Self-caught methodologies for measuring mind wandering with meta-awareness: A systematic review.

Mind wandering, also known as task-unrelated thought, refers to the drift of attention from a focal task or train of thought. Because self-caught measures of mind wandering require participants to spontaneously indicate when they notice their attention drift, self-caught methodologies provide a way to measure mind wandering with meta-awareness. Given the proposed role of meta-awareness in mental health and psychological interventions, an overview of existing self-caught methodologies would help clinicians and researchers make informed decisions when choosing or adapting a mind wandering or meta-awareness measure. This systematic review included 39 studies after 790 studies were assessed for eligibility. All studies operationalised mind wandering as instances of attention drift from a primary task. Three types of primary task were identified: (1) tasks adapted from computerised continuous performance tests (CPT) of sustained attention, (2) tasks involving focusing on the breath or a stream of aural beats, akin to in-vivo mindfulness meditation, (3) tasks involving an everyday life activity such as reading. Although data on mind wandering without meta-awareness (e.g., measured with probe-caught measures) was also obtained in many studies, such data was not always used in conjunction with self-caught mind wandering data to determine level of mind wandering meta-awareness. Few studies reported both reliability and validity of the measures used. This review shows that considerable methodological heterogeneity exists in the literature. Methodological variants of self-caught mind wandering methodologies are documented and examined, and suggestions for future research and clinical work are suggested.

The Causes and Consequences of Nonenzymatic Protein Acylation.

Thousands of protein acyl modification sites have now been identified in vivo. However, at most sites the acylation stoichiometry is low, making functional enzyme-driven regulation in the majority of cases unlikely. As unmediated acylation can occur on the surface of proteins when acyl-CoA thioesters react with nucleophilic cysteine and lysine residues, slower nonenzymatic processes likely underlie most protein acylation. Here, we review how nonenzymatic acylation of nucleophilic lysine and cysteine residues occurs; the factors that enhance acylation at particular sites; and the strategies that have evolved to limit protein acylation. We conclude that protein acylation is an unavoidable consequence of the central role of reactive thioesters in metabolism. Finally, we propose a hypothesis for why low-stoichiometry protein acylation is selected against by evolution and how it might contribute to degenerative processes such as aging.

The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae.

Toxicity of metals to aquatic organisms is dependent on both external factors, such as exposure concentration and water quality parameters, and intracellular processes including specific metal-binding sites and detoxification. Current models used to predict copper toxicity in microalgae do not adequately consider these intracellular processes. This study compared the copper-binding proteins from four species of marine microalgae, Dunaliella tertiolecta, Tetraselmis sp., Phaedactylum tricornutum and Ceratoneis closterium, in controls (no added copper) and following a 72-h exposure to copper (sufficient to inhibit growth by approximately 50%). Cells were lysed by sonication, which was optimised to obtain 54-94% cell rupture for the different algae. Cell lysates were processed by immobilised metal affinity chromatography (IMAC) using Cu(2+) as the bound metal (i.e. Cu-IMAC). Bound proteins were subsequently analysed by SDS-PAGE, comparing proteins recovered from algae that were exposed to copper versus untreated control cells. Individual proteins for which copper exposure resulted in changes to proteins present were excised from gels and further analysed by nano LC ESI-MS/MS; proteins were identified using the Mascot database. Proteins identified in this way included heat-shock proteins, rubisco, α- and ß-tubulins and ATP synthase (ß subunit). The results established that Cu-IMAC is a useful approach to identify proteins involved in copper binding in algae. This study identified several proteins that may play an active role in responses to copper toxicity in marine microalgae.

Evolving methods for single nucleotide polymorphism detection: Factor V Leiden mutation detection.

