Identification of Two Linear Epitopes on MGF_110-13L Protein of African Swine Fever Virus with Monoclonal Antibodies.

African swine fever caused by African swine fever virus (ASFV) is an acute, highly contagious swine disease with high mortality. To facilitate effective vaccine development and find more serodiagnostic targets, fully exploring the ASFV antigenic proteins is urgently needed. In this study, the MGF_110-13L was identified as an immunodominant antigen among the seven transmembrane proteins. The main outer-membrane domain of MGF_110-13L was expressed and purified. Two monoclonal antibodies (mAbs; 8C3, and 10E4) against MGF_110-13L were generated. The epitopes of two mAbs were preliminary mapped with the peptide fusion proteins after probing with mAbs by enzyme-linked immunosorbent assay (ELISA) and Western blot. And the two target epitopes were fine-mapped using further truncated peptide fusion protein strategy. Finally, the core sequences of mAbs 8C3 and 10E4 were identified as 48WDCQDGICKNKITESRFIDS67, and 122GDHQQLSIKQ131, respectively. The peptides of epitopes were synthesized and probed with ASFV antibody positive pig sera by a dot blot assay, and the results showed that epitope 10E4 was an antigenic epitope. The epitope 10E4 peptide was further evaluated as a potential antigen for detecting ASFV antibodies. To our knowledge, this is the first report of antigenic epitope information on the antigenic MGF_110-13L protein of ASFV.

Mammalian Cell-Line-Expressed CD2v Protein of African Swine Fever Virus Provides Partial Protection against the HLJ/18 Strain in the Early Infection Stage.

A cell line expressing the CD2v protein of ASFV was generated. The efficient expression of CD2v protein was determined by immunofluorescence and Western blotting. The CD2v protein was Ni-affinity purified from the supernatant of cell cultures. The CD2v-expressing cells showed properties of hemadsorption, and the secreted CD2v protein exhibited hemagglutinating activity. The antigenicity and immunoprotection ability of CD2v were evaluated by immunizing pigs alone, combined with a cell-line-expressed p30 protein or triple combined with p30 and K205R protein. Immunized pigs were challenged with the highly virulent ASFV strain HLJ/18. Virus challenge results showed that CD2v immunization alone could provide partial protection at the early infection stage. Protein p30 did not show synergistic protection effects in immunization combined with CD2v. Interestingly, immunization with the triple combination of CD2V, p30 and K205R reversed the protection effect. The viremia onset time was delayed, and one pig out of three recovered after the challenge. The pig recovered from ASFV clinical symptoms, the rectal temperature returned to normal levels and the viremia was cleared. The mechanism of this protection effect warrants further investigation.

Sodium glucose cotransporter-2-inhibitor dapagliflozin improves nonalcoholic fatty liver disease by ameliorating dipeptidyl-peptidase-4 protein expression in diabetic mice.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. It can progress from simple steatosis to nonalcoholic steatohepatitis and may even develop into liver fibrosis, hepatocirrhosis, or hepatocellular carcinoma, but there is no effective treatment. MATERIAL AND METHODS: Wild-type (wt) and diabetic (db/db) mouse NAFLD-induced models were used to investigate the hepatoprotective effects and potential mechanisms of dapagliflozin (a new oral hypoglycaemic drug) on type 2 diabetes mellitus (T2DM) complicated with NAFLD, and to establish wt and db/db mouse NAFLD-induced and dapagliflozin treatment models. RESULTS: Dapagliflozin reduces blood glucose, glycosylated haemoglobin, blood lipids, and serum transaminase levels in db/db mice and improves T2DM-related liver injury accompanied by NAFLD; the mechanism may be related to the decrease in dipeptidyl-peptidase-4 (DPP4) protein expression and improvement in liver enzymes. Further mechanism-related studies by our team revealed that dapagliflozin can also downregulate the expression of DPP4 proteins in the liver and reduce serum soluble DPP4 enzyme levels, thereby improving the hepatic steatosis and insulin resistance of NAFLD. CONCLUSION: Dapagliflozin may be an effective drug for the treatment of T2DM-induced NAFLD and NAFLD, providing a reliable laboratory basis and new treatment methods for the clinical treatment of NAFLD.

