My Recent Publications
A systems approach points to a therapeutic role for retinoids in asparaginase-associated pancreatitis
Sci Transl Med. 2023 Mar 15;15(687):eabn2110. doi: 10.1126/scitranslmed.abn2110. Epub 2023 Mar 15.
Among drug-induced adverse events, pancreatitis is life-threatening and results in substantial morbidity. A prototype example is the pancreatitis caused by asparaginase, a crucial drug used to treat acute lymphoblastic leukemia (ALL). Here, we used a systems approach to identify the factors affecting asparaginase-associated pancreatitis (AAP). Connectivity Map analysis of the transcriptomic data showed that asparaginase-induced gene signatures were potentially reversed by retinoids (vitamin A and its analogs). Analysis of a large electronic health record database (TriNetX) and the U.S. Federal Drug Administration Adverse Events Reporting System demonstrated a reduction in AAP risk with concomitant exposure to vitamin A. Furthermore, we performed a global metabolomic screening of plasma samples from 24 individuals with ALL who developed pancreatitis (cases) and 26 individuals with ALL who did not develop pancreatitis (controls), before and after a single exposure to asparaginase. Screening from this discovery cohort revealed that plasma carotenoids were lower in the cases than in controls. This finding was validated in a larger external cohort. A 30-day dietary recall showed that the cases received less dietary vitamin A than the controls did. In mice, asparaginase administration alone was sufficient to reduce circulating and hepatic retinol. Based on these data, we propose that circulating retinoids protect against pancreatic inflammation and that asparaginase reduces circulating retinoids. Moreover, we show that AAP is more likely to develop with reduced dietary vitamin A intake. The systems approach taken for AAP provides an impetus to examine the role of dietary vitamin A supplementation in preventing or treating AAP.
PMID:36921036 | DOI:10.1126/scitranslmed.abn2110
BRD9-SMAD2/3 orchestrates stemness and tumorigenesis in pancreatic ductal adenocarcinoma
bioRxiv. 2023 Mar 2:2023.03.02.530770. doi: 10.1101/2023.03.02.530770. Preprint.
The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodelling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFβ/Activin-SMAD2/3 signalling pathway. Inhibition and genetic ablation of BDR9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumours from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.
PMID:36909530 | PMC:PMC10002796 | DOI:10.1101/2023.03.02.530770
Transcriptome analysis in acute gastrointestinal graft-versus host disease reveals a unique signature in children and shared biology with pediatric inflammatory bowel disease
Haematologica. 2023 Feb 2. doi: 10.3324/haematol.2022.282035. Online ahead of print.
We performed transcriptomic analyses on freshly frozen (n=21) and paraffin embedded (n=35) gastrointestinal (GI)biopsies from children with and without acute GI graft versus host disease (GVHD) to study differential gene expressions. We identified 164 significant genes, 141 upregulated and 23 downregulated, in acute GVHD from freshy frozen biopsies. CHI3L1 was the top differentially expressed gene in acute GVHD, involved in macrophage recruitment and bacterial adhesion. Mitochondrial genes were among the top downregulated genes. Immune deconvolution identified a macrophage cellular signature. Weighted gene co-expression network analysis showed enrichment of genes in the ERK1/2 cascade. Transcriptome data from 206 ulcerative colitis (UC) patients were included to uncover genes and pathways shared between GVHD and UC. Comparison with the UC transcriptome showed both shared and distinct pathways. Both UC and GVHD transcriptomes shared an innate antimicrobial signature and FCγ1RA/CD64 was upregulated in both acute GVHD (log fold increase 1.7, p=0.001) and UC. Upregulation of the ERK1/2 cascade pathway was specific to GVHD. We performed additional experiments to confirm transcriptomics. Firstly, we examined phosphorylation of ERK (pERK) by immunohistochemistry on GI biopsies (acute GVHD n=10, no GVHD n=10). pERK staining was increased in acute GVHD biopsies compared to biopsies without acute GVHD (p= 0.001). Secondly, plasma CD64, measured by ELISA (n=85) was elevated in acute GI GVHD (p.
PMID:36727399 | DOI:10.3324/haematol.2022.282035
Combination of esomeprazole and pirfenidone enhances antifibrotic efficacy in vitro and in a mouse model of TGFβ-induced lung fibrosis
Sci Rep. 2022 Nov 30;12(1):20668. doi: 10.1038/s41598-022-24985-x.
