The use of a minimally invasive integrated endoscopic system to perform hemilaminectomies in chondrodystrophic dogs with thoracolumbar intervertebral disc extrusions.

Introduction: The objective was to evaluate the use of a minimally invasive surgical (MIS) approach to perform hemilaminectomies in chondrodystrophic dogs with thoracolumbar intervertebral disc extrusions (IVDE). Additionally, we aimed to evaluate the degree of soft tissue trauma using the endoscopic procedure compared to the standard open approach. Methods: Eight client-owned dogs presented to the Colorado State University Veterinary Teaching Hospital with acute onset thoracolumbar IVDE were included in this study. This was a prospective, randomized case-series. Patients were assigned to undergo an endoscopic (group 1; n = 4) or a standard open approach (group 2; n = 4) for a hemilaminectomy. A post-operative MRI was performed in all cases. Results: Conversion to an open approach was not necessary for any case in group 1. All cases had adequate spinal cord decompression on post-operative MRI. There was no significant difference in soft tissue changes noted on post-operative MRI between the two groups. Discussion: The MIS approach to hemilaminectomies in chondrodystrophic dogs with thoracolumbar IVDE can successfully be performed to decompress the neural tissue and appears to lead to similar clinical outcomes in the early postoperative period compared to the standard open approach. Larger studies are needed to determine the potential advantages of the MIS technique compared to the standard open approach in veterinary medicine.

Constrictive physiology is not present in all dogs with idiopathic chylothorax.

OBJECTIVE: To determine whether subtotal pericardectomy affects recurrence and long-term outcomes in dogs with idiopathic chylothorax (IC). ANIMALS: 12 client-owned dogs diagnosed with IC between July 26, 2016, and March 23, 2023. METHODS: The diagnosis of constrictive physiology (CP) was established with cardiac catheterization and defined as elevated and equal diastolic pressures in all 4 cardiac chambers. Dogs were then entered into the constrictive physiology (CP) group or non-CP (NCP) group. All dogs received at least a thoracic duct ligation (TDL). The dogs in the CP group had a subtotal pericardectomy performed in addition to TDL. Repeated surgical interventions, recurrence, long-term outcomes, and survival times were recorded. RESULTS: 8 dogs were entered into the CP group and underwent TDL and subtotal pericardectomy. Four dogs were entered in the NCP group and underwent only a TDL. Four dogs in the CP group and 1 in the NCP group required multiple surgeries for recurrent chylothorax. The 1-, 2-, and 3-year disease-free rates were, respectively, 100%, 100%, and 50% for the NCP group and 87.5%, 72.9%, and 72.9% for the CP group (P = .935). The 1-, 2-, and 3-year survival rates were, respectively, 100%, 100%, and 100% for the NCP group and 87.5%, 72.9%, and 72.9% for the CP group (P = .317). CLINICAL RELEVANCE: Constrictive physiology should be evaluated by cardiac catheterization before surgical treatment of IC in dogs. If CP is not diagnosed, subtotal pericardectomy may not be required.

Dedifferentiated early postnatal lung myofibroblasts redifferentiate in adult disease.

Alveolarization ensures sufficient lung surface area for gas exchange, and during bulk alveolarization in mice (postnatal day [P] 4.5-14.5), alpha-smooth muscle actin (SMA)+ myofibroblasts accumulate, secrete elastin, and lay down alveolar septum. Herein, we delineate the dynamics of the lineage of early postnatal SMA+ myofibroblasts during and after bulk alveolarization and in response to lung injury. SMA+ lung myofibroblasts first appear at ∼ P2.5 and proliferate robustly. Lineage tracing shows that, at P14.5 and over the next few days, the vast majority of SMA+ myofibroblasts downregulate smooth muscle cell markers and undergo apoptosis. Of note, ∼8% of these dedifferentiated cells and another ∼1% of SMA+ myofibroblasts persist to adulthood. Single cell RNA sequencing analysis of the persistent SMA- cells and SMA+ myofibroblasts in the adult lung reveals distinct gene expression profiles. For instance, dedifferentiated SMA- cells exhibit higher levels of tissue remodeling genes. Most interestingly, these dedifferentiated early postnatal myofibroblasts re-express SMA upon exposure of the adult lung to hypoxia or the pro-fibrotic drug bleomycin. However, unlike during alveolarization, these cells that re-express SMA do not proliferate with hypoxia. In sum, dedifferentiated early postnatal myofibroblasts are a previously undescribed cell type in the adult lung and redifferentiate in response to injury.

