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Supplementary Materials and Methods describing 16S rRNA sequencing analysis. Figure S1. PCR detection of F. nucleatum adhesins. Figure S2. ApcMin/+;Il10-/- mice have increased colon proliferation. Figure S3. Bacteria do not promote colon inflammation and tumorigenesis in ApcMin/+ mice. Figure S4. Microbiota promote proximal colon proliferation in ApcMin/+;Il10-/- mice. Figure S5. Colibactin promotes CRC development in AOM/Il10-/- mice. Table S1. Spearman correlations and corresponding p values from linear models used to generate Figure 3. Table S2. V1-V3 MiSeq Primers. Table S3. Accession numbers for 16S rRNA sequenced ApcMin/+;Il10-/- stool samples from Figure 3.

Inflammation and microbiota are critical components of intestinal tumorigenesis. To dissect how the microbiota contributes to tumor distribution, we generated germ-free (GF) ApcMin/+and ApcMin/+;Il10−/− mice and exposed them to specific-pathogen-free (SPF) or colorectal cancer-associated bacteria. We found that colon tumorigenesis significantly correlated with inflammation in SPF-housed ApcMin/+;Il10−/−, but not in ApcMin/+mice. In contrast, small intestinal neoplasia development significantly correlated with age in both ApcMin/+;Il10−/− and ApcMin/+ mice. GF ApcMin/+;Il10−/− mice conventionalized by an SPF microbiota had significantly more colon tumors compared with GF mice. Gnotobiotic studies revealed that while Fusobacterium nucleatum clinical isolates with FadA and Fap2 adhesins failed to induce inflammation and tumorigenesis, pks+Escherichia coli promoted tumorigenesis in the ApcMin/+;Il10−/− model in a colibactin-dependent manner, suggesting colibactin is a driver of carcinogenesis. Our results suggest a distinct etiology of cancers in different locations of the gut, where colon cancer is primarily driven by inflammation and the microbiome, while age is a driving force for small intestine cancer. Cancer Res; 77(10); 2620–32. ©2017 AACR.

Lipopolysaccharide (LPS), also known as endotoxin, can trigger septic shock, a severe form of inflammation-mediated sepsis with a very high mortality rate. However, the precise mechanisms underlying this endotoxin remain to be defined and detoxification of LPS is yet to be established. Macrophages, a type of immune cells, initiate a key response responsible for the cascade of events leading to the surge in inflammatory cytokines and immunopathology of septic shock. This study was undertaken to determine whether the LPS-induced inflammation in macrophage cells could be ameliorated via CDDO-IM (2-cyano-3,12 dioxooleana-1,9 dien-28-oyl imidazoline), a novel triterpenoid compound. Data from this study show that gene expression levels of inflammatory cytokine genes such as interleukin-1 beta (IL-1β), interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) were considerably increased by treatment with LPS in macrophages differentiated from ML-1 monocytes. Interestingly, LPS-induced increase in expression of pro-inflammatory cytokine levels is reduced by CDDO-IM. In addition, endogenous upregulation of a series of antioxidant molecules by CDDO-IM provided protection against LPS-induced cytotoxicity in macrophages. LPS-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) transcriptional activity was also noted to decrease upon treatment with CDDO-IM in macrophages suggesting the involvement of the NF-κB signaling. This study would contribute to improve our understanding of the detoxification of endotoxin LPS by the triterpenoid CDDO-IM.

Epidemiological studies have exhibited a strong correlation between exposure to air pollution and deaths due to vascular diseases such as atherosclerosis. Benzo-a-pyrene-1,6-quinone (BP-1,6-Q) is one of the components of air pollution. This study was to examine the role of GSH in BP-1,6-Q mediated cytotoxicity in human EA.hy96 endothelial cells and demonstrated that induction of cellular glutathione by a potent triterpenoid, CDDO-Im (1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole), protects cells against BP-1,6-Q induced protein and lipid damage. Incubation of EA.hy926 endothelial cells with BP-1,6-Q caused a significant increase in dose-dependent cytotoxicity as measured by LDH release assay and both apoptotic and necrotic cell deaths as measured by flow cytometric analysis. Incubation of EA.hy926 endothelial cells with BP-1,6-Q also caused a significant decrease in cellular GSH levels. The diminishment of cellular GSH by buthionine sulfoximine (BSO) potentiated BP-1,6-Q-induced toxicity significantly suggesting a critical involvement of GSH in BP-1,6-Q induced cellular toxicity. GSH-induction by CDDO-Im significantly protects cells against BP-1,6-Q induced protein and lipid damage as measured by protein carbonyl (PC) assay and thiobarbituric acid reactive substances (TBARS) assay, respectively. However, the co-treatment of cells with CDDO-Im and BSO reversed the cytoprotective effect of CDDO-Im on BP-1,6-Q-mediated lipid peroxidation and protein oxidation. These results suggest that induction of GSH by CDDO-Im might be the important cellular defense against BP-1,6-Q induced protein and lipid damage. These findings would contribute to better understand the action of BP-1,6-Q and may help to develop novel therapies to protect against BP-1,6-Q-induced atherogenesis.

Deoxycholic acid (DCA) is a naturally occurring secondary bile acid that originates from intestinal bacterial metabolic conversion of cholate, a primary bile acid. Deoxycholic acid was shown to have antihistomonal properties in vitro, leading to our hypothesis that DCA inclusion within the feed might prevent histomoniasis. Selected dietary concentrations of DCA were evaluated for effects on body weight gain (BWG), lesions, and mortality of turkeys challenged with wild-type Histomonas meleagridis (WTH). Treatments consisted of non-challenged control (NC; basal diet), 0.25% DCA diet + challenge, 0.5% DCA diet + challenge, 1% DCA diet + challenge, and a positive-challenged control (PC; basal diet). All groups were fed a basal starter diet until day 7, at which time DCA diets were administered to the respective groups. On day 14, 2 × 105 WTH cells/turkey were intracloacally administered. H. meleagridis-related lesions were evaluated on day 13 post-challenge. Pre-challenge day 0 to 14 BWG was higher (P ≤ 0.05) in the 0.25% DCA group than in the 1% DCA group. There were no significant differences in pre-challenge day 0 to 14 BWG between any of the other groups. No significant differences in mortalities from histomoniasis occurred in the DCA groups as compared to the PC group. No H. meleagridis lesions or mortalities were observed at any time in the NC group. Presence of H. meleagridis-related liver lesions was higher (P ≤ 0.05) in the 0.5% DCA group as compared to the PC group. Using the same controls and experimental timeline, an additional group was included to evaluate a biliogenic diet formulated with 20% whole egg powder to encourage endogenous bile acid production. The biliogenic diet had no statistical impact on pre-challenge day 0 to 14 BWG, but did not reduce H. meleagridis-related mortalities or lesions after the challenge. Taken together, these data suggest that DCA inclusion within the feed at these concentrations and under these experimental conditions does not prevent histomoniasis.

