OBJECTIVETo investigate the main factors that influence the results of sperm alkaline single-cell gel electrophoresis (SCGE), optimize the conditions, and standardize its procedures.

METHODSUsing alkaline SCGE, we detected the DNA fragments of sperm treated with different concentrations of H2O2 and determined the influences of the number of agarose gel layers, pH during DNA unwinding and electrophoresis, the time of DNA unwinding and electrophoresis, and cumulative sperm number on the results of sperm alkaline SCGE. Then we optimized the procedures, analyzed the repeatability of the optimized method, and examined 40 semen samples using the method.

RESULTSThree agarose gel layers could reduce the background. The optimal pH during DNA unwinding and electrophoresis was 10, and the best times for DNA unwinding and electrophoresis were 40 min and 30 min, respectively. Fifty sperm were adequate to ensure the reliability of the results. Based on the percentage of tail DNA, the intra- and inter-assay repeatabilities of the optimized sperm alkaline SCGE were 3.12% and 7.13%, and by the DNA damage score, they were 2.38% and 6.09%, respectively. Sperm DNA fragments were significantly increased in the infertile patients with oligoasthenoteratozoospermia as compared with healthy fertile males (P <0.05).

CONCLUSIONThe optimized sperm alkaline SCGE, highly repeatable and easy to be standardized, can be applied to the clinical detection of sperm DNA fragmentation in infertile men.

Asthenozoospermia ; genetics ; Comet Assay ; standards ; DNA Damage ; DNA Fragmentation ; Humans ; Hydrogen Peroxide ; toxicity ; Male ; Oligospermia ; genetics ; Oxidants ; toxicity ; Reproducibility of Results ; Sperm Count ; Spermatozoa ; drug effects ; enzymology ; Time Factors

In order to solve the problem of resistance of Pseudomonas aeruginosa to multiple antibiotics, it is an effective way to find inhibitors of P. aeruginosa efflux pump. In this study, 15 new ornithine peptidomimetic derivatives were designed and synthesized by changing the side chain structure of natural amino acids with PAβN, a dipeptide efflux pump inhibitor, and their synergic activity with aztreonam, a monocyclic β-lactam antibiotic, against P. aeruginosa was evaluated. Among them, the representative compound 12b not only enhanced the antibacterial activity of β-lactam antibiotics aztreonam, ceftazidime and meropenem, but also significantly enhanced the antibacterial action of macrolide antibiotics clarithromycin, showing a broad-spectrum synergic sensitization effect. In addition, compound 12b also has a good safety. Preliminary mechanisms suggest that 12b works by directly targeting the efflux transporter MexB. These results provide a new lead compound for the development of a new class of efflux pump inhibitors against P. aeruginosa.

OBJECTIVETo investigate the effect of new bone formation in sinus augmentation with guided bone regeneration (GBR) using collagen membranes.

METHODSThe first maxillary molars of 18 adult female Beagle dogs were extracted and the sinus floors of both sides were lifted with simultaneous implantation. A combination of autografts and Bio-Oss in a 2:1 ratio was placed in the space under the membrane. On the experimental side in each dog, the collagen membrane was folded at the lateral osteotomy window, the apex of the implants and a certain part of palatal bone. On the contralateral control side, the collagen membrane only covered the osteotomy window. Six animals were sacrificed at 4, 12, and 24 weeks respectively after surgery. Gross observation, biomechanical testing and histological examinations were performed.

RESULTSThe translocation of grafted materials and bone absorption were found on the top of implants in the control side, and the grafted materials kept original shape at the experimental side at 4th week. The granule of Bio-oss absorbed obviously at 12th and 24th week. The pull-out force increased with time. At 24th week, the force of pull out was 558.1 ± 37.4 N at the study side, and 471.4 ± 31.5 N at the control side. There was a significant difference in the pull-out force was noted between the two groups (P < 0.01). Histological examination showed new bone formation on the sinus floor, and the grafted materials gradually reduced with time

CONCLUSIONSGBR with the enfolded-coverage of the membrane can effectively decrease absorption of the grafted materialon the apical surface of implants and stimulate new bone formation in the sinus augmentation.

Animals ; Collagen ; Dental Implantation ; Dental Implants ; Dogs ; Female ; Guided Tissue Regeneration ; Male ; Maxilla ; Maxillary Sinus ; surgery ; Minerals ; Molar ; Osteogenesis ; physiology ; Sinus Floor Augmentation ; methods ; Tooth Extraction

OBJECTIVETo develop a tight tetracycline-controlled HCV-C double transgenic mouse model.

METHODSBy crossbreeding of ApoE-rtTA-tTS transgenic mice with TRE-HCV-C transgenic mice, the double transgenic mice were produced in the F1 generation. The presence of HCV-C and tTS gene in the F1 generation was confirmed by PCR, followed by further identification and quantification of the transgene using Southern blot hybridization. The expression of HCV-C in the liver of the mouse model was detected immunohistochemically.

