[Weidiao-3 Mixture Improves the Clinical Efficacy of Immunotherapy for Advanced Gastric Cancer by Regulating Intestinal Flora].

Objective To investigate the effects of Weidiao-3(WD-3)Mixture on the clinical efficacy of immunotherapy for advanced gastric cancer and the intestinal flora.Methods Fifty-one patients with advanced gastric cancer treated in Wuxi Traditional Chinese Medicine Hospital from January 2020 to December 2021 were randomized into a WD-3 group(immunotherapy + WD-3 Mixture,one dose per day)(n=25)and a gastric cancer(GC) group(only immunotherapy)(n=26)according to the admission time.Ten healthy volunteers were included as the healthy control group.The Karnofsky score and the Quality of Life Questionnare-Core score were evaluated before and after treatment,and the clinical efficacy was compared after treatment.After treatment,the stool samples were collected for 16SrRNA gene high-throughput sequencing and targeted metabolomics.The α and ß diversity and structure of the intestinal flora and the content of short-chain fatty acids were compared between groups.Results The quality of life in both groups improved after treatment and was better in the WD-3 group than in the GC group(P=0.035).The dry mouth(P=0.038)and altered taste(P=0.008)were mitigated in the WD-3 group after treatment,and the reflux(P=0.001)and dry mouth(P=0.022)were mitigated in the GC group after treatment.After treatment,the WD-3 group outperformed the GC group in terms of dysphagia(P=0.047)and dry mouth(P=0.045).The WD-3 group was superior to the GC group in terms of objective remission rate and disease control rate,with prolonged median progression-free survival and median overall survival(P=0.039,P=0.043).The α and ß diversity indexes of the intestinal flora showed no significant differences between WD-3 and GC groups(all P>0.05).At the phylum level,WD-3 and GC groups had lower relative abundance of Firmicutes(P=0.038,P=0.042)and higher relative abundance of Proteobacteria(P=0.016,P=0.015)than the healthy control group.The relative abundance of Actinomycetes in the GC group was lower than that in the healthy control group(P=0.035)and the WD-3 group(P=0.046).At the genus level,the GC group had lower relative abundance of Bifidobacteria and Coprococcus than the healthy control group and the WD-3 group(all P<0.001).LEfSe revealed the differences in the relative abundance of 6 intestinal bacterial taxa between the WD-3 group and the GC group.At the genus level,Saccharopolyspora had higher relative abundance in the WD-3 group than in the healthy control group and only existed in the WD-3 group.The content of isobutyric acid and isovaleric acid in the WD-3 group was higher than that in the healthy control group(P=0.037,P=0.004).Conclusion WD-3 Mixture may increase the relative abundance of Bifidobacteria and Coprococcus and the content of isobutyric acid and isovaleric acid to alter the intestinal microecology,thereby improving the efficacy of immunotherapy for gastric cancer.

Aetiology of superficial fungal infections of the foot in urban outpatients in mainland China: A multicentre, prospective case study.

BACKGROUND: In China, the prevalence of superficial fungal infections of the foot is high and recurrence is common. However, a prospective, large-scale and multicentre study on the aetiology of superficial fungal infections of the foot is still lacking. OBJECTIVES: To study the epidemiology of aetiological agents of superficial fungal infections of the foot in urban outpatients in mainland China, as well as to understand the aetiology features of the pathogenic agent. METHODS: The study was designed as a multicentre, prospective epidemiological survey. A total of 1704 subjects were enrolled from seven geographical areas in mainland China. For each subject, one mycological sample and one bacterial sample were collected. KOH wet mount examination and culture were performed at local laboratories. The bacterial results were only reported in those with positive mycology. Further morphological identification and, if necessary, molecular biological identification were conducted in a central laboratory. RESULTS: Of 1704 enrolled subjects, 1327 (77.9%) subjects had positive fungal culture results. The incidence of dermatophytes, yeasts and moulds was 90.1%, 8.1% and 1.1%, respectively. The most frequently isolated aetiological agent (fungus) was Trichophyton rubrum. Moccasin form was the most commonly reported clinical diagnosis of superficial fungal infections. The most frequently isolated bacterial genus in patients was Staphylococcus. CONCLUSION: This study prospectively investigated the clinical and mycological features of human dermatophytosis in mainland China. T rubrum was the most frequently isolated fungus, and moccasin form was the most commonly reported clinical diagnosis of superficial fungal infections.

