fMRI BOLD and MEG theta power reflect complementary aspects of activity during lexicosemantic decision in adolescents with ASD.

Neuroimaging studies of autism spectrum disorder (ASD) have been predominantly unimodal. While many fMRI studies have reported atypical activity patterns for diverse tasks, the MEG literature in ASD remains comparatively small. Our group recently reported atypically increased event-related theta power in individuals with ASD during lexicosemantic processing. The current multimodal study examined the relationship between fMRI BOLD signal and anatomically-constrained MEG (aMEG) theta power. Thirty-three adolescents with ASD and 23 typically developing (TD) peers took part in both fMRI and MEG scans, during which they distinguished between standard words (SW), animal words (AW), and pseudowords (PW). Regions-of-interest (ROIs) were derived based on task effects detected in BOLD signal and aMEG theta power. BOLD signal and theta power were extracted for each ROI and word condition. Compared to TD participants, increased theta power in the ASD group was found across several time windows and regions including left fusiform and inferior frontal, as well as right angular and anterior cingulate gyri, whereas BOLD signal was significantly increased in the ASD group only in right anterior cingulate gyrus. No significant correlations were observed between BOLD signal and theta power. Findings suggest that the common interpretation of increases in BOLD signal and theta power as 'activation' require careful differentiation, as these reflect largely distinct aspects of regional brain activity. Some group differences in dynamic neural processing detected with aMEG that are likely relevant for lexical processing may be obscured by the hemodynamic signal source and low temporal resolution of fMRI.

Algorithmic assessment of cellular senescence in experimental and clinical specimens.

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies-pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells-that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies.

Dynamic time warping outperforms Pearson correlation in detecting atypical functional connectivity in autism spectrum disorders.

Resting state fMRI (rsfMRI) is frequently used to study brain function, including in clinical populations. Similarity of blood-oxygen-level-dependent (BOLD) fluctuations during rsfMRI between brain regions is thought to reflect intrinsic functional connectivity (FC), potentially due to history of coactivation. To quantify similarity, studies have almost exclusively relied on Pearson correlation, which assumes linearity and can therefore underestimate FC if the hemodynamic response function differs regionally or there is BOLD signal lag between timeseries. Here we show in three cohorts of children, adolescents and adults, with and without autism spectrum disorders (ASDs), that measuring the similarity of BOLD signal fluctuations using non-linear dynamic time warping (DTW) is more robust to global signal regression (GSR), has higher test-retest reliability and is more sensitive to task-related changes in FC. Additionally, when comparing FC between individuals with ASDs and typical controls, more group differences are detected using DTW. DTW estimates are also more related to ASD symptom severity and executive function, while Pearson correlation estimates of FC are more strongly associated with respiration during rsfMRI. Together these findings suggest that non-linear methods such as DTW improve estimation of resting state FC, particularly when studying clinical populations whose hemodynamics or neurovascular coupling may be altered compared to typical controls.

Auditory time perception in Huntington's disease.

BACKGROUND: Huntington's disease (HD) is characterized by early involvement of the striatum. It affects the pace of repetitive motor activity, as motor timing depends on basal ganglia activity. However, data are lacking on the impact of this process on auditory time perception in motor non-affected gene carriers. OBJECTIVE: This work aims to test the performance in time perception of a group of mutation carriers, either without motor symptoms or at an early stage of motor involvement. This should allow designing therapies targeting compensation strategies and possibly be used as a disease progression marker. METHOD: Time was assessed using two different tasks. An absolute, duration-based time perception was assessed in a first task and a relative, beat-based time perception was assessed in a second one. HD-mutation carriers with low-to-middle grades of motor involvement (HD-motor, n = 10) or without motor signs (HD-premotor n = 21), were compared with age- and sex-matched healthy controls (control (n = 27)). Thresholds of time difference perception where assessed. RESULTS: For both tasks, poorer performances were found in HD-motor patients as compared with HD-premotor and controls. Thresholds of time difference perception correlated positively with the CAP score for the whole group of HD-gene carriers in both tasks. In a post-hoc exploratory analysis performed by a multiple regression, a negative correlation was found between the thresholds in both tasks and the Stroop interference test. Furthermore, in the first task, a positive correlation was found between thresholds and a trail making B test and a negative one with a total functional score. CONCLUSION: Our data confirm that the impairment in time perception in persons affected by HD correlates with the advancing disease. They also suggest that time perception depends on similar cognitive mechanisms as the ones sub-serving the Stroop interference test.

