Regulation of Energy Expenditure by Brainstem GABA Neurons.

Homeostatic control of core body temperature is essential for survival. Temperature is sensed by specific neurons, in turn eliciting both behavioral (i.e., locomotion) and physiologic (i.e., thermogenesis, vasodilatation) responses. Here, we report that a population of GABAergic (Vgat-expressing) neurons in the dorsolateral portion of the dorsal raphe nucleus (DRN), hereafter DRNVgat neurons, are activated by ambient heat and bidirectionally regulate energy expenditure through changes in both thermogenesis and locomotion. We find that DRNVgat neurons innervate brown fat via a descending projection to the raphe pallidus (RPa). These neurons also densely innervate ascending targets implicated in the central regulation of energy expenditure, including the hypothalamus and extended amygdala. Optogenetic stimulation of different projection targets reveals that DRNVgat neurons are capable of regulating thermogenesis through both a "direct" descending pathway through the RPa and multiple "indirect" ascending pathways. This work establishes a key regulatory role for DRNVgat neurons in controlling energy expenditure.

Engineering a Model Cell for Rational Tuning of GPCR Signaling.

G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.

Excess Translation of Epigenetic Regulators Contributes to Fragile X Syndrome and Is Alleviated by Brd4 Inhibition.

Fragile X syndrome (FXS) is a leading genetic cause of intellectual disability and autism. FXS results from the loss of function of fragile X mental retardation protein (FMRP), which represses translation of target transcripts. Most of the well-characterized target transcripts of FMRP are synaptic proteins, yet targeting these proteins has not provided effective treatments. We examined a group of FMRP targets that encode transcriptional regulators, particularly chromatin-associated proteins. Loss of FMRP in mice results in widespread changes in chromatin regulation and aberrant gene expression. To determine if targeting epigenetic factors could reverse phenotypes associated with the disorder, we focused on Brd4, a BET protein and chromatin reader targeted by FMRP. Inhibition of Brd4 function alleviated many of the phenotypes associated with FXS. We conclude that loss of FMRP results in significant epigenetic misregulation and that targeting transcription via epigenetic regulators like Brd4 may provide new treatments for FXS.

Dynamic changes to lipid mediators support transitions among macrophage subtypes during muscle regeneration.

Muscle damage elicits a sterile immune response that facilitates complete regeneration. Here, we used mass spectrometry-based lipidomics to map the mediator lipidome during the transition from inflammation to resolution and regeneration in skeletal muscle injury. We observed temporal regulation of glycerophospholipids and production of pro-inflammatory lipid mediators (for example, leukotrienes and prostaglandins) and specialized pro-resolving lipid mediators (for example, resolvins and lipoxins) that were modulated by ibuprofen. These time-dependent profiles were recapitulated in sorted neutrophils and Ly6Chi and Ly6Clo muscle-infiltrating macrophages, with a distinct pro-resolving signature observed in Ly6Clo macrophages. RNA sequencing of macrophages stimulated with resolvin D2 showed similarities to transcriptional changes found during the temporal transition from Ly6Chi macrophage to Ly6Clo macrophage. In vivo, resolvin D2 increased Ly6Clo macrophages and functional improvement of the regenerating muscle. These results reveal dynamic lipid mediator signatures of innate immune cells and provide a proof of concept for their exploitable effector roles in muscle regeneration.

Altered differentiation is central to HIV-specific CD4+ T cell dysfunction in progressive disease.

Dysfunction of virus-specific CD4+ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4+ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cell)-like profile characterized HIV-specific CD4+ T cells in viremic infection. HIV-specific CD4+ T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the TH1, TH17 and TH22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with TFH cells, but persistently low expression of genes associated with TH1, TH17 and TH22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4+ T cells and involves both gain of function and loss of function.

Nuclear RNF2 inhibits interferon function by promoting K33-linked STAT1 disassociation from DNA.

Prolonged activation of interferon-STAT1 signaling is closely related to inflammatory autoimmune disorders, and therefore the identification of negative regulators of these pathways is important. Through high-content screening of 115 mouse RING-domain E3 ligases, we identified the E3 ubiquitin ligase RNF2 as a potent inhibitor of interferon-dependent antiviral responses. RNF2 deficiency substantially enhanced interferon-stimulated gene (ISG) expression and antiviral responses. Mechanistically, nuclear RNF2 directly bound to STAT1 after interferon stimulation and increased K33-linked polyubiquitination of the DNA-binding domain of STAT1 at position K379, in addition to promoting the disassociation of STAT1/STAT2 from DNA and consequently suppressing ISG transcription. Our study provides insight into the regulation of interferon-dependent responses via a previously unrecognized post-translational modification of STAT1 in the nucleus.

