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

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.

Principles for the design of multicellular engineered living systems.

Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell-cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics.

Monitoring reactivation of latent HIV by label-free gradient light interference microscopy.

Human immunodeficiency virus (HIV) can infect cells and take a quiescent and nonexpressive state called latency. In this study, we report insights provided by label-free, gradient light interference microscopy (GLIM) about the changes in dry mass, diameter, and dry mass density associated with infected cells that occur upon reactivation. We discovered that the mean cell dry mass and mean diameter of latently infected cells treated with reactivating drug, TNF-α, are higher for latent cells that reactivate than those of the cells that did not reactivate.

Synergistic Chromatin-Modifying Treatments Reactivate Latent HIV and Decrease Migration of Multiple Host-Cell Types.

Upon infection of its host cell, human immunodeficiency virus (HIV) establishes a quiescent and non-productive state capable of spontaneous reactivation. Diverse cell types harboring the provirus form a latent reservoir, constituting a major obstacle to curing HIV. Here, we investigate the effects of latency reversal agents (LRAs) in an HIV-infected THP-1 monocyte cell line in vitro.

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 cure patients. It remains unknown whether reactivation of individual latent cells occurs stochastically in response to latency reversal agents (LRAs) or is a deterministic outcome of an underlying cell state. 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 colony expansion.

Screening for gene expression fluctuations reveals latency-promoting agents of HIV.

Upon treatment removal, spontaneous reactivation of latently infected T cells remains a major barrier toward curing HIV. Therapies that reactivate and clear the latent reservoir are only partially effective, while latency-promoting agents (LPAs) used to suppress reactivation and stabilize latency are understudied and lack diversity in their mechanisms of action. Here, we identify additional LPAs using a screen for gene-expression fluctuations (or "noise") that drive cell-fate specification and control HIV reactivation from latency.

Internetwork connectivity of molecular networks across species of life.

Molecular interactions are studied as independent networks in systems biology. However, molecular networks do not exist independently of each other. In a network of networks approach (called multiplex), we study the joint organization of transcriptional regulatory network (TRN) and protein-protein interaction (PPI) network.

Cell size dependent migration of T-cells latently infected with HIV.

Human immunodeficiency virus (HIV) preferentially infects T-lymphocytes by integrating into host DNA and forming a latent transcriptionally silent provirus. As previously shown, HIV-1 alters migration modes of T-lymphocytes by co-regulating viral gene expression with human C-X-C chemokine receptor-4 (CXCR4). Here, we show that motility of infected T-lymphocytes is cell size dependent.

Perspective: The promise of multi-cellular engineered living systems.

Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of "multi-cellular engineered living systems," which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered.

Fine-tuning of noise in gene expression with nucleosome remodeling.

Engineering stochastic fluctuations of gene expression (or "noise") is integral to precisely bias cellular-fate decisions and statistical phenotypes in both single-cell and multi-cellular systems. Epigenetic regulation has been shown to constitute a large source of noise, and thus, engineering stochasticity is deeply intertwined with epigenetics. Here, utilizing chromatin remodeling, we report that Caffeic acid phenethyl ester (CA) and Pyrimethamine (PYR), two inhibitors of BAF250a, a subunit of the Brahma-associated factor (BAF) nucleosome remodeling complex, enable differential and tunable control of noise in transcription and translation from the human immunodeficiency virus long terminal repeat promoter in a dose and time-dependent manner.

Perspective: Engineering noise in biological systems towards predictive stochastic design.

Significant progress has been made towards engineering both single-cell and multi-cellular systems through a combination of synthetic and systems biology, nanobiotechnology, pharmaceutical science, and computational approaches. However, our ability to engineer systems that begin to approach the complexity of natural pathways is severely limited by important challenges, e.g.

Cell Size-Based Decision-Making of a Viral Gene Circuit.

Latently infected T cells able to reinitiate viral propagation throughout the body remain a major barrier to curing HIV. Distinguishing between latently infected cells and uninfected cells will advance efforts for viral eradication. HIV decision-making between latency and active replication is stochastic, and drug cocktails that increase bursts of viral gene expression enhance reactivation from latency.

A Post-Transcriptional Feedback Mechanism for Noise Suppression and Fate Stabilization.

Diverse biological systems utilize fluctuations ("noise") in gene expression to drive lineage-commitment decisions. However, once a commitment is made, noise becomes detrimental to reliable function, and the mechanisms enabling post-commitment noise suppression are unclear. Here, we find that architectural constraints on noise suppression are overcome to stabilize fate commitment.

Similarity in viral and host promoters couples viral reactivation with host cell migration.

Viral-host interactomes map the complex architecture of an evolved arms race during host cell invasion. mRNA and protein interactomes reveal elaborate targeting schemes, yet evidence is lacking for genetic coupling that results in the co-regulation of promoters. Here we compare viral and human promoter sequences and expression to test whether genetic coupling exists and investigate its phenotypic consequences.

