Effects of plant- and animal-based high-fat diets on lipid storage and distribution in environmental bacteria-colonized gnotobiotic mice.

Different edible oils such as lard and soybean oil have been reported to interact with the gut microbiota, affecting host lipid metabolism. However, whether bacteria derived from the environment influence host lipid metabolism remains unclear. This study aimed to clarify the roles of environmental bacteria in host lipid storage and distribution with various edible oils. Gnotobiotic C57BL/6JNarl mice were inoculated with Lysinibacillus xylanilyticus and Paenibacillus azoreducens and then fed either a normal diet (LabDiet 5010, control group) or a diet containing 60% lard (L-group) or soybean oil (S-group) for 18 months. Interestingly, the S-group accumulated massive amounts of white adipose tissue compared to the L- and control groups, while the L-group displayed more hepatic steatosis and fatty droplets than the other groups. The expression of fatty acid synthase (FAS), hydroxymethylglutaryl-coenzyme A reductase (HMGCR), sterol regulatory element-binding protein 2 (SREBP2), and peroxisome proliferator-activated receptor gamma (PPARγ) in the livers of the L-group were markedly elevated compared to the S-group. FAS and PPARγ protein levels were also markedly elevated. However, there were no differences in the expression of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-α between the groups. Our results suggest that environmental bacteria may affect host hepatic inflammation and lipid distribution in the presence of high-fat diets, with different effects depending on the fat type consumed.

Klebsiella pneumoniae translocates across the intestinal epithelium via Rho GTPase- and phosphatidylinositol 3-kinase/Akt-dependent cell invasion.

Klebsiella pneumoniae is an important pathogen that causes hospital-acquired septicemia and is associated with the recent emergence of community-acquired pyogenic liver abscess (PLA). Clinical typing suggests that K. pneumoniae infections originate from the gastrointestinal reservoir. However, the underlying mechanism remains unknown. Here, we have sought to determine how K. pneumoniae penetrates the intestinal barrier. We identified that bacteremia and PLA clinical isolates adhered to and invaded intestinal epithelial cells. Internalization of K. pneumoniae in three different human colonic cell lines was visualized by confocal microscopy and three-dimensional (3D) imaging. Using a Transwell system, we demonstrated that these K. pneumoniae isolates translocated across a polarized Caco-2 monolayer. No disruptions of transepithelial electrical resistance and altered distribution of tight junction protein ZO-1 or occludin were observed. Therefore, K. pneumoniae appeared to penetrate the intestinal epithelium via a transcellular pathway. Using specific inhibitors, we characterized the host signaling pathways involved. Inhibition by cytochalasin D and nocodazole suggested that actin and microtubule cytoskeleton were both important for K. pneumoniae invasion. A Rho inhibitor, ML141, LY294002, and an Akt1/2 inhibitor diminished K. pneumoniae invasion in a dose-dependent manner, indicating that Rho family GTPases and phosphatidylinositol 3-kinase (PI3K)/Akt signaling were required. By a mouse model of gastrointestinal colonization, in vivo invasion of K. pneumoniae into colonic epithelial cells was demonstrated. Our results present evidence to describe a possible mechanism of gastrointestinal translocation for K. pneumoniae. Cell invasion by manipulating host machinery provides a pathway for gut-colonized K. pneumoniae cells to penetrate the intestinal barrier and access extraintestinal locations to cause disease.

Klebsiella pneumoniae peptidoglycan-associated lipoprotein and murein lipoprotein contribute to serum resistance, antiphagocytosis, and proinflammatory cytokine stimulation.

