Effects of DDT and DDE on placental cholinergic receptors.

The placental cholinergic system; known as an important factor in intracellular metabolic activities, regulation of placental vascular tone, placental development, and neurotransmission; can be affected by persistent organic pesticides, particularly organochlorine pesticides(OCPs), which can influence various epigenetic regulations and molecular pathways. Although OCPs are legally prohibited, trace amounts of the persistent dichlorodiphenyltrichloroethane(DDT) are still found in the environment, making prenatal exposure inevitable. In this study, the effects of 2,4'-DDT and 4,4'-DDT; and its breakdown product 4,4'-DDE in the environment on placental cholinergic system were evaluated with regards to cholinergic genes. 40 human placentas were screened, where 42,50% (17 samples) were found to be positive for the tested compounds. Average concentrations were 10.44 µg/kg; 15.07 µg/kg and 189,42 µg/kg for 4,4'-DDE; 2,4'-DDT and 4,4'-DDT respectively. RNA-Seq results revealed 2396 differentially expressed genes in positive samples; while an increase in CHRM1,CHRNA1,CHRNG and CHRNA2 genes at 1.28, 1.49, 1.59 and 0.4 fold change were found(p<0028). The increase for CHRM1 was also confirmed in tissue samples with immunohistochemistry. In vitro assays using HTR8/SVneo cells; revealed an increase in mRNA expression of CHRM1, CHRM3 and CHRN1 in DDT and DDE treated groups; which was also confirmed through western blot assays. An increase in the expression of CHRM1,CHRNA1, CHRNG(p<0001) and CHRNA2(p<0,05) were found from the OCPs exposed and non exposed groups.The present study reveals that intrauterine exposure to DDT affects the placental cholinergic system mainly through increased expression of muscarinic receptors. This increase in receptor expression is expected to enhance the sensitivity of the placental cholinergic system to acetylcholine.

Differentiation and molecular characterization of endothelial progenitor and vascular smooth muscle cells from induced pluripotent stem cells.

Introduction: Pluripotent stem cells have been used by various researchers to differentiate and characterize endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) for the clinical treatment of vascular injuries. Studies continue to differentiate and characterize the cells with higher vascularization potential and low risk of malignant transformation to the recipient. Unlike previous studies, this research aimed to differentiate induced pluripotent stem (iPS) cells into endothelial progenitor cells (EPCs) and VSMCs using a step-wise technique. This was achieved by elucidating the spatio-temporal expressions of the stage-specific genes and proteins during the differentiation process. The presence of highly expressed oncogenes in iPS cells was also investigated during the differentiation period. Methods: Induced PS cells were differentiated into lateral mesoderm cells (Flk1+). The Flk1+ populations were isolated on day 5.5 of the mesodermal differentiation period. Flk1+ cells were further differentiated into EPCs and VSMCs using VEGF165 and platelet-derived growth factor-BB (PDGF-BB), respectively, and then characterized using gene expression levels, immunocytochemistry (ICC), and western blot (WB) methods. During the differentiation steps, the expression levels of the marker genes and proto-oncogenic Myc and Klf4 genes were simultaneously studied. Results: The optimal time for the isolation of Flk1+ cells was on day 5.5. EPCs and VSMCs were differentiated from Flk1+ cells and characterized with EPC-specific markers, including Kdr, Pecam1, CD133, Cdh5, Efnb2, Vcam1; and VSMC-specific markers, including Acta2, Cnn1, Des, and Myh11. Differentiated cells were validated based on their temporal gene expressions, protein synthesis, and localization at certain time points. Significant decreases in Myc and Klf4 gene expression levels were observed during the EPCs and VSMC differentiation period. Conclusion: EPCs and VSMCs were successfully differentiated from iPS cells and characterized by gene expression levels, ICC, and WB. We observed significant decreases in oncogene expression levels in the differentiated EPCs and VSMCs. In terms of safety, the described methodology provided a better safety margin. EPCs and VSMC obtained using this method may be good candidates for transplantation and vascular regeneration.

