Targeting MFGE8 secreted by cancer-associated fibroblasts blocks angiogenesis and metastasis in esophageal squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) play vital roles in establishing a suitable tumor microenvironment. In this study, RNA sequencing data revealed that CAFs could promote cell proliferation, angiogenesis, and ECM reconstitution by binding to integrin families and activating PI3K/AKT pathways in esophageal squamous cell carcinoma (ESCC). The secretions of CAFs play an important role in regulating these biological activities. Among these secretions, we found that MFGE8 is specifically secreted by CAFs in ESCC. Additionally, the secreted MFGE8 protein is essential in CAF-regulated vascularization, tumor proliferation, drug resistance, and metastasis. By binding to Integrin αVß3/αVß5 receptors, MFGE8 promotes tumor progression by activating both the PI3K/AKT and ERK/AKT pathways. Interestingly, the biological function of MFGE8 secreted by CAFs fully demonstrated the major role of CAFs in ESCC and its mode of mechanism, showing that MFGE8 could be a driver factor of CAFs in remodeling the tumor environment. In vivo treatment targeting CAFs-secreting MFGE8 or its receptor produced significant inhibitory effects on ESCC growth and metastasis, which provides an approach for the treatment of ESCC.

Enhanced mitophagy driven by ADAR1-GLI1 editing supports the self-renewal of cancer stem cells in HCC.

BACKGROUND AND AIMS: Deregulation of adenosine-to-inosine editing by adenosine deaminase acting on RNA 1 (ADAR1) leads to tumor-specific transcriptome diversity with prognostic values for HCC. However, ADAR1 editase-dependent mechanisms governing liver cancer stem cell (LCSC) generation and maintenance have remained elusive. APPROACH AND RESULTS: RNA-seq profiling identified ADAR1-responsive recoding editing events in HCC and showed editing frequency of GLI1 , rather than transcript abundance was clinically relevant. Functional differences in LCSC self-renewal and tumor aggressiveness between wild-type (GLI1 wt ) and edited GLI1 (GLI1 edit ) were elucidated. We showed that overediting of GLI1 induced an arginine-to-glycine (R701G) substitution, augmenting tumor-initiating potential and exhibiting a more aggressive phenotype. GLI1 R701G harbored weak affinity to SUFU, which in turn, promoted its cytoplasmic-to-nuclear translocation to support LCSC self-renewal by increased pluripotency gene expression. Moreover, editing predisposed to stabilize GLI1 by abrogating ß-TrCP-GLI1 interaction. Integrative analysis of single-cell transcriptome further revealed hyperactivated mitophagy in ADAR1-enriched LCSCs. GLI1 editing promoted a metabolic switch to oxidative phosphorylation to control stress and stem-like state through PINK1-Parkin-mediated mitophagy in HCC, thereby conferring exclusive metastatic and sorafenib-resistant capacities. CONCLUSIONS: Our findings demonstrate a novel role of ADAR1 as an active regulator for LCSCs properties through editing GLI1 in the highly heterogeneous HCC.

Loss to gain: pseudogenes in microorganisms, focusing on eubacteria, and their biological significance.

Pseudogenes are defined as "non-functional" copies of corresponding parent genes. The cognition of pseudogenes continues to be refreshed through accumulating and updating research findings. Previous studies have predominantly focused on mammals, but pseudogenes have received relatively less attention in the field of microbiology. Given the increasing recognition on the importance of pseudogenes, in this review, we focus on several aspects of microorganism pseudogenes, including their classification and characteristics, their generation and fate, their identification, their abundance and distribution, their impact on virulence, their ability to recombine with functional genes, the extent to which some pseudogenes are transcribed and translated, and the relationship between pseudogenes and viruses. By summarizing and organizing the latest research progress, this review will provide a comprehensive perspective and improved understanding on pseudogenes in microorganisms. KEY POINTS: • Concept, classification and characteristics, identification and databases, content, and distribution of microbial pseudogenes are presented. • How pseudogenization contribute to pathogen virulence is highlighted. • Pseudogenes with potential functions in microorganisms are discussed.

A live attenuated DIVA vaccine affords protection against Listeria monocytogenes challenge in sheep.

