Fucoidan, a group of sulfated heteropolysaccharide, was extracted from Laminaria japonica, an important economic alga species in China. Three sulfated polysaccharide fractions (F1, F2, and F3) were successfully isolated through anion-exchange column chromatography and had their antioxidant activities investigated employing various established in vitro systems, including superoxide and hydroxyl radical scavenging activity, chelating ability, and reducing power. Chemical analysis suggested that F1 and F3 were heteropolysaccharide in which galactose was the major component, while F2 was a typical fucoidan. All fractions possessed considerable antioxidant activity, and F1, F2 and F3 had stronger antioxidant ability than fucoidan in certain tests. The correlation between the sulfate content and scavenging superoxide radical ability was positive. Available data obtained with in vitro models suggested that the ratio of sulfate content/fucose was an effective indicator to antioxidant activity of the samples.

The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation.

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling, which is implicated in MPD pathogenesis. We developed TG101209, an orally bioavailable small molecule that potently inhibits JAK2 (IC(50)=6 nM), FLT3 (IC(50)=25 nM) and RET (IC(50)=17 nM) kinases, with significantly less activity against other tyrosine kinases including JAK3 (IC(50)=169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC(50) of approximately 200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line, TG101209-induced cell cycle arrest and apoptosis, and inhibited phosphorylation of JAK2V617F, STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore, TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations.

BACKGROUND

The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT.

RESULTS

In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC(50)) of 200+/-40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC(50) of 150+/-80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant.

CONCLUSIONS

Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.

OBJECTIVE

To compare shaping ability and cleaning effectiveness of two reciprocating single-file systems with Mtwo and ProTaper rotary instruments during the preparation of curved root canals in extracted teeth.

METHODS

A total of 80 root canals with curvatures ranging between 25° and 39° were divided into four groups of 20 canals. Based on radiographs taken prior to instrumentation, the groups were balanced with respect to the angle and the radius of canal curvature. Canals were prepared to the following apical sizes: Mtwo: size 35 using the single-length technique; ProTaper: F3, instruments were used in a modified crown-down manner; Reciproc and WaveOne: size 25. Using pre- and post-instrumentation radiographs, straightening of the canal curvatures was determined with a computer image analysis program. Preparation time and instrument failures were also recorded. These data were analysed statistically using anova and Student-Newman-Keuls test. The amounts of debris and smear layer were quantified on the basis of a numerical evaluation scale and were analysed statistically using the Kruskal-Wallis test.

RESULTS

During preparation no file fractured. All instruments maintained the original canal curvature well with no significant differences between the different files (P = 0.382). Instrumentation with Reciproc was significantly faster than with all other instruments (P < 0.05), while WaveOne was significantly faster than Mtwo and ProTaper (P < 0.05). For debris removal, Mtwo and Reciproc instruments achieved significantly better results (P < 0.05) than the other instruments in the apical third of the canals. In the middle and coronal parts, no significant differences were obtained between Mtwo, Reciproc and WaveOne (P>> 0.05), while ProTaper showed significantly more residual debris (P < 0.05). The results for remaining smear layer were similar and not significantly different for the different parts of the canals (P>> 0.05).

CONCLUSIONS

Under the conditions of this study, all instruments maintained the original canal curvature well and were safe to use. The use of Mtwo and Reciproc instruments resulted in better canal cleanliness in the apical part compared with ProTaper and WaveOne.

Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non-small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal-regulated kinase (ERK)-1/2 in human 293T cells and to growth factor-independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma.

OBJECTIVE

The current study aims to evaluate the performance of a new elastographic method (ARFI) in noninvasive fibrosis assessment and to compare it to another validated technology (transient elastography, TE).

METHODS

112 consecutive chronic hepatitis C patients (histologically proven according to the Metavir scoring system: 12.5% F0, 26.6% F1, 16.1% F2, 7.1% F3, 37.5% F4) were prospectively included in this study. They were examined on the same day, using both ARFI (with shear wave velocity--SWV-quantification) and TE (with liver stiffness quantification).

