Characterization of Gut Microbiota Compositions along the Intestinal Tract in CD163/pAPN Double Knockout Piglets and Their Potential Roles in Iron Absorption.

The gut microbiota is known to play a role in regulating host metabolism, yet the mechanisms underlying this regulation are not well elucidated. Our study aimed to characterize the differences in gut microbiota compositions and their roles in iron absorption between wild-type (WT) and CD163/pAPN double-gene-knockout (DKO) weaned piglets. A total of 58 samples along the entire digestive tract were analyzed for microbial community using 16S rRNA gene sequencing. The colonic microbiota and their metabolites were determined by metagenomic sequencing and untargeted liquid chromatography-mass spectrometry (LC-MS), respectively. Our results showed that no alterations in microbial community structure and composition were observed between DKO and WT weaned piglets, with the exception of colonic microbiota. Interestingly, the DKO piglets had selectively increased the relative abundance of the Leeia genus belonging to the Neisseriaceae family and decreased the Ruminococcaceae_UCG_014 genus abundance. Functional capacity analysis showed that organic acid metabolism was enriched in the colon in DKO piglets. In addition, the DKO piglets showed increased iron levels in important tissues compared with WT piglets without any pathological changes. Pearson's correlation coefficient indicated that the specific bacteria such as Leeia and Ruminococcaceae_UCG_014 genus played a key role in host iron absorption. Moreover, the iron levels had significantly (P < 0.05) positive correlation with microbial metabolites, particularly carboxylic acids and their derivatives, which might increase iron absorption by preventing iron precipitation. Overall, this study reveals an interaction between colonic microbiota and host metabolism and has potential significance for alleviating piglet iron deficiency. IMPORTANCE Iron deficiency is a major risk factor for iron deficiency anemia, which is among the most common nutritional disorders in piglets. However, it remains unclear how the gut microbiota interacts with host iron absorption. The current report provides the first insight into iron absorption-microbiome connection in CD163/pAPN double knockout piglets. The present results showed that carboxylic acids and their derivatives contributed to the absorption of nonheme iron by preventing ferric iron precipitation.

Multi-Omic Analysis in a Metabolic Syndrome Porcine Model Implicates Arachidonic Acid Metabolism Disorder as a Risk Factor for Atherosclerosis.

Background: The diet-induced gut microbiota dysbiosis has been suggested as a major risk factor for atherothrombosis, however, the detailed mechanism linking these conditions is yet to be fully understood. Methods: We established a long-term excessive-energy diet-induced metabolic syndrome (MetS) inbred Wuzhishan minipig model, which is characterized by its genetic stability, small size, and human-like physiology. The metabolic parameters, atherosclerotic lesions, gut microbiome, and host transcriptome were analyzed. Metabolomics profiling revealed a linkage between gut microbiota and atherothrombosis. Results: We showed that white atheromatous plaque was clearly visible on abdominal aorta in the MetS model. Furthermore, using metagenome and metatranscriptome sequencing, we discovered that the long-term excessive energy intake altered the local intestinal microbiota composition and transcriptional profile, which was most dramatically illustrated by the reduced abundance of SCFAs-producing bacteria including Bacteroides, Lachnospiraceae, and Ruminococcaceae in the MetS model. Liver and abdominal aorta transcriptomes in the MetS model indicate that the diet-induced gut microbiota dysbiosis activated host chronic inflammatory responses and significantly upregulated the expression of genes related to arachidonic acid-dependent signaling pathways. Notably, metabolomics profiling further revealed an intimate linkage between arachidonic acid metabolism and atherothrombosis in the host-gut microbial metabolism axis. Conclusions: These findings provide new insights into the relationship between atherothrombosis and regulation of gut microbiota via host metabolomes and will be of potential value for the treatment of cardiovascular diseases in MetS.

Enantioselective H-bond-directed vinylogous iminium ion strategy for the functionalization of vinyl-substituted heteroaryl aldehydes.