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease of unknown etiology. Currently, pirfenidone and nintedanib are the only FDA-approved drugs for the treatment of IPF and are now the standard of care. This is a significant step in slowing down the progression of the disease. However, the drugs are unable to stop or reverse established fibrosis. Several retrospective clinical studies indicate that proton pump inhibitors (PPIs; FDA-approved to treat gastroesophageal reflux) are associated with favorable outcomes in patients with IPF, and emerging preclinical studies report that PPIs possess antifibrotic activity. In this study, we evaluated the antifibrotic efficacy of the PPI esomeprazole when combined with pirfenidone in vitro and in vivo. In cell culture studies of IPF lung fibroblasts, we assessed the effect of the combination on several fibrosis-related biological processes including TGFβ-induced cell proliferation, cell migration, cell contraction, and collagen production. In an in vivo study, we used mouse model of TGFβ-induced lung fibrosis to evaluate the antifibrotic efficacy of esomeprazole/pirfenidone combination. We also performed computational studies to understand the molecular mechanisms by which esomeprazole and/or pirfenidone regulate lung fibrosis. We found that esomeprazole significantly enhanced the anti-proliferative effect of pirfenidone and favorably modulated TGFβ-induced cell migration and contraction of collagen gels. We also found that the combination significantly suppressed collagen production in response to TGFβ in comparison to pirfenidone monotherapy. In addition, our animal study demonstrated that the combination therapy effectively inhibited the differentiation of lung fibroblasts into alpha smooth muscle actin (αSMA)-expressing myofibroblasts to attenuate the progression of lung fibrosis. Finally, our bioinformatics study of cells treated with esomeprazole or pirfenidone revealed that the drugs target several extracellular matrix (ECM) related pathways with esomeprazole preferentially targeting collagen family members while pirfenidone targets the keratins. In conclusion, our cell biological, computational, and in vivo studies show that the PPI esomeprazole enhances the antifibrotic efficacy of pirfenidone through complementary molecular mechanisms. This data supports the initiation of prospective clinical studies aimed at repurposing PPIs for the treatment of IPF and other fibrotic lung diseases where pirfenidone is prescribed.
PMID:36450789 | DOI:10.1038/s41598-022-24985-x
Targeting of Cdc42 GTPase in regulatory T cells unleashes antitumor T-cell immunity
J Immunother Cancer. 2022 Nov;10(11):e004806. doi: 10.1136/jitc-2022-004806.
BACKGROUND: Cancer immunotherapy has taken center stage in cancer treatment. However, the current immunotherapies only benefit a small proportion of patients with cancer, necessitating better understanding of the mechanisms of tumor immune evasion and improved cancer immunotherapy strategies. Regulatory T (Treg) cells play an important role in maintaining immune tolerance through inhibiting effector T-cell function. In the tumor microenvironment, Treg cells are used by tumor cells to counteract effector T cell-mediated tumor suppression. Targeting Treg cells may thus unleash the antitumor activity of effector T cells. While systemic depletion of Treg cells can cause excessive effector T-cell responses and subsequent autoimmune diseases, controlled targeting of Treg cells may benefit patients with cancer.
METHODS: Treg cells from Treg cell-specific heterozygous Cdc42 knockout mice, C57BL/6 mice treated with a Cdc42 inhibitor CASIN, and control mice were examined for their homeostasis and stability by flow cytometry. The autoimmune responses in Treg cell-specific heterozygous Cdc42 knockout mice, CASIN-treated C57BL/6 mice, and control mice were assessed by H&E staining and ELISA. Antitumor T-cell immunity in Treg cell-specific heterozygous Cdc42 knockout mice, CASIN-treated C57BL/6 mice, humanized NSGS mice, and control mice was assessed by challenging the mice with MC38 mouse colon cancer cells, KPC mouse pancreatic cancer cells, or HCT116 human colon cancer cells.
RESULTS: Treg cell-specific heterozygous deletion or pharmacological targeting of Cdc42 with CASIN does not affect Treg cell numbers but induces Treg cell instability, leading to antitumor T-cell immunity without detectable autoimmune reactions. Cdc42 targeting causes an additive effect on immune checkpoint inhibitor anti-programmed cell death protein-1 antibody-induced T-cell response against mouse and human tumors. Mechanistically, Cdc42 targeting induces Treg cell instability and unleashes antitumor T-cell immunity through carbonic anhydrase I-mediated pH changes.
CONCLUSIONS: Rational targeting of Cdc42 in Treg cells holds therapeutic promises in cancer immunotherapy.
