Multifunctional Gold Nanozyme-Engineered Amphotericin B for Enhanced Antifungal Infection Therapy.

Chronic wounds of significant severity and acute injuries are highly vulnerable to fungal infections, drastically impeding the expected wound healing trajectory. The clinical use of antifungal therapeutic drug is hampered by poor solubility, high toxicity and adverse reactions, thereby necessitating the urgent development of novel antifungal therapy strategy. Herein, this study proposes a new strategy to enhance the bioactivity of small-molecule antifungal drugs based on multifunctional metal nanozyme engineering, using amphotericin B (AmB) as an example. AmB-decorated gold nanoparticles (AmB@AuNPs) are synthesized by a facile one-pot reaction strategy, and the AmB@AuNPs exhibit superior peroxidase (POD)-like enzyme activity, with maximal reaction rates (Vmax ) 3.4 times higher than that of AuNPs for the catalytic reaction of H2 O2 . Importantly, the enzyme-like activity of AuNPs significantly enhanced the antifungal properties of AmB, and the minimum inhibitory concentrations of AmB@AuNPs against Candida albicans (C. albicans) and Saccharomyces cerevisiae (S. cerevisiae) W303 are reduced by 1.6-fold and 50-fold, respectively, as compared with AmB alone. Concurrent in vivo studies conducted on fungal-infected wounds in mice underscored the fundamentally superior antifungal ability and biosafety of AmB@AuNPs. The proposed strategy of engineering antifungal drugs with nanozymes has great potential for enhanced therapy of fungal infections and related diseases.

Establishment and evaluation of detection methods for process-specific residual host cell protein and residual host cell DNA in biological preparation.

To establish accurate detection methods of process-specific Escherichia coli residual host cell protein (HCP) and residual host cell DNA (rcDNA) in recombinant biological preparations. Taking the purification process of GLP expressed by E. coli as a specific-process model, the HCP of empty E. coli was intercepted to immunize mice and rabbits. Using IgG from immunized rabbits as the coating antibody and mouse immune serum as the second sandwich antibody, a process-specific enzyme-linked immunosorbent assay (ELISA) for E. coli HCP was established. Targeting the 16S gene of E. coli, ddPCR was used to obtain the absolute copies of rcDNA in samples. Non-process-specific commercial ELISA kit and the process-specific ELISA established in this study were used to detect the HCP in GLP preparation. About 62% of HCPs, which should be process-specific HCPs, could not be detected by the non-process-specific commercial ELISA kit. The sensitivity of established ELISA can reach 338 pg/mL. The rcDNA could be absolutely quantitated by ddPCR, for the copies of rcDNA in three multiple diluted samples showed a reduced gradient. While the copies of rcDNA in three multiple diluted samples could not be distinguished by the qPCR. Process-specific ELISA has high sensitivity in detecting process-specific E. coli HCP. The absolutely quantitative ddPCR has much higher accuracy than the relatively quantitative qPCR, it is a nucleic acid quantitative method that is expected to replace qPCR in the future.

Construction and Mechanism of IL-15-Based Coactivated Polymeric Micelles for NK Cell Immunotherapy.

Natural killer (NK) cells are an important contributor to cancer immunotherapy, but their antitumor efficacy remains suboptimal. While cytokine-based priming shows promise in enhancing NK-cell activity, its clinical translation faces many challenges, including coactivation of multiple cytokines, poor pharmacokinetics, and limited mechanistic understanding. Here, this work develops a polymeric micelle-based IL-15/IL-2 codelivery system (IL-15/2-PEG-PTMC) for NK-cell activation. In vivo studies demonstrate that half-life of IL-15 and IL-2 and the recruitment of NK cell within tumor tissue are significantly increased after PEG-PTMC loading. Coupled with the coactivation effect of IL-15 and IL-2 conferred by this system, it noticeably delays the growth of tumors compared to conventional NK-cell activation approach, that is free IL-15 and IL-2. It is also surprisingly found that cholesterol metabolism is highly involved in the NK cell activation by IL-15/2-PEG-PTMC. Following stimulation with IL-15/2-PEG-PTMC or IL-15, NK cells undergo a series of cholesterol metabolism reprogramming, which elevates the cholesterol levels on NK cell membrane. This in turn promotes the formation of lipid rafts and activates immune synapses, effectively contributing to the enhancement of NK cell's antitumor activity. It is believed that it will open a new avenue for improving the efficacy of NK cell immunotherapy by regulating cholesterol metabolism.

