Search

Children with severe motor and intellectual disabilities (SMIDs) frequently and continuously receive enteral nutrition and medications and lack adequate exercise, which may lead to dysbiosis, an imbalance in the composition of the gut microbiota. However, studies on the composition of gut microbiota in children with SMIDs are limited. Therefore, we aimed to examine the characteristics of the gut microbiota in children with SMIDs.16S rRNA gene sequencing was performed using fecal samples of 10 children with SMIDs, who received enteral nutrition through a gastric fistula or gastric tube (SMID group: median age, 10.0 years), and 19 healthy children (healthy control [HC] group: median age, 9.0 years). Microbial diversity, microbial composition, and abundance of butyric acid-producing bacteria were compared between the groups. Daily dietary fiber intake in the SMID group was evaluated using questionnaires.The Shannon and Simpson indices (alpha diversity indices) were significantly lower in the SMID group than those in the HC group. Beta diversity analysis identified different clusters. Compared with the HC group, Clostridiales and butyric acid-producing bacteria were less abundant and Bacteroidales were more abundant in the SMID group. Dietary fiber intake in the SMID group was approximately two-thirds of the estimated average requirement for healthy Japanese children.Children with SMIDs showed dysbiosis with alteration in the microbial diversity, which could partly be attributed to their low dietary fiber intake. Further studies, with the intervention of prebiotics, probiotics, and synbiotics, are warranted to improve dysbiosis in children with SMIDs.

The gut microbiota was reported to differ between children with autism spectrum disorder (ASD) and typically developing (TD) children, and dysbiosis of the gut microbiota in preterm infants is common. Here, we explored the characteristics of gut microbiota in children born preterm with ASD. We performed 16S rRNA gene sequencing using stool samples from ASD children born preterm and TD children born preterm. Alpha diversity was significantly greater in the ASD group. A comparison of beta diversity showed different clusters. Linear discriminant analysis effect size analysis revealed significantly more Firmicutes in the ASD group compared with the TD group. In conclusion, the gut microbiota in children born preterm differs between children with ASD and TD.

In human identification methods that target short tandem repeats (STRs), massively parallel sequencing (MPS) technology has made it possible to genotype at the level of the specific sequence itself. This allows for the detection of repeat unit variants and single nucleotide polymorphisms (SNPs) adjacent to the STRs. Using the GlobalFiler™ NGS STR Panel v2, Ion S5, and Converge software, this study constructed a Japanese database of 31 autosomal STRs (auSTRs) and two sex markers from 322 individuals. After excluding some sequence errors and stutters, a total of 31 novel alleles were identified. Additionally, using the allele frequencies of 31 auSTR loci, the match probabilities for the length-based and sequence-based data were calculated to be 1.433 × 10

Spastic paraplegia type 4 (SPG4) is caused by mutations of the SPAST gene and is the most common form of autosomal-dominantly inherited pure hereditary spastic paraplegia (HSP). We herein report a Japanese patient with SPG4 with a confirmed de novo mutation of SPAST. On exome sequencing and Sanger sequencing, we identified the heterozygous missense mutation p.R460L in the SPAST gene. This mutation was absent in the parents, and the paternity and maternity of the parents were both confirmed. The patient showed a pure SPG4 phenotype with an infantile onset. This study may expand the clinical and genetic findings for SPG4.

BACKGROUND We investigated whether an association exists between regulatory T cells (Tregs) during initial presentation in children with idiopathic nephrotic syndrome (INS) and later development of frequently relapsing INS. METHODS Blood samples were obtained at onset and at remission from 25 patients (median age, 4.0 years) with INS; eight did not show relapse after initial response (non-relapsing [NR]), whereas 17 showed frequent relapses (frequently relapsing [FR]). Tregs were measured by flow cytometry; increases were compared between groups. Fecal samples were obtained at onset from 20 patients with INS, as well as from 20 age-matched healthy children. Gut microbiota composition was assessed using 16S ribosomal RNA (rRNA) sequencing (ion PGM). RESULTS The rate of increase in Tregs from onset to remission was significantly lower in the FR group (124.78%) than in the NR group (879.16%; P