Resveratrol, a natural compound in grapes and red wine, has drawn attention due to potential cardiovascular-related health benefits. However, its effect on vascular inflammation at physiologically achievable concentrations is largely unknown. In this study, resveratrol in concentrations as low as 1 μm suppressed TNF-α-induced monocyte adhesion to human EA.hy926 endothelial cells (ECs), a key event in the initiation and development of atherosclerosis. Low concentrations of resveratrol (0.25–2 μm) also significantly attenuated TNF-α-stimulated mRNA expressions of MCP-1/CCL2 and ICAM-1, which are vital mediators of EC-monocyte adhesion molecules and cytokines for cardiovascular plaque formation. Additionally, resveratrol diminished TNF-α-induced IκB-α degradation and subsequent nuclear translocation of NF-κB p65 in ECs. In the animal study, resveratrol supplementation in diet significantly diminished TNF-α-induced increases in circulating levels of adhesion molecules and cytokines, monocyte adhesion to mouse aortic ECs, F4/80-positive macrophages and VCAM-1 expression in mice aortas and restored the disruption in aortic elastin fiber caused by TNF-α treatment. The animal study also confirmed that resveratrol blocks the activation of NF-κB In Vivo. In conclusion, resveratrol at physiologically achievable concentrations displayed protective effects against TNF-α-induced vascular endothelial inflammation in vitro and In Vivo. The ability of resveratrol in reducing inflammation may be associated with its role as a down-regulator of the NF-κB pathway. Published version

Mucus-invasive bacterial biofilms are identified on the colon mucosa of approximately 50% of colorectal cancer (CRC) patients and approximately 13% of healthy subjects. Here, we test the hypothesis that human colon biofilms comprise microbial communities that are carcinogenic in CRC mouse models. Homogenates of human biofilm-positive colon mucosa were prepared from tumor patients (tumor and paired normal tissues from surgical resections) or biofilm-positive biopsies from healthy individuals undergoing screening colonoscopy; homogenates of biofilm-negative colon biopsies from healthy individuals undergoing screening colonoscopy served as controls. After 12 weeks, biofilm-positive, but not biofilm-negative, human colon mucosal homogenates induced colon tumor formation in 3 mouse colon tumor models (germ-free ApcMinΔ850/+;Il10-/- or ApcMinΔ850/+ and specific pathogen-free ApcMinΔ716/+ mice). Remarkably, biofilm-positive communities from healthy colonoscopy biopsies induced colon inflammation and tumors similarly to biofilm-positive tumor tissues. By 1 week, biofilm-positive human tumor homogenates, but not healthy biopsies, displayed consistent bacterial mucus invasion and biofilm formation in mouse colons. 16S rRNA gene sequencing and RNA-Seq analyses identified compositional and functional microbiota differences between mice colonized with biofilm-positive and biofilm-negative communities. These results suggest human colon mucosal biofilms, whether from tumor hosts or healthy individuals undergoing screening colonoscopy, are carcinogenic in murine models of CRC.

Enterobacteriaceae are among the first colonizers of neonate intestine. Members of this family, such as Escherichia and Klebsiella, are considered pathobionts and as such are capable of inducing local and systemic disease under specific colonization circumstances. Interplay between developing microbiota and pathogenic function of pathobionts are poorly understood. In this study, we investigate the functional interaction between various colonization patterns on an early colonizer, K. pneumoniae. K. pneumoniae 51-5 was isolated from stool of a healthy, premature infant, and found to contain the genotoxin island pks associated with development of colorectal cancer. Using intestinal epithelial cells, macrophages, and primary splenocytes, we demonstrate K. pneumoniae 51-5 upregulates expression of proinflammatory genes in vitro. Gnotobiotic experiments in Il10−/− mice demonstrate the neonate isolate induces intestinal inflammation in vivo, with increased expression of proinflammatory genes. Regulation of microbiota assembly revealed K. pneumoniae 51-5 accelerates onset of inflammation in Il10−/− mice, most significantly when microbiota is naturally acquired. Furthermore, K. pneumoniae 51-5 induces DNA damage and cell cycle arrest. Interestingly, K. pneumoniae 51-5 induced tumors in ApcMin/+; Il10−/− mice was not significantly affected by absence of colibactin activating enzyme, ClbP. These findings demonstrate pathogenicity of infant K. pneumoniae isolate is sensitive to microbial colonization status.

Despite the high efficiency of self-regeneration in liver, liver stem cell organoid culture is way behind other cell types such as intestinal organoid (Enteroid). The contributing factor is the lack of effi¬cient technique to isolate liver cells and to culture liver stem cell organoid. Here we reported a new, eco¬nomical, and simple method to generate 3D hepatocyte organoid (Hepatoid) and cholangiocyte organoid (Cholangoid). We have isolated liver cells using ice-cold 3mM EDTA PBS medium. The isolated cells were cultured on 50% Conditional Medium. After 1 or 4 days post isolation and culture, Cholangoid and Hepatoid were readily to be detected under light microscope and they continued to grow well after several passages. This method could be used to isolate and culture human liver organoids. This feasible protocol will greatly facilitate more research on liver function and diseases using liver cell organoids. Keywords:Organoid; Stem cell; 3D culture; Liver

Million cases of campylobacteriosis and complications of post-Campylobacter jejuni infection occur every year around the world with huge life losses and economic burdens of billions of dollars. Few therapy options, such as antibiotics, are available to relieve severe cases of the enteritis. The slow progression on new intervention discovery and application is partially resulted from limited mechanistic understanding on campylobacteriosis pathogenesis. As a type of intestinal disorders, campylobacteriosis shares many common features with other intestinal diseases such as inflammatory bowel diseases (IBD) and Clostridium difficile infection. In pace with the advancement of the gastroenterology field, a large body of knowledge is accumulating on the factors influencing campylobacteriosis onset, development, and outcomes, including host immune response, intestinal microbiota, and its metabolites. In this chapter, we review the intestinal immune system, intestinal microbiome, and microbiome modulation of inflammation in the development of campylobacteriosis. The interplay between immunity, microbiota, and its metabolites may play essential roles on campylobacteriosis pathogenesis and the finding on the interaction may lead to new prevention and treatment options. The purpose of this chapter is to provide updated knowledge on the role of host–microbe interaction and the therapeutic potential on campylobacteriosis.

