Long-Term Epigenetic Regulation of Foxo3 Expression in Neonatal Valproate-Exposed Rat Hippocampus with Sex-Related Differences.

Perinatal exposure to valproic acid is commonly used for autism spectrum disorder (ASD) animal model development. The inhibition of histone deacetylases by VPA has been proposed to induce epigenetic changes during neurodevelopment, but the specific alterations in genetic expression underlying ASD-like behavioral changes remain unclear. We used qPCR-based gene expression and epigenetics tools and Western blotting in the hippocampi of neonatal valproic acid-exposed animals at 4 weeks of age and conducted the social interaction test to detect behavioral changes. Significant alterations in gene expression were observed in males, particularly concerning mRNA expression of Foxo3, which was significantly associated with behavioral changes. Moreover, notable differences were observed in H3K27ac chromatin immunoprecipitation, quantitative PCR (ChIP-qPCR), and methylation-sensitive restriction enzyme-based qPCR targeting the Foxo3 gene promoter region. These findings provide evidence that epigenetically regulated hippocampal Foxo3 expression may influence social interaction-related behavioral changes. Furthermore, identifying sex-specific gene expression and epigenetic changes in this model may elucidate the sex disparity observed in autism spectrum disorder prevalence.

Measuring people's views on health system performance: Design and development of the People's Voice Survey.

Todd Lewis and co-authors discuss development and use of the People's Voice Survey for health system assessment.

Non-coding de novo mutations in chromatin interactions are implicated in autism spectrum disorder.

Three-dimensional chromatin interactions regulate gene expressions. The significance of de novo mutations (DNMs) in chromatin interactions remains poorly understood for autism spectrum disorder (ASD). We generated 813 whole-genome sequences from 242 Korean simplex families to detect DNMs, and identified target genes which were putatively affected by non-coding DNMs in chromatin interactions. Non-coding DNMs in chromatin interactions were significantly involved in transcriptional dysregulations related to ASD risk. Correspondingly, target genes showed spatiotemporal expressions relevant to ASD in developing brains and enrichment in biological pathways implicated in ASD, such as histone modification. Regarding clinical features of ASD, non-coding DNMs in chromatin interactions particularly contributed to low intelligence quotient levels in ASD probands. We further validated our findings using two replication cohorts, Simons Simplex Collection (SSC) and MSSNG, and showed the consistent enrichment of non-coding DNM-disrupted chromatin interactions in ASD probands. Generating human induced pluripotent stem cells in two ASD families, we were able to demonstrate that non-coding DNMs in chromatin interactions alter the expression of target genes at the stage of early neural development. Taken together, our findings indicate that non-coding DNMs in ASD probands lead to early neurodevelopmental disruption implicated in ASD risk via chromatin interactions.

Neonatal Exposure to Valproate Induces Long-Term Alterations in Steroid Hormone Levels in the Brain Cortex of Prepubertal Rats.

Valproic acid (VPA) is a known drug for treating epilepsy and mood disorders; however, it is not recommended for pregnant women because of its possible teratogenicity. VPA affects neurotransmission and gene expression through epigenetic mechanisms by acting as a histone deacetylase inhibitor and has been used to establish animal models of autism spectrum disorder (ASD). However, studies on the long-term effects of early exposure to VPA on glucocorticoid and neurosteroid synthesis in the brain are lacking. Therefore, this study aimed to investigate the long-term changes in metabolic alterations and gene expression regulation according to sex, using metabolic steroid profiling data from cerebral cortex samples of rats four weeks after VPA exposure (400 mg/kg). In neonatal VPA-exposed models, estradiol levels decreased, and cytochrome P450 19A1 gene (Cyp19a1) expression was reduced in the prepubertal male cortex. Progesterone and allopregnanolone levels decreased, and 3ß-hydroxysteroid dehydrogenase 1 gene (Hsd3b1) expression was also downregulated in the prepubertal female cortex. Furthermore, cortisol levels increased, and mRNA expression of the nuclear receptor subfamily 3 group C member 1 gene (Nr3c1) was downregulated in the cortices of both sexes. Unlike the neonatal VPA-exposed models, although a decrease in progestin and estradiol levels was observed in females and males, respectively, no differences were observed in cortisol levels in the cortex tissues of 8-week-old adult rats administered VPA for four weeks. These results indicate that early environmental chemical exposure induces long-term neurosteroid metabolic effects in the brain, with differences according to sex.

