Systemic Pharmacology Reveals the Potential Targets and Signaling Mechanisms in the Adjuvant Treatment of Brucellosis with Traditional Chinese Medicine.

Human brucellosis is one of the world's most common zoonoses, caused by Brucella infection and characterized by induced inflammation, which in severe cases can lead to abortion and sterility in humans and animals. There is growing evidence that traditional Chinese medicine (TCM) is beneficial as an adjunct to the treatment of brucellosis. However, its specific targets of action and molecular mechanisms remain unclear. In this study, a systematic pharmacological approach was applied to demonstrate pharmacological targets, biological functions, and signaling pathways of TCM as an adjunct to the treatment of brucellosis (TCMTB). The results of network pharmacology were further verified by in vitro experiments. Network analysis revealed that 133 active ingredients and 247 targets were screened in TCMTB. Further data analysis identified 21 core targets and 5 core compounds in TCMTB, including beta-sitosterol, quercetin, kaempferol, luteolin, and paeoniflorin. Gene ontology and the Kyoto Encyclopedia of Gene and Genome analysis showed that TCMTB might actively treat brucellosis by regulating inflammatory response, enhancing immune function, and targeting signaling pathways such as tuberculosis and TNF. Molecular docking results showed that multiple compounds could bind to multiple targets. Further, in vitro experiments confirmed that quercetin, among the active compounds screened, induced the strongest immunomodulatory and pro-inflammatory cytokine production during Brucella abortus infection. Further, quercetin induced nitric oxide production, which attenuated the ability of B. abortus to internalize THP-1 cells as well as intracellular survival. This study reveals the mechanism by which TCMTB aids in the treatment of brucellosis through a synergistic multicomponent, multipathway, and multitarget action. The contribution of quercetin treatment to B. abortus infection was demonstrated for the first time, which may be related to the quercetin-induced production of nitric oxide and immunomodulatory and inflammatory cytokines. These predictions of the core compounds and targets may be used in the future for the clinical treatment of brucellosis.

Brucella abortus modulates macrophage polarization and inflammatory response by targeting glutaminases through the NF-κB signaling pathway.

Objectives: The mechanism of Brucella infection regulating macrophage phenotype has not been completely elucidated until now. This study aimed to determine the mechanism of Brucella abortus in the modulation of macrophage phenotype using RAW264.7 cells as a model. Materials and methods: RT-qPCR, ELISA and flow cytometry were used to detect the inflammatory factor production and phenotype conversion associated with M1/M2 polarization of macrophages by Brucella abortus infection. Western blot and immunofluorescence were used to analyze the role of nuclear factor kappa B (NF-κB) signaling pathway in regulation of Brucella abortus-induced macrophage polarization. Chromatin immunoprecipitation sequencing (Chip-seq), bioinformatics analysis and luciferase reporter assay were used to screen and validate NF-κB target genes associated with macrophage polarization and further verify its function. Results: The results demonstrate that B. abortus induces a macrophage phenotypic switch and inflammatory response in a time-dependent manner. With the increase of infection time, B. abortus infection-induced M1-type increased first, peaked at 12 h, and then decreased, whereas the M2-type decreased first, trough at 12 h, and then increased. The trend of intracellular survival of B. abortus was consistent with that of M2 type. When NF-κB was inhibited, M1-type polarization was inhibited and M2-type was promoted, and the intracellular survival of B. abortus increased significantly. Chip-seq and luciferase reporter assay results showed that NF-κB binds to the glutaminase gene (Gls). Gls expression was down-regulated when NF-κB was inhibited. Furthermore, when Gls was inhibited, M1-type polarization was inhibited and M2-type was promoted, the intracellular survival of B. abortus increased significantly. Our data further suggest that NF-κB and its key target gene Gls play an important role in controlling macrophage phenotypic transformation. Conclusions: Taken together, our study demonstrates that B. abortus infection can induce dynamic transformation of M1/M2 phenotype in macrophages. Highlighting NF-κB as a central pathway that regulates M1/M2 phenotypic transition. This is the first to elucidate the molecular mechanism of B. abortus regulation of macrophage phenotype switch and inflammatory response by regulating the key gene Gls, which is regulated by the transcription factor NF-κB.