Salmonella Enteritidis is an intracellular foodborne pathogen that has developed multiple mechanisms to alter poultry intestinal physiology and infect the gut. Short chain fatty acid butyrate is derived from microbiota metabolic activities, and it maintains gut homeostasis. There is limited understanding on the interaction between S. Enteritidis infection, butyrate, and host intestinal response. To fill this knowledge gap, chicken macrophages (also known as HTC cells) were infected with S. Enteritidis, treated with sodium butyrate, and proteomic analysis was performed. A growth curve assay was conducted to determine sub-inhibitory concentration (SIC, concentration that do not affect bacterial growth compared to control) of sodium butyrate against S. Enteritidis. HTC cells were infected with S. Enteritidis in the presence and absence of SIC of sodium butyrate. The proteins were extracted and analyzed by tandem mass spectrometry. Our results showed that the SIC was 45 mM. Notably, S. Enteritidis-infected HTC cells upregulated macrophage proteins involved in ATP synthesis through oxidative phosphorylation such as ATP synthase subunit alpha (ATP5A1), ATP synthase subunit d, mitochondrial (ATP5PD) and cellular apoptosis such as Cytochrome-c (CYC). Furthermore, sodium butyrate influenced S. Enteritidis-infected HTC cells by reducing the expression of macrophage proteins mediating actin cytoskeletal rearrangements such as WD repeat-containing protein-1 (WDR1), Alpha actinin-1 (ACTN1), Vinculin (VCL) and Protein disulfide isomerase (P4HB) and intracellular S. Enteritidis growth and replication such as V-type proton ATPase catalytic subunit A (ATPV1A). Interestingly, sodium butyrate increased the expression of infected HTC cell protein involving in bacterial killing such as Vimentin (VIM). In conclusion, sodium butyrate modulates the expression of HTC cell proteins essential for S. Enteritidis invasion.

Salmonella Enteritidis (SE) is a facultative intracellular pathogen that colonizes the chicken gut leading to contamination of carcasses during processing. A reduction in intestinal colonization by SE could result in reduced carcass contamination thereby reducing the risk of illnesses in humans. Short chain fatty acids such as butyrate are microbial metabolites produced in the gut that exert various beneficial effects. However, its effect on SE colonization is not well known. The present study investigated the effect of sub-inhibitory concentrations (SICs) of sodium butyrate on the adhesion and invasion of SE in primary chicken enterocytes and chicken macrophages. In addition, the effect of sodium butyrate on the expression of SE virulence genes and selected inflammatory genes in chicken macrophages challenged with SE were investigated. Based on the growth curve analysis, the two SICs of sodium butyrate that did not reduce SE growth were 22 and 45 mM, respectively. The SICs of sodium butyrate did not affect the viability and proliferation of chicken enterocytes and macrophage cells. The SICs of sodium butyrate reduced SE adhesion by ∼1.7 and 1.8 Log CFU/mL, respectively. The SE invasion was reduced by ∼2 and 2.93 Log CFU/mL, respectively in chicken enterocytes (P < 0.05). Sodium butyrate did not significantly affect the adhesion of SE to chicken macrophages. However, 45 mM sodium butyrate reduced invasion by ∼1.7 Log CFU/mL as compared to control (P < 0.05). Exposure to sodium butyrate did not change the expression of SE genes associated with motility (flgG, prot6E), invasion (invH), type 3 secretion system (sipB, pipB), survival in macrophages (spvB, mgtC), cell wall and membrane integrity (tatA), efflux pump regulator (mrr1) and global virulence regulation (lrp) (P > 0.05). However, a few genes contributing to type-3 secretion system (ssaV, sipA), adherence (sopB), macrophage survival (sodC) and oxidative stress (rpoS) were upregulated by at least twofold. The expression of inflammatory genes (Il1β, Il8, and Mmp9) that are triggered by SE for host colonization was significantly downregulated (at least 25-fold) by sodium butyrate as compared to SE (P < 0.05). The results suggest that sodium butyrate has an anti-inflammatory potential to reduce SE colonization in chickens.

Campylobacter jejuni is a prevalent foodborne pathogen mainly transmitting through poultry. It remains unknown how chicken-transmitted C. jejuni and microbiota impact on human campylobacteriosis. Campylobacter jejuni AR101 (Cj-P0) was introduced to chickens and isolated as passage 1 (Cj-P1). Campylobacter jejuni Cj-P1-DCA-Anaero was isolated from Cj-P0-infected birds transplanted with DCA-modulated anaerobic microbiota. Specific pathogen free Il10−/− mice were gavaged with antibiotic clindamycin and then infected with Cj-P0, Cj-P1, or Cj-P1-DCA-Anaero, respectively. After 8 days post infection, Il10−/− mice infected with Cj-P1 demonstrated severe morbidity and bloody diarrhea and the experiment had to be terminated. Cj-P1 induced more severe histopathology compared to Cj-P0, suggesting that chicken transmission increased C. jejuni virulence. Importantly, mice infected with Cj-P1-DCA-Anaero showed attenuation of intestinal inflammation compared to Cj-P1. At the cellular level, Cj-P1 induced more C. jejuni invasion and neutrophil infiltration into the Il10−/− mouse colon tissue compared to Cj-P0, which was attenuated with Cj-P1-DCA-Anaero. At the molecular level, Cj-P1 induced elevated inflammatory mediator mRNA accumulation of Il17a, Il1β, and Cxcl1 in the colon compared to Cj-P0, while Cj-P1-DCA-Anaero showed reduction of the inflammatory gene expression. In conclusion, our data suggest that DCA-modulated anaerobes attenuate chicken-transmitted campylobacteriosis in mice and it is important to control the elevation of C. jejuni virulence during chicken transmission process.

