Carbon ion irradiation combined with PD-1 inhibitor trigger abscopal effect in Lewis lung cancer via a threshold dose.

Background and goal: Carbon ion beam is radio-biologically more efficient than photons and is beneficial for treating radio-resistant tumors. Several animal experiments with tumor-bearing suggest that carbon ion beam irradiation in combination with immunotherapy yields better results, especially in controlling distant metastases. This implies that carbon ion induces a different anti-tumor immune response than photon beam. More complex molecular mechanisms need to be uncovered. This in vivo and in vitro experiment was carried out in order to examine the radio-immune effects and the mechanism of action of carbon ion beam versus X-ray in combination with PD-1 inhibitors. Methods and Materials: Lewis lung adenocarcinoma cells and C57BL/6 mice were used to create a tumor-bearing mouse model, with the non-irradiated tumor growing on the right hind leg and the irradiated tumor on the left rear. 10Gy carbon ion beam or X-ray radiation, either alone or in combination with PD-1 inhibitor, were used to treat the left back tumor. The expression of molecules linked to immunogenicity and the infiltration of CD8+ T lymphocytes into tumor tissues were both identified using immunohistochemistry. IFN-ß in mouse serum was measured using an ELISA, while CD8+ T cells in mouse peripheral blood were measured using flow cytometry. Lewis cells were exposed to different dose of X-ray and carbon ion. TREX1, PD-L1, and IFN-ß alterations in mRNA and protein levels were identified using Western blot or RT-PCR, respectively. TREX1 knockdown was created by siRNA transfection and exposed to various radiations. Using the CCK8 test, EdU assay, and flow cytometry, changes in cell viability, proliferation, and apoptosis rate were discovered. Results: Bilateral tumors were significantly inhibited by the use of carbon ion or X-ray in combination with PD-1, particularly to non-irradiated tumor(p<0.05). The percentage of infiltrating CD8+ T cells and the level of IFN-ß expression were both raised by 10Gy carbon ion irradiation in the irradiated side tumor, although PD-L1 and TREX1 expression levels were also elevated. Lewis cell in vitro experiment further demonstrated that both X-ray and carbon ion irradiation can up-regulate the expression levels of PD-L1 and TREX1 with dose-dependent in tumors, particularly the trend of up-regulation TREX1 is more apparent at a higher dose in carbon ion, i.e. 8 or 10Gy, while the level of IFN-ß is decreased. IFN-ß levels were considerably raised under hypofractionated doses of carbon ion radiation by gene silencing TREX1. Conclusions: By enhancing tumor immunogenicity and increasing CD8+T infiltration in TME through a threshold dosage, X-ray or carbon ion radiation and PD-1 inhibitors improve anti-tumor activity and cause abscopal effect in Lewis lung adenocarcinoma-bearing mice. TREX1 is a possible therapeutic target and prognostic marker.

Regulatory role of RGMb in lung injury promoted by the combination of carbon ion irradiation and anti-PD-1 antibody through Erk1/2 and p38 MAPK pathways.

