Systemic Administration of Porphyromonas Gingivalis Lipopolysaccharide Induces Glial Activation and Depressive-Like Behavior in Rats.

BACKGROUND: Periodontitis is one of the most common chronic inflammatory disorders in adults. Although clinical studies have suggested a causal relationship between periodontitis and major depression (MD), the biological mechanisms by which periodontitis instigates MD are unknown. We investigated whether a systemic administration of lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg), a major Gram-negative pathogen of periodontitis, causes depressive-like behavior and glial activation in the hippocampus and the prefrontal cortex (PFC), which are MD-related brain regions. MATERIALS AND METHODS: Eight-week-old male Sprague Dawley rats were randomly divided into a behavioral test group and an immunohistochemistry group. The rats in each group were further assigned to the sham injection (saline) and Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) injection protocols. The rats received an intraperitoneal injection of saline or Pg-LPS with gradually increasing doses (day 1: 0.5, day 2: 0.5, day 3: 0.75, day 4: 0.75, day 5: 1.0, day 6: 1.0, and day 7: 1.0 mg/kg of body weight) for seven consecutive days. After the systemic administration, the behavior test group underwent the forced swimming test (FST) and Y-maze test. For the immunohistochemistry group, we quantified the immunoreactivity for microglial Iba-1 (ionized calcium-binding adapter molecule 1) and astrocytic glial fibrillary acidic protein (GFAP) in the hippocampus (dentate gyrus [DG], cornu ammonis [CA1 and CA3]) and PFC (prelimbic [PrL] and the infralimbic [IL]) areas. RESULTS: The FST immobility time in the Pg-LPS group was significantly longer than that in the sham group. In the Y-maze test, a significant decline in spontaneous alternation behavior was observed in the Pg-LPS group compared to the sham group. The peripheral administration of Pg-LPS significantly increased the immunoreactivity for Iba-1 in the CA3 and PrL. Pg-LPS injection significantly increased the immunoreactivity for GFAP in the DG, CA1, and CA3. CONCLUSIONS: The major result of this study is that a repeated systemic administration of Pg-LPS caused depressive-like behavior and both microglial and astrocytic activation in rats. This finding may comprise biological evidence of a causal relationship between periodontitis and MD.

Cell-free DNA Release in the Plasma of Patients with Cardiac Disease is Associated with Cell Death Processes.

Cell-free DNA (cfDNA) is released into the plasma of patients with cardiac disease. Here, the source and mechanism of plasma cfDNA release in patients with myocardial infarction (MI) and other cardiac diseases (n = 59) were investigated. Plasma levels of various markers including M30 (apoptosis), M65 (apoptosis and necrosis), cyclophilin A (CyPA) (necrosis), and myeloperoxidase (MPO) (neutrophil activation) were assayed. The plasma cfDNA concentrations in MI and other cardiac diseases were significantly higher than that in the healthy control subjects. Significant differences were not observed among the cardiac disease patients (MI and other cardiac diseases) and healthy control subjects in M30, M65, and CyPA levels. In contrast,the MPO levels were significantly elevated in cardiac disease patients when compared to control groups, and MPO levels in MI patients were significantly higher than other cardiac diseases patients. These results suggest that cfDNA is mainly released by neutrophils via NETosis in addition to apoptosis except for epithelial apoptosis in patients with cardiac disease and the degree is greater in MI patients. The results from this study provide basic information for diagnosis marker of MI.

RUNX1 mutation in a patient with myelodysplastic syndrome and decreased erythrocyte expression of blood group A antigen.