Background Clostridium perfringens-induced chicken necrotic enteritis (NE) is responsible for substantial economic losses worldwide annually. Recently, as a result of antibiotic growth promoter prohibition, the prevalence of NE in chickens has reemerged. This study was aimed to reduce NE through titrating dietary deoxycholic acid (DCA) as an effective antimicrobial alternative. Materials and methods Day-old broiler chicks were assigned to six groups and fed diets supplemented with 0 (basal diet), 0.8, 1.0 and 1.5 g/kg (on top of basal diet) DCA. The birds were challenged with Eimeria maxima (20,000 oocysts/bird) at d 18 and C. perfringens (109 CFU/bird per day) at d 23, 24, and 25 to induce NE. The birds were sacrificed at d 26 when ileal tissue and digesta were collected for analyzing histopathology, mRNA accumulation and C. perfringens colonization by real-time PCR, targeted metabolomics of bile acids, fluorescence in situ hybridization (FISH), or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results At the cellular level, birds infected with E. maxima and C. perfringens developed subclinical NE and showed shortening villi, crypt hyperplasia and immune cell infiltration in ileum. Dietary DCA alleviated the NE-induced ileal inflammation in a dose-dependent manner compared to NE control birds. Consistent with the increased histopathological scores, subclinical NE birds suffered body weight gain reduction compared to the uninfected birds, an effect attenuated with increased doses of dietary DCA. At the molecular level, the highest dose of DCA at 1.5 g/kg reduced C. perfringens luminal colonization compared to NE birds using PCR and FISH. Furthermore, the dietary DCA reduced subclinical NE-induced intestinal inflammatory gene expression and cell apoptosis using PCR and TUNEL assays. Upon further examining ileal bile acid pool through targeted metabolomics, subclinical NE reduced the total bile acid level in ileal digesta compared to uninfected birds. Notably, dietary DCA increased total bile acid and DCA levels in a dose-dependent manner compared to NE birds. Conclusion These results indicate that DCA attenuates NE-induced intestinal inflammation and bile acid reduction and could be an effective antimicrobial alternative against the intestinal disease.

In the present work, the multiaxial 3D (M3D) woven composites and 3D layer-to-layer (3D LTL) interlock woven composites were designed and manufactured. Both unnotched and notched specimens of two composites were tested in quasi-static tension, and the macro mechanical behavior characteristics of all specimens were monitored using the digital image correlation (DIC) technique. Based on the meso-structure characteristics of the M3D woven composites, a macro-meso coupling and full-scale analysis model for the centrally notched specimens was proposed. Furthermore, the progressive damage analysis was performed to simulate the damage evolution processes of the open-hole (OH) specimens, and the failure mechanisms were revealed in cooperation with the CT measurement. The study concluded that the reduction in tensile strength for the OH-M3D composites was about 19.8% compared to the unnotched strength, which was significantly lower than the 3D LTL case. Besides, the strain field in the loading direction uniformly distributed on the surface of the OH-M3D specimen, and the notched plate failed along the + 45° and −45° directions, exhibiting a diamond like fracture behavior. The interwoven structure of M3D specimen with the + 45° and −45° yarns influenced the stress distributions in the vicinity of the hole and damage mode.

Numerous studies conducted in the past have reported deaths in the human population due to cardiovascular diseases (CVD) on exposure to air particulate matter (APM). BP-1,6-quinone (BP-1,6-Q) is one of the significant components of APM. However, the mechanism(s) by which it can exert its toxicity in endothelial cells is not yet completely understood. NAD(P)H: quinone oxidoreductase-1 (NQO1) is expressed highly in myocardium and vasculature tissues of the heart and plays a vital role in maintaining vascular homeostasis. This study, demonstrated that BP-1,6-Q diminishes NQO1 enzyme activity in a dose-dependent manner in human EA.hy926 endothelial cells. The decrease in the NQO1 enzyme causes potentiation in BP-1,6-Q-mediated toxicity in EA.hy926 endothelial cells. The enhancement of NQO1 in endothelial cells showed cytoprotection against BP-1,6-Q-induced cellular toxicity, lipid, and protein damage suggesting an essential role of NQO1 in cytoprotection against BP-1,6-Q toxicity. Using various biochemical assays and genetic approaches, results from this study further demonstrated that NQO1 also plays a crucial role in BP-1,6-Q-induced production of reactive oxygen species (ROS). These findings will contribute to elucidating BP-1,6-Q mediated toxicity and its role in the development of atherosclerosis.

This paper reports an innovative multiaxial 3D orthogonal-interlock woven architecture and weaving process, associated with the compressive properties of its composites. Three types of multiaxial 3D woven composite plates with different bias yarn contents (S1: 22.7%, S2: 27.1%, and S3: 33.2%) are prepared, compressive are loads applied in both the warp and weft directions, and the failure mechanism is evaluated using micro-computed tomography (micro-CT). The experimental results show that the compressive stress–strain curves of the specimens S1 and S2 under warp loading follow a linear increase prior to the peak stress, while that of the specimen S3 increases nonlinearly up to failure. Additionally, the stress–strain curves of all specimens loaded in the weft direction show a linear response within the full strain range. S1 and S3 withstand the maximum compressive strengths in the warp and weft directions accordingly. The compressive properties of multiaxial 3D woven composites mainly depend on the directional fiber content along the loading direction. CT image slices show that matrix cracks, fiber/matrix interface debonding and fiber breakage/splitting are the major failure patterns of multiaxial 3D woven composites subjected to compression loads.

