Method of isolation and purification of recombinant hepcidin was described, and the bioactivity of the protein was assayed in this paper.The oxidation of his-hepcidin was carried out in the cysteine-cystine system, and the multimers were removed through gel filtration under denaturation condition.Then the protein was refolded by continuous dilution and digested by enterokinase.The total yield of his-hepcidin before enterokinase cleavage is 50%, and the purity is above 95%.Through agar diffusion assay, the recombinant hepcidin displayed obvious antibacterial activity against B.subtilis.The LC-ESI-MS analysis of recombinant hepcidin showed that the measured molecular weight accorded with the calculated molecular weight, and the CD spectrum indicated that the secondary structure of recombinant hepcidin is similar with native hepcidin.

OBJECTIVE: To explore the feasibility of detecting of Y-STR of fetal DNA in maternal plasma using Ion Torrent PGM™ System. METHODS: A total of 16 fetal DNA samples from maternal plasmas (8 cases from 38 weeks gestational age and 8 ones from 12 weeks) were prepared and a multiplex assay with 7 STR loci (DYS390, DYS391, DYS393, DYS438, DYS437, DYS456, DYS635) was designed for multiplex-PCR amplification. Using Ion Torrent PGM™ System, the results of Y-STR sequences and capillary electrophoresis were obtained and compared. RESULTS: Y-STR specific alleles were detected in the maternal plasma of all the pregnant women having male babies of second and third trimester, which were higher than that detected by capillary electrophoresis. Consistent Y-STR genotypes were observed between fetal DNA from maternal plasma and genomic DNA from the newborn babies. CONCLUSION Based on Ion Torrent PGM™ System, the prenatal Y-STR detection method may provide a high-sensitive and high-throughput choice for prenatal STR detection in forensic testing.
Alleles ; Chromosomes, Human, Y/genetics* ; DNA/blood* ; Family ; Female ; Fetal Blood/chemistry* ; Genotype ; Haplotypes ; Humans ; Male ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Pregnancy ; Sensitivity and Specificity ; Sex Determination Analysis ; Tandem Repeat Sequences/genetics*

OBJECTIVE: To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluorescence labeling for mitochondrial DNA (mtDNA) SNP typing. METHODS: Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided into 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood samples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three random samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. RESULTS: Distinct electropherograms of 200 blood samples were obtained successfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10 microL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. CONCLUSION AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.
Alleles ; DNA ; DNA Primers ; DNA, Mitochondrial/analysis* ; Electrophoresis, Capillary ; Haplotypes ; Humans ; Mitochondria ; Polymerase Chain Reaction/methods* ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

AIMTo explore the effect of cerebellar fastigial nuclei (FN)on lymphocyte function and the pathway mediating the effect.

METHODSKainic acid (KA) was microinjected into bilateral FN of rats to destroy neuronal bodies in the FN. On the eighth day after the surgery, lymphocyte percentage in the peripheral blood and level of sheep red blood cell(SRBC)-specific IgM antibody in the serum were measured by using blood corpuscle counter and enzyme-linked immunosorbent assay (ELISA), respectively.A technology of electrolytic lesion was used to destroy the projections of cerebellar FN neurons to hypothalamus in decussation of superior cerebellar peduncle(xscp).

RESULTSOn the eighth day after the microinjection of KA into the bilateral FN of rats, the Nissl-stained neuronal bodies in the FN disappeared and glia could proliferated within the damaged FN. In the nuclei close to FN, the interposed nuclei and the dentate nuclei, Nissl-stained neurons still could be seen. On the control cerebellar sections, in which FN was infused with saline, we could see the normal Nissl-stained neurons in the FN and the other two nuclei.On day 8 following the effective FN lesions, both the lymphocyte percentage in the peripheral blood and the level of anti-SRBC IgM antibody in the serum were significantly increased in comparison with those of control rats infused with saline in the FN. On the eighth day after electrolytic lesion of the fibres in xscp, the FN-hypothalamic projections were damaged and there were no visible BDA-positive endings in hypothalamus. Meanwhile, both the lymphocyte percentage in the peripheral blood and the level of anti-SRBC IgM antibody in the serum were remarkably enhanced relative to those of control rats with sham lesion of xscp.

