Recurrence quantification analysis of resting state EEG signals in autism spectrum disorder - a systematic methodological exploration of technical and demographic confounders in the search for biomarkers.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a worldwide prevalence of 1-2%. In low-resource environments, in particular, early identification and diagnosis is a significant challenge. Therefore, there is a great demand for 'language-free, culturally fair' low-cost screening tools for ASD that do not require highly trained professionals. Electroencephalography (EEG) has seen growing interest as an investigational tool for biomarker development in ASD and neurodevelopmental disorders. One of the key challenges is the identification of appropriate multivariate, next-generation analytical methodologies that can characterise the complex, nonlinear dynamics of neural networks in the brain, mindful of technical and demographic confounders that may influence biomarker findings. The aim of this study was to evaluate the robustness of recurrence quantification analysis (RQA) as a potential biomarker for ASD using a systematic methodological exploration of a range of potential technical and demographic confounders. METHODS: RQA feature extraction was performed on continuous 5-second segments of resting state EEG (rsEEG) data and linear and nonlinear classifiers were tested. Data analysis progressed from a full sample of 16 ASD and 46 typically developing (TD) individuals (age 0-18 years, 4802 EEG segments), to a subsample of 16 ASD and 19 TD children (age 0-6 years, 1874 segments), to an age-matched sample of 7 ASD and 7 TD children (age 2-6 years, 666 segments) to prevent sample bias and to avoid misinterpretation of the classification results attributable to technical and demographic confounders. A clinical scenario of diagnosing an unseen subject was simulated using a leave-one-subject-out classification approach. RESULTS: In the age-matched sample, leave-one-subject-out classification with a nonlinear support vector machine classifier showed 92.9% accuracy, 100% sensitivity and 85.7% specificity in differentiating ASD from TD. Age, sex, intellectual ability and the number of training and test segments per group were identified as possible demographic and technical confounders. Consistent repeatability, i.e. the correct identification of all segments per subject, was found to be a challenge. CONCLUSIONS: RQA of rsEEG was an accurate classifier of ASD in an age-matched sample, suggesting the potential of this approach for global screening in ASD. However, this study also showed experimentally how a range of technical challenges and demographic confounders can skew results, and highlights the importance of probing for these in future studies. We recommend validation of this methodology in a large and well-matched sample of infants and children, preferably in a low- and middle-income setting.

Localization of the gene for familial partial lipodystrophy (Dunnigan variety) to chromosome 1q21-22.

Obesity is strongly implicated in the pathophysiology of insulin resistance, diabetes mellitus and dyslipidemia. The mechanisms, however, by which obesity causes these complications are not known. The study of single-gene disorders affecting adipose tissue may elucidate some of the mechanisms involved in these processes. Familial partial lipodystrophy, Dunnigan variety, (FPLD, OMIM 308980) is an autosomal-dominant condition characterized by marked loss of subcutaneous adipose tissue affecting the trunk and extremities but with excess fat deposition in the head and neck areas. Affected individuals show an increased preponderance of insulin resistance, diabetes mellitus, dyslipidemia and acanthosis nigricans. The genetic basis of FPLD is unknown. We carried out a genome-wide scan with a set of highly polymorphic short tandem-repeats (STR) in individuals from five well-characterized pedigrees and mapped the FPLD locus to chromosome 1q21-22. The maximum two-point lod score obtained with a highly polymorphic microsatellite at D1S2624 at theta(max)=0 was 5.84. Multipoint-linkage analysis yielded a peak lod score of 8.25 between D1S305 and D1S1600. There was no evidence for genetic heterogeneity (alpha=1) in the pedigrees.

Diabetic retinopathy screening: perspectives of people with diabetes, screening intervals and costs of attending screening.

