A genome-wide transposon mutagenesis screening identifies LppB as a key factor associated with Mycoplasma bovis colonization and invasion into host cells.

Mycoplasma spp., the smallest self-replicating and genome-reduced organisms, have raised a great concern in both the medical and veterinary fields due to their pathogenicity. The molecular determinants of these wall-less bacterium efficiently use their limited genes to ensure successful infection of the host remain unclear. In the present study, we used the ruminant pathogen Mycoplasma bovis as a model to identify the key factors for colonization and invasion into host cells. We constructed a nonredundant fluorescent transposon mutant library of M. bovis using a modified transposon plasmid, and identified 34 novel adhesion-related genes based on a high-throughput screening approach. Among them, the ΔLppB mutant exhibited the most apparent decrease in adhesion to embryonic bovine lung (EBL) cells. The surface-localized lipoprotein LppB, which is highly conserved in Mycoplasma species, was then confirmed as a key factor for M. bovis adhesion with great immunogenicity. LppB interacted with various components (fibronectin, vitronectin, collagen IV, and laminin) of host extracellular matrix (ECM) and promoted plasminogen activation through tPA to degrade ECM. The 439-502 amino acid region of LppB is a critical domain, and F465 and Y493 are important residues for the plasminogen activation activity. We further revealed LppB as a key factor facilitating internalization through clathrin- and lipid raft-mediated endocytosis, which helps the Mycoplasma invade the host cells. Our study indicates that LppB plays a key role in Mycoplasma infection and is a potential new therapeutic and vaccine target for Mycoplasma species.

The Dual Roles of Activating Transcription Factor 3 (ATF3) in Inflammation, Apoptosis, Ferroptosis, and Pathogen Infection Responses.

Transcription factors are pivotal regulators in the cellular life process. Activating transcription factor 3 (ATF3), a member of the ATF/CREB (cAMP response element-binding protein) family, plays a crucial role as cells respond to various stresses and damage. As a transcription factor, ATF3 significantly influences signal transduction regulation, orchestrating a variety of signaling pathways, including apoptosis, ferroptosis, and cellular differentiation. In addition, ATF3 serves as an essential link between inflammation, oxidative stress, and immune responses. This review summarizes the recent advances in research on ATF3 activation and its role in regulating inflammatory responses, cell apoptosis, and ferroptosis while exploring the dual functions of ATF3 in these processes. Additionally, this article discusses the role of ATF3 in diseases related to pathogenic microbial infections. Our review may be helpful to better understand the role of ATF3 in cellular responses and disease progression, thus promoting advancements in clinical treatments for inflammation and oxidative stress-related diseases.

LppA is a novel plasminogen receptor of Mycoplasma bovis that contributes to adhesion by binding the host extracellular matrix and Annexin A2.

Mycoplasma bovis is responsible for various inflammatory diseases in cattle. The prevention and control of M. bovis are complicated by the absence of effective vaccines and the emergence of multidrug-resistant strains, resulting in substantial economic losses worldwide in the cattle industry. Lipoproteins, vital components of the Mycoplasmas cell membrane, are deemed potent antigens for eliciting immune responses in the host upon infection. However, the functions of lipoproteins in M. bovis remain underexplored due to their low sequence similarity with those of other bacteria and the scarcity of genetic manipulation tools for M. bovis. In this study, the lipoprotein LppA was identified in all examined M. bovis strains. Utilizing immunoelectron microscopy and Western blotting, it was observed that LppA localizes to the surface membrane. Recombinant LppA demonstrated dose-dependent adherence to the membrane of embryonic bovine lung (EBL) cells, and this adhesion was inhibited by anti-LppA serum. In vitro binding assays confirmed LppA's ability to associate with fibronectin, collagen IV, laminin, vitronectin, plasminogen, and tPA, thereby facilitating the conversion of plasminogen to plasmin. Moreover, LppA was found to bind and enhance the accumulation of Annexin A2 (ANXA2) on the cell membrane. Disrupting LppA in M. bovis significantly diminished the bacterium's capacity to adhere to EBL cells, underscoring LppA's function as a bacterial adhesin. In conclusion, LppA emerges as a novel adhesion protein that interacts with multiple host extracellular matrix proteins and ANXA2, playing a crucial role in M. bovis's adherence to host cells and dissemination. These insights substantially deepen our comprehension of the molecular pathogenesis of M. bovis.

