Search

Background: Exploring the epigenetic regulations, such as microRNA, in newborns holds significant promise for enhancing our ability to address and potentially prevent early-life developmental delays. Objectives: Hence, this research seeks to investigate if the expression of miRNA in the umbilical cord blood of infants can forecast their developmental outcomes as they grow older. Design and method: We enrolled 143 full-term newborns, delivered either via cesarean section (CS) or through natural spontaneous delivery (NSD). We then analyzed the profiles of specific miRNAs (miR-486-5p, miR-126-5p, miR-140-3p, miR-151a-3p, miR-142-5p, and miR-30e-5p) in the umbilical cord blood of these infants. Subsequently, we performed follow-up assessments using Bayley-III scores when the cohort reached 1 year of age. Furthermore, we conducted pathway-enrichment analyses on the target genes associated with these examined miRNAs. Results: When comparing newborns delivered via cesarean section (CS) to those born via natural spontaneous delivery (NSD), we observed notable differences. Specifically, newborns through NSD displayed significantly higher ΔCt values for miR-486-5p, alongside lower ΔCt values for miR-126-5p and miR-151a-3p in their cord blood. At 1 year of age, cognitive development was significantly linked to the ΔCt values of miR-140-3p and miR-142-5p, while language development showed a significant association with the ΔCt values of miR-140-3p. Moreover, our pathway enrichment analyses revealed that the target genes of these miRNAs were consistently involved in the pathways related to neurons, such as axon guidance and the neurotrophin signaling pathway. Conclusion: In summary, this study represents a pioneering effort in elucidating the potential connections between miRNA levels in cord blood and the health indicators and neurodevelopment of newborns at 1 year of age. Our findings underscore the significance of miRNA levels at birth in influencing mechanisms related to neurodevelopment.

Abstract Background Acanthamoeba castellanii is a free-living protist that feeds on diverse bacteria. A. castellanii has frequently been utilized in studies on microbial interactions. Grazing bacteria also exhibit diverse effects on the physiological characteristics of amoebae, such as their growth, encystation, and cytotoxicity. Since the composition of amoebae amino acids is closely related to cellular activities, it can indicate the overall responses of A. castellanii to various stimuli. Method A. castellanii was exposed to different culture conditions in low-nutrient medium with heat-killed DH5α to clarify their effects. A targeted metabolomic technique was utilized to evaluate the concentration of cellular amino acids. The amino acid composition and pathways were analyzed by two web-based tools: MetaboAnalyst and Pathview. Then, long-term exposure to A. castellanii was investigated through in silico and in vitro methods to elucidate the homeostasis of amino acids and the growth of A. castellanii. Results Under short-term exposure, all kinds of amino acids were enriched in all exposed groups. In contrast to the presence of heat-killed bacteria, the medium exhibited obvious effects on the amino acid composition of A. castellanii. After long-term exposure, the amino acid composition was more similar to that of the control group. A. castellanii may achieve amino acid homeostasis through pathways related to alanine, aspartate, citrulline, and serine. Discussion Under short-term exposure, compared to the presence of bacteria, the type of medium exerted a more powerful effect on the amino acid composition of the amoeba. Previous studies focused on the interaction of the amoeba and bacteria with effective secretion systems and effectors. This may have caused the effects of low-nutrient environments to be overlooked. Conclusion When A. castellanii was stimulated in the coculture system through various methods, such as the presence of bacteria and a low-nutrient environment, it accumulated intracellular amino acids within a short period. However, different stimulations correspond to different amino acid compositions. After long-term exposure, A. castellanii achieved an amino acid equilibrium by downregulating the biosynthesis of several amino acids.

