Biology subjects

Sembrat, J.

Publications and source records attributed to Sembrat, J..

10 recordsLinked to original sources

bioRxiv2026

A lethal human H5N5 influenza virus isolate exhibits low pandemic risk traits

In fall of 2025, a fatal infection of highly pathogenic avian influenza (HPAI) virus H5N5 occurred. To define the risk of this emerging virus to humans, we performed a comprehensive analysis based on our established triage. Serological analysis revealed that humans across all birth years had no detectable neutralizing antibodies to this H5N5 isolate. Further characterization revealed a lack of phenotypic signatures associated with epidemiologically successful influenza viruses in humans, including reduced replication in human airway cells and an avian-like pH of inactivation. Additionally, assessment of H5N5 in ferrets revealed a lack of direct contact transmission and moderate disease severity. H5N5 infection in ferrets with prior immunity against the 2009 H1N1 pandemic strain resulted in fewer clinical signs and reduced viral shedding. Together our data suggest that the current H5N5 HPAI lineage poses a low pandemic risk. ImportanceHPAI H5N5 viruses have caused widespread infection and death in avian species, and characterizing their pandemic risk traits is critical to understanding the threat posed to humans. In this work we analyzed an isolate that resulted in a human fatality in 2025. We found that this strain lacks many key features of influenza viruses with epidemiological success in humans including reduced growth in human lung cultures, a pH of inactivation less than 5.0, and lack of transmission to cohoused recipient ferrets. Prior immunity with seasonal H1N1 strain also reduced the viral load and disease burden of the virus. Taken together, these data suggest that currently circulating H5N5 poses a low risk to humans but highlights the importance of phenotypic characterizations for future risk assessments as the virus evolves in wild birds.

microbiology↗

bioRxiv2026

Alveolar Epithelial Cell Loss of the Mitochondrial Regulator TFAM Drives Progressive Lung Fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by failed alveolar epithelial repair and progressive fibrotic remodeling. Although aberrant reprogramming of alveolar type 2 (AT2) cells and accumulation of transitional AT2 states are increasing recognized as central features of IPF, the epithelial-intrinsic mechanisms that initiate these pathogenic states remain incompletely understood. Here, we identify mitochondrial transcription factor A (TFAM), a regulator of mitochondrial DNA maintenance, as a critical regulator of AT2 cell homeostasis. TFAM expression was reduced in AT2 cells from human IPF lungs. Inducible AT2 cell-specific Tfam deletion in mice caused spontaneous fibrotic remodeling and increased susceptibility to bleomycin-induced lung injury. TFAM-deficient AT2 cells acquired KRT8+ transitional and p21+ senescence-associated features before the onset of fibrotic transformation, accompanied by impaired oxidative phosphorylation, redox imbalance, mitochondrial superoxide accumulation, repression of mtDNA-encoded respiratory genes, and disrupted mitochondrial ultrastructure. TFAM-deficient AT2 cells developed a profibrotic secretory program that promoted extracellular matrix deposition and fibroblast activation. We further identified insulin-like growth factor-binding protein 2 (IGFBP2) as a secreted mediator induced in TFAM-deficient AT2 cells. IGFBP2 was elevated in AT2 cells in human IPF lung tissue and bronchoalveolar lavage fluid (BALF) from patients with IPF. IGFBP2 was detected in supernatants from fibrotic human precision-cut lung slices (hPCLS). IGFBP2 neutralization attenuated profibrotic remodeling in fibrotic hPCLS. Collectively, our findings identify TFAM-dependent mitochondrial homeostasis as an epithelial checkpoint linking AT2 cell-state stability to impaired epithelial-mesenchymal crosstalk driving pulmonary fibrosis.

pathology↗

bioRxiv2024

Altered AP-1, RUNX, and EGR chromatin dynamics drive fibrotic lung disease

Pulmonary fibrosis, including systemic sclerosis-associated interstitial lung disease (SSc-ILD), involves myofibroblasts and SPP1hi macrophages as drivers of fibrosis. Single-cell RNA sequencing has delineated fibroblast and macrophages transcriptomes, but limited insight into transcriptional control of profibrotic gene programs. To address this challenge, we analyzed multiomic snATAC/snRNA-seq on explanted SSc-ILD and donor control lungs. The neural network tool ChromBPNet inferred increased TF binding at single base pair resolution to profibrotic genes, including CTHRC1 and ADAM12, in fibroblasts and SPP1 and CCL18 in macrophages. The novel algorithm HALO confirmed AP-1, RUNX, and EGR TF activity controlling profibrotic gene programs and established TF-regulatory element-gene networks. This TF action atlas provides comprehensive insights into the transcriptional regulation of fibroblasts and macrophages in healthy and fibrotic human lungs.