Dendritic Cell - Fibroblast Crosstalk via TLR9 and AHR Signaling Drives Lung Fibrogenesis.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring and loss of lung function. With limited treatment options, patients succumb to the disease within 2-5 years. The molecular pathogenesis of IPF regarding the immunologic changes that occur is poorly understood. We characterize a role for non-canonical aryl-hydrocarbon receptor signaling (ncAHR) in dendritic cells (DCs) that leads to production of IL-6 and IL-17, promoting fibrosis. TLR9 signaling in myofibroblasts is shown to regulate production of TDO2 which converts tryptophan into the endogenous AHR ligand kynurenine. Mice with augmented ncAHR signaling were created by crossing floxed AHR exon-2 deletion mice (AHR Δex2 ) with mice harboring a CD11c-Cre. Bleomycin was used to study fibrotic pathogenesis. Isolated CD11c+ cells and primary fibroblasts were treated ex-vivo with relevant TLR agonists and AHR modulating compounds to study how AHR signaling influenced inflammatory cytokine production. Human datasets were also interrogated. Inhibition of all AHR signaling rescued fibrosis, however, AHR Δex2 mice treated with bleomycin developed more fibrosis and DCs from these mice were hyperinflammatory and profibrotic upon adoptive transfer. Treatment of fibrotic fibroblasts with TLR9 agonist increased expression of TDO2. Study of human samples corroborate the relevance of these findings in IPF patients. We also, for the first time, identify that AHR exon-2 floxed mice retain capacity for ncAHR signaling.

Surgical interventions and outcome in a population of canine trauma patients.

OBJECTIVE: To determine signalment, injury type, trauma severity score, and outcome of canine trauma patients undergoing surgical (emergency room [ER] or operating room [OR]) and nonsurgical treatment in addition to time to surgery, specialty services involved, and cost in the OR surgery population. DESIGN: Retrospective evaluation of medical record and hospital trauma registry data on canine trauma cases. SETTING: University teaching hospital. ANIMALS: One thousand six hundred and thirty dogs presenting for traumatic injury between May 2017 and July 2020. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Demographics and outcome were compared for canine trauma patients undergoing OR surgery (12.8%, 208/1630), ER surgery (39.1%, 637/1630), or no surgical intervention (48.2%, 785/1630). Among the 2 surgical groups, 98.9% (836/845) survived to discharge compared with 92.2% (724/785) of the nonsurgical group (P < 0.0001). The OR surgical group had significantly higher median Animal Trauma Triage scores (2 vs 1, P < 0.0001) and median days in hospital (2 vs < 1, P < 0.0001) compared with the other groups. For the OR surgical cohort, electronic medical records were reviewed to determine the specialty surgery service involved, time to and duration of anesthesia and surgery, and visit cost. The most common surgery services involved were orthopedics (45.2%, 94/208) and general surgery (26.9%, 56/208). Neurology and general surgery cases required the longest median length of stay in hospital, and ophthalmology and dentistry cases required the shortest. The median cost of visit was highest in neurology ($10,032) and lowest in ophthalmology ($2305) and dentistry ($2404). CONCLUSIONS: Surgical intervention in canine trauma patients appears to be associated with higher survival rates, and among the surgery groups, mortality was highest in the ER and general surgery groups. OR surgical intervention, in particular general surgery and neurology, was associated with increased length of hospitalization, increased cost, and higher Animal Trauma Triage scores.

Organ Boundary Circuits Regulate Sox9+ Alveolar Tuft Cells During Post-Pneumonectomy Lung Regeneration.

