Idiopathic pulmonary fibrosis is a progressive lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. Yes-associated protein (YAP) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. We previously identified HMG-CoA reductase inhibitors (statins) as YAP inhibitors based on a high-throughput small-molecule screen in primary human lung fibroblasts. Here we report that several Aurora kinase inhibitors were also identified from the top hits of this screen. MK-5108, a highly selective inhibitor for Aurora kinase A (AURKA), induced YAP phosphorylation and cytoplasmic retention, and significantly reduced profibrotic gene expression in human lung fibroblasts. The inhibitory effect on YAP nuclear translocation and profibrotic gene expression is specific to inhibition of AURKA, but not Aurora kinase B or C, and is independent of the Hippo pathway kinases LATS1 and LATS2. Further characterization of the effects of MK-5108 demonstrate that it inhibits YAP nuclear localization indirectly via effects on actin polymerization and TGFβ signaling. In addition, MK-5108 treatment reduced lung collagen deposition in the bleomycin mouse model of pulmonary fibrosis. Our results reveal a novel role for AURKA in YAP-mediated profibrotic activity in fibroblasts and highlight the potential of small-molecule screens for YAP inhibitors for identification of novel agents with anti-fibrotic activity.

BACKGROUND: Several epigenome-wide association studies (EWAS) of ambient particulate matter with aerodynamic diameter ≤ 2.5 µm (PM2.5) have been reported. However, EWAS of PM2.5 elements (PEs), reflecting different emission sources, are very limited. OBJECTIVES: We performed EWAS of short- and intermediate-term exposure to PM2.5 and 13 PEs. We hypothesized that significant changes in DNAm may vary by PM2.5 mass and its elements. METHODS: We repeatedly collected blood samples in the Normative Aging Study and measured leukocyte DNA methylation (DNAm) with the Illumina HumanMethylation450K BeadChip. We collected daily PM2.5 and 13 PEs at a fixed central site. To estimate the associations between each PE and DNAm at individual cytosine-phosphate-guanine (CpG) sites, we incorporated a distributed-lag (0-27 d) term in the setting of median regression with subject-specific intercept and examined cumulative lag associations. We also accounted for selection bias due to loss to follow-up and mortality prior to enrollment. Significantly differentially methylated probes (DMPs) were identified using Bonferroni correction for multiple testing. We further conducted regional and pathway analyses to identify significantly differentially methylated regions (DMRs) and pathways. RESULTS: We included 695 men with 1,266 visits between 1999 and 2013. The subjects had a mean age of 75 years. The significant DMPs, DMRs, and pathways varied by to PM2.5 total mass and PEs. For example, PM2.5 total mass was associated with 2,717 DMPs and 10,470 DMRs whereas Pb was associated with 3,173 DMPs and 637 DMRs. The identified pathways by PM2.5 mass were mostly involved in mood disorders, neuroplasticity, immunity, and inflammation, whereas the pathways associated with motor vehicles (BC, Cu, Pb, and Zn) were related with cardiovascular disease and cancer (e.g., "PPARs signaling"). CONCLUSIONS: PM2.5 and PE were associated with methylation changes at multiple probes and along multiple pathways, in ways that varied by particle components.

Human embryonic stem cells (hESCs) provide opportunities for cell replacement therapy of insulin-dependent diabetes. Therapeutic quantities of human stem cell-derived islets (SC-islets) can be produced by directed differentiation. However, preventing allo-rejection and recurring autoimmunity, without the use of encapsulation or systemic immunosuppressants, remains a challenge. An attractive approach is to transplant SC-islets, genetically modified to reduce the impact of immune rejection. To determine the underlying forces that drive immunogenicity of SC-islets in inflammatory environments, we performed single-cell RNA sequencing (scRNA-seq) and whole-genome CRISPR screen of SC-islets under immune interaction with allogeneic peripheral blood mononuclear cells (PBMCs). Data analysis points to "alarmed" populations of SC-islets that upregulate genes in the interferon (IFN) pathway. The CRISPR screen in vivo confirms that targeting IFNγ-induced mediators has beneficial effects on SC-islet survival under immune attack. Manipulating the IFN response by depleting chemokine ligand 10 (CXCL10) in SC-islet grafts confers improved survival against allo-rejection compared with wild-type grafts in humanized mice. These results offer insights into the nature of immune destruction of SC-islets during allogeneic responses and provide targets for gene editing.