Behçet syndrome: The disturbed balance between anti- (CLEC12A, CLC) and proinflammatory (IFI27) gene expressions.

INTRODUCTION: Behçet syndrome (BS) is a chronic, multisystemic inflammatory condition with unanswered questions regarding its pathogenesis and rational therapeutics. A microarray-based comparative transcriptomic analysis was performed to elucidate the molecular mechanisms of BS and identify any potential therapeutic targets. METHODS: Twenty-nine BS patients (B) and 15 age and sex-matched control subjects (C) were recruited. Patients were grouped as mucocutaneous (M), ocular (O), and vascular (V) according to their clinical phenotypes. GeneChip Human Genome U133 Plus 2.0 arrays were used for expression profiling on peripheral blood samples of the patients and the control subjects. Following documentation of the differentially expressed gene (DEG) sets, the data were further evaluated with bioinformatics analysis, visualization, and enrichment tools. Validation of the microarray data was performed using quantitative reverse transcriptase polymerase chain reaction. RESULTS: When p ≤ 0.05 and fold change ≥2.0 were chosen, the following numbers of DEGs were obtained; B versus C: 28, M versus C: 20, O versus C: 8, V versus C: 555, M versus O: 6, M versus V: 324, O versus V: 142. Venn diagram analysis indicated only two genes, CLEC12A and IFI27, in the intersection of M versus C ∩ O versus C ∩ V versus C. Another noteworthy gene appeared as CLC in the DEG sets. Cluster analyses successfully clustered distinct clinical phenotypes of BS. While innate immunity-related processes were enriched in the M group, adaptive immunity-specific processes were significantly enriched in the O and V groups. CONCLUSIONS: Distinct clinical phenotypes of BS patients displayed distinct expression profiles. In Turkish BS patients, expression differences regarding the genes CLEC12A, IFI27, and CLC seemed to be operative in the disease pathogenesis. Based on these findings, future research should consider the immunogenetic heterogeneity of BS clinical phenotypes. Two anti-inflammatory genes, namely CLEC12A and CLC, may be valuable as therapeutic targets and may also help design an experimental model in BS.

Molecular Signatures of Human Chronic Atrial Fibrillation in Primary Mitral Regurgitation.

OBJECTIVES: Transcriptomics of atrial fibrillation (AFib) in the setting of chronic primary mitral regurgitation (MR) remains to be characterized. We aimed to compare the gene expression profiles of patients with degenerative MR in AFib and sinus rhythm (SR) for a clearer picture of AFib pathophysiology. METHODS: After transcriptomic analysis and bioinformatics (n = 59), differentially expressed genes were defined using 1.5-fold change as the threshold. Additionally, independent datasets from GEO were included as meta-analyses. RESULTS: QRT-PCR analysis confirmed that AFib persistence was associated with increased expression molecular changes underlying a transition to heart failure (NPPB, P = 0.002; ANGPTL2, P = 0.002; IGFBP2, P = 0.010), structural remodeling including changes in the extracellular matrix and cellular stress response (COLQ, P = 0.003; COMP, P = 0.028; DHRS9, P = 0.038; CHGB, P = 0.038), and cellular stress response (DNAJA4, P = 0.038). Furthermore, AFib persistence was associated with decreased expression of the targets of structural remodeling (BMP7, P = 0.021) and electrical remodeling (CACNB2, P = 0.035; MCOLN3, P = 0.035) in both left and right atrial samples. The transmission electron microscopic analysis confirmed ultrastructural atrial remodeling and autophagy in human AFib atrial samples. CONCLUSIONS: Atrial cardiomyocyte remodeling in persistent AFib is closely linked to alterations in gene expression profiles compared to SR in patients with primary MR. Study findings may lead to novel therapeutic targets. This trial is registered with ClinicalTrials.gov identifier: NCT00970034.

SLCO1B1 Polymorphisms are Associated With Drug Intolerance in Childhood Leukemia Maintenance Therapy.