Listeria monocytogenes (Lm) is a deadly foodborne pathogen that comprises 14 serotypes, among which, serotype 4b Lm is the primary cause of listeriosis outbreaks in humans and animals. Here, we evaluated the safety, immunogenicity, and protective efficacy of a serotype 4b vaccine candidate Lm NTSNΔactA/plcB/orfX in sheep. The infection dynamics, clinical features, and pathological observation verified that the triple genes deletion strain has adequate safety for sheep. Moreover, NTSNΔactA/plcB/orfX significantly stimulated humoral immune response and provided 78% immune protection to sheep against lethal wild-type strain challenge. Notably, the attenuated vaccine candidate could differentiate infected and vaccinated animals (DIVA) via serology determination of the antibody against listeriolysin O (LLO, encoded by hly) and phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB). These data suggest that the serotype 4b vaccine candidate has high efficacy, safety, and DIVA characteristics, and may be used to prevent Lm infection in sheep. Our study provides a theoretical basis for its future application in livestock and poultry breeding.

Genomic Insights into Pangenome and Antimicrobial Resistance in Campylobacter spp. Isolated from Chickens at Specific Growth Stages.

Improved understanding of the genetic basis of Campylobacter spp. colonization of poultry at specific growth stage is the key to developing a farm-based strategy to prevent flock colonization. In this study, 39 Campylobacter spp. strains (chicken isolates, n = 29; environmental isolates, n = 10) were collected from six marked chickens at the growth stage from week 7 to week 13. Then, we use comparative genomics techniques to analyze the temporal genomic characteristics of Campylobacter spp. in individual chickens across a production cycle. Genotype, average nucleotide identity (ANI), and phylogenetic trees all indicated the evolutionary relationships between the strains from different sampling weeks. The clustering of isolates was not dependent on sampling time and sample source, indicating that strains could persist over several weeks in a flock. Notably, 10 antimicrobial resistance (AMR) genes were identified in the genome of Campylobacter coli isolates, and the genomes of isolates sampled at week 11 harbored fewer AMR genes and insertion sequences (IS) than the isolates from other weeks. Consistent with this, pangenome-wide association analysis demonstrated that gene acquisition and loss could happen at week 11 and week 13. These genes were mainly associated with cell membrane biogenesis, ion metabolism, and DNA replication, suggesting that genomic change may be related to Campylobacter adaptive response. This is a novel study focused on the genetic changes occurring in Campylobacter spp. isolates in a particular space and time; it highlights that accessory genes and AMR genes were overall stable at chicken farm, which will help us understand the survival and the transmission route of Campylobacter spp. better, and have the potential to inform the strategy on the safety control of market-ready chickens.

CrisprVi: a software for visualizing and analyzing CRISPR sequences of prokaryotes.

BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR) and their spacers are important components of prokaryotic CRISPR-Cas systems. In order to analyze the CRISPR loci of multiple genomes more intuitively and comparatively, here we propose a visualization analysis tool named CrisprVi. RESULTS: CrisprVi is a Python package consisting of a graphic user interface (GUI) for visualization, a module for commands parsing and data transmission, local SQLite and BLAST databases for data storage and a functions layer for data processing. CrisprVi can not only visually present information of CRISPR direct repeats (DRs) and spacers, such as their orders on the genome, IDs, start and end coordinates, but also provide interactive operation for users to display, label and align the CRISPR sequences, which help researchers investigate the locations, orders and components of the CRISPR sequences in a global view. In comparison to other CRISPR visualization tools such as CRISPRviz and CRISPRStudio, CrisprVi not only improves the interactivity and effects of the visualization, but also provides basic statistics of the CRISPR sequences, and the consensus sequences of DRs/spacers across the input strains can be inspected from a clustering heatmap based on the BLAST results of the CRISPR sequences hitting against the genomes. CONCLUSIONS: CrisprVi is a convenient tool for visualizing and analyzing the CRISPR sequences and it would be helpful for users to inspect novel CRISPR-Cas systems of prokaryotes.

PICALM exerts a role in promoting CRC progression through ERK/MAPK signaling pathway.

