Objective To explore the expression of HCK gene during the cardiomyocyte differentiation of mouse embryonic stem cells and analyze the role of HCK gene in maintenance of pluripotency of embryonic stem cells.Methods Mouse embryonic stem cells were cultured,then induced to differentiate into cardiomyocytes.Total RNAs were isolated from mouse embryonic stem cells in the differentiation days:0 day(D0),the second day(D2),the fourth day(D4),the sixth day(D6),the eighth day(D8),respectively.The levels of HCK mRNAs were assessed by the method of semi-quantitive reverse transcriptase-polymerase chain reaction(RT-PCR).In the meanwhile,Total proteins were also isolated from mouse embryonic stem cells in the differentiation D0,D2,D4,D6,D8,and the levels of HCK proteins were evaluated by Western-blot.Results HCK mRNAs could be detected in the mouse embryonic stem cells in D0 and D2,however,they were undetectable from D4 to D8.The expression of HCK mRNAs was rapidly down-regulated during cardiomyocyte differentiation of mouse embryonic stem cells.Expression of HCK proteins,which coincided with HCK mRNAs,down-regulated during differentiation and couldn't be detected in D4.Conclusions With the cardiomyocyte differentiation of mouse embryonic stem cells,the expression of HCK in the levels of mRNA and proteins are sharply down-regulated;HCK may play an important role in maintaining the pluripotency of embryonic stem cell.

OBJECTIVETo investigate the differences in semen quality at different times of reanalysis and the correlation of sperm DNA fragmentation index (DFI) with sperm motility alteration using semen samples completely liquefied and normal in initial examination.

METHODSWe analyzed 127 semen samples up to the inclusion criteria with the computer-assisted semen analysis (CASA) system at 15, 30 and 60 min after semen collection, and obtained sperm morphology parameters and DFI by Shorr staining and acridine orange test (AOT) , respectively.

RESULTSSperm concentration, and the percentages of grades a and b sperm showed no statistically significant differences at the three time points (P > 0.05). The percentages of grades a + b and a + b + c sperm were significantly higher at 15 min than at 30 and 60 min after semen collection (P < 0.05), but with no significant difference between the latter two time points (P > 0.05). The incidence of alternation from normal to abnormal in at least one index of sperm motility at different times was 25.2%, but there were no significant differences in sperm DFI and morphology between the normal and abnormal groups (P > 0.05). Among the altered parameters of sperm motility from 15 to 60 min, the percentages of grades a, a + b and a + b + c sperm were all positively correlated with sperm DFI (P < 0.05).

CONCLUSIONSemen samples completely liquefied within 15 min after collection and normal in initial examination, when reanalyzed at 30 and 60 min, showed significant decreases in the percentages of grades a + b and a + b + c sperm, but not in the percentages of grades a and b sperm, and the parameters of sperm motility might be abnormal. Thus, at least 2 sperm analyses are required for a comprehensive evaluation of fertility. Significant difference between the results of the two analyses, and particularly a markedly reduced percentage of rapidly progressive sperm, might indicate sperm DNA damage, and thus the necessity of sperm DNA damage detection.

Adult ; DNA Damage ; DNA Fragmentation ; Diagnosis, Computer-Assisted ; Fertility ; genetics ; Humans ; Male ; Middle Aged ; Semen Analysis ; Sperm Count ; Sperm Motility ; Spermatozoa ; Young Adult

Objective To generate a P19-αMHC-EGFP reporter line and induce cardiomyocyte differentiation of this reporter line. Methods The P19 cells were transfected with pαMHC-EGFP, a P19- αMHC-EGFP reporter line was obtained after G418 selection and limited dilution of recombinant clones, The reporter line was induced to differentiate into cardiomyocytes which would beat and express green fluorescent protein. A comparison of cardiomyocyte differentiation rate and cTnI expression amount between the reporter line and the untransfected P19 cells was also performed. The ultrastructure was observed under transmission electron microscope. Results The ultrastructure characteristics indicated cardiomyocytes-like changes on induction day 10. The beating cardiomyocytes which express GFP appear in the seventh induction day. The cardiomyocyte differentiation rate and cTnI expression amount of P19-αMHC-EGFP reporter line were similar as those in untransfected P19 cells (P > 0. 05). Conclusion The P19-αMHC-EGFP reporter line is of great benefit for identifying and purifying cardiomyocytes from undifferentiated P19 cells without influencing the differentiation of P19 cells. This feature makes P19-αMHC-EGFP reporter line a promising cell source for clinical cardiomyocyte replacement therapy.

