Clostridium butyricum upregulates GPR109A/AMPK/PGC-1α and ameliorates acute pancreatitis-associated intestinal barrier injury in mice.

Acute pancreatitis frequently causes intestinal barrier damage, which aggravates pancreatitis. Although Clostridium butyricum exerts anti-inflammatory and protective effects on the intestinal barrier during acute pancreatitis, the underlying mechanism is unclear. The G protein-coupled receptors 109 A (GPR109A) and adenosine monophosphate-activated protein kinase (AMPK)/ peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) signaling pathways can potentially influence the integrity of the intestinal barrier. Our study generated acute pancreatitis mouse models via intraperitoneal injection of cerulein and lipopolysaccharides. After intervention with Clostridium butyricum, the model mice showed reduced small intestinal and colonic intestinal barrier damage, dysbiosis amelioration, and increased GPR109A/AMPK/PGC-1α expression. In conclusion, Clostridium butyricum could improve pancreatic and intestinal inflammation and pancreatic injury, and relieve acute pancreatitis-induced intestinal barrier damage in the small intestine and colon, which may be associated with GPR109A/AMPK/PGC-1α.

Machine learning-based integration develops a stress response stated T cell (Tstr)-related score for predicting outcomes in clear cell renal cell carcinoma.

BACKGROUND: Establishment of a reliable prognostic model and identification of novel biomarkers are urgently needed to develop precise therapy strategies for clear cell renal cell carcinoma (ccRCC). Stress response stated T cells (Tstr) are a new T-cell subtype, which are related to poor disease stage and immunotherapy response in various cancers. METHODS: 10 machine-learning algorithms and their combinations were applied in this work. A stable Tstr-related score (TCs) was constructed to predict the outcomes and PD-1 blockade treatment response in ccRCC patients. A nomogram based on TCs for personalized prediction of patient prognosis was constructed. Functional enrichment analysis and TimiGP algorithm were used to explore the underlying role of Tstr in ccRCC. The key TCs-related gene was identified by comprehensive analysis, and the bioinformatics results were verified by immunohistochemistry using a tissue microarray. RESULTS: A robust TCs was constructed and validated in four independent cohorts. TCs accurately predicted the prognosis and PD-1 blockade treatment response in ccRCC patients. The novel nomogram was able to precisely predict the outcomes of ccRCC patients. The underlying biological process of Tstr was related to acute inflammatory response and acute-phase response. Mast cells were identified to be involved in the role of Tstr as a protective factor in ccRCC. TNFS13B was shown to be the key TCs-related gene, which was an independent predictor of unfavorable prognosis. The protein expression analysis of TNFSF13B was consistent with the mRNA analysis results. High expression of TNFSF13B was associated with poor response to PD-1 blockade treatment. CONCLUSIONS: This study provides a Tstr cell-related score for predicting outcomes and PD-1 blockade therapy response in ccRCC. Tstr cells may exert their pro-tumoral role in ccRCC, acting against mast cells, in the acute inflammatory tumor microenvironment. TNFSF13B could serve as a key biomarker related to TCs.

Exploring the effect of Clostridium butyricum on lung injury associated with acute pancreatitis in mice by combined 16S rRNA and metabolomics analysis.