Comprehensive mapping of antigenic linear B-cell epitopes on K205R protein of African swine fever virus with monoclonal antibodies.

African swine fever virus causes an acute, highly contagious swine disease with high mortality, leading to enormous losses in the pig industry. The K205R, a nonstructural protein of African swine fever virus, is abundantly expressed in the cytoplasm of infected cells at the early stage of infection and induces a strong immune response. However, to date, the antigenic epitopes of this immunodeterminant have not been characterized. In the present study, the K205R protein was expressed in a mammalian cell line and purified using Ni-affinity chromatography. Furthermore, three monoclonal antibodies (mAbs; 5D6, 7A8, and 7H10) against K205R were generated. Indirect immunofluorescence assay and western blot results showed that all three mAbs recognized native and denatured K205R in African swine fever virus (ASFV)-infected cells. To identify the epitopes of the mAbs, a series of overlapping short peptides were designed and expressed as fusion proteins with maltose-binding protein. Subsequently, the peptide fusion proteins were probed with monoclonal antibodies using western blot and enzyme-linked immunosorbent assay. The three target epitopes were fine-mapped; the core sequences of recognized by the mAbs 5D6, 7A8, and 7H10 were identified as 157FLTPEIQAILDE168, 154REKFLTP160, and 136PTNAMFFTRSEWA148, respectively. Probing with sera from ASFV-infected pigs in a dot blot assay demonstrated that epitope 7H10 was the immunodominant epitope of K205R. Sequence alignment showed that all epitopes were conserved across ASFV strains and genotypes. To our knowledge, this is the first study to characterize the epitopes of the antigenic K205R protein of ASFV. These findings may serve as a basis for the development of serological diagnostic methods and subunit vaccines.

COL12A1 rs970547 Polymorphism Does Not Alter Susceptibility to Anterior Cruciate Ligament Rupture: A Meta-Analysis.

Objective: Currently available evidence regarding the association between collagen type XII α1 (COL12A1) polymorphism and risk of anterior cruciate ligament rupture (ACLR) remains elusive. The aim of our present study was to assess the association between COL12A1 rs970547 polymorphism and ACLR risk. Methods: Five online databases, namely, PubMed, EMBASE, ISI Web of Science, CENTRAL, and CNKI, were searched from their inception data up to December 2020 to identify relative observational studies. The methodological quality of each individual study was evaluated using the Newcastle-Ottawa Scale (NOS). The "model-free approach" was employed to estimate the magnitude of effect of COL12A1 rs970547 polymorphism on ACLR, and the association was expressed using odds ratio (OR) and its associated 95% confidence interval (95% CI). Subgroup analysis was performed by ethnicity and sex of included subjects. Results: Eight studies involving 1,477 subjects with ACLR and 100,439 healthy controls were finally included in our study. The methodological quality of included studies was deemed moderate to high based on NOS scores. The "model-free" approach suggested no genotype differences between ACLR and healthy control for the rs970547 polymorphism, but we still used the allele model to present the combined data. Under the random-effect model, there was no significant difference in the frequency of effecting allele between ACLR and control (OR: 0.91, 95% CI 0.77, 1.08; p = 0.28). Stratified analysis by sex and ethnicity also showed no difference in allele frequency. Conclusion: The findings of this current meta-analysis suggested that rs970547 was not associated with ACLR risk in male, female, and the overall population among Asians or Caucasians.

Role of Mutant TBP in Regulation of Myogenesis on Muscle Satellite Cells.