BACKGROUND: Peptidoglycan-associated lipoprotein (Pal), murein lipoprotein (LppA), and outer membrane protein A (OmpA) are dominant outer membrane proteins (OMPs) that are released by gram-negative bacteria during sepsis. OMPs are implicated in the maintenance of cell envelope integrity. Here, we characterize the roles of these OMPs in pathogenesis during bacteremia caused by Klebsiella pneumoniae. METHODS: pal-, lppA-, and ompA-deficient K. pneumoniae strains were constructed using an unmarked deletion method. Serum sensitivity, antiphagocytosis activity, outer membrane permeability, and sensitivity to anionic detergents and antimicrobial polypeptides were determined for these OMP gene deletion mutants. The ability of these OMP gene deletion mutants to induce immune responses was compared with that of the wild-type strain in a bacteremic mouse model. RESULTS: Klebsiella pneumoniae strains deleted for pal or lppA exhibited reduced protection from serum killing and phagocytosis; perturbation to the outer membrane permeability barrier and hypersensitivity to bile salts and sodium dodecyl sulfate. The strain mutated for lppA had reduced ability to activate Toll-like receptor 4. Immunization of mice with the pal or lppA mutant provided protection against infection by the wild-type strain. CONCLUSIONS: Our findings indicate that K. pneumoniae Pal and LppA proteins are important in the maintenance of cell integrity, contribute to virulence, and could be used as attenuated vaccines.

Decolonization of carbapenem-resistant Klebsiella pneumoniae from the intestinal microbiota of model mice by phages targeting two surface structures.

Background: Klebsiella pneumoniae is a normal component of the human gastrointestinal tract microbiota. However, in some cases, it can cause disease. Over the past 20 years, the prevalence of antibiotic-resistant bacteria, such as carbapenem-resistant K. pneumoniae (CRKP), has been increasing. Materials and methods: We attempted to specifically eliminate CRKP from a mouse model with the human intestinal microbiota. To establish humanized microbiota-colonized mice, we administered K64 CRKP-containing human microbiota to germ-free mice by fecal microbiota transplantation. Then, we used two phages, one targeting the capsule (φK64-1) and one targeting O1 lipopolysaccharide (φKO1-1) of K64 K. pneumoniae, to eliminate CRKP. Results: In untreated control and φKO1-1-treated K64-colonized mice, no change in CRKP was observed, while in mice treated with φK64-1, a transient reduction was observed. In half of the mice treated with both φKO1-1 and φK64-1, CRKP was undetectable in feces by PCR and culture for 60 days. However, in the other 50% of the mice, K. pneumoniae was transiently reduced but recovered 35 days after treatment. Conclusion: Combination treatment with φK64-1 and φKO1-1 achieved long-term decolonization in 52.3% of mice carrying CRKP. Importantly, the composition of the intestinal microbiota was not altered after phage treatment. Therefore, this strategy may be useful not only for eradicating drug-resistant bacterial species from the intestinal microbiota but also for the treatment of other dysbiosis-associated diseases.

Circulating gut microbiota-related metabolites influence endothelium plaque lesion formation in ApoE knockout rats.

Atherosclerosis is the main cause of cardiac and peripheral vessel infarction in developed countries. Recent studies have established that gut microbiota and their metabolites play important roles in the development and progression of cardiovascular disease; however, the underlying mechanisms remain unclear. The present study aimed to investigate endothelium plaque lesion formation in ApoE-deficient rats fed a normal chow diet under germ-free (GF) and specific-pathogen-free (SPF) conditions at various time points. There was no difference in serum cholesterol and triglyceride levels between SPF-rats and GF-rats. Histological studies revealed that the GF-rats developed endothelium plaques in the aorta from 26 to 52 weeks, but this was not observed in SPF-rats. GF-rat coronary arteries had moderate-to-severe endothelium lesions during this time period, but SPF-rat coronary arteries had only mild lesion formation. Immunohistochemical staining showed higher accumulation of CD68-positive and arginase-negative foamy-like macrophages on the arterial walls of GF-rats, and expression of TNF-α and IL-6 in foam cells was only observed in GF-rats. In addition, microbial metabolites, including equol derivatives, enterolactone derivatives, indole-3-propionate, indole-3-acrylic acid, cholic acid, hippuric acid, and isoquinolone, were significantly higher in the SPF group than in the GF group. In conclusion, our results indicate that gut microbiota may attenuate atherosclerosis development.

PPP2R2B CAG repeat length in the Han Chinese in Taiwan: Association analyses in neurological and psychiatric disorders and potential functional implications.

