Bacitracin-resistant Staphylococcus aureus induced in chicken gut and in vitro under bacitracin exposure.

It is common knowledge that prolonged and excessive use of antibiotics can lead to antimicrobial resistance. However, the characteristics and mechanism of resistant-bacteria induced by clinically recommended and prophylactic dose drugs remain largely unclear. This study aimed to observe the trends of drug resistance of the bacitracin-susceptible Staphylococcus aureus strain FS127 under exposure to bacitracin (BAC), which were induced in vitro and in chicken gut. Antimicrobial susceptibility testing was used to detect the susceptibility of S. aureus induced in vitro and in the chicken gut to gentamicin, chloramphenicol, tetracycline, doxycycline, penicillin and chloramphenicol. The research results showed that bacitracin could induce drug resistance in S. aureus both in vitro and in vivo. The bacitracin-resistance rate of S. aureus isolated from chicken gut was positively correlated with the dose and time of bacitracin administration. The findings revealed that bacitracin-resistant S. aureus induced in vivo had enhanced susceptibility to chloramphenicol but no such change in vitro. Meanwhile, RT-qPCR assay was used to detect the expression levels of vraD, braD, braR and bacA in typical strains with different bacitracin-resistance levels. It was found that BacA may play a key role in the bacitracin resistance of S. aureus. In conclusion, this work reveals the characteristics and mechanism of bacitracin-resistant S. aureus induced by bacitracin in vivo and in vitro respectively.

Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin).

Streptococcus pneumoniae (S. pneumoniae) represents a major human bacterial pathogen leading to high morbidity and mortality in children and the elderly. Recent research emphasizes the role of extracellular vesicles (EVs) in bacterial pathogenicity. However, the contribution of S. pneumoniae EVs (pEVs) to host-microbe interactions has remained unclear. Here, we observed that S. pneumoniae infections in mice led to severe lung injuries and alveolar epithelial barrier (AEB) dysfunction. Infections of S. pneumoniae reduced the protein expression of tight junction protein OCLN (occludin) and activated macroautophagy/autophagy in lung tissues of mice and A549 cells. Mechanically, S. pneumoniae induced autophagosomal degradation of OCLN leading to AEB impairment in the A549 monolayer. S. pneumoniae released the pEVs that could be internalized by alveolar epithelial cells. Through proteomics, we profiled the cargo proteins inside pEVs and found that these pEVs contained many virulence factors, among which we identified a eukaryotic-like serine-threonine kinase protein StkP. The internalized StkP could induce the phosphorylation of BECN1 (beclin 1) at Ser93 and Ser96 sites, initiating autophagy and resulting in autophagy-dependent OCLN degradation and AEB dysfunction. Finally, the deletion of stkP in S. pneumoniae completely protected infected mice from death, significantly alleviated OCLN degradation in vivo, and largely abolished the AEB disruption caused by pEVs in vitro. Overall, our results suggested that pEVs played a crucial role in the spread of S. pneumoniae virulence factors. The cargo protein StkP in pEVs could communicate with host target proteins and even hijack the BECN1 autophagy initiation pathway, contributing to AEB disruption and bacterial pathogenicity.Abbreviations: AEB: alveolarepithelial barrier; AECs: alveolar epithelial cells; ATG16L1: autophagy related 16 like 1; ATP:adenosine 5'-triphosphate; BafA1: bafilomycin A1; BBB: blood-brain barrier; CFU: colony-forming unit; co-IP: co-immunoprecipitation; CQ:chloroquine; CTRL: control; DiO: 3,3'-dioctadecylox-acarbocyanineperchlorate; DOX: doxycycline; DTT: dithiothreitol; ECIS: electricalcell-substrate impedance sensing; eGFP: enhanced green fluorescentprotein; ermR: erythromycin-resistance expression cassette; Ery: erythromycin; eSTKs: eukaryotic-like serine-threoninekinases; EVs: extracellular vesicles; HA: hemagglutinin; H&E: hematoxylin and eosin; HsLC3B: human LC3B; hpi: hours post-infection; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LC/MS: liquid chromatography-mass spectrometry; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MVs: membranevesicles; NC:negative control; NETs:neutrophil extracellular traps; OD: optical density; OMVs: outer membrane vesicles; PBS: phosphate-buffered saline; pEVs: S.pneumoniaeextracellular vesicles; protK: proteinase K; Rapa: rapamycin; RNAi: RNA interference; S.aureus: Staphylococcusaureus; SNF:supernatant fluid; sgRNA: single guide RNA; S.pneumoniae: Streptococcuspneumoniae; S.suis: Streptococcussuis; TEER: trans-epithelium electrical resistance; moi: multiplicity ofinfection; TEM:transmission electron microscope; TJproteins: tight junction proteins; TJP1/ZO-1: tight junction protein1; TSA: tryptic soy agar; WB: western blot; WT: wild-type.

