[Application of high-throughput next-generation sequencing technology in clinical laboratory diagnosis of infectious diseases].

With advances in sequencing technologies, high-throughput next-generation sequencing (NGS) has triggered increased attention on its application in clinical laboratories and facilitates the molecular diagnosis and treatment of infectious diseases. Compared with conventional microbiology laboratory methods, NGS has greatly increased the sensitivity and accuracy of diagnosis, and reduced detection time for infectious pathogens, especially for diagnosis of complex and mixed infections. However, there are still some problems that hamper the NGS application in infections diagnosis, including lack of standardization, cost, and variation in data interpretation, etc. In recent years, with the developing of policies and legislation, and guidance and supports from Chinese government, the sequencing industry has gained continuously healthy development and sequencing application market gradually becomes mature. Meanwhile, worldwide microbiology experts are striving to develop the standards and reach the consensus, there are more and more clinical laboratories equipped with sequencing instruments and personnel with expertise. All of these measures would certainly promote the clinical application of NGS, and making full use of high-throughput NGS could contribute to the accurate clinical diagnosis and appropriate treatment. The current article describes the application of high-throughput next-generation sequencing technology in the laboratory diagnosis of clinical microbial infectious diseases, as well as the policy system support and development direction.

Correlation research of serum substance P, CCK-8, and 5-HT values with depression levels in stroke survivors.

OBJECTIVE: This study aimed to investigate the correlation of serum octapeptide cholecystokinin-8 (CCK-8), substance P (SP), and 5-hydroxytryptryptamine (5-HT) values with depression levels in patients with post-stroke depression (PSD). It also aimed to explore the potential approach for the early diagnosis of PSD. PATIENTS AND METHODS: A correlation research between patients' biochemical indicators and depression levels was performed among 70 stroke patients during hospitalization from June 2021 to February 2022. The 70 stroke patients were selected and divided into post-stroke depression and non-depression groups according to the Hamilton Depression Scale (HAMD) score. The concentrations of CCK-8, SP, and 5-HT in both groups were measured, and the relationship between the values of CCK-8, SP, 5-HT and the depression levels was analyzed. RESULTS: Among the 70 stroke survivors, 35 were in the depression group and 35 were in the non-depression group. Significant differences were observed in the concentration of CCK-8, SP, and 5-HT between the patients in the depression and non-depression group (p < 0.05). Accompanied by an increase in the depression level, the SP value gradually increased, but the CCK-8 and 5-HT values gradually decreased. Spearman correlation analysis indicated that the order of the correlation between CCK-8, 5-HT, SP, and the depression levels was CCK-8 > SP > 5-HT. CONCLUSIONS: All the CCK-8, SP and 5-HT values were correlated with the depression levels in stroke survivors. Furthermore, the correlation between CCK-8, SP, and post-stroke depression levels was higher than that of 5-HT, suggesting that the early diagnosis of PSD may be reflected more precisely through the detection of CCK-8, and SP values, thus providing potential priority for biochemical detection in the diagnosis of PSD.

[Establishment and application of quantitative immunoassay method based on stable element labeling and inductively coupled plasma mass spectrometry].

