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The Community Multiscale Air Quality Model (CMAQ) is a local- to hemispheric-scale numerical air quality modeling system developed by the U.S. Environmental Protection Agency (USEPA) and supported by the Community Modeling and Analysis System (CMAS) center. CMAQ is used for regulatory purposes by the USEPA program offices and state and local air agencies and is also widely used by the broader global research community to simulate and understand complex air quality processes and for computational environmental fate and transport and climate and health impact studies. Leveraging state-of-the-science cloud computing resources for high-performance computing (HPC) applications, CMAQ is now available as a fully tested, publicly available technology stack (HPC cluster and software stack) for two major cloud service providers (CSPs). Specifically, CMAQ configurations and supporting materials have been developed for use on their HPC clusters, including extensive online documentation, tutorials and guidelines to scale and optimize air quality simulations using their services. These resources allow modelers to rapidly bring together CMAQ, cloud-hosted datasets, and visualization and evaluation tools on ephemeral clusters that can be deployed quickly and reliably worldwide. Described here are considerations in CMAQ version 5.3.3 cloud use and the supported resources for each CSP, presented through a benchmark application suite that was developed as an example of a typical simulation for testing and verifying components of the modeling system. The outcomes of this effort are to provide findings from performing CMAQ simulations on the cloud using popular vendor-provided resources, to enable the user community to adapt this for their own needs, and to identify specific areas of potential optimization with respect to storage and compute architectures.

Branched chain amino acids (BCAAs) are essential for protein homeostasis, energy balance, and signaling pathways. Changes in BCAA homeostasis have emerged as pivotal contributors in the pathophysiology of several cardiometabolic diseases, including type 2 diabetes, obesity, hypertension, atherosclerotic cardiovascular disease, and heart failure. In this review, we provide a detailed overview of BCAA metabolism, focus on molecular mechanisms linking disrupted BCAA homeostasis with cardiometabolic disease, summarize the evidence from observational and interventional studies investigating associations between circulating BCAAs and cardiometabolic disease, and offer valuable insights into the potential for BCAA manipulation as a novel therapeutic strategy for cardiometabolic disease.

Abstract Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare neurological disorder caused by the mutations in the DARS2 gene, which encodes the mitochondrial aspartyl-tRNA synthetase. The objective of this study was to understand the impact of DARS2 mutations on cell processes through evaluation of LBSL patient stem cell derived cerebral organoids and neurons. We generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) of seven LBSL patients and three healthy controls using an unguided protocol. Single cells from 70-day-old hCOs were subjected to SMART-seq2 sequencing and bioinformatic analysis to acquire high-resolution gene and transcript expression datasets. Global gene expression analysis demonstrated dysregulation of a number of genes involved in mRNA metabolism and splicing processes within LBSL hCOs. Importantly, there were distinct and divergent gene expression profiles based on the nature of the DARS2 mutation. At the transcript level, pervasive differential transcript usage and differential spliced exon events that are involved in protein translation and metabolism were identified in LBSL hCOs. Single-cell analysis of DARS2 (exon 3) showed that some LBSL cells exclusively express transcripts lacking exon 3, indicating that not all LBSL cells can benefit from the “leaky” nature common to splice site mutations. At the gene- and transcript-level, we uncovered that dysregulated RNA splicing, protein translation and metabolism may underlie at least some of the pathophysiological mechanisms in LBSL. To confirm hCO findings, iPSC-derived neurons (iNs) were generated by overexpressing Neurogenin 2 using lentiviral vector to study neuronal growth, splicing of DARS2 exon 3 and DARS2 protein expression. Live cell imaging revealed neuronal growth defects of LBSL iNs, which was consistent with the finding of downregulated expression of genes related to neuronal differentiation in LBSL hCOs. DARS2 protein was downregulated in iNs compared to iPSCs, caused by increased exclusion of exon 3. The scope and complexity of our data imply that DARS2 is potentially involved in transcription regulation beyond its canonical role of aminoacylation. Nevertheless, our work highlights transcript-level dysregulation as a critical, and relatively unexplored, mechanism linking genetic data with neurodegenerative disorders.

Background Genome-wide association studies for glycemic traits have identified hundreds of loci associated with these biomarkers of glucose homeostasis. Despite this success, the challenge remains to link variant associations to genes, and underlying biological pathways. Methods To identify coding variant associations which may pinpoint effector genes at both novel and previously established genome-wide association loci, we performed meta-analyses of exome-array studies for four glycemic traits: glycated hemoglobin (HbA1c, up to 144,060 participants), fasting glucose (FG, up to 129,665 participants), fasting insulin (FI, up to 104,140) and 2hr glucose post-oral glucose challenge (2hGlu, up to 57,878). In addition, we performed network and pathway analyses. Results Single-variant and gene-based association analyses identified coding variant associations at more than 60 genes, which when combined with other datasets may be useful to nominate effector genes. Network and pathway analyses identified pathways related to insulin secretion, zinc transport and fatty acid metabolism. HbA1c associations were strongly enriched in pathways related to blood cell biology. Conclusions Our results provided novel glycemic trait associations and highlighted pathways implicated in glycemic regulation. Exome-array summary statistic results are being made available to the scientific community to enable further discoveries.