RESULTS AND CONCLUSIONTwo transgenic mice were obtained, which contained ApoE-rtTA-tTS and TRE-HCV-C genes in the genome. Five founders contained HCV-C gene as confirmed by PCR and Southern blot hybridization. The tight tetracycline-controlled system may facilitate further study of HCV-C gene expression and gene therapy of hepatic cellular carcinoma.

Animals ; Apolipoproteins E ; genetics ; Blotting, Southern ; Breeding ; Crosses, Genetic ; Female ; Gene Expression Regulation, Viral ; drug effects ; Hepacivirus ; genetics ; immunology ; Hepatitis C Antigens ; genetics ; immunology ; Male ; Mice ; Mice, Transgenic ; Polymerase Chain Reaction ; Tetracycline ; pharmacology ; Trans-Activators ; genetics ; Viral Core Proteins ; genetics

Objective To investigate the profiles of resistance following continuous treatment with TM208 using pharmacokinetics/pharmacodynamic (PK/PD) model,as well as to characterize the estrogen levels in hibition in human breast cancer xenograft mice.Methods MCF-7 xenograftnudemice were given TM208 (150 mg · kg-1 · d-1) or vehicle orally for 18 days.The concentrations of estrogen were measured using the human estrogen ELISA kit.The pharmacokinetics and pharmacodynamics of TM208 were evaluated after administration.A one-compartment model with first order absorption was developed to describe the pharmacokinetics of TM208,and the time course of estrogen degradation was characterized by the indirect response model.Tolerance model was used to describe drug resistance of tumor.Results The simulation results showed that the inhibitory effects of TM208 on estrogen may decrease after continuous administration,and the KTO was 0.68 h-1 Conclusion The established PK/PD model allowed a better understanding of the role of estrogen inhibition in breast cancer treatment with TM208.Simulation based on the estrogen model allowed prediction of the detailed processes of TM208 effects on estrogen.Furthermore,the tolerance model-based modeling and simulation methods provided an option for the description and prediction of estrogen resistance.

OBJECTIVETo explore a method for the treatment of the skin defects at the distal phalanges of 2-5th fingers.

METHODSThe island flap at the dorsum of the middle phalange was designed with the pedicle of dorsal branches from the digital proper artery. When the flap was used to repair defect at finger pulp, the dorsal branch of the digital proper nerve in the flap was kept to be anastomosed to the digital proper nerve at the recipient finger. From Feb. 2005 to May. 2010, 54 cases with skin defects at the distal phalanges of 61 fingers were treated with the flap, including 35 defects at finger pulp and 26 defects at finger tip.

RESULTSThe maximum size of defects and flaps was 2.2 cm x 2.5 cm and 2.4 cm x 2.7 cm, respectively. 61 flaps survived completely. Blister was happened in 3 flaps 2 days after operation, which healed spontaneously without necrosis. 54 cases were followed up for 5 to 22 months (average, 11 months). The flaps had good texture and color match with normal sensation (grade S4). The 2-point discrimination distance was 6-9 mm. The interphalangeal joint had normal movement.

CONCLUSIONSThe island flap at the dorsum of the middle phalange is an ideal method for the skin defect at the distal phalange of finger.

Adolescent ; Adult ; Aged ; Arteries ; Female ; Fingers ; blood supply ; surgery ; Humans ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; methods ; Skin Transplantation ; methods ; Surgical Flaps ; blood supply ; Young Adult

OBJECTIVETo investigate the pathological changes of liver in children with hepatitis B virus associated glomerulonephritis (HBV-GN).

METHODSThirteen children with HBV-GN (aged from 2-14 years) underwent renal and liver biopsy. The biopsy findings were analyzed.

RESULTSDifferent degrees of hepatic lesions were seen in all of the 13 patients, mild lesions accounting for 69.2% (9/13). HBSAg positive was the most common in the liver tissue [76.9% (10/13)]. Among the renal lesions, membranous glomerulopathy accounted for 69.2%( 9/13), followed by membranoproliferative glomerulonephritis 30.8% (4/13). HBsAg and HBcAg positive were presented in all patients' kidney tissues. HBV antigens were detected in stroma between nephric tubule in all samples. Four patients presented with HBcAg positive in both live and kidneys.

CONCLUSIONSThe children with HBV-GN couple with liver lesions. The severity of the renal lesions is not always accord with that of the liver lesions. The appearance of HBcAg in both kidneys and liver indicates severe lesions of the two organs. It is suggested that a liver-kidney holistic treatment is necessary for children with HBV-GN.