Campylobacter jejuni, a prevalent foodborne bacterial pathogen, is mainly transmitted from poultry with few effective prevention approaches. In this study, we aimed to investigate the role of microbiota on C. jejuni chicken colonization. Microbiota from specific pathogen-free (SPF) mouse stools were collected as SPF-Aerobe and SPF-Anaerobe. Birds were colonized with SPF-Aerobe or SPF-Anaerobe at day 0 and infected with C. jejuni AR101 at day 12. Notably, C. jejuni AR101 colonized at 5.3 and 5.6 log10&nbsp, C. jejuni CFU/g chicken cecal digesta at days 21 and 28, respectively, while both SPF-Aerobe and SPF-Anaerobe microbiota reduced pathogen colonization. Notably, SPF-Aerobe and SPF-Anaerobe increased cecal phylum Bacteroidetes and reduced phylum Firmicutes compared to those in the nontransplanted birds. Interestingly, microbiota from noninfected chickens, SPF-Aerobe, or SPF-Anaerobe inhibited AR101 in vitro growth, whereas microbiota from infected birds alone failed to reduce pathogen growth. The bacterium Enterobacter102 isolated from infected birds transplanted with SPF-Aerobe inhibited AR101 in vitro growth and reduced pathogen gut colonization in chickens. Together, SPF mouse microbiota was able to colonize chicken gut and reduce C. jejuni chicken colonization. The findings may help the development of effective strategies to reduce C. jejuni chicken contamination and campylobacteriosis.

The open-hole tension (OHT) properties and failure processes of three-dimensional (3D) woven composite plates with circular, racetrack, and square holes were investigated in this study through experiments and finite element (FE) modeling. Tensile tests were performed on unnotched and notched specimens and measured via digital image correlation (DIC) to record the full-field strain fields, while micro-computed tomography (micro-CT) was employed to obtain the fracture morphologies. To predict the OHT performance, stress concentration factor (SCF), and damage propagation processes of the notched specimens, progressive damage analysis based on the FE model of a macro-meso coupling model was implemented on three open-hole plates. It was found that the OHT strength of 3D woven composites was reduced by approximately 35% compared with that of the unnotched specimen, whereas the modulus remained almost unchanged. The hole shape significantly influenced the strain distributions but had no perceptible effect on the OHT strength. The SCF of the specimen with a circular hole was 36% higher than those cases of the racetrack and square holes. Moreover, the hole-edge stress concentrations were mainly distributed on the warp yarns and became a cause for early failure. The major failure modes near the hole edge were warp breakage and pull-out.

Necrotic enteritis (NE), mainly induced by the pathogens of Clostridium perfringens and coccidia, causes huge economic losses with limited intervention options in the poultry industry. This study investigated the role of specific bile acids on NE development. Day-old broiler chicks were assigned to six groups: noninfected, NE, and NE with four bile diets of 0.32% chicken bile, 0.15% commercial ox bile, 0.15% lithocholic acid (LCA), or 0.15% deoxycholic acid (DCA). The birds were infected with Eimeria maxima at day 18 and C. perfringens at day 23 and 24. The infected birds developed clinical NE signs. The NE birds suffered severe ileitis with villus blunting, crypt hyperplasia, epithelial line disintegration, and massive immune cell infiltration, while DCA and LCA prevented the ileitis histopathology. NE induced severe body weight gain (BWG) loss, while only DCA prevented NE-induced BWG loss. Notably, DCA reduced the NE-induced inflammatory response and the colonization and invasion of C. perfringens compared to NE birds. Consistently, NE reduced the total bile acids in the ileal digesta, while dietary DCA and commercial bile restored it. Together, this study showed that DCA and LCA reduced NE histopathology, suggesting that secondary bile acids, but not total bile acid levels, play an essential role in controlling the enteritis.

Despite reducing the prevalent foodborne pathogenCampylobacter jejuniin chickens decreases campylobacteriosis, few effective approaches are available. The aim of this study was to use microbial metabolic product bile acids to reduceC. jejunichicken colonization. Broiler chicks were fed with deoxycholic acid (DCA), lithocholic acid (LCA), or ursodeoxycholic acid (UDCA). The birds were also transplanted with DCA modulated anaerobes (DCA-Anaero) or aerobes (DCA-Aero). The birds were infected with human clinical isolateC. jejuni81-176 or chicken isolateC. jejuniAR101. Notably,C. jejuni81-176 was readily colonized intestinal tract at d16 and reached an almost plateau at d21. Remarkably, DCA excludedC. jejunicecal colonization at 100, 99.997, and 100% at 16, 21, and 28 days of age. Neither chicken ages of infection nor LCA or UDCA alteredC. jejuniAR101 chicken colonization level, while DCA reduced 91% of the bacterium in chickens at d28. Notably, DCA diet reduced phylum Firmicutes but increased Bacteroidetes compared to infected control birds. Importantly, DCA-Anaero attenuated 93% ofC. jejunicolonization at d28 compared to control infected birds. In conclusion, DCA shapes microbiota composition againstC. jejunicolonization in chickens, suggesting a bidirectional interaction between microbiota and microbial metabolites.

Necrotic enteritis (NE) caused by Clostridium perfringens infection has reemerged as a prevalent poultry disease worldwide due to reduced usage of prophylactic antibiotics under consumer preferences and regulatory pressures. The lack of alternative antimicrobial strategies to control this disease is mainly due to limited insight into the relationship between NE pathogenesis, microbiome, and host responses. Here we showed that the microbial metabolic byproduct of secondary bile acid deoxycholic acid (DCA), at as low as 50 µM, inhibited 82.8% of C. perfringens growth in Tryptic Soy Broth (P Eimeria maxima and C. perfringens challenges significantly induced NE, severe intestinal inflammation, and body weight (BW) loss in broiler chickens. These negative effects were diminished (P C. perfringens invasion into ileum (P Infγ, Litaf, Il1β, and Mmp9 mRNA accumulation in ileal tissue. Mechanism studies revealed that C. perfringens induced (P Infγ, Litaf, and Ptgs2 (Cyclooxygenases-2 (COX-2) gene) in chicken splenocytes. Inhibiting the COX signaling by aspirin significantly attenuated INFγ-induced inflammatory response in the splenocytes. Consistent with the in vitro assay, chickens fed 0.12 g/kg aspirin diet protected the birds against NE-induced BW loss, ileal inflammation, and intestinal cell apoptosis. In conclusion, microbial metabolic product DCA prevents NE-induced BW loss and ileal inflammation through attenuating inflammatory response. These novel findings of microbiome protecting birds against NE provide new options on developing next generation antimicrobial alternatives against NE.