Abstract Neonates who are born preterm (PT) are usually characterized by immature physiological development, and preterm birth (PTB) is the leading cause of neonatal morbidity and mortality if intensive medical care is not available to PTB neonates. Early prediction of a PTB enables medical personnel to make preparations in advance, protecting the neonate from the subsequent health risks. Therefore, many studies have worked on identifying invasive or noninvasive PT biomarkers. In this study, we collected amniocentesis-derived (at the second trimester of gestation) amniotic fluid (AF) samples. At delivery, AF samples were classified into PTB or full-term birth (FTB). We first applied protein mass spectrometry technology to globally screen AF proteins, followed by specific protein validation with ELISA. We identified four protein biomarkers of PTB, including lactotransferrin (LTF), glutathione-disulfide reductase (GSR), myeloperoxidase (MPO) and superoxide dismutase 2 (SOD2). Further analyses demonstrated that their abundances were negatively correlated with neonatal weight and gestational age. In addition, by mimicking survival rate analysis widely used in tumor biology, we found that LTF and SOD2 were prognostic factors of gestational age, with higher levels denoting shorter gestational age. Finally, using the abundances of the four protein biomarkers, we developed a prediction model of PTB with an auROC value of 0.935 (sensitivity = 0.94, specificity = 0.89, p value = 0.0001). This study demonstrated that the abundances of specific proteins in amniotic fluid were not only the prognostic factors of gestational age but also the predictive biomarkers of PTB. These four AF proteins enable identification of PTB early in the second trimester of gestation, facilitating medical intervention to be applied in advance.

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis primarily affecting peripheral and axial joints. The osteolytic effect in the damaged joint is mediated by osteoclast activation. We aimed to investigate differential gene expression in peripheral CD14+ monocytes between patients with psoriatic arthritis (n = 15) and healthy controls (HCs; n = 15). Circulating CD14+ monocytes were isolated from peripheral blood mononuclear cells using CD14+ magnetic beads. Cell apoptosis was measured via Annexin V using flow cytometry. The gene expression profiling was analyzed via microarray (available in the NCBI GEO database; accession number GSE261765), and the candidate genes were validated using PCR. The results showed a higher number of peripheral CD14+ monocytes in patients with PsA than in the HCs. By analyzing the microarray data, identifying the differentially expressed genes, and conducting pathway enrichment analysis, we found that the apoptosis signaling pathway in CD14+ cells was significantly impaired in patients with PsA compared to the HCs. Among the candidate genes in the apoptotic signaling pathway, the relative expression level of cathepsin L was confirmed to be significantly lower in the PsAs than in the HCs. We concluded that the numbers of peripheral CD14+ monocytes increased, and their apoptosis activity was impaired in patients with PsA, which could lead to enhanced macrophage maturation and osteoclast activation. The resistance of apoptotic death in peripheral CD14+ monocytes may contribute to active joint inflammation in PsA.

Abstract Introduction Autoimmune encephalitis (AE) is caused by autoantibodies attacking neuronal cell surface antigens and/or synaptic antigens. We previously demonstrated that S100A6 was hypomethylated in patients with AE and that it promoted B lymphocyte infiltration through the simulated blood–brain barrier (BBB). In this study, we focused on the epigenetic regulation of S100A6, the process by which S100A6 affects B lymphocyte infiltration, and the therapeutic potential of S100A6 antibodies. Methods We enrolled and collected serum from 10 patients with AE and 10 healthy control (HC) subjects. Promoter methylation and 5‐azacytidine treatment assays were conducted to observe the methylation process of S100A6. The effect of S100A6 on B lymphocytes was analyzed using an adhesion assay and leukocyte transendothelial migration (LTEM) assay. A LTEM assay was also used to compare the effects of the serum of HCs, serum of AE patients, S100A6 recombinant protein, and S100A6 antibodies on B lymphocytes. Result The promoter methylation and 5‐azacytidine treatment assays confirmed that S100A6 was regulated by DNA methylation. The adhesion study demonstrated that the addition of S100A6 enhanced adhesion between B lymphocytes and a BBB endothelial cell line in a concentration‐dependent manner. The LTEM assay showed that the serum of AE patients, as well as S100A6, promoted B lymphocyte infiltration and that this effect could be attenuated by S100A6 antibodies. Conclusion We clarified that S100A6 was under epigenetic regulation in patients with AE and that it helped B lymphocytes to adhere to and infiltrate the BBB endothelial layer, which could be counteracted by S100A6 antibodies. Therefore, the methylation profile of S100A6 could be a marker of the activity of AE, and countering the effect of S100A6 may be a potential treatment target for AE.

