Morphological Observation and Comparative Transcriptomic Analysis of Clostridium perfringens Biofilm and Planktonic Cells.

Bacterial biofilms can enhance survival in adverse environments and promote infection. However, little is known about biofilm formation by Clostridium perfringens. To better characterize this process, we used SEM to observe the surfaces of C. perfringens biofilms after 12, 24, 48, and 72 h of incubation. Biofilm cells appeared to be encased in a dense matrix material, and the total biomass of the biofilm increased with incubation time. To gain insight into the differentially expressed genes (DEGs) between biofilm and planktonic cells, we carried out comparative transcriptomic analysis using RNA sequencing. In total, 91 genes were significantly differentially expressed, with 40 being up-regulated and 51 down-regulated. In particular, genes encoding sortase, ribosomal proteins, and ATP synthase were up-regulated in biofilms, while genes coding for clostripain and phospholipase C were down-regulated. To validate the RNA sequencing results, qRT-PCR analysis was performed using five randomly selected DEGs. Results showed that all five genes were up-regulated, which was in accordance with the RNA sequencing results. To examine the functional differences, the DEGs were characterized by GO and KEGG pathway enrichment analyses. Results showed that the up-regulated genes were divided into 32 significantly enriched GO terms, with "macromolecular complex" being the most common. Oxidative phosphorylation was the only significantly enriched pathway, suggesting that ATP is required for biofilm stability. This study provides valuable insights into the morphology and transcriptional regulation of C. perfringens during biofilm formation, and will be useful for understanding and developing biofilm-based processes.

Development and application of microsatellite markers in Hippophae rhamnoides subsp. sinensis Rousi (Hippophae rhamnoides L.) based on transcriptome sequencing.

Hippophae rhamnoides subsp. sinensis Rousi is a cold- and drought-tolerant pioneer species with significant economic and ecological value. Evaluating its genetic diversity and population structure is of great importance for guiding the development and utilization of resources. In this study, a total of 41,804 SSRs were generated by transcriptome sequencing of Hippophae rhamnoides subsp. sinensis Rousi. Among the different SSR motif types, mononucleotide repeats (26,972) were the most abundant, followed by trinucleotides, tetranucleotides, and pentanucleotides. 200 pairs of SSR primers were selected to detect polymorphisms, of which 15 pairs primers were selected as validated polymorphic SSRs used for genetic diversity and population structure analysis. A total of 63 alleles were identified with 15 pairs primers, with Nei's genetic diversity index ranged from 0.27 to 0.83 (average: 0.54), and the expected heterozygosity ranged from 0.16 to 0.73 (average: 0.46). The polymorphism information content ranged from 0.23 to 0.81 (average: 0.48). Genetic structure analyses showed that the 10 populations could be broadly categorized into two groups. AMOVA denoted that genetic variations primarily originated from within the populations, with minimal differences observed between the groups, accounting for only 7% of the total genetic variation. This implies that mutation in H. rhamnoides subsp. sinensis Rousi mainly occurred within the populations. The results showed that the 10 populations of H. rhamnoides subsp. sinensis Rousi are rich in genetic diversity, with low levels of population differentiation and a high degree of gene exchange, which should be taken into consideration for the future work of germplasm resource preservation and seedling breeding.

Exploring effect of herbal monomers in treating gouty arthritis based on nuclear factor-kappa B signaling: A review.

Gouty arthritis (GA) is an inflammatory disease caused by disorders of the purine metabolism. Although increasing number of drugs have been used to treat GA with the deepening of relevant research, GA still cannot be cured by simple drug therapy. The nuclear factor-kappa B (NF-κB) signaling pathway plays a key role in the pathogenesis of GA. A considerable number of Chinese herbal medicines have emerged as new drugs for the treatment of GA. This article collected relevant research on traditional Chinese medicine monomers in the treatment of GA using NF-κB, GA, etc. as keywords; and conducted a systematic search of relevant published articles using the PubMed database. In this study, we analyzed the therapeutic effects of traditional Chinese medicine monomers on GA in the existing literature through in vivo and in vitro experiments using animal and cell models. Based on this review, we believe that traditional Chinese medicine monomers that can treat GA through the NF-κB signaling pathway are potential new drug development targets. This study provides research ideas for the development and application of new drugs for GA.

Effects of Tibetan Sheep-Derived Compound Probiotics on Growth Performance, Immune Function, Intestinal Tissue Morphology, and Intestinal Microbiota in Mice.