PMID:36427906 | DOI:10.1136/jitc-2022-004806
Personalized medicine approaches in cystic fibrosis related pancreatitis
Am J Transl Res. 2022 Oct 15;14(10):7612-7620. eCollection 2022.
We report a rare case of a patient with cystic fibrosis suffering from debilitating abdominal pain due to chronic pancreatitis. This 13-year-old patient was evaluated for surgical intervention to relieve pain from chronic pancreatitis and to improve quality of life. The patient carried two mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene; the most common ΔF508 variant and a second variant, p.Glu1044Gly, which has not been previously described. The patient's condition did not improve despite medical management and multiple endoscopic interventions, and therefore total pancreatectomy with islet autotransplantation and a near-total duodenectomy was offered for definitive management. Patient-derived duodenal crypts were isolated and cultured from the resected duodenum, and duodenal organoids were generated to test CFTR function. Our studies demonstrate that this novel mutation (ΔF508/p.Glu1044Gly) caused severely impaired CFTR function in vitro. The Food and Drug Administration (FDA)-approved drug ivacaftor, a CFTR potentiator, was identified to robustly improve CFTR function in the context of this novel mutation. Herein, we describe a personalized medicine approach consisting of performing drug testing on individual patient derived organoids that has potential to guide management of patients with novel CFTR genetic mutations. Identified effective medical therapeutics using this approach may avoid irreversible surgical treatments such as total pancreatectomy with islet autotransplantation in the future.
PMID:36398272 | PMC:PMC9641468
<em>gene2gauss</em>: A multi-view gaussian gene embedding learner for analyzing transcriptomic networks
AMIA Annu Symp Proc. 2022 May 23;2022:206-215. eCollection 2022.
Analyzing gene co-expression networks can help in the discovery of biological processes and regulatory mechanisms underlying normal or perturbed states. Unlike standard differential analysis, network-based approaches consider the interactions between the genes involved leading to biologically relevant results. Applying such network-based methods to jointly analyze multiple transcriptomic networks representing independent disease cohorts or studies could lead to the identification of more robust gene modules or gene regulatory networks. We present gene2gauss, a novel feature learning framework that is capable of embedding genes as multivariate gaussian distributions by taking into account their long-range interaction neighborhoods across multiple transcriptomic studies. Using multiple gene co-expression networks from idiopathic pulmonary fibrosis, we demonstrate that these multi-dimensional gaussian features are suitable for identifying regulons of known transcription factors (TF). Using standard TF-target libraries, we demonstrate that the features from our method are highly relevant in comparison with other feature learning approaches on transcriptomic data.
PMID:35854722 | PMC:PMC9285176
Heterogeneous Distribution of Genetic Mutations in Myosin Binding Protein-C Paralogs
Front Genet. 2022 Jun 27;13:896117. doi: 10.3389/fgene.2022.896117. eCollection 2022.
Myosin binding protein-C (MyBP-C) is a sarcomeric protein which regulates the force of contraction in striated muscles. Mutations in the MYBPC family of genes, including slow skeletal (MYBPC1), fast skeletal (MYBPC2) and cardiac (MYBPC3), can result in cardiac and skeletal myopathies. Nonetheless, their evolutionary pattern, pathogenicity and impact on MyBP-C protein structure remain to be elucidated. Therefore, the present study aimed to systematically assess the evolutionarily conserved and epigenetic patterns of MYBPC family mutations. Leveraging a machine learning (ML) approach, the Genome Aggregation Database (gnomAD) provided variants in MYBPC1, MYBPC2, and MYBPC3 genes. This was followed by an analysis with Ensembl's variant effect predictor (VEP), resulting in the identification of 8,618, 3,871, and 3,071 variants in MYBPC1, MYBPC2, and MYBPC3, respectively. Missense variants comprised 61%-66% of total variants in which the third nucleotide positions in the codons were highly altered. Arginine was the most mutated amino acid, important because most disease-causing mutations in MyBP-C proteins are arginine in origin. Domains C5 and C6 of MyBP-C were found to be hotspots for most mutations in the MyBP-C family of proteins. A high percentage of truncated mutations in cMyBP-C cause cardiomyopathies. Arginine and glutamate were the top hits in fMyBP-C and cMyBP-C, respectively, and tryptophan and tyrosine were the most common among the three paralogs changing to premature stop codons and causing protein truncations at the carboxyl terminus. A heterogeneous epigenetic pattern was identified among the three MYBP-C paralogs. Overall, it was shown that databases using computational approaches can facilitate diagnosis and drug discovery to treat muscle disorders caused by MYBPC mutations.