In polyglutamine (PolyQ) diseases, mutant proteins cause not only neurological problems but also peripheral tissue abnormalities. Among all systemic damages, skeletal muscle dystrophy is the severest. Previously by studying knock-in (KI) mouse models of spinal cerebellar ataxia 17 (SCA17), it was found that mutant TATA box binding protein (TBP) decreases its interaction with myogenic differentiation antigen, thus reducing the expression of skeletal muscle structural proteins and resulting in muscle degeneration. In this paper, the role of mutant TBP in myogenesis was investigated. Single myofibers were isolated from tibialis anterior muscles of wild type (WT) and SCA17KI mice. The 1TBP18 staining confirmed the expression of mutant TBP in muscle satellite cells in SCA17KI mice. In the BaCl2-induced TA muscle injury, H&E cross-section staining showed no significant change in myofibril size before and after BaCl2 treatment, and there was no significant difference in centralized nuclei between WT and SCA17KI mice, suggesting that mutant TBP had no significant effect on muscle regeneration. In the cultured primary myoblasts from WT and SCA17KI mice in vitro, representative BrdU immunostaining showed no significant difference in proliferation of muscle satellite cells. The primary myoblasts were then induced to differentiate and immunostained for eMyHC, and the staining showed there was no significant difference in differentiation of primary myoblasts between WT and SCA1KI mice. Our findings confirmed that mutant TBP had no significant effect on myogenesis.

Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B.

Signal peptidase complex subunit 1 (SPCS1) is a newly identified host factor that regulates flavivirus replication, but the molecular mechanism is not fully understood. Here, using Japanese encephalitis virus (JEV) as a model, we investigated the mechanism through which the host factor SPCS1 regulates the replication of flaviviruses. We first validated the regulatory function of SPCS1 in JEV propagation by knocking down and knocking out endogenous SPCS1. The loss of SPCS1 function markedly reduced intracellular virion assembly and the production of infectious JEV particles but did not affect cell entry, RNA replication, or translation of the virus. SPCS1 was found to interact with nonstructural protein 2B (NS2B), which is involved in posttranslational protein processing and virus assembly. Serial deletion mutation of the JEV NS2B protein revealed that two transmembrane domains, NS2B(1-49) and NS2B(84-131), interact with SPCS1. Further mutagenesis analysis of conserved flavivirus residues in two SPCS1 interaction domains of NS2B demonstrated that G12A, G37A, and G47A in NS2B(1-49) and P112A in NS2B(84-131) weakened the interaction with SPCS1. Deletion mutation of SPCS1 revealed that SPCS1(91-169), which contains two transmembrane domains, was involved in interactions with both NS2B(1-49) and NS2B(84-131). Taken together, these results demonstrate that SPCS1 affects viral replication by interacting with NS2B, thereby influencing the posttranslational processing of JEV proteins and the assembly of virions.IMPORTANCE Understanding virus-host interactions is important for elucidating the molecular mechanisms of virus propagation and identifying potential antiviral targets. Previous reports demonstrated that SPCS1 is involved in the flavivirus life cycle, but the mechanism remains unknown. In this study, we confirmed that SPCS1 participates in the posttranslational protein processing and viral assembly stages of the JEV life cycle but not in the cell entry, genome RNA replication, or translation stages. Furthermore, we found that SPCS1 interacts with two independent transmembrane domains of the flavivirus NS2B protein. NS2B also interacts with NS2A, which is proposed to mediate virus assembly. Therefore, we propose a protein-protein interaction model showing how SPCS1 participates in the assembly of JEV particles. These findings expand our understanding of how host factors participate in the flavivirus replication life cycle and identify potential antiviral targets for combating flavivirus infection.

Valproate attenuates diabetic nephropathy through inhibition of endoplasmic reticulum stress­induced apoptosis.

Previous studies have suggested that endoplasmic reticulum stress (ERS) is one of the mechanisms responsible for the pathogenesis of diabetic nephropathy (DN). Histone acetylation modification can regulate the transcription of genes and is involved in the regulation of ERS. Valproate (VPA), a nonselective histone deacetylase inhibitor, has been reported to have a protective role in kidney tissue injury, however, whether VPA can prevent DN remains to be elucidated. In the present study, it was found that VPA increases the expression of glucose­regulated protein (GRP78) and reduces the protein expression of C/EBP­homologous protein (CHOP), growth arrest and DNA­damage­inducible gene 153 and caspase­12 in a rat model of DN. VPA can reduce renal cell apoptosis and alleviate proteinuria and alterations in serum creatinine. VPA also upregulates the acetylation level of histone H4 in the promoter of GRP78 and downregulates the acetylation level of histone H4 in the promoter of CHOP. Collectively, the data indicate that VPA can relieve ERS and reduce renal cell apoptosis, and thus attenuate renal injury in a rat model of DN by regulating the acetylation level of histone H4 in ERS­associated protein promoters.