BACKGROUND: Loss of blood group ABO antigens on red blood cells (RBCs) is well known in patients with leukemias, and such decreased ABO expression has been reported to be strongly associated with hypermethylation of the ABO promoter. We investigated the underlying mechanism responsible for A-antigen reduction on RBCs in a patient with myelodysplastic syndrome. STUDY DESIGN AND METHODS: Genetic analysis of ABO was performed by PCR and sequencing using peripheral blood. RT-PCR were carried out using cDNA prepared from total bone marrow (BM) cells. Bisulfite genomic sequencing was performed using genomic DNA from BM cells. Screening of somatic mutations was carried out using a targeted sequencing panel with genomic DNA from BM cells, followed by transient transfection assays. RESULTS: Genetic analysis of ABO did not reveal any mutation in coding regions, splice sites, or regulatory regions. RT-PCR demonstrated reduction of A-transcripts when the patient's RBCs were not agglutinated by anti-A antibody and did not indicate any significant increase of alternative splicing products in the patient relative to the control. DNA methylation of the ABO promoter was not obvious in erythroid cells. Targeted sequencing identified somatic mutations in ASXL1, EZH2, RUNX1, and WT1. Experiments involving transient transfection into K562 cells showed that the expression of ABO was decreased by expression of the mutated RUNX1. CONCLUSION: Because the RUNX1 mutation encoded an abnormally elongated protein without a transactivation domain which could act as dominant negative inhibitor, this frame-shift mutation in RUNX1 may be a genetic candidate contributing to A-antigen loss on RBCs.

Measurement of Serum Tenascin-X in Joint Hypermobility Syndrome Patients.

Joint hypermobility syndrome (JHS) (also termed hypermobility type Ehlers-Danlos syndrome, hEDS) is a heritable connective tissue disorder that is characterized by generalized joint hypermobility, chronic pain, fatigue, and minor skin changes. Initially, it was reported that there is a small subset of patients with JHS/hEDS who have haploinsufficiency of tenascin-X (TNX). However, the relationship between TNXB and JHS/hEDS has not been reported at all afterwards. EDS was reclassified into thirteen types in 2017, and the causative gene of JHS/hEDS remained to be identified. Therefore, in this study in order to determine whether JHS/hEDS can be diagnosed by the concentrations of serum form of TNX (sTNX), we measured the concentrations of sTNX in 17 JHS/hEDS patients. The sTNX concentrations in half of the JHS/hEDS patients were significantly lower than those in healthy individuals. No mutations, insertions or deletions were detected in the TNX exon sequence of the JHS/hEDS patients except for one in patient. That patient has a heterozygous mutation. A correlation between sTNX concentration and mutation of the TNXB genomic sequence was not found in the JHS/hEDS patients. These results indicate that the decrease in sTNX concentration could be used as a risk factor for JHS/hEDS.

High Serum Cortisol Levels as a Potential Indicator for Changes in Well-Regulated Daily Life among Junior High School Students.

Problematic smartphone use among adolescents has become a social concern and is associated with poor sleep quality. The relationship between life habits, such as smartphone use and sleep duration, and levels of immunological and neuroendocrine biomarkers, including the stress hormone cortisol, in adolescents seems to be important to objectively comprehend their health and well-being in school life. However, such a relationship has not been well documented. We therefore studied rural junior high school students in Japan to elucidate the relationship between serum cortisol (SC) levels and their life habits. A total of 155 students in the seventh grade in 2016 were recruited as subjects. Of them, 140 students with eligible responses and blood samples (12-13 years; 80 boys, 60 girls) were finally included in the study (response rate 90.3%). A questionnaire survey concerning wake-up time, sleep duration, and the length of time using a smartphone per day was conducted. Blood samples were collected from peripheral veins of participants under fasting conditions between 8:30 and 11:00 a.m. The Spearman rank correlation coefficients were as follows: between SC and wake-up time, 0.199 (p = 0.018); between SC and sleep duration, 0.185 (p = 0.029); and between SC and time spent on smartphones, 0.172 (p = 0.042). The multiple regression analysis showed that high SC levels were significantly associated with late wake-up time and with short sleep duration. We therefore propose that measuring SC levels is useful for early detection of the change in the well-regulated daily life among junior high school students.

Epithelial Expression of Human ABO Blood Group Genes Is Dependent upon a Downstream Regulatory Element Functioning through an Epithelial Cell-specific Transcription Factor, Elf5.