Therapeutic effect of iturin A on Candida albicans oral infection and its pathogenic factors.

Candida albicans is responsible for conditions ranging from superficial infections such as oral or vaginal candidiasis to potentially fatal systemic infections. It produces pathogenic factors contributing to its virulence. Iturin A, a lipopeptide derived from Bacillus sp., exhibits a significant inhibitory effect against C. albicans. However, its exact mechanism in mitigating the pathogenic factors of C. albicans remains to be elucidated. This study aimed to explore the influence of iturin A on several pathogenic attributes of C. albicans, including hypha formation, cell membrane permeability, cell adhesion, biofilm formation, and therapeutic efficacy in an oral C. albicans infection model in mice. The minimal inhibitory concentration of iturin A against C. albicans was determined to be 25 µg/mL in both YEPD and RPMI-1640 media. Iturin A effectively inhibited C. albicans hyphal formation, decreased cell viability within biofilms, enhanced cell membrane permeability, and disrupted cell adhesion in vitro. Nonetheless, iturin A did not significantly affect the phospholipase activity or hydrophobicity of C. albicans. A comparative study with nystatin demonstrated the superior therapeutic efficacy of iturin A in a mouse model of oral C. albicans infection, significantly decreasing C. albicans count and inhibiting both fungal hypha formation and tongue surface adhesion. High-dose iturin A treatment (25 µg/mL) in mice had no significant effects on blood indices, tongue condition, or body weight, indicating the potential for iturin A in managing oral infections. This study confirmed the therapeutic potential of iturin A and provided valuable insights for developing effective antifungal therapies targeting C. albicans pathogenic factors.

Effects of ultra-strong static magnetic field on the gut microbiota of humans and mice.

To explore the effect of ultra-strong static magnetic field on gut microbiota, 16 T static magnetic field was used to study the changes in the structure and composition of human and mouse gut microbiota in this environment. In the mouse gut microbiota, at the genus level, the magnetic field significantly decreased the relative abundances of Escherichia-Shigella, Lactobacillus, Enterococcus, Burkholderia-Caballeronia-Paraburkholderia, Parasutterella, and Ralstonia and significantly increased those of Parabacteroides, Alloprevotella, Alistipes, Odoribacter, Bacteroides, Mucispirillum, Sutterella, and Prevotellaceae_UCG-001. Similarly, at the genus level, the relative abundances of Bacteroides, Parabacteroides, Romboutsia, and Streptococcus significantly decreased in the human gut microbiota. Contrary to the changing trend of the abundance in the mouse gut, the abundances of Bacteroides and Parabacteroides in the human gut were significantly reduced under magnetic field. The BugBase phenotypic prediction analysis showed that the relative abundances of five phenotypes, including anaerobism, mobile elements, potential pathogenicity, stress-tolerant, and biofilm formation, changed significantly in the mouse gut microbiota, while the relative abundances of two phenotypes, including Gram-positive and Gram-negative phenotypes, changed significantly in the human gut microbiota. The 16 T magnetic field could differently affect the composition, structure, and phenotypes of gut microbiota in human and mice, suggesting the importance of model selection in studying the biological effects of magnetic field.

Modifications of Ganoderma lucidum spores into digestive-tissue highly adherent porous carriers with selective affinity to hydrophilic or hydrophobic drugs.