We examined the ability of hydrogen peroxide plasma (HPP) to remove DNA contamination, to evaluate whether it is a suitable forensic-grade treatment under ISO 18385. HPP treatment was compared to ethylene-oxide gas (EOG) treatment, which is required by ISO 18385. For the evaluation, commercial control DNA solution and cultured cells sprinkled on Petri dishes were used, and the DNA fragments (214 and 80 bp autosomal DNA fragments and 75 bp Y chromosome fragment) were quantified. HPP treatment was performed up to four times and EOG treatment was performed once. Performing HPP treatment three times was as effective as EOG treatment, with all fragments decreasing to below 1/1,000 in DNA solution. With STR and Y-STR typing, no alleles were detected for three HPP treatments of control DNA using the original amount, i.e., 1 ng. Therefore, HPP appears useful for removing DNA contamination. For cells sprinkled on Petri dishes, the DNA degradation abilities of the HPP and EOG were comparable. However, less DNA was degraded with the HPP and EOG and neither met the ISO criteria. Although the current version of ISO 18385 recommends an evaluation method using cultured cells sprinkled on Petri dishes, it needs to be revised. These findings should be considered when revising ISO 18385.

Butyric acid produced in the intestine by butyric acid-producing bacteria (BAPB) is known to suppress excessive inflammatory response and may prevent chronic disease development. We evaluated whether fiber-rich barley intake increases BAPB in the gut and concomitantly butyric acid in feces. Eighteen healthy adults received granola containing functional barley (BARLEYmax®) once daily for four weeks. Fecal DNA before intake, after intake, and one month after intake was analyzed using 16S rRNA gene sequencing to assess microbial diversity, microbial composition at the order level, and the proportion of BAPB. Fecal butyric acid concentration was also measured. There were no significant differences in diversities and microbial composition between samples. The proportion of BAPB increased significantly after the intake (from 5.9% to 8.2%). However, one month after stopping the intake, the proportion of BAPB returned to the original value (5.4%). Fecal butyric acid concentration increased significantly from 0.99 mg/g feces before intake to 1.43 mg/g after intake (p = 0.028), which decreased significantly to 0.87 mg/g after stopping intake (p = 0.008). As BAPB produce butyric acid by degrading dietary fiber, functional barley may act as a prebiotic, increasing BAPB and consequently butyric acid in the intestine.

© 2019 The Authors Current forensic ancestry-informative panels are limited in their ability to differentiate populations in the Asia-Pacific region. MAPlex (Multiplex for the Asia-Pacific), a massively parallel sequencing (MPS) assay, was developed to improve differentiation of East Asian, South Asian and Near Oceanian populations found in the extensive cross-continental Asian region that shows complex patterns of admixture at its margins. This study reports the development of MAPlex; the selection of SNPs in combination with microhaplotype markers; assay design considerations for reducing the lengths of microhaplotypes while preserving their ancestry-informativeness; adoption of new population-informative multiple-allele SNPs; compilation of South Asian-informative SNPs suitable for forensic AIMs panels; and the compilation of extensive reference and test population genotypes from online whole-genome-sequence data for MAPlex markers. STRUCTURE genetic clustering software was used to gauge the ability of MAPlex to differentiate a broad set of populations from South and East Asia, the West Pacific regions of Near Oceania, as well as the other globally distributed population groups. Preliminary assessment of MAPlex indicates enhanced South Asian differentiation with increased divergence between West Eurasian, South Asian and East Asian populations, compared to previous forensic SNP panels of comparable scale. In addition, MAPlex shows efficient differentiation of Middle Eastern individuals from Europeans. MAPlex is the first forensic AIM assay to combine binary and multiple-allele SNPs with microhaplotypes, adding the potential to detect and analyze mixed source forensic DNA.