In this work the first H-bond-directed vinylogous iminium ion strategy has been developed as a convenient strategy for the γ,δ-functionalization of vinyl-substituted heteroaromatic aldehydes. Their reaction with α-mercaptoketones proceeds in a cascade manner involving 1,6-addition followed by intramolecular aldol reaction. Excellent stereoselectivities have been obtained as a result of the H-bond interactions controlling the outcome of the cyclization step. The application of the strategy for the synthesis of tricyclic compounds bearing furan, tetrahydrothiophene and dihydropyran moieties has also been demonstrated.

Doubly vinylogous and doubly rearomative functionalization of 2-alkyl-3-furfurals.

The manuscript describes a straightforward functionalization of 2-alkyl-3-furfurals via simple aminocatalytic conjugate addition. The reaction proceeds through the formation of dearomatized dienamine-like intermediate that undergoes 1,6-addition to 4-alkylidene-2,6-dialkylcyclohexa-2,5-dienones. This process can be described as doubly rearomative as it proceeds with the re-formation of both furan and phenyl aromatic moieties. Target products have been obtained in a highly stereoselective manner, providing an interesting example of 2-alkyl-3-furfural functionalization via doubly vinylogous Michael addition. The mechanism of the reaction has been studied by means of computational methods.

Whole-exome sequencing identified genetic risk factors for asparaginase-related complications in childhood ALL patients.

Allergy, pancreatitis and thrombosis are common side-effects of childhood acute lymphoblastic leukemia (ALL) treatment that are associated with the use of asparaginase (ASNase), a key component in most ALL treatment protocols. Starting with predicted functional germline variants obtained through whole-exome sequencing (WES) data of the Quebec childhood ALL cohort we performed exome-wide association studies with ASNase-related toxicities. A subset of top-ranking variants was further confirmed by genotyping (N=302) followed by validation in an independent replication group (N=282); except for thrombosis which was not available for that dataset. SNPs in 12 genes were associated with ASNase complications in discovery cohort including 3 that were associated with allergy, 3 with pancreatitis and 6 with thrombosis. The risk was further increased through combined SNPs effect (p≤0.002), suggesting synergistic interactions between the SNPs identified in each of the studied toxicities. Interestingly, rs3809849 in the MYBBP1A gene was associated with allergy (p= 0.0006), pancreatitis (p=0.002), thrombosis (p=0.02), event-free survival (p=0.02) and overall survival (p=0.003). Furthermore, rs11556218 in IL16 and rs34708521 in SPEF2 were both associated with thrombosis (p=0.01 and p=0.03, respectively) and pancreatitis (p=0.02). The association of SNPs in MYBBP1A, SPEF2 and IL16 geneswith pancreatitis was replicated in the validation cohort (p ≤0.05) as well as in combined cohort (p=0.0003, p=0.008 and p=0.02, respectively). The synergistic effect of combining risk loci had the highest power to predict the development of pancreatitis in both cohorts and was further potentiated in the combined cohort (p=1x10-8).The present work demonstrates that using WES data is a successful "hypothesis-free" strategy for identifying significant genetic markers modulating the effect of the treatment in childhood ALL.

[Construction of BAC Library of Fagopyrum tataricum and Analysis of BAC End Sequences].

Objective: To further study Fagopyrum tataricum genome and to screen the functional genes. Methods: The variety JINQIAO No. 2( Fagopyrum tataricum) native to Shanxi, was used for BAC library construction. The high molecular weight DNA( HMWDNA) was isolated from Fagopyrum tataricum leaves used the methods of Peterson. The HMW-DNA was cut by Hind Ⅲ and ligated to BAC vectors; the ligations were transformed into Escherichia coli DH10 B. Then 48 BAC clones were randomly selected and sequenced BAC end sequences. Results: The library consisted of 30 000 clones with an average insert size of about 123 kb and the empty clone ratio was less than 1%. The library represented an equivalent about 6. 9 fold size of Fagopyrum tataricum genome. Which obtained 89 BAC end sequences,among which 7 sequences( 8%) had alignment results when comparing with NCBI Gen Bank database. The blast hits contained some genes with known function,such as rpo Tm2,Trrap and MDR1 genes,etc. Those genes were associated with DNA binding, transmembrane transport and phosphotransferase activity function. And it was also found that the sequence of 40G19-F was probably repetitive sequences in Fagopyrum tataricum genome. Conclusion: The BAC library will be helpful for the gene cloning and whole genome sequencing of Fagopyrum tataricum.