Analysis of Oral Microbiota Revealed High Abundance of Prevotella Intermedia in Gout Patients.

BACKGROUND/AIMS: Microbes reside in a number of body sites, including the oral cavity, and are associated with the progression of many systemic diseases. In this study, we aimed to investigate the effects of gout and hyperuricemia (HUA) on the composition of oral microbiomes. METHODS: Analysis of the oral microbiota from 12 gout patients, 11 HUA patients, and 19 healthy control subjects was performed using a deep sequencing approach, and validation of significant changes in Prevotella intermedia and Serratia marcescens in new patient cohorts was performed using quantitative PCR (qPCR). RESULTS: Our analysis indicated that both gout and HUA significantly altered the composition of the oral microbiome in patients. Patients with gout or HUA had significantly greater levels of salivary Prevotella intermedia but significantly lower levels of Serratia marcescens than healthy control subjects. CONCLUSION: We demonstrated the association between the oral microbiome and gout and HUA for the first time. In particular, 16S sequencing and qPCR analysis revealed significantly higher levels of oral Prevotella intermedia in gout/HUA patients, which suggests that these patients might be at risk for the development of periodontitis.

Discovery and Validation of Salivary Extracellular RNA Biomarkers for Noninvasive Detection of Gastric Cancer.

BACKGROUND: Biomarkers are needed for noninvasive early detection of gastric cancer (GC). We investigated salivary extracellular RNA (exRNA) biomarkers as potential clinical evaluation tools for GC. METHODS: Unstimulated whole saliva samples were prospectively collected from 294 individuals (163 GC and 131 non-GC patients) who underwent endoscopic evaluation at the Samsung Medical Center in Korea. Salivary transcriptomes of 63 GC and 31 non-GC patients were profiled, and mRNA biomarker candidates were verified with reverse transcription quantitative real-time PCR (RT-qPCR). In parallel, microRNA (miRNA) biomarkers were profiled and verified with saliva samples from 10 GC and 10 non-GC patients. Candidate biomarkers were validated with RT-qPCR in an independent cohort of 100/100 saliva samples from GC and non-GC patients. Validated individual markers were configured into a best performance panel. RESULTS: We identified 30 mRNA and 15 miRNA candidates whose expression pattern associated with the presence of GC. Among them, 12 mRNA and 6 miRNA candidates were verified with the discovery cohort by RT-qPCR and further validated with the independent cohort (n = 200). The configured biomarker panel consisted of 3 mRNAs (SPINK7, PPL, and SEMA4B) and 2 miRNAs (MIR140-5p and MIR301a), which were all significantly down-regulated in the GC group, and yielded an area under the ROC curve (AUC) of 0.81 (95% CI, 0.72-0.89). When combined with demographic factors, the AUC of the biomarker panel reached 0.87 (95% CI, 0.80-0.93). CONCLUSIONS: We have discovered and validated a panel of salivary exRNA biomarkers with credible clinical performance for the detection of GC. Our study demonstrates the potential utility of salivary exRNA biomarkers in screening and risk assessment for GC.

Identification of Metabolite Biomarkers for Gout Using Capillary Ion Chromatography with Mass Spectrometry.