Zinc Deficiency Induces Autophagy in HT-22 Mouse Hippocampal Neuronal Cell Line.

Zinc is a trace metal vital for various functions in nerve cells, although the effect of zinc deficiency on neuronal autophagy remains unclear. This study aimed to elucidate whether zinc deficiency induced by treatment with N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a zinc chelator, affects and alters autophagy activity. In cell viability assays, TPEN showed cytotoxicity in HT-22 cells. TPEN treatment also increased LC3-II levels and the ratio of LC3-II to LC3-I. Western blot analysis showed that phospho-AMP-activated protein kinase levels and the ratio of phospho-AMP-activated protein kinase to total AMP-activated protein kinase increased. Protein levels of the mammalian target of rapamycin and sirtuin 1 decreased following TPEN treatment. When TPEN-treated HT-22 cells were cotreated with autophagy inhibitors, 3-methyladenine (1 mM), or bafilomycin A1 (3 nM), the TPEN-induced decrease in cell viability was exacerbated. Cotreatment with chloroquine (10 µM) partially restored cell viability. The study showed that zinc deficiency induces autophagy and may be cytoprotective in neurons. We expect our results to add a new perspective to our understanding of the neuronal pathology related to zinc deficiency.

Prenatal Trimethyltin Exposure Induces Long-Term DNA Methylation Changes in the Male Mouse Hippocampus.

Trimethyltin (TMT) is an irreversible neurotoxicant. Because prenatal TMT exposure has been reported to induce behavioral changes, this study was conducted to observe gender differences and epigenetic changes using a mouse model. In behavioral testing of offspring at 5 weeks of age, the total times spent in the center, corner, or border zones in the male prenatal TMT-exposed mice were less than those of control unexposed mice in the open-field test. Female TMT-exposed mice scored lower on total numbers of arm entries and percentages of alternations than controls in the Y-maze test with lower body weight. We found that only TMT-exposed males had fewer copies of mtDNA in the hippocampus and prefrontal cortex region than controls. Additional epigenetic changes, including increased 5-methyl cytosine/5-hydroxymethyl cytosine levels in the male TMT hippocampus, were observed. After methylation binding domain (MBD) sequencing, multiple signaling pathways related to metabolism and neurodevelopment, including FoxO signaling, were identified by pathway analysis for differentially methylated regions (DMRs). Increased FOXO3 and decreased ASCL1 expression were also observed in male TMT hippocampi. This study suggests that sex differences and epigenetics should be more carefully considered in prenatal toxicology studies.

Acute Valproate Exposure Induces Sex-Specific Changes in Steroid Hormone Metabolism in the Cerebral Cortex of Juvenile Mice.

Valproic acid (VPA), an antiepileptic and mood stabilizer, modulates neurotransmission and gene expression by inhibiting histone deacetylase activity. It is reported that VPA may affects the steroid hormone level. In this study, VPA-induced acute metabolic alterations were investigated using liquid chromatography-tandem mass spectrometry in prepubertal mice brain. In VPA-treated (400 mg/kg in saline solution, intraperitoneal) mice, cortisol levels were increased (female: P < 0.004, male: P < 0.003) and 17ß-estradiol levels were decreased (Both P < 0.03). Furthermore, in the VPA-treated male mice, dihydrotestosterone levels were increased (P < 0.02) and testosterone were decreased (P < 0.002). The 4-hydroxylase activity was upregulated in the female VPA-treated mice (P < 0.01) and the 5α-reductase activity was increased in the male VPA-treated mice (P < 0.003). These results indicate sex specific differences in VPA-induced steroid metabolism in the brain cortex.

Anti-Proliferative Effect of Allium senescens L. Extract in Human T-Cell Acute Lymphocytic Leukemia Cells.

