Establishment of qRT-PCR for absolute quantitative detection of Chikungunya virus / 中国热带医学

@#Abstract: Objective　To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results　A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions　The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.

Stimulation quantitation of saponins and sterones from Achyranthis Bidentatae Radix by high-performance liquid chromatography with double external standards calibration method / 药学学报

Saponins and sterones are two main characteristic components in Achyranthis Bidentatae Radix. In order to control the quality of Achyranthis Bidentatae Radix more effectively, a high-performance liquid chromatography (HPLC) method was established by using double external standards calibration method (DESCM) for simultaneous determination of the contents of achyranthoside C, achyranthoside D, β-ecdysterone, 25R-inokosterone and 25S-inokosterone in Achyranthis Bidentatae Radix. Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 (150 mm × 4.6 mm, 2.7 µm) using 0.1% phosphoric acid in water and 0.1% phosphoric acid in acetonitrile as mobile phase. The flow rate was 0.8 mL·min-1 and the column temperature was set as 35 ℃, the injection volume was 5 μL and the total analytical time was 30 min. β-Ecdysterone was used as the reference to calculate the relative correction factors (RCF) and relative retention time (RRT) of 25R-inokosterone and 25S-inokosterone, achyranthoside D was used for achyranthoside C. The RCFs of 25R-inokosterone, 25S-inokosterone, and achyranthoside C were 1.116, 1.056, and 0.888 1, respectively. The double external standards calibration method (DESCM) and external standard method (ESM) were used to calculate the contents of five ingredients in Achyranthis Bidentatae Radix samples from different sources and the variation between the results was within acceptable limits (RE ≤ 5%). The results showed that the contents of two saponins and three sterones of Achyranthis Bidentatae Radix were 0.597%-1.916% and 0.044%-0.150% respectively. The total content of saponins was about 10 times that of sterones. In conclusion, the established DESCM allowed simultaneous determination of five ingredients (achyranthoside C, achyranthoside D, β-ecdysterone, 25R-inokosterone, and 25S-inokosterone) in Achyranthis Bidentatae Radix, providing a scientific and feasible overall quality evaluation method for Achyranthis Bidentatae Radix.

Protective effect of serum containing ginseng and Moutan Cortex on HUVEC injured by H_2O_2 / 中国中药杂志

This study aimed to investigate the effect of serum containing ginseng and Moutan Cortex on human umbilical vein endothelial cells(HUVEC) injured with hydrogen peroxide(H_2O_2). HUVEC injured with H_2O_2 were divided into 6 groups, namely blank group, model group, ginsenoside(TGG) group, total glucosides of Moutan Cortex(TGM) group, paeonol(P) group and TGG+TGM+P group. After 24 hours of co-culture with H_2O_2, the activities of succinate dehydrogenase(SDH) and Ca~(2+)-Mg~(2+)-ATP were detected by microenzyme labeling. The apoptosis rate, intracellular Ca~(2+) concentration, reactive oxygen species(ROS) and mitochondrial membrane potential(JC-1) were detected by flow cytometry. The expressions of mitochondrial apoptosis pathway-related proteins Bax, Bcl-2, cytochrome C, caspase-3 and caspase-9 were detected by Western blot. The results showed that H_2O_2 could significantly damage HUVEC, decrease the activity of SDH and Ca~(2+)-Mg~(2+)-ATP(P<0.01), while could increase the apoptosis+necrosis rate, JC-1 decline rate, ROS increase rate and Ca~(2+) concentration increase rate(P<0.01). Serum containing ginseng and Moutan Cortex could increase the activities of SDH and Ca~(2+)-Mg~(2+)-ATP to different degrees, decrease the apoptosis+necrosis rate, JC-1 decline rate, ROS increase rate and Ca~(2+) concentration increase rate(P<0.05 or P<0.01), and down-regulate the protein expressions of Bax, caspase-3, caspase-9, cytochrome C, and up-regulate the protein expression of Bcl-2. The results showed that serum containing ginseng and Moutan Cortex has a protective effect on vascular endothelial cell injury induced by ROS, and its mechanism may be related to the improvement of mitochondrial function and the inhibition of the activation of mitochondrial apoptosis pathway.

Effect of α7nAChR agonists on acute lung injury caused by cardiopulmonary bypass in rats / 中华麻醉学杂志

