to 200 Hz) and assessed several features, e.g. depression, recovery, latency, temporal precision, quantal size and content, readily releasable pool size, release probability, and replenishment rate. Overall, LL-ICc inputs performed less robustly and temporally precisely than CN-LSO and MNTB-LSO inputs. When stimulated at ≥50 Hz, the former depressed completely within a few seconds. In contrast, CN-LSO and MNTB-LSO inputs transmitted faithfully up to 200 Hz, indicative of very efficient replenishment mechanisms. LSO inputs also displayed considerably lower latency jitter than LL-ICc inputs. The latter behaved similarly to two types of input in the hippocampus for which we performed a meta-analysis. Mechanistically, the high-fidelity behavior of LSO inputs, particularly MNTB-LSO synapses, is based on exceptional release properties not present at auditory midbrain or hippocampal inputs. We conclude that robustness and temporal precision are hallmarks of auditory synapses in the medullary brainstem. These key features are less eminent at higher stations, such as the ICc, and they are also absent outside the central auditory system, namely the hippocampal formation.
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http://dx.doi.org/10.1016/j.heares.2019.07.008DOI Listing
September 2019

Understanding the liver under heat stress with statistical learning: an integrated metabolomics and transcriptomics computational approach.

BMC Genomics 2019 Jun 17;20(1):502. Epub 2019 Jun 17.

Animal and Food Sciences, University of Delaware, Newark, Delaware, USA.

Background: We present results from a computational analysis developed to integrate transcriptome and metabolomic data in order to explore the heat stress response in the liver of the modern broiler chicken. Heat stress is a significant cause of productivity loss in the poultry industry, both in terms of increased livestock morbidity and its negative influence on average feed efficiency. This study focuses on the liver because it is an important regulator of metabolism, controlling many of the physiological processes impacted by prolonged heat stress. Using statistical learning methods, we identify genes and metabolites that may regulate the heat stress response in the liver and adaptations required to acclimate to prolonged heat stress.

Results: We describe how disparate systems such as sugar, lipid and amino acid metabolism, are coordinated during the heat stress response.

Conclusions: Our findings provide more detailed context for genomic studies and generates hypotheses about dietary interventions that can mitigate the negative influence of heat stress on the poultry industry.
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http://dx.doi.org/10.1186/s12864-019-5823-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580474PMC
June 2019

Chandipura Virus Utilizes the Prosurvival Function of RelA NF-κB for Its Propagation.

J Virol 2019 07 28;93(14). Epub 2019 Jun 28.

Systems Immunology Laboratory, National Institute of Immunology, New Delhi, India

Chandipura virus (CHPV), a cytoplasmic RNA virus, has been implicated in several outbreaks of acute encephalitis in India. Despite the relevance of CHPV to human health, how the virus interacts with the host signaling machinery remains obscure. In response to viral infections, mammalian cells activate RelA/NF-κB heterodimers, which induce genes encoding interferon beta (IFN-β) and other immune mediators. Therefore, RelA is generally considered to be an antiviral transcription factor. However, RelA activates a wide spectrum of genes in physiological settings, and there is a paucity of direct genetic evidence substantiating antiviral RelA functions. Using mouse embryonic fibroblasts, we genetically dissected the role of RelA in CHPV pathogenesis. We found that CHPV indeed activated RelA and that RelA deficiency abrogated the expression of IFN-β in response to virus infections. Unexpectedly, infection of fibroblasts led to a decreased CHPV yield. Our investigation clarified that RelA-dependent synthesis of prosurvival factors restrained infection-inflicted cell death and that exacerbated cell death processes prevented multiplication of CHPV in RelA-deficient cells. Chikungunya virus, a cytopathic RNA virus associated also with epidemics, required RelA, and Japanese encephalitis virus, which produced relatively minor cytopathic effects in fibroblasts, circumvented the need of RelA for their propagation. In sum, we documented a proviral function of the pleiotropic factor RelA linked to its prosurvival properties. RelA promoted the growth of cytopathic RNA viruses by extending the life span of infected cells, which serve as the replicative niche of intracellular pathogens. We argue that our finding bears significance for understanding host-virus interactions and may have implications for antiviral therapeutic regimes. RelA/NF-κB participates in a wide spectrum of physiological processes, including shaping immune responses against invading pathogens. In virus-infected cells, RelA typically induces the expression of IFN-β, which restrains viral propagation in neighboring cells involving paracrine mechanisms. Our study suggested that RelA might also play a proviral role. A cell-autonomous RelA activity amplified the yield of Chandipura virus, a cytopathic RNA virus associated with human epidemics, by extending the life span of infected cells. Our finding necessitates a substantial revision of our understanding of host-virus interactions and indicates a dual role of NF-κB signaling during the course of RNA virus infections. Our study also bears significance for therapeutic regimes which alter NF-κB activities while alleviating the viral load.
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http://dx.doi.org/10.1128/JVI.00081-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600208PMC
July 2019

