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Congenital cleft palate causes a serious obstacle to children with regard to language and eating function. The aim of the current study was to examine the clinical application of a type of palatoplasty that has a reduced impact on the maxillary growth and good function in velopharyngeal competence. A total of 37 patients with cleft palate were treated with levator veli palatini retropositioning combined with Buccinator myomucosal island flap. The patients were successfully treated in the first phase and were followed up for 1-3 years. Speech intelligibility was satisfactory and no fistula occurred. In conclusion, the results suggested that levator veli palatini retropositioning combined with the Buccinator myomucosal island flap may restore normal anatomic structure and location of the levator veli palatini , obtain good velopharyngeal competence, and decrease the incidence rate thereof. Thus, levator veli palatini retropositioning combined with the Buccinator myomucosal island flap is a functional procedure for cleft palate repair.

Aim: To investigate Fusobacterium nucleatum (F. nucleatum) abundance in colorectal cancer (CRC) tissues and its association with CRC invasiveness in Chinese patients., Methods: The resected cancer and adjacent normal tissues (10 cm beyond cancer margins) from 101 consecutive patients with CRC were collected. Fluorescent quantitative polymerase chain reaction (FQ-PCR) was applied to detect F. nucleatum in CRC and normal tissues. The difference of F. nucleatum abundance between cancer and normal tissues and the relationship of F. nucleatum abundance with clinical variables were evaluated. Fluorescence in situ hybridization (FISH) analysis was performed on 22 CRC tissues with the highest F. nucleatum abundance by FQ-PCR testing to confirm FQ-PCR results., Results: The median abundance of F. nucleatum in CRC tissues [0.242 (0.178-0.276)] was significantly higher than that in normal controls [0.050 (0.023-0.067)] (P < 0.001). F. nucleatum was over-represented in 88/101 (87.1%) CRC samples. The abundance of F. nucleatum determined by 2(-ΔCT) was significantly greater in tumor samples [0.242 (0.178, 0.276)] than in normal controls [0.050 (0.023, 0.067)] (P < 0.001). The frequency of patients with lymph node metastases was higher in the over-abundance group [52/88 (59.1%)] than in the under-abundance group [0/13 (0%)] (P < 0.005). No significant association of F. nucleatum with other clinico-pathological variables was observed (P > 0.05). FISH analysis also found more F. nucleatum in CRC than in normal tissues (median number 6, 25(th) 3, 75(th) 10 vs 2, 25(th) 1, 75(th) 5) (P < 0.01)., Conclusion: F. nucleatum was enriched in CRC tissues and associated with CRC development and metastasis.

Background & Aims: The pathways regulating liver regeneration have been extensively studied within the liver. However, the signaling contribution derived from the gut microbiota to liver regeneration is poorly understood., Methods: Microbiota and expression of hepatic genes in regenerating livers obtained from mice at 0h to 9days post 2/3 partial hepatectomy were temporally profiled to establish their interactive relationships., Results: Partial hepatectomy led to rapid changes in gut microbiota that was reflected in an increased abundance of Bacteroidetes S24-7 and Rikenellaceae and decreased abundance of Firmicutes Clostridiales, Lachnospiraceae, and Ruminococcaceae. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was used to infer biological functional changes of the shifted microbiota. RNA-sequencing data revealed 6125 genes with more than a 2-fold difference in their expression levels during regeneration. By analyzing their expression pattern, six uniquely expressed patterns were observed. In addition, there were significant correlations between hepatic gene expression profiles and shifted bacterial populations during regeneration. Moreover, hepatic metabolism and immune function were closely associated with the abundance of Ruminococcacea, Lachnospiraceae, and S24-7. Bile acid profile was analyzed because bacterial enzymes produce bile acids that significantly impact hepatocyte proliferation. The data revealed that specific bacteria were closely associated with the concentration of certain bile acids and expression of hepatic genes., Conclusions: The presented data established, for the first time, an intimate relationship between intestinal microbiota and the expression of hepatic genes in regenerating livers., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Fibrosis is the result of the abnormal accumulation of the extracellular matrix and ineffective clearance of fibroplasia. CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are immunosuppressive lymphocytes that are highly expressed in the fibrotic tissues and peripheral blood of patients with cirrhosis or hepatocellular carcinoma. The role of Tregs in the progression of liver fibrosis is not well understood. Our experiments reveal that abundant of Tregs was scattered around sites of fibroplasia. Conversely, the depletion of Tregs promoted the resolution of liver fibrosis. As a consequence of Tregs depletion, the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) was altered; mmp9 and timp1 were reduced, whereas mmp2 and mmp14 were enhanced. The mmp9/timp1, mmp13/timp1, and mmp14/timp2 ratios were significantly increased in association with fibrosis resolution. Kupffer cells (KCs) are the main source of MMP. We observed that when KCs were cocultured with Tregs, the Tregs were able to inhibit MMP expression of KCs even at a low ratio; and anti-transforming growth factor-β (TGF-β) significantly reversed the inhibition of Tregs on MMP. Meanwhile, we also found that after Tregs depletion, TGF-β levels decreased in the mice liver, unlike in fibrosis. Furthermore, double depletion of both KCs and Tregs did not cause fiber resolution in mice. Thus, our results demonstrate that the persistence of liver cirrhosis is maintained by increased Tregs in the sites of fibroplasia and the subsequent regulation of the MMP/TIMP balance and that the suppression of KC-mediated MMP expression contributed to the regulatory process., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Genomic imprinting is an important epigenetic mechanism that has vital effects on fetal growth and development. We observed the differences in four tissues (heart, spleen, liver, and kidney) from dead transgenic cloned goats using hematoxylin and eosin (H&E) staining. Eight imprinted genes in the tissues of dead transgenic cloned and normal goats were analyzed using reverse transcription polymerase chain reaction. H&E staining results from the abortion group indicated the lack of obvious morphological changes in heart and spleen tissues, while inflammatory cell infiltration and glomerular nephritis characteristics were observed in liver and kidney tissues, respectively. Compared to the control group, CDKN1C, H19, IGF2R, and SNRPN were significantly (P < 0.05) overexpressed in the heart tissue of the abortion group, while XIST was significantly reduced. In the liver tissues, CDKN1C and DLK1 expression decreased, while GNAS, H19, IGF2R, PEG3, and XIST expression increased significantly. In the spleen tissues, DLK1 expression increased, while GNAS, H19, IGF2R, PEG3, SNRPN, and XIST expression decreased. In the kidney tissues, CDKN1C, DLK1, GNAS, IGF2R, and PEG3 expression increased, while H19 and XIST expression decreased. The overall expression of imprinted genes was abnormal in different tissues of transgenic cloned goats, and the degree of abnormal genomic imprinting was more severe in the abortion group compared to the death and control groups. These results suggest that abnormal expression of imprinted genes may cause developmental defects in transgenic cloned goats. Moreover, abnormal epigenetic modifications may affect the reprogramming of transgenic donor cells.

Background & Aims: All-trans Retinoic acid (RA) regulates hepatic lipid and bile acid homeostasis. Similar to bile acid (BA), RA accelerates partial hepatectomy (PHx)-induced liver regeneration. Because there is a bidirectional regulatory relationship between gut microbiota and BA synthesis, we examined the effect of RA in altering the gut microbial population and BA composition and established their relationship with hepatic biological processes during the active phases of liver regeneration., Methods: C57BL/6 mice were treated with RA orally followed by 2/3 PHx. The roles of RA in shifting gut microbiota and BA profiles as well as hepatocyte metabolism and proliferation were studied., Results: RA-primed mice exhibited accelerated hepatocyte proliferation revealed by higher numbers of Ki67-positive cells compared to untreated mice. Firmicutes and Bacteroidetes phyla dominated the gut microbial community (>85%) in both control and RA-primed mice after PHx. RA reduced the ratio of Firmicutes to Bacteroidetes, which was associated with a lean phenotype. Consistently, RA-primed mice lacked transient lipid accumulation normally found in regenerating livers. In addition, RA altered BA homeostasis and shifted BA profiles by increasing the ratio of hydrophilic to hydrophobic BAs in regenerating livers. Accordingly, metabolic regulators fibroblast growth factor 21, Sirtuin1, and their downstream targets AMPK and ERK1/2 were more robustly activated in RA-primed than unprimed regenerating livers., Conclusions: Priming mice with RA resulted in a lean microbiota composition and hydrophilic BA profiles, which were associated with facilitated metabolism and enhanced cell proliferation.

