[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

Serum hepatitis B surface antigen correlates with fibrosis and necroinflammation: A multicentre perspective in China.

The kinetics of serum hepatitis B surface antigen (HBsAg) during the natural history of hepatitis B virus (HBV) infection has been studied, but the factors affecting them remain unclear. We aimed to investigate the factors affecting HBsAg titres, using data from multicentre, large-sized clinical trials in China. The baseline data of 1795 patients in 3 multicentre trials were studied, and the patients were classified into 3 groups: hepatitis B early antigen (HBeAg)-positive chronic HBV infection (n = 588), HBeAg-positive chronic hepatitis B (n = 596), and HBeAg-negative chronic hepatitis B (n = 611). HBsAg titres in the different phases were compared, and multiple linear progression analyses were performed to investigate the implicated factors. HBsAg titres varied significantly in different phases (P = .000), with the highest (4.60 log10 IU/mL [10%-90% confidence interval: 3.52 log10 IU/mL-4.99 log10 IU/mL]) in patients with HBeAg-positive chronic HBV infection. In all phases, age and HBV DNA were correlated with serum HBsAg level. In HBeAg-positive chronic hepatitis B patients, a negative correlation between HBsAg titres and fibrosis stage was observed. Alanine amonitransferase or necroinflammatory activity was also correlated with HBsAg titres in HBeAg-negative chronic hepatitis B patients. In conclusion, decreased HBsAg titres may be associated with advancing fibrosis in HBeAg-positive chronic hepatitis B patients or increased necroinflammation in those with HBeAg-negative chronic hepatitis B. Our findings may help clinicians better understand the kinetics of HBsAg and provide useful insights into the management of this disease.

[Response and prognosis of neoadjuvant dose-dense or standard schedule chemotherapy with anthracyclines and taxanes for Luminal B breast cancer].

Objective: To evaluate the efficacy and safety of neoadjuvant dose-dense or standard schedule chemotherapy with anthracyclines and taxanes for Luminal B (HER2-)Breast Cancer. Methods: From January 2010 to December 2014, 168 Luminal B (HER2-) breast cancer patients with stageⅡA-ⅢC confirmed by pathology were randomly assigned to receive one of the following regimens: (group A) concurrent TEC× 4 every 3 weeks, ( group B ) sequential EC× 4-T × 4 every 3 weeks, (group C ) dose-dense TEC× 4 every 2 weeks with G-CSF, (group D) sequential EC× 4(dose-dense)-T × 4 with dose-dense every 2 weeks . Results: A total of 168 patients completed the neoadjuvant chemotherapy as planned. The pathologic complete response (pCR) was 16.8% in the 4 groups.The pCR were 30.9% and 26.1% in the group C and group D respectively, significantly higher than patients with group A and group B(9.5%and 7.1%) ( P<0.05). Median follow-up was 43 months (IQR 3-63). The 3-year disease free survival (DFS) rate was 64.7%, 55.5%, 87.8% and 92.1% and the 3-year overall survival(OS)rate was 79.4%, 77.7%, 95.1%, 97.3% in the 4 groups respectively. Patients in the dose-dense group had better 3-year DFS and 3-year OS than those with the regular group.The side-effects could be evaluated in 154 patients.The incidence of neutropenia was 29.2% and 21.9% in the group C and group D versus 65.7%and 51.3% in the regular group(P<0.05), the incidence of nervous toxicity was 54.2%, 18.9%, 60.0%, 26.8% in the 4 groups respectively. The incidence of nervous toxicity in the dose-dense group was lower than that in the regular regimen group(P<0.05). Conclusion: Neoadjuvant dose-dense chemotherapy with anthracyclines and taxanes for Luminal B (HER2-)Breast Cancer was effective and can improve the pCR, DFS and OS.Comparing the two dose dense regimens, sequentially with anthracyclines and taxanes, the incidence of nervous toxicity were lower.

[The diagnostic value of plasma fibrin related markers in perioperation of femoral fracture].

