The dynamic clustering of insulin receptor underlies its signaling and is disrupted in insulin resistance.

Insulin receptor (IR) signaling is central to normal metabolic control and is dysregulated in metabolic diseases such as type 2 diabetes. We report here that IR is incorporated into dynamic clusters at the plasma membrane, in the cytoplasm and in the nucleus of human hepatocytes and adipocytes. Insulin stimulation promotes further incorporation of IR into these dynamic clusters in insulin-sensitive cells but not in insulin-resistant cells, where both IR accumulation and dynamic behavior are reduced. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue IR accumulation and the dynamic behavior of these clusters. This rescue is associated with metformin's role in reducing reactive oxygen species that interfere with normal dynamics. These results indicate that changes in the physico-mechanical features of IR clusters contribute to insulin resistance and have implications for improved therapeutic approaches.

Endometrial cytokines in patients with and without endometriosis evaluated for infertility.

OBJECTIVE: To evaluate whether endometrial molecular profiles distinguish subsets of patients according to clinical characteristics, and to infer dysregulated immune networks, by measuring cytokines, chemokines, and growth factors in endometrial biopsy specimens from a cohort of infertile women with a high incidence of endometriosis. DESIGN: Prospective cohort study. SETTING: Department of Gynecology at a university hospital. PATIENT(S): Patients undergoing laparoscopy for infertility assessment (n = 103). INTERVENTION(S): Endometrial biopsies were performed during surgery. Fertility outcome and clinical parameters were registered preoperatively and after 6 months. MAIN OUTCOME MEASURE(S): The concentrations of 48 factors in endometrial biopsy specimens were analyzed with respect to clinical status in univariate and multivariate frameworks. RESULT(S): The concentrations of 44 factors from endometrial tissues of 74 patients were suitable for analysis. Although the tissue concentrations of interleukin (IL)15, IL-7, and interferon γ-induced protein (IP)-10 were individually lower in patients with endometriosis than in those without endometriosis, the differences were not significant after multiple comparison. However, multivariate modeling incorporating covariation showed separation between subsets of endometriotic and nonendometriotic patients, based predominantly on IP-10, monocyte chemoattractant protein-1, IL-16, and IL-18; this result was independent of cycle and fertility status. Analysis restricted to endometrial tissues from the secretory phase separated endometriotic and nonendometriotic patients by a combination of IL-15, IP-10, monocyte chemoattractant protein-1, IL-16, and IL-18. This combination suggests a uterine natural killer cell defect. We found no significant correlations between endometrial cytokines and fertility outcome. CONCLUSION(S): A molecular signature in endometrial tissue was able to distinguish endometriotic from nonendometriotic patients, implicating uterine natural killer cells in endometriosis.

Regulatory T Cells Control Effector T Cell Inflammation in Human Prediabetes.

A disparate array of plasma/serum markers provides evidence for chronic inflammation in human prediabetes, a condition that is most closely replicated by standard mouse models of obesity and metaflammation. These remain largely nonactionable and contrast with our rich understanding of inflammation in human type 2 diabetes. New data show that inflammatory profiles produced by CD4+ T cells define human prediabetes as a unique inflammatory state. Regulatory T cells (Treg) control mitochondrial function and cytokine production by CD4+ effector T cells (Teff) in prediabetes and type 2 diabetes by supporting T helper (Th)17 or Th1 cytokine production, respectively. These data suggest that Treg control of Teff metabolism regulates inflammation differentially in prediabetes compared with type 2 diabetes. Queries of genes that impact mitochondrial function or pathways leading to transcription of lipid metabolism genes identified the fatty acid importer CD36 as highly expressed in Treg but not Teff from subjects with prediabetes. Pharmacological blockade of CD36 in Treg from subjects with prediabetes decreased Teff production of the Th17 cytokines that differentiate overall prediabetes inflammation. We conclude that Treg control CD4+ T cell cytokine profiles through mechanisms determined, at least in part, by host metabolic status. Furthermore, Treg CD36 uniquely promotes Th17 cytokine production by Teff in prediabetes.

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. Cells with mean dry mass and diameter less than approximately 10 pg and 8 µm, respectively, remain exclusively in the latent state. Also, cells with mean dry mass greater than approximately 28-30 pg and mean diameter greater than 11-12 µm 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.

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. Here, we show that a larger host-cell size provides a natural cellular mechanism for enhancing burst size of viral expression and is necessary to destabilize the latent state and bias viral decision-making. Latently infected Jurkat and primary CD4+ T cells reactivate exclusively in larger activated cells, while smaller cells remain silent. In addition, reactivation is cell-cycle dependent and can be modulated with cell-cycle-arresting compounds. Cell size and cell-cycle dependent decision-making of viral circuits may guide stochastic design strategies and applications in synthetic biology and may provide important determinants to advance diagnostics and therapies.

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. CA conserves noise levels while increasing mean abundance, resulting in direct tuning of the transcriptional burst size, while PYR strictly increases transcriptional initiation frequency while conserving a constant transcriptional burst size. Time-dependent treatment with CA reveals non-continuous tuning with noise oscillating at a constant mean abundance at early time points and the burst size increasing for treatments after 5 h. Treatments combining CA and Protein Kinase C agonists result in an even larger increase of abundance while conserving noise levels with a highly non-linear increase in variance of up to 63× untreated controls. Finally, drug combinations provide non-antagonistic combinatorial tuning of gene expression noise and map a noise phase space for future applications with viral and synthetic gene vectors. Active remodeling of nucleosomes and BAF-mediated control of gene expression noise expand a toolbox for the future design and engineering of stochasticity in living systems.

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. We show that viral-host co-evolution is imprinted within promoter gene sequences before transcript or protein interactions. Co-regulation of human immunodeficiency virus (HIV) and human C-X-C chemokine receptor-4 (CXCR4) facilitates migration of infected cells. Upon infection, HIV can actively replicate or remain dormant. Migrating infected cells reactivate from dormancy more than non-migrating cells and exhibit differential migration-reactivation responses to drugs. Cells producing virus pose a risk for reinitiating infection within niches inaccessible to drugs, and tuning viral control of migration and reactivation improves strategies to eliminate latent HIV. Viral-host genetic coupling establishes a mechanism for synchronizing transcription and guiding potential therapies.