systems biology↗

bioRxiv2024

Post-transcriptional regulation of IFI16 promotes inflammatory endothelial pathophenotypes observed in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease driven by endothelial cell inflammation and dysfunction, resulting in the pathological remodeling of the pulmonary vasculature. Innate immune activation has been linked to PAH development; however, the regulation, propagation, and reversibility of the induction of inflammation in PAH is poorly understood. Here, we demonstrate a role for interferon inducible protein 16 (IFI16), an innate immune sensor, as a modulator of endothelial inflammation in pulmonary hypertension, utilizing human pulmonary artery endothelial cells (PAECs). Inflammatory stimulus of PAECs with IL-1{beta} up-regulates IFI16 expression, inducing proinflammatory cytokine up-regulation and cellular apoptosis. IFI16 mRNA stability is regulated by post-transcriptional m6A modification, mediated by Wilms tumor 1-associated protein (WTAP), a structural stabilizer of the methyltransferase complex, via regulation of m6A methylation of IFI16. Additionally, m6A levels are increased in the peripheral blood mononuclear cells of PAH patients compared to control, indicating that quantifying this epigenetic change in patients may hold potential as a biomarker for disease identification. In summary, our study demonstrates IFI16 mediates inflammatory endothelial pathophenotypes seen in pulmonary arterial hypertension.

cell biology↗

bioRxiv2023

GCN5L1-mediated acetylation prevents Rictor degradation in cardiac cells after hypoxic stress

Cardiomyocyte apoptosis and cardiac fibrosis are the leading causes of mortality in patients with ischemic heart disease. As such, these processes represent potential therapeutic targets to treat heart failure resulting from ischemic insult. We previously demonstrated that the mitochondrial acetyltransferase protein GCN5L1 regulates cardiomyocyte cytoprotective signaling in ischemia- reperfusion injury in vivo and hypoxia-reoxygenation injury in vitro. The current study investigated the mechanism underlying GCN5L1-mediated regulation of the Akt/mTORC2 cardioprotective signaling pathway. Rictor protein levels in cardiac tissues from human ischemic heart disease patients were significantly decreased relative to non-ischemic controls. Rictor protein levels were similarly decreased in cardiac AC16 cells following hypoxic stress, while mRNA levels remained unchanged. The reduction in Rictor protein levels after hypoxia was enhanced by the knockdown of GCN5L1, and was blocked by GCN5L1 overexpression. These findings correlated with changes in Rictor lysine acetylation, which were mediated by GCN5L1 acetyltransferase activity. Rictor degradation was regulated by proteasomal activity, which was antagonized by increased Rictor acetylation. Finally, we found that GCN5L1 knockdown restricted cytoprotective Akt signaling, in conjunction with decreased mTOR abundance and activity. In summary, these studies suggest that GCN5L1 promotes cardioprotective Akt/mTORC2 signaling by maintaining Rictor protein levels through enhanced lysine acetylation.

cell biology↗

bioRxiv2022

Non-canonical Telomerase Reverse Transcriptase Controls Osteogenic Differentiation of Aortic Valve Cells Through STAT5