BACKGROUND: Colorectal cancer (CRC) is a common malignant tumor in gastrointestinal tract with high incidence and mortality. In this study, the functions and potential mechanism of phosphatidylinositol-binding clathrin assembly protein (PICALM) in CRC were preliminarily explored. METHODS: Based on the Cancer Genome Atlas database and immunohistochemistry staining, revealing that the expression level of PICALM in CRC tissues was higher than that in adjacent normal tissues. RESULTS: Moreover, loss-of-function and gain-of-function assays in HCT 116 and RKO cells found that PICALM promotes proliferation and migration of CRC cells and inhibits apoptosis. Consistently, knockdown of PICALM inhibited tumorigenicity of CRC cells in vivo. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that knockdown of PICALM resulted in the enrichment of MAPK signaling pathway. Treatment of CRC cells with MAPK inhibitor reversed the effects of PICALM overexpression on proliferation and apoptosis. In addition, overexpression of PICALM upregulated the protein levels of ERK1/2 (p-ERK1/2), MEK1/2 (p-MEK1/2), p38 (p-p38) and JNK (p-JNK), and these effects were partially alleviated by the treatment of MAPK inhibitor. CONCLUSIONS: In summary, the study presented the new discovery that PICALM promoted CRC progression through ERK/MAPK signaling pathway, which drew further interest regarding its clinical application as a promising therapeutic target.

A balanced gut microbiota is essential to maintain health in captive sika deer.

Certain animals harbor a high proportion of pathogens, particular the zoonotic pathogens, in their gut microbiome but are usually asymptomic; however, their carried pathogens may seriously threaten the public health. By understanding how the microbiome overcomes the negative effects of pathogens to maintain host health, we can develop novel solutions to control animal-mediated pathogen transmission including identification and application of beneficial microbes. Here, we analyzed the gut microbiota of 10 asymptomic captive sika deer individuals by full-length 16S rDNA sequencing. Twenty-nine known pathogens capable of infecting humans were identified, and the accumulated proportions of the identified pathogens were highly variable among individuals (2.33 to 39.94%). The relative abundances of several beneficial bacteria, including Lactobacillus and Bifidobacterium, were found to be positively correlated with the relative abundances of accumulated pathogens. Whole-genome metagenomic analysis revealed that the beneficial- and pathogenic-associated functions, such as genes involved in the synthesis of short chain fatty acids and virulence factors, were also positively correlated in the microbiome, indicating that the beneficial and pathogenic functions were maintained at a relatively balanced ratio. Furthermore, the bacteriophages that target the identified pathogens were found to be positively correlated with the pathogenic content in the microbiome. Several high-quality genomes of beneficial bacteria affiliated with Lactobacillus and Bifidobacterium and bacteriophages were recovered from the metagenomic data. Overall, this study provides novel insights into the interplay between beneficial and pathogenic content to ensure maintenance of a healthy gut microbiome, and also contributes to discovery of novel beneficial microbes and functions that control pathogens. KEY POINTS: • Certain asymptomic captive sika deer individuals harbor relatively high amounts of zoonotic pathogens. • The beneficial microbes and the beneficial functions are balanced with the pathogenic contents in the gut microbiome. • Several high-quality genomes of beneficial bacteria and bacteriophages are recovered by metagenomics.

Prevalence of Campylobacter spp. in Pig Slaughtering Line in Eastern China: Analysis of Contamination Sources.

Aiming at investigating the potential sources of Campylobacter spp. contamination in pig slaughterhouse in eastern China, a total of 2056 samples were collected in pork production chain stretching from the upstream farm to the slaughterhouse, including 54 cloacal swabs from 54 live pigs on farm, 1726 samples from 214 pig carcasses along the eight main steps in the slaughtering line, and 276 samples from slaughtering environment. Campylobacter spp. were found, may be propagated in each slaughtering step, with an average prevalence of 19.3% (333/1726). The isolation rate of Campylobacter spp. in samples collected before the slaughter (42.5%, 4.87 ± 1.23 log colony-forming units [CFU]/g), dehairing (28%, 2.40 ± 0.49 log CFU/500 cm2), and evisceration (29.4%, 2.50 ± 0.54 log CFU/500 cm2) were significantly higher than other slaughter processes (p < 0.05). The prevalence of Campylobacter spp. of pigs on farm was 18.5% (10/54), compared to the slaughtering environment, which was 27.9% (77/276). Campylobacter spp. isolates were obtained from a batch of samples in slaughterhouse and farm belonged to ST-828 complex. Interventions are required to minimize Campylobacter spp. contamination in slaughtering line, especially during dehairing and evisceration. The upstream farm was an additional and usually neglected source of contamination. These findings may provide a new perspective regarding the safety provision of Campylobacter spp. contamination in pork meat production.

