Coxsackievirus and adenovirus receptor inhibits tilapia lake virus infection via binding to viral segment 8 and 10 encoded protein.

The global aquaculture industry of tilapia (Oreochromis niloticus) has been significantly impacted by the emergence of tilapia lake virus (TiLV). However, effective prevention and control measures are still not available due to a lack of unclear pathogenesis of TiLV. Our previous transcriptome found that coxsackievirus and adenovirus receptor (CAR) was in response to TiLV infection in tilapia. To explore the potential function of OnCAR, the effect of OnCAR on TiLV proliferation was analyzed in this study. The OnCAR open reading frame (ORF) sequence of tilapia was 516 bp in length that encoded 171 amino acids with an Ig-like domain and transmembrane region. The OnCAR gene showed widespread expression in all investigated tissues, with the highest levels in the heart. Moreover, the OnCAR gene in the liver and muscle of tilapia exhibited dynamic expression levels upon TiLV challenge. Subcellular localization analysis indicated that OnCAR protein was mainly localized on the membrane of tilapia brain (TiB) cells. Importantly, the gene transcripts, genome copy number, S8-encoded protein, cytopathic effect, and internalization of TiLV were obviously decreased in the TiB cells overexpressed with OnCAR, indicating that OnCAR could inhibit TiLV replication. Mechanically, OnCAR could interact with viral S8 and S10-encoded protein. To the best of our knowledge, OnCAR is the first potential anti-TiLV cellular surface molecular receptor discovered for inhibiting TiLV infection. This finding is beneficial for better understanding the antiviral mechanism of tilapia and lays a foundation for establishing effective prevention and control strategies against tilapia lake virus disease (TiLVD).

Construction of Streptococcus agalactiae sialic acid mutant and evaluation of its potential as a live attenuated vaccine in Nile tilapia (Oreochromis niloticus).

AIMS: This study aimed to develop a live attenuated vaccine as an effective approach to prevent streptococcosis in tilapia (Oreochromis niloticus). METHODS AND RESULTS: We eliminated the virulence factor, sialic acid (Sia) encoded by the neuA-D gene cluster from the Group B Streptococcus (Streptococcus agalactiae, GBS) strain WC1535, to construct Sia-deficient S. agalactiae (ΔSia) mutant by homologous recombination. Results showed that the ΔSia mutant had higher adherence to HEp-2 cells and lower resistance to RAW264.7 cell phagocytosis than the wild-type S. agalactiae. The virulence of the ΔSia mutant to tilapia dramatically decreased with no virulence recovery. The relative percent survivals (RPSs) were 50.00% and 54.50% at 30 days when challenged at the wild-type WC1535 doses of 1.0 × 107 and 5.0 × 107  CFU fish-1 , respectively, via intraperitoneal (IP) injection. The tilapia vaccinated via IP injection with the ΔSia mutant induced strong antibody agglutination titers. The expression of IL-1ß, TNF-α, MHC-Iα, and MHC-IIß could be enhanced in the intestine, spleen, and head kidney for tilapia administered with the ΔSia mutant. CONCLUSIONS: GBS Sia plays a critical role in adherence to HEp-2 cells and resistance to the immune clearance of RAW264.7 cells. Moreover, the ΔSia mutant is a safe, stable, and immunogenic live attenuated vaccine candidate to protect tilapia against GBS infection. SIGNIFICANCE AND IMPACT OF STUDY: The results offer more evidence of the importance of Sia in GBS and may be instructive in the control of tilapia streptococcosis.

Flow-through flap with wrist epithelial branch of ulnar artery for repair of finger soft tissue defect: a case series.

