ObjectiveTo explore the effect of precocious puberty on glucose metabolism and lipid metabolism in female rats. MethodsSixty two-day-old female rats were randomly divided into 2 groups. When aged 5 days， the precocious puberty group and normal group were given a single subcutaneous injection of danazol and solvent soybean oil respectively. The vaginal opening of rats was monitored from their 21 days of age. After 12 hours of fasting， all successful modeling rats were randomly executed within 3 days after vaginal opening， when aged 7 and 12 weeks. Then we measured the rats’ body weight and length， determined the concentrations of glucose， insulin， blood lipids， estradiol， leptin and adiponectin with enzyme-linked immunosorbent assay and observed the pathological changes of perirenal fat， uterus and ovary. ResultsFor body weight and length， rats in the precocious puberty group were smaller than those in the normal group within 3 days after vaginal opening， but which did not affect their subsequent growth and development， and there was no significant difference between the two groups at 7 and 12 weeks of age. Within 3 days after vaginal opening， insulin levels had significant difference between the two groups （P = 0.001）， the precocious group showed hyperinsulinemia and increased number of perirenal adipocytes. At three execution times， no significant difference was noted in estradiol， leptin and adiponectin levels between the two groups. The same was true in the ratios of ovary or uterus to body weight between the two groups. ConclusionsPrecocious puberty makes earlier onset of pubertal development and allows body maladaptation to the sudden changes of the internal environment. However， the changes due to precocious puberty are temporary and reversible， and they may become normal in adulthood.

AIM:The paper is to explore a rapid and simple method for the culture of mouse primary thyroid cells.METHODS:Mouse thyroid cells were isolated by enzyme digestion and cultured with improved medium,and their morphology,characteristics and secretory function were observed within 14 d.RESULTS:In the cultures,the active pri-mary cells were obtained from the thyroid tissue after digestion for 25 min;adherent growth was observed on the 2nd day.And secondary follicles appeared from the 5th to 7th day.Over 95%cells were detected with thyroglobulin.The secretion of total triiodothyronine and total thyroxine maintains over 60%in 7 d.The expression levels of specific genes can still maintain more than 50%in 10 d.CONCLUSION:Mouse thyroid primary cells can be rapidly cultured by this method,and the cells can be used for studying thyroid endocrine secretion within 7 d and studying thyroid genes within 10 d.

Objective To establish and optimize a method for simultaneous determination for the contents of sodium,potassium,calci-um and magnesium ions in serum using microwave digestion combined with ion chromatography,and evaluate its performance.Methods Serum samples were pretreated by microwave digestion,and four cations were quantitatively determined using ion chromatography.The linearity,precision,recovery and accuracy of the established method were verified with reference to the EP6-A and EP15-A3 docu-ments issued by the Clinical and Laboratory Standards Institute(CLSI).Results The imprecisions of determinations for sodium,po-tassium,calcium and magnesium ions expressed as coefficient of variation(CV)were 0.10％to 0.36％,0.15％to 0.48％,0.22％to 0.87％and 0.21％to 0.73％,respectively.The recoveries with adding standard of the four cations were between 97.3％and 103.0％.Referred to RELA2020 and RELA2021,the deviations of the analysis results were-0.57％to-0.14％,-0.84％to-0.16％,-1.82％to-0.37％,and-0.60％to 0.34％for sodium,potassium,calcium,and magnesium,respectively,and all the comparisons were pas-sed.The limits of detection/quantification were 0.017 5/0.058 3 mmol/L,0.000 5/0.001 7 mmol/L,0.009 9/0.032 9 mmol/L and 0.001 1/0.003 5 mmol/L for the four cations,indicating that after pretreatment,no significantly interfere from other impurities for the results of determination was found.Conclusion A method for simultaneous determination of sodium,potassium,calcium and magnesi-um ions in serum was successfully established and optimized using microwave digestion combined with ion chromatography.The method showed good precision and accuracy,simple operation,short time consumption and low cost,thus it could be used for quality improve-ment of clinical electrolyte testing in serum.