Objective To evaluate the effect of α7 nicotinic acetylcholine receptor (α7nAChR)agonists on lung injury caused by cardiopulmonary bypass (CPB) in rats.Methods Eighteen healthy clean-grade adult male Sprague-Dawley rats,weighing 350-400 g,were divided into 3 groups (n =6 each)using a random number table method:sham operation group (group S),CPB group and α7nAChR agonist PHA568487 group (group PHA).The rats underwent no CPB and were mechanically ventilated for 60 min in group S.PHA568487 0.8 mg/kg (diluted to 2 ml in normal saline) was intraperitoneally injected at 30 min before CPB,and then CPB was performed for 60 min in group PHA.Normal saline 2 ml was intraperitoneally injected at 30 min before CPB,and then CPB was performed for 60 min in group CPB.Blood samples were collected from the internal jugular vein for determination of serum interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) concentrations by enzyme-linked immunosorbent assay.Lung tissues were obtained for microscopic examination of the pathologic changes and for determination of wet/dry weight ratio (W/D ratio) and matrix metalloproteinase-9 (MMP-9) expression (by Western blot).Results Compared with group S,the W/D ratio and serum concentrations of TNF-α and IL-6 were significantly increased,and the expression of MMP-9 was up-regulated in CPB and PHA groups (P＜0.05).Compared with group CPB,the W/D ratio and serum concentrations of TNF-α and IL-6 were significantly decreased,the expression of MMP-9 was down-regulated (P＜0.05),and the pathological changes of lung tissues were significantly attenuated in group PHA (P＜0.05).Conclusion α7nAChR agonists can reduce the acute lung injury caused by CPB in rats,and the mechanism may be related to down-regulating MMP-9 expression and inhibiting systemic inflammatory responses.

Clinical application of abdominal lifting and compression cardiopulmonary resuscitation method for treatment of patients with cardiac arrest / 中华危重病急救医学

Objective To investigate the clinical application of rhythmic abdominal lifting and compression cardiopulmonary resuscitation (ALC-CPR) in cardiac arrest (CA) patients with conventional cardiopulmonary resuscitation (CPR) contraindication (chest rib fracture, chest trauma, etc.).Methods The clinical data of patients with CA in emergency department of Hefei Second People's Hospital from June 2015 to December 2016 were analyzed retrospectively, and the patients with weights 40-150 kg and CA≤20 minutes, and resuscitated with ALC-CPR were selected. The vital signs and blood gas analysis indexes of patients before resuscitation and 30 minutes after return of spontaneous circulation (ROSC, after successful resuscitation) were recorded.Results A total of 19 patients with CA were treated with CPR-LW1000 type ALC-CPR apparatus for resuscitation, and with 12 males, 7 females; the age ranged from 43 to 87 years, and the average age was (70.32±13.49) years; there were 4 cases of heart disease, and 15 cases of non-heart disease. Compared with before resuscitation, the heart rate [HR (bpm): 115.05±21.70 vs. 0], mean arterial pressure [MAP (mmHg, 1 mmHg = 0.133 kPa): 66.05±11.69 vs. 0], pulse oxygen saturation (SpO2: 0.928±0.057 vs. 0.417±0.118), pH value (7.05±0.23 vs. 6.80±0.28), arterial oxygen partial pressure [PaO2 (mmHg): 65.42±19.11 vs. 42.42±10.78], HCO3- (mmol/L: 19.22±2.77 vs. 17.18±3.76) were significantly improved after successful resuscitation (allP 0.05).Conclusion Rhythmic ALC-CPR has obvious clinical value for CA patients with conventional CPR contraindication.

Analysis and Management of Off-label Use of Tamoxifen for Ovulation Induction / 中国药房

OBJECTIVE:To analyze the rationality of tamoxifen for ovulation induction,and to provide reference for the stan-dardization of off-label use of tamoxifen. METHODS:Takingtamoxifen,ovulation inductionandpolycystic ovarian syn-dromeas keywords,relevant literatures about off-label use of tamoxifen for ovulation induction were analyzed and summarized by a search of CNKI,Wanfang database,PubMed database up to July 2015. RESULTS:58 literatures were searched,among which clinical trial or systematic review about the effect of tamoxifen on ovulation induction were selected. 11 valid literatures were col-lected to confirm clinical efficacy of tamoxifen for ovulation induction. Referring to Diagnosis and Treatment of Polycystic Ovary Syndrome:An Expert Consensus,pharmaceutical affairs council of our hospital had agreed to off-label use of tamoxifen for induc-tion ovulation,and formulated the standard of off-label use of tamoxifen. CONCLUSIONS:Standard management of off-label use of tamoxifen could be better protect the rights both of patient and doctor.

Nutrition in Chinese-Korean Children and Adolescents / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the nutrition habits among Chinese-Korean children and adolescents in Yanbian Autonomous Prefecture, Jilin, China.</p><p><b>METHODS</b>Data were obtained from the Chinese National Survey on Students' Constitution and Health in 1995, 2000, 2005, and 2010 for Chinese-Korean children and adolescents aged 7-18 years. The number of the subjects included was 4789, 4704, 5875, and 5315, respectively.</p><p><b>RESULTS</b>The rate of the occurrence of stunting showed a declining trend from 1995 to 2010 (for boys: urban, 6.3%; rural, 12.7% in 1995 and 3.5% for both in 2010. For girls: urban, 7.8%; rural, 13.4% in 1995 and 4.2% and 5.5%, respectively, in 2010). Although the ratio of wasting did not show significant differences between the urban and rural children and adolescents in 1995, 2000, 2005, and 2010 respectively, the ratio of occurrence of overweight or obesity increased (for boys: urban, 7.3% and 1.3% in 1995, 17.6% and 12.9% in 2010; rural, 7.0% and 1.3% in 1995, 14.6% and 12.8% in 2010, respectively. For girls: urban, 8.1% and 1.0% in 1995, 17.3% and 8.6% in 2010; rural 5.7% and 0.7% in 1995, 16.4% and 7.4% in 2010, respectively).</p><p><b>CONCLUSION</b>The ratio of malnutrition in children and adolescents in Chinese-Korean areas declined from 1995 to 2010, and the distinction in malnutrition between the urban and rural areas was negligible in 2010. Further, the ratio of overweight and obesity increased over this period.</p>