OBJECTIVES: Acute lung injury is a critical complication of severe acute pancreatitis (SAP). The gut microbiota and its metabolites play an important role in SAP development and may provide new targets for AP-associated lung injury. Based on the ability to reverse AP injury, we proposed that Clostridium butyricum may reduce the potential for AP-associated lung injury by modulating with intestinal microbiota and related metabolic pathways. METHODS: An AP disease model was established in mice and treated with C. butyricum. The structure and composition of the intestinal microbiota in mouse feces were analyzed by 16 S rRNA gene sequencing. Non-targeted metabolite analysis was used to quantify the microbiota derivatives. The histopathology of mouse pancreas and lung tissues was examined using hematoxylin-eosin staining. Pancreatic and lung tissues from mice were stained with immunohistochemistry and protein immunoblotting to detect inflammatory factors IL-6, IL-1ß, and MCP-1. RESULTS: C. butyricum ameliorated the dysregulation of microbiota diversity in a model of AP combined with lung injury and affected fatty acid metabolism by lowering triglyceride levels, which were closely related to the alteration in the relative abundance of Erysipelatoclostridium and Akkermansia. In addition, C. butyricum treatment attenuated pathological damage in the pancreatic and lung tissues and significantly suppressed the expression of inflammatory factors in mice. CONCLUSIONS: C. butyricum may alleviate lung injury associated with AP by interfering with the relevant intestinal microbiota and modulating relevant metabolic pathways.

Corrigendum: Clostridium butyricum protects against pancreatic and intestinal injury after severe acute pancreatitis via downregulation of MMP9.

[This corrects the article DOI: 10.3389/fphar.2022.919010.].

Clostridium butyricum Protects Against Pancreatic and Intestinal Injury After Severe Acute Pancreatitis via Downregulation of MMP9.

Background: Evidence have shown that gut microbiota plays an important role in the development of severe acute pancreatitis (SAP). In addition, matrix metalloproteinase-9 (MMP9) plays an important role in intestinal injury in SAP. Thus, we aimed to determine whether gut microbiota could regulate the intestinal injury during SAP via modulating MMP9. Methods: In this study, the fecal samples of patients with SAP (n = 72) and healthy controls (n = 32) were analyzed by 16S rRNA gene sequencing. In addition, to investigate the association between gut microbiota and MMP9 in intestinal injury during SAP, we established MMP9 stable knockdown Caco2 and HT29 cells in vitro and generated a MMP9 knockout (MMP9-/-) mouse model of SAP in vivo. Results: We found that the abundance of Clostridium butyricum (C. butyricum) was significantly decreased in the SAP group. In addition, overexpression of MMP9 notably downregulated the expressions of tight junction proteins and upregulated the expressions of p-p38 and p-ERK in Caco2 and HT29 cells (p < 0.05). However, C. butyricum or butyrate treatment remarkably upregulated the expressions of tight junction proteins and downregulated the expressions of MMP9, p-p38 and p-ERK in MMP9-overexpressed Caco2 and HT29 cells (p < 0.05). Importantly, C. butyricum or butyrate could not affect the expressions of tight junction proteins, and MMP9, p-p38 and p-ERK proteins in MMP9-knockdown cells compared with MMP9-knockdown group. Consistently, C. butyricum or butyrate could not attenuate pancreatic and intestinal injury during SAP in MMP9-/- mice compared with the SAP group. Conclusion: Collectively, C. butyricum could protect against pancreatic and intestinal injury after SAP via downregulation of MMP9 in vitro and in vivo.

A bispecific nanobody targeting the dimerization interface of epidermal growth factor receptor: Evidence for tumor suppressive actions in vitro and in vivo.

Targeting the dimer interface for the epidermal growth factor receptor (EGFR) that is highly conserved in the structure and directly involved in dimerization may solve the resistance problem that plagues anti-EGFR therapy. Heavy chain single domain antibodies have promising prospects as therapeutic antibodies. A bispecific nanobody was constructed based on previously screened humanized nanobodies that target the ß-loop at the EGFR dimer interface, an anti-FcγRIIIa (CD16) of natural killer cells (NK) nanobodies and anti-human serum albumin (HSA) nanobodies. The target gene was effectively expressed and secreted while controlled by promoter GAP in Pichia pastoris X33, and the expressed product was purified with a cation exchange and nickel chelation chromatography. The bispecific nanobody specifically bound to the surfaces of EGFR-overexpressed human epidermal carcinoma A431 cells and effectively inhibited tumor cell growth both in vitro and in vivo. In the A431 cell nude mouse xenograft model, the growth inhibition effect from the bispecific nanobody was significantly increased with the assistance of peripheral blood mononuclear cells (PBMCs), which was consistent with the results obtained in vitro, suggesting that there was an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. In addition, the intraperitoneal administration of bispecific nanobodies effectively reached tumor tissues in the shoulder dorsal region, but in significantly less distributed quantities than EGFR Dimer Nb77. To conclude, a bispecific nanobody targeting the EGFR dimer interface with ADCC effect was successfully constructed.

