Cadherin adhesion and mechanotransduction.

Cadherins are the principal adhesion proteins at intercellular junctions and function as the biochemical Velcro that binds cells together. Besides this mechanical function, cadherin complexes are also mechanotransducers that sense changes in tension and trigger adaptive reinforcement of intercellular junctions. The assembly and regulation of cadherin adhesions are central to their mechanical functions, and new evidence is presented for a comprehensive model of cadherin adhesion, which is surprisingly more complex than previously appreciated. Recent findings also shed new light on mechanisms that regulate cadherin junction assembly, adhesion, and mechanotransduction. We further describe recent evidence for cadherin-based mechanotransduction, and the rudiments of the molecular mechanism, which involves α-catenin and vinculin as key elements. Potential roles of a broader cast of possible force-sensitive partners are considered, as well as known and speculative biological consequences of adhesion and force transduction at cadherin-mediated junctions.

Genetic and metabolic determinants of methotrexate-induced mucositis in pediatric acute lymphoblastic leukemia.

Methotrexate (MTX) is an effective and toxic chemotherapeutic drug in the treatment of pediatric acute lymphoblastic leukemia(ALL). In this prospective study, we aimed to identify metabolic and genetic determinants of MTX toxicity. One hundred and thirty-four Dutch pediatric ALL patients were treated with four high infusions MTX (HD-MTX: 5 g m(-2)) every other week according to the DCOG-ALL-10 protocol. Mucositis (National Cancer Institute grade ⩾ 3) was the most frequent occurring toxicity during the HD-MTX phase (20%) and occurred especially after the first MTX course. Mucositis was not associated with plasma MTX, plasma folate or plasma homocysteine levels. Patients with mucositis had higher erythrocyte folate levels at the start of protocol M than patients without mucositis (median 1.4 vs 1.2 µmol l(-1), P<0.008), this could reflect an increased MTX uptake in mucosal cells of patients with mucositis. From 17 single-nucleotide polymorphisms in the MTX pathway, only patients with the wild-type variant of rs7317112 SNP in the ABCC4 gene had more mucositis (AA (39%) vs AG/GG (15%), P=0.016). We found no evidence that erythrocyte folate levels mediate in the association between the rs7317112 and mucositis.

No evidence of benefit from class-II compression stockings in the prevention of lower-limb lymphedema after inguinal lymph node dissection: results of a randomized controlled trial.

Graduated compression stockings have been advocated for prevention of lymphedema after inguinal lymph node dissection (ILND) although scientific evidence of their efficacy in preventing lymphedema is lacking. The primary objective of this study was to assess the efficacy of class II compression stockings for the prevention of lymphedema in cancer patients following ILND. Secondary objectives were to investigate the influence of stockings on the occurrence of wound complications and genital edema, health-related quality of life (HRQoL) and body image. Eighty patients (45 with melanoma, 35 with urogenital tumors) who underwent ILND at two specialized cancer centers were randomly allocated to class II compression stocking use for six months or to a usual care control group. Lymphedema of the leg and genital area, wound complications, HRQoL, and body image were assessed at regular intervals prior to and up to 12 months after ILND. No significant differences were observed between groups in the incidence of edema, median time to the occurrence of edema, incidence of genital edema, frequency of complications, HRQoL, or body image. Based on the results of the current study, routine prescription of class II graduated compression stockings after ILND should be questioned and alternative prevention strategies should be considered.

First-in-human study of the pharmacokinetics and antiviral activity of IDX375, a novel nonnucleoside hepatitis C virus polymerase inhibitor.