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. The ABO system is composed of complex carbohydrate structures that are biosynthesized by A- and B-transferases encoded by the ABO gene. However, the mechanisms regulating ABO gene expression in epithelial cells remain obscure. On the basis of DNase I-hypersensitive sites in and around ABO in epithelial cells, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays and histone modifications indicated a novel positive regulatory element, designated the +22.6-kb site, downstream from ABO, and this was shown to enhance ABO promoter activity in an epithelial cell-specific manner. Expression of ABO and B-antigen was reduced in gastric cancer KATOIII cells by biallelic deletion of the +22.6-kb site using the CRISPR/Cas9 system. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the site bound to an epithelial cell-specific transcription factor, Elf5. Mutation of the Ets binding motifs to abrogate binding of this factor reduced the regulatory activity of the +22.6-kb site. Furthermore, ELF5 knockdown with shRNA reduced both endogenous transcription from ABO and B-antigen expression in KATOIII cells. Thus, Elf5 appeared to be involved in the enhancer potential of the +22.6-kb site. These results support the contention that ABO expression is dependent upon a downstream positive regulatory element functioning through a tissue-restricted transcription factor, Elf5, in epithelial cells.

Whole brain analysis of postmortem density changes of grey and white matter on computed tomography by statistical parametric mapping.

OBJECTIVES: This study examined the usefulness of statistical parametric mapping (SPM) for investigating postmortem changes on brain computed tomography (CT). METHODS: This retrospective study included 128 patients (23 - 100 years old) without cerebral abnormalities who underwent unenhanced brain CT before and after death. The antemortem CT (AMCT) scans and postmortem CT (PMCT) scans were spatially normalized using our original brain CT template, and postmortem changes of CT values (in Hounsfield units; HU) were analysed by the SPM technique. RESULTS: Compared with AMCT scans, 58.6 % and 98.4 % of PMCT scans showed loss of the cerebral sulci and an unclear grey matter (GM)-white matter (WM) interface, respectively. SPM analysis revealed a significant decrease in cortical GM density within 70 min after death on PMCT scans, suggesting cytotoxic brain oedema. Furthermore, there was a significant increase in the density of the WM, lenticular nucleus and thalamus more than 120 min after death. CONCLUSIONS: The SPM technique demonstrated typical postmortem changes on brain CT scans, and revealed that the unclear GM-WM interface on early PMCT scans is caused by a rapid decrease in cortical GM density combined with a delayed increase in WM density. SPM may be useful for assessment of whole brain postmortem changes. KEY POINTS: • The original brain CT template achieved successful normalization of brain morphology. • Postmortem changes in the brain were independent of sex. • Cortical GM density decreased rapidly after death. • WM and deep GM densities increased following cortical GM density change. • SPM could be useful for assessment of whole brain postmortem changes.

Functional Single Nucleotide Polymorphisms (SNPs) in the Genes Encoding the Human Deoxyribonuclease (DNase) Family Potentially Relevant to Autoimmunity.

OBJECTIVE: To continue our previous investigations, we have extensively investigated the function of the 61, 41, and 35 non-synonymous single nucleotide polymorphisms (SNPs) in the human genes encoding DNASE1, DNASE1L3, and DNASE2, respectively, potentially relevant to autoimmune diseases. METHODS: The site-directed mutagenesis was employed to amino acid-substituted constructs corresponding to each SNP. The COS-7 cells were transfected with each vector and DNase activity was assayed by the single radial enzyme diffusion method. By using PolyPhen-2, changes in the DNase function of each non-synonymous SNP were predicted. Genotyping of all the non-synonymous SNPs was performed in 14 different populations including 3 ethnic groups using the polymerase chain reaction followed by the restriction fragment length polymorphism method. RESULTS: Expression analysis demonstrated these SNPs to be classified into four categories with regard to the effect on DNase activity: SNPs not affecting the activity level, ones reducing it, ones abolishing it, and ones elevating it. In particular, 9, 5, and 4 SNPs producing a loss-of-function variant of the enzymes in DNASE1, DNASE1L3, and DNASE2, respectively, were confirmed. SNPs producing DNase loss of function can be estimated by PolyPhen-2 to be "probably damaging" with a high accuracy of prediction. Almost all of these functional SNPs producing a loss of function or substantially low activity-harboring forms exhibited a mono-allelic distribution in all of the populations. CONCLUSION: A minor allele of functional SNPs, despite the remarkably low genetic heterogeneity of the SNPs, might be a genetic risk factor for autoimmune diseases.

