This study reports the whole-genome sequence of Lactiplantibacillus plantarum cqf-43 isolated from healthy sow feces. Based on genomic analysis, we performed a comprehensive safety assessment of strain cqf-43, using both in vitro and in vivo experiments, and explored its probiotic potential. The total genome length measures 3,169,201 bp, boasting a GC content of 44.59%. Through phylogenetic analyses, leveraging both 16S rRNA gene and whole-genome sequences, we confidently categorize strain cqf-43 as a member of Lactiplantibacillus. Genome annotation using Prokka unveiled a total of 3141 genes, encompassing 2990 protein-coding sequences, 71 tRNAs, 16 rRNAs, and 1 tmRNA. Functional annotations derived from COG and KEGG databases highlighted a significant abundance of genes related to metabolism, with a notable emphasis on carbohydrate utilization. The genome also revealed the presence of prophage regions and CRISPR-Cas regions while lacking virulence and toxin genes. Screening for antibiotic resistance genes via the CARD database yielded no detectable transferable resistance genes, effectively eliminating the potential for harmful gene transfer. It is worth highlighting that the virulence factors identified via the VFDB database primarily contribute to bolstering pathogen resilience in hostile environments. This characteristic is particularly advantageous for probiotics. Furthermore, the genome is devoid of menacing genes such as hemolysin, gelatinase, and biogenic amine-producing genes. Our investigation also unveiled the presence of three unannotated secondary metabolite biosynthetic gene clusters, as detected by the online tool antiSMASH, suggesting a great deal of unknown potential for this strain. Rigorous in vitro experiments confirmed tolerance of strain cqf-43 in the intestinal environment, its antimicrobial efficacy, sensitivity to antibiotics, absence of hemolysis and gelatinase activity, and its inability to produce biogenic amines. In addition, a 28-day oral toxicity test showed that the strain cqf-43 did not pose a health hazard in mice, further establishing it as a safe strain.

Melittin, a natural antimicrobial peptide, has broad-spectrum antimicrobial activity. This has resulted in it gaining increasing attention as a potential antibiotic alternative; however, its practical use has been limited by its weak antimicrobial activity, high hemolytic activity, and low proteolytic stability. In this study, N-terminal fatty acid conjugation was used to develop new melittin-derived lipopeptides (MDLs) to improve the characteristics of melittin. Our results showed that compared with native melittin, the antimicrobial activity of MDLs was increased by 2 to 16 times, and the stability of these MDLs against trypsin and pepsin degradation was increased by 50 to 80%. However, the hemolytic activity of the MDLs decreased when the length of the carbon chain of fatty acids exceeded 10. Among the MDLs, the newly designed analog Mel-C8 showed optimal antimicrobial activity and protease stability. The antimicrobial mechanism studied revealed that the MDLs showed a rapid bactericidal effect by interacting with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) and penetrating the bacterial cell membrane. In conclusion, we designed and synthesized a new class of MDLs with potent antimicrobial activity, high proteolytic stability, and low hemolytic activity through N-terminal fatty acid conjugation.

Background: Ulcerative colitis is an emerging global health concern that poses a significant threat to human health and can progress to colorectal cancer if not diagnosed and treated promptly. Currently, the biomarkers used clinically for diagnosis and dynamic severity monitoring lack disease specificity.Methods: Mouse models induced with 2%, 2.5%, and 3% DSS were utilized to simulate human UC with varying severities of inflammation. Transcriptome sequencing technology was employed to identify differentially expressed genes (DEGs) between the control group and each treatment group. Functional enrichment analysis of the KEGG database was performed for shared DEGs among the three treatment groups. DEGs that were significantly and strongly correlated with DSS concentrations were identified using Spearman correlation analysis. Human homologous genes of the interested DEGs were searched in the HomoloGene database, and their regulation patterns in UC patients were validated using the GSE224758 dataset. These genes were then submitted to the DisGeNET database to identify their known associations with human diseases. Online tools, including SignalP 6.0 and DeepTMHMM 1.0, were used to predict signal peptides and transmembrane helices in the amino acid sequences of human genes homologous to the DEGs of interest.Results: A total of 1,230, 995, and 2,214 DEGs were identified in the 2%, 2.5%, and 3% DSS-induced groups, respectively, with 668 DEGs common across all three groups. These shared DEGs were primarily associated with signaling transport, pathogenesis, and immune response. Through extensive screening, LGI2 and PRSS22 were identified as potentially novel biomarkers with higher specificity and ease of detection for the early diagnosis and dynamic severity monitoring of human UC, respectively.Conclusion: We have identified two potentially novel biomarkers, LGI2 and PRSS22, which are easy of detection and more specific for human UC. These findings provide new insights into the accurate diagnosis and dynamic monitoring of this persistent disease.