Ganoderma lucidum spores (GLSs) have been suggested to provide optimal structures for transporting orally bioavailable drugs. However, the double-layer wall and cavities of GLSs are naturally closed. This study aimed to modify GLSs into porous carriers by opening the layers and internal cavity with iturin A (IA) followed by potassium hydroxide (KOH) or hydrochloric acid (HCl). The (IA + KOH)- and (IA + HCl)-treated GLS carriers exhibited a high loading rate of 301.50 ± 2.33 and 268.18 ± 7.72 mg/g for the hydrophilic methylene blue (MB) and hydrophobic rifampicin (RF), respectively. The mechanisms underlying the modification involved the enhancement of the specific surface area with IA and the exposure of hydrophilic groups or hydrophobic groups of the GLSs with KOH or HCl. The sustained 48-h molecule-release profiles of the MB- and RF-loaded GLS carriers were best fitted using a first-order kinetics model in simulated gastric (or intestinal) fluid compared with other models. In mice, the designed GLS carriers had high adhesion capacities onto the mucosa of the digestive tract and long retention times (120 h), and even promoted the secretion of mucus and expression of several key intestinal barrier proteins. This study provided a new method to modify GLSs into oral carriers with selective drug affinity, high loading capacity, sustained drug release, and high adhesion to the digestive tract.

Energy metabolic mechanisms for high altitude sickness: Downregulation of glycolysis and upregulation of the lactic acid/amino acid-pyruvate-TCA pathways and fatty acid oxidation.

Hypobaric hypoxia is often associated with the plateau environment and can lead to altitude sickness or death. The underlying cause is a lack of oxygen, which limits energy metabolism and leads to a compensatory stress response. Although glycolysis is commonly accepted as the primary energy source during clinical hypoxia, our preliminary experiments suggest that hypobaric hypoxia may depress glycolysis. To provide a more comprehensive understanding of energy metabolism under short-term hypobaric hypoxia, we exposed mice to a simulated altitude of 5000 m for 6 or 12 h. After the exposure, we collected blood and liver tissues to quantify the substrates, enzymes, and metabolites involved in glycolysis, lactic acid metabolism, the tricarboxylic acid cycle (TCA), and fatty acid ß-oxidation. We also performed transcriptome and enzymatic activity analyses of the liver. Our results show that 6 h of hypoxic exposure significantly increased blood glucose, decreased lactic acid and triglyceride concentrations, and altered liver enzyme activities of mice exposed to hypoxia. The key enzymes in the glycolytic, TCA, and fatty acid ß-oxidation pathways were primarily affected. Specifically, the activities of key glycolytic enzymes, such as glucokinase, decreased significantly, while the activities of enzymes in the TCA cycle, such as isocitrate dehydrogenase, increased significantly. Lactate dehydrogenase, pyruvate carboxylase, and alanine aminotransferase were upregulated. These changes were partially restored when the exposure time was extended to 12 h, except for further downregulation of phosphofructokinase and glucokinase. This study demonstrates that acute high altitude hypoxia upregulated the lactic acid/amino acid-pyruvate-TCA pathways and fatty acid oxidation, but downregulated glycolysis in the liver of mice. The results obtained in this study provide a theoretical framework for understanding the mechanisms underlying the pathogenesis of high-altitude sickness in humans. Additionally, these findings have potential implications for the development of prevention and treatment strategies for altitude sickness.

Microgravity and immune cells.

The microgravity environment experienced during spaceflight severely impaired immune system, making astronauts vulnerable to various diseases that seriously threaten the health of astronauts. Immune cells are exceptionally sensitive to changes in gravity and the microgravity environment can affect multiple aspects of immune cells through different mechanisms. Previous reports have mainly summarized the role of microgravity in the classification of innate and adaptive immune cells, lacking an overall grasp of the laws that microgravity effects on immune cells at different stages of their entire developmental process, such as differentiation, activation, metabolism, as well as function, which are discussed and concluded in this review. The possible molecular mechanisms are also analysed to provide a clear understanding of the specific role of microgravity in the whole development process of immune cells. Furthermore, the existing methods by which to reverse the damage of immune cells caused by microgravity, such as the use of polysaccharides, flavonoids, other natural immune cell activators etc. to target cell proliferation, apoptosis and impaired function are summarized. This review will provide not only new directions and ideas for the study of immune cell function in the microgravity environment, but also an important theoretical basis for the development of immunosuppression prevention and treatment drugs for spaceflight.