Mitochondrial protein 18 is a positive apoptotic regulator in cardiomyocytes under oxidative stress.

Accumulation of reactive oxygen species is a common phenomenon in cardiac stress conditions, for instance, coronary artery disease, aging-related cardiovascular abnormalities, and exposure to cardiac stressors such as hydrogen peroxide (H2O2). Mitochondrial protein 18 (Mtp18) is a novel mitochondrial inner membrane protein, shown to involve in the regulation of mitochondrial dynamics. Although Mtp18 is abundant in cardiac muscles, its role in cardiac apoptosis remains elusive. The present study aimed to detect the role of Mtp18 in H2O2-induced mitochondrial fission and apoptosis in cardiomyocytes. We studied the effect of Mtp18 in cardiomyocytes by modulating its expression with lentiviral construct of Mtp18-shRNA and Mtp18 c-DNA, respectively. We then analyzed mitochondrial morphological dynamics with MitoTracker Red staining; apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) and cell death detection assays; and protein expression with immunoblotting. Here, we observed that Mtp18 could regulate oxidative stress- mediated mitochondrial fission and apoptosis in cardiac myocytes. Mechanistically, we found that Mtp8 induced mitochondrial fission and apoptosis by enhancing dynamin-related protein 1 (Drp1) accumulation. Conversely, knockdown of Mtp18 interfered with Drp1-associated mitochondrial fission and subsequent activation of apoptosis in both HL-1 cells and primary cardiomyocytes. However, overexpression of Mtp18 alone was not sufficient to execute apoptosis when Drp1 was minimally expressed, suggesting that Mtp18 and Drp1 are interdependent in apoptotic cascade. Together, these data highlight the role of Mtp18 in cardiac apoptosis and provide a novel therapeutic insight to minimize cardiomyocyte loss via targetting mitochondrial dynamics.

MicroRNA-381 Favors Repair of Nerve Injury Through Regulation of the SDF-1/CXCR4 Signaling Pathway via LRRC4 in Acute Cerebral Ischemia after Cerebral Lymphatic Blockage.

BACKGROUND/AIMS: Acute cerebral ischemia is a manifestation of cerebral vascular insufficiency and has a high mortality. However, the therapy for acute cerebral ischemia is still limited. This study aimed to investigate the effect of microRNA-381 (miR-381) on the repair of nerve injury in rats with acute cerebral ischemia after cerebral lymphatic blockage (CLB) by targeting leucine-rich repeat C4 protein (LRRC4) through the Stromal cell-derived factor-1/CXC chemokine receptor-4 signaling pathway. METHODS: Rat models of CLB and middle cerebral artery occlusion (MCAO) were established, and 56 Wistar rats were divided into sham, MCAO, CLB + MCAO, CLB + MCAO + miR-381 inhibitor, CLB + MCAO + miR-381 mimic, CLB + MCAO + AMD3100 and CLB + MCAO + miR-381 mimic + AMD3100 groups. Modified neurological severity score (mNSS was used to determine nerve injury, TTC staining to measure infarction volume, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and flow cytometry to evaluate cell apoptosis, immunofluorescence to measure BrdU-positive cell number, enzyme-linked immunosorbent assay (ELISA) to determine contents of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10), nerve growth factor (NGF) and neurite outgrowth inhibitor -A (Nogo-A), Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting to evaluate expression of miR-381, LRRC4, SDF-1, CXCR4, pERK, Slit2 and vascular endothelial growth factor (VEGF). RESULTS: LRRC4 was a target gene of miR-381. Compared with the results in the CLB + MCAO group, mNSS, infarction volume, apoptosis rate and TNF-α, IL-1ß, IL-6 and Nogo-A contents as well as LRRC4 expression in the CLB + MCAO + miR-381 inhibitor and CLB + MCAO + AMD3100 groups were increased (those in the CLB + MCAO + AMD3100 group > those in the CLB + MCAO + miR-381 mimic + AMD3100 group), while BrdU-positive cell number, contents of NGF and IL-10, and expression of SDF-1, CXCR4, pERK, Slit2 and VEGF in brain tissues were decreased (those in the CLB + MCAO + AMD3100 group < those in the CLB + MCAO + miR-381 mimic + AMD3100 group). The results in the CLB + MCAO + mimic group were opposite of those in the CLB + MCAO + miR-381 inhibitor and CLB + MCAO + AMD3100 groups. CONCLUSION: Taken together, we concluded that up-regulation of miR-381 promoted nerve injury repair in acute cerebral ischemia rats after CLB by negatively regulating LRRC4 through activating the SDF-1/CXCR4 signaling pathway.