OBJECTIVE: To evaluate the surgical technique and the efficacy of flow-through flap with a wrist epithelial branch of the ulnar artery to repair a finger soft tissue defect. METHODS: Between June 2015 and December 2017, 12 cases of soft tissue defects of fingers and injured digital artery were repaired by flow-through flap with wrist epithelial branch of the ulnar artery, including 7 males and 5 females (age range: 18-45 years old, average age: 23.6 years old). The causes of injury included electric saw injury in 7 cases, and machine crush injury in 5 cases. 5 cases were combined with tendon injury, 4 cases with fracture, 12 cases with vessel injury and 2 cases with nerve injury. The area range of the flap was 3.0 cm ×1.8 cm to 6.0 cm ×3.0 cm. The length of the pedicles of the flaps ranged from 2.3 cm to 4.7 cm, with an average length of 3.7 cm. The donor sites were sutured directly in 10 cases, and 2 cases were repaired with a full-thickness skin graft from the ilioinguinal region. Flow-through anastomoses of the distal and proximal end of the wrist epithelial branch of the ulnar artery to the distal and proximal end of the digital artery were created, so as to connect the vessels and reach the physiologic state of blood supply. RESULTS: All flaps and skin grafts survived after operation, and all wounds healed at I phase. All patients were followed up 6-12 months (mean: 9 months). The flaps exhibited smooth appearance and soft texture, similar to that of the normal surrounding skin. At last follow-up, the two-point distance of flaps was 9-15 mm (mean: 11 mm). According to the assessment of upper limb function issued by the Hand Surgery Society of Chinese Medical Association, the hand function was excellent in 10 cases, and good in 2 cases. The ulnar wrist donor areas only had linear scar. CONCLUSION: Flow-through flap with wrist epithelial branch of ulnar artery exhibits strength in a concealed donor site, reliable blood supply, and simple operation. Flow-through method can be used to repair a broken or defective digital artery in I stage. It is a good method to repair a soft tissue defect of fingers.

Downregulation of miR-20b-5p facilitates Mycobacterium tuberculosis survival in RAW 264.7 macrophages via attenuating the cell apoptosis by Mcl-1 upregulation.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). The interaction between Mtb and macrophages, which is regulated by microRNAs, determines the development of TB. However, the function of microRNA-20b-5p (miR-20b-5p) in RAW 264.7 macrophages against Mtb remains unknown. In this study, we analyzed the expression level of miR-20b-5p in macrophage responses to Mtb infection and exosomes derived from macrophages after Mtb infection. MiR-20b-5p mimics and inhibitor were, respectively, transfected to evaluate the effect of miR-20b-5p on Mtb and macrophages. In addition, the targets of miR-20b-5p were predicted by a bioinformatics analysis. The macrophages were respectively transfected with miR-20b-5p mimics and inhibitor to determine the messenger RNA expression levels of the targets by reverse transcription-polymerase chain reaction assay. The results revealed that the miR-20b-5p expression level was decreased in the infected macrophages at different times. MiR-20b-5p was shown in the exosomes released from macrophages infected with Mtb. Upregulation of the miR-20b-5p level suppressed the survival of Mtb in macrophages, while downregulation of the miR-20b-5p level enhanced the survival of Mtb in macrophages. Overexpression of miR-20b-5p decreased the cell viability and induced apoptosis in Mtb-infected macrophages, while underexpression of miR-20b-5p increased the cell vitality and attenuated apoptosis in Mtb-infected macrophages. The bioinformatics analysis revealed that Mcl-1 was a target of miR-20b-5p. MiR-20b-5p negatively regulated the expression of Mcl-1. Overall, this study is the first to demonstrate the effect of miR-20b-5p on Mtb infection and present miR-20b-5p and exosomes as the potential therapeutic targets of TB.

Molecular characterization and function analysis of three RIG-I-like receptor signaling pathway genes (MDA5, LGP2 and MAVS) in Oreochromis niloticus.