Gout is a common form of inflammatory arthritis, and the detailed pathogenic mechanisms for this metabolic disorder remain largely unknown. In this study, we first profiled the salivary metabolites in 8 patients with gout, 15 patients with hyperuricaemia (HUA), and 15 healthy individuals using capillary ion chromatography (CIC) with tandem mass spectrometry (MS/MS). Forty-nine salivary metabolites were found to be significantly changed between gout patient and healthy control groups, and 26 salivary metabolites were significantly different between gout and HUA patient groups. Three metabolite biomarkers, uric acid, oxalic acid, and l-homocysteic acid (HCA), were selected for validation in the saliva samples of 30 patients with gout, 30 patients with HUA, and 30 healthy control subjects. By using commercial assay kits for the measurements, salivary uric acid and oxalic acid levels were found to be significantly higher in gout patients than healthy controls, whereas salivary HCA level was significantly higher in gout patients than both HUA patients and healthy controls. These assay measurements were in line with those obtained by CIC-MS/MS. In conclusion, we have demonstrated a new application of CIC-MS/MS for the discovery of novel metabolite biomarkers of gout. Validated biomarkers may be used for noninvasive, diagnostic and prognostic applications in gout. Future studies are warranted to investigate the clinical utility of these identified biomarkers for monitoring gout flare and/or treatment efficacy.

Development of duplex PCR for differential detection of goatpox and sheeppox viruses.

BACKGROUND: Clinically, sheeppox and goatpox have the same symptoms and cannot be distinguished serologically. A cheaper and easy method for differential diagnosis is important in control of this disease in endemic region. METHODS: A duplex PCR assay was developed for the specific differential detection of Goatpox virus (GTPV) and Sheeppox virus (SPPV), using two sets of primers based on viral E10R gene and RPO132 gene. RESULTS: Nucleic acid electrophoresis results showed that SPPV-positive samples appear two bands, and GTPV-positive samples only one stripe. There were no cross-reactions with nucleic acids extracted from other pathogens including foot-and-mouth disease virus, Orf virus. The duplex PCR assay developed can specially detect SPPV or GTPV present in samples (n = 135) collected from suspected cases of Capripox. CONCLUSIONS: The duplex PCR assay developed is a specific and sensitive method for the differential diagnosis of GTPV and SPPV infection, with the potential to be standardized as a detection method for Capripox in endemic areas.

Activin A can induce definitive endoderm differentiation from human parthenogenetic embryonic stem cells.

OBJECTIVES: As activin/nodal signaling plays a key role in definitive endoderm (DE) differentiation, we have explored activin A-induced differentiation of DE from human parthenogenetic embryonic stem cells (hPESCs). RESULTS: Administration of 5 ng activin A/ml had no effect on the expression of markers of DE differentiation. However, higher concentrations of activin A (50 and 100 ng/ml) upregulated Sox17 and Cxcr4, as well upregulating the mesendodermal precursor marker, Brachyury. CONCLUSIONS: These findings demonstrate that low dose activin A can maintain the undifferentiated potency of hPESCs, whereas higher doses induce DE differentiation; 50 ng/ml is the optimal concentration for inducing DE from hPESCs.

Fatty acid epoxyisoprostane E2 stimulates an oxidative stress response in endothelial cells.

Atherosclerosis is the main underlying cause of major cardiovascular diseases such as stroke and heart attack. Oxidized phospholipids such as oxidized 1-palmitoyl-2-arachidonoyl-sn-Glycero-3-phosphorylcholine (OxPAPC) accumulate in lesions of and promote atherosclerosis. OxPAPC activates endothelial cells, a critical early event of atherogenesis. Epoxyisoprostane E2 (EI) is an oxidized fatty acid contained at the sn-2 position of 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine (PEIPC), the most active component of OxPAPC in regulating inflammation. OxPAPC and its components including PEIPC activate endothelial cells to express an array of genes in different categories including oxidative stress response genes such as tumor suppressor gene OKL38 and Heme oxygenase-1 (HO-1). EI can be released by lipase from PEIPC. In this study, we examined the ability of EI to stimulate oxidative stress response in endothelial cells. EI released from OxPAPC and synthetic EI stimulated the expression of oxidative stress response gene OKL38 and antioxidant gene HO-1. Treatment of endothelial cells with EI increased the production of superoxide. NADPH oxidase inhibitor Apocynin and superoxide scavenger N-acetyl-cysteine (NAC) significantly attenuated EI-stimulated expression of OKL38 and HO-1. We further demonstrated that EI activated oxidative stress-sensitive transcription factor Nrf2. Silencing of Nrf2 with siRNA significantly reduced EI stimulated expression of OKL38 and HO-1. Thus, we demonstrated that EI induced oxidative stress in endothelial cells leading to increased expression of oxidative stress response gene OKL38 and HO-1 via Nrf2 signaling pathway relevant to atherosclerosis.

