DNA damage and repair of leukocytes from Fanconi anaemia patients, carriers and healthy individuals as measured by the alkaline comet assay

Fanconi anaemia (FA) patients show cellular sensitivity to a variety of clastogens and prominently to cross-linking agents. Although there is a long-standing clinical impression of radiosensitivity, in vitro studies have yielded conflicting results. In this study, initial radiation-induced DNA damage and kinetics of DNA repair in (60)Co gamma-irradiated leukocytes from healthy volunteers, FA patients and heterozygotes were assessed using alkaline comet assay. Results showed higher levels of baseline DNA damage in leukocytes of patients and heterozygotes than in controls. Gamma-ray-induced initial DNA damage in leukocytes of FA cases was not significantly different from that of healthy donors and heterozygotes. However, after a repair time of 4 h, following irradiation, samples from the healthy individuals and carriers showed less residual DNA damage in their leukocytes, whereas FA patients revealed more DNA damages than their baseline. Although similar initial induced DNA damage was observed for all groups, the repair kinetics of radiation-induced DNA damage of leukocytes from FA patients was statistically different from healthy and carrier subjects. These findings may suggest that hypersensitivity of FA cells to cross-linking and clastogenic agents might be due to inefficient and delayed repair machinery of these cells. Also, the amount of residual DNA damage after irradiation could be used as a putative predictor of FA screening and cellular radiosensitivity.     

Complex mosaic structural variations in human fetal brains

Somatic mosaicism, manifesting as single nucleotide variants (SNVs), mobile element insertions, and structural changes in the DNA, is a common phenomenon in human brain cells, with potential functional consequences. Using a clonal approach, we previously detected 200–400 mosaic SNVs per cell in three human fetal brains (15–21 wk postconception). However, structural variation in the human fetal brain has not yet been investigated. Here, we discover and validate four mosaic structural variants (SVs) in the same brains and resolve their precise breakpoints. The SVs were of kilobase scale and complex, consisting of deletion(s) and rearranged genomic fragments, which sometimes originated from different chromosomes. Sequences at the breakpoints of these rearrangements had microhomologies, suggesting their origin from replication errors. One SV was found in two clones, and we timed its origin to ∼14 wk postconception. No large scale mosaic copy number variants (CNVs) were detectable in normal fetal human brains, suggesting that previously reported megabase-scale CNVs in neurons arise at later stages of development. By reanalysis of public single nuclei data from adult brain neurons, we detected an extrachromosomal circular DNA event. Our study reveals the existence of mosaic SVs in the developing human brain, likely arising from cell proliferation during mid-neurogenesis. Although relatively rare compared to SNVs and present in ∼10% of neurons, SVs in developing human brain affect a comparable number of bases in the genome (∼6200 vs. ∼4000 bp), implying that they may have similar functional consequences.