The combination of carbon ion radiotherapy and anti-PD-1 antibody represents a new approach to treating thoracic tumors. However, the lung damage caused by this combination therapy may limit its use, and the potential mechanisms for this are worthy of investigation. The objective of this research was to examine the potential involvement of repulsive guidance molecule b (RGMb) in lung damage promoted by the utilization of carbon ion irradiation combined with an anti-PD-1 antibody. The C57BL/6 mice have been randomly separated into four distinct groups: control, anti-PD-1, whole thorax carbon ion irradiation, and irradiation in combination with anti-PD-1 treatment groups (combination group). Detection of pathological changes in lung tissue using HE staining. Detection of pulmonary fibrosis by Masson staining and the hydroxyproline assay. ELISA to detect TNF-α, TGF-ß, IL-6, and IL-1ß expression levels within lung homogenates. The expression of RGMb, p38 MAPK, and Erk1/2 pathways was detected using a fully automated digital Western blotting system WES (ProteinSimple, USA). Flow cytometry was employed to analyze tissue-resident memory T cells (TRM) within the lung. Subsequently, the siRNA gene was employed to induce the downregulation of RGMb in mice in order to validate the involvement of RGMb in radiation-immune lung injury. The present study observed a significant increase in both inflammatory and fibrotic indicators within the mice group's lung tissue that received the combination treatment. The combination group exhibited elevated levels of TGF-ß, TNF-α, IL-6, and IL-1ß in lung homogenates. Anti-PD-1 antibody and carbon ion irradiation, upregulated RGMb, phospho-p38 MAPK and phospho-Erk1/2. The results obtained from the flow cytometry analysis indicated that the combination group was significantly higher in the number of clonal expansion TRMs, which were predominantly characterized by the expression of CD8+CD103+CD69-TRMs. The downregulate of RGMb via siRNA in mice resulted in a decrease in phospho-p38 MAPK and phospho-Erk1/2. The combination group exhibited a reduction in TNF-α, TGF-ß, IL-6, and IL-1ß in their lung tissues, and the number of CD8+CD103+CD69-TRM was significantly reduced. The combination group exhibited a significant improvement in inflammatory and fibrotic indicators within the lung tissues. Anti-PD-1 antibody and carbon ion irradiation synergistically regulate RGMb, leading to strong clonal expansion of lung TRM through the p38 MAPK and Erk1/2 pathways. The present study offers valuable insights into the treatment of lung injury due to the combined administration of carbon ion radiotherapy and anti-PD-1 antibody therapy.

Multi-phase-combined CECT radiomics models for Fuhrman grade prediction of clear cell renal cell carcinoma.

Objective: This study aimed to evaluate the effectiveness of multi-phase-combined contrast-enhanced CT (CECT) radiomics methods for noninvasive Fuhrman grade prediction of clear cell renal cell carcinoma (ccRCC). Methods: A total of 187 patients with four-phase CECT images were retrospectively enrolled and then were categorized into training cohort (n=126) and testing cohort (n=61). All patients were confirmed as ccRCC by histopathological reports. A total of 110 3D classical radiomics features were extracted from each phase of CECT for individual ccRCC lesion, and contrast-enhanced variation features were also calculated as derived radiomics features. These features were concatenated together, and redundant features were removed by Pearson correlation analysis. The discriminative features were selected by minimum redundancy maximum relevance method (mRMR) and then input into a C-support vector classifier to build multi-phase-combined CECT radiomics models. The prediction performance was evaluated by the area under the curve (AUC) of receiver operating characteristic (ROC). Results: The multi-phase-combined CECT radiomics model showed the best prediction performance (AUC=0.777) than the single-phase CECT radiomics model (AUC=0.711) in the testing cohort (p value=0.039). Conclusion: The multi-phase-combined CECT radiomics model is a potential effective way to noninvasively predict Fuhrman grade of ccRCC. The concatenation of first-order features and texture features extracted from corticomedullary phase and nephrographic phase are discriminative feature representations.

Purification identification and function analysis of ACE inhibitory peptide from Ulva prolifera protein.

In the present study, Ulva prolifera, an edible alga, was used to prepare angiotensin-I converting enzyme (ACE) inhibitory peptide. The algae protein was isolated and later hydrolyzed by five commercial enzymes (alcalase, papain, pepsin, trypsin, neutral protease), either individually or in combination. Hydrolysate, with the highest in vitro ACE inhibitory activity, was processed using the Sephadex-G100, ultrafiltration, HPLC-Q-TOF-MS, ADMET screening and molecular docking, respectively. The ACE inhibitory peptide DIGGL with a IC50 value of 10.32 ± 0.96 µM was then identified. The peptide against ACE by a non-competitive mode and mainly attributable to the three Conventional Hydrogen Bonds. It could activate Endothelial nitric oxide synthase activity in NO generation and reduce Endothelin-1 secretion induced by Angiotensin II in Human umbilical vein endothelial cells. Meanwhile, DIGGL could promote mice splenocytes proliferation, which was also effective when co-incubated with Con A or LPS, respectively. Besides, the anti-ACE peptide could remain active during the digestion of gastrointestinal proteases (pepsin-trypsin) in vitro.

The Global research of protein post-translational modifications in the cancer field: A bibliometric and visualized study.

