Development of a real-time loop-mediated isothermal amplification assay for detection of porcine circovirus 3.

BACKGROUND: Porcine circovirus type 3 (PCV3) is an emerging circovirus species, that has been reported in major pig-raising countries including the United States, China, South Korea, Brazil, Spain, and Poland. RESULTS: A real-time loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of porcine circovirus 3 (PCV3). The method had a detection limit of 1 × 101 copies/µL with no cross-reactions with classical swine fever virus (CSFV) C strain, foot-and-mouth disease virus (FMDV), porcine circovirus 2 (PCV2) LG vaccine strain, porcine epidemic diarrhoea virus (PEDV), porcine respiratory and reproductive syndrome virus (PRRSV), or pseudorabies virus (PRV). The PCV3 positive detection rate of 203 clinical samples for the real-time LAMP assay was 89.66% (182/203). CONCLUSIONS: The real-time LAMP assay is highly sensitive, and specific for use in epidemiological investigations of PCV3.

Levels of Peripheral Th17 Cells and Th17-Related Cytokines in Patients with Ankylosing Spondylitis: A Meta-analysis.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic inflammatory disease. Several proinflammatory cytokines produced by T helper 17 (Th17) cells are involved in the pathogenesis of AS. We performed a meta-analysis to determine the levels of Th17 cells and serum Th17-associated cytokines in patients with AS. METHODS: We determined the levels of Th17 cells and Th17 cytokines in patients with AS using data extracted from published articles retrieved from the PubMed, Embase, Web of Science, Cochrane Library, MEDLINE, Web of Knowledge, Clinical Trials.gov, and FDA.gov. DATABASES: The effect estimates were pooled using a random-effects model. The review protocols were registered on PROSPERO (reference: CRD42021255741) and followed the PRISMA guideline. RESULTS: This meta-analysis included 138 studies. Compared to healthy controls (HCs), patients with AS had a higher proportion of Th17 cells (standardized mean difference [SMD] 2.23, 95% confidence interval [CI] 1.78-2.68; p < 0.001) and levels of proinflammatory cytokines, such as interleukin (IL)-17 (SMD 2.04, 95% CI 1.70-2.38; p < 0.001), IL-21 (SMD 1.77, 95% CI 0.95-2.59; p < 0.001), and IL-23 (SMD 1.11, 95% CI 0.78-1.44; p < 0.001). The subgroup analysis showed higher levels of IL-17+ Th17 cells among peripheral blood mononuclear cells (PBMCs) and CD4+ T cells in patients with AS compared to HCs (SMD 2.26, 95% CI 1.58-2.94 [p < 0.001] and SMD 1.61, 95% CI 0.55-2.67 [p = 0.003], respectively). Patients with AS had higher levels of CD4+IL-17+IFN-γ- Th17 in PBMCs and of CD4+CCR6+CCR4+Th17 in CD4+ T cells compared to HCs (SMD 1.85, 95% CI 1.06-2.64 [p < 0.001] and SMD 7.72, 95% CI 6.55-8.89 [p < 0.001], respectively). No significant differences were observed in the proportions of CD4+IL-17+IFN-γ- Th17 in CD4+ T cells and CD4+CCR6+CCR4+ Th17 in PBMCs (SMD - 0.11, 95% CI - 0.61 to 0.38 [p = 0.650] and SMD 1.32, 95% CI - 0.54 to 3.19 [p = 0.165], respectively). In addition, compared to stable AS, the levels of Th17 cells and IL-17 and IL-23 were significantly higher in active AS (SMD 1.58, 95% CI 0.30-2.85 [p = 0.016], SMD 3.52, 95% CI 0.72-6.33 [p = 0.014], and SMD 5.10, 95% CI 1.83-8.36 [p = 0.002], respectively). CONCLUSIONS: The levels of Th17 cells and serum IL-17, IL-21, and IL-23 were higher in patients with AS than in HCs and, compared with stable AS, they increased more significantly in active AS. These results suggest that Th17 cells and Th17-related cytokines play major roles in AS pathogenesis and are an important target for treatment.

An m6A-Related lncRNA Signature Predicts the Prognosis of Hepatocellular Carcinoma.

Objective: The purpose of this study was to establish an N6-methylandenosine (m6A)-related long non-coding RNA (lncRNA) signature to predict the prognosis of hepatocellular carcinoma (HCC). Methods: Pearson correlation analysis was used to identify m6A-related lncRNAs. We then performed univariate Cox regression analysis and least absolute shrinkage and selection operator (LASSO) Cox regression analysis to construct an m6A-related lncRNA signature. Based on the cutoff value of the risk score determined by the X-title software, we divided the HCC patients into high -and low-risk groups. A time-dependent ROC curve was used to evaluate the predictive value of the model. Finally, we constructed a nomogram based on the m6A-related lncRNA signature. Results: ZEB1-AS1, MIR210HG, BACE1-AS, and SNHG3 were identified to comprise an m6A-related lncRNA signature. These four lncRNAs were upregulated in HCC tissues compared to normal tissues. The prognosis of patients with HCC in the low-risk group was significantly longer than that in the high-risk group. The M6A-related lncRNA signature was significantly associated with clinicopathological features and was established as a risk factor for the prognosis of patients with HCC. The nomogram based on the m6A-related lncRNA signature had a good distinguishing ability and consistency. Conclusion: We identified an m6A-related lncRNA signature and constructed a nomogram model to evaluate the prognosis of patients with HCC.