CRISPR-Cas systems target endogenous genes to impact bacterial physiology and alter mammalian immune responses.

CRISPR-Cas systems are an immune defense mechanism that is widespread in archaea and bacteria against invasive phages or foreign genetic elements. In the last decade, CRISPR-Cas systems have been a leading gene-editing tool for agriculture (plant engineering), biotechnology, and human health (e.g., diagnosis and treatment of cancers and genetic diseases), benefitted from unprecedented discoveries of basic bacterial research. However, the functional complexity of CRISPR systems is far beyond the original scope of immune defense. CRISPR-Cas systems are implicated in influencing the expression of physiology and virulence genes and subsequently altering the formation of bacterial biofilm, drug resistance, invasive potency as well as bacterial own physiological characteristics. Moreover, increasing evidence supports that bacterial CRISPR-Cas systems might intriguingly influence mammalian immune responses through targeting endogenous genes, especially those relating to virulence; however, unfortunately, their underlying mechanisms are largely unclear. Nevertheless, the interaction between bacterial CRISPR-Cas systems and eukaryotic cells is complex with numerous mysteries that necessitate further investigation efforts. Here, we summarize the non-canonical functions of CRISPR-Cas that potentially impact bacterial physiology, pathogenicity, antimicrobial resistance, and thereby altering the courses of mammalian immune responses.

Phenotypic and Genotypic Characterization of Multidrug-Resistant Enterobacter hormaechei Carrying qnrS Gene Isolated from Chicken Feed in China.

Multidrug resistance (MDR) in Enterobacteriaceae including resistance to quinolones is rising worldwide. The plasmid-mediated quinolone resistance (PMQR) gene qnrS is prevalent in Enterobacteriaceae. However, the qnrS gene is rarely found in Enterobacter hormaechei (E. hormaechei). Here, we reported one multidrug resistant E. hormaechei strain M1 carrying the qnrS1 and blaTEM-1 genes. This study was to analyze the characteristics of MDR E. hormaechei strain M1. The E. hormaechei strain M1 was identified as Enterobacter cloacae complex by biochemical assay and 16S rRNA sequencing. The whole genome was sequenced by the Oxford Nanopore method. Taxonomy of the E. hormaechei was based on multilocus sequence typing (MLST). The qnrS with the other antibiotic resistance genes were coexisted on IncF plasmid (pM1). Besides, the virulence factors associated with pathogenicity were also located on pM1. The qnrS1 gene was located between insertion element IS2A (upstream) and transposition element ISKra4 (downstream). The comparison result of IncF plasmids revealed that they had a common plasmid backbone. Susceptibility experiment revealed that the E. hormaechei M1 showed extensive resistance to the clinical antimicrobials. The conjugation transfer was performed by filter membrane incubation method. The competition and plasmid stability assays suggested the host bacteria carrying qnrS had an energy burden. As far as we know, this is the first report that E. hormaechei carrying qnrS was isolated from chicken feed. The chicken feed and poultry products could serve as a vehicle for these MDR bacteria, which could transfer between animals and humans through the food chain. We need to pay close attention to the epidemiology of E. hormaechei and prevent their further dissemination. IMPORTANCE Enterobacter hormaechei is an opportunistic pathogen. It can cause infections in humans and animals. Plasmid-mediated quinolone resistance (PMQR) gene qnrS can be transferred intergenus, which is leading to increase the quinolone resistance levels in Enterobacteriaceae. Chicken feed could serve as a vehicle for the MDR E. hormaechei. Therefore, antibiotic-resistance genes (ARGs) might be transferred to the intestinal flora after entering the gastrointestinal tract with the feed. Furthermore, antibiotic-resistant bacteria (ARB) were also excreted into environment with feces, posing a huge threat to public health. This requires us to monitor the ARB and antibiotic-resistant plasmids in the feed. Here, we demonstrated the characteristics of one MDR E. hormaechei isolate from chicken feed. The plasmid carrying the qnrS gene is a conjugative plasmid with transferability. The presence of plasmid carrying antibiotic-resistance genes requires the maintenance of antibiotic pressure. In addition, the E. hormaechei M1 belonged to new sequence type (ST). These data show the MDR E. hormaechei M1 is a novel strain that requires our further research.