Allium species are well known plants distributed throughout the world, and they contain various bioactive components with different biological activities including anti-cancer effects. In this study, we investigated the inhibitory effect of Allium senescens L. (A.S.) extract on cell survival and IL-2-mediated inflammation in human T cell acute lymphocytic leukemia (T-ALL) Jurkat cells. Our results showed that A.S. extract induced caspase-dependent apoptosis of Jurkat cells with no significant cytotoxicity in the normal peripheral blood mononuclear cells. A.S. extract induced ROS generation through the activation of MAPK p38 phosphorylation. It also inhibited IL-2 mRNA expression and NF-κB signaling mediated by phorbol 12-myristate 13-acetate, and phytohemagglutinin. Combined treatment with A.S. extract and axitinib/dovitinib exerted enhanced inhibitory effects on T-ALL cell growth and IL-2 production. These results provide novel information on the potential use of A.S. extract as a therapeutic herbal agent for the treatment and prevention of T-ALL.

Plumbagin exerts an immunosuppressive effect on human T-cell acute lymphoblastic leukemia MOLT-4 cells.

Of the hematological disorders typified by poor prognoses and survival rates, T-cell acute lymphoblastic leukemia (T-ALL) is one of the most commonly diagnosed. Despite the development of new therapeutic agents, the treatment options for this cancer remain limited. In this manuscript, we investigated the anti-proliferative effects of plumbagin, mediated by the activation of mitogen-activated protein kinase (MAPK) pathways, and inhibition of NF-κB signaling; the human T-ALL MOLT-4 cell line was used as our experimental system. Plumbagin is a natural, plant derived compound, which exerts an anti-proliferative activity against many types of human cancer. Our experiments confirm that plumbagin induces a caspase-dependent apoptosis of MOLT-4 cells, with no significant cytotoxicity seen for normal peripheral blood mononuclear cells (PBMCs). Plumbagin also inhibited LPS-induced phosphorylation of p65, and the transcription of NF-κB target genes. Our results now show that plumbagin is a potent inhibitor of the NF-κB signaling pathway, and suppressor of T-ALL cell proliferation.

Exosome cargo reflects TGF-ß1-mediated epithelial-to-mesenchymal transition (EMT) status in A549 human lung adenocarcinoma cells.

It has been suggested that tumor cells secrete exosomes to modify the local microenvironment, which then promotes intercellular communication and metastasis. Although exosomes derived from cancer cells may contribute to the epithelial-mesenchymal transition (EMT) in untransformed cells, few studies have defined exosome cargo upon induction of EMT. In this study, we investigated the changes in exosomal cargo from the epithelial to mesenchymal cell phenotype by inducing EMT with transforming growth factor (TGF)-ß1 in A549 human lung adenocarcinoma cells. The protein content of the exosomes reflects the change in the cell phenotype. In addition, miR-23a was significantly enriched in the exosomes after mesenchymal transition. Following treatment of exosomes from mesenchymal cells via EMT induction with TGF-ß1 to the epithelial cell type, phenotypic changes in protein expression level and cell morphology were observed. Autologous treatment of exosomes enhanced the transcriptional activity and abundance of ß-catenin. Our results suggest that the exosomal protein and miRNA content reflects the physiological condition of its source and that exosomes induce phenotypic changes via autocrine signaling.

Traf2- and Nck-interacting kinase (TNIK) is involved in the anti-cancer mechanism of dovitinib in human multiple myeloma IM-9 cells.

Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family. TNIK was first identified as a kinase that is involved in regulating cytoskeletal organization in many types of cells, and it was recently proposed as a novel therapeutic target in several types of human cancers. Although previous studies suggest that TNIK plays a pivotal role in cancer cell survival and prognosis, its function in hematological cancer cell survival has not been investigated. Here we investigated the relationship between TNIK function and cell viability in multiple myeloma IM-9 cells using TNIK small interfering RNA (siRNA) transfection and dovitinib treatment. Treatment of IM-9 cells with TNIK siRNA and dovitinib treatment reduced cell proliferation. The ATP competing kinase assay and western blot analysis showed that dovitinib strongly inhibited both the interaction of TNIK with ATP (K i, 13 nM) and the activation of Wnt signaling effectors such as ß-catenin and TCF4. Dovitinib also induced caspase-dependent apoptosis in IM-9 cells without significant cytotoxicity in PBMCs. Our results provide new evidence that TNIK may be involved in the proliferation of multiple myeloma IM-9 cells and in the anti-cancer activity of dovitinib via inhibition of the endogenous Wnt signaling pathway.

