Antibiotic-induced microbiome depletion promotes intestinal colonization by Campylobacter jejuni in mice.

BACKGROUND: To establish a method to induce Campylobacter jejuni colonization in the intestines of C57BL/6 mice through antibiotic-induced microbiome depletion. RESULTS: Fifty-four female C57BL/6 mice were divided into the normal, control, and experimental groups. The experimental group was administered intragastric cefoperazone sodium and sulbactam sodium (50 mg/mL) for 2 days; then, the experimental and control mice were intragastrically administered 200 µL C. jejuni, which was repeated once more after 2 days. Animal feces were collected, and the HipO gene of C. jejuni was detected using TaqMan qPCR from day 1 to day 14 after modeling completion. Immunofluorescence was used to detect intestinal C. jejuni colonization on day 14, and pathological changes were observed using hematoxylin and eosin staining. Additionally, 16S rDNA analyses of the intestinal contents were conducted on day 14. In the experimental group, C. jejuni was detected in the feces from days 1 to 14 on TaqMan qPCR, and immunofluorescence-labeled C. jejuni were visibly discernable in the intestinal lumen. The intestinal mucosa was generally intact and showed no significant inflammatory-cell infiltration. Diversity analysis of the colonic microbiota showed significant inter-group differences. In the experimental group, the composition of the colonic microbiota differed from that in the other 2 groups at the phylum level, and was characterized by a higher proportion of Bacteroidetes and a lower proportion of Firmicutes. CONCLUSIONS: Microbiome depletion induced by cefoperazone sodium and sulbactam sodium could promote long-term colonization of C. jejuni in the intestines of mice.

Growth differentiation factor 7 pretreatment enhances the therapeutic capacity of bone marrow-derived mesenchymal stromal cells against cerebral ischemia-reperfusion injury.

Bone marrow-derived mesenchymal stem cells (BMSCs) transplantation is a promising therapeutic strategy for cerebral ischemia/reperfusion (I/R) injury; however, the clinical outcome is barely satisfactory and demands further improvement. The present study aimed to investigate whether preconditioning of BMSCs by recombinant human growth differentiation factor 7 (rhGDF7) could enhance its therapeutic capacity against cerebral I/R injury. Mouse BMSCs and primary neurons were co-cultured and exposed to oxygen glucose deprivation/reperfusion (OGD/R) stimulation. To investigate the role of exosomal microRNA-369-3p (miR-369-3p), inhibitors, RNAi and the miR-369-3p antagomir were used. Meanwhile, mice were intravenously injected with rhGDF7-preconditioned BMSCs and then received cerebral I/R surgery. Markers of inflammation, oxidative stress and neural damage were evaluated. To inhibit AMP-activated protein kinase (AMPK), compound C was used in vivo and in vitro. Compared with cell-free transwell or vehicle-preconditioned BMSCs, rhGDF7-preconditioned BMSCs significantly prevented OGD/R-induced inflammation, oxidative stress and neural damage in vitro. Meanwhile, rhGDF7-preconditioned BMSCs could prevent I/R-induced cerebral inflammation and oxidative stress in vivo. Mechanistically, rhGDF7 preconditioning significantly increased exosomal miR-369-3p expression in BMSCs and then transferred exosomal miR-369-3p to primary neurons, where it bound to phosphodiesterase 4 D (Pde4d) 3'-UTR and downregulated PDE4D expression, thereby preventing I/R-induced inflammation, oxidative stress and neural damage through activating AMPK pathway. Our study identify GDF7 pretreatment as a promising adjuvant reagent to improve the therapeutic potency of BMSCs for cerebral I/R injury and ischemic stroke.

Smoking Cessation, Weight Gain, and Risk for Type 2 Diabetes: A Prospective Study.