Rejection is the primary barrier to broader implementation of vascularized composite allografts (VCAs), including face and limb transplants. The immunologic pathways activated in face transplant rejection have not been fully characterized.METHODSUsing skin biopsies prospectively collected over 9 years from 7 face transplant patients, we studied rejection by gene expression profiling, histology, immunostaining, and T cell receptor sequencing.RESULTSGrade 1 rejection did not differ significantly from nonrejection, suggesting that it does not represent a pathologic state. In grade 2, there was a balanced upregulation of both proinflammatory T cell activation pathways and antiinflammatory checkpoint and immunomodulatory pathways, with a net result of no tissue injury. In grade 3, IFN-γ-driven inflammation, antigen-presenting cell activation, and infiltration of the skin by proliferative T cells bearing markers of antigen-specific activation and cytotoxicity tipped the balance toward tissue injury. Rejection of VCAs and solid organ transplants had both distinct and common features. VCA rejection was uniquely associated with upregulation of immunoregulatory genes, including SOCS1; induction of lipid antigen-presenting CD1 proteins; and infiltration by T cells predicted to recognize CD1b and CD1c.CONCLUSIONOur findings suggest that the distinct features of VCA rejection reflect the unique immunobiology of skin and that enhancing cutaneous immunoregulatory networks may be a useful strategy in combatting rejection.Trial registrationClinicalTrials.gov NCT01281267.FUNDINGAssistant Secretary of Defense and Health Affairs, through Reconstructive Transplant Research (W81XWH-17-1-0278, W81XWH-16-1-0647, W81XWH-16-1-0689, W81XWH-18-1-0784, W81XWH-1-810798); American Society of Transplantation's Transplantation and Immunology Research Network Fellowship Research Grant; Plastic Surgery Foundation Fellowship from the American Society of Plastic Surgeons; Novo Nordisk Foundation (NNF15OC0014092); Lundbeck Foundation; Aage Bangs Foundation; A.P. Moller Foundation for the Advancement of Medical Science; NIH UL1 RR025758.

BACKGROUND: Despite a number of known health hazards of welding fume exposure, it is unclear how exposure affects the human metabolome. OBJECTIVE: We assessed the metabolic profiles of welders before and after a 6-hour welding shift, controlling for circadian rhythm of metabolism on a non-welding day. METHODS: Welders were recruited from a training centre in Quincy, Massachusetts, in 2006 and 2010-2012 and donated blood samples on a welding shift day before and after work, as well as on a non-welding day spent in an adjacent classroom. In total, we collected 509 samples from 74 participants. Liquid chromatography-mass spectrometry quantified 665 metabolites from thawed plasmas. Metabolites with significant time (afternoon compared with morning) and day (welding/classroom) interactions were identified by two-way analysis of variance, and the overnight changes were evaluated. RESULTS: Sphingosine 1-phosphate (S1P) and sphingasine 1-phosphate (SA1P) exhibited significant interaction effects between day and time with false discovery rate-adjusted p values of 0.03 and <0.01, respectively. S1P, SA1P and sphingosine shared similar trends over time: high relative levels in the morning of a non-welding day declining by afternoon, but with lower starting levels on a welding day and no decline. There was no obvious pattern related to current smoking status. CONCLUSION: S1P and SA1P profiles were different between welding day and classroom day. The S1P pathway was disrupted on the day of welding exposure. The levels of S1P, SA1P and sphingosine were highly correlated over time. S1P is a signalling lipid with many vital roles; thus, the underlying mechanism and clinical implications of this alteration need further investigation.

Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify nongenetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.

Introduction: Human metabolism and inflammation are closely related modulators of homeostasis and immunity. Metabolic profiling is a useful tool to understand the association between metabolism and inflammation at a systemic level. Objective: To investigate the longitudinal associations between the concentration of plasma metabolites and biomarkers related to inflammation and oxidative stress. Methods: We conducted a repeated cross-sectional analysis consisting of 8 short-term panels that included 88 healthy adult male welders in Massachusetts, USA. In each panel, we collected 1-6 repeated measurements of blood and urine. We used a human vascular injury panel assay and custom cytokine/chemokine assay to quantify inflammatory biomarker plasma levels, liquid chromatography-mass spectrometry to quantify the concentrations of 665 plasma metabolites, and a competitive enzyme-linked immunoassay to quantify urinary 8-OHdG and 8-isoprostane levels. We used linear mixed effects models to estimate the longitudinal association between each inflammatory and oxidative stress biomarker and each metabolite. Results: At a 5% FDR threshold, we detected ≥1metabolite association for 8 unique inflammatory and oxidative stress biomarkers: urinary 8-isoprostane, plasma C-reactive protein (CRP), serum amyloid A (SAA), intercellular adhesion molecule 1, circulating vascular cell adhesion molecule-1, interleukin 8 (IL-8), interleukin 10 (IL-10) and vascular endothelial growth factor. Specifically, 3 metabolites in the androgenic steroids pathway were negatively associated with SAA; 3 dihydrosphingomyelins metabolites were positively associated with 1 or more of CRP, SAA, IL-8 and IL-10; 4 metabolites in acyl choline metabolism pathways were negatively associated with IL-8; 7 lysophospholipid metabolites were negatively associated with 1 or more of CRP, SAA and IL-8; 4 sphingomyelins were positively associated with CRP and/or SAA; and 10 metabolites in the xanthine pathway were positively associated with urinary 8-isoprostane. Conclusion: We found that metabolites in phospholipid groups had strong associations with multiple inflammatory biomarkers, especially CRP, SAA and IL-8. The mechanism of these associations warrants further investigation.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