Objective: To establish a quantitative immunoassay method based on stable element labeling and inductively coupled plasma mass spectrometry (ICP-MS) for the detection of serum amyloid A (SAA) and evaluate its performance. Methods: An immunoassay system based on sandwich method was established with magnetic bead as carrier and holmium (Ho) as element tags. The binding ratio of hydrophilic streptavidin magnetic beads and biotinylated antibody, the amount of elemental antibody, and the reaction time were optimized to choose the optimal reaction conditions. According to the documents of Clinical and Laboratory Standards Institute (CLSI), the analytical performance was evaluated, including the limit of blank (LOB), linearity, accuracy, specificity, imprecision and interference test. Finally, 82 SAA plasma samples were collected after the turbidimetric inhibition immunoassay, and the newly established method was used for detection. Moreover, the detection results of the two methods were analyzed by Pearson correlation analysis. Results: The optimal binding ratio of hydrophilic streptavidin and biotinylated antibody was 1∶0.15, the amount of Ho-labeled antibody was 3 µl and the incubation time of the two reaction steps was 40 min and 30 min, respectively. The LOB was 0.6 ng/ml. The linearity was good within the range of 0-1 200 µg/L (R2=0.998 9, P<0.001). The inter-batch precision of high-value samples and low-value samples was 9.42% and 7.95%, respectively, and the intra-batch precision was 14.56% and 13.56%, respectively. The recovery was 96.01%-104.76%. The cross-reaction rates with procalcitonin (PCT) and C-reactive protein (CRP) were 0.45% and 0.015%, respectively. When the concentration of triglyceride≤35.5 mg/L, bilirubin≤0.52 mg/L and hemoglobin≤2.4 g/L, the interference bias was less than 10%. The results of 82 SAA plasma samples were 12.65 (4.45, 59.03) mg/L by ICP-MS immunoassay and 18.23 (9.33, 68.72) mg/L by turbidimetric inhibition immunoassay, respectively. The newly established system was well correlated with turbidimetric inhibition immunoassay (R2=0.983, P<0.001). Conclusion: The quantitative immunoassay for SAA with Ho as marker established in this study has high precision, good accuracy, high specificity, and wide linear range, which can meet the clinical testing requirements.

Phyllosticta rizhaoensis sp. nov. causing leaf blight of Ophiopogon japonicus in China.

Ophiopogon japonicus (Asparagaceae) is a perennial grass species which can be cultivated as an ornamental and medicinal plant. From April 2021 to September 2022, a serious leaf blight disease of O. japonicus was discovered in Rizhao City, Shandong Province, China. The initial disease symptoms were small yellow spots, finally developing as tip blight, often associated with many small, black, semi-immersed pycnidial conidiomata formed in lesions. To obtain isolates of the causal agent for this disease, samples were randomly collected from O. japonicus diseased leaves in Rizhao City. In total 97 Phyllosticta isolates were obtained from samples, and studied using morphological features and multi-locus phylogenetic analyses of a combined dataset using the internal transcribed spacers (ITS), the 28S large subunit of ribosomal RNA (LSU), and partial translation elongation factor 1-alpha (tef), actin (act) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) loci. Phylogenetically, these Phyllosticta isolates formed a clade in the P. concentrica species complex, and clustered with P. pilospora and P. spinarum. Morphologically, isolates in this clade differed from P. pilospora and P. spinarum by the size of conidiogenous cells and conidia, and the absence of an apical conidial appendage. As a result, these isolates were described as a novel species Phyllosticta rizhaoensis. Pathogenicity was confirmed using Koch's postulates, which showed that P. rizhaoensis could induce leaf blight symptoms on O. japonicus in China. Citation: Wang C-B, Wang T-T, Ma C-Y, Xue H, Li Y, Piao C-G, Jiang N (2023). Phyllosticta rizhaoensis sp. nov. causing leaf blight of Ophiopogon japonicus in China. Fungal Systematics and Evolution 11: 43-50. doi: 10.3114/fuse.2023.11.03.

[Clinical features, risk factors and prognosis of idiopathic dilated cardiomyopathy complicated by ischemic stroke].

