A Re-Emerging Marker for Prognosis in Hepatocellular Carcinoma: The Add-Value of FISHing c-myc Gene for Early Relapse

Hepatocellular carcinoma is one leading cause of cancer-related death and surgical resection is still one of the major curative therapies. Recently, there has been a major effort to find mechanisms involved in carcinogenesis and early relapse. c-myc gene abnormality is found in hepatocarcinogenesis. Our aim was to analyze the role of c-myc as prognostic factor in terms of overall survival and disease-free survival and to investigate if c-myc may be an important target for therapy. We studied sixty-five hepatocellular carcinomas submitted to surgical resection with curative intent. Size, macro-microvascular invasion, necrosis, number of nodules, grading and serum alfa-fetoprotein level were registered for all cases. We evaluated the c-myc aberrations by using break-apart FISH probes. Probes specific for the centromeric part of chromosome 8 and for the locus specific c-myc gene (8q24) were used to assess disomy, gains of chromosomes (polysomy due to polyploidy) and amplification. c-myc gene amplification was scored as 8q24/CEP8 > 2. Statistical analysis for disease-free survival and overall survival were performed. At molecular level, c-myc was amplified in 19% of hepatocellular carcinoma, whereas showed gains in 55% and set wild in 26% of cases. The 1- and 3-year disease-free survival and overall survival for disomic, polysomic and amplified groups were significantly different (p=0.020 and p=.018 respectively). Multivariate analysis verified that the AFP and c-myc status (amplified vs. not amplified) were significant prognostic factors for overall patients survival. c-myc gene amplification is significantly correlated with disease-free survival and overall survival in patients with hepatocellular carcinoma after surgical resection and this model identifies patients with risk of early relapse (≤12 months). We suggest that c-myc assessment may be introduced in the clinical practice for improving prognostication (high and low risk of relapse) routinely and may have be proposed as biomarker of efficacy to anti-c-myc targeted drugs in clinical trials.     

Somatic Mutations in Exocrine Pancreatic Tumors: Association with Patient Survival

KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.     

Random CNT network and regioregular poly(3-hexylthiophen) FETs for pH sensing applications: A comparison

Background

Nowadays, there is a tremendous need for cheap disposable sensing devices for medical applications. Materials such as Carbon Nanotubes (CNTs) and regioregular P3HT are proven to offer a huge potential as cost-effective and solution processable semiconductors for (bio)sensing applications.

Methods

CNT-based field-effect transistors (CNT-FETs) as well as regioregular P3HT-based ones (P3HT-FETs) are fabricated and operated in the so-called electrolyte-gated configuration. The active layer of the P3HT-FETs consists of a spin-coated regioregular P3HT layer, which serves on one hand as the active sensing element and on the other hand as passivation layer for the transistor's metal contacts. The active layer of the nanotube transistors consists of a randomly distributed single walled CNT-network (> 90% semiconducting tubes) deposited from a CNT-ink solution by spin-coating.

Results

We compare both devices concerning their stability in aqueous environment and their response when exposed to buffers with different pH. We found that even if P3HT shows lower stability its pH sensitivity is reproducible even after long-term measurements.

Conclusion

CNT-FETs and P3HT-FETs offer different advantages and drawbacks concerning their stability in solution and the ease of fabrication. A discussion of their different sensing mechanisms as well as sensitivity is given here.

General Significance

This work reports on fast and cost-effective production of solution processable thin-film transistors based on carbon nanotubes and regioregular P3HT and demonstrates their suitability as reliable pH sensors. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.     

An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin

The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.     