IDX375 is a potent and selective palm-binding nonnucleoside inhibitor of the hepatitis C virus (HCV) genotype 1 polymerase. This first-in-human study evaluated the safety, tolerability, and pharmacokinetics of IDX375 in healthy volunteers, as well as its antiviral activity in HCV-infected patients. IDX375, as a choline salt, was administered for 1 day to 40 healthy male volunteers (25- to 200-mg IDX375-equivalent single ascending doses and a 200-mg twice-daily [BID] dose) and three patients chronically infected with HCV genotype 1 (200 mg BID only). IDX375 was well absorbed and well tolerated by all of the study participants. A single-day 200-mg BID dose resulted in exposure-related anti-HCV activity with maximal 0.5 to 1.1 log(10) reductions in plasma HCV RNA. These observations support further clinical investigations of IDX375.

Intermediate-term survival of robot-assisted versus open radical cystectomy for muscle-invasive and high-risk non-muscle invasive bladder cancer in The Netherlands.

BACKGROUND: Radical cystectomy with pelvic lymph node dissection is the recommended treatment in non-metastatic muscle-invasive bladder cancer (MIBC). In randomised trials, robot-assisted radical cystectomy (RARC) showed non-inferior short-term oncological outcomes compared with open radical cystectomy (ORC). Data on intermediate and long-term oncological outcomes of RARC are limited. OBJECTIVE: To assess the intermediate-term overall survival (OS) and recurrence-free survival (RFS) of patients with MIBC and high-risk non-MIBC (NMIBC) who underwent ORC versus RARC in clinical practice. METHODS AND MATERIALS: A nationwide retrospective study in 19 Dutch hospitals including patients with MIBC and high-risk NMIBC treated by ORC (n = 1086) or RARC (n = 386) between January 1, 2012 and December 31, 2015. Primary and secondary outcome measures were median OS and RFS, respectively. Survival outcomes were estimated using Kaplan-Meier curves. A multivariable Cox regression model was developed to adjust for possible confounders and to assess prognostic factors for survival including clinical variables, clinical and pathological disease stage, neoadjuvant therapy and surgical margin status. RESULTS: The median follow-up was 5.1 years (95% confidence interval ([95%CI] 5.0-5.2). The median OS after ORC was 5.0 years (95%CI 4.3-5.6) versus 5.8 years after RARC (95%CI 5.1-6.5). The median RFS was 3.8 years (95%CI 3.1-4.5) after ORC versus 5.0 years after RARC (95%CI 3.9-6.0). After multivariable adjustment, the hazard ratio for OS was 1.00 (95%CI 0.84-1.20) and for RFS 1.08 (95%CI 0.91-1.27) of ORC versus RARC. Patients who underwent ORC were older, had higher preoperative serum creatinine levels and more advanced clinical and pathological disease stage. CONCLUSION: ORC and RARC resulted in similar intermediate-term OS and RFS in a cohort of almost 1500 MIBC and high-risk NMIBC.

Requirement of the Caenorhabditis elegans RapGEF pxf-1 and rap-1 for epithelial integrity.

The Rap-pathway has been implicated in various cellular processes but its exact physiological function remains poorly defined. Here we show that the Caenorhabditis elegans homologue of the mammalian guanine nucleotide exchange factors PDZ-GEFs, PXF-1, specifically activates Rap1 and Rap2. Green fluorescent protein (GFP) reporter constructs demonstrate that sites of pxf-1 expression include the hypodermis and gut. Particularly striking is the oscillating expression of pxf-1 in the pharynx during the four larval molts. Deletion of the catalytic domain from pxf-1 leads to hypodermal defects, resulting in lethality. The cuticle secreted by pxf-1 mutants is disorganized and can often not be shed during molting. At later stages, hypodermal degeneration is seen and animals that reach adulthood frequently die with a burst vulva phenotype. Importantly, disruption of rap-1 leads to a similar, but less severe phenotype, which is enhanced by the simultaneous removal of rap-2. In addition, the lethal phenotype of pxf-1 can be rescued by expression of an activated version of rap-1. Together these results demonstrate that the pxf-1/rap pathway in C. elegans is required for maintenance of epithelial integrity, in which it probably functions in polarized secretion.

Protein kinase B activation and lamellipodium formation are independent phosphoinositide 3-kinase-mediated events differentially regulated by endogenous Ras.

Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events.

