Fibronectin is an important component of the extracellular matrix and is involved in a diverse range of physiological processes. It is a mosaic protein composed almost entirely of three types of module, F1, F2 and F3. Although the structures of single F1, F2 and F3 modules have been available for a number of years, in many cases the key to understanding the structure-function relationships in fibronectin and other proteins containing these modules lies in studies of module pairs and larger domains. This review focuses on recent advances in the understanding of the structure and function of fibronectin modules.

The IL-7 receptor (IL-7R) plays critical roles in expansion and V(D)J recombination during lymphocyte development. Here we demonstrate that cytokine stimulation rapidly recruits Stat5 and transcriptional coactivators to the Jgamma germline promoter and induces histone acetylation, germline transcription, and accessibility in Ba/F3 cells. We also show that histone acetylation of the TCRgamma locus is significantly reduced in IL-7R-deficient thymocytes and that the introduction of active Stat5 restores the histone acetylation and accessibility of the locus. Furthermore, treatment with histone deacetylase inhibitor recovers the histone acetylation and accessibility in IL-7R-deficient thymocytes. Therefore, these results suggest that Stat5 may recruit the transcriptional coactivators to the Jgamma germline promoter and control the accessibility of the TCRgamma locus by histone acetylation.

STAT5A and STAT5B proteins belong to the family of signal transducers and activators of transcription. They are encoded by two separate genes with 91% identity in their amino acid sequences. Despite their high degree of conservation, STAT5A and STAT5B exert non-redundant functions, resulting at least in part from differences in target gene activation. To better characterize the differential contribution of STAT5A and STAT5B in gene regulation, we performed single or double knockdown of STAT5A and STAT5B using small interfering RNA. Subsequent gene expression profiling and RT-qPCR analyses of IL-3-stimulated Ba/F3-beta cells led to the identification of putative novel STAT5 target genes. Chromatin immunoprecipitation assays analyzing the corresponding gene loci identified unusual STAT5 binding sites compared to conventional STAT5 responsive elements. Some of the STAT5 targets identified are upregulated in several human cancers, suggesting that they might represent potential oncogenes in STAT5-associated malignancies.

Pathogen-activated and damage-associated molecular patterns activate the inflammasome in macrophages. We report that mouse macrophages release IL-1β while co-incubated with pro-B (Ba/F3) cells dying, as a result of IL-3 withdrawal, by apoptosis with autophagy, but not when they are co-incubated with living, apoptotic, necrotic or necrostatin-1 treated cells. NALP3-deficient macrophages display reduced IL-1β secretion, which is also inhibited in macrophages deficient in caspase-1 or pre-treated with its inhibitor. This finding demonstrates that the inflammasome is activated during phagocytosis of dying autophagic cells. We show that activation of NALP3 depends on phagocytosis of dying cells, ATP release through pannexin-1 channels of dying autophagic cells, P(2)X(7) purinergic receptor activation, and on consequent potassium efflux. Dying autophagic Ba/F3 cells injected intraperitoneally in mice recruit neutrophils and thereby induce acute inflammation. These findings demonstrate that NALP3 performs key upstream functions in inflammasome activation in mouse macrophages engulfing dying autophagic cells, and that these functions lead to pro-inflammatory responses.

BACKGROUND

Chronic social instability during adolescence and early adulthood is known to produce a variety of long-lasting effects that may contribute to future psychiatric disorders. However, its potential to affect future generations has not been tested.

METHODS

Female and male mice were exposed to chronic social stress involving social instability and disruption of social hierarchy from postnatal day 27 to 76. After treatment, a group of animals was used to evaluate long-term behavioral effects of the stress exposure, and other mice were used to generate F1, F2, and F3 offspring, to test for behavioral effects across generations.

RESULTS

Chronic social instability during adolescence and early adulthood induces persistent behavioral alterations, including enhanced anxiety and social deficits that are transmitted predominantly to females across at least three generations. Both mothers and fathers can transmit all of these altered behaviors to their F1 offspring. However, only F1 fathers transmit all of them to their F2 and F3 daughters. In the F1 generation, enhanced anxiety and social deficits are associated with elevated serum corticosterone levels; however, in the F2 and F3 generations, they are not.

CONCLUSIONS

These findings support the idea that individual risk for psychiatric disorders that involve enhanced anxiety and/or social dysfunction may be dependent not only on the specific alleles of genes that are inherited from one's parents and on one's own experiences, but also on the experiences of one's parents when they were young.