Purpose DNA methylation is thought to play a role in exercise-induced gene expression. We aimed to examine changes in muscular strength and body composition in elderly patients with end-stage knee osteoarthritis before and after artificial knee arthroplasty and exercise therapy. We aimed to confirm the relationship between DNA methylation and body composition, using the methylation rate of the pyruvate dehydrogenase kinase 4 (PDK4) gene that regulates skeletal muscle and fat metabolism. Patients and methods Patients underwent artificial knee arthroplasty between April 2017 and June 2017 at Kansai Medical University Hospital. Six patients (seven knees) were included in the analysis (four males/two females; average age, 75.7 years; body mass index, 25.1 kg/m2). Body composition and knee extension muscle strength were measured before surgery and 5 months after surgery. Rehabilitation was performed for 3 months after surgery. In the remaining 2 months, patients performed resistance training and aerobic exercise using an ergometer for 20 mins, twice a week. A biopsy of the vastus medialis was taken during surgery and 5 months post-surgery. Biopsy samples were treated with bisulfite after DNA extraction, and DNA methylation rate was calculated. Results Body weight (P=0.046), total weight (P=0.027), and total fat mass (P=0.028) were significantly lower 5 months postoperatively than preoperatively. Five months post-surgery, the PDK4 gene was significantly more hypomethylated at eight sites in the CpG island, compared to pre-surgery. There was a significant correlation (r=0.88, P=0.02) between promoter region hypomethylation and weight loss. Total methylation rate and weight loss were significantly correlated (r=0.829, P=0.042). Total methylation rate and decrease in total fat mass showed a positive trending relationship (r=0.812, P=0.05). Conclusion Rehabilitative exercise resulted in significant decreases in weight and body fat. Hypomethylation of the PDK4 gene promoter region signified the effect of postoperative management focus on exercise therapy on weight and fat loss.

As one of external visible characteristics (EVCs) in forensic phenotyping, age estimation is essential to providing additional information about a sample donor. With the development of epigenetics, age-related DNA methylation may be used as a reliable predictor of age estimation. With the aim of building a feasible age estimation model for Japanese individuals, 53 CpG sites distributed between 11 candidate genes were selected from previous studies. The DNA methylation level of each target CpG site was identified and measured on a massive parallel platform (synthesis by sequencing, Illumina, California, United States) from 60 forensic blood samples during the initial training phase. Multiple linear regression and quantile regression analyses were later performed to build linear and quantile age estimation models, respectively. Four CpG sites on four genes— ASPA, ELOVL2, ITGA2B, and PDE4C —, were found to be highly correlated with chronological age in DNA samples from Japanese individuals (|R| > 0.75). Subsequently, an independent validation dataset (n = 30) was used to verify and evaluate the performance of the two models. Comparison of mean absolute deviation (MAD) with other indicators showed that both models provide accurate age predictions (MAD: linear = 6.493 years; quantile = 6.243 years). The quantile model, however, can provide the changeable prediction intervals that grow wider with increasing age, and this tendency is consistent with the natural aging process in humans. Hence, the quantile model is recommended in this study.

We previously reported that a decrease in butyrate-producing bacteria in the gut is a potential cause of regulatory T cell (Treg) abnormalities in children with idiopathic nephrotic syndrome (INS). Therefore, we hypothesized that administration of butyrate-producing bacteria might reduce INS relapse and the need for immunosuppressants in these patients. Twenty patients in remission from INS (median age 5.3 years, 15 boys) were enrolled in the study and assigned to receive either daily oral treatment with a preparation of 3 g Clostridium butyricum or no probiotic treatment. The number of relapses and requirement for immunosuppressive agents were compared between the two groups. In the probiotic treatment group, analyses of the gut microbiota and Treg measurements were also performed. Probiotic-treated patients experienced fewer INS relapses per year compared with non-probiotic-treated patients (p = 0.016). Further, administration of rituximab in the probiotic treatment group was significantly less frequent compared with the non-probiotic-treated group (p = 0.025). In the probiotic treatment group, analyses before and after probiotic treatment revealed the significant increases in the relative abundance of butyrate-producing bacteria (p = 0.017) and blood Treg counts (p = 0.0065). Thus, oral administration of butyrate-producing bacteria during INS remission may reduce the frequency of relapse and the need for immunosuppressive agents.

