Interpreting the gene expression microarray results: a user-based experience.

In recent years many tools have been developed to cope with the interpretation of gene expression results from microarray experiments. The effectiveness of these tools largely depends on their ease of use by biomedical researchers. Tools based on effective computational methods, indeed, cannot be fully exploited by users if they are not supported by an intuitive interface, a large set of utilities and effective outputs. In this paper, 10 tools for the interpretation of gene expression microarray results have been tested on 11 microarray datasets and evaluated according to eight assessment criteria: 1. interface design and usability, 2. easiness of input submission, 3. effectiveness of output representation and 4. of the downloaded outputs, 5. possibility to submit multiple gene IDs, 6. sources of information, 7. provision of different statistical tests and 8. of multiple test correction methods. Strengths and weaknesses of each tool are highlighted to: a. provide useful tips to users dealing with the biological interpretation of microarray results; b. draw the attention of software developers on the usability of their tools.

Effects on human transcriptome of mutated BRCA1 BRCT domain: a microarray study.

BACKGROUND: BRCA1 (breast cancer 1, early onset) missense mutations have been detected in familial breast and ovarian cancers, but the role of these variants in cancer predisposition is often difficult to ascertain. In this work, the molecular mechanisms affected in human cells by two BRCA1 missense variants, M1775R and A1789T, both located in the second BRCT (BRCA1 C Terminus) domain, have been investigated. Both these variants were isolated from familial breast cancer patients and the study of their effect on yeast cell transcriptome has previously provided interesting clues to their possible role in the pathogenesis of breast cancer. METHODS: We compared by Human Whole Genome Microarrays the expression profiles of HeLa cells transfected with one or the other variant and HeLa cells transfected with BRCA1 wild-type. Microarray data analysis was performed by three comparisons: M1775R versus wild-type (M1775RvsWT-contrast), A1789T versus wild-type (A1789TvsWT-contrast) and the mutated BRCT domain versus wild-type (MutvsWT-contrast), considering the two variants as a single mutation of BRCT domain. RESULTS: 201 differentially expressed genes were found in M1775RvsWT-contrast, 313 in A1789TvsWT-contrast and 173 in MutvsWT-contrast. Most of these genes mapped in pathways deregulated in cancer, such as cell cycle progression and DNA damage response and repair. CONCLUSIONS: Our results represent the first molecular evidence of the pathogenetic role of M1775R, already proposed by functional studies, and give support to a similar role for A1789T that we first hypothesized based on the yeast cell experiments. This is in line with the very recently suggested role of BRCT domain as the main effector of BRCA1 tumor suppressor activity.

Genes and Aggressive Behavior: Epigenetic Mechanisms Underlying Individual Susceptibility to Aversive Environments.

Over the last two decades, the study of the relationship between nature and nurture in shaping human behavior has encountered a renewed interest. Behavioral genetics showed that distinct polymorphisms of genes that code for proteins that control neurotransmitter metabolic and synaptic function are associated with individual vulnerability to aversive experiences, such as stressful and traumatic life events, and may result in an increased risk of developing psychopathologies associated with violence. On the other hand, recent studies indicate that experiencing aversive events modulates gene expression by introducing stable changes to DNA without modifying its sequence, a mechanism known as "epigenetics". For example, experiencing adversities during periods of maximal sensitivity to the environment, such as prenatal life, infancy and early adolescence, may introduce lasting epigenetic marks in genes that affect maturational processes in brain, thus favoring the emergence of dysfunctional behaviors, including exaggerate aggression in adulthood. The present review discusses data from recent research, both in humans and animals, concerning the epigenetic regulation of four genes belonging to the neuroendocrine, serotonergic and oxytocinergic pathways-Nuclear receptor subfamily 3-group C-member 1 (NR3C1), oxytocin receptor (OXTR), solute carrier-family 6 member 4 (SLC6A4) and monoamine oxidase A (MAOA)-and their role in modulating vulnerability to proactive and reactive aggressive behavior. Behavioral genetics and epigenetics are shedding a new light on the fine interaction between genes and environment, by providing a novel tool to understand the molecular events that underlie aggression. Overall, the findings from these studies carry important implications not only for neuroscience, but also for social sciences, including ethics, philosophy and law.

