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Background Antimicrobial peptides (AMPs) have attracted extensive attention in various fields. Gloverin is a group of AMPs derived from Lepidoptera insects. In this study, a novel gloverin CpGlv was identified through sequence alignment. The open reading frame (ORF) of mature CpGlv, devoid of its signal peptide region, was recombinantly expressed in Escherichia coli . The antibacterial activity of the fusion protein CpGlv against Staphylococcus aureus ( S. aureus ) was then assessed by determining the minimum inhibitory concentration (MIC) and using scanning electron microscopy (SEM) to observe the ultrastructure of S. aureus cells. Furthermore, the effects of CpGlv on the cell wall and membrane permeability of S. aureus were evaluated by detecting the leakage of intracellular nucleic acids, proteins, alkaline phosphatase (ALP), and β-galactosidase, as well as through the propidium iodide (PI) staining experiment. Finally, the influence of CpGlv on the S. aureus biofilm formation was investigated using the crystal violet staining assay. Results The results showed that the ORF of CpGlv contained a nucleotide sequence of 591 bp. The expressed fusion protein CpGlv had a molecular weight of approximately 24 kDa. The purified CpGlv exhibited significant antibacterial activity against S. aureus , with a MIC of 40 µg/mL. 2×MIC of CpGlv caused S. aureus cells to exhibit adhesion, swelling, deformation, and even rupture. Further, CpGlv resulted in the leakage of intracellular nucleic acids, proteins, ALP, and β-galactosidase in S. aureus . After PI staining, there was intracellular fluorescence intensity in S. aureus treated by CpGlv. Additionally, 1/4×MIC of CpGlv significantly reduced the biofilm formation of S. aureus . Conclusion A novel gloverin CpGlv was identified, expressed, and purified, and the recombinant CpGlv exhibited antimicrobial activity against S. aureus by increasing the cell membrane and wall permeability, and inhibiting the biofilm formation, laying a foundation for in-depth research on the antibacterial mechanism and application of CpGlv.

Transmissible gastroenteritis (TGE), caused by the TGE virus (TGEV), is a highly contagious enteric disease characterized by vomiting, dehydration, and watery diarrhea. It mainly endangers piglets within two weeks of age, with a 100% mortality rate, inflicting severe economic losses on the global swine industry. Since enteric tropism of the virus and mucosa serves as the first line of defense against viral invasion, an oral vaccine inducing sufficient secretory immunoglobulin A (SIgA) antibodies in animals should be developed. Being a generally recognized as safe (GRAS) microorganism, Bacillus subtilis can form endospores under extreme environmental conditions, which confer resistance to the hostile gastric environment and have been widely employed as delivery vehicles for oral vaccines owing to their immunoadjuvant activity and non-specific antidiarrheal effects. In this study, the AD antigenic epitope of the TGEV S protein was selected as the immunogen. The mature peptide of the B subunit of the heat-labile enterotoxin from enterotoxigenic Escherichia coli served as a mucosal adjuvant, and B. subtilis WB800N was used as the delivery host to construct the recombinant strain pHT43-LTB-AD/WB800N. After confirming the successful expression of the target protein, oral immunization was performed using mice as a model. The results demonstrated that this recombinant strain induced robust mucosal, humoral, and cellular immunity, along with considerable levels of neutralizing antibodies. These findings indicate that recombinant B. subtilis could serve as an oral vaccine candidate to combat TGEV infections.