Unlabelled: This study aimed to elucidate the associations between interleukin-4 (IL-4) single nucleotide polymorphisms (SNPs), 590C/T and 589C/T, serum IL-4 levels, and atopic dermatitis (AD) in children., Methods: A total of 82 children with AD were randomly selected as the case group and divided into mild group (15 cases), moderate group (46 cases), and severe group (21 cases). Additionally, 100 healthy children were selected as the control group. Genotype frequencies of IL-4 SNPs were detected by PCR-RFLP. Serum IL-4 levels were measured by ELISA., Results: Significant differences were shown in genotype distributions and allele frequencies of 589C/T and allele frequencies of 590C/T (all P < 0.05). Serum IL-4 levels in the mild, moderate, and severe groups were significantly higher than those in the control group; significant differences were found among these three groups with increased severity of AD. Serum IL-4 levels of heterozygote and mutant homozygote carriers in the mild, moderate, and severe groups were higher than wild homozygote carriers in those three groups and the control group (all P < 0.05)., Conclusion: 590T and 589T alleles of IL-4 gene may be associated with high levels of serum IL-4, which may increase the risk of AD in children.

Studies examining the mechanisms by which the liver incurs injury and then regenerates usually focus on factors and pathways directly within the liver, neglecting the signaling derived from the gut-liver axis. The intestinal content is rich in microorganisms as well as metabolites generated from both the host and colonizing bacteria. Through the gut-liver axis, this complex "soup" exerts an immense impact on liver integrity and function. This review article summarizes data published in the past 30 years demonstrating the signaling derived from the gut-liver axis in relation to liver injury and regeneration. Due to the intricate networks of implicated pathways as well as scarcity of available mechanistic data, it seems that nutrigenomic, metabolomics, and microbiota profiling approaches are warranted to provide a better understanding regarding the interplay and impact between nutrition, bacteria, and host response in influencing liver function and healing. Therefore elucidating the possible molecular mechanisms that link microbiota alteration to host physiological response and vice versa., (Published by Elsevier B.V.)

Dairy goat is a good model for production of transgenic proteins in milk using somatic cell nuclear transfer (SCNT). However, animals produced from SCNT are often associated with lung deficiencies. We recently produced six transgenic cloned dairy goats harboring the human lactoferrin gene, including three live transgenic clones and three deceased transgenic clones that died from respiratory failure during the perinatal period. Imprinted genes are important regulators of lung growth, and may be subjected to faulty reprogramming. In the present study, first, microsatellite analysis, PCR, and DNA sequence identification were conducted to confirm that these three dead kids were genetically identical to the transgenic donor cells. Second, the CpG island methylation profile of the imprinted insulin-like growth factor receptor (IGF2R) gene was assessed in the lungs of the three dead transgenic kids and the normally produced kids using bisulfite sequencing PCR. In addition, the relative mRNA level of IGF2R was also determined by real-time PCR. Results showed that the IGF2R gene in the lungs of the dead cloned kids showed abnormal hypermethylation and higher mRNA expression levels than the control, indicating that aberrant DNA methylation reprogramming is one of the important factors in the death of transgenic cloned animals.

This research aimed to define the energy requirement of Dorper and Hu Hybrid F1 ewes 20 to 50 kg of body weight, furthermore to study energy requirement changes with age and evaluate the effect of age on energy requirement parameters. In comparative slaughter trial, thirty animals were divided into three dry matter intake treatments (ad libitum, n = 18; low restricted, n = 6; high restricted, n = 6), and were all slaughtered as baseline, intermediate, and final slaughter groups, to calculate body chemical components and energy retained. In digestibility trial, twelve ewes were housed in individual metabolic cages and randomly assigned to three feeding treatments in accordance with the design of a comparative slaughter trial, to evaluate dietary energetic values at different feed intake levels. The combined data indicated that, with increasing age, the net energy requirement for maintenance (NEm) decreased from 260.62±13.21 to 250.61±11.79 kJ/kg(0.75) of shrunk body weight (SBW)/d, and metabolizable energy requirement for maintenance (MEm) decreased from 401.99±20.31 to 371.23±17.47 kJ/kg(0.75) of SBW/d. Partial efficiency of ME utilization for maintenance (km, 0.65 vs 0.68) and growth (kg, 0.42 vs 0.41) did not differ (p>0.05) due to age; At the similar condition of average daily gain, net energy requirements for growth (NEg) and metabolizable energy requirements for growth (MEg) for ewes during late fattening period were 23% and 25% greater than corresponding values of ewes during early fattening period. In conclusion, the effect of age upon energy requirement parameters in the present study were similar in tendency with previous recommendations, values of energy requirement for growth (NEg and MEg) for Dorper and Hu crossbred female lambs ranged between the NRC (2007) recommendation for early and later maturating growing sheep.

Because of the significant tumor-suppressive role of microRNA-22 (miR-22), the current study was designed to understand the regulation of miR-22 and to identify additional downstream miR-22 targets in liver and colon cells. The data showed that miR-22 was transcriptionally regulated by bile acid receptor farnesoid X receptor (FXR) through direct binding to an invert repeat 1 motif located at -1012 to -1025 bp upstream from miR-22. Among the studied primary and secondary bile acids, chenodeoxycholic acid, which has the highest binding affinity to FXR, induced miR-22 level in both Huh7 liver and HCT116 colon cells in a dose- and time-dependent manner. In addition, cyclin A2 (CCNA2) was identified as a miR-22 novel target in liver and colon cancer cells. The sequence of miR-22, which is conserved in mice, rats, humans, and other mammalians, aligns with the sequence of 3'-UTR of CCNA2. Chenodeoxycholic acid treatment and miR-22 mimics reduced CCNA2 protein and increased the number of G0/G1 Huh7 and HCT116 cells. In FXR KO mice, reduction of miR-22 was accompanied by elevated hepatic and ileal CCNA2 protein, as well as an increased number of hepatic and colonic Ki-67-positive cells. In humans, the expression levels of miR-22 and CCNA2 are inversely correlated in liver and colon cancers. Taken together, our data showed that bile acid-activated FXR stimulates miR-22-silenced CCNA2, a novel pathway for FXR to exert its protective effect in the gastrointestinal tract., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: Bile acids (BA) are endogenous agents capable of causing cancer throughout the gastrointestinal (GI) tract. To uncover the mechanism by which BAs exert carcinogenic effects, both human liver and colon cancer cells as well as mouse primary hepatocytes were treated with BAs and assayed for viability, genotoxic stress, and transcriptional response. BAs induced both Nur77 (NR4A1) and proinflammatory gene expression. The intracellular location of BA-induced Nur77 was time dependent; short-term (1-3 hours) exposure induced nuclear Nur77, whereas longer (1-2 days) exposure also increased cytosolic Nur77 expression and apoptosis. Inhibiting Nur77 nuclear export with leptomycin B decreased lithocholic acid (LCA)-induced apoptosis. Extended (7 days) treatment with BA generated resistance to BA with increased nuclear Nur77, viability, and mobility. While, knockdown of Nur77 in BA-resistant cells increased cellular susceptibility to LCA-induced apoptosis. Moreover, in vivo mouse xenograft experiments demonstrated that BA-resistant cells form larger tumors with elevated Nur77 expression compared with parental controls. DNA-binding and gene expression assays identified multiple survival genes (CDK4, CCND2, MAP4K5, STAT5A, and RBBP8) and a proapoptosis gene (BID) as Nur77 targets. Consistently, BA-induced upregulation of the aforementioned genes was abrogated by a lack of Nur77. Importantly, Nur77 was overexpressed in high percentage of human colon and liver cancer specimens, and the intracellular location of Nur77 correlated with elevated serum total BA levels in patients with colon cancer. These data show for the first time that BAs via Nur77 have a dual role in modulating cell survival and death., Implications: These findings establish a direct link between Nur77 and the carcinogenic effect of BAs., (©2014 American Association for Cancer Research.)

The purpose of this study was to observe the hemodynamic changes of unexplained syncope patients in the head-up tilt test and their correlations with age and gender. Eighty-six patients with unexplained syncope were administered the basic head-up test and nitroglycerin provocation test with continuous monitoring and recording of electrocardiogram and blood pressure changes. Basic characteristics of the patients and their hemodynamic responses throughout the tests were analyzed. All 86 patients tolerated and completed the head-up test. Forty-nine (56.98%) of the patients displayed a positive reaction, 37 (43.02%) patients displayed a negative reaction. Patients were divided into groups as follows: Group A, age ≤ 35 years; Group B, age 36-45 years; and Group C, age ≥ 46 years. Older patients were more prone to chronotropic incompetence, and younger patients were more prone to an excessive increase in heart rate. Older age correlated with the occurrence of autonomic nerve reaction disorder and mixed vasovagal syncope, whereas younger age was related to the occurrence of vasodepressor type vasovagal syncope (P < 0.01). Gender did not significantly correlate with negative or positive head-up test results (P = 0.184). During the head-up test, younger patients mainly manifested an excessive heart rate increase, whereas older patients did not have significant heart rate changes. Analyzing the hemodynamic changes in the head-up test and studying the relationships between age, gender, and hemodynamic responses are crucial to determine etiologies of syncope and select appropriate treatment.