The use of 16S next generation sequencing (NGS) technology to identify the relative abundance of microbial communities have become the standard when studying the intestinal microbiome. The increased use is due to the ability to identify a proportion of bacteria that cannot be observed with culture-based methods. However, culture-based techniques are acceptable to identify key bacterial groups, yet may grossly underestimate the microbial community in question. Since there is limited research comparing NGS results to colony forming units (CFU), the objective of this study was to compare total Enterobacteriaceae and lactic acid bacteria (LAB) recovery with culture techniques (CFU/g ceca) to total number of reads from operational taxonomic units (OTU) categorized as Enterobacteriaceae or LAB from Illumina MiSeq platform from matched chick cecal samples at three and 10 days of age. Both CFU recovery (1.09x109 ± 2.42x108; 1.37x108 ± 5.57x107) and reads (5460 ± 1164 ; 282 ± 163) belonging to Enterobacteriaceae decreased by 10 days of age (p < 0.001). Similarly, LAB reads decreased over time (21,128 ± 2262; 6220 ± 817, respectively p < 0.0001). However, LAB CFU recovery increased by 10 days (1.18x108 ± 1.91x107; 1.62x109 ± 5.00x108, respectively p < 0.01). At three days the Pearson’s correlation was -0.082 between CFU of culturable Enterobacteriaceae to reads and culturable LAB CFU to reads at 0.097, showing no correlation (p = 0.606, 0.551; respectively). By 10 days, no correlation of reads and CFU occurred with Enterobacteriaceae (r=-0.049; p-value = 0.769) while with LAB the correlation was 0.290 (p = 0.066) at 10 days. The CFU may be appropriate to identify a few families that change due to treatment or product. Without identifying viable cells to DNA recovered from NGS, there will always be the question whether the reads within the binned OTU in the intestinal tract is accurate.

A novel multiaxial 3D angle-interlock woven composites (MAWC) was developed in this paper. Two MAWCs with different weave patterns and 3D angle-interlock woven composites (3DAWC) were manufactured to identify the particular influence of bias yarns on the in-plane shear performance and failure mechanism of the composites. Based on the variable cross-sections, a realistic meso-model of MAWC was proposed. The in-plane shear tests were carried out and the digital image correlation (DIC) was used to record the full-field strain distribution of the specimens. Furthermore, a progressive damage model and X-ray micro-computed tomography (Micro-CT) were implemented to reveal the damage evolution and mechanisms of composites. Results indicated that all load–displacement curves presented nonlinear behavior, and the in-plane shear modulus and strength were significantly improved due to the bias yarns. The fiber slippage, tow splitting and interfacial debonding were the primary failure modes of bias yarns in MAWC architectures.

For 3D woven composites used in the structural applications, the open-hole mechanical performance is of critical concern for the components in service. In the present study, the circular notched 3D woven composite (N-3DWC) plates with three different apertures were prepared, and the effects of notch size on the tensile properties were investigated by experimental and numerical methods. Digital image correlation technique was used to record the full-field strain distributions, and the micro computed tomography was applied to evaluate the failure fractures. Based on the developed macro-meso coupling model of N-3DWC, the open-hole tensile strength and failure mechanisms were predicted and analyzed, and the model was validated with the experimental results. The results showed that the stress-strain curves of all N-3DWC specimens were basically linear in the initial loading stage. The notch size had little effect on the tensile stiffness of notched plates, while the tensile strength decreased with the increasing aperture. The stress concentration mostly occurred at the hole edge, and the distribution width was approximately equal to the notch diameter. As the aperture increased, the main failure mode of weft yarn changed from fiber pull-out to breakage, and the contour of damaged region changed from to convex to concave.

Clostridium perfringens-induced necrotic enteritis (NE) has reemerged as a prevalent chicken disease worldwide due to reduced usage of prophylactic antibiotics. The lack of antimicrobial alternative strategies to control NE is mainly due to limited insight into the disease pathogenesis. The aim of this study is to investigate the role of microbiota metabolic product secondary bile acid deoxycholic acid (DCA) on preventing NE.C. perfringensgrowth was inhibited by 82.8% in 50 μM DCA Tryptic Soy Broth. SequentialEimeria maximaandC. perfringenschallenges induced acute NE showed as severe intestinal inflammation and body weight (BW) loss in broiler chickens, while 1.5 g/kg DCA diet dramatically reduced the disease. At the cellular level, DCA alleviated NE-associated ileal epithelial death and reduced lamina propria cell apoptosis. Interestingly, DCA reducedC. perfringensinvasion into ileum without altering the bacterial ileal luminal colonization. Molecular analysis showed that DCA reduced inflammatory mediators ofInfγ,Litaf, andMmp9mRNA accumulation in ileal tissue. Mechanism studies revealed thatC. perfringensinduced elevated expression of inflammatory mediators ofInfγ,Litaf,Mmp9,andPtgs2(Cyclooxygenase- 2 (COX-2) gene) in chicken splenocytes. Blocking COX signaling by pharmacological inhibitor aspirin attenuated INFγ-induced inflammatory response in the splenocytes. Consistent with thein vitroassay, chickens fed 0.12 g/kg aspirin diet protected the birds against NE-induced ileal inflammation, intestinal cell apoptosis, and BW loss. In conclusion, microbial metabolic product DCA prevents NE-induced ileal inflammation and BW loss through attenuating inflammatory response. These novel findings offer new strategies againstC. perfringens-induced diseases.Significance StatementWidespread antimicrobial resistance has become a serious challenge to both agricultural and healthcare industries. Withdrawing antimicrobials without effective alternatives exacerbates chicken productivity loss at billions of dollars every year, caused by intestinal diseases, such as coccidiosis-andC. perfringens-induced necrotic enteritis. This study revealed that microbial metabolic product secondary bile acid DCA preventsC. perfringens-induced intestinal disease in chickens through modulating inflammatory COX signaling pathways. Therefore, microbiome and its downstream targets of host inflammatory responses could be used to control NE. These findings have opened new avenues for developing novel antimicrobial free alternatives to prevent or treatC. perfringens-induced diseases.