Controlling organism size by regulating constituent cell numbers.

Proc IEEE Conf Decis Control 2018 Dec 21;2018:2685-2690. Epub 2019 Jan 21.

Department of Electrical and Computer Engineering, Biomedical Engineering, Mathematical Sciences, Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE USA 19716.

How living cells employ counting mechanisms to regulate their numbers or density is a long-standing problem in developmental biology that ties directly with organism or tissue size. Diverse cells types have been shown to regulate their numbers via secretion of factors in the extracellular space. These factors act as a proxy for the number of cells and function to reduce cellular proliferation rates creating a negative feedback. It is desirable that the production rate of such factors be kept as low as possible to minimize energy costs and detection by predators. Here we formulate a stochastic model of cell proliferation with feedback control via a secreted extracellular factor. Our results show that while low levels of feedback minimizes random fluctuations in cell numbers around a given set point, high levels of feedback amplify Poisson fluctuations in secreted-factor copy numbers. This trade-off results in an optimal feedback strength, and sets a fundamental limit to noise suppression in cell numbers with short-lived factors providing more efficient noise buffering. We further expand the model to consider external disturbances in key physiological parameters, such as, proliferation and factor synthesis rates. Intriguingly, while negative feedback effectively mitigates disturbances in the proliferation rate, it amplifies disturbances in the synthesis rate. In summary, these results provide unique insights into the functioning of feedback-based counting mechanisms, and apply to organisms ranging from unicellular prokaryotes and eukaryotes to human cells.
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http://dx.doi.org/10.1109/CDC.2018.8619546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420813PMC
December 2018

Single-molecule dynamics of the P granule scaffold MEG-3 in the Caenorhabditis elegans zygote.

Mol Biol Cell 2019 02 12;30(3):333-345. Epub 2018 Dec 12.

Department of Biological Sciences, Dartmouth College, Hanover, NH 03755.

During the asymmetric division of the Caenorhabditis elegans zygote, germ (P) granules are disassembled in the anterior cytoplasm and stabilized/assembled in the posterior cytoplasm, leading to their inheritance by the germline daughter cell. P granule segregation depends on MEG (maternal-effect germline defective)-3 and MEG-4, which are enriched in P granules and in the posterior cytoplasm surrounding P granules. Here we use single-molecule imaging and tracking to characterize the reaction/diffusion mechanisms that result in MEG-3::Halo segregation. We find that the anteriorly enriched RNA-binding proteins MEX (muscle excess)-5 and MEX-6 suppress the retention of MEG-3 in the anterior cytoplasm, leading to MEG-3 enrichment in the posterior. We provide evidence that MEX-5/6 may work in conjunction with PLK-1 kinase to suppress MEG-3 retention in the anterior. Surprisingly, we find that the retention of MEG-3::Halo in the posterior cytoplasm surrounding P granules does not appear to contribute significantly to the maintenance of P granule asymmetry. Rather, our findings suggest that the formation of the MEG-3 concentration gradient and the segregation of P granules are two parallel manifestations of MEG-3's response to upstream polarity cues.
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http://dx.doi.org/10.1091/mbc.E18-06-0402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589573PMC
February 2019

Identifying mechanisms of regulation to model carbon flux during heat stress and generate testable hypotheses.