Chromatin dependencies in cancer and inflammation.

Multiple cell-signalling pathways converge on chromatin to induce gene expression programmes. The inducible transcriptional programmes that are established as a result of inflammatory or oncogenic signals are controlled by shared chromatin regulators. Therapeutic targeting of such chromatin dependencies has proved effective for controlling tumorigenesis and for preventing immunopathologies that are driven by overt inflammation. In this Review, we discuss how chromatin dependencies are established to regulate the expression of key oncogenes and inflammation-promoting genes and how a better mechanistic understanding of such chromatin dependencies can be leveraged to improve the magnitude, timing, duration and selectivity of cell responses with the aim of minimizing unwanted cellular and systemic effects. Recently, exciting progress has been made in cancer immunotherapy and in the development of drugs that target chromatin regulators. We discuss recent advances in clinical trials and the challenge of combining immune-cell-based therapies and epigenetic therapies to improve human health.

Interferon-λ and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection.

The epithelium is the main entry point for many viruses, but the processes that protect barrier surfaces against viral infections are incompletely understood. Here we identified interleukin 22 (IL-22) produced by innate lymphoid cell group 3 (ILC3) as an amplifier of signaling via interferon-λ (IFN-λ), a synergism needed to curtail the replication of rotavirus, the leading cause of childhood gastroenteritis. Cooperation between the receptor for IL-22 and the receptor for IFN-λ, both of which were 'preferentially' expressed by intestinal epithelial cells (IECs), was required for optimal activation of the transcription factor STAT1 and expression of interferon-stimulated genes (ISGs). These data suggested that epithelial cells are protected against viral replication by co-option of two evolutionarily related cytokine networks. These data may inform the design of novel immunotherapy for viral infections that are sensitive to interferons.

Novel RNA molecular bioengineering technology efficiently produces functional miRNA agents.

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNAGly and BioRNALeu, respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.

Bcl-6 directly represses the gene program of the glycolysis pathway.

Despite the increasing knowledge of the molecular events that induce the glycolysis pathway in effector T cells, very little is known about the transcriptional mechanisms that dampen the glycolysis program in quiescent cell populations such as memory T cells. Here we found that the transcription factor Bcl-6 directly repressed genes encoding molecules involved in the glycolysis pathway, including Slc2a1, Slc2a3, Pkm and Hk2, in type 1 helper T cells (TH1 cells) exposed to low concentrations of interleukin 2 (IL-2). Thus, Bcl-6 had a role opposing the IL-2-sensitive glycolytic transcriptional program that the transcription factors c-Myc and HIF-1α promote in effector T cells. Additionally, the TH1 lineage-specifying factor T-bet functionally antagonized the Bcl-6-dependent repression of genes encoding molecules in the glycolysis pathway, which links the molecular balance of these two factors to regulation of the metabolic gene program.

Diversification of TAM receptor tyrosine kinase function.

The clearance of apoptotic cells is critical for both tissue homeostasis and the resolution of inflammation. We found that the TAM receptor tyrosine kinases Axl and Mer had distinct roles as phagocytic receptors in these two settings, in which they exhibited divergent expression, regulation and activity. Mer acted as a tolerogenic receptor in resting macrophages and during immunosuppression. In contrast, Axl was an inflammatory response receptor whose expression was induced by proinflammatory stimuli. Axl and Mer differed in their ligand specificities, ligand-receptor complex formation in tissues, and receptor shedding upon activation. These differences notwithstanding, phagocytosis by either protein was strictly dependent on receptor activation triggered by bridging of TAM receptor-ligand complexes to the 'eat-me' signal phosphatidylserine on the surface of apoptotic cells.

PPARγ is a nexus controlling alternative activation of macrophages via glutamine metabolism.

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is known to regulate lipid metabolism in many tissues, including macrophages. Here we report that peritoneal macrophage respiration is enhanced by rosiglitazone, an activating PPARγ ligand, in a PPARγ-dependent manner. Moreover, PPARγ is required for macrophage respiration even in the absence of exogenous ligand. Unexpectedly, the absence of PPARγ dramatically affects the oxidation of glutamine. Both glutamine and PPARγ have been implicated in alternative activation (AA) of macrophages, and PPARγ was required for interleukin 4 (IL4)-dependent gene expression and stimulation of macrophage respiration. Indeed, unstimulated macrophages lacking PPARγ contained elevated levels of the inflammation-associated metabolite itaconate and express a proinflammatory transcriptome that, remarkably, phenocopied that of macrophages depleted of glutamine. Thus, PPARγ functions as a checkpoint, guarding against inflammation, and is permissive for AA by facilitating glutamine metabolism. However, PPARγ expression is itself markedly increased by IL4. This suggests that PPARγ functions at the center of a feed-forward loop that is central to AA of macrophages.