PMID:35832193 | PMC:PMC9272480 | DOI:10.3389/fgene.2022.896117
Defining the Dynamic Regulation of O-GlcNAc Proteome in the Mouse Cortex---the O-GlcNAcylation of Synaptic and Trafficking Proteins Related to Neurodegenerative Diseases
Front Aging. 2021 Sep 29;2:757801. doi: 10.3389/fragi.2021.757801. eCollection 2021.
O-linked conjugation of ß-N-acetyl-glucosamine (O-GlcNAc) to serine and threonine residues is a post-translational modification process that senses nutrient availability and cellular stress and regulates diverse biological processes that are involved in neurodegenerative diseases and provide potential targets for therapeutics development. However, very little is known of the networks involved in the brain that are responsive to changes in the O-GlcNAc proteome. Pharmacological increase of protein O-GlcNAcylation by Thiamet G (TG) has been shown to decrease tau phosphorylation and neurotoxicity, and proposed as a therapy in Alzheimer's disease (AD). However, acute TG exposure impairs learning and memory, and protein O-GlcNAcylation is increased in the aging rat brain and in Parkinson's disease (PD) brains. To define the cortical O-GlcNAc proteome that responds to TG, we injected young adult mice with either saline or TG and performed mass spectrometry analysis for detection of O-GlcNAcylated peptides. This approach identified 506 unique peptides corresponding to 278 proteins that are O-GlcNAcylated. Of the 506 unique peptides, 85 peptides are elevated by > 1.5 fold in O-GlcNAcylation levels in response to TG. Using pathway analyses, we found TG-dependent enrichment of O-GlcNAcylated synaptic proteins, trafficking, Notch/Wnt signaling, HDAC signaling, and circadian clock proteins. Significant changes in the O-GlcNAcylation of DNAJC6/AUXI, and PICALM, proteins that are risk factors for PD and/or AD respectively, were detected. We compared our study with two key prior O-GlcNAc proteome studies using mouse cerebral tissue and human AD brains. Among those identified to be increased by TG, 15 are also identified to be increased in human AD brains compared to control, including those involved in cytoskeleton, autophagy, chromatin organization and mitochondrial dysfunction. These studies provide insights regarding neurodegenerative diseases therapeutic targets.
PMID:35822049 | PMC:PMC9261315 | DOI:10.3389/fragi.2021.757801
Is There a Benefit From Islet Autotransplantation in Patients With Type 1 Diabetes Mellitus Undergoing Total Pancreatectomy?
Pancreas. 2022 Apr 1;51(4):399-403. doi: 10.1097/MPA.0000000000002017.
Children with acute recurrent and chronic pancreatitis (CP) experience abdominal pain that leads to hospitalizations, opioid dependence, and poor quality of life. Total pancreatectomy with islet autotransplantation (TPIAT) is offered as a surgical option in management of debilitating pancreatitis that fails medical and endoscopic therapy to reduce or eliminate pain. Given that patients with type 1 diabetes mellitus (T1DM) lack insulin-producing β cells, the outcomes from autotransplanting islet isolates back into total pancreatectomy patients with T1DM are not fully known.We performed TPIAT in 2 CP patients who also had a diagnosis of T1DM for at least 6 years before the operation and evaluated the clinical and laboratory outcomes before and after the operation. Postoperatively both patients' abdominal pain had significantly subsided, they were weaned off opioid medications, and they were able to return to full-time school attendance. In addition, total daily dose of insulin in 1 patient was able to be slightly reduced at 12 months post-TPIAT. We observed in vitro that residual α cells and β cells in T1DM islets were able to secrete a small amount of glucagon and insulin, respectively.
PMID:35775640 | DOI:10.1097/MPA.0000000000002017
Targeted Assessment of Mucosal Immune Gene Expression Predicts Clinical Outcomes in Children with Ulcerative Colitis
J Crohns Colitis. 2022 Jun 4:jjac075. doi: 10.1093/ecco-jcc/jjac075. Online ahead of print.
BACKGROUND AND AIMS: We aimed to determine whether a targeted gene expression panel could predict clinical outcomes in pediatric UC and investigated putative pathogenic roles of predictive genes.