Transcriptional Bursting Explains the Noise-Versus-Mean Relationship in mRNA and Protein Levels.

Recent analysis demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-to-cell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds.

The Low Noise Limit in Gene Expression.

Protein noise measurements are increasingly used to elucidate biophysical parameters. Unfortunately noise analyses are often at odds with directly measured parameters. Here we show that these inconsistencies arise from two problematic analytical choices: (i) the assumption that protein translation rate is invariant for different proteins of different abundances, which has inadvertently led to (ii) the assumption that a large constitutive extrinsic noise sets the low noise limit in gene expression.

Screening for noise in gene expression identifies drug synergies.

Stochastic fluctuations are inherent to gene expression and can drive cell-fate specification. We used such fluctuations to modulate reactivation of HIV from latency-a quiescent state that is a major barrier to an HIV cure. By screening a diverse library of bioactive small molecules, we identified more than 80 compounds that modulated HIV gene-expression fluctuations (i.

An endogenous accelerator for viral gene expression confers a fitness advantage.

Many signaling circuits face a fundamental tradeoff between accelerating their response speed while maintaining final levels below a cytotoxic threshold. Here, we describe a transcriptional circuitry that dynamically converts signaling inputs into faster rates without amplifying final equilibrium levels. Using time-lapse microscopy, we find that transcriptional activators accelerate human cytomegalovirus (CMV) gene expression in single cells without amplifying steady-state expression levels, and this acceleration generates a significant replication advantage.

BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism.

The therapeutic potential of pharmacologic inhibition of bromodomain and extraterminal (BET) proteins has recently emerged in hematological malignancies and chronic inflammation. We find that BET inhibitor compounds (JQ1, I-Bet, I-Bet151 and MS417) reactivate HIV from latency. This is evident in polyclonal Jurkat cell populations containing latent infectious HIV, as well as in a primary T-cell model of HIV latency.

Transcriptional burst frequency and burst size are equally modulated across the human genome.

Gene expression occurs either as an episodic process, characterized by pulsatile bursts, or as a constitutive process, characterized by a Poisson-like accumulation of gene products. It is not clear which mode of gene expression (constitutive versus bursty) predominates across a genome or how transcriptional dynamics are influenced by genomic position and promoter sequence. Here, we use time-lapse fluorescence microscopy to analyze 8,000 individual human genomic loci and find that at virtually all loci, episodic bursting--as opposed to constitutive expression--is the predominant mode of expression.

Dynamics of protein noise can distinguish between alternate sources of gene-expression variability.

Within individual cells, two molecular processes have been implicated as sources of noise in gene expression: (i) Poisson fluctuations in mRNA abundance arising from random birth and death of individual mRNA transcripts or (ii) promoter fluctuations arising from stochastic promoter transitions between different transcriptional states. Steady-state measurements of variance in protein levels are insufficient to discriminate between these two mechanisms, and mRNA single-molecule fluorescence in situ hybridization (smFISH) is challenging when cellular mRNA concentrations are high. Here, we present a perturbation method that discriminates mRNA birth/death fluctuations from promoter fluctuations by measuring transient changes in protein variance and that can operate in the regime of high molecular numbers.

Model for biological communication in a nanofabricated cell-mimic driven by stochastic resonance.

Cells offer natural examples of highly efficient networks of nanomachines. Accordingly, both intracellular and intercellular communication mechanisms in nature are looked to as a source of inspiration and instruction for engineered nanocommunication. Harnessing biological functionality in this manner requires an interdisciplinary approach that integrates systems biology, synthetic biology, and nanofabrication.

Noise in biological circuits.

Noise biology focuses on the sources, processing, and biological consequences of the inherent stochastic fluctuations in molecular transitions or interactions that control cellular behavior. These fluctuations are especially pronounced in small systems where the magnitudes of the fluctuations approach or exceed the mean value of the molecular population. Noise biology is an essential component of nanomedicine where the communication of information is across a boundary that separates small synthetic and biological systems that are bound by their size to reside in environments of large fluctuations.

Using noise to probe and characterize gene circuits.

Stochastic fluctuations (or "noise") in the single-cell populations of molecular species are shaped by the structure and biokinetic rates of the underlying gene circuit. The structure of the noise is summarized by its autocorrelation function. In this article, we introduce the noise regulatory vector as a generalized framework for making inferences concerning the structure and biokinetic rates of a gene circuit from its noise autocorrelation function.

Transient-mediated fate determination in a transcriptional circuit of HIV.

Steady-state behavior and bistability have been proposed as mechanisms for decision making in gene circuits. However, transient gene expression has also been proposed to control cell fate, with the decision arbitrated by the duration of a transient gene expression pulse. Here, using an HIV-1 model system, we directly quantify transcriptional feedback strength and its effects on both the duration of HIV-1 Tat transcriptional pulses and the fate of HIV-infected cells.