To gain a better understanding of the genetic diversity and evolution of PRRSV in the Ningxia Hui Nationality Autonomous Region (Ningxia) of China, the nsp2 genes from a series of PRRSV strains collected from the region in 2007 were partially sequenced. These sequences were then analyzed along with the classical strain (ch-la) and two other epidemic strains SD (3) and SD2006. Comparison of the nucleotide sequence with ch-la indicated that nsp2 genes of seventeen Ningxia isolates (NX strain) have deletions of 87 nucleotides. Sequence analysis indicated that homology between the Ningxia strain and ch-la was 60.3%-79.9% in the nucleotide sequence, and homology between the NX strains and SD strains was 80.3%-98.8% in the nucleotide sequence. The nsp2 genes of the seventeen isolates had 74.9%-100% nucleotide sequence identities with each other. This study was undertaken to assess the regional variation of prevalent PRRSV and to establish a sequence database for PRRSV molecular epidemiological studies.

Objective To investigate the effect of microRNA(miR)-21 on proliferation and differentiation of murine pulmonary fibroblasts. Methods C57BL/6 mice of SPF grade (n=24) were randomly divided into Sham group and Pulmo?nary fibrosis model group with 12 mice in each group. Pulmonary fibrosis model was established by trans-tracheal jet ventila?tion of bleomycin into mice. The transcription levels of miR-21 were examined by quantitative real-time PCR in various pul?monary fibrosis tissues. Primary fibroblast were isolated and digested by Trypsin then inoculated into 6 well plate to reach confluence of 30%-50%. PBS (2.5μL), negative control stock solution and miR-21 mimic stock solution (20μmol/L) were added into Opti-MEM (50μL) as control group, blank group and miR-21 mimic group respectively.The cell viability was as?sessed by CCK-8. Expressions of ADAMTS-1 and TGF-β1 in the pulmonary fibroblasts were tested using Western blot. Re?sults The expression of miR-21 was significantly increased in lungs of mice in pulmonary fibrosis model group than that in sham group. Expression of miR-21 was higher in miR-21 mimic group than that in control group and blank group. Expres?sion of miR-21 was significantly higher with better cell viability in miR-21 mimic group than that in control group and blank group. The expression of ADAMTS-1 was significantly decreased in miR-21mimic group, while the expression of TGF-β1, a target gene of miR-21, was significantly increased in miR-21 mimic group compared with the other two groups. There is no significant different in expressions of ADAMTS-1 and TGF-β1 between control group and blank group. Conclusion Over?expression of miR-21 in pulmonary fibroblasts disrupts TGF-β1 signaling pathway by reducing expression of ADAMTS-1, which promotes the proliferation and differentiation of pulmonary fibroblast.

STUDY DESIGN: Retrospective series. PURPOSE: Assess results of decompression-only surgery for low-grade degenerative spondylolisthesis with consideration of instability. OVERVIEW OF LITERATURE: There is no consensus on whether fusion or decompression-only surgery leads to better outcomes for patients with low-grade degenerative spondylolisthesis. Current trends support fusion but many studies are flawed due to over-generalization without consideration of radiological instability and their variable presentations and natural history. METHODS: Patients with surgically treated degenerative spondylolisthesis from 1990-2013 were included. Clinical and radiological instability measures were included. Any residual or recurrence of symptoms, revision surgery performed and functional outcome scores including the numerical global rate of change scale, visual analogue scale, and modified Barthel index were measured. Follow-up periods for patients were divided into short-term (<5 years), mid-term (5-10 years) and long-term (>10 years). RESULTS: A total of 64 patients were recruited. Mechanical low back pain was noted in 48 patients and most (85.4%) had relief of back pain postoperatively. Radiological instability was noted in 4 subjects by flexion-extension radiographs and 12 subjects with prone traction radiographs by increased disc height and reduction of olisthesis and slip angle. From the results of the short-term, mid-term and long-term follow-up, reoperation only occurred within the first 5-year follow-up period. All functional scores improved from preoperative to postoperative 1-year follow-up. CONCLUSIONS: Decompression-only for low-grade degenerative spondylolisthesis has good long-term results despite instability. Further higher-level studies should be performed on this patient group with radiological instability to suggest the superior surgical option.
Back Pain ; Consensus ; Decompression* ; Follow-Up Studies ; Humans ; Low Back Pain ; Natural History ; Recurrence ; Reoperation ; Retrospective Studies ; Spondylolisthesis* ; Traction