Objectives: Protein post-translational modifications (PTMs) are closely associated with tumorigenesis, targeting PTMs of key proteins might be the focus of antitumor drug discovery. This study aimed to analyze the research progress on protein PTMs in tumorigenesis by performing qualitative and quantitative evaluations. Methods: The Web of Science Core Collection was selected as the database, and Science Citation Index Expanded was selected as the citation index. Visualization tools such as VOSviewer, CiteSpace, HistCite, and Online Analysis Platform of Bibliometrics were used to deeply explore the information of the retrieved research papers and analyze them in terms of research trends and main aspects of research. Results: The search yielded 3777 relevant articles. The results showed that the total number of PTMs related papers in cancer field has been increasing annually, with the largest number of papers published in the United States of America. The co-word cluster analysis showed that the research on PTMs and tumorigenesis was primarily focused on the following four areas, mechanism, histone, P53, key Technologies. Tumor metabolism, autophagy, and protein-protein interaction, histone ubiquitination have become new research topics. Conclusion: This study provides an important reference for the research direction and selection of topics of interest in the PTMs of cancer field.

Searching for the methylation sites involved in human papillomavirus type 16 and 18­positive women with cervical cancer.

It has been reported that >90% of women with cervical cancer are human papillomavirus (HPV)-positive, with HPV16 and 18 being the most 'highest-risk' HPV genotypes. However, in numerous women, HPV infection will not progress to cervical cancer. Accordingly, more appropriate screening markers need to be explored. In the present study, genome-wide DNA methylomic differences between cervical cancer tissues with HPV-16 or HPV-18 infection and normal cervical tissues were detected by using an Illumina Human Methylation 850 K BeadChip. The Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted in order to define the nearest neighbouring genes of differentiated methylation sites. Moreover, differentiated methylation sites were verified using pyrosequencing. KEGG analyses suggested that the focal adhesion pathway and pathways in cancer were highly enriched. Bioinformatics and statistical analysis indicated that the nine CpG loci had the most significant differences amongst the genes involved in these pathways. Among these, six CpG sites in the CHRM2, LAMA4, COL11A1, FGF10, IGF1 and TEK genes were highly associated with HPV-16-positive cervical cancer, as validated using pyrophosphate sequencing. Additionally, 10 significantly different CpG sites of the HPV-18-positive group were selected and verified in The Cancer Genome Atlas, indicating their possible diagnostic roles in cervical cancer development and determination. In addition, eight hypermethylated CpG island sites that were associated with HPV-16-positive cervical cancer tissues and 10 hypermethylated CpG island sites that were associated with HPV-18-positive cervical cancer tissues were identified, highlighting their potential roles in screening and evaluating targeted therapy efficacy and prognosis. The main focus of the present study was to identify the genetic variability in HPV-16- and HPV-18-positive samples and to elucidate possible methylation biomarkers in HPV-positive women with a risk of developing cervical cancer.

Antibiotics Resistance Prevalence of Helicobacter pylori Strains in Northwest China.

Purpose: This study aims to estimate the resistance rate of Helicobacter pylori (HP) to commonly used antibiotics and analyze the potential influencing factors in northwest regions of China. Patients and Methods: HP-positive patients visiting the outpatient department of multiple hospitals were enrolled in the study. Then, gastric mucosal biopsy specimens were collected for HP isolation, culture, and investigation of the resistance rate of HP to amoxicillin, metronidazole, tetracycline, levofloxacin, and clarithromycin by Epsilometer test (E-test) antibiotic susceptibility testing. In addition, multi-drug resistance, the influence of HP eradication history, age, and region of residence on drug resistance rate were analyzed. Results: In total, 198 HP clinical strains were successfully isolated and cultured. The resistance rates of amoxicillin, metronidazole, tetracycline, levofloxacin, and clarithromycin were 16.16%, 85.86%, 7.58%, 46.46%, and 55.05%, respectively. The multi-drug resistance rates demonstrated that dual and triple resistances were 30.30% and 22.73%, respectively. The quadruple resistance rate reached 9.60%. Our results revealed that the prior eradication history of HP significantly increased levofloxacin and clarithromycin resistance. Metronidazole and levofloxacin resistances significantly differed among different age groups, which presented an upward trend with increasing age. Drug resistance rates varied with geographic regions, especially amoxicillin and clarithromycin resistance, which were highest in Hexi Corridor and Longnan regions. Conclusion: The current situation of HP resistance to common antibiotics is severe. Tetracycline is the most sensitive antibiotic, followed by amoxicillin, the first choice for HP eradication. However, the eradication failure of HP may lead to an increase in the resistance rate. Therefore, it is necessary to strengthen the standardized diagnosis and treatment of HP to improve the primary eradication rate.