Objective: To analyze the clinical features, risk factors and prognosis of idiopathic dilated cardiomyopathy (DCM) complicated with ischemic stroke (IS) (DCM-IS). Methods: The clinical data of patients with idiopathic DCM (n=613) in Beijing Anzhen Hospital, Liangxiang Hospital and Fuxing Hospital from January 2016 to December 2020 were retrospectively collected, and among them, 123 cases were DCM-IS. Clinical features of patients with DCM-IS were summarized and multivariate logistic regression model was utilized to analyze the independent risk factors of DCM-IS. Furthermore, 1-year follow-up was conducted and Kaplan-Meier curve was adopted to analyze the prognosis of DCM, using all-cause death and heart transplantation as adverse outcomes. Results: Among the 70 patients with DCM-IS, 6 patients (8.6%, 6/70) were in accordance with the subtype of large artery atherosclerosis, and 47 patients (67.1%, 47/70) were in line with the subtype of cardiogenic embolism, and small artery occlusion subtype (ie, lacunar infarction) were detected in 17 cases (24.3%, 17/70). Hypertension [odds ratio (OR)=1.617, 95% confidence interval (CI): 1.049-2.491, P=0.029], hyperlipidemia (OR=1.918, 95%CI: 1.198-3.073, P=0.007), atrial fibrillation (AF) (OR=1.617, 95%CI: 1.016-2.572, P=0.043), lower estimated glomerular filtration rate (eGFR) (OR=0.986, 95%CI: 0.977-0.996, P=0.005) and a higher incidence of intracardiac thrombus (OR=6.127, 95%CI: 3.174-11.827, P<0.001) were risk factors for DCM-IS. The overall 1-year survival rate was lower in DCM-IS patients (70.7%) than DCM patients without stroke (83.6%, P=0.004), and the main causes of death included obstinate heart failure (3 cases of DCM-IS, and 5 cases of non-DCM-IS) and malignant arrhythmia (DCM-IS) (22 cases of DCM-IS, and 18 cases of non-DCM-IS). Conclusions: Among IS patients with idiopathic DCM, cardioembolism is the most common, followed by lacunar infarction, and the large-artery atherosclerotic subtype is the least common.Hypertension, hyperlipidemia, AF, lower eGFR value and higher incidence of intracardiac thrombus are risk factors for DCM-IS. DCM patients complicated with IS have poor short-term prognosis, and obstinate heart failure and malignant arrhythmia are their main causes of death.

Characteristics of abnormal subjects in screening of tumor markers among middle-aged and elderly people in Weihai area.

OBJECTIVE: Based on the Huimin policy of Weihai Social Security Bureau, this project aims to investigate feasibility of screening through screening of serum tumor markers (TM) in the middle-aged and elderly population in Weihai area. According to the joint mode of examination institution and clinical department (oncology), we provide dynamic follow-up for subjects for early intervention of abnormal tumor screening and high-risk population, through which we can cut off pathogenic pathway of cancer and improve early diagnosis of cancer and enhance the cure rate. PATIENTS AND METHODS: We continued to track subjects whose TM was not reduced to normal until it was normal or diagnosed, collecting the blood samples of eligible patients that we removed high-risk factors from the subjects so that TM could be lowered to normal. RESULTS: A total of 83,049 blood samples were detected in our hospital, and 89 patients were diagnosed with newly diagnosed tumor. The positive expression rate of CEA in lung cancer patients was 91.4%. CONCLUSIONS: The diagnosis of tumor relies not only on TM, but also on observation of clinical symptoms, continuous observation of TM dynamic changes and individualized comprehensive analysis.  The main purpose of this policy is not only to find patients with "early tumor", but also to cut off the pathogenic pathway of cancer, achieve purpose of tertiary prevention and significantly save medical costs. The examination mechanism and the clinical-related departments are connected, and the pattern of screening, tracking and analysis of abnormal results in large samples is in line with the present situation of China and is worthy of clinical promotion.

[Analysis of the diagnostic efficiency of combining multiple laboratory hematological indicators in alpha-fetoprotein-negative hepatocellular carcinoma].