Probiotics play an important role in animal growth, immunity, and gut microbial balance and are now widely used in agriculture, food, and medicine. This study analysed the effects of different concentrations of Tibetan sheep compound probiotics on the immunity, tissue morphology, and intestinal microbiota of mice using histological, molecular, and 16S rRNA techniques. The results showed that the composite probiotics sourced from Tibetan sheep improved the growth performance of mice, increased the length of small intestinal villi and mucosal thickness, and enhanced the intestinal barrier function of mice. DZ-L and DZ-M significantly increased the mRNA expression levels of ZO-1, Occludin, and Claudin-1 mRNA. They also up-regulated IL-10 and TNF-ß, and down-regulated TNF-α, IL-1ß, and IL-8. The immune function of mice was enhanced, with DZ-M treatment having an extremely significant effect, while the effect of DZ-H treatment was slightly lower compared to DZ-L and DZ-M. In addition, the composition and diversity of the intestinal microbiota were modulated, and at the phylum level, the relative abundance of Firmicutes was higher in the DZ-M group, the relative abundance of Desulfobacterota, Actinobacteriota, and Patescibacteria was reduced in the probiotic complex group, and the relative abundance of Verrucomicrobiota was higher. At the genus level, the relative abundance of Muribaculaceae was higher in the DZ-L and DZ-M groups, and the relative abundance of Lachnospiraceae_NK4A136_group in the DZ-H group; and the relative abundance of Bacteroides and Roseburia in the composite probiotic group. This study can improve the reference for the development of new green feed additives instead of antibiotics, which will also further promote the development of the livestock industry.

Effects of three different probiotics of Tibetan sheep origin and their complex probiotics on intestinal damage, immunity, and immune signaling pathways of mice infected with Clostridium perfringens type C.

Tibetan sheep have unique intestinal microorganisms in their intestines that are adapted to the highland alpine and anoxic environment. To further clarify the probiotic properties of Tibetan sheep-derived probiotics, we selected three Tibetan sheep-derived probiotic isolates (Enterococcus faecalis EF1-mh, Bacillus subtilis BS1-ql, and Lactobacillus sakei LS-ql) to investigate the protective mechanisms of monocultures and their complex strains against Clostridium perfringens type C infection in mice. We established a model of C. perfringens type C infection and used histology and molecular biology to analyze the effects and mechanisms of different probiotic treatments on mice after C. perfringens type C infection. After supplementation with either probiotics or complex probiotics, mice were improved in terms of weight reduction and reduced the levels of cytokines in serum and increased the levels of intestinal sIgA, and supplementation with complex probiotics was effective. In addition, both probiotic and complex probiotic supplementation effectively improved the damage of intestinal mucosa and spleen tissue. The relative expressions of Muc 2, Claudin-1, and Occludin genes were increased in the ileum. The three probiotics and the compound probiotics treatment significantly reduced the relative mRNA expression of toll-like/MyD88/NF-κB/MAPK. The effect of probiotic treatment was similar to the results of engramycin treatment, but the effect of engramycin treatment on intestinal sIgA was not significant. Our results clarify the immunomodulatory effects of the three probiotic isolates and the complex probiotics on C. perfringens infection, and the repair of the intestinal mucosal barrier.

Ultrathin ZIF-8 wrapping on Au-dotted Ag-nanowires for highly selective SERS-based CO2 gas detection.

Ultrathin ZIF-8 wrapping was constructed on Au-dotted Ag-nanowires to obtain Ag@Au@ZIF-8. A thin film (Ag@Au@ZIF-8 NWs/TF) was constructed to selectively adsorb and enrich CO2 molecules, which solved the problem that SERS signals could not be detected due to the poor affinity of the metal surface and the low concentration of gas molecules.

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis.

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Transcriptomic analysis of drought stress responses of sea buckthorn (Hippophae rhamnoidessubsp. sinensis) by RNA-Seq.