Background Porcine Transmissible Gastroenteritis Virus (TGEV) is widespread and frequently involved in co-infections, leading to high mortality rates in piglets and significantly constraining the swine industry. Based on the genetic sequence of TGEV strain HB-1, we designed and constructed a eukaryotic expression plasmid, pCAGGS-TGEV-CTD, encoding the CTD gene of TGEV. The recombinant CTD protein was successfully expressed and purified using the HEK-293 F suspension cell expression system. Balb/c mice were immunized with the purified CTD protein. Results Upon achieving a serum antibody titer of 10⁵, antigen-specific memory B cells were isolated using single-cell flow cytometry sorting. A total of 83 matched antibody light- and heavy-chain gene pairs were obtained via single-cell RT-PCR. These gene pairs were cloned into pCAGGS eukaryotic expression vectors containing murine constant light (CL) and heavy (CH) chain regions, resulting in the construction of 42 recombinant plasmids for antibody expression. Following transfection into HEK-293T cells, antibody supernatants were harvested at 48 h post-transfection. ELISA screening identified 18 antibodies reactive to the TGEV CTD protein, five high-titer antibodies were confirmed to cross-react with TGEV virions through indirect immunofluorescence assay (IFA). Further neutralization assays demonstrated that all five monoclonal antibodies exhibited in vitro neutralizing activity, effectively inhibiting TGEV infection and replication in swine testicular (ST) cells. Conclusion This study successfully generated TGEV-specific antibodies, providing critical support for fundamental research on TGEV research and the development of clinical diagnostic reagents.

Objective: This study aimed to identify genes regulated by cyclin dependent kinases 5 (CDK5) that participate in hair pigmentation in mice.Methods: The mRNA expression profiles of skin samples from CDK5-knockdown mice were constructed using high-throughput RNA sequencing and compared with those of wild-type mice.Results: In total, 8,002 known genes were differentially expressed between CDK5-knockdown and wild-type mice. Of these, 3,658 were upregulated and 4,344 were downregulated in the skin of CDK5-knockdown mice. An additional 318 previously unknown genes were also differentially expressed, with 171 downregulated and 147 upregulated genes in the skin of CDK5-knockdown mice. Of the known genes expressed in mouse skin, 80 were associated with hair color, with 61 showing lower expression and 19 exhibiting higher expression in skin of CDK5-knockdown mice. Importantly, the expression of the tyrosinase-related protein 1 (TYRP1) and the calcium signaling pathway were also found to be regulated by CDK5, suggesting that pigmentation is regulated by CDK5 via the calcium signaling pathway and TYRP1.Conclusion: The transcriptome profiles obtained from the skin of CDK5-knockdown mice compared to wild-type mice provide a valuable resource to help understand the mechanism by which CDK5 regulates melanogenesis in mice and other animals.

A novel circovirus called porcine circovirus type 4 (PCV4) was recently detected in pigs suffering from severe clinical diseases in Hunan province, China. There are few reports on the origin and evolution of PCV4, although some researchers have conducted epidemiological investigations of PCV4 and found that PCV4 is widespread in pigs. Based on the previous study, we detected PCV2 in farmed foxes and raccoon dogs with reproductive failure. To explore whether the PCV4 genome also exists in fur animals, we detected 137 cases admitted from fur animal farms in Hebei China between 2015 and 2020, which were characterized by inappetence, lethargy, depression, abortion, and sterility. The overall infection rate of PCV4 was 23.36% (32/137), including 20.37% (22/108) for raccoon dogs, 18.75% (3/16) for foxes, and 53.85% (7/13) for minks. Finally, five raccoon dog-origin PCV4 strains and one fox-origin PCV4 strain were sequenced in our study, whose nucleotide identities with other representative PCV4 strains varied from 96.5% to 100%. Phylogenetic analysis based on the complete genomes of PCV4 strains indicated a close relationship with those of PCV4 strains identified from pigs. To our knowledge, this is the first study to detect PCV4 in fur animals. Interestingly, we also identified PCV4 in a mixed farm (feeding pigs and raccoon dogs at the same time). In summary, our findings extend the understanding of the molecular epidemiology of PCV4 and provide new evidence for its cross-species transmission.