Objective: To establish a diagnostic model for alpha-fetoprotein-negative hepatocellular carcinoma (AFP-NHCC) by combining multiple laboratory hematological indicators and explore its clinical diagnostic efficiency. Methods: A total of 124 inpatients, including 110 males and 14 females, aged 57 (51, 66) years, who were first diagnosed with AFP-NHCC in the PLA General Hospital were included from December 2011 to June 2017. Meanwhile, 331 cases of non-HCC were enrolled as the control group, including 279 males and 52 females, aged 58 (51, 63) years old, with 47 cases of hepatitis B virus (HBV) infection, 40 cases of liver cirrhosis, 64 cases of hepatic hemangioma or cysts, 7 cases of liver nodules, 8 cases of fatty liver, 146 cases of non-liver disease and 19 health controls. Subjects in the AFP-NHCC group and the control group were divided into a training group and a validation group. A total of 196 subjects were involved in the training group, including 103 AFP-NHCC patients and 93 non-HCC patients (19 healthy controls, 25 patients with HBV infection, 22 patients with liver cirrhosis, 23 patients with hepatic hemangioma or cyst, and 4 patients with liver nodules). The differences in laboratory parameters were analyzed, and a diagnostic model of AFP-NHCC under different AFP levels was established. Likewise, 259 subjects, including 113 patients with liver disease, were involved in the validation group to verify the diagnostic efficiency of the model for AFP-NHCC. The receiver operating characteristic (ROC) curve was used to analyze the sensitivity and specificity of different models, and the area under the curve (AUC) was calculated to evaluate the diagnostic performance of different models. Results: In the training group, the indicators of AFP-NHCC diagnostic model included platelet (PLT), prothrombin activity (PTA), serum albumin (ALB), prothrombin time (PT) and carbohydrate antigen 19-9 (CA19-9), and the AUC of the model was 0.848 (95%CI: 0.786-0.911) when AFP≤5 µg/L. Similarly, the indicators of AFP-NHCC diagnostic model included PLT, PTA, ALB, PT and hematocrit (HCT), and the AUC of the model was 0.839 (95%CI: 0.780-0.897) when AFP≤10 µg/L. When AFP≤20 µg/L, the indicators of AFP-NHCC diagnostic model contained PLT, PTA, ALB, PT, HCT and AFP, and the AUC of the model was 0.866 (95%CI: 0.815-0.917). The AUC values of these three models were higher than those of AFP and CA19-9 alone for the diagnosis of AFP-NHCC [0.634 (95%CI: 0.560-0.709), 0.691 (95%CI:0.620-0.761), all P<0.05]. The indicators screened by these three models were combined to establish the final diagnostic model, and the AUC of the model was 0.873 (95%CI: 0.824-0.923), with the sensitivity of 78.6% (81/103) and the specificity of 81.7% (76/93). In the validation group, the predictive AUC of the final model in liver disease patients was 0.892 (95%CI: 0.832-0.951), with the sensitivity of 100% (21/21) and the specificity of 71.7% (66/92), while in the total validation population, the predictive AUC was 0.931 (95%CI: 0.890-0.972), with the sensitivity of 100.0% (21/21) and the specificity of 75.6% (180/238). Conclusion: The final diagnostic model includes PLT, PTA, ALB, PT, HCT, CA19-9 and AFP, which has higher sensitivity and specificity, and has good diagnostic efficiency for the clinical diagnosis of AFP-NHCC.

[Progress in treatment of PD-1/PD-L1 inhibitors for bladder cancer].

Because of the limited effect of traditional treatment methods such as surgical treatment, radiotherapy and chemotherapy,the emergence of immunotherapy has brought new hope for the treatment of patients with bladder cancer. As an immune checkpoint inhibitor, programmed death receptor 1/programmed death receptor-ligand 1 (PD-1/PD-L1) inhibitor has shown good anti-tumor activity and safety in the treatment of advanced bladder cancer, and has been recommended for advanced bladder cancer as second-line treatment by NCCN guidelines. PD-1/PD-L1 inhibitor for the treatment of bladder cancer has covered the first-line and second-line treatment, as well as maintenance therapy after first-line chemotherapy of locally advanced or metastatic bladder cancer, adjuvant and neoadjuvant therapy of muscle-invasive bladder cancer, treatment of high-risk non-muscle invasive bladder cancer failed by Bacille Calmette-Guérin vaccine perfusion, and bladder preservation therapy of muscle-invasive bladder cancer. Some of related studies have achieved certain results, and some are in progress, both of which need to be further examined. Maybe it can provide new guidance and ideas for clinical treatment of bladder cancer.