O-Phenylenediamine: a privileged pharmacophore of ferrostatins for radical-trapping reactivity in blocking ferroptosis.

Ferroptosis is a non-apoptotic, iron dependent form of regulated cell death that is characterized by the accumulation of lipid hydroperoxides. It has drawn considerable attention owing to its putative involvement in diverse neurodegenerative diseases. Ferrostatins are the first identified inhibitors of ferroptosis and they inhibit ferroptosis by efficiently scavenging free radicals in lipid bilayers. However, their further medicinal application has been limited due to the deficient knowledge of the lipid peroxyl radical-trapping mechanism. In this study, experimental and theoretical methods were performed to illustrate the possible lipid hydroperoxide inhibition mechanism of ferrostatins. The results show that an ortho-amine (-NH) moiety from ferrostatins can simultaneously interact with lipid radicals, and then form a planar seven-membered ring in the transition state, and finally present greater reactivity. NBO analysis shows that the formed planar seven-membered ring forces ortho-amines into better alignment with the aromatic π-system. It significantly increases the magnitudes of amine conjugation and improves spin delocalization in the transition state. Additionally, a classical H-bond type interaction was discovered between a radical and an o-NH group as another transition state stabilizing effect. This type of radical-trapping mechanism is novel and has not been found in diphenylamine or traditional polyphenol antioxidants. It can be said that o-phenylenediamine is a privileged pharmacophore for the design and development of ferroptosis inhibitors.

Isolation and determination of four potential antimicrobial components from Pseudomonas aeruginosa extracts.

Background:Pseudomonas aeruginosa can cause disease and also can be isolated from the skin of healthy people. Additionally, it exhibits certain antimicrobial effects against other microorganisms.Methods: We collected 60 strains of P. aeruginosa and screened their antimicrobial effects against Staphylococcus aureus (ATCC 25923) using the filter paper-disk method, the cross-streaking method and the co-culture method and then evaluated the antimicrobial activity of the chloroform-isolated S. aureus extracts against methicillin-resistant S. aureus (MRSA, Gram-positive cocci), vancomycin intermediate-resistant S. aureus (VISA, Gram-positive cocci), Corynebacterium spp. (CS, Gram-positive bacilli), Acinetobacter baumannii (AB, Gram-negative bacilli), Moraxella catarrhalis (MC, Gram-negative diplococcus), Candida albicans (CA, fungi), Candida tropicalis (CT, fungi), Candida glabrata (CG, fungi) and Candida parapsilosis (CP, fungi). Results: The PA06 and PA46 strains have strong antimicrobial effects. High-performance liquid chromatography (HPLC) analysis revealed that the major components of PA06 and PA46 that exhibit antimicrobial activity are functionally similar to phenazine-1-carboxylic acid (PCA) and pyocyanin. Preparative HPLC was performed to separate and isolate the 4 major potential antimicrobial components: PA06ER10, PA06ER16, PA06ER23 and PA06ER31. Further, the molecular masses of PA06ER10 (260.1), PA06ER16 (274.1), PA06ER23 (286.1) and PA06ER31 (318.2) were determined by electrospray ionization (ESI) mass spectrometry. Conclusion:P. aeruginosa can produce small molecules with potential antimicrobial activities against MRSA, VISA, CS, MC, CA, CT, CG and CP but not against AB.

Calreticulin protects rat microvascular endothelial cells against microwave radiation-induced injury by attenuating endoplasmic reticulum stress.

OBJECTIVE: This study was designed to evaluate whether exogenous CRT was beneficial for alleviating MR-induced injury by suppressing ER stress in rat MMECs. METHODS: MMECs were pretreated with CRT (25 pg/mL) for 12 hours, followed by the exposure to 2.856 GHz radiation at a mean power density of 30 mW/cm(2) for six minutes. MR-induced injury in MMECs was evaluated by LDH leakage, apoptosis, and cell viability analysis. The expression of GRP78, CRT, CHOP, Bcl-2, and Bax were examined by Western blot analysis to reflect ER stress response and ER stress-related apoptosis. RESULTS: MR induced marked MMECs injury, as shown by increased LDH leakage and apoptosis rate and decreased cell viability. MR also induced excessive ER stress, characterized by increased expression of GRP78 and CRT, and ER stress-related apoptotic signaling as well, as shown by the upregulation of CHOP and Bax and the downregulation of Bcl-2. Exogenous CRT pretreatment remarkably attenuated MR-induced cell apoptosis and LDH leakage, ER stress, and activation of the ER stress-related apoptotic signaling. CONCLUSIONS: Exogenous CRT attenuates MR-induced ER stress-related apoptosis by suppressing CHOP-mediated apoptotic signaling pathways in MMECs.