A Pan-Cancer Analysis of the Oncogenic Role of Cell Division Cycle-Associated Protein 4 (CDCA4) in Human Tumors.

PURPOSE: To unravel the oncogenic role of CDCA4 in different cancers from the perspective of tumor immunity. METHODS: Raw data on CDCA4 expression in tumor samples and paracancerous samples were obtained from TCGA and GTEX databases. In addition, we investigated pathological stages and the survival analysis of CDCA4 in pan-cancer across Gene Expression Profiling Interactive Analysis (GEPIA) database. Cox Proportional Hazards Model shows that high CDCA4 levels are associated with several vital indicators in oncology. On the one hand, we explored the correlation between CADA4 expression and tumor immune infiltration by the TIMER tool; On the other hand, we utilized the methods of CIBERSORT and ESTIMATE computational to evaluate the proportion of tumor infiltrating immune cells (TIIC) and the amounts of stromal and immune components based on TCGA database. The use of antineoplastic drugs and the expression of CDCA4 also showed a high correlation via linear regression. Protein-Protein Interaction analysis was performed in the GeneMANIA database, and enrichment analysis was performed and predicted signaling pathways were identified by using Gene Ontology and Kyoto Encyclopedia of Genes. The correlation between CDCA4 expression with Copy number variations (CNV) and methylation is detailed, respectively. Molecular biology experiments including Western blotting, flow cytometry, EDU staining, Transwell and Wound Healing assay to validate the cancer promoting role of CDCA4 in hepatocellular carcinoma (HCC). RESULTS: Most tumors highly expressed CDCA4. Elevated CDCA4 expression was associated with poor OS and DFS. There was a significant correlation between CDCA4 expression and TITCs. Moreover, markers of TIICs exhibited distinct patterns of CDCA4 associated immune infiltration. In addition, we pay attention to the association between the expression of CDCA4 and the use of the anti-tumor drugs. CDCA4 is related to biological progress (BP), cellular component (CC) and molecular function (MF). Dopaminergic Synapse, AMPK, Sphingolipid, Chagas Disease, mRNA Surveillance were significantly enriched pathways in positive and negative correlation genes with CDCA4. CNV is thought to be a positive correlation with CDCA4 expression. Conversely, methylation is negative correlation with CDCA4 expression. Molecular biology experiments confirm a cancer promoting role for CDCA4 in HCC. CONCLUSION: CDCA4 may serve as a biomarker for cancer immunologic infiltration and poor prognosis, providing a new way of thinking for cancer treatment.

Development of a Recombinase Polymerase Amplification Fluorescence Assay for the Detection of Canine Adenovirus 2.

Canine adenovirus type 2 (CAdV-2) is often found in co-infections with other pathogens causing canine infectious respiratory disease (CIRD). Rapid, efficient, and convenient pathogen detection is the best approach for early confirmatory diagnosis. In this study, we developed and evaluated a rapid real-time recombinase polymerase amplification (RPA) assay for detection of canine adenovirus 2 (CAV), which can detect CAV within 15 min at 39°C. The detection limit that assay was 214 copies/µl DNA molecules per reaction. The specificity was indicated by a lack of cross-reaction with canine distemper virus (CDV), canine coronavirus (CCV), and canine parvovirus (CPV). Field and clinical applicability of this assay were evaluated using 86 field samples. The coincidence rate of the detection results for clinical samples between CAV-RPA and qPCR was 97.7%. In summary, the real-time CAV-RPA analysis provides an efficient, rapid and sensitive detection method for CAV.

A Serological Survey of Borrelia burgdorferi Infection in Sheep in Northeast China Regions Through Outer Surface Protein C-Based Enzyme-Linked Immunosorbent Assay.

