Ribosomal DNA Abundance in the Patient’s Genome as a Feasible Marker in Differential Diagnostics of Autism and Childhood-Onset Schizophrenia

Introduction: Differential diagnostics of early-onset schizophrenia and autism spectrum disorders (ASD) are a problem of child psychiatry. The prognosis and relevant treatment are to a large degree determined by the correctness of diagnosis. We found earlier that leucocyte DNA of adult schizophrenia patients contained significantly larger copy numbers of ribosomal repeats (rDNA) coding for rRNA, than DNA of mentally healthy controls. Aim: To compare the contents of ribosomal repeats in the leucocyte DNA of children with schizophrenia, children with ASD, and healthy age-matched controls to estimate the possibility of using this genetic trait in the differential diagnostics of the two types of disorders. Patients and methods: Blood samples of patients with infantile autism (A—F84.0 according to ICD-10, N = 75) and with childhood-onset schizophrenia (SZ—F20.8 according to ICD-10, N = 43) were obtained from the Child Psychiatry Department of the Mental Health Research Center. The healthy control blood samples (HC, N = 86) were taken from the Research Centre for Medical Genetics collection. The recruitment of cases was based on the clinical psychopathologic approach. DNA was extracted from blood leukocytes with organic solvents. Nonradioactive quantitative hybridization technique was applied for determining the abundance of ribosomal repeats in the genomes. Statistical processing was performed using StatPlus, Statgraphics and MedCalc. Findings: DNA derived from SZ cases contained 565 ± 163 rDNA copies, which is significantly (p < 10−6) higher than the rDNA content in ASD cases (405 ± 109 copies) and controls (403 ± 86 copies). The HC and A groups did not differ by rDNA copy number (p > 0.4). The genetic trait “rDNA copy number in patient’s genome” can potentially be applied as an additional marker in differential diagnostics of childhood-onset schizophrenia and autism spectrum disorders

In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls

Schizophrenia is associated with low-grade systemic inflammation. Circulating cell-free DNA (c-cfDNA) belongs to the DAMP class. The major research question was: can the c-cfDNA of schizophrenic patients (sz-cfDNA) stimulate the DNA sensor genes, which control the innate immunity? We investigated the in vitro response of ten human skin fibroblast (HSF) lines to five DNA probes containing different amounts of a GC-rich marker (the ribosomal repeat) and a DNA oxidation marker (8-oxodG) including sz-cfDNA and healthy control c-cfDNA (hc-cfDNA) probes. After 1 h, 3 h, and 24 h of incubation, the expression of 6 protein genes responsible for cfDNA transport into the cell (EEA1 and HMGB1) and the recognition of cytosolic DNA (TLR9, AIM2, STING and RIG-I) was analyzed at the transcriptional (RT-qPCR) and protein level (flow cytometry and fluorescence microscopy). Additionally, we analyzed changes in the RNA amount of 32 genes (RT-qPCR), which had been previously associated with different cellular responses to cell-free DNA with different characteristics. Adding sz-cfDNA and hc-cfDNA to the HSF medium in equal amounts (50 ng/mL) blocked endocytosis and stimulated TLR9 and STING gene expression while blocking RIG-I and AIM2 expression. Sz-cfDNA and hc-cfDNA, compared to gDNA, demonstrated much stronger stimulated transcription of genes that control cell proliferation, cytokine synthesis, apoptosis, autophagy, and mitochondrial biogenesis. No significant difference was observed in the response of the cells to sz-cfDNA and hc-cfDNA. Sz-cfDNA and hc-cfDNA showed similarly high biological activity towards HSFs, stimulating the gene activity of TLR9 and STING DNA sensor proteins and blocking the activity of the AIM2 protein gene. Since the sz-cfDNA content in the patients’ blood is several times higher than the hc-cfDNA content, sz-cfDNA may upregulate pro-inflammatory cytokines in schizophrenia

The Psychoemotional Stress-Induced Changes in the Abundance of SatIII (1q12) and Telomere Repeats, but Not Ribosomal DNA, in Human Leukocytes

