Reverse transcription recombinase-aided amplification assay for rapid detection of the influenza A(H1N1)pdm09 H275Y mutation that confers oseltamivir resistance.

The emergence of the influenza A(H1N1)pdm09 virus with the NA-H275Y mutation, which confers oseltamivir resistance, must be monitored, especially in patients undergoing neuraminidase inhibitor treatment. In this study, we developed a reverse transcription recombinase-aided amplification assay that has high sensitivity (detection limit: 1.0 × 101 copies/µL) and specificity for detecting the oseltamivir-resistant H275Y mutation; the assay is performed within 30 min at a constant temperature of 39° Celsius using an isothermal device. This method is suitable for the clinical application of targeted testing, thereby providing technical support for precision medicine in individual drug applications for patients with severe infection or immunosuppression.

The peripheral blood transcriptome identifies dysregulation of inflammatory response genes in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women, resulting in ovulation failure and other metabolic problems. However, the underlying mechanisms of it remain largely uncertain due to the complexity of clinical manifestations. This systemic disorder is involved in endocrine, metabolism, immune system and many organs, and few studies have explored peripheral blood transcriptome in patients with PCOS. We performed gene expression profiling of peripheral blood from 8 PCOS patients and eight healthy women with microarray. The significance analysis of microarray (SAM) software was employed to screen the differentially expressed genes (DEGs) and gene ontology (GO) was used for functional enrichment analysis. In total, 181 DEGs with fold-changes >2.0 and q-values <0.05 were identified between the two groups. Among them, 149 were up-regulated and 32 down-regulated in PCOS. Unsupervised clustering of expressed genes could readily differentiate PCOS from control. More importantly, inflammatory response pathway including 14 dysregulated genes was highly enriched in PCOS. Furthermore, 10 DEGs were validated using quantitative reverse-transcription PCR (qRT-PCR) assays. Our study provides independent evidence for the involvement of systemic inflammatory response in PCOS and it may facilitate a greater understanding of this complex disease.

[Time interval from the end of sperm processing to artificial intrauterine in semination with husband's sperm correlates to the rate of clinical pregnancy].

OBJECTIVE: To investigate the influence of the time interval from the end of semen processing to artificial intrauterine in semination with husband's sperm (AIH-IUI) on the rate of clinical pregnancy. METHODS: This study involved 191 AIH-IUI cycles with the same ovulation induction protocol. After Percoll density gradient centrifugation, we divided the sperm into four groups based on the incubation time: 0-19, 20-39, 40-59, and 60-80 min, and again into another four groups according to the total progressively motile sperm count (TPMC): (0-9), (10-20), (21-30), and > 30 x 10(6). We analyzed the correlation of the clinical pregnancy rate with the time interval from the end of sperm processing to AIH-IUI and with other influencing factors, such as maternal age, infertility duration, and semen quality. RESULTS: The rate of clinical pregnancy was significantly higher in the 20-39 min group (18.3%) than in the 0-19, 40-59, and 60-80 min groups (12.7, 11.4 and 9.1%) (all P < 0.05). The (10-20) x 10(6) group achieved a remarkably higher pregnancy rate (16.7%) than the (0-9), (21-30), and > 30 x 10(6) groups (0, 11.4, and 8.3%) (all P < 0.05). Logistic multivariate analysis showed that the rate of clinical pregnancy was decreased with the increased age of the women (OR 0.89, 95% CI 0.83-0.94) but significantly elevated in the 20-39 min group (OR 2.11, 95% CI 1.34-3.13) and of (10-20) x 10(6) group (OR 2.06, 95% CI 1.32-3.46). CONCLUSION: The time interval from the end of sperm processing to AIH-IUI is a most significant factor influencing the rate of clinical pregnancy of AIH-IUI.

Epidemiologic characterization of 30 confirmed cases of human infection with avian influenza A(H7N9) virus in Hangzhou, China.