Somatic mosaicism, the presence of more than one genotype in the somatic cells of an individual, is a prominent phenomenon in the human central nervous system. Forms of mosaicism include aneuploidies and smaller copy number variants (CNVs), structural variants (SVs), mobile element insertions, indels, and single nucleotide variants (SNVs). The developing human brain exhibits high levels of aneuploidy compared to other tissues, generating genetic diversity in neurons (Pack et al. 2005; Yurov et al. 2007; Bushman and Chun 2013). Such aneuploidy was suggested to be a natural feature of neurons, rather than a distinctive feature of neurodegeneration. However, the frequency of aneuploidy in neurons has been debated, with a separate study suggesting that aneuploidies occur in only about 2.2% of mature adult neurons (Knouse et al. 2014). They hence infer that such aneuploidy could have adverse effects at the cellular and organismal levels. Additionally, analysis of single cells from normal and pathological human brains identified large, private, and likely clonal somatic CNVs in both normal and diseased brains (Gole et al. 2013; McConnell et al. 2013; Cai et al. 2014; Knouse et al. 2016; Chronister et al. 2019; Perez-Rodriguez et al. 2019), with 3%–25% of human cerebral cortical nuclei carrying megabase-scale CNVs (Chronister et al. 2019) and deletions being twice as common as duplications (McConnell et al. 2013). Given that CNVs often arise from nonhomologous recombination and replication errors, their likely time of origin is during brain development. However, when CNVs first arise in human brain development has not yet been investigated. The present work is the first to examine this question using clonal populations of neuronal progenitor cells (NPCs) obtained from fetal human brains.Detection of CNVs in single neurons is challenging, given the need to amplify DNA. Such amplification may introduce artifacts that could, in turn, be misinterpreted as CNVs. In order to address this technical limitation, Hazen et al. reprogrammed adult postmitotic neurons using somatic cell nuclear transfer (SCNT) of neuronal nuclei into enucleated oocytes (Hazen et al. 2016). These oocytes then made sufficient copies of the neuronal genome allowing for whole-genome sequencing (WGS), thus eliminating the need for amplification in vitro. Using this method, they identified a total of nine structural variants in six neurons from mice, three of which were complex rearrangements. However, it is not possible to extend such studies to humans, given the ethical issues involved, besides the technical challenges in obtaining and cloning adult neurons. To circumvent the need of single-cell DNA amplification or nuclear cloning, we examined clonal cell populations obtained from neural progenitor cells from the frontal region of the cerebral cortex (FR), parietal cortex (PA) and basal ganglia (BG) and describe here the discovery and analysis of mosaic SVs in these NPCs (Bae et al. 2018). These clones were sequenced at 30× coverage (much higher than most previous single-cell studies), allowing identification of SVs other than large deletions and duplications as well as precise breakpoint resolution.     

Parental Determinants of Overweight and Obesity in Iranian Adolescents: A National Study

Objective

Overweight among adolescents is not only an important public health problem but also a problem affecting economic growth in developing countries. The aim of this study was to investigate the parental determinants of overweight and obesity in Iranian adolescents at national level.

Methods

This cross sectional study was conducted within the framework of the Comprehensive Study on Household Food Consumption Patterns and Nutritional Status of IR Iran during 2001-2003. In adolescents, anthropometric indices were defined based on the CDC 2000 cut-off points for age and gender-specific body mass index (BMI). Parental characteristics were collected by questionnaire. Among the 7158 participating households, data on 7908 adolescents aged 11-19 years (3750 girls and 4158 boys) was analyzed.

Findings

The prevalence of overweight (85–94th percentile) in boys and girls was 6.2%, and 8.7%, respectively. The prevalence of obesity (≥95th percentile) among boys and girls was similar (3.3%). Parents’ weight status, father''s job and parents’ education showed significant association with weight status in adolescents. Logistic regression analysis showed that parental overweight and obesity, parental education and father''s job were the main parental determinants of overweight and obesity in Iranian adolescents.

Conclusion

Parental overweight and obesity, parental education and father''s job seem to be the major parental determinants of overweight in Iranian adolescents. Future prevention programs must take these risk factors into account.     

Occlusal caries: Evaluation of direct microscopy versus digital imaging used for two histological classification systems

Objective

Histology is frequently used as a gold standard to validate caries detection devices. Poor assessment consistency could lead to apparent changes in diagnostic accuracy. In multi-center, multi-examiner studies electronic transfer of information would be convenient, provided there is no deteriation in quality. This study tested the hypothesis that examiner reproducibility in the assessment of caries lesion depth when viewing photographic images of histological sections on a computer monitor, is comparable with viewing the same sections under a microscope using two histological classification systems.

Methods

166 investigation sites (96 teeth) were selected for visual examination (ICDAS-II) and sections made using a novel technique which reduced risk of section damage and allowed accurate allocation of section to each investigation site. Digital images of the sections were produced and four examiners viewed the sections under a microscope and on a separate occasion corresponding digital images on a computer monitor. Presence and extent of caries was scored according to two histological classification systems (Downer, ERK).

Results

The inter- and intra-examiner reproducibility for both histological classification systems and both examination techniques was substantial to almost perfect (weighted kappa = 0.63–0.90). Comparing the kappa values between microscopy and viewing digital images, there was no effect or only a small effect between both examination techniques (effect size 0.00–0.28). There was also a strong relationship between the two viewing techniques (r_s = 0.748–0.844).

Conclusions

Viewing digital images of tooth sections produces results comparable to viewing images directly under a microscope and therefore has potential benefits for multi-centre studies.     