METHODS: 313 rectal RNA samples from a cohort of newly diagnosed pediatric UC patients (PROTECT) were analyzed by a real-time PCR microfluidic array for expression of type 1, 2, and 17 inflammation genes. Associations between expression and clinical outcomes were assessed by logistic regression. Identified prognostic markers were further analyzed using existing RNA sequencing (RNA-seq) data sets and tissue immunostaining.
RESULTS: IL13RA2 was associated with lower likelihood of corticosteroid-free remission (CSFR) on mesalamine at week 52 (P= .002). A model including IL13RA2 and only baseline clinical parameters was as accurate as an established clinical model, which requires week 4 remission status. RORC was associated with lower likelihood of colectomy by week 52. A model including RORC and PUCAI predicted colectomy by 52 weeks (AUC 0.71). Bulk RNA-seq identified IL13RA2 and RORC as hub genes within UC outcome-associated expression networks related to extracellular matrix and innate immune response, and lipid metabolism and microvillus assembly, respectively. Adult UC single-cell RNA-seq data revealed IL13RA2 and RORC co-expressed genes were localized to inflammatory fibroblasts and undifferentiated epithelial cells, respectively, which was supported by protein immunostaining.
CONCLUSION: Targeted assessment of rectal mucosal immune gene expression predicts 52-week CSFR in treatment-naïve pediatric UC patients. Further exploration of IL-13Rɑ2 as a therapeutic target in UC, and future studies of the epithelial-specific role of RORC in UC pathogenesis are warranted.
PMID:35665804 | DOI:10.1093/ecco-jcc/jjac075
Consensus Gene Co-Expression Network Analysis Identifies Novel Genes Associated with Severity of Fibrotic Lung Disease
Int J Mol Sci. 2022 May 13;23(10):5447. doi: 10.3390/ijms23105447.
Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease characterized by irreversible scarring of the lung parenchyma leading to dyspnea, progressive decline in lung function, and respiratory failure. We analyzed lung transcriptomic data from independent IPF cohorts using weighted gene co-expression network analysis (WGCNA) to identify gene modules based on their preservation status in these cohorts. The consensus gene modules were characterized by leveraging existing clinical and molecular data such as lung function, biological processes, pathways, and lung cell types. From a total of 32 consensus gene modules identified, two modules were found to be significantly correlated with the disease, lung function, and preserved in other IPF datasets. The upregulated gene module was enriched for extracellular matrix, collagen metabolic process, and BMP signaling while the downregulated module consisted of genes associated with tube morphogenesis, blood vessel development, and cell migration. Using a combination of connectivity-based and trait-based significance measures, we identified and prioritized 103 "hub" genes (including 25 secretory candidate biomarkers) by their similarity to known IPF genetic markers. Our validation studies demonstrate the dysregulated expression of CRABP2, a retinol-binding protein, in multiple lung cells of IPF, and its correlation with the decline in lung function.
PMID:35628257 | DOI:10.3390/ijms23105447
Rewiring of 3D Chromatin Topology Orchestrates Transcriptional Reprogramming and the Development of Human Dilated Cardiomyopathy
Circulation. 2022 Apr 11. doi: 10.1161/CIRCULATIONAHA.121.055781. Online ahead of print.