Cytosolic calreticulin inhibits microwave radiation-induced microvascular endothelial cell injury through the integrin-focal adhesion kinase pathway.

OBJECTIVE: To determine the effects of cytosolic CRT on MR-induced MMEC injury, and the underlying mechanism. METHODS: MMECs were randomized into eight groups: control, AdCRT (infected with pAdCMV/V5-DEST-CRT adenovirus), stCRT (transfected with rCRT-siRNAs), Mock (transfected with scrambled siRNAs), MR (exposed to MR for six minutes), AdCRT + MR, stCRT + MR, and Mock + MR. The magnitude of cell injury were assessed by Annexin V-PI staining, LDH activity in culture medium, MMEC migration ability, ultrastructure and cytoskeletal stability. Subcellular colocalization of CRT and ConA or integrin were evaluated by immunocytochemistry. The mRNA and protein expression levels of target genes were examined by qRT-PCR and western blotting, respectively. RESULTS: MR-induced cytotoxicity was dose-dependent. Overexpression of cytosolic CRT suppressed MR injury, shown as decreased cell apoptosis, reduced LDH activity, enhanced cell migration capability, and maintenance of ultrastructure and cytoskeleton integrity. Conversely, CRT deficiency aggravated MR-induced injury. Exposure of AdCRT MMECs to MR promoted membrane translocation of CRT and the interaction of CRT-integrin-α. Correlation analysis revealed that integrin-α expression or FAK phosphorylation was positively associated with cytosolic CRT expression. CONCLUSIONS: Cytosolic CRT inhibits MR-induced MMEC injury through activation of the integrin-FAK pathway.

Hypoxic preconditioning protects microvascular endothelial cells against hypoxia/reoxygenation injury by attenuating endoplasmic reticulum stress.

Endothelial cells (ECs) are directly exposed to hypoxia and contribute to injury during myocardial ischemia/reperfusion. Hypoxic preconditioning (HPC) protects ECs against hypoxia injury. This study aimed to explore whether HPC attenuates hypoxia/reoxygenation (H/R) injury by suppressing excessive endoplasmic reticulum stress (ERS) in cultured microvascular ECs (MVECs) from rat heart. MVECs injury was measured by lactate dehydrogenase (LDH) leakage, cytoskeleton destruction, and apoptosis. Expression of glucose regulating protein 78 (GRP78) and C/EBP homologous protein (CHOP), activation of caspase-12 (pro-apoptosis factors) and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) were detected by western blot analysis. HPC attenuated H/R-induced LDH leakage, cytoskeleton destruction, and cell apoptosis, as shown by flow cytometry, Bax/Bcl-2 ratio, caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. HPC suppressed H/R-induced ERS, as shown by a decrease in expression of GRP78 and CHOP, and caspase-12 activation. HPC enhanced p38 MAPK phosphorylation but decreased that of protein kinase R-like ER kinase (PERK, upstream regulator of CHOP). SB202190 (an inhibitor of p38 MAPK) abolished HPC-induced cytoprotection, downregulation of GRP78 and CHOP, and activation of caspase-12, as well as PERK phosphorylation. HPC may protect MVECs against H/R injury by suppressing CHOP-dependent apoptosis through p38 MAPK mediated downregulation of PERK activation.

[Pharmacological postconditioning with lactic acid and low dose edaravone could attenuate myocardial reperfusion injury through mitochondrial pathway].