Eliminating prophylactic antibiotics in food animal production has exerted pressure on discovering antimicrobial alternatives (e.g. microbiome) to reduce elevated intestinal diseases. Intestinal tract is a complex ecosystem coupling host cells with microbiota. The microbiota and its metabolic activities and products are collectively called microbiome. Intestinal homeostasis is reached through dynamic and delicate crosstalk between host immunity and microbiome. However, this balance can be occasionally broken, which results in intestinal inflammatory diseases such as human Inflammatory Bowel Diseases, chicken necrotic enteritis, and swine postweaning diarrhea. In this review, we introduce the intestinal immune system, intestinal microbiome, and microbiome modulation of inflammation against intestinal diseases. The purpose of this review is to provide updated knowledge on host-microbe interaction and to promote using microbiome as new antimicrobial strategies to reduce intestinal diseases.

Necrotic enteritis (NE) caused by Clostridium perfringens infection has reemerged as a prevalent poultry disease worldwide due to reduced usage of prophylactic antibiotics. The lack of alternative antimicrobial strategies to control this disease is mainly due to limited insight into NE pathogenesis, microbiome relationships, and host responses. Here we reported that the metabolic byproduct of microbial metabolism of bile acids to deoxycholic acid (DCA), at as low as 50 μM, inhibited 82.8% of C. perfringens growth in Tryptic Soy Broth (P < 0.05). Sequential Eimeria maxima and C. perfringens challenge strongly induced NE, severe intestinal inflammation, and body weight (BW) loss in broiler chickens. These negative effects were diminished by 1.5 g/kg DCA diet. At the cellular level, DCA alleviated NE-associated ileal epithelial death and lamina propria immune cell apoptosis. Interestingly, DCA reduced C. perfringens invasion into villi without significantly altering the bacterial luminal colonization. Molecular analysis showed that DCA reduced inflammatory mediators of Infγ, Litaf (Tnfα), Il1β, and Mmp9 mRNA accumulation in ileal tissue. Mechanically, C. perfringens induced elevated expression of inflammatory cytokines of Infγ, Litaf, and Ptgs2 (COX-2 gene) in chicken splenocytes. Inhibiting the COX signaling by aspirin attenuated INFγ- or TNFa-induced inflammatory response in the splenocytes. Consistently, chickens fed 0.12 g/kg aspirin diet resisted against NE-induced BW loss, ileal inflammation, and villus apoptosis. In conclusion, microbial metabolic product DCA prevents NE-induced BW loss and ileal inflammation through curbing inflammatory response. These novel findings could serve as a stepping-stone for developing next generation antimicrobial alternatives against NE.

Substantial evidence suggests that overproduction of reactive oxygen species (ROS) and its associated oxidative stress and inflammatory responses play a crucial role in the pathogenesis of various human chronic diseases including diabetes, cardiovascular diseases, obesity, neurodegenerative diseases, and cancer. Enhanced ROS production can cause oxidative damage to the cellular macromolecules including lipids, proteins, and DNA. In addition, oxidative stress activates nuclear factor kappa B (NF-?B), nuclear factor-erythroid 2-related factor 2 (Nrf2), and antioxidant-responsive elements (ARE) to regulate anti-inflammatory and antioxidative genes involved in inflammatory responses and cellular defense mechanisms. Recent studies have showed that certain natural compounds and/or their derived small molecules have the ability to protect cells from oxidative stress and ameliorate various oxidative stress-related diseases. However, a large number of natural products are underutilized and unexploited because their bioactivities are unknown and the molecular mechanisms involved are yet to be identified. Understanding the mechanisms of actions of natural products would shed further light into the application of these compounds in the prevention and treatment of oxidative stress-related diseases in humans. This special issue includes two invited reviews and twelve original research articles that are focused on the antioxidative/anti-inflammatory effects of various natural compounds and their safety in vitro and in vivo. Further, these studies identified the cellular and molecular mechanisms underlying the bioactivities of these natural compounds.

Background & Aims Campylobacter jejuni, a prevalent foodborne bacterial pathogen, exploits the host innate response to induce colitis. Little is known about the roles of microbiota in C jejuni–induced intestinal inflammation. We investigated interactions between microbiota and intestinal cells during C jejuni infection of mice. Methods Germ-free C57BL/6 Il10−/− mice were colonized with conventional microbiota and infected with a single dose of C jejuni (109 colony-forming units/mouse) via gavage. Conventional microbiota were cultured under aerobic, microaerobic, or anaerobic conditions and orally transplanted into germ-free Il10−/− mice. Colon tissues were collected from mice and analyzed by histology, real-time polymerase chain reaction, and immunoblotting. Fecal microbiota and bile acids were analyzed with 16S sequencing and high-performance liquid chromatography with mass spectrometry, respectively. Results Introduction of conventional microbiota reduced C jejuni–induced colitis in previously germ-free Il10−/− mice, independent of fecal load of C jejuni, accompanied by reduced activation of mammalian target of rapamycin. Microbiota transplantation and 16S ribosomal DNA sequencing experiments showed that Clostridium XI, Bifidobacterium, and Lactobacillus were enriched in fecal samples from mice colonized with microbiota cultured in anaerobic conditions (which reduce colitis) compared with mice fed microbiota cultured under aerobic conditions (susceptible to colitis). Oral administration to mice of microbiota-derived secondary bile acid sodium deoxycholate, but not ursodeoxycholic acid or lithocholic acid, reduced C jejuni–induced colitis. Depletion of secondary bile acid–producing bacteria with antibiotics that kill anaerobic bacteria (clindamycin) promoted C jejuni–induced colitis in specific pathogen-free Il10−/− mice compared with the nonspecific antibiotic nalidixic acid; colitis induction by antibiotics was associated with reduced level of luminal deoxycholate. Conclusions We identified a mechanism by which the microbiota controls susceptibility to C jejuni infection in mice, via bacteria-derived secondary bile acids.