Tissue homeostasis is controlled by cellular circuits governing cell growth, organization, and differentation. In this study we identify previously undescribed cell-to-cell communication that mediates information flow from mechanosensitive pleural mesothelial cells to alveolar-resident stem-like tuft cells in the lung. We find mesothelial cells to express a combination of mechanotransduction genes and lineage-restricted ligands which makes them uniquely capable of responding to tissue tension and producing paracrine cues acting on parenchymal populations. In parallel, we describe a large population of stem-like alveolar tuft cells that express the endodermal stem cell markers Sox9 and Lgr5 and a receptor profile making them uniquely sensitive to cues produced by pleural Mesothelium. We hypothesized that crosstalk from mesothelial cells to alveolar tuft cells might be central to the regulation of post-penumonectomy lung regeneration. Following pneumonectomy, we find that mesothelial cells display radically altered phenotype and ligand expression, in a pattern that closely tracks with parenchymal epithelial proliferation and alveolar tissue growth. During an initial pro-inflammatory stage of tissue regeneration, Mesothelium promotes epithelial proliferation via WNT ligand secretion, orchestrates an increase in microvascular permeability, and encourages immune extravasation via chemokine secretion. This stage is followed first by a tissue remodeling period, characterized by angiogenesis and BMP pathway sensitization, and then a stable return to homeostasis. Coupled with key changes in parenchymal structure and matrix production, the cumulative effect is a now larger organ including newly-grown, fully-functional tissue parenchyma. This study paints Mesothelial cells as a key orchestrating cell type that defines the boundary of the lung and exerts critical influence over the tissue-level signaling state regulating resident stem cell populations. The cellular circuits unearthed here suggest that human lung regeneration might be inducible through well-engineered approaches targeting the induction of tissue regeneration and safe return to homeostasis.

Mast-cell expressed membrane protein-1 is expressed in classical monocytes and alveolar macrophages in idiopathic pulmonary fibrosis and regulates cell chemotaxis, adhesion, and migration in a TGFß-dependent manner.

Mast-cell expressed membrane protein-1 (MCEMP1) is higher in patients with idiopathic pulmonary fibrosis (IPF) with an increased risk of death. Here we aimed to establish the mechanistic role of MCEMP1 in pulmonary fibrosis. We identified increased MCEMP1 expression in classical monocytes and alveolar macrophages in IPF compared with controls. MCEMP1 is upregulated by transforming growth factor beta (TGFß) at the mRNA and protein levels in monocytic leukemia THP-1 cells. TGFß-mediated MCEMP1 upregulation results from the cooperation of SMAD3 and SP1 via concomitant binding to SMAD3/SP1 cis-regulatory elements within the MCEMP1 promoter. We also found that MCEMP1 regulates TGFß-mediated monocyte chemotaxis, adhesion, and migration. Our results suggest that MCEMP1 may regulate the migration and transition of monocytes to monocyte-derived alveolar macrophages during pulmonary fibrosis development and progression.NEW & NOTEWORTHY MCEMP1 is highly expressed in circulating classical monocytes and alveolar macrophages in IPF, is regulated by TGFß, and participates in the chemotaxis, adhesion, and migration of circulating monocytes by modulating the effect of TGFß in RHO activity.

Single-cell transcriptomic analysis of human pleura reveals stromal heterogeneity and informs in vitro models of mesothelioma.

The pleural lining of the thorax regulates local immunity, inflammation and repair. A variety of conditions, both benign and malignant, including pleural mesothelioma, can affect this tissue. A lack of knowledge concerning the mesothelial and stromal cells comprising the pleura has hampered the development of targeted therapies. Here, we present the first comprehensive single-cell transcriptomic atlas of the human parietal pleura and demonstrate its utility in elucidating pleural biology. We confirm the presence of known universal fibroblasts and describe novel, potentially pleural-specific, fibroblast subtypes. We also present transcriptomic characterisation of multiple in vitro models of benign and malignant mesothelial cells, and characterise these through comparison with in vivo transcriptomic data. While bulk pleural transcriptomes have been reported previously, this is the first study to provide resolution at the single-cell level. We expect our pleural cell atlas will prove invaluable to those studying pleural biology and disease. It has already enabled us to shed light on the transdifferentiation of mesothelial cells, allowing us to develop a simple method for prolonging mesothelial cell differentiation in vitro.

Increased expression of CXCL6 in secretory cells drives fibroblast collagen synthesis and is associated with increased mortality in idiopathic pulmonary fibrosis.

