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Over the past decade, the use of human stem cell-derived ß cells (SC-ß cells) to model pancreatic ß cell development, function and disease has become increasingly common. Though protocols are rapidly improving, current directed differentiation strategies do not yield a pure population of insulin-positive SC-ß cells in vitro. Therefore, it is experimentally advantageous to have reporter lines that allow for live sorting of insulin-positive populations. To aid in these studies, we have knocked mNeonGreen fluorescent protein into the endogenous insulin locus of the commonly used H1 (WA01) human embryonic stem cell line.

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Alpha-1 antitrypsin (AAT) is an acute-phase reactant with immunomodulatory properties that mainly inhibits neutrophil elastase. Low serum levels cause AAT deficiency (AATD), an underdiagnosed condition that predisposes to pulmonary and hepatic diseases. The SERPINA1 gene, which encodes AAT, contains more than 500 variants. PI*Z and PI*S alleles are the most diagnosed causes of AATD, but the role of the SERPINA1 haplotypes in AAT function remains unknown. SERPINA1 gene was PCR amplified from 94 asthma patients, using primers with tails for indexing. Sequencing libraries were loaded into a MinION-Mk1C, and MinKNOW was used for basecalling and demultiplexing. Nanofilt and Minimap2 were used for filtering and mapping/alignment. Variant calling/phasing were performed with PEPPER-Margin-DeepVariant. SERPINA1 gene was 100% covered for all samples, with a minimum sequencing depth of 500X. 75 single nucleotide variants and 4 indels were detected, with 45 and 2 of them highly polymorphic (MAF>0.1), respectively. Nine of the SNVs showed differences in allele frequencies when compared with the overall Spanish population. More than 90% of heterozygous SNVs were phased, yielding 91 and 58 different haplotypes for each SERPINA1 amplified region. Haplotype-based Linkage Disequilibrium (LD) analysis suggests that a recombination hotspot could generate variation in the SERPINA1 gene. The proposed workflow enables haplotype-aware genotyping of the SERPINA1 gene by nanopore sequencing, which will allow the development of novel AATD diagnostic strategies.

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The Bacillus Calmette Guerin (BCG) vaccine has been shown to induce non-specific protection against diseases other than tuberculosis in vaccinated individuals, attributed to the induction of trained immunity. We have previously demonstrated that BCG administration induces innate immune training in mixed peripheral blood mononuclear cells and monocytes in calves. Gamma Delta (γδ) T cells are non-conventional T cells that exhibit innate and adaptive immune system features. They are in higher proportion in the peripheral blood of cattle than humans or rodents and play an essential role in bovine immune response to pathogens. In the current study, we determined if BCG administration induced innate immune training in bovine γδ T cells. A group of 16 pre-weaned Holstein calves (2-4 d age) were enrolled in the study and randomly assigned to vaccine and control groups (n=8/group). The vaccine group received two doses of 106 colony forming units (CFU) BCG Danish strain subcutaneously, separated by 2 weeks. The control group remained unvaccinated. Gamma delta T cells were purified from peripheral blood using magnetic cell sorting three weeks after receiving the 1st BCG dose. We observed functional changes in the γδ T cells from BCG-treated calves shown by increased IL-6 and TNF-α cytokine production in response to in vitro stimulation with Escherichia coli LPS and PAM3CSK4. ATAC-Seq analysis of 78,278 regions of open chromatin (peaks) revealed that γδ T cells from BCG-treated calves had an altered epigenetic status compared to cells from the control calves. Differentially accessible peaks (DAP) found near the promoters of innate immunity-related genes like Siglec14, Irf4, Ifna2, Lrrfip1, and Tnfrsf10d were 1 to 4-fold more accessible in cells from BCG-treated calves. MOTIF enrichment analysis of the sequences within DAPs, which explores transcription factor binding motifs (TFBM) upstream of regulatory elements, revealed TFBM for Eomes and IRF-5 were among the most enriched transcription factors. GO enrichment analysis of genes proximal to the DAPs showed enrichment of pathways such as regulation of IL-2 production, T-cell receptor signaling pathway, and other immune regulatory pathways. In conclusion, our study shows that subcutaneous BCG administration in pre-weaned calves can induce innate immune memory in the form of trained immunity in γδ T cells. This memory is associated with increased chromatin accessibility of innate immune response-related genes, thereby inducing a functional trained immune response evidenced by increased IL-6 and TNF-α cytokine production.

