To construct secretory expression vector of PPV NS1 gene, the fragment of PPV NS1 gene coding for major antigen region of the NS1 protein was amplified by PCR and inserted into multiple clone site of eukaryotic expression vector pPICZalpha-A. The recombinant pPICZalpha-A-NS1 plasmid was transferred into P. pastoris strain GS115 mediated by electro transform. Recombinant P. pastoris strain GS115 was induced to express the fusion protein by methanol. The expressed and purified protein was analyzed by SDS-PAGE and Western Blot. The recombinant protein was highly-expressed and showed a good immunoreactivity. The indirect ELISA method was developed for detecting antibodies against PPV by checkerboard titration assay. The result showed that the optimal concentration of coated antigen was 3.2 microg/mL and the best dilution of serum was 1 : 80. The positive cut-off value of the ELISA assay was OD450 > 0.4 and OD450 positive serum/OD450 negative serum > 2.0. Compared with HI and commercial ELISA kits, the assay revealed 94.2% and 92.1% agreement respectively. The assay demonstrates good specificity and sensitivity, and can be applied in the detection of porcine parvovirus.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Parvoviridae Infections ; diagnosis ; immunology ; veterinary ; virology ; Parvovirus, Porcine ; genetics ; immunology ; isolation & purification ; Recombinant Proteins ; genetics ; immunology ; Swine ; Swine Diseases ; diagnosis ; immunology ; virology ; Viral Nonstructural Proteins ; genetics ; immunology

MicroRNAs (miRNAs) constitute a novel, extensive class of small RNAs (approximately 21 nucleotides), and play important gene-regulation roles during growth and development in various organisms. Here we conducted a homology search to identify homologs of previously validated miRNAs from silkworm genome. We identified 24 potential miRNA genes, and gave each of them a name according to the common criteria. Interestingly, we found that a great number of newly identified miRNAs were conserved in silkworm and Drosophila, and family alignment revealed that miRNA families might possess single nucleotide polymorphisms. miRNA gene clusters and possible functions of complement miRNA pairs are discussed.
Animals ; Base Sequence ; Bombyx ; Cluster Analysis ; Computational Biology ; methods ; Drosophila melanogaster ; Genetic Complementation Test ; Genome ; MicroRNAs ; metabolism ; Molecular Sequence Data ; Multigene Family ; Polymorphism, Single Nucleotide ; Sequence Homology, Nucleic Acid ; Software ; Thermodynamics

OBJECTIVETo evaluate the clinical effectiveness of transurethral seminal vesiculoscopy (TUSV) combined with finasteride in the treatment of recurrent hemospermia.

METHODSThis study included 32 patients with recurrent hematospermia, with the disease course of 3 months to 4 years. After administration of finasteride at 5 mg/d for 2 weeks, the patients underwent TUSV for both exploration of the causes and treatment, followed by medication with finasteride at the same dose for another 2 weeks. Postoperative follow-up was conducted for observation of the outcomes and complications.

RESULTSTUSV was successfully accomplished in all the 32 cases, which revealed 16 cases of seminal vesiculitis, 10 seminal calculi, 1 seminal vesicle cyst, 2 seminal vesicle polyps, and 3 seminal vesicle abscess. The operative time was 20 to 51 (31.0 +/- 5.2) minutes. Postoperative complications included 1 case of acute epididymitis and 3 cases of breast discomfort within the first 4 weeks. No incontinence, urethral stricture, rectal injury, retrograde ejaculation, and sexual dysfunction occurred postoperatively. All the patients but 1 were followed up for 6 months to 2 years. Twenty-nine of the cases were cured, and 2 experienced recurrence.

CONCLUSIONTransurethral seminal vesiculoscopy combined with finasteride is safe and effective for the treatment of recurrent hemospermia.

Adult ; Endoscopy ; methods ; Finasteride ; therapeutic use ; Follow-Up Studies ; Hemospermia ; therapy ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome

The 1H-NMR fingerprints of three different species tibetan medicine sea buckthorn were established by 1H-HMR metabolomics to find out different motablism which could provide a new method for the quality evaluation of sea buckthorn. The obtained free induction decay (FID) signal will be imported into MestReNova software and into divide segments. The data will be normalized and processed by principal component analysis and.partial least squares discriminant analysis to perform pattern recognition. The results showed that 25 metabolites belonging to different chemical types were detected from sea buckthorn,including flavonoids, triterpenoids, amino acids, carbohydrates, fatty acids, etc. PCA and PLS-DA analysis showed three different varietiest of sea buckthorn that can be clearly separated by the content of L-quebrachitol, malic acid and some unidentified sugars, which can be used as the differences metabolites of three species of sea buckthorn. 1H-NMR-based metabonomies method had a holistic characteristic with sample preparation and handling. The results of this study can offer an important reference for the species identification and quality control of sea buckthorn.
Hippophae ; metabolism ; Magnetic Resonance Spectroscopy ; methods ; Medicine, Tibetan Traditional ; Metabolomics

OBJECTIVETo study, the high polar media-water extraction process of Herba epimedii with the microwave technology.