Epigenetic Modifications of the α-Synuclein Gene and Relative Protein Content Are Affected by Ageing and Physical Exercise in Blood from Healthy Subjects.

Epigenetic regulation may contribute to the beneficial effects of physical activity against age-related neurodegeneration. For example, epigenetic alterations of the gene encoding for α-synuclein (SNCA) have been widely explored in both brain and peripheral tissues of Parkinson's disease samples. However, no data are currently available about the effects of physical exercise on SNCA epigenetic regulation in ageing healthy subjects. The present paper explored whether, in healthy individuals, age and physical activity are related to blood intron1-SNCA (SNCAI1 ) methylation, as well as further parameters linked to such epigenetic modification (total, oligomeric α-synuclein and DNA methyltransferase concentrations in the blood). Here, the SNCAI1 methylation status increased with ageing, and consistent with this result, low α-synuclein levels were found in the blood. The direct relationship between SNCAI1 methylation and α-synuclein levels was observed in samples characterized by blood α-synuclein concentrations of 76.3 ng/mg protein or lower (confidence interval (CI) = 95%). In this selected population, higher physical activity reduced the total and oligomeric α-synuclein levels. Taken together, our data shed light on ageing- and physical exercise-induced changes on the SNCA methylation status and protein levels of α-synuclein.

α-Synuclein Aggregates with ß-Amyloid or Tau in Human Red Blood Cells: Correlation with Antioxidant Capability and Physical Exercise in Human Healthy Subjects.

Neurodegenerative disorders (NDs) are characterized by abnormal accumulation/misfolding of specific proteins, primarily α-synuclein (α-syn), ß-amyloid1-42 (Aß), and tau, in both brain and peripheral tissue. In addition to homo-oligomers, the role of α-syn interactions with Aß or tau has gradually emerged. The altered protein accumulation has been related to both oxidative stress and physical activity; nevertheless, no correlation among the presence of peripheral α-syn hetero-aggregates, antioxidant capacity, and physical exercise has been discovered as of yet. Herein, the content of α-syn, Aß, tau, and of their heterocomplexes was determined in red blood cells (RBCs) of healthy subjects (sedentary and athletes). Such parameters were related to the extent of the antioxidant capability (AOC), a key marker of oxidative stress in aging-related pathologies, and to physical exercise, which is known to play an important preventive role in NDs and to modulate oxidative stress. Tau content and plasma AOC toward hydroxyl radicals were both reduced in older or sedentary subjects; in contrast, α-syn and Aß accumulated in elderly subjects and showed an inverse correlation with both hydroxyl AOC and the level of physical activity. For the first time, α-syn heterocomplexes with Aß or tau were quantified and demonstrated to be inversely related to hydroxyl AOC. Furthermore, α-syn/Aß aggregates were significantly reduced in athletes and inversely correlated with physical activity level, independent of age. The positive correlation between antioxidant capability/physical activity and reduced protein accumulation was confirmed by these data and suggested that peripheral α-syn heterocomplexes may represent new indicators of ND-related protein misfolding.

α-Synuclein Aggregated with Tau and ß-Amyloid in Human Platelets from Healthy Subjects: Correlation with Physical Exercise.

The loss of protein homeostasis that has been associated with aging leads to altered levels and conformational instability of proteins, which tend to form toxic aggregates. In particular, brain aging presents characteristic patterns of misfolded oligomers, primarily constituted of ß-amyloid (Aß), tau, and α-synuclein (α-syn), which can accumulate in neuronal membranes or extracellular compartments. Such aging-related proteins can also reach peripheral compartments, thus suggesting the possibility to monitor their accumulation in more accessible fluids. In this respect, we have demonstrated that α-syn forms detectable hetero-aggregates with Aß or tau in red blood cells (RBCs) of healthy subjects. In particular, α-syn levels and its heteromeric interactions are modulated by plasma antioxidant capability (AOC), which increases in turn with physical activity. In order to understand if a specific distribution of misfolded proteins can occur in other blood cells, a cohort of human subjects was enrolled to establish a correlation among AOC, the level of physical exercise and the concentrations of aging-related proteins in platelets. The healthy subjects were divided depending on their level of physical exercise (i.e., athletes and sedentary subjects) and their age (young and older subjects). Herein, aging-related proteins (i.e., α-syn, tau and Aß) were confirmed to be present in human platelets. Among such proteins, platelet tau concentration was demonstrated to decrease in athletes, while α-syn and Aß did not correlate with physical exercise. For the first time, α-syn was shown to directly interact with Aß and tau in platelets, forming detectable hetero-complexes. Interestingly, α-syn interaction with tau was inversely related to plasma AOC and to the level of physical activity. These results suggested that α-syn heterocomplexes, particularly with tau, could represent novel indicators to monitor aging-related proteins in platelets.