Virulence Comparison of Salmonella enterica Subsp. enterica Isolates from Chicken and Whole Genome Analysis of the High Virulent Strain S. Enteritidis 211.

BACKGROUND: Salmonellaenterica is one of the common pathogens in both humans and animals that causes salmonellosis and threatens public health all over the world. METHODS AND RESULTS: Here we determined the virulence phenotypes of nine Salmonellaenterica subsp. enterica (S. enterica) isolates in vitro and in vivo, including pathogenicity to chicken, cell infection, biofilm formation and virulence gene expressions. S. Enteritidis 211 (SE211) was highly pathogenic with notable virulence features among the nine isolates. The combination of multiple virulence genes contributed to the conferring of the high virulence in SE211. Importantly, many mobile genetic elements (MGEs) were found in the genome sequence of SE211, including a virulence plasmid, genomic islands, and prophage regions. The MGEs and CRISPR-Cas system might function synergistically for gene transfer and immune defense. In addition, the neighbor joining tree and the minimum spanning tree were constructed in this study. CONCLUSIONS: This study provided both the virulence phenotypes and genomic features, which might contribute to the understanding of bacterial virulence mechanisms in Salmonella enterica subsp. enterica. The first completed genomic sequence for the high virulent S. Enteritidis isolate SE211 and the comparative genomics and phylogenetic analyses provided a preliminary understanding of S. enterica genetics and laid the foundation for further study.

Longitudinal Surveillance and Risk Assessment of Resistance in Escherichia coli to Enrofloxacin from A Large-Scale Chicken Farm in Hebei, China.

The purpose of this study was to investigate the changes of resistance phenotype and plasmid-mediated quinolone resistance genes (PMQRs) in Escherichia coli (E. coli) during enrofloxacin (ENR) administration in different breeding cycles. In 2020, 983 strains of E. coli were isolated from different samples in different cycles at the broiler farm with the largest single batch of slaughter capacity in Hebei Province, China. All samples were from chicken, environmental, and human sources. The sensitivity of the isolates to various antibiotics was determined by broth microdilution method. The findings of this study include: (1) the total isolation rate of E. coli in the four cycles was 63.83% (983/1540); (2) the average resistance rate of E. coli from 1-day-old chickens to enrofloxacin was as high as 75% in each cycle, and with the use of enrofloxacin, the resistance rate of E. coli from chickens gradually increased to 100%; (3) 107 strains of E. coli randomly selected from different cycles and sources demonstrated the multi-drug resistance phenotypes. The highest resistance rate was doxycycline (100%), and the lowest was erythromycin (54.21%); (4) the detection rate of PMQRs of E. coli from chickens in different cycles were always higher than that from environmental and human. In particular, the PMQRs pollution rate of chicken seedlings in each cycle were generally higher than that of other sources; (5) We used SPSS software to analyze the Kendall rank correlation of the experimental data. The resistance of E. coli isolated from this farm to ciprofloxacin (CIP) may increase along with the increase of resistance to enrofloxacin (Kendall's tau-b = 0.190, p = 0.021). All these data highlight the serious problem of bacterial resistance in this farm. Therefore, it is urgent to provide guidance for the prevention and control of colibacillosis and drug resistance in this farm.

Enrofloxacin Promotes Plasmid-Mediated Conjugation Transfer of Fluoroquinolone-Resistance Gene qnrS.