Vacuolating Cytotoxin A Triggers Mitophagy in Helicobacter pylori-Infected Human Gastric Epithelium Cells.

Helicobacter pylori (H. pylori)-derived vacuolating cytotoxin A (VacA) causes damage to various organelles, including mitochondria, and induces autophagy and cell death. However, it is unknown whether VacA-induced mitochondrial damage can develop into mitophagy. In this study, we found that H. pylori, H. pylori culture filtrate (HPCF), and VacA could activate autophagy in a gastric epithelial cell line (GES-1). VacA-caused mitochondrial depolarization retards the import of PINK1 into the damaged mitochondria and evokes mitophagy. And, among mass spectrometry (LC-MS/MS) identified 25 mitochondrial proteins bound with VacA, Tom20, Tom40, and Tom70, TOM complexes responsible for PINK1 import, were further identified as having the ability to bind VacA in vitro using pull-down assay, co-immunoprecipitation, and protein-protein docking. Additionally, we found that the cell membrane protein STOM and the mitochondrial inner membrane protein PGAM5 also interacted with VacA. These findings suggest that VacA captured by STOM forms endosomes to enter cells and target mitochondria. Then, VacA is transported into the mitochondrial membrane space through the TOM complexes, and PGAM5 aids in inserting VacA into the inner mitochondrial membrane to destroy the membrane potential, which promotes PINK1 accumulation and Parkin recruitment to induce mitophagy. This study helps us understand VacA entering mitochondria to induce the mitophagy process.

Effects of Ginsenoside Rg1 on the Biological Activity of Human Periodontal Ligament Cells.

, Objective. To investigate the effect of ginsenoside Rg1 on the biological activity of primary cultured human periodontal ligament cells (PDLC). Methods. The effects of ginsenoside Rg1 on the proliferation activity, protein synthesis, and alkaline phosphatase (ALP) activity of primary cultured human periodontal ligament cells were investigated by thiazole blue (MTT) colorimetric method, Coomassie brilliant blue method, and enzyme kinetics method. The effect of ginsenoside Rg1 on cell cycle was detected by flow cytometry, and the cells were labeled with calcium ion-sensitive fluorescent probe Fluo3/AM, and the effect of ginsenoside Rg1 on intracellular free calcium concentration was detected by laser scanning confocal microscope. Results. Compared with the control group, the experimental groups of ginsenoside Rg1 at various concentrations could significantly promote cell proliferation, and the effect time was the longest in the concentration range of 0.01-0.05 µmol/L;, Rg1 0.01umol/L and 0.05umol/L. The protein content in the 72-hour cell culture medium of the µmol/L group was significantly higher than that of the control group; the ALP activity in the 72-hour cell culture medium of the Rg1 0.01 µmol/L, 0.05 µmol/L, and 0.1 µmol/L groups was significantly higher than that of the control group; FCM assay showed that after 0.1 µmol/L Rg1 for 48 hours, compared with the control group, the proportion of cells in the early stage of DNA synthesis (G1%) of PDLC was significantly reduced, while the proportion of cells in the DNA synthesis stage (S%) and the value of cell proliferation index PrI (S + G2M)% were significantly increased; Rg1 increased intracellular calcium in PDLC cells at first and then decreased and finally maintained at a slightly higher resting calcium level than before drug addition. Conclusion. Ginsenoside Rg1 can increase the proliferation activity, protein synthesis, and alkaline phosphatase activity of periodontal ligament cells within a certain concentration range; Rg1 reduces the cells in G1 phase and increases cells in S phase of periodontal ligament fibroblasts. Change the concentration of free calcium ions in cells and promote more cells to enter a proliferative state.