Parthenolide (PTL) is a sesquiterpene lactone isolated from feverfew and exhibits potent antitumor activity against various cancers. Many studies indicate that PTL treatment leads to apoptosis, however, the mechanism has not been defined. Here, we observed that cells underwent autophagy shortly after PTL treatment. Inhibition of autophagy by knocking out autophagy associated gene atg5 blocked PTL-induced apoptosis. Surprisingly, PTL decreased the level of translation initiation factor eIF4E binding protein 1 (4E-BP1) in correlation with autophagy. Ectopic expression or shRNA knockdown of 4E-BP1 further verified the effect of 4E-BP1 on PTL-induced autophagy. Meanwhile, PTL elevated the cellular reactive oxygen species (ROS) which located upstream of the depletion of 4E-BP1, and contributed to the consequent autophagy. This study revealed 4E-BP1 as a trigger for PTL-induced autophagy and may lead to therapeutic strategy to enhance the efficacy of anticancer drugs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Peroxisome proliferator activated receptor α (PPARα) stimulates hepatocellular proliferation is species-specific. Activation of mouse, but not human, PPARα induces hepatocellular proliferation, hepatomegaly, and liver cancer. Here we tested the hypothesis that human and mouse PPARα affects liver regeneration differentially. PPARα-humanized mice (hPPARα(PAC)) were similar to wild type mice in responding to fasting-induced PPARα signaling. However, these mouse livers failed to regenerate in response to partial hepatectomy (PH). The liver-to-body weight ratios did not recover even 3 months after PH in hPPARα(PAC). The mouse PPARα-mediated down-regulation of let-7c was absent in hPPARα(PAC), which might partially be responsible for impaired proliferation. After PH, hPPARα(PAC) displayed steatosis, necrosis, and inflammation mainly in periportal zone 1, which suggested bile-induced toxicity. Quantification of hepatic bile acids (BA) revealed BA overload with increased hydrophobic BA in hPPARα(PAC). Forced FGF21 expression in partial hepatectomized hPPARα(PAC) reduced hepatic steatosis, prevented focal necrosis, and restored liver mass. Compared to mouse PPARα, human PPARα has a reduced capacity to regulate metabolic pathways required for liver regeneration. In addition, FGF21 can compensate for the reduced ability of human PPARα in stimulating liver regeneration, which suggests the potential application of FGF21 in promoting hepatic growth in injured and steatotic livers in humans.

Nur77, encoded by Nr4a1 (alias Nur77), plays roles in cell death, survival, and inflammation. To study the role of Nur77 in liver regeneration, wild-type (WT) and Nur77 knockout (KO) mice were subjected to standard two-thirds partial hepatectomy (PH). Nur77 mRNA and protein levels were markedly induced at 1 hour after PH in WT livers, coinciding with ERK1/2 activation. Surprisingly, Nur77 KO mice exhibited a higher liver-to-body weight ratio than WT mice at 24, 48, and 72 hours after PH. Nur77 KO livers exhibited increase in Ki-67-positive hepatocytes at 24 hours, with early induction of cell-cycle genes. Despite accelerated regeneration, Nur77 KO livers paradoxically incurred necrosis, hepatocyte apoptosis, elevated serum alanine aminotransferase activity, and Kupffer cell accumulation. Microarray analysis revealed up-regulation of genes modulating inflammation, cell proliferation, and apoptosis but down-regulation (due to Nur77 deficiency) of glucose and lipid homeostasis genes. Levels of proinflammatory cytokines IL-6, IL-12, IL-23, and CCL2 were increased and levels of anti-inflammatory IL-10 were decreased, compared with WT. Activated NF-κB and STAT3 and mRNA levels of target genes Myc and Bcl2l1 were elevated in Nur77 KO livers. Overall, Nur77 appears essential for regulating early signaling of liver regeneration by modulating cytokine-mediated inflammatory, apoptotic, and energy mobilization processes. The accelerated liver regeneration observed in Nur77 KO mice is likely due to a compensatory effect caused by injury., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: To clone and express autophagy-related protein 3 (TgAtg3) gene of Toxoplasma gondii, and obtain the specific polyclonal antibody against TgAtg3., Methods: TgAtg3 cDNA was inserted into prokaryotic expression vector pET28a. After identification, the constructed plasmid pET28a-TgAtg3 was transformed into E. coli Rosetta cells, and induced by special induction medium for expression of the protein. The recombinant protein was purified via Ni-NTA affinity chromatography. Western blotting assay was performed with anti-His tag mouse monoclonal antibody as the primary antibody. Rabbits were immunized with 125 μg purified TgAtg recombinant protein. Each rabbit received 4 immunizations at 2-week intervals with the same dose of antigen. The specific anti-TgAtg3 polyclonal antibody was obtained, and analyzed by Western blotting and indirect immunofluorescence assay (IFA)., Results: pET28a-TgAtg3 plasmid was identified by restriction enzyme digestion, PCR amplification and sequencing. SDS-PAGE and Western blotting analysis showed that the recombinant TgAtg3 protein (about Mr 44,000) was expressed in E. coli Rosetta cells. TgAtg3 protein from tachyzoite lysates was recognized by the specific anti-TgAtg3 polyclonal antibody. IFA assay determined that the specific polyclonal antibody bound to TgAtg3 protein from the cytoplasm of tachyzoites., Conclusion: The obtained soluble polyclonal antibody against TgAtg3 can specifically react to the endogenous TgAtg3 protein.

Aim: To assess three different methods in treating patients with cesarean scar pregnancy (CSP)., Methods: We evaluated pre-, intra- and postoperative conditions of 124 CSP patients in one of the three treatment groups, of which 37 patients underwent uterine curettage by hysteroscopy under ultrasound monitoring (group 1), 28 patients were treated with methotrexate followed by hysteroscopy (group 2) and 59 cases underwent uterine arterial embolization followed by hysteroscopy (group 3). The treatment options were determined based on the patients' conditions., Results: Among all three groups, group 3 (uterine arterial embolization followed by hysteroscopy) had the least intraoperative blood loss and the highest success rate with curettage, but the highest hospitalization cost. Group 1 (only hysteroscopy) had the shortest length of hospitalization and the lowest cost, but the highest intraoperative blood loss and slowest recovery. Group 2 (methotrexate followed by hysteroscopy) had the longest period of hospitalization, and other indexes had fallen in between the other two groups., Conclusion: Among the three methods, uterine arterial embolization followed by hysteroscopy is the safest and most efficient method without considering the cost of hospitalization. Patients with a low level of β-hCG may consider choosing hysteroscopy under ultrasound monitoring or methotrexate followed by hysteroscopy. The advantage is low cost of hospitalization; however, patients may be under relatively higher surgical risks and lower first time surgical success rate, especially for patients treated by hysteroscopy under ultrasound monitoring., (© 2014 The Authors. Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology.)

Because of increasingly widespread sedentary lifestyles and diets high in fat and sugar, the global diabetes and obesity epidemic continues to grow unabated. A substantial body of evidence has been accumulated which associates diabetes and obesity to dramatically higher risk of cancer development, particularly in the liver and gastrointestinal tract. Additionally, diabetic and obese individuals have been shown to suffer from dysregulation of bile acid (BA) homeostasis and dysbiosis of the intestinal microbiome. Abnormally elevated levels of cytotoxic secondary BAs and a pro-inflammatory shift in gut microbial profile have individually been linked to numerous enterohepatic diseases including cancer. However, recent findings have implicated a detrimental interplay between BA dysregulation and intestinal dysbiosis that promotes carcinogenesis along the gut-liver axis. This review seeks to examine the currently investigated interactions between the regulation of BA metabolism and activity of the intestinal microbiota and how these interactions can drive cancer formation in the context of diabesity. The precarcinogenic effects of BA dysregulation and gut dysbiosis including excessive inflammation, heightened oxidative DNA damage, and increased cell proliferation are discussed. Furthermore, by focusing on the mediatory roles of BA nuclear receptor farnesoid x receptor, ileal transporter apical sodium dependent BA transporter, and G-coupled protein receptor TGR5, this review attempts to connect BA dysregulation, gut dysbiosis, and enterohepatic carcinogenesis at a mechanistic level. A better understanding of the intricate interplay between BA homeostasis and gut microbiome can yield novel avenues to combat the impending rise in diabesity-related cancers., (© 2014 by the Society for Experimental Biology and Medicine.)