RESULTS

SWV is correlated only with fibrosis (r=0.717, p less than 0.0001) and necroinflammatory activity (r=0.328, p=0.014), but not with steatosis (r=0.122, p=0.321). There is a significant increase of SWV in parallel with the increase in the fibrosis stage: 1.079+/-0.150 (F0-F1), 1.504+/-0.895 (F2), 1.520+/-0.575 (F3), 2.552+/-0.782 (F4), p<0.0001, but there is a certain degree of overlap between the consecutive stages F1-F2 (p=0.072), F2-F3 (p=0.965). SWV cut-off values (m/s) that were predictive for each fibrosis stage were: 1.19 (F more or equal to 1), 1.34 (F>or=2), 1.61 (F more or equal to 3) and 2.00 (F4). AUROC for ARFI vs TE were: 0.709 vs 0.902, p=0.006 (F>or=1), 0.851 vs 0.941, p=0.022 (F>or=2), 0.869 vs 0.926, p=0.153 (F>or=3) and 0.911 vs 0.945, p=0.331 (F4).

CONCLUSIONS

ARFI allows SWV quantification, in strong correlation with the fibrosis stage. Steatosis does not influence SWV. The maximal performance of the method consists of the prediction in severe fibrosis and cirrhosis. The diagnostic accuracy is strongly comparable to TE only for the prediction of severe fibrosis and cirrhosis, whereas for earlier stages, TE performs better.

Bisphenol A (BPA) was evaluated at concentrations of 0, 0.015, 0.3, 4.5, 75, 750, and 7500 ppm ( approximately 0.001, 0.02, 0.3, 5, 50, and 500 mg/kg/day of BPA) administered in the diet ad libitum to 30 CD((R)) Sprague-Dawley rats/sex/dose for 3 offspring generations, 1 litter/generation, through F3 adults. Adult systemic toxicity at 750 and 7500 ppm in all generations included: reduced body weights and body weight gains, reduced absolute and increased relative weanling and adult organ weights (liver, kidneys, adrenals, spleen, pituitary, and brain), and female slight/mild renal and hepatic pathology at 7500 ppm. Reproductive organ histopathology and function were unaffected. Ovarian weights as well as total pups and live pups/litter on postnatal day (PND) 0 were decreased at 7500 ppm, which exceeded the adult maximum tolerated dose (MTD). Mating, fertility, gestational indices; ovarian primordial follicle counts; estrous cyclicity; precoital interval; gestational length; offspring sex ratios; postnatal survival; nipple/areolae retention in preweanling males; epididymal sperm number, motility, morphology; daily sperm production (DSP), and efficiency of DSP were all unaffected. At 7500 ppm, vaginal patency (VP) and preputial separation (PPS) were delayed in F1, F2, and F3 offspring, associated with reduced body weights. Anogenital distance (AGD) on PND 0 was unaffected for F2 and F3 males and F3 females (F2 female AGD was increased at some doses, not at 7500 ppm, and was considered not biologically or toxicologically relevant). Adult systemic no observed adverse effect level (NOAEL) = 75 ppm (5 mg/kg/day); reproductive and postnatal NOAELs = 750 ppm (50 mg/kg/day). There were no treatment-related effects in the low-dose region (0.001-5 mg/kg/day) on any parameters and no evidence of nonmonotonic dose-response curves across generations for either sex. BPA should not be considered a selective reproductive toxicant, based on the results of this study.

Mutations in codon D816 of the KIT gene represent a recurrent genetic alteration in acute myeloid leukemia (AML). To clarify the biologic implication of activation loop mutations of the KIT gene, 1940 randomly selected AML patients were analyzed. In total, 33 (1.7%) of 1940 patients were positive for D816 mutations. Of these 33 patients, 8 (24.2%) had a t(8;21), which was significantly higher compared with the subgroup without D816 mutations. Analyses of genetic subgroups showed that KIT-D816 mutations were associated with t(8;21)/AML1-ETO and other rare AML1 translocations. In contrast, other activating mutations like FLT3 and NRAS mutations were very rarely detected in AML1-rearranged leukemia. KIT mutations had an independent negative impact on overall (median 304 vs 1836 days; P = .006) and event-free survival (median 244 vs 744 days; P = .003) in patients with t(8;21) but not in patients with a normal karyotype. The KIT-D816V receptor expressed in Ba/F3 cells was resistant to growth inhibition by the selective PTK inhibitors imatinib and SU5614 but fully sensitive to PKC412. Our findings clearly indicate that activating mutations of receptor tyrosine kinases are associated with distinct genetic subtypes in AML. The KIT-D816 mutations confer a poor prognosis to AML1-ETO-positive AML and should therefore be included in the diagnostic workup. Patients with KIT-D816-positive/AML1-ETO-positive AML might benefit from early intensification of treatment or combination of conventional chemotherapy with KIT PTK inhibitors.