Objective:To develop a candidate reference method for cortisol in human serum by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS).Methods:An isotope-labeled internal standard was added to samples, followed by alkalizing with sodium carbonate solution and liquid-liquid extraction with n-hexane/ethyl acetate. Isoelution with a methanol aqueous solution was employed for liquid chromatographic separation, while ESI in the positive ion and multiple reaction monitoring mode were used for mass spectrometry. The volume of sample, standard, and internal standard solutions were all controlled by weighing, and the results were calculated by bracketing method. The accuracy, precision, specificity, linearity, LOD and LOQ of this method were evaluated referring to the CLSI C62-A and C50-A guidelines and the domestic expert consensus documents.Results:The method demonstrated excellent accuracy and precision with the bias of ERM-DA192, ERM-DA193 and RELA 2021-2023 all being less than 2%. The recovery of added cortisol ranged from 98.2% to 101.1%. Both intra- and inter-assay imprecisions was <2%. The method was free from interference by structural analogue and showed a good linearity in the range of 25-1 600 nmol/L. The LOD and LOQ were 0.5 nmol/L and 1.0 nmol/L, respectively. A cortisol assay kit (chemiluminescence immunoassay) traced to this candidate reference method was used to determine 46 clinical serum samples concurrently, and the two methods exhibited good correlation.Conclusions:A candidate reference method for the determination of cortisol in serum was established, demonstrating high sensitivity, good repeatability and accuracy. This method can serve as a reference for the measurement traceability and accuracy evaluation of routine methods.

AIM:To investigate the activation of Cl-channels by sodium taurocholate(NaTC)in mouse pan-creatic acinar cells.METHODS:The single isolated pancreatic acinar cells from FVB/N mice were prepared using colla-genase digestion method.Whole-cell patch clamp technique was performed to record the currents.Intracellular adenosine triphosphate(ATP)dependence of the channels was examined via eliminating ATP from the pipette solution.Anion per-meability of the channels was investigated with ion-exchange method.The pharmacological characteristics of the channels was confirmed by two Cl-channel blockers.The volume sensitivity of the channels was detected using 47%hypertonic bathing solution.Extracellular Ca2+dependence of activating the channels was examined through eliminating Ca2+from the bathing solution.Intracellular reactive oxygen species(ROS)level was detected by an oxidation-sensitive fluorescent probe,2',7'-dichlorofluorescin diacetate.The experiment was repeated 6 times in each group.RESULTS:Extracellular application of 5 mmol/L sodium taurocholate induced a Cl-current,exhibiting the properties of outward-rectification,a se-lectivity sequence of I->Br-≥Cl->gluconate-and intracellularATP dependence(P<0.01).The currents were inhibited by chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate(DIDS)and tamoxifen and by 47%hypertonicity stimulation(P<0.01).When ROS production was scavenged by N-acetyl-L-cysteine,the sodi-um taurocholate-induced Cl-currents were unaffected.The effect of sodium taurocholate on ROS production did not alter with the treatment with DIDS.Sodium taurocholate failed to induce Cl-currents when Ca2+was absent in extracellular bath-ing solution(P>0.05).CONCLUSION:Sodium taurocholate activates Cl-channels in mouse pancreatic acinar cells,which is dependent on extracellular Ca2+but not ROS pathway.

Electroencephalogram (EEG) has been an important tool for scientists to study epilepsy and evaluate the treatment of epilepsy for half a century, since epilepsy seizures are caused by the diffusion of excessive discharge of brain neurons. This paper reviews the clinical application of scalp EEG in the treatment of intractable epilepsy with vagus nerve stimulation (VNS) in the past 30 years. It mainly introduces the prediction of the therapeutic effect of VNS on intractable epilepsy based on EEG characteristics and the effect of VNS on EEG of patients with intractable epilepsy, and expounds some therapeutic mechanisms of VNS. For predicting the efficacy of VNS based on EEG characteristics, EEG characteristics such as epileptiform discharge, polarity of slow cortical potential changes, changes of EEG symmetry level and changes of EEG power spectrum are described. In view of the influence of VNS treatment on patients' EEG characteristics, the change of epileptiform discharge, power spectrum, synchrony, brain network and amplitude of event-related potential P300 are described. Although no representative EEG markers have been identified for clinical promotion, this review paves the way for prospective studies of larger patient populations in the future to better apply EEG to the clinical treatment of VNS, and provides ideas for predicting VNS efficacy, assessing VNS efficacy, and understanding VNS treatment mechanisms, with broad medical and scientific implications.
Drug Resistant Epilepsy ; Electroencephalography ; Humans ; Prospective Studies ; Scalp ; Treatment Outcome ; Vagus Nerve Stimulation