Overexpression of splicing factor poly(rC)-binding protein 1 elicits cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells.

PURPOSE: Splicing factor poly(rC)-binding protein 1 (PCBP1) is a novel tumor suppressor that is downregulated in several cancers thereby regulating tumor formation and metastasis. However, the involvement of PCBP1 in apoptosis of cancer cells and the molecular mechanism remains elusive. On this basis, we sought to investigate the role of splicing factor PCBP1 in the apoptosis in human cervical cancer cells. METHODS: To investigate PCBP1 functions in vitro, we overexpressed PCBP1 in human cervical cancer cells. A series of cytological function assays were employed to study to the role of PCBP1 in cell proliferation, cell cycle arrest and apoptosis. RESULTS: Overexpression of PCBP1 was found to greatly repress proliferation of HeLa cells in a time-dependent manner. It also induced a significant increase in G2/M phase arrest and apoptosis. Furthermore, overexpressed PCBP1 favored the production of long isoforms of p73, thereby inducing upregulated ratio of Bax/Bcl-2, the release of cytochrome c and the expression of caspase-3. CONCLUSION: Our results revealed that PCBP1 played a vital role in p73 splicing, cycle arrest and apoptosis induction in human cervical carcinoma cells. Targeting PCBP1 may be a potential therapeutic strategy for cervical cancer therapy.

Ultrasound Radiomics-Guided Iliac Fascia Block on Postoperative Cognitive Dysfunction in Elderly Patients Undergoing Hip Surgery.

Objective: Elderly patients with hip surgery are prone to postoperative cognitive dysfunction (POCD), leading to health management difficulties. This study is aimed at investigating the effect of ultrasound radiomics-guided iliac fascia block on POCD. Methods: A total of 67 cases of patients who had undergone hip joint surgery were divided into a training set (n = 47) and a validation set (radiomics-guided group, n = 20). The patients were intervened with ultrasound radiomics-guided iliac fascia block, and the maximum relevance minimum redundancy sifts out the image omics features obtained from 2D ultrasound images of patients. Another 20 patients undergone general anesthesia served as control. The incidence of POCD, the total amount of fentanyl, the visual analogue score (VAS) at different time points, and the levels of CRP and NSE in plasma were compared between the two groups. Results: The AUC on the training and validation sets were higher than 0.940. The incidence of POCD in the radiomics-guided and general anesthesia group was 5% and 30%, respectively (P = 0.037). Compared with the general anesthesia group, the dosage of fentanyl in the radiomics-guided was lower, the VAS score at 6 h, 1 d, and 2 d after operation was smaller, and the levels of CRP and NSE were lower (all P < 0.05). Conclusions: For elderly patients with hip surgery, the ultrasound radiomics-guided iliac fascia block can reduce the incidence of POCD and improve the effect of nerve block.

Lactobacillus plantarum strains attenuated DSS-induced colitis in mice by modulating the gut microbiota and immune response.

Gut microbiota has become a new therapeutic target in the treatment of inflammatory Bowel Disease (IBD). Probiotics are known for their beneficial effects and have shown good efficacy in the clinical treatment of IBD and animal models of colitis. However, how these probiotics contribute to the amelioration of IBD is largely unknown. In the current study, the DSS-induced mouse colitis model was treated with oral administration of Lactobacillus plantarum strains to investigate their effects on colitis. The results indicated that the L. plantarum strains improved dysbiosis and enhanced the abundance of beneficial bacteria related to short-chain fatty acids (SCFAs) production. Moreover, L. plantarum strains decreased the level of pro-inflammatory cytokines, i.e., IL-17A, IL-17F, IL-6, IL-22, and TNF-α and increased the level of anti-inflammatory cytokines, i.e., TGF-ß, IL-10. Our result suggests that L. plantarum strains possess probiotic effects and can ameliorate DSS colitis in mice by modulating the resident gut microbiota and immune response.

Agmatine and glycolipid metabolism. / 胍丁胺与糖脂代谢.

