Differentiation expression of toll-like receptor4 (TLR4) caused by the dysregulation of microRNA-140-5p is responsible for the development of postoperation infection.

BACKGROUND: Toll-like receptor4 (TLR4) has proven to be an important factor that's responsible for the development of postoperation infection. MicroRNAs (miRNAs) are widely regarded as key mediators of gene expression. The objectives of our study were to identify miRNA(s) and the target genes differentially expressed in monocytes in the individuals with postoperation infection. METHODS: MiRNA microarrays were performed to identify and compare miRNA expression in monocytes from those with or without postoperative infection. In-silico analysis was used to further investigate the target miRNAs and finally, luciferase assay and real-time polymerase chain reaction (PCR) were performed to confirm the target miRNA identified. Enzyme-linked immunosorbent assay, real-time PCR and Western-blot were performed to explore the role of miR-140 involved in postoperation infection. RESULTS: MiRNA microarray results showed that ten miRNAs were upregulated in the postoperation infection group, while six miRNAs were downregulated, compared with those in the postoperation group without infection. Computational analysis was further performed to reveal that four miRNAs (miR-140, miR-7, miR-448, and miR-217) targeted the 3'-untranslated region (UTR) of TLR4 mRNA. The luciferase assay showed that only miR-140 inhibited luciferase activity of wild-type TLR4 3'-UTR and the luciferase activity of the cells cotransfected with miR-7, miR-448 or miR-217 and wild-type or mutant TLR4 3'-UTR was comparable with the control. Furthermore, only miR-140 levels were significantly lower in the postoperation infection group, while levels of miR-217, miR-7, and miR-448 showed no obvious difference between the postoperation infection and postoperation without infection groups. TLR4, tumor necrosis factor-α (TNF-α), and IL-6 levels were much higher in the postoperation infection group. In comparison with the control group, TLR4, TNF-α and Interleukin 6 (IL-6) levels in cells were decreased following transfection with miR-140 mimics and TLR4 small interfering RNA. However, the cells treated with lipopolysaccharides increased TLR4, TNF-α, and IL-6 levels. CONCLUSION: This study demonstrates that miR-140 is differentially expressed in monocytes collected from patients diagnosed with postoperation infection. The downregulation of miR-140 cause upregulation of toll-like receptor4 (TLR4), a proinflammatory factor, and is associated with infection risk in patients received surgery.

Cecropin P1 inhibits porcine reproductive and respiratory syndrome virus by blocking attachment.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pig industry, causing high economic losses worldwide. Current vaccines have specific limitations in terms of their safety and efficacy, so the development of novel antiviral drugs is urgently required. The aim of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of the antimicrobial peptide cecropin P1 (CP1) against PRRSV infection in vitro. RESULTS: CP1 not only displayed extracellular virucidal activity against PRRSV, but also exerted a potent inhibitory effect when added either before, simultaneously with, or after viral inoculation. The inhibitory effect of CP1 occurred during viral attachment, but not at viral entry into Marc-145 cells. CP1 also inhibited viral particle release and attenuated virus-induced apoptosis during the late phase of infection. CP1 exerted similar inhibitory effects against PRRSV infection in porcine alveolar macrophages, the cells targeted by the virus in vivo during its infection of pigs. The expression of interleukin 6 was elevated by CP1 in porcine alveolar macrophages, which might contribute to its inhibition of PRRSV infection. CONCLUSIONS: Collectively, our findings provide a new direction for the development of potential therapeutic drugs against PRRSV infection.

Production and immunogenicity of VP2 protein of porcine parvovirus expressed in Pichia pastoris.