BACKGROUND: The many techniques used to diagnose the Factor V Leiden (FVL) mutation, the most common hereditary hypercoagulation disorder in Eurasians, and the most frequently requested genetic test reflect the evolving strategies in protein and DNA diagnosis. METHODS: Here, molecular methods to diagnose the FVL mutation are discussed. RESULTS: Protein-based detection assays include the conventional functional activated protein C resistance coagulation test and the recently reported antibody-mediated sensor detection; and DNA-based assays include approaches that use electrophoretic fractionation e.g., restriction fragment length polymorphism, denaturing gradient gel electrophoresis, and single-stranded conformational PCR analysis, DNA hybridization (e.g., microarrays), DNA polymerase-based assays, e.g., extension reactions, fluorescence polarization template-directed dye-terminator incorporation, PCR assays (e.g., amplification-refractory mutation system, melting curve analysis using real-time quantitative PCR, and helicase-dependent amplification), DNA sequencing (e.g., direct sequencing, pyrosequencing), cleavase-based Invader assay and ligase-based assays (e.g., oligonucleotide ligation assay and ligase-mediated rolling circle amplification). CONCLUSION: The method chosen by a laboratory to diagnose FVL not only depends on the available technical expertise and equipment, but also the type, variety, and extent of other genetic disorders being diagnosed.

Advanced paternal age is a risk factor for schizophrenia in Iranians.

BACKGROUND: Since 1958 many, but not all studies have demonstrated that paternal age is a risk factor for schizophrenia. There may be many different explanations for differences between studies, including study design, sample size, collection criteria, heterogeneity and the confounding effects of environmental factors that can for example perturb epigenetic programming and lead to an increase in disease risk. The small number of children in Western families makes risk comparisons between siblings born at different paternal ages difficult. In contrast, more Eastern families have children both at early and later periods of life. In the present study, a cross-sectional population study in an Iranian population was performed to compare frequency of schizophrenia in younger offspring (that is, older paternal age) versus older offspring. METHODS: A total of 220 patients with the diagnosis of schizophrenia (cases) from both psychiatric hospitals and private clinics and 220 individuals from other hospital wards (controls), matched for sex and age were recruited for this study. Patients with neurological problem, substance abuse, mental retardation and mood disorder were excluded from both groups. RESULTS: Birth rank comparisons revealed that 35% vs 24% of the cases vs the controls were in the third or upper birth rank (P = 0.01). Also, the mean age of fathers at birth in case group (30 ± 6.26 years) was significantly more than the control group (26.45 ± 5.64 years; P = 0.0001). The age of 76 fathers at birth in case group was over 32 versus 33 fathers in control group. Individuals whose fathers' age was more than 32 (at birth) were at higher risk (2.77 times) for schizophrenia versus others (P < 0.0001, 95% CI 1.80 to 4.27). The maternal age at parturition of the case versus controls groups was 26.1 ± 5.41 vs 25.07 ± 4.47 (P = 0.02). Logistic regression analysis suggests that maternal age is less likely to be involved in the higher risk of schizophrenia than advanced parental age. DISCUSSION: This study demonstrates a relationship between paternal age and schizophrenia in large families of an Iranian population. Arguments have been put forth that DNA bases changes or epigenetic changes in sperm account for the increased risk associated with older fathers. However, it would not be surprising that both de novo germline mutations and epigenetic changes contribute to disease occurrence because DNA replication and DNA methylation are closely linked at both the macromolecular level (that is, methylation closely follows replication), and at the metabolic level (both processes require folate), and susceptible to modulation by the environment. Further research on samples such as those collected here are needed to sort out the contributions of de novo mutations versus epigenetic changes to schizophrenia.

Epigenetic alterations of the dopaminergic system in major psychiatric disorders.