The time-dependent manner of sinusoidal electromagnetic fields on rat bone marrow mesenchymal stem cells proliferation, differentiation, and mineralization.

Electromagnetic fields (EMFs) are used clinically to promote fracture healing and slow down osteoporosis without knowledge of optimal parameters and underlying principles. In the present study, we investigate the effects of irritation for different durations with 15 Hz 1 mT sinusoidal EMFs (SEMFs) on rat bone marrow mesenchymal stem cells (BMSCs) proliferation, differentiation, and mineralization potentials. Our results show that SEMFs irritation promote rat BMSCs proliferation in a time-dependent manner, and the expression of osteogenic gen [Cbfa 1/RUNX2, bone sialoprotein (BSP), osteopontin (OPN)], alkaline phosphatase activity, and calcium deposition were enhanced after SEMFs treatment depending on the time duration of treatment. To determine the role of MEK/ERK signaling pathway, U0126, a MEK/ERK inhibitor was used. It can suppress rat BMSCs' proliferation with or without SEMF exposure, and partly attenuate the expression of osteogenesis related proteins (RUNX2, BSP, OPN) which were improved by SEMF. This finding suggests that the effects of SEMF on rat BMSCs' proliferation differentiation and mineralization are time duration dependent and MEK/ERK signaling pathway plays important role.

Retrospective comparison of H-graft and posterior vertebral graft procedures for the treatment of thoracolumbar burst fractures.

A retrospective cohort study was conducted to compare fusion rates achieved by H-grafts, posterior vertebral grafts, and no graft for the surgical treatment of thoracolumbar fractures. Ninety-two patients were included in this study. The patients fell into 1 of 3 groups: those who received H-grafts (n=36), posterior vertebral grafts (n=30), and no graft (n=26). Mean follow-up was 38 months (range, 24-51 months). All operations were performed by a single senior surgeon. All patients underwent operative treatment with posterior reduction and instrumentation. Radiographic parameters, estimated blood loss, operative time, and length of hospital stay were compared among patients in the 3 graft groups. Differences were assessed using unpaired t tests. P values <.05 were considered significant. We found no significant difference among groups in age, fracture location, or type of fracture. Patients who received H-grafts or posterior vertebral grafts achieved solid fusion, but spontaneous fusion occurred in only 2 patients who received no bone graft. Most patients with neurological deficits showed significant neurological improvement. Operative time and estimated blood loss were significantly lower in the no-graft group than in the H-graft and posterior vertebral graft groups (P<.05). Mean loss of correction, operative time, and estimated blood loss were lower for patients who received H-grafts than for those who received posterior vertebral grafts (P<.05). The use of an atlas fixation system in combination with a posterior H-graft for the treatment of thoracolumbar fracture is a stable and reliable method that effectively prevents inner fixation failure and reduces bone loss and anisotropy.

Effects of mutant huntingtin on mGluR5-mediated dual signaling pathways: implications for therapeutic interventions.

Glutamate excitotoxicity is thought to play an important role in Huntington's disease (HD), which is caused by a polyglutamine expansion in the HD protein huntingtin (htt). Overactivation of group I metabotropic glutamate receptors (mGluRs), which include mGluR1 as well as mGluR5 and are coupled via phospholipase C to the inositol phosphate pathway, is found to be involved in mutant htt-mediated neurotoxicity. However, activation of mGluR5 also leads to neuronal protection. Here, we report that mutant htt can activate both mGluR5-mediated ERK and JNK signaling pathways. While increased JNK signaling causes cell death, activation of ERK signaling pathway is protective against cell death. Expression of mutant htt in cultured cells causes greater activation of JNK than ERK. These findings suggest that selective inhibition of the JNK signaling pathway may offer an effective therapeutic approach for reducing htt-mediated excitotoxicity.