[Prevalence and correlates of HIV infection among cross-border families in Dehong Dai and Jingpo Autonomous Prefecture].

Objective: To study the prevalence and correlations of HIV infection among cross-border couples in the Dehong prefecture. Methods: A cross-sectional mass screening study with questionnaire interview and HIV testing was conducted among 17 594 registered cross-border couples from May 2017 through June 2018. Results: Among 32 400 participants, the overall prevalence of HIV infection was 2.27% (736/32 400), 2.44% (375/15 372) for Chinese citizens, and 2.12% (361/17 028) for foreign spouses. Among all the 13 853 couples with both spouses receiving HIV testing, 13 415(96.84%) were seroconcordant-negative couples, 142(1.03%) were serocondordant-positive couples, and 296(2.13%) were serodiscordant couples, including 167(1.20%) couples with positive husband and negative wife and 129(0.93%) couples with positive wife and negative husband. Multiple logistic regression analyses indicated that HIV infection was associated with drug use and risky sexual behaviors for male spouses. In contrast, HIV infection was associated with risky sexual behaviors for female spouses. Conclusion: The prevalence of HIV among cross-border couples in Dehong prefecture is high, underscoring the urgent need to scale up HIV testing, prevention, and behavioral intervention.

[Prognosis analysis of local recurrence after excision of breast phyllodes tumors].

Objective: To examine treatment outcomes of breast phyllodes tumors and the prognosis factors of local recurrence. Methods: This retrospective cohort study included 276 patients who underwent surgical resection at Breast Center, Peking University People's Hospital from January 2011 to December 2019. Tumor subtype and histopathological features were determined from pathology reports, and the deadline of follow-up was September 30th, 2020. All 276 patients underwent open surgery, including 17 patients of mastectomy, and 259 patients of lumpectomy. The enrolled patients were all female, with age of (41.5±11.3) years (rang: 11 to 76 years), and tumor diameter of 35(28) mm (M(QR)). The Kaplan-Meier method and Log-rank test were used for survival analysis. The multivariate analysis was implemented using the Cox proportional hazard model. Results: According the pathologic test, there were 191 patients of benign phyllodes tumor, 67 patients of borderline tumor and 18 patients of malignant tumor. There were 249 patients with a follow-up of more than 6 months, and 14.1% (35/249) had local recurrence. The time-to-recurrence was (28.6±22.2) months (range: 2 to 96 months), (29.1±18.1) months (range: 2 to 80 months), (32.1±30.1) months (range: 5 to 96 months) and (12.0±6.9) months (range: 8 to 20 months) for benign, borderline and malignant phyllodes tumors. Tumor diameter (≥100 mm vs.<50 mm, HR=3.968, 95%CI: 1.550 to 10.158, P=0.004) and malignant heterologous element (yes vs. no, HR=26.933, 95%CI: 3.105 to 233.600, P=0.003) were prognosis factors of local recurrence. One death from malignant phyllodes occurred after distant metastasis. The 3-year disease-free survival rates of benign, borderline and malignant phyllodes tumor were 88.2%, 81.7% and 81.4% (P=0.300). Conclusion: Phyllodes tumors have a considerable local recurrence rate, which may be associated with tumor diameter and malignant heterologous element.

[Establishment of mouse model and preliminary screening of serum biomarkers for Candidemia].