Objective: To investigate potential value of fibrin related markers in patients with femoral fracture during perioperative period. Methods: Ninety-five patients were enrolled, including 39 males with (53±24) years old and 56 females with (73±13) years old, of which 44 fracture on caput femoris, 34 collum femoris and 17 shaft of femur. Sampling on the day before operation and 1(st,) 3(rd,) 5(th) days after operation, fibrin monomer (FM), D-dimer(DD), fibrinogen degradation product (FDP) and other coagulation assays were detected by reagents from Stago. Difference in day-to-day and between surgical sites were analyzed with general linear model (repeated measures). Results: FDP level on pre-operation, 1(st,) 3(rd,) 5(th) day after operation were 7.88(5.19, 12.12), 15.68(9.84, 29.48), 8.44 (6.27, 12.49) and 10.28 (7.56, 14.00) mg/L, the value of fibrin monomer were 5.00 (5.00, 6.03), 9.89(5.04, 30.12), 5.00 (5.00, 6.04) and 5.02(5.00, 5.76) mg/L. The value of D-Dimer were 2.24(1.41, 3.60), 4.78(2.74, 9.18), 2.60(1.79, 3.88) and 2.91(2.20, 3.85) mg/L, respectively, each parameter changs statistically during observation (Z=4.758, 6.027, 3.238 respectively, P<0.05). On the 5th day after surgery, fibrin monomer in patients with venous thrombus embolism (VTE) were higher than that in patients without VTE, 10.18(7.24, 28.11) mg/L vs 5.10(5.00, 6.73) mg/L (Z=-1.580, P<0.05), which showed potential value evaluating of post-operative VTE. No statistical changes were found in prothrombin time or thrombin time (TT) (P>0.05), but activated partial thromboplastin time (APTT) varied from day to day in (38.1±4.9), (40.8±5.2), (45.1±6.2) and (41.9±6.3)s with statistically difference (F=7.127, P<0.05). Similarly, fibrinogen changed statistically in (5.01±0.94), (4.99±1.35), (6.00±1.75), (5.81±1.38)g/L (F=8.927, P<0.05). Conclusion: Fibrin monomer, additional to markers as D-dimer, shows its value on activated coagulation associated to post-operative thromboembolism for patients receiving femoral surgery.

Relationship between adipoq gene polymorphism and lipid levels and diabetes.

Metabolic syndrome (MS), a series of physiological and metabolic disorders caused by insulin resistance, combines clinical syndrome of abdominal obesity, diabetes or impaired glucose regulation, dyslipidemia, hypertension and other metabolic diseases. Several studies have found that multiple single nucleotide polymorphism (SNP) exists in adiponectin gene (ADIPOQ) and some unusual mutations might be related to hypoadiponectinemia and MS. This study aims to explore the relationship between ADIPOQ gene polymorphism and lipid levels and diabetes. A total of 1,049 confirmed MS cases were selected for research. From the perspective of potential functional SNPs of ADIPOQ gene, tag SNPs, combined with environmental factors, we studied the relationship between ADIPOQ gene polymorphism and phenotype (serum adiponectin level) and further analyzed the correlation of ADIPOQ gene polymorphism and metabolic syndrome components so as to clear the relationship between ADIPOQ gene polymorphism and lipid levels and diabetes and at the same time provide a scientific basis for preventing primarily MS etiology and screening high-risk groups.

Effects of changes in serum endostatin and fibroblast growth factor 19 on the chemotherapeutic sensitivity in acute myeloid leukemia patients.

The present study aimed to explore the changes in serum endostatin and fibroblast growth factor 19 (FGF-19) in acute myeloid leukemia patients, and to determine their effects on chemotherapeutic sensitivity. Sixty acute myeloid leukemia patients and 30 healthy controls were included in the study. Patient serum endostatin and FGF-19 levels were measured on admission, and then, standard chemotherapy was administered. The patients were divided into 2 groups according to chemotherapeutic effects: 21 patients in the chemotherapeutic sensitivity group (complete remission + partial remission) and 39 in the chemotherapeutic resistance group (no remission + degradation). A receiver operating characteristic (ROC) curve was used to analyze the relationship of serum endostatin and FGF-19 levels with chemotherapeutic sensitivity in acute myeloid leukemia patients. The levels of serum endostatin and FGF-19 in acute myeloid leukemia patients before chemotherapy were significantly higher than those in the control group. Moreover, these levels significantly decreased after chemotherapy (P < 0.01). The levels of serum endostatin and FGF-19 in the chemotherapeutic sensitivity group were lower than those in the chemotherapeutic resistance group, both before and after chemotherapy (P < 0.05 and P < 0.01, respectively). ROC curve analysis showed that the predictive values of endostatin and FGF-19 were good, and there was no significant difference between these results. In conclusion, serum endostatin and FGF-19 can be used as predictors of chemotherapeutic sensitivity for acute myeloid leukemia patients, and may be important for determining prognosis.

Autophagy in drug resistance of the multiple myeloma cell line RPMI8226 to doxorubicin.

We investigated the effect of autophagy on drug resistance of multiple myeloma (MM) to doxorubicin (DOX). A DOX-resistant MM cell line (RPMI8226/DOX) was developed by progressively increasing the DOX concentration gradient. The drug resistance index was determined using the MTT method. Transmission electron microscopy, anti-light chain 3-fluorescein isothiocyanate immunofluorescence, and Western blotting were used to detect autophagy of MM cells. Flow cytometry was applied to detect changes in apoptosis of RPMI8226/DOX cells (stained with annexin-V/propidium iodide) caused by inhibition by hydroxychloroquine and 3-methyladenine on autophagy. The drug resistance index of RPMI8226/DOX to DOX was 10.8, and autophagy/lysosomal was clearly observed in RPMI8226/DOX cells under transmission electron microscopy, while immunofluorescence showed granular immunofluorescence in cells. Western blot analysis showed that light chain 3-II protein expression level was higher in RPMI8226/DOX cells than in RPMI8226/S cells. The apoptosis test showed that hydroxychloroquine or 3-methyladenine partially reversed the drug resistance of RPMI8226/DOX cells by inhibiting autophagy. Activation of autophagy in MM cells may explain the drug resistance of myeloma.