Borrelia burgdorferi as a causative agent of Lyme disease is transmitted by Ixodes spp. ticks to humans and animals. Sheep is considered a natural reservoir for B. burgdorferi and plays a pivotal role in disease transmission and the expansion of natural foci. An epidemiological investigation of B. burgdorferi in sheep is essential for prevention and control of Lyme disease. In this study, we developed a recombinant outer surface protein C (OspC)-based ELISA for serological study of B. burgdorferi in sheep with a specificity and sensitivity of 84.4% and 86.2%, respectively. A total of 972 collected serum samples from the Northeast China regions in 2015 and 2016 were determined with positive rates of 5.8% and 12.2%, respectively. Thus, specific pathogen-free sheep were infected with B. burgdorferi SZ strain to study on the secretion of specificity antibody against OspC. It revealed that specific antibody was detected on day 5 postinoculation and sustained in a high level for ∼28 days, the peak occurred at ∼13 days. Taken together, the result indicated that the established ELISA is capable for clinical diagnosis and epidemiological study on B. burgdorferi in sheep at the early stage of infection and detecting the specific antibody during the secretion period.

A controlled synthesis method of alkyl methacrylate block copolymers via living anionic polymerization at ambient temperature.

A controlled synthesis method of alkyl methacrylate block copolymers such as poly(methyl methacrylate)-b-poly(ethyl methacrylate) (PMMA-b-PEMA), poly(methyl methacrylate)-b-poly(butyl methacrylate) (PMMA-b-PBMA) and poly(ethyl methacrylate)-b-poly(butyl methacrylate) (PEMA-b-PBMA) via living anionic polymerization was innovated with potassium tert-butoxide (t-BuOK) as initiator in tetrahydrofuran(THF) solvent. The sequential anionic copolymerization could be smoothly conducted at 0 °C and the conversion of all monomers reached up to almost 100%. The copolymers were characterized by gel permeation chromatography (GPC), proton nuclear magnetic resonance (1H-NMR), fourier transform infrared spectroscopy (FTIR) and dynamic mechanical analysis (DMA). It was found that all block copolymers were in a narrow MWD while M w and weight ratio of each block were coincided with the theoretical values and feed ratio. DMA measurement indicated that all the block copolymers have two glass transition temperatures which have proved the certain microphase separation and the partial compatibility of the blocks. The similar results were achieved after changing feed order or addition amount. Furthermore, the reactivity ratio was also studied and confirmed that reactivity ratio of MMA was the largest among alkyl methacrylate. Based on these results, the anionic block copolymerization containing polar alkyl methacrylate monomers at a commercial scale starts to become possible.

Simultaneous detection of 4 prototypic rat parvoviruses using the luminex xTAG assay in laboratory animal health monitoring.

There are currently four rat parvoviruses including Kilham rat virus (KRV), Toolans H-1 parvovirus (H-1virus), rat parvovirus type 1a (RPV-1a) and rat minute virus (RMV). Virus detection methods are commonly based on conventional PCR - agarose gel electrophoresis or serological assay methods These methods are both time-consuming and lack specificity. In this study, we developed a bead array xTAG assay for the simultaneous detection and discrimination of four rat parvoviruses. The detection limits ranged from 100 to 1000 copies/µL of input purified plasmid DNA. We examined 50 clinical specimens and 15 facal samples by xTAG assay and conventional PCR. The results showed a high consistency except for several weak positive infections. It demonstrated that the xTAG-multiplex PCR method is specific, sensitive and suitable for high throughput platforms for rat parvovirus screening of clinical samples and contaminated biological materials.

The effects of thoracic sympathotomy on heart rate variability in patients with palmar hyperhidrosis.

PURPOSE: To observe the evolution of heart rate variability (HRV) in patients with palmar hyperhidrosis before and after endoscopic thoracic sympathotomy and to evaluate the effects of the surgery on the autonomic nervous system. MATERIALS AND METHODS: Endoscopic thoracic sympathotomy was performed on 20 patients with palmar hyperhidrosis. The thoracic sympathetic chain at the level of the third to fourth rib (R3-R4) was transected, but the ganglia were left in position without removal. A slightly larger ramus, in comparison to the other rami, that arose laterally from the sympathetic chain was interrupted to achieve adequate sympathetic denervation of the upper extremity. Before and on the day after the surgery, 24-hour Holter Electrocardiograph was performed, obtaining time domain and frequency domain parameters. RESULTS: Compared with preoperative variables, there was a significant increase in the number of adjacent normal R wave to R wave (R-R) intervals that differed by more than 50 ms, as percent of the total number of normal RR intervals (pNN50); root mean square difference, the square root of the mean of the sum of squared differences between adjacent normal RR intervals over the entire 24-hour recording; standard deviation of the average normal RR interval for all 5-minute segments of a 24-hour recording (SDANN) after thoracic sympathotomy. Low frequencies (LF, 0.04 to 0.15 Hz) decreased significantly. There was no statistical difference in high frequencies (HF, 0.15 to 0.40 Hz), LF/HF ratio (LF/HF), or standard deviation for all normal RR intervals for the entire 24-h recording (SDNN) before and after thoracic sympathotomy. CONCLUSION: There was a significant improvement in HRV in patients with palmar hyperhidrosis after thoracic sympathotomy. This may be attributable to an improvement autonomic nervous system balance and parasympathetic predominance in the early postoperative stage.