We evaluated the effect of long-term low dose antibiotic prophylaxis on children's gut microbiota.We conducted 16S ribosomal RNA gene sequencing using stool samples from 35 patients younger than 3 years old (median age 5.2 months; male-to-female ratio 17:18) who underwent antibiotic treatment during the acute phase of febrile urinary tract infection. Samples were collected at 5 time points, ie before, during and at 1 to 2, 3 to 4, and 5 to 6 months after febrile urinary tract infection onset and antibiotic treatment. Continuous antibiotic prophylaxis using trimethoprim-sulfamethoxazole was initiated in 23 patients with grade III or higher vesicoureteral reflux and was not administered in 12 patients without reflux.Within 2 weeks after initiation of treatment for febrile urinary tract infection almost all enteric bacteria belonged to the order Lactobacillales, and gut microbiota diversity decreased compared to the pretreatment level (average Shannon index 2.9 before treatment, 1.4 during treatment). The diversity recovered within 1 to 2 months after febrile urinary tract infection onset in both groups. Diversity was maintained during the study period in both groups (p=0.43). A smaller proportion of gut microbiota component belonged to the order Enterobacteriales (p=0.002) in the antibiotic prophylaxis group.Our results revealed that patients receiving continuous antibiotic prophylaxis had normal gut microbiota diversity, indicating that the effect of trimethoprim-sulfamethoxazole on gut microbiota was insignificant. Furthermore, prophylaxis with trimethoprim-sulfamethoxazole might selectively suppress the growth of bacteria belonging to the order Enterobacteriales, such as Escherichia coli and Klebsiella species, which are the main causative bacteria of febrile urinary tract infections.

Spastic paraplegia type 4 (SPG4) is caused by mutations of the SPAST gene and is the most common form of autosomal-dominantly inherited pure hereditary spastic paraplegia (HSP). We herein report a Japanese patient with SPG4 with a confirmed de novo mutation of SPAST. On exome sequencing and Sanger sequencing, we identified the heterozygous missense mutation p.R460L in the SPAST gene. This mutation was absent in the parents, and the paternity and maternity of the parents were both confirmed. The patient showed a pure SPG4 phenotype with an infantile onset. This study may expand the clinical and genetic findings for SPG4.

Background: While the etiology of idiopathic nephrotic syndrome (idiopathic nephrotic syndrome [INS]; characterized by repeated relapses and comorbid allergic conditions) remains unknown, recent evidence suggests that dysfunction in regulatory T cells (Tregs) plays an important role in the development of INS as well as allergic diseases. We hypothesized that dysbiosis involving decreased butyric acid-producing gut microbiota leads to defective induction and differentiation of peripherally induced Tregs, resulting in INS relapse. Methods: Study subjects were 12 children with INS, 8 classified as relapsing (R group; median age: 3.0 years) and 4 as non-relapsing (NR group; median age: 4.3 years), and 11 healthy children (HC group; median age: 5.1 years) serving as normal controls. Measurement of microbiota was performed using 16S ribosomal RNA metagenomic analysis, and fecal butyric acid was measured using high performance liquid chromatography. Flow-cytometric analysis of Tregs and CD4-positive (CD4+) cells in peripheral blood was also performed. Results: Metagenomic analysis of gut microbiota using feces showed that the proportion of butyric acid-producing bacteria was significantly lower in R (median 6.36%) than HC (median 18.84%; p = 0.0013), but no different between NR (median 16.71%) and HC (p = 0.29). Fecal organic acid analysis revealed significantly lower butyric acid quantities in R than HC (medians: 0.48 vs. 0.99 mg/g, p = 0.042). Circulating Tregs as a proportion of CD4+ cells were decreased in 75% of R and NR. Conclusion: Pediatric relapsing INS patients show gut microbiota dysbiosis, characterized by a decreased proportion of butyric acid-producing bacteria and lower fecal butyric acid quantities, concomitant with reduced circulatory Tregs.