AIMS: To obtain the views of people with diabetes about the provision of diabetic retinopathy screening services; and the interval of screening. METHODS: Between October 2009 and January 2010, people with diabetes attending diabetic retinopathy screening clinics across Wales were asked to complete a questionnaire comprising of two parts: the first asking about their health, diabetes history, demographic characteristics and views about the diabetic retinopathy screening service, and the second asking about the costs of attending the screening. RESULTS: The response rate was 40% (n = 621) from 1550 questionnaires distributed at diabetic retinopathy clinics, with 600 complete responses analysed. Respondents had a mean known duration of diabetes of 8.5 years (sd 7.8) and had attended for screening on average 3.2 times (sd 1.6) in the last 5 years. Sixty-eight per cent (n = 408) of respondents reported having their eyes screened approximately once a year. Eighty-five per cent (n = 507) felt that they should have their eyes screened every year. However, 65% (n = 390) of respondents would accept screening at 2- or 3-year intervals if medical evidence showed that it was safe. The reported personal costs incurred by respondents attending diabetic retinopathy screening were low. CONCLUSION: Our study suggests that people with diabetes undergoing diabetic retinopathy screening would accept a greater screening interval, provided that adequate clinical evidence and medical reassurance were given.

SCF and APC: the Yin and Yang of cell cycle regulated proteolysis.

Progression through the cell cycle requires the activity of two ubiquitination complexes, the Skp1-cullin-F-box-protein complex (SCF) and the anaphase-promoting complex/cyclosome (APC). Observations in the past year have revealed unexpected similarities between the SCF and the APC and have allowed detailed insight into the regulation of their activities. Both complexes are now known to exist in different forms that target different substrates for ubiquitin-dependent proteolysis.

Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells.

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.

Glutathione-S-transferase (GST) P1, GSTM1, exercise, ozone and asthma incidence in school children.

BACKGROUND: Because asthma has been associated with exercise and ozone exposure, an association likely mediated by oxidative stress, we hypothesised that glutathione-S-transferase (GST)P1, GSTM1, exercise and ozone exposure have interrelated effects on the pathogenesis of asthma. METHODS: Associations of the well characterised null variant of GSTM1 and four single nucleotide polymorphisms (SNPs) that characterised common variation in the GSTP1 locus with new onset asthma in a cohort of 1610 school children were examined. Children's exercise and ozone exposure were classified using participation in team sports and community annual average ozone levels, respectively. RESULTS: A two SNP model involving putatively functional variants (rs6591255, rs1695 (Ile105Va)) best captured the association between GSTP1 and asthma. The risk of asthma was lower for those with the Val allele of Ile105Val (hazard ratio (HR) 0.60, 95% CI 0.4 to 0.8) and higher for the variant allele of rs6591255 (HR 1.40, 95% CI 1.1 to 1.9). The risk of asthma increased with level of exercise among ile(105) homozygotes but not among those with at least one val(105) allele (interaction p value = 0.02). The risk was highest among ile(105) homozygotes who participated in >or=3 sports in the high ozone communities (HR 6.15, 95% CI 2.2 to 7.4). GSTM1 null was independently associated with an increased risk of asthma and showed little variation with air pollution or GSTP1 genotype. These results were consistent in two independent fourth grade cohorts recruited in 1993 and 1996. CONCLUSION: Children who inherit a val(105) variant allele may be protected from the increased risk of asthma associated with exercise, especially in high ozone communities. GSTM1 null genotype was associated with an increased risk of asthma.

Characterization of vertebrate cohesin complexes and their regulation in prophase.

In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint.

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice.

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.

Splitting the chromosome: cutting the ties that bind sister chromatids.

In eukaryotic cells, sister DNA molecules remain physically connected from their production at S phase until their separation during anaphase. This cohesion is essential for the separation of sister chromatids to opposite poles of the cell at mitosis. It also permits chromosome segregation to take place long after duplication has been completed. Recent work has identified a multisubunit complex called cohesin that is essential for connecting sisters. Proteolytic cleavage of one of cohesin's subunits may trigger sister separation at the onset of anaphase.

Cohesin cleavage by separase required for anaphase and cytokinesis in human cells.

Cell division depends on the separation of sister chromatids in anaphase. In yeast, sister separation is initiated by cleavage of cohesin by the protease separase. In vertebrates, most cohesin is removed from chromosome arms by a cleavage-independent mechanism. Only residual amounts of cohesin are cleaved at the onset of anaphase, coinciding with its disappearance from centromeres. We have identified two separase cleavage sites in the human cohesin subunit SCC1 and have conditionally expressed noncleavable SCC1 mutants in human cells. Our results indicate that cohesin cleavage by separase is essential for sister chromatid separation and for the completion of cytokinesis.

How proteolysis drives the cell cycle.