BACKGROUND: Therapy discontinuations and toxicities occur because of significant interindividual variations in 6-mercaptopurine (6-MP) and methotrexate (MTX) response during maintenance therapy of childhood acute lymphoblastic leukemia (ALL). 6-MP/MTX intolerance in some of the patients cannot be explained by thiopurine S-methyl transferase (TPMT) gene variants. In this study, we aimed to investigate candidate pharmacogenetic determinants of 6-MP and MTX intolerance in Turkish ALL children. METHODS: In total, 48 children with ALL who had completed or were receiving maintenance therapy according to Children's Oncology Group (COG) protocols were enrolled. Fifteen single-nucleotide polymorphisms in 8 candidate genes that were related to drug toxicity or had a role in the 6-MP/MTX metabolism (TPMT, ITPA, MTHFR, IMPDH2, PACSIN2, SLCO1B1, ABCC4, and PYGL) were genotyped by competitive allele-specific PCR (KASP). Drug doses during maintenance therapy were modified according to the protocol. RESULTS: The median drug dose intensity was 50% (28% to 92%) for 6-MP and 58% (27% to 99%) for MTX in the first year of maintenance therapy, which were lower than that scheduled in all patients. Among the analyzed polymorphisms, variant alleles in SLCO1B1 rs4149056 and rs11045879 were found to be associated with lower 6-MP/MTX tolerance. CONCLUSIONS: SLCO1B1 rs4149056 and rs11045879 polymorphisms may be important genetic markers to individualize 6-MP/MTX doses.

Treatment of plasminogen deficiency patients with fresh frozen plasma.

Congenital plasminogen (Plg) deficiency leads to the development of ligneous membranes on mucosal surfaces. Here, we report our experience with local and intravenous fresh frozen plasma (FFP). We retrospectively reviewed medical files of 17 patients and their eight first-degree relatives. Conjunctivitis was the main complaint. Thirteen patients were treated both with intravenous and conjunctival FFP. Venous thrombosis did not develop in any. Genetic evaluation revealed heterogeneous mutations as well as polymorphisms. Diagnosis and treatment of Plg deficiency is challenging; topical and intravenous FFP may be an alternative treatment.

The effect of biological heterogeneity on R-CHOP treatment outcome in diffuse large B-cell lymphoma across five international regions.

Addressing the global burden of cancer, understanding its diverse biology, and promoting appropriate prevention and treatment strategies around the world has become a priority for the United Nations and International Atomic Energy Agency (IAEA), the WHO, and International Agency for Research on Cancer (IARC). The IAEA sponsored an international prospective cohort study to better understand biology, treatment response, and outcomes of diffuse large B-cell lymphoma (DLBCL) in low and middle-income countries across five UN-defined geographical regions. We report an analysis of biological variation in DLBCL across seven ethnic and environmentally diverse populations. In this cohort of 136 patients treated to a common protocol, we demonstrate significant biological differences between countries, characterized by a validated prognostic gene expression score (p < .0001), but International Prognostic Index (IPI)-adjusted survivals in all participating countries were similar. We conclude that DLBCL treatment outcomes in these populations can be benchmarked to international standards, despite biological heterogeneity.

Protocol for qRT-PCR analysis from formalin fixed paraffin embedded tissue sections from diffuse large b-cell lymphoma: Validation of the six-gene predictor score.

As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting.

From RNA isolation to microarray analysis: Comparison of methods in FFPE tissues.

BACKGROUND: Genome-wide gene expression profiling analysis of FFPE tissue samples is indispensable for cancer research and provides the opportunity to evaluate links between molecular and clinical information, however, working with FFPE samples is challenging due to extensive cross-linking, fragmentation and limited quantities of nucleic acid. Thus, processing of FFPE tissue samples from RNA extraction to microarray analysis still needs optimization. MATERIALS AND METHODS: In this study, a modified deparaffinization protocol was conducted prior to RNA isolation. Trizol, Qiagen RNeasy FFPE and Arcturus PicoPure RNA Isolation kits were used in parallel to compare their impact on RNA isolation. We also evaluated the effect of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) on the percentage of present calls. RESULTS: Our optimization study shows that the Qiagen RNeasy FFPE kit with modified deparaffinization step gives better results (RNA quantity and quality) than the other two isolation kits. The Ribo-SPIA protocol gave a significantly higher percentage of present calls than the 3' IVT cDNA amplification and labeling system. However, no significant differences were found between the two array platforms. CONCLUSION: Our study paves the way for future high-throughput transcriptional analysis by optimizing FFPE tissue sample processing from RNA isolation to microarray analysis.