BACKGROUND: We examined the clinical and epidemiological characteristics of 30 cases of human infection with avian influenza A(H7N9) virus in Hangzhou and investigated their external environments to provide evidence for contact tracing and disease prevention and control. METHODS: The cases confirmed from April 1 through May 1, 2013 were studied. Field epidemiologic surveys were conducted to collect the clinical and epidemiologic data. Case-related and environmental specimens were collected for etiologic detection. RESULTS: Thirty cases of human infection with avian influenza A(H7N9) virus were confirmed in Hangzhou from April 1 through May 1, 2013, including one pregnant woman and three deaths. The median age of the patients was 62 years (range: 38-86 years). Twenty-three of the patients were men (76.67%). The median duration between disease onset and occurrence of respiratory failure and confirmed diagnosis was 5 and 6 days, respectively. Maximum medical observation of 666 close contacts of the patients revealed no irregularity. Of 314 external environmental specimens, the overall positive detection rate of H7N9 nucleic acid was 28.98%. Eight districts of Hangzhou city had positive detections in the external environments, the highest rate being in Yuhang District (78.13%). Statistical analysis of the specimen collection locations indicates a significant difference between the case-linked locations and the non-case locations (χ2 = 16.563, p < 0.05) in terms of H7N9 viral nucleic acid detection rate. No epidemiologic link has been found among the 30 cases. CONCLUSIONS: Most of the infected were retired individuals aged 60 years or older. Men made the majority. The cases are sporadic at present, with no evidence of human-to-human transmission. Exposures to poultry and live poultry markets may be important sources of infection.

Pandemic influenza A(H1N1) 2009 virus in pregnancy.

Two hundred fourteen abstracts and 87 full texts regarding pregnant women infected with pandemic influenza A(H1N1) 2009 virus were systematically reviewed by using a PubMed search and assessing pandemic, clinical, laboratory test, vaccine, and control experiences. Both policy and health education were excluded. This review counted the total number of pregnant cases from different countries and analyzed their epidemic features, including trimester distribution, morbidity, hospitalization, intensive care unit admissions, maternal mortality, underlying diseases, complications, high-risk factors for death, pregnancy outcome, and clinical symptoms compared with the previous pandemic seasonal influenza A/H1N1 as compared with the general population. Early identification and treatment were the most important factors in different countries and areas examined. The vaccine and antiviral drugs that have been the most efficient means to control the novel virus appear to be safe but require more extensive study. In the future, the focus should be placed on understanding vertical transmission and the severe mechanisms.

Fibronectin promotes tumor progression through integrin αvß3/PI3K/AKT/SOX2 signaling in non-small cell lung cancer.

The tumor microenvironment, especially the extracellular matrix (ECM), is strongly associated with tumor cell proliferation and metastasis. Numerous studies have provided evidence suggesting that fibronectin (FN) in ECM supports cancer cell escape and contributes to cell migration, resulting in distant cancer metastasis and poor outcomes in patients. In our study, it was demonstrated that FN expression was elevated in tumor tissues from highly malignant NSCLC patients, compared to those with low malignancy (p = 0.0076). Importantly, FN promoted proliferative phenotypes and strengthened tumorigenesis capabilities in NSCLC cells, including A549 and Lewis cells, leading to sustained tumor growth in vivo. Mechanistically, it was identified that FN facilitated the activation of the integrin αvß3/PI3K/AKT signaling pathway, which subsequently upregulated tumor stemness through the downstream transcription factor SOX2. Blockade of integrin αvß3 signal efficiently suppressed NSCLC proliferation and tumorigenesis both in vitro and in vivo. In conclusion, our study demonstrated that extracellular FN could facilitate NSCLC development through the integrin αvß3/PI3K/AKT/SOX2 signaling pathway. Blockade of integrin αvß3 could efficiently enhance the anticancer effects of chemotherapy, offering an innovative approach for clinical NSCLC therapy.

Two adenovirus serotype 3 outbreaks associated with febrile respiratory disease and pharyngoconjunctival fever in children under 15 years of age in Hangzhou, China, during 2011.