With the prohibition of antibiotics in animal feed, the livestock industry faces significant challenges, including increased morbidity and mortality rates and reduced farming efficiency. Developing green, natural, and safe antibiotic alternatives has become a research hotspot. This study evaluated the effects of octapeptin as a feed additive on growth performance, diarrhea incidence, serum biochemistry, serum immune factors, and gut microbiota of weaned piglets. Seventy-two weaned piglets were randomly assigned to three groups based on body weight and sex, with each group receiving different dietary treatments: a negative control group (CON, basal diet), a positive control group (MC, basal diet + 5 mg/kg Microcin C7), and an octapeptin supplement group (OP, basal diet + 40 mg/kg octapeptin). After 28 days of feeding experimental diets, the results demonstrated that supplementing the diet of weaned piglets with octapeptin significantly improved the feed conversion ratio compared to the control group (p < 0.05) over the entire experimental period. Furthermore, a reduction in diarrhea incidence was observed during the late nursery period (14–28 d), resulting in an overall improvement in diarrhea compared to the other two groups (p < 0.01). Serum biochemical analysis results revealed a trend towards decreased alanine aminotransferase level in the octapeptin group, with no significant differences in other indicators, suggesting potential improvements in liver function without causing liver damage. In addition, compared to the control group, octapeptin enhanced mucosal immunity by decreasing TNF-α level (p < 0.05). Fecal microbiota analysis results showed a significant increase in beneficial bacteria such as Collinsella and Olsenella in the octapeptin group compared to the other two groups (p < 0.05), indicating a positive impact on gut health. These findings supported the potential of octapeptin as an alternative to antibiotic growth promoters in weaned piglets’ diets.

The immunity-related functions of defensins seem to be dependent on environmental stimuli, the cell type, and the concentration of peptides. However, the function and mechanism of porcine β-defensin 114 (pBD114) in regulating the inflammatory response to macrophages are unclear. Therefore, the modulatory effects of porcine pBD114 on the inflammatory response were investigated by treating the mouse monocyte macrophage cell line RAW264.7 with different concentrations of pBD114 with or without lipopolysaccharide (LPS). RNA-seq analysis was performed to investigate the mechanisms underlying pBD114’s regulation of inflammatory responses in macrophages. In addition, the inflammatory response-modulating effects of pBD114 were also further verified with a mouse assay. The results showed that 100 μg/mL of pBD114 significantly promoted the secretion of TNF-α and IL-10 in RAW264.7. However, the LPS-induced increase in TNFα in the RAW264.7 cell cultures was significantly decreased with 10 μg/mL of pBD114. These results suggest that pBD114 can exhibit pro-inflammatory activities under normal physiological conditions with 100 μg/mL of pBD114, and anti-inflammatory activities during an excessive inflammatory response with 10 μg/mL of pBD114. RNA-seq analysis was performed to gain further insights into the effects of pBD114 on the inflammatory response. Among the pBD114-promoting RAW264.7 pro-inflammatory responses, pBD114 significantly up-regulated 1170 genes and down-regulated 724 genes. KEGG enrichment showed that the differentially expressed genes (DEGs) were significantly enriched in the immune- and signal-transduction-related signaling pathways. Protein-Protein Interaction (PPI) and key driver analysis (KDA) analyses revealed that Bcl10 and Bcl3 were the key genes. In addition, pBD114 significantly up-regulated 12 genes and down-regulated 38 genes in the anti-inflammatory response. KEGG enrichment analysis revealed that the DEGs were mainly enriched in the “Cytokine–cytokine receptor interaction” signaling pathway, and PPI and KDA analyses showed that Stat1 and Csf2 were the key genes. The results of qRT-PCR verified those of RNA-seq. In vivo mouse tests also confirmed the pro- or anti-inflammatory activities of pBD114. Although the inflammatory response is a rapid and complex physiological reaction to noxious stimuli, this study found that pBD114 plays an essential role mainly by acting on the genes related to immunity, signal transduction, signaling molecules, and interactions. In conclusion, this study provides a certain theoretical basis for the research and application of defensins.

The interaction forces and mechanical properties of the interaction between melittin (Mel) and lipopolysaccharide (LPS) are considered to be crucial driving forces for Mel when killing Gram-negative bacteria (GNB). However, how their interaction forces perform at the single-molecule level and the dissociation kinetic characteristics of the Mel/LPS complex remain poorly understood. In this study, the single-molecule-level interaction forces between Mel and LPSs from E. coli K-12, O55:B5, O111:B4, and O128:B12 were explored using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS). AFM-based dynamic force spectroscopy (DFS) and an advanced analytical model were employed to investigate the kinetic characteristics of the Mel/LPS complex dissociation. The results indicated that Mel could interact with both rough (R)-form LPS (E. coli K-12) and smooth (S)-form LPSs (E. coli O55:B5, O111:B4, and O128:B12). The S-form LPS showed a more robust interaction with Mel than the R-form LPS, and a slight difference existed in the interaction forces between Mel and the diverse S-form LPS. Mel interactions with the S-form LPSs showed greater specific and non-specific interaction forces than the R-form LPS (p < 0.05), as determined by AFM-based SMFS. However, there was no significant difference in the specific and non-specific interaction forces among the three samples of S-form LPSs (p > 0.05), indicating that the variability in the O-antigen did not affect the interaction between Mel and LPSs. The DFS result showed that the Mel/S-form LPS complexes had a lower dissociation rate constant, a shorter energy barrier width, a longer bond lifetime, and a higher energy barrier height, demonstrating that Mel interacted with S-form LPS to form more stable complexes. This research enhances the existing knowledge of the interaction micromechanics and kinetic characteristics of Mel and LPS at the single-molecule level. Our research may help with the design and evaluation of new anti-GNB drugs.