RATIONALE: Recent data suggest that the localisation of airway epithelial cells in the distal lung in idiopathic pulmonary fibrosis (IPF) may drive pathology. We set out to discover whether chemokines expressed in these ectopic airway epithelial cells may contribute to the pathogenesis of IPF. METHODS: We analysed whole lung and single-cell transcriptomic data obtained from patients with IPF. In addition, we measured chemokine levels in blood, bronchoalveolar lavage (BAL) of IPF patients and air-liquid interface cultures. We employed ex vivo donor and IPF lung fibroblasts and an animal model of pulmonary fibrosis to test the effects of chemokine signalling on fibroblast function. RESULTS: By analysis of whole-lung transcriptomics, protein and BAL, we discovered that CXCL6 (a member of the interleukin-8 family) was increased in patients with IPF. Elevated CXCL6 levels in the BAL of two cohorts of patients with IPF were associated with poor survival (hazard ratio of death or progression 1.89, 95% CI 1.16-3.08; n=179, p=0.01). By immunostaining and single-cell RNA sequencing, CXCL6 was detected in secretory cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased collagen I levels in donor and IPF fibroblasts 4.4-fold and 1.7-fold, respectively. Both silencing of and chemical inhibition of CXCR1/2 blocked the effects of CXCL6 on collagen, while overexpression of CXCR2 increased collagen I levels 4.5-fold in IPF fibroblasts. CONCLUSIONS: CXCL6 is expressed in ectopic airway epithelial cells. Elevated levels of CXCL6 are associated with IPF mortality. CXCL6-driven collagen synthesis represents a functional consequence of ectopic localisation of airway epithelial cells in IPF.

Posterior Tibiotalar Impingement After Pediatric Ankle Fracture Surgical Fixation: A Case Report.

CASE: A 13-year-old healthy, nearly skeletally mature, female patient presented to an outpatient clinic after sustaining a bimalleolar ankle fracture-dislocation, which was subsequently treated with open reduction and internal fixation and casting. Postoperatively, the patient had significant limitations to ankle range of motion. Imaging revealed posterior tibiotalar impingement. The patient underwent arthroscopic debridement and osteoplasty, and she was able to return to previous levels of activity. CONCLUSIONS: Complications from pediatric ankle fractures are rare, so further diagnostic workup is warranted for patients with persistent pain and limitations.

Circulating Mitochondrial DNA Is Associated With High Levels of Fatigue in Two Independent Sarcoidosis Cohorts.

BACKGROUND: Patients with sarcoidosis who develop severe clinical phenotypes of pulmonary fibrosis or multiorgan disease experience debilitating symptoms, with fatigue being a common chief complaint. Studies that have investigated this patient-related outcome measure (PROM) have used the Fatigue Assessment Scale (FAS), a self-reported questionnaire that reflects mental and physical domains. Despite extensive work, its cause is unknown and treatment options remain limited. Previously, we showed that the plasma of patients with sarcoidosis with extrapulmonary disease endorsing fatigue was enriched for mitochondrial DNA (mtDNA), a ligand for the innate immune receptor toll-like receptor 9 (TLR9). Through our cross-disciplinary platform, we investigated a relationship between sarcoidosis-induced fatigue and circulating mtDNA. RESEARCH QUESTION: Is there a psychobiologic mechanism that connects sarcoidosis-induced fatigue and mtDNA-mediated TLR9 activation? STUDY DESIGN AND METHODS: Using a local cohort of patients at Yale (discovery cohort) and the National Institutes of Health-sponsored Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis study (validation cohort), we scored the FAS and quantified in the plasma, mtDNA concentrations, TLR9 activation, and cytokine levels. RESULTS: Although FAS scores were independent of corticosteroid use and Scadding stage, we observed a robust association between FAS scores, which included mental and physical domains, and multiorgan sarcoidosis. Subsequently, we identified a significant correlation between plasma mtDNA concentrations and all domains of fatigue. Additionally, we found that TLR9 activation is associated with all aspects of the FAS and partially mediates this PROM through mtDNA. Last, we found that TLR9-associated soluble mediators in the plasma are independent of all facets of fatigue. INTERPRETATION: Through our cross-disciplinary translational platform, we identified a previously unrecognized psychobiologic connection between sarcoidosis-induced fatigue and circulating mtDNA concentrations. Mechanistic work that investigates the contribution of mtDNA-mediated innate immune activation in this PROM and clinical studies with prospective cohorts has the potential to catalyze novel therapeutic strategies for this patient population and those with similar conditions.

