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Biology subjects

Dar, R. D.

Publications and source records attributed to Dar, R. D..

7 recordsLinked to original sources

Variational Autoencoder-enabled High-throughput Drug Screening for HIV Latency Modulators predicted through Noise in Gene Expression

Due to its ability to establish a pool of undetectable and latently infected cells that can initiate viral production through random reactivation, a cure to human immunodeficiency virus (HIV) infections has remained elusive. Many approaches have been proposed, including the "shock and kill" method where latency reversing agents (LRAs) are administered to reactivate latently infected cells out of latency and remove them through immune targeting and clearance, and the "block and lock" method where latency promoting agents (LPAs) are administered to inhibit reactivation and potentially induce a "deep latency" state where infected cells can no longer reactivate. Previous large scale drug screen studies have demonstrated a correlation between a compounds capability to modulate the fluctuations (or "noise") in HIV gene expression and its potential to modulate HIV latency. However, measurements of gene expression noise are labor-and cost-intensive. To circumvent these drawbacks, we trained a variational autoencoder (VAE) on a previously published large scale time-lapse fluorescence microscopy dataset, and performed an in silico screening of [~]175,000 compounds for HIV latency modulators. Out of the top 113 predicted modulators that were experimentally tested, 16 latency reversing agent (LRA) synergizers and 2 latency promoting agents (LPAs) were confirmed, yielding an overall experimental hit rate of 15.9%. Our work demonstrates that in silico drug screening modalities, guided by existing large-scale experimental datasets, can yield high experimental hit rates, reducing costs incurred from labor-intensive wet lab-focused methodologies.

biochemistry↗

Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation

Human Immunodeficiency Virus (HIV) latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to- macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed PKC signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing pro-inflammatory M1 macrophages suppressed HIV reactivation while M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive- oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and discovered that inhibitors of the thioredoxin (Trx) system acted as latency promoting agents (LPAs) in T-cells and monocytes, but opposingly acted as latency reversing agents (LRAs) in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase (TrxR) inhibition led to ROS induced NF-{kappa}B activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.

microbiology↗

Relative stability of mRNA and protein severely limits inference of gene networks from single-cell mRNA measurements

Inference of gene regulatory networks from single-cell expression data, such as single-cell RNA sequencing, is a popular problem in computational biology. Despite diverse methods spanning information theory, machine learning, and statistics, it is unsolved. This shortcoming can be attributed to measurement errors, lack of perturbation data, or difficulty in causal inference. Yet, it is not known if kinetic properties of gene expression also cause an issue. We show how the relative stability of mRNA and protein hampers inference. Available inference methods perform benchmarking on synthetic data lacking protein species, which is biologically incorrect. We use a simple model of gene expression, incorporating both mRNA and protein, to show that a more stable protein than mRNA can cause loss in correlation between the mRNA of a transcription factor and its target gene. This can also happen when mRNA and protein are on the same timescale. The relative difference in timescales affects true interactions more strongly than false positives, which may not be suppressed. Besides correlation, we find that information-theoretic nonlinear measures are also prone to this problem. Finally, we demonstrate these principles in real single-cell RNA sequencing data for over 1700 yeast genes.

systems biology↗

Monitoring Reactivation of Latent HIV by Label-Free Gradient Light Interference Microscopy

Latent human immunodeficiency virus (HIV) reservoirs in infected individuals present the largest barrier to a cure. The first step towards overcoming this challenge is to understand the science behind latency-reactivation interplay. Fluorescence imaging of GFP-tagged HIV has been the main method for studying reactivation of latent HIV in individually infected cells. In this paper, we report insights provided by label-free, gradient light interference microscopy (GLIM) about the changes in measures including dry mass, diameter, and dry mass density associated with infected cells that occur upon reactivation. We discovered that mean cell dry mass and mean diameter of latently infected cells treated with reactivating drug, TNF-, are higher for cells with reactivated HIV as compared to those with latent disease. Results also indicate that cells with mean dry mass and diameter less than 10pg and 8{micro}m, respectively, remain exclusively in the latent state. Also, cells with mean dry mass greater than 23pg and mean diameter greater than 11{micro}m have a higher probability of reactivating. This study is significant as it presents a new label-free approach to quantify latent reactivation of a virus in single cells based on changes in cell morphology.