To determine whether aberrantly activated tyrosine kinases other than FLT3 and c-KIT contribute to acute myeloid leukemia (AML) pathogenesis, we used high-throughput (HT) DNA sequence ana-lysis to screen exons encoding the activation loop and juxtamembrane domains of 85 tyrosine kinase genes in 188 AML patients without FLT3 or c-KIT mutations. The screen identified 30 nonsynonymous sequence variations in 22 different kinases not previously reported in single-nucleotide polymorphism (SNP) databases. These included a novel FLT3 activating allele and a previously described activating mutation in MET (METT1010I). The majority of novel sequence variants were stably expressed in factor-dependent Ba/F3 cells. Apart from one FLT3 allele, none of the novel variants showed constitutive phosphorylation by immunoblot analysis and none transformed Ba/F3 cells to factor-independent growth. These findings indicate the majority of these alleles are not potent tyrosine kinase activators in this cellular context and that a significant proportion of nonsynonymous sequence variants identified in HT DNA sequencing screens may not have functional significance. Although some sequence variants may represent SNPs, these data are consistent with recent reports that a significant fraction of such sequence variants are "passenger" rather than "driver" alleles and underscore the importance of functional assessment of candidate disease alleles.

The dorsolateral prefrontal cortex (DLPFC) has been implicated in the pathophysiology of several psychiatric illnesses including major depressive disorder and schizophrenia. In this regard, the DLPFC has been targeted in repetitive transcranial magnetic stimulation (rTMS) studies as a form of treatment to those patients who are resistant to medications. The '5-cm method' and the '10-20 method' for positioning the transcranial magnetic stimulation (TMS) coil over DLPFC have been scrutinised due to poor targeting accuracies attributed to inter-subject variability. We evaluated the accuracy of such methods to localise the DLPFC on the scalp in 15 healthy subjects and compared them with our novel neuronavigational method, which first estimates the DLPFC position in the cortex based on a standard template and then determines the most appropriate position on the scalp in which to place the TMS coil. Our neuronavigational method yielded a scalp position for the left DLPFC between electrodes F3 and F5 in standard space and was closest to electrode F5 in individual space. Further, we found that there was significantly less inter-subject variability using our neuronavigational method for localising the DLPFC on the scalp compared with the '5-cm method' and the '10-20 method'. Our findings also suggest that the '10-20 method' is superior to the '5-cm method' in reducing inter-subject variability and that electrode F5 should be the stimulation location of choice when MRI co-registration is not available.

OBJECTIVE

The aim of this study was to assess the accuracy of liver stiffness measurement (LSM) for the diagnosis of extensive fibrosis and cirrhosis in patients with alcoholic liver disease (ALD).

METHODS

One hundred and seventy-four patients with ALD were enrolled in four liver units and underwent concomitant liver biopsy and LSM. Fibrosis was assessed using the Brunt et al. and the Chevallier et al. scoring systems. Steatosis and histological alcoholic hepatitis (HAH) were quoted in classes.

RESULTS

Twenty-seven patients had inadequate biopsy or LSM. Distribution in 147 patients according to the Brunt score (median LSM) was: F1: n=13 (5.7kPa); F2: n=24 (8.3kPa); F3: n=31 (17.5kPa) and F4: n=79 (40.9kPa) (P<0.0001). LSM was correlated with the amount of fibrosis according to the Chevallier score (r=0.70, P<0.0001). LSM was correlated to fibrosis stage (tau beta, 0.53; P<0.0001) and HAH (tau beta, 0.30; P<0.0001). In multivariate analysis, fibrosis was the only parameter correlated with LSM. The areas under the ROC curve were 0.94 and 0.87 for the diagnosis of extensive fibrosis (Brunt et al. score>> or =3) and cirrhosis, respectively (threshold-values: 12.9 and 22.6kPa).

CONCLUSIONS

LSM accurately assesses extensive fibrosis and cirrhosis in alcoholic patients.

