Enhanced oxidative stress is associated with tissue neutrophilia and poor steroid response in chronic rhinosinusitis with nasal polyps.

Objective: To analyze the oxidative stress status and its association with tissue neutrophilia and oral steroid response in chronic rhinosinusitis with nasal polyps (CRSwNP) patients. Methods: The levels of total oxidant status (TOS) were detected in the sinonasal tissues by using specific assay kits. Tissue neutrophil was examined by immunohistochemical staining, and oxidant status index (OSI) was evaluated in polyps tissues, and the messenger RNA (mRNA) levels of superoxide dismutase 2 (SOD2), aldehyde dehydrogenase 1 (ALDH1A1), and microsomal glutathione S-transferase 1 (MGST1) were examined using quantitative real-time polymerase chain reaction in the sinonasal tissues. The receiver operating characteristics (ROCs) curve of ALDH1A1, MGST1, and SOD2 mRNA levels were evaluated to determine the steroid response of CRSwNP patients. Results: The levels of TOS and OSI were significantly higher in CRSwNP and CRSsNP than in normal controls, and OSI in polyps tissues was positively associated with tissue neutrophilia and poor steroid response. The ALDH1A1, MGST1, and SOD2 mRNA levels showed comparable accuracy as predictors of poor steroid response indicated by the area under the curve. Conclusion: These findings provided evidence that the increased level of oxidative stress contributes to enhanced tissue neutrophilia and poor steroid response in CRSwNP patients.

Major Venous Repair or Reconstruction During Laparoscopic Pancreatic Surgery: A Single Center's Experience.

Background: In pancreatic cancer surgery, tumor violation of blood vessels is often considered a contraindication to surgery, especially laparoscopic surgery. We have completed 17 cases of major venous repair or reconstruction during laparoscopic pancreatic surgery, and we believe that this surgical method may be safe and feasible based on the skilled laparoscopic techniques. Materials and Methods: Between January 2014 and March 2022, a prospective cohort of 17 patients underwent major venous repair or reconstruction in our department. Among them, 15 cases underwent laparoscopic pancreaticoduodenectomy, 1 case underwent laparoscopic distal pancreatectomy, and 1 case underwent laparoscopic central pancreatectomy. In all of these cases, the pancreatic tumor invaded either portal veins (PV) or superior mesenteric veins. Given these clinical situations, 13 cases accepted laparoscopic venous resection and reconstruction, and 4 cases underwent venous repair. Results: Ten of 17 patients (58.8%) were male. The mean age was 67.1 (range 57-81). All patients' operations were successfully completed without transit to open. The average blocking time of venous resection and reconstruction was 30.1 (range 15-41) minutes and the average time of venous wedge resection and stitching was 24.0 (range 18-30) minutes. After surgeries, there were no complications such as PV stenosis, bleeding, thrombosis, and liver failure. Thirteen patients died within 2 years because of the tumor recurrence, and 4 patients are currently followed by outpatient visits, with no obvious signs of tumor recurrence. Conclusion: Studies have shown that the reconstruction or repair of the major veins under laparoscopic surgery is safe and effective. We recommended that surgeons need to have the basics of open surgery in case laparoscopic surgery cannot be continued, and have proficient laparoscopic surgery techniques combined with extensive training to achieve a learning curve for vascular anastomosis. Clinical Trial Registration number: KY2021SL152-01.

DENR controls JAK2 translation to induce PD-L1 expression for tumor immune evasion.

RNA-binding proteins (RBPs) can recognize thousands of RNAs that help to maintain cell homeostasis, and RBP dysfunction is frequently observed in various cancers. However, whether specific RBPs are involved in tumor immune evasion by regulating programmed death ligand-1 (PD-L1) is unclear. Here, we perform targeted RBP CRISPR/Cas9 screening and identify density regulated re-initiation and release factor (DENR) as a PD-L1 regulator. DENR-depleted cancer cells exhibit reduced PD-L1 expression in vitro and in vivo. DENR depletion significantly suppresses tumor growth and enhances the tumor-killing activity of CD8+ T cells. Mechanistically, DENR antagonizes the translational repression of three consecutive upstream open reading frames (uORFs) upstream of Janus kinase 2 (Jak2); thus, DENR deficiency impairs JAK2 translation and the IFNγ-JAK-STAT signaling pathway, resulting in reduced PD-L1 expression in tumors. Overall, we discover an RBP DENR that could regulate PD-L1 expression for tumor immune evasion, and highlight the potential of DENR as a therapeutic target for immunotherapy.

m6A mRNA modification maintains colonic epithelial cell homeostasis via NF-κB-mediated antiapoptotic pathway.