Five RecA-like proteins of Schizosaccharomyces pombe are involved in meiotic recombination.

The genome of Schizosaccharomyces pombe contains five genes that code for proteins with sequence similarity to the Escherichia coli recombination protein RecA: rad51+, rhp55+, rhp57+, rlp1+, and dmc1+. We analyzed the effect of deletion of each of these genes on meiotic recombination and viability of spores. Meiotic recombination levels were different from wild type in all recA-related mutants in several genetic intervals, suggesting that all five RecA homologs of S. pombe are required for normal levels of meiotic recombination. Spore viability was reduced in rad51, rhp55, and rhp57 mutants, but not in rlp1 and dmc1. It is argued that reduction of crossover is not the only cause for the observed reduction of spore viability. Analysis of double and triple mutants revealed that Rad51 and Dmc1 play major and partially overlapping roles in meiotic recombination, while Rhp55, Rhp57, and Rlp1 play accessory roles. Remarkably, deletion of Rlp1 decreases the frequency of intergenic recombination (crossovers), but increases intragenic recombination (gene conversion). On the basis of our results, we present a model for the involvement of five RecA-like proteins of S. pombe in meiotic recombination and discuss their respective roles.

The osseofasciocutaneous flap: a new method to transfer fibula along with a sufficient amount of skin.

We describe a new technique of bone transfer, using a deep fascial blood supply to transfer bone together with a large area of skin. The viability of this flap and further confirmation by isotope scanning have established that the bone transfer is vascularised. This provides a simple solution to the complex problem of leg trauma with massive skin and bone loss.

Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair.

To study the role of Rad50 in the DNA damage response, we cloned and deleted the Schizosaccharomyces pombe RAD50 homologue. The deletion is sensitive to a range of DNA-damaging agents and shows dynamic epistatic interactions with other recombination-repair genes. We show that Rad50 is necessary for recombinational repair of the DNA lesion at the mating-type locus and that rad50Delta shows slow DNA replication. We also find that Rad50 is not required for slowing down S phase in response to hydroxy urea or methyl methanesulfonate (MMS) treatment. Interestingly, in rad50Delta cells, the recombination frequency between two homologous chromosomes is increased at the expense of sister chromatid recombination. We propose that Rad50, an SMC-like protein, promotes the use of the sister chromatid as the template for homologous recombinational repair. In support of this, we found that Rad50 functions in the same pathway for the repair of MMS-induced damage as Rad21, the homologue of the Saccharomyces cerevisiae Scc1 cohesin protein. We speculate that Rad50 interacts with the cohesin complex during S phase to assist repair and possibly re-initiation of replication after replication fork collapse.

Live observation of fission yeast meiosis in recombination-deficient mutants: a study on achiasmate chromosome segregation.

Regular segregation of homologous chromosomes during meiotic divisions is essential for the generation of viable progeny. In recombination-proficient organisms, chromosome disjunction at meiosis I generally occurs by chiasma formation between the homologs (chiasmate meiosis). We have studied meiotic stages in living rec8 and rec7 mutant cells of fission yeast, with special attention to prophase and the first meiotic division. Both rec8 and rec7 are early recombination mutants, and in rec7 mutants, chromosome segregation at meiosis I occurs without any recombination (achiasmate meiosis). Both mutants showed distinct irregularities in nuclear prophase movements. Additionally, rec7 showed an extended first division of variable length and with single chromosomes changing back and forth between the cell poles. Two other early recombination deficient mutants (rec14 and rec15) showed very similar phenotypes to rec7 during the first meiotic division, and the fidelity of achiasmate chromosome segregation slightly exceeded the expected random level. We discuss possible regulatory mechanisms of fission yeast to deal with achiasmate chromosome segregation.

Biochemical characterization of the structure-specific DNA-binding protein Cmb1 from Schizosaccharomyces pombe.