BackgroundCalcific aortic valve disease (CAVD) is the pathological remodeling of valve leaflets. The initial steps in valve leaflet osteogenic reprogramming are not fully understood. As telomerase reverse transcriptase (TERT) overexpression primes mesenchymal stem cells to differentiate into osteoblasts, we investigated whether TERT contributes to the osteogenic reprogramming of valve interstitial cells (VICs). MethodsHuman control and CAVD aortic valve leaflets and patient-specific hVICs were used in in vivo and in vitro calcification assays. Loss of function experiments in hVICs and cells isolated from Tert-/-and Terc-/- mice were used for mechanistic studies. Calcification was assessed in Tert+/+ and Tert-/- mice ex vivo and in vivo. In silico modeling, proximity ligation and co-immunoprecipitation assays defined novel TERT interacting partners. Chromatin immunoprecipitation and CUT&TAG sequencing defined protein-DNA interactions. ResultsTERT protein was highly expressed in calcified valve leaflets without changes in telomere length, DNA damage, or senescence markers, and these features were retained in isolated primary hVICs. TERT expression increased with osteogenic or inflammatory stimuli, and knock-down or genetic deletion of TERT prevented calcification in vitro and in vivo. Mechanistically, TERT was upregulated via NF-{kappa}B and required to initiate osteogenic reprogramming, independent of its canonical reverse transcriptase activity and the lncRNA TERC. TERT exerts non-canonical osteogenic functions via binding with Signal Transducer and Activator of Transcription 5 (STAT5). Depletion or inhibition of STAT5 prevented calcification. STAT5 was found to bind the promoter region of Runt-Related Transcription Factor 2 (RUNX2), the master regulator of osteogenic reprogramming. Lastly, we demonstrate that TERT and STAT5 are upregulated and colocalized in CAVD tissue compared to control tissue. ConclusionsTERTs non-canonical activity is required to initiate calcification. TERT is upregulated via inflammatory signaling pathways and partners with STAT5 to bind the RUNX2 gene promoter. These data identify a novel mechanism and potential therapeutic target to decrease vascular calcification. Novelty and SignificanceWhat is known? Calcific aortic valve disease (CAVD) is the most prevalent form of aortic valve pathology. CAVD strongly correlates with age and leads to heart failure and a high risk of stroke. Currently, the only therapeutic option is valve replacement, which comes with significant healthcare costs and additional risks to patients. Runt-related transcription factor 2 (RUNX2) is the master transcription factor required for osteogenic differentiation of stem cells to osteoblasts and osteogenic reprogramming of cardiovascular cells. Yet, the early events driving its activity in aortic valve cells are poorly defined. In addition to its reverse transcriptase enzymatic activity, TERT exhibits non-canonical transcriptional regulatory functions and overexpression of TERT primes mesenchymal stem cells to differentiate down the osteoblast lineage. What new information does this article contribute? TERT protein levels in calcified aortic leaflets and valve interstitial cells, and its non-canonical osteogenic activity are independent of changes in telomere length and cell senescence. Genetic loss or depletion of TERT prevented calcification in valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells in vitro and the vasculature in vivo. Early in the osteogenic reprogramming inflammatory signaling promotes TERT to co-localize with SMARCA4 and STAT5, and this TERT-tethered STAT5 binds to the RUNX2 gene promoter, the master regulator of osteogenic transcriptional programs. STAT5 depletion and pharmacological inhibition prevent calcification of human valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells. What are the clinical implications? We have identified TERT-STAT5 as a novel signaling axis that orchestrates the early steps in the osteogenic reprogramming of aortic valve cells. Inhibiting TERT/STAT5 interaction or their activity may be leveraged for the development of therapeutic strategies to halt or prevent calcification in the aortic valve, bioprosthetic valves, and or perhaps other cardiovascular tissues. Invasive and expensive surgical procedures are currently the only treatment option for patients with CAVD. The discovery and defining of the early events driving vascular calcification identifies novel and druggable targets for developing non-surgical therapies.

pathology↗

bioRxiv2022

Early events marking lung fibroblast transition to profibrotic state in idiopathic pulmonary fibrosis

BackgroundIdiopathic Pulmonary Fibrosis (IPF) is an age-associated progressive lung disease with accumulation of scar tissue impairing gas exchange. Previous high-throughput studies elucidated the role of cellular heterogeneity and molecular pathways in advanced disease. However, critical pathogenic pathways occurring in the transition of fibroblasts from normal to profibrotic have been largely overlooked. MethodsWe used single cell transcriptomics (scRNA-seq) from lungs of healthy controls and IPF patients (lower and upper lobes). We identified fibroblast subclusters, genes and pathways associated to early disease. Immunofluorescence assays validated the role of MOXD1 early in fibrosis. FindingsWe identified four distinct fibroblast subgroups, including one marking the normal-to-profibrotic state transition. Our results show for the first time that global downregulation of ribosomal proteins and significant upregulation of the majority of copper-binding proteins, including MOXD1, mark the IPF transition. We find no significant differences in gene expression in IPF upper and lower lobe samples, which were selected to have low and high degree of fibrosis, respectively. InterpretationEarly events during IPF onset in fibroblasts include dysregulation of ribosomal and copper-binding proteins. Fibroblasts in early stage IPF have already acquired a profibrotic phenotype while hallmarks of advanced disease, including fibroblast foci and honeycomb formation, are still not evident. The new transitional fibroblasts we discover could prove very important for studying the role of fibroblast plasticity in disease progression and help develop early diagnosis tools and therapeutic interventions targeting earlier disease states.