Background Porcine circovirus type 2 (PCV2) is a major emerging virus of porcine circovirus-associated disease (PCVAD), which has brought huge economic losses to the global pig industry. Pigs are well known as the natural reservoir of PCV2. Recently, many researchers have revealed PCV2 could infect many other mammals like mice, calves, minks, dogs and goats. In 2018, our laboratory has admitted six cases of raccoon dogs from Qinhuangdao city of China, which were characterized by inappetence, lethargy, depression, abortion, and sterility. Results At last, six raccoon dog-origin PCV2 strains were isolated in this study. Pairwise-sequence comparisons demonstrated that the six raccoon dog-origin PCV2 strains shared a nucleotide similarity of 92.1–99.8% among 40 PCV2 representative strains. Phylogenetic analysis indicated these PCV2 isolates belonged to Chinese epidemic genotypes PCV2b and PCV2d. And aborted or sterile symptom was significantly associated with PCV2 infection in raccoon dogs by the chi-square test (χ 2  = 87.3, p  < 0.001). The retrospective study revealed that raccoon dog-origin PCV2 strains shared 100% sequence similarity with the PCV2 stains isolated from pig farms around these raccoon dog farms, respectively. Conclusion In this study, the first supported evidence of PCV2 prevalence in raccoon dog farms of China was documented. PCV2 may be one of the most significant causative agents resulting in the reproductive failure of farmed raccoon dogs, implying that PCV2 could transmit from pigs to raccoon dogs. That indicated that PCV2 cross-species transmission will be a serious threat to China’s fur animal farming industry.

Canine distemper virus (CDV) is a major pathogen not only in raccoon dogs but also in a variety of carnivorous animals, including domesticated animals, particularly if they have not been vaccinated. In this study, a wild-type strain of CDV was isolated from lung tissue from a raccoon dog kept at a fur farm in Jilin Province, China. Cytopathic effects typical of CDV infection were observed after three blind passages in Vero cells, yielding a virus titer of 10 4.6 TCID 50 /mL. Virus identification was carried out by RT-PCR, immunofluorescence, electron microscopy, and genome sequencing. The results showed that the isolated virus, termed the SY strain, corresponded to the Asia-1 genotype of CDV and has a genome of 15,690 nucleotides. This represents the first complete nucleotide sequence of a CDV strain circulating in raccoon dogs in China.

Objective: This study aimed to identify genes regulated by cyclin dependent kinases 5 (CDK5) that participate in hair pigmentation in mice.Methods: The mRNA expression profiles of skin samples from CDK5-knockdown mice were constructed using high-throughput RNA sequencing and compared with those of wild-type mice.Results: In total, 8,002 known genes were differentially expressed between CDK5-knockdown and wild-type mice. Of these, 3,658 were upregulated and 4,344 were downregulated in the skin of CDK5-knockdown mice. An additional 318 previously unknown genes were also differentially expressed, with 171 downregulated and 147 upregulated genes in the skin of CDK5-knockdown mice. Of the known genes expressed in mouse skin, 80 were associated with hair color, with 61 showing lower expression and 19 exhibiting higher expression in skin of CDK5-knockdown mice. Importantly, the expression of the tyrosinase-related protein 1 (TYRP1) and the calcium signaling pathway were also found to be regulated by CDK5, suggesting that pigmentation is regulated by CDK5 via the calcium signaling pathway and TYRP1.Conclusion: The transcriptome profiles obtained from the skin of CDK5-knockdown mice compared to wild-type mice provide a valuable resource to help understand the mechanism by which CDK5 regulates melanogenesis in mice and other animals.

Objective: This study aimed to identify genes regulated by cyclin dependent kinases 5 (CDK5) that participate in hair pigmentation in mice. Methods: The mRNA expression profiles of skin samples from CDK5-knockdown mice were constructed using high-throughput RNA sequencing and compared with those of wild-type mice. Results: In total, 8,002 known genes were differentially expressed between CDK5-knockdown and wild-type mice. Of these, 3,658 were upregulated and 4,344 were downregulated in the skin of CDK5-knockdown mice. An additional 318 previously unknown genes were also differentially expressed, with 171 downregulated and 147 upregulated genes in the skin of CDK5-knockdown mice. Of the known genes expressed in mouse skin, 80 were associated with hair color, with 61 showing lower expression and 19 exhibiting higher expression in skin of CDK5-knockdown mice. Importantly, the expression of the tyrosinase-related protein 1 (TYRP1) and the calcium signaling pathway were also found to be regulated by CDK5, suggesting that pigmentation is regulated by CDK5 via the calcium signaling pathway and TYRP1. Conclusion: The transcriptome profiles obtained from the skin of CDK5-knockdown mice compared to wild-type mice provide a valuable resource to help understand the mechanism by which CDK5 regulates melanogenesis in mice and other animals.

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysac- charide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles exist- ing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psi matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in im- proved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disas- sembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.