PPP2R2B, a protein widely expressed in neurons throughout the brain, regulates the protein phosphatase 2A (PP2A) activity for the microtubule-associated protein tau and other substrates. Altered PP2A activity has been implicated in spinocerebellar ataxia 12, Alzheimer's disease (AD), and other tauopathies. Through a case-control study and a reporter assay, we investigated the association of PPP2R2B CAG repeat polymorphism with Taiwanese AD, essential tremor (ET), Parkinson's disease (PD), and schizophrenia and clarified the functional implication of this polymorphism. The distribution of the alleles was not significantly different between patients and controls, with 68.6-76.1% alleles at lengths of 10, 13, and 16 triplets. No expanded alleles were detected in either group. However, the frequency of the individuals carrying the short 5-, 6-, and 7-triplet alleles was notably higher in patients with AD (5/180 [2.8%], Fisher's exact test, P = 0.003; including 2 homozygotes) and ET (4/132 [3.0%], Fisher's exact test, P < 0.001) than in the controls (1/625 [0.2%]). The PPP2R2B transcriptional activity was significantly lower in the luciferase reporter constructs containing the (CAG)(5-7) allele than in those containing the common 10-, 13-, and 16-triplet alleles in both neuroblastoma and embryonic kidney cells. Therefore, our preliminary results suggest that the PPP2R2B gene CAG repeat polymorphism may be functional and may, in part, play a role in conferring susceptibility to AD and ET in Taiwan.

Gnotobiotic mice inoculated with Firmicutes, but not Bacteroidetes, deteriorate nonalcoholic fatty liver disease severity by modulating hepatic lipid metabolism.

Nonalcoholic fatty liver disease (NAFLD) is a serious liver disorder and characterized by the hepatic accumulation of excess fatty acids. Clinical studies and animal models have shown a shift of gut microbiota from bacteroidetes to firmicutes in NAFLD patients and a diet-induced NAFLD mouse model. Therefore, we hypothesized that these 2 groups of bacteria may have differential effects on lipid metabolism in the liver, which further contributed to pathogenesis of NAFLD. To elucidate these effects, we inoculated two species of Bacteroidetes (B-group) or five species of Firmicutes (F-group) which were isolated from healthy individuals into germ-free mice. We found that the F-group induced elevated body weight, liver weight, and hepatic steatosis compared to the B-group under high-fat diet (HFD) conditions. The mRNA expression level of cluster of differentiation 36 (CD36) was elevated in the F-group compared to that in the B-group. Increased mRNA expression levels of fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD1), and diacylglycerol O-acyltransferase 2 (DGAT2) were also seen under HFD conditions in the F-group compared to that in the B-group. In addition, the expression level of miR802-5p was only elevated in the F-group under HFD conditions. Taken together, our results suggested that these specific species of Firmicutes may induce more hepatic steatosis by modulating fatty acid influx and lipogenesis compared to those of Bacteroidetes. These results may provide more understanding of the effects of gut microbiota on NAFLD.

Antifungal activity of cinnamaldehyde and eugenol congeners against wood-rot fungi.

In this study, the antifungal activities of cinnamaldehyde and eugenol congeners against white-rot fungus Lenzites betulina and brown-rot fungus Laetiporus sulphureus were evaluated and the relationships between the antifungal activity and the chemical structures were also examined. Results from antifungal tests revealed that cinnamaldehyde, alpha-methyl cinnamaldehyde, (E)-2-methylcinnamic acid, eugenol and isoeugenol exhibited strong antifungal activity against all fungi tested. Results derived from the chemical structure-antifungal activity relationship study suggested that compounds with an aldehyde group or an acid group, a conjugated double bond and a length of CH chain outside the ring affect their antifungal properties. Furthermore, the presence of the methyl moiety in the ortho position may have a considerable influence on the inhibitory action against L. betulina and L. sulphureus. In addition, the lipophilicity may play, in part, a crucial role in determining the toxicity of phenylpropenes.

Terminating red imported fire ants using Cinnamomum osmophloeum leaf essential oil.