Although there is evidence to link schizophrenia (SCZ) and bipolar disorder (BD) to genetic and environmental factors, specific individual or groups of genes/factors causative of the disease have been elusive to the research community. An understanding of the molecular aberrations that cause these mental illnesses requires comprehensive approaches that examine both genetic and epigenetic factors. Because of the overwhelming evidence for the role of environmental factors in the disease presentation, our initial approach involved deciphering how epigenetic changes resulting from promoter DNA methylation affect gene expression in SCZ and BD. Apparently, the central reversible but covalent epigenetic modification to DNA is derived from methylation of the cytosine residues that is potentially heritable and can affect gene expression and downstream activities. Environmental factors can influence DNA methylation patterns and hence alter gene expression. Such changes can be especially problematic in individuals with genetic susceptibilities to specific diseases. Recent reports from our laboratory provided compelling evidence that both hyper- and hypo-DNA methylation changes of the regulatory regions play critical roles in defining the altered functionality of genes in major psychiatric disorders such as SCZ and BD. In this chapter, we outline the technical details of the methods that could help to expand this line of research to assist with compiling the differential methylation-mediated epigenetic alterations that are responsible for the pathogenesis of SCZ, BD, and other mental diseases. We use the genes of the extended dopaminergic (DAergic) system such as membrane-bound catechol-O-methyltransferase (MB-COMT), monoamine oxidase A (MAOA), dopamine transporter 1 (DAT1), tyrosine hydroxylase (TH), dopamine (DA) receptors1 and 2 (DRD1/2), and related genes (e.g., reelin [RELN] and brain-derived neurotrophic factor [BDNF]) to illustrate the associations between differential promoter DNA methylations and disease phenotype. It is our hope that comprehensive analyses of the DAergic system as the prototype could provide the impetus and molecular basis to uncover early markers for diagnosis, help in the understanding of differences in disease severity in individuals with similar or identical genetic makeup, and assist with the identification of novel targets for therapeutic applications.

Proximal Cysteines that Enhance Lysine N-Acetylation of Cytosolic Proteins in Mice Are Less Conserved in Longer-Living Species.

Acetyl-coenzyme A (CoA) is an abundant metabolite that can also alter protein function through non-enzymatic N-acetylation of protein lysines. This N-acetylation is greatly enhanced in vitro if an adjacent cysteine undergoes initial S-acetylation, as this can lead to S→N transfer of the acetyl moiety. Here, using modeled mouse structures of 619 proteins N-acetylated in mouse liver, we show lysine N-acetylation is greater in vivo if a cysteine is within ∼10 Å. Extension to the genomes of 52 other mammalian and bird species shows pairs of proximal cysteine and N-acetylated lysines are less conserved, implying most N-acetylation is detrimental. Supporting this, there is less conservation of cytosolic pairs of proximal cysteine and N-acetylated lysines in species with longer lifespans. As acetyl-CoA levels are linked to nutrient supply, these findings suggest how dietary restriction could extend lifespan and how pathologies resulting from dietary excess may occur.

Targeted cDNA differential display (TcDD).

Targeted cDNA differential display (TcDD) was developed to study expression of a different selected gene families especially those at low copy numbers per cell. This method is an adaptation of our previously described targeted genomic differential display method (TGDD). In TcDD, the expression of genes containing target sequences such as CAG repeating sequences or genes encoding for zinc-finger binding proteins were followed in an experimental rat model with salt-induced hypertension. DNA sequencing experiments demonstrated that the effectiveness of targeting was greater than 99%.

Cloneless genomic DNA analysis: an efficient and simple methods for de novo genomic sequencing projects and gap filling.

The utility of using genomic DNA directly in agarose, i.e. cloneless libraries, in place of large clone libraries, radiation hybrid panels, or chromosome dissection was demonstrated. The advantage of the cloneless library approach is that, in principle, a targeted genomic resource can be developed rapidly for any genomic region using any genomic DNA sample. Here, a human chromosome 20 Not I fragment library was generated by slicing a pulsed field gel lane containing fractionating Not I cleaved DNA from a monosomic hybrid cell line into 2 mm pieces. A reliable PCR method using agarose embedded DNA was developed. InterAlu PCR generated unique patterns of products from adjacent slices (e.g. fractions). Further, the specificity of the interAlu products was demonstrated by FISH analysis and in other hybridization experiments to arrayed interAlu products. STS content mapping was used to order the fractions and also demonstrate the unique content of the library fractions.

Mild conditions for releasing mono and bis-biotinylated macromolecules from immobilized streptavidin.

The high affinity (kd= approximately 10(-15)M) of streptavidin and avidin for biotin is key to a large number of biological applications and is essentially irreversible unless the complex is exposed to harsh conditions (e.g. heat (100 degrees C for 10 min)), detergents, and/or denaturants which damage macromolecules. Thus, high binding affinity becomes a disadvantage when a biotinylated target must be released for further processing. This work describes relatively mild conditions that release biotin and mono- and bis-biotinylated macromolecules from immobilized streptavidin on monodispersed magnetic beads.