Objectives: To examine the association between smoking cessation and risk of type 2 diabetes with emphasis on post-cessation weight gain. Methods: In total, 8,951 participants from the China Health and Retirement Longitudinal Study at the baseline (2011) were included. Diabetes incidence was accessed at the third survey (2015). Current smokers were treated as the reference and odds ratios (OR) of type 2 diabetes for never smokers, recent, and long-term quitters were computed using multivariable logistic regression. Stratified analysis was further conducted by weight gain after smoking cessation. Results: There were 712 cases of type 2 diabetes identified. Compared with current smokers, the fully multivariable-adjusted ORs were 1.55 (1.02, 2.36) for recent quitters, 0.88 (0.61, 1.28) for long-term quitters, and 0.75 (0.59, 0.95) for never smokers. Stratified analysis showed recent quitters with weight gain of ≥2.0 kg had a significantly higher odds of type 2 diabetes [2.25 (1.02, 4.95)]. Conclusion: The present study of the Chinese population suggested recent quitters with weight gain of ≥2.0 kg, compared with current smokers, had a significantly increased odds of type 2 diabetes.

Identifying Potential Gene Defect Patterns Related to COVID-19 Based on Pharmacological and Bioinformatics Analysis for Lung Adenocarcinoma.

Background: Coronavirus disease 2019 (COVID-19) greatly affects cancer patients, especially those with lung cancer. This study aimed to identify potential drug targets for lung adenocarcinoma (LUAD) patients with COVID-19. Methods: LUAD samples were obtained from public databases. Differentially expressed genes (DEGs) related to COVID-19 were screened. Protein-protein interactions among COVID-19-related genes, the traditional Chinese medicine (TCM) and TCM target genes were analyzed by CytoScape. The correlation between tumor microenvironment and COVID-19 target genes were assessed by Pearson correlation analysis. Unsupervised consensus clustering was conducted to categorize molecular subtypes. Results: We filtered 26 COVID-19 target genes related to TCM for LUAD. Interleukin (IL)-17 signaling pathway and tumor necrosis factor (TNF) signaling pathway were significantly enriched in these 26 genes. A strong correlation was found between COVID-19 target genes and tumor microenvironment (TME), cell death. Importantly, interleukin-1beta (IL1B) was identified as a core gene in the protein-protein interactions (PPI) network. Based on the 26 target genes, two molecular subtypes showing distinct overall survival, TME and response to target therapy were developed. Conclusions: This study explored 26 COVID-19 target genes, which could serve as potential therapeutic drug targets for LUAD. IL1B was verified as a critical target for developing new molecular drugs. Furthermore, two novel molecular subtypes showed the potential to guide personalized therapies in clinical practice.

Five-gene signature predicts acute kidney injury in early kidney transplant patients.

Patients with acute kidney injury (AKI) show high morbidity and mortality, and a lack of effective biomarkers increases difficulty in its early detection. Weighted gene co-expression network analysis (WGCNA) detected a total of 22 gene modules and 6 miRNA modules, of which 4 gene modules and 3 miRNA modules were phenotypically co-related. Functional analysis revealed that these modules were related to different molecular pathways, which mainly involved PI3K-Akt signaling pathway and ECM-receptor interaction. The brown modules related to transplantation mainly involved immune-related pathways. Finally, five genes with the highest AUC were used to establish a diagnosis and prediction model of AKI. The model showed a high area under curve (AUC) in the training set and validation set, and their prediction accuracy for AKI was as high as 100%. Similarly, the prediction accuracy of AKI after 24 h in the 0 h transplant sample was 100%. This study may provide new features for the diagnosis and prediction of AKI after kidney transplantation, and facilitate the diagnosis and drug development of AKI in kidney transplant patients.

Draft Genome Sequence of a Campylobacter coli WL22 Isolate Possessing erm(B) with gyrA Mutations, Isolated from Poultry in China.

Campylobacter coli is a major foodborne pathogen worldwide that causes campylobacteriosis cases in humans and is an emerging threat in developing countries. The rapid dissemination of the macrolide resistance gene erm(B) poses a significant threat to the clinical therapy of campylobacteriosis. Here, we report the draft genome sequences of one Campylobacter coli strain possessing erm(B), isolated from the cecal contents of poultry in Jinhua, China.