Sea buckthorn is one of the most important eco-economic tree species in China due to its ability to grow and produce acceptable yields under limited water and fertilizer availability. In this study, the differentially expressed genes under drought stress (DS) of sea buckthorn were identified and compared with control (CK) by RNA-Seq. A total of 122,803 unigenes were identified in sea buckthorn, and 70,025 unigenes significantly matched a sequence in at least one of the seven databases. A total of 24,060 (19.59%) unigenes can be assigned to 19 KEGG pathways, and 1,644 unigenes were differentially expressed between DS and CK, of which 519 unigenes were up-regulated and 1,125 unigenes down-regulated. Of the 47 significantly enriched GO terms, 14, 7 and 26 items were related to BP, CC and MF, respectively. KEGG enrichment analysis showed 398 DEGs involved in 97 different pathways, of which 119 DEGs were up-regulated and 279 DEGs were down-regulated under drought stress. In addition, we found 4438 transcriptor factors (TFs) in sea buckthorn, of which 100 were differentially expressed between DS and CK. These results lay a first foundation for further investigations of the very specific functions of these unigenes in sea buckthorn in response to drought stress.

Cytopathic effect of classical swine fever virus NS3 protein on PK-15 cells.

In order to further research the relationship between classical swine fever virus' (CSFV) NS3 protein and the cytopathic effect (CPE) in cells infected with the CSFV, and to reveal the effect of protein NS3 on the host cells, the NS3 of CSFV Shimen strain amplified by RT-PCR was subcloned into the pEGFP-C1, named pEGFP-C1-NS3. The insert position, the size and the reading frame were correct for restriction enzyme digestion and sequence analysis. The pEGFP-C1-NS3 and pEGFP-C1 were transfected into PK-15 cells by liposome, and positive cell clones were gained by G418. The NS3-EGFP fusion protein expressed in pEGFP-C1-NS3 cells was observed by inverted fluorescence microscopy and identified by Western blot. The CPE appeared in positive pEGFP-C1-NS3 cells 72 h after passaging, apoptosis detection was also performed on positive pEGFP-C1-NS3 cells and pEGFP-C1 cells 72 h after passaging by TUNEL assay. The apoptosis rates in the positive pEGFP-C1-NS3 and pEGFP-C1 cells were 43.4 and 13.1%, respectively (p < 0.05). The results suggest that the CPE in positive pEGFP-C1-NS3 cells was induced by apoptosis and there is a relationship between the expression of NS3 and apoptosis.

[Phylogenetic relationships of seabuckthorn based on ITS sequences].

Sequences of the internal transcribed spacer (ITS) was compared in three seabuckthrons (Hippophae rhamnoides subsp. sinensis, H. tibetana and H. neurocarpa) distributed in Qinghai Province, then the systematic positions of 15 seabuckthron samples were analyzed with Elaeagnaceae angustifolia being outgroup. The results showed that ITS sequences in three seabuckthron species varied in length from 600 to 605 bp. ITS-1, 5.8S and ITS-2 varied from 201 to 203 bp, 166 to 167 bp and 232 to 236 bp, respectively. The sequence divergence among three seabuckthorn species was also remarkably high; Cluster analysis based on ITS indicated that H. gonicocarpa subsp. litangensis and H. gonicocarpa subsp. goniocarpa were distinct and could be classified as H. gonicocarpa and H. litangensis. H. gyantsensis had the closest genetic relationship with H. salicifolia and the distant relationship with H. rhamnoides subsp. sinensis. The positions of nine subspecies of H. rhamnoides based on morphological classification were different from the results of ITS.

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Oral administration of tepoxalin in the horse: a PK/PD study.

Tepoxalin is a non-steroidal anti-inflammatory drug with analgesic, anti-inflammatory, and antipyretic properties and has been recently introduced into veterinary medicine. The aim of this study was to evaluate the pharmacokinetic/pharmacodynamic (PK/PD) profile of tepoxalin to assess whether it would be suitable for clinical use in horses. Six female fasting/fed horses were given 10mg/kg tepoxalin orally in a cross-over study. After administration, tepoxalin underwent rapid and extensive hydrolytic conversion to its carboxylic acid metabolite RWJ-20142. In animals that had been fed, the plasma concentrations of tepoxalin were undetectable, whereas in fasting animals they were close to the limit of quantification of the method. No differences between the fasting/fed groups in RWJ-20142 plasma concentrations were shown. Tepoxalin showed a strong and long-lasting ex vivo inhibitory activity against cyclooxygenase (COX)-1, mainly due to its main metabolite RWJ-20142. Tepoxalin and RWJ-20142 do not seem to possess either COX-2 or 5-lipoxygenase inhibitory activity in the horse. These features suggest that the drug is a selective COX-1 inhibitor in horses, with no significant anti-inflammatory activity. Thus, its long term use in equine practice could be of concern.