Description: Objective The total enteroscopy rate of single-balloon enteroscopy (SBE) using air insufflation is not satisfactory, and whether carbon dioxide (CO 2 ) insufflation increases the total enteroscopy rate of SBE is unknown. This randomised controlled trial aimed to determine whether CO 2 insufflation facilitates the intubation depth and total enteroscopy rate of SBE. Design A total of 214 eligible patients referred for SBE were randomised to receive either air or CO 2 insufflation, and included in the intention-to-test (ITT) analysis. In addition, 199 patients in whom enteroscopy was completed were included in the per-protocol (PP) analysis. Both the patients and endoscopists were blinded, and the intubation depth and total enteroscopy rate were defined as the primary outcomes. Results The CO 2 group showed a superiority of intubation in the ITT analysis (oral route: 323.8±64.2 vs 238.3±68.6 cm; anal route: 261.6±74.2 vs 174.7±62.1 cm, both p〈0.001), and the total enteroscopy rate (34.9% vs 17.6%, p=0.006). Similar results were obtained in a PP analysis for both outcomes. In addition, in the PP analysis, the addition of circumference after the procedure was less in the CO 2 group (0.8±0.6 vs 3.3±1.8 cm, p=0.005) for the oral route. No serious complications were reported. The overall percentage of procedures with significant pathological findings was 52.8%; the rates were 58.5% and 47.2% (p=0.100, ITT analysis) in the CO 2 and air groups, respectively. Conclusions CO 2 insufflation improves the intubation depth and total enteroscopy rate in SBE with a good safety profile and acceptability compared with that of air, and thus is recommended for clinical utilisation. Trial registration number ClinicalTrial.gov identifier: NCT01758900.

Description: Background Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication. Objective To identify recurrent somatic mutations with prognostic significance in patients with CRC. Method Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases. Results Seven significantly mutated genes, including four reported ( APC , TP53 , KRAS and SMAD4 ) and three novel recurrently mutated genes ( CDH10 , FAT4 and DOCK2 ), exhibited high mutation prevalence (6–14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature ( CDH10 , COL6A3 , SMAD4 , TMEM132D , VCAN ) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study. Conclusions The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging.

Description: Objective Using whole genome sequencing, we identified gene amplification of solute carrier family 12 member 5 ( SLC12A5 ) located at 20q13.12 in colorectal cancer (CRC). We analysed its amplification, overexpression, biological effects and prognostic significance in CRC. Design SLC12A5 amplification status was evaluated by fluorescence in situ hybridisation (FISH). The effects of SLC12A5 re-expression or knockdown were determined in proliferation, apoptosis, invasion and metastasis assays. SLC12A5 target genes and related pathways were identified by reporter activity and cDNA microarray analyses. Clinical impact of SLC12A5 overexpression was assessed in 195 patients with CRC. Results Amplification of SLC12A5 was verified in 78 out of 191 (40.8%) patients with primary CRC by FISH, which was positively correlated with its protein overexpression (p〈0.001). Biofunctional investigation of SLC12A5 revealed that SLC12A5 significantly increased cell proliferation, G1-S cell cycle transition, invasion/migration abilities, but suppressed apoptosis in vitro and promoted xenograft tumour growth as well as lung metastasis in vivo. The antiapoptosis effect by SLC12A5 was mediated through inhibiting apoptosis-inducing factor and endonuclease G-dependent apoptotic signalling pathway; and the pro-metastasis role was by regulating key elements of the matrix architecture, including matrix metallopeptidase and fibronectin. After a median follow-up of 50.16 months, multivariate analysis revealed that patients with SLC12A5 protein overexpression had a significant decrease in overall survival. Kaplan–Meier survival curves showed that SLC12A5 overexpression was significantly associated with shortened survival in patients with CRC. Conclusions SLC12A5 plays a pivotal oncogenic role in colorectal carcinogenesis; its overexpression is an independent prognostic factor of patients with CRC.

Description: Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease characterized by massive enlargement of fluid-filled cysts in the kidney. However, there is no effective therapy yet for this disease. To examine whether ginkgolide B, a natural compound, inhibits cyst development, a Madin-Darby canine kidney (MDCK) cyst model, an embryonic kidney cyst model, and a PKD mouse model were used. Interestingly, ginkgolide B significantly inhibited MDCK cyst formation dose dependently, with up to 69% reduction by 2 μM ginkgolide B. Ginkgolide B also significantly inhibited cyst enlargement in the MDCK cyst model, embryonic kidney cyst model, and PKD mouse model. To determine the underlying mechanisms, the effect of ginkgolide B on MDCK cell viability, proliferation, apoptosis, chloride transporter CFTR activity, and intracellular signaling pathways were also studied. Ginkgolide B did not affect cell viability, proliferation, and expression and activity of the chloride transporter CFTR that mediates cyst fluid secretion. Ginkgolide B induced cyst cell differentiation and altered the Ras/MAPK signaling pathway. Taken together, our results demonstrate that ginkgolide B inhibits renal cyst formation and enlargement, suggesting that ginkgolide B might be developed into a novel candidate drug for ADPKD.