Aeromonas spp. from hospital sewage act as a reservoir of genes resistant to last-line antibiotics.

BACKGROUND: Aeromonas species are opportunistic pathogens distributed widely in the ecosystem. They are known to be capable of acquiring antibiotic resistance genes, including those encoding proteins against last-line antibiotics, such as the tmexCD-toprJ, mcr and carbapenemase genes. We investigated the genomic and phenotypic characteristics of tmexCD-toprJ-positive Aeromonas strains collected from human, animals, and water samples, particularly those from hospital wastewater in China. METHODS: Samples were collected from living animals, meat, water and human. Aeromonas strains in these samples were isolated in selective media. Antimicrobial resistance profiles of all Aeromonas strains were tested by the broth microdilution method. The presence of tmexCD-toprJ was verified by polymerase chain reaction (PCR). All tmexCD-toprJ-positive (n = 36) and selected tmexCD-toprJ-negative (n = 18) Aeromonas strains were subjected to whole genome sequencing. Carriage of antimicrobial resistance genes, the genetic environment of tmexCD-toprJ and genetic diversity of tmexCD-toprJ-positive Aeromonas strains were determined by bioinformatics analysis. Phylogenetic tree of the Aeromonas strains was built by using the Harvest Suite. FINDINGS: Among the 636 Aeromonas strains isolated from different sources, 36 were positive for tmexCD-toprJ, with the highest prevalence of tmexCD-toprJ being found in fishes (8.8%, 95 CI% 3.6-17.2%), followed by hospital wastewater (6.5%, 95 CI% 4.3-9.3%), river water (2.0%, 0.1-10.9) and duck (1.2%, 95 CI% 3.6-17.2%). All tmexCD-toprJ-positive Aeromonas strains carried multiple antimicrobial resistance genes and exhibited resistance to different classes of antibiotics. Co-existence of tmexCD-toprJ, mcr and blaKPC-2 were identified in 21 strains. The tmexCD-toprJ-positive Aeromonas strains were genetically diverse and found to belong to four different species that could be clustered into three major lineages. The tmexCD-toprJ gene clusters were predominantly located in the chromosome (35/36) of Aeromonas spp., with only one strain carrying the plasmid-borne tmexCD-toprJ cluster. The tmexCD-toprJ genes were associated with seven different types of genetic environments, each of which carried distinct types of mobile elements that may be responsible for mediating transmission of this gene cluster.

Potential role of selenium in alleviating obesity-related iron dyshomeostasis.

Obesity is a serious health problem in modern life and increases the risk of many comorbidities including iron dyshomeostasis. In contrast to malnourished anemia, obesity-related iron dyshomeostasis is mainly caused by excessive fat accumulation, inflammation, and disordered gut microbiota. In obesity, iron dyshomeostasis also induces disorders associated with gut microbiota, neurodegenerative injury, oxidative damage, and fat accumulation in the liver. Selenium deficiency is often accompanied by obesity or iron deficiency, and selenium supplementation has been shown to alleviate obesity and overcome iron deficiency. Selenium inhibits fat accumulation and exhibits anti-inflammatory activity. It regulates gut microbiota, prevents neurodegenerative injury, alleviates oxidative damage to the body, and ameliorates hepatic fat accumulation. These effects theoretically meet the requirements for the inhibition of factors underlying obesity-related iron dyshomeostasis. Selenium supplementation may have a potential role in the alleviation of obesity-related iron dyshomeostasis. This review verifies this hypothesis in theory. All the currently reported causes and results of obesity-related iron dyshomeostasis are reviewed comprehensively, together with the effects of selenium. The challenges and strategies of selenium supplementation are also discussed. The findings demonstrate the possibility of selenium-containing drugs or functional foods in alleviating obesity-related iron dyshomeostasis.

Genomic Shift in Population Dynamics of mcr-1-positive Escherichia coli in Human Carriage.

