An essential function for autocrine Hedgehog signaling in epithelial proliferation and differentiation in the trachea

The tracheal epithelium is a primary target for pulmonary diseases as it provides a conduit for air flow between the environment and the lung lobes. The cellular and molecular mechanisms underlying airway epithelial cell proliferation and differentiation remain poorly understood. Hedgehog (Hh) signaling orchestrates communication between epithelial and mesenchymal cells in the lung, where it modulates stromal cell proliferation, differentiation and signaling back to the epithelium. Here, we reveal a new, autocrine function of Hh signaling in airway epithelial cells. Epithelial cell depletion of the ligand Sonic hedgehog (SHH) or its effector Smoothened (SMO) causes defects in both epithelial cell proliferation and differentiation. In cultured primary human airway epithelial cells, Hh signaling inhibition also hampers cell proliferation and differentiation. Epithelial Hh function is mediated, at least in part, through transcriptional activation as Hh signaling inhibition leads to downregulation of cell-type specific transcription factor genes in both the mouse trachea and human airway epithelial cells. These results provide new insights into the role of Hh signaling in epithelial cell proliferation and differentiation during airway development.

Transcriptome Analysis of Pterygium and Pinguecula Reveals Evidence of Genomic Instability Associated with Chronic Inflammation

Solar damage due to ultraviolet radiation (UVR) is implicated in the development of two proliferative lesions of the ocular surface: pterygium and pinguecula. Pterygium and pinguecula specimens were collected, along with adjacent healthy conjunctiva specimens. RNA was extracted and sequenced. Pairwise comparisons were made of differentially expressed genes (DEGs). Computational methods were used for analysis. Transcripts from 18,630 genes were identified. Comparison of two subgroups of pterygium specimens uncovered evidence of genomic instability associated with inflammation and the immune response; these changes were also observed in pinguecula, but to a lesser extent. Among the top DEGs were four genes encoding tumor suppressors that were downregulated in pterygium: C10orf90, RARRES1, DMBT1 and SCGB3A1; C10orf90 and RARRES1 were also downregulated in pinguecula. Ingenuity Pathway Analysis overwhelmingly linked DEGs to cancer for both lesions; however, both lesions are clearly still benign, as evidenced by the expression of other genes indicating their well-differentiated and non-invasive character. Pathways for epithelial cell proliferation were identified that distinguish the two lesions, as well as genes encoding specific pathway components. Upregulated DEGs common to both lesions, including KRT9 and TRPV3, provide a further insight into pathophysiology. Our findings suggest that pterygium and pinguecula, while benign lesions, are both on the pathological pathway towards neoplastic transformation.

Comparative analysis of therapeutic effects between medium cut-off and high flux dialyzers using metabolomics and proteomics: exploratory, prospective study in hemodialysis

In this single-center prospective study of 20 patients receiving maintenance hemodialysis (HD), we compared the therapeutic effects of medium cut-off (MCO) and high flux (HF) dialyzers using metabolomics and proteomics. A consecutive dialyzer membrane was used for 15-week study periods: 1st HF dialyzer, MCO dialyzer, 2nd HF dialyzer, for 5 weeks respectively. 1H-nuclear magnetic resonance was used to identify the metabolites and liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis was used to identify proteins. To compare the effects of the HF and MCO dialyzers, orthogonal projection to latent structure discriminant analysis (OPLS-DA) was performed. OPLS-DA showed that metabolite characteristics could be significantly classified by 1st HF and MCO dialyzers. The Pre-HD metabolites with variable importance in projection scores ≥ 1.0 in both 1st HF versus MCO and MCO versus 2nd HF were succinate, glutamate, and histidine. The pre-HD levels of succinate and histidine were significantly lower, while those of glutamate were significantly higher in MCO period than in the HF period. OPLS-DA of the proteome also substantially separated 1st HF and MCO periods. Plasma pre-HD levels of fibronectin 1 were significantly higher, and those of complement component 4B and retinol-binding protein 4 were significantly lower in MCO than in the 1st HF period. Interestingly, as per Ingenuity Pathway Analysis, an increase in epithelial cell proliferation and a decrease in endothelial cell apoptosis occurred during the MCO period. Overall, our results suggest that the use of MCO dialyzers results in characteristic metabolomics and proteomics profiles during HD compared with HF dialyzers, which might be related to oxidative stress, insulin resistance, complement-coagulation axis, inflammation, and nutrition.