STK/RON tyrosine kinase, a member of the hepatocyte growth factor (HGF) receptor family, is a receptor for macrophage-stimulating protein (MSP). To examine the STK/RON signalling pathway, we generated STK/ RON transfectants showing opposite features in growth. STK/RON-expressing Ba/F3 pro-B cells (BaF/STK) exhibited MSP-dependent growth, whereas STK/ RON-expressing mouse erythroleukaemia cells (MEL/ STK) displayed MSP-induced apoptosis. This apoptosis was accompanied by the prolonged activation of c-Jun N-terminal kinase (JNK), which has recently been implicated in the initiation of apoptosis. Co-immunoprecipitation analyses showed that autophosphorylated STK/RON associated with PLC-gamma, P13-kinase, Shc and Grb2 in both transfectants. However, major tyrosine-phosphorylated proteins, p61 and p65, specifically associated with STK/RON in MEL/STK cells. Mutations at two C-terminal tyrosine residues, Y1330 and Y1337, in the counterpart of the multifunctional docking site of the HGF receptor abolished both MSP-induced growth and apoptosis. Analyses of these mutants and in vitro association revealed that signalling proteins including p61 and p65 directly bound to the phosphotyrosines in the multifunctional docking site. These results demonstrate that positive or negative signals toward cell growth are generated through the multifunctional docking site and suggest the involvement of p61 and p65 as well as JNK in apoptosis. Our findings provide the first evidence for apoptosis via a receptor tyrosine kinase.

OBJECTIVE

Fibrotest (FT) and Actitest (AT) are biochemical markers of fibrosis and activity for use as a non-invasive alternative to liver biopsy in patients with chronic hepatitis C virus (HCV). The aim of this study was to perform an external validation of FT and AT and to study the discordances between FT/AT and liver biopsy in patients with chronic hepatitis C.

METHODS

A total of 519 consecutive patients with chronic HCV were prospectively included in five centers, with liver biopsy and biochemical markers taken at the same day. Fifteen patients were excluded because their biopsies could not be interpreted. Diagnostic accuracies were assessed by receiver operating characteristic (ROC) curve analysis.

RESULTS

Median biopsy size was 15 mm (range: 2-58), with 9 portal tracts (1-37) and 1 fragment (1-12). 46% (230/504) were classified F2-F4 in fibrosis and 39% A2-A3 in activity. FT area under ROC curve for diagnosis of activity (A2-A3), significant fibrosis (F2-F4), and severe fibrosis (F3-F4) were 0.73 [0.69-0.77], 0.79 [0.75-0.82], and 0.80 [0.76-0.83], respectively. Among the 92 patients (18%) with 2 fibrosis stages of discordance between FT and biopsy, the discordance was attributable to FT in 5% of cases, to biopsy in 4%, and undetermined in 9%.

CONCLUSIONS

This prospective independent and multicenter study confirms the diagnostic value of FT and AT found in the princeps study and suggests that FT and AT can be an alternative to biopsy in most patients with chronic HCV.

Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

A study was performed in 10 European health care centers in which 914 patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) who had elevated serum alanine aminotransferase (ALT) levels underwent liver biopsy during the period of 1992 through 2002. Overall, the METAVIR liver fibrosis stage was F0 in 10% of patients, F1 in 33%, F2 in 22%, F3 in 22%, and F4 in 13%. Predictors of severe liver fibrosis (METAVIR stage, F3 or F4) in multivariate analysis were age of >35 years (odds ratio [OR], 2.95; 95% confidence interval [CI], 2.08-4.18), alcohol consumption of >50 g/day (OR, 1.61; 95% CI, 1.1-2.35), and CD4+ T cell count of <500 cells/mm3 (OR, 1.43; 95% CI, 1.03-1.98). Forty-six percent of patients aged >40 years had severe liver fibrosis, compared with 15% of subjects aged <30 years. The use of antiretroviral therapy was not associated with the severity of liver fibrosis. In summary, severe liver fibrosis is frequently found in HCV-HIV-coinfected patients with elevated serum ALT levels, and its severity increases significantly with age. The rate of complications due to end-stage liver disease will inevitably increase in this population, for whom anti-HCV therapy should be considered a priority.