Copper-induced changes in intracellular thiols in two marine diatoms: Phaeodactylum tricornutum and Ceratoneis closterium.

Phytochelatins and glutathione (reduced (GSH) and oxidised (GSSG)) are important intracellular ligands involved in metal sequestration and detoxification in algae. Intracellular ratios of GSH:GSSG are sensitive indicators of metal stress in algae, and like phytochelatin production are influenced by metal speciation, concentration, exposure time and the biological species. This study investigated the effect of copper exposure on phytochelatin and glutathione content in two marine diatoms Phaeodactylum tricornutum and Ceratoneis closterium at various time intervals between 0.5 and 72h. Liberation of cellular glutathione and phytochelatins was optimised using freeze/thaw cycles and chemical extraction, respectively. Extracted phytochelatins were derivatised (by fluorescent tagging of thiol compounds), separated and quantified using HPLC with fluorescence detection. Glutathione ratios were determined using a commercially available kit, which uses the enzyme glutathione reductase to measure total and oxidised glutathione. Despite similarities in size and shape between the two diatoms, differences in internalised copper, phytochelatin production (both chain length and quantity) and reduced glutathione concentrations were observed. P. tricornutum maintained reduced glutathione at between 58 and 80% of total glutathione levels at all time points, which would indicate low cellular stress. In C. closterium reduced glutathione constituted <10% of total glutathione after 48h. P. tricornutum also produced more phytochelatins and phytochelatins of longer chain length than C. closterium despite the latter species internalising significantly more copper.

Conference scene: 2nd cancer epigenetics conference.

The GTC Cancer Summit: Novel Approaches to Drug Discovery was divided into two parallel tracks: the 2nd Cancer Epigenetics Conference, and the Protein Kinases and Drug Design Conference. The 2nd Cancer Epigenetics Conference focused on exciting changes in drug discovery that include an unprecedented private and public collaboration on drug discovery in epigenetics through the Structural Genomics Consortium (SGC), which has led to several major breakthroughs including: the development of small-molecule inhibitors that interfere with protein interactions, especially bromodomain-containing protein acetylation readers; the indirect but successful targeting of the elusive MYC oncogene; and the identification of epigenetic drugs that are disease-specific. Also reported were the development of clinically useful DNA methylation assays; cell, peptide and protein arrays for testing antibody- and protein-binding specificity; and tools for chromatin capture and DNA modification analysis. Several groups reported on the lack of specificity of some commercial, but unnamed, antibodies used for epigenetic studies.

Genomic and epigenomic instability, fragile sites, schizophrenia and autism.

Increasing evidence links genomic and epigenomic instability, including multiple fragile sites regions to neuropsychiatric diseases including schizophrenia and autism. Cancer is the only other disease associated with multiple fragile site regions, and genome and epigenomic instability is a characteristic of cancer. Research on cancer is far more advanced than research on neuropsychiatric disease; hence, insight into neuropsychiatric disease may be derived from cancer research results. Towards this end, this article will review the evidence linking schizophrenia and other neuropsychiatric diseases (especially autism) to genomic and epigenomic instability, and fragile sites. The results of studies on genetic, epigenetic and environmental components of schizophrenia and autism point to the importance of the folate-methionine-transulfuration metabolic hub that is diseases also perturbed in cancer. The idea that the folate-methionine-transulfuration hub is important in neuropsychiatric is exciting because this hub present novel targets for drug development, suggests some drugs used in cancer may be useful in neuropsychiatric disease, and raises the possibility that nutrition interventions may influence the severity, presentation, or dynamics of disease.

Epigenetic and pharmacoepigenomic studies of major psychoses and potentials for therapeutics.