CONCLUSIONThe electrolytic lesion of the FN-hypothalamic projections in xscp causes an enhancement of lymphocyte function similar to that of KA lesions of neuronal soma in the FN. These findings suggest that the cerebellohypothalamic projections participate in mediating the modulation of lymphocyte function by the cerebellum.

Animals ; Cerebellar Nuclei ; immunology ; injuries ; Female ; Hypothalamus ; immunology ; physiology ; Kainic Acid ; Lymphocyte Count ; Lymphocytes ; cytology ; immunology ; Male ; Neural Pathways ; immunology ; physiology ; Neuroimmunomodulation ; physiology ; Rats ; Rats, Sprague-Dawley

Objective To investigate the effects of transcranial direct current stimulation (tDCS) on motor function of upper limbs of stroke patients. Methods 80 stroke patients were randomly divided into experimental group and control group. Both groups accepted routine rehabilitation, while the experimental group accepted anodal stimulation, and the control group received sham stimulation. They were assessed with Brunnstrom stages of arms and hands, Fugl-Meyer Assessment (FMA) of upper extremities, Action Research Arm Test (ARAT), Motor Assessment Scale (MAS) and modified Barthel Index (MBI) before and 1 month after treatment. Results All the scores improved in both groups after treatment (P<0.05), and improved more in the Brunnstrom stages of arms and hands, FMA, ARAT in the experimental group than in the control group (P<0.05). Conclusion tDCS may promote the recovery of arms and hands function of stroke patients.

OBJECTIVESTo explore intervention with electromagnetic noise for co-suppression effect on gap-junctional intercellular communication (GJIC) induced or strengthened by low intensity magnetic field with carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA).

METHODSFibroblast cells from NIH 3T3 mice were exposed to extremely low intensity magnetic field (MF) 0.2 mT, 0.2 mT + TPA or/and electromagnetic noise with the same intensity of MF for 24 h, and GJIC was determined using fluorescence recovery analysis after photobleaching (FRAP) with a laser-scanning confocal microscope (Leica, Germany).

RESULTSGJIC function could be co-suppressed by MF of 0.2 mT with TPA, with fluorescence recovery of (23 +/- 11)%, lower than that in the control group [(46 +/- 19)%] and in the group with TPA only [(34 +/- 17) %] (P < 0.01), indicating 0.2 mT MF plus TPA could co-inhibit GJIC (P < 0.01). Superposition of 0.2 mT noise MF could get a fluorescence recovery of (35 +/- 19)% and significantly antagonize its co-suppression by TPA.

CONCLUSIONElectromagnetic noise of 0.2 mT could block the intensifying effect of power frequency magnetic field on TPA-induced GJIC inhibition.

Animals ; Cell Communication ; drug effects ; physiology ; radiation effects ; Cell Line ; Electromagnetic Fields ; adverse effects ; Fluorescence Recovery After Photobleaching ; Gap Junctions ; drug effects ; physiology ; radiation effects ; Mice ; NIH 3T3 Cells ; Noise ; adverse effects ; Tetradecanoylphorbol Acetate ; pharmacology