Objective: To establish mouse models of Candidemia, and investigates statistically significant polypeptide peaks to provide auxiliary diagnosis of this disease. Methods: A total of 170 specific pathogen free adult male ICR mice with body mass of 27-30 g were completely randomly divided into Candida albicans infection group (n=80), Candida parapsilosis infection group (n=80) and the normal control group (n=10), and the two kinds of Candidemia mouse models were established via tail vein injection. The serum samples were analyzed by Matrix-assisted laser desorption-ionization time of flight mass spectrometry and relevant software, and the polypeptide peaks with significant differences were screened to establish diagnostic models. Results: A total of 65 differential polypeptide peaks were obtained compared with the Candida albicans infection group and the normal control group. Combined with m/z 1 100.4, 1 581.0, 3 808.0 as differential polypeptide peaks to established the diagnostic model, the sensitivity was 95.24%(40/42), the specificity was 90.63%(29/32), the accuracy rate was 93.24%(69/74), and the AUC value of the ROC curve was 0.972(95%CI: 0.941-1.000). A total of 73 differential polypeptide peaks were obtained compared with Candida parapsilosis infection group and the normal control group. Combined with m/z 1 433.2, 1 148.5, 4 093.5, 4 522.2, 8 140.9, 8 234.6 as differential polypeptide peaks to established the diagnostic model, the sensitivity was 95%(38/40), the specificity was 81.25%(26/32), the accuracy rate was 88.89%(64/72), and the AUC value of the ROC curve was 0.953(95%CI: 0.903-1.000). A total of 78 differential polypeptide peaks were obtained compared with Candida albicans infection group and Candida parapsilosis infection group. Combined with m/z 2 736.9, 8 091.5, 8 153.7 as differential polypeptide peaks to established the diagnostic model, the accuracy of distinguishing C. albicans infection from C. parapsilosis infection was 98.78%(81/82). Conclusions: Successfully screened the differential polypeptides and established the related diagnostic models. Which is helpful to find serum biomarkers for the auxiliary diagnosis of Candidemia, and provides a basis for the early diagnosis and the rational use of drugs.

[Clinical factors of pathological complete response after neoadjuvant chemoradiotherapy in rectal cancer].

Objective: To explore the feasibility of clinical factors to predict the pathological complete response after neoadjuvant chemoradiotherapy in rectal cancer. Methods: A retrospective analysis was performed on clinical factors of 162 patients with rectal cancer, who underwent neoadjuvant chemoradiotherapy in the General Hospital of People's Liberation Army from January 2011 to December 2018.According to the postoperative pathological results, the patients were divided into pathological complete response (pCR) group and non-pathological complete response group (non-pCR group) to check the predictive clinical factors for pCR. Results: Twenty-eight cases achieved pCR after neoadjuvant chemoradiation (17.3%, 28/162). Univariate analysis showed that patients with higher differentiation (P=0.024), tumor occupation of the bowel lumen≤1/2 (P=0.006), earlier clinical T stage (P=0.013), earlier clinical N stage (P=0.009), the time interval between neoadjuvant chemoradiotherapy and surgery>49 days (P=0.006), and maximum tumor diameter≤5 cm (P=0.019) were more likely to obtain pCR, and the differences werestatistically significant. Multivariate analysis showed that tumor occupation of the bowel lumen≤1/2 (P=0.01), maximum tumor diameter≤5 cm (P=0.035), and the interval>49 days (P=0.009) were independent factors in predicting pCR after neoadjuvant therapy. Conclusion: Tumor occupation of the bowel lumen, maximum tumor diameter, and the time interval between neoadjuvant chemoradiotherapy and surgery can predict the pCR in rectal cancer.

LINC00662 triggers malignant progression of chordoma by the activation of RNF144B via targeting miR-16-5p.

