Knocking-down long non-coding RNA LINC01094 prohibits chondrocyte apoptosis via regulating microRNA-577/metal-regulatory transcription factor 1 axis.

PURPOSE: The abnormal function and survival of chondrocytes result in articular cartilage failure, which may accelerate the onset and development of osteoarthritis (OA). This study is aimed to investigate the role of LINC01094 in chondrocyte apoptosis. METHODS: The viability and apoptosis of lipopolysaccharide (LPS)-induced chondrocytes were evaluated through CCK-8 assay and flow cytometry analysis, respectively. The expression levels of LINC01094, miR-577 and MTF1 were detected by qRT-PCR. Dual luciferase reporter tests were implemented for the verification of targeted relationships among them. Western blotting was employed to measure the levels of pro-apoptotic proteins (Caspase3 and Caspase9). RESULTS: The viability of LPS-induced chondrocytes was overtly promoted by loss of LINC01094 or miR-577 upregulation, but could be repressed via MTF1 overexpression. The opposite results were observed in apoptosis rate and the levels of Caspase3 and Caspase9. LINC01094 directly bound to miR-577, while MTF1 was verified to be modulated by miR-577. Both LINC01094 and MTF1 were at high levels, whereas miR-577 was at low level in OA synovial fluid and LPS-induced chondrocytes. Furthermore, the highly expressed miR-577 abolished the influences of MTF1 overexpression on LPS-induced chondrocytes. CONCLUSIONS: Silencing of LINC01094 represses the apoptosis of chondrocytes through upregulating miR-577 expression and downregulating MTF1 levels, providing a preliminary insight for the treatment of OA in the future.

Downregulation of lncRNA NEAT1 interacts with miR-374b-5p/PGAP1 axis to aggravate the development of osteoarthritis.

BACKGROUND: Osteoarthritis (OA), characterized by inflammation and articular cartilage degradation, is a prevalent arthritis among geriatric population. This paper was to scrutinize the novel mechanism of long noncoding RNA (lncRNA) NEAT1 in OA etiology. METHODS: A total of 10 OA patients and 10 normal individuals was included in this study. Cell model of OA was built in human normal chondrocytes induced by lipopolysaccharide (LPS). An OA Wistar rat model was established through intra-articular injection of L-cysteine and papain mixtures (proportion at 1:2) into the right knee. Quantitative reverse transcription-polymerase chain reaction was employed to ascertain the expression levels of NEAT1, microRNA (miR)-374b-5p and post-GPI attachment to protein 1 (PGAP1), while dual-luciferase reporter experiments were used for the validation of target relationship among them. Cell cycle and apoptosis were calculated by flow cytometry analysis. CCK-8 assay was done to evaluate the proliferative potentials of chondrocytes. The levels of cell cycle-related proteins (Cyclin A1, Cyclin B1 and Cyclin D2) and pro-apoptotic proteins (Caspase3 and Caspase9) were measured by western blotting. Tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß) and IL-6 levels were determined via ELISA. Hematoxylin & eosin (HE) Staining was used for pathological examination in OA rats. RESULTS: Pronounced downregulation of NEAT1 and PGAP1 and high amounts of miR-374b-5p were identified in OA patients, LPS-induced chondrocytes and OA rats. NEAT1 targeted miR-374b-5p to control PGAP1 expression. Loss of NEAT1 or upregulation of miR-374b-5p dramatically accelerated apoptosis, led to the G1/S arrest and promoted the secretion of inflammatory cytokines in LPS-induced chondrocytes, while ectopic expression of PGAP1 exhibited the opposite influences on chondrocytes. Additionally, we further indicated that upregulation of miR-374b-5p attenuated the effects of PGAP1 overexpression on LPS-induced chondrocytes. CONCLUSIONS: Reduced NEAT1 induces the development of OA via miR-374b-5p/PGAP1 pathway. This suggests that the regulatory axis NEAT1/miR-374b-5p/PGAP1 is a novel and prospective target for OA treatment.

The analgesic evaluation of gabapentin for arthroscopy: A meta-analysis of randomized controlled trials.