Oscillations in the activity of cyclin-dependent kinases (CDKs) promote progression through the eukaryotic cell cycle. This review examines how proteolysis regulates CDK activity-by degrading CDK activators or inhibitors-and also how proteolysis may directly trigger the transition from metaphase to anaphase. Proteolysis during the cell cycle is mediated by two distinct ubiquitin-conjugation pathways. One pathway, requiring CDC34, initiates DNA replication by degrading a CDK inhibitor. The second pathway, involving a large protein complex called the anaphase-promoting complex or cyclosome, initiates chromosome segregation and exit from mitosis by degrading anaphase inhibitors and mitotic cyclins. Proteolysis therefore drives cell cycle progression not only by regulating CDK activity, but by directly influencing chromosome and spindle dynamics.

Identification of BIME as a subunit of the anaphase-promoting complex.

The initiation of anaphase and exit from mitosis require the activation of a proteolytic system that ubiquitinates and degrades cyclin B. The regulated component of this system is a large ubiquitin ligase complex, termed the anaphase-promoting complex (APC) or cyclosome. Purified Xenopus laevis APC was found to be composed of eight major subunits, at least four of which became phosphorylated in mitosis. In addition to CDC27, CDC16, and CDC23, APC contained a homolog of Aspergillus nidulans BIME, a protein essential for anaphase. Because mutation of bimE can bypass the interphase arrest induced by either nimA mutation or unreplicated DNA, it appears that ubiquitination catalyzed by APC may also negatively regulate entry into mitosis.

APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle.

The molecular mechanisms that link cell-cycle controls to the mitotic apparatus are poorly understood. A component of the Saccharomyces cerevisiae spindle, Ase1, was observed to undergo cell cycle-specific degradation mediated by the cyclosome, or anaphase promoting complex (APC). Ase1 was degraded when cells exited from mitosis and entered G1. Inappropriate expression of stable Ase1 during G1 produced a spindle defect that is sensed by the spindle assembly checkpoint. In addition, loss of ASE1 function destabilized telophase spindles, and expression of a nondegradable Ase1 mutant delayed spindle disassembly. APC-mediated proteolysis therefore appears to regulate both spindle assembly and disassembly.

Identification of a cullin homology region in a subunit of the anaphase-promoting complex.

The anaphase-promoting complex is composed of eight protein subunits, including BimE (APC1), CDC27 (APC3), CDC16 (APC6), and CDC23 (APC8). The remaining four human APC subunits, APC2, APC4, APC5, and APC7, as well as human CDC23, were cloned. APC7 contains multiple copies of the tetratrico peptide repeat, similar to CDC16, CDC23, and CDC27. Whereas APC4 and APC5 share no similarity to proteins of known function, APC2 contains a region that is similar to a sequence in cullins, a family of proteins implicated in the ubiquitination of G1 phase cyclins and cyclin-dependent kinase inhibitors. The APC2 gene is essential in Saccharomyces cerevisiae, and apc2 mutants arrest at metaphase and are defective in the degradation of Pds1p. APC2 and cullins may be distantly related members of a ubiquitin ligase family that targets cell cycle regulators for degradation.

Proteasomes: protein degradation machines of the cell.

20 S proteasomes are cylinder-shaped protein complexes that play an important role in intracellular protein degradation in eukaryotes and certain archaebacteria. Although 20 S proteasomes were first described many years ago, it has been discovered only recently that these particles can assemble with other protein complexes, presumably of regulatory function. One of the macromolecular assemblies formed, the 26 S proteasome complex, functions as an ATP-dependent protease in the ubiquitin pathway, which has been implicated in the degradation of abnormal proteins, degradation of short-lived regulatory proteins and antigen presentation.

PPARbeta/delta selectively induces differentiation and inhibits cell proliferation.

Peroxisome proliferator-activated receptor (PPAR) beta-null mice exhibit exacerbated epithelial cell proliferation and enhanced sensitivity to skin carcinogenesis, suggesting that ligand activation of PPARbeta will inhibit keratinocyte proliferation. By using of a highly specific ligand (GW0742) and the PPARbeta-null mouse model, activation of PPARbeta was found to selectively induce keratinocyte terminal differentiation and inhibit keratinocyte proliferation. Additionally, GW0742 was found to be anti-inflammatory due to inhibition of myeloperoxidase activity, independent of PPARbeta. These data suggest that ligand activation of PPARbeta could be a novel approach to selectively induce differentiation and inhibit cell proliferation, thus representing a new molecular target for the treatment of skin disorders resulting from altered cell proliferation such as psoriasis and cancer.

Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation.

BACKGROUND: The destruction of cyclin B is required for exit from mitosis, and is mediated by the ubiquitin pathway. Recently, a 20S complex, termed the anaphase-promoting complex (APC) or the cyclosome, has been genetically and biochemically identified as the cyclin-specific ubiquitin ligase (E3). In addition, a ubiquitin-conjugating enzyme (E2), UBC4, was shown to be involved in cyclin ubiquitination in Xenopus egg extracts. Another E2 activity, designated UBCx, can independently support cyclin ubiquitination in Xenopus. A similar activity (E2-C) has also been observed in clams. However, the molecular identity of Xenopus UBCx or clam E2-C has not been established. RESULTS: We have purified and cloned Xenopus UBCx. Sequence comparisons with known E2s reveal that UBCx is a novel ubiquitin-conjugating enzyme. Purified recombinant UBCx is sufficient to complement purified APC and E1 in destruction box-dependent cyclin ubiquitination. UBCx and UBC4 are active in a similar concentration range and with similar kinetics. At saturating enzyme concentrations, UBCx converts twice as much substrate into ubiquitin conjugates, but generates conjugates of lower molecular mass than UBC4. CONCLUSIONS: UBCx is a novel ubiquitin-conjugating enzyme involved in cyclin ubiquitination in Xenopus. Like UBC4, ubiquitination catalyzed by UBCx is dependent on both the destruction box and the APC, suggesting that these E2s function through a similar mechanism. However, as the patterns of conjugates generated by these E2s are distinct, these enzymes may play different roles in promoting cyclin proteolysis in mitosis.

Activation of the human anaphase-promoting complex by proteins of the CDC20/Fizzy family.

The initiation of anaphase and exit from mitosis depend on the activation of the cyclosome/anaphase-promoting complex (APC) that ubiquitinates regulatory proteins such as anaphase inhibitors and mitotic cyclins [1-4]. Genetic experiments have demonstrated that two related WD40-repeat proteins--called Cdc20p and Hct1p/Cdh1p in budding yeast and Fizzy and Fizzy-related in Drosophila--are essential for APC--dependent proteolysis [5-11]. Human orthologs of these proteins--hCDC20/p55CDC [12] and hCDH1--have recently been found to associate with APC in a cell-cycle-dependent manner [13,14]. Here, we show that the amount of hCDC20 and hCDH1 bound to APC correlates with a high ubiquitination activity of APC and that binding of recombinant hCDC20 and hCDH1 can activate APC in vitro. Our results suggest that the association between hCDH1 and APC is regulated by post-translational mechanisms, whereas the amount of hCDC20 bound to APC may in addition be controlled by hCDC20 synthesis and destruction [15]. The temporally distinct association of hCDC20 and hCDH1 with APC suggests that these proteins are, respectively, mitosis-specific and G1-specific activating subunits of APC.

Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.

Prolonged deprivation of food induces dramatic changes in mammalian metabolism, including the release of large amounts of fatty acids from the adipose tissue, followed by their oxidation in the liver. The nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPARalpha) was found to play a role in regulating mitochondrial and peroxisomal fatty acid oxidation, suggesting that PPARalpha may be involved in the transcriptional response to fasting. To investigate this possibility, PPARalpha-null mice were subjected to a high fat diet or to fasting, and their responses were compared with those of wild-type mice. PPARalpha-null mice chronically fed a high fat diet showed a massive accumulation of lipid in their livers. A similar phenotype was noted in PPARalpha-null mice fasted for 24 hours, who also displayed severe hypoglycemia, hypoketonemia, hypothermia, and elevated plasma free fatty acid levels, indicating a dramatic inhibition of fatty acid uptake and oxidation. It is shown that to accommodate the increased requirement for hepatic fatty acid oxidation, PPARalpha mRNA is induced during fasting in wild-type mice. The data indicate that PPARalpha plays a pivotal role in the management of energy stores during fasting. By modulating gene expression, PPARalpha stimulates hepatic fatty acid oxidation to supply substrates that can be metabolized by other tissues.