Dietary Zinc Supplementation in Steers Modulates Labile Zinc Concentration and Zinc Transporter Gene Expression in Circulating Immune Cells.

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.

An injury-induced tissue niche shaped by mesenchymal plasticity coordinates the regenerative and disease response in the lung.

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.

TGF-ß controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription in mice.

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.

Enabling the recycling of metals from the shredder light fraction derived from waste of electrical and electronic equipment via continuous pyrolysis process.

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.

Modeling Human Respiratory Syncytial Virus (RSV) Infection: Recent Contributions and Future Directions Using the Calf Model of Bovine RSV Disease.

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.

DOT1L regulates lung developmental epithelial cell fate and adult alveolar stem cell differentiation after acute injury.

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.

Human genetics influences microbiome composition involved in asthma exacerbations despite inhaled corticosteroid treatment.

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 

TGFß controls alveolar type 1 epithelial cell plasticity and alveolar matrisome gene transcription.

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.

Alpha-1 antitrypsin deficiency and Pi*S and Pi*Z SERPINA1 variants are associated with asthma exacerbations.

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.

Temporal and spatial staging of lung alveolar regeneration is determined by the grainyhead transcription factor Tfcp2l1.

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.

Biophysical forces mediated by respiration maintain lung alveolar epithelial cell fate.

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.

Multi-omic approach associates blood methylome with bronchodilator drug response in pediatric asthma.

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.

Admixture mapping of severe asthma exacerbations in Hispanic/Latino children and youth.

BACKGROUND: In the USA, genetically admixed populations have the highest asthma prevalence and severe asthma exacerbations rates. This could be explained not only by environmental factors but also by genetic variants that exert ethnic-specific effects. However, no admixture mapping has been performed for severe asthma exacerbations. OBJECTIVE: We sought to identify genetic variants associated with severe asthma exacerbations in Hispanic/Latino subgroups by means of admixture mapping analyses and fine mapping, and to assess their transferability to other populations and potential functional roles. METHODS: We performed an admixture mapping in 1124 Puerto Rican and 625 Mexican American children with asthma. Fine-mapping of the significant peaks was performed via allelic testing of common and rare variants. We performed replication across Hispanic/Latino subgroups, and the transferability to non-Hispanic/Latino populations was assessed in 1001 African Americans, 1250 Singaporeans and 941 Europeans with asthma. The effects of the variants on gene expression and DNA methylation from whole blood were also evaluated in participants with asthma and in silico with data obtained through public databases. RESULTS: Genomewide significant associations of Indigenous American ancestry with severe asthma exacerbations were found at 5q32 in Mexican Americans as well as at 13q13-q13.2 and 3p13 in Puerto Ricans. The single nucleotide polymorphism (SNP) rs1144986 (C5orf46) showed consistent effects for severe asthma exacerbations across Hispanic/Latino subgroups, but it was not validated in non-Hispanics/Latinos. This SNP was associated with DPYSL3 DNA methylation and SCGB3A2 gene expression levels. CONCLUSIONS: Admixture mapping study of asthma exacerbations revealed a novel locus that exhibited Hispanic/Latino-specific effects and regulated DPYSL3 and SCGB3A2.

Effect of bovine oocyte vitrification with EGTA and post-warming recovery with resveratrol on meiotic spindle, mitochondrial function, reactive oxygen species, and developmental competence.

The present study aimed to determine the effects of the addition of EGTA to vitrification solutions and a post-warming recovery period supplemented with 1 µM resveratrol on meiotic spindle integrity, mitochondrial activity, ATP content, reactive oxygen species (ROS) levels, and developmental potential of partially denuded, vitrified-warmed bovine oocytes. Results of microtubule distribution and chromosomal arrangement indicated that resveratrol supplementation, irrespective to EGTA addition, reduced the incidence of abnormal meiotic spindles to similar levels of the control group. Mitochondrial membrane potential was similar in all groups, but ATP content was negatively affected by the vitrification-warming procedure and failed to recover after 4 h of post-warming culture. Resveratrol caused the reduction of ROS to lower levels of the control group, and showed the lowest ROS levels when combined with EGTA treatment. Oocytes in all vitrification groups presented lower developmental potential when compared to fresh oocytes. However, oocytes that underwent vitrification supplemented with EGTA and post-warming culture along with resveratrol showed higher developmental competence compared with vitrified-warmed oocytes not supplemented with resveratrol. The results of our study indicate that submitting vitrified-warmed, partially denuded bovine oocytes to a post-warming recovery period supplemented with 1 µM resveratrol improves vitrification outcomes. However, the benefits of EGTA on vitrification and warming of bovine oocytes need to be further investigated.