Inflammation and microbiota are critical components of intestinal tumorigenesis. To dissect how the microbiota contributes to tumor distribution, we generated germ-free (GF) ApcMin/+and ApcMin/+;Il10−/− mice and exposed them to specific-pathogen-free (SPF) or colorectal cancer-associated bacteria. We found that colon tumorigenesis significantly correlated with inflammation in SPF-housed ApcMin/+;Il10−/−, but not in ApcMin/+mice. In contrast, small intestinal neoplasia development significantly correlated with age in both ApcMin/+;Il10−/− and ApcMin/+ mice. GF ApcMin/+;Il10−/− mice conventionalized by an SPF microbiota had significantly more colon tumors compared with GF mice. Gnotobiotic studies revealed that while Fusobacterium nucleatum clinical isolates with FadA and Fap2 adhesins failed to induce inflammation and tumorigenesis, pks+Escherichia coli promoted tumorigenesis in the ApcMin/+;Il10−/− model in a colibactin-dependent manner, suggesting colibactin is a driver of carcinogenesis. Our results suggest a distinct etiology of cancers in different locations of the gut, where colon cancer is primarily driven by inflammation and the microbiome, while age is a driving force for small intestine cancer. Cancer Res; 77(10); 2620–32. ©2017 AACR.

Cardiovascular diseases are the most prominent circulation disorders around the world. Biomarkers are characteristic biological properties that can be objectively measured as an indicator to evaluate a variety of health or disease characteristics. Cardiac biomarkers are a valuable tool for assessing the pathogenesis and diagnosis of cardiovascular diseases. In this review, we will focus on the major biomarkers used in recent clinical research for the diagnosis of cardiovascular diseases, which include mean platelet volume, hyperhomocysteinemia, serum magnesium, microalbuminuria, and prolongation of QT interval and dispersion. We also highlight the key findings of clinical case report based studies presented in this issue of JCDR.

Inflammatory bowel diseases (IBDs), including ulcerative colitis and Crohn disease, afflict 1.4 million people in the United States alone. The exact etiology of IBD remains to be defined, but the host genetic susceptibility, microbiota, and environmental factors play critical roles in the development of the diseases [1]. Nucleotide-binding oligomerization domain (NOD) proteins are members of a large family of proteins named “NOD-like receptors” whose functions are essential in innate/adaptive host responses to various commensal and pathogenic bacteria [2–5]. NOD2 senses the bacterial cell wall component peptidoglycan and its derivative byproduct, muramyl dipeptide [4, 5]. In addition, NOD2 senses live intracellular pathogenic microorganisms such as Salmonella enterica, Listeria monocytogenes, Mycobacterium tuberculosis, and Streptococcus pneumoniae [6–9]. This host response is central to the elimination of the damaging agents and to the reestablishment of homeostasis. For example, defective NOD2 signaling impaired intestinal epithelial cells in their clearance of Salmonella Typhimurium in vitro and decreased host responses to L. monocytogenes infection in vivo [7, 8]. Similarly, NOD2-deficient mice are more susceptible than wild-type mice to S. Typhimurium infection [10]. The molecular mechanism by which NOD2 controls host response to bacteria is unclear, but its role in autophagy and microbial killing has recently been documented in vitro [11, 12]. For example, NOD2-mediated autophagy is required for bacterial handling and antigen presentation in dendritic cells [11]. Importantly, patients with IBD are often subject to a relapsing episode following infection with enteric bacterial pathogens such as Salmonella or Campylobacter species [13]. Campylobacter jejuni is one of the most prevalent food-borne bacterial pathogens in the world. Millions of people are infected with C. jejuni every year in the United States [14]. Clinical features of acute campylobacteriosis include bloody diarrhea, abdominal cramps, and severe intestinal inflammation. Despite the prevalence of campylobacteriosis, its pathogenesis remains largely unknown because of the limited availability of experimental animal models mimicking human disease. Interleukin 10 (IL-10) is secreted by a variety of immune cells, including monocytes/macrophages and T cells, upon stimulation with various agents, such as lipopolysaccharide and bacteria [15]. IL-10 plays an essential role in maintaining intestinal homeostasis by regulating expression of proinflammatory cytokines, chemokines, and costimulatory molecules [16]. Indeed, 129SvEv Il10−/− mice born and raised in specific-pathogen-free (SPF) conditions developed intestinal inflammation after a couple of months but remained healthy when raised in germ-free conditions, showing the key role of bacteria in intestinal inflammation. Interestingly, C57BL/6 Il10−/− mice raised in SPF conditions and infected with C. jejuni strain 11168 displayed mild intestinal inflammation at day 35 after infection [17]. More recently, we reported that germ-free C57BL6/129SvEv and SPF 129 Il10−/− mice infected with C. jejuni 84–176 showed acute campylobacteriosis [18–20]. In addition, Haag et al showed that C. jejuni induces acute intestinal inflammation in C57BL/10 mice pretreated with antibiotics [21]. Using germ-free 129SvEv Il10−/−; Rag2−/− mice, we recently showed that innate immune cells are essential in mediating the early phase of campylobacteriosis [20], suggesting an important role for innate immunity in controlling C. jejuni infection. These findings illustrate the benefit of using Il10−/− mice for studying mechanisms implicated in campylobacteriosis. Reactive nitrogen species (RNS) such as nitric oxide (·NO) and superoxide (O2·−) are a group of antimicrobial intermediate molecules produced by macrophages and are involved in eliminating intercellular and intracellular pathogens [22, 23]. The formation of RNS molecules is mediated by inducible nitric oxide synthase 2 (iNOS) and nicotinamide adenine dinucleotide phosphate oxidase. Interestingly, C. jejuni survival in vitro can be reduced by chemically generated RNS, using an acidified nitrite solution [24]. However, the role of host-generated NO in defense against C. jejuni infection remains unknown. Furthermore, no studies have directly assessed the relationship between NOD2 and RNS in the host response to bacterial infection in vivo. In this study, we investigated the role of NOD2 in C. jejuni–induced intestinal inflammation. Using genetic manipulation, we showed that NOD2 enhances myeloid cell–induced bactericidal capacity and attenuates C. jejuni–induced intestinal inflammation through RNS generation. These findings provide a basis for developing novel approaches with the potential to control campylobacteriosis by targeting NOD2-mediated RNS.