Variations outside the conserved motifs of PB1 catalytic active site may affect replication efficiency of the RNP complex of influenza A virus.

PB1 functions as the catalytic subunit of influenza virus RNA polymerase complex and plays an essential role in viral RNA transcription and replication. To determine plasticity in the PB1 enzymatic site and map catalytically important residues, 658 mutants were constructed, each with one to seven mutations in the enzymatic site of PB1. The polymerase activities of these mutants were quantified using a minigenome assay, and polymerase activity-associated residues were identified using sparse learning. Results showed that polymerase activities are affected by the residues not only within the conserved motifs, but also across the inter-motif regions of PB1, and the latter are primarily located at the base of the palm domain, a region that is conserved in avian PB1 but with high sequence diversity in swine PB1. Our results suggest that mutations outside the PB1 conserved motifs may affect RNA replication and could be associated with influenza virus host adaptation.

Effect of luteolin on apoptosis, MAPK and JNK signaling pathways in guinea pig chondrocyte with osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease usually seen in the elderly, which incidence increases with age. Its pathogenesis and underlying mechanism are still unclear. The disease severely affects the physical health and life quality of patients, thereby constituting a huge economic burden to family and society. Luteolin (LUT) is a natural flavonoid with multiple pharmacological properties. Many plants containing LUT have been applied in the treatment of several inflammation-related diseases due the relatively strong anti-inflammatory effects of LUT. The present study investigated the influence of LUT on cell apoptosis and inflammatory reactions in cartilage of OA guinea pigs, and its underlying mechanism. It was found that LUT effectively inhibited proliferation of OA cartilage cells, down-regulated the expressions of JNK and p38MAPK in cartilage cells of OA, and downregulated NO, TNF-α and IL-6. Thus, it alleviated inflammatory reactions, protected cartilage cells, and delayed cartilage degeneration.

Effects of ginsenoside Rb1 on spinal cord ischemia-reperfusion injury in rats.

BACKGROUND: The aim of this study was to evaluate the effects of different doses of ginsenoside Rb1 (GRb1) pretreatment on spinal cord ischemia-reperfusion (SCII) in rats and explore the potential mechanisms about the expression of survivin protein after the intervention. METHODS: A total of 90 healthy adult Sprague-Dawley (SD) rats were randomly divided into six groups: sham-operated (n = 15), SCII model (n = 15), and GRb1-treated groups (n = 60). The GRb1-treated group was divided into four subgroups: 10 mg/kg, 20 mg/kg, 40 mg/kg, and 80 mg/kg (n = 15). The corresponding dose of GRb1 was injected intraperitoneally 30 min before operation and every day after operation. Forty-eight hours after model establishment, the neurological function of hind limbs was measured with Basso, Beattie, and Bresnahan (BBB) scale. The superoxide dismutase (SOD) and malondialdehyde (MDA) levels in serum and spinal cord tissue were detected respectively. The expression of survivin protein was observed by immunofluorescence staining. HE and TUNEL staining were used to observe neural cell injury and apoptosis, respectively, in the spinal cord of rats with SCII. RESULTS: The intervention of different doses of GRb1 could increase SOD activity and decrease MDA content in serum and spinal cord tissue, increase survivin protein expression, and decrease neuronal apoptosis. It was dose-dependent, but there was no significant change between 40 mg/kg and 80 mg/kg. CONCLUSIONS: GRb1 could reduce the cell apoptosis induced by SCII through inhibiting oxidative stress. It can also inhibit apoptosis by promoting the expression of Survivin protein. Ginsenoside Rb1 had a dose-dependent protective effect on SCII in the dose range of 10 mg/kg-40 mg/kg.

The hsa-miR-181a-5p reduces oxidation resistance by controlling SECISBP2 in osteoarthritis.