Non-alcoholic fatty liver disease (NAFLD) is very common in the general population, but identifying patients with increased risk of mortality and liver-specific morbidity remains a challenge. Non-alcoholic steatohepatitis (NASH) is thought to enhance this risk; therefore, resolution of NASH is a major endpoint in current pharmacologic studies. Herein, we aim to investigate the long-term prognosis of a large cohort of NAFLD patients, and to study the specific effect of NASH and fibrosis stage on prognosis.

We conducted a retrospective cohort study of 646 biopsy-proven NAFLD patients. Each case was matched for age, sex and municipality to ten controls. Outcomes on mortality and severe liver disease, defined as cirrhosis, liver decompensation/failure or hepatocellular carcinoma, were evaluated using population-based registers. Cox regression models adjusted for age, sex and type 2 diabetes were used to examine the long-term risk according to fibrosis stage. Likelihood ratio tests were used to assess whether adding NASH to these models increased the predictive capacity. Laplace regression was used to estimate the time to severe liver disease according to stage of fibrosis.

During a follow-up of mean 20years (range 0-40) equivalent to 139,163 person-years, 12% of NAFLD patients and 2.2% of controls developed severe liver disease (p<0.001). Compared to controls, the risk of severe liver disease increased per stage of fibrosis (hazard ratio ranging from 1.9 in F0 to 104.9 in F4). Accounting for the presence of NASH did not change these estimates significantly (likelihood ratio test >0.05 for all stages of fibrosis). Similar results were seen for overall mortality. The lower end of the 95% confidence interval for the 10th percentile of time to development of severe liver disease was 22-26years in F0-1, 9.3years in F2, 2.3years in F3, and 0.9years to liver decompensation in F4.

In this, the largest ever study of biopsy-proven NAFLD, the presence of NASH did not increase the risk of liver-specific morbidity or overall mortality. Knowledge of time to development of severe liver disease according to fibrosis stage can be used in individual patient counselling and for public health decisions.

Non-alcoholic fatty liver disease (NAFLD) is very common in the general population, but reaching an accurate prognosis remains challenging. We investigate the long-term prognosis of a large cohort of NAFLD patients. In this, the largest ever study of biopsy-proven NAFLD, the presence of NASH did not increase the risk of liver-specific morbidity or overall mortality. Knowledge of time to development of severe liver disease according to fibrosis stage can be used in individual patient counselling and for public health decisions.

The laminar pattern of cytochrome oxidase activity was studied in the agranular frontal cortex (area 4-6 complex) of the macaque monkey. The cortex, stained with this method, showed 6 stripes of different enzymatic activity. On the basis of their characteristics and of the presence of highly active cells, the agranular frontal cortex could be parcellated in 5 areas (F1-F5). F1 very likely corresponds to area FA of von Bonin and Bailey. Rostral to F1 two large regions could be distinguished, one located medial to the spur of the arcuate sulcus and its imaginary caudal extension, the other laterally. The superior region was formed by areas F2 and F3. The first was located on the dorsomedial cortical surface, the other on the mesial surface. F3 possibly corresponds to the supplementary motor area. The inferior region was formed by areas F4 and F5. The rostral area (F5) showed transition characteristics that rendered it somehow similar to the prefrontal areas. It may correspond to the cytoarchitectonic area FCBm. The cytocrome oxidase technique is a useful means of parcellating the agranular frontal cortex and may greatly help in physiological and behavioral experiments.

