Specific detection of amatoxins and phallotoxins in body fluids is necessary for an early diagnosis of an intoxication with mushrooms.In this study, a rapid method for the simultaneous determination of α-, (β-and γ-amanitin, phalloidin and phallacidin in human urine and plasma was first developed by ultra-perform ance liquid chromatography-tandem mass spectrometry.Urine sample was directly injected into the separation system and plasma sample was initially prepared by precipitation of proteins with 1% acetic acid in acetoni trile.The toxin was analyzed on an ACQUITY UPLC HSS T3 column using a gradient program with a cycle time of 9 min, and detected by positive electrospray ionization tandem mass spectrometry in the MRM mode, and quantified by matrix-match standard solution.The detection limits (S/N = 3) of the toxins were within 0.2-1 μg/L and 0.1-0.5 μg/L for urine and plasma, respectively.The standard curves were linear in the range of 2-100 μg/L for urine and 1-100 μg/L for plasma.The average recoveries were 92.0%-108.0% and 85.0%-100.0% for the toxins spiked in urine and plasma, with RSDs of 1.0%-22.0% and 2.0%-22.0% (n = 6), respectively.The method was simple, selective and sensitive to detect the amatoxins and phallotoxins in urine and plasma for both clinical and forensic purposes.

A rapid method for the detection of tetrodotoxin(TTX) in human plasma and urine was developed by hydrophilic interaction liquid chromatography-tandem mass spectrometry. After a simple protein precipitation step was undertaken, the subsequent analysis of TTX was achieved on a TSK-gel amide-80 column using an ammonium formate-methanol-acetonitrile gradient with a cycle time of 13 min, and detected by positive electrospray ionization tandem mass spectrometry in the MRM mode, and quantified by matrix-match standard solution. It was found that linearity in urine was observed within concentration ranged from 3 μg/L to 500 μg/L, that in plasma 1 μg/L to 200 μg/L and that limits of detection(S/N=3) for urine and plasma were 1 and 0.3 μg/L, respectively. The average recoveries were 96%-108% and 100%-105% for TTX spiked in urine and plasma, respectively, with relative standard deviations of 1.7%-8.6% and 8.9%-16%(n=6). This method was simple, selective and sensitive to detect TTX in urine and plasma for both clinical and forensic purposes.

The high mutation rate during hepatitis B virus (HBV)replication leads to HBV quasispecies.The study of HBV quasispecies has an important significance for the hepatitis B pathogenesis,prognosis,and outcome prediction.Recently,the next-generation sequen-cing (NGS)is extensively used in many biological and medical fields due to its high throughput,ultra-deep coverage,and high sensitivity, which also brings new strategies to HBV quasispecies studies.This article describes the working principles and features of several commonly used NGS technologies,reviews the application of NGS technologies in HBV quasispecies detection in recent years,and particularly discus-ses the variations in different regions of HBV genome and the population characteristics of HBV quasispecies.For now,NGS technologies used in HBV variation detection mainly presents the advantages of high sensitivity and high throughput.However,how to lower the cost,in-crease the accuracy of sequencing,and standardize the procedure of massive sequencing data management and personalized analysis are still challenging problems.The issue about NGS that matters at present and urgently needs to be solved is how to overcome its limitations,and then put it into HBV-related studies and ultimately clinical application.

Acupuncture Points ; Acupuncture Therapy ; Adolescent ; Alopecia ; physiopathology ; therapy ; Female ; Hair ; growth & development ; Humans

Objective To investigate the value of bronchoscopy in the etiologic diagnosis and therapy of right middle lobe atelectasis. Methods Clinical data of 45 cases of right middle lobe atelectasis under fiberoptic bronchoscopy from January 2012 to February 2016 were analyze retrospectively. Results 28 cases (62.2 %) were determined inflammation, 9 cases (20.0 %) of tumor, 4 cases (8.9 %) of tuberculosis, 1 case (2.2%) of foreign body, 3 cases (6.7 %) unexplained. After treatment, 30 cases (66.7 %) were cured, 8 cases (17.8 %) improved while 7 cases (15.5 %) invalid. Conclusions The bronchoscopy is a critical technology for diagnosis and therapy of right middle lobe atelectasis.