Objective:To investigate the effects of osimertinib on proliferation, migration and invasion of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) overexpressing HCC827 cells and explore the potential mechanism of PLOD2 induced osimertinib resistance.Methods:We transfected HCC827 cells with LV-vector and LV-over/PLOD2. The expression of PLOD2 was detected by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. The effects of osimertinib on the proliferation of HCC827-vector and HCC827-PLOD2 cells were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The effects of osimertinib on the migration and invasion of HCC827-vector and HCC827-PLOD2 cells were determined by Transwell assays. The expressions of E-cadherin and vimentin in cells were detected by immunofluorescence (IF). The expressions of epithelial-mesenchymal transition (EMT), FAK-PI3K/AKT and MAPK signal pathway related proteins were detected by western blotting.Results:The MTT assay showed that HCC827-PLOD2 cells were hyposensitive to osimertinib. The 50% inhibitory concentration (IC 50) and resistance index of osimertinib for HCC827-PLOD2 cells was over 1 000 nmol/L and over 100, respectively. The result of wound healing assay showed that the migration distance of HCC827-PLOD2 was about (2.13±0.21) fold changes as that of HCC827-vector cells. The result of Transwell assay showed that the numbers of HCC827-PLOD2 passing through the matrix membrane were (212.78±10.43), significantly higher than (101.32±12.52) of HCC827-vector cells ( P<0.01). The result of IF showed that compared with HCC827-vector cells, the expression of E-cadherin was down-regulated while vimentin was up-regulated in HCC827-PLOD2 cells. Osimertinb downregulated E-cadherin and upregulated vimentin expression in HCC827-vector cells but had limited effect in HCC827-PLOD2 cells. The result of western blotting showed that PLOD2 significantly increased vimentin expression level while decreased E-cadherin expression level. Osimertinib inhibited the expression of p-EGFR, but did not affect the expressions of PLOD2, p-FAK, p-AKT, p-ERK, vimentin and E-cadherin in HCC827-PLOD2 cells. Conclusion:PLOD2 confers resistance to osimertinib in HCC827 cells by regulating EMT, FAK-PI3K/AKT and MAPK signal pathways.

OBJECTIVE: To explore the effect of clinical pathway of emergency respiratory and cardiac arrest on management of patients with sudden respiratory and cardiac arrest. METHODS: The clinical data of patients with sudden respiratory and cardiac arrest admitted to Huashan North Hospital Affiliated to Fudan University from 1 year before to 1 year (started in July 2017) after the implementation of clinical pathway of emergency respiratory and cardiac arrest were retrospectively analyzed. The patients who managed by clinical pathway of emergency respiratory and cardiac arrest (from July 2017 to June 2018) were served as observational group, and those manually managed by 2015 American Heart Association cardiopulmonary resuscitation and cardiovascular emergency guide update and the procession in the management of emergency key diseases (from July 2016 to June 2017) were set as control group. The gender, age, underlying disease, the initiation time of cardiopulmonary resuscitation (CPR), the first time of defibrillation, the completion time of endotracheal intubation, the time of venous access, the usage time of the first dose of adrenaline, the usage time of vasoactive drugs, the completion rate of high quality CPR, the success rate of return of spontaneous circulation (ROSC) and the table procedure of clinical pathway were compared between the two groups. RESULTS: There was no statistically significant difference in gender, age or underlying disease between the two groups. Compared with the control group, the clinical pathway could effectively guide the decision-making of the emergency medical staffs, significantly reduce the variation in the procession of the resuscitation, the first time of defibrillation, the completion time of the deep vein catheterization, the usage time of norepinephrine and the completion time of the blood specimen delivery were significantly shortened [the first time of defibrillation (minutes): 1.28±0.86 vs. 2.93±1.61, the completion time of deep vein catheter (minutes): 15.13±2.73 vs. 17.25±3.02, the usage time of norepinephrine (minutes): 15.43±2.80 vs. 17.88±1.67, the completion time of blood specimen delivery (minutes): 7.81±1.42 vs. 9.00±1.03, all P < 0.05]. There was no statistically significant difference in the initiation time of CPR, the completion time of tracheal intubation, the time of peripheral venous access, the usage time of the first dose of adrenaline or sodium bicarbonate, or the success rate of ROSC between the two groups. However, the usage time of the first dose of adrenaline and sodium bicarbonate was shortened in the observation group [the usage time of the first dose of adrenaline (minutes): 3.81±1.22 vs. 4.00±1.32, the usage time of the first does of sodium bicarbonate (minutes): 8.94±3.49 vs. 11.19±3.54, both P > 0.05], and the success rate of ROSC was relatively increased as compared with those in the control group [15.04% (17/113) vs. 12.50% (12/96), P > 0.05]. CONCLUSIONS The clinical pathway of emergency respiratory and cardiac arrest could effectively guide the clinical decision-making of the emergency medical staffs, significantly reduce the variation in the procession of the resuscitation, improve the quality of the resuscitation, and ensure medical safety of emergency department.
Cardiopulmonary Resuscitation ; Case-Control Studies ; Critical Pathways ; Electric Countershock ; Emergency Service, Hospital ; Female ; Health Services Research ; Heart Arrest/therapy* ; Humans ; Male ; Retrospective Studies