Emergence of the colistin resistance gene, mcr-1, has attracted worldwide attention. Despite the prevalence of mcr-1-positive Escherichia coli (MCRPEC) strains in human carriage showing a significant decrease between 2016 and 2019, genetic differences in MCRPEC strains remain largely unknown. We therefore conducted a comparative genomic study on MCRPEC strains from fecal samples of healthy human subjects in 2016 and 2019. We identified three major differences in MCRPEC strains between these two time points. First, the insertion sequence ISApl1 was often deleted and the percentage of mcr-1-carrying IncI2 plasmids was increased in MCRPEC strains in 2019. Second, the antibiotic resistance genes (ARGs), aac(3)-IVa and blaCTX-M-1, emerged and coexisted with mcr-1 in 2019. Third, MCRPEC strains in 2019 contained more virulence genes, resulting in an increased proportion of extraintestinal pathogenic E. coli (ExPEC) strains (36.1%) in MCRPEC strains in 2019 compared to that in 2016 (10.5%), implying that these strains could occupy intestinal ecological niches by competing with other commensal bacteria. Our results suggest that despite the significant reduction in the prevalence of MCRPEC strains in humans from 2016 to 2019, MCRPEC exhibits increased resistance to other clinically important ARGs and contains more virulence genes, which may pose a potential public health threat.

Simulated Microgravity Accelerates Alloy Corrosion by Aspergillus sp. via the Enhanced Production of Organic Acids.

Metal corrosion caused by Aspergillus sp. was shown to be significantly enhanced on a space station, but its mechanism is still unknown. To simulate this on earth, the corrosion capability of A. carbonarius on five metal sheets was investigated under simulated microgravity. Also, the effect of metal ions on growth and organic acid production was determined. Results showed that A. carbonarius could corrode all five types of metal, including Ti alloy, aluminum alloy, iron, and aluminum and copper sheet, and the corrosion was intensified under simulated microgravity. Energy dispersive X-ray spectrometry (EDS) analysis showed that metal ions enriched on A. carbonarius spores, especially iron, aluminum ions, and copper ions, indicating that A. carbonarius can use these metal ions. In particular, the content of oxalic acid was significantly increased after A. carbonarius cocultured with five metal materials under simulated microgravity. Al3+, Fe3+, and Cu2+ at the concentration of 0.3 mg/mL and Mg2+ at 0.8 mg/mL significantly promoted the growth and oxalic acid and citric acid production of A. carbonarius and A. niger under normal gravity and simulated microgravity. Comparing the impact of metal ions and metal sheets on the production of organic acids, it can be inferred that oxalic acid may dominate in the corrosion process of A. carbonarius. In summary, molds promoted metal corrosion by producing organic acids, and the released metal ions will further promote the growth of mold and the accumulation of organic acids. This may be an important reason for the intensification of mold corrosion under microgravity. IMPORTANCE The space station and other long-term manned spacecrafts will experience the risk of microbial corrosion, especially mold, which will be harmful to the platform system and astronauts. Aspergillus sp. has been widely reported to produce organic acids that corrode and destroy materials, and the ability of these crafts to fly through space can be significantly affected. Research on the mechanism that causes enhanced corrosion ability of fungi in space stations is important to control their growth. Our research focuses on the interaction between mold and metals. In particular, it is found that metal ions promote mold growth and produce organic acids, thus accelerating mold corrosion of metals. Our results provide a new perspective for the control of fungal corrosion under simulated microgravity.

Fecal Carriage of Escherichia coli Harboring the tet(X4)-IncX1 Plasmid from a Tertiary Class-A Hospital in Beijing, China.