We determined contrast thresholds for lesion detection as a function of lesion size in both mammograms and filtered noise backgrounds with the same average power spectrum, P(f)=B/f3. Experiments were done using hybrid images with digital images of tumors added to digitized normal backgrounds, displayed on a monochrome monitor. Four tumors were extracted from digitized specimen radiographs. The lesion sizes were varied by digital rescaling to cover the range from 0.5 to 16 mm. Amplitudes were varied to determine the value required for 92% correct detection in two-alternative forced-choice (2AFC) and 90% for search experiments. Three observers participated, two physicists and a radiologist. The 2AFC mammographic results demonstrated a novel contrast-detail (CD) diagram with threshold amplitudes that increased steadily (with slope of 0.3) with increasing size for lesions larger than 1 mm. The slopes for prewhitening model observers were about 0.4. Human efficiency relative to these models was as high as 90%. The CD diagram slopes for the 2AFC experiments with filtered noise were 0.44 for humans and 0.5 for models. Human efficiency relative to the ideal observer was about 40%. The difference in efficiencies for the two types of backgrounds indicates that breast structure cannot be considered to be pure random noise for 2AFC experiments. Instead, 2AFC human detection with mammographic backgrounds is limited by a combination of noise and deterministic masking effects. The search experiments also gave thresholds that increased with lesion size. However, there was no difference in human results for mammographic and filtered noise backgrounds, suggesting that breast structure can be considered to be pure random noise for this task. Our conclusion is that, in spite of the fact that mammographic backgrounds have nonstationary statistics, models based on statistical decision theory can still be applied successfully to estimate human performance.

Environmental compounds can promote epigenetic transgenerational inheritance of adult-onset disease in subsequent generations following ancestral exposure during fetal gonadal sex determination. The current study examined the ability of dioxin (2,3,7,8-tetrachlorodibenzo[p]dioxin, TCDD) to promote epigenetic transgenerational inheritance of disease and DNA methylation epimutations in sperm. Gestating F0 generation females were exposed to dioxin during fetal day 8 to 14 and adult-onset disease was evaluated in F1 and F3 generation rats. The incidences of total disease and multiple disease increased in F1 and F3 generations. Prostate disease, ovarian primordial follicle loss and polycystic ovary disease were increased in F1 generation dioxin lineage. Kidney disease in males, pubertal abnormalities in females, ovarian primordial follicle loss and polycystic ovary disease were increased in F3 generation dioxin lineage animals. Analysis of the F3 generation sperm epigenome identified 50 differentially DNA methylated regions (DMR) in gene promoters. These DMR provide potential epigenetic biomarkers for transgenerational disease and ancestral environmental exposures. Observations demonstrate dioxin exposure of a gestating female promotes epigenetic transgenerational inheritance of adult onset disease and sperm epimutations.

OBJECTIVE

Pediatric gastrointestinal stromal tumors (GIST) are rare and occur preferentially in females as multifocal gastric tumors, typically lacking mutations in KIT and PDGFRA. As KIT oncoprotein is consistently overexpressed in pediatric GIST, we sought to investigate the activation of KIT downstream targets and alterations of KIT/PDGFRA gene copy number, mine novel therapeutic targets by gene expression, and test tyrosine kinase receptor activation by proteomic profiling.

METHODS

Seventeen pediatric GISTs were investigated for KIT/PDGFRA genotype and biochemical activation of KIT downstream targets. The transcriptional profile of 13 nodules from 8 pediatric patients was compared with 8 adult wild-type (WT) GISTs, including 3 young adults. The drug sensitivity of second-generation kinase inhibitors was tested in murine Ba/F3 cells expressing human WT KIT, as well as in short-term culture of explants of WT GIST cells.

RESULTS

A KIT/PDGFRA WT genotype was identified in all 12 female patients, whereas two of five males had either a KIT exon 11 or PDGFRA exon 18 mutation. KIT downstream targets were consistently activated. Pediatric GISTs showed a distinct transcriptional signature, with overexpression of BAALC, PLAG1, IGF1R, FGF4, and NELL1. In vitro studies showed that nilotinib, sunitinib, dasatinib, and sorafenib are more effective than imatinib against WT KIT.