Development of loop-mediated isothermal amplification assay for specific and rapid detection of differential goat pox virus and sheep pox virus.

BACKGROUND: Capripox viruses are economically important pathogens in goat and sheep producing areas of the world, with specific focus on goat pox virus (GTPV), sheep pox virus (SPPV) and the Lumpy Skin Disease virus (LSDV). Clinically, sheep pox and goat pox have the same symptoms and cannot be distinguished serologically. This presents a real need for a rapid, inexpensive, and easy to operate and maintain genotyping tool to facilitate accurate disease diagnosis and surveillance for better management of Capripox outbreaks. RESULTS: A LAMP method was developed for the specific differential detection of GTPV and SPPV using three sets of LAMP primers designed on the basis of ITR sequences. Reactions were performed at 62°C for either 45 or 60 min, and specificity confirmed by successful differential detection of several GTPV and SPPV isolates. No cross reactivity with Orf virus, foot-and-mouth disease virus (FMDV), A. marginale Lushi isolate, Mycoplasma mycoides subsp. capri, Chlamydophila psittaci, Theileria ovis, T. luwenshuni, T. uilenbergi or Babesia sp was noted. RFLP-PCR analysis of 135 preserved epidemic materials revealed 48 samples infected with goat pox and 87 infected with sheep pox, with LAMP test results showing a positive detection for all samples. When utilizing GTPV and SPPV genomic DNA, the universal LAMP primers (GSPV) and GTPV LAMP primers displayed a 100% detection rate; while the SPPV LAMP detection rate was 98.8%, consistent with the laboratory tested results. CONCLUSIONS: In summary, the three sets of LAMP primers when combined provide an analytically robust method able to fully distinguish between GTPV and SPPV. The presented LAMP method provides a specific, sensitive and rapid diagnostic tool for the distinction of GTPV and SPPV infections, with the potential to be standardized as a detection method for Capripox viruses in endemic areas.

Development of transcriptomic biomarker signature in human saliva to detect lung cancer.

Lung cancer is the leading cause of cancer death for both men and women worldwide. Since most of the symptoms found for lung cancer are nonspecific, diagnosis is mostly done at late and progressed stage with the consecutive poor therapy outcome. Effective early detection techniques are sorely needed. The emerging field of salivary diagnostics could provide scientifically credible, easy-to-use, non-invasive and cost-effective detection methods. Recent advances have allowed us to develop discriminatory salivary biomarkers for a variety of diseases from oral to systematic diseases. In this study, salivary transcriptomes of lung cancer patients were profiled and led to the discovery and pre-validation of seven highly discriminatory transcriptomic salivary biomarkers (BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and LZTS1). The logistic regression model combining five of the mRNA biomarkers (CCNI, EGFR, FGF19, FRS2, and GREB1) could differentiate lung cancer patients from normal control subjects, yielding AUC value of 0.925 with 93.75 % sensitivity and 82.81 % specificity in the pre-validation sample set. These salivary mRNA biomarkers possess the discriminatory power for the detection of lung cancer. This report provides the proof of concept of salivary biomarkers for the non-invasive detection of the systematic disease. These results poised the salivary biomarkers for the initiation of a multi-center validation in a definitive clinical context.

[Current status and implication of research on Bardet-Biedl syndrome].

OBJECTIVE: Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disease initially reported by Bardet and Biedl in the 1920s. BBS is a pleiotropic and genetically heterogeneous disorder characterized by retinopathy, obesity, polydactyly, renal malformations and functional abnormalities, learning disabilities and hypogenitalism. BBS patients are also prone to diabetes mellitus, hypertension and congenital heart disease. To date, 16 BBS genes (BBS1-BBS16) have been identified. However, the molecular etiology of BBS is not yet entirely clear. In this article, we have reviewed recent research on BBS and discussed its implications for understanding of ciliopathology.