The emergence of the mobile tigecycline-resistance gene, tet(X4), poses a significant threat to public health. To investigate the prevalence and genetic characteristics of the tet(X4)-positive Escherichia coli in humans, 1101 human stool samples were collected from a tertiary class-A hospital in Beijing, China, in 2019. Eight E. coli isolates that were positive for tet(X4) were identified from clinical departments of oncology (n = 3), hepatology (n = 2), nephrology (n = 1), urology (n = 1), and general surgery (n = 1). They exhibited resistance to multiple antibiotics, including tigecycline, but remained susceptible to meropenem and polymyxin B. A phylogenetic analysis revealed that the clonal spread of four tet(X4)-positive E. coli from different periods of time or departments existed in this hospital, and three isolates were phylogenetically close to the tet(X4)-positive E. coli from animals and the environment. All tet(X4)-positive E. coli isolates contained the IncX1-plasmid replicon. Three isolates successfully transferred their tigecycline resistance to the recipient strain, C600, demonstrating that the plasmid-mediated horizontal gene transfer constitutes another critical mechanism for transmitting tet(X4). Notably, all tet(X4)-bearing plasmids identified in this study had a high similarity to several plasmids recovered from animal-derived strains. Our findings revealed the importance of both the clonal spread and horizontal gene transfer in the spread of tet(X4) within human clinics and between different sources.

Response of human gut microbiota under simulated microgravity.

The present study was conducted to investigate the influence of microgravity on human gut microbiota using 16S rRNA gene sequencing in vitro. The diamagnetic material magnetic levitation method was used to simulate weightless environment. The human gut microbiota was cultured under two different conditions: normal gravity (1 g), and simulated microgravity (0 g), which showed that both the richness (P = 0.04) and diversity (P = 0.0002) of human gut microbiota were significantly altered. As compared to the normal gravity, the simulated microgravity significantly reduced abundance of bacteria related to anti-inflammatory effects, such as Subdoligranulum, Faecalibacterium, Fusicatenibacter, Butyricicoccus, and Lachnospiraceae-NK4A136-0 group (P < 0.05), while significantly increased that of Alistipes and Eubacterium-Ventriosum-group (P < 0.05). Moreover, the Spearman's correlation analysis showed that there were more significantly correlated species (|r|≥ 0.5, P < 0.05) in normal gravity than that in the simulated microgravity. KEGG pathway analysis revealed that the microgravity significantly (P < 0.05) affected the metabolism of gut microbiota, such as the metabolism of pyrimidine, fatty acids, glyoxylate and dicarboxylate, peptidoglycan biosynthesis, and carbon fixation in photosynthetic organisms. These results suggested that the exposure to a microgravity environment might induce disturbances in human gut microbiota. KEY POINTS: • Using 16sRNA gene sequencing technology, it was found that magnetic levitation-simulated microgravity had varying degrees of influence on the abundance, diversity, species correlation, and KEGG pathways of human intestinal microbes. • Digital PCR can improve the detection rate of microorganisms with low abundance.

Potentials of orally supplemented selenium-enriched Lacticaseibacillus rhamnosus to mitigate the lead induced liver and intestinal tract injury.

Lead is a metal that exists naturally in the Earth's crust and is a ubiquitous environmental contaminant. The alleviation of lead toxicity is important to keep human health under lead exposure. Biosynthesized selenium nanoparticle (SeNPs) and selenium-enriched Lactobacillus rhamnosus SHA113 (Se-LRS) were developed in this study, and their potentials in alleviating lead-induced injury to the liver and intestinal tract were evaluated in mice by oral administration for 4 weeks. As results, oral intake of lead acetate (150 mg/kg body weight per day) caused more than 50 times and 100 times lead accumulation in blood and the liver, respectively. Liver function was seriously damaged by the lead exposure, which is indicated as the significantly increased lipid accumulation in the liver, enhanced markers of liver function injury in serum, and occurrence of oxidative stress in liver tissues. Serious injury in intestinal tract was also found under lead exposure, as shown by the decrease of intestinal microbiota diversity and occurrence of oxidative stress. Except the lead content in blood and the liver were lowered by 52% and 58%, respectively, oral administration of Se-LRS protected all the other lead-induced injury markers to the normal level. By the comparison with the effects of normal L. rhamnosus SHA113 and the SeNPs isolated from Se-LRS, high protective effects of Se-LRS can be explained as the extremely high efficiency to promote lead excretion via feces by forming insoluble mixture. These findings illustrate the developed selenium-enriched L. rhamnosus can efficiently protect the liver and intestinal tract from injury by lead.