Adenovirus serotype 3 and 7 outbreaks have occurred periodically in northern, eastern, and southern China since 1955, but there has been no report since the adenovirus serotype 7 outbreak first occurred in Hangzhou, China, in 1991. Here we explored the epidemiology and etiology of two adenovirus serotype 3 outbreaks in Hangzhou in 2011. One acute respiratory outbreak was found in Chun'an County, where a total of 371 cases were confirmed in 5 of 23 towns from 4 to 31 May 2011. The outbreak affected 18.57% (13/70) of schools and 14.49% (90/621) of classes. The incidence was 5.18% (371/7,163). The population was distributed among individuals ages 7 to 15 years. No parents or teachers were infected. Another pharyngoconjunctival fever outbreak was discovered in the Chenjinglun Swimming Center located in the Xihu District between 1 and 15 July 2011. A total of 134 cases were confirmed in 900 amateur swimmers, with an incidence of 14.89% (134/900). The ages ranged from 4 to 9 years. The two outbreaks had no severe complications or death. The viruses in 66.67% (10/15) of throat swabs from children with acute respiratory infections and 100% (10/10) of the swabs from children with pharyngoconjunctival fever were confirmed to be adenovirus serotype 3 with 100% homology by PCR. Of these samples, 60.0% (12/20) had a classical characteristic cytopathic effect, presented as grape-like clusters at 72 h after infection in HEp-2 cells. In conclusion, the acute respiratory infection and pharyngoconjunctival fever outbreak in Hangzhou were caused by the completely homologous type 3 adenovirus in subgenus B. Moreover, these outbreaks demonstrated rapid transmission rates, possibly due to close contact and droplet transmission.

Ginsenoside-Rg1 mediates microenvironment-dependent endothelial differentiation of human mesenchymal stem cells in vitro.

Bone marrow-derived mesenchymal stem cells (MSCs) possess a multi-lineage differentiation potential and have the ability to repair and rebuild injured vessels. The autologous differentiated MSC transplantation also makes possible the tissue-engineered grafts. Therefore, the efficient endothelial differentiation of MSCs could be beneficial in the successful injured vessel repair and engraftment. Ginsenoside-Rg1, the most prevalent active constituent of ginseng, is a potent proangiogenic factor of vascular endothelial cells and also has the ability to enhance the proliferation of bone marrow cells. The aim of this study is to investigate the role of ginsenoside-Rg1 in the microenvironment-dependent endothelial differentiation of human MSCs (hMSCs) in vitro. The endothelial differentiation environment was established by co-culturing hMSCs with mature endothelial cells (human umbilical vein endothelial cells) indirectly in vitro. Reverse transcriptase-polymerase chain reaction analysis and fluorescence immunocytochemistry showed a strong expression of endothelial-specific markers such as CD31, Von Willebrand factor, and VE-cadherin. Electron microscopy showed the endothelial characteristic Weibel-Palade bodies of differentiated hMSCs. The increased expression of CD31 demonstrated that Rg1 promoted the endothelial differentiation of hMSCs. The findings here show the differentiation of hMSCs into cells with phenotypic features of endothelial cells using indirect co-culture with mature endothelial cells and provide the evidence that ginsenoside-Rg1 can promote the milieu-dependent endothelial differentiation of hMSCs in vitro.

2009 pandemic characteristics and controlling experiences of influenza H1N1 virus 1 year after the inception in Hangzhou, China.