Abstract Background The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases. Methods Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing. Results Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes. Conclusions This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.

Abstract Background The pathology of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and most lung cancers involves the small airway epithelium (SAE), the single continuous layer of cells lining the airways ≥ 6th generations. The basal cells (BC) are the stem/progenitor cells of the SAE, responsible for the differentiation into intermediate cells and ciliated, club and mucous cells. To facilitate the study of the biology of the human SAE in health and disease, we immortalized and characterized a normal human SAE basal cell line. Methods Small airway basal cells were purified from brushed SAE of a healthy nonsmoker donor with a characteristic normal SAE transcriptome. The BC were immortalized by retrovirus-mediated telomerase reverse transcriptase (TERT) transduction and single cell drug selection. The resulting cell line (hSABCi-NS1.1) was characterized by RNAseq, TaqMan PCR, protein immunofluorescence, differentiation capacity on an air-liquid interface (ALI) culture, transepithelial electrical resistance (TEER), airway region-associated features and response to genetic modification with SPDEF. Results The hSABCi-NS1.1 single-clone-derived cell line continued to proliferate for > 200 doubling levels and > 70 passages, continuing to maintain basal cell features (TP63+, KRT5+). When cultured on ALI, hSABCi-NS1.1 cells consistently formed tight junctions and differentiated into ciliated, club (SCGB1A1+), mucous (MUC5AC+, MUC5B+), neuroendocrine (CHGA+), ionocyte (FOXI1+) and surfactant protein positive cells (SFTPA+, SFTPB+, SFTPD+), observations confirmed by RNAseq and TaqMan PCR. Annotation enrichment analysis showed that “cilium” and “immunity” were enriched in functions of the top-1500 up-regulated genes. RNAseq reads alignment corroborated expression of CD4, CD74 and MHC-II. Compared to the large airway cell line BCi-NS1.1, differentiated of hSABCi-NS1.1 cells on ALI were enriched with small airway epithelial genes, including surfactant protein genes, LTF and small airway development relevant transcription factors NKX2–1, GATA6, SOX9, HOPX, ID2 and ETV5. Lentivirus-mediated expression of SPDEF in hSABCi-NS1.1 cells induced secretory cell metaplasia, accompanied with characteristic COPD-associated SAE secretory cell changes, including up-regulation of MSMB, CEACAM5 and down-regulation of LTF. Conclusions The immortalized hSABCi-NS1.1 cell line has diverse differentiation capacities and retains SAE features, which will be useful for understanding the biology of SAE, the pathogenesis of SAE-related diseases, and testing new pharmacologic agents.

Abstract Background Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects different end organs, including skin and brain. We and others have previously shown the importance of macrophages in the pathogenesis of cutaneous and neuropsychiatric lupus. Additionally, autoantibodies produced by autoreactive B cells are thought to play a role in both the skin and central nervous system pathologies associated with SLE. Methods We used a novel inhibitor of Bruton’s tyrosine kinase (BTK), BI-BTK-1, to target both macrophage and B cell function in the MRL-lpr/lpr murine model of SLE, and examined the effect of treatment on skin and brain disease. Results We found that treatment with BI-BTK-1 significantly attenuated the lupus associated cutaneous and neuropsychiatric disease phenotypes in MRL/lpr mice. Specifically, BI-BTK-1 treated mice had fewer macroscopic and microscopic skin lesions, reduced cutaneous cellular infiltration, and diminished inflammatory cytokine expression compared to control mice. BTK inhibition also significantly improved cognitive function, and decreased accumulation of T cells, B cells, and macrophages within the central nervous system, specifically the choroid plexus. Conclusions Directed therapies may improve the response rate in lupus-driven target organ involvement, and decrease the dangerous side effects associated with global immunosuppression. Overall, our results suggest that inhibition of BTK may be a promising therapeutic option for cutaneous and neuropsychiatric disease associated with SLE.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrotic lung disease with an irreversible decline of lung function. "Bronchiolization", characterized by ectopic appearance of airway epithelial cells in the alveolar regions, is one of the characteristic features in the IPF lung. Based on the knowledge that club cells are the major epithelial secretory cells in human small airways, and their major secretory product uteroglobin (SCGB1A1) is significantly increased in both serum and epithelial lining fluid of IPF lung, we hypothesize that human airway club cells contribute to the pathogenesis of IPF. By assessing the transcriptomes of the single cells from human lung of control donors and IPF patients, we identified two SCGB1A1+ club cell subpopulations, highly expressing MUC5B, a significant genetic risk factor strongly associated with IPF, and SCGB3A2, a marker heterogeneously expressed in the club cells, respectively. Interestingly, the cellular proportion of SCGB1A1+MUC5B+ club cells was significantly increased in IPF patients, and this club cell subpopulation highly expressed genes related to mucous production and immune cell chemotaxis. In contrast, though the cellular proportion did not change, the molecular phenotype of the SCGB1A1+SCGB3A2high club cell subpopulation was significantly altered in IPF lung, with increased expression of mucins, cytokine and extracellular matrix genes. The single cell transcriptomic analysis reveals the cellular and molecular heterogeneity of club cells, and provide novel insights into the biological functions of club cells in the pathogenesis of IPF.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrotic lung disease with an irreversible decline of lung function. “Bronchiolization”, characterized by ectopic appearance of airway epithelial cells in the alveolar regions, is one of the characteristic features in the IPF lung. Based on the knowledge that club cells are the major epithelial secretory cells in human small airways, and their major secretory product uteroglobin (SCGB1A1) is significantly increased in both serum and epithelial lining fluid of IPF lung, we hypothesize that human airway club cells contribute to the pathogenesis of IPF. By assessing the transcriptomes of the single cells from human lung of control donors and IPF patients, we identified two SCGB1A1+ club cell subpopulations, highly expressing MUC5B, a significant genetic risk factor strongly associated with IPF, and SCGB3A2, a marker heterogeneously expressed in the club cells, respectively. Interestingly, the cellular proportion of SCGB1A1+MUC5B+ club cells was significantly increased in IPF patients, and this club cell subpopulation highly expressed genes related to mucous production and immune cell chemotaxis. In contrast, though the cellular proportion did not change, the molecular phenotype of the SCGB1A1+SCGB3A2high club cell subpopulation was significantly altered in IPF lung, with increased expression of mucins, cytokine and extracellular matrix genes. The single cell transcriptomic analysis reveals the cellular and molecular heterogeneity of club cells, and provide novel insights into the biological functions of club cells in the pathogenesis of IPF.

Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD) and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE), we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10th to 12th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p1.5), with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/β-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.

The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFβ was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.

The identification and validation of biomarkers to support the assessment of novel therapeutics for COPD continues to be an important area of research. The aim of the current study was to identify systemic protein biomarkers correlated with measures of COPD severity, as well as specific protein signatures associated with comorbidities such as metabolic syndrome. 142 protein analytes were measured in serum of 140 patients with stable COPD, 15 smokers without COPD and 30 non-smoking controls. Seven analytes (sRAGE, EN-RAGE, NGAL, Fibrinogen, MPO, TGF-α and HB-EGF) showed significant differences between severe/very severe COPD, mild/moderate COPD, smoking and non-smoking control groups. Within the COPD subjects, univariate and multivariate analyses identified analytes significantly associated with FEV(1), FEV(1)/FVC and DLCO. Most notably, a set of 5 analytes (HB-EGF, Fibrinogen, MCP-4, sRAGE and Sortilin) predicted 21% of the variability in DLCO values. To determine common functions/pathways, analytes were clustered in a correlation network by similarity of expression profile. While analytes related to neutrophil function (EN-RAGE, NGAL, MPO) grouped together to form a cluster associated with FEV(1) related parameters, analytes related to the EGFR pathway (HB-EGF, TGF-α) formed another cluster associated with both DLCO and FEV(1) related parameters. Associations of Fibrinogen with DLCO and MPO with FEV(1)/FVC were stronger in patients without metabolic syndrome (r = -0.52, p = 0.005 and r = -0.61, p = 0.023, respectively) compared to patients with coexisting metabolic syndrome (r = -0.25, p = 0.47 and r = -0.15, p = 0.96, respectively), and may be driving overall associations in the general cohort. In summary, our study has identified known and novel serum protein biomarkers and has demonstrated specific associations with COPD disease severity, FEV(1), FEV(1)/FVC and DLCO. These data highlight systemic inflammatory pathways, neutrophil activation and epithelial tissue injury/repair processes as key pathways associated with COPD.

The kinetics of metabolic and inflammatory parameters associated with obesity were evaluated in a murine diet-induced obesity (DIO) model using a diet high in fat and cholesterol. Cellular infiltration and mediator production were assessed and shown to be therapeutically modulated by the PPARgamma agonist rosiglitazone. C57BL/6 mice were maintained on a 45% fat/0.12% cholesterol (HF/CH) or Chow diet for 3, 6, 16, or 27 weeks. Flow cytometry was employed to monitor peripheral blood monocytes and adipose tissue macrophages (ATM). Gene expression and protein analysis methods were used to evaluate mediator production from total epididymal fat (EF), stromal vascular fraction (SVF), and sorted SVF cells. To investigate therapeutic intervention, mice were fed a HF/CH diet for 12 weeks and then a diet formulated with rosiglitazone (5 mg/kg) for an additional 6 weeks. A HF/CH diet correlated with obesity and a dramatic proinflammatory state. Therapeutic intervention with rosiglitazone attenuated the HF/CH induced inflammation. In addition, a novel population was found that expressed the highest levels of the pro-inflammatory mediators CCL2 and IL-6.