The chelating reaction between glutathione peptides and divalent cadmium ion was used as a typical model for investigating the coordination chemistry of SH-containing peptides and heavy metal ions, which was essential to understand the mechanism of intracellular cadmium detoxification.Here, the mutant (CM113R)7 αHL protein nanopore equipped with a first synthesized per-6-quaternary ammonium-β-cyclodextrin (p-QABCD) was used as nanoreactor and detector to investigate the single-molecule reaction of GSH molecules and Cd2+ ions.Different reaction pathways, intermediates, and products could be recognized.Cd2+-GSH reaction was highly dependent on the solution pH.The Cd(GSH)2 was formed at pH 7.4, while the Cd(GSH)2 and Cd2(GSH)2 were formed at pH 9.0.Cd2(GSH)2 was formed by two possible pathways: (1) A Cd2+ ion primarily coordinated with the thiol group of two GSH molecules to form Cd(GSH)2, and then the second Cd2+ ion quickly incorporated with the deprotonated amino group of Cd(GSH)2 to form Cd2(GSH)2;(2) Two Cd2+ ions separately coordinated with the thiol and deprotonated amino group of one GSH molecule to form Cd2(GSH)1, and the second GSH molecule quickly bounded to Cd2+ ions to form Cd2(GSH)2.This work studied the chelating reaction between metal ions and bioactive glutathione at single-molecule level without labeling and chemical modification, and would be important to further understand intracellular mechanisms of detoxification of heavy metals and greatly expand the research field of nanopore single-molecule technique.

OBJECTIVE:To establish the method for the simultaneous determination of contents of 4 heavy metal elements in cultured Cordyceps militaris. METHODS:The samples were digested by nitric acid heating method and then determined by ICP-MS including radio-frequency power of 1.15 kW,sampling depth of 65 nm,auxiliary gas flow rate of 1.0 L/min,cooling gas flow of 13.0 L/min,temperature of 25 ℃,automatic plasma. RESULTS:The linear ranges of arsenic(As),cadmium(Cd),mer-cury (Hg) and lead (Pb) were 0-20 μg/mL(r=0.9970),0-10 μg/mL(r=0.9995),0-5 μg/mL(r=0.9955),0-20 μg/mL(r=0.9960),respectively. Detectio limit were 0.128,0.003,0.002,0.004 mg. RSDs of precision,stability and reproducibility tests were all lower than 6.5%. Recoveries of them were 90.4%-100.6%(RSD=3.45%,n=9),94.3%-101.3%(RSD=2.93%,n=9), 90.0%-102.3%(RSD=4.03%,n=9),92.3%-103.0%(RSD=3.53%,n=9),respectively. CONCLUSIONS:The method is sim-ple,precise,stable,reproducible,and can be used for simultaneous determination of contents of 4 heavy metal elements in cul-tured C. militaris. The contents of As,Cd,Hg and Pb in 10 batches of sample are all in line with related national standards.

OBJECTIVE:To establish a method for simultaneous determination of 5 nucleoside from different parts of cultured Cordyceps militaris. METHODS:HPLC method was adopted. The determination was performed on InertSustain C18 column with mobile phase consisting of methanol-0.02 mol/L monobasic potassium phosphate solution (gradient elution) at the flow rate of 0.6 mL/min. The detection wavelength was set at 254 nm,the column temperature was 30 ℃. RESULTS:The linear ranges of uridine, inosine,guanosine,adenosine and cordycepin were 0.568-3.408 μg(r=0.9999),0.284-1.704 μg(r=0.9999),0.264-1.584 μg(r=0.9999),0.232-1.392 μg(r=0.9999)and 1.672-10.032 μg(r=0.9998),respectively. RSDs of precision,stability and repeatabili-ty tests were all lower than 3.0%. The recoveries were 98.2%-103.9%(RSD=1.97%,n=9),96.2%-101.6%(RSD=1.76%,n=9),96.7%-102.0%(RSD=1.94%,n=9),95.1%-99.4%(RSD=1.43%,n=9) and 95.6%-101.3%(RSD=1.82%,n=9). CON-CLUSIONS:The method is simple,precise,stable and repeatable,and can be used for simultaneous determination of 5 nucleoside from cultured C. militaris,the content of nucleosides are different in different parts of C. militaris.