Adolescent ; Child ; Child, Preschool ; Female ; Glomerulonephritis ; pathology ; Hepatitis B ; complications ; pathology ; Hepatitis B Core Antigens ; analysis ; Humans ; Kidney ; pathology ; Liver ; pathology ; Male

In order to establish a real-time RT-PCR based on SYBR Green Ⅱ for detection of hepatitis E virus (HEV),a pair of special primers was designed according to the conserved sequences of ORF2 in GenBank.Result showed that the standard curve of established SYBR Green Ⅱ real-time RT-PCR had a wide dynamic range from 4.10 × 102-4.10 × 108 copies/μL with a linear correlation(r2) of 0.996.The sensitivity could reach 1.00 × 102 copies/μL.The melting curve analysis using SYBR Green Ⅱ dye showed one specific peak with a melting temperature(Tm) of 84.0 C ±0.1 C.No amplification was detected from the RNA samples of porcine reproductive and respiratory syndrome virus,classial swine fever virus,transmissible gastroenteritis virus,porcine bocavirus,porcine epidemic dearrhoea virus porcine kobuvirus and porcine rotavirus by this PCR,respectively.Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.83 ％-0.94 ％ and inter-assay of 0.83％-0.94％.Further detection of 61 specimens showed that 9 of them were HEV positive,and the results of the quantitative RT-PCR were the same as that of the conventional RT-PCR.In conclusion,the real-time quantitative RT-PCR for HEV is feasible,the real-time RT-PCR established in this study will be useful for earlier rapid laboratory diagnosis and pathogenesis of HEV.

Objective To summarize the clinical features,natural history and causes of death of mitochondrial encephalomyopathy,lactic acidosis and stroke-like episodes (MELAS).Methods We retrospectively evaluated the clinical findings of 64 patients diagnosed as MELAS more than 3 years (death cases excluded) in Huashan Hospital from January 2005 to March 2017 and analyzed the natural course and the causes of death of the disease.Results Among 64 patients,the male-to-female ratio was 1.3 ∶ 1.Median onset age was 20.5 (16.8) years.The peak of incidence age was from 14 to 22 years.The most common features of MELAS in acute phase were seizures (48/64,75.0％),headache (41/64,64.1％),blurred vision (37/64,57.8％),nausea and vomiting (27/64,42.1％),fever (25/64,39.1％),mental and behavioral disorder (24/64,37.5％).Lactate dehydrogenase (31/60,51.6％),resting blood lactic acid (43/58,74.1％) and cerebral spinal fluid lactic acid (9/9) were elevated.Abnormal findings in electroencephalogram (36/40,90.0％),electrocardiogram (37/47,78.7％),electromyography (25/41,61.0％) were detected.In this cohort,20 patients (20/64,31.3％) with MELAS were dead.A Kaplan-Meier survival curve showed the estimated overall median survival time was 12 years.The median survival time of the group onset before sex maturity (≤ 14 years) was 8 years and that in the group onset after sex maturity (＞ 14 years) was 21 years.The causes of death were cardiogenic incidence (4/20,20.0％),pulmonary infection (4/20,20.0％),lactic acidosis (2/20,10.0％) and status epilepticus (2/20,10.0％).Conclusions MELAS is usually presented in young people associated with high mortality rate.The leading causes of death are cardiogenic,pulmonary infection and lactic acidosis.

OBJECTIVETo investigate fibronectin synthesis in SD rat mesangial cells after transforming growth factor-beta1 (TGF-beta1) is silenced by the short interfering RNA (siRNA) expressed by reconstructed pGEFP-C1 vectors.

METHODSDepending upon the 538th - 556th (A) and 895th - 913th (B) nucleotides of rat TGF-beta1 gene, a nucleotide (A or B) was constructed into a small hairpin nucleotide which was separately (A or B) or together (A plus B) inserted into a pGEFP-C1 vector with three reconstructed pGEFP-C1 vectors separately expressing the siRNAs for A or/and B. TGF-beta1 and fibronectin were dynamically investigated for their interrelationship by ELISA in the supernatant and RT-PCR in their extracted total RNA.

RESULTSThe siRNA hairpin-like molecules were constructed according to the 538th - 556th nucleotides of rat TGF-beta1 gene were able to markedly silence the expression of TGF-beta1 mRNA (P < 0.01) and protein (P < 0.01) at 48 h. Lipfectamin 2000 transfection stimulated the peak secretion of fibronectin at 24 h in the control and the experimental group whose TGF-beta1 was not silenced, but the silence of TGF-beta1 in both experimental groups delayed the top values of fibronectin to 48 h (P < 0.01).

CONCLUSIONThe silence of TGF-beta1 by siRNA decreased the fibronectin expression, but the latter was possibly not completely TGF-dependent.

Animals ; Cells ; Cells, Cultured ; Fibronectins ; metabolism ; Mesangial Cells ; drug effects ; metabolism ; RNA Interference ; drug effects ; physiology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; chemistry ; genetics