Association of XRCC1 polymorphisms with arsenic methylation.

The associations of four single nucleotide polymorphisms (p.Arg194Trp, p.Arg280His, p.Pro206Pro, and p.Arg399Gln) in X-ray repair cross-complementing group 1 with urinary arsenic metabolites and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were investigated in a Vietnamese population (n = 100). Individuals with genotype AA in p.Pro206Pro showed significantly higher urinary monomethylarsonic acid (MMA(V)) and lower dimethylarsinic acid (DMA(V))/MMA(V) ratio than genotype AG. As for p.Arg399Gln, both Arg/Arg homozygous subjects and Arg/Gln heterozygous individuals showed a significantly higher urinary inorganic As percentage and lower 8-OHdG concentrations than Gln/Gln homozygous. Our results suggested that Arg399Gln is a functional SNP that may be related to DNA repair activity.

Expression of ABO blood-group genes is dependent upon an erythroid cell-specific regulatory element that is deleted in persons with the B(m) phenotype.

The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase I-hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cell-specific manner. Electrophoretic mobility-shift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element. Furthermore, we found that a partial deletion in intron 1 involving the element was associated with B(m) phenotypes. Therefore, it is plausible that deletion of the erythroid cell-specific regulatory element could down-regulate transcription in the B(m) allele, leading to reduction of B-antigen expression in cells of erythroid lineage, but not in mucus-secreting cells. These results support the contention that the enhancer-like element in intron 1 of ABO has a significant function in erythroid cells.

Association of a single nucleotide polymorphism (rs27434) in the ERAP1 gene with plural tissue weight.

Our study was designed to examine the correlation between single nucleotide polymorphism (SNP) in the endoplasmic reticulum aminopeptidase 1 (ERAP1) gene, specifically focusing on rs27434, and plural tissue weight. We conducted this investigation using autopsy samples from the Japanese population. Blood samples were collected from 178 Japanese subjects who had undergone autopsies in Shimane Prefecture. Genomic DNA was subsequently extracted from these samples. SNP (rs27434, G＞A substitution) was analyzed by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. In the present study, rs27434 exhibited a statistically significant association with brain weight (g) in both female and male individuals. Among males, rs27434 displayed significant relationships with liver weight (g), and body surface area (m2). In females, rs27434 was significantly related to the length of the appendix. Across both genders, individuals with GA and AA genotypes tended to exhibit higher levels in these respective measurements compared to those with the GG genotype. These results suggest that genetic variant of ERAP1 gene may influence the weight of the organs. To the best of our knowledge, this is the first study investigating the interaction between the association of rs27434 in the ERAP1 gene and data routinely measured at autopsy, such as tissue weight. However, conducting further investigations with larger population samples could provide more comprehensive insights to clarify this issue.

The relationship between a comprehensive index of prosperity and suicide rates in former Soviet countries, including a comparison with European and Asian countries: Discussion from a new perspective.

Investigations of suicide in countries of the former Soviet Union, which broke into 15 different countries in the early 1990s, require examinations of a combination of economic, social, and health factors. It is important to address these factors individually and to examine the various composite indicators for each. Moreover, it would be worthwhile to explore the potential applicability of a comprehensive worldwide index. We analyzed data from nine of the former Soviet countries for which both the annual suicide rate and the Global Competitiveness Index (GCI) were available for the years 2006-2017. We determined the precise relationships between the suicide rate and the GCI during this period in these nine countries as well as in nine countries with high suicide rates in Europe and Asia. The results indicated the following: (i) In six of the nine former Soviet countries with complete data, the suicide rate showed a relationship with the GCI. Notably, this relationship was inverse in all but one country. (ii) Among the nine European and Asian countries with high suicide rates, three exhibited a correlation between the suicide rate and the GCI. Measures to prevent suicide should be devised especially in countries of the former Soviet Union through collaboration among multiple fields and organizations, as necessary, with particular attention paid to countries with worse or worsening GCI values.