Structural diversity of the ISCR2-mediated rolling-cycle transferable unit carrying tet(X4).

BACKGROUND: Mobile tigecycline-resistance gene tet(X) variants have emerged as diverse pathogens from animal, human as well as their associated environments, which could potentially threaten public health. The insertion sequence, ISCR2, carries tet(X4) for horizontal transfer by rolling-cycle (RC) transposition. However, the diversity of ISCR2 and tet(X4) isolated from different sources is largely unknown. METHODS: The tet(X4)-carrying isolates were collected from human and livestock in several multiple regions of China. The whole genomic sequences of these isolates were either obtained from NCBI GenBank or determined by Illumina Hiseq 2500 and the MinION platform. The intact transposon region, ISCR2-tet(X4)-ISCR2, observed in a small number of isolates as the reference sequence to construct the transposon phylogeny. The diversity of the genetic environments of all ISCR2-tet(X4) elements were analyzed. RESULTS: A 2760-bp element encompassing the tet(X4)-hydrolase-encoding gene, catD, located between two ISCR2 elements was highly conserved in all isolates and could form an RC transposable unit (RC-TU). ISCR2 could also capture more resistance genes and formed a larger RC-TU base on RC transposition. However, the ISCR2-mediated RC-TUs were constantly truncated and inserted by other IS elements, indicating frequent recombination events. Of these elements, IS26 disrupted both the upstream and downstream ISCR2-mediated RC-TUs, indicating that IS26 captured tet(X4), thus leading to a wider spread of tet(X4). CONCLUSIONS: These results confirmed the critical role of ISCR2 for dissemination and co-transmission of tet(X4) and other resistance genes. More effort is needed to monitor the variation tendencies of tet(X4)-carrying mobile elements and determine the driving factors for disseminating transferable tigecycline resistance.

Distinct increase in antimicrobial resistance genes among Escherichia coli during 50 years of antimicrobial use in livestock production in China.

Antimicrobial use in livestock production is linked to the emergence and spread of antimicrobial resistance (AMR), but large-scale studies on AMR changes in livestock isolates remain scarce. Here we applied whole-genome sequence analysis to 982 animal-derived Escherichia coli samples collected in China from the 1970s to 2019, finding that the number of AMR genes (ARGs) per isolate doubled-including those conferring resistance to critically important agents for both veterinary (florfenicol and norfloxacin) and human medicine (colistin, cephalosporins and meropenem). Plasmids of incompatibility groups IncC, IncHI2, IncK, IncI and IncX increased distinctly in the past 50 years, acting as highly effective vehicles for ARG spread. Using antimicrobials of the same class, or even unrelated classes, may co-select for mobile genetic elements carrying multiple co-existing ARGs. Prohibiting or strictly curtailing antimicrobial use in livestock is therefore urgently needed to reduce the growing threat from AMR.

The Role of Vasoactive Intestinal Peptide and Mast Cells in the Regulatory Effect of Lactobacillus casei ATCC 393 on Intestinal Mucosal Immune Barrier.

Vasoactive intestinal peptide (VIP) plays an important role in the neuro-endocrine-immune system. Mast cells (MCs) are important immune effector cells. This study was conducted to investigate the protective effect of L. casei ATCC 393 on Enterotoxigenic Escherichia coli (ETEC) K88-induced intestinal mucosal immune barrier injury and its association with VIP/MC signaling by in vitro experiments in cultures of porcine mucosal mast cells (PMMCs) and in vivo experiments using VIP receptor antagonist (aVIP) drug. The results showed that compared with the ETEC K88 and lipopolysaccharides (LPS)-induced model groups, VIP pretreatment significantly inhibited the activation of MCs and the release of ß-hexosaminidase (ß-hex), histamine and tryptase. Pretreatment with aVIP abolished the protective effect of L. casei ATCC 393 on ETEC K88-induced intestinal mucosal immune barrier dysfunction in C57BL/6 mice. Also, with the blocking of VIP signal transduction, the ETEC K88 infection increased serum inflammatory cytokines, and the numbers of degranulated MCs in ileum, which were decreased by administration of L. casei ATCC 393. In addition, VIP mediated the regulatory effect of L. casei ATCC 393 on intestinal microbiota in mice. These findings suggested that VIP may mediate the protective effect of L.casei ATCC 393 on intestinal mucosal immune barrier dysfunction via MCs.