Eleven compounds from indigenous cinnamon (Cinnamomum osmophloeum) leaf essential oil were identified by GC-MS and the dominant constituent was trans-cinnamaldehyde (79.85%). The toxicity of leaf essential oil and trans-cinnamaldehyde were then determined to study their effectiveness in controlling the red imported fire ant, Solenopsis invicta Buren. The results of the toxicity tests indicated that both the indigenous cinnamon leaf essential oil and trans-cinnamaldehyde had an excellent inhibitory effect in controlling the red imported fire ant. The LT(50) values for both 2% leaf essential oil and 2% trans-cinnamaldehyde after open exposure were 105.0min and 32.2min; after close exposure were 18.5min and 21.2min, respectively.

Nonalcoholic Fatty Liver Disease Is Exacerbated in High-Fat Diet-Fed Gnotobiotic Mice by Colonization with the Gut Microbiota from Patients with Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is a serious liver disorder associated with the accumulation of fat and inflammation. The objective of this study was to determine the gut microbiota composition that might influence the progression of NAFLD. Germ-free mice were inoculated with feces from patients with nonalcoholic steatohepatitis (NASH) or from healthy persons (HL) and then fed a standard diet (STD) or high-fat diet (HFD). We found that the epididymal fat weight, hepatic steatosis, multifocal necrosis, and inflammatory cell infiltration significantly increased in the NASH-HFD group. These findings were consistent with markedly elevated serum levels of alanine transaminase, aspartate transaminase, endotoxin, interleukin 6 (IL-6), monocyte chemotactic protein 1 (Mcp1), and hepatic triglycerides. In addition, the mRNA expression levels of Toll-like receptor 2 (Tlr2), Toll-like receptor 4 (Tlr4), tumor necrosis factor alpha (Tnf-α), Mcp1, and peroxisome proliferator-activated receptor gamma (Ppar-γ) significantly increased. Only abundant lipid accumulation and a few inflammatory reactions were observed in group HL-HFD. Relative abundance of Bacteroidetes and Firmicutes shifted in the HFD-fed mice. Furthermore, the relative abundance of Streptococcaceae was the highest in group NASH-HFD. Nevertheless, obesity-related Lactobacillaceae were significantly upregulated in HL-HFD mice. Our results revealed that the gut microbiota from NASH Patients aggravated hepatic steatosis and inflammation. These findings might partially explain the NAFLD progress distinctly was related to different compositions of gut microbiota.

TLR2 and interleukin-10 are involved in Bacteroides fragilis-mediated prevention of DSS-induced colitis in gnotobiotic mice.

BACKGROUND AND AIMS: Bacteroides fragilis (BF) are Gram-negative anaerobe symbionts present in the colon. Recent studies have reported the beneficial role of BF in maintaining intestinal homeostasis, stimulating host immunologic development, and preventing infectious colitis caused by pathogenic bacteria. Our previous studies showed that monocolonization of germ-free mice with BF significantly reduced colon inflammations and damage. METHODS: In order to investigate the Toll-like receptor-2 (TLR2), TLR4, and interleukin 10 (IL-10) molecular signaling pathways involved in BF-mediated prevention of dextran sulfate sodium (DSS)-induced colitis. The wild-type (WT), TLR4, TLR2, and IL-10 knockout (-/-) germ-free mice grown were with or without BF colonization for 28 days, and then administered 1% DSS in drinking water for 7 day to induce acute ulcerative colitis. RESULTS: We compared phenotypes such as weight loss, disease activity, intestinal histological scores, and immunohistochemistry for inflammatory cells. Unlike WT and TLR4-/- mice, the severity of DSS-colitis did not improve in TLR2-/- animals after BF colonization. The BF enhanced anti-inflammatory cytokines IL-10 expression and inhibited pro-inflammatory-related tumor necrosis factor (TNF-α) and IL-6 mRNA expression in both WT and TLR4-/- mice. In contrast, the failed to up-regulated IL-10 and down-regulated the TNF-α and IL-6 in BF colonization TLR2-/- mice. In addition, we further perform IL-10-/- mice to clarify whether the BF through TLR2 /IL-10 pathway to alleviate DSS-colitis. There were no significant differences in colitis severity and pro-inflammatory related genes expression in the IL-10-/- mice with or without BF colonization. CONCLUSIONS: These results indicate the disease-preventing effects of BF in acute DSS-induced colitis may occur through the TLR2/IL-10 signal pathway.