Five non-synonymous SNPs in the gene encoding human deoxyribonuclease I-like 2 implicated in terminal differentiation of keratinocytes reduce or abolish its activity.

Several non-synonymous SNPs in the human deoxyribonuclease I-like 2 (DNase 1L2) gene responsible for DNA degradation during terminal differentiation of epidermal keratinocytes have been identified. However, only limited population data are available, and furthermore the effect of these SNPs on the DNase 1L2 activity remains unknown. Genotyping of all of the 17 SNPs was performed using the PCR-RFLP method in three ethnic groups including 14 different populations. A series of amino acid-substituted DNase 1L2 corresponding to each SNP was expressed, and its activity was measured. All of the six non-synonymous SNPs exhibited a mono-allelic distribution, whereas the distribution of some SNPs other than exonic ones was ethnicity-dependent. Each of the minor alleles in SNPs, p.Ala20Asp, p.Val104Leu, p.Asp197Ala, p.Glu274Lys and p.Asp287Asn, among the non-synonymous SNPs produced low or no activity-harbouring DNase 1L2. DNase 1L2 is well conserved, retaining full levels of enzymatic activity, with regard to these exonic SNPs in human populations. It seems plausible to assume that these SNPs affecting the activity may be one of the factors responsible for a genetic pre-disposition for failure of differentiation-associated cell death in various keratinocyte lineages, thereby leading to the development of parakeratosis. Our results may have clinical implications in relation to the pathogenesis of parakeratosis.

Seven nonsynonymous SNPs in the gene encoding human deoxyribonuclease II may serve as a functional SNP potentially implicated in autoimmune dysfunction.

Many nonsynonymous SNPs in the human DNase II gene (DNASE2), potentially relevant to autoimmunity in conditions such as rheumatoid arthritis, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all the 15 nonsynonymous human DNase II SNPs was performed in three ethnic groups including 16 different populations using the PCR-restriction fragment length polymorphism technique. A series of constructs corresponding to each SNP was examined. Fifteen nonsynonymous SNPs in the gene, except for p.Val206Ile in a Korean population, exhibited a mono-allelic distribution in all of the populations. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, four activity-abolishing and five activity-reducing SNPs were confirmed to be functional. The amino acid residues in activity-abolishing SNPs were conserved in animal DNase II. All the nonsynonymous SNPs that affected the catalytic activity of human DNase II showed extremely low genetic heterogeneity. However, a minor allele of seven SNPs producing a loss-of-function or extremely low activity-harboring variant could serve as a genetic risk factor for autoimmune dysfunction. These functional SNPs in DNASE2 may have clinical implications in relation to the prevalence of autoimmune diseases.

Mutation of the GATA site in the erythroid cell-specific regulatory element of the ABO gene in a Bm subgroup individual.

BACKGROUND: The ABO blood group is important in blood transfusion. Recently, an erythroid cell-specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell-specific regulatory activity of the element was dependent upon GATA-1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes. STUDY DESIGN AND METHODS: In this study, further investigation was performed to reveal the underlying mechanism responsible for reduction of B antigen expression in the exceptional Bm individual. Peptide nucleic acid-clamping polymerase chain reaction was carried out to amplify the B-related allele, followed by sequence determination. Electrophoretic mobility assays and promoter assays were performed to examine whether a nucleotide substitution reduced the binding of a transcription factor and induced loss of function of the element. RESULTS: Sequence determination revealed one point mutation of the GATA motif in the element. The electrophoretic mobility shift assays showed that the mutation abolished the binding of GATA transcription factors, and the promoter assays demonstrated complete loss of enhancer activity of the element. CONCLUSION: These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the Bm individual.