Role Stress and Turnover Intention of Front-Line Hotel Employees: The Roles of Burnout and Service Climate.

High turnover rate represents one of the most significant challenges the hotel industry faces. High turnover rates mean labor shortages, resulting in high costs of recruiting, staffing and training. Turnover also has a negative impact on service quality. Scholars continue to search for the root causes of turnover and propose solutions. To further understand employees' turnover intention, this study reveals the role of stress on hotel front-line employees' turnover intention through the mediation of burnout. Moreover, the study examines the moderating effect of service climate on the underlying mechanism that links role stress with turnover intention. Using a sample of 583 questionnaires from front-line hotel employees in South China, this study reveals that role stress as a four-dimensional construct (i.e., conflict, ambiguity, qualitative overload and quantitative overload) has a statistically significant impact on burnout, which leads to turnover intention. Burnout completely mediates the relationship between role stress and turnover intention, that is, employees under role stress do not resign immediately unless they experience high levels of burnout. In addition, service climate moderates the influence of role stress on burnout, suggesting a moderated mediation relationship. The study contributes to the organizational management literature by confirming the four dimensions of role stress and demonstrating how role stress impacts employees' turnover intention. Furthermore, the critical effect of service climate is further investigated. Theoretical contributions and managerial implications are discussed based on the findings. the study also investigates the moderating effect of service climate on role stress (challenge-hindrance stressors) and burnout.

Nanobodies targeting the interaction interface of programmed death receptor 1 (PD-1)/PD-1 ligand 1 (PD-1/PD-L1).

Targeting the interaction interface is an effective strategy to obtain programmed death receptor 1 (PD-1)/PD-1 ligand 1 (PD-L1) nanobody blockers. To validate this strategy, the interaction interface between PD-1 and the PD-L1 extracellular domain were analyzed using Cn3D 4.1. The peptide PD-1125-136 located at the interface of PD-1 was selected as the antigen to screen nanobodies from a humanized nanobody phage display library. Six different nanobodies were screened, with molecular weights of 12 ∼ 13 kDa, excluding a single basic protein. The nanobody with the longest CDR3 region, termed PD-1-Nb-B20, was selected for further analysis. For mass production, the C-terminal His6-tagged nanobody coding sequence was optimized and cloned into pET-21b for over-expression under the T7 promoter in Escherichia coli BL21 (DE3). PD-1-Nb-B20 was expressed and pancreatic adenocarcinoma cells BxPC-3 over-expressing PD-L1 were selected for nanobody competitive inhibition assays. The purified nanobodies significantly inhibited PD-1 binding to the surface of target cells, indicating their ability to block the PD-1/PD-L1 interaction.

[Preparation and identification of single-chain fragment variable against epidermal growth factor receptor 3].

Objective To express, purify and identify the single-chain fragment variable (scFv) against human epidermal growth factor receptor 3 (HER3). Methods We searched NCBI for the light chain sequence and heavy chain sequence of anti-HER3 mAb LJM716 to construct the gene of scFv against HER3. The recombinant expression vector pGAPZαA-anit-HER3-scFv was constructed using the constitutive expression vector pGAPZαA and then electro-transformed into Pichia Pastoris X-33 to screen the strains with high expression of the protein of interest. After shaking flask fermentation, the supernatant was purified by hydrophobic chromatography and metal ion affinity chromatography. The purified product was identified by Western blotting and ELISA. Results The anti-HER3-scFv gene was successfully constructed and the strains with high expression of anti-HER3-scFv were obtained. The anti-HER3-scFv was purified to a purity of more than 95% by two-step chromatography, and the purified yield was 192 mg/L. Western blotting showed that the anti-HER3-scFv was correctly expressed and ELISA indicated that anti-HER3-scFv could specifically recognize HER3. Conclusion The anti-HER3-scFv has been successfully prepared.