Functionally relevant neuronal connections are often organized within discrete layers of neuropil to ensure proper connectivity and information processing. While layer-specific assembly of neuronal connectivity is a dynamic process involving stepwise interactions between different neuron types, the mechanisms underlying this critical developmental process are not well understood. Here, we investigate the role of the transcription factor dFezf in layer selection within the Drosophila visual system, which is important for synaptic specificity. Our findings show that dFezf functions as a transcriptional repressor governing the precise temporal expression pattern of downstream genes, including other transcription factors required for proper connectivity. Layer-specific assembly of neuronal connectivity in the fly visual system is thus orchestrated by precise, temporally controlled transcriptional cascades.The layered compartmentalization of synaptic connections, a common feature of nervous systems, underlies proper connectivity between neurons and enables parallel processing of neural information. However, the stepwise development of layered neuronal connections is not well understood. The medulla neuropil of the Drosophila visual system, which comprises 10 discrete layers (M1 to M10), where neural computations underlying distinct visual features are processed, serves as a model system for understanding layered synaptic connectivity. The first step in establishing layer-specific connectivity in the outer medulla (M1 to M6) is the innervation by lamina (L) neurons of one of two broad, primordial domains that will subsequently expand and transform into discrete layers. We previously found that the transcription factor dFezf cell-autonomously directs L3 lamina neurons to their proper primordial broad domain before they form synapses within the developing M3 layer. Here, we show that dFezf controls L3 broad domain selection through temporally precise transcriptional repression of the transcription factor slp1 (sloppy paired 1). In wild-type L3 neurons, slp1 is transiently expressed at a low level during broad domain selection. When dFezf is deleted, slp1 expression is up-regulated, and ablation of slp1 fully rescues the defect of broad domain selection in dFezf-null L3 neurons. Although the early, transient expression of slp1 is expendable for broad domain selection, it is surprisingly necessary for the subsequent L3 innervation of the M3 layer. DFezf thus functions as a transcriptional repressor to coordinate the temporal dynamics of a transcriptional cascade that orchestrates sequential steps of layer-specific synapse formation.All raw data for RNA-seq and ATAC-seq have been deposited in the Gene Expression Omnibus, https://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE163311) (47).

BACKGROUND & AIMS: Patients with simple steatosis (SS) and nonalcoholic steatohepatitis can develop progressive liver fibrosis, which is associated with liver-related mortality. The mechanisms contributing to liver fibrosis development in SS, however, are poorly understood. SS is characterized by hepatocellular free fatty acid (FFA) accumulation without lobular inflammation seen in nonalcoholic steatohepatitis. Because the Hippo signaling transcriptional coactivator YAP1 (YAP) has previously been linked with nonalcoholic fatty liver disease (NAFLD)-related fibrosis, we sought to explore how hepatocyte FFAs activate a YAP-mediated profibrogenic program. METHODS: We analyzed RNA sequencing data from a GEO DataSet (accession: GSE162694) consisting of 143 patients with NAFLD. We also performed immunohistochemical, immunofluorescence, immunoblot, and quantitative reverse-transcription polymerase chain reaction analyses (qRT-PCR) in liver specimens from NAFLD subjects, from a murine dietary NAFLD model, and in FFA-treated hepatic spheroids and hepatocytes. RESULTS: YAP-target gene expression correlated with increasing fibrosis stage in NAFLD patients and was associated with fibrosis in mice fed a NAFLD-inducing diet. Hepatocyte-specific YAP deletion in the murine NAFLD model attenuated diet-induced fibrosis, suggesting a causative role of YAP in NAFLD-related fibrosis. Likewise, in hepatic spheroids composed of Huh7 hepatoma cells and primary human hepatic stellate cells, Huh7 YAP silencing reduced FFA-induced fibrogenic gene expression. Notably, inhibition of p38 mitogen-activated protein kinase could block YAP activation in FFA-treated Huh7 cells. CONCLUSIONS: These studies provide further evidence for the pathological role of YAP in NAFLD-associated fibrosis and that YAP activation in NAFLD may be driven by FFA-induced p38 MAPK activation.