Insights into the impact of flhF inactivation on Campylobacter jejuni colonization of chick and mice gut.

BACKGROUND: Campylobacter jejuni (C. jejuni) is a leading cause of foodborne gastroenteritis worldwide. This bacterium lacks many of the classical virulence factors, and flagellum-associated persistent colonization has been shown to be crucial for its pathogenesis. The flagellum plays a multifunctional role in C. jejuni pathogenesis, and different flagellar elements make diverse contributions. The flhF gene encodes the flagellar biosynthesis regulator, which is important for flagellar biosynthesis. In this study, the influence of flhF on C. jejuni colonization was systematically studied, and the possible mechanisms were also analyzed. RESULTS: The flhF gene has a significant influence on C. jejuni colonization, and its inactivation resulted in severe defects in the commensal colonization of chicks, with approximately 104- to 107-fold reductions (for NCTC 11168 and a C. jejuni isolate respectively) observed in the bacterial caecal loads. Similar effects were observed in mice where the flhF mutant strain completely lost the ability to continuously colonize mice, which cleared the isolate at 7 days post inoculation. Characterization of the phenotypic properties of C. jejuni that influence colonization showed that the adhesion and invasion abilities of the C. jejuni flhF mutant were reduced to approximately 52 and 27% of that of the wild-type strain, respectively. The autoagglutination and biofilm-formation abilities of the flhF mutant strain were also significantly decreased. Further genetic investigation revealed that flhF is continuously upregulated during the infection process, which indicates a close association of this gene with C. jejuni pathogenesis. The transcription of some other infection-related genes that are not directly involved in flagellar assembly were also influenced by its inactivation, with the flagellar coexpressed determinants (Feds) being apparently affected. CONCLUSIONS: Inactivation of flhF has a significant influence on C. jejuni colonization in both birds and mammals. This defect may be caused by the decreased adhesion, invasion, autoagglutination and biofilm-formation abilities of the flhF mutant strain, as well as the influence on the transcription of other infection related genes, which provides insights into this virulence factor and the flagellum mediated co-regulation of C. jejuni pathogenesis.

Occurrence and genotypes of Campylobacter species in broilers during the rearing period.

Poultry are the main source of Campylobacter infection worldwide. To obtain information on Campylobacter-infected flocks and create a reference for preventing and controlling Campylobacter at farm level, Campylobacter isolates were recovered from broilers and the environments of nine chicken flocks in two farms during growth. The genetic relationship between the Campylobacter isolates was determined using multilocus sequence typing. Flocks were colonized as early as 3 weeks after introduction to the farm. The highest colonization rate was more than 90% and occurred 4-6 weeks after introduction to the farm. Quantitative data showed that the highest Campylobacter loads appeared at 1-2 weeks after initial colonization. Campylobacter loads in cloacal swabs in four flocks were significantly higher at 5 weeks than at 3 weeks (P < 0.05). Multilocus sequence typing of 171 selected isolates revealed 20 sequence types (STs), which consisted of 12 STs for Campylobacter jejuni and eight for Campylobacter coli isolates. The STs of the Campylobacter isolates recovered from farm 1 were more diversified than those from farm 2. The STs of environmental samples were highly consistent with those of the cloacal swab samples. The consistency between Campylobacter STs in the environmental and cloacal swab samples suggested that the environment might be one of the main sources of infection. Thus, our study highlights the prevalence and contamination load of Campylobacter in broilers during their rearing period and emphasizes the need for control and prevention measures for Campylobacter infection in broilers, which is also important for human health.

Syringe Injectable Electronics: Precise Targeted Delivery with Quantitative Input/Output Connectivity.