OBJECTIVETo compare the difference in the clinical efficacy on endometriosis (EM) between electroacupuncture (EA) and western medication.

METHODSSeventy-two EM patients were divided into an EA group and a western medication group according to visiting departments, 36 cases in each one. In the EA group, acupuncture was applied to Qihai (CV 6), Guanyuan (CV 4), Zhongji (CV 3), Zigong (EX-CA 1), Diji (SP 8), Sanyinjiao (SP 6), Hegu (LI 4) and Taichong (LR 3). After qi arrival, G6805-I pulse electronic apparatus was attached to bilateral Zigong (EX-CA 1), Guanyuan (CV 4) and Zhongji (CV 3), with continuous wave, 70 Hz in frequency, 3 mA in intensity. The EA was given once every two days. In the western medication group, mifepristone tablets were prescribed for oral administration, 12. 5 mg per treatment, once a day, for 6 months. The pain degree was observed before and after treatment and the clinical efficacy and recurrence rate were evaluated in the two groups. The enzyme-linked immunoassay (ELISA) was adopted to determine the tumor marker serum CA125 before and after treatment in the two groups.

RESULTSThe total effective rate was 94. 4% (34/36) in the EA group and was 91. 7% (33/36) in the western medication group, without significant difference (P>0. 05). The pain score after treatment was lower than that before treatment in the two groups (both P< 0. 01), but the score after treatment in the EA group was lower than that in the western medication group (P<0. 05). Serum CA125 was reduced after treatment in the patients of the two groups (both P<0. 01), and serum CA125 after treatment in the EA group was lower than that in the western medication group (P<0. 05). In the follow-up visit of one year, the reoccurrence rate was 17. 6% (6/34) in the EA group and was 33. 3% (11/33) in the western medication group, indicating the significant difference (P<0. 05).

CONCLUSIONEA achieves the significant clinical efficacy and the reoccurrence rate in 1 year in the EA group is obviously lower than that in the western medication group. This therapy could be promoted in clinical practice of acupuncture and moxibustion.

Acupuncture Points ; Adult ; Electroacupuncture ; Endometriosis ; therapy ; Female ; Humans ; Middle Aged ; Treatment Outcome ; Young Adult

Objective To clone and express hypervariable region 1 (HVR1) fragments of different genotypes of HCV strains from different cities of China.Then detect the reactivity of the expressed HVR1 fusion proteins with sera of chronic he-patitis C patients to analyzing it's signification in clinical utilization. Methods HVR1 genes of four HCV strains (genotype 1b and 2a) were amplified from pGEMT-E2 plasmids and cloned into pQE40 vectors, respectively to construct four recombinant prokaryotic expression plasmids which expressed HVR1 as fusion proteins with DHFR in Escherichia coli strain TG1. Then we used the purified DHFR-HVR1 proteins to detect the anti-HVR1 antibodies in 70 serum samples of chronic hepatitis C patients. Results Four DHFR- HVR1 fusion proteins were successfully expressed in E.coli (320～ 800 ?g fusion proteins per 100 ml culture). Each fusion protein (SH1b, BJ1b, SD1b and SD2a) reacted with 72.8%(51/70), 60%(42/70), 48.6%(34/70), and 58.6%(41/30) of the anti-HCV positive patients' sera, respectively by ELISA. 57% (4/7) of the non responders' sera taken before therapy reacted with all four HVR1 fusion proteins, while only 15.3% (2/13) of the sera from responders reacted with all of them. The A values of sera from IFN therapy responders were significantly higher than non responders (P