Dihydroartemisinin Ameliorates Chronic Nonbacterial Prostatitis and Epithelial Cellular Inflammation by Blocking the E2F7/HIF1α Pathway

Objective: Chronic nonbacterial prostatitis (CNP) has remained one of the most prevalent urological diseases, particularly in older men. Dihydroartemisinin (DHA) has been identified as a semi-synthetic derivative of artemisinin that exhibits broad protective effects. However, the role of DHA in inhibiting CNP inflammation and prostatic epithelial cell proliferation remains largely unknown. Materials and Methods: CNP mice model was induced by carrageenan and Haemotoxylin Eosin (HE) ,immunofluorescence and immunochemistry staining were used to confirm CNP and E2F7 expression. Human prostatic epithelial cells (HPECs) and RWPE-1 was induced by lipopolysaccharide (LPS) to mimic CNP model in vitro. Real-time quantitative PCR and Western blot were used to detect proliferation and inflammatory genes expression. Cell proliferation was determined using MTT assay.Results: DHA significantly alleviated the rough epithelium and inhibited multilamellar cell formation in the prostatic gland cavity and prostatic index induced by carrageenan. In addition, DHA decreased the expression of TNF-α and IL-6 inflammatory factors in prostatitis tissues and in LPS-induced epithelial cells. Upregulation of transcription factor E2F7, which expression was inhibited by DHA, was found in CNP tissues, human BPH tissues and LPS-induced epithelial cells inflammatory response. Mechanically, we found that depletion of E2F7 by shRNA inhibited epithelial cell proliferation and LPS-induced inflammation while DHA further enhance these effects. Furthermore, HIF1α was transcriptional regulated by E2F7 and involved in E2F7-inhibited CNP and cellular inflammatory response. Interestingly, we found that inhibition of HIF1α blocks E2F7-induced cell inflammatory response but does not obstruct E2F7-promoted cell growth.Conclusion: The results revealed that DHA inhibits the CNP and inflammation by blocking the E2F7/HIF1α pathway. Our findings provide new evidence for the mechanism of DHA and its key role in CNP, which may provide an alternative solution for the prevention and treatment of CNP.

SARS-CoV-2 ORF8 Forms Intracellular Aggregates and Inhibits IFNγ-Induced Antiviral Gene Expression in Human Lung Epithelial Cells

Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a disease that involves significant lung tissue damage. How SARS-CoV-2 infection leads to lung injury remains elusive. The open reading frame 8 (ORF8) protein of SARS-CoV-2 (ORF8SARS-CoV-2) is a unique accessory protein, yet little is known about its cellular function. We examined the cellular distribution of ORF8SARS-CoV-2 and its role in the regulation of human lung epithelial cell proliferation and antiviral immunity. Using live imaging and immunofluorescent staining analyses, we found that ectopically expressed ORF8SARS-CoV-2 forms aggregates in the cytosol and nuclear compartments of lung epithelial cells. Using in silico bioinformatic analysis, we found that ORF8SARS-CoV-2 possesses an intrinsic aggregation characteristic at its N-terminal residues 1-18. Cell culture did not reveal any effects of ORF8SARS-CoV-2 expression on lung epithelial cell proliferation and cell cycle progression, suggesting that ORF8SARS-CoV-2 aggregates do not affect these cellular processes. Interestingly, ectopic expression of ORF8SARS-CoV-2 in lung epithelial cells suppressed basal expression of several antiviral molecules, including DHX58, ZBP1, MX1, and MX2. In addition, expression of ORF8SARS-CoV-2 attenuated the induction of antiviral molecules by IFNγ but not by IFNβ in lung epithelial cells. Taken together, ORF8SARS-CoV-2 is a unique viral accessory protein that forms aggregates when expressing in lung epithelial cells. It potently inhibits the expression of lung cellular anti-viral proteins at baseline and in response to IFNγ in lung epithelial cells, which may facilitate SARS-CoV-2 escape from the host antiviral innate immune response during early viral infection. In addition, it seems that formation of ORF8SARS-CoV-2 aggregate is independent from the viral infection. Thus, it would be interesting to examine whether any COVID-19 patients exhibit persistent ORF8 SARS-CoV-2 expression after recovering from SARS-CoV-2 infection. If so, the pathogenic effect of prolonged ORF8SARS-CoV-2 expression and its association with post-COVID symptoms warrant investigation in the future.