INTRODUCTION. As shown earlier, copy number variations (CNV) in the human satellite III (1q12) fragment (f-SatIII) and the telomere repeat (TR) reflects the cell’s response to oxidative stress. The contents of f-SatIII and TR in schizophrenic (SZ) patients were found to be lower than in healthy controls (HC) in previous studies. The major question of this study was: ‘What are the f-SatIII and TR CNV dynamic changes in human leukocytes, depending on psychoemotional stress?’ MATERIALS AND METHODS. We chose a model of psychoemotional stress experienced by second-year medical students during their exams. Blood samples were taken in stressful conditions (exams) and in a control non-stressful period. Biotinylated probes were used for f-SatIII, rDNA, and TR quantitation in leukocyte DNA by non-radioactive quantitative hybridization in SZ patients (n = 97), HC (n = 97), and medical students (n = 17, n = 42). A flow cytometry analysis was used for the oxidative stress marker (NOX4, 8-oxodG, and γH2AX) detection in the lymphocytes of the three groups. RESULTS. Oxidative stress markers increased significantly in the students’ lymphocytes during psychoemotional stress. The TR and f-SatIII, but not the rDNA, contents significantly changed in the DNA isolated from human blood leukocytes. After a restoration period (post-examinational vacations), the f-SatIII content decreased, and the TR content increased. Changes in the blood cells of students during examinational stress were similar to those in SZ patients during an exacerbation of the disease. CONCLUSIONS. Psychoemotional stress in students during exams triggers a universal mechanism of oxidative stress. The oxidative stress causes significant changes in the f-SatIII and TR contents, while the ribosomal repeat content remains stable. A hypothesis is proposed to explain the quantitative polymorphisms of f-SatIII and TR contents under transient (e.g., students’ exams) or chronic (in SZ patients) stress. The changes in the f-SatIII and TR copy numbers are non-specific events, irrespective of the source of stress. Thus, our findings suggest that the psychoemotional stress, common in SZ patients and healthy students during exams, but not in a schizophrenia-specific event, was responsible for the changes in the repeat contents that we observed earlier in SZ patients

Copy Number of Human Ribosomal Genes With Aging: Unchanged Mean, but Narrowed Range and Decreased Variance in Elderly Group

Introduction: The multi-copied genes coding for the human 18, 5.8, and 28S ribosomal RNA (rRNA) are located in five pairs of acrocentric chromosomes forming so-called rDNA. Human genome contains unmethylated, slightly methylated, and hypermethylated copies of rDNA. The major research question: What is the rDNA copy number (rDNA CN) and the content of hypermethylated rDNA as a function of age?Materials and Methods: We determined the rDNA CN in the blood leukocyte genomes of 651 subjects aged 17 to 91 years. The subjects were divided into two subgroups: “elderly” group (E-group, N = 126) – individuals over 72 years of age (the age of the population’s mean lifetime for Russia) and “non-elderly” group (NE-group, N = 525). The hypermethylated rDNA content was determined in the 40 DNA samples from the each group. The change in rDNA during replicative cell senescence was studied for the cultured skin fibroblast lines of five subjects from NE-group. Non-radioactive quantitative dot- and blot-hybridization techniques (NQH) were applied.Results: In the subjects from the E-group the mean rDNA CN was the same, but the range of variation was narrower compared to the NE-group: a range of 272 to 541 copies in E-group vs. 200 to 711 copies in NE-group. Unlike NE-group, the E-group genomes contained almost no hypermethylated rDNA copies. A case study of cultured skin fibroblasts from five subjects has shown that during the replicative senescence the genome lost hypermethylated rDNA copies only.Conclusion: In the elderly group, the mean rDNA CN is the same, but the range of variation is narrower compared with the younger subjects. During replicative senescence, the human fibroblast genome loses hypermethylated copies of rDNA. Two hypotheses were put forward: (1) individuals with either very low or very high rDNA content in their genomes do not survive till the age of the population’s mean lifetime; and/or (2) during the aging, the human genome eliminates hypermethylated copies of rDNA

Effects of Functionalized Fullerenes on ROS Homeostasis Determine Their Cytoprotective or Cytotoxic Properties

Background: Functionalized fullerenes (FF) can be considered regulators of intracellular reactive oxygen species (ROS) homeostasis; their direct oxidative damage—as well as regulation of oxidant enzymes and signaling pathways—should be considered. Methods: Uptake of two water-soluble functionalized C70 fullerenes with different types of aromatic addends (ethylphenylmalonate and thienylacetate) in human fetal lung fibroblasts, intracellular ROS visualization, superoxide scavenging potential, NOX4 expression, NRF2 expression, oxidative DNA damage, repair genes, cell proliferation and cell cycle were studied. Results & conclusion: The intracellular effects of ethylphenylmalonate C70 derivative (FF1) can be explained in terms of upregulated NOX4 activity. The intracellular effects of thienylacetate C70 derivative (FF2) can be probably resulted from its superoxide scavenging potential and inhibition of lipid peroxidation. FF1 can be considered a NOX4 upregulator and potential cytotoxicant and FF2, as a superoxide scavenger and a potential cytoprotector

Neurophysiology and Psychopathology Underlying PTSD and Recent Insights into the PTSD Therapies—A Comprehensive Review