Clinical trials including an analytical treatment interruption (ATI) are vital for evaluating the efficacy of novel strategies for HIV remissions. We briefly describe an interactive tool for predicting viral rebound timing in ATI trials and the impact of posttreatment controller (PTC) definitions on PTC frequency estimates. A 4-week viral load threshold of 1000 cps/ml provides both high specificity and sensitivity for PTC detection. PTC frequency varies greatly based on the definition of a PTC.

OBJECTIVE: The mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism. METHODS: ATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor, rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4KO) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4KO mice. RESULTS: We demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver, in a hepatocyte-intrinsic manner by insulin in response to feeding. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of nonessential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine. CONCLUSIONS: The results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in the liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.

PURPOSE: Acute respiratory distress syndrome (ARDS) is accompanied by a dysfunctional immune-inflammatory response following lung injury, including during coronavirus disease 2019 (COVID-19). Limited causal biomarkers exist for ARDS development. We sought to identify novel genetic susceptibility targets for ARDS to focus further investigation on their biological mechanism and therapeutic potential. METHODS: Meta-analyses of ARDS genome-wide association studies were performed with 1250 cases and 1583 controls in Europeans, and 387 cases and 387 controls in African Americans. The functionality of novel loci was determined in silico using multiple omics approaches. The causality of 114 factors potentially involved in ARDS development was assessed using Mendelian Randomization analysis. RESULTS: There was distinct genetic heterogeneity in ARDS between Europeans and African Americans. rs7967111 at 12p13.2 was functionally associated with ARDS susceptibility in Europeans (odds ratio = 1.38; P = 2.15 × 10-8). Expression of two genes annotated at this locus, BORCS5 and DUSP16, was dynamic but ultimately decreased during ARDS development, as well as downregulated in immune cells alongside COVID-19 severity. Causal inference implied that comorbidity of inflammatory bowel disease and elevated levels of C-reactive protein and interleukin-10 causally increased ARDS risk, while vitamin D supplementation and vasodilator use ameliorated risk. CONCLUSION: Our findings suggest a novel susceptibility locus in ARDS pathophysiology that implicates BORCS5 and DUSP16 as potentially acting in immune-inflammatory processes. This locus warrants further investigation to inform the development of therapeutic targets and clinical care strategies for ARDS, including those induced by COVID-19.

While mitochondrial function is essential for life in all multicellular organisms, a mild impairment of mitochondrial function can extend longevity in model organisms. By understanding the molecular mechanisms involved, these pathways might be targeted to promote healthy aging. In studying two long-lived mitochondrial mutants in C. elegans, we found that disrupting subunits of the mitochondrial electron transport chain results in upregulation of genes involved in innate immunity, which is driven by the mitochondrial unfolded protein response (mitoUPR) but also dependent on the canonical p38-mediated innate immune signaling pathway. Both of these pathways are required for the increased resistance to bacterial pathogens and extended longevity of the long-lived mitochondrial mutants, as is the FOXO transcription factor DAF-16. This work demonstrates that both the p38-mediated innate immune signaling pathway and the mitoUPR act in concert on the same innate immunity genes to promote pathogen resistance and longevity and that input from the mitochondria can extend longevity by signaling through these pathways. This indicates that multiple evolutionarily conserved genetic pathways controlling innate immunity also function to modulate lifespan.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide. In adults with NAFLD, fibrosis can develop and progress to liver cirrhosis and liver failure. However, the underlying molecular mechanisms of fibrosis progression are not fully understood. Using total RNA-Seq, we investigated the molecular mechanisms of NAFLD and fibrosis. We sequenced liver tissue from 143 adults across the full spectrum of fibrosis stage including those with stage 4 fibrosis (cirrhosis). We identified gene expression clusters that strongly correlate with fibrosis stage including four genes that have been found consistently across previously published transcriptomic studies on NASH i.e. COL1A2, EFEMP2, FBLN5 and THBS2. Using cell type deconvolution, we estimated the loss of hepatocytes versus gain of hepatic stellate cells, macrophages and cholangiocytes with advancing fibrosis stage. Hepatocyte-specific functional analysis indicated increase of pro-apoptotic pathways and markers of bipotent hepatocyte/cholangiocyte precursors. Regression modelling was used to derive predictors of fibrosis stage. This study elucidated molecular and cell composition changes associated with increasing fibrosis stage in NAFLD and defined informative gene signatures for the disease.

[Figure: see text].