Effect of difference in training skills on stress in horses trained by Kazakh trainers.

This study aimed to evaluate the effect of differences in trainer skills on horse training during the early stages of riding habituation by measuring the levels of stress and changes in stress levels. Among nine untrained horses employed, five in Group A were trained by two low-skilled trainers, whereas the remaining four in Group B were trained by two high-skilled trainers using the traditional Kazakh method. Salivary α-amylase concentration was measured as a biomarker of stress immediately before and after each riding session during the training period. In the duration of riding and mooring times to the total riding habituation time for each horse, no significant difference was observed between the two groups. In contrast, the mean total stress and mean final stress were significantly lower in Group B than in Group A, and the mean total change in stress before and after riding habituation was significantly higher in Group B. Differences in trainer skills were evidenced as differences in training methods to suppress the total stress levels through differences in the application of stress burden during the training of individual horses.

Survival characteristics and transcriptome profiling reveal the adaptive response of the Brucella melitensis 16M biofilm to osmotic stress.

Brucella can inhabit hostile environments, including osmotic stress. How Brucella responds collectively to osmotic stress is largely unexplored, particularly in spatially structured communities such as a biofilm. To gain insight into this growth mode, we set out to characterize the Brucella melitensis 16M biofilm, describe its phenotype, and carry out a comparative transcriptomic analysis between biofilms under osmotic stress and control conditions. We determined that the bacteria challenged with 1.5 M NaCl had a reduced ability to aggregate and form clumps and develop a biofilm; however, the salt stress promoted the release of the outer membrane vesicles from the biofilm. Together with the genotypical response to osmotic stress, we identified 279 differentially expressed genes in B. melitensis 16M grown under osmotic conditions compared with control conditions; 69 genes were upregulated and 210 downregulated. Under osmotic stress, the main changed genes of biofilm were predicted to be involved in flagellar assembly, cell envelope, translation, small RNA regulation, transport and binding proteins, and energy metabolism. In addition, the ABC transporter was enriched in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. We highlight 12 essential ABC transporter genes associated with a bacterial response to osmotic stress at the biofilm stage, including one specific locus, BME_RS12880, mediating betaine accumulation in biofilms to eliminate osmotic stress. The current study results can help researchers gain insights into B. melitensis 16M biofilm adaptation to osmotic stress and provide information for developing intervention strategies to control Brucella.

Genetic characterization of Kushum horses in Kazakhstan based on haplotypes of mtDNA and Y chromosome, and genes associated with important traits of the horses.

The Kushum is a relatively new breed of horses in Kazakhstan that was established in the middle of the 20th century through a cross between mares of Kazakhstan local horses and stallions of Thoroughbred, Trotter, and Russian Don breeds to supply military horses. To reveal the genetic characteristics of this breed, we investigated haplotypes of mitochondrial DNA (mtDNA) and single-nucleotide polymorphisms of the Y chromosome, as well as genotypes of five functional genes associated with coat color, body composition, and locomotion traits. We detected 10 mtDNA haplotypes that fell into 8 of the 17 major haplogroups of horse mtDNA, indicating a unique haplotype composition with high genetic diversity. We also found two Y-chromosomal haplotypes in Kushum horses, which likely originated from Trotter and/or Don breeds. The findings regarding the mtDNA and Y-chromosomal haplotypes are concordant with the documented maternal and paternal origins of the Kushum horses. The allele frequencies of ASIP, MC1R, and MATP associated with coat color were consistent with the coat color variations of Kushum horses. The allele frequencies of MSTN associated with endurance performance and those of DMRT3 associated with gait suggested that the observed allele frequencies of these genes were the result of selective breeding for these traits. As a result of this study, we were able to obtain useful information for a better understanding of the origin and breeding history of the Kushum horse breed using molecular markers.