Plasma cell-free DNA in patients with acute promyelocytic leukemia treated with arsenic trioxide.

BACKGROUND: Cell-free DNA (cfDNA) is free DNA found in circulating blood that originates from apoptosis or necrosis, and elevated cfDNA concentrations have been reported in cancers and other diseases. METHODS: In this study, the concentrations and fragment distributions of plasma cfDNA were preliminary investigated in elderly (n = 1) and pediatric (n = 1) patients with acute promyelocytic leukemia (APL) treated with arsenic trioxide (ATO). RESULTS: A slight increase in cfDNA concentrations was observed in the APL patients compared with healthy controls. The change in plasma cfDNA concentrations corresponded to the change in plasma arsenic concentrations during ATO treatment. The fragment distribution pattern did not differ before and during treatment. Three ladder fragments were observed in part of the cfDNA in the second consolidation therapy in an elderly APL patient and the first consolidation therapy of a pediatric APL patient, while two fragments were observed in all other treatment periods. Moreover, APL-related gene mutations were successfully genotyped from plasma cfDNA by using polymerase chain reaction-based methods and these results are consistent with those from leukocytes. CONCLUSION: This study is the first to report the concentrations and fragment patterns of cfDNA from APL patients treated with ATO. The results suggested that plasma cfDNA concentration in APL patients increased with ATO treatment and that cfDNA is released mainly via neutrophil extracellular traps (and/or necrosis) in addition to apoptosis. To confirm whether cfDNA concentrations and fragment patterns can be used as a biomarker for APL treated with ATO, further accumulative data are needed.

Implementation of a personal identification system using alveolar bone images.

OBJECTIVE: In recent years, personal identification has been performed using antemortem panoramic X-ray images and postmortem-CT images. Using these, we have developed a personal identification method that focuses on the alveolar bone. This study examined the effectiveness of this method and aimed to implement a reproducible system. MATERIALS AND METHODS: For personal identification, a total of 633 CT images and panoramic X-ray images belonging to three groups with different conditions were used.　These images were 160 sets in the same person group and 96,820 in the other groups. The similarity of alveolar bone images was calculated using the landmark method of Procrustes analysis. The processes were system implemented and the methodology was validated. RESULTS: The ability to identify between the same person group and other person groups showed 0.9769 as the area under the curve (AUC: ROC curve). At the cutoff value of 4.978, there was no false rejection rate, but false acceptance rate was slightly higher. CONCLUSION: This method was useful as a screening method for personal identification. In addition, system implementation was efficient and reduced human error. In the future, we aim to realize a more efficient personal identification method using distortion-corrected images and including auto-detective landmarks using deep learning.

Genetic and expression analysis of SNPs in the human deoxyribonuclease II: SNPs in the promoter region reduce its in vivo activity through decreased promoter activity.

Five SNPs in the human DNase II gene have been reported to be associated with rheumatoid arthritis (RA). Genotype and haplotype analysis of 14 SNPs, nine SNPs of which reported in the NCBI dbSNP database in addition to these five SNPs, was performed in healthy subjects. The enzymatic activities of the amino acid substituted DNase II corresponding to each SNP and serum DNase II in healthy Japanese, and promoter activities derived from each haplotype of the RA-related SNPs were measured. Significant correlations between genotype in each RA-related SNP and enzymatic activity levels were found; alleles associated with RA exhibited a reduction in serum DNase II activity. Furthermore, the promoter activities of each reporter construct corresponding to predominant haplotypes in three SNPs in the promoter region of the gene exhibited significant correlation with levels of serum DNase II activity. These findings indicate these three SNPs could alter the promoter activity of DNASE2, leading to a decline in DNase II activity in the serum through gene expression. Since the three SNPs in the promoter region of the DNase II gene could affect in vivo DNase II activity through reduction of the promoter activity, it is feasible to identify these SNPs susceptible to RA.