The recognition of microbial pathogens, which is mediated by pattern recognition receptors (PRRs), is critical to the initiation of innate immune responses. In the present study, we isolated the full-length cDNA and genomic DNA sequences of the MDA5, LGP2 and MAVS genes in Nile tilapia, termed OnMDA5, OnLGP2 and OnMAVS. The OnMDA5 gene encodes 974 amino acids and contains two caspase-associated recruitment domains (CARDs), a DExDc domain (DExD/H box-containing domain), a HELICc (helicase superfamily C-terminal) domain and a C-terminal regulatory domain (RD). The OnLGP2 gene encodes 679 amino acids and contains a DExDc, a HELICc and an RD. The OnMAVS gene encodes 556 amino acids and contains a CARD, a proline-rich domain, a transmembrane helix domain and a putative TRAF2-binding motif (269PVQDT273). Phylogenetic analyses showed that all three genes from Nile tilapia were clustered together with their counterparts from other teleost fishes. Real-time PCR analyses showed that all three genes were constitutively expressed in all examined tissues in Nile tilapia. OnMDA5 presented the highest expression level in the blood and the lowest expression level in the liver, while OnMAVS presented the highest expression level in the kidney. The highest expression level of OnLGP2 was detected in the liver. An examination of the expression patterns of these RIG-I-like receptors (RLRs) during embryonic development showed that the highest expression levels of OnMDA5 occurred at 2 days postfertilization (dpf), and the expression significantly decreased from 3 to 8 dpf. The expression levels of OnLGP2 significantly increased from 4 to 8 dpf. The expression levels of OnMAVS mRNA were stable from 2 to 8 dpf. Upon stimulation by intraperitoneal injection of Streptococcus agalactiae, the expression levels of OnMDA5 were first downregulated and then upregulated in the blood, gill and spleen. In the intestine and kidney, the expression of OnMDA5 was first upregulated, then downregulated, and then upregulated again. The expression of OnLGP2 was upregulated in the kidney and intestine, and the expression of OnMAVS was upregulated in the spleen. Overexpression of OnMAVS increased NF-κB activation in 293 T cells (p < 0.05), and after cotransfection with OnMDA5, the OnMAVS-dependent NF-κB activation was slightly increased (p > 0.05), after cotransfection with OnLGP2, the OnMAVS-dependent NF-κB activation was significantly decreased (p < 0.05). These findings suggest that, although the deduced protein structure of OnMDA5 is evolutionarily conserved with the structures of other RLR members, its signal transduction function is markedly different. The results also suggest that OnLGP2 has a negative regulatory effect on the OnMAVS gene. OnMDA5 and OnMAVS were uniformly distributed throughout the cytoplasm in 293 T cells, whereas OnLGP2 was distributed throughout the cytoplasm and nucleus. These results are helpful for clarifying the innate immune response against bacterial infection in Nile tilapia.

miR-192 suppresses T follicular helper cell differentiation by targeting CXCR5 in childhood asthma.

The aim of this study was to investigate the role of miR-192 in differentiation of T follicular helper cells in childhood asthma. Blood samples were taken from eighteen children with acute asthma attacks and fifteen healthy children (HC). Quantitative real-time PCR and Western blotting were used to detect the expression levels of miR-192, C-X-C chemokine receptor type 5 (CXCR5), B-cell lymphoma 6 (BCL-6) and inducible T-cell costimulator (ICOS). The flow cytometry was performed to detect the proportion of CD4 + CXCR5+ Tfh cells on CD4 + T lymphocytes. The enzyme-linked immunosorbent assay (ELISA) was carried out to determine the plasma concentrations of total IgE and IL-21. The effect of miR-192 on the T follicular helper cells differentiation by targeting CXCR5 was determined by dual-luciferase reporter assay. Children with asthma had lower levels of miR-192 than HC. The proportion of CD4 + CXCR + Tfh cells was significantly higher in the acute asthma group than HC. Similarly, the plasma concentration of total IgE and IL-21 in the acute group markedly increased compared with the HC, and IgE concentration was positively correlated with the proportion of CD4 + CXCR5 + Tfh cells. Furthermore, the expression levels of CXCR5, Bcl-6 and ICOS were significantly higher in the acute group than in the HC. While the proportion of CD4 + CXCR5 + Tfh cells, IL-21, CXCR5, Bcl-6 and ICOS were obviously lower in the CD4 + T cells transfected with miR-192 plasmid than that in miR-192 + CXCR5 group and control group. In conclusion, miR-192 blocks the activation pathway of Tfh cells by targeting CXCR5, which is a reasonable cellular target for therapeutic intervention.

Antibacterial effects of bacteriocins isolated from Lactobacillus rhamnosus (ATCC 53103) in a rabbit model of knee implant infection.

Infection following orthopedic surgery is a major complication that can have serious implications on patient health. The present study aimed to investigate the antibacterial effects of bacteriocins obtained from Lactobacillus rhamnosus on a rabbit model of Staphylococcus aureus infection following knee replacement surgery. Blood samples were collected 1, 2, 3, 4 and 5 days after bacteriocin injection, and C-reactive protein (CRP) and interleukin (IL)-6 levels were measured using commercial ELISA kits. In addition, biofilm formation was evaluated by fluorescence microscopy. Bacteriocins were identified to exhibit significant inhibitory effects on Staphylococcus aureus biofilm formation, and on CRP and IL-6 levels in the serum, following surgery and infection (all P<0.05 vs. the control group). The results of the present study indicate that bacteriocins are a potential agent for the prevention of orthopedic postoperative infections.