OBJECTIVE: To test the hypothesis that pharmacological postconditioning with lactic acid and low dose edaravone could mimic the upper trigger of mechanical postconditioning and relieve reperfusion injury through mitochondrial pathway. METHODS: Rats were randomly divided into 6 groups (n = 18 each): sham, reperfusion/injury(I/R), postconditioning (IP), lactic acid (Lac, 60 µl), low dose edaravone (Eda, 3 µg/kg), and Lac+Eda. After 45 min myocardial ischemia, different drugs or saline were administrated around the infarct border according to different groups using micro syringe at the time of reperfusion. After 10 min reperfusion, right atrial plasma pH value was determined in all rats. Then the rats were sacrificed at 1, 6 and 24 h (n = 6 each), apoptotic index was measured by TUNEL, infarct area and ischemic area were measured through Evans blue-TTC double staining, mitochondrial absorbance, the contents of MDA and SOD in ischemic myocardium were detected by spectrophotometry, and the expression of apoptotic pathway molecules, such as Bcl-2, Bax and Cytochrome c (Cyt-c) , were detected by Western blot. RESULTS: Right atrial plasma pH value was significantly lower, the content of MDA was significantly lower, and the content of SOD was significantly higher in IP and Lac+Eda groups than in I/R group (all P < 0.05). The mitochondrial absorbance in Lac+Eda group at all time points were all significantly higher than those in I/R group (all P < 0.05). The level of Bcl-2 in ischemic myocardium in Lac+Eda group was significantly higher than in I/R group (1.02 ± 0.19 vs.0.02 ± 0.01, P < 0.05), the level of Bax (0.38 ± 0.07 vs.2.40 ± 0.45, P < 0.05) and Cyt-c(0.78 ± 0.05 vs.6.54 ± 1.86, P < 0.05) were all lower than those in I/R group. The content of CK[(849 ± 228) vs.(1249 ± 211) U/L, P < 0.05] and CK-MB[(470 ± 266) vs. (966 ± 263) U/L, P < 0.05] in Lac+Eda group were all significantly lower than in I/R group, apoptotic index [(10.51 ± 1.52)% vs. (15.00 ± 1.90) %, P < 0.05] and infarct area [(27.12 ± 5.55)% vs. (45.66 ± 10.81)%, P < 0.05] in Lac+Eda group were all significantly lower than those in I/R group. CONCLUSION: Pharmacological postconditioning with lactic acid and low dose edaravone could mimic the upper triggers of mechanical postconditioning and attenuate myocardial reperfusion injury through mitochondrial pathway.

Novel mandelic acid derivatives suppress virulence of Ralstonia solanacearum via type III secretion system.

BACKGROUND: Bacterial wilt induced by Ralstonia solanacearum is regarded as one of the most devastating diseases. However, excessive and repeated use of the same bactericides has resulted in development of bacterial resistance. Targeting bacterial virulence factors, such as type III secretion system (T3SS), without inhibiting bacterial growth is a possible assay to discover new antimicrobial agents. RESULTS: In this work, identifying new T3SS inhibitors, a series of mandelic acid derivatives with 2-mercapto-1,3,4-thiazole moiety was synthesized. One of them, F-24, inhibited the transcription of hrpY gene significantly. The presence of this compound obviously attenuated hypersensitive response (HR) without inhibiting bacterial growth of R. solanacearum. The transcription levels of those typical T3SS genes were reduced to various degrees. The test of the ability of F-24 in protecting plants demonstrated that F-24 protected tomato plants against bacterial wilt without restricting the multiplication of R. solanacearum. The mechanism of this T3SS inhibition is through the PhcR-PhcA-PrhG-HrpB pathway. CONCULSION: The screened F-24 could inhibit R. solanacearum T3SS and showed better inhibitory activity than previously reported inhibitors without affecting the growth of the strain, and F-24 is a compound with good potential in the control of R. solanacearum. © 2023 Society of Chemical Industry.

Lack of association of conjunctival MALT lymphoma with Chlamydiae or Helicobacter pylori in a cohort of Chinese patients.

BACKGROUND: This study was conducted to detect microbial pathogens in conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma specimens in an attempt to determine possible associations between conjunctival MALT lymphoma and microbial infections. MATERIAL/METHODS: Using PCR technique, freshly obtained tumor specimens from 16 cases of conjunctival MALT lymphoma, as confirmed by postoperative pathology, were analyzed for DNA of Chlamydia psittaci (C. psittaci), Chlamydia trachomatis (C. trachomatis), Chlamydia pneumoniae (C. pneumoniae) and Helicobacter pylori (H. pylori). Synthetic C. psittaci, C. trachomatis, C. pneumoniae and H. pylori DNA were used as positive control, and blank plasmid DNA as negative control. RESULTS: Electrophoresis showed that no bands corresponding to the positive control were observed in the specimens, indicating that no DNA of the 4 microorganisms was detected in the specimens of the 16 cases of conjunctival MALT lymphoma. CONCLUSIONS: The PCR technique was able to detect the positive control quickly and accurately, but the results of PCR in analyzing the 16 specimens were negative, indicating that there is no association between conjunctival MALT lymphoma and the 4 microorganisms in Chinese patients.