Objective:To the effects of testosterone replacement therapy on muscle strength and function among the elderly (≥65 years old).Methods:We searched English and Chinese databases of PubMed, SinoMed, etc, upto July 4, 2020. All retrieved literatures were reviewed according to inclusion and exclusion criteria in accordance with PICOS principles. The Cochrane risk bias assessment tool was used for quality evaluation. Two researchers extracted data and evaluated literature quality independently. The outcomes included muscle strength (assessed by handgrip strength, leg extension strength, etc) and function (assessed by walking speed according to 6-minute walking test, 6MWT). Review Manager 5.3 software was used for statistical analysis. The fixed or random effects model was used to merge data of upper-and lower-extremity strength or 6MWT to produce forest plot and funnel plot. The subgroup analyses were conducted based on the characteristics of included studies. The sensitivity analyses were conducted for excluding literatures with small sample size, etc.Results:A total of 15 relatively high quality researches (14 English literatures and 1 Chinese literature) were included. The results of this meta analyses showed TRT could improve upper-extremity (0.21[0.11, 0.32]) and lower-extremity (0.34[0.12, 0.55]) muscle strength while not physical function (17.62[-13.06, 48.31]) among the elderly men. Subgroup analyses showed that region, source of participants, administration route and intervention period, while not the baseline testosterone level had effect on the pooled effect size. Funnel plot suggested a certain degree of publication bias. Sensitivity analyses revealed that the meta analyses were robust.Conclusion:TRT can improve muscle strength while not physical function among elderly men.

Objective To construct and screen effective shRNA expression vectors targeting human AQP1 gene ,and evaluate the interference efficiency of the AQP1 shRNA recombinant plasmids ,thus provide basis for further exploration on the effect and mechanism of AQP1 gene on human breast cancer cells .Methods Four pairs of shRNA sequences targeting human AQP1 gene were designed and synthesized ,and then inserted into the GV115 vector .AQP1 shRNA and control shRNA plasmids were trans‐fected into human breast cancer MCF‐7 cells .The expression of AQP1 mRNA and protein were detected by real time PCR(RT‐PCR) and Western blot to evaluate the interfering efficiency .Results RT‐PCR demonstrated that AQP1 was expressed in human breast cancer MCF‐7 cells .Sequencing showed that the shRNA vectors targeting AQP1 were successfully constructed .48 h after the AQP1 shRNA transfection ,AQP1 mRNA and protein expression levels in MCF‐7 cells were reduced to a significant degree ,and the AQP1 shRNA 4 plasmid vector could inhibit the AQP1 most efficiently .Conclusion The AQP1 shRNA recombinant plasmids vectors were successfully constructed and can significantly inhibit the expression of AQP1 in MCF‐7 human breast cancer cells .

Objective To isolate and identify exosomes from human serum , explore the feasibility of detecting exosomal miRNA in human serum.Methods Retrospective study.Serum samples from 10 healthy individuals in January 2013 were randomly selected.Besides, from January 2013 to December 2014, serum samples from prostate cancer(PCa) patients (n=20), benign prostatic hyperplasia(BPH) patients ( n=20 ) and healthy controls ( n=20 ) were selected.Exosomes were isolated from these serum samples using ExoQuick , and then identified by using transmission electron microscopy , NanoSight nano particle analyzer and Western Blot for morphology and molecular phenotype.The quality of exosomal RNA was analyzed using Agilent 2100 Bioanalyser.Then quantificational real-time polymerase chain reaction ( qRT-PCR) was carried out to detect miRNAs in different components of human serum ,and nonparametric tests were used for difference analysis.Results Exosomes isolated from human serum showed round or oval vesicles, mainly in diameter 40-100 nm, and with maximum peak distribution of 58 nm.Moreover, they expressed HSP70 and four transmembrane protein CD 63.Agilent 2100 Bioanalyzer results showed that the major RNA component of exosome was about 25nt small RNA.qRT-PCR confirmed that 4 normal miRNAs were expressed in human serum exosome , and the expression of miRNAs in exosome pellets were higher than the whole serum (miR-21, U =16,P =0.007 2; miR-16, U =3,P<0.000 1; miR-20a, U =2,P <0.000 1;let-7a, U=13,P=0.003 2) and exosome-depleted supernatant ( miR-21, U=15,P=0.006 5;miR-16, U=2,P<0.000 1;miR-20a, U=1,P<0.000 1;let-7a, U=10,P=0.002 8).miR-141, the molecular marker of prostate cancer ,were analyzed by qRT-PCR in whole serum samples and serum exosome pellets isolated from the same serum in a cohort of 20 PCa patients , 20 BPH patients and 20 healthy control people.The results showed that , in three groups , exosomal miR-141 expression were all significantly higher than serum circulating miR-141 (Control group, U=66,P=0.000 3; BPH group, U=83,P=0.001 6;PCa group, U=54,P<0.000 1).In addition, the expreession of exosomal miR-141 in PCa patients was significantly higher than BPH patients or healthy controls (3.85 fold, U=74,P=0.000 7 and 4.06 fold, U=70,P=0.000 5).Conclusion Exosome can be efficiently isolated from human serum.Compared with the whole serum , isolation of serum exosome may helpful to improve the detection of circulating miRNA.