Inhibition of the Mitochondrial Protease ClpP as a Therapeutic Strategy for Human Acute Myeloid Leukemia.

From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.

Comparison of ginsenosides Rg1 and Rb1 for their effects on improving scopolamine-induced learning and memory impairment in mice.

Rg1 and Rb1 are two major active compounds of ginseng that facilitate learning and memory. The present study aimed to compare the nootropic effects of Rg1 and Rb1 in a scopolamine induced dementia mice model. After 6 and 12 mg/kg of Rg1 and Rb1 intraperitoneal administration to mice for 7 days, their effects were assessed using the step-down passive avoidance (SD) and the Morris water maze (MWM) tests, the acetylcholinesterase (AChE) activity, acetylcholine (ACh) content and serotonin (5-HT) level in the hippocampus were analysed after SD and MWM tests. The results showed that Rg1 and Rb1 ameliorated cognition-deficiency in mice with dementia. Rg1 showed stronger effects than Rb1 on escape acquisition in MWM. Both Rg1 and Rb1 increased ACh levels in the hippocampus, but Rg1 inhibited AChE activity while Rb1 had no effect on AChE activity. Both Rg1 and Rb1 inhibited the decrease of 5-HT induced by scopolamine, but Rb1 was more active than the same dose of Rg1. These results demonstrate that multiple administrations of Rg1 and Rb1 are effective in improving memory deficiency induced by scopolamine. Rg1 appears to be more potent than Rb1 in improving acquisition impairment, and the two ginsenosides may act through different mechanisms.

[Inhibitory Effect of CCL20 on CD4+ CD25+ regulatory T cell development in mouse thymus].

The aim of this study was to investigate the roles of chemokine CCL20 in development of CD4(+)CD25(+) thymocytes by means of fetal thymus organ culture. Fetal mouse thymus lobes were removed at the fetus age of 14.5 days and cultured in complete RPMI 1640 with 20% FBS in vitro. Phenotypes of the thymocytes were analyzed by FACS and the number of cells per lobe was counted. The results revealed that from day 14.5 to day 19, the absolute and relative numbers of the CD4(+)CD25(+) thymocytes varied similarly as their development as in vitro culture at 6 days. Data showed that during the 6 days in vitro culture the CD4(+)CD25(+) cell percentage out of CD4(+) cells was 58.29%, 12.14%, 6.08%, 17.78%, 9.06%, 4.04% and the CD4(+)CD25(+) cell percentage out of CD25(+) cells was 3.75%, 10.81%, 17.20%, 51.93%, 61.64%, 80.06%. All these data indicated similar characters to their development in vivo. Moreover, at interference with CCL20, the percentage of CD4(+)CD25(+) T cells in thymocytes significantly decreased at the 3 and 6 days from 3.24+/-0.18 and 3.96+/-0.24 to 1.27+/-0.11 (p<0.001) and 1.76+/-0.22 (p<0.001) respectively. It is concluded that the development of CD4(+)CD25(+) thymocytes is similar both in vitro and in vivo, interfering with CCL20 significantly downregulate the expression of CD4(+)CD25(+) T cells. The above data may help to understand the development of naturally arising CD4(+)CD25(+) regulatory T cells.

Modulation of activator protein-1 (AP-1) and MAPK pathway by flavonoids in human prostate cancer PC3 cells.