Description: Objective Cortactin acts as a prominent substrate of histone deacetylases (HDACs) and plays important roles in tumour progression in several human cancers. However, the clinical significance of its expression in human prostate cancer (PCa) has not been determined. We aimed to identify the potential role of cortactin expression in tumour progression and prognosis in PCa and the association with HDACs. Methods 256 foci with distinctive lesions in 110 prostate specimens were collected to identify the status of among cortactin, SIRT2, histone deacetylase 6 (HDAC6) by immunohistochemistry and its relationship with clinicopathological and follow-up data were analysed. Results The results showed that cortactin expression was significantly higher (79.1%), and SIRT2 expression was lower (37.3%) in PCa foci, when it was compared with high-grade prostatic intraepithelial neoplasia foci and benign foci, respectively. HDAC6 expression was low and had no statistical significance in PCa. High intensity of cortactin staining was significantly and independently associated with a high prostate-specific antigen (PSA) level, high Gleason score, clinical stage progression and shortened survival time in patients with PCa. High intensity of SIRT2 staining was significantly and independently associated with a high PSA level, old age, high Gleason score and clinical stage progression. Multivariable Cox regression analysis showed cortactin expression was a significant prognostic factor for survival of patients with PCa (β, 0.736; 95% CI 1.371 to 3.181; p=0.001). Conclusions The results suggested that cortactin seems to be a satisfactory marker to predict tumour progression and survival in cases of PCa. And it may be SIRT2 rather than HADAC6 is responsible for tumour occurrence and the progression of PCa.

Description: Inflammation-dominated sympathetic sprouting adjacent to the necrotic region following myocardial infarction (MI) has been implicated in the etiology of arrhythmias resulting in sudden cardiac death; however, the mechanisms responsible remain to be elucidated. Although being a key immune mediator, the role of Notch has yet to be explored. We investigated whether Notch regulates macrophage responses to inflammation and affects cardiac sympathetic reinnervation in rats undergoing MI. MI was induced by coronary artery ligation. A high level of Notch intracellular domain was observed in the macrophages that infiltrated the infarct area at 3 days post-MI. The administration of the Notch inhibitor N-N-(3,5-difluorophenacetyl- l -alanyl)-S-phenylglycine-t-butyl ester (DAPT) (intravenously 30 min before MI and then daily until death) decreased the number of macrophages and significantly increased the M2 macrophage activation profile in the early stages and attenuated the expression of nerve growth factor (NGF). Eventually, NGF-induced sympathetic hyperinnervation was blunted, as assessed by the immunofluorescence of tyrosine hydroxylase. At 7 days post-MI, the arrhythmia score of programmed electric stimulation in the vehicle-treated infarcted rats was higher than that in rats treated with DAPT. Further deterioration in cardiac function and decreases in the plasma levels of TNF-α and IL-1β were also detected. In vitro studies revealed that LPS/IFN- upregulated the surface expression of NGF in M1 macrophages in a Notch-dependent manner. We concluded that Notch inhibition during the acute inflammatory response phase is associated with the downregulation of NGF, probably through a macrophage-dependent pathway, thus preventing the process of sympathetic hyperinnervation.

Description: Bisphenol A diglycidyl ether (BADGE), a PPAR2 antagonist, has been shown to inhibit marrow adipogenesis and promote bone formation in intact animals. We investigated the impact of BADGE on a new and more clinically relevant physiological model, the ovariectomized (OVX) rat model. Forty female Wistar rats were divided into four treatment groups for 12 wk ( n = 10/group): sham+vehicle, sham+BADGE, OVX+vehicle, and OVX+BADGE. Postmortem analyses included MRI, micro-CT, serological test, histomorphometry, biomechanical tests, RT-PCR, and Western blot. Overall, OVX induced a sequential marrow fat expansion accompanied by bone deterioration. Compared with OVX controls, BADGE reduced fat fraction of the distal femur by 36.3%, adipocyte density by 33.0%, adipocyte size by 28.6%, adipocyte volume percentage by 57.8%, and adipogenic markers PPAR2 and C/EBPα by ~50% in OVX rats. Similar results were observed in sham rats vs. vehicle. BADGE could promote bone quality in sham rats; however, BADGE did not significantly improve trabecular microarchitecture, biomechanical strength, and dynamic histomorphometric parameters except for trabecular separation in OVX rats. We concluded that early BADGE treatment at a dose of 30 mg/kg attenuates marrow adiposity in ovary-intact and OVX rats and stimulates bone formation in ovary-intact rats but does not significantly rescue bone quality in OVX rats.