Cmb1, a novel HMG box protein from Schizosaccharomyces pombe, has been characterized biochemically using glutaraldehyde cross-linking, gel-filtration and analytical ultracentrifugation. It was identified as a monomeric, non-spherical protein, with a tendency to aggregate in solution. Limited proteolysis with trypsin and chymotrypsin showed that the C-terminal HMG box was a compact, proteolytically stable domain and the N-terminal region of Cmb1 was relatively unstructured and more easily digested. As Cmb1 was previously identified as a potential mismatch-binding protein, the binding constants and stoichiometry for both homoduplex and heteroduplex DNA were determined using an IASys resonant mirror biosensor. Cmb1 indeed demonstrated a tighter association with mismatched DNA, especially with the C/Delta-mismatch. Expression constructs of Cmb1 were made to study the sections of the protein involved in DNA binding. Constructs with the N-terminal region absent revealed that the C-terminal HMG box was the primary DNA-binding region. The presence of the N-terminal region did, however, facilitate tighter binding to both homoduplex and heteroduplex DNA. The amino acid residues isoleucine 14 and leucine 39 were located as putative intercalating residues using structure guided homology modelling. The model templates were derived from two distinct HMG:DNA complexes: HMG-D bound to homoduplex DNA and HMG 1 bound to cisplatin DNA. Binding studies using the Cmb1 HMG box with point mutations in these residues showed that isoleucine 14 was important for the binding of Cmb1 to homoduplex DNA, but affected binding to mismatches to a lesser extent. In contrast, leucine 39 appeared to have a more significant function in binding to mismatched DNA.

Characterization of rec7, an early meiotic recombination gene in Schizosaccharomyces pombe.

rec7 is involved in intra- and intergenic meiotic recombination in all tested regions of the genome of the fission yeast Schizosaccharomyces pombe. Segregational analysis in a rec7 gene disruption mutant revealed frequent occurrence of two-spored asci. Spores giving rise to diploid colonies were shown to derive from skipping of the second meiotic division. Nondisjunction of homologous chromosomes at the first meiotic division was also frequent. The cytological structures and processes, such as formation of linear elements, pairing of homologous chromosomes, and clustering of telomeres and centromeres, are regular in the mutant. Northern blot experiments revealed meiosis-specific expression of rec7. Screening of a meiotic cDNA library also identified transcripts from the opposite strand in the rec7 region. A Rec7-GFP fusion protein was localized in the nucleus of whole cells before karyogamy, during prophase, and after meiosis I. On spreads of prophase nuclei approximately 50 foci of Rec7-GFP were counted. Some of the observed phenotypes of the disruption mutant and the N-terminal sequence homology suggest that Rec7p is a functional homolog of Rec114p of Saccharomyces cerevisiae. The observed phenotypes of the disruption and the appearance of Rec7-GFP in mating haploid cells and after meiosis I are consistent with Rec7p functions before, during, and after meiotic prophase.

Counteracting regulation of chromatin remodeling at a fission yeast cAMP response element-related recombination hotspot by stress-activated protein kinase, cAMP-dependent kinase and meiosis regulators.

In fission yeast, an ATF/CREB-family transcription factor Atf1-Pcr1 plays important roles in the activation of early meiotic processes via the stress-activated protein kinase (SAPK) and the cAMP-dependent protein kinase (PKA) pathways. In addition, Atf1-Pcr1 binds to a cAMP responsive element (CRE)-like sequence at the site of the ade6-M26 mutation, which results in local enhancement of meiotic recombination and chromatin remodeling. Here we studied the roles of meiosis-inducing signal transduction pathways in M26 chromatin remodeling. Chromatin analysis revealed that persistent activation of PKA in meiosis inhibited M26 chromatin remodeling, suggesting that the PKA pathway represses M26 chromatin remodeling. The SAPK pathway activated M26 chromatin remodeling, since mutants lacking a component of this pathway, the Wis1 or Spc1/Sty1 kinases, had no M26 chromatin remodeling. M26 chromatin remodeling also required the meiosis regulators Mei2 and Mei3 but not the subsequently acting regulators Sme2 and Mei4, suggesting that induction of M26 chromatin remodeling needs meiosis-inducing signals before premeiotic DNA replication. Similar meiotic chromatin remodeling occurred meiotically around natural M26 heptamer sequences. These results demonstrate the coordinated action of genetic and physiological factors required to remodel chromatin in preparation for high levels of meiotic recombination and eukaryotic cellular differentiation.

No mutagenic or recombinogenic effects of mobile phone fields at 900 MHz detected in the yeast Saccharomyces cerevisiae.