In last couple of decades the use of natural compounds like flavonoids as chemopreventive agents has gained much attention. Our current study focuses on identifying chemopreventive flavonoids and their mechanism of action on human prostate cancer cells. Human prostate cancer cells (PC3), stably transfected with activator protein 1 (AP-1) luciferase reporter gene were treated with four main classes of flavonoids namely flavonols, flavones, flavonones, and isoflavones. The maximum AP-1 luciferase induction of about 3 fold over control was observed with 20 microM concentrations of quercetin, chrysin and genistein and 50 microM concentration of kaempferol. At higher concentrations, most of the flavonoids demonstrated inhibition of AP-1 activity. The MTS assay for cell viability at 24 h showed that even at a very high concentration (500 microM), cell death was minimal for most of the flavonoids. To determine the role of MAPK pathway in the induction of AP-1 by flavonoids, Western blot of phospho MAPK proteins was performed. Four out of the eight flavonoids namely kaempferol, apigenin, genistein and naringenin were used for the Western Blot analysis. Induction of phospho-JNK and phospho-ERK activity was observed after two hour incubation of PC3-AP1 cells with flavonoids. However no induction of phospho-p38 activity was observed. Furthermore, pretreating the cells with specific inhibitors of JNK reduced the AP-1 luciferase activity that was induced by genistein while pretreatment with MEK inhibitor reduced the AP-1 luciferase activity induced by kaempferol. The pharmacological inhibitors did not affect the AP-1 luciferase activity induced by apigenin and naringenin. These results suggest the possible involvement of JNK pathway in genistein induced AP-1 activity while the ERK pathway seems to play an important role in kaempferol induced AP-1 activity.

Induction of liver cytochrome P450 1A2 expression by flutamide in rats.

AIM: To investigate the modulation of liver cytochrome P450 1A2 (CYP1A2) expression by giving flutamide to adult rats. METHODS: Rats were given 50, 100, and 200 mg/kg po of flutamide for 2 weeks. Liver CYP1A2 mRNA was measured using reverse transcription-polymerase chain reaction. CYP1A2 protein was detected using immunoblotting. CYP1A2 activity was assayed using high performance liquid chromatography, with caffeine as the CYP1A2 substrate. RESULTS: CYP1A2 mRNA levels after flutamide treatment at 100 mg/kg and 200 mg/kg were, respectively, 1.86 and 3.11-fold higher than those of the control. Correspondingly, CYP1A2 protein increased 1.78 and 2.89-fold and CYP1A2 activity increased approximately 1.65 and 2.83-fold, respectively, relative to controls. Flutamide treatment at 50 mg/kg had no significant effect on CYP1A2 mRNA, protein, or enzyme activity. CONCLUSION: Giving rats flutamide induced liver CYP1A2 expression in a dose-dependent manner.

Pharmacokinetic difference between S-(+)- and R-(-)-etodolac in rats.

AIM: To study whether etodolac enantiomers have pharmacokinetic difference after oral administration. METHODS: Fourteen rats, divided into two groups randomly, were orally given S-(+)- or R-(-)-etodolac at a single dose of 20 mg/kg, respectively. Blood samples were collected before and at 5, 10, 20, 30 min and 1, 3, 6, 12, 24, 48, 72 h after treatment. The plasma samples were analyzed with a high-performance liquid chromatographic method. RESULTS: The calibration curves were linear in the range of 0.5-50.0 mg/L (r=0.9999) to S-(+)-etodolac and 2.0-200.0 mg/L (r=0.9999) to R-(-)-etodolac, respectively. The main pharmacokinetic parameters of S-(+)- and R-(-)-etodolac were as follows: t1/2(lambdaz) 18+/-4 h vs 19.4+/-2.2 h, tmax 3.3+/-2.6 h vs 4+/-4 h; Cmax 29+/-6 mg/L vs 97+/-14 mg/L, AUC0-t 706+/-100 h.mg.L(-1) vs 2940+/-400 h mg.L(-1), CL(s) 0.030+/-0.006 L.kg(-1).h(-1) vs 0.0065+/-0.0010 L.kg(-1).h(-1) and V/F 0.25+/-0.22 L.kg(-1) vs 0.03+/-0.05 L.kg(-1). There was no significant difference in t1/2(lambdaz), and tmax between S-(+)- and R-(-)-etodolac (P>>0.05). The Cmax, and AUC0-t of R-(-)-etodolac were markedly higher (P<0.05), while the CL(s) and V/F were markedly lower than those of S-etolodac (P<0.05). CONCLUSION: There is pharmacokinetic difference between S-(+)- and R-(-)- etodolac enantiomers in rats after oral administration.

