This study aims to analyse the value of CODEHOP RT-PCR in the detection of Flavivirus. According to the amino acid sequences of polyproteins of different flaviviruses published in GenBank, a pair of primers was designed using the CODEHOP method. One-step RT-PCR was used to detect Japanese encephalitis virus strain JEV1201, Dengue virus strain JKD001, and yellow fever virus vaccine YV6161. BLAST analysis and phylogenetic analysis were performed after the RT-PCR products of nucleocapsid genes were sequenced. The results showed that this method could amplify Flavivirus specifically, and the size and sequence of the target fragment accorded with the anticipated result. JEV1201 had the highest homology to Japanese encephalitis virus strain YL2009-4/YC2009-3, belonging to the branch of the phylogenetic tree of Japanese encephalitis virus strains. JKD001 had the highest homology to Dengue virus strain DENV-2/ID/1022DN/1975, belonging to the branch of the phylogenetic tree of Dengue virus strains. YV6161 had the highest homology to Yellow fever virus strain 17D, belonging to the branch of the phylogenetic tree of Yellow fever virus strains. In conclusion, the method of CODEHOP RT-PCR can be effectively used to detect, identify, and phylogenetically analyse Flavivirus.
DNA Primers ; genetics ; Flavivirus ; classification ; genetics ; isolation & purification ; Flavivirus Infections ; virology ; Humans ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Nonstructural Proteins ; genetics

Objective To investigate the clinical significance in MRP1/CD_9 expression in cervical squamous cancer tissues and normal cervical tissues.Methods The expression of MRP1/CD_9 were assayed by SABC immunohistochemical methods in 53 cases of cervical cancer tissues and 13 cases of normal cervical tissues.Results Positive expression of MRP1/CD_9 was detected in 13 normal cervical tissue.MRP1/D_9 ex- pression is down-regulated in cervical carcinoma(P

Objective To investigate the clinical characteristics of neurosyphilis presenting clinically with cerebral infarction and study its treatment and prognosis. Methods A retrospective analysis was conducted in 41 patients with neurosyphilis who presented clinically with cerebral infarction. The clinical manifestations, neurological deficit scores, activity of daily life (ADL) scores, mental scale scores, and laboratory and imaging examination results of the patients were analyzed. Results Of the 41 patients, 23 exhibited reduced intelligence, 30 had limb paralysis, 8 had limb numbness, 2 complained of dizziness, and 1 experienced epileptic attacks. Head magnetic resonance imaging (MRI) revealed the presence of brain atrophy and multiple infarction foei without obvious space-occupying effect of cerebral edema in these patients. All the patients showed positive results of serum and cerebrospinal fluid Treponema pallidum hemagglutination assay (TPHA). After treatment with high-dose penicillin, 95.1% of the patients had improvements of the clinical symptoms. Conclusion The diverse clinical manifestations of neurosyphilis often result in misdiagnosis of the disease. Patients with neurosyphilis show multiple cerebral infarction foci and cerebral atrophy without obvious space-occupying effect on MRI. Penicillin can be an effective treatment in such cases, which generally have low rate of negative TPHA within 3 months. Such patients have more favorable clinical outcomes than those with common cerebral infarction, but poor prognosis is indicated in cases with concurrent extensive cerebral infarction.

Objective:To determine whether heme oxygenase-2(HO-2) gene deletion can attenuate oxidative injury induced by free Fe2+. Methods:Stereotactic injection of 10 μl sterile FeCl2 (10 mmol/L) was made into the right striata of HO-2 knockout mice and wild-type mice. Brain edema severity was measured at 24 h. Cell viability, protein oxidation, and lipid oxidation of the basal ganglia were determined at 72 h. Western blot analysis was applied for heme oxygenase-1 (HO-1) measurement.Results: Brain water content significantly decreased in HO-2 knockout mice at 24 h compared with wild-type mice. Protein oxidation and lipid oxidation significantly decreased in HO-2 knockout mice at 72 h compared with wild-type mice, while the striatal cell viability increased significantly. HO-1 expression at baseline and 72 h was also similar to that in wild-type mice. Conclusion:These results show that HO-2 gene deletion can protect basal ganalia cells from free Fe2+ -mediated oxidative stress injury,suggesting that selective inhibition of HO-2 may have a protective effect on brain oxidative injury.

Adolescent ; Adult ; Athletic Performance ; psychology ; Back ; physiology ; Electromyography ; Exercise ; physiology ; Humans ; Imagery (Psychotherapy) ; Male ; Young Adult

OBJECTIVE: To evaluate the clinical effects and characteristics of combined transperineal and transpubic urethroplasty for patients with complex pelvic fracture urethral distraction defect (PFUDD). METHODS: We retrospectively reviewed the clinical data of 17 male patients with complex posterior PFUDD from January 2010 to December 2019. The complications included urethrorectal fistulas in 2 patients (11.8%), urethroperineal fistula in 1 patient (5.9%). Ten patients had undergone previous treatments: dilatation in 3 patients (17.6%), internal urethrotomy in 1 patient, failed urethroplasty in 6 patients (35.3%), of whom 2 patients had two times of failed urethroplasties. All the patients were performed with urethroplasty by combined transperineal and transpubic approach with removing the entire pubic bone followed by the anastomosis. RESULTS: The mean age of the patients included in this study was 35.5 (range: 21-62) years. The mean length of stricture was 5.5 (range: 4.5-7.0) cm, the mean follow-up was 27 (range: 7-110) months, the mean time of operation was 190 (range: 150-260) min, the mean evaluated blood loss was 460 (range: 200-1 200) mL. There were 5 patients who needed blood transfusion intraoperatively or postoperatively. Wound infection was seen in 4 out of 17 patients and thrombosis of lower extremities in 1 out of 17 patients. The last follow-up showed that the mean postoperative maximum urinary flow rate was 22.7 (range: 15.5-40.7) mL/s. After removing the catheter, one patient presented with decreased urinary flow and symptoms of urinary infection. Cystoscopy showed the recurrent anastomotic stricture, which was cured by internal urethrotomy. In our series, the success rate of the combined transperineal and transpubic urethroplasty was 94.1% (16/17). CONCLUSION Combined transperineal and transpubic urtheroplasty can achieve a tension free anastomosis after removing the entire wedge of pubis in some patients with complex PFUDD. However, this procedure should be completed in a regional referral hospital due to the complexity of the operation and the high percentage of complications.
Adult ; Anastomosis, Surgical ; Fractures, Bone/surgery* ; Humans ; Male ; Middle Aged ; Pelvic Bones ; Retrospective Studies ; Treatment Outcome ; Urethra ; Urethral Stricture ; Young Adult

Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.

OBJECTIVETo construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E. coli, and to explore their values in measuring ADAMTS13 activity as substrates.

METHODSThe DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1, a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 (rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay (ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GST-vWF114-H were used to measure plasma ADAMTS13 activity as substrates.

RESULTSTwo small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified, which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GST-vWF73-H and GST-vWF114-H as substrates.

CONCLUSIONSTwo GST fusion proteins in vWF A2 domain, vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.

ADAM Proteins ; blood ; genetics ; ADAMTS13 Protein ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Glutathione Transferase ; metabolism ; Humans ; Male ; Purpura, Thrombotic Thrombocytopenic ; blood ; genetics ; metabolism ; von Willebrand Factor ; genetics ; metabolism

OBJECTIVETo investigate the susceptibility of von Willebrand factor (VWF) type 2A mutant A1500E to proteolysis by metalloprotease ADAMTS13 and to provide the direct supports for the pathogenesis of VWF mutation A1500E responsible for von Willebrand disease (VWD) type 2A.

METHODSRecombinant wild-type VWF (WT-VWF) and A1500E mutant VWF transiently expressed on transfected HeLa cell lines. Expression media were collected and concentrated, then cleaved directly by recombinant ADAMTS13 (rADAMTS13). Compared with WT-VWF, the susceptibility of A1500E mutant VWF to proteolysis by ADAMTS13 was analyzed using SDS-agarose gel VWF multimers analysis.

RESULTSIn vitro the expression of VWF:Ag in the supernatants of WT-VWF and A1500E mutant VWF were 1.10 U/ml and 0.78 U/ml, respectively, while VWF:Ag in cells lysates of A1500E mutant VWF was 90.6% of that of WT-VWF. The SDS-agarose gel VWF multimers analysis showed that there were no differences between WT-VWF and A1500E mutant VWF. The A1500E mutant VWF could be efficiently cleaved by ADAMTS13 under static condition without denaturants such as urea and guanidine HCl. VWF multimeric analysis showed that high and intermediate molecular weight multimers dramatically decreased while low molecular weight multimers obviously increased. Conversely, WT-VWF could not be cleaved by ADAMTS13 under the same condition.

CONCLUSIONThe A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.

ADAM Proteins ; genetics ; metabolism ; ADAMTS13 Protein ; Genotype ; HeLa Cells ; Humans ; Hydrolysis ; Mutation ; Recombinant Proteins ; genetics ; metabolism ; von Willebrand Disease, Type 2 ; genetics ; metabolism ; von Willebrand Factor ; genetics

BACKGROUNDCarotid stenosis is one of the common reasons for patients with ischemic stroke, and the two invasive options carotid endarterectomy (CEA) and carotid artery stenting (CAS) are the most popular treatments. But the relative efficacy and safety of the methods are not clear.

METHODSAbout 521 articles related to CAS and CEA for carotid stenosis published in 1995 - 2011 were retrieved from MEDLINE, Cochrane Library (CL), and China National Knowledge Infrastructure (CNKI) China Journal Full-Test database. Of them, eight articles were chosen. Meta-analysis was used to assess the relative risks.

RESULTSThe eight studies included 3873 patients with symptomatic carotid artery stenosis, including 1941 cases in the carotid stent angioplasty group, and 1932 cases in the carotid endarterectomy group. Fixed effect model analysis showed that within 30 days of incidence of all types of strokes, surgery was significantly highly preferred in CAS patients (CAS group) than the CEA patients (CEA group), and the difference was statistically significant (relative ratio (RR) = 1.80, 95% confidence interval (CI): 1.380 - 2.401, P < 0.0001). But the incidence of death in the two groups is not showed and is not statistically significant after 30 days (RR = 1.52, 95%CI: 0.82 - 2.82, P = 0.18). The rate of cranial nerve injury in the CAS group is lower than the CEA group (RR = 0.14, 95%CI: 0.05 - 0.43, P = 0.0005). The incidence of CAS patients with myocardial infarction is lower than the CEA group after 30 days, but statistically meaningless (RR = 0.22, 95%CI: 0.05 - 1.02, P = 0.05). The stroke or death in CAS patients were higher than the CEA group after 1 year of treatment (RR = 2.58, 95%CI: 1.03 - 6.48, P = 0.04).

CONCLUSIONSCompared to CAS, carotid endarterectomy is still the preferred treatment methodology of symptomatic carotid artery stenosis. Future meta-analyses should then be performed in long-term follow-up to support this treatment recommendation.

Carotid Stenosis ; surgery ; therapy ; Endarterectomy, Carotid ; Humans ; Stents