Overexpression of exogenous lineage-determining factors succeeds in directly reprogramming fibroblasts to various cell types. Several studies have reported reprogramming of fibroblasts into induced cardiac progenitor cells (iCPCs). CRISPR/Cas9-mediated gene activation is a potential approach for cellular reprogramming due to its high precision and multiplexing capacity. Here we show lineage reprogramming to iCPCs through a dead Cas9 (dCas9)-based transcription activation system. Targeted and robust activation of endogenous cardiac factors, including GATA4, HAND2, MEF2C and TBX5 (G, H, M and T; GHMT), can reprogram human fibroblasts toward iCPCs. The iCPCs show potentials to differentiate into cardiomyocytes, smooth muscle cells and endothelial cells . Addition of MEIS1 to GHMT induces cell cycle arrest in G2/M and facilitates cardiac reprogramming. Lineage reprogramming of human fibroblasts into iCPCs provides a promising cellular resource for disease modeling, drug discovery and individualized cardiac cell therapy.

Objective To investigate the association of serum magnesium (Mg) level with all-cause mortality in maintenance hemodialysis patients. Methods A multicenter retrospective cohort study was conducted in seven hemodialysis centers of Guizhou province. The adult outpatients who underwent hemodialysis for more than 3 months were included from June 2015 to June 2016. Demographics, baseline clinical and laboratory test results were collected. All patients were followed up until June 30, 2018. Patients were divided into 4 groups according to their baseline serum Mg levels (interquartile range). Kaplan-Meier method was used to compare the survival rates of the four group. Cox regression model was used to analyze the association of Mg with all-cause mortality. Logistic regression was used to analyze the influencing factors of low Mg level. Results A total of 868 hemodialysis dialysis patients with baseline Mg data were enrolled in this study, with age of (55.47± 16.17) years old, among whom 59.4% were male. There were 11 (1.3%) patients with hypomagnesemia (Mg＜0.7 mmol/L), 432(49.8% ) patients with hypermagnesemia (Mg＞1.05 mmol/L), and 16(1.8% ) patients with Mg＞2.0 mmol/L. Median Mg was 1.05 mmol/L and interquartile range was 0.95-1.24 mmol/L. The comparison between Mg quartile groups showed that the difference in age, hemoglobin, serum albumin, serum calcium, parathyroid hormone (PTH), serum creatinine, uric acid and urea nitrogen was statistically significant (all P＜0.05). After a median follow-up of 24 months, 207 patients died. Kaplan-Meier curves showed higher all-cause mortality in patients with Mg≤0.95 mmol/L (Q1 group) (Log - rank test χ2=15.11, P=0.002). However, after adjusting for age, comorbidities and biochemical indicators(especially albumin), there was no statistically significant difference in the hazard ratio for all-cause death among the four groups. Multiple logistic regression analysis results showed that low serum albumin (OR=0.946, 95%CI 0.913-0.979, P=0.002) and low serum uric acid (OR=0.994, 95% CI 0.992-0.997, P＜0.001) were the risk factors for baseline Mg≤0.95 mmol/L. Conclusions Hypomagnesemia is rare in MHD patients, while hypermagnesemia is more common. Baseline serum Mg≤0.95 mmol/L in MHD patients is correlated with increased risk of all-cause death, but it may be not an independent risk factor. Baseline serum Mg≤0.95 mmol/L that occurred is associated with low levels of albumin and serum uric acid.