Melanoma Detection Using XGB Classifier Combined with Feature Extraction and K-Means SMOTE Techniques.

Melanoma, a very severe form of skin cancer, spreads quickly and has a high mortality rate if not treated early. Recently, machine learning, deep learning, and other related technologies have been successfully applied to computer-aided diagnostic tasks of skin lesions. However, some issues in terms of image feature extraction and imbalanced data need to be addressed. Based on a method for manually annotating image features by dermatologists, we developed a melanoma detection model with four improvement strategies, including applying the transfer learning technique to automatically extract image features, adding gender and age metadata, using an oversampling technique for imbalanced data, and comparing machine learning algorithms. According to the experimental results, the improved strategies proposed in this study have statistically significant performance improvement effects. In particular, our proposed ensemble model can outperform previous related models.

Effect of miR-184 and miR-205 on the tumorigenesis of conjunctival mucosa associated lymphoid tissue lymphoma through regulating RasL10B and TNFAIP8.

AIM: To explore the effect of miR-184 and miR-205 on the proliferation and metastasis of conjunctival mucosa associated lymphoid tissue (MALT) lymphoma. METHODS: Tissue of tumor and adjacent normal control from 5 patients with conjunctival MALT was included. RPMI8226 cell line was selected to verify the effect of miRNAs in B cells. The function of microRNA on the RPMI8226 cell apoptosis, migration and invasion was evaluated by apoptosis assay and Transwell assay. The mRNA and protein expression were examined by quantitative RT-PCR and Western blotting. The effect of microRNA on regulation of downstream gene expression was evaluated by luciferase report assay. RESULTS: A decreased level of miR-184 and miR-205 was observed in MALT lymphoma tissue. Exogenous miR-184 and miR-205 analogues promoted apoptosis, and inhibited the survival, migration, and invasion of RPMI8226 cells. miR-184 and miR-205 inhibitor reversed the process. The RNA and protein level of RasL10B and TNFAIP8 were downregulated in MALT lymphoma tissue. The exogenous of miR-184 and miR-205 promoted the expression of RasL10B and TNFAIP8. Meanwhile, inhibition of miR-184 and miR-205 repressed the expression of target gene, RasL10B and TNFAIP8. CONCLUSION: miR-184 and miR-205 suppresses the tumorigenesis of conjunctival MALT lymphoma through regulating RasL10B and TNFAIP8.

miR-664b-3p inhibits colon cell carcinoma via negatively regulating Budding uninhibited by benzimidazole 3.

MiR-664b-3p has been reported to play a crucial role in cancer progression. This research explores the biological effect and molecular mechanisms of miR-664b-3p in cell proliferation, apoptosis, migration, and invasion of colon cancer. The expression level of miR-664b-3p and Budding uninhibited by benzimidazole 3 (Bub3) in colon cancer cell lines and tissues were detected and analyzed using quantitative real-time PCR and bioinformatics method. The Western blot measured the expression level of proliferation-related, migration-related, and apoptosis-related proteins. CCK-8 assessed cell viability, and the cell proliferation, migration, and invasion were detected by the Edu assay, wound-healing assay, and transwell assay, respectively. Annexin/propidium iodide (PI) assays detected apoptosis of cells. The target of miR-664b-3p was predicted by bioinformatics methods and then validated by gene engineering technology. MiR-664b-3p was downregulated in colon cancer tissues and cells. The cell proliferation, migration, and invasion of cells were inhibited after transfecting by miR-664b-3p mimics, whereas apoptosis was promoted. Over-expression of miR-664b-3p could reduce the expression of proliferation-promoted proliferating cell nuclear antigen (PCNA), proliferation marker protein Ki-67 (Ki-67), migration-promoted Cyclooxygenase-2 (COX-2), Matrix Metallopeptidase 2 (MMP-2), and Matrix Metallopeptidase 9 (MMP-9), and apoptosis-inhibited protein (Bcl-2) while increasing the expression of apoptosis-promoted BCL2-Associated X Protein (Bax), caspase-3, and caspase-9 proteins. The study indicated that miR-664b-3p plays a significant role in colon cancer and could regulate the progression of colon cancer tumor growth by suppressing the expression of BUB3 protein. These findings provide a novel strategy to screen and treat colon cancer.