Colonic mucosal barrier dysfunction is one of the major causes of inflammatory bowel disease (IBD). However, the mechanisms underlying mucosal barrier dysfunction are poorly understood. N6-methyladenosine (m6A) mRNA modification is an important modulator of epitranscriptional regulation of gene expression, participating in multiple physiological and pathological processes. However, the function of m6A modification in colonic epithelial cells and stem cells is unknown. Here, we show that m6A modification is essential for maintaining the homeostatic self-renewal in colonic stem cells. Specific deletion of the methyltransferase 14 (Mettl14) gene in mouse colon resulted in colonic stem cell apoptosis, causing mucosal barrier dysfunction and severe colitis. Mechanistically, we revealed that Mettl14 restricted colonic epithelial cell death by regulating the stability of Nfkbia mRNA and modulating the NF-κB pathway. Our results identified a previously unidentified role for m6A modification in colonic epithelial cells and stem cells, suggesting that m6A modification may be a potential therapeutic target for IBD.

CRISPR-based RNA-binding protein mapping in live cells.

RNA-binding proteins (RBPs) are involved in all aspects of RNA metabolism, and RNA-RBP interactions are important for cell homeostasis and viral replication. The global RNA-binding proteome was recently reported; however, little is known about the proteins that bind to specific RNAs. In this study, we describe a novel CRISPR-based RNA interaction proteomics method in live cells. In brief, dCas13a with an HA tag was expressed in cells and bound to an RNA of interest with the help of gRNA. The RNA-protein complexes physically bound to dCas13a-HA were crosslinked using UV light and captured using anti-HA beads, after which the proteins were purified and identified using mass spectrometry. We optimized this system and subsequently applied it to U1 small nuclear RNA, which revealed 226 proteins.

DAPT suppresses proliferation and migration of hepatocellular carcinoma by regulating the extracellular matrix and inhibiting the Hes1/PTEN/AKT/mTOR signaling pathway.

BACKGROUND: The aim of the present study was to investigate the antitumor properties of N-(N-[3,5-difluorophenacetyl]-1-alanyl)-S-phenylglycine t-butyl ester (DAPT) against hepatocellular carcinoma (HCC), as well as the underlying mechanism. METHODS: Immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay were used to determine the expression of Notch1 in HCC tissues. The expression of Notch1 in 3 HCC cell lines was evaluated by qRT-PCR and Western blot. The proliferation ability of cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Flow cytometry and Transwell assay were used to check the apoptosis and migration of HepG2 cells, respectively. Western blot was used to determine the expression level of Notch1, Hes1, Phosphatase and tensin homolog (PTEN), protein kinase B1 (AKT1), phosphorylated AKT1, mammalian target of rapamycin (mTOR), phosphorylated mTOR, intracellular adhesion molecule-1, vascular cell adhesion protein 1, matrix metalloproteinase (MMP)-2, MMP-9, and focal adhesion kinase in cells and tumor tissues. A HepG2 xenograft experiment was conducted to evaluate the in vivo antitumor properties of DAPT. RESULTS: Notch1 was found to be significantly upregulated in both HCC tissues and cell lines. DAPT significantly inhibited the proliferation and migration of HepG2 cells in a dose-dependent manner, accompanied by the suppression of Notch1/Hes1 signaling, inactivation of AKT/mTOR signaling, downregulation of MMPs, and decreased expression of adhesion molecules. The activation of Notch1/Hes1 or AKT/mTOR signaling removed the inhibitory effect of DAPT on the proliferation and migration of HepG2 cells, as well as the inhibitory properties of DAPT on the expression of MMPs and adhesion molecules. The antitumor properties and regulatory effect of DAPT against the extracellular matrix (ECM) and Hes1/PTEN/AKT/mTOR signaling were verified by the HepG2 xenograft experiments. CONCLUSIONS: DAPT could suppress the proliferation and migration of HCC by regulating the ECM and inhibiting the Hes1/PTEN/AKT/mTOR signaling pathway.

miR-3677-5p promotes the proliferation, migration and invasion of hepatocellular carcinoma cells and is associated with prognosis.

MicroRNA (miRNA/miR)-3677 has been indicated to be negatively associated with the survival of patients with hepatocellular carcinoma (HCC) based on The Cancer Genome Atlas database. However, as a novel miRNA, the role of miR-3677-5p in HCC has remained to be elucidated. In the present study, the expression of miR-3677-5p was assessed in HCC tissues and cell lines using reverse transcription-quantitative PCR. Survival analysis was performed using Kaplan-Meier curves. Furthermore, the prognostic significance of miR-3677-5p was evaluated using Cox regression analysis. The effects of miR-3677-5p on cell proliferation, as well as migration and invasion capacities, were analyzed using Cell Counting Kit-8, crystal violet and Transwell assays. The results demonstrated that the level of miR-3677-5p expression was upregulated in human HCC tissues and cell lines and that miR-3677-5p expression was closely associated with tumor size, TNM stage and vascular invasion. Furthermore, high miR-3677-5p expression was significantly associated with unfavorable clinical prognosis for patients with HCC. Overexpression of miR-3677-5p was indicated to significantly promote the proliferation, migration and invasion of HCC cells, whereas knockdown of miR-3677-5p was observed to have an inhibitory effect. In conclusion, the present study demonstrated that miR-3677-5p acts as an oncogene that has a critical role in the regulation of HCC proliferation and progression. Hence, miR-3677-5p may serve as a valuable prognostic biomarker and may be developed as a promising therapeutic target for HCC.

The effect of breathing exercises on patients with GERD: a meta-analysis.

