A novel glycine-rich peptide from Zophobas atratus, coleoptericin B, targets bacterial membrane and protects against Klebsiella pneumoniae-induced mastitis in mice.

OBJECTIVES: The growing occurrence of bacterial resistance has spawned the development of novel antimicrobial agents. Antimicrobial peptides, a class of small molecules with antimicrobial activity, have been regarded as the ideal alternatives to antibiotics. METHODS: In this study, we amplified a new type of Zophobas atratus coleoptericin (denoted coleoptericin B) through rapid amplification of cDNA ends (RACE) PCR and expressed recombinant Z. atratus coleoptericin B (rZA-col B) by prokaryotic expression. Subsequently, we evaluated the antimicrobial effect and biocompatibility of rZA-col B in vivo, investigated its antimicrobial mechanism, and assessed its therapeutic effect in a murine model of mastitis caused by MDR Klebsiella pneumoniae. RESULTS: The in vivo studies demonstrated that rZA-col B possesses broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. It exhibited less than 1.5% haemolysis and 10% cytotoxicity, even at a concentration of 128 µM. Additionally, rZA-col B had a minimal risk of inducing drug resistance. Furthermore, rZA-col B could disrupt the integrity of bacterial membranes, induce membrane permeabilization and ultimately lead to bacterial death. Importantly, rZA-col B also alleviated mastitis caused by MDR K. pneumoniae in a murine model by enhancing bacterial clearance, reducing neutrophil infiltration, decreasing TNF-α and IL-1ß expression, and protecting the mammary barrier. CONCLUSIONS: rZA-col B may be a promising antibacterial agent to combat MDR bacterial infection.

Non-invasive assessment of liver fibrosis by serum metabolites in non-human primates and human patients.

Liver fibrosis, a rising cause of chronic liver diseases, could eventually develop into cirrhosis and liver failure. Current diagnosis of liver fibrosis relies on pathological examination of hepatic tissues acquired from percutaneous biopsy, which may produce invasive injuries. Here, for non-invasive assessment of liver fibrosis, we applied comparative multi-omics in non-human primates (rhesus macaques) and subsequent serum biopsy in human patients. Global transcriptomics showed significant gene enrichment of metabolism process, in parallel with oxidative stress and immune responses in fibrotic primates. Targeted metabolomics were concordant with transcriptomic patterns, identifying elevated lipids and porphyrin metabolites during hepatic fibrosis. Importantly, liquid biopsy results validated that specific metabolites in the serum (e.g., biliverdin) were highly diagnostic to distinguish human patients from healthy controls. Findings describe the interconnected transcriptional and metabolic network in primate liver fibrosis and provide potential indices for non-invasive detection of liver fibrosis in humans.

A Novel ß-Hairpin Peptide Z-d14CFR Enhances Multidrug-Resistant Bacterial Clearance in a Murine Model of Mastitis.

The widespread prevalence of antimicrobial resistance has spawned the development of novel antimicrobial agents. Antimicrobial peptides (AMPs) have gained comprehensive attention as one of the major alternatives to antibiotics. However, low antibacterial activity and high-cost production have limited the applications of natural AMPs. In this study, we successfully expressed recombinant Zophobas atratus (Z. atratus) defensin for the first time. In order to increase the antimicrobial activity of peptide, we designed 5 analogues derived from Z. atratus defensin, Z-d13, Z-d14C, Z-d14CF, Z-d14CR and Z-d14CFR. Our results showed that Z-d14CFR (RGCRCNSKSFCVCR-NH2) exhibited a broad-spectrum antimicrobial activity to both Gram-positive bacteria and Gram-negative bacteria, including multidrug-resistant bacteria. It possessed less than 5% hemolysis and 10% cytotoxicity, even at a high concentration of 1 mg/mL. Antimicrobial mechanism studies indicated that Z-d14CFR performed antimicrobial effect via inhibiting biofilm formation, disrupting bacterial membrane integrity and inducing cellular contents release. Furthermore, Z-d14CFR showed a great therapeutic effect on the treatment of multidrug-resistant Escherichia coli (E. coli) infection by enhancing bacterial clearance, decreasing neutrophils infiltration and the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) in a murine model of mastitis. Our findings suggest that Z-d14CFR could be a promising candidate against multidrug-resistant bacteria.