Viral and host mediators of non-suppressible HIV-1 viremia.

Non-suppressible HIV-1 viremia (NSV) is defined as persistent low-level viremia on antiretroviral therapy (ART) without evidence of ART non-adherence or significant drug resistance. Unraveling the mechanisms behind NSV would broaden our understanding of HIV-1 persistence. Here we analyzed plasma virus sequences in eight ART-treated individuals with NSV (88% male) and show that they are composed of large clones without evidence of viral evolution over time in those with longitudinal samples. We defined proviruses that match plasma HIV-1 RNA sequences as 'producer proviruses', and those that did not as 'non-producer proviruses'. Non-suppressible viremia arose from expanded clones of producer proviruses that were significantly larger than the genome-intact proviral reservoir of ART-suppressed individuals. Integration sites of producer proviruses were enriched in proximity to the activating H3K36me3 epigenetic mark. CD4+ T cells from participants with NSV demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, participants with NSV showed significantly lower HIV-specific CD8+ T cell responses compared with untreated viremic controllers with similar viral loads. We identified potential critical host and viral mediators of NSV that may represent targets to disrupt HIV-1 persistence.

Peripheral Blood Single-Cell Sequencing Uncovers Common and Specific Immune Aberrations in Fibrotic Lung Diseases.

Rationale and Objectives: The extent and commonality of peripheral blood immune aberrations in fibrotic interstitial lung diseases are not well characterized. In this study, we aimed to identify common and distinct immune aberrations in patients with idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (FHP) using cutting-edge single-cell profiling technologies. Methods: Single-cell RNA sequencing was performed on patients and healthy controls' peripheral blood and bronchoalveolar lavage samples using 10X Genomics 5' gene expression and V(D)J profiling. Cell type composition, transcriptional profiles, cellular trajectories and signaling, and T and B cell receptor repertoires were studied. The standard Seurat R pipeline was followed for cell type composition and differential gene expression analyses. Transcription factor activity was imputed using the DoRothEA-VIPER algorithm. Pseudotime analyses were conducted using Monocle3, while RNA velocity analyses were performed with Velocyto, scVelo, and CellRank. Cell-cell connectomics were assessed using the Connectome R package. V(D)J analyses were conducted using CellRanger and Immcantation frameworks. Across all analyses, disease group differences were assessed using the Wilcoxon rank-sum test. Measurements and Main Results: 327,990 cells from 83 samples were profiled. Overall, changes in monocytes were common to IPF and FHP, whereas lymphocytes exhibited disease-specific aberrations. Both diseases displayed enrichment of CCL3 hi /CCL4 hi CD14+ monocytes (p<2.2e-16) and S100A hi CD14+ monocytes (p<2.2e-16) versus controls. Trajectory and RNA velocity analysis suggested that pro-fibrotic macrophages observed in BAL originated from peripheral blood monocytes. Lymphocytes exhibited disease-specific aberrations, with CD8+ GZMK hi T cells and activated B cells primarily enriched in FHP patients. V(D)J analyses revealed unique T and B cell receptor complementarity-determining region 3 (CDR3) amino acid compositions (p<0.05) in FHP and significant IgA enrichment in IPF (p<5.2e-7). Conclusions: We identified common and disease-specific immune mechanisms in IPF and FHP; S100A hi monocytes and SPP1 hi macrophages are common to IPF and FHP, whereas GMZK hi T lymphocytes and T and B cell receptor repertoires were unique in FHP. Our findings open novel strategies for the diagnosis and treatment of IPF and FHP.

Mast-Cell Expressed Membrane Protein-1 (MCEMP1) is expressed in classical monocytes and alveolar macrophages in Idiopathic Pulmonary Fibrosis and regulates cell chemotaxis, adhesion, and migration in a TGFß dependent manner.