Background: Malnutrition is a common and modifiable risk factor for postoperative complications and adverse outcomes in orthopedics. The purpose of this study was to identify biomarkers of malnutrition in patients undergoing elective total knee arthroplasty (TKA) that are predictive of adverse in-hospital postoperative complications, to facilitate the identification of at-risk patients for nutritional optimization before surgery. Methods: A total of 624 patients who underwent elective TKA between 2013 and 2017 were evaluated; potential biomarkers of preoperative malnutrition, including hypoalbuminemia (serum albumin < 3.5 g/dL), total lymphocyte count (TLC < 1500 cells/mm3), and body mass index (BMI), were assessed for any association with in-hospital postoperative complications. Results: The prevalence of hypoalbuminemia, low TLC, overweight, obesity class I, and obesity class II were, respectively 2.72%, 33.4%, 14.8%, 44.5%, and 26.9%. There was a significant association between hypoalbuminemia and obesity class II (BMI ≥ 30.0 kg/m2) with rates of peri-prosthetic joint infection, and no significant association between such complications and low TLC, overweight, or obesity class I. Logistic regression analysis showed that patients with hypoalbuminemia or being in obesity class II with gouty arthritis were more likely to suffer from peri-prosthetic joint infection. Conclusions Hypoalbuminemia and obesity class II together is a reliable biomarker of preoperative malnutrition for predicting peri-prosthetic joint infection after elective TKA, whereas low TLC, overweight, and obesity class I were not significantly associated with an increased risk of such complications.

OBJECTIVETo study the effects of insulin on the proteolysis of cultured rabbit skeletal muscular myotubes in vitro, and their possible mechanisms.

METHODSMuscles of lower limbs of juvenile rabbits were isolated for tissue-block culture. After passage, myoblasts were formed and fused into myotubes. Then the protein in myotubes was radiolabelled with L-[ 3,5-3H] tyrosine. The myotubes were cultured in DMEM medium containing 100 nmol/L insulin (n = 24, group B) , 100 nmol/L dexamethasone (n = 24, group C) , 100 nmol/L insulin and 100 nmol/L dexamethasone (n = 24, group D) , no insulin or dexamethasone (n =24, group A), respectively. Twenty-four hours after culture, the L-[3,5-3H] tyrosine content in culture medium and cells were determined, and the degradation rates of protein were calculated. The mRNA expressions of ubiquitin and protease C2 subunit were determined by Northern blot.

RESULTSThe degradation rates of myotube protein in group A(0. 38+/-0.04) was obviously lower than that in group C (0.50+/-0.03, P <0.01), but it was obviously higher than that in group B(0. 35+/-0.03, P <0.05). Though the degradation rates of myotube protein in group D (0.41+/-0. 03) was evidently lower than that in group C ( P < 0.01) , it was still higher than that in group A( P < 0.05 ). The mRNA expressions of ubiquitin and protease C2 subunit in group A ( the scale: 2. 4 kb ubiquitin was 0. 82+/-0. 15, 1. 2 kb ubiquitin was 0. 60+/-0. 10, C2 subunit was 0. 75+/-0. 16) was obviously lower than that in group C ( the scale: 2.4 kb ubiquitin was 2. 15+/-0. 23, 1.2 kb ubiquitin was 1.50+/-0. 14,C2 subunit was 1.50+/-0. 13 , P <0. 01) , but it in group D was lower than that in group C (the scale: 2. 4 kb ubiquitin was 1. 25+/-0. 17, 1. 2 kb ubiquitin was 0. 85+/-0. 09, C2 subunit was 0. 90+/-0. 15, P <0. 01) , and it was similar to that in group B (the scale: 2.4 kb ubiquitin was 0. 85+/-0.07, 1.2 kb ubiquitin was 0. 65+/- 0. 12, C2 subunit was 0. 76 +/-0. 09, P > 0. 05).