Development of a Trivalent Construct Omp18/AhpC/FlgH Multi Epitope Peptide Vaccine Against Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is one of the major pathogens contributing to the enteritis in humans. Infection can lead to numerous complications, including but not limited to Guillain-Barre syndrome, reactive arthritis, and Reiter's syndrome. Over the past two decades, joint efforts have been made toward developing a proper strategy of limiting the transmission of C. jejuni to humans. Nevertheless, except for biosecurity measures, no available vaccine has been developed so far. Judging from the research findings, Omp18, AhpC outer membrane protein, and FlgH flagellin subunits of C. jejuni could be adopted as surface protein antigens of C. jejuni for screening dominant epitope thanks to their strong antigenicity, expression of varying strains, and conservative sequence. In this study, bioinformatics technology was adopted to analyze the T-B antigenic epitopes of Omp18, AhpC, and FlgH in C. jejuni strain NCTC11168. Both ELISA and Western Blot methods were adopted to screen the dominant T-B combined epitope. GGS (GGCGGTAGC) sequence was adopted to connect the dominant T-B combined epitope peptides and to construct the prokaryotic expression system of tandem repeats of antigenic epitope peptides. The mouse infection model was adopted to assess the immunoprotective effect imposed by the trivalent T-B combined with antigen epitope peptide based on Omp18/AhpC/FlgH. In this study, a tandem epitope AhpC-2/Omp18-1/FlgH-1 was developed, which was composed of three epitopes and could effectively enhance the stability and antigenicity of the epitope while preserving its structure. The immunization of BALB/c mice with a tandem epitope could induce protective immunity accompanied by the generation of IgG2a antibody response through the in vitro synthesis of IFN-γ cytokines. Judging from the results of immune protection experiments, the colonization of C. jejuni declined to a significant extent, and it was expected that AhpC-2/Omp18-1/FlgH-1 could be adopted as a candidate antigen for genetic engineering vaccine of C. jejuni MAP.

Development and Validation of an Individualized Immune Prognostic Signature for Recurrent Prostate Cancer.

BACKGROUND: Immune-related genes possess promising prognostic potential in multiple cancer types. Here, we describe the development of an immune-related prognostic signature for predicting prostate cancer recurrence. METHODS: Prostate cancer gene expression profiles for 477 prostate cases, as well as accompanying follow-up information were downloaded from The Cancer Genome Atlas (TCGA) and GEO. The samples were divided into 3 groups and immune gene sets significantly associated with prognosis were identified by evaluating the relationship between the expression of 1039 immune genes and prognosis in the training set. Relative expression levels of these genes were used to identify prognostic gene pairs. LASSO was used for feature selection and robust biomarkers selected. Finally, the identified immune prognostic markers were validated using dataset and GEO validation dataset and their performance compared with existing prognostic models. RESULTS: In total, 87 immune genes, significantly associated with prognosis, were identified and 2447 immune gene pairs (IRGPs) established. Univariate survival analysis identified 641 prognosis-associated immune gene pairs. 8-IRGPs were obtained via LASSO feature selection and an 8-IRGPs signature established. The 8-IRGPs signature exhibited an independent prognosis value in prostate cancer of the training set, test set, and external validation set (p = <0.001). The 5- year survival AUC in both the training set and the validation set was >0.7. The 8-IRGPs outperformed clinical tumor classification features, including T, N, radiation therapy (RT) and targeted molecular therapy (TMT) (p <0.01). In addition, we compared the prognostic characteristics of 8-IRGPs with 3 reported prostate cancers and found that 8-IRGPs achieved a high C index (0.85) and had the highest predictive performance within 10 years of follow-up (HR: 10.5). Finally, we integrated T, N, RT, TMT, and 8-IRGPs and generated a novel alignment chart to aid the prediction of prostate cancer recurrence in individual patients (p <0.01). CONCLUSION: Here, we identified an 8-IRGP novel prognostic signature for the prediction of prostate cancer recurrence.