Syringe-injectable mesh electronics with tissue-like mechanical properties and open macroporous structures is an emerging powerful paradigm for mapping and modulating brain activity. Indeed, the ultraflexible macroporous structure has exhibited unprecedented minimal/noninvasiveness and the promotion of attractive interactions with neurons in chronic studies. These same structural features also pose new challenges and opportunities for precise targeted delivery in specific brain regions and quantitative input/output (I/O) connectivity needed for reliable electrical measurements. Here, we describe new results that address in a flexible manner both of these points. First, we have developed a controlled injection approach that maintains the extended mesh structure during the "blind" injection process, while also achieving targeted delivery with ca. 20 µm spatial precision. Optical and microcomputed tomography results from injections into tissue-like hydrogel, ex vivo brain tissue, and in vivo brains validate our basic approach and demonstrate its generality. Second, we present a general strategy to achieve up to 100% multichannel I/O connectivity using an automated conductive ink printing methodology to connect the mesh electronics and a flexible flat cable, which serves as the standard "plug-in" interface to measurement electronics. Studies of resistance versus printed line width were used to identify optimal conditions, and moreover, frequency-dependent noise measurements show that the flexible printing process yields values comparable to commercial flip-chip bonding technology. Our results address two key challenges faced by syringe-injectable electronics and thereby pave the way for facile in vivo applications of injectable mesh electronics as a general and powerful tool for long-term mapping and modulation of brain activity in fundamental neuroscience through therapeutic biomedical studies.

Multilocus Sequence Types of Campylobacter jejuni Isolates from Different Sources in Eastern China.

Campylobacter jejuni is a major food-borne pathogen that causes human gastroenteritis in many developed countries. In our study, we applied multilocus sequence typing (MLST) technology to 167 C. jejuni isolates from diverse sources in Eastern China to examine their genetic diversity. MLST defined 94 sequence types (STs) belonging to 18 clonal complexes (CCs). Forty-five STs from 60 isolates (36%) and 22 alleles have not been previously documented in an international database. One hundred and two isolates, accounting for 61.1% of all isolates, belonged to eight clonal complexes. The eight major CCs were also the most common complexes from different sources. The most common ST type of isolates from human and food was ST-353. The dominant ST type in chicken and foods was ST-354. Among 21 STs that contained two or more different sources isolates, 15 STs contained human isolates and isolates from other sources, suggesting that potentially pathogenic strains are not restricted to specific lineages.

[Epidemic condition and molecular subtyping of ciprofloxacin and cefotaxime co-resistant Salmonella Indiana isolated from retail chicken carcasses in six provinces, China].

OBJECTIVE: To elucidate the epidemic condition and molecular subtyping of ciprofloxacin and cefotaxime co-resistant Salmonella Indiana (S. Indiana) isolated from retail chicken carcasses in six provinces of China. METHODS: A total of 2 647 Salmonella strains isolated from retail chicken carcasses collected from six provinces of China were subjected to antimicrobial susceptibility testing. All Salmonella isolates co-resistant to ciprofloxacin and cefotaxime were further characterized by serotyping, extended-spectrum beta-lactamases (ESBLs) producing strains screening and pulsed field gel electrophoresis (PFGE) typing. RESULTS: Among 2 629 Salmonella isolates tested, 227 (8.52%) isolates were co-resistant to ciprofloxacin and ceftazidime/cefotaxime (Beijing: 11.67% (99/874), Jilin: 8.20% (60/726), Guangdong: 1.39% (7/502), Jiangsu: 15.61% (42/260), Shaanxi: 8.56% (16/186), Inner Mongolia: 0 (0/81)), and 224 of them were identified as S. Indiana. 213 (95.10%) isolates of S. Indiana were ESBLs producing strains. All ciprofloxacin and cefotaxime co-resistant S. Indiana isolates developed a multi-drug resistant profile and 17.86% (40/224) of them were resistant to all antibiotics tested except carbapenems, and 50.89% (114/224) of them resistant to 9 antibiotics, additionally, 25.45% (57/224) of them showed multi-drug resistance to 8 antibiotics. All ciprofloxacin and cefotaxime co-resistant S. Indiana isolates were divided into 32 PFGE clusters and 150 PFGE patterns. Strains of S. Indiana from same or different sampling site and time seemed to either share the same PFGE patterns or be differential to each other in different regions. CONCLUSION: The results indicated that chicken carcasses collected from parts of China were heavily contaminated by ciprofloxacin and cefotaxime co-resistant S. Indiana and could serve as an important reservoir of ciprofloxacin and cefotaxime co-resistant Salmonella. Molecular subtyping results indicated that cross contamination or common pollution source might be in these strains.

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients.