As DNA typing systems have become increasingly sensitive in recent years, probability distribution models for back, forward, double-back, and minus 2-nt stutter ratios have been desired to be considered in DNA evidence interpretation using specific software programs. However, experimental investigations have been insufficient, especially for forward, double-back, and minus 2-nt stutters. In this study, we experimentally reevaluated the probability distribution models for each stutter ratio in the typing systems of GlobalFiler™ PCR Amplification Kit and 3500xL Genetic Analyzer from Thermo Fisher Scientific. In addition, to enhance the reliability of longest uninterrupted stretch (LUS) values and corrected allele numbers used in previously developed models for stutter ratios using sequence information (i.e., LUS model and multi-seq model), we propose the weighted average of LUS values and corrected allele numbers based on the number of observations in sequence-based population data. Back stutter ratios demonstrated a positive correlation with allele numbers (allele model) in eight loci, LUS values (LUS model) in eight loci, and corrected allele numbers (multi-seq model) in five loci. The forward stutter ratios (FSRs) of D22S1045 followed the LUS model. FSRs other than D22S1045 and double-back stutter ratios followed the LUS model by considering multiple loci together. Minus 2-nt stutter ratios observed in SE33 and D1S1656 did not increase with the increase in the allele numbers. The adopted models for each stutter ratio can be implemented in software programs for DNA evidence interpretation and enable a reliable interpretation of crime stain profiles in forensic caseworks.

Introduction MicroRNA (miR) is non-coding small RNA that regulate mRNA at the post-transcriptional level by degradation or inhibition. To find physical stress markers, we developed a rat model involving a simple and complicated stress and measured serum miR levels. Materials and methods To demonstrate changes in serum miR levels when physical stress is applied, we constructed three stress modalities using rats: alcohol intake, treadmill running and restraint. After alcohol administration, the rats were made to run on a treadmill and some of the rats were further stressed by restraining with a 2 kg water bag immediately after the treadmill run. The rats were grouped as follows: control, run for 20 min, run for 90 min, run and restrained for 20 min, run and restrained for 90 min. Using total RNA extracted from sera, expression levels of eight miRs were measured by real-time PCR. Results The level of miR-199a was increased by 20 min stress procedures and the levels of miR-1, miR-24a and miR-133a/b were increased by 90 min stress procedures. No change in the levels of miR-208, miR-212 or miR-296-5p was seen under any stress conditions. There was no significant difference between a treadmill run only and a combination of treadmill run and being restrained by a 2 kg water bag. Discussion We demonstrated that a combination of these serum miRs might indicate the intensity of stress experienced.

When full STR profiles cannot be obtained, further DNA analyses targeting single nucleotide polymorphisms (SNPs) may occasionally yield valuable information. Although the discrimination power of each SNP is relatively low, combined analysis of many SNPs can improve the personal identification ability to a level as high as that of commercial STR typing kits. In this study, we developed a new SNP typing method, named the amplified-product length polymorphism (APLP) 48-ID assay, for genotyping of 47 autosomal SNPs and two X and Y chromosomal markers for sex typing. Forty-seven SNPs were selected from all 22 autosomes, showing high diversity in European, Nigerian, Han Chinese, and Japanese population in the HapMap data. PCR primers were designed to generate amplicons 40-100 bp in length to increase the robustness of the PCR. The APLP 48-ID assay consisted of four independent PCR reactions followed by electrophoretic run on four lanes in a polyacrylamide gel. Complete profiles were obtained when more than 1.2 ng of DNA was used. We applied this assay for genotyping of 236 Japanese individuals. The random matching probability was 3.3E-20, and the power of exclusion was greater than 0.9999999. This method is a rapid, robust, and cost-effective approach for human identification and paternity testing.