BACKGROUND: The phenotypes of osteoarthritis (OA) consist of cartilage extracellular matrix (ECM) metabolism disorder and the breakdown of cartilage homeostasis, which are induced by pro-inflammatory factors and oxidative stress. Selenoproteins regulated by selenocysteine insertion sequence binding protein 2 (SBP2) are highly effective antioxidants, but their regulatory mechanisms, particularly the involvement of miRNAs, are not fully understood. METHODS: To explore whether miR-181a-5p and SBP2 are involved in OA pathogenesis, we established an IL-1ß model using the chondrocyte SW1353 cell line. Next, we up- or down-regulated SBP2 and miRNA-181a-5p expression in the cells. Finally, we measured the expression of miRNA-181a-5p, SBP2 and three selenoproteins in OA cartilage and peripheral blood. RESULTS: The results showed that IL-1ß increased hsa-miR-181a-5p and decreased SBP2 in a time- and dose-dependent manner. GPX1 and GPX4, which encode crucial glutathione peroxidase antioxidant enzymes, were up-regulated along with SBP2 and miR-181a-5p. Furthermore, SBP2 showed a significant negative correlation with miR-181a-5p during induced ATDC5 cell differentiation. There was lower GPX1 and GPX4 mRNA expression and SBP2 protein expression in damaged cartilage than in smooth cartilage from the same OA sample, and hsa-miR-181a-5p expression on the contrary. Similar results were observed in peripheral blood. In conclusion, we have reported a novel pathway in which pro-inflammatory factors, miRNA, SBP2 and selenoproteins are associated with oxidation resistance in cartilage. CONCLUSION: Overall, this study provides the first comprehensive evidence that pro-inflammatory factors cause changes in the cartilage antioxidant network and describes the discovery of novel mediators of cartilage oxidative stress and OA pathophysiology. Our data suggest that miR-181a-5p may be used to develop novel early-stage diagnostic and therapeutic strategies for OA.

Management of patellar fracture with titanium cable cerclage.

Early rehabilitation after surgery for patellar fracture is challenging. The purpose of this study was to evaluate the surgical outcome of titanium cable cerclage for patellar fracture in early functional activity.We reviewed a series of 24 patients treated at our hospital with titanium cable. Functional exercises were started early. Patients were followed up for at least 12 months.Fifteen were males and 9 were females. Fracture occurred in the right knee in 13 patients and in the left knee in 11 patients. The most common mode of injury involves a tumble. None of the patients presented with any postoperative complications. The management resulted in satisfactory outcomes.Titanium cable cerclage offers a new strategy in treating patellar fracture.

The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).

Identification of the source of A (H10N8) virus causing human infection.

A novel H10N8 influenza A virus has been detected in three humans in China since December 2013. Although this virus was hypothesized to be a novel reassortant among influenza viruses from wild birds and domestic poultry, its evolutionary path leading to human infection is unknown. Sporadic surveillance at the live poultry market (LPM) suspected to be the source of infection for the first H10N8 patient has shown a gradual increase in influenza virus prevalence culminating with a predominance of H10N8 viruses. Influenza viruses detected in the LPM up to 8 months prior to human infection contributed genetic components to the zoonotic virus. These H10N8 viruses have continued to evolve within this LPM subsequent to the human infection, and continuous assessments of these H10N8 viruses will be necessary. Serological surveillance showed that the virus appears to have been present throughout the LPM system in Nanchang, China. Reduction of the influenza virus burden in LPMs is essential in preventing future emergence of novel influenza viruses with zoonotic and pandemic potential.

Comparative analysis of a highly variable region within the genomes of Spodoptera frugiperda ascovirus 1d (SfAV-1d) and SfAV-1a.

The recently discovered ascoviruses have a worldwide distribution. Here we report a new member of the family Ascoviridae, Spodoptera frugiperda ascovirus 1d (SfAV-1d) with a variable region in the genome. Restriction fragment length polymorphism, Southern hybridization and genome sequencing analyses confirmed that SfAV-1d and the earlier reported SfAV-1a are closely related but are not identical. The genome size of SfAV-1d is approximately 100 kbp, which is about 57 kbp smaller than SfAV-1a. The SfAV-1d genome has a major deletion of 14 kbp that corresponds to one of the inverted repeat (IR) regions of SfAV-1a. Cloning and sequencing revealed that the region flanking the deletion within the SfAV-1d genome is highly variable. In all the variants of this region, the whole IR region is missing, with 88.2 % of the variants missing part of or the whole adjacent SfAV-1a ORF71, 94.1 % missing part of or the whole of adjacent ORF72 and 64.6 % missing part of or the whole of ORF73.

Using host 28S ribosomal RNA as a housekeeping gene for quantitative real-time reverse transcription-PCR (qRT-PCR) in virus-infected animal cells.

The use of quantitative real-time reverse transcription-PCR (qRT-PCR) for studying regulation of gene transcription requires an internal template-loading control or a housekeeping gene to guarantee the validity of the data collection, analysis, and interpretation. Analysis of gene transcription in virus-infected animal cells is problematic because virus infection often results in modified or fluctuating gene transcription patterns of conventionally used housekeeping genes, such as the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene and the ß-actin gene. It has been demonstrated that the host 28S ribosomal gene can be used as a housekeeping gene in qRT-PCR in virus-infected insect cells. The stability of the human 28S rRNA gene transcription during the infection of HeLa cells with adenovirus has been confirmed, and this method has been extended to the use of the human 28S rRNA gene as a housekeeping gene in adenovirus-infected HeLa cells. Step-by-step instructions are described for use of this control in analysis of gene transcription in both types of virus-infected animal cells.