A unique characteristic of neural stem cells is their capacity to track glioma cells that have migrated away from the main tumor mass into the normal brain parenchyma. PEX, a naturally occurring fragment of human metalloproteinase-2, acts as an inhibitor of glioma and endothelial cell proliferation, migration, and angiogenesis. In the present study, we evaluated the antitumor activity of PEX-producing human neural stem cells against malignant glioma. The HB1.F3 cell line (immortalized human neural stem cell) was transfected by a pTracer vector with PEX. The retention of the antiproliferative activity and migratory ability of PEX-producing HB1.F3 cells (HB1.F3-PEX) was confirmed in vitro. For the in vivo studies, DiI-labeled HB1.F3-PEX cells were stereotactically injected into established glioma tumor in nude mice. Tumor size was subsequently measured by magnetic resonance imaging and at the termination of the studies by histologic analysis including tumor volume, microvessel density, proliferation, and apoptosis rate. Histologic analysis showed that DiI-labeled HB1.F3-PEX cells migrate at the tumor boundary and cause a 90% reduction of tumor volume (P < 0.03). This reduction in tumor volume in animals treated with HB1.F3-PEX was associated with a significant decrease in angiogenesis (44.8%, P < 0.03) and proliferation (23.6%, P < 0.03). These results support the use of neural stem cells as delivery vehicle for targeting therapeutic genes against human glioma.

The mechanisms by which air pollution has multiple systemic effects in humans are not fully elucidated, but appear to include inflammation and thrombosis. This study examines whether concentrations of ozone and components of fine particle mass are associated with changes in methylation on tissue factor (F3), interferon gamma (IFN-γ), interleukin 6 (IL-6), toll-like receptor 2 (TLR-2), and intercellular adhesion molecule 1 (ICAM-1). We investigated associations between air pollution exposure and gene-specific methylation in 777 elderly men participating in the Normative Aging Study (1999-2009). We repeatedly measured methylation at multiple CpG sites within each gene's promoter region and calculated the mean of the position-specific measurements. We examined intermediate-term associations between primary and secondary air pollutants and mean methylation and methylation at each position with distributed-lag models. Increase in air pollutants concentrations was significantly associated with F3, ICAM-1, and TLR-2 hypomethylation, and IFN-γ and IL-6 hypermethylation. An interquartile range increase in black carbon concentration averaged over the four weeks prior to assessment was associated with a 12% reduction in F3 methylation (95% CI: -17% to -6%). For some genes, the change in methylation was observed only at specific locations within the promoter region. DNA methylation may reflect biological impact of air pollution. We found some significant mediated effects of black carbon on fibrinogen through a decrease in F3 methylation, and of sulfate and ozone on ICAM-1 protein through a decrease in ICAM-1 methylation.

H60, originally described as a dominant minor histocompatibility Ag, is an MHC class I-like molecule that serves as a ligand for the NKG2D receptor. In the present study, we identified two novel mouse chromosome 10-encoded NKG2D ligands structurally resembling H60. These ligands, which we named H60b and H60c, encode MHC class I-like molecules with two extracellular domains. Whereas H60b has a transmembrane region, H60c is a GPI-anchored protein. Recombinant soluble H60b and H60c proteins bound to NKG2D with affinities typical of cell-cell recognition receptors (K(d) = 310 nM for H60b and K(d) = 8.7 muM for H60c). Furthermore, expression of H60b or H60c rendered Ba/F3 cells susceptible to lysis by NK cells, thereby establishing H60b and H60c as functional ligands for NKG2D. H60b and H60c transcripts were detected only at low levels in tissues of healthy adult mice. Whereas H60b transcripts were detectable in various tissues, H60c transcripts were detected mainly in the skin. Infection of mouse embryonic fibroblasts with murine cytomegalovirus induced expression of H60b, but not H60c or the previously known H60 gene, indicating that transcriptional activation of the three types of H60 genes is differentially regulated. The present study adds two new members to the current list of NKG2D ligands.

OBJECTIVE

The c-kit gene encodes a receptor tyrosine kinase (KIT). Recently, we found gain-of-function mutations of the c-kit gene in gastrointestinal stromal tumors (GISTs). All mutations were confined within the 11 amino acids (Lys-550 to Val-560) in the juxtamembrane domain, but one GIST showed a novel deletion-type mutation at codon 579 (Asp) in the juxtamembrane domain. The aim of this study was to clarify whether the mutation is activating.

METHODS

Mutant c-kit cDNA was transfected into an interleukin 3 (IL-3)-dependent Ba/F3 murine lymphoid cell line, and the magnitude of autophosphorylation of the mutant KIT was examined with or without stem cell factor (SCF), a ligand of KIT. An in vitro kinase assay was also performed. The biological behavior of the transfectant was estimated by both an in vitro proliferation assay and in vivo transplantation to nude mice.