PLoS One 2018 26;13(10):e0205824. Epub 2018 Oct 26.

Department of Animal and Food Sciences, University of Delaware, Newark, Delaware, United States of America.

Understanding biological response to stimuli requires identifying mechanisms that coordinate changes across pathways. One of the promises of multi-omics studies is achieving this level of insight by simultaneously identifying different levels of regulation. However, computational approaches to integrate multiple types of data are lacking. An effective systems biology approach would be one that uses statistical methods to detect signatures of relevant network motifs and then builds metabolic circuits from these components to model shifting regulatory dynamics. For example, transcriptome and metabolome data complement one another in terms of their ability to describe shifts in physiology. Here, we extend a previously described linear-modeling based method used to identify single nucleotide polymorphisms (SNPs) associated with metabolic changes. We apply this strategy to link changes in sulfur, amino acid and lipid production under heat stress by relating ratios of compounds to potential precursors and regulators. This approach provides integration of multi-omics data to link previously described, discrete units of regulation into functional pathways and identifies novel biology relevant to the heat stress response, in addition to generating hypotheses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205824PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203350PMC
April 2019

Ploidy and Size at Multiple Scales in the Arabidopsis Sepal.

Plant Cell 2018 10 24;30(10):2308-2329. Epub 2018 Aug 24.

Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York 14853

Ploidy and size phenomena are observed to be correlated across several biological scales, from subcellular to organismal. Two kinds of ploidy change can affect plants. Whole-genome multiplication increases ploidy in whole plants and is broadly associated with increases in cell and organism size. Endoreduplication increases ploidy in individual cells. Ploidy increase is strongly correlated with increased cell size and nuclear volume. Here, we investigate scaling relationships between ploidy and size by simultaneously quantifying nuclear size, cell size, and organ size in sepals from an isogenic series of diploid, tetraploid, and octoploid plants, each of which contains an internal endopolyploidy series. We find that pavement cell size and transcriptome size increase linearly with whole-organism ploidy, but organ area increases more modestly due to a compensatory decrease in cell number. We observe that cell size and nuclear size are maintained at a constant ratio; the value of this constant is similar in diploid and tetraploid plants and slightly lower in octoploid plants. However, cell size is maintained in a mutant with reduced nuclear size, indicating that cell size is scaled to cell ploidy rather than to nuclear size. These results shed light on how size is regulated in plants and how cells and organisms of differing sizes are generated by ploidy change.
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http://dx.doi.org/10.1105/tpc.18.00344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6241276PMC
October 2018

Synaptic transmission may provide an evolutionary benefit to HIV through modulation of latency.

J Theor Biol 2018 10 23;455:261-268. Epub 2018 Jul 23.

Department of Electrical and Computer Engineering, Biomedical Engineering, Mathematical Sciences, University of Delaware, Newark DE 19716, USA. Electronic address:

Transmission of HIV is known to occur by two mechanisms in vivo: the free virus pathway, where viral particles bud off an infected cell before attaching to an uninfected cell, and the cell-cell pathway, where infected cells form virological synapses through close contact with an uninfected cell. It has also been shown that HIV replication includes a positive feedback loop controlled by the viral protein Tat, which may act as a stochastic switch in determining whether an infected cell enters latency. In this paper, we introduce a simple mathematical model of HIV replication containing both the free virus and cell-cell pathways. Using this model, we demonstrate that the high multiplicity of infection in cell-cell transmission results in a suppression of latent infection, and that this modulation of latency through balancing the two transmission mechanisms can provide an evolutionary benefit to the virus. This benefit increases with decreasing overall viral fitness, which may provide a within-host evolutionary pressure toward more cell-cell transmission in late-stage HIV infection.
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http://dx.doi.org/10.1016/j.jtbi.2018.07.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187919PMC
October 2018