Background: Mast-Cell Expressed Membrane Protein-1 (MCEMP1) is higher in Idiopathic Pulmonary Fibrosis (IPF) patients with increased risk of death and poor outcomes. Here we seek to establish the mechanistic role of MCEMP1 in pulmonary fibrosis. Methods: MCEMP1 expression was analyzed by single-cell RNA sequencing, immunofluorescence in Peripheral Blood Mononuclear Cells (PBMC) as well as in lung tissues from IPF patients and controls. Chromatin Immunoprecipitation (ChiP) and Proximity Ligation Assay (PLA) were used to study the transcriptional regulation of MCEMP1 . Transient RNA interference and lentivirus transduction were used to knockdown and knock-in MCEMP1 in THP-1 cells to study chemotaxis, adhesion, and migration. Bulk RNA sequencing was used to identify the mechanisms by which MCEMP1 participates in monocyte function. Active RHO pull-down assay was used to validate bulk RNA sequencing results. Results: We identified increased MCEMP1 expression in classical monocytes and alveolar macrophages in IPF compared to controls. MCEMP1 was upregulated by TGFß at the mRNA and protein levels in THP-1. TGFß-mediated MCEMP1 upregulation results from the cooperation of SMAD3 and SP1 via concomitant binding to SMAD3/SP1 cis -regulatory elements within the MCEMP1 promoter. In terms of its function, we found that MCEMP1 regulates TGFß-mediated monocyte chemotaxis, adhesion, and migration. 400 differentially expressed genes were found to increase after TGFß stimulation of THP-1, further increased in MCEMP1 knock-in cells treated with TGFß and decreased in MCEMP1 knockdown cells treated with TGFß. GO annotation analysis of these genes showed enrichment for positive regulation of RHO GTPase activity and signal transduction. While TGFß enhanced RHO GTPase activity in THP-1 cells, this effect was attenuated following MCEMP1 knockdown. Conclusion: MCEMP1 is highly expressed in circulating classical monocytes and alveolar macrophages in IPF. MCEMP1 is regulated by TGFß and participates in the chemotaxis, adhesion, and migration of circulating monocytes by modulating the effect of TGFß in RHO activity. Our results suggest that MCEMP1 may regulate the migration and transition of monocytes to monocyte-derived alveolar macrophages during pulmonary fibrosis development and progression.

A hybrid machine learning and regression method for cell type deconvolution of spatial barcoding-based transcriptomic data.

Spatial barcoding-based transcriptomic (ST) data require cell type deconvolution for cellular-level downstream analysis. Here we present SDePER, a hybrid machine learning and regression method, to deconvolve ST data using reference single-cell RNA sequencing (scRNA-seq) data. SDePER uses a machine learning approach to remove the systematic difference between ST and scRNA-seq data (platform effects) explicitly and efficiently to ensure the linear relationship between ST data and cell type-specific expression profile. It also considers sparsity of cell types per capture spot and across-spots spatial correlation in cell type compositions. Based on the estimated cell type proportions, SDePER imputes cell type compositions and gene expression at unmeasured locations in a tissue map with enhanced resolution. Applications to coarse-grained simulated data and four real datasets showed that SDePER achieved more accurate and robust results than existing methods, suggesting the importance of considering platform effects, sparsity and spatial correlation in cell type deconvolution.

iDESC: identifying differential expression in single-cell RNA sequencing data with multiple subjects.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) technology has enabled assessment of transcriptome-wide changes at single-cell resolution. Due to the heterogeneity in environmental exposure and genetic background across subjects, subject effect contributes to the major source of variation in scRNA-seq data with multiple subjects, which severely confounds cell type specific differential expression (DE) analysis. Moreover, dropout events are prevalent in scRNA-seq data, leading to excessive number of zeroes in the data, which further aggravates the challenge in DE analysis. RESULTS: We developed iDESC to detect cell type specific DE genes between two groups of subjects in scRNA-seq data. iDESC uses a zero-inflated negative binomial mixed model to consider both subject effect and dropouts. The prevalence of dropout events (dropout rate) was demonstrated to be dependent on gene expression level, which is modeled by pooling information across genes. Subject effect is modeled as a random effect in the log-mean of the negative binomial component. We evaluated and compared the performance of iDESC with eleven existing DE analysis methods. Using simulated data, we demonstrated that iDESC had well-controlled type I error and higher power compared to the existing methods. Applications of those methods with well-controlled type I error to three real scRNA-seq datasets from the same tissue and disease showed that the results of iDESC achieved the best consistency between datasets and the best disease relevance. CONCLUSIONS: iDESC was able to achieve more accurate and robust DE analysis results by separating subject effect from disease effect with consideration of dropouts to identify DE genes, suggesting the importance of considering subject effect and dropouts in the DE analysis of scRNA-seq data with multiple subjects.