Both actively growing and resting cells of the yeast Saccharomyces cerevisiae were exposed to 900-MHz fields that closely matched the Global System for Mobile Communication (GSM) pulsed modulation format signals for mobile phones at specific absorption rates (SAR) of 0.13 and 1.3 W/kg. Two identical anechoic test chambers were constructed to perform concurrent control and test experiments under well-controlled exposure conditions. Using specific test strains, we examined the genotoxic potential of mobile phone fields, alone and in combination, with a known genotoxic compound, the alkylating agent methyl methansulfonate. Mutation rates were monitored by two test systems, a widely used gene-specific forward mutation assay at CAN1 and a wide-range assay measuring the induction of respiration-deficient (petite) clones that have lost their mitochondrial function. In addition, two further assays measured the recombinogenic effect of mobile phone fields to detect possible effects on genomic stability: First, an intrachromosomal, deletion-formation assay previously developed for genotoxic screening; and second, an intragenic recombination assay in the ADE2 gene. Fluctuation tests failed to detect any significant effect of mobile phone fields on forward mutation rates at CAN1, on the frequency of petite formation, on rates of intrachromosomal deletion formation, or on rates of intragenic recombination in the absence or presence of the genotoxic agent methyl methansulfonate.

Large transverse fasciocutaneous leg flap: whole leg flap.

Large defects of leg and sole often need massive tissue transfer. As an alternative to microvascular transfer, we have developed a fasciocutaneous flap in which almost the whole of the skin of the opposite leg is transferred based just on the septocutaneous perforators of the posterior tibial artery. The flap has been used as a cross-leg fasciocutaneous flap with potential for use as a free flap by taking a segment of the posterior tibial artery. Our experience with 11 cases is presented. Two patients suffered marginal necrosis while donor site problems were seen in another two patients. We have found this flap to be safe, technically easy and with minimal donor-site morbidity.

Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fission yeast to humans.

Our work and that of others defined mitosis-specific (Rad21 subfamily) and meiosis-specific (Rec8 subfamily) proteins involved in sister chromatid cohesion in several eukaryotes, including humans. Mutation of the fission yeast Schizosaccharomyces pombe rec8 gene was previously shown to confer a number of meiotic phenotypes, including strong reduction of recombination frequencies in the central region of chromosome III, absence of linear element polymerization, reduced pairing of homologous chromosomes, reduced sister chromatid cohesion, aberrant chromosome segregation, defects in spore formation, and reduced spore viability. Here we extend the description of recombination reduction to the central regions of chromosomes I and II. We show at the protein level that expression of rec8 is meiosis specific and that Rec8p localizes to approximately 100 foci per prophase nucleus. Rec8p was present in an unphosphorylated form early in meiotic prophase but was phosphorylated prior to meiosis I, as demonstrated by analysis of the mei4 mutant blocked before meiosis I. Evidence for the persistence of Rec8p beyond meiosis I was obtained by analysis of the mutant mes1 blocked before meiosis II. A human gene, which we designate hrec8, showed significant primary sequence similarity to rec8 and was mapped to chromosome 14. High mRNA expression of mouse and human rec8 genes was found only in germ line cells, specifically in testes and, interestingly, in spermatids. hrec8 was also expressed at a low level in the thymus. Sequence similarity and testis-specific expression indicate evolutionarily conserved functions of Rec8p in meiosis. Possible roles of Rec8p in the integration of different meiotic events are discussed.

Involvement of nucleotide-excision repair in msh2 pms1-independent mismatch repair.

Nucleotide-excision repair (NER) and mismatch repair (MMR) are prominent examples of highly conserved DNA repair systems which recognize and replace damaged and/or mispaired nucleotides in DNA. In humans, inheritable defects in components of the NER system are associated with severe diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS), whereas inactivation of MMR is accompanied by predisposition to certain types of cancer. In Schizosaccharomyces pombe, the msh2- and pms1-dependent long-patch MMR system efficiently corrects small insertion/deletion loops and all base-base mismatches, except C/C. Up to 70% of C/C mismatches generated in recombination intermediates, and to a lesser extent also other base-base mismatches, are thought to undergo correction by a minor, short-patch excision repair system. We identify here the NER genes rhpl4, swi10 and rad16 as components of this repair pathway and show that they act independently of msh2 and pms1.

The msh2 gene of Schizosaccharomyces pombe is involved in mismatch repair, mating-type switching, and meiotic chromosome organization.

We have identified in the fission yeast Schizosaccharomyces pombe a MutS homolog that shows highest homology to the Msh2 subgroup. msh2 disruption gives rise to increased mitotic mutation rates and increased levels of postmeiotic segregation of genetic markers. In bandshift assays performed with msh2Delta cell extracts, a general mismatch-binding activity is absent. By complementation assays, we showed that S. pombe msh2 is allelic with the previously identified swi8 and mut3 genes, which are involved in mating-type switching. The swi8-137 mutant has a mutation in the msh2 gene which causes a truncated Msh2 peptide lacking a putative DNA-binding domain. Cytological analysis revealed that during meiotic prophase of msh2-defective cells, chromosomal structures were frequently formed; such structures are rarely found in the wild type. Our data show that besides having a function in mismatch repair, S. pombe msh2 is required for correct termination of copy synthesis during mating-type switching as well as for proper organization of chromosomes during meiosis.