CONCLUSIONThe effects of insulin on the activity of ubiquitin-proteasome pathway and the proteolytic rate in normal myotubes were relatively weak. However, insulin can significantly inhibit the effects of dexamethasone on the gene expressions of ubiquitin system and the proteolytic rate in myotubes, but the mechanism needs further research.

Animals ; Cells, Cultured ; In Vitro Techniques ; Insulin ; pharmacology ; Male ; Muscle Fibers, Skeletal ; drug effects ; metabolism ; Muscle Proteins ; metabolism ; Rabbits ; Ubiquitin ; metabolism

BACKGROUND:Previous studies on immunosuppression and anti-rejection after organ transplantation mainly focused on effects of T lymphocytes-mediated immune response and immunosuppressive agents on T lymphocytes. Effects of dendritic cel s were unclear. The manifestation and mechanism of immunosuppressive agent effects on dendritic cel s are not identical. OBJECTIVE:To compare the effects of different immunosuppressive agents on expression and function of costimulatory molecules of dendritic cel s, and to explore the mechanism of action of immunosuppressive agents. METHODS:20μg/L rapamycin, 0.04 mg/L mycophenolate, 10μg/L tacrolimus and 1 mg/L cyclosporine A were separately added during bone marrow cel s of C57BL/6 mice were differentiated into dendritic cel s. RESULTS AND CONCLUSION:Flow cytometry results revealed that CD40 expression in each group:rapamycin0.05). One-way mixed lymphocyte reaction results displayed dendritic cel costimulatory T cel proliferation in each group:rapamycin

Background/Aims: Hepatitis B surface antigen (HBsAg) seroclearance remains uncommon in chronic hepatitis B (CHB) infection. During acute flares of CHB (AFOCHB), alanine aminotransferase elevation reflects a mounting immune response toward viral clearance. We hypothesized that severe AFOCHB is associated with a greater quantitative HBsAg (qHBsAg) decline and HBsAg seroclearance rate. Methods: A total of 75 patients with severe AFOCHB with alanine aminotransferase 10× the upper limit of normal were matched to a control group by age and sex in a 1:2 ratio. qHBsAg levels were measured at the time of flare and annually (for both cases and controls) until the last follow-up. Results: The median follow-up times for patients with severe AFOCHB and controls were 8.8 and 10.5 years, respectively. The cumulative rate of HBsAg seroclearance was higher in the severe AFOCHB group than in the control group (11.8% vs 5.0%, p=0.04) despite the former group having a trend of a higher baseline median qHBsAg (3,127 IU/mL vs 1,178 IU/mL, p=0.076). Compared with the control group, the severe AFOCHB group had a greater annual qHBsAg reduction (–242.4 IU/mL/yr vs –47.3 IU/mL/yr, p=0.002). Increasing age (p=0.049), lower baseline qHBsAg (p=0.002), and severe AFOCHB (p=0.014) were independently associated with HBsAg seroclearance. However, the cumulative rate of hepatocellular carcinoma was significantly higher in the severe AFOCHB group than in the control group (15.8% vs 1.9%, p<0.001). Conclusions Severe AFOCHB was associated with a greater incidence of HBsAg seroclearance and qHBsAg decline. However, it was associated with a higher incidence of hepatocellular carcinoma.

OBJECTIVEChanges of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum (ER). This ER function disorder is called endoplasmic reticulum stress (ERS). Severe long-term ERS can trigger the ER apoptosis signaling pathway, resulting in cell apoptosis and organism injury. Recent researches revealed that ERS-induced cell death was involved in the neurocyte retrogradation in the progress of neuron degenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease and so on. Therefore, the protection effect of the traditional Chinese drug-Tiantai No. 1 (1) on the ERS injury of AD was investigated at the molecular gene level in this study with a view to explore the gene pharmacodynamic actions and mechanisms of this drug.

METHODSPrimarily cultured marrow mesenchymal stem cells (MSCs) of rats were treated by tunicamycin (TM) in order to induce ERS. RT-PCR, fluorescence immunocytochemistry and Western blot techniques were used to determine the mRNA and protein expression levels of the protective stress protein-ER molecular chaperones GRP78 and GRP94 (which would assist cells to resist cellular stress injury), and to determine the mRNA and protein expression levels of apoptosis promoting molecule Caspase-12 on the membrane of the ER, respectively.