Diagnosis and treatment of mammary duct ectasia:a report of 59 cases / 中国医师进修杂志

Objective To explore the diagnosis and treatment strategy of mammary duct ectasia.Methods The clinical data of 59 cases with mammary duct ectasia from January 2006 to December 2013 were analyzed retrospectively.Results The main clinical manifestations of mammary duct ectasia were mammary inflammatory mass in 46 cases,nipple discharge in 21 cases,nipple retraction in 18 cases,mammary abscess and mammary fistula in 8 cases.Definite diagnosis of mammary duct ectasia depended on pathology.All the patients were treated by operation,followed up for 3 months to 6 years,and none of them had recurrence.Conclusions Operation is the main method of curing mammary duct ectasia.To select proper operation time and method according to disease type,lesion size,location and scope.Thorough resection,repeat rinsing,wound clearance and immediate breast shape can not only cure disease,but also reserve breast configuration as possible.Therapeutic effect is satisfactory.

Research progress of effect of anti-diabetic traditional Chinese medicines based on regulation of glucose metabolic enzyme / 中国中药杂志

Diabetes is a global threat threatening human health in the world, with an increasing incidence rate in recent years. The disorder of glucose metabolism is one of the major factors. As relevant glucose metabolic enzymes such as alpha-glucosidase, glucose-6-phosphatase (G-6-P), glycogen phosphorylase (GP) and glycogen synthase kinase-3 (GSK-3) get involved in and control the process of glucose metabolism, the regulation of the activity of glucose metabolic enzymes is of significance to the treatment of diabetes. Traditional Chinese medicines (TCMs) have been widely researched because of their low toxicology and high efficiency, and many extracts and components from TCMs have been proven to be regulators of glucose metabolic enzymes. Compared with anti-diabetic western medicines, anti-diabetic TCMs feature safety, reliability and low price. This essay summarizes the anti-diabetic effect of TCMs on regulating glucose metabolic enzymes.

Quantitative assessment of hepatic fibrosis by contrast-enhanced ultrasonography / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To explore the contrast-enhanced ultrasonographic features for quantitative assessment of hepatic fibrosis.</p><p><b>METHODS</b>86 patients with chronic viral hepatitis B were enrolled in this study from March 2007 to August 2009. The patients were classified into 5 groups (S(0)-S(4)) according to fibrosis stage evaluated with ultrasound guided liver biopsy. New contrast-enhanced ultrasonography (CEUS) features including area under the time-intensity curve (TIC) of portal venous phase/hepatic arterial phase (Qp/Qa) and intensity of portal venous phase/hepatic arterial phase (Ip/Ia) were used to detect the blood supply ratio (portal vein/hepatic artery) in each group. Arrival time of portal vein trunk (Tp) and decreasing rate of TIC (β) were also analyzed.</p><p><b>RESULTS</b>Qp/Qa and Ip/Ia decreased from S(0) to S(4), while Tp and β increased. These 4 features were significantly correlated with the degree of fibrosis (P<0.001) and were significantly different among the five groups (P<0.001). Sensitivity and specificity of Ip/Ia were 80% and 86% for groups ≥S(1), 75% and 86% for groups ≥ S(2), 71% and 84% for groups ≥ S(3), and 76% and 80% for group S(4), respectively. Sensitivity and specificity of Qp/Qa were 70% and 88% for groups ≥ S(1), 80% and 76% for groups ≥ S(2), 74% and 70% for groups ≥ S(3), and 81% and 95% for group S(4), respectively.</p><p><b>CONCLUSION</b>Ip/Ia and Qp/Qa could be adopted as reliable, non-invasive features for quantitative assessment of hepatic fibrosis.</p>

Proteomic analysis of cell lines to identify the irinotecan resistance proteins.

Chemotherapeutic drug resistance is a frequent cause of treatment failure in colon cancer patients. Several mechanisms have been implicated in drug resistance. However, they are not suffi cient to exhaustively account for this resistance emergence. In this study, two-dimensional gel electrophoresis (2-DE) and the PDQuest software analysis were applied to compare the differential expression of irinotecan-resistance-associated protein in human colon adenocarcinoma LoVo cells and irinotecan-resistant LoVo cells (LoVo/irinotecan). The differential protein dots were excised and analysed by ESI-Q-TOF mass spectrometry (MS). Fifteen proteins were identifi ed, including eight proteins with decreased expression and seven proteins with increased expression. The identifi ed known proteins included those that function in diverse biological processes such as cellular transcription, cell apoptosis, electron transport/redox regulation, cell proliferation/differentiation and retinol metabolism pathways. Identifi cation of such proteins could allow improved understanding of the mechanisms leading to the acquisition of chemoresistance.