Individuals with neuropsychiatric diseases have epigenetic programming disturbances, both in the brain, which is the primary affected organ, and in secondary tissues. Epigenetic modulations are molecular modifications made to DNA, RNA and proteins that fine-tune genotype into phenotype and do not include DNA base changes. For instance, gene-expression modulation is linked to epigenetic codes in chromatin that consist of post-replication DNA methylation and histone protein modifications (e.g., methylation, acetylation and so on), particularly in gene-promoter regions. Epigenetic coding is modulated globally, and in a gene-specific manner by environmental exposures that include nutrition, toxins, drugs and so on. Analysis of epigenetic aberrations in diseases helps to identify dysfunctional genes and pathways, establish more robust cause-effect relationships than genetic studies alone, and identify new pharmaceutical targets and drugs, including nucleic acid reagents such as inhibitory RNAs. The emerging science of pharmacoepigenomics can impact the treatment of psychiatric and other complex diseases. In fact, some therapeutics now in use target epigenetic programming. In the near future, epigenetic interventions should help stabilize affected individuals and lead to prevention strategies.

Hypomethylation of MB-COMT promoter is a major risk factor for schizophrenia and bipolar disorder.

The variability in phenotypic presentations and the lack of consistency of genetic associations in mental illnesses remain a major challenge in molecular psychiatry. Recently, it has become increasingly clear that altered promoter DNA methylation could play a critical role in mediating differential regulation of genes and in facilitating short-term adaptation in response to the environment. Here, we report the investigation of the differential activity of membrane-bound catechol-O-methyltransferase (MB-COMT) due to altered promoter methylation and the nature of the contribution of COMT Val158Met polymorphism as risk factors for schizophrenia and bipolar disorder by analyzing 115 post-mortem brain samples from the frontal lobe. These studies are the first to reveal that the MB-COMT promoter DNA is frequently hypomethylated in schizophrenia and bipolar disorder patients, compared with the controls (methylation rate: 26 and 29 versus 60%; P=0.004 and 0.008, respectively), particularly in the left frontal lobes (methylation rate: 29 and 30 versus 81%; P=0.003 and 0.002, respectively). Quantitative gene-expression analyses showed a corresponding increase in transcript levels of MB-COMT in schizophrenia and bipolar disorder patients compared with the controls (P=0.02) with an accompanying inverse correlation between MB-COMT and DRD1 expression. Furthermore, there was a tendency for the enrichment of the Val allele of the COMT Val158Met polymorphism with MB-COMT hypomethylation in the patients. These findings suggest that MB-COMT over-expression due to promoter hypomethylation and/or hyperactive allele of COMT may increase dopamine degradation in the frontal lobe providing a molecular basis for the shared symptoms of schizophrenia and bipolar disorder.

Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: a preliminary report.

DNA methylation changes could provide a mechanism for DNA plasticity and dynamism for short-term adaptation, enabling a type of cell memory to register cellular history under different environmental conditions. Some environmental insults may also result in pathological methylation with corresponding alteration of gene expression patterns. Evidence from several studies has suggested that in schizophrenia and bipolar disorder, mRNA of the reelin gene (RELN), which encodes a protein necessary for neuronal migration, axonal branching, synaptogenesis, and cell signaling, is severely reduced in post-mortem brains. Therefore, we investigated the methylation status of the RELN promoter region in schizophrenic patients and normal controls as a potential mechanism for down regulation of its expression. Ten post-mortem frontal lobe brain samples from male schizophrenic patients and normal controls were obtained from the Harvard Brain Tissue Resources Center. DNA was extracted using a standard phenol-chloroform DNA extraction protocol. To evaluate differences between patients and controls, we applied methylation specific PCR (MSP) using primers localized to CpG islands flanking a potential cyclic AMP response element (CRE) and a stimulating protein-1 (SP1) binding site located in the promoter region. For each sample, DNA extraction, bisulfite treatment, and MSP were independently repeated at least four times to accurately determine the methylation status of the target region. Forty-three PCR trials were performed on the test and control samples. MSP analysis of the RELN promoter revealed an unmethylated signal in all reactions (43 of 43) using DNA from the frontal brain tissue, derived from either the schizophrenic patients or normal controls indicating that this region of the RELN promoter is predominantly unmethylated. However, we observed a distinct methylated signal in 73% of the trials (16 of 22) in schizophrenic patients compared with 24% (5 of 21) of controls. Thus, the hypermethylation of the CpG islands flanking a CRE and SP1 binding site observed at a significantly higher level (t = -5.07, P = 0.001) may provide a mechanism for the decreased RELN expression, frequently observed in post-mortem brains of schizophrenic patients. We also found an inverse relationship between the level of DNA methylation using MSP analysis and the expression of the RELN gene using semi-quantitative RT-PCR. Despite the small sample size, these studies indicate that promoter hypermethylation of the RELN gene could be a significant contributor in effecting epigenetic alterations and provides a molecular basis for the RELN gene hypoactivity in schizophrenia. Further studies with a larger sample set would be required to validate these preliminary observations.