New diagnostic EEG markers of the Alzheimer’s disease using visibility graph

A new chaos–wavelet approach is presented for electroencephalogram (EEG)-based diagnosis of Alzheimer’s disease (AD) employing a recently developed concept in graph theory, visibility graph (VG). The approach is based on the research ideology that nonlinear features may not reveal differences between AD and control group in the band-limited EEG, but may represent noticeable differences in certain sub-bands. Hence, complexity of EEGs is computed using the VGs of EEGs and EEG sub-bands produced by wavelet decomposition. Two methods are employed for computation of complexity of the VGs: one based on the power of scale-freeness of a graph structure and the other based on the maximum eigenvalue of the adjacency matrix of a graph. Analysis of variation is used for feature selection. Two classifiers are applied to the selected features to distinguish AD and control EEGs: a Radial Basis Function Neural Network (RBFNN) and a two-stage classifier consisting of Principal Component Analysis (PCA) and the RBFNN. After comprehensive statistical studies, effective classification features and mathematical markers were discovered. Finally, using the discovered features and a two-stage classifier (PCA-RBFNN), a high diagnostic accuracy of 97.7% was obtained.     

The Frequency Following Response (FFR) May Reflect Pitch-Bearing Information But is Not a Direct Representation of Pitch

The frequency following response (FFR), a scalp-recorded measure of phase-locked brainstem activity, is often assumed to reflect the pitch of sounds as perceived by humans. In two experiments, we investigated the characteristics of the FFR evoked by complex tones. FFR waveforms to alternating-polarity stimuli were averaged for each polarity and added, to enhance envelope, or subtracted, to enhance temporal fine structure information. In experiment 1, frequency-shifted complex tones, with all harmonics shifted by the same amount in Hertz, were presented diotically. Only the autocorrelation functions (ACFs) of the subtraction-FFR waveforms showed a peak at a delay shifted in the direction of the expected pitch shifts. This expected pitch shift was also present in the ACFs of the output of an auditory nerve model. In experiment 2, the components of a harmonic complex with harmonic numbers 2, 3, and 4 were presented either to the same ear (“mono”) or the third harmonic was presented contralaterally to the ear receiving the even harmonics (“dichotic”). In the latter case, a pitch corresponding to the missing fundamental was still perceived. Monaural control conditions presenting only the even harmonics (“2 + 4”) or only the third harmonic (“3”) were also tested. Both the subtraction and the addition waveforms showed that (1) the FFR magnitude spectra for “dichotic” were similar to the sum of the spectra for the two monaural control conditions and lacked peaks at the fundamental frequency and other distortion products visible for “mono” and (2) ACFs for “dichotic” were similar to those for “2 + 4” and dissimilar to those for “mono.” The results indicate that the neural responses reflected in the FFR preserve monaural temporal information that may be important for pitch, but provide no evidence for any additional processing over and above that already present in the auditory periphery, and do not directly represent the pitch of dichotic stimuli.     

Metabolic syndrome and leptin concentrations in obese children

Objective Obesity and its complications including metabolic syndrome has been increased in children and adolescents recently. Leptin is known to play an important role in the pathogenesis of obesity. Methods The objective of this study was to evaluate the relationship of leptin and metabolic syndrome in obese Iranian children. A cross sectional study was carried out in 65 primary schools in Tehran. The children with waist circumferences equal or above 90^th percentile for their height and age were chosen for further evaluations. 505 children aged 7–12 years participated in the study. Anthropometric variables measurements, blood pressure, fasting plasma glucose, triglyceride, high-density lipoprotein cholesterol and serum leptin were obtained from the study sample. Results Serum leptin levels were significantly higher in girls in comparison to the boys (with median 11.0 Vs 8.25 ng/dl; P value =0.007). Serum level of leptin were higher in children with metabolic syndrome (median 11.3 Vs 8.9 ng/dl; P value=0.045). However, after adjustment for sex, this association was removed. Conclusion Leptin did not appear to have a major role in metabolic syndrome, even though it was strongly associated with obesity parameters. More studies evaluating the relationship between leptin and metabolic syndrome in various ethnic groups are recommended.