Post-traumatic stress disorder (PTSD) is a well-known psychiatric disorder that affects millions of people worldwide. Pharmacodynamic and cognitive-behavioral therapies (CBT) have been used to treat patients with PTSD. However, it remains unclear whether there are concurrent changes in psychopathological and neurophysiological factors associated with PTSD patients. Past reports described those PTSD patients with efficient fatty acid metabolism, neurogenesis, mitochondrial energy balance could improve ability to cope against the conditioned fear responses and traumatic memories. Furthermore, cognitive, behavioral, cellular, and molecular evidence can be combined to create personalized therapies for PTSD sufferers either with or without comorbidities such as depression or memory impairment. Unfortunately, there is still evidence lacking to establish a full understanding of the underlying neurophysiological and psychopathological aspects associated with PTSD. This review has extensively discussed the single nucleotide polymorphism (SNPs) of genetic factors to cause PTSD, the implications of inflammation, neurotransmitter genomics, metabolic alterations, neuroendocrine disturbance (hypothalamus-pituitary-adrenal (HPA) axis), mitochondrial dynamics, neurogenesis, and premature aging related to PTSD-induced psychopathology and neurophysiology. In addition, the review delineated the importance of CBT and several pharmacodynamic therapies to mitigate symptomatology of PTSD

The Phosphonate Derivative of C60 Fullerene Induces Differentiation towards the Myogenic Lineage in Human Adipose-Derived Mesenchymal Stem Cells

Inductors of myogenic stem cell differentiation attract attention, as they can be used to treat myodystrophies and post-traumatic injuries. Functionalization of fullerenes makes it possible to obtain water-soluble derivatives with targeted biochemical activity. This study examined the effects of the phosphonate C60 fullerene derivatives on the expression of myogenic transcription factors and myogenic differentiation of human mesenchymal stem cells (MSCs). Uptake of the phosphonate C60 fullerene derivatives in human MSCs, intracellular ROS visualization, superoxide scavenging potential, and the expression of myogenic, adipogenic, and osteogenic differentiation genes were studied. The prolonged MSC incubation (within 7–14 days) with the C60 pentaphoshonate potassium salt promoted their differentiation towards the myogenic lineage. The transcription factors and gene expressions determining myogenic differentiation (MYOD1, MYOG, MYF5, and MRF4) increased, while the expression of osteogenic differentiation factors (BMP2, BMP4, RUNX2, SPP1, and OCN) and adipogenic differentiation factors (CEBPB, LPL, and AP2 (FABP4)) was reduced or did not change. The stimulation of autophagy may be one of the factors contributing to the increased expression of myogenic differentiation genes in MSCs. Autophagy may be caused by intracellular alkalosis and/or short-term intracellular oxidative stress

Une âme de jeune fille [ : Gabrielle***] / Jean Vaudon,...

<p>A. Distribution of CpG-motifs within rDNA (transcribed region of human ribosomal repeat). The digits indicate the nucleotide order number, the vertical bar shows the motif location. Red color is used to mark region A and region B, that were analyzed for the presence of methylated CCGG sites. B. Determination of methylation index of three genes in DNA from cells treated with 20 μM DBP(1–4), 72 h (description is given in Methods).</p

DNA methylation in nuclei of the control MCF-7 cells.

<p>A. Cells were processed for immunofluorescence staining with anti 5-mC antibody. (1)—Magnification X60; (2)—For analysis of the nuclei the photo was enlarged by an order using computer processing. The arrow points to the methylated DNA block close to the nucleolus. B. (1)—DNA methylation in nuclei of the control MCF-7 cells and the cells treated with 10 μM AzaC or 20μM DBP(1–4), 72 h. Background: Control cells were only treated with secondary antibodies conjugated with FITC. (2) The photo was enlarged considerably for the analysis of nuclei size. The nucleus of the cell cultivated with DBP(4) is shown for an example. C. An example of the different DNA methylation level in cells’ nuclei that were cultivated in the presence of 20μM DBP(4), 72 h. A,B(1) and C—Cell nuclei were additionally colored with DAPI.</p

The influence of DBP(1–4) on the MCF-7mitochondria.

<p>A. (1)—control cells (stained with MitoTracker TMRM) in visible light (VIS), in λex = 520 nm (TMRM) and in λex = 380 nm (background). (2)—control cells stained with MitoTracker TMRM and Hoechst 33258 (10μM, 0.5 h). Specially selected field shows single cells with a weak mitochondria staining. Particularly these cells accumulate Hoechst 33258 in the nuclei. B. MCF-7 cells treated with 20μM DBP(2) for 0.5–48 h after incubation with MitoTracker TMRM (15 min). For 0.5 h time point we specially selected a field that shows cells with a weak mitochondria staining. DBP(2) quickly penetrates into these cells, similar to Hoechst 33258. Magnification X 60. The cell in a yellow square is enlarged several times for demonstration.</p