Abstract Attention-deficit/hyperactivity disorder (ADHD) is a common childhood mental disorder with undetermined pathophysiological mechanisms. The gut microbiota and immunological dysfunction may influence brain functions and social behaviours. In the current study, we aimed to explore the correlation of gut microbiome imbalance and inflammation in the pathophysiology of ADHD. Forty-one children with ADHD and thirty-nine healthy-control (HC) individuals were recruited. Faecal samples from all participants were collected and submitted for 16 S rRNA V3–V4 amplicon microbiome sequencing analysis. The plasma levels of 10 cytokines, including TNF-α, IL-6, IL-1β, IL-2, IL-10, IL-13, IL-17A, IFN-α2, IFN-γ, and MCP-1, were determined using a custom-made sandwich enzyme-linked immunosorbent assay (ELISA) developed by Luminex Flowmetrix. There was no significant difference between the ADHD and HC groups in species diversity in the faeces, as determined with α-diversity and β-diversity analysis. In the ADHD group, three differentially abundant taxonomic clades at the genus level were observed, namely Agathobacter, Anaerostipes, and Lachnospiraceae. Top differentially abundant bacteria and representative biological pathways were identified in children with ADHD using linear discriminant analysis (LDA) effect size (LEfSe), and the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis, respectively. The plasma levels of TNF-α were significantly lower in children with ADHD than in HCs. Within the ADHD group, the levels of TNF-α were negatively correlated with ADHD symptoms and diversity of the gut microbiome. Our study provides new insights into the association between gut microbiome dysbiosis and immune dysregulation, which may contribute to the pathophysiology of ADHD.

Abstract Attention-deficit/hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder. This study aimed to examine whether miRNA expression abundance in total white blood cells (WBCs) facilitated the identification of ADHD and reflected its response to treatment. Furthermore, whether miRNA markers facilitated the growth of the human cortical neuronal (HCN-2) cells was also investigated. Total WBC samples were collected from 145 patients and 83 controls, followed by RNA extraction and qPCR assays. Subsequently, WBC samples were also collected at the endpoint from ADHD patients who had undergone 12 months of methylphenidate treatment. The determined ΔCt values of 12 miRNAs were applied to develop an ADHD prediction model and to estimate the correlation with treatment response. The prediction model applying the ΔCt values of 12 examined miRNAs (using machine learning algorithm) demonstrated good validity in discriminating ADHD patients from controls (sensitivity: 96%; specificity: 94.2%). Among the 92 ADHD patients completing the 12-month follow-up, miR-140-3p, miR-27a-3p, miR-486-5p, and miR-151-5p showed differential trends of ΔCt values between treatment responders and non-responders. In addition, the in vitro cell model revealed that miR-140-3p and miR-126-5p promoted the differentiation of HCN-2 cells by enhancing the length of neurons and the number of junctions. Microarray and flow cytometry assays confirmed that this promotion was achieved by repressing apoptosis and/or necrosis. The findings of this study suggest that the expression levels of miRNAs have the potential to serve as both diagnostic and therapeutic biomarkers for ADHD. The possible biological mechanisms of these biomarker miRNAs in ADHD pathophysiology were also clarified.

ABSTRACT Aims/Introduction Identifying diabetes‐susceptible genetic variants will help to provide personalized therapy for the management of type 2 diabetes. Previous studies have reported a genetic risk score (GRS), computed by the sum of nuclear DNA (nDNA) risk alleles, that may predict the future requirement for insulin therapy. Although mitochondrial dysfunction has a close association with insulin resistance (IR), there are few studies investigating whether genetic variants of mitochondrial DNA (mtDNA) will affect the clinical characteristics of type 2 diabetes. Materials and Methods Mitochondrial haplogroups were determined using mtDNA whole genome next generation sequencing and 13 single nucleotide polymorphisms (SNPs) in nDNA susceptibility loci of 13 genes in 604 Taiwanese subjects with type 2 diabetes. A GRS of nDNA was computed by summation of the number of risk alleles. The correlation between the mtDNA haplogroup and the clinical characteristics of type 2 diabetes was assessed by logistic regression analysis. The results were compared with the GRS subgroups for the risk of insulin requirement. Results Mitochondrial haplogroups modulate the clinical characteristics of type 2 diabetes, in which patients harboring haplogroup D4, compared with those harboring non‐D4 haplotypes, were less prone to require insulin treatment, after adjusting for age, gender, and diabetes duration. However, there was no association between insulin requirement and GRS calculated from nuclear genetic variants. Conclusions Mitochondrial haplogroups, but not nuclear genetic variants, have a better association with the insulin requirement. The results highlight the role of mitochondria in the management of common metabolic diseases.