Carnosol controls the human glioblastoma stemness features through the epithelial-mesenchymal transition modulation and the induction of cancer stem cell apoptosis.

A high cell proliferation rate, invasiveness and resistance to chemotherapy are the main features of glioblastoma (GBM). GBM aggressiveness has been widely associated both with a minor population of cells presenting stem-like properties (cancer stem-like cells, CSCs) and with the ability of tumor cells to acquire a mesenchymal phenotype (epithelial-mesenchymal transition, EMT). Carnosol (CAR), a natural inhibitor of MDM2/p53 complex, has been attracted attention for its anti-cancer effects on several tumor types, including GBM. Herein, the effects of CAR on U87MG-derived CSC viability and stemness features were evaluated. CAR decreased the rate of CSC formation and promoted the CSC apoptotic cell death through p53 functional reactivation. Moreover, CAR was able to control the TNF-α/TGF-ß-induced EMT, counteracting the effects of the cytokine on EMT master regulator genes (Slug, Snail, Twist and ZEB1) and modulating the activation of miR-200c, a key player in the EMT process. Finally, CAR was able to increase the temozolomide (TMZ) anti-proliferative effects. These findings demonstrate that CAR affected the different intracellular mechanism of the complex machinery that regulates GBM stemness. For the first time, the diterpene was highlighted as a promising lead for the development of agents able to decrease the stemness features, thus controlling GBM aggressiveness.

Genetically-Driven Enhancement of Dopaminergic Transmission Affects Moral Acceptability in Females but Not in Males: A Pilot Study.

Moral behavior has been a key topic of debate for philosophy and psychology for a long time. In recent years, thanks to the development of novel methodologies in cognitive sciences, the question of how we make moral choices has expanded to the study of neurobiological correlates that subtend the mental processes involved in moral behavior. For instance, in vivo brain imaging studies have shown that distinct patterns of brain neural activity, associated with emotional response and cognitive processes, are involved in moral judgment. Moreover, while it is well-known that responses to the same moral dilemmas differ across individuals, to what extent this variability may be rooted in genetics still remains to be understood. As dopamine is a key modulator of neural processes underlying executive functions, we questioned whether genetic polymorphisms associated with decision-making and dopaminergic neurotransmission modulation would contribute to the observed variability in moral judgment. To this aim, we genotyped five genetic variants of the dopaminergic pathway [rs1800955 in the dopamine receptor D4 (DRD4) gene, DRD4 48 bp variable number of tandem repeat (VNTR), solute carrier family 6 member 3 (SLC6A3) 40 bp VNTR, rs4680 in the catechol-O-methyl transferase (COMT) gene, and rs1800497 in the ankyrin repeat and kinase domain containing 1 (ANKK1) gene] in 200 subjects, who were requested to answer 56 moral dilemmas. As these variants are all located in genes belonging to the dopaminergic pathway, they were combined in multilocus genetic profiles for the association analysis. While no individual variant showed any significant effects on moral dilemma responses, the multilocus genetic profile analysis revealed a significant gender-specific influence on human moral acceptability. Specifically, those genotype combinations that improve dopaminergic signaling selectively increased moral acceptability in females, by making their responses to moral dilemmas more similar to those provided by males. As females usually give more emotionally-based answers and engage the "emotional brain" more than males, our results, though preliminary and therefore in need of replication in independent samples, suggest that this increase in dopamine availability enhances the cognitive and reduces the emotional components of moral decision-making in females, thus favoring a more rationally-driven decision process.

Human Neural Stem Cell Aging Is Counteracted by α-Glycerylphosphorylethanolamine.