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Asthma is a leading worldwide biomedical concern. Patients can experience life-threatening worsening episodes (exacerbations) usually controlled by anti-inflammatory and bronchodilator drugs. However, substantial heterogeneity in treatment response exists, and a subset of patients with unresolved asthma carry the major burden of this disease. The study of the epigenome and microbiome might bridge the gap between human genetics and environmental exposure to partially explain the heterogeneity in drug response. This review aims to provide a critical examination of the existing literature on the microbiome and epigenetic studies examining associations with asthma treatments and drug response, highlight convergent pathways, address current challenges, and offer future perspectives. Current epigenetic and microbiome studies have shown the bilateral relationship between asthma pharmacologic interventions and the human epigenome and microbiome. These studies, focusing on corticosteroids and to a lesser extent on bronchodilators, azithromycin, immunotherapy, and mepolizumab, have improved the understanding of the molecular basis of treatment response and identified promising biomarkers for drug response prediction. Immune and inflammatory pathways (eg, IL-2, TNF-α, NF-κB, and C/EBPs) underlie microbiome-epigenetic associations with asthma treatment, representing potential therapeutic pathways to be targeted. A comprehensive evaluation of these omics biomarkers could significantly contribute to precision medicine and new therapeutic target discovery.

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Seventy-two Angus-cross steers (261 ±â€…14 kg) were utilized to determine the effects of supplemental Zn sulfate on growth, trace mineral status, circulating immune cells, and functional innate immune responses. Steers were stratified by weight and implanted with a Component E-S with Tylan implant (Elanco Animal Health, Greenfield, IN) on day 0. Dietary treatments included: control (CON; no supplemental Zn), Zn100 (100 mg supplemental Zn/kg DM), and Zn150 (150 mg supplemental Zn/kg DM). Analyzed dietary concentrations of Zn were 58, 160, and 207 mg Zn/kg DM, respectively. On days 13 and 57, blood from nine steers per treatment was collected for immune analyses (cell phenotyping and response to stimulus). On day 16, implant abscesses were evaluated by palpation and visual appraisal. Sixty percent of steers had abscesses; however, there were no differences in abscess prevalence due to treatment (P = 0.67). Data were analyzed as a split-plot design using the Mixed procedure of SAS 9.4 (Cary, NC) with effects of dietary treatment, abscess, and their interaction. There was a tendency (treatment × abscess; P ≤ 0.09) for steers without abscesses to have greater average daily gain (ADG; treatment × abscess P = 0.06) and gain:feed (G:F; treatment × abscess P = 0.09) from d 14 to 27 in CON and Zn100 while within Zn150 steers without abscesses tended to have lesser ADG and G:F than abscessed steers. There were no other treatment × abscess effects for growth performance, but steers with abscesses tended to have decreased final body weight (P = 0.10) and overall G:F (days 0 to 57; P = 0.08). There was no interaction of treatment and abscess on immune cell populations on days 13 or 58 (treatment × abscess P ≥ 0.11). On day 13, Zn150 steers had increased CD45RO + gamma delta (P = 0.04) T cells. Abscessed steers had increased CD21 + B cells (P = 0.03) and tended to have increased CD21 + (P = 0.07) and CD21 + MHCIIhi (P = 0.07) B cells in circulation. This study shows zinc supplementation and implant abscesses can alter the immune system and growth performance of growing beef steers.

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Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease disrupting lung health throughout the life of an individual and that is increasing in incidence. The TGF-ß superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that TGFbr2 is critical for alveolar epithelial (AT1) cell fate maintenance and function. Loss of TGFbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analyses reveal the necessity of TGFbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGF-ß signaling regulates integrin transcription to alter AT1 cell morphology, which further impacts ECM expression through changes in mechanotransduction. These data reveal the cell intrinsic necessity of TGF-ß signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome.