RESULTSProtein expression levels of GRP78 and GRP94 were significantly increased in the TM-induced MSCs, and the mRNA level of Caspase-12 was also remarkably increased in the TM-induced MSCs (P<0.05). All these proved that the ERS model was successfully established by TM in MSC. Meanwhile, the mRNA and protein levels of GRP78 and GRP94 were all significantly increased compared with the model group (P<0.05 or P<0.01) after MSCs were treated with Tiantai No.1 while the mRNA and protein expression levels of Caspase-12 were significantly decreased compared with the model group (P<0.05 or P<0.01). This effect showed a dose dependent manner.

CONCLUSIONTiantai No.1 might attenuate the cell apoptosis induced by ERS injury, and thus protect the neurons against AD.

Animals ; Anti-Bacterial Agents ; antagonists & inhibitors ; pharmacology ; Cells, Cultured ; Drug Antagonism ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Heat-Shock Proteins ; genetics ; metabolism ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; RNA ; analysis ; drug effects ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological ; drug effects ; genetics ; Tunicamycin ; antagonists & inhibitors ; pharmacology

To investigate the non-viral vector mediating human coagulation factor VIII gene expression in mouse 32D cell line, a recombinant plasmid vector, pRC/RSV-hFVIIIBDcDNA, was constructed by cloning B-domain-deleted (Delta760aa-1639aa) human factor VIII cDNA (hFVIIIBDcDNA) into plasmid vector, pRC/RSV. The plasmid RC/RSV-hFVIIIBDcDNA was then transfected by means of SuperFect Transfection Reagent into mouse 32D cell line. After screening with G418, the procoagulant activity (hFVIII:C) and antigen (hFVIII:Ag) of human factor VIII in the culture medium were detected using one-stage method and ELISA, respectively. Furthermore, RT-PCR was performed to observe the transcription of hFVIIIBDcDNA. The results showed that human coagulation factor VIII protein existed in culture medium with hFVIII:C up to 2.01 U/(10(6) cell x 24 hours) and hFVIII:Ag to 450.08 ng/(10(6) cell x 24 hours). RT-PCR displayed mRNA of hFVIIIBDcDNA in 32D cells. It is concluded that the recombinant plasmid RC/RSV-hFVIIIBDcDNA can successfully express human FVIII in mouse 32D cell line, and hFVIII expressed in vitro presents the similar coagulant activity to the native hFVIII existing in normal human plasma.
Animals ; Cell Line ; DNA, Complementary ; genetics ; Enzyme-Linked Immunosorbent Assay ; Factor VIII ; genetics ; metabolism ; Gene Expression Regulation ; Humans ; Mice ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVEElectron microscopical study of infected cells to identify the pathogenic agent of SARS.

METHODSVero E6 cells infected with lung autopsy samples or nasopharyngeal swabs from SARS patients of Beijing and Guangzhou were inoculated. The supernatant and cultured cells exhibiting identifiable cytopathic effect (CPE) were prepared for electron microscopic study.

RESULTSExamination of CPE cells on thin-section revealed characteristic coronavirus particles within the cisternae of endoplasmic reticulum, Golgi apparatus, vesicles and extracellular space. They were mainly spherical or oval in shape, annular or dense, about 80 nm in diameter. Negative-stain electron microscopy identified coronavirus particles in culture supernatant, 80 - 120 nm in diameter, with club-shaped surface projections. Elongated, rod-, kidney- or other irregular shaped virons with the size of 100 - 200 nm by 60 - 90 nm were also found in the cultured cells infected with the lung samples from the Guangdong patients. Infectious virons entered cells by endocytosis or membrane fusion and released through a budding process.

CONCLUSIONThese data indicate a novel coronavirus as the causative agent of SARS. Most viral particles showed typical characteristics of coronavirus. The potential role of special shape viruses is expected to be further investigated.

Animals ; Cercopithecus aethiops ; Humans ; Microscopy, Electron ; SARS Virus ; ultrastructure ; Severe Acute Respiratory Syndrome ; virology ; Vero Cells