bioengineering↗

Internetwork connectivity of molecular networks across species of life

BackgroundMolecular interactions have been studied as independent complex networks in systems biology. However, molecular networks dont exist independently of each other. In a network of networks approach (called multiplex), we uncover the design principles for the joint organization of transcriptional regulatory network (TRN) and protein-protein interaction (PPI) network. ResultsWe find that TRN and PPI networks are non-randomly coupled in the TRN-PPI multiplex across five different eukaryotic species. Gene degrees in TRN (number of downstream genes) are positively correlated with protein degrees in PPI (number of interacting protein partners). Gene-gene interactions in TRN and protein-protein interactions in PPI also non-randomly overlap in the multiplex. These design principles are conserved across the five eukaryotic species. We show that the robustness of the TRN-PPI multiplex is dependent on these design principles. Further, functionally important genes and proteins, such as essential, disease-related and those involved in host-pathogen PPI networks, are preferentially situated in essential parts of the human multiplex with highly overlapping interactions. ConclusionWe unveil the multiplex architecture of TRN and PPI networks across different species. Multiplex architecture may thus define a general framework for studying molecular networks across the different species of life. This approach may uncover the building blocks of the hierarchical organization of molecular interactions.

systems biology↗

A transient heritable memory regulates HIV reactivation from latency

Reactivation of human immunodeficiency virus 1 (HIV-1) from latently infected T cells is a critical barrier to successfully eradicate HIV-1 from patients. Latency models in Jurkat T-cells reveal that individual cells reactivate in an all-or-none fashion when exposed to latency reversal agents (LRAs). Remarkably, this heterogeneity arises within a clonal cell population of genetically identical cells containing a single copy of the provirus integrated at the same genomic site. To characterize these single-cell responses, we leverage the classical Luria-Delbrück fluctuation test where single cells are isolated from a clonal population, and exposed to LRAs after a period of colony expansion. If cellular responses are purely random, then the fraction of reactivating cells should have minimal colony-to-colony fluctuations given the large number of cells present after weeks of colony growth. In contrast, data shows considerable colony-to-colony fluctuations with the fraction of reactivating cells following a skewed distribution. Systematic measurements of fluctuations over time in combination with mathematical modeling uncovers the existence of a heritable memory that regulates HIV-1 reactivation, where single cells are in a LRA-responsive state for a few weeks before switching back to an irresponsive state. These results have enormous implications for designing therapies to purge the latent reservoir and illustrate the utility of fluctuation-based assays to uncover hidden transient cellular states underlying phenotypic heterogeneity.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

HIVs Feedback Circuit Breaks The Fundamental Limit On Noise Suppression To Stabilize Fate

Diverse biological systems utilize gene-expression fluctuations ( noise) to drive lineage-commitment decisions1-5. However, once a commitment is made, noise becomes detrimental to reliable function6,7 and the mechanisms enabling post-commitment noise suppression are unclear. We used time-lapse imaging and mathematical modeling, and found that, after a noise-driven event, human immunodeficiency virus (HIV) strongly attenuated expression noise through a non-transcriptional negative-feedback circuit. Feedback is established by serial generation of RNAs from post-transcriptional splicing, creating a precursor-product relationship where proteins generated from spliced mRNAs auto-deplete their own precursor un-spliced mRNAs. Strikingly, precursor auto-depletion overcomes the theoretical limits on conventional noise suppression--minimizing noise far better than transcriptional auto-repression--and dramatically stabilizes commitment to the active-replication state. This auto-depletion feedback motif may efficiently suppress noise in other systems ranging from detained introns to non-sense mediated decay.

cell biology↗