This study aimed to determine the effect of enrofloxacin (ENR) on the transfer of the plasmid-mediated quinolone resistance (PMQR) gene qnrS from opportunistic pathogen Escherichia coli (E2) to Salmonella Enteritidis (SE211) and to analyze the resistance characteristics of SE211-qnrS isolates. The plasmid carrying qnrS gene of E2 was sequenced by Oxford Nanopore technology. The plasmid carrying qnrS gene belonged to incompatibility group IncY. In vitro, the transfer experiment of IncY plasmid was performed by the liquid medium conjugation method. The conjugation transfer frequency of the IncY plasmid was 0.008 ± 0.0006 in the absence of ENR, 0.012 ± 0.003 in 1/32 MICENR, 0.01 ± 0.008 in 1/8 MICENR, and 0.03 ± 0.015 (Mean±SD) in 1/2 MICENR, respectively. After inoculation of E. coli E2 and SE211, chickens were treated with different doses of ENR (3.03, 10, and 50 mg/kg b.w.) for 7 days consecutively. To screen the SE211-qnrS strains from intestinal tract of chickens, the resistance genes and susceptibility of isolates were identified. The amount of E. coli E2 and the copy number of qnrS gene in the chicken intestinal tract were determined by colony counting and qPCR, respectively. In vivo, more SE211-qnrS strains were isolated from the treated group compared with the untreated group. SE211-qnrS strains not only obtained IncY plasmid, but also showed similar resistance phenotype as E2. In conclusion, ENR treatment can promote the spread of a IncY-resistance plasmid carrying the qnrS fluoroquinolone-resistance gene in Escherichia coli and the development of drug-resistant bacteria.

Transcriptional regulation of seven cyadox-related genes mainly activated by PI3K and NF-кB signaling pathways in PK-15 cells.

Cyadox, a new antibacterial agent as the quinoxaline-1, 4-dioxides, has a good antibacterial and growth-promoting effect, and has the advantages of lower toxicity, adequate safety and faster absorption. Seven differential expressed genes (DEGs) induced by cyadox were screened in swine liver tissues, including Insulin-like Growth Factor-1 (IGF-1), Epidermal Growth Factor (EGF), Poly ADP-ribose polymerase (PARP), the Defender Against Apoptotic Death 1 (DAD1), Complement Component 3 (C3), Transketolase (TK) and cyadox-related novel gene (CRNG). To elucidate the signal mechanism that cyadox altered these genes expression, the time-effect relationship and signaling pathways related to 7 DEGs induced by cyadox were determined in Porcine Kidney-15 (PK-15) cells by RT-qPCR and the application of various signal pathway inhibitors. The phosphorylation levels of signal factors in PK-15 cells were detected by Western blot. The analyses demonstrated that, the mRNA expressions of 7 DEGs were significantly enhanced by cyadox mainly through the phosphoinositide 3-kinase (PI3K) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) signaling pathways in PK-15 cells. Furthermore, EGF might be the early response gene of cyadox to activate downstream signaling pathways and regulates the expression of other related genes or directly exerting biological effects. In brief, cyadox mainly regulates the expression of these 7 genes by PI3K and NF-кB signaling pathways to exert it's antibacterial and growth-promoting activity in PK-15 cells.

CRISPR-cas3 of Salmonella Upregulates Bacterial Biofilm Formation and Virulence to Host Cells by Targeting Quorum-Sensing Systems.

Salmonella is recognized as one of the most common microbial pathogens worldwide. The bacterium contains the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, providing adaptive immunity against invading foreign nucleic acids. Previous studies suggested that certain bacteria employ the Cas proteins of CRISPR-Cas systems to target their own genes, which also alters the virulence during invasion of mammals. However, whether CRISPR-Cas systems in Salmonella have similar functions during bacterial invasion of host cells remains unknown. Here, we systematically analyzed the genes that are regulated by Cas3 in a type I-E CRISPR-Cas system and the virulence changes due to the deletion of cas3 in Salmonella enterica serovar Enteritidis. Compared to the cas3 gene wild-type (cas3 WT) Salmonella strain, cas3 deletion upregulated the lsrFGBE genes in lsr (luxS regulated) operon related to quorum sensing (QS) and downregulated biofilm-forming-related genes and Salmonella pathogenicity island 1 (SPI-1) genes related to the type three secretion system (T3SS). Consistently, the biofilm formation ability was downregulated in the cas3 deletion mutant (Δcas3). The bacterial invasive and intracellular capacity of Δcas3 to host cells was also reduced, thereby increasing the survival of infected host cells and live chickens. By the transcriptome-wide screen (RNA-Seq), we found that the cas3 gene impacts a series of genes related to QS, the flagellum, and SPI-1-T3SS system, thereby altering the virulence phenotypes. As QS SPI-1-T3SS and CRISPR-Cas systems are widely distributed in the bacteria kingdom, our findings extend our understanding of virulence regulation and pathogenicity in mammalian hosts for Salmonella and potentially other bacteria.