Neural stem cells (NSCs) represent a subpopulation of cells, located in specific regions of the adult mammalian brain, with the ability of self-renewing and generating neurons and glia. In aged NSCs, modifications in the amount and composition of membrane proteins/lipids, which lead to a reduction in membrane fluidity and cholinergic activities, have been reported. In this respect, molecules that are effective at normalizing the membrane composition and cholinergic signaling could counteract stem cell aging. α-Glycerylphosphorylethanolamine (GPE), a nootropic drug, plays a role in phospholipid biosynthesis and acetylcholine release. Herein, GPE was assayed on human NSC cultures and on hydroxyurea-aged cells. Using cell counting, colorimetric, and fluorimetric analyses, immunoenzymatic assays, and real time PCR experiments, NSC culture proliferation, senescence, reactive oxygen species, and ADP/ATP levels were assessed. Aged NSCs exhibited cellular senescence, decreased proliferation, and an impairment in mitochondrial metabolism. These changes included a substantial induction in the nuclear factor NF-κB, a key inflammatory mediator. GPE cell treatment significantly protected the redox state and functional integrity of mitochondria, and counteracted senescence and NF-κB activation. In conclusion, our data show the beneficial properties of GPE in this model of stem cell aging.

Expression status of candidate genes in mesothelioma tissues and cell lines.

In order to broaden knowledge on the pathogenesis of malignant pleural mesothelioma (MPM), we reviewed studies on the MPM-transcriptome and identified 119 deregulated genes. However, there was poor consistency among the studies. Thus, the expression of these genes was further investigated in the present work using reverse transcriptase-quantitative PCR (RT-qPCR) in 15 MPM and 20 non-MPM tissue samples. Fifty-nine genes showed a statistically significant deregulation and were further evaluated in two epithelioid MPM cell lines (compared to MET-5A, a non-MPM cell line). Nine genes (ACSL1, CCNO, CFB, PDGFRB, SULF1, TACC1, THBS2, TIMP3, XPOT) were deregulated with statistical significance in both cell lines, 12 (ASS1, CCNB1, CDH11, COL1A1, CXADR, EIF4G1, GALNT7, ITGA4, KRT5, PTGIS, RAN, SOD1) in at least one cell line, whereas 7 (DSP, HEG1, MCM4, MSLN, NME2, NMU, TNPO2) were close but did not reach the statistical significance in any of the cell line. Patients whose MPM tissues expressed elevated mRNA levels of BIRC5, DSP, NME2, and THBS2 showed a statistically significant shorter overall survival. Although MPM is a poorly studied cancer, some features are starting to emerge. Novel cancer genes are suggested here, in particular those involved in cell-cell and cell-matrix interactions.

Molecular genetics and antisocial behavior: where do we stand?

Over the last two decades, it has become increasingly evident that control of aggressive behavior is modulated by the individual genetic profile as well. Several candidate genes have been proposed to play a role in the risk to develop antisocial behavior, and distinct brain imaging studies have shown that specific cortical areas may be functionally and/or structurally impaired in impulsive violent subjects on the basis of their genotypes. In this paper, we review the findings regarding four polymorphisms-MAOA (Monoamine oxidase A) uVNTR, SLC6A4 (solute carrier family 6 (neurotransmitter transporter), member 4) 5HTTLPR, COMT (Catechol-O-methyltransferase) Val158Met and DRD4 (dopamine D4 receptor) VNTR 1-11-that all have been found to be associated with an increased vulnerability for antisocial and impulsive behavior in response to aversive environmental conditions. These results, however, have not been replicated by other studies, likely because of crucial methodological discrepancies, including variability in the criteria used to define antisocial behavior and assessment of environmental factors. Finally, it has been recently proposed that these genetic variants may actually increase the individual susceptibility not merely to the negative environmental factors, but to the positive ones as well. In this view, such alleles would play a wider modulatory role, by acting as "plasticity" rather than "vulnerability" genes. Overall, these findings have potential important implications that span well outside of neuroscience and psychiatry, to embrace ethics, philosophy, and the law itself, as they pose new challenges to the very notion of Free Will. Novel properly controlled studies that examine multi-allelic genetic profiles, rather than focusing on distinct single variants, will make it possible to achieve a clearer understanding of the molecular underpinnings of the nature by nurture interaction.

Modulation of gene expression by 3-iodothyronamine: genetic evidence for a lipolytic pattern.