Viral protein 2 (VP2) of porcine parvovirus (PPV) is the major viral structural protein and is responsible for eliciting neutralizing antibodies in immunized animals. In this study, we constructed and characterized a recombinant yeast vector encoding the VP2 protein, designated as pGAPZαA-VP2. The construct was confirmed by restriction enzyme digestion, PCR, and sequencing and then introduced into P. pastoris strain SMD1168 by electroporation. The expressed VP2 protein was analyzed by SDS-PAGE and western blot. Immunization of mice with the VP2 protein elicited a PPV-specific humoral immune response. Notably, a preparation of VP2 protein containing adjuvant induced a much better antibody response than VP2 alone. Clearly, the adjuvant strongly enhanced the immunogenicity of VP2. This study provides a foundation for the application of the VP2 protein in the clinical diagnosis of PPV and in vaccination against PPV in the future.

Establishment and validation of a prediction model for myocarditis in Chinese children below 14 years old: a protocol for a retrospective cohort study.

INTRODUCTION: Paediatric myocarditis, a rare inflammatory disease, often presents without clear early symptoms. Although cardiac troponin I levels can aid in diagnosing myocarditis, they are not definitive indicators. Troponin I levels frequently fluctuate within and outside the reference range, potentially causing misinterpretations by clinicians. Although a negative troponin I result is valuable for excluding myocarditis, its specificity is low. Moreover, the clinical diagnosis of paediatric myocarditis is exceptionally challenging, and accurate early-stage diagnosis and treatment pose difficulties. Currently, the Dallas criteria, involving cardiac biopsy, serves as the gold standard for myocarditis diagnosis. However, this method has several drawbacks and is unsuitable for children, resulting in its limited use. METHODS AND ANALYSIS: In this study, we will employ multiple logistic regression analysis to develop a predictive model for early childhood myocarditis. This model will assess the patient's condition at onset and provide the probability of a myocarditis diagnosis. Model performance will be evaluated for accuracy and calibration, and the results will be presented through receiver operating characteristic (ROC) curves and calibration plots. Clinical decision curve analysis, in conjunction with ROC curve analysis, will be employed to determine the optimal cut-off value and calculate the net clinical benefit value for assessing clinical effectiveness. Finally, internal model validation will be conducted using bootstrapping. ETHICS AND DISSEMINATION: Approval from the Clinical Research Ethics Committee of The Third Affiliated Hospital of Wenzhou Medical University has been obtained. The research findings will be disseminated through presentations at scientific conferences and publication in peer-reviewed journals.

Genetic diversity analysis of the ORF5 gene in porcine reproductive and respiratory syndrome virus samples from South China.

To understand the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in South China, we collected 231 clinical samples from pigs with suspected PRRSV infection in Guangdong between 2007 and 2009. We found that 74 of 231 samples were positive by RT-PCR. The PCR products of the ORF5 gene of 35 isolates from different farms were sequenced and their DNA sequences were compared to 23 other PRRSV isolates in the GenBank. We found that the nucleotide similarity among all South China isolates ranged from 87.6% to 100%, and all belonged to the North American genotype. Most of them were classified into subgenotype I, but the rest mapped to subgenotypes III, V or VI. Those in subgenotypes I and III were found to be highly variable in the primary neutralising epitope (PNE) with a specific amino acid mutation (F39/L39→I39), and a few isolates in subgenotypes I and III isolates also had a mutation at L41 (L41→S41). PRRSV isolates in subgenotypes III, V and VI had less potential glycosylation sites than those in subgenotype I. Our data contribute to the understanding of molecular variation of PRRSV in South China.

Biologically active 1,25-dihydroxyvitamin D3 protects against experimental sepsis by negatively regulating the Toll-like receptor 4/myeloid differentiation primary response gene 88/Toll-IL-1 resistance-domain-containing adapter-inducing interferon-ß signaling pathway.