Interaction among inflammasome, autophagy and non-coding RNAs: new horizons for drug.

Autophagy and inflammasomes are shown to interact in various situations including infectious disease, cancer, diabetes and neurodegeneration. Since multiple layers of molecular regulators contribute to the interplay between autophagy and inflammasome activation, the detail of such interplay remains largely unknown. Non-coding RNAs (ncRNAs), which have been implicated in regulating an expanding list of cellular processes including immune defense against pathogens and inflammatory response in cancer and metabolic diseases, may join in the crosstalk between inflammasomes and autophagy in physiological or disease conditions. In this review, we summarize the latest research on the interlink among ncRNAs, inflammasomes and autophagy and discuss the emerging role of these three in multiple signaling transduction pathways involved in clinical conditions. By analyzing these intriguing interconnections, we hope to unveil the mechanism inter-regulating these multiple processes and ultimately discover potential drug targets for some refractory diseases.

Molecular Characterization and Biological Function of a Novel LncRNA CRNG in Swine.

Our previous study has showed that a novel gene is differentially expressed in the liver of cyadox-fed piglets, but its sequence and function are unknown. Here, rapid amplification of cDNA ends (RACE) and bioinformatics analysis showed that the novel gene is 953 bp without protein-coding ability and locates in chromosome 11. Hence, we identified the novel gene as long non-coding RNA (lncRNA) and named it cyadox-related novel gene (CRNG). Fluorescence in situ hybridization (FISH) showed that CRNG mainly distributes in cytoplasm. Moreover, microarray assay in combination with CRNG interference and overexpression showed that the differential genes such as ANPEP, KITLG, STAT5A, FOXP3, miR-451, IL-2, IL-10, IL-6, and TNF-α are mainly involved in viral and pathogens infection and the immune-inflammatory responses in PK-15 cells. This work reveals that CRNG might play a role in preventing the host from being infected by pathogens and viruses and exerting immune regulatory effects in the cytoplasm, which may be involved in prophylaxis of cyadox in piglets.

The epigenetic mechanisms in Fusarium mycotoxins induced toxicities.

Fusarium mycotoxins are the most economically important fungal toxins. Fumonisins, zearalenone and trichothecenes (T-2 toxin, HT-2 toxin, deoxynivalenol, nivalenol etc) are the major representatives and most studied of Fusarium mycotoxins. The Fusarium mycotoxins contaminate cereal grains, animal feeds and human food products, and cause huge economic losses and pose a threat to animal and human health globally. Depending on the type, the toxicity of Fusarium mycotoxins and the mechanisms have been well investigated. Epigenetic modifications (DNA methylation, histone modifications and regulation of non-coding RNA) have been implicated in various human diseases and the toxicities in animals caused by Fusarium mycotoxins, including carcinogenesis, genotoxicity and reproductive disorders. On the basis of recently documented data, this review discussed the relationship between the epigenetic modifications and Fusarium mycotoxins-induced toxicities.

An overview of epigenetic agents and natural nutrition products targeting DNA methyltransferase, histone deacetylases and microRNAs.

Several humans' diseases such as; cancer, heart disease, diabetes retain an etiology of epigenetic, and a new therapeutic option termed as "epigenetic therapy" can offer a potential way to treat these diseases. A numbers of epigenetic agents such as; inhibitors of DNA methyltransferase (DNMT) and histone deacetylases (HDACs) have grew an intensive investigation, and many of these agents are currently being tested in a clinical trial, while some of them have been approved for the use by the authorities. Since miRNAs can act as tumor suppressors or oncogenes, the miRNA mimics and molecules targeted at miRNAs (antimiRs) have been designed to treat some of the diseases. Much naturally occurring nutrition were discovered to alter the epigenetic states of cells. The nutrition, including polyphenol, flavonoid compounds, and cruciferous vegetables possess multiple beneficial effects, and some can simultaneously change the DNA methylation, histone modifications and expression of microRNA (miRNA). This review mainly summarizes the information of epigenetic agents of DNMTs and HDACs inhibitors, miRNA mimics and antimiRs, as well as the natural nutrition. In addition, some future perspectives related to the epigenetic therapy are also included.

Cyadox regulates the transcription of different genes by activation of the PI3K signaling pathway in porcine primary hepatocytes.