CONCLUSIONS

Rare cases of pediatric GIST may occur in male patients and harbor activating KIT/PDGFRA mutations. Pediatric GISTs show distinct transcriptional signature, suggesting a different biology than WT GIST in adults. In vitro drug screening showed that second-generation kinase inhibitors may provide greater clinical benefit in pediatric GIST.

OBJECTIVE

While several risk factors for the histological progression of chronic hepatitis C have been identified, the contribution of HCV genotypes to liver fibrosis evolution remains controversial. The aim of this study was to assess independent predictors for fibrosis progression.

METHODS

We identified 1189 patients from the Swiss Hepatitis C Cohort database with at least one biopsy prior to antiviral treatment and assessable date of infection. Stage-constant fibrosis progression rate was assessed using the ratio of fibrosis Metavir score to duration of infection. Stage-specific fibrosis progression rates were obtained using a Markov model. Risk factors were assessed by univariate and multivariate regression models.

RESULTS

Independent risk factors for accelerated stage-constant fibrosis progression (>0.083 fibrosis units/year) included male sex (OR=1.60, [95% CI 1.21-2.12], P<0.001), age at infection (OR=1.08, [1.06-1.09], P<0.001), histological activity (OR=2.03, [1.54-2.68], P<0.001) and genotype 3 (OR=1.89, [1.37-2.61], P<0.001). Slower progression rates were observed in patients infected by blood transfusion (P=0.02) and invasive procedures or needle stick (P=0.03), compared to those infected by intravenous drug use. Maximum likelihood estimates (95% CI) of stage-specific progression rates (fibrosis units/year) for genotype 3 versus the other genotypes were: F0->>F1: 0.126 (0.106-0.145) versus 0.091 (0.083-0.100), F1->>F2: 0.099 (0.080-0.117) versus 0.065 (0.058-0.073), F2->>F3: 0.077 (0.058-0.096) versus 0.068 (0.057-0.080) and F3->>F4: 0.171 (0.106-0.236) versus 0.112 (0.083-0.142, overall P<0.001).

CONCLUSIONS

This study shows a significant association of genotype 3 with accelerated fibrosis using both stage-constant and stage-specific estimates of fibrosis progression rates. This observation may have important consequences for the management of patients infected with this genotype.

The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease.

The actions of environmental toxicants and relevant mixtures in promoting the epigenetic transgenerational inheritance of ovarian disease was investigated with the use of a fungicide, a pesticide mixture, a plastic mixture, dioxin and a hydrocarbon mixture. After transient exposure of an F0 gestating female rat during embryonic gonadal sex determination, the F1 and F3 generation progeny adult onset ovarian disease was assessed. Transgenerational disease phenotypes observed included an increase in cysts resembling human polycystic ovarian disease (PCO) and a decrease in the ovarian primordial follicle pool size resembling primary ovarian insufficiency (POI). The F3 generation granulosa cells were isolated and found to have a transgenerational effect on the transcriptome and epigenome (differential DNA methylation). Epigenetic biomarkers for environmental exposure and associated gene networks were identified. Epigenetic transgenerational inheritance of ovarian disease states was induced by all the different classes of environmental compounds, suggesting a role of environmental epigenetics in ovarian disease etiology.

Conducting gramicidin channels form predominantly by the transmembrane association of monomers, one from each side of a lipid bilayer. In single-channel experiments in planar bilayers the two gramicidin analogs, [Val1]gramicidin A (gA) and [4,4,4-F3-Val1]gramicidin A (F3gA), form dimeric channels that are structurally equivalent and have characteristically different conductances. When these gramicidins were added asymmetrically, one to each side of a preformed bilayer, the predominant channel type was the hybrid channel, formed between two chemically dissimilar monomers. These channels formed by the association of monomers residing in each half of the membrane. These results also indicate that the hydrophobic gramicidins are surprisingly membrane impermeant, a conclusion that was confirmed in experiments in which gA was added asymmetrically and symmetrically to preformed bilayers.