The hormonally active form of vitamin D (VD), 1,25­dihydroxyvitamin D3, has been reported to be a key immunoregulator in the reduction of inflammation. In this study, we investigated the effects of VD in an experimental sepsis cell model, and the underlying mechanisms. The sepsis cell model was first established in monocytes, isolated from newborns and healthy adults, which were stimulation with lipopolysaccharide (LPS). We observed that cell viability was significantly impaired in the monocytes after LPS stimulation, using a Cell Counting Kit­8 and trypan blue assays. Additionally, ELISA revealed that LPS stimulation significantly elevated the expression of interleukin 6 (IL­6), IL­10 and tumor necrosis factor­α (TNF­α). The expression levels of Toll­like receptor (TLR4), myeloid differentiation primary response gene 88 (MyD88), and Toll­IL­1 resistance­domain­containing adapter­inducing interferon­ß (TRIF) mRNA were also significantly elevated under LPS stimulation using reverse transcription­quantitative PCR and western blot analysis. VD treatment could significantly suppress the effects of LPS simulation on monocytes by negatively regulating inflammatory cytokines and TLR4/MyD88/TRIF signaling. Furthermore, a regulatory feedback mechanism was proposed to involve TLR4, MyD88 and TRIF in the sepsis cell model. In conclusion, VD may effectively decrease the release of inflammatory cytokines by inhibiting the TLR4/MyD88/TRIF signaling pathway, could be considered as a potential therapeutic agent for the treatment of sepsis.

Efficacy evaluation of three modified-live PRRS vaccines against a local strain of highly pathogenic porcine reproductive and respiratory syndrome virus.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is characterized by high fever and high mortality in pigs of all ages and has severely affected the pork industry of China in the last decades. This study evaluated the differences in protection conferred by of three MLV PRRS vaccines derived from classical PRRSV (C-PRRSV, VR2332) and HP-PRRSV (TJM-F92 and JXA1-R) against the field challenge of HP-PRRSV TP strain (JXA1-R like). Compared to the experiment pigs in control group which were vaccinated with normal saline, the MLV PRRS vaccinated pigs had milder clinical symptoms, fewer pathological changes in the lung, and higher body weight gain at the end of the study. However, piglets vaccinated with VR2332 had higher body temperature, higher viral loads and lower body weight gain when compared with piglets vaccinated with TJM-F92 or JXA1-R vaccines at the end of the study. The results demonstrated that VR2332 vaccine provided a limited cross-protection against the HP-PRRSV TP strain infection, while in contrast the TJM-F92 and JXA1-R vaccines provided more efficacious protection. The findings of this study could serve as a valuable reference guide for the pig producers and veterinarians when considering the choice of which type of MLV PRRS vaccines to protect their pig herds against field challenge by HP-PRRSV TP strain.

MicroRNA-135a is up-regulated and aggravates myocardial depression in sepsis via regulating p38 MAPK/NF-κB pathway.

MicroRNA-135a (miR-135a) is implicated in the pathological processes of several cancers. However, the roles and regulatory mechanism of miR-135a in sepsis-induced myocardial depression (MD) remain largely unknown. In this study, the serum of patients with sepsis and healthy controls was obtained. The miR-135a expression was then measured. Then lentiviruses (miR-135a mimic, inhibitor and scramble control) were transfected into BALB/c mice. After 4days of transfection, polymicrobial sepsis model was established by cecal ligation and puncture (CLP) surgery. The serum tumor-necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 were detected. Cardiac function was assessed. In addition, the protein expressions of p38 MAPK/NF-κB pathway-related proteins were determined. Besides, SB203580 and JSH-23, the inhibitors of p38 MAPK and NF-κB respectively, were used to treat the isolated ventricular myocytes in vitro. MiR-135a was significantly up-regulated in the serum of patients with sepsis. In comparison with CLP group, the concentrations of TNF-α, IL-1ß and IL-6 and the expressions of p-p38 and p-p65 in CLP+miR-135a mimic group were significantly increased, while markedly decreased in CLP+miR-135a inhibitor group. Moreover, EF, FS, LVdP/dt (max), LVdP/dt (min) and LVDP of CLP+miR-135a mimic group were all significantly decreased, while markedly increased in CLP+miR-135a inhibitor group. Besides, the increased expressions of p-p38 and p-p65, decreased expression of p-IKBα and the decreased percentage of contraction amplitude in miR-135a mimic group were markedly reversed by SB203580 or JSH-23 treatments. Up-regulation of miR-135a could aggravate sepsis-induced inflammation and myocardial dysfunction via activation of p38 MAPK/NF-κB pathway.