This paper described the epidemiology and controlling experiences of influenza H1N1 in Hangzhou in the past 1 year. A total of 2,078 cases confirmed by real-time quantitative PCR till March 31, 2010, were analyzed by SPSS 12.0 software. During the early pandemic stage, a patient must be tested for H1N1 nucleic acid once he/she had influenza-like symptoms with the epidemiological history in 7 days, and be diagnosed if it was positive. But in the pandemic peak, we made efforts to identify and save severe cases combined with pneumonia or hypoxemia or respiratory failure or septic shock or multiple organ dysfunctions and failure. In general, the prevalence was 2.77/100,000 (2,078/7,510,844); severity rate, 10.44% (217/2,078); fatality rate, 0.48% (10/2,078). The carrier and secondary attack rate were 9.52% (58/612) and 8.66% (53/612), respectively. About 50% of serious cases and 100% of deaths had the basic underlying diseases: cardiovascular diseases, 13.66% (25/217); chronic lung disease, 12.02% (22/217); pregnant, 7.1% (13/217). Of all cases aged from 1 month to 89 years, 52.99% (1,435/2,708) were in the 10-29 years, with most of them distributed in downtown area. The timeline showed two epidemic peaks occurred in September and November 2009, respectively. Furthermore, the hemagglutinin gene remained 99% identical with the American and vaccine strains, but only 70% with the 1947-2008 Chinese strains. In conclusion, Hangzhou pandemic influenza H1N1 was caused by the highly conserved virus, with low prevalence, transmission, and mortality, because we took efficient controlling methods.

[Effect of ginsenoside Rg1 on the microenvironment dependent differentiation of human bone marrow mesenchymal stem cell to vaso-endothelioid formative cells in vitro].

OBJECTIVE: To investigate the effect of ginsenoside Rg1 on the microenvironment dependent differentiation of human mesenchymal stem cells (hMSCs) to vaso-endothelioid cells (VECs) in vitro. METHODS: The in vitro differentiation of hMSCs to VECs were established adopting the in vivo environment simulated semi-permeable membrane separated non-contact co-culturing method. The mRNA expressions of endothelial markers, such as platelet endothelial adhesive factor-1 (CD31), vascular hemophillia factor (vWF) and vascular endothelial cadherin (VE-cadherin) were analyzed by RT-PCR; the protein expressions of CD31 and vascular endothelial adhesive factor-1 (VCAM1) were detected by fluorescence immunohistochemistry; structural identification for the endothelial characteristics of differentiated hMSCs were made under electron microscopy; and the percentage of CD31 expression in differentiated hMSCs was determined by flow cytometry to explore the effect of ginsenoside Rg1 on the differentiation. RESULTS: The bone marrow mesenchymal stem cells co-cultured with mature endothelial membrane showed a microenvironment dependent capacity for differentiating to endothelium, with the morphological changes revealed starting from the 2nd week, showing cell body contraction, polygonal-shaped change; and at the 3rd week, the markedly speedily cell proliferation with elliptic or slabstone-like change of cells. High levels of classic endothelial cell markers, such as mRNA expressions of CD31, vWF, VE-cadherin, and protein expressions of CD31 and VCAM1, were shown; the typical weibel-palade body of endothelial cell was found in the differentiated cells. Moreover, percentage of CD31 expression in the differentiated hMSCs was increased after Rg1 treatment dose-dependently. CONCLUSION: Under the microenvironment of co-culture, hMSCs could differentiate into cells presenting the characteristics of endothelial cell in aspects of the morphology and ultrastructure of cells, as well as the gene and protein expressions of cell markers; ginsenoside Rg1 can promote the microenvironment dependent differentiation of hMSCs to VECs system in vitro.

RNAi-mediated human Nestin silence inhibits proliferation and migration of malignant melanoma cells by G1/S arrest via Akt-GSK3ß-Rb pathway.