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Zinc (Zn) is critical for immune function, and marginal Zn deficiency in calves can lead to suboptimal growth and increased disease susceptibility. However, in contrast to other trace minerals such as copper, tissue concentrations of Zn do not change readily in conditions of supplementation or marginal deficiency. Therefore, the evaluation of Zn status remains challenging. Zinc transporters are essential for maintaining intracellular Zn homeostasis, and their expression may indicate changes in Zn status in the animal. Here, we investigated the effects of dietary Zn supplementation on labile Zn concentration and Zn transporter gene expression in circulating immune cells isolated from feedlot steers. Eighteen Angus crossbred steers (261 ± 14 kg) were blocked by body weight and randomly assigned to two dietary treatments: a control diet (58 mg Zn/kg DM, no supplemental Zn) or control plus 150 mg Zn/kg DM (HiZn; 207 mg Zn/kg DM total). After 33 days, Zn supplementation increased labile Zn concentrations (as FluoZin-3 fluorescence) in monocytes, granulocytes, and CD4 T cells (P < 0.05) but had the opposite effect on CD8 and γδ T cells (P < 0.05). Zn transporter gene expression was analyzed on purified immune cell populations collected on days 27 or 28. ZIP11 and ZnT1 gene expression was lower (P < 0.05) in CD4 T cells from HiZn compared to controls. Expression of ZIP6 in CD8 T cells (P = 0.02) and ZnT7 in B cells (P = 0.01) was upregulated in HiZn, while ZnT9 tended (P = 0.06) to increase in B cells from HiZn. These results suggest dietary Zn concentration affects both circulating immune cell Zn concentrations and Zn transporter gene expression in healthy steers.

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Severe lung injury causes basal stem cells to migrate and outcompete alveolar stem cells resulting in dysplastic repair and a loss of gas exchange function. This "stem cell collision" is part of a multistep process that is now revealed to generate an injury-induced tissue niche (iTCH) containing Keratin 5+ epithelial cells and plastic Pdgfra+ mesenchymal cells. Temporal and spatial single cell analysis reveals that iTCHs are governed by mesenchymal proliferation and Notch signaling, which suppresses Wnt and Fgf signaling in iTCHs. Conversely, loss of Notch in iTCHs rewires alveolar signaling patterns to promote euplastic regeneration and gas exchange. The signaling patterns of iTCHs can differentially phenotype fibrotic from degenerative human lung diseases, through apposing flows of FGF and WNT signaling. These data reveal the emergence of an injury and disease associated iTCH in the lung and the ability of using iTCH specific signaling patterns to discriminate human lung disease phenotypes.

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The surge in Waste Electrical and Electronic Equipment (WEEE) generation, reaching 53.6 million metric tons (Mt) in 2019, demands efficient recycling solutions. This study focuses on the Shredder Light Fraction (SLF), a material stream derived from the mechanical pre-processing of WEEE, which is considered "municipal waste". SLF constitutes 4.2% of the output material and is rich in metals like copper, tin, lead, zinc, silver, and gold. Pyrolysis treatment was applied to SLF, enabling recyclability. Both batch and continuous setups were employed for materials flow analysis and technical evaluation of the resource potential. The research evaluates the impact of pyrolysis technology on solid fraction metal content and pyrolysis gas/oil energy potential. Scaling up the process addressed material heterogeneity and increased the reliability of the obtained results. An innovative pyrometallurgical extraction approach was suggested, to recover valuable metals in SLF which otherwise could be lost via energy recovery methods. The resulting solid product after pyrolysis showed enriched concentrations of copper, zinc, lead, and precious metals with concentrations acceptable for industrial use. Additionally, it displayed reduced mass and diminished hazardous constituents. The non-condensable gas, rich in hydrogen, carbon monoxide, and methane, exhibited potential as an alternative energy source or reducing agent in the metallurgical sector. This research advances metal recycling from SLF, offering valuable insights for environmental impact mitigation as waste was transformed into a valuable by-product for potential use in the copper industry.