Schizosaccharomyces pombe exo1 is involved in the same mismatch repair pathway as msh2 and pms1.

Besides the MutLS-like system, Schizosaccharomyces pombe has an additional pathway of mismatch repair. This minor pathway, producing short excision tracts, repairs C/C and, with lower efficiency, other mismatches also. We investigated the involvement of the exo1+, msh2+ and pms1+ genes in the two pathways. The exo1+ gene encodes a 5' to 3' exonuclease, while msh2+ and pms1+ are homologs of Escherichia coli mutS and mutL, respectively. Intragenic two-factor crosses showed that exo1+, msh2+ and pms1+ are involved in the major, but not in the C/C-correcting, pathway. Post-meiotic segregation frequencies and mitotic mutation rates in single and double mutants supported this finding. Furthermore, msh2 delta was epistatic over exo1 delta, and the ExoI enzyme is likely to be redundant with other exonucleases.

The high mobility group domain protein Cmb1 of Schizosaccharomyces pombe binds to cytosines in base mismatches and opposite chemically altered guanines.

The mismatch-binding activity Cmb1 of Schizosaccharomyces pombe was enriched from wild type cells, and N-terminal sequencing enabled cloning of the respective gene. The deduced amino acid sequence of cmb1(+) contains a high mobility group domain, a motif that is common to a heterogeneous family of DNA-binding proteins. In crude protein extracts of a cmb1 gene-disruption strain, specific binding to C/T, C/A, and C/Delta was abolished. Weak binding to C/C revealed the presence of a second mismatch-binding activity, Cmb2. Cmb1, enriched from S. pombe and purified from Escherichia coli, bound specifically to C/C, C/T, C/A, T/T, and C/Delta but showed little or no affinity to other mismatches and small loops. Cmb1 recognizes 1,2 GpG intrastrand cross-links, produced by the chemotherapeutic drug cisplatin, when two cytosines are opposite the cross-linked guanines but not when other bases are present. Consistently, O6-methylguanine:C but not O6-methylguanine/T lesions were bound. Thus, cytosines in mismatches and opposite chemically modified guanines are the preferred target of Cmb1 recognition. cmb1 mutant cells are more sensitive to cisplatin than wild type cells, indicating a role of Cmb1 in repair of cisplatin-induced DNA damage.

The role of topoisomerase II in meiotic chromosome condensation and segregation in Schizosaccharomyces pombe.

Topoisomerase II is able to break and rejoin double-strand DNA. It controls the topological state and forms and resolves knots and catenanes. Not much is known about the relation between the chromosome segregation and condensation defects as found in yeast top2 mutants and the role of topoisomerase II in meiosis. We studied meiosis in a heat-sensitive top2 mutant of Schizosaccharomyces pombe. Topoisomerase II is not required until shortly before meiosis I. The enzyme is necessary for condensation shortly before the first meiotic division but not for early meiotic prophase condensation. DNA replication, prophase morphology, and dynamics of the linear elements are normal in the top2 mutant. The top2 cells are not able to perform meiosis I. Arrested cells have four spindle pole bodies and two spindles but only one nucleus, suggesting that the arrest is nonregulatory. Finally, we show that the arrest is partly solved in a top2 rec7 double mutant, indicating that topoisomerase II functions in the segregation of recombined chromosomes. We suggest that the inability to decatenate the replicated DNA is the primary defect in top2. This leads to a loss of chromatin condensation shortly before meiosis I, failure of sister chromatid separation, and a nonregulatory arrest.

Opioid receptor mediated anticonvulsant effect of pentazocine.

Intraperitoneal (i.p.) administration of (+/-) pentazocine (10, 30 & 50 mg/kg), a Sigma opioid agonist, resulted in a dose dependent anticonvulsant action against maximal electroshock seizures in mice. This anticonvulsant effect of pentazocine was not antagonized by both the doses of naloxone (1 and 10 mg/kg) suggesting thereby that its anticonvulsant action is probably mediated by Sigma opiate binding sites. Its anticonvulsant effect was potentiated by both the anticonvulsant drugs viz. diazepam and diphenylhydantoin. Morphine, mu opioid agonist, on the other hand, failed to protect the animals against maximal electroshock seizures when it was given in doses of 10-40 mg/kg body wt.