METHODThe single parameters of extraction process was investigated with dry extract yield and extraction efficiency of icariin as content index.

RESULTThrough studying the single parameter, the optimal conditions are as follows: 70% ethanol as wetting solvent, volume of solvent to weight of material equal to 6:1 in wetting, 30 min wetting time, 2.5 min microwave irradiation time, volume of solvent to weight of material equal to 50:1 in extraction process, 10 min microwave-assisted extraction time.

CONCLUSIONCompared with conventional methods, microwave-assisted extraction (MAE) after Microwave Pretreament (MP) of raw material was higher extraction efficiency and time-saving.

Drugs, Chinese Herbal ; analysis ; isolation & purification ; Epimedium ; chemistry ; Flavonoids ; analysis ; isolation & purification ; Microwaves ; Plants, Medicinal ; chemistry ; Solvents ; Technology, Pharmaceutical ; methods

OBJECTIVETo compare the differences in the clinical function and lumbar and abdominal myodynamia in patiants of lumbar disc herniation treated with moxibustion at Dazhui (GV 14) and Guanyuan (CV 4) and acupuncture.

METHODSForty cases were randomized into a moxibustion group and an acupuncture group, 20 cases in each group. In the moxibustion group, the warm moxibustion was applied alternatively at Dazhui (GV 14) and Guanyuan (CV 4) once every other day, 1 h each time, once every day. In the acupuncture group, acupuncture was applied to the corresponding acupoints based on the affected lumbar vertebras, such as Jiaji (EX-B 2), Shens-hu (BL 23), Dachangshu (BL 25) and Huantiao (GB 30), etc. , once evey day 30 min each time. The treatment for 3 weeks was taken as one session in each group. Totally, one session treatment was required. Surface electromyography (SEMG) of bilateral paraspinal muscle and rectus muscle, and JOA score of low back pain were observed in the two groups.

RESULTS(1) JOA score: the score of subjective symptoms, score of activity of daily living (ADL) and total score were improved obviously as compared with those before treatment in the two groups (P<0.01, P<0.05). The results of subjective symptoms score, score of ADL and total score in the acupuncture group were superior to those in the moxibustion group after treatment (6.95+/-0.94 vs 5.50 +/-0.89,10. 90+/-1.86 vs 8.90+/- 1. 92,22.50 +/- 2.82 vs 19.35 +/- 2. 70, all P<0. 05). (2) SEMG comparison: root-mean-square value (RMS) was all reduced in SEMG of the anteflexion, rear protraction, orthostatism, bilateral bending and neck and leg rear flexion for strengthening lumbar muscle as compared with those before treatment in the two groups (P< 0.05, P<0. 01). RMS of the anteflexion and bilateral bending in the acupunture group were reduced much obviously as compared with the moxibustion group. In terms of sitting position anteflexion, rear protraction, orthostatism, bilateral bending and neck and leg rear flexion for strengthening lumbar muscle, median frequency (MF) after treatment was all improved as compared with that before treatment in the two groups (P<0. 05, P<0. 01). In terms of anteflexion, the electrode MF after treatment was improved much obviously in the acupuncture group(P<0. 05).

CONCLUSIONMoxibustion at Dazhui (GV 14) and Guanyuan (CV 4) and conventional acupuncture all improve muscle function, relieve muscle fatigue, increase the ability of anti-muscle fatigue, strengthen lumbar vertebral stability, release subjective symptoms and improve ADL. But, the effects of moxibustion are slightly lower than those of acupuncture.

Acupuncture Therapy ; Adult ; Aged ; Electromyography ; Female ; Humans ; Intervertebral Disc Displacement ; physiopathology ; therapy ; Lumbar Vertebrae ; physiopathology ; Male ; Middle Aged ; Moxibustion ; Young Adult

OBJECTIVELower eyelid ectropion is one of the most severe complications following lower eyelid blepharoplasty. Combination of multiple procedures was used to repair this deformity and the results were observed.

METHODSSevere lower eyelid ectropion following blepharoplasty was repaired with the combination method of multiple procedures, including transferring the island forehead flap, buried guiding suture of the lower eyelid skin to orbital periosteum and horizontal lid-shortening. 10 patients (15 eyelids) with lower eyelid ectropion after blepharoplasty were treated with the above method from Mar 2001 to Mar 2004.

RESULTSThe lower eyelid ectropion was repaired effectively. All the patients were satisfied with the results.

CONCLUSIONSThe combination method of multiple procedures was an effective method for severe lower eyelid ectropion.