[The epidemiological study of work-related musculoskeletal disorders and related factors among automobile assembly workers].

OBJECTIVE: To investigate the work-related musculoskeletal disorders among automobile assembly workers, to discusses the related risk factors and their relationship. METHOD: The selected 1508 automobile assembly workers from a north car manufacturing company were regarded as the study object. The hazard zone jobs checklist, Nordic musculoskeletal symptom questionnaire (NMQ) and pain questionnaire were used to perform the epidemiological cross-sectional and retrospective survey and study for the General status, awkward ergonomics factors and related influencing factors, and musculoskeletal disorders of workers. RESULTS: The predominant body sites of occurring WMSDs among automobile assembly workers were mainly low back, wrist, neck and shoulders, the predominant workshop section of occurring WMSDs were mostly concentrated in engine compartment, interior ornament, door cover, chassis and debugging section. The predominant body site of WMSDs among engine compartment and chassis section workers was low back, interior ornament workers were low back and wrist, door cover workers was wrist, chassis workers was low back, debugging workers were neck and low back. Neck musculoskeletal disorders had the trend with the increase of a body height; Smoking may increase the occurrence of musculoskeletal disorders. CONCLUSION: The WMSDs appears to be a serious ergonomic proble assem among automobile assembly workers, predominant occurring site of WMSDs is with different workshop section, its characteristics is quite obvious, probably related to its existing awkward work position or activities. The worker height and smoking habits may be important factors which affect musculoskeletal disorders happen.

SnRK2.6 interacts with phytochrome B and plays a negative role in red light-induced stomatal opening.

Light is an important environmental factor for plant growth and development. Phytochrome B (phyB), a classical red/far-red light receptor, plays vital role in controlling plant photomorphogenesis and light-induced stomatal opening. Phytohormone abscisic acid (ABA) accumulates rapidly and triggers a series of physiological and molecular events during the responses to multiple abiotic stresses. Recent studies showed that phyB mutant synthesizes more ABA and exhibits improved tolerance to salt and cold stress, suggesting that a crosstalk exists between light and ABA signaling pathway. However, whether ABA signaling components mediate responses to light remains unclear. Here, we showed that SnRK2.6 (Sucrose Nonfermenting 1-Related Protein Kinase 2.6), a key regulator in ABA signaling, interacts with phyB and participates in light-induced stomatal opening. First, we checked the interaction between phyB and SnRK2s, and found that SnRK2.2/2.3/2.6 kinases physically interacted with phyB in yeast and in vitro. We also performed co-IP assay to support that SnRK2.6 interacts with phyB in plant. To investigate the role of SnRK2.6 in red light-induced stomatal opening, we obtained the snrk2.6 mutant and overexpression lines, and found that snrk2.6 mutant exhibited a significantly larger stomatal aperture under red light treatment, while the two independent overexpression lines showed significantly smaller stomatal aperture, indicative of a negative role for SnRK2.6 in red light-induced stomatal opening. The interaction of SnRK2.6 with red light receptor and the negative role of SnRK2.6 in red light-induced stomatal opening provide new evidence for the crosstalk between ABA and red light in guard cell signaling.

Carbachol inhibits TNF-α-induced endothelial barrier dysfunction through alpha 7 nicotinic receptors.

AIM: To test whether carbachol can influence endothelial barrier dysfunction induced by tumor necrosis factor (TNF)-α and whether the alpha 7 nicotinic receptor can mediate this process. METHODS: Rat cardiac microvascular endothelial cells were exposed to carbachol followed by TNF-α treatment in the presence or the absence of α-bungarotoxin (an antagonist of the alpha 7 nicotinic receptor). Permeability of endothelial cells cultured on Transwell filters was assayed using FITC-albumin. F-actin was stained with FITC- phalloidin. Expression of vascular endothelial cadherin, intercellular adhesion molecule 1 (ICAM-1), phosphor-ERK1/2 and phosphor-JNK was detected using Western blot. RESULTS: Carbachol (2 µmol/L-2 mmol/L) prevented increase in endothelial cell permeability induced by TNF-α (500 ng/mL) in a dose-dependent manner. Further, it attenuated the down-regulation of vascular endothelial cadherin and the up-regulation of ICAM-1 induced by TNF-α. In addition, treatment of endothelial cells with carbachol decreased phosphor-ERK1/2 and phosphor-JNK. These effects of carbachol were blocked by α-bungarotoxin 3 µg/mL. CONCLUSION: These data suggest that the inhibitory effect of carbachol on TNF-α-induced endothelial barrier dysfunction mediated by the alpha 7 nicotinic receptor.