Functionally conserved effects of rapamycin exposure on zebrafish.

Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase important in cell proliferation, growth and protein translation. Rapamycin, a well­known anti­cancer agent and immunosuppressant drug, inhibits mTOR activity in different taxa including zebrafish. In the present study, the effect of rapamycin exposure on the transcriptome of a zebrafish fibroblast cell line, ZF4, was investigated. Microarray analysis demonstrated that rapamycin treatment modulated a large set of genes with varying functions including protein synthesis, assembly of mitochondrial and proteasomal machinery, cell cycle, metabolism and oxidative phosphorylation in ZF4 cells. A mild however, coordinated reduction in the expression of proteasomal and mitochondrial ribosomal subunits was detected, while the expression of numerous ribosomal subunits increased. Meta­analysis of heterogeneous mouse rapamycin microarray datasets enabled the comparison of zebrafish and mouse pathways modulated by rapamycin, using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology pathway analysis. The analyses demonstrated a high degree of functional conservation between zebrafish and mice in response to rapamycin. In addition, rapamycin treatment resulted in a marked dose­dependent reduction in body size and pigmentation in zebrafish embryos. The present study is the first, to the best of our knowledge, to evaluate the conservation of rapamycin­modulated functional pathways between zebrafish and mice, in addition to the dose­dependent growth curves of zebrafish embryos upon rapamycin exposure.

Optimization of gene expression microarray protocol for formalin-fixed paraffin-embedded tissues.

Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples.

Behçet's: A Disease or a Syndrome? Answer from an Expression Profiling Study.

Behçet's disease (BD) is a chronic, relapsing, multisystemic inflammatory disorder with unanswered questions regarding its etiology/pathogenesis and classification. Distinct manifestation based subsets, pronounced geographical variations in expression, and discrepant immunological abnormalities raised the question whether Behçet's is "a disease or a syndrome". To answer the preceding question we aimed to display and compare the molecular mechanisms underlying distinct subsets of BD. For this purpose, the expression data of the gene expression profiling and association study on BD by Xavier et al (2013) was retrieved from GEO database and reanalysed by gene expression data analysis/visualization and bioinformatics enrichment tools. There were 15 BD patients (B) and 14 controls (C). Three subsets of BD patients were generated: MB (isolated mucocutaneous manifestations, n = 7), OB (ocular involvement, n = 4), and VB (large vein thrombosis, n = 4). Class comparison analyses yielded the following numbers of differentially expressed genes (DEGs); B vs C: 4, MB vs C: 5, OB vs C: 151, VB vs C: 274, MB vs OB: 215, MB vs VB: 760, OB vs VB: 984. Venn diagram analysis showed that there were no common DEGs in the intersection "MB vs C" ∩ "OB vs C" ∩ "VB vs C". Cluster analyses successfully clustered distinct expressions of BD. During gene ontology term enrichment analyses, categories with relevance to IL-8 production (MB vs C) and immune response to microorganisms (OB vs C) were differentially enriched. Distinct subsets of BD display distinct expression profiles and different disease associated pathways. Based on these clear discrepancies, the designation as "Behçet's syndrome" (BS) should be encouraged and future research should take into consideration the immunogenetic heterogeneity of BS subsets. Four gene groups, namely, negative regulators of inflammation (CD69, CLEC12A, CLEC12B, TNFAIP3), neutrophil granule proteins (LTF, OLFM4, AZU1, MMP8, DEFA4, CAMP), antigen processing and presentation proteins (CTSS, ERAP1), and regulators of immune response (LGALS2, BCL10, ITCH, CEACAM8, CD36, IL8, CCL4, EREG, NFKBIZ, CCR2, CD180, KLRC4, NFAT5) appear to be instrumental in BS immunopathogenesis.