Integrative bioinformatics analysis of a prognostic index and immunotherapeutic targets in renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the most common malignancies. The immunogenomic landscape signature significantly correlates with the progression and prognosis of RCC. Novel therapeutic targets and prognostic indices in RCC are highly desirable. The TCGA database enables comprehensive immunogenomic landscape analysis. Differentially expressed immune-related genes (IRGs) were obtained from TCGA and GO analyses, and KEGG pathway analyses were performed to explore their functions and molecular mechanisms. Multivariable Cox analysis was utilized to calculate the risk score of each patient and locate survival-associated IRGs, thereby constructing a novel immune-related gene-based prognostic index (IRGPI). The correlation between IRGPI and immune cell infiltration was also investigated. A total of 41 differentially expressed IRGs were notably related to prognosis in RCC. GO functions and KEGG pathway analyses demonstrated that these genes were primarily associated with the tumour immune response and cytokine-cytokine receptor interaction pathway. An IRGPI based on seventeen survival-associated differentially expressed IRGs was constructed and exhibited a moderate predictive value in the prognosis of RCC patients and a powerful identification ability in refining the risk stratification of RCC patients. A close correlation was found between IRGPI and specific clinicopathological parameters, including age, gender, pathological stage, tumour stage, lymph node metastasis and distant metastasis. A positive correlation was found between IRGPI and the infiltration levels of neutrophils, dendritic cells, CD8+ T cells and B cells. Our results demonstrated the clinical significance and potential function of IRGs, providing additional data for prognostic risk prediction and immunotherapeutic target selection in RCC.

Evaluations of Alkyl hydroperoxide reductase B cell antigen epitope as a potential epitope vaccine against Campylobacter jejuni.

OBJECTIVE: The present study aimed to screen and find alkyl hydroperoxide reductase (AhpC) B cell dominant epitope of Campylobacter jejuni (C. jejuni). MATERIALS AND METHODS: Bio-informatic algorithms were used to predict B cell epitopes of AhpC. The AhpC protein and chemically synthesized antigenic epitopes of C. jejuni were considered as antigens, and the AhpC antibody was used as the primary antibody, ELISA and dot blot were used to analyze and screen the dominant epitope. The specific IgG of mice serum and IL-4 in splenocyte culture supernatant were detected by ELISA. The protective efficacy was evaluated by animal disease index and tissue histopathological staining of the jejunum. RESULTS: Seven epitopes of AhpC were predicted, one epitope (AhpC4-16) was found to recognize the antibodies of AhpC and had strong antigenicity by ELISA and dot blot analysis. In epitope AhpC4-16 immunized mice, specific IgG of serum and IL-4 in splenocyte culture supernatant were significantly higher. The illness index decreased significantly, the protective rate was 66.67%. Histopathology displayed that the jejunum morphology was better than the control group. CONCLUSIONS: These findings suggested that epitope AhpC4-16 showed effective protective role against C. jejuni and is a candidate epitope of vaccine against this pathogen.

Effects of Campylobacter jejuni lipopolysaccharide on axonal injury in the spinal cord in rats.

To explore the effects of Campylobacter jejuni lipopolysaccharide (Cj-LPS) on axonal injury in the spinal cord. Wistar rats were divided into the control (NC) group, model group (Cj-LPS), and LPS antibody group (Anti-LPS). Rats in the NC group were injected with a mixture of normal saline and complete Freund's adjuvant (CFA) while those in Cj-LPS group were injected with Cj-LPS, composed of LPS, CFA, and saline. Rats were sacrificed at 4th week and 6th week after injection, and hematoxylin and eosin (HE) staining was performed on the spinal cord sections. Real time-reverse transcription(RT-PCR) was used to detect mRNA expression of the axonal nutrition factor neurotrophin-3 (NT-3) with its receptor tropomyosin receptor kinase C (TrkC) and axon inhibitory factor of NogoA/NgR (Nogo receptor). The results indicated that Cj-LPS induce axonal injury in the rat spinal cord, decreased the mRNA expression of the axonal nutrition factor NT-3/TrkC, and increased the mRNA expression of the inhibitory factor NogoA/NgR. However, anti-LPS ameliorated axonal injury in the rat spinal cord induced by Cj-LPS.