BACKGROUND: Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence. METHODS: In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni. RESULTS: Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI-TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro. CONCLUSION: We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients. GENERAL SIGNIFICANCE: This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.

BTB/POZ domain-containing protein 7: epithelial-mesenchymal transition promoter and prognostic biomarker of hepatocellular carcinoma.

UNLABELLED: Epithelial-mesenchymal transition (EMT) is a critical step in the metastasis of hepatocellular carcinoma (HCC). BTB/POZ domain-containing protein 7 (BTBD7) regulates EMT-associated proteins implicated in HCC progression. However, the role(s) of BTBD7 in HCC have not been identified. Using highly metastatic HCC HCCLM3 cells, immortalized L02 hepatocytes, metastatic HCC animal models, and three independent cohorts of HCC patient specimens, we aimed to determine the involvement of BTBD7 in HCC metastasis. We show that BTBD7 messenger RNA and protein was highly expressed in HCC cells and tumor tissues, with such expression being associated with: enhanced cell motility, venous invasion, and poor prognosis. BTBD7 promoted HCC angiogenesis and metastasis in vitro and in vivo, but did not influence cell proliferation or colony formation. BTBD7 enhancement of HCC invasion and EMT phenotype occurred through activation of a RhoC-Rock2-FAK-signaling pathway, resulting in matrix metalloproteinase-2/9 production and microvessel formation. Applying a predictive risk score model, Cox regression analysis revealed that high BTBD7 expression integrated with high microvessel density was a powerful independent predictive factor of HCC clinical outcome. CONCLUSION: The present study identifies BTBD7 as a novel candidate prognostic factor and a potential therapeutic target of HCC. (HEPATOLOGY 2013; 57:2326-2337).

Intraperitoneal administration of fetuin-A attenuates D-galactosamine/lipopolysaccharide-induced liver failure in mouse.

BACKGROUND: Fulminant hepatic failure (FHF) is a devastating syndrome, which sometimes results in death or liver transplantation, in which inflammation would aggravate the development of fetuin-A which would act as an anti-inflammatory factor and may be an available approach to attenuate FHF. AIMS: The purpose of this study was to investigate the effects of fetuin-A on D-galactosamine/lipopolysaccharide (D-GalN/LPS)-induced liver failure in mice. METHODS: A mouse model of FHF induced by D-GalN/LPS was established and fetuin-A was injected intraperitoneally prior to D-GalN/LPS treatment. At different time points after D-GalN/LPS intervention, serum TNF-α and IL-6 levels were measured by ELISA. Fetuin-A mRNA and protein expression in liver tissues was assessed by RT-PCR, Western blotting and immunohistochemical staining. Besides, an observation of liver tissue injury, the apoptosis of hepatocytes, was analyzed by TUNEL assay. RESULTS: Expression of fetuin-A mRNA and protein in liver tissue were significantly and gradually decreased after D-GalN/LPS administration. A pre-intervention of exogenous fetuin-A significantly improved the liver function, decreased TNF-α and IL-6 expression in peripheral blood, and liver tissue inhibited hepatocyte apoptosis responded to D-GalN/LPS induction so as to decrease the mortality rates of FHF mouse. Meanwhile, fetuin-A was negatively correlated with the hepatic pathological score and TNF-α protein staining in FHF mouse. CONCLUSIONS: An intraperitoneal injection of fetuin-A attenuates D-GalN/LPS-induced FHF in mice. Fetuin-A might be a protective agent of liver damage partly through inhibiting liver inflammatory response and hepatocyte apoptosis.

[Preparation and characterization of monoclonal antibodies against cytolethal distending toxin protein of Campylobacter jejuni].