Development of EST-SSR markers related to disease resistance and their application in genetic diversity and evolution analysis in Gossypium.

Cotton (Gossypium spp) is one of the most economically important crops that provide the world's most widely used natural fiber. Diseases such as Fusarium wilt and particularly Verticillium wilt seriously affect cotton production, and thus breeding for disease resistance is one of the most important goals of cotton breeding programs. Currently, potential exists to improve disease resistance in cultivated cotton. Increasing the understanding of the distribution, structure, and organization of genes or quantitative trait loci for disease resistance will help the breeders improve crop yield even in the event of disease. To facilitate the mapping of disease-resistance quantitative trait loci to achieve disease-resistant molecular breeding in cotton, it is necessary to develop polymorphic molecular markers. The objective of this study was to develop simple sequence repeat markers based on cotton expressed sequence tags for disease resistance. The efficacy of these simple sequence repeat markers, their polymorphisms, and cross-species transferability were evaluated. Their value was further investigated based on genetic diversity and evolution analysis. In this study, the unique sequences used to develop markers were compared with the G. arboretum and G. raimondii genome sequences to investigate their position, homology, and collinearity between G. arboretum and G. raimondii.

Phylogeography of Cephalotaxus oliveri (Cephalotaxaceae) in relation to habitat heterogeneity, physical barriers and the uplift of the Yungui Plateau.

Habitat heterogeneity, physical barriers, and the uplift of the Yungui Plateau were found to deeply affect the phylogeographic pattern and evolutionary history of Cephalotaxus oliveri, a perennial conifer endemic to China. In this study, we explored the phylogeography using three chloroplast sequences (trnL-trnF, trnT-trnD and atpB-rbcL) in 22 natural populations of C. oliveri distributed throughout its range. The Yungui Plateau populations of C. oliveri were revealed to origin ca. 9.15Ma by molecular clock estimation, which is consistent with rapid uplift of the Qinghai-Tibetan Plateau (QTP) ca. 8-10Ma. Additionally, geological effects of the Yungui Plateau were suggested to promote the rapid intra-specific differentiation of C. oliveri in the Pliocene and Early Pleistocene. The relatively low level of genetic diversity (h=0.719, θ=1.17×10(-3)) and high population differentiation (NST=0.771 and GST=0.642) implied restricted gene flow among populations, which was confirmed by the Nested Clade Analysis (NCA). Mismatch distribution and haplotypes network provided evidences of recent demographic population expansion. Furthermore, the statistical dispersal-vicariance analysis indicated that the center of origin was in Central China. The comparison of haplotype distribution patterns indicated that the regions of HNHPS and HBLD were the potential refugia during the Pleistocene ice ages. Our results highlighted that habitat heterogeneity and physical barriers presenting in a species range can predict genetic patterns.

Timing of xenon-induced delayed postconditioning to protect against spinal cord ischaemia-reperfusion injury in rats.

BACKGROUND: This study was designed to assess the neuroprotective effect of xenon-induced delayed postconditioning on spinal cord ischaemia-reperfusion injury (IRI) and to determine the time of administration for best neuroprotection in a rat model of spinal cord IRI. METHODS: Fifty male rats were randomly divided equally into a sham group, control group, and three xenon postconditioning groups (n=10 per group). The control group underwent spinal cord IRI and immediately inhaled 50% nitrogen/50% oxygen for 3 h at the initiation of reperfusion. The three xenon postconditioning groups underwent the same surgical procedure and immediately inhaled 50% xenon/50% oxygen for 3 h at the initiation of reperfusion or 1 and 2 h after reperfusion. The sham operation group underwent the same surgical procedure without aortic occlusion, and inhaled 50% nitrogen/50% oxygen. Neurological function was assessed using the Basso, Beattie, and Bresnahan score at 4, 24, and 48 h of reperfusion. Histological examination was performed using Nissl staining and immunohistochemistry, and apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling staining. RESULTS: Compared with the control group, the three xenon postconditioning groups showed improvements in neurological outcomes, and had more morphologically normal neurones at 48 h of reperfusion. Apoptotic cell death was reduced and the ratio of Bcl-2/Bax immunoreactivity increased in xenon-treated rats compared with controls. CONCLUSIONS: Xenon postconditioning up to 2 h after reperfusion provided protection against spinal cord IRI in rats, but the greatest neuroprotection occurred with administration of xenon for 1 h at reperfusion.