Objective To investigate the therapeutic effects and the possible mechanism of fecal transplantation on rats with Clostridium difficile-associated pseudomembranous colitis. Methods A total of 40 Sprague-Dawley rats were divided into four groups including the healthy control group, model group, fecal transplant treatment group and vancomycin treatment group. Rats in three experimental groups were subcuta-neously injected with clindamycin phosphate (10 mg), followed by treatment with toxin producing Clostridi-um difficile (ACTT43255) enema 24 hours later. The rats in fecal transplant treatment group and vancomy-cin treatment group were respectively treated with fecal suspension and vancomycin one day after modeling. The rats were fasted for one day after the last administration and then executed. The levels of potassium ion ( K) , sodium ion ( Na) , albumin ( ALB) , white blood cells ( WBC) , C-reaction protein ( CRP) , interleu-kin-1β ( IL-1β) , interleukin-10 ( IL-10 ) , interleukin-12 ( IL-12 ) and interleukin-17 ( IL-17 ) as well as the percentage of neutrophils ( N%) in serum samples were detected. The colon tissue samples were collect-ed for pathology examination. Results The rat model of pseudomembranous colitis was successfully estab-lished by subcutaneous injection of clindamycin in combination with toxin-producing Clostridium difficile (ACTT43255) enema. The signs of intestinal inflammation including serious weight loss, remarkably short-ened colon length and significantly increased colon wet weight index were observed in rats from the model group (P<0. 05). Compared with the rats from model group, the rats received fecal transplant showed sig-nificantly increased levels of K, ALB, IL-10 and IL-10/IL-12 in serum and decreased levels of WBC, N%, CRP, IL-1β and IL-17 (P<0. 05). Conclusion Fecal transplantation was proved to be an effective ap-proach for the treatment of pseudomembranous colitis. The therapeutic mechanism might due to its impacts on serum inflammatory factors.

Blue laser imaging (BLI) is a new endoscopic system equipped with the laser beam emitting two different wavelengths.It produces bright and high resolution images for observation of microvascular and microsurface patterns of esophageal and gastric mucosa, helping the diagnosis of early upper gastrointestinal cancer.Compared with the existed endoscopic techniques, BLI shows its unique advantages.In this article, advances in application of BLI in diagnosis of early upper gastrointestinal cancer were reviewed.

Objective To study the relationship between gene polymorphism of apolipoprotein E (apolipoproteinE,ApoE)of peripheral blood and Mycoplasma pneumoniae infection in children.Methods Collected 236 cases serum of inpatient and outpatient screening in children with Mycoplasma pneumoniae infection and healthy children between March 2011 and March 2014 in the First Affiliated Hospital of Xi’an Medical University and Xi’an Children’s Hospital,at the age of 3~8 years old,divided into two groups:110 cases of control group and 126 cases of Mycoplasma pneumoniae infection in chil-dren.Used multiple allele-specific PCR (multi-AS PCR)to detect gene polymorphism of ApoE in each group.Results ApoE gene was polymorphic and 6 genotypes:3 homozygous (ε2/2,ε3/3,ε4/4)and 3 heterozygote (ε3/2,ε3/4,ε4/2).Theε3/2 had four bands,ε3/3,ε3/4 and 4/2 had three bands,ε2/2 andε4/4 had two bands.ε3/3 of ApoE genotype distribution in two groups was the most common,control group was 66.7%,infection group was 46.4%.Allele frequencies ofε3 and genotype frequencies ofε3/3 inMycoplasmapneumoniae infection of children were lower than those in control group (P<0.05).But allele frequencies ofε4 and genotype frequency ofε4/4 in Mycoplasma pneumoniae infection of children were increased, which were compared with those in control group (P<0.05).Conclusion There were an association between ApoE gene polymorphism and the incidence of Mycoplasma pneumoniae infection in children.Allelesε3 seems to be a protective factor and allelesε4 may contribute to the development of Mycoplasma pneumoniae infection of children.

Objective To investigate the correlation between the serum ghrelin levels and nutrition state in the patients with chronic obstructive pulmonary disease(COPD) ,as well as to explore the role of ghxelin in the nutri-tion metabolism. Methods Fifty-three COPD patients were observed, thirty-one of them were with malnutrition (group A), twenty-two COPD patients with normal nutrition status(group B), and twenty were healthy controls.Serum ghrelin, and nutritional parameters such as body mass index(BMI), ideal body weitht( % IBW), mid-upper arm cricumference(MAC), serum albumin(ALB), total lymphocyte counts(LYM) were determined. The correlation between ghrelin and nutritional parameters was analysed. Resuits The level of serum ghrelin in group A were sig-nificantly higher than those in group B and in healthy controls. And the serum ghrelin showed a negative correlation with BMI, % IBW, MAC, but there was no correlation between serum ghrelin level, ALB and LYM. Conclusion Ghrelin participated in the nutrition metabolism in patients with COPD, it would become much higher because of malnutrition in COPD.