ASPN Synergizes with HAPLN1 to Inhibit the Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells and Extracellular Matrix Mineralization of Osteoblasts.

OBJECTIVE: Bone marrow mesenchymal stromal cells (BMSCs) are major sources of osteogenic precursor cells in bone remodeling, which directly participate in osteoporosis (OP) progression. However, the involved specific mechanisms of BMSCs in OP warrant mass investigations. Initially, our bioinformatics analysis uncovered the prominent up-regulation of Asporin (ASPN) and proteoglycan link protein 1 (HAPLN1) in osteoblasts (OBs) of OP patients and their possible protein interaction. Hence, this study aimed to explore the effects of ASPN and HAPLN1 on osteogenic differentiation of BMSCs, extracellular matrix (ECM) mineralization of OBs, and osteoclastogenesis, hoping to offer research basis for OP treatment. METHODS: GSE156508 dataset was used for analysis and screening to acquire the differentially expressed genes in OBs of OP patients, followed by the predicative analysis via STRING. OP mouse models were induced by ovariectomy (OVX), and ASPN and HAPLN1 expression was determined. BMSCs and bone marrow macrophages (BMMs) were isolated from OVX mice and induced for osteogenic differentiation and osteoclastogenesis, respectively. After knockdown experiments, we assessed adipogenic differentiation and osteogenic differentiation in BMSCs. Osteogenic (OPN, OCN, and COL1A1) and osteoclast (Nfatc1 and c-Fos) marker protein expression was determined. The binding of ASPN to HAPLN1 was analyzed. RESULTS: High expression of ASPN and HAPLN1 and their protein interaction were observed in OBs of OP patients via bioinformatics and in bone tissues of OVX mice. ASPN interacted with HAPLN1 in BMSCs of OVX mice. ASPN/HAPLN1 knockdown increased ALP, OPN, OCN, and COL1A1 protein expression and ECM mineralization in BMSCs while decreasing Nfatc1 and c-Fos expression in BMMs. These effects were aggravated by the simultaneous knockdown of ASPN and HAPLN1. CONCLUSION: Our results indicate that ASPN synergises with HAPLN1 to suppress the osteogenic differentiation of BMSCs and ECM mineralization of OBs and promote the osteoclastogenesis in OP.

Analysis of Correlation Between Coronary Heart Disease and Genetic Polymorphism Detected by Gold Magnetic Nanoparticles Chromatography.

It aimed to explore the correlation of Glu504Lys locus mutation of aldehyde dehydrogenase-2 (ALDH2) with coronary heart disease (CHD) based on gold magnetic nanoparticles (GMNPs) chromatography and amplification refractory mutation system-PCR (ARMS-PCR). 120 CHD patients admitted to the cardiovascular Department of Wenling First People's Hospital affiliated to Wenzhou Medical University from December 2020 to December 2021 were selected as Case group and 80 non-CHD patients admitted during the same period were selected as Ctrl group. The venous blood and indexes of Total Cholesterol (TC), Triglyceride (TG), Low Density Lipoprotein Cholesterol (LDL-C), High Density Lipoprotein Cholesterol (HDL-C), and Fasting Blood Glucose (FBS) were collected. The ARMS-PCR GMNPs chromatography based on ARMS-PCR and immunochromatography assay was adopted to detect gene polymorphism of ALDH2. Correlation between ALDH2 gene polymorphism and risk factors of CHD was analyzed via logistic regression. In contrast to Ctrl group, the genotypes of GG, GA, and AA in Case group were evidently different (P < 0.05), and the frequency of A allelic gene was obviously increased (P < 0.05). Under the dominant model, frequency of GA + AA genotype in Case group was remarkably higher in contrast to Ctrl group (P < 0.05). Under the recessive model, there was no obvious difference in genotype frequency between two groups. In contrast to Ctrl group, TC, LDL-C, and FBS in Case group were notably increased (P < 0.05), while HDL-C was notably decreased (P < 0.05). The distribution frequency of abnormal LDL-C, HDL-C, and FBS in Case group was notably higher in contrast to Ctrl group (P < 0.05). LDL-C and FBS had no obvious effect on the genotypes and frequency distribution of alleles in CHD patients. However, the frequency distribution of genotypes of GA and AA and A allelic gene in patients with abnormal HDL-C was notably lower in contrast to those with normal HDL-C (P < 0.05). Logistic regression analysis showed that abnormal HDC-C with A allelic gene were independent risk factors for CHD (P = 0.001, OR = 1.934). The gene polymorphism of Glu504Lys locus of ALDH2 was closely related to the pathogenesis of CHD, A allelic gene may be a susceptibility gene for CHD, and patients with abnormal HDC-C and carried A allelic gene had relatively higher incidence of CHD.