FOXS1 is increased in liver fibrosis and regulates TGFß responsiveness and proliferation pathways in human hepatic stellate cells.

Liver fibrosis commences with liver injury stimulating transforming growth factor beta (TGFß) activation of hepatic stellate cells (HSCs), causing scarring and irreversible damage. TGFß induces expression of the transcription factor Forkhead box S1 (FOXS1) in hepatocytes and may have a role in the pathogenesis of hepatocellular carcinoma (HCC). To date, no studies have determined how it affects HSCs. We analyzed human livers with cirrhosis, HCC, and a murine fibrosis model and found that FOXS1 expression is significantly higher in fibrotic livers but not in HCC. Next, we treated human LX2 HSC cells with TGFß to activate fibrotic pathways, and FOXS1 mRNA was significantly increased. To study TGFß-FOXS1 signaling, we developed human LX2 FOXS1 CRISPR KO and scrambled control HSCs. To determine differentially expressed gene transcripts controlled by TGFß-FOXS1, we performed RNA-seq in the FOXS1 KO and control cells and over 400 gene responses were attenuated in the FOXS1 KO HSCs with TGFß-activation. To validate the RNA-seq findings, we used our state-of-the-art PamGene PamStation kinase activity technology that measures hundreds of signaling pathways nonselectively in real time. Using our RNA-seq data, kinase activity data, and descriptive measurements, we found that FOXS1 controls pathways mediating TGFß responsiveness, protein translation, and proliferation. Our study is the first to identify that FOXS1 may serve as a biomarker for liver fibrosis and HSC activation, which may help with early detection of hepatic fibrosis or treatment options for end-stage liver disease.

Protein dynamics in drug combinations: a linear superposition of individual-drug responses.

Drugs and drug combinations have complex biological effects on cells and organisms. Little is known about how drugs affect protein dynamics that determine these effects. Here, we use a dynamic proteomics approach to accurately follow 15 protein levels in human cells in response to 13 different drugs. We find that protein dynamics in response to combinations of drugs are described accurately by a linear superposition (weighted sum) of their response to individual drugs. The weights in this superposition describe the relative impact of each drug on each protein. Using these weights, we show that one can predict the dynamics in a three-drug or four-drug combination on the basis of the dynamics in drug pairs. Our approach might eliminate the need to increase the number of experiments exponentially with the number of drugs and suggests that it might be possible to rationally control protein dynamics with specific drug combinations.

Neutralizing antibodies with neurotropic factor treatment maintain neurodevelopmental gene expression upon exposure to human cytomegalovirus.

IMPORTANCE: Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development.

The p110δ isoform of the kinase PI(3)K controls the subcellular compartmentalization of TLR4 signaling and protects from endotoxic shock.

Lipopolysaccharide activates plasma-membrane signaling and endosomal signaling by Toll-like receptor 4 (TLR4) through the TIRAP-MyD88 and TRAM-TRIF adaptor complexes, respectively, but it is unclear how the signaling switch between these cell compartments is coordinated. In dendritic cells, we found that the p110δ isoform of phosphatidylinositol-3-OH kinase (PI(3)K) induced internalization of TLR4 and dissociation of TIRAP from the plasma membrane, followed by calpain-mediated degradation of TIRAP. Accordingly, inactivation of p110δ prolonged TIRAP-mediated signaling from the plasma membrane, which augmented proinflammatory cytokine production while decreasing TRAM-dependent endosomal signaling that generated anti-inflammatory cytokines (interleukin 10 and interferon-ß). In line with that altered signaling output, p110δ-deficient mice showed enhanced endotoxin-induced death. Thus, by controlling the 'topology' of TLR4 signaling complexes, p110δ balances overall homeostasis in the TLR4 pathway.

Copper-mediated siRNA activation for conditional control of gene expression.

Copper plays a crucial role in maintaining biological redox balance in living organisms, with elevated levels observed in cancer cells. Short interfering RNAs (siRNAs) are effective in gene silencing and find applications as both research tools and therapeutic agents. A method to regulate RNA interference using copper is especially advantageous for cancer-specific therapy. We present a chemical approach of selective siRNA activation triggered by intracellular copper ions. We designed and synthesized nucleotides containing copper-responsive moieties, which were incorporated into siRNAs. These copper-responsive siRNAs effectively silenced the target cyclin B1 mRNA in living cells. This pioneering study introduces a novel method for conditionally controlling gene silencing using biologically relevant metal ions in human cells, thereby expanding the repertoire of chemical knockdown tools.

Protective effects of butyric acid during heat stress on the survival, immune response, histopathology, and gene expression in the hepatopancreas of juvenile pacific shrimp (L. Vannamei).