RESULTS

The mutant KIT exhibited constitutive phosphorylation and strong kinase activity without SCF. The transfectant grew autonomously without IL-3 and SCF, and it formed tumors in nude mice.

CONCLUSIONS

Deletion at codon 579 (Asp) in the juxtamembrane domain of the c-kit gene is a novel gain-of-function mutation other than the region between Lys-550 and Val-560.

Transcranial direct current stimulation (tDCS) is a brain stimulation technique that has the potential to improve working memory (WM) deficits in many clinical disorders. The aim of this study was to investigate the role of current strength on the ability of anodal tDCS to improve WM, and secondly to investigate the time course of effects. Twelve healthy participants underwent three stimulation sessions consisting of 20 min of either 1 mA anodal tDCS, 2 mA anodal tDCS, or sham tDCS to the left dorsolateral prefrontal cortex (DLPFC) localized via F3, all whilst completing a WM task. Intra-stimulation and post-stimulation WM performances were measured using the n-back and Sternberg tasks respectively. Results revealed no significant improvements in participants' accuracy, but a significant interaction was found with respect to current strength and time for accurate reaction time. The finding provides partial support for the hypothesis, in that it appears current strength may affect aspects of WM performance. However, more research is needed, and a higher difficulty level of WM tasks is one of the suggestions discussed for future research.

BACKGROUND

Oncolytic adenoviral virotherapy (OV) is a highly promising approach for the treatment of glioblastoma multiforme (GBM). In practice, however, the approach is limited by poor viral distribution and spread throughout the tumor mass.

METHODS

To enhance viral delivery, replication, and spread, we used a US Food and Drug Administration-approved neural stem cell line (NSC), HB1.F3.CD, which is currently employed in human clinical trials. HB1.F3.CD cells were loaded with an oncolytic adenovirus, CRAd-Survivin-pk7, and mice bearing various human-derived GBMs were assessed with regard to NSC migration, viral replication, and therapeutic efficacy. Survival curves were evaluated with Kaplan-Meier methods. All statistical tests were two-sided.

RESULTS

Antiglioma activity of OV-loaded HB1.F3.CD cells was effective against clinically relevant human-derived glioma models as well as a glioma stem cell-enriched xenograft model. Median survival was prolonged by 34% to 50% compared with mice treated with OV alone (GBM43FL model median survival = 19.5 days, OV alone vs NSC + OV, hazard ratio of survival = 2.26, 95% confidence interval [CI] = 1.21 to 12.23, P = .02; GBM12 model median survival = 43.5 days, OV alone vs NSC + OV, hazard ratio of survival = 2.53, 95% CI = 1.21 to 10.38, P = .02). OV-loaded HB1.F3.CD cells were shown to effectively migrate to the contralateral hemisphere and hand off the therapeutic payload of OV to targeted glioma cells. In vivo distribution and migratory kinetics of the OV-loaded HB1.F3.CD cells were successfully monitored in real time by magnetic resonance imaging. OV-loaded NSCs retained their differentiation fate and were nontumorigenic in vivo.

CONCLUSIONS

HB1.F3.CD NSCs loaded with CRAd-Survivin-pk7 overcome major limitations of OV in vivo and warrant translation in a phase I human clinical trial for patients with GBM.

Bisphenol A (BPA) is a man-made endocrine disrupting compound used to manufacture polycarbonate plastics. It is found in plastic bottles, canned food linings, thermal receipts and other commonly used items. Over 93% of people have detectable BPA levels in their urine. Epidemiological studies report correlations between BPA levels during pregnancy and activity, anxiety, and depression in children. We fed female mice control or BPA-containing diets that produced plasma BPA concentrations similar to concentrations in humans. Females were mated and at birth, pups were fostered to control dams to limit BPA exposure to gestation in the first generation. Sibling pairs were bred to the third generation with no further BPA exposure. First (F1) and third (F3) generation juveniles were tested for social recognition and in the open field. Adult F3 mice were tested for olfactory discrimination. In both generations, BPA exposed juvenile mice displayed higher levels of investigation than controls in a social recognition task. In F3 BPA exposed mice, dishabituation to a novel female was impaired. In the open field, no differences were noted in F1 mice, while in F3, BPA lineage mice were more active than controls. No impairments were detected in F3 mice, all were able to discriminate different male urine pools and urine from water. No sex differences were found in any task. These results demonstrate that BPA exposure during gestation has long lasting, transgenerational effects on social recognition and activity in mice. These findings show that BPA exposure has transgenerational actions on behavior and have implications for human neurodevelopmental behavioral disorders.