[Clinical features of liver failure in children].

OBJECTIVE: To analyze the etiology, clinical and laboratory characteristics of hepatic failure in 105 children. METHODS: The clinical data of 105 children with hepatic failure treated in our hospital from January 1986 to June 2003 were retrospectively analyzed by EXCELL 2000 and t test. RESULTS: (1)Of the 105 children with hepatic failure, 9 were cases with fulminant hepatic failure, 38 with subacute hepatic failure and 58 with chronic hepatic failure. (2)Morbidity was the highest in 7-12 years old children (43/105, 41.0%) followed by infants (30/105, 28.6%). (3)CMV infection could be confirmed in 9 infants (30.0%), etiological diagnosis was not possible in 13 infants (43.3%). Etiological diagnosis could be confirmed in children over 1 year of age, which included hepatitis B (n=22, 29.3%), Wilson's disease (n=15, 20.0%), hepatitis A (n=10,13.3%). Etiology in 21 cases (28.0%) could not be confirmed. (4)Seventy-one cases (67.6%) had ascites, 34 of them (47.9%) had spontaneous peritonitis. Thirty-five cases were complicated with other infections. The commonest complication was pulmonary infection and sepsis was the next. Fifty-one cases (48.6%) had hydroelectrolyte imbalance. Forty-eight cases (46.2%) had hepatic encephalopathy, which may be subclinical in children under three years of age. (5)The incidence of hypoglycemia was 77.2%(71/92). CONCLUSION: The etiology of liver failure was related to age. CMV infection was the commonest in infants. HBV, HAV infection was the commonest in children over 1 year of age and Wilson?s disease was the next. It is necessary to prevent and manage the associated complications as early as possible such as spontaneous peritonitis, hepatic encephalopathy, hydroelectrolyte imbalance and hypoglycemia etc.

[Sex-difference on flutamide metabolism in rat liver microsomal cytochrome P450 1A2].

AIM: To assess the sex-difference on flutamide metabolism in rat liver microsomes useing rat cytochrome P450, 1A2, inhibitory monoclonal antibody. METHODS: Liver microsomes were prepared from male or female rats. Protein concentration and total cytochrome P450 content were determined. Incubation mixture included liver microsomes (1.0 nmol.L-1), reduced form of nicotinamide adenine dinucleotide phosphate (NADPH, 0.1 nmol.L-1), CYP1A2 (1:400) and flutamide (2 mg.L-1). The incubation time was 30 min. The concentration of flutamide and its major metabolite 2-hydroxyflutamide were analyzed by reverse high-performance liquid chromatography. The mobile phase was a mixture of methanol-acetonitrile-water-diethylether (40:20:35:1) with methyltestosterone as internal standard. The detection wavelength was 234 nm. The reaction mixture was extracted with acetic ether 4 mL. Sex-difference on flutamide metabolism was expressed as the difference between the concentration ratio of 2-hydroxyflutamide to flutamide in male and female rat liver microsomes. RESULTS: The recoveries of flutamide and 2-hydroxyflutamide for the proposed method were more than 75%. The formation of 2-hydroxyflutamide from flutamide was inhibited by CYP1A2 antibodies (1:400) in male and female rat liver microsome for 30 min of incubation time, but the inhibition of flutamide metabolism in female rat was stronger than that in male. The concentration ratios of 2-hydroxyflutamide to flutamide were (1.5 +/- 0.6) and (0.9 +/- 0.4) in male and female rat liver microsomes, respectively (P < 0.01). CONCLUSION: The results indicate that the activity of male rat CYP1A2 is higher than that of the female rat. There is difference in sex-related rate of flutamide metabolism in rat liver microsomes.