Background: SK Hep-1 cells (SK cells) derived from a patient with liver adenocarcinoma have been considered a human hepatoma cell line with mesenchymal origin characteristics, however, SK cells do not express liver genes and exhibit liver function, thus, we hypothesized whether mesenchymal cells might contribute to human liver primary cancers. Here, we characterized SK cells and its tumourigenicity., Methods and Principal Findings: We found that classical mesenchymal stem cell (MSC) markers were presented on SK cells, but endothelial marker CD31, hematopoietic markers CD34 and CD45 were negative. SK cells are capable of differentiate into adipocytes and osteoblasts as adipose-derived MSC (Ad-MSC) and bone marrow-derived MSC (BM-MSC) do. Importantly, a single SK cell exhibited a substantial tumourigenicity and metastatic capacity in immunodefficient mice. Metastasis not only occurred in circulating organs such as lung, liver, and kidneys, but also in muscle, outer abdomen, and skin. SK cells presented greater in vitro invasive capacity than those of Ad-MSC and BM-MSC. The xenograft cells from subcutaneous and metastatic tumors exhibited a similar tumourigenicity and metastatic capacity, and showed the same relatively homogenous population with MSC characteristics when compared to parental SK cells. SK cells could unlimitedly expand in vitro without losing MSC characteristics, its tumuorigenicity and metastatic capacity, indicating that SK cells are oncogenic MSC with enhanced self-renewal capacity. We believe that this is the first report that human MSC appear to be transformed into cancer stem cells (CSC), and that their derivatives also function as CSCs., Conclusion: Our findings demonstrate that SK cells represent a transformation mechanism of normal MSC into an enhanced self-renewal CSC with metastasis capacity, SK cells and their xenografts represent a same relative homogeneity of CSC with substantial metastatic capacity. Thus, it represents a novel mechanism of tumor initiation, development and metastasis by CSCs of non-epithelial and endothelia origin.

Retinoic acid (RA) and bile acids share common roles in regulating lipid homeostasis and insulin sensitivity. In addition, the receptor for RA (retinoid x receptor) is a permissive partner of the receptor for bile acids, farnesoid x receptor (FXR/NR1H4). Thus, RA can activate the FXR-mediated pathway as well. The current study was designed to understand the effect of all-trans RA on bile acid homeostasis. Mice were fed an all-trans RA-supplemented diet and the expression of 46 genes that participate in regulating bile acid homeostasis was studied. The data showed that all-trans RA has a profound effect in regulating genes involved in synthesis and transport of bile acids. All-trans RA treatment reduced the gene expression levels of Cyp7a1, Cyp8b1, and Akr1d1, which are involved in bile acid synthesis. All-trans RA also decreased the hepatic mRNA levels of Lrh-1 (Nr5a2) and Hnf4α (Nr2a1), which positively regulate the gene expression of Cyp7a1 and Cyp8b1. Moreover, all-trans RA induced the gene expression levels of negative regulators of bile acid synthesis including hepatic Fgfr4, Fxr, and Shp (Nr0b2) as well as ileal Fgf15. All-trans RA also decreased the expression of Abcb11 and Slc51b, which have a role in bile acid transport. Consistently, all-trans RA reduced hepatic bile acid levels and the ratio of CA/CDCA, as demonstrated by liquid chromatography-mass spectrometry. The data suggest that all-trans RA-induced SHP may contribute to the inhibition of CYP7A1 and CYP8B1, which in turn reduces bile acid synthesis and affects lipid absorption in the gastrointestinal tract., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Vav proteins are guanine nucleotide exchange factors (GEFs) that activate a group of small G proteins (GTPases). Vav1 is predominantly expressed in hematopoietic cells, whereas Vav2 and Vav3 are ubiquitously distributed in almost all human tissues. All three Vav proteins contain conserved structural motifs and associate with a variety of cellular activities including proliferation, migration, and survival. Previous observation with Jurkat leukemia T cells showed that Vav1 possessed anti-apoptotic activity by enhancing Bcl-2 transcription. However the mechanism has not been unveiled. Here, we explored the effectors of Vav1 in promoting Bcl-2 expression in Jurkat cells and revealed that Rac2-Akt was specifically evoked by the expression of Vav1, but not Vav2 or Vav3. Although all three Vav isoforms existed in Jurkat cells, Rac2 was distinguishably activated by Vav1 and that led to enhanced Bcl-2 expression and cell survival. Akt was modulated downstream of Vav1-Rac2, and the activation of Akt was indispensable in the enhanced transcription of Bcl-2. Intriguingly, neither Vav2 nor Vav3 was able to activate Rac2-Akt pathway as determined by gene silencing approach. Our data illustrated a unique role of Vav1 in T leukemia survival by selectively triggering Rac2-Akt axis and elevating the expression of anti-apoptotic Bcl-2., (Copyright © 2014 Elsevier Inc. All rights reserved.)

All-trans retinoic acid (RA) is a potent inducer of regeneration. Because the liver is the principal site for storage and bioactivation of vitamin A, the current study examines the effect of RA in mouse hepatocyte proliferation and liver regeneration. Mice that received a single dose of RA (25μg/g) by oral gavage developed hepatomegaly with increased number of Ki67-positive cells and induced expression of cell cycle genes in the liver. DNA binding data revealed that RA receptors retinoic acid receptor β (RARβ) and retinoid x receptor α (RXRα) bound to cell cycle genes Cdk1, Cdk2, Cyclin B, Cyclin E, and Cdc25a in mice with and without RA treatment. In addition, RA treatment induced novel binding of RARβ/RXRα to Cdk1, Cdk2, Cyclin D, and Cdk6 genes. All RARβ/RXRα binding sites contained AGGTCA-like motifs. RA treatment also promoted liver regeneration after partial hepatectomy (PH). RA signaling was implicated in normal liver regeneration as the mRNA levels of RARβ, Aldh1a2, Crabp1, and Crbp1 were all induced 1.5 days after PH during the active phase of hepatocyte proliferation. RA treatment prior to PH resulted in early up-regulation of RARβ, Aldh1a2, Crabp1, and Crbp1, which was accompanied by an early induction of cell cycle genes. Western blotting for RARβ, c-myc, Cyclin D, E, and A further supported the early induction of retinoid signal and cell proliferation by RA treatment. Taken together, our data suggest that RA may regulate cell cycle progression and accelerates liver regeneration. Such effect is associated with an early induction of RA signaling, which includes increased expression of the receptor, binding proteins, and processing enzyme for retinoids., (Published by Elsevier Inc.)

Background & Aims: Farnesoid X receptor (FXR, NR1H4) is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing (ChIP-seq), with results indicating that FXR may be involved in regulating diverse pathways in liver. However, limited information exists for the functions of human FXR and the suitability of using murine models to study human FXR functions., Methods: In the current study, we performed ChIP-seq in primary human hepatocytes (PHHs) treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq) and RNA microarray were performed for GW4064 or control treated PHHs and wild type mouse livers, respectively., Results: ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment., Conclusions: In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions.

Retinoic acid (RA) has diverse biological effects. The liver stores vitamin A, generates RA, and expresses receptors for RA. The current study examines the hepatic binding profile of two RA receptor isoforms, RARA (RARα) and RARB (RARβ), in response to RA treatment in mouse livers. Our data uncovered 35,521, and 14,968 genomic bindings for RARA and RARB, respectively. Each expressed unique and common bindings, implying their redundant and specific roles. RARB has higher RA responsiveness than RARB. RA treatment generated 18,821 novel RARB bindings but only 14,798 of RARA bindings, compared with the control group. RAR frequently bound the consensus hormone response element [HRE; (A/G)G(G/T)TCA], which often contained the motifs assigned to SP1, GABPA, and FOXA2, suggesting potential interactions between those transcriptional factors. Functional annotation coupled with principle component analysis revealed that the function of RAR target genes were motif dependent. Taken together, the cistrome of RARA and RARB revealed their extensive biological roles in the mouse liver. RAR target genes are enriched in various biological processes. The hepatic RAR genome-wide binding data can help us understand the global molecular mechanisms underlying RAR and RA-mediated gene and pathway regulation., (Copyright © 2014 the American Physiological Society.)

Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17β-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be α form, not β. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ERα might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.

Pyrrolizidine alkaloids (PAs) are among the most hepatotoxic natural products that produce irreversible injury to humans via the consumption of herbal medicine and honey, and through tea preparation. Toxicity and death caused by PA exposure have been reported worldwide. Metabolomics and genomics provide scientific and systematic views of a living organism and have become powerful techniques for toxicology research. In this study, senecionine hepatotoxicity on rats was determined via a combination of metabolomic and genomic analyses. From the global analysis generated from two omics data, the compromised bile acid homeostasis in vivo was innovatively demonstrated and confirmed. Serum profiling of bile acids was altered with significantly elevated conjugated bile acids after senecionine exposure, which was in accordance with toxicity. Similarly, the hepatic mRNA levels of several key genes associated with bile acid metabolism were significantly changed. This process included cholesterol 7-α hydroxylase, bile acid CoA-amino acid N-acetyltransferase, sodium taurocholate cotransporting polypeptide, organic anion-transporting polypeptides, and multidrug-resistance-associated protein 3. In conclusion, a cross-omics study provides a comprehensive analysis method for studying the toxicity caused by senecionine, which is a hepatotoxic PA. Moreover, the change in bile acid metabolism and the respective transporters may provide a new PA toxicity mechanism.