Methyl-accepting chemotaxis proteins 3 and 4 are responsible for Campylobacter jejuni chemotaxis and jejuna colonization in mice in response to sodium deoxycholate.

Methyl-accepting chemotaxis proteins (MCPs), also termed transducer-like proteins (Tlps), serve as sensors in bacterial chemotactic signalling, and detect attractants and promote bacterial movement towards suitable sites for colonization. Campylobacter jejuni is a leading cause of human enteritis, but the mechanisms responsible for bacterial chemotaxis and early colonization in the jejunum of hosts are poorly understood. In the present study, we identified several types of bile and sodium deoxycholate (SDC) acting as chemotactic attractants of C. jejuni strain NCTC 11168-O in vitro, in which SDC was the most efficient chemoattractant. In mice with bile duct ligation, the wild-type strain displayed a markedly attenuated ability for colonization. Blockage of Tlp3 or Tlp4 protein with antibody or disruption of the tlp3 or tlp4 gene (Δtlp3 or Δtlp4) caused a significant inhibition of SDC-induced chemotaxis and attenuation for colonization on jejunal mucosa in mice of the bacterium. Disruption of both the genes (Δtlp3/Δtlp4) resulted in the absence of bacterial chemotaxis and colonization, while the tlp-gene-complemented mutants (CΔtlp3 and CΔtlp4) reacquired these abilities. The results indicate that SDC is an effective chemoattractant for C. jejuni, and Tlp3 and Tlp4 are the SDC-specific sensor proteins responsible for the bacterial chemoattraction.

[Role of fliY gene in pathogenicity-associated chemotaxis and colonization of Campylobacter jejuni].

OBJECTIVE: To construct a knockout fliY gene mutant strain of Campylobacter jejuni for determining the role of FliY protein in flagellar movement related to bacterial motility, chemotaxis and colonization. METHODS: The plasmid pBluescript-II-SK was used to construct the suicide plasmid; according to homologous exchange principle, the suicide plasmid was utilized to generate fliY gene knockout mutant(fliY) in Campylobacter jejuni strain NCTC11168. The fliY mutant strain was identified by PCR, sequencing and Western blotting. The chemotactic and colonizing abilities of fliY mutant were determined by colony migration test and bacterial chemotactic test in vitro, and colonization test in jejunum of mice. RESULTS: The fliY(-)mutant strain showed a growth curve in medium similar to that of wild-type strain. PCR, sequencing and Western blotting assay confirmed that the fliY gene in fliY(-)mutant was deleted. Compared to the wild-type strain, the colonies of fliY-mutant on semisolid plate were much smaller (P <0.05), the chemotactic ability of fliY mutant towards sodium deoxycholate and bovine bile was significantly attenuated (P <0.05), and the number of fliY mutant (CFU) in jejunal tissue specimens of the infected mice was significantly decreased (P<0.05). CONCLUSION: The function of C.jejuni fliY gene refers to controlling flagellar movement, which is involved in bacterial chemotaxis and colonization.

[Investigation on the hosts with natural Paragonimus infection and species identification in Jinhua Prefecture of Zhejiang Province].