The prevalence of abnormal glucose and lipid metabolism and its relevant diseases has increased year by year, and it has become a problem that threatens human health. Therefore, finding a more effective way to prevent and treat diseases related to abnormal glucose and lipid metabolism has become an urgent public problem. Agmatine is a polyamine substance which widely presents in mammals.It is a metabolite produced by decarboxylation of L-arginine under the action of arginine decarboxylase, hence also known as decarboxylated arginine. Its biological effects have been confirmed. Previous studies have shown that agmatine possesses anti-diabetic effects in diabetic animals. Agmatine not only increases the insulin secretion form ß-pancreatic cells to inhibit the hyperglycemia, but also attenuates insulin resistance in rats. Agmatine also plays a positive role in lipid metabolism disorders and related diseases by modulating lipid metabolism and fatty acid oxidation.

Protein acetylation and deacetylation: An important regulatory modification in gene transcription (Review).

Cells primarily rely on proteins to perform the majority of their physiological functions, and the function of proteins is regulated by post-translational modifications (PTMs). The acetylation of proteins is a dynamic and highly specific PTM, which has an important influence on the functions of proteins, such as gene transcription and signal transduction. The acetylation of proteins is primarily dependent on lysine acetyltransferases and lysine deacetylases. In recent years, due to the widespread use of mass spectrometry and the emergence of new technologies, such as protein chips, studies on protein acetylation have been further developed. Compared with histone acetylation, acetylation of non-histone proteins has gradually become the focus of research due to its important regulatory mechanisms and wide range of applications. The discovery of specific protein acetylation sites using bioinformatic tools can greatly aid the understanding of the underlying mechanisms of protein acetylation involved in related physiological and pathological processes.

Fertility preservation in BRCA mutation carriers-efficacy and safety issues: a review.

BRCA mutation carriers face various situations that influence their fertility potential. There is still a lack of guideline or expert consensus on Fertility Preservation (FP) in BRCA mutation carriers and the necessity and safety of FP in BRCA mutation carriers is still in dispute. This review aims to focus on the population of BRCA mutation carriers by analyzing the existing FP strategies, comprehensively comparing the pros and cons of each strategy and its applicability.FP is a suggestion for BRCA mutation carriers with birth planning. Different FP strategies have different characteristics. Considering the particularity of BRCA mutation carriers, multiple factors need to be carefully considered. This review focuses on the applicability of each FP method for carriers under various circumstances. Available FP strategies including oocyte cryopreservation, ovarian tissue cryopreservation, preimplantation genetic diagnosis, and egg/embryo donation are analyzed by comparing existing methods comprehensively. In the attempt to provide an up-to-date decision-making guidance. Conditions taking into consideration were the carrier's age, the risk of breast and ovarian metastasis, plans for oncotherapy, FP outcome, time available for FP intervention and accessibility.Overall, FP is necessary and safe for BRCA mutation carriers. Among all available FP methods, oocyte cryopreservation is the most reliable procedure; ovarian tissue cryopreservation is the only way for preserving both fertility and endocrine function, recommended for pre-pubertal carriers and when time is limited for oocyte stimulation. A clear framework provides frontline clinical practitioners a new thought and eventually benefit thousands of BRCA mutation carriers.

Molecular epidemiology of Chinese Han deaf patients with bi-allelic and mono-allelic GJB2 mutations.

BACKGROUND: Recessive mutations in GJB2 is the most common cause of genetic hearing loss worldwide. The aim of this study is to determine the spectrum and frequency of GJB2 variants in Chinese Han deaf patients and to investigate the underlying causative genes in patients with mono-allelic GJB2 mutations. METHODS: We analyzed the mutation screening results of GJB2 in 1852 Chinese Han probands with apparently autosomal-recessive hearing loss in our laboratory. Targeted next-generation sequencing of 139 known deafness-related genes were performed in 44 probands with mono-allelic GJB2 mutations. RESULTS: Bi-allelic GJB2 mutations was identified in 25.65% of patients, in which the c.235delC (p.L79Cfs*3) mutation is the most frequent cause for both severe-to-profound (84.93%) and mild-to-moderate hearing loss (54.05%), while the c.109G > A (p.V37I) mutation is another frequent cause for mild-to-moderate hearing loss (40.54%). In 3.89% of patients only one mutant allele can be identified in GJB2. Targeted next generation sequencing in 44 such probands revealed digenic heterozygous mutations in GJB2/GJB6 and GJB2/GJB3 as the likely pathogenic mechanism in three probands. In 13 probands, on the other hand, pathogenic mutations in other deafness-associated genes (STRC, EYA1, MITF, PCDH15, USH2A, MYO15A, CDH23, OTOF, SLC26A4, SMPX, and TIMM8A) can be identified as the independent genetic cause, suggesting that the mono-allelic GJB2 mutations in those probands is likely co-incidental. CONCLUSIONS: Our results demonstrated that GJB2 should be a primary target for mutation screening in Chinese Han deaf patients, and those with mono-allelic GJB2 mutations should be further screened by next generation sequencing.