Collagen-rich stroma in aggressive colon tumors induces mesenchymal gene expression and tumor cell invasion.

Gene expression-based classification systems have identified an aggressive colon cancer subtype with mesenchymal features, possibly reflecting epithelial-to-mesenchymal transition (EMT) of tumor cells. However, stromal fibroblasts contribute extensively to the mesenchymal phenotype of aggressive colon tumors, challenging the notion of tumor EMT. To separately study the neoplastic and stromal compartments of colon tumors, we have generated a stroma gene filter (SGF). Comparative analysis of stromahigh and stromalow tumors shows that the neoplastic cells in stromahigh tumors express specific EMT drivers (ZEB2, TWIST1, TWIST2) and that 98% of differentially expressed genes are strongly correlated with them. Analysis of differential gene expression between mesenchymal and epithelial cancer cell lines revealed that hepatocyte nuclear factor 4α (HNF4α), a transcriptional activator of intestinal (epithelial) differentiation, and its target genes are highly expressed in epithelial cancer cell lines. However, mesenchymal-type cancer cell lines expressed only part of the mesenchymal genes expressed by tumor-derived neoplastic cells, suggesting that external cues were lacking. We found that collagen-I dominates the extracellular matrix in aggressive colon cancer. Mimicking the tumor microenvironment by replacing laminin-rich Matrigel with collagen-I was sufficient to induce tumor-specific mesenchymal gene expression, suppression of HNF4α and its target genes, and collective tumor cell invasion of patient-derived colon tumor organoids. The data connect collagen-rich stroma to mesenchymal gene expression in neoplastic cells and to collective tumor cell invasion. Targeting the tumor-collagen interface may therefore be explored as a novel strategy in the treatment of aggressive colon cancer.

Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras.

Ras is a small GTPase that cycles between an inactive GDP-bound and an active GTP-bound form. A large variety of ligands that stimulate cell surface receptors induce the activation of Ras. Thus far, this activation could only be measured by the increase of GTP bound to Ras, which was precipitated from radio-labelled cell extract. We have used the minimal Ras-binding domain (RBD) of Raf1 (aa 51-131) to identify in vivo activated Ras. This novel method is based on the observation that RBD binds RasGTP in vitro with a Kd of 20 nM whereas the affinity between RBD and RasGDP is three orders of magnitude lower. Here we show that the Gst-RBD fusion protein precipitates transfected RasL61 (RasGTP) but not RasN17 (RasGDP) from cell lysates. In addition, we demonstrate for two different cell lines that the increase in RasGTP is reflected by an increase in Ras bound to Gst-RBD. From these results we conclude that the minimal Ras-binding domain of Raf1 is an excellent activation specific-probe for Ras.

Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1.

Epac1 is a Rap-specific guanine-nucleotide exchange factor (GEF) which is activated by the binding of cAMP to a cyclic nucleotide monophosphate (cNMP)-binding domain. We investigated the equilibrium and dynamics of the interaction of cAMP and Epac1 using a newly designed fluorescence analogue of cAMP, 8-MABA-cAMP. We observed that the interaction of cAMP, measured by competition with 8-MABA-cAMP, with an isolated cNMP binding domain of Epac1 has an overall equilibrium constant (Kd) of 4 microM and that the kinetics of the interaction are highly dynamic. The binding properties of cAMP are apparently not affected when the catalytic domain is present, despite the fact that binding of cAMP results in activation of Epac1. This indicates that for the activation process, no appreciable binding energy is required. However, when bound to Rap1b, the apparent Kd of Epac to cAMP was about fivefold lower, suggesting that substrate interaction stabilizes cAMP binding. Since the fluorescent analogues used here were either less able or unable to induce activation of Epac1, we concluded that the binding of nucleotide to Epac and the activation of GEF activity are uncoupled processes and that thus appropriate cAMP analogues can be used as inhibitors of the Epac1-mediated signal transduction pathway of Rap.

The formation and remineralization of artificial white spot lesions: a constant composition approach.