Novel plasminogen gene mutations in Turkish patients with type I plasminogen deficiency.

The plasminogen (Plg) protein is the inactive proenzyme form of plasmin that dissolves fibrin thrombi by a process called fibrinolysis. It has been shown that homozygous or compound-heterozygous deficiency of this protein is a major cause of a rare inflammatory disease affecting mainly mucous membranes found in different body sites. In this study, five individual Turkish patients and nine Turkish families with type 1 Plg deficiency were investigated for PLG gene mutations. All of the coding regions of the PLG gene mutations were screened for mutations using denaturing high-pressure liquid chromatography (DHPLC). Samples showing a different DHPLC profile were subjected to DNA sequencing analysis. Here, we described five novel mutations namely, Cys49Ter, +1 IVS6 G>A, Gly218Val, Tyr283Cys, and Gly703Asp. Previously identified five nonsynonymous (Lys38Glu, Glu180Lys, Gly420Asp, Asp453Asn, Pro763Ser), five synonymous (330 C>T, 582 C>T, 771 T>C, 1083 A>G, 2286 T>G), and a 3' untranslated region (3' UTR) mutation (c.*45 A>G) were also reported in this present study. In this study, we have identified a total of eight mutations, five of which are novel. The mutations/polymorphisms identified in eight of the patients do not explain the disease phenotype. These cases probably carry other pathological mutations (homozygous or compound heterozygous) that cannot be detected by DHPLC.

C-type lectin domain family 12, member A: A common denominator in Behçet's syndrome and acute gouty arthritis.

C-type lectin domain family 12, member A (CLEC12A) is a C-type lectin-like pattern recognition receptor capable of recognizing monosodium urate crystals. Monosodium urate crystals, the causative agents of gout are also among the danger-associated molecular patterns reflecting cellular injury/cell death. In response to monosodium urate crystals, CLEC12A effectively inhibits granulocyte and monocyte/macrophage functions and hence acts as a negative regulator of inflammation. Behçet's syndrome and gout are autoinflammatory disorders sharing certain pathological (neutrophilic inflammation), clinical (exaggerated response to monosodium urate crystals) and therapeutic (colchicine) features. We propose the hypothesis that decreased expression of CLEC12A is a common denominator in the hyperinflammatory responses observed in Behçet's syndrome and gout. Major lines of evidence supporting this hypothesis are: (1) Downregulation/deficiency of CLEC12A is associated with hyperinflammatory responses. (2) CLEC12A polymorphisms with functional and clinical implications have been documented in other inflammatory diseases. (3) Colchicine, a fundamental therapeutic agent used both in Behçet's syndrome and gout is shown to oppose the downregulation of CLEC12A. (4) Behçet's syndrome and gout are characterized by a hyperinflammatory response to monosodium urate crystals and other than gout, Behçet's syndrome is the only inflammatory condition exhibiting this exaggerated response. (5) Genomewide linkage and association studies of Behçet's syndrome collectively point to 12p12-13, the chromosomal region harboring CLEC12A. (6) Patients with severe forms of Behçet's syndrome underexpress CLEC12A with respect to patients with mild forms of the disease. If supported by well-designed, rigorous experiments, the forementioned hypothesis pertinent to CLEC12A will carry important implications for therapy, designing experimental models, and uncovering immunopathogenic mechanisms in Behçet's syndrome and gout.

Comparison of high-resolution melting analysis to denaturing high performance liquid chromatography in the detection of point mutations in MEFV, F5, and F2 genes.

BACKGROUND/AIM: Sensitive and cost-effective detection of point mutations is important in genetics research. Denaturing high-performance liquid chromatography (DHPLC) is known to be one of the most sensitive techniques for point mutation detection. A more recent technique, high-resolution melting (HRM), is based on the melting behavior of PCR products. In this study, the efficiency and sensitivity of HRM and DHPLC for the detection of MEFV, F5, and F2 gene point mutations were evaluated. MATERIALS AND METHODS: We studied 15 patients with MEFV mutations (E148Q, M680I, M694V, or V726A), 7 patients with the F51691G>A mutation, and 12 patients with the F220210G>A mutation. All mutations were screened by HRM and DHPLC. RESULTS: All mutations were successfully detected by HRM. However, only 4 (MEFVE148Q and M680I, F51691G>A, and F220210G>A) of 6 mutations were successfully detected with DHPLC. CONCLUSION: Our study showed that HRM is more sensitive than DHPLC for detection of the studied point mutations.