OBJECTIVE: Chordoma is a rare malignant tumor difficult to diagnose and treat. Long non-coding RNAs acting as novel biomarkers are frequently reported in numerous cancers. The purpose of this study was to investigate the role of long intergenic non-coding RNA 00662 (LINC00662) and its associated action mechanisms in chordoma. MATERIALS AND METHODS: The expression of LINC00662, Ring finger protein 144B (RNF144B), and microRNA-16-5p (miR-16-5p) was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The protein levels of RNF144B, cell proliferation markers (Cyclin D1 and Ki67), epithelial-mesenchymal transition (EMT) markers (E-cadherin, Vimentin and N-cadherin), and glycolysis markers [glucose transporter 1 (GLUT1), hexokinase II (HK2), and lactic dehydrogenase A (LDHA)] were determined by Western blot. Cell proliferation, the number of colonies, migration, and invasion were investigated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation, and transwell assays, respectively. Glycolysis progress was evaluated by the glycolysis stress test, glucose consumption, lactate production, and ATP production. The relationship between miR-16-5p and LINC00662 or RNF144B was predicted by the online bioinformatics tool starBase and verified by Dual-Luciferase reporter assay. Xenograft tumor model was established to monitor the role of LINC00662 in vivo. RESULTS: LINC00662 and RNF144B were aberrantly upregulated in chordoma tissues. Knockdown of LINC00662 or RNF144B impeded proliferation, colony formation, invasion, migration, EMT, and glycolysis in chordoma cells. Besides, RNF144B overexpression reversed the role of LINC00662 knockdown. It was confirmed that miR-16-5p was a target of LINC00662, and miR-16-5p could target RNF144B. The relationship between LINC00662 and RNF144B was established by miR-16-5p. In addition, LINC00662 stable knockdown inhibited tumor growth in vivo. CONCLUSIONS: LINC00662 participated in the malignant progression of chordoma by the promotion of RNF144B by acting as a sponge of miR-16-5p, suggesting that LINC00662 was a promising therapeutic target for chordoma.

MicroRNA-26a regulates cerebral ischemia injury through targeting PTEN.

OBJECTIVE: MicroRNA-26a (miR-26a) exhibits diverse functions in different human disease. However, further research is needed to investigate the potential role of miR-26a in cerebral ischemia injury. MATERIALS AND METHODS: The expressions of miR-26a and PTEN (phosphatase and tensin homolog) were detected via Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) assay. The protein expression of Bcl-2 and Bax was detected by Western blot assay. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to observe the cell viability of SH-SY5Y cells. The relationship between miR-26a and PTEN was confirmed by Dual-Luciferase assay and human SH-SY5Y cells were treated with oxygen-glucose deprivation (OGD)/reperfusion to mimic I/R injury. RESULTS: The expression of miR-26a was increased in the OGDR model. Moreover, upregulation of miR-26a promoted cell viability and inhibited OGDR-induced apoptosis. PTEN was confirmed as a direct target gene for miR-26a. Under OGDR conditions, the expression of PTEN was significantly decreased. Moreover, overexpression of PTEN inhibited cell viability, promoted cell apoptosis and deepened the effect of the OGDR model. CONCLUSIONS: MiR-26a promoted the viability of SH-SY5Y cells and suppressed apoptosis under OGDR conditions by targeting PTEN.

[Influence of low dose perfluorooctanoate acid exposure to the cell proliferation, migration and invasion of the human muscle rhabdomyosarcoma cell line].