BACKGROUND: Breathing exercises can improve the symptoms of patients with gastroesophageal reflux disease (GERD), but their specific effect and function are disputed. To evaluate and conduct a meta-analysis on the effect of breathing exercises on patients with GERD. METHODS: A literature search for randomized controlled trials (RCTs) and prospective studies on the effects of employing breathing exercises on patients with GERD was conducted of all major online English databases (PubMed, Embase, the Cochrane library, CENTRAL, Web of Science, AMED, and CINAHL). After the systematic review of all the studies according to inclusion and exclusion criteria, we analyzed the extracted data through meta-analysis by using RevMan 5.3 software. RESULTS: This thesis analyzes 7 studies (including three RCTs), which together involved 194 patients and 16 healthy volunteers. The primary outcomes of these studies included GERD symptoms, esophageal manometry, esophageal pH monitoring, laryngoscopic findings, and acid suppression usage. The results of meta-analysis indicate that breathing exercises can improve pressure generated by the lower oesophageal sphincter (LES), and a statistically significant difference was observed. The possible mechanism behind this is the enhancement of the anti-regurgitation barrier [especially crural diaphragm (CD) tension]. CONCLUSIONS: To some extent, breathing exercises can relieve the symptoms of patients with GERD.

The effects of resiquimod in an ovalbumin-induced allergic rhinitis model.

Growing evidence indicates that the Toll-like receptor7/8(TLR7/8) agonist resiquimod (R848) is a potential inhibitor of type-2 immunity. However, the mechanisms mediating its therapeutic effects are not fully understood. This study investigated the effects of R848 on OVA-induced allergic rhinitis(AR) mice and the expression of IL-25, IL-33, TSLP, T-cell immunoglobulin mucin1 (TIM1) and T-cell immunoglobulin mucin3 (TIM3). BALB/c mice were intranasally sensitized and challenged with ovalbumin (OVA), and R848 was intraperitoneally injected into AR mice. Histological changes in the nasal mucosa were evaluated by hematoxylin and eosin (H & E) and Periodic Acid-Schiff (PAS) staining; cytokine levels in serum were measured with enzyme-linked immunosorbent assays (ELISAs);the mRNA expression levels of IFN-γ, IL-17 and Foxp3 in the spleen determined by quantitative real-time RT-PCR (qRT-PCR); the proportions of Th1, Th2, Th17, Treg and TIM3 + IFN-γ + Th1 cells in the spleen were assessed with flow cytometry; TIM1, TIM3 and IL-33 expression levels in the nasal mucosa were evaluated with immunofluorescence staining(IF).R848 alleviated the nasal allergic symptoms; reduced eosinophil cell infiltration, goblet cell hyperplasia in the nasal mucosa; reduced IL-13, IL-17, IL-25 and IL-33 levels in serum; upregulated the relative mRNA expression of IFN-γ and Foxp3, and downregulated the relative mRNA expression of IL-17 in the spleen; decreased Th2, Th17 and TIM3 + IFN-γ + Th1 cells ratios, increased the proportion of Th1 and Treg cells in the spleen; suppressed TIM1 and TIM3,but increased IL-33 expression in the nasal mucosa in OVA-induced AR mice. R848 suppresses IL-25, IL-33 released and TIM1, TIM3 expression, which may contribute to its anti-allergic effects.

Effects of 1,25-dihydroxyvitamin D3 in an ovalbumin-induced allergic rhinitis model.

IL-17-producing Th17 cells play an important role in allergic airway diseases, but their local expression and regulation in allergic rhinitis (AR) is not well understood. This study investigated the effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on T-bet expression, Th1 cells, Th2 cells, Th17 cells and IL-33-positive epithelial cells in AR. C57BL/6 mice were intranasally sensitized and challenged with ovalbumin (OVA), and 1,25-(OH)2D3 was intraperitoneally injected into AR mice. Cytokine levels were measured with enzyme-linked immunosorbent assays, phenotypic analysis of Th1, Th2 and Th17 cells in the spleen was completed with flow cytometry, and the CD4+IL-17+ cells in the Nasopharynx-associated lymphoid tissue (NALT) and IL-33-positive cells in nasal mucosa was evaluated with immunofluorescence microscopy. AR mice shown significantly increased Th2 and Th17 cell ratio in spleen, IL-17 level in serum, IL-5 and IL-13 levels in NALF but a lower number of IL-33-positive epithelial cells and Th1 response (Th1 and Tbet+Th1 cell ratio in the spleen and serum IFN-γ level) than the control mice.1,25-(OH)2D3 treatment significantly decreased the number of sneezing, nasal rubbing, OVA-sIgE and IL-17 in serum, IL-5 and IL-13 levels in NALF, Th17 cell ratio in the spleen and the histological of nasal mucosal but increased the number of IL-33-positive epithelial cells in AR mice. However, 1,25-(OH)2D3 treatment did not significantly influence IFN-γ level in serum, and Th1, Tbet+Th1 and Th2 cell ratio in spleen. Thus, 1,25-(OH)2D3 may exert anti-allergic effects by suppressing Th17 responses and local production of IL-5 and IL-13 cytokines.