Preparation and characterization of humanized nanobodies targeting the dimer interface of epidermal growth factor receptor (EGFR).

Epidermal growth factor receptor (EGFR) is an effective target for the treatment of many epithelial cancers. However, EGFR inhibitors have low clinical response rates and are prone to drug resistance arising from mutations and heterodimerization of EGFR. Therefore, targeting the highly conserved dimer interface of EGFR may be an effective strategy for improving the clinical response of anti-EGFR therapies. Nanobodies have significant advantages over conventional antibodies in terms of size, solubility, stability and cost-effectiveness. To investigate the feasibility of nanobodies targeting the dimer interface of EGFR as novel anticancer drugs, four nanobodies were screened from a commercial humanized nanobody phage antibody library using the EGFR237-267 peptide from the ß-hairpin loop of the dimer interface of EGFR as the antigen. A nanobody with an isoelectric point (pI) of 8.6, named EGFR dimer Nb77, was selected for further analysis of anticancer activities. EGFR dimer Nb77 was expressed in Escherichia coli Shuffle T7-B as a soluble (His)6-tagged protein and purified by a CM Sepharose column and a nickel-nitrilotriacetic acid (Ni-NTA) column. Purified EGFR dimer Nb77 could specifically bind to the surface of EGFR-overexpressing A431 cells in a dose-dependent and ligand-dependent manner, and this nanobody could effectively inhibit the growth of the tumour cells, with an inhibition rate similar to that of the monoclonal antibody EGFR dimer 5G9, which also targets the dimer interface of EGFR. This work is the first to prove that nanobodies targeting the dimer interface of EGFR have promising prospects as anticancer agents.

Application of a nanotechnology antimicrobial spray to prevent lower urinary tract infection: a multicenter urology trial.

BACKGROUND: Catheter-associated urinary tract infection (CAUTI) is a common nosocomial device-associated infection. It is now recognized that the high infection rates were caused by the formation of biofilm on the surface of the catheters that decreases the susceptibility to antibiotics and results in anti-microbial resistance.In this study, we performed an in vitro test to explore the mechanism of biofilm formation and subsequently conducted a multi-center clinical trial to investigate the efficacy of CAUTI prevention with the application of JUC, a nanotechnology antimicrobial spray. METHODS: Siliconized latex urinary catheters were cut into fragments and sterilized by autoclaving. The sterilized sample fragments were randomly divided into the therapy and control group, whereby they were sprayed with JUC and distilled water respectively and dried before use.The experimental standard strains of Escherichia coli (E. coli) were isolated from the urine samples of patients. At 16 hours and 7 days of incubation, the samples were extracted for confocal laser scanning microscopy.A total of 1,150 patients were accrued in the clinical study. Patients were randomized according to the order of surgical treatment. The odd array of patients was assigned as the therapy group (JUC), and the even array of patients was assigned as the control group (normal saline). RESULTS: After 16 hours of culture, bacterial biofilm formed on the surface of sample fragments from the control group. In the therapy group, no bacterial biofilm formation was observed on the sample fragments. No significant increase in bacterial colony count was observed in the therapy group after 7 days of incubation.On the 7th day of catheterization, urine samples were collected for bacterial culture before extubation. Significant difference was observed in the incidence of bacteriuria between the therapy group and control group (4.52% vs. 13.04%, p < 0.001). CONCLUSIONS: In this study, the effectiveness of JUC in preventing CAUTI in a hospital setting was demonstrated in both in vitro and clinical studies.