Abstract The diagnostic peripheral biomarkers are still lacking for the bipolar II disorder (BD-II). We used isobaric tags for relative and absolute quantification technology to identify five upregulated candidate proteins [matrix metallopeptidase 9 (MMP9), phenylalanyl-tRNA synthetase subunit beta (FARSB), peroxiredoxin 2 (PRDX2), carbonic anhydrase 1 (CA-1), and proprotein convertase subtilisin/kexin type 9 (PCSK9)] for the diagnosis of BD-II. We analysed the differences in the plasma levels of these candidate proteins between BD-II patients and controls (BD-II, n = 185; Controls, n = 186) using ELISA. To establish a diagnostic model for the prediction of BD-II, the participants were divided randomly into a training group (BD-II, n = 149; Controls, n = 150) and a testing group (BD-II, n = 36; Controls, n = 36). Significant increases were found in all five protein levels between BD-II and controls in the training group. Logistic regression was analysed to form the composite probability score of the five proteins in the training group. Receiver-operating characteristic curve analysis revealed the diagnostic validity of the probability score [area under curve (AUC) = 0.89, P

Attention-deficit/hyperactivity disorder (ADHD) is a commonly seen mental disorder in children. Intestinal permeability may be associated with the pathogenesis of ADHD. The study herein investigated the role of gut leakage biomarkers in the susceptibility of ADHD. A total of 130 children with ADHD and 73 healthy controls (HC) individuals were recruited. Serum concentrations of zonulin, occludin, and defensin (DEFA1) were determined. Visual attention was assessed with Conners’ continuous performance test (CPT). In order to rate participants’ ADHD core symptoms at home and school, their parents and teachers completed the Swanson, Nolan, and Pelham—Version IV Scale (SNAP-IV), respectively. We found significantly lower DEFA1 levels in the ADHD group compared to that in the HC group (p = 0.008), but not serum levels of zonulin and occludin. The serum levels of DEFA1 showed an inverse correlation with the inattention scores in the SNAP-IV parent form (p = 0.042) and teacher form (p = 0.010), and the hyperactivity/impulsivity scores in the SNAP-IV teacher form (p = 0.014). The serum levels of occludin showed a positive correlation with the subtest of detectability in the CPT (p = 0.020). Our study provides new reference into the relation between gut leakage markers and cognition, which may advance research of the pathophysiology of ADHD.

Background: Myocardial infarction (MI) is a life-threatening disease, often leading to heart failure. Defining therapeutic targets at an early time point is important to prevent heart failure. Methods: MicroRNA screening was performed at early time points after MI using paired samples isolated from the infarcted and remote myocardium of pigs. We also examined the microRNA expression in plasma of MI patients and pigs. For mechanistic studies, AAV9-mediated microRNA knockdown and overexpression were administrated in mice undergoing MI. Findings: MicroRNAs let-7a and let-7f were significantly downregulated in the infarct area within 24 h post-MI in pigs. We also observed a reduction of let-7a and let-7f in plasma of MI patients and pigs. Inhibition of let-7 exacerbated cardiomyocyte apoptosis, induced a cardiac hypertrophic phenotype, and resulted in worsened left ventricular ejection fraction. In contrast, ectopic let-7 overexpression significantly reduced those phenotypes and improved heart function. We then identified TGFBR3 as a target of let-7, and found that induction of Tgfbr3 in cardiomyocytes caused apoptosis, likely through p38 MAPK activation. Finally, we showed that the plasma TGFBR3 level was elevated after MI in plasma of MI patients and pigs. Interpretation: Together, we conclude that the let-7-Tgfbr3-p38 MAPK signalling plays an important role in cardiomyocyte apoptosis after MI. Furthermore, microRNA let-7 and Tgfbr3 may serve as therapeutic targets and biomarkers for myocardial damage. Fund: Ministry of Science and Technology, National Health Research Institutes, Academia Sinica Program for Translational Innovation of Biopharmaceutical Development-Technology Supporting Platform Axis, Thematic Research Program and the Summit Research Program, Taiwan. Keywords: MicroRNA let-7, TGFBR3, Gene therapy, Biomarkers