Background: Transcriptional reconfiguration is central to heart failure, the common cause of which is dilated cardiomyopathy (DCM). However, the impact of three-dimensional (3D) chromatin topology on transcriptional dysregulation and pathogenesis in human DCM remains elusive. Methods: We generated a compendium of 3D-epigenome and transcriptome maps from 101 biobanked human DCM and non-failing heart tissues through HiChIP (H3K27ac), in situ Hi-C, ChIP-seq, ATAC-seq and RNA-seq profiling. We employed human iPSC-derived cardiomyocytes (hiPSC-CMs) and mouse models to further interrogate the key transcription factor implicated in 3D chromatin organization and transcriptional regulation in DCM pathogenesis. Results: We discovered that the active regulatory elements (H3K27ac peaks) and their connectome (H3K27ac loops) were extensively reprogrammed in DCM hearts and contributed to transcriptional dysregulation implicated for DCM development. For example, we identified that non-transcribing NPPA-AS1 promoter functions as an enhancer and physically interacts with the NPPA and NPPB promoters, leading to the co-transcription of NPPA and NPPB in DCM hearts. We uncovered that DCM-enriched H3K27ac loops largely resided in conserved high-order chromatin architectures (Compartments, Topologically Associating Domains) and unexpectedly their anchors had equivalent chromatin accessibility. Intriguingly, we discovered that the DCM-enriched H3K27ac loop anchors exhibited a strong enrichment for Heart and Neural Crest Derivatives Expressed 1 (HAND1), a key transcription factor involved in early cardiogenesis. In line with this, its protein expression was upregulated in human DCM and mouse failing hearts. To further validate whether HAND1 is a causal driver for the reprogramming of enhancer/promoter connectome in DCM hearts, we performed comprehensive 3D epigenome mappings in hiPSC-CMs. We found that forced overexpression of HAND1 in hiPSC-CM induced a distinct gain of enhancer/promoter connectivity and, correspondingly, increased the expression of their connected genes implicated in DCM etiology, thus recapitulating the transcriptional signature in human DCM hearts. Moreover, electrophysiology analysis demonstrated that forced overexpression of HAND1 in hiPSC-CM induced abnormal calcium handling. Furthermore, cardiomyocyte-specific overexpression of Hand1 in the mouse hearts resulted in a dilated cardiac remodeling with impaired contractility/Ca2+ handling in cardiomyocytes, increased ratio of heart weight/body weight and compromised cardiac function, which were ascribed to recapitulation of transcriptional reprogramming in DCM. Conclusions: This study provided novel chromatin topology insights into DCM pathogenesis and illustrated a model whereby a single transcription factor (HAND1) reprograms the genome-wide enhancer/promoter connectome to drive DCM pathogenesis.
PMID:35400201 | DOI:10.1161/CIRCULATIONAHA.121.055781
Eicosatetraynoic Acid and Butyrate Regulate Human Intestinal Organoid Mitochondrial and Extracellular Matrix Pathways Implicated in Crohn's Disease Strictures
Inflamm Bowel Dis. 2022 Mar 8:izac037. doi: 10.1093/ibd/izac037. Online ahead of print.
BACKGROUND: Perturbagen analysis of Crohn's disease (CD) ileal gene expression data identified small molecules including eicosatetraynoic acid (ETYA), which may exert an antifibrotic effect. We developed a patient-specific human intestinal organoid (HIO) model system to test small molecule regulation of mitochondrial and wound-healing functions implicated in stricturing behavior.
METHODS: HIOs were made from CD induced pluripotent stem cells with and without a loss-of-function haplotype in the DUOX2 gene implicated in ileal homeostasis and characterized under basal conditions and following exposure to butyrate and ETYA using RNA sequencing, flow cytometry, and immunofluorescent and polarized light microscopy. Mitochondrial activity was measured using high-resolution respirometry and tissue stiffness using atomic force microscopy.
RESULTS: HIOs expressed core mitochondrial and extracellular matrix (ECM) genes and enriched biologic functions implicated in CD ileal strictures; ECM gene expression was suppressed by both butyrate and ETYA, with butyrate also suppressing genes regulating epithelial proliferation. Consistent with this, butyrate, but not ETYA, exerted a profound effect on HIO epithelial mitochondrial function, reactive oxygen species production, and cellular abundance. Butyrate and ETYA suppressed HIO expression of alpha smooth muscle actin expressed by myofibroblasts, type I collagen, and collagen protein abundance. HIOs exhibited tissue stiffness comparable to normal human ileum; this was reduced by chronic ETYA exposure in HIOs carrying the DUOX2 loss-of-function haplotype.
CONCLUSIONS: ETYA regulates ECM genes implicated in strictures and suppresses collagen content and tissue stiffness in an HIO model. HIOs provide a platform to test personalized therapeutics, including small molecules prioritized by perturbagen analysis.
PMID:35259271 | DOI:10.1093/ibd/izac037
Computational workflow for functional characterization of COVID-19 through secondary data analysis
STAR Protoc. 2021 Sep 24;2(4):100873. doi: 10.1016/j.xpro.2021.100873. eCollection 2021 Dec 17.
Standard transcriptomic analyses cannot fully capture the molecular mechanisms underlying disease pathophysiology and outcomes. We present a computational heterogeneous data integration and mining protocol that combines transcriptional signatures from multiple model systems, protein-protein interactions, single-cell RNA-seq markers, and phenotype-genotype associations to identify functional feature complexes. These feature modules represent a higher order multifeatured machines collectively working toward common pathophysiological goals. We apply this protocol for functional characterization of COVID-19, but it could be applied to many other diseases. For complete details on the use and execution of this protocol, please refer to Ghandikota et al. (2021).