Whole exome sequencing for a patient with Rubinstein-Taybi syndrome reveals de novo variants besides an overt CREBBP mutation.

Rubinstein-Taybi syndrome (RSTS) is a rare condition with a prevalence of 1 in 125,000-720,000 births and characterized by clinical features that include facial, dental, and limb dysmorphology and growth retardation. Most cases of RSTS occur sporadically and are caused by de novo mutations. Cytogenetic or molecular abnormalities are detected in only 55% of RSTS cases. Previous genetic studies have yielded inconsistent results due to the variety of methods used for genetic analysis. The purpose of this study was to use whole exome sequencing (WES) to evaluate the genetic causes of RSTS in a young girl presenting with an Autism phenotype. We used the Autism diagnostic observation schedule (ADOS) and Autism diagnostic interview revised (ADI-R) to confirm her diagnosis of Autism. In addition, various questionnaires were used to evaluate other psychiatric features. We used WES to analyze the DNA sequences of the patient and her parents and to search for de novo variants. The patient showed all the typical features of Autism, WES revealed a de novo frameshift mutation in CREBBP and de novo sequence variants in TNC and IGFALS genes. Mutations in the CREBBP gene have been extensively reported in RSTS patients, while potential missense mutations in TNC and IGFALS genes have not previously been associated with RSTS. The TNC and IGFALS genes are involved in central nervous system development and growth. It is possible for patients with RSTS to have additional de novo variants that could account for previously unexplained phenotypes.

No association of the norepinephrine transporter gene (SLC6A2) and cognitive and behavioural phenotypes of patients with autism spectrum disorder.

We examined the association between the norepinephrine transporter (SLC6A2) gene and autism spectrum disorder (ASD) in a Korean population. In addition, we investigated which phenotypes of ASD are best attributed to the genotype of SLC6A2. A total of 184 subjects with ASD, their 156 unaffected siblings and both biological parents were recruited through university hospitals. We used the Autism Diagnostic Interview-Revised, the Aberrant Behaviour Checklist (ABC), the Child Behaviour Checklist (CBCL), the Stroop Colour-Word Interference Test and the Wisconsin Card Sorting Test (WCST) as quantitative measures of the ASD phenotypes. The associations between the quantitative measures and specific single-nucleotide polymorphisms (SNPs) were tested with linear regression analyses. We did not find any evidence of the over-transmission of either allele of the 10SLC6A2 SNPs in the DFAM test. At an empirical p value <0.05, findings that were consistent between the linear regression analyses and the QFAM tests were the positive associations between the A allele of rs36020 and attention problems on the CBCL and stereotypical behaviours on the ABC and between the C allele of rs1814270 and the number of trials required to complete the first WCST category. However, these associations did not remain after correction for multiple testing. The study results of this study do not support the association between the SLC6A2 and the diagnosis or phenotype of ASD. However, the study must be replicated in larger populations and with using more genetic markers.

Factors influencing healthcare utilization among children with pneumonia in Muntinlupa City, the Philippines.

We evaluated the factors influencing healthcare utilization among children aged < 5 years with pneumonia symptoms in Muntinlupa City, the Philippines. We interviewed 1,330 caregivers from 190 households per district in seven districts from March to April, 2009 using a standardized questionnaire to obtain information about demographic characteristics, access to healthcare facilities, and potential barriers to healthcare utilization. The mean age of the children was 32 months; 54.7% were boys. Seventy-four point three percents (n = 972) of caregivers stated when their child had pneumonia symptoms they brought them to a government health center, 14.1% (n = 184) went to a private clinic, 8.0% (n = 104) went to a hospital, 3% (n = 40) were treated at home and 0.5% (n = 7) received traditional medicine from a local healer. Caregivers required an average of 12 minutes to reach a healthcare facility. Reasons given by caregivers for their choice of healthcare facility were low cost (47.5%), availability and accessibility to transportation (29.6%) and perceived good quality of care (16.5%). In conclusion, nearly three quarters of caregivers interviewed brought their child with pneumonia symptoms to a government health center for treatment. Cost was the main factor influencing choice, followed by transportation availability and quality of care.