Accumulating evidence supports the role of miR-122 in fatty liver disease. We investigated miR-122 expression in a steatotic hepatocyte model, the effect of miR-122 over-expression and inhibition in the pathogenesis. Human hepatic cell line L02 was induced with oleic acid to establish the steatotic hepatocyte model. Intracellular lipid content was observed with laser scanning confocal microscope (LSCM), and triglyceride content was determined with kits. Total RNA was extracted and reversely transcribed into cDNA. miR-122 expression was measured using qRT-PCR. Subsequently, miR-122 mimic and miR-122 inhibitor were transfected into steatotic hepatocytes to observe their effect on intracellular lipid content. The lipid fluorescence intensity and triglyceride content within the steatotic hepatocytes were significantly higher than those in normal control (860.01 ± 26.52 vs. 257.77 ± 29.69 and 3.47 ± 0.12 vs. 1.85 ± 0.02 at 24 h) (P < 0.01). miR-122 expression in steatotic hepatocytes was down-regulated compared with that in control (2-ΔCt value: 0.0286 ± 0.0078 vs. 0.0075 ± 0.0012) (P ≪ 0.01). After transfection, miR-122 expression (2-ΔCt value) in the miR-122 mimic group increased 2.96-fold compared with that in control, and its lipid fluorescence intensity was significantly lower than that in control (790.92 ± 46.72 vs. 1,022.16 ± 49.66) (P < 0.01). Nevertheless, miR-122 expression decreased 3.45-fold in the miR-122 inhibitor group compared with that in control, and its fluorescence intensity was significantly higher than that in control (1,386.49 ± 40.34 vs 1,022.16 ± 49.66)(P ≪ 0.01). We concluded that miR-122 was down-regulated in steatotic hepatocytes model. The pathogenesis of hepatocyte steatosis was enhanced by miR-122 mimic and reduced with miR-122 inhibitor., (© 2013 Wiley Periodicals, Inc.)

Unlabelled: The rs738409 G>C single nucleotide polymorphism occurring in the patatin-like phospholipase 3 gene has been identified as a novel genetic marker for hepatic steatosis. Recent studies also associated rs738409 with fibrosis in hepatitis C (HCV). Therefore, we sought to determine the impact of donor and recipient rs738409 genotype on the progression of fibrosis after liver transplantation for HCV. This cohort study included 101 patients infected with HCV who underwent liver transplantation between January 2008, and June 2011. Donor and recipient rs738409 genotypes were determined from donor wedge biopsies and recipient explants. The time to Ishak stage 3 fibrosis, or HCV-related mortality/graft loss was analyzed by the Cox model adjusting for HCV-Donor Risk Index, warm ischemic time, pretransplant Model for Endstage Liver Disease (MELD) and viral load. The rs738409 CC variant was present in 56% of donors and 57% of recipients. The median follow-up period was 620 days. A total of 39 patients developed the primary outcome of ≥stage 3 fibrosis or HCV-related mortality/graft loss, the time to which differed by donor (P = 0.019) but not recipient (P = 0.89) genotype. In the multivariate model, donor GC or GG variants had 2.53 times the risk (95% confidence interval [CI] 1.25-5.02, P = 0.008) compared to CC variants. In the alternative endpoint: stage 3 fibrosis or all-cause mortality/graft loss, the effect of donor genotype was attenuated but remained significant at 1.98 (95% CI 1.11-3.53)., Conclusions: The rs738409 genotype is an important predictor of posttransplant outcome in HCV. Liver, and not adipocytes, is the site at which this effect occurs. Our finding may be useful in donor selection for liver transplantation with HCV, and may guide decisions regarding early antiviral treatment., (© 2013 by the American Association for the Study of Liver Diseases.)

Somatic cell nuclear transfer (SCNT) is a promising technique to produce mammalian transgenic clones. Only a small proportion of manipulated embryos, however, can develop into viable offspring. The abnormal growth and development of cloned animals, furthermore, are accompanied by aberrant lung development. Our objective was to investigate molecular background of lung developmental problems in transgenic (random insertion of exogenous DNA) cloned goats. We examined expression of 15 genes involved in growth regulation, imprinting, and epigenetic transcription in lung tissue of deceased transgenic cloned and normal goats of various ages. Compared with normal goats of the same age from conventional reproduction, expression of 13 genes (BMP4, FGF10, GHR, HGFR, PDGFR, RABP, VEGF, H19, CDKNIC, PCAF, MeCP2, HDAC1, and Dnmt3b) decreased in transgenic cloned goats that died at or shortly after birth; Expression of eight genes (FGF10, PDGFR, RABP, VEGF, PCAF, HDAC1, MeCP2, and Dnmt3b) decreased in fetal death of transgenic cloned goats. Expression of two epigenetic transcription genes (PCAF and Dnmt3b) decreased in disease death of transgenic cloned goats (1-4 months old). Disruptions in gene expression might be associated with the high neonatal mortality in transgenic cloned animals. These findings have implications in understanding the low efficiency of transgenic cloning., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The surface soil on sewage outfall and effluent of farmer household septic tank were collected in situ from the typical region of plain river network areas in Taihu Lake Basin, and the typical rainfall (summer 30 mm . times-1, winter 5 mm times -1), temperature (summer 27 degrees C, winter 5 degrees C ) condition and pollutant load were artificial simulated by indoor simulation soil column experiments for estimating nitrogen abatement rate of rural sewage treated by the outfall soil and exploring the abatement rule in different seasons and weather process (7 days before the rain, 3 rainy days, 7 days after the rain). Results showed that: there was the significant difference (P <0.05) in abatement/increase rate of outfall soil on nitrogen between summer and winter. The TN abatement rate, NO-3 -N increase rate of summer showed a significant difference (P <0.01) among different weather processes, but the NH+4 -N abatement rate of summer and the TN, NH+4 -N abatement rate, NO -N increase rate of winter were not significant (P > 0. 05). Therefore, the TN, NH+4 -N abatement rate, NO-3 -N increase rate need to be divided by seasons, TN abatement rate, NO-3 -N increase rate of summer need to be divided by the weather process, which were 38.5% , - 25.0% , 46. 0% and 478. 1%, 913.8%, 382. 0% , before the rain, in rainy day, after the rain, respectively; while the NH+4 -N abatement rate of summer and the TN, NH+4 -N abatement rate, NO-3 -N increase rate of winter do not need to be divided by weather process, were 91.7% , 50.4% , 85.5% and 276.0% , respectively. In the summer, the TN abatement rate in different weather processes was not correlated with NH+4 -N abatement rate, but significantly negative correlated with NO-3 -N increase rate. In the winter, the stable accumulation of TN in soil was an important reason of the TN abatement rate which had no significant difference and kept a high level among different weather processes, and it was closely related to the stable accumulation of NH+4 -N in soil.