Objective　Diabetic cardiomyopathy (DCM) is one of the complications of diabetes, which is closely related to the change of miRNA. In this study, we observed the characteristic expression of miR-26b in the tissues of the C57BL/6J mouse and in the heart, adipose tissue and liver of the ob/ob mouse, and investigated the effect of high glucose (Glu) on the expression of miR-26b in H9C2 cardiomyocytes.　Methods　Using RT-PCR, we measured the levels of miR-26b in the heart, adipose tissue, liver and other tissues of C57BL/6J and ob/ob mice. H9C2 cardiomyocytes were treated with Glu at 5.5, 15, 25 and 35 mmol/L for 0, 24, 48, 72, 96 and 120 hours, followed by detection of the proliferation of cardiomyocytes by CCK-8 and determination of thelevels miR-26b.　Results　The expression of miR-26b was the highest in the heart of the C57BL/6J mice, significantly higher than in the cardiac and white adipose tissues of the ob/ob mice (P < 0.05). The proliferation of cardiomyocytes was markedly increased in the 15, 25 and 35 mmol/L Glu groups at 24, 48, 72, 96 and 120 hours as compared with that in the 5.5 mmol/L Glu group (P < 0.05), higher in the 25 than in the 15 mmol/L Glu group at 24 hours (0.74±0.02 vs 0.72±0.01, P<0.05), but lower in the 35 than in the 15 mmol/L Glu group at 48 hours (0.92±0.01 vs 0.94±0.01, P<0.05), in the 25 and 35 mmol/L Glu groups at 96 hours (P < 0.05), in the 35 mmol/L Glu group at 120 hours (1.12±0.02 vs 1.19±0.05, P<0.05), in the 35 than in the 25 mmol/L Glu group at 24 and 48 hours (P<0.05). The expression of miR-26b in the H9C2 cardiomyocytes was significantly down-regulated in the 25 and 35 mmol/L Glu groups in comparison with that in the 5.5 mmol/L Glu group (P<0.05), remarkably lower in the 25 mmol/L Glu group at 96 and 120 hours than at 0 hour (P<0.05).　Conclusion　High glucose can down-regulate the expression of miR-26b in H9C2 cardiomyocytes, which suggests that miR-26b may participate in the pathogenesis of DCM.

Background: Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype–phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. Methods: In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results: Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. Conclusions We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.

Objective::To observe the intervention effect of Yiqi Huoxue recipe (YQHX) on ventricular remodeling in rats with chronic heart failure, in order to explore its mechanism. Method::Among 40 male SD rats, 10 were randomly selected as the sham operation group. The left anterior descending coronary artery ligation was performed to construct the chronic heart failure(CHF) rat model. After modeling, they were randomly divided into model group, captopril group(13.5 mg·kg^-1·d^-1) and YQHX group (20 g·kg^-1·d^-1), and orally given the corresponding drugs. After 8 weeks of intervention, cardiac tissues were collected, body mass and heart mass were weighed, and echocardiography were performed to detect the changes in cardiac structure. Masson staining was performed to determine the myocardial interstitial collagen volume fraction. Western blot was used to detect the expression levels of mitochondrial fusion protein optic atrophy 1 (Opa1) and cleavage protein dynamic-related protein 1 (Drpl). The quantitative real-time fluorescence polymerase chain reaction(Real-time PCR)was applied to detect the expressions of Wnt/β-catenin pathway-related factors such as lipoprotein receptor-related protein 6 (LRP6), glycogen synthase kinase-3β (GSK-3β) and β-catenin. Result::Compared with the sham group, the left ventricular wall of the model group was significantly thickened (P<0.05), the cardiac cavity was significantly enlarged, and the content of collagen in the myocardial interstitium was increased (P<0.01). The expression level of Opal decreased, the expression level of Drp1 increased (P<0.05), the mRNA expression level of LRP6, GSK-3, and β-catenin increased (P<0.01). Compared with the model group, YQHX group can reduce ventricular wall thickening, heart chamber enlargement, myocardial interstitial collagen content, up-regulate the low expression of Opa1, but down-regulate the high expressions of Drpl, LRP6, GSK-3β, β-catenin(P<0.05, P<0.01). Conclusion::YQHX can effectively alleviate ventricular remodeling and improve mitochondrial energy metabolism in rats with CHF. The mechanism may be related to the inhibition of Wnt/β-catenin related factors.