BACKGROUND

Colistin-carbapenem combinations are synergistic in vitro against carbapenem-resistant Gram-negative bacteria. We aimed to test whether combination therapy improves clinical outcomes for adults with infections caused by carbapenem-resistant or carbapenemase-producing Gram-negative bacteria.

METHODS

A randomised controlled superiority trial was done in six hospitals in Israel, Greece, and Italy. We included adults with bacteraemia, ventilator-associated pneumonia, hospital-acquired pneumonia, or urosepsis caused by carbapenem-non-susceptible Gram-negative bacteria. Patients were randomly assigned (1:1) centrally, by computer-generated permuted blocks stratified by centre, to intravenous colistin (9-million unit loading dose, followed by 4·5 million units twice per day) or colistin with meropenem (2-g prolonged infusion three times per day). The trial was open-label, with blinded outcome assessment. Treatment success was defined as survival, haemodynamic stability, improved or stable Sequential Organ Failure Assessment score, stable or improved ratio of partial pressure of arterial oxygen to fraction of expired oxygen for patients with pneumonia, and microbiological cure for patients with bacteraemia. The primary outcome was clinical failure, defined as not meeting all success criteria by intention-to-treat analysis, at 14 days after randomisation. This trial is registered at ClinicalTrials.gov, number NCT01732250, and is closed to accrual.

RESULTS

Between Oct 1, 2013, and Dec 31, 2016, we randomly assigned 406 patients to the two treatment groups. Most patients had pneumonia or bacteraemia (355/406, 87%), and most infections were caused by Acinetobacter baumannii (312/406, 77%). No significant difference between colistin monotherapy (156/198, 79%) and combination therapy (152/208, 73%) was observed for clinical failure at 14 days after randomisation (risk difference -5·7%, 95% CI -13·9 to 2·4; risk ratio [RR] 0·93, 95% CI 0·83-1·03). Results were similar among patients with A baumannii infections (RR 0·97, 95% CI 0·87-1·09). Combination therapy increased the incidence of diarrhoea (56 [27%] vs 32 [16%] patients) and decreased the incidence of mild renal failure (37 [30%] of 124 vs 25 [20%] of 125 patients at risk of or with kidney injury).

CONCLUSIONS

Combination therapy was not superior to monotherapy. The addition of meropenem to colistin did not improve clinical failure in severe A baumannii infections. The trial was unpowered to specifically address other bacteria.

BACKGROUND

EU AIDA grant Health-F3-2011-278348.

Ellagitannins (ETs) are dietary polyphenols, containing ellagic acid (EA) subunits, with antioxidant and cancer chemopreventive activities that might contribute to health benefits in humans. However, little is known about their metabolic fate. We investigate here the metabolism of different dietary ETs and EA derivatives in humans. Forty healthy volunteers were distributed in four groups. Each group consumed, in a single dose, a different ET-containing foodstuff, i.e., strawberries (250 g), red raspberries (225 g), walnuts (35 g), and oak-aged red wine (300 mL). After the intake, five urine fractions (F) were collected at 8 (F1), 16 (F2), 32 (F3), 40 (F4), and 56 (F5) h. Neither ETs nor EA were detected in urine after LC-MS/MS analysis. However, the microbial metabolite 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one (urolithin B) conjugated with glucuronic acid was detected along the fractions F3-F5 in all of the subjects, independently of the consumed foodstuff. The mean percentage of metabolite excretion ranged from 2.8 (strawberries) to 16.6% (walnuts) regarding the ingested ETs. Considerable interindividual differences were noted, identifying "high and low metabolite excreters" in each group, which supported the involvement of the colonic microflora in ET metabolism. These results indicate that urolithin B (a previously described antiangiogenic and hyaluronidase inhibitor compound) is a biomarker of human exposure to dietary ETs and may be useful in intervention studies with ET-containing products. The antioxidant and anticarcinogenic effects of dietary ETs and EA should be considered in the gastrointestinal tract whereas the study of potential systemic activities should be focused on the bioavailable urolithin B derivatives.

OBJECTIVE

To assess how intrahepatic fat and insulin resistance relate to daily fructose and energy intake during short-term overfeeding in healthy subjects.