Objective: This study aimed to explore the effect of perfluorooctanoate acid (PFOA) on the proliferation, migration and invasion of the human muscle rhabdomyosarcoma RD cell line and its related mechanisms. Methods: RD cells were cultured and exposed to PFOA of different concentrations with 6-72 hours. The cell viability was assessed by cell counting kit-8 (CCK-8) assay. Wound healing and transwell filter assay were used to evaluated the migration and invasion ability of the RD cells respectively. The cell cycles were detected by Flow cytometry. Quantitative real-time PCR and Western blot were used to quantify the mRNA and protein expression difference of related genes, respectively. Results: CCK-8 assay showed that, after treated the RD cell with different dose of PFOA for 72 h, low dose PFOA (1,10,50, 100 µmol/L) promotes the proliferation of RD cells while high dose PFOA (250, 500 mol/L) inhibits the proliferation (P<0.001). Flow cytometry showed that compared with the control group, there was no significant difference in G0/G1 phase, while cells in S phase deceased and G2/M phase cells increased after treated with PFOA (50 µmol/L) for 72 h. The relative proportions of S and G2/M were significantly different between the two groups (P<0.01). The results of qPCR showed that the mRNA relative expression of CDK2 of the control group and the PFOA (50 µmol/L) group were 0.97±0.07 and 2.64±0.11 respectively, and there was a significant difference (t=12.60, P<0.001); The mRNA relative expression of cyclin E2 of the control group and the PFOA (50 µmol/L) group were 1.33±0.17 and 3.35±0.22 respectively, and there was a significant difference (t=7.42, P<0.001); The results of Western blot showed that the protein relative expression of CDK2 of the control group and the PFOA (50 µmol/L) group were 0.35±0.01 and 0.84±0.03 respectively, and there was a significant difference (t=14.60, P<0.001); The protein relative expression of cyclin E2 of the control group and the PFOA (50 µmol/L) group were 0.67±0.04 and 0.86±0.01 respectively, and there was a significant difference (t=4.88, P<0.01); There was no significant difference in the mRNA and protein expression of p21 and p53 between the PFOA and control group (P>0.05). The wound healing rate of the PFOA (50 µmol/L) group was faster than that of the control group, and the relative migration area of the PFOA group was larger accordingly (P<0.001). After PFOA (50 µmol/L) treated, the number of the cell through the membranes was much more than the control group (t=54.40, P<0.001), which means PFOA significantly stimulated the invasion ability of the RD cells. The results of qPCR showed that the mRNA relative expression of vimentin of the control group and the PFOA (50 µmol/L) group were 0.71±0.03 and 2.53±0.16 respectively, and there was a significant difference (t=11.00, P<0.001); The mRNA relative expression of MMP2 of the control group and the PFOA (50 µmol/L) group were 1.09±0.04 and 10.73±1.20 respectively, and there was a significant difference (t=8.04, P<0.001). The results of Western blot showed that the protein relative expression of vimentin of the control group and the PFOA (50 µmol/L) group were 0.55±0.06 and 0.81±0.01 respectively, and there was a significant difference (t=4.50, P<0.05). The protein relative expression of cyclin E2 of the control group and the PFOA (50 µmol/L) group were 0.64±0.04 and 1.03±0.13 respectively, and there was a significant difference (t=2.94, P<0.05). Conclusions: Low dose PFOA (50 µmol/L) exposure promotes cell proliferation, migration and invasion in the human muscle rhabdomyosarcoma cell line through inducing the expressions of MMP2, vimentin and cell cycle related genes.

Postconditioning Protection Against Myocardiocyte Anoxia/Reoxygenation Injury From Penehyclidine Hydrochloride.

BACKGROUND/AIMS: To investigate the postconditioning protective effect of penehyclidine hydrochloride (PHC) against anoxia/reoxygenation (A/R) injury in H9c2 cells along with the involved mechanism and timing effect. METHODS: We divided H9c2 cells into 7 groups: control group, A/R group and PHC+A/R groups at 0 min, 5 mins, 10 mins, 20 mins, 30 mins, respectively (treated with 0.1 µm/L PHC at 0 min, 5 mins, 10 mins, 20 mins, 30 mins after the reoxygenation procedure began). Cell apoptosis, oxidative stress, intracellular Ca2+ concentration, mitochondrial membrane potential and mitochondrial permeability transition pore (MPTP) opening were explored. Bcl-2, Bax, Cyt C, caspase-3 and caspase-9 levels were measured. RESULTS: A/R significantly increased both cell injury and cell apoptosis. PHC showed postconditioning protective effect by attenuating superoxide production, decreasing Ca2+ overload, restraining MPTP activities, restoring mitochondrial membrane potential, regulating cell apoptosis proteins and modulation of mitochondrial pathway. Earlier administration of PHC offered greater postconditioning protective effect. CONCLUSION: H9c2 cells were protected by PHC from A/R injury regardless of timing of PHC administration (0 min, 5 mins, 10 mins, 20 mins, 30 mins). However, earlier administration of PHC resulted in better PHC postconditioning protection.