Erythroid differentiation and regulatory gene expression are modulated by adenosine derivatives interfering with S-adenosylmethionine metabolic pathway

The differentiation of murine erythroleukemia cells and the expression of SCL, Id1 and c-myc regulatory genes were studied. The first gene is a positive regulator of differentiation, while the other two are both negative regulators of differentiation and positive regulators of proliferation. Accordingly, our data show that when differentiation is stimulated SCL is upregulated while Id1 and c-myc are, coordinately, downregulated. The cultures were treated with two adenosine derivatives, 3-deazaadenosine and 3-deazaaristeromycin, known to act on the metabolic pathway of the methyl donor S-adenosylmethionin, in order to assess the possibility of a coordinated modulation, by these drugs, of regulatory gene expression and erythroid cell differentiation. 3-Deazaaristeromycin caused the simultaneous downregulation of Id1 and c-myc, whereas 3-deazaadenosine caused their upregulation; both drugs produced a transient increase in SCL expression. The use of these drugs evidenced a predominant regulatory effect of negative regulators in the control of erythroid differentiation. The distinct effects of the two drugs on regulatory gene expression led to an increased differentiation induced by 3-deazaaristeromycin and to a reduced differentiation induced by 3-deazaadenosine, if compared with controls. Southern analysis of DNA digested with methylation-specific restriction endonucleases showed that the administration of 3-deazaaristeromycin resulted in hypomethylation of SCL and c-myc, thus evidencing, in these cells, a clear correlation between DNA hypomethylation and differentiation but no straightforward correlation between DNA methylation and gene expression.     

The expression of native and oxidized LDL receptors in brain microvessels is specifically enhanced by astrocytes-derived soluble factor(s)

Based on the functional characterization of sucrose biosynthesis related protiens[SBP: sucrose-phosphate synthase (SPS), sucrose-phosphate phosphatase (SPP), and sucrose synthase (SuS)] in Anabaena sp. PCC7120 and sequence analysis, we have shown that SBP are restricted to cyanobacterium species and plants, and that they are multidomain proteins with modular architecture. Anabaena SPS, a minimal catalytic SPS unit, defines a glucosyltransferase domain present in all SPSs and SuSs. Similarly, Anabaena SPP defines a phosphohydrolase domain characteristic of all SPPs and some SPSs. Phylogenetic analysis points towards the evolution of modern cyanobacterial and plant SBP from a bidomainal common ancestral SPS-like gene.     

Hormone-stimulated Mg(2+) accumulation into rat hepatocytes: a pathway for rapid Mg(2+) and Ca(2+) redistribution

Many diseases such as cardiac arrhythmia, diabetes, and chronic alcoholism are associated with a marked decrease of plasma and parenchymal Mg(2+), and Mg(2+) administration is routinely used therapeutically. This study uses isolated rat hepatocytes to ascertain if and under which conditions increases in extracellular Mg(2+) result in an increase in intracellular Mg(2+). In the absence of stimulation, changing extracellular Mg(2+) had no effect on total cellular Mg(2+) content. By contrast, carbachol or vasopressin administration promoted an accumulation of Mg(2+) that increased cellular Mg(2+) content by 13.2 and 11.8%, respectively, and stimulated Mg(2+) uptake was unaffected by the absence of extracellular Ca(2+). Mg(2+) efflux resulting from stimulation of alpha- or beta-adrenergic receptors operated with a Mg(2+):Ca(2+) exchange ratio of 1. These data indicate that cellular Mg(2+) uptake can occur rapidly and in large amounts, through a process distinct from Mg(2+) release, but operating only upon specific hormonal stimulation.     

TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion.

We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport. To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex ( approximately 800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.     

Bayesian Hierarchical Functional Data Analysis Via Contaminated Informative Priors

Summary .  A variety of flexible approaches have been proposed for functional data analysis, allowing both the mean curve and the distribution about the mean to be unknown. Such methods are most useful when there is limited prior information. Motivated by applications to modeling of temperature curves in the menstrual cycle, this article proposes a flexible approach for incorporating prior information in semiparametric Bayesian analyses of hierarchical functional data. The proposed approach is based on specifying the distribution of functions as a mixture of a parametric hierarchical model and a nonparametric contamination. The parametric component is chosen based on prior knowledge, while the contamination is characterized as a functional Dirichlet process. In the motivating application, the contamination component allows unanticipated curve shapes in unhealthy menstrual cycles. Methods are developed for posterior computation, and the approach is applied to data from a European fecundability study.