OBJECTIVE: To investigate the natural hosts infected with Paragonimus sp. and identify the species of the parasite in selected counties/districts of Jinhua prefecture in Zhejiang Province. METHODS: Three townships/towns were randomly sampled from each of the 9 counties/districts in Jinhua as pilot spots for the survey. Fresh-water snails were collected from the fields for examining cercariae. Crabs were collected and detected for metacercariae by routine technique and the metacercariae were fed to dogs purchased in areas free from paragonimiasis. Fecal materials of dogs and cats around the villages and streams where crabs were found infected were collected for examining eggs. The artificially infected dogs were sacrificed 55 d after infection to receive adult worms. The size of cercariae, metacercariae, eggs and adult worms was measured. After the DNA of the adult worm was extracted, PCR was used to amplify the COI gene and ITS2 gene of the mitochondria from the worms. Homology with relative strains/isolates was analyzed and phylogenetic tree constructed. RESULTS: The survey demonstrated that the snail Semisulcospira libertina and the crab Sinopotamon chekiangense served as the first and second intermediate hosts respectively. Natural infection was found in Wucheng District with an infection rate of 0.2% (2/1,088) in snails and 76.7% (46/60) in crabs in Shafan township, and an infection index (II) of 2.0 in crabs, 0.1% (1/1,683) in snails and 53.0% (46/60) in crabs with an II of 0.9 in Langya town. The infection rate was 0 (0/575) in snails and 30.0% (18/60) in crabs with an II of 0.1 in Baimu township of Wuyi County. Paragonimus eggs were detected in feces of stray cats with a positive rate of 8.3% (1/12) in Shafan and 0.6% (1/17) in Langya. The size and morphology of the cercariae, metacercariae, eggs and adult worms were similar to those of Paragonimus westermani. The sequences of the COI and ITS2 genes were with 390 bp and 363 bp respectively, indicating a homology of 88.2%-98.2% and 86.5%-88.1% to the 11 strains/isolates of P. westermani recorded in the GenBank. The phylogenetic trees revealed that the parasite in Jinhua was in between the Minchin (Minqing, Fujian) strain and the Japanese Mie and Chiba strains. CONCLUSION: Natural paragonimus infection has been detected in snails, The parasite has been identified as P. westermani which is phylogenetically close to those crabs and cats in Jinhua. reported from Fujian Province and from Mie and Chiba of Japan.

[CRTH2 antagonist ameliorates airway inflammation in rats with asthma].

OBJECTIVE: To investigate the effect of prostaglandin D2 receptor antagonists on the airway inflammation in rats with asthma. METHODS: Forty male SD rats were randomly divided into four groups: Group A (normal control), Group B (asthma group), Group C (CRTH2 antagonist BAYu3405 treatment group), Group D (DP1 antagonist BWA868C treatment group). Asthma was induced by ovalbumin (OVA) challenge. The rats in each group were sacrificed 24 h after the last challenge of OVA.DP1/CRTH2 receptors on eosinophils (EOS) were measured by radiological binding assay (RBA). The left lungs were used for histological examinations and bronchoalveolar lavage fluid (BALF) was collected from the right lungs. The total cell numbers, EOS absolute count and differential cell counts in BALF were performed. Serum concentrations of IL-4, 5 and IFN-gamma were measured by ELISA. RESULTS: Rats in BAYu3405 treatment group showed profoundly decreased infiltrates of EOS and lymphocytes in the wall of bronchus when compared with those of asthma group and BWA868C treatment group. Serum concentrations of IFN-gamma in rats of BAYu3405 treatment group increased, but IL-4 and IL-5 decreased significantly when compared with those in rats of asthma group and BWA868C treatment group (P<0.01), and BALF EOS count was decreased significantly (P<0.01). Peripheral blood EOS count was higher than that in rats of normal control group, but was not significantly different from that in rats of asthma group and BWA868C treatment group. The combining capacity of CRTH2 and DP total combining capacity on EOS in asthma group, BAYu3405 treatment group and BWA868C treatment group were significantly higher than those in Group A (P<0.01). There was no significant difference in DP1 among all the groups (P>0.05). CONCLUSION: CRTH2, but not DP1 antagonist can effectively ameliorate airway inflammation in rats with asthma.