Oocyte aging severely decreases the quality of oocytes, which hampers fertilization and subsequent embryo development. In the present study, age-dependent molecular changes in goat oocytes were investigated. First, the quality of goat oocytes with various in vitro culture times (24, 30, 36, 48, and 60 hours) was evaluated on the basis of developmental rates of parthenogenetically activated embryos and apoptosis of cumulus cells (CCs). Second, relative gene expression of six genes (mitochondrial genes: PGC-1α and NRF-1; epigenetic modification genes: SNRPN and HAT1; mitotic spindle checkpoint protein: SMAD2; and hyaluronan synthase gene: HAS3) were analyzed during oocyte aging. Third, we further studied the changes of seven genes (PGC-1α and NRF-1; apoptotic-related genes: BAX and BCL2; hyaluronan synthase gene: HAS2; metabolism-related gene: STAR; and superoxide dismutase gene: SOD1) in CCs during oocyte aging. In these studies, the blastocyst rate gradually decreased and the number of apoptotic cells significantly increased as the culture time increased (P < 0.05). Moreover, relative gene expressions of PGC-1α, NRF-1 and SMAD2 significantly decreased from 24 to 36 hours (P < 0.05), whereas the levels of HAT1 and HAS3 slowly increased as culture was prolonged. Furthermore, the levels of PGC-1α, BCL2, HAS2 and SOD1 quickly reduced, and BAX significantly increased from 24 to 36 hours in aged CCs (P < 0.05). In conclusion, goat oocytes started to age at 30 hours in vitro culture, and gene expression patterns of oocytes and CCs significantly changed as the oocytes aged. Gene expression pattern changes in CCs may provide a convenient and effective way to detect oocyte aging without compromising oocyte integrity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims/hypothesis: Perinatal exposure to bisphenol A (BPA), a widely distributed environmental endocrine disruptor, is associated with insulin resistance and diabetes in offspring. The underlying molecular mechanisms could involve epigenetics, as adverse effects induced by environmental exposure in early life are suggested through DNA methylation. In this study we sought to elucidate the relationship between perinatal BPA exposure and alteration of hepatic DNA methylation., Methods: Pregnant Wistar rats were administered BPA (50 μg/kg/day) or corn oil by oral gavage throughout gestation and lactation. Variables associated with insulin resistance and hepatic DNA methylation were examined at postnatal week 3 and week 21 in male offspring., Results: In BPA-treated offspring, serum insulin and HOMA-insulin resistance were increased, and the insulin sensitivity index and hepatic glycogen storage were decreased compared with controls at week 21. At week 3, none of these variables were significantly changed. However, hepatic global DNA methylation was decreased, accompanied by overexpression of DNA methyltransferase 3B mRNA at week 3. Meanwhile, perinatal exposure to BPA induced promoter hypermethylation and a reduction in gene expression of hepatic glucokinase. Moreover, increased promoter hypermethylation of Gck became more pronounced in BPA-treated offspring at week 21., Conclusions/interpretation: Abnormal DNA methylation in hepatic tissue precedes development of insulin resistance induced by perinatal BPA exposure. These findings support the potential role of epigenetics in fetal reprogramming by BPA-induced metabolic disorders.

Background: The eyes and skin are obvious retinoid target organs. Vitamin A deficiency causes night blindness and retinoids are widely used to treat acne and psoriasis. However, more than 90% of total body retinol is stored in liver stellate cells. In addition, hepatocytes produce the largest amount of retinol binding protein and cellular retinoic acid binding protein to mobilize retinol from the hepatic storage pool and deliver retinol to its receptors, respectively. Furthermore, hepatocytes express the highest amount of retinoid x receptor alpha (RXRα) among all the cell types. Surprisingly, the function of endogenous retinoids in the liver has received very little attention., Results: Based on the data generated from chromatin immunoprecipitation followed by sequencing, the global DNA binding of transcription factors including retinoid x receptor α (RXRα) along with its partners i.e. retinoic acid receptor α (RARα), pregnane x receptor (PXR), liver x receptor (LXR), farnesoid x receptor (FXR), and peroxisome proliferator-activated receptor α (PPARα) has been established. Based on the binding, functional annotation illustrated the role of those receptors in regulating hepatic lipid homeostasis. To correlate the DNA binding data with gene expression data, the expression patterns of 576 genes that regulate lipid homeostasis were studied in wild type and liver RXRα-null mice treated with and without RA. The data showed that RA treatment and RXRα-deficiency had opposite effects in regulating lipid homeostasis. A subset of genes (114), which could clearly differentiate the effect of ligand treatment and receptor deficiency, were selected for further functional analysis. The expression data suggested that RA treatment could produce unsaturated fatty acids and induce triglyceride breakdown, bile acid secretion, lipolysis, and retinoids elimination. In contrast, RXRα deficiency might induce the synthesis of saturated fatty acids, triglyceride, cholesterol, bile acids, and retinoids. In addition, DNA binding data indicated extensive cross-talk among RARα, PXR, LXR, FXR, and PPARα in regulating those RA/RXRα-dependent gene expression levels. Moreover, RA reduced serum cholesterol, triglyceride, and bile acid levels in mice., Conclusions: We have characterized the role of hepatic RA for the first time. Hepatic RA mediated through RXRα and its partners regulates lipid homeostasis.

The field of lipidomics is providing nutritional science a more comprehensive view of lipid intermediates. Lipidomics research takes advantage of the increase in accuracy and sensitivity of mass detection of MS with new bioinformatics toolsets to characterize the structures and abundances of complex lipids. Yet, translating lipidomics to practice via nutritional interventions is still in its infancy. No single instrumentation platform is able to solve the varying analytical challenges of the different molecular lipid species. Biochemical pathways of lipid metabolism remain incomplete and the tools to map lipid compositional data to pathways are still being assembled. Biology itself is dauntingly complex and simply separating biological structures remains a key challenge to lipidomics. Nonetheless, the strategy of combining tandem analytical methods to perform the sensitive, high-throughput, quantitative, and comprehensive analysis of lipid metabolites of very large numbers of molecules is poised to drive the field forward rapidly. Among the next steps for nutrition to understand the changes in structures, compositions, and function of lipid biomolecules in response to diet is to describe their distribution within discrete functional compartments lipoproteins. Additionally, lipidomics must tackle the task of assigning the functions of lipids as signaling molecules, nutrient sensors, and intermediates of metabolic pathways., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background & Aims: Both polymorphisms in the IL28B gene locus and ISG expression levels are associated with the outcome of hepatitis C virus (HCV) infection. The two are also interrelated, although the mechanism is unknown. Favourable CC genotype at rs12979860 expresses lower baseline ISG levels and responds better to treatment than unfavourable CT and TT genotypes. Little is known about this relationship in normal, uninfected liver. This study sought to explore this relationship., Methods: Normal human liver specimens (64) and HCV positive human liver specimens (95) were genotyped for IL28B rs12979860 C > T. mRNA levels of ISGs and other relevant genes were studied by qPCR., Results: Most studied ISGs had significantly different expression by IL28B genotype in normal liver. CC genotype expressed the highest levels, CT intermediate and TT the lowest. This is opposite to the pattern seen in HCV patients. Principal component analysis of IL28B genotype and ISG expression further revealed a distinct set of genes correlated with the C allele (ISG15, HTATIP2, LGALS3BP, IRF2 and BCL2) and T allele (IFNα, β, γ, λ3 and CD80)., Conclusion: A subset of ISGs was found to be differentially expressed in normal liver by IL28B genotype. This suggests a relationship between IL28B genotype and gene expression before HCV infection., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Beauveria bassiana is an important entomopathogenic fungus that not only often causes infection and epidemics of wild insects but some strains also show pathogenicity to the silkworm, Bombyx mori. The present study is about diversity of B. bassiana isolated from the silkworm in southwest China. Five strains of B. bassiana were isolated from infected silkworm. Two isolates, GXtr1009 and GXtr1010, were isolated from infected silkworms treated with two kinds of biological pesticides applied in Guangxi province, and three isolates, SCsk1006, YNsk1106 and GXsk1011, were collected from naturally infected silkworms from different geographical locations in Yunnan and Sichuan. All of the isolates showed highly similar conidia and conidial fructification, but the colony characteristics demonstrated great differences among the isolates. The ITS and 18S rDNA sequence analysis was sufficient to identify all five isolates as B. bassiana. However, the dendrogram, based on the ISSR data, produced two large genetic groups. GXtr1009 and GXtr1010 comprised one group, and SCsk1006, YNsk1106 and GXsk1011 converged in a different large group. The results suggested that, although all of these five B. bassiana strains were pathogenic to silkworms, strains of biological pesticides could be differentiated from strains of naturally infected silkworm via ISSR analysis.

The current study tests the hypothesis that peroxisome proliferator-activated receptor β (PPARβ) has a role in liver regeneration due to its effect in regulating energy homeostasis and cell proliferation. The role of PPARβ in liver regeneration was studied using two-third partial hepatectomy (PH) in Wild-type (WT) and PPARβ-null (KO) mice. In KO mice, liver regeneration was delayed and the number of Ki-67 positive cells reached the peak at 60 hr rather than at 36-48 hr after PH shown in WT mice. RNA-sequencing uncovered 1344 transcriptomes that were differentially expressed in regenerating WT and KO livers. About 70% of those differentially expressed genes involved in glycolysis and fatty acid synthesis pathways failed to induce during liver regeneration due to PPARβ deficiency. The delayed liver regeneration in KO mice was accompanied by lack of activation of phosphoinositide-dependent kinase 1 (PDK1)/Akt. In addition, cell proliferation-associated increase of genes encoding E2f transcription factor (E2f) 1-2 and E2f7-8 as well as their downstream target genes were not noted in KO livers 36-48 hr after PH. E2fs have dual roles in regulating metabolism and proliferation. Moreover, transient steatosis was only found in WT, but not in KO mice 36 hr after PH. These data suggested that PPARβ-regulated PDK1/Akt and E2f signaling that controls metabolism and proliferation is involved in the normal progression of liver regeneration.