A Multi-omic Analysis of the Human Lung Reveals Distinct Cell Specific Aging and Senescence Molecular Programs.

Age is a major risk factor for lung disease. To understand the mechanisms underlying this association, we characterized the changing cellular, genomic, transcriptional, and epigenetic landscape of lung aging using bulk and single-cell RNAseq (scRNAseq) data. Our analysis revealed age-associated gene networks that reflected hallmarks of aging, including mitochondrial dysfunction, inflammation, and cellular senescence. Cell type deconvolution revealed age-associated changes in the cellular composition of the lung: decreased alveolar epithelial cells and increased fibroblasts and endothelial cells. In the alveolar microenvironment, aging is characterized by decreased AT2B cells and reduced surfactant production, a finding that was validated by scRNAseq and IHC. We showed that a previously reported senescence signature, SenMayo, captures cells expressing canonical senescence markers. SenMayo signature also identified cell-type specific senescence-associated co-expression modules that have distinct molecular functions, including ECM regulation, cell signaling, and damage response pathways. Analysis of somatic mutations showed that burden was highest in lymphocytes and endothelial cells and was associated with high expression of senescence signature. Finally, aging and senescence gene expression modules were associated with differentially methylated regions, with inflammatory markers such as IL1B, IL6R, and TNF being significantly regulated with age. Our findings provide new insights into the mechanisms underlying lung aging and may have implications for the development of interventions to prevent or treat age-related lung diseases.

Maternal Feeding Beliefs and Behaviors Relate to Infant Diet and Appetite.

INTRODUCTION: Maternal feeding practices may be linked to infant obesogenic outcomes, but research to date has focused primarily on infant growth as an outcome of maternal feeding practices rather than exploring additional obesogenic outcomes like infant appetite and diet. Therefore, the current study examined the association between maternal feeding practices and beliefs and infant growth, diet, and appetite simultaneously at a critical timepoint for obesity risk development (i.e., 3-months-old). METHODS: Thirty-two 3-month-old infants and their mothers participated in this cross-sectional study. Infant anthropometrics were collected by trained staff and mothers completed questionnaires regarding maternal feeding practices and beliefs and infant diet and appetite. The data were analyzed by Spearman correlations. RESULTS: Statistically significant correlations were identified between maternal feeding practices (e.g., using food to calm, concern about infant weight) and infant satiety, appetite, food responsiveness, slow eating, and kilocalories consumed. Infant weight-for-length was related to maternal concern about infant underweight and mother-infant social interaction during feeding. DISCUSSION: These findings highlight the importance of the mother-infant feeding relationship and how these associations may influence responsive feeding practices and infant weight-related outcomes.

Viral and Host Mediators of Non-Suppressible HIV-1 Viremia.

Non-suppressible HIV-1 viremia (NSV) can occur in persons with HIV despite adherence to combination antiretroviral therapy (ART) and in the absence of significant drug resistance. Here, we show that plasma NSV sequences are comprised primarily of large clones without evidence of viral evolution over time. We defined proviruses that contribute to plasma viremia as "producer", and those that did not as "non-producer". Compared to ART-suppressed individuals, NSV participants had a significantly larger producer reservoir. Producer proviruses were enriched in chromosome 19 and in proximity to the activating H3K36me3 epigenetic mark. CD4+ cells from NSV participants demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, NSV participants showed no elevation in HIV-specific CD8+ cell responses and producer proviruses were enriched for HLA escape mutations. We identified critical host and viral mediators of NSV that represent potential targets to disrupt HIV persistence and promote viral silencing.