Complete Genome Sequences of Mycoplasma ovipneumoniae Strains 150 and 274, Isolated from Different Regions in Bosnia and Herzegovina.

Mycoplasma ovipneumoniae is an important pathogen in sheep, goats, and wild ruminants. We sequenced M. ovipneumoniae strains 150 and 274 from Bosnia and Herzegovina. Strain 150 has a circular genome of 1,053,380 bp with 29.15% GC content while strain 274 has 1,081,404 bp with 28.82% GC content.

Comparative untargeted and targeted metabonomics reveal discriminations in metabolite profiles between Mycoplasma capricolum subsp. capripneumoniae and Mycoplasma capricolum subsp. capricolum.

Background: Mycoplasmas are among the smallest prokaryotic microbes that can grow and proliferate on non-living media. They have reduced genomes, which may be associated with a concomitant reduction in their metabolic capacity. Mycoplasma capricolum subsp. capripneumoniae (Mccp) and Mycoplasma capricolum subsp. capricolum (Mcc), both belong to the Mycoplasma mycoides cluster, are significant important pathogenic Mycoplasma species in veterinary research field. They share high degree of genome homology but Mcc grows markedly faster and has higher growth titer than Mccp. Methods: This study investigated the metabolites of these two pathogenic bacteria from the middle and late stages of the logarithmic growth phase through liquid chromatography-mass spectrometry-based metabolomics and targeted energy metabolomics. The multivariate analysis was conducted to identify significant differences between the two important Mycoplasma species. Results: A total of 173 metabolites were identified. Of them, 33 and 34 metabolites involved in purine and pyrimidine, pyruvate metabolism, and amino acid synthesis were found to significantly differ in the middle and late stages, respectively. The abundance of fructose 1,6-bisphosphate, ADP, and pyruvate was higher in Mcc than in Mccp during the whole logarithmic period. Lactate was upregulated in slow-growing Mccp. The pH buffering agent N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] added to media effectively prevented pH reduction and increase bacterial viability and protein biomass. The multivariate analysis revealed that the two Mycoplasma species significantly differed in glucose metabolism, growth factor transport and metabolism, cholesterol utilization, and environmental regulation. Conclusion: The study data are beneficial for understanding the metabolomic characteristics of these two crucial Mycoplasma species and shedding more light on mycoplasma metabolism, and serve as a resource for the pathogenesis and development of related vaccines.

Involvement of non-structural proteins (NS) in influenza A infection and viral tropism.

Hemagglutinin (HA) of influenza A has been reported as the key protein in viral infection. Therefore, the density and the dynamic pattern of this protein in viral envelope will affect the virus to infect target cells. We used a lentiviral system to study the influenza A H1N1 viral infection. Herein we demonstrate that the influenza non-structural proteins (NS) significantly promote viral infection. By substituting NS gene segment from an H1N1 genome set of A/WSN/1933 with the NS segment isolated from another H1N1 substrain genome set, China246, we found that viral infection tropism was significantly altered. The reassortant H1N1 shows almost identical infectivity compared with its parental virus, A/WSN/1933, for the human epithelial cell line HOT, but shows only 1/100 infectivity of its parental virus when infecting the Madin-Darby canine kidney (MDCK) cell line. These results suggest that not only is NS important in the infectivity of human influenza virus, but that it may play a critical role in viral tropism, allowing the virus to mutate and spread to other species.

RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells.

BACKGROUND: Goatpox is an economically important disease in goat and sheep-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. Hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses. However, none of these studies has been attempted to study GTPV. In the interest of exploiting improved methods to control goat pox, it is participated that RNAi may provide effective protection against GTPV. In this study we show the suppression of Goatpox virus (GTPV) replication via knockdown of virion core protein using RNA interference. RESULTS: Four short interfering RNA (siRNA) sequences (siRNA-61, siRNA-70, siRNA-165 and siRNA-296) against a region of GTPV ORF095 were selected. Sense and antisense siRNA-encoding sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes under the control of a human U6 promoter. ORF095 amplicon was generated using PCR, and then cloned into pEGFP-N1 vector, named as p095/EGFP. p095/EGFP and each of the siRNA expression cassettes (p61, p70, p165 and p296) were co-transfected into BHK-21 cells. Fluorescence detection, flow cytometric analysis, retro transcription PCR (RT-PCR) and real time PCR were used to check the efficiency of RNAi. The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095. When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells. CONCLUSIONS: This study demonstrates that vector-based shRNA methodology can effectively inhibit GTPV replication on Vero cells. Simultaneously, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of GTPV infection.

Direct saliva transcriptome analysis.

BACKGROUND: Current standard operating procedures for salivary transcriptomic analysis require low temperatures and lengthy mRNA isolation, which substantially hamper its use in the clinic. We developed a streamlined, ambient-temperature processing, stabilization, and storage protocol for clinical analysis of salivary RNA. METHODS: The direct saliva transcriptome analysis (DSTA) used cell-free saliva supernatant instead of isolated mRNA for saliva transcriptomic detection, and all procedures, including processing, stabilization, and storage of saliva samples, were performed at ambient temperature without a stabilizing reagent. We evaluated this streamlined protocol by comparing the mRNA expression levels of 3 saliva internal reference genes [glyceraldehyde-3-phosphate dehydrogenase (GAPDH); actin, beta (ACTB); and ribosomal protein S9 (RPS9)] to levels measured with standard procedures, and detecting the variation of their expression levels under long-term ambient temperature storage. The clinical utility of DSTA was assessed by use of 7 oral cancer salivary mRNA biomarkers in a clinical study. RESULTS: Each saliva internal reference gene mRNA showed similar expression levels when assayed by the DSTA or standard procedures, and remained stable under ambient temperature storage for at least 10 weeks without significant degradation (P = 0.918, 0.288, and 0.242 for GAPDH, ACTB, and RPS9, respectively). Compared with standard procedures, the performance characteristics of oral cancer salivary transcriptomic markers were retained as assayed by DSTA after 10 weeks of storage at ambient temperature. These results indicate that the DSTA is a suitable alternative method for saliva transcriptomic analysis and is feasible for use in clinical cancer research applications. CONCLUSIONS: The streamlined DSTA protocol can impact the saliva-handling method and improve the standard operating procedures for clinical saliva transcriptomic diagnostics.

[Establishment of B lymphoblastoid cell lines of Miao pedigree with Bardet-Biedl syndrome].

OBJECTIVE: To establish immortalized lymphoblastoid cell lines of a Miao core pedigree with Bardet-Biedl syndrome (BBS), in order to provide a long-term source of material for research. METHODS: With Epstein-Barr virus transformation of B cells and addition of cyclosporine A to inhibit the activity of T cells, fresh anticoagulated blood samples with heparin were collected from 12 members of the core pedigree, and were used to establish the immortalized lymphoblastoid cell lines of B lymphocytes. RESULTS: Twelve immortalized lymphoblastoid cell lines of the core BBS pedigree were obtained successfully. CONCLUSION: The immortalized B lymphoblastoid cell lines of the Miao pedigree with BBS can preserve the whole genome information and provide long-term research materials for BBS study.