Artificial white spot lesions have been prepared on bovine enamel surfaces by the controlled addition of lactate buffer containing methylhydroxydiphosphonate ions. The rates of remineralization have been measured using a constant composition method in solutions of calcium phosphate of low supersaturation. Deposition of new hydroxyapatite solid phase takes place exclusively within the white spot lesion at a rate appreciably slower than that of an acid-etched enamel surface, probably due to the presence of methylhydroxydiphosphonate located within the intact surface region of the lesion.

An econometric model of an episode of mental health care for patients with mild conditions: implications for caregiver substitution.

This article presents a model of the number of hours of mental health care, the concurrent improvement in the patient's condition, the probability the patient will receive medications, and the reasons for treatment termination. The variables related to these aspects of mental health care are analyzed separately for patients of psychiatrists, psychologists, and social workers. Estimates of the average length of treatment, the average price and income elasticities, and the average cost of treatment are obtained from the model. The major conclusions from this study are that psychiatrists do not have a benefit-cost advantage in the treatment of relatively mild conditions, and that consumer responsiveness to variations in price appear to be largely confined to the decision to seek treatment. These and other findings provide a basis for making tentative recommendations about personnel substitution and reimbursement policies in mental health.

Prediction scores or gastroenterologists' Gut Feeling for triaging patients that present with acute upper gastrointestinal bleeding.

INTRODUCTION: Several prediction scores for triaging patients with upper gastrointestinal (GI) bleeding have been developed, yet these scores have never been compared to the current gold standard, which is the clinical evaluation by a gastroenterologist. The aim of this study was to assess the added value of prediction scores to gastroenterologists' Gut Feeling in patients with a suspected upper GI bleeding. METHODS: WE PROSPECTIVELY EVALUATED GUT FEELING OF SENIOR GASTROENTEROLOGISTS AND ASKED THEM TO ESTIMATE: (1) the risk that a clinical intervention is needed; (2) the risk of rebleeding; and (3) the risk of mortality in patients presenting with suspected upper GI bleeding, subdivided into low, medium, or high risk. The predictive value of the gastroenterologists' Gut Feeling was compared to the Blatchford and Rockall scores for various outcomes. RESULTS: We included 974 patients, of which 667 patients (68.8%) underwent a clinical intervention. During the 30-day follow up, 140 patients (14.4%) developed recurrent bleeding and 44 patients (4.5%) died. Gut Feeling was independently associated with all studied outcomes, except for the predicted mortality after endoscopy. Predictive power, based on the AUC of the Blatchford and Rockall prediction scores, was higher than the Gut Feeling of the gastroenterologists. However, combining both the Blatchford and Rockall scores and the Gut Feeling yielded the highest predictive power for the need of an intervention (AUC 0.88), rebleeding (AUC 0.73), and mortality (AUC 0.71 predicted before and 0.77 predicted after endoscopy, respectively). CONCLUSIONS: Gut Feeling is an independent predictor for the need of a clinical intervention, rebleeding, and mortality in patients presenting with upper GI bleeding; however, the Blatchford and Rockall scores are stronger predictors for these outcomes. Combining Gut Feeling with the Blatchford and Rockall scores resulted in the most optimal prediction.

Randomized, double-blind, placebo-controlled, dose-escalating phase I, healthy subjects study of intravenous OPN-305, a humanized anti-TLR2 antibody.

Upregulation of Toll-like receptor 2 (TLR2) plays a critical role in inflammation associated with ischemia/reperfusion-induced tissue damage. OPN-305 is the first humanized IgG4 monoclonal antibody against TLR2 in development and is intended for the prevention of reperfusion injury following renal transplantation and other indications. A phase I, single-center, prospective randomized, double-blind, placebo-controlled study was performed to evaluate single ascending doses of OPN-305 in 41 healthy male subjects (age range: 19-58 years) randomized to OPN-305 or placebo across six cohorts. OPN-305 was well tolerated across all doses, with no elevations in endogenous cytokines. A dose-proportional increase in maximum serum concentration (Cmax) was observed, with area under the curve increasing in a greater-than-dose-proportional manner with increasing elimination half-life. OPN-305 produced full TLR2 receptor blockade on CD14(+)CD45(+) cells (monocytes), from 14 (0.5 mg/kg) to >90 (10 mg/kg) days, with a linear effect on the duration of inhibition of interleukin-6 release after TLR2 stimulation.