Hypoxia is a critical aspect of the microenvironment in glioma and generally signifies unfavorable clinical outcome. Effective targeting of hypoxic areas in gliomas remains a significant therapeutic challenge. New therapeutic platforms using neural stem cells (NSC) for tumor-targeted drug delivery show promise in treatment of cancers that are refractory to traditional therapies. However, the molecular mechanisms of NSC targeting to hypoxic tumor areas are not well understood. Therefore, we investigated the role of hypoxia in directed migration of NSCs to glioma and identified the specific signaling molecules involved. Our data showed that hypoxia caused increased migration of human HB1.F3 NSCs to U251 human glioma-conditioned medium in vitro. In HB1.F3 NSCs, hypoxia led to up-regulation of CXCR4, urokinase-type plasminogen activator receptor (uPAR), vascular endothelial growth factor receptor 2 (VEGFR2), and c-Met receptors. Function-inhibiting antibodies to these receptors inhibited the migration of HB1.F3 cells to glioma-conditioned medium. Small interfering RNA knockdown of hypoxia-inducible factor-1alpha in glioma cells blocked the hypoxia-induced migration of NSCs, which was due to decreased expression of stromal cell-derived factor-1 (SDF-1), uPA, and VEGF in glioma cells. Our in vivo data provided direct evidence that NSCs preferentially distributed to hypoxic areas inside intracranial glioma xenografts, as detected by pimonidazole hypoxia probe, as well as to the tumor edge, and that both areas displayed high SDF-1 expression. These observations indicate that hypoxia is a key factor in determining NSC tropism to glioma and that SDF-1/CXCR4, uPA/uPAR, VEGF/VEGFR2, and hepatocyte growth factor/c-Met signaling pathways mediate increased NSC-to-glioma tropism under hypoxia. These results have significant implications for development of stem cell-mediated tumor-selective gene therapies.

The erythropoietin (EPO) receptor (EPO-R), a member of a large cytokine receptor superfamily, has a 236-amino-acid cytoplasmic region which contains no obvious tyrosine kinase or other catalytic domain. In order to delineate the linear functional domains of the cytoplasmic tail, we generated truncated mutant cDNAs which were transfected into a murine interleukin-3-dependent cell line, Ba/F3, and the EPO-dependent growth characteristics of the stable transfectants were assayed. We identified two unique domains of the cytoplasmic tail. A membrane-proximal positive signal transduction domain of less than or equal to 103 amino acids, in a region highly similar to the interleukin-2 receptor beta chain, was sufficient for EPO-mediated signal transduction. A carboxy-terminal negative-control domain, a serine-rich region of approximately 40 amino acids, increased the EPO requirement for the Ba/F3 transfectants without altering EPO-R cell surface expression, affinity for EPO, receptor oligosaccharide processing, or receptor endocytosis. Truncation of this negative-control domain allowed the Ba/F3 transfectants to grow maximally in only 1 pM EPO, 1/10 the concentration required for growth of cells expressing the wild-type EPO-R. All truncated EPO-R mutants which retained the transmembrane region of the EPO-R polypeptide bound to the gp55 envelope protein of Friend spleen focus-forming virus. Only the functional EPO-R mutants were activated by the gp55, however, suggesting that gp55- and EPO-mediated signaling occur via a similar mechanism.

Recurrence of hepatitis C after liver transplantation (LT) is the main cause of graft loss and retransplantation. Frequent liver biopsies are essential to follow-up hepatitis C virus (HCV)-induced liver damage. However, liver biopsy is an invasive and expensive procedure. We evaluated prospectively the diagnostic accuracy of noninvasive measurement of liver stiffness (by transient elastography) to assess the severity of hepatitis C recurrence after LT. For this purpose, we included 124 HCV-infected liver transplant recipients who underwent 169 liver biopsies and 129 hepatic hemodynamic studies with determination of hepatic venous pressure gradient (HVPG). Simultaneously, patients underwent measurement of liver stiffness. Liver fibrosis was mild (F0-F1) in 96 cases (57%) and significant (F2-F4) in 73 (43%). HVPG was normal (<6 mm Hg) in 69 cases (54%) and elevated >>or=6 mm Hg) in 60 (46%). Using a liver stiffness cutoff value of 8.5 kilopascals, the sensitivity, specificity, negative predictive value, and positive predictive value for diagnosis of fibrosis>>or=F2 were 90%, 81%, 79%, and 92%, respectively. The area under the curve (AUC) for diagnosis of fibrosis>>or=F2,>>or=F3 and F4 were 0.90, 0.93, and 0.98, respectively. There was a close direct correlation between liver stiffness and HVPG (Pearson coefficient, 0.84; P < 0.001) and the AUC for diagnosis of portal hypertension (HVPG>>or=6 mm Hg) was 0.93. Importantly, none of the individuals with liver stiffness below the cutoff value had either bridging fibrosis (F3) or cirrhosis (F4) or significant portal hypertension (HVPG>>or=10 mm Hg). In conclusion, determination of liver stiffness is an extremely valuable tool to assess the severity of HCV recurrence after LT and in reducing the need of follow-up liver biopsies.