Adult ; Blepharoplasty ; adverse effects ; Ectropion ; etiology ; surgery ; Female ; Humans ; Male ; Middle Aged ; Postoperative Complications ; surgery ; Reconstructive Surgical Procedures ; methods

OBJECTIVETo investigate the effects of the reconstruction of the mentum with cervical tissue flaps in the surgical management of postburn cervical scar contracture.

METHODSThirteen postburn patients with severe cervical scar contracture and mental deformity were enrolled in the study. The platysma flap (3 cases), scar tissue flap (6 cases) and cervical deep fascia tissue flap (3 cases), with the pedicle containing the facial and the superior thyroidal artery, were turned upwards to replenish mental soft tissue, and to form new mentum and sulcus mentolabialis. The cervical scars were repaired with expanded flap (11 cases), free flap (1 case) or trapezius muscular flap.

RESULTSThe post-operative cervical configuration was found to be good, and the tissue flaps for mental reconstruction healed satisfactorily. Ten patients were followed-up for 6 to 24 months. All flaps were successful and achieved good results, except that scar formation was found in the cervical linear incision in 4 patients.

CONCLUSIONTransfer of the platysma flaps, scar tissue flaps, or deep cervical fascial flaps for the reconstruction of cervical scar were simple and effective. The use of cervical scar tissue flaps could not only loose the constructed scar, but also beneficial in rectifying the configuration of the chin.

Adolescent ; Adult ; Burns ; complications ; Chin ; surgery ; Cicatrix ; etiology ; surgery ; Contracture ; etiology ; surgery ; Female ; Follow-Up Studies ; Humans ; Male ; Neck ; surgery ; Reconstructive Surgical Procedures ; methods ; Treatment Outcome ; Young Adult

The aim of this study is to establish the method of loop-mediated indirect PCR assay for detection of Reproductive and Respiratory Syndrome Virus (PRRSV) infection and differentiation of highly pathogenic PRRSV (HP-PRRSV) and lowly pathogenic PRRSV (LP-PRRSV). Based on the alignments of ORF2 gene sequences and ORFla gene sequences of PRRSV Chinese isolates deposited in GenBank, two pairs of specific probes were designed and labeled to both ends of the soybean Lectin gene fragment by PCR, respectively. The probe-labeled soybean Lectin genes were used to be reporter genes for detection and differentiation of PRRSV. After one round strand displacement reaction, the reporter genes were amplified by reverse PCR. The specific PCR products were 193bp, 355bp for HP-PRRSV and 193bp, 442bp for LP-PRRSV, respectively. The method could detect 5. 6 TCID50/mL LP-PRRSV RNA and 18 TCIDs0/ mL HP-PRRSV RNA, and co-infection did not affect detection sensitivity. No amplification was observed with other porcine originated pathogens including CSFV, PPV, PRV, PCV2, ETEC and Haemophilus parasui. Twenty clinical samples were used for comparative testing with conventional PCR. Fourteen samples were found positive for PRRSV by the loop-mediated indirect PCR, of which 4 were LP-PRRSV, 9 HP-PRRSV and 1 LP/HP-PRRSV co-infection, consistent with the conventional PCR test results. In conclusion, the loop-mediated indirect PCR is a simple, rapid, sensitive and specific etiologic diagnosis tool, and suitable for the differential diagnosis of HP/LP-PRRSV, especially for identification of mixed infection of HP/LP-PRRSV.
Animals ; Coinfection ; veterinary ; DNA Primers ; genetics ; DNA, Complementary ; genetics ; Diagnosis, Differential ; Genes, Reporter ; Genetic Markers ; genetics ; Humans ; Porcine Reproductive and Respiratory Syndrome ; diagnosis ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; isolation & purification ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; veterinary ; Sensitivity and Specificity ; Swine ; Time Factors ; Viral Proteins ; genetics

Objective:This study aimed to construct a lentiviral expression vector for microRNA-194 and investigate its effect on the metastasis of human osteosarcoma cell line U2-OS. Methods:Pri-and mature miR-194 amplified by PCR were inserted into the plenty-GFP vector and identified by restriction endonuclease digestion and nucleotide sequencing. The osteosarcoma cell line U2-OS was transfected with the lentivirus. Then, the stable transfected cells were used in Transwell and wound healing assay. Results:Restric-tion analysis and sequencing showed that the recombinant lentiviral expression vector was constructed correctly. The titers of obtained overexpression and suppression expression recombinant lentivirus were 1.5*108 and 4*108 TU/ml. Cell metastasis ability was signifi-cantly different in different experimental groups (P<0.01). Conclusion:The lentiviral expression vector for microRNA-194 was suc-cessfully constructed. MicroRNA-194 could influence the metastasis of the osteosarcoma cell line U2-OS;thus, it could be further ex-plored as a potential target in osteosarcoma therapy.