NOS cofactor tetrahydrobiopterin contributes to anesthetic preconditioning induced myocardial protection in the isolated ex vivo rat heart.

Anesthetic preconditioning (APC) may decrease the myocardium injury nearly 50% following ischemia/reperfusion (I/R) by enhancing recovery of cardiac function, reducing myocardial enzyme release and lowering infarct size when utilized as pretreatment or posttreatment agents. I/R increases nitric oxide (NO) production through endothelial NO synthase (NOS3) and heat shock protein 90 (HSP90). The present study aimed to observe the role of BH4 availability and the association of HSP90 with NOS3 in APC­mediated cardioprotection against I/R injury. Isolated rat hearts were subjected to no­flow ischemia for 30 min and reperfusion for 120 min. Sevoflurane (3.5%) was administered for 15 min followed by a 15 min washout prior to ischemia. 2,4-Diamino-6-hydroxypyrimidine (DAHP) or sepiapterin (SP) was administered for 40 min until the onset of ischemia. The results revealed that compared with pre­ischemic basal levels, BH4 levels decreased and BH2 levels increased following I/R. BH4 levels were significantly increased and BH2 levels were significantly decreased in the APC + I/R hearts compared with the I/R group hearts. The BH4:BH2 ratio in the APC­treated hearts was also increased compared with that in the I/R group hearts. SP increased the recovery of contractile function and the production of NO, and decreased the production of superoxide anion (O2·­) in I/R heart, but did not elicit these effects in APC­treated hearts. DAHP treatment inhibited the APC­mediated recovery of contractile function, increased O2·­ levels and decreased NO production, but had no effect in I/R hearts. The cardioprotection of APC was demonstrated to be modulated by the BH4 precursor SP, which increased BH4 levels, or DAHP, which inhibited GTP cyclohydrolase I. Both APC and SP treatments increased the combination of HSP90 and NOS3, which improved the NOS3 activity and function. The results suggested that BH4, which servesas a cofactor for NOS, mediated the resistance of APC to I/R injury by promoting the binding of HSP90 and NOS3.

Effects of isoflurane on complex II­associated mitochondrial respiration and reactive oxygen species production: Roles of nitric oxide and mitochondrial KATP channels.

Volatile anesthetics may protect the heart against ischemia­reperfusion injury via the direct action on mitochondrial complexes and by regulating the production of reactive oxygen species (ROS). Recently, we reported that isoflurane induced the attenuation of mitochondrial respiration caused by complex I substrates. This process was not associated with endogenous production of mitochondrial nitric oxide (NO). In the present study, we investigated the effects of isoflurane on mitochondrial respiration and ROS production using complex II substrates. The detailed mechanism of these effects was explored with regards to NO production and the expression of mitochondrial ATP­dependent K+ (mKATP) channels. Mitochondria were isolated from the heart of Sprague­Dawley rats. The respiratory rates of mitochondria (0.5 mg/ml) were measured via polarography at 28˚C with computer­controlled Clark­type O2 electrodes. The complex II substrate succinate (5 mM) was used; 0.25 mM of isoflurane was administered prior to ADP­initiated state 3 respiration. The mitochondrial membrane potential (ΔΨm) was measured under treatment with the substrate succinate, or succinate in the presence of the complex I inhibitor rotenone. The detection was achieved in a cuvette­based spectrophotometer operating at wavelengths of 503 nm (excitation) 527 nm (emission) in the presence of 50 nM of the fluorescent dye rhodamine 123. The H2O2 release rates in the mitochondria were measured spectrophotometrically with succinate, or succinate and rotenone using the fluorescent dye Amplex red (12.5­25 µM). The results indicated that isoflurane increased the state 3 and 4 respiration rates caused by succinate, which were higher than those noted in the control group in the presence of succinate alone. The NOS inhibitor L­NIO or the NO­sensitive guanylyl cyclase 1H­[1,2,4]oxadiazolo[4,3­a]quinoxalin­1­one did not inhibit the increase in the respiration rate (state 3) induced by isoflurane. The ROS scavengers SPBN and manganese (III) tetrakis (4­benzoic acid) porphyrin chloride inhibited the increase in the respiration rate (state 3 and 4) induced by isoflurane. This effect was not noted for the putative KATP channel blockers 5­hydroxydecanoic acid and glibenclamide. Isoflurane caused a greater decrease in the concentration of H2O2 during ADP­initiated state 3 respiration, and L­N5­(1­Iminoethyl)­ornithine did not inhibit this effect. In conclusion, isoflurane was determined to modulate mitochondrial respiration and ROS production caused by the complex II substrate succinate. These effects were independent of endogenous mitochondrial NO generation and mitochondrial KATP channel opening.