Introduction: Several environmental stimuli may influence lupus, particularly viral infections. In this study, we used an imiquimod-induced lupus mouse model focused on the TLR7 pathway and proteomics analysis to determine the specific pathway related to viral infection and the related protein expressions in splenic B cells to obtain insight into B-cell responses to viral infection in the lupus model. Materials and Methods: We treated FVB/N wild-type mice with imiquimod for 8 weeks to induce lupus symptoms and signs, retrieved splenocytes, selected B cells, and conducted the proteomic analysis. The B cells were co-cultured with CD40L+ feeder cells for another week before performing Western blot analysis. Panther pathway analysis was used to disclose the pathways activated and the protein–protein interactome was analyzed by the STRING database in this lupus murine model. Results: The lupus model was well established and well demonstrated with serology evidence and pathology proof of lupus-mimicking organ damage. Proteomics data of splenic B cells revealed that the most important activated pathways (fold enrichment > 100) demonstrated positive regulation of the MDA5 signaling pathway, negative regulation of IP-10 production, negative regulation of chemokine (C-X-C motif) ligand 2 production, and positive regulation of the RIG-I signaling pathway. A unique protein–protein interactome containing 10 genes was discovered, within which ISG15, IFIH1, IFIT1, DDX60, and DHX58 were demonstrated to be downstream effectors of MDA5 signaling. Finally, we found B-cell intracellular cytosolic proteins via Western blot experiment and continued to observe MDA5-related pathway activation. Conclusion: In this experiment, we confirmed that the B cells in the lupus murine model focusing on the TLR7 pathway were activated through the MDA5 signaling pathway, an important RNA sensor implicated in the detection of viral infections and autoimmunity. The MDA5 agonist/antagonist RNAs and the detailed molecular interactions within B cells are worthy of further investigation for lupus therapy.

Abstract Background Kawasaki disease (KD) is a prevalent pediatric disease worldwide and can cause coronary artery aneurysm as a severe complication. Typically, DNA methylation is thought to repress the expression of nearby genes. However, the cases in which DNA methylation promotes gene expression have been reported. In addition, globally, to what extent DNA methylation affects gene expression and how it contributes to the pathogenesis of KD are not yet well understood. Methods To address these important biological questions, we enrolled subjects, collected DNA and RNA samples from the subjects’ total white blood cells, and performed DNA methylation (M450K) and gene expression (HTA 2.0) microarray assays. Results By analyzing the variation ratios of CpG beta values (methylation percentage) and gene expression intensities, we first concluded that the CpG markers close (− 1500 bp to + 500 bp) to the transcription start sites had higher variation ratios, reflecting significant regulation capacities. Next, we observed that, globally speaking, gene expression was modestly negatively correlated (correlation rho ≈ − 0.2) with the DNA methylation status of both upstream and downstream CpG markers in the promoter region. Third, we found that specific CpG markers were hypo-methylated in disease samples compared with healthy samples and hyper-methylated in convalescent samples compared with disease samples, promoting and repressing S100A genes’ expressions, respectively. Finally, using an in vitro cell model, we demonstrated that S100A family proteins enhanced leukocyte transendothelial migration in KD. Conclusions This is the first study to integrate genome-wide DNA methylation with gene expression assays in KD and showed that the S100A family plays important roles in the pathogenesis of KD.

Abstract Background Kawasaki disease (KD) patients could develop coronary artery lesion (CAL) which threatens children's life. A previous study identified KD biomarker miRNAs that could discriminate KD patients from febrile non‐KD patients. We wonder whether these KD prediction biomarkers could be further applied to predict CAL formation in KD patients. Methods To examine this hypothesis, we conducted a meta‐analysis, miRNA mimic transfection, in vitro cell model and microarray assays. Results We first showed that miR‐182‐5p and miR‐183‐5p kept higher levels in the KD patients with CAL than those without CAL (p