INTRODUCTION: The efficacy of gabapentin for pain management of arthroscopy remains controversial. We conduct a systematic review and meta-analysis to explore the influence of gabapentin versus placebo on the postoperative pain intensity of arthroscopy. METHODS: We search PubMed, EMbase, Web of science, EBSCO, and Cochrane library databases through April 2020 for randomized controlled trials assessing the effect of gabapentin versus placebo on pain control of arthroscopy. This meta-analysis is performed using the random-effect model. RESULTS: Five randomized controlled trials are included in the meta-analysis. Overall, compared with control group for arthroscopy, gabapentin remarkably decreases pain scores at 24 hour (standard mean difference [SMD]=-0.68; 95% confidence interval [CI]=-1.15 to -0.02; P = .21), analgesic consumption (SMD = -18.24; 95% CI=-24.61 to -11.88; P < .00001), nausea and vomiting (OR = 0.42; 95% CI = 0.21 to 0.84; P = .01), but has no obvious influence on pain scores at 6 h (SMD = -1.30; 95% CI = -2.92 to 0.31; P = .11) or dizziness (OR = 1.12; 95% CI = 0.56 to 2.24; P = .75). CONCLUSIONS: Gabapentin is effective for pain control after arthroscopy.

Identification of Novel Compound Heterozygous Mutations in the GAN Gene of a Chinese Patient Diagnosed With Giant Axonal Neuropathy.

Giant axonal neuropathy (GAN) is a very rare autosomal recessive disorder characterized by abnormally large and dysfunctional neuronal axons. Mutations in the GAN gene have been identified as the cause of this disorder. In this report, we performed a detailed phenotypic assessment of a Chinese patient with GAN. An array-based exon capture test and targeted next-generation sequencing were used to detect the suspected mutation sites. Compound heterozygous mutations of p.S79L (c.236C > T) in the BTB domain and p.T489S (c.1466C > G) in the kelch domain were identified in the proband's genome. S79L was a known mutation, and T489S was reported for the first time. The p.S79L and p.T489S were confirmed in the proband's mother and father, respectively. Both mutations were located in highly conserved regions and affected the predicted protein crystal structures. The proband's sural biopsy revealed the classical GAN phenotype of swollen axons filled with closely packed neurofilaments. The combined application of the next-generation sequencing platform and bioinformatics analyses was an effective method for diagnosing GAN. The novel compound mutations of S79L and T489S in the GAN gene were likely the cause of the patient's GAN symptoms. Our findings enrich the spectrum of mutations associated with this rare type of axonopathy.

The efficacy of intra-articular fentanyl supplementation for knee arthroscopy: A meta-analysis of randomized controlled studies.

INTRODUCTION: The efficacy of intra-articular fentanyl supplementation for pain control after knee arthroscopy remains controversial. We conduct a systematic review and meta-analysis to explore the influence of intra-articular fentanyl supplementation for pain intensity after arthroscopic knee surgery. METHODS: We searched PubMed, EMbase, Web of Science, EBSCO, and Cochrane Library databases through May 2019 for randomized controlled trials (RCTs) assessing the efficacy and safety of intra-articular fentanyl supplementation for arthroscopic knee surgery. This meta-analysis is performed using the random-effects model. RESULTS: Four RCTs are included in the meta-analysis. Overall, compared with control group after knee arthroscopy, intra-articular fentanyl supplementation is associated with reduced pain scores at 1 h (standard mean difference (Std MD) = -3.50; 95% confidence interval (CI) = -5.68 to -1.32; p = 0.002), 2 h (Std MD = -4.73; 95% CI = -8.75 to -0.71; p = 0.02), and 8 h (Std MD = -5.02; 95% CI = -9.73 to -0.30; p = 0.04) but shows no substantial impact on pain scores at 4 h (Std MD = -3.94; 95% CI = -7.93 to 0.05; p = 0.05) or the supplementary analgesia (risk ratio = 0.56; 95% CI = 0.09-3.59; p = 0.54). CONCLUSIONS: Intra-articular fentanyl supplementation does benefit in pain control after knee arthroscopy.

A Novel AURKA Mutant-Induced Early-Onset Severe Hepatocarcinogenesis Greater than Wild-Type via Activating Different Pathways in Zebrafish.

Aurora A kinase (AURKA) is an important regulator in mitotic progression and is overexpressed frequently in human cancers, including hepatocellular carcinoma (HCC). Many AURKA mutations were identified in cancer patients. Overexpressing wild-type Aurka developed a low incidence of hepatic tumors after long latency in mice. However, none of the AURKA mutant animal models have ever been described. The mechanism of mutant AURKA-mediated hepatocarcinogenesis is still unclear. A novel AURKA mutation with a.a.352 Valine to Isoleucine (V352I) was identified from clinical specimens. By using liver-specific transgenic fish overexpressing both the mutant and wild-type AURKA, the AURKA(V352I)-induced hepatocarcinogenesis was earlier and much more severe than wild-type AURKA. Although an increase of the expression of lipogenic enzyme and lipogenic factor was observed in both AURKA(V352I) and AURKA(WT) transgenic fish, AURKA(V352I) has a greater probability to promote fibrosis at 3 months compared to AURKA(WT). Furthermore, the expression levels of cell cycle/proliferation markers were higher in the AURKA(V352I) mutant than AURKA(WT) in transgenic fish, implying that the AURKA(V352I) mutant may accelerate HCC progression. Moreover, we found that the AURKA(V352I) mutant activates AKT signaling and increases nuclear ß-catenin, but AURKA(WT) only activates membrane form ß-catenin, which may account for the differences. In this study, we provide a new insight, that the AURKA(V352I) mutation contributes to early onset hepatocarcinogenesis, possibly through activation of different pathways than AURKA(WT). This transgenic fish may serve as a drug-screening platform for potential precision medicine therapeutics.