Strategy of the use of 28S rRNA as a housekeeping gene in real-time quantitative PCR analysis of gene transcription in insect cells infected by viruses.

Quantitative real-time reverse transcription-PCR (qRT-PCR) has been used widely to measure gene transcription regulation in cells. qRT-PCR must include one or more internal housekeeping genes to normalize data collection. A strategy to use the host cell 28S rRNA as a housekeeping gene in qRT-PCR analysis of gene transcription of insect cells infected by baculovirus and ascovirus was developed. It has been found that the 28S rRNA reverse primer can be incorporated in the oligo-dT-primed cDNA synthesis reaction. In such a way, amplification of 28S cDNA showed lower and less variable cycle thresholds in cells infected by viruses than by using only oligo-dT and other published housekeeping genes such as the TATA box binding protein (TBP) gene, the peptidyl prolyl isomerase A (PPI) gene and the ribosomal protein 13 (L13) gene. Incorporation of the 28S reverse primer in oligo-dT-primed cDNA synthesis also does not interfere with the detection of other polymerase II transcribed genes.

[Operative outcomes of complex acetabular fractures and its influence factors].

OBJECTIVE: To evaluate the results of operative treatment of complex acetabular fractures and to investigate its influence factors. METHODS: From June 2000 to August 2006, 54 patients with complex acetabular fractures were treated, including 44 males and 10 females aged 20-75 years old (average 39.1 years old). Fractures were due to traffic accident in 40 cases, falling from high places in 8 cases and crush by heavy objects in 6 cases. All cases were fresh and close fractures and the time from injury to operation was 5-72 days. There were 5 cases of posterior column and posterior wall fracture, 25 of transverse and posterior wall fracture, 2 of T-type fracture, and 22 of double column fracture. During operation, Kocker-Lagenbach approach was used in 23 cases, anterior ilioinguinal approach was applied for 3 cases and the combination of anterior and posterior approaches was performed on 28 cases. AO reconstructive plate and screw internal fixation were used in all the cases. RESULTS: Fifty-two cases were followed up for 12-74 months (average 31.3 months). Anatomical reduction was achieved in 23 cases, satisfactory reduction in 19 cases, poor reduction in 10 cases, and the excellent and good rate reached 80.77%. During operation, 1 case suffered from a tear in the external iliac vein and healed after vein repair; 2 cases had sciatic nerve injury and took mecobalamin as oral administration, one of them fully recovered, and the other had incomplete recovery at 18-month follow-up. At the final follow-up, there were 6 cases of severe heterotopic ossification, one of them received heterotopic bone resection and the rest 5 patients received conservative treatment; there were 9 cases of traumatic osteoarthritis, one of them received total hip replacement and the rest 8 patients received conservative treatment; there were 5 cases of avascular necrosis of the femoral head, two of them received total hip replacement, 1 received no further treatment because the femoral head didn't collapse, and the rest 2 patients gave up total hip replacement; 75.00% patients were graded as excellent and good according to the modified Merled'Aubigné-Postel hip score system. Patients' quality of life was compared with local population norms matched for age and sex by using SF-36 scales, their overall score were below the local population norms, and their general health, vitality, role limitation due to emotional problems and mental health were comparable to the local population norms. Logistic regression analysis revealed the time to reduce hip dislocation, quality of fracture reduction and traumatic arthritis were independent risk factors affecting postoperative functional outcomes. CONCLUSION: Applying open reduction and internal fixation in the treatment of displaced complex acetabular fractures has a satisfying therapeutic effect. Time to reduce hip dislocation, quality of fracture reduction as well as traumatic arthritis are independent risk factors affecting postoperative functional outcomes.

Sequence and organization of the Trichoplusia ni ascovirus 2c (Ascoviridae) genome.

The complete Trichoplusia ni ascovirus 2c (TnAV-2c) genome sequence was determined. The circular genome contains 174,059 bp with 165 open reading frames (ORFs) of greater than 180 bp and two major homologous regions (hrs). The genome is quite A+T rich at 64.6%. Fifty-four ORFs had homologues in other insect viruses, such as ascoviruses, iridoviruses, baculoviruses and entomopoxviruses; 30 ORFs showed low identities with those from different parasitic protozoa and 12 ORFs were unique to TnAV-2c. TnAV-2c has 15 ORFs that could be grouped into six gene families. Three major conserved repeating sequences were identified and were interspersed in two regions. BLAST analyses revealed that there were 16 enzymes involved in gene transcription, DNA replication, and nucleotide metabolism. TnAV-2c has 12 and 25 ORFs sharing high identities with ascovirus and iridovirus homologues, respectively. The codon usage bias appears to be more similar to Spodoptera frugiperda ascovirus 1a than to iridoviruses.