Effects of Bacillus subtilis iturin A on HepG2 cells in vitro and vivo.

Iturin A with cyclic peptide and fatty acid chain isolated from Bacillus subtilis fermentation shows a variety of biological activities. Among them, the anticancer activity attracted much attention. However, the molecular mechanism of its inhibitory effect on hepatocellular carcinoma was still unclear. Thus its effect on hepatocellular carcinoma was tested in this research. It was found that iturin A could enter HepG2 cells immediately and cause reactive oxygen species burst, disrupt cell cycle and induce apoptosis, paraptosis and autophagy in vitro. The iturin A without fatty acid chain showed no antitumor activity. Amphiphilic is critical to the activity of iturin A. The anticancer activity of iturin A to hepatocellular carcinoma was also verified in mice models carrying xenograft tumors constructed by HepG2 cells. At a dosage of 3 mg/kg/day, iturin A significantly inhibited the further increase of the tumor weight by 58.55%, and reduced the expression of Ki67 in tumor. In the tumor treated with iturin A, lymphocyte infiltration was found, and the expressions of TGF-ß1and PD-L1 were decreased, which indicated that the tumor immune microenvironment was improved. Besides, iturin A showed no significant harm on the health of mice except slight disturbance of liver function. These results suggested that iturin A had significant antitumor effect in vitro and vivo, and provide a basis for the application of iturin A as anticancer agent.

Lactobacillus rhamnosus from human breast milk ameliorates ulcerative colitis in mice via gut microbiota modulation.

Gut microbiota imbalance is one of the major causes of ulcerative colitis (UC). L. rhamnosus SHA113 (LRS), a strain isolated from healthy human milk, influences the regulation of gut flora. This study aims to determine whether this strain can ameliorate UC by modulating gut microbiota. Mouse models of UC were established using C57BL/6Cnc mice with intragastric administration of 3.0% (w/v) dextran sodium sulfate (DSS). LRS was used to treat the mouse models of UC with 109 cfu mL-1 cell suspension via intragastric administration. To verify the effect of gut microbiota on UC, fecal microbiota collected from the mice after the treatment with LRS were also used to treat the UC mouse models (FMT). The severity of UC was evaluated based on body weight, colon length, disease activity index (DAI), and hematoxylin-eosin staining. The microbial composition was analyzed by 16S rRNA sequencing. The mRNA expression levels of cytokines, mucins, tight junction proteins, and antimicrobial peptides in the gastrointestinal tract were detected by quantitative real-time polymerase chain reaction. The short-chain fatty acid (SCFAs) in the cecal contents of all mice were quantitatively detected by gas chromatography and mass spectrometry. Both LRS and FMT exerted excellent therapeutic effects on UC, as evidenced by the reduction in body weight loss, colon length, and colon structural integrity, as well as the increase in the DAI (disease activity index). LRS and FMT treatments showed similar effects: (1) an increase of total SCFA production in the cecal contents and the abundance of gut microbial diversity and flora composition; (2) decreases in two genera (Parabacteroides and Escherichia/Shigella) related to the DAI and the enhancement of SCFAs and IL-10 positively related genera in the gut microbiota (Bilophila, Roseburia, Akkermansia, and Bifidobacterium); (3) downregulation of the expression of tumor necrosis factor-α, interleukin IL-6, and IL-1ß, and upregulation of the expression of the anti-inflammatory cytokine IL-10; and (4) upregulation of the expression of mucins (Muc1-4) and tight junction protein ZO-1. Overall, L. rhamnosus SHA113 relieves UC via the regulation of gut microbiota: increases in SCFA-producing genera and decreases in UC-related genera. In addition, a single strain is sufficient to induce a significant change in the gut microbiota and exert therapeutic effects on UC.