Background/Aims: The mode of delivery (vaginal or cesarean section) and feeding type (breastfeeding or formula feeding) of neonates are considered the most influential factors in the development of gut microbiota. Objectives: This study investigated the effect of prebiotic-rich breast milk on overcoming gut microbiota dysbiosis. Method: Stool samples from 36 healthy Japanese neonates were obtained at 4 days and 1 month of age, and divided into 4 groups based on mode of delivery and feeding type. The gut microbiota composition and bacterial diversity were assessed using 16S rRNA sequencing. Results: At 4 days old, vaginally delivered neonates had a significantly higher diversity of bacteria than those born by cesarean section. Bacteroidales and Enterobacteriales were overrepresented in vaginally delivered neonates (p = 0.0031 and p = 0.011), while Bacillales and Lactobacillales were overrepresented in caesarean section delivered neonates (p = 0.012 and p = 0.0016). However, there was little difference in bacterial diversity and bacterial relative abundance at 1 month of age between groups. Conclusions: Cesarean section delivery appeared to reduce the diversity of neonate gut microbiota, resulting in dysbiosis, but this improved to the equivalent level seen in vaginally delivered infants by 1 month of age. Breastfeeding, even for short periods, may therefore improve neonate gut dysbiosis.

Many genomic variants associated with sudden unexpected death (SUD) have been found with the use of the next generation sequencing (NGS), and registered in the clinical pathogenetic variant databases such as SIFT, Poly-Phen2 and Grantham. In this study, we search additional variants by NGS analysis of genomic DNA extracted from the cadavers in the sudden unexpected infant death (SID) cases. Genomic DNA was extracted from the ventricular septum of three infants of SID cases, three infants died of myocarditis, asphyxia and Down syndrome, a 15 year-old boy died of SUD, and an adult died of intracerebral bleeding. After bisulfite treatment of DNA samples, methylation levels were measured using the Infinium MethylationEPIC BeadChip Kit and iSCAN system (Illumina). This system can check over 860,000 methylation sites per sample. Methylation levels were compared among SID and other cases by GenomeStudio software (Illumina), and top 50 sites with increasing or decreasing methylation levels were obtained. When SID cases and a SUD case were compared, SID cases showed severe decrease in methylation level at cg16032134 site. This site exists in ATP10A gene coding ATPase class V type 10A enzyme and is related to several diseases such as asthma and chronic obstructive pulmonary disease (COPD).

Non-coding microRNAs (miRNAs) hold enormous potential as a postmortem biomarker to quantify the physical stress applied before the death. Excited delirium syndrome causes a sudden unexpected death under agitated and excited situation. The forensic autopsies and laboratory tests can't reveal the mechanism of the sudden death. Then, we constructed a rat model of excited delirium syndrome-like situation by applying three stress modalities, alcohol intake for 3.8g/kg body weight, treadmill running for 90min, and suppression by 2kg water bag for 90min. The rats were divided into three groups: the control, the stress group A with alcohol intake and running, and the stress group B with alcohol intake, running and suppression. After the rats were euthanized by sevoflurane over-dose, total RNA was extracted from 20mg left ventricle tissues. The small RNA library and templates were prepared for the next generation sequencer (NGS) analysis with Ion PGM system and Ion 318Chip (Thermo Fisher Scientific). The obtained data were analyzed by Torrent Suite software with reference to the rat miRNA databases. The top 30 miRNAs in expression level were selected and analyzed by statistical methods between the control and stress groups. The seven miRNAs (miR-126a-3p, miR-143-3p, miR-16-5p, miR-22-3p, miR-26a-5p, miR-29a-3p, and miR-29b-3p) showed the significant difference in expression level among the groups. Especially, miR-143-3p and miR-22-3p might reflect the physical stress on rat hearts well.