Genome Sequence of the Boron-Tolerant and -Requiring Bacterium Bacillus boroniphilus.

Bacillus boroniphilus is a highly boron-tolerant bacterium that also requires this element for its growth. The complete genome sequence of B. boroniphilus was determined by a combination of shotgun sequencing and paired-end sequencing using 454 pyrosequencing technology. A total of 84,872,624 reads from shotgun sequencing and a total of 194,092,510 reads from paired-end sequencing were assembled using Newbler 2.3. The estimated size of the draft genome is 5.2 Mb.

Genome-wide transcriptional reorganization associated with senescence-to-immortality switch during human hepatocellular carcinogenesis.

Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal") by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene hepatocellular immortality signature test that discriminated HCC from cirrhosis with high accuracy. Our findings demonstrate that senescence bypass plays a central role in hepatocellular carcinogenesis engendering systematic changes in the transcription of genes regulating DNA repair, proliferation, differentiation and metabolism.

Biologic tumor behavior in pilocytic astrocytomas.

PURPOSE: The aim is to describe the behavior of pilocytic astrocytoma (PAs) and its effects on patient prognosis by using flow cytometric, immunohistochemical and cytogenetic methods. We also aim to find out whether there is any difference between differently localized tumors by the above mentioned analyses. METHODS: We studied DNA index, expression of p53, p16, pRb, MMAC/PTEN1, VEGF, MIB-1 index and chromosomal anomalies which can be detected by array comparative genomic hybridization (CGH) technique. We analyzed the association of the results of these studies with clinical prognosis and tumor localization. We included 53 patients (18 cerebellar, 20 chiasmatic/hypothalamic and 15 hemispheric). Samples were studied from paraffin embedded tumors. RESULTS: We found that PAs are mostly diploid and ploidy pattern does not affect the prognosis. The expression of p53, p16, pRb, MMAC/PTEN1 and VEGF was not significantly different between different localizations and could not predict the prognosis. Frequently seen copy number aberrations (CNAs) are: amplification in 1p36.33, 2p11.2, 9p11.2, 9q12, 16p11.2, 19q13.12-q13.2, Xp22.2-p21.3, Xp11.3-p11.22, Xq11.1-q12, Xq13.1, Xq21.1-q21.31, Xq22.3, Xq26.3 and homozygous deletion in 2p11.2, 8p23.1, 16p12.3. Among them, 2p11.2 amp, 9p11.2 amp and 1p36.21 hom del were correlated with prognosis. Moreover, we found a significant correlation between 16p11.2 amp and tumor localization. CONCLUSIONS: Differently localized PAs have different properties which make them behave with different biological aggressiveness. PAs demonstrate a significant amount of CNAs that can be detected by a high-resolution study. However, tumor suppressor genes p53, p16, pRb, MMAC/PTEN1 and expression patterns do not play a significant role in PAs.

A novel approach for small sample size family-based association studies: sequential tests.

In this paper, we propose a sequential probability ratio test (SPRT) to overcome the problem of limited samples in studies related to complex genetic diseases. The results of this novel approach are compared with the ones obtained from the traditional transmission disequilibrium test (TDT) on simulated data. Although TDT classifies single-nucleotide polymorphisms (SNPs) to only two groups (SNPs associated with the disease and the others), SPRT has the flexibility of assigning SNPs to a third group, that is, those for which we do not have enough evidence and should keep sampling. It is shown that SPRT results in smaller ratios of false positives and negatives, as well as better accuracy and sensitivity values for classifying SNPs when compared with TDT. By using SPRT, data with small sample size become usable for an accurate association analysis.