genomics↗

bioRxiv2022

The Role of EBP50 in Regulating Endothelial-To-Mesenchymal Transition in Pulmonary Hypertension

ObjectivePulmonary hypertension (PH) is a cardiopulmonary disease manifesting in increased pulmonary arterial pressure and right ventricular dysfunction. PH pathogenesis involves extensive pulmonary vascular remodeling precipitated, at least in part, by endothelial reprogramming. Mounting evidence points to endothelial-to-mesenchymal transition (EndMT) as an important potentiator of endothelial reprogramming in PH, yet progress in dissecting these processes remains limited. Approach and ResultsLung samples from pulmonary arterial hypertension (PAH) patients and two rodent models of PH were used. Expression of the scaffolding protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50, or NHERF1) was downregulated in PAH patient pulmonary arteries and isolated pulmonary arterial endothelial cells (PAECs), and in PH animal lung tissue and mouse isolated PAECs. In human PAECs in vitro, EBP50 was downregulated by PH-relevant stimuli, hypoxia and proinflammatory cytokine interleukin-1 beta (IL-1{beta}). Phenocopy of EBP50 reduction in PAECs time-dependently increased expression and nuclear abundance of EndMT transcription factors Snail and Zeb1, and potentiated hypoxia-driven upregulation of Slug. Loss of EBP50 also drove expression of mesenchymal markers S100A4, fibronectin, N-cadherin, and transgelin (SM22), and inhibited cell proliferation and barrier function. In vivo studies on female EBP50+/- mice demonstrated that downregulation of EBP50 exacerbated the chronic hypoxia-induced rise in RV maximum pressure. ConclusionsThese data identify EBP50 as a key regulator of EndMT in PH whose expression is downregulated in PH patient pulmonary endothelium and whose partial deletion exacerbates PH disease manifestations in rodents, opening doors for future therapeutic strategies to target EBP50 restoration to reverse PH.

cell biology↗

bioRxiv2022

Single-nucleus chromatin accessibility identifies a critical role for TWIST1 in idiopathic pulmonary fibrosis myofibroblast activity

In idiopathic pulmonary fibrosis (IPF) myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise. We performed and integrated snATAC-seq and scRNA-seq from human IPF and donor control explants to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. TWIST1 and other E-box transcription factor motifs were significantly enriched in IPF myofibroblasts compared to both IPF non-myogenic and control fibroblasts. TWIST1 expression was also selectively upregulated in IPF myofibroblasts. Overexpression of Twist1 in lung COL1A2-expressing fibroblasts in bleomycin-injured mice was associated with increased collagen synthesis. Our studies utilizing human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box TF motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.

molecular biology↗

bioRxiv2021

High CXCL6 drives matrix expression and correlate with markers of poor outcome in IPF

Signaling via G protein-coupled receptors (GPCRs) can modulate levels of cyclic adenosine monophosphate (cAMP) and shape the functions of fibroblasts in idiopathic pulmonary fibrosis (IPF). We have identified Chemokine (C-X-C) Motif Ligand 6 (CXCL6) as a potential pro-fibrotic GPCR ligand. We tested the function of CXCL6 in ex vivo human donor and fibrotic lung fibroblasts and in an animal model of pulmonary fibrosis. We also measured levels of CXCL6 in the blood and bronchoalveolar lavage (BAL) of patients with IPF. CXCL6 decreased cAMP levels in a dose-dependent manner in Donor and IPF Fibroblasts. CXCL6 mRNA and protein were localized to epithelial cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased Collagen I and -SMA levels in Donor and IPF Fibroblasts. Silencing of CXCR1/2 as well as Reparixin, a CXCR1/2 inhibitor, blocked effects of CXCL6. Treprostinil blocked effects of CXCL6 only on levels of -SMA but not on Collagen I. CXCL6 levels in the BAL of two separate cohorts of patients with IPF was associated with poor survival. We conclude that high CXCL6 drives fibroblast function and correlates with poor outcomes in IPF.

cell biology↗