The upper-airway microbiome as a biomarker of asthma exacerbations despite inhaled corticosteroid treatment.

BACKGROUND: The response to inhaled corticosteroids (ICS) in asthma is affected by the interplay of several factors. Among these, the role of the upper-airway microbiome has been scarcely investigated. We aimed to evaluate the association between the salivary, pharyngeal, and nasal microbiome with asthma exacerbations despite receipt of ICS. METHODS: Samples from 250 asthma patients from the Genomics and Metagenomics of Asthma Severity (GEMAS) study treated with ICS were analyzed. Control/case subjects were defined by the absence/presence of asthma exacerbations in the past 6 months despite being treated with ICS. The bacterial microbiota was profiled by sequencing the V3-V4 region of the 16S rRNA gene. Differences between groups were assessed by PERMANOVA and regression models adjusted for potential confounders. A false discovery rate (FDR) of 5% was used to correct for multiple comparisons. Classification models of asthma exacerbations despite ICS treatment were built with machine learning approaches based on clinical, genetic, and microbiome data. RESULTS: In nasal and saliva samples, case subjects had lower bacterial diversity (Richness, Shannon, and Faith indices) than control subjects (.007 ≤ P ≤ .037). Asthma exacerbations accounted for 8% to 9% of the interindividual variation of the salivary and nasal microbiomes (.003 ≤ P ≤ .046). Three, 4, and 11 bacterial genera from the salivary, pharyngeal, and nasal microbiomes were differentially abundant between groups (4.09 × 10-12 ≤ FDR ≤ 0.047). Integrating clinical, genetic, and microbiome data showed good discrimination for the development of asthma exacerbations despite receipt of ICS (AUCtraining: 0.82 and AUCvalidation: 0.77). CONCLUSION: The diversity and composition of the upper-airway microbiome are associated with asthma exacerbations despite ICS treatment. The salivary microbiome has a potential application as a biomarker of asthma exacerbations despite receipt of ICS.

SARS-CoV-2 disrupts host epigenetic regulation via histone mimicry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the devastating global pandemic of coronavirus disease 2019 (COVID-19), in part because of its ability to effectively suppress host cell responses1-3. In rare cases, viral proteins dampen antiviral responses by mimicking critical regions of human histone proteins4-8, particularly those containing post-translational modifications required for transcriptional regulation9-11. Recent work has demonstrated that SARS-CoV-2 markedly disrupts host cell epigenetic regulation12-14. However, how SARS-CoV-2 controls the host cell epigenome and whether it uses histone mimicry to do so remain unclear. Here we show that the SARS-CoV-2 protein encoded by ORF8 (ORF8) functions as a histone mimic of the ARKS motifs in histone H3 to disrupt host cell epigenetic regulation. ORF8 is associated with chromatin, disrupts regulation of critical histone post-translational modifications and promotes chromatin compaction. Deletion of either the ORF8 gene or the histone mimic site attenuates the ability of SARS-CoV-2 to disrupt host cell chromatin, affects the transcriptional response to infection and attenuates viral genome copy number. These findings demonstrate a new function of ORF8 and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Further, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.

Recovery of spindle morphology and mitochondrial function through extended culture after vitrification-warming of bovine oocytes.

The present study aimed to determine the effects of vitrification on the meiotic spindle and mitochondrial function of bovine oocytes submitted to different times of post-warming culture. Partially denuded cumulus-oocyte complexes were vitrified at different maturation times (18-, 20-, and 24-h) using a two-step cryoprotectant addition protocol and submitted to 6-, 4-, or 0-h of post-warming extended culture in maturation medium. Microtubule configuration and chromosomal arrangement were analyzed after 0- and 6-h of extended culture, whereas mitochondrial membrane potential and ATP content were measured at 0-, 4-, and 6-h of post-warming recovery. Results of meiotic spindle integrity revealed that vitrified-warmed oocytes that underwent 6-h of culture had similar incidence of normal microtubule configuration and chromosomal arrangement as compared to fresh oocytes, but higher than oocytes in the vitrification control group (no culture). Mitochondrial membrane potential was not different in all the vitrification groups, but the oocytes that were cultured for 4-h after warming had similar levels compared to fresh oocytes. ATP concentration in all vitrification groups was lower than the control group. However, oocytes cultured for 6-h had the lowest rate of ATP depleted oocytes among the vitrification groups. The results of this study indicate that extended culture after warming promotes the recovery of the meiotic spindle and, to some extent, mitochondrial function of vitrified-warmed metaphase II bovine oocytes.