3-Iodothyronamine (T1AM) is an endogenous biogenic amine, structurally related to thyroid hormone, which is regarded as a novel chemical messenger. The molecular mechanisms underlying T1AM effects are not known, but it is possible to envisage changes in gene expression, since delayed and long-lasting phenotypic effects have been reported, particularly with regard to the modulation of lipid metabolism and body weight. To test this hypothesis we analysed gene expression profiles in adipose tissue and liver of eight rats chronically treated with T1AM (10 mg/Kg twice a day for five days) as compared with eight untreated rats. In vivo T1AM administration produced significant transcriptional effects, since 378 genes were differentially expressed in adipose tissue, and 114 in liver. The reported changes in gene expression are expected to stimulate lipolysis and beta-oxidation, while inhibiting adipogenesis. T1AM also influenced the expression of several genes linked to lipoprotein metabolism suggesting that it may play an important role in the regulation of cholesterol homeostasis. No effect on the expression of genes linked to toxicity was observed. The assay of tissue T1AM showed that in treated animals its endogenous concentration increased by about one order of magnitude, without significant changes in tissue thyroid hormone concentration. Therefore, the effects that we observed might have physiological or pathophysiological importance. Our results provide the basis for the reported effectiveness of T1AM as a lipolytic agent and gain importance in view of a possible clinical use of T1AM in obesity and/or dyslipidaemia.

Serotonin transporter gene: a new polymorphism may affect response to antidepressant treatments in major depressive disorder.

BACKGROUND AND OBJECTIVES: Several gene variants have been related to major depressive disorder (MDD) treatment outcomes; however, few studies have investigated a possible different effect on pharmacotherapy and brief psychotherapy response. METHODS: A total of 137 MDD patients were randomized to either interpersonal counseling (IPC; n = 40) or antidepressant pharmacological treatment (n = 97). Outcomes were remission, response, and symptom improvement at week 8. Five genetic variants were investigated (5HTR2A rs6314, BDNF rs6265, SLC6A4 rs8076005, CREB1 rs2253206, and TPH2 rs11179023) as possible modulators of outcomes. RESULTS: The LC6A4 rs8076005 AA genotype and A allele were associated with response rate in the antidepressant group (p = 0.015 and 0.005, respectively) and in the whole sample (p = 0.03 and 0.02, respectively). In the IPC group a non-significant trend in the same direction was observed. The TPH2 rs11179023 A allele showed a marginal association with symptom improvement in the IPC group only. Other gene variants did not impact on outcomes in any treatment group. CONCLUSION: Our study suggests that rs8076005 in the SLC6A4 gene may be a modulator of antidepressant response, especially when pharmacological treatment is used.

Characterisation of gene expression profiles of yeast cells expressing BRCA1 missense variants.

Germline mutations in breast cancer susceptibility gene 1 (BRCA1) confer high risk of developing breast and ovarian cancers. Even though most BRCA1 cancer-predisposing mutations produce a non-functional truncated protein, 5-10% of them cause single amino acid substitutions. This second type of mutations represents a useful tool for examining BRCA1 molecular functions. Human BRCA1 inhibits cell proliferation in transformed Saccharomyces cerevisiae cells and this effect is abolished by disease-associated mutations in the BRCT domain. Moreover, BRCA1 mutations located both inside and outside the BRCT domain may induce an increase in the homologous recombination frequency in yeast cells. Here we present a microarray analysis of gene expression induced in yeast cells transformed with five BRCA1 missense variants, in comparison with gene expression induced by wild-type BRCA1. Data analysis was performed by grouping the BRCA1 variants into three sets: Recombination (R)-set (Y179C and S1164I), Recombination and Proliferation (RP)-set (I1766S and M1775R) and Proliferation (P)-set (A1789T), according to their effects on yeast cell phenotype. We found 470, 740 and 1136 differentially expressed genes in R-, P- and RP-set, respectively. Our results point to some molecular mechanisms critical for the control of cell proliferation and of genome integrity providing support to a possible pathogenic role of the analysed mutations. They also confirm that yeast, despite the absence of a BRCA1 homologue, represents a valid model system to examine BRCA1 molecular functions, as the molecular pathways activated by BRCA1 variants are conserved in humans.