Objective:To investigate the effects of osimertinib on proliferation, migration and invasion of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) overexpressing HCC827 cells and explore the potential mechanism of PLOD2 induced osimertinib resistance.Methods:We transfected HCC827 cells with LV-vector and LV-over/PLOD2. The expression of PLOD2 was detected by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. The effects of osimertinib on the proliferation of HCC827-vector and HCC827-PLOD2 cells were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The effects of osimertinib on the migration and invasion of HCC827-vector and HCC827-PLOD2 cells were determined by Transwell assays. The expressions of E-cadherin and vimentin in cells were detected by immunofluorescence (IF). The expressions of epithelial-mesenchymal transition (EMT), FAK-PI3K/AKT and MAPK signal pathway related proteins were detected by western blotting.Results:The MTT assay showed that HCC827-PLOD2 cells were hyposensitive to osimertinib. The 50% inhibitory concentration (IC 50) and resistance index of osimertinib for HCC827-PLOD2 cells was over 1 000 nmol/L and over 100, respectively. The result of wound healing assay showed that the migration distance of HCC827-PLOD2 was about (2.13±0.21) fold changes as that of HCC827-vector cells. The result of Transwell assay showed that the numbers of HCC827-PLOD2 passing through the matrix membrane were (212.78±10.43), significantly higher than (101.32±12.52) of HCC827-vector cells ( P<0.01). The result of IF showed that compared with HCC827-vector cells, the expression of E-cadherin was down-regulated while vimentin was up-regulated in HCC827-PLOD2 cells. Osimertinb downregulated E-cadherin and upregulated vimentin expression in HCC827-vector cells but had limited effect in HCC827-PLOD2 cells. The result of western blotting showed that PLOD2 significantly increased vimentin expression level while decreased E-cadherin expression level. Osimertinib inhibited the expression of p-EGFR, but did not affect the expressions of PLOD2, p-FAK, p-AKT, p-ERK, vimentin and E-cadherin in HCC827-PLOD2 cells. Conclusion:PLOD2 confers resistance to osimertinib in HCC827 cells by regulating EMT, FAK-PI3K/AKT and MAPK signal pathways.

BACKGROUNDReactive oxygen species are thought to contribute to the development of renal damage. The P22phox subunit of nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase, encoded by the cytochrome b245a polypeptide gene, CYBA, plays a key role in superoxide anion production. We investigated the association of CYBA rs7195830 polymorphism with estimated glomerular filtration rate (eGFR) and the role it plays in the pathogenesis of chronic kidney disease (CKD) in a Han Chinese sample.

METHODSThe Gaoyou study enrolled 4473 participants. Serum levels of creatinine were measured and eGFR was estimated using the Chronic Kidney Disease Epidemiology Collaboration equations. The CYBA polymorphisms were genotyped. Then we investigated the association between eGFR and the rs7195830 polymorphism in the recessive model.