Population pharmacokinetics of rhTNFR-Fc in healthy Chinese volunteers and in Chinese patients with Ankylosing spondylitis.

AIM: To investigate the population pharmacokinetics of recombinant human tumor necrosis factor receptor-Fc fusion protein (rhTNFR-Fc) administered via subcutaneous (SC) injection in healthy Chinese volunteers and in Chinese patients with ankylosing spondylitis (AS). METHODS: Thirty-two healthy volunteers were randomly assigned to receive a single SC injection of 12.5, 25, 37.5, or 50 mg of rhTNFR-Fc. Twenty male patients with moderate AS were randomly assigned to receive seven consecutive SC injections of rhTNFR-Fc at either 25 mg twice a week (BIW) or 50 mg once a week (QW). Population pharmacokinetic (PK) analysis was applied to obtain PK parameters of rhTNFR-Fc by the NONMEM method. RESULTS: The data were best described by a one-compartment model with lag time. We found that gender had a significant effect on the apparent clearance (CL/F), with the male CL/F ratio being only 0.665 times the female ratio; the absorption coefficient (F) of multiple dosages of rhTNFR-Fc was only 0.674 times that of a single dosage. The outcome parameters were CL/F (female: 0.168 L/h, male: 0.110 L/h), the apparent volume of distribution (Vd/F: 15.5 L), the absorption rate constant (Ka) (single dosage: 0.0605 h⁻¹, multiple dosage: 0.0408 h⁻¹), and the lag time (T(lag): 1.03 h). The inter-individual variability in the CL/F, Vd/F, Ka, and T(lag) were 33.3%, 42.7%, 55.6%, and 81.8%, respectively. CONCLUSION: Chinese females have a higher CL/F than Chinese males, and multiple dosings can significantly decrease the absorption of rhTNFR-Fc (SC). The population PK parameters of rhTNFR-Fc in healthy Chinese volunteers and patients with AS were similar to those reported for subjects in published American studies.

Postconditioning with Calreticulin Attenuates Myocardial Ischemia/Reperfusion Injury and Improves Autophagic Flux.

BACKGROUND: Impaired autophagic flux contributes to cardiomyocyte death in ischemia/reperfusion (I/R) injury. Restoring the impaired autophagic flux by using agents may be a promising strategy that alleviates myocardial I/R injury. The present study aimed to evaluate the effect of exogenous calreticulin (CRT) postconditioning on impaired autophagic flux induced by hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: Rat myocardial I/R injury model was prepared. CRT postconditionging was fulfilled by an intraperitoneal injection of CRT (0.5 mg/kg body weight) 5 min before reperfusion. Hemodynamics, serum lactate dehydrogenase (LDH) activity and Cardiac troponin T (TnT) content, and infarct size were measured. The H/R injury model of H9c2 cells was prepared. CRT postconditioning was performed by adding 25 pg/mL CRT to the medium at the onset of reoxygenation. Cell death rate, lactate dehydrogenase (LDH) leakage, intracellular reactive oxygen species (ROS), and malondialdehyde (MDA) were assessed. Autophagic flux was monitored by mRFP-GFP-LC3 adenovirus infection. The number of autophagosomes and autolysosomes in cells were determined by counting the fluorescence dots. Western blot assay was used to determine the expression of autophagy-related proteins. RESULTS: CRT postconditionging improved cardiac function, reduced serum LDH activity and TnT content, and limited myocardial infarct size after myocardial I/R injury in rat. H/R induced H9c2 cells injury and autophagosomes accumulation in cells. CRT postconditioning attenuated H/R-induced cell death, LDH leakage, and the increase of intracellular ROS and MDA. Meanwhile, CRT postconditioning suppressed H/R-induced excessive formation of autophagosomes, as shown by a decrease of autophagosomes and the ratio of LC3-II/LC3-I, LC3-II, and Beclin1. It also improved H/R-induced impaired autophagosomes clearance, as shown by an increase of autolysosomes and the level of LAMP-2, and a decrease of the level of p62. CONCLUSION: These findings suggested that CRT postconditioning reduced myocardial I/R injury. CRT postconditioning also inhibited the excessive formation of autophagosomes, promoted the clearance of autophagosomes, and resorted the autophagic flux, consequently reduced the H/R injury in H9c2 cells.