PMID:34746856 | PMC:PMC8551262 | DOI:10.1016/j.xpro.2021.100873
Interstitial Lung Disease in Children with Rubinstein-Taybi Syndrome
Pediatr Pulmonol. 2021 Sep 29. doi: 10.1002/ppul.25709. Online ahead of print.
INTRODUCTION: Rubinstein-Taybi syndrome (RSTS) is a rare genetic syndrome caused primarily by a mutation in the CREBBP gene found on chromosome 16. Patients with RSTS are at greater risk for a variety of medical problems, including upper airway obstruction and aspiration. Childhood interstitial lung disease (ILD) thus far has not been definitively linked to RSTS. Here we present three patients with RSTS who developed ILD and discuss possible mechanisms by which a mutation in CREBBP may be involved in the development of ILD.
METHODS: Routine hematoxylin and eosin staining was performed on lung biopsy tissue for histological analysis. Immunofluorescent staining was performed on lung biopsy tissue for markers of fibrosis, surfactant deficiency and histone acetylation. Cases 1 and 2 had standard clinical microarray analysis. Case 3 had whole exome sequencing. Bioinformatics analyses were performed to identify possible causative genes using ToppGene.
RESULTS: CT images in all cases showed consolidated densities overlying ground glass opacities. Lung histopathology revealed accumulation of proteinaceous material within alveolar spaces, evidence of fibrosis, and increased alveolar macrophages. Immunofluorescent staining showed increase in surfactant protein C staining, patchy areas of increased aSMA staining, and increased staining for acetylated histone 2 and histone 3 lysine 9.
DISCUSSION: Clinical characteristics, radiographic imaging, lung histopathology, and immunofluorescent staining results shared by all cases demonstrated findings consistent with ILD. Immunofluorescent staining suggests two possible mechanisms for the development of ILD: abnormal surfactant metabolism and/or persistent activation of myofibroblasts. These two pathways could be related to dysfunctional CREBBP protein. This article is protected by copyright. All rights reserved.
PMID:34585851 | DOI:10.1002/ppul.25709
Dysregulated overexpression of Sox9 induces fibroblast activation in pulmonary fibrosis
JCI Insight. 2021 Sep 14:152503. doi: 10.1172/jci.insight.152503. Online ahead of print.
Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease associated with unremitting fibroblast activation including fibroblast-to-myofibroblast transformation (FMT), migration, resistance to apoptotic clearance, and excessive deposition of extracellular matrix (ECM) proteins in the distal lung parenchyma. Aberrant activation of lung-developmental pathways is associated with severe fibrotic lung disease; however, the mechanisms through which these pathways activate fibroblasts in IPF remain unclear. Sox9 is a member of the HMG box family of DNA-binding transcription factors that are selectively expressed by epithelial cell progenitors to modulate branching morphogenesis during lung development. We demonstrate that Sox9 is upregulated via MAPK/PI3K-dependent signaling and by the transcription factor Wilms' tumor 1 in distal lung-resident fibroblasts in IPF. Mechanistically, using fibroblast activation assays, we demonstrate that Sox9 functions as a positive regulator of FMT, migration, survival, and ECM production. Importantly, our in vivo studies demonstrate that fibroblast-specific deletion of Sox9 is sufficient to attenuate collagen deposition and improve lung function during TGFα-induced pulmonary fibrosis. Using a mouse model of bleomycin-induced pulmonary fibrosis, we show that myofibroblast-specific Sox9 overexpression augments fibroblast activation and pulmonary fibrosis. Thus, Sox9 functions as a profibrotic transcription factor in activating fibroblasts, illustrating the potential utility of targeting Sox9 in IPF treatment.
PMID:34520400 | DOI:10.1172/jci.insight.152503
Inhibition of PDIA3 in club cells attenuates osteopontin production and lung fibrosis
Thorax. 2021 Aug 16:thoraxjnl-2021-216882. doi: 10.1136/thoraxjnl-2021-216882. Online ahead of print.
BACKGROUND: The role of club cells in the pathology of idiopathic pulmonary fibrosis (IPF) is not well understood. Protein disulfide isomerase A3 (PDIA3), an endoplasmic reticulum-based redox chaperone required for the functions of various fibrosis-related proteins; however, the mechanisms of action of PDIA3 in pulmonary fibrosis are not fully elucidated.
OBJECTIVES: To examine the role of club cells and PDIA3 in the pathology of pulmonary fibrosis and the therapeutic potential of inhibition of PDIA3 in lung fibrosis.