Human Nestin (hNestin) has been found to express in melanoma, and its expression is positively correlated with the advanced stage of melanoma. However, the precise role of hNestin in the development of melanoma has not been fully understood. The present study aimed to explore the role of hNestin in the proliferation and invasion of melanoma cells. The lentivirus vector carrying a short hairpin RNAs (shRNAs) targeting hNestin (hNestin-shRNA-LV) was stably infected into human melanoma cells UACC903, which expressed high levels of hNestin. The effects of hNestin knockdown on the proliferation, apoptosis, migration of melanoma cells and the related signaling pathways were investigated by immunofluorence, Western blotting and reverse transcription polymerase chain reaction (RT-PCR), respectively. The results showed that hNestin was expressed in most melanoma specimens and the melanoma cells studied. Knockdown of hNestin expression significantly inhibited the proliferation of melanoma cells, blocked the formation of cell colony, arrested cell cycle at G1/S stage and suppressed the activation of Akt and GSK3ß. hNestin-silent cells also showed a sheet-like appearance with tight cell-cell adhesion, decreased membrane expression of N-cadherin and ß-catenin, and attenuated migration. Furthermore, hNestin silence resulted in the inhibition of tumor growth in vivo. Our study indicates that hNestin knockdown suppresses the proliferation of melanoma cells, which might be through affecting Akt-GSK3ß-Rb pathway-mediated G1/S arrest, and hNestin silence inhibits the migration by selectively modulating the expression of cell adhesion molecules in the process of epithelial-mesenchymal transition.

Interim estimates of divergence date and vaccine strain match of human influenza A(H3N2) virus from systematic influenza surveillance (2010-2015) in Hangzhou, southeast of China.

OBJECTIVES: In the post-pandemic period 2010-2015, seasonal influenza A(H3N2) virus predominated in Hangzhou, southeast of China, with an increased activity and semi-annual seasons. This study utilized HA virus gene segment sequences to analyze the divergence date and vaccine strain match of human influenza A(H3N2) virus from systematic influenza surveillance in Hangzhou. METHODS: Virological and serological analyses of 124 representative A(H3N2) viruses from prospective studies of systematic surveillance samples were conducted to quantify the genetic and antigenic characteristics and their vaccine strain match. RESULTS: Bayesian phylogenetic inference showed that two separate subgroups 3C.3 and 3C.2 probably diverged from group 3C in early 2012 and then evolved into groups 3C.3a and 3C.2a, respectively, in the 2014/15 influenza season. Furthermore, high amino acid substitution rates of the HA1 subunit were found in A(H3N2) group 3C.2a variants, indicating that increased antigenic drift of A(H3N2) group 3C.2a virus is associated with a vaccine mismatch to the 2015/16 vaccine reference strain Switzerland/9715293/2013 (group 3C.3a). CONCLUSIONS: A portion of the group 3C.2a isolates are not covered by the current A(H3N2) vaccine strain. These findings offer insights into the emergence of group 3C.2a variants with epidemic potential in the imminent influenza seasons.

Retrospective study of risk factors for mortality in human avian influenza A(H7N9) cases in Zhejiang Province, China, March 2013 to June 2014.

BACKGROUND: The influenza A(H7N9) virus causes a serious disease that threatens human health. Fatalities associated with human infections caused by this virus are of great public health concern; however, the possible risk factors are not yet fully known. METHODS: A stratified sampling method, incorporating household income levels and a random number table method, was used to select laboratory-confirmed A(H7N9) cases for this study. Eighty-five patients were selected randomly from 139 laboratory-confirmed A(H7N9) cases occurring in Zhejiang Province between March 1, 2013 and June 30, 2014. Data were collected using a standard method. To test the statistical significance among discrete variables, univariate analyses were used to compare two groups. The Kaplan-Meier product-limit method was used to analyze the patient survival fraction. The Cox proportional hazards regression model was used to analyze all variables with p ≤ 0.05 in the univariate analysis. Lastly, a stepwise procedure was used to construct a final model with a significance level of p > 0.10 for removal and p<0.05 for re-entry. RESULTS: A total of 85 patients with H7N9 virus infection were identified. Among these, 30 (35.29%) died. In the univariate analysis, the following factors were associated with a high risk of influenza A(H7N9) case fatality: age ≥ 60 years (p=0.008), low education level (p=0.030), chronic diseases (p=0.029), poor hand hygiene (p=0.010), time from illness onset to the first medical visit (p=0.029) and to intensive care unit admission (p=0.008), an incubation period of ≤ 5 days (p=0.039), a peak C-reactive protein ≥ 120 mg/l (p=0.012), increased initial neutrophil count (p=0.020), decreased initial lymphocyte count (p=0.021), and initial infection of both lungs (p=0.003). Multivariate analysis confirmed that the independent predictors of H7N9 virus infection mortality in Zhejiang, China were hand hygiene (hazard ratio (HR) 5.163, 95% confidence interval (CI) 1.164-22.661), age (HR 1.042, 95% CI 1.007-1.076), and peak CRP (HR 1.009, 95% CI 1.002-1.016). CONCLUSIONS: Improvements in immunity, early case identification and treatment, and personal protection measures are key to addressing the high human avian influenza A(H7N9) case fatality rate.