Differential expression of long non-coding RNAs (lncRNAs) plays critical roles in hepatocarcinogenesis. Considerable attention has focused on the antitumor effect of histone deacetylase inhibitor (Trichostatin A, TSA) as well as the coding gene expression-induced apoptosis of cancer cells. However, it is not known whether lncRNA has a role in TSA-induced apoptosis of human hepatocellular carcinoma (HCC) cells. The global expression of lncRNAs and coding genes was analyzed with the Human LncRNA Array V2.0 after 24 h treatment. Expression was verified in cell lines and tissues by quantitative real-time PCR. The data showed that 4.8% (959) of lncRNA and 6.1% (1849) of protein coding gene were significantly differentially expressed. The differential expressions of lncRNA and protein coding genes had distinguishable hierarchical clustering expression profiling pattern. Among these differentially expressed lncRNAs, the greatest change was noted for uc002mbe.2, which had more than 300 folds induction upon TSA treatment. TSA selectively induced uc002mbe.2 in four studied HCC cell lines. Compared with normal human hepatocytes and adjacent noncancerous tissues, uc002mbe.2 expression level was significantly lower in the HCC cell lines and liver cancer tissues. The TSA-induced uc002mbe.2 expression was positively correlated with the apoptotic effect of TSA in HCC cells. In addition, knockdown the expression of uc002mbe.2 significantly reduced TSA-induced apoptosis of Huh7cells. Therefore, TSA-induced apoptosis of HCC cells is uc002mbe.2 dependent and reduced expression of uc002mbe.2 may be associated with liver carcinogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Complex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes have been limited. Deciphering the molecular mechanism that differentiate between normal and disease state may lead to identification of biomarkers for carcinoma., Results: Many proteins displayed differential expression when nuclear membrane proteome of hepatocellular carcinoma (HCC), fibrotic liver, and HepG2 cell line were assessed using 2-DE and ESI-Q-TOF MS/MS. From the down regulated set in HCC, we have identified for the first time a 15 KDa cytochrome b5A (CYB5A), ATP synthase subunit delta (ATPD) and Hemoglobin subunit beta (HBB) with 11, 5 and 22 peptide matches respectively. Furthermore, nitrosylation studies with S-nitrosocysteine followed by immunoblotting with anti SNO-cysteine demonstrated a novel and biologically relevant post translational modification of thiols of CYB5A in HCC specimens only. Immunofluorescence images demonstrated increased protein S-nitrosylation signals in the tumor cells and fibrotic region of HCC tissues. The two other nuclear membrane proteins which were only found to be nitrosylated in case of HCC were up regulated ATP synthase subunit beta (ATPB) and down regulated HBB. The decrease in expression of CYB5A in HCC suggests their possible role in disease progression. Further insight of the functional association of the identified proteins was obtained through KEGG/ REACTOME pathway analysis databases. String 8.3 interaction network shows strong interactions with proteins at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor of liver pathology., Conclusion: These findings may have broader implications for understanding the mechanism of development of carcinoma. However, large scale studies will be required for further verification of their critical role in development and progression of HCC.

Aims: Ethnic differences in genotype frequency provide a natural condition for assessing the contribution of gene variations to the causes and treatments of disease. Accordingly, the purpose of this study was to determine whether ethnic variations in allele frequencies of the serotonin transporter gene-linked polymorphic region (5-HTTLPR) of the SLC6A4 gene were related to the response to the treatment of depression., Main Methods: African-Americans (n=101) and Caucasians (n=100) with major depressive disorder were treated with the antidepressant citalopram (20-60mg/day) for 8weeks. Genotyping for the long (L) and short (s) alleles (LL, Ls, and ss) of the SLC6A4 gene was performed and the association between genotype and treatment response was assessed., Key Findings: Subjects in both ethnic groups showed a significant reduction in depression scores over time (p<.0001). However, in spite of a significantly greater frequency of the L allele in African-Americans as compared to Caucasians, a comparable clinical response between the two groups was found with 5-HTTLPR polymorphism not significantly associated with clinical response in either ethnic group., Significance: The results are consistent with a previous finding and in accord with most of the results obtained in Caucasian subjects that SLC6A4 genotype is not related, at least by itself, to a response to treatment in either ethnic group to any clinically significant degree., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In this study, cellobiose, a reducing disaccharide was used to produce succinic acid by Actinobacillus succinogenes NJ113. A final succinic acid concentration of 30.3g/l with a yield of 67.8% was achieved from an initial cellobiose concentration of 50 g/l via batch fermentation in anaerobic bottles. The cellobiose uptake mechanism was investigated and the results of enzyme assays revealed that the phosphoenolpyruvate phosphotransferase system (PEP-PTS) played an important role in the cellobiose uptake process. In batch fermentation with 18 g/l of cellobiose and 17 g/l of other sugars from sugarcane bagasse cellulose hydrolysates, a succinic acid concentration of 20.0 g/l was obtained, with a corresponding yield of 64.7%. This study found that cellobiose from incomplete hydrolysis of cellulose could be a potential carbon source for economical and efficient succinic acid production by A. succinogenes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Fenretinide is significantly more effective in inducing apoptosis in cancer cells than all-trans retinoic acid (ATRA). The current study uses a genome-wide approach to understand the differential role fenretinide and ATRA have in inducing apoptosis in Huh7 cells. Fenretinide and ATRA-induced gene expressions and DNA bindings were profiled using microarray and chromatin immunoprecipitation with anti-RXRα antibody. The data showed that fenretinide was not a strong transcription regulator. Fenretinide only changed the expressions of 1 093 genes, approximately three times less than the number of genes regulated by ATRA (2 811). Biological function annotation demonstrated that both fenretinide and ATRA participated in pathways that determine cell fate and metabolic processes. However, fenretinide specifically induced Fas/TNFα-mediated apoptosis by increasing the expression of pro-apoptotic genes i.e., DEDD2, CASP8, CASP4, and HSPA1A/B; whereas, ATRA induced the expression of BIRC3 and TNFAIP3, which inhibit apoptosis by interacting with TRAF2. In addition, fenretinide inhibited the expression of the genes involved in RAS/RAF/ERK-mediated survival pathway. In contrast, ATRA increased the expression of SOSC2, BRAF, MEK, and ERK genes. Most genes regulated by fenretinide and ATRA were bound by RXRα, suggesting a direct effect. This study revealed that by regulating fewer genes, the effects of fenretinide become more specific and thus has fewer side effects than ATRA. The data also suggested that fenretinide induces apoptosis via death receptor effector and by inhibiting the RAS/RAF/ERK pathway. It provides insight on how retinoid efficacy can be improved and how side effects in cancer therapy can be reduced., (Published by Elsevier Inc.)

Objective: To establish a detection method based on gas chromatography-mass spectrometry (GC-MS) for concentrations of volatile nitrosamine compounds in urine, and apply it to the test of real samples., Methods: Target compounds dichloromethane in urine samples was extracted with dichloromethane through liquid-liquid extraction, then the dichloromethane extract was filtrated, evaporated with nitrogen at 40°C to dryness, and the volume was set with 0.2 ml dichloromethane. Analysis of nine volatile nitroso-compounds were performed with GC-MS under selected ion monitoring mode, external reference method was used for quantification, and the detection limit, repeatability and sensitivity were evaluated. In addition, nine volatile nitroso-compounds of 92 urine samples in a town of Anhui province were measured., Results: A good linear range of 2 - 200 ng/ml (with correlation coefficient 0.9985 - 0.9999) were obtained for the above mentioned nine kinds of analyte, and the lowest examination concentration was 0.05 - 0.50 ng/ml. The addition standard recoveries were 68%-102% with the RSD of 0.4% - 5.5% (n = 3). The detection limits were 0.001 - 0.013 ng/ml urine. The detection rate of N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-propylamine (NDPA), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR), N-nitrosopiperidine (NPIP), N-nitrosodi-n-butylamine (NDBA) and N-nitrosodiphenylamine (NDPhA) were 71% (65), 74% (68), 65% (60), 80% (73), 92% (85), 78% (72), 76% (70), 87% (80), 98% (90), respectively, with the results (0.27 ± 0.12), (0.75 ± 0.29), (0.06 ± 0.02), (0.16 ± 0.07), (23.66 ± 5.18), (1.01 ± 0.35), (0.38 ± 0.11), (2.47 ± 0.52) and (15.13 ± 3.48) nmol/g creatinine., Conclusions: A gas chromatography-mass spectrometry detect method was developed for low level volatile nitrosamines in urine samples.