Chemical polymorphism and antifungal activity of essential oils from leaves of different provenances of indigenous cinnamon (Cinnamomum osmophloeum).

The essential oils isolated from nine geographical provenances of indigenous cinnamon (Cinnamomum osmophloeum Kaneh.) leaves were examined by GC-MS and their chemical constituents were compared. According to GC-MS and cluster analyses the leaf essential oils of the nine provenances and their relative contents were classified into six chemotypes-cinnamaldehyde type, cinnamaldehyde/cinnamyl acetate type, cinnamyl acetate type, linalool type, camphor type and mixed type. In addition, the antifungal activities of leaf essential oils and their constituents from six chemotypes of indigenous cinnamon were investigated in this study. Results from the antifungal tests demonstrated that the leaf essential oils of cinnamaldehyde type and cinnamaldehyde/cinnamyl acetate type had an excellent inhibitory effect against white-rot fungi, Trametes versicolor and Lenzites betulina and brown-rot fungus Laetiporus sulphureus. The antifungal indices of leaf essential oils from these two chemotypes at the level of 200 micro/ml against T. versicolor, L. betulina and L. sulphureus were all 100%. Among them, the IC(50) (50% of inhibitory concentrations) value of the essential oil of cinnamaldehyde type leaf against L. sulphureus was 52-59microg/ml. Cinnamaldehyde possessed the strongest antifungal activities in comparison with other constituents of the essential oils from cinnamaldehyde type leaf, at the level of 100microg/ml its antifungal indices against T. versicolor, L. betulina and L. sulphureus were 100%. The IC50 values of cinnamaldehyde against T. versicolor, L. betulina and L. sulphureus were 73, 74 and 73microg/ml, respectively.

Study on the antiinflammatory activity of essential oil from leaves of Cinnamomum osmophloeum.

The leaf essential oil from indigenous cinnamon (Cinnamomum osmophloeum Kaneh.) was investigated by gas chromatography-mass spectrometry, and 21 compounds were identified. The major constituents of leaf essential oil were the monoterpenes 1,8-cineole (17.0%) and santolina triene (14.2%) and the sesquiterpenes spathulenol (15.7%) and caryophyllene oxide (11.2%). In the antiinflammatory activity assay, we demonstrated that the essential oil has a higher capacity to inhibit proIL-1beta protein expression induced by LPS-treated J774A.1 murine macrophage. At dosages of 60 microg/mL, essential oil clearly inhibited proIL-1beta protein expression. Furthermore, a dose of 60 microg/mL of essential oil was effectively inhibitory for IL-1beta and IL-6 production but not for TNF-alpha, suggesting that essential oil was bioactive in antiinflammation in vitro. This study is the first to report antiinflammatory activity of extracts obtained from the leaf essential oil of C. osmophloeum.

Chemical composition and mosquito larvicidal activity of essential oils from leaves of different Cinnamomum osmophloeum provenances.

Chemical compositions of leaf essential oils from eight provenances of indigenous cinnamon (Cinnamomum osmophloeum Kaneh.) were compared. According to GC-MS and cluster analyses, the leaf essential oils of the eight provenances and their relative contents were classified into five chemotypes-cinnamaldehyde type, linalool type, camphor type, cinnamaldehyde/cinnamyl acetate type, and mixed type. The larvicidal activities of leaf essential oils and their constituents from the five chemotypes of indigenous cinnamon trees were evaluated by mosquito larvicidal assay. Results of larvicidal tests demonstrated that the leaf essential oils of cinnamaldehyde type and cinnamaldehyde/cinnamyl acetate type had an excellent inhibitory effect against the fourth-instar larvae of Aedes aegypti. The LC(50) values for cinnamaldehyde type and cinnamaldehyde/cinnamyl acetate type against A. aegypti larvae in 24 h were 36 ppm (LC(90) = 79 ppm) and 44 ppm (LC(90) = 85 ppm), respectively. Results of the 24-h mosquito larvicidal assays also showed that the effective constituents in leaf essential oils were cinnamaldehyde, eugenol, anethole, and cinnamyl acetate and that the LC(50) values of these constituents against A. aegypti larvae were <50 ppm. Cinnamaldehyde had the best mosquito larvicidal activity, with an LC(50) of 29 ppm (LC(90) = 48 ppm) against A. aegypti. Comparisons of mosquito larvicidal activity of cinnamaldehyde congeners revealed that cinnamaldehyde exhibited the strongest mosquito larvicidal activity.