Cyadox, a new derivative of quinoxalines, has been ascertained as an antibiotic with significant growth promoting, low poison, quick absorption, swift elimination, brief residual period, and noncumulative effect. Seven differential expressed genes, including Insulin-like Growth Factor-1 ( IGF-1), Epidermal Growth Factor ( EGF), Poly ADP-ribose polymerase ( PARP), the Defender Against Apoptotic Death 1 ( DAD1), Complement Component 3 ( C3), Transketolase ( TK) and a New gene, were induced by cyadox in swine liver tissues by messenger RNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) in our laboratory. However, the signal mechanism that cyadox altered these genes expression is not completely elucidated. The signaling pathways involved in the expressions of seven genes induced by cyadox were determined in porcine primary hepatocytes by RT-qPCR and the application of various signal pathway inhibitors. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that cyadox could stimulate proliferation of porcine primary hepatocytes in a time-dependent manner. In porcine primary cultured hepatocytes, phosphoinositide 3-kinase (PI3K) and transforming growth factor-ß (TGF-ß) signal pathways were the main signal pathways involved in the expressions of seven genes induced by cyadox. Taken together, these results demonstrate for the first time that seven cyadox-related genes expressions in porcine primary hepatocytes treated with cyadox are mediated mainly through the PI3K signaling pathway, potentially leading to enhanced cell growth and cell immunity. EGF might be the early response gene of cyadox, and a primary regulator of the other gene expressions such as IGF-1 and DAD1, playing an important role in cell proliferation promoted by cyadox.

Signaling pathways involved in the expression of SZNF and the target genes binding with SZNF related to cyadox.

SZNF (Sus scrofa zinc finger CCHC domain containing 3) is a post-transcription regulation factor, belonging to CCHC zinc finger proteins. Cyadox is a novel quinoxaline drug with antibacterial and growth promotion effects. In this study, we investigated the pharmacological mechanism of cyadox mediated by SZNF. Firstly, signaling pathways related to cyadox-induced SZNF expression were studied. The results showed that the mRNA level of SZNF reached the peak as early as 4 h after 2 µM cyadox treatment in swine hepatocytes. Several signaling pathways, including JAK2/STAT1, PI3K/Akt, TGF-ß/Smad3 and p38, might play critical roles in regulation of SZNF. The JAK2/STAT1, JAK2/PI3K/Akt, PI3K/Akt and myD88 & TAK1 & ASK1 /P38 signaling pathways were firstly activated after cyadox treatment in swine hepatocyte, the TGF-ß/Smad3 signaling pathway was activated later. Then given the characteristic of RNA binding of CCHC zinc finger proteins, the target mRNAs binding with SZNF were detected by RNA immunoprecipitation coupled to sequencing (RIP-seq) in PK-15 cells treated with cyadox. The RIP-Seq results showed that the bound mRNAs of 45 genes and 93 genes by SZNF protein were increased and decreased, respectively in cyadox-treated PK-15 cells compared with blank sample. With bioinformatics analysis, we showed that cyadox might exert its antibacterial and growth promotion effect by regulating SZNF-associated target genes in post-transcriptional level, such as genes related to growth (MLXIP, CKS2) and inflammation (LGALS3, PLAU). Thus, our results indicated that SZNF can post-transcriptionally regulate its target genes related to growth and inflammatory in cyadox-treated cells, which may explain the pharmacological mechanism of this drug.

Differentially expressed genes in response to cyadox in swine liver analyzed by DDRT-PCR.

Cyadox is a good antimicrobial growth-promoter of quinoxalines. However, the molecular mechanism of action remains unclear. A growth performance study and mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) in combination with Northern dot-blot and reverse Northern dot-blot analysis were conducted to determine the differentially expressed genes in liver tissues of piglets after treatment with cyadox. Transcription levels of the differentially expressed genes were quantificated by realtime RT-PCR in porcine primary hepotocytes. Cyadox could significantly promote body weight of piglets via feed with average daily gain (ADG) improved by 24.7% and 64.8% in 100 and 500mg/kg group, compared with control. A total of eight differentially expressed genes were found, of which the expression levels of five genes had positive correlation with cyadox dose. One gene expression had a negative correlation with cyadox dose and it was a new gene. The other two genes were up-regulated by cyadox, but the expression quantity was invariably when the cyadox doses were increased. Among the up-regulated genes, one was transcriptional regulating factor, two were growth-related factors, one was involved in immune defense and immune-regulation and three might be involved in the maintenance of normal development. In primary cultured pig hepatocytes, cyadox treatment evoked a significant time-dependent effect of eight genes expression. The results suggest, at the transcriptional level in vitro and in vivo, that growth factor and metabolism may be associated with cyadox growth-promoting activity, whereas immune defense and immune-regulation could play major roles in prophylaxis of cyadox in piglets.