Autoimmune encephalitis (AE) is a severe neurological disease. The brain of the AE patient is attacked by a dysregulated immune system, which is caused by the excessive production of autoantibodies against neuronal receptors and synaptic proteins. AE is also characterized by the uncontrolled B lymphocyte infiltration through the blood–brain barrier (BBB) layer, and the investigation of the underlying mechanism involved in this infiltration may facilitate the discovery of novel therapies for AE. However, few AE-related studies have focused on this issue. In this study, we aimed to identify the factors involved in B lymphocyte infiltration in AE. For this purpose, we first enrolled four healthy control and five AE subjects, collecting their serum and/or total white blood cell samples. The white blood cell samples were further used for collecting RNA and DNA. Then, we simulated the in vivo B lymphocyte infiltration with an in vitro leukocyte transendothelial migration model. It turned out that AE serum treatment significantly and specifically promoted B cells to penetrate the BBB endothelial layer without affecting neutrophils. Next, through genome-wide DNA methylation assays on bisulfite-conversion DNA samples, we identified S100A6 and S100A11 as potential hypo-methylated disease genes in the AE samples. Further qPCR assays demonstrated their upregulation in AE samples, reflecting the negative correlations between gene expression and DNA methylation. Finally, recombinant S100A6 protein treatment significantly increased B lymphocyte infiltration through the BBB endothelial layer, which partially recapitulated the effect of AE serum. In summary, by using an in vitro leukocyte transendothelial migration model, we confirmed that S100A6 promoted B lymphocyte to penetrate the BBB endothelial layer, which is similar to the in vivo clinical manifestations of AE. Therefore, further studies on how the S100A6 protein facilitates B lymphocyte infiltration and on whether other factors in serum also contribute to this phenomenon are likely to improve our understanding of AE and hopefully to reveal novel therapeutic targets for this emerging treatable neurological disorder.

A quick prediction method may help confirm the diagnosis of Kawasaki disease (KD), and reduce the risk of coronary artery lesions. The purpose of this study was to evaluate potential candidate diagnostic serum proteins in KD using isobaric tagging for relative and absolute quantification (iTRAQ) gel-free proteomics. Ninety two subjects, including 68 KD patients (1.6 ± 1.2 years, M/F 36/32) and 24 fever controls with evident respiratory tract infection (2.1 ± 1.2 years, M/F 13/11) were enrolled. Medical records were reviewed for demographic and laboratory data. The iTRAQ gel-free proteomics was used to screen serum proteins completely and compare the difference between two groups followed by specific validation with ELISA. The candidate proteins and conventional laboratory items were selected for the prediction model of KD diagnosis by support vector machine. Five selected candidate proteins, including protein S100-A8, protein S100-A9, protein S100-A12, neutrophil defensin 1, and alpha-1-acid glycoprotein 1 were identified for developing the prediction model of KD diagnosis. They were used to develop an efficient KD prediction model with an area under receiver operating characteristic (auROC) value of 0.92 (95% confidence interval: 0.84, 0.98). These protein biomarkers were significantly correlated with the conventional laboratory items as follows: C-reactive protein, glutamic pyruvic transaminase, white blood count, platelet, segment and hemoglobin. These conventional laboratory items were used to develop a prediction model of KD diagnosis with an auROC value of 0.88 (95% confidence interval: 0.80, 0.96). Our result demonstrated that the prediction model with combined five selected candidate protein levels may be a good diagnostic tool of KD. Further prediction model with combined six conventional laboratory data is also an acceptable alternative method for KD diagnosis.

Acanthamoeba castellanii is a free-living protozoan pathogen capable of causing a blinding keratitis and fatal granulomatous encephalitis. Current treatment generally involves an hourly application of polyhexamethylene biguanide (PHMB) over a period of several days but this is not entirely effective against all strains/isolates. The tolerance mechanisms of PHMB in Acanthamoeba cells remain unclear. In this study, we found that the mRNA expression level of disulfideisomerase domain containing protein (PDI) increased rapidly in surviving cells of the highly PHMB-tolerant Acanthamoeba castellanii strain, NCKH_D, during PHMB treatment, but not in the ATCC standard strain. After PDI-specific silencing, NCKH_D was found to be more vulnerable to PHMB treatment. The results described above show that PDI is an important gene for PHMB tolerance ability in a highly PHMB-tolerant strain of Acanthamoeba and provide a new insight for more efficient medicine development for Acanthamoeba keratitis.