Vav1 is a guanine nucleotide exchange factor (GEF) specifically expressed in hematopoietic cells. It consists of multiple structural domains and plays important roles in T cell activation. The other highly conserved isoforms of Vav family, Vav2 and Vav3, are ubiquitously expressed in human tissues including lymphocytes. All three Vav proteins activate Rho family small GTPases, which are involved in a variety of biological processes during T cell activation. Intensive studies have demonstrated that Vav1 is indispensable for T cell receptor (TCR)-mediated signal transduction, whereas Vav2 and Vav3 function as GEFs that overlap with Vav1 on TCR-induced cytoskeleton reorganization. T cells lacking Vav1 exhibited severe defect in TCR-mediated calcium elevation, indicating that the co-existing Vav2 and Vav3 did not compensate Vav1 in calcium signaling. What is the functional particularity of Vav1 in lymphocytes? In this study, we identified the N-terminal 20 amino acids of Vav1 in the calponin homology (CH) domain to be essential for its interaction with calmodulin (CaM) that leads to TCR-induced calcium mobilization. Substitution of the 1-20 amino acids of Vav1 with those of Vav2 or Vav3 abolished the association with CaM, and the N-terminal mutations of Vav1 failed to potentiate normal TCR-induced calcium mobilization, that in turn, suspended nuclear factor of activated T cells (NFAT) activation and IL-2 production. This study highlights the importance of the N-terminal 20 aa of Vav1 for CaM binding, and provides new insights into the distinguished and irreplaceable role of Vav1 in T cell activation and signal transduction.

Summary: Nuclear receptors (NRs) are a class of transcription factors playing important roles in various biological processes. An NR often impacts numerous genes and different NRs share overlapped target networks. To fulfil the need for a database incorporating binding sites of different NRs at various conditions for easy comparison and visualization to improve our understanding of NR binding mechanisms, we have developed NURBS, a database for experimental and predicted nuclear receptor binding sites of mouse (NURBS). NURBS currently contains binding sites across the whole-mouse genome of 8 NRs identified in 40 chromatin immunoprecipitation with massively parallel DNA sequencing experiments. All datasets are processed using a widely used procedure and same statistical criteria to ensure the binding sites derived from different datasets are comparable. NURBS also provides predicted binding sites using NR-HMM, a Hidden Markov Model (HMM) model., Availability: The GBrowse-based user interface of NURBS is freely accessible at http://shark.abl.ku.edu/nurbs/. NR-HMM and all results can be downloaded for free at the website., Contact: jwfang@ku.edu

Myostatin, a member of the transforming growth factor-β family, acts as a negative regulator of skeletal muscle mass. In this study, myostatin-targeted caprine fibroblasts were obtained and subjected to SCNT to determine whether myostatin-knockout goats could be created. Fibroblasts from a 2-mo-old goat were transfected with a myostatin-targeted vector to prepare transgenic donor cells for nuclear transfer. After serum-starvation (for synchronization of the cell cycle), the percentage of transgenic fibroblasts in the G(0)/G(1) phase increased (66.2% vs. 82.9%; P < 0.05) compared with that in the control group, whereas the apoptosis rate and mitochondrial membrane potential were unaffected (P > 0.05). There were no significant differences between in vivo- and in vitro-matured oocytes as recipient cytoplasts for rates of fusion (86.5% vs. 78.4%), pregnancy (21.6% vs. 16.7%), or kidding (2.7% vs. 0%). One female kid from an in vivo-matured oocyte was born, but died a few hours later. Microsatellite analysis and polymerase chain reaction identification confirmed that this kid was genetically identical to the donor cells. Based on Western blot analysis, myostatin of the cloned kid was not expressed compared with that of nontransgenic kids. In conclusion, SCNT using myostatin-targeted 2-mo-old goat fibroblasts as donors has potential as a method for producing myostatin-targeted goats., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: To evaluate the hepatoprotective effect of the root and rhizome of Rhubarb (Rhei Radix et Rhizoma) against α-naphthylisothiocyanate (ANIT)-induced liver injury using metabolic profile of bile acids (BAs) along with biochemical parameters and histological alterations., Materials and Methods: Ultra-performance liquid chromatography coupled with quadrupole mass spectrometry (UPLC-MS) was applied to determinate the concentration of BAs, which was followed by multivariate statistical analysis of Principal Component Analysis (PCA) and Partial Least Squares Discriminate Analysis (PLS-DA)., Results: Based on PCA results, three groups (Vehicle group, ANIT group and RhO+ANIT group) were clearly distinguished. Tauro-cholic acid (TCA), tauro-hyodesoxycholic acid (THDCA), glyco-cholic acid (GCA), and glyco-chenodeoxycholic acid (GCDCA) were proved to be the most important markers corresponding to ANIT-induced liver injury and protection provided by Rhubarb, which is further confirmed by PLS-DA. A correlation was found between the foregoing BAs and biochemical parameters including serum aspartate aminotransferase (ALT) and aspartate aminotransferase (AST), which confirmed that TCA, THDAC, GCA, and GCDCA could be considered as sensitive biomarkers., Conclusion: The variance of the BAs contents can be used to evaluate ANIT-induced hepatotoxicity caused by ANIT and protective effects of Rhubarb. It also lays the foundation for the further research on the mechanisms of cholestasis as well as the therapeutic effect of Rhubarb., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Aim: To investigate the association of polymorphisms of nur-related receptor 1 (Nurr1) and development of alcohol dependence in Mexican Americans., Methods: Peripheral blood samples were collected from 374 alcoholic and 346 nonalcoholic Mexican Americans; these two groups were sex- and age-matched. Sample DNA was extracted and genomic DNA was amplified by polymerase chain reaction. The -2922(C) 2-3 polymerase chain reaction products were digested with Sau96I, alleles of 1345(G/C), and -1198(C/G) in the regulatory region as well as Ex+132 (G/T/A/C) and Ex+715(T/-) in exon 3 were studied by sequencing., Results: The C2/C2, C2/C3, C3/C3 genotype distribution of -2922(C) 2-3 was 34.4%, 38.2% and 27.5% in the nonalcoholic group compared to 23.3%, 51.2% and 25.4% in the alcoholic group (P = 0.001). The C/C, C/G, G/G genotype distribution of -1198(C/G) was 23.5%, 46.1% and 30.3% in the nonalcoholic group compared to 13.9%, 50.9% and 35.3% in the alcoholic group (P = 0.007). However, the -1345 (G/C), Ex3+132(G/T/A/C) and Ex3+715(T/-) alleles were not polymorphic in Mexican Americans, and all those studied had G/G, G/G and T/T genotype for these three alleles, respectively. The -2922(C) 2-3 did not show allele level difference between alcoholic and nonalcoholic individuals, but -1198 (C/G) showed a significant allele frequency difference between alcoholic (39.3%) and nonalcoholic (46.6%) populations (P = 0.005). Excluding obese individuals, significant differences were found at both genotypic and allelic levels for the -2922(C) 2-3 polymorphism (P = 0.000 and P = 0.049) and the -1198 (C/G) polymorphism (P = 0.008 and P = 0.032) between nonobese alcoholics and nonobese controls. Excluding smokers, a significant difference was found only at the genotypic level for the -2922(C) 2-3 polymorphism (P = 0.037) between nonsmoking alcoholics and nonsmoking controls, but only at the allelic level for the -1198(C/G) polymorphism (P = 0.034)., Conclusion: Polymorphisms in the regulatory region of Nurr1 are implicated in pathogenesis of alcohol dependence and the Nurr1/dopamine signaling pathway might be important for this dependence development in Mexican Americans.

Non-alcoholic fatty liver disease (NAFLD) is defined as excessive accumulation of fatty acid in the liver, a common disease in the world. The research of single nucleotide polymorphisms (SNPs) provides a new approach for managing NAFLD. SNPs may increase or decrease the functions of the target genes and their encoding proteins. Peroxisome proliferator-activated receptor (PPAR) plays a key role in modulating metabolism of hepatic triglycerides and consequently magnitude of NAFLD. In this study, we investigated the effect of three SNPs in the PPAR-γ gene i.e. rs10865710 (C-681G), rs7649970 (C-689T) and rs1801282 (C34G, also termed Pro12Ala) on susceptibility to NAFLD. The participants were selected from our epidemiological survey. Totally 169 participants were enrolled in NAFLD group, and 699 healthy subjects were included as controls. PCR-RFLP was applied to detect the SNPs. The G allele frequency of rs10865710 in NAFLD group (41.1%) was significantly higher than that (34.8%) in controls (p = 0.03). Differences in other two loci (rs7649970 and rs1801282) were not statistically significant between the two groups (p > 0.05). This result was confirmed by haplotype analysis. The GCC haplotype (a set of 3 adjacent SNPs in linkage disequilibrium, corresponding to the three alleles of above polymorphisms in order) was a risk factor for the susceptibility to NAFLD (p = 0.03). This study has revealed that the G allele of rs10865710 in the PPAR-γ gene is associated with the increased susceptibility to NAFLD. Our findings may provide novel diagnostic biomarkers and therapeutic targets for NAFLD.