[Expression and characterization of porcine epidermal growth factor in Lactobacillus plantarum].

Epidermal growth factor (EGF) is an epithelial cell growth factor that can stimulate intestinal development, repair the damage of epidermal cells as well as reduce the incidence of pathogen infection and diarrhea. In order to produce a recombinant Lactobacillus plantarum (L. plantarum) expressing porcine epidermal growth factor (pEGF), we constructed a recombinant vector stably expressing pEGF in L. plantarum strains. First, L. plantarum strain Lp-1 was isolated from intestinal contents of piglets. Then the functional domain of pEGF, M6 precursor protein signal peptide (SP) and super strong constitutive promoter (SCP) were connected with the backbone plasmid pIAß8 to construct the recombinant vector that was transformed into Lp-1 by electroporation. Afterwards, pEGF was expressed in Lp-1 and detected by Tricine-SDS-PAGE and ELISA. After orally irrigated early-weaned BALB/c mice with the recombinant L. plantarum every morning and late afternoon for 10 consecutive days, body weight, villous height and crypt depth in the intestine were measured to examine the influence of the recombinant bacteria on the intestinal development of early-weaned mice in vivo. Finally, the results of our experiments demonstrated that pEGF was successfully expressed in Lp-1 and the molecular weight of pEGF was 6 kDa. In addition, the recombinant pEGF can enhanced the daily gain and exerted significance influence (P < 0.05) to the small intestinal morphology of early-weaned BALB/c mice. In conclusion, pEGF could be expressed in L. plantarum and the recombinant pEGF possesses good biological activity.

Inhibition of porcine reproductive and respiratory syndrome virus by Cecropin D in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Although vaccines are commercially available for the control of PRRSV infection, no vaccination regimen has been proved sustained success in terms of generating a protective immune response. Therefore, the development of novel antivirals is urgently needed. Antimicrobial peptides display broad-spectrum antimicrobial activities against bacteria, fungi, and viruses and play an important role in host innate immune response. Here, we tested whether Cecropin D (CD) could inhibit PRRSV infection and replication in vitro. The inhibitory effect of CD occurred during viral attachment and the early period of viral entry into Marc-145 cells. CD also attenuated virus-induced apoptosis during the late phase of PRRSV infection and suppressed virus release in Marc-145 cells, which might contribute to the inhibition of PRRSV infection. Similar inhibitory effects on PRRSV infection were also found with CD treatment in porcine alveolar macrophages, the major target cell type of PRRSV infection in pigs in vivo. These findings suggest that CD has the potential to develop a new therapeutic agent against PRRSV infection.

Recombinant adenovirus expression of FMDV P1-2A and 3C protein and its immune response in mice.

The purpose of this research was to develop a new type of recombinant adenovirus vaccine against foot-and-mouth disease (FMD) virus and confirm whether both 2A and 3C proteases can fully process FMDV P1 polyprotein into VP1 protein. We constructed and characterized recombinant adenoviruses, designated as rAd-P1, rAd-P1-2A, rAd-L-P1, rAd-L-2A, and rAd-3C. The construct was further confirmed by the enzyme digestion, polymerase chain reaction (PCR) testing, sequencing, and transfection. The infective replication-defective adenovirus rAd-3C was used to co-infect Vero cells with rAd-P1, rAd-P1-2A, rAd-L-P1, and rAd-L-2A, respectively; the supernatant of cracked cells was then collected to immunize mice. Notably, the supernatant of the Vero cells co-infected with rAd-L-2A/rAd-3C or rAd-P1-2A/rAd-3C was shown to induce specific humoral and cellular immune responses in the study animals. Most importantly, we confirmed that 3C protease successfully processed P1 polyprotein into VP1 protein without L protein only under the precondition of 2A protease; however, P1-2A and L-2A polyprotein were not fully processed. Our study highlighted that P1 polyprotein cannot be fully processed into structural protein VP1 only under the condition of 2A and 3C proteases. The results also suggest that the recombinant adenovirus may be an attractive candidate as a vaccine for preventing the disease associated with FMD virus infection.

Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris.

To express the antimicrobial peptide cecropin D in Pichia pastoris and determine the activity of the expressed product, four oligonucleotide fragments were synthesized in accordance with the available cecropin D sequences and a codon bias suitable for Pichia pastoris. Sequence fragments were phosphorylated, annealed, linked and cloned into the expression vector pGAPZαA and the yeast α-mating factor signal peptide was used as the signal sequence. The P. pastoris SMD1168 cells were transformed by electroporation using the constructed recombinant plasmid pGAPZαA-cecropin D. We were able to demonstrate by PCR that the cecropin D sequence had integrated into the P. pastoris genome. The expressed and secreted product was identified using Tricine-SDS-PAGE. Antibacterial activity was demonstrated using an agarose diffusion test and turbidimetry. The molecular mass of the recombinant cecropin D was estimated to be 3,900 Da. The recombinant cecropin D exhibited antibacterial activity for both Gram-positive and Gram-negative bacteria, suggesting that cecropin D was successfully expressed in P. pastoris. This approach holds great promise for antibacterial drug development.

Isolation and Phylogenetic Analysis of H1N1 Swine Influenza Virus from Sick Pigs in Southern China.

Two swine influenza (SI) H1N1 virus was isolated from a pig during a severe outbreak of respiratory disease in south China. The two H1N1 influenza viruses were classical SI virus. A/swine/Guangdong/L6/09 is classical SI virus of recent years, which is of the main SI virus in China. Howere, A/swine/Guangdong/L3/09 was closet to A/swine/Iowa/1931, which was the first isolated SI virus and had demonstrated significant pathogenicity in animal models. The results of phylogenetic analysis of A/swine/Guangdong/L3/09 showed a close relationship with the 1918 pandemic virus. The results suggested that the previous SI virus appeared again. Whether, it brought a new pandemic to pigs deserves more attention.

Targeted correction of single-base-pair mutations with adeno-associated virus vectors under nonselective conditions.

Recombinant adeno-associated virus (rAAV) vectors possess the unique ability to introduce genetic alterations at sites of homology in genomic DNA through a mechanism thought to predominantly involve homologous recombination. We have investigated the efficiency of this approach using a mutant enhanced green fluorescent protein (eGFP) fluorescence recovery assay that facilitates detection of gene correction events in living cells under nonselective conditions. Our data demonstrate that rAAV infection can correct a mutant eGFP transgene at an efficiency of 0.1% in 293 cells, as determined by fluorescence-activated cell-sorting analysis. Gene repair was also confirmed using clonal expansion of GFP-positive cells and sequencing of the eGFP transgene. These results support previous findings demonstrating the efficacy of rAAV for gene targeting. In an effort to improve gene-targeting efficiencies, we evaluated several agents known to increase rAAV transduction (i.e., expression of an expressed gene), including genotoxic stress and proteasome inhibitors, but observed no correlation between the level of gene repair and rAAV transduction. Interestingly, however, our results demonstrated that enrichment of G(1)/S-phase cells in the target population through the addition of thymidine moderately (approximately 2-fold) increased gene correction compared to cells in other cell cycle phases, including G(0)/G1, G(1), and G(2)/M. These results suggest that the S phase of the cell cycle may more efficiently facilitate gene repair by rAAV. Transgenic mice expressing the mutant GFP were used to evaluate rAAV targeting efficiencies in primary fetal fibroblast and tibialis muscles. However, targeting efficiencies in primary mouse fetal fibroblasts were significantly lower (approximately 0.006%) than in 293 cells, and no correction was seen in tibialis muscles following rAAV infection. To evaluate the molecular structures of rAAV genomes that might be responsible for gene repair, single-cell injection studies were performed with purified viral DNA in a mutant eGFP target cell line. However, the failure of direct cytoplasm- or nucleus-injected rAAV DNA to facilitate gene repair suggests that some aspect of intracellular viral processing may be required to prime recombinant viral genomes for gene repair events.