METHODS: Role of PDIA3 and aberrant club cells in lung fibrosis was studied by analyses of human transcriptome dataset from Lung Genomics Research Consortium, other public resources, the specific deletion or inhibition of PDIA3 in club cells and blocking SPP1 downstream of PDIA3 in mice.
RESULTS: PDIA3 and club cell secretory protein (SCGB1A1) signatures are upregulated in IPF compared with control patients. PDIA3 or SCGB1A1 increases also correlate with a decrease in lung function in patients with IPF. The bleomycin (BLM) model of lung fibrosis showed increases in PDIA3 in SCGB1A1 cells in the lung parenchyma. Ablation of Pdia3, specifically in SCGB1A1 cells, decreases parenchymal SCGB1A1 cells along with fibrosis in mice. The administration of a PDI inhibitor LOC14 reversed the BLM-induced parenchymal SCGB1A1 cells and fibrosis in mice. Evaluation of PDIA3 partners revealed that SPP1 is a major interactor in fibrosis. Blocking SPP1 attenuated the development of lung fibrosis in mice.
CONCLUSIONS: Our study reveals a new relationship with distally localised club cells, PDIA3 and SPP1 in lung fibrosis and inhibition of PDIA3 or SPP1 attenuates lung fibrosis.
PMID:34400514 | DOI:10.1136/thoraxjnl-2021-216882
Ileal derived organoids from Crohn's disease patients show unique transcriptomic and secretomic signatures
Cell Mol Gastroenterol Hepatol. 2021 Jul 13:S2352-345X(21)00133-8. doi: 10.1016/j.jcmgh.2021.06.018. Online ahead of print.
BACKGROUND: We used patient derived organoids (PDOs) to study the epithelial-specific transcriptional and secretome signatures of the ileum during CD with varying phenotypes to screen for disease profiles and potential druggable targets.
METHODS: RNA sequencing was performed on isolated intestinal crypts and 3-week-old PDOs derived from ileal biopsies of CD patients (n= 8 B1, inflammatory; n= 8 B2, stricturing disease) and non-IBD controls (n= 13). Differentially expressed (DE) genes were identified by comparing CD vs control, B1 vs B2, and inflamed vs non-inflamed. DE genes were used for computational screening to find candidate small molecules that could potentially reverse B1and B2 gene signatures. The secretome of a second cohort (n= 6 non-IBD controls, n=7 CD; 5 non-inflamed, 2 inflamed) was tested by Luminex using cultured organoid conditioned media.
RESULTS: We found a 90% similarity in both the identity and abundance of protein coding genes between PDOs and intestinal crypts (15,554 transcripts of 19,900 genes). DE analysis identified 814 genes among disease group (CD vs non-IBD control), 470 genes different between the CD phenotypes, and 5 FDR significant genes between inflamed and non-inflamed CD. The PDOs showed both similarity and diversity in the levels and types of soluble cytokines and growth factors they released. Perturbagen analysis revealed potential candidate compounds to reverse B2 disease phenotype to B1 in PDOs.
CONCLUSION: PDOs are similar at the transcriptome level with the in vivo epithelium and retain disease-specific gene expression for which we have identified secretome products, druggable targets and corresponding pharmacological agents. Targeting the epithelium could reverse a stricturing phenotype and improve outcomes.
PMID:34271224 | DOI:10.1016/j.jcmgh.2021.06.018
MGATRx: Discovering Drug Repositioning Candidates Using Multi-view Graph Attention
IEEE/ACM Trans Comput Biol Bioinform. 2021 May 20;PP. doi: 10.1109/TCBB.2021.3082466. Online ahead of print.
In-silico drug repositioning or predicting new indications for approved or late-stage clinical trial drugs is a resourceful and time-ecient strategy in drug discovery. However, inferring novel candidate drugs for a disease is challenging, given the heterogeneity and sparseness of the underlying biological entities and their relationships (e.g., disease/drug annotations). By integrating drug-centric and disease-centric annotations as multiviews, we propose a multi-view graph attention network for indication discovery (MGATRx). Unlike most current similaritybased methods, we employ graph attention network on the heterogeneous drug and disease data to learn the representation of nodes and identify associations. MGATRx outperformed four other state-of-art methods used for computational drug repositioning. Further, several of our predicted novel indications are either currently investigated or are supported by literature evidence, demonstrating the overall translational utility of MGATRx.
PMID:34014830 | DOI:10.1109/TCBB.2021.3082466