BACKGROUND

Neurons containing proopiomelanocortin (POMC)-derived peptides, known to control stress axis, metabolic, and immune functions, have a lower function in patients with a family history of alcoholism, raising the possibility that alcohol effects on the POMC system may transmit through generations. Here we describe epigenetic modifications of Pomc gene that transmit through generation via male germline and may be critically involved in alcoholism-inherited diseases.

METHODS

Whether an epigenetic mechanism is involved in causing a Pomc expression deficit in fetal alcohol-exposed rats is studied by determining Pomc gene methylation, expression, and functional abnormalities and their normalization following suppression of DNA methylation or histone acetylation. Additionally, transgenerational studies were conducted to evaluate the germline-transmitted effect of alcohol.

RESULTS

Fetal alcohol-exposed male and female rat offspring showed a significant deficit in POMC neuronal functions. Associated with this was an increased methylation status of several CpG dinucleotides in the proximal part of the Pomc promoter region and altered level of histone-modifying proteins and DNA methyltransferases levels in POMC neurons. Suppression of histone deacetylation and DNA methylation normalized Pomc expression and functional abnormalities. Fetal alcohol-induced Pomc gene methylation, expression, and functional defects persisted in the F2 and F3 male but not in female germline. Additionally, the hypermethylated Pomc gene was detected in sperm of fetal alcohol-exposed F1 offspring that was transmitted through F3 generation via male germline.

CONCLUSIONS

Trangenerational epigenetic studies should spur new insight into the biological mechanisms that influence the sex-dependent difference in genetic risk of alcoholism-inherited diseases.

OBJECTIVE

The purpose of our study was to compare the utility of MR elastography (MRE) and diffusion-weighted imaging (DWI) in characterizing fibrosis and chronic hepatitis in patients with chronic liver diseases.

METHODS

Seventy-six patients with chronic liver disease underwent abdominal MRI, MRE, and DWI. Severities of liver fibrosis and chronic hepatitis were graded by histopathologic analysis according to standard disease-specific classifications. The overall predictive ability of MRE and DWI in assessment of fibrosis was compared by constructing a receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC) on the basis of histopathologic analysis.

RESULTS

Using ROC analysis, MRE showed greater capability than DWI in discriminating stage 2 or greater (≥ F2), stage 3 or greater (≥ F3), and cirrhosis (≥ F4), shown as significant differences in AUC (p = 0.003, p = 0.001, and p = 0.001, respectively). Higher sensitivity and specificity were shown by MRE in predicting fibrosis scores ≥ F2 (91% and 97%), scores ≥ F3 (92% and 95%), and scores F4 (95% and 87%) compared with DWI (84% and 82%, 88% and 76%, and 85% and 68%, respectively). Although MRE had higher ability in identification of liver with fibrosis scores ≥ F1 than DWI, a significant difference was not seen (p = 0.398). Stiffness values on MRE increased in relation to increasing severity of fibrosis confirmed by histopathology scores; however, a consistent relationship between apparent diffusion coefficient (ADC) values and stage of fibrosis was not shown. In addition, liver tissue with chronic hepatitis preceding fibrosis may account for mild elevation of liver stiffness.

CONCLUSIONS

MRE had greater predictive ability in distinguishing the stages of liver fibrosis than DWI.