Exosome Secretion and Cellular Signaling Change in a Fabry Disease Cell Model Induced by Gene-silencing.

BACKGROUND/AIM: Fabry disease (FD) is caused by α-galactosidase A (AGA) deficiency, which ultimately leads to the intracellular accumulation of globotriaosylceramide (Gb3). Exosomes play a role in maintaining cellular homeostasis by clearing damaged or toxic materials, including proteins. In the process of excessive accumulation of intracellular Gb3 in Fabry disease, it may be suggested that exosomal secretion of Gb3 increases to preserve cell homeostasis. This study sought to determine how exosomal secretion and cell signaling change in an FD cell model produced by gene silencing. MATERIALS AND METHODS: HEK293T cells were transfected with plasmids carrying shRNA against the GLA gene to produce the FD cell model. A recombinant AGA, agalsidase-beta, was used to evaluate the effect of enzyme replacement therapy (ERT) on exosomal secretion and cell signaling. RESULTS: Exosome secretion was significantly increased in the Fabry disease cell model compared to the control vector cell model, and significantly decreased after agalsidase-beta treatment. The FD cell model showed higher reactive oxygen species (ROS) production and p53 protein expression compared to the control vector cell model. CONCLUSION: Increased exosomal secretion in Fabry disease may be a cellular mechanism to avoid excessive and cytotoxic accumulation of Gb3 in lysosomes through intracellular signaling, including increased p53 expression.

Increasing Natural Killer Cell Activity of Mineral Nanomaterial ALP1018 in Healthy Adults: A Randomized, Double-Blind, Placebo Comparative Clinical Trial.

This randomized, double-blind, placebo comparative clinical trial aimed to determine the immune-enhancing effects and safety of a nanomaterial with iron and zinc (ALP1018) in healthy adults. Participants who met the inclusion criteria were recruited for this study (n = 80) and randomly assigned to either the test group (n = 40), which was given Alp1018 in capsule form, or the placebo group (n = 40), which was given crystal cellulose capsules of identical appearance, weight, and flavor for 8 weeks. Compared to baseline, natural killer (NK) cell activity (%) increased in the test group after 8 weeks, although there were no changes in the placebo group. Furthermore, in the subgroup analysis of Coronavirus disease 2019 (COVID-19) affected participants, significantly increased NK cell activity was observed in the test group at 4 (p < 0.05) and 8 weeks (p < 0.05). No significant differences were observed in cytokine levels between the two groups. ALP1018 supplementation appeared to enhance immune function by improving NK cell activity without adverse effects in healthy adults.

In vitro and in vivo genotoxicity evaluation of ALP1018, a nanomineral food supplement.

The use of nano-based dietary supplements is increasing around the world, as nanotechnology can help enhance nutrient bioavailability. ALP1018 is a newly developed iron-zinc complex supplement designed as a nanoformulation to improve the efficacy of iron and zinc supplementation. However, safety concerns have been raised, as there is no clear evaluation of ALP1018 toxicity. The goal of this study was to determine the potential mutagenicity and genotoxicity of ALP1018 through three standard screenings: the Ames test, which evaluates bacterial reverse mutations; the in vitro test of chromosomal aberration in Chinese hamster lung cells; and the in vivo micronucleus assay using ICR mice. ALP1018 showed no mutagenic effect, as no increase was observed in the presence or absence of metabolic activation (S9 mix) in revertant colonies on all the bacterial strains used in the Ames test. No structural chromosomal abnormalities were observed in the presence or absence of the S9 mix in mammalian cells used in the chromosomal aberration assay. In the micronucleus test, the frequency of micronucleated polychromatic erythrocytes was not significantly increased in mouse bone marrow cells. Based on these findings, we can conclude that ALP1018 is safe to use and has no mutagenic or genotoxic potential.