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The human orthopneumovirus (human respiratory syncytial virus [RSV]) is a leading cause of respiratory disease in children worldwide and a significant cause of infant mortality in low- and middle-income countries. The natural immune response to the virus has a preponderant role in disease progression, with a rapid neutrophil infiltration and dysbalanced T cell response in the lungs associated with severe disease in infants. The development of preventive interventions against human RSV has been difficult partly due to the need to use animal models that only partially recapitulate the immune response as well as the disease progression seen in human infants. In this brief review, we discuss the contributions of the calf model of RSV infection to understanding immunity to RSV and in developing vaccine and drug candidates, focusing on recent research areas. We propose that the bovine model of RSV infection is a valuable alternative for assessing the translational potential of interventions aimed at the human population.

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AT2 cells harbor alveolar stem cell activity in the lung and can self-renew and differentiate into AT1 cells during homeostasis and after injury. To identify epigenetic pathways that control the AT2-AT1 regenerative response in the lung, we performed an organoid screen using a library of pharmacological epigenetic inhibitors. This screen identified DOT1L as a regulator of AT2 cell growth and differentiation. In vivo inactivation of Dot1l leads to precocious activation of both AT1 and AT2 gene expression during lung development and accelerated AT1 cell differentiation after acute lung injury. Single-cell transcriptome analysis reveals the presence of a new AT2 cell state upon loss of Dot1l, characterized by increased expression of oxidative phosphorylation genes and changes in expression of critical transcription and epigenetic factors. Taken together, these data demonstrate that Dot1l controls the rate of alveolar epithelial cell fate acquisition during development and regeneration after acute injury.

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BACKGROUND: The upper-airway microbiome is involved in asthma exacerbations despite inhaled corticosteroid (ICS) treatment. Although human genetics regulates microbiome composition, its influence on asthma-related airway bacteria remains unknown. OBJECTIVE: We sought to identify genes and biological pathways regulating airway-microbiome traits involved in asthma exacerbations and ICS response. METHODS: Saliva, nasal, and pharyngeal samples from 257 European patients with asthma were analyzed. The association of 6,296,951 genetic variants with exacerbation-related microbiome traits despite ICS treatment was tested through microbiome genome-wide association studies. Variants with 1 × 10-4 

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Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease increasing in incidence which disrupts lung health throughout the lifespan. The TGFß superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that Tgfbr2 is critical for AT1 cell fate maintenance and function. Loss of Tgfbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analysis reveal the necessity of Tgfbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGFß signaling regulates integrin transcription to alter AT1 cell morphology, which further impacts ECM expression through changes in mechanotransduction. These data reveal the cell intrinsic necessity of TGFß signaling in maintaining AT1 cell fate and reveal this cell lineage as a major orchestrator of the alveolar matrisome.

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INTRODUCTION AND OBJECTIVES: Asthma is a chronic inflammatory disease of the airways. Asthma patients may experience potentially life-threatening episodic flare-ups, known as exacerbations, which may significantly contribute to the asthma burden. The Pi*S and Pi*Z variants of the SERPINA1 gene, which usually involve alpha-1 antitrypsin (AAT) deficiency, had previously been associated with asthma. The link between AAT deficiency and asthma might be represented by the elastase/antielastase imbalance. However, their role in asthma exacerbations remains unknown. Our objective was to assess whether SERPINA1 genetic variants and reduced AAT protein levels are associated with asthma exacerbations. MATERIALS AND METHODS: In the discovery analysis, SERPINA1 Pi*S and Pi*Z variants and serum AAT levels were analyzed in 369 subjects from La Palma (Canary Islands, Spain). As replication, genomic data from two studies focused on 525 Spaniards and publicly available data from UK Biobank, FinnGen, and GWAS Catalog (Open Targets Genetics) were analyzed. The associations between SERPINA1 Pi*S and Pi*Z variants and AAT deficiency with asthma exacerbations were analyzed with logistic regression models, including age, sex, and genotype principal components as covariates. RESULTS: In the discovery, a significant association with asthma exacerbations was found for both Pi*S (odds ratio [OR]=2.38, 95% confidence interval [CI]= 1.40-4.04, p-value=0.001) and Pi*Z (OR=3.49, 95%CI=1.55-7.85, p-value=0.003)Likewise, AAT deficiency was associated with a higher risk for asthma exacerbations (OR=5.18, 95%CI=1.58-16.92, p-value=0.007) as well as AAT protein levels (OR= 0.72, 95%CI=0.57-0.91, p-value=0.005). The Pi*Z association with exacerbations was replicated in samples from Spaniards with two generations of Canary Islander origin (OR=3.79, p-value=0.028), and a significant association with asthma hospitalizations was found in the Finnish population (OR=1.12, p-value=0.007). CONCLUSIONS: AAT deficiency could be a potential therapeutic target for asthma exacerbations in specific populations.