This study looked at the effects of adding butyric acid (BA) to the diets of juvenile Pacific shrimp and how it affected their response to survival, immunity, histopathological, and gene expression profiles under heat stress. The shrimp were divided into groups: a control group with no BA supplementation and groups with BA inclusion levels of 0.5 %, 1 %, 1.5 %, 2 %, and 2.5 %. Following the 8-week feeding trial period, the shrimp endured a heat stress test lasting 1 h at a temperature of 38 °C. The results showed that the control group had a lower survival rate than those given BA. Interestingly, no mortality was observed in the group receiving 1.5 % BA supplementation. Heat stress had a negative impact on the activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the control group. Still, these activities were increased in shrimp fed the BA diet. Similar variations were observed in AST and ALT fluctuations among the different groups. The levels of triglycerides (TG) and cholesterol (CHO) increased with high temperatures but were reduced in shrimp-supplemented BA. The activity of an antioxidant enzyme superoxide dismutase (SOD) increased with higher BA levels (P < 0.05). Moreover, the groups supplemented with 1.5 % BA exhibited a significant reduction in malondialdehyde (MDA) content (P < 0.05), suggesting the potential antioxidant properties of BA. The histology of the shrimp's hepatopancreas showed improvements in the groups given BA. Conversely, the BA significantly down-regulated the HSPs and up-regulated MnSOD transcript level in response to heat stress. The measured parameters determine the essential dietary requirement of BA for shrimp. Based on the results, the optimal level of BA for survival, antioxidant function, and immunity for shrimp under heat stress is 1.5 %.

Structure-activity relationship studies of pyrogallol as a calcineurin/NFAT signaling suppressor.

Previously, we have shown that pyrogallol alleviated nasal symptoms and suppressed IL-9 gene up-regulation in allergy model rats by inhibiting calcineurin/NFAT signaling. As pyrogallol has antioxidative activity, it may be responsible for inhibiting calcineurin/NFAT signaling-mediated IL-9 gene expression. However, the relationship between antioxidative activity and suppression of IL-9 gene expression has not been elucidated yet. Here, we conducted the structure-activity relationship studies of pyrogallol and its structurally related compounds to understand the mechanism of IL-9 gene suppression by pyrogallol. 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging assay showed that the antioxidative activity of catechol, resorcinol, phloroglucinol, and gallic acid is 60.1%, 10.4%, 18.8%, and 113.5% of pyrogallol, respectively. Catechol, resorcinol, and phloroglucinol did not suppress NFAT dephosphorylation. Gallic acid suppressed dephosphorylation of NFAT. Gallic acid also suppressed ionomycin-induced up-regulation of IL-9 gene expression with the IC50 value of 82.6 µM. However, catechol, resorcinol and phloroglucinol showed no suppressive activity. In addition, using gallic acid-immobilized beads, we isolated and identified Poly(U)-binding-splicing factor 60 (PUF60) as a pyrogallol binding protein. These results suggest that the antioxidative activity of pyrogallol is not likely to be the mechanism of IL-9 gene suppression. Data also suggest that PUF60 is one of its target molecules responsible for the suppression of calcineurin/NFAT signaling by pyrogallol.

Glucocorticoid-induced acute diuresis in rats in relation to the reduced renal expression of sodium-dependent cotransporter genes.

Although several studies have shown that glucocorticoids exert diuretic effects in animals and humans, the underlying mechanism responsible for the acute diuretic effect remains obscure. Here we examined the mechanism in terms of gene-expression. We observed that glucocorticoids, including dexamethasone (Dex) and prednisolone (PSL), acutely induced diuresis in rats in a dose-dependent manner. Free water clearance values were negative after Dex or PSL treatment, similar to those observed after treatment with osmotic diuretics (furosemide and acetazolamide). Dex significantly increased the urinary excretion of sodium, potassium, chloride, glucose, and inorganic phosphorus. Renal microarray analysis revealed that Dex significantly altered the renal expression of genes related to transmembrane transport activity. The mRNA levels of sodium/phosphate (NaPi-2a/Slc34a1, NaPi-2b/Slc34a2, and NaPi-2c/Slc34a3) and sodium/glucose cotransporters (Sglt2/Slc5a2) were significantly reduced in the Dex-treated kidney, being negatively correlated with the urinary excretion of their corresponding solutes. Dex did not affect renal expression of the natriuretic peptide receptor 1 (Npr1) gene, or the expression, localization, and phosphorylation of aquaporin-2 (AQP2), a water channel protein. These findings suggest that the acute diuretic effects of glucocorticoids might be mediated by reduced expression of sodium-dependent cotransporter genes.