RESULTSThe AA genotype of rs7195830 was associated with significantly lower values of eGFR compared with the GG and AG genotypes ((102.76 ± 17.07) ml×min(-1)×1.73 m(-2) vs. (105.08 ± 16.30) ml×min(-1)± 1.73 m(-2)). The association remained significant in the recessive model after adjusting for age, gender, body mass index, smoking, hypertension, diabetes mellitus, uric acid, triglyceride, low density lipoprotein cholesterol and high density lipoprotein cholesterol (β=1.666, P=0.031). The rs7195832 AA genotype was an independent risk factor for CKD: eGFR <60 ml×min(-1)×1.73 m(-2) (odds ratio=3.32; 95% CI=1.21-9.13).

CONCLUSIONThe AA genotype of rs7195830 is independently associated with lower estimated glomerular filtration rate and is significantly associated with CKD.

Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Female ; Glomerular Filtration Rate ; genetics ; Humans ; Male ; Middle Aged ; NADPH Oxidases ; genetics ; Polymorphism, Genetic ; genetics ; Renal Insufficiency, Chronic ; epidemiology ; genetics ; Young Adult

Objective To investigate the relationship between c-reactive protein (CRP) and blood pressure in a general population. Methods We randomly selected 3889 subjects aged 18 -74 years stratified by gender and age in Baqiao, a rural area of Jiangsu Province. A standardized questionnaire was used to collect information on medical history, smoking, alcohol intake and use of medications. Blood pressure was measured by mercury sphygmomanometer. Serum CRP (hCRP) concentration was measured using a high sensitivity BNprosec immunonephelometric assay. Subjects were divided into 4 groups according to their interquartile range of CRP levers: group Q1 ( men hCRP ＜2. 04 mg/L;women hCRP ＜ 1.80 mg/L);group Q2 ( men 2. 04 mg/L ≤ hCRP ＜ 3.01 mg/L; women 1.80 mg/L ≤ hCRP ＜ 2. 76 mg/L); group Q3(men 3.01 mg/L≤ hCRP ＜4. 14 mg/L;women 2.76 mg/L ≤ hCRP ＜3. 84 mg/L);and group Q4(men 4. 14 mg/L≤hCRP;women 3. 84 mg/L ≤ hCRP). Results Systolic blood pressure ( SBP, adjusted P =0. 016 ) and pulse pressure ( PP, adjusted P =0. 003 ) of men and PP ( adjusted P =0. 002) of women were increased in proportion to increased CRP levels. Diastolic blood pressure was not associated with CRP levels. Multiple stepwise regression analysis showed that logCRP was independently associated with SBP and PP in men and PP in women. hCRP was independently associated with hypertension in men. Compared with group Q1, male people in group Q4 faced a40. 4% (95% confidence interval:4. 9% -87.9% ) higher risk of hypertension. Conclusions hCRP was independently associated with PP in men and women, and SBP in men. hCRP was independently associated with hypertension in men but not in women in this study population.

Several Orobanche medicinal plants sometimes served as alternative sources of Cistanches Herba, attributing to the benefits such as tonifying kidney, strengthening tendons and bones. Among them, O. coerulescens, O. cernua and O. pycnostachya have been widely utilized in northern China for treatments of pains in the loins and knees, impotence, and spermatorrhea. However, their chemical profiles haven't been elucidated. In the present study, UHPLC-IT-TOF-MS was implemented to conduct in-depth chemome profiling of O. coerulescens, O. cernua and O. pycnostachya, aiming to achieve a comprehensive chemical characterization and to provide pronounced information for the quality control and clinical applications. An ACE Ultra-Core 2.5 Super C_(18)(3.0 mm×150 mm, 2.5 μm) column was deployed for chromatographic separations, and high-resolution MS~n spectra were recorded by IT-TOF-MS. Forty-eight components, in total, were observed, and thirty-eight ones were structurally annotated according to proposing mass fragmentation patterns, matching with relevant databases. Particularly, nine ones were confirmed by reference compounds. Overall, the chemical compositions of O. coerulescens and O. cernua are quite similar, and differences occur between O. pycnostachya and the prior two ones; primary chemical family is phenylethanoid glycosides, and several lignan glycosides as well as iridoid glycosides are also observed; the primary components include acteoside, isoacteoside, crenatoside and 2'-acetylacteoside, etc.
China ; Cistanche ; Glycosides ; Male ; Orobanche ; Plants, Medicinal