NUP98/JARID1A is a novel recurrent abnormality in pediatric acute megakaryoblastic leukemia with a distinct HOX gene expression pattern.

Cytogenetic abnormalities and early response to treatment are the main prognostic factors in acute myeloid leukemia (AML). Recently, NUP98/NSD1 (t(5; 11)(q35; p15)), a cytogenetically cryptic fusion, was described as recurrent event in AML, characterized by dismal prognosis and HOXA/B gene overexpression. Using split-signal fluorescence in situ hybridization, other NUP98-rearranged pediatric AML cases were identified, including several acute megakaryoblastic leukemia (AMKL) cases with a cytogenetically cryptic fusion of NUP98 to JARID1A (t(11;15)(p15;q35)). In this study we screened 105 pediatric AMKL cases to analyze the frequency of NUP98/JARID1A and other recurrent genetic abnormalities. NUP98/JARID1A was identified in 11/105 patients (10.5%). Other abnormalities consisted of RBM15/MKL1 (n=16), CBFA2T3/GLIS2 (n=13) and MLL-rearrangements (n=13). Comparing NUP98/JARID1A-positive patients with other pediatric AMKL patients, no significant differences in sex, age and white blood cell count were found. NUP98/JARID1A was not an independent prognostic factor for 5-year overall (probability of overall survival (pOS)) or event-free survival (probability of event-free survival (pEFS)), although the 5-year pOS for the entire AMKL cohort was poor (42 ± 6%). Cases with RBM15/MLK1 fared significantly better in terms of pOS and pEFS, although this was not independent from other risk factors in multivariate analysis. NUP98/JARID1A cases were characterized by HOXA/B gene overexpression, which is a potential druggable pathway. In conclusion, NUP98/JARID1A is a novel recurrent genetic abnormality in pediatric AMKL.

Differential suppression of dendritic cell cytokine production by anti-inflammatory drugs.

BACKGROUND: Various anti-inflammatory drugs are available for the treatment of skin disorders. In these diseases, untoward immune responses to endogenous and/or environmental antigens are initiated by maturation and polarization of dendritic cells (DC). OBJECTIVE: To explore the suppressive effects of anti-inflammatory drugs on DC maturation and, in particular, polarization. METHODS: Exposure of DC to nickel in vitro results in DC maturation and secretion of both type 1 and type 2 cytokines, thereby providing a model to study the effects of anti-inflammatory drugs on DC responses. The inhibitory effects of anti-inflammatory drugs (ciclosporin, dexamethasone, diclofenac, dimethylfumarate, hydrocortisone, lactoferrin, 1-alpha,25-dihydroxyvitamin D3) on DC maturation (CD83, CD86, HLA-DR, CXCL8) and polarization (type 1: IL-12p70, TNF-alpha; type 2: IL-10, CCL17) were studied. RESULTS: All anti-inflammatory drugs, except for lactoferrin, had inhibitory effects on DC maturation. Hydrocortisone and dexamethasone exclusively suppressed the release of type 1 cytokines. A less pronounced, but similar profile was observed for dimethylfumarate and 1-alpha,25-dihydroxyvitamin D3. Ciclosporin suppressed both type 1 and 2 cytokines. In contrast, diclofenac suppressed only type 2 DC cytokine secretion. CONCLUSION: The present results give more insight into the pharmacological effects of immunosuppressive drugs on the immune system, and can thereby contribute to a more rational selection of anti-inflammatory drugs for the treatment of inflammatory skin disorders.