Therapeutic Effect of Darkling Beetle (Zophobas morio) Hemolymph on Skin Thermal Injury in Mice Infected by Staphylococcus haemolyticus.

Staphylococci are the most common pathogens isolated from skin infections in livestock or companion animals. Antibiotic therapy is the best treatment for infections, but local or systemic use of antimicrobials increases the risk of bacterial resistance. Insects are rich in antimicrobial peptides, which can reduce bacterial resistance and can be used to treat bacterial infections after skin burns. We propose that the use of the darkling beetle (Z. morio) hemolymph to treat skin infections in mice by Staphylococcus haemolyticus is one of the alternatives. Z. morio hemolymph alleviated the increase in wound area temperature in mice with a skin infection, reduced the bacterial load of the wound, and accelerated the wound healing speed significantly. Pathological sections showed that Z. morio hemolymph can significantly reduce inflammatory cell infiltration, and promote skin tissue repair. Real-time fluorescent quantitative polymerase chain reaction (PCR) revealed that the Z. morio hemolymph can significantly reduce the levels of pro-inflammatory cytokines, including interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and chemokine interleukin-8 (IL-8). Our findings suggest that Z. morio antibacterial hemolymph can promote wound contraction, relieve local inflammatory responses and promote wound healing in mice infected with a heat injury, which has a positive therapeutic effect and enormous potential for skin thermal injury.

MNDR v3.0: mammal ncRNA-disease repository with increased coverage and annotation.

Many studies have indicated that non-coding RNA (ncRNA) dysfunction is closely related to numerous diseases. Recently, accumulated ncRNA-disease associations have made related databases insufficient to meet the demands of biomedical research. The constant updating of ncRNA-disease resources has become essential. Here, we have updated the mammal ncRNA-disease repository (MNDR, http://www.rna-society.org/mndr/) to version 3.0, containing more than one million entries, four-fold increment in data compared to the previous version. Experimental and predicted circRNA-disease associations have been integrated, increasing the number of categories of ncRNAs to five, and the number of mammalian species to 11. Moreover, ncRNA-disease related drug annotations and associations, as well as ncRNA subcellular localizations and interactions, were added. In addition, three ncRNA-disease (miRNA/lncRNA/circRNA) prediction tools were provided, and the website was also optimized, making it more practical and user-friendly. In summary, MNDR v3.0 will be a valuable resource for the investigation of disease mechanisms and clinical treatment strategies.

Recombinant Erns-E2 protein vaccine formulated with MF59 and CPG-ODN promotes T cell immunity against bovine viral diarrhea virus infection.

To obtain an effective vaccine candidate against bovine viral diarrhea virus (BVDV) disease which causes great economical loss in cattle industries, recombinant Erns-E2 protein vaccine containing MF59 and CPG-ODN adjuvants was prepared and assessed in this study. The recombinant plasmid (pET32a-Erns-E2) was constructed and transformed into BL21 (DE3) cells to produce Erns-E2 protein. We immunized mice with the MF59-and CPG-ODN-adjuvanted recombinant Erns-E2 protein, E2 protein, or Erns protein, respectively. To evaluate immunogenicity and efficacy of a vaccine-adjuvant combination, mice were challenged with BVDV BJ175170 strain after immunization. All adjuvanted vaccines elicited detectable humoral and cellular immune responses, the BVDV-specific antibody titers as well as interleukin 4 (IL-4) levels in sera of mice immunized with the recombinant Erns-E2 protein were higher than in those of mice immunized with either the recombinant Erns or E2 protein. Besides, immunization with the Erns-E2 vaccines induced higher percentage of CD4+IFN-γ+, CD8+IFN-γ+ T cells and CD3+TNF-α+ T cells compared with the other vaccines. More protective efficacy against BVDV infection was acquired in the mice treated with the recombinant Erns-E2 protein, as shown by a reduction of viremia and slight pathological changes compared with both the control mice and the other vaccinated mice. Our findings suggest that the use of the recombinant Erns-E2 protein vaccine formulated with MF59 and CPG-ODN adjuvants enhances T cell responses and viral control, which warrants the Erns-E2 protein vaccine-adjuvant combination could be as a vaccine strategy to against BVDV.