Monocolonization of germ-free mice with Bacteroides fragilis protects against dextran sulfate sodium-induced acute colitis.

Ulcerative colitis is inflammatory conditions of the colon caused by interplay of genetic and environmental factors. Previous studies indicated that the gut microflora may be involved in the colonic inflammation. Bacteroides fragilis (BF) is a Gram-negative anaerobe belonging to the colonic symbiotic. We aimed to investigate the protective role of BF in a colitis model induced in germ-free (GF) mice by dextran sulfate sodium (DSS). GF C57BL/6JNarl mice were colonized with BF for 28 days before acute colitis was induced by DSS. BF colonization significantly increased animal survival by 40%, with less reduction in colon length, and decreased infiltration of inflammatory cells (macrophages and neutrophils) in colon mucosa following challenge with DSS. In addition, BF could enhance the mRNA expression of anti-inflammatory-related cytokine such as interleukin 10 (IL-10) with polymorphism cytokine IL-17 and diminish that of proinflammatory-related tumor necrosis factor α with inducible nitric oxide synthase in the ulcerated colon. Myeloperoxidase activity was also decreased in BF-DSS mice. Taking these together, the BF colonization significantly ameliorated DSS-induced colitis by suppressing the activity of inflammatory-related molecules and inducing the production of anti-inflammatory cytokines. BF may play an important role in maintaining intestinal immune system homeostasis and regulate inflammatory responses.

Insecticidal activities of leaf essential oils from Cinnamomum osmophloeum against three mosquito species.

The larvicidal activities of leaf essential oils and their constituents from six chemotypes of indigenous cinnamon (Cinnamomum osmophloeum Kaneh.) trees were evaluated against three mosquito species. Results of larvicidal tests demonstrated that the leaf essential oils of cinnamaldehyde type and cinnamaldehyde/cinnamyl acetate type had an excellent inhibitory effect against Aedes albopictus larvae, and their LC(50) values in 24h were 40.8 microg/ml (LC(90)=81.7 microg/ml) and 46.5 microg/ml (LC(90)=83.3 microg/ml), respectively. Results of the 24-h mosquito larvicidal assays also showed that the effective constituents in leaf essential oils were trans-cinnamaldehyde and benzaldehyde and that the LC(50) values of these constituents against A. albopictus larvae were below 50 mug/ml. In addition, cinnamaldehyde type leaf essential oil and trans-cinnamaldehyde have also exhibited great larvicidal performance against Culex quinquefasciatus and Armigeres subalbatus larvae. Comparisons of mosquito larvicidal activity of trans-cinnamaldehyde congeners revealed that alpha-methyl cinnamaldehyde, benzaldehyde, and trans-cinnamaldehyde exhibited strong mosquito larvicidal activity.

Polysaccharides from Dioscorea batatas induce tumor necrosis factor-alpha secretion via Toll-like receptor 4-mediated protein kinase signaling pathways.

The bioactive polysaccharides (named ZPF1) from yam ( Dioscorea batatas) were chemically determined, suggesting repeating beta-1,4-mannan as mainly having a feature of acetylation on C2-OH and C3-OH, around 28%. The ZPF1 participated in the stimulation of murine wild-type macrophages predominantly in tumor necrosis factor-alpha (TNFalpha). Toll-like receptor 4 is proved to be one of the cellular receptors in ZPF1-mediated TNFalpha secretion. Reactive oxygen species transmission and PI3-kinase are found necessary for regulating TNFalpha secretion by ZPF1 stimulation. Moreover, we found that extracellular signal-regulated kinase 1/2, Jun N-terminal kinase 1/2, and p38 mitogen-activated protein kinase play important roles in the regulation of TNFalpha secretion in ZPF1-stimulated macrophages.