METHODS

The analysis of the data collected in several studies in which fasting hepatic glucose production (HGP), hepatic insulin sensitivity index (HISI), and intrahepatocellular lipids (IHCL) had been measured after both 6-7 days on a weight-maintenance diet (control, C; n = 55) and 6-7 days of overfeeding with 1.5 (F1.5, n = 7), 3 (F3, n = 17), or 4 g fructose/kg/day (F4, n = 10), with 3 g glucose/kg/day (G3, n = 11), or with 30% excess energy as saturated fat (fat30%, n = 10).

RESULTS

F3, F4, G3, and fat30% all significantly increased IHCL, respectively by 113 ± 86, 102 ± 115, 59 ± 92, and 90 ± 74% as compared to C (all P < 0.05). F4 and G3 increased HGP by 16 ± 10 and 8 ± 11% (both P < 0.05), and F3 and F4 significantly decreased HISI by 20 ± 22 and 19 ± 14% (both P < 0.01). In contrast, there was no significant effect of fat30% on HGP or HISI.

CONCLUSIONS

Short-term overfeeding with fructose or glucose decreases hepatic insulin sensitivity and increases hepatic fat content. This indicates short-term regulation of hepatic glucose metabolism by simple carbohydrates.

A general feature of the cell adhesion molecules belonging to the immunoglobulin family (Ig-CAMs) is to display a modular structure that provides a framework for multiple binding sites for other recognition molecules. Among this family, F3/contactin is a glycan phosphatidyl-inositol (GPI)-anchored molecule expressed by neurons that displays the distinctiveness to exert heterophilic but no homophilic binding activities. The Ig domains of F3/contactin were shown to interact with the L1 family of Ig-CAMs, including L1, NrCAM, and neurofascin. Binding between F3/contactin and NrCAM is known to modulate axonal elongation of the cerebellar granule cells and to control sensory axon guidance. F3/contactin mediates neuron-glial contacts through its association with extracellular matrix components (tenascin-R, tenascin-C) and RPTPbeta/phosphacan, influencing axonal growth and fasciculation. Another major role of F3/contactin is to organize axonal subdomains at the node of Ranvier of myelinated fibers in interplay with other Ig-CAMs, through its binding with caspr/paranodin at paranodes and the voltage-gated sodium channels in the nodal region. The F3/contactin deficient mice display a severe ataxia correlated with defects in axonal and dendritic projections in the cerebellum. These mice also display defects in nerve influx conduction due to the disruption of the axo-glial contacts at paranodes. Finally, the recent identification of a Drosophila homologue of F3/contactin indicated that this family of GPI-anchored CAMs plays a conserved function in axonal insulation.

Thrombopoietin (TPO) acts through its receptor, Mpl, to stimulate the proliferation and maturation of megakaryocytes and their progenitors. The Mpl cytoplasmic domain controls this process through assembly of an active signaling complex using various receptor docking sites. In this report, eight carboxyl truncations of the 121-aa murine Mpl cytoplasmic domain were tested for the ability to support growth of a cytokine-dependent cell line (Ba/F3) and for their capacity to induce TPO-stimulated tyrosine phosphorylation of specific signaling proteins. Point mutations of the five tyrosine residues in the cytoplasmic domain of the receptor were subsequently used to confirm our conclusions. From these studies we demonstrate that: (i) TPO-induced proliferation is moderately reduced by truncation of as many as 53 C-terminal amino acids of Mpl, including the sites of receptor tyrosine phosphorylation; (ii) truncation/mutation of residues 69-83 of the Mpl cytoplasmic domain enhances proliferative signaling, perhaps mediated by a decrease in receptor-driven cellular differentiation; (iii) Mpl can be phosphorylated at either Y112 or Y117 but not at the three proximal cytoplasmic tyrosine residues (Y8, Y29, and Y78); (iv) Y112 of Mpl is necessary for tyrosine phosphorylation of Shc and Shc-associated p145 (SHIP); and (v) unlike STAT3, STAT5 is partially phosphorylated in the absence of any tyrosine residues in the Mpl cytoplasmic domain. These studies identify subdomains of Mpl necessary for activation of several critical signaling pathways and point to two potentially novel mechanisms of TPO-induced signal transduction, an indirect pathway to STAT5 activation and a differentiation domain that acts by limiting proliferation.