OBJECTIVE: To express Campylobacter jejuni cytolethal distending toxin B protein (CdtB) in a prokaryote to prepare monoclonal antibodies (mAbs) against the protein, and to study their antitoxic effects. METHODS: The C. jejuni cdtB gene was amplified and inserted into the expression plasmids pET-30a( + ) and pGEX-6p-1. The purified rGST-CdtB protein was used as the immunogen to screen hybridoma cells for mAbs against the protein. The mAb titers were determined with an indirect enzyme-linked immunosorbent assay (ELISA), and their specificity with a Dot-ELISA and western blotting analysis. We determined the antitoxic properties of the mAbs in CaCo-2 and HD-11 cells. RESULTS: Recombinant expression plasmids pET-30a (+)-cdtB and pGEX-6p-l-cdtB were successfully constructed, and fusion proteins rHis-CdtB and rGST-CdtB expressed, respectively. Five hybridoma cell lines, designated 1F3, IF5, 2E4, 2E11, and 2F2, were screened for the stable secretion of mAbs against CdtB. The immunoglobulin subclass of 2E11 was IgG2b and that of the other mAbs was IgG1. The mAb titers in the ascites fluids were 1:1 x 10(8) on indirect ELISA. Dot-ELISA demonstrated that the five mAbs reacted specifically with C. jejuni. Western blotting analysis confirmed that the five mAbs reacted well with the rGST-CdtB fusion protein. The mAbs significantly reduced the adhesion and invasion capacities of the bacterium in CaCo-2 cells (P < 0.01). CONCLUSION: The successful preparation of five mAbs specific for the CdtB protein will allow further study of the biological characteristics of CdtB and the pathogenesis of C. jejuni.

Study on vibration characteristics of the dike crossing pipeline based on EWT and CWT.

The dike crossing pipeline is an important part of urban water transmission and supply projects. Identifying effective vibration analysis methods to determine the primary vibration sources and coupling vibration characteristics of pipelines is crucial for targeted vibration reduction and reinforcement of vulnerable pipeline sections. Therefore, this paper proposes a coupled vibration characteristics analysis method for the dike crossing pipeline based on EWT and CWT, taking the 6# dike crossing pipeline of Yang'er water plant in Foshan city, Guangdong province as the research object, firstly, the main vibration sources of the dike crossing pipeline are analyzed with the help of the prototype observation data using the mutual correlation power spectral, and the characteristics of the main vibration sources of the pipe are extracted using the empirical wavelet transform (EWT); then, focusing on the main vibration source, wavelet transform is used to analyze the source characteristics of the dike crossing pipeline; finally, the pipeline coupled vibration characteristics are analyzed using the cross wavelet transform (CWT). The research results show that: 1) The vibration of the 6# dike crossing pipeline of Yang'er water plant is mainly caused by the multiple rotational frequency such as 29.5 Hz, 36.5 Hz and the leaf frequency 59.0 Hz; 2) The EWT method can effectively remove the interference signal and extract the characteristic frequencies 29.5 Hz, 36.5 Hz and 59.0 Hz; 3) Analyzing the coupled vibration characteristics of the dike crossing pipeline based on the CWT, the peak energy of the coupled vibration of the 6# pipeline is generally concentrated at the frequency of 29.5 Hz, and the source of the coupled vibration is the multiple rotational frequency of the 6# pipeline unit. The results of this study can offer new insights into the identification of vibration characteristics of the dike crossing pipeline, and can provide technical support for the analysis of vibration characteristics and reduction needs of similar projects.

Microbiological safety assessment of restaurants and HACCP-certified kitchens in hotels: A study in eastern China.

The Hazard Analysis and Critical Control Point (HACCP) system plays a crucial role in ensuring food safety within food service establishments, effectively reducing the risk of foodborne diseases. This study focused on assessing the risk of microbe contamination in poultry-based cook-served food during meal preparation in four restaurants and five selected HACCP-certified hotels in eastern China. We examined samples collected from 26 poultry-based cooked dishes, 248 food contact surfaces, 252 non-food contact surfaces, and 121 hand swabs. Our findings indicated a favorable trend of compliance with Chinese national standards, as Escherichia coli and Campylobacter were not detected in any cooked food samples. However, the microbiological assessments revealed non-compliance with total plate count standards in 7 % of the cooked samples from restaurants. In contrast, both dine-in hotels and restaurants exhibited significant non-compliance with guidance concerning food and non-food contact surfaces. Furthermore, our study found that chefs' hand hygiene did not meet microbiological reference standards, even after washing. Notably, Campylobacter persisted at 27 % and 30 % on chefs' hands, posing a significant risk of cross-contamination and foodborne diseases. These findings emphasize the urgent necessity for enhanced supervision of hygiene procedures and process monitoring in the HACCP-certified establishments engaged in the preparation and serving of food. Targeted interventions and food safety education for different chef subgroups can enhance food handling practices and reduce the risk of foodborne diseases in independent food establishments.