Simultaneous determination of hydroquinone and catechol at PASA/MWNTs composite film modified glassy carbon electrode.

A poly-amidosulfonic acid and multi-wall carbon nanotubes composite (PASA/MWNTs) modified electrode has been constructed by electropolymerization on glassy carbon electrode (GCE). The electrochemical behaviors of hydroquinone (HQ) and catechol (CC) were investigated using cyclic and differential pulse voltammetries (DPVs) at the prepared electrode. Separation of the reductive peak potentials for HQ and CC was about 120 mV in pH 6.0 phosphate buffer solution (PBS), which makes it suitable for simultaneous determination of these compounds. In the presence of 1.0 x 10(-4)mol L(-1) isomer, the reductive peak currents of DPV are proportional to the concentration of HQ in the range of 6.0 x 10(-6) to 4.0 x 10(-4)mol L(-1), and to that of CC in the range of 6.0 x 10(-6) to 7.0 x 10(-4)mol L(-1). When simultaneously changing the concentration of both HQ and CC, the linear concentration range of HQ (or CC) is 6.0 x 10(-6) to 1.0 x 10(-4)mol L(-1) (or 6.0 x 10(-6) to 1.8 x 10(-4)mol L(-1)), and the corresponding detection limits are 1.0 x 10(-6)mol L(-1). The proposed method has been applied to simultaneous determination of HQ and catechol in water sample, and the results are satisfactory.

[Effects of exercise training during 21 d -6 degrees head down bed rest on dynamic posture equilibrium and motor coordination].

OBJECTIVE: To study the effects of 21 d -6 degrees head down bed rest (HDBR) with exercise training on dynamic posture equilibrium and motor coordination. METHOD: Ten healthy young men were randomly divided into HDBR control groups and HDBR exercise groups, with 5 in each group. The subjects in the exercise group performed exercise with a bicycle ergometer in the supine position 1 h/d during bed rest. Dynamic posture equilibrium as well as isokinetic concentric muscular strength were determined before and after 21 d HD-BR. RESULT: After HDBR, average dynamic proprioception score and dynamic motor coordination decreased significantly, relative peak torque (peak torque /body weights PT/BW) also decreased significantly and accompanied with increase of the peak torque ratio between hamstrings and quadriceps (H/Q) in HDBR control groups. Average dynamic proprioception score and dynamic motor coordination in the HDBR exercise groups remained above the HDBR control groups, but the H/Q ratio showed no significant change. CONCLUSION: The results showed that exercise training during 21 d -6 degrees head-down tilt bed-rest do have beneficial effects on retaining the dynamic posture equilibrium and dynamic motor coordination of the subjects after bed rest.

[Enhancing hGH expression level in insect cells by shortening the 5'-UTR of hGH cDNA].

The regulation of foreign gene expression in Insect-Baculovirus Expression System is very complex. In this report, the effect of 5'-UTR in the expression of hGH gene in cultured Sf9 cells was examined. A 18 bp length in the end of 5'-UTR of hGH (human Growth Hormone, hGH) cDNA including a stem-loop structure was deleted by PCR. The truncated hGH cDNA, delta 1hGH was cloned in pFastBac1, named pFast-Bac-delta 1hGH. After transforming into E. coli. DH10Bac, which have a shuttle vetor-Bacmid, the delta 1hGH was integrated into Bacmid by site-specific transposition, and an expression vector, rBacmid-delta 1hGH DNA was acquired. By transfecting the cultured Sf9 cells with the recombinant expression vector DNA, pure recombinant virus, rAcV-Bac-delta 1hGH was obtained, and hGH gene was expressed. Immuno-blot and Chemiluminescent assay revealed that the expressed hGH had normal immunological activity, the amount of hGH expression level in Sf9 cell supernatant infected with rAcV-Bac-delta 1hGH containing the truncated 5'UTR was four to five times higher than that infected with rAcV-Bac-hGH.