Alteration of Gut Microbiota in Inflammatory Bowel Disease (IBD): Cause or Consequence? IBD Treatment Targeting the Gut Microbiome.

Inflammatory bowel disease (IBD) is a chronic complex inflammatory gut pathological condition, examples of which include Crohn's disease (CD) and ulcerative colitis (UC), which is associated with significant morbidity. Although the etiology of IBD is unknown, gut microbiota alteration (dysbiosis) is considered a novel factor involved in the pathogenesis of IBD. The gut microbiota acts as a metabolic organ and contributes to human health by performing various physiological functions; deviation in the gut flora composition is involved in various disease pathologies, including IBD. This review aims to summarize the current knowledge of gut microbiota alteration in IBD and how this contributes to intestinal inflammation, as well as explore the potential role of gut microbiota-based treatment approaches for the prevention and treatment of IBD. The current literature has clearly demonstrated a perturbation of the gut microbiota in IBD patients and mice colitis models, but a clear causal link of cause and effect has not yet been presented. In addition, gut microbiota-based therapeutic approaches have also shown good evidence of their effects in the amelioration of colitis in animal models (mice) and IBD patients, which indicates that gut flora might be a new promising therapeutic target for the treatment of IBD. However, insufficient data and confusing results from previous studies have led to a failure to define a core microbiome associated with IBD and the hidden mechanism of pathogenesis, which suggests that well-designed randomized control trials and mouse models are required for further research. In addition, a better understanding of this ecosystem will also determine the role of prebiotics and probiotics as therapeutic agents in the management of IBD.

A ß-glucosidase-producing M-2 strain: Isolation from cow dung and fermentation parameter optimization for flaxseed cake.

Flaxseed cake contains cyanogenic glucosides, which can be metabolized into hydrocyanic acid in an animal's body, leading to asphyxia poisoning in cells. Beta-glucosidase is highly efficient in degrading cyanogenic glucosides. The Cattle may have ß-glucosidase-producing strains in the intestinal tract after eating small amounts of flaxseed cake for a long time. This study aimed to isolate of a strain from cow dung that produces ß-glucosidase with high activity and can significantly reduce the amount of cyanogenic glucosides. We used cow dung as the microflora source and an esculin agar as the selective medium. After screening with 0.05% esculin and 0.01% ferric citrate, we isolated 5 strains producing high amounts of ß-glucosidase. In vitro flaxseed cake fermentation was fermented by these 5 strains, in which the strain M-2 exerted the best effect (P < 0.05). The strain M-2 was identified as Lichtheimia ramosa and used as the fermentation strain to optimize the fermentation parameters by a single factor analysis and orthogonal experimental design. The optimum condition was as follows: inoculum size 3%, water content 60%, time 144 h, and temperature 32 °C. Under this condition, the removal rate of cyanogenic glucosides reached 89%, and crude protein increment reached 44%. These results provided a theoretical basis for the removal of cyanogenic glucosides in flaxseed and the comprehensive utilization of flaxseed cake.