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Alveolar epithelial type 2 (AT2) cells harbor the facultative progenitor capacity in the lung alveolus to drive regeneration after lung injury. Using single-cell transcriptomics, software-guided segmentation of tissue damage, and in vivo mouse lineage tracing, we identified the grainyhead transcription factor cellular promoter 2-like 1 (Tfcp2l1) as a regulator of this regenerative process. Tfcp2l1 loss in adult AT2 cells inhibits self-renewal and enhances AT2-AT1 differentiation during tissue regeneration. Conversely, Tfcp2l1 blunts the proliferative response to inflammatory signaling during the early acute injury phase. Tfcp2l1 temporally regulates AT2 self-renewal and differentiation in alveolar regions undergoing active regeneration. Single-cell transcriptomics and lineage tracing reveal that Tfcp2l1 regulates cell fate dynamics across the AT2-AT1 differentiation and restricts the inflammatory program in murine AT2 cells. Organoid modeling shows that Tfcp2l1 regulation of interleukin-1 (IL-1) receptor expression controlled these cell fate dynamics. These findings highlight the critical role Tfcp2l1 plays in balancing epithelial cell self-renewal and differentiation during alveolar regeneration.

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Lungs undergo mechanical strain during breathing, but how these biophysical forces affect cell fate and tissue homeostasis are unclear. We show that biophysical forces through normal respiratory motion actively maintain alveolar type 1 (AT1) cell identity and restrict these cells from reprogramming into AT2 cells in the adult lung. AT1 cell fate is maintained at homeostasis by Cdc42- and Ptk2-mediated actin remodeling and cytoskeletal strain, and inactivation of these pathways causes a rapid reprogramming into the AT2 cell fate. This plasticity induces chromatin reorganization and changes in nuclear lamina-chromatin interactions, which can discriminate AT1 and AT2 cell identity. Unloading the biophysical forces of breathing movements leads to AT1-AT2 cell reprogramming, revealing that normal respiration is essential to maintain alveolar epithelial cell fate. These data demonstrate the integral function of mechanotransduction in maintaining lung cell fate and identifies the AT1 cell as an important mechanosensor in the alveolar niche.

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BACKGROUND: Albuterol is the drug most widely used as asthma treatment among African Americans despite having a lower bronchodilator drug response (BDR) than other populations. Although BDR is affected by gene and environmental factors, the influence of DNA methylation is unknown. OBJECTIVE: This study aimed to identify epigenetic markers in whole blood associated with BDR, study their functional consequences by multi-omic integration, and assess their clinical applicability in admixed populations with a high asthma burden. METHODS: We studied 414 children and young adults (8-21 years old) with asthma in a discovery and replication design. We performed an epigenome-wide association study on 221 African Americans and replicated the results on 193 Latinos. Functional consequences were assessed by integrating epigenomics with genomics, transcriptomics, and environmental exposure data. Machine learning was used to develop a panel of epigenetic markers to classify treatment response. RESULTS: We identified 5 differentially methylated regions and 2 CpGs genome-wide significantly associated with BDR in African Americans located in FGL2 (cg08241295, P = 6.8 × 10-9) and DNASE2 (cg15341340, P = 7.8 × 10-8), which were regulated by genetic variation and/or associated with gene expression of nearby genes (false discovery rate < 0.05). The CpG cg15341340 was replicated in Latinos (P = 3.5 × 10-3). Moreover, a panel of 70 CpGs showed good classification for those with response and nonresponse to albuterol therapy in African American and Latino children (area under the receiver operating characteristic curve for training, 0.99; for validation, 0.70-0.71). The DNA methylation model showed similar discrimination as clinical predictors (P > .05). CONCLUSIONS: We report novel associations of epigenetic markers with BDR in pediatric asthma and demonstrate for the first time the applicability of pharmacoepigenetics in precision medicine of respiratory diseases.

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