Association of the catechol-o-methyltransferase gene polymorphisms with Korean autism spectrum disorders.

This study evaluated the family-based genetic association between autism spectrum disorders (ASDs) and 5 single-nucleotide polymorphisms (SNPs) in the catechol-o-methyltransferase gene (COMT), which was found among 151 Korean ASDs family trios (dominant model Z = 2.598, P = 0.009, P FDR = 0.045). We found a statistically significant allele transmission or association in terms of the rs6269 SNP in the ASDs trios. Moreover, in the haplotype analysis, the haplotypes with rs6269 demonstrated significant evidence of an association with ASDs (additive model rs6269-rs4818-rs4680-rs769224 haplotype P = 0.004, P FDR = 0.040). Thus, an association may exist between the variants of the COMT gene and the occurrence of ASDs in Koreans.

Acute valproate exposure affects proneural factor expression by increasing FOXO3 in the hippocampus of juvenile mice with a sex-based difference.

Valproic acid (VPA), an anticonvulsant and mood stabilizer, may affect Notch signaling and mitochondrial function. In a previous study, acute VPA exposure induced increased expression of FOXO3, a transcription factor that shares common targets with pro-neuronal ASCL1. In this study, intraperitoneal acute VPA (400 mg/kg) administration in 4-week-old mice increased and decreased FOXO3 and ASCL1 expression, respectively, in the hippocampus, associated with sex-based differences. Treatment of Foxo3 siRNA increased the mRNA expression levels of Ascl1, Ngn2, Hes6, and Notch1 in PC12 cells. Furthermore, VPA exposure induced significant expression changes of mitochondria-related genes, including COX4 and SIRT1, in hippocampal tissues, associated with sex-based differences. This study suggests that acute VPA exposure differently affects proneural gene expression via FOXO3 induction in the hippocampus based on sex.

An expanded age range for meningococcal meningitis: molecular diagnostic evidence from population-based surveillance in Asia.

BACKGROUND: To understand epidemiologic patterns of meningococcal disease in Asia, we performed a retrospective molecular analysis of cerebrospinal fluid (CSF) specimens collected in prospective surveillance among children aged < 5 years of age in China, South Korea, and Vietnam. METHODS: A total of 295 isolates and 2,302 CSFs were tested by a meningococcal species- and serogroup-specific polymerase chain reaction (PCR) assay targeting the Neisseria meningitidis (Nm) ctrA gene. Multi-locus sequence typing (MLST) was performed in Nm gene amplification analysis and incidence rates for meningococcal meningitis were estimated. RESULTS: Among 295 isolates tested, 10 specimens from Vietnam were confirmed as serogroup B and all were Sequence Type (ST) 1576 by MLST. Among the 2,032 CSF specimen tested, 284 (14%) were confirmed by PCR (ctrA gene), including 67 (23.6%) from China, 92 (32.4%) from Korea, and 125 (44.0%) from Vietnam. Neonates and infants aged < 6 months of age accounted for more than 50% of Nm-PCR positive CSF. Two CSF specimens from Vietnam were identified as serogroup B using MLST. In addition, 44 specimens underwent sequencing to confirm meningococcal serogroup; of these, 21 (48%) were serogroup C, 12 (27%) were serogroup X, 9 (20%) were serogroup Y and 2 (5%) were serogroup B. The incidence rates of meningococcal meningitis among children < 5 years of age was highest in Vietnam (7.4/100,000 [95% CI, 3.6-15.3] followed by Korea (6.8/100,000 [95% CI, 3.5-13.5] and China (2.1/100,000) [95% CI, 0.7-6.2]). CONCLUSIONS: These results suggest that there is a previously undetected, yet substantial burden of meningococcal meningitis among infants and young children. Standardized, sensitive and specific molecular diagnostic assays with Nm serogrouping capacity are needed throughout Asia to understand the true burden of N. meningitidis disease.