[A survey on eperythrozoon infection in the population from Hangzhou city, Zhejiang province].

OBJECTIVE: To understand the situation of eperythrozoon (EPE) infection in the population from Hangzhou. METHODS: According to the situation in Hangzhou, a questionnaire was designed to investigate the study population. Venous blood specimens from the studied objects were collected before an Improved Wright-Giemsa Fast Dyeing method was used. Microscopic examination was applied to test their condition of infection. SPSS 13.0 software was applied for statistical analysis. RESULTS: Totally, 580 persons were under investigation in this study, including 111 with jobs ('occupational') and another 118 served as internal controls of them. The rest 351 were considered as external controls. Finally, 54 people were detected as having EPE infection with a total positive rate as 9.31%. The infection rates were 20.72% (23/111), 7.12% (25/351) and 5.08% (6/118) among the 'occupational' population, external controls and the internal controls, respectively. The difference among these three rates was statistically significant (χ² = 21.60, P < 0.05). There was also significant difference found in the infection rate between people who washed their hands promptly after being exposed to animal coat, raw meat or animal excrements and those who did not wash their hands in time. The infection rate of the population who washed hands with soap or other cleaning products was lower than that of those who washed hands with only water (χ² = 6.76, P < 0.05). We found that residential area, pet feeding, exposure to animal coat/raw meat/animal excrement, trauma were not risk factors of EPE infection. People with higher education degree had lower risk to EPE infection than those with low education. The infection rate was not different between the populations with different eating habits. The Improved Wright-Giemsa Fast Dyeing method used in this study was good in detecting the positive rates and easy to handle. CONCLUSION: The risk factors to EPE infection were livestock contacting, washing their hands not promptly or washing hands without soap or other cleaning products after contacting.

The study of early application with Dixiong Decoction (地芎汤) for non-small cell lung cancer to decrease the incidence and severity of radiation pneumonitis: A prospective, randomized clinical trial.

OBJECTIVE: To evaluate the efficacy of compound Dixiong Decoction (地芎汤, a Chinese herbal decoction) on early prevention of radiation pneumonitis. METHODS: Forty-six patients with non-small cell lung cancer who were planning to receive radiotherapy were randomly assigned to the treatment group treated with the compound Dixiong Decoction and the control group treated with a commonly used herbal decoction which has the effects of supplementing qi and nourishing yin, clearing heat and detoxifying at the time of radiotherapy. Primary measure was the incidence of radiation pneumonitis after radiotherapy. Secondary outcomes included Watters clinical radiographic physiologic (CRP) dyspnea score, the Radiation Therapy Oncology Group (RTOG) grading score, Karnofsky Performance Status (KPS) score, and the application of corticosteroids. RESULTS: The incidence of radiation pneumonitis in the treatment group was 10.0%, while that in the control group was 26.3% (P=0.0032). The Watters CRP dyspnea score and RTOG grading score in the treatment group were significantly =lower than those in the control group (P<0.05). The KPS score in the treatment group was significantly higher than that in the control group (P<0.01). The dosage of corticosteroids was smaller with a shorter duration of therapy in the treatment group than that in the control group. CONCLUSION: The early application of the Chinese herbal decoction compound Dixiong Decoction can decrease the incidence of radiation pneumonitis, reduce the injury of the lung, and improve the life quality of the patients.