Objective:To investigate the dose-effect relationship of Xianfu ointment and its decomposed recipes the 1-chloro-2,4-dini-trochlorobenzene(DNCB) induced chronic eczema in mice, and confirm the median effective dose (ED50) of each formula and the synergetic effect by compatibility. Methods:DNCB was used to induce chronic eczema in C57 mice. The mice were treated with gradient dosages of the Xianfu ointment (11.71-11 662.50 mg?kg-1?d-1,k = 0.316), Anemone flaccid (0.53-530.12 mg?kg-1?d-1,k = 0.316), Xianfu ointment without Anemone flaccid (11.18-11 132.40 mg?kg-1?d-1,k =0.316),respectively. The pathological features were observed after hematoxylin-eosin staining. The volume ratio of epidermides and the number of lymphocyte infiltrated in dermis were analyzed with morphometry. The serum levels of IL-2,IFN-γ,IL-4,and IL-13 were detected by ELISA assay. The ED50was calculated by non-linear regression with various slope using Prism-5.0 software.Results:The effects of Xianfu ointment and its decomposed recipes on chronic eczema showed a dose-dependent tendency. The dose-response curves showed"S"shape. The efficacy of Xianfu ointment on chronic eczema was the most significant among the three formulas, which was demonstrated by decreased epidemical thicknes (ED50= 377.90 mg?kg-1?d-1), reduced infiltrated lymphocyte number(ED50= 153.20 mg?kg-1?d-1), increased serum IL-2(ED50=608.90 mg?kg-1?d-1) and IFN-γ (ED50= 205.50 mg?kg-1?d-1) levels, and decreased serum IL-4(ED50= 198.70 mg?kg-1?d-1) and IL-13 levels (ED50= 117.60 mg?kg-1?d-1). And the dose-effect curves of Anemone flaccid and Xianfu ointment without Anemone flaccid groups were both right shift when compared with that of Xianfu ointment. Conclusion:Xianfu ointment and its decomposed recipes can effectively treat chronic eczema. Anemone flaccid has obvious compatibility synergy in the whole formula. The effects of Xianfu ointment is most significant.

Objective: To study the dose-effect relationship of Yuning ointment and its decomposed recipes in the treatment of oleic acid induced acne in mice. Methods: Oleic acid was administrated to the back (2 cm ×2 cm) of the mice (once a day) for 21 days to induce acne. At d22, the gradient dosage of Anemone flaccida crude drug (1. 06-1 060. 23 mg?kg-1?d-1,k=3. 16), Yuning oint-ment without Anemone flaccida crude drug (4. 73-1 767. 75 mg?kg-1?d-1, k=3. 16) and Yuning ointment (2. 84-2 827. 28 mg?kg-1?d-1, k=3. 16) was respectively administrated to the back of mice for 14 days. The pathological changes of skin were observed by hematoxylin-eosin (HE) staining. The diameter of sebaceous glands and the ratio of follicular keratinization area were morphomet-rically analyzed. The serum levels of IL-1, IL-6 and TNF-α were detected by ELISA assay. The median effective dosages (ED50) of A-nemone flaccida in the three prescriptions were regressed by Prism 5. 01 software to determine the prescription dose-effect. Results: All the therapy groups were with significantly relieved pathological changes of sebaceous glands hypertrophy and follicular keratinization, and decreased serum levels of IL-1, IL-6 and TNF-α in a dose-dependent manner. The dose-response curves showed an "S" shape. A-mong the three therapy groups, the effect of Yuning ointment was the best. The ED50of Yuning ointment regressed by Anemone flaccida dose was 0. 28-fold for improving sebaceous glands hypertrophy, 0. 14-fold for inhibiting follicular keratinization, and 0. 15-, 0. 49-and 0. 24-fold for decreasing serum levels of IL-1, IL-6 and TNF-α. . Regressed by Yuning ointment without Anemone flaccida, the ED50of Yuning ointment was lower than Yuning ointment without Anemone flaccid in terms of improving pathological changes and inhibiting the secretion of cytokines. Conclusion: Yuning ointment can prevent and treat acne through regulating immune function. And the prescrip-tion compatibility can enhance the effects of Anemone flaccida.