Description: Glucagon-like peptides (GLP-1/2) are cosecreted from endocrine L cells in the gut and preproglucagonergic neurons in the brain. Peripheral GLP-2 action is essential for maintaining intestinal homeostasis, improving absorption efficiency and blood flow, promoting immune defense, and producing efficacy in treatment of gastrointestinal diseases. However, it is unknown if CNS GLP-2 plays a physiological role in the control of energy homeostasis. Since GLP-1/2 are cotranslated from preproglucagongene and coproduced by prohormone convertase-1, it is challenging to knockout GLP-2 only. Instead, our laboratory has generated a Glp2r -floxed mouse line to dissect cell-specific GLP-2 receptor GLP-2R) action in the regulation of energy balance. Our objective was to determine if GLP-2R in the hypothalamus modulates feeding behavior and gastric emptying. We show that Glp2r mRNA and protein are highly expressed in the arcuate nucleus and dorsomedial nucleus of the mouse hypothalamus. Using the Cre-LoxP system, we generated mice that lack Glp2r expression in POMC neurons (KO; mainly in the hypothalamus). The KO mice showed hyperphagic behavior (such as increases in food intake and meal frequency), accelerated gastric emptying (assessed by [ 13 C]octanoic acid breath test), and late-onset obesity, yet there was no decrease in basal metabolic rate. Infusion of GLP-2 (2.5 nmol into the 4th ventricle) suppressed food intake and gastric emptying, while GLP-2-mediated effects were abolished in the melanocortin receptor-4 (MC4R) KO mice. We conclude that Glp2r deletion in POMC neurons enhances feeding behavior and gastric motility, whereas icv GLP-2R activation suppresses food intake and gastric emptying through the MC4R signaling pathway. This study indicates that CNS GLP-2R plays a physiological role in the control of feeding behavior and gastric emptying and that this is mediated probably through the melanocortin system.

Description: A balance between alveolar liquid absorption and secretion is critical for maintaining optimal alveolar subphase liquid height and facilitating gas exchange in the alveolar space. However, the role of cystic fibrosis transmembrane regulator protein (CFTR) in this homeostatic process has remained elusive. Using a newly developed porcine model of cystic fibrosis, in which CFTR is absent, we investigated ion transport properties and alveolar liquid transport in isolated type II alveolar epithelial cells (T2AECs) cultured at the air-liquid interface. CFTR was distributed exclusively to the apical surface of cultured T2AECs. Alveolar epithelia from CFTR –/– pigs failed to increase liquid absorption in response to agents that increase cAMP, whereas cAMP-stimulated liquid absorption in CFTR +/– epithelia was similar to that in CFTR +/+ epithelia. Expression of recombinant CFTR restored stimulated liquid absorption in CFTR –/– T2AECs but had no effect on CFTR +/+ epithelia. In ex vivo studies of nonperfused lungs, stimulated liquid absorption was defective in CFTR –/– alveolar epithelia but similar between CFTR +/+ and CFTR +/– epithelia. When epithelia were studied at the air-liquid interface, elevating cAMP levels increased subphase liquid height in CFTR +/+ but not in CFTR –/– T2AECs. Our findings demonstrate that CFTR is required for maximal liquid absorption under cAMP stimulation, but it is not the rate-limiting factor. Furthermore, our data define a role for CFTR in liquid secretion by T2AECs. These insights may help to develop new treatment strategies for pulmonary edema and respiratory distress syndrome, diseases in which lung liquid transport is disrupted.

Description: Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to β-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca 2+ channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca 2+ channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca 2+ current ( I Ca,L ) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α 1C subunit of voltage-gated Ca 2+ channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α 1C were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased I Ca,L in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized I Ca,L stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted β-adrenergic stimulation of I CaL in failing hearts results from hyperphosphorylation of Ca 2+ channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca 2+ channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca 2+ channel remodeling of the failing heart.