Postoperative plasma 8-iso-prostaglandin F2α levels are associated with delirium and cognitive dysfunction in elderly patients after hip fracture surgery.

BACKGROUND: Oxidative stress may be involved in occurrence of postoperative delirium (POD) and cognitive dysfunction (POCD). 8-iso-Prostaglandin F2α (8-iso-PGF2α), an isoprostane derived from arachidonic acid via lipid peroxidation, is considered a gold standard for measuring oxidative stress. The present study aimed to investigate the ability of postoperative plasma 8-iso-PGF2α levels to predict POD and POCD in elderly patients undergoing hip fracture surgery. METHODS: Postoperative plasma 8-iso-PGF2α levels of 182 patients were measured by an enzyme-linked immunosorbent assay. We assessed the relationships between plasma 8-iso-PGF2α levels and the risk of POD and POCD using a multivariate analysis. RESULTS: Plasma 8-iso-PGF2α levels and age were identified as the independent predictors for POD and POCD. Based on areas under receiver operating characteristic curve, the predictive values of 8-iso-PGF2α were obviously higher than those of age for POD and POCD. In a combined logistic-regression model, 8-iso-PGF2α significantly enhanced the areas under curve of age for prediction of POD and POCD. CONCLUSIONS: Postoperative plasma 8-iso-PGF2α levels may have the potential to predict POD and POCD in elder patients undergoing hip fracture surgery.

A novel approach to cloning and expression of human thymidylate synthase.

Thymidylate synthase (TS) catalyzes the transfer of a methyl group from methylenetetrahydrofolate to dUMP to form dTMP. It is a primary target in the chemotherapy of colorectal cancers and some other neoplasms. In order to obtain pure protein for analysis of structure and biological function, an expression vector TS-pET28b (+) was constructed by inserting wild-type human thymidylate synthase (hTS) cDNA into pET28b (+). Then an expression strain was selected after transformation of the recombined plasmid into Rosetta (DE3). Fusion protein with His-tag was efficiently expressed in the form of inclusion bodies after IPTG induction and the content was approximately 40.0% of total bacteria proteins after optimizing expression conditions. When inclusion bodies were washed, dissolved and purified by Ni-NTA under denatured conditions, the purity was up to 90%. On SDS-PAGE and West-blotting, the protein band was found to match well with the predicted relative molecular mass-36kDa. Bioactivity was 0.1 U/mg. The results indicated that high-level expression of wild-type hTS cDNA can be achieved in prokaryotes with our novel method, facilitating research into related chemotherapy.

The expression of classical swine fever virus structural protein E2 gene in tobacco chloroplasts for applying chloroplasts as bioreactors.

It has been reported that genes encoding antigens of bacterial and viral pathogens can be expressed in plants and are shown to induce protection antibodies. The structural protein E2 of classical swine fever virus (CSFV), which has been shown to carry critical epitopes, has been expressed in different systems. Here, we report the expression of CFSV E2 gene in tobacco chloroplasts. Mice immunized with leaf extracts elicited specific antibodies. This indicated that the expressed E2 proteins had a certain degree of immunogenicity. To our knowledge, this is the first report showing induction of protective antibody in response to classical swine fever virus (CSFV) by immunization with antigen protein E2 expressed in tobacco chloroplasts, which will open a new way to protection from CSFV by plant chloroplasts as bioreactors.

Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas reinhardtii chroloplasts.

The expression of classical swine fever virus (CSFV) structural protein E2 in different vectors, which has been shown to carry critical epitopes, has been established. Here, we reported a Chlamydomonas reinhardtii chloroplast expression vector, P64E2, containing classical swine fever virus structural protein E2 gene, which was constructed and transferred to C. reinhardtii by biolistic bombardment method. The transformants were identified by PCR, Southern blotting, Western blotting after selecting on resistant media. ELISA quantification assay showed that the expressed E2 protein accumulated up to 1.5-2% of the total soluble protein. The results of the study on the immunological activity indicated that the protein E2 expressed in C. reinhardtii chloroplasts could elicit animal bodies to produce antibodies against protein E2.