Objective To construct Hepatitis B Virus (HBV) Chinese-strain wild type and Lamivudine-resistant variant recombinant baculovirus. Methods 1.2-unit length HBV construct (adr serotype, C genotype) was cloned into the multiple cloning region of the pFastBac Ⅰ vector and then transformed into DH10Bac, in which homologous recombination occurred between the pFastBac vector and the inside bacmid with the help of helper plasmid. So HBV recombinant bacmid was produced and transfected into sf9 cells to generate a Recombinant HBV baculovirus called vAcHBVc. The viral titers were detected by immune plaque assay and TCID 50. Lamivudine resistance mutation (YVDD) was introduced by site-directed mutagenesis using continuous PCR. HBV YVDD recombinant baculovirus (vAcHBVcYVDD) was generated and titered as above. Meanwhile, the recombinant HBV baculovirus containing GFP reporter was constructed for evaluating the efficacy of transfection and titration of virus. Results Recombinant wild and YMDD variant HBV baculovirus were successfully developed and the titer was between 10~6～10~8pfu/ml through plaque assay. Conclusion Different kinds of recombinant HBV baculovirus could be generated rapidly and efficiently with Bac to Bac expression system. The developed recombinant HBV baculovirus could be further used in the study of anti-viral resistances.

A method for the determination of methylmercury ( MeHg ) and ethylmercury ( EtHg ) in aquatic products was developed using gas chromatography-mass spectrometry with stable isotope-labelled internal standard. After ultrasonication assisted hydrochloric acid extraction, MeHg and EtHg in samples were extracted into toluene under the presence of sodium chloride and then back-extracted into cysteine aqueous solution. The MeHg and EtHg were released from their complexes with cysteine by adding cupric ions, and then derived with sodium tetraphenylborate. Under the optimal chromatographic conditions, MeHgPh and EtHgPh, the resulting derivatives, were separated completely on a DB-5MS capillary column and detected by electron impact ionization mass spectrometry in the selective ion monitoring ( SIM) mode, and quantified by a stable isotope dilution method using the d3-methylmercury as internal standard. The calibration curves were linear in the range of 1-500 μg/L of MeHg and EtHg. Concentration of 0. 828 mg Hg/kg with relative standard deviation ( RSD ) of 3 . 2% ( n=6 ) was obtained for MeHg in GBW 10029 . This was in good agreement with the certified values of (0. 84±0. 03) mg Hg/kg. The average recoveries were 94%-101% and 81%-104% for MeHg and EtHg spiked in aquatic samples, with RSDs of 1. 9%-4. 7% and 3. 1%-8. 2%(n=6), respectively. The limits of detection (S/N=3) of the two targets were 0. 1-0. 3μg/kg. This method was sensitive, accurate and could meet the demand of the determination of methylmercury and ethylmercury in aquatic products.

Objective To further determine the inflammation?associated cytokine changes during endotoxin(LPS)tolerance. Methods Kun?ming mice were divided into groups. The endotoxin tolerance mouse model was established with repeated injection of LPS. Serum levels of tumor ne?crosis factor?α(TNF?α),interleukin?10(IL?10)and IL?17 were determined by ELISA assay. Results The serum concentrations of TNF?α,IL?10 and IL?17 were 2 261.49±1 090.28 pg/mL,7 780.42±2 320.04 pg/mL,and 27.28±13.76 pg/mL in the LPS control group,which were increased significantly compared to the normal healthy control group. The serum concentrations of TNF?α,IL?10 and IL?17 were 1 174.19±450.91 pg/mL, 1 293.26±520.85 pg/mL,and 22.26±11.44 pg/mL in the endotoxin tolerance group. Levels of TNF?αand IL?17 were further decreased by dexameth?asone treatment during LPS tolerance inducing,while the IL?10 level did not change significantly. Conclusion Compared to the status of endotoxin inflammation,levels of TNF?αand IL?10 decreased significantly,while levels of IL?17 were not significantly changed in the status of endotoxin toler?ance. Dexamethasone could further suppress TNF?αand IL?17 levels during endotoxin tolerance inducing.