Methylomics in psychiatry: Modulation of gene-environment interactions may be through DNA methylation.

Fine-tuning of neuronal connections during development is regulated through environmental interactions. Some fine-tuning occurs through changes in gene expression and/or epigenetic gene-specific DNA methylation states. DNA methylation occurs by transfer of a methyl group from S-adenosyl methionine to cytosine residues in the dinucleotide sequence CpG. Although CpG sequences spread throughout the genome are usually heavily methylated, those occurring in CpG islands in the promoter regions of genes are less methylated. In most cases, the extent of DNA methylation correlates with the extent of gene inactivation. Other known epigenetic mechanisms include histone deacetylation and chromatin remodeling, RNA inhibition, RNA modification, and DNA rearrangement. Exposure memory expressed as epigenetic DNA modifications allows genomic plasticity and short-term adaptation of each generation to their environment. Environmental factors that affect DNA methylation include diet, proteins, drugs, and hormones. Induced methylation changes may produce altered gene response upon subsequent hormonal stimulation. The gene-specific DNA methylation state may be preserved upon transmission through mitosis and meiosis. An increasing amount of data implicates a role for DNA methylation in multi-factorial psychiatric disorders. For example, L-methionine treatment can exacerbate psychosis; while valproate, a drug producing hypomethylated DNA, reduces such symptoms. Hypermethylation of the promoter region of the RELN gene correlates with reduced gene expression. This gene's protein Reelin, which is necessary for neuronal migration and synaptogenesis, is reduced in schizophrenia and bipolar disorder, suggesting hypermethylation of the promoter region in these disorders. Some evidence implicates methylation of the promoter regions of the DRD2 and HTR2A genes in schizophrenia and mood disorders as well. DNA methylation usually increases with age, although hypomethylation of the promoter region of the amyloid A4 precursor gene during aging may play a role in Alzheimer's disease. More studies are needed to define the role of methylomics and other epigenetic phenomena in the nervous system.

DNA stability and schizophrenia in twins.

The goal of these experiments was to understand DNA changes relevant to schizophrenia. This work compared DNA of monozygotic (MZ) twins surrounding (CAG)(n) repeating sequences, and characterized the relationship between fragile sites and schizophrenia. Twelve twin-pairs, previously classified as MZ and 18 unrelated sib-pairs, from seven families were studied. Eight twin-pairs were affected by schizophrenia, four concordantly and four discordantly. DNA comparisons were made using profiles of electrophoretic size fractionations of PCR amplified (CAG)(n) containing genomic fragments. These profiles were generated by a new method, developed by us, called targeted genomic differential display (TGDD). Surprisingly, the number of peak profile differences in MZ twin-pairs discordant for schizophrenia was greater than the concordantly ill twins and the well twins and, in some cases, overlapped the range of sib-pairs. These results might mean that some twins were not MZ but it was not possible to definitively test these samples for zygosity. Alternatively, the results might be explained as an increased mutation rate (or genomic instability) around (CAG)(n) sites in individuals afflicted with schizophrenia. Also, we uncovered an association of schizophrenia (i.e., a linkage of chromosomal abnormalities and gene localizations) with fragile sites spread throughout the genome (chi(2), P = 0.001). Furthermore, it appears that an increasing number of genes linked to schizophrenia are associated with (CAG)(n) sequences. Fragile sites and (CAG)(n) repeat sequences are known to be unstable. We speculate the association of genomic instability with schizophrenia accounts for seemingly disparate biological and environmental factors that influence disease occurrence.