Preparation and characterization of chitosan membranes.

The present study investigates a new solvent system for the dissolution of chitosan and a new method for preparing chitosan membranes. First, aqueous tartaric acid was used to pretreat chitosan. Then, the chitosan was precipitated with ethanol or other regenerating agents, and 1.5 mL of 1-ethyl-3-methylimidazolium acetate ([EMIM]AC) was added to obtain translucent suspensions. The chitosan membranes were prepared by casting the suspensions on glass plates and allowing solvent evaporation. The structure and properties of the films were investigated by SEM, FT-IR, XRD and TGA. Also, the mechanical properties, as well as physical and chemical characteristics, of the chitosan films were evaluated. The results indicated that the optimum dissolution time was 10 min and the most suitable drying temperature was 60 °C. The thus-prepared film was moderately thick (about 0.02 mm) and had a smooth surface, without curling. The chitosan film prepared by ethanol regeneration had a tensile strength of up to 24 MPa, a minimum swelling degree of 78%, and a water vapor transmission rate of 270 g m-2 d-1 without the addition of plasticizer.

Nitric oxide mediates apoptosis and mitochondrial dysfunction and plays a role in growth hormone deficiency by nivalenol in GH3 cells.

Nivalenol (NIV), a type B trichothecenes commonly found in cereal crops, can cause growth impairment in animals. However, limited information about its mechanisms is available. Trichothecenes have been characterized as an inhibitor of protein synthesis and induce apoptosis in cells. Oxidative stress is considered an underlying mechanism. However, whether NIV can induce oxidative stress and apoptosis in rat pituitary cells line GH3 is unclear. The present study showed that NIV significantly reduced the viability of cells and caused oxidative stress in GH3 cells. Further experiments showed that nitric oxide (NO), but not ROS, mediated NIV-induced oxidative stress. Additionally, NIV induced caspase-dependent apoptosis, decrease in mitochondrial membrane potential and mitochondrial ultrastructural changes. However, NIV-induced caspase activation, mitochondrial damage and apoptosis were partially alleviated by Z-VAD-FMK or NO scavenger hemoglobin. Finally, NIV changed the expression of growth-associated genes and pro-inflammatory cytokines. NIV also reduced the GH secretion in GH3 cells, which was reversed by hemoglobin. Taken together, these results suggested that NIV induced apoptosis in caspase-dependent mitochondrial pathway in GH3 cells, which might be an underlying mechanism of NIV-induced GH deficiency. Importantly, NO played a critical role in the induction of oxidative stress, apoptosis and GH deficiency in NIV-treated GH3 cells.

A two-year dietary carcinogenicity study of cyadox in Sprague-Dawley rats.

To investigate the potential carcinogenicity of cyadox, an antimicrobial agent, four groups of Sprague-Dawley rats (50 rats/sex/group) were fed diets containing cyadox (0, 200, 600 or 2000 mg/kg) for up to two years. There were significant decreases in body weight, feed intake and feed efficiency in both genders during most of the period in the 2000 mg/kg group. Significant decreases in serum ALT were observed in the 2000 mg/kg group at weeks 52, 78 and 104. For the control, 200, 600, and 2000 mg/kg groups, the tumor incidence in females was 33.3%, 37.2%, 40.0% and 19.0%, while it in males it was 18.9%, 2.6%, 17.1% and 13.6%, respectively. At histopathology, no increases in tumor incidence were attributed to treatment with cyadox. The mild swelling and fatty degeneration in hepatocytes, and mild swelling and tubular necrosis in the kidney were observed in 2000 mg/kg group. The no-observed-effect-level (NOEL) for carcinogenicity of cyadox fed to rats was 2000 mg/kg diet (132.18-156.28 mg/kg b.w./day). In conclusion, cyadox was not carcinogenic to rats with the liver and kidney as the target organs, and the side chain may be involved in toxicity and carcinogenicity mediated by QdNOs.