Acanthamoeba act as hosts for various microorganisms and pathogens, causing Acanthamoeba Keratitis (AK). To investigate the association between endosymbionts and AK progression, we performed a metagenomics study to characterize the intracellular microbiome from five lenses associated with AK isolates and standard strains to characterize the role of ocular flora in AK progression. The used clinical isolates were axenic cultured from lenses associated with AK patients. AK isolates and standard controls such as 16S ribosomal RNA sequencing techniques were used for analysis. The microbiome compositions and relative abundance values were compared. The orders of Clostridiales and Bacteroidales presented major populations of intracellular microbes belonging to all isolates. Comparison of the different source isolates showed that most of the abundance in keratitis isolates came from Ruminococcus gnavus (121.0 folds), Eubacterium dolichum (54.15 folds), Roseburia faecis (24.51 folds), and Blautia producta (3.15 folds). Further analysis of the relative abundance data from keratitis isolates showed that Blautia producta was positively correlated with the disease course. In contrast, Bacteroides ovatus was found to be abundant in early-stage keratitis isolates. This study reveals the abundant anaerobic Gram-positive rods present in severe keratitis isolate and characterize the association between Acanthamoeba and ocular flora in AK progression.

DNA methylation levels are associated with neurodevelopment. Attention-deficit/hyperactivity disorder (ADHD), characterized by attention deficits, is a common neurodevelopmental disorder. We used methylation microarray and pyrosequencing to detect peripheral blood DNA methylation markers of ADHD. DNA methylation profiling data from the microarray assays identified potential differentially methylated CpG sites between 12 ADHD patients and 9 controls. Five candidate CpG sites (cg00446123, cg20513976, cg07922513, cg17096979, and cg02506324) in four genes (LIME1, KCNAB2, CAPN9, and SPTBN2) were further examined with pyrosequencing. The attention of patients were tested using the Conners’ Continuous Performance Test (CPT). In total, 126 ADHD patients with a mean age of 9.2 years (78.6% males) and 72 healthy control subjects with a mean age of 9.3 years (62.5% males) were recruited. When all participants were categorized by their CPT performance, the DNA methylation levels in LIME1 (cg00446123 and cg20513976) were found to be significantly higher and those in SPTBN2 (cg02506324) were significantly lower in children with worse CPT performance. Therefore, DNA methylation of two CpG sites in LIME1 and one CpG site in SPTBN2 is associated with attention deficits in children. DNA methylation biomarkers may assist in identifying attention deficits of children in clinical settings.

Kawasaki disease (KD) is the most common cause of heart disease acquired in childhood. Even if treated with high-dose intravenous immunoglobulin G (IVIG) at the early stage; children are still at risk of developing coronary artery lesions. Accumulating evidence suggests that autophagy is enhanced in various heart diseases. Evaluating the pathogenic role of autophagy in KD and coronary artery lesions (CAL) may aid in identifying a potential therapeutic target for the treatment or prevention of the disease. Blood samples were obtained from 20 children with KD at the onset of disease and 21 days after IVIG therapy. Twenty children with other causes of febrile disease and 20 healthy children were included as controls. Total RNA was extracted from white blood cells; and autophagy-related gene mRNA expression levels were measured using real-time polymerase chain reaction. The patients with KD had downregulated levels of LC3B mRNA (0.50 ± 0.06 vs. 1.67 ± 0.15; p < 0.001), BECN1 mRNA (0.70 ± 0.08 vs. 1.43 ± 0.23; p < 0.05), and ATG16L1 mRNA (0.28 ± 0.04 vs. 0.96 ± 0.16; p < 0.01) compared to the febrile control group. The values of these parameters all increased significantly 21 days after the IVIG therapy as follows: LC3B mRNA (1.77 ± 0.29 vs. 0.50 ± 0.06; p < 0.001), BECN1 mRNA (1.67 ± 0.36 vs. 0.70 ± 0.08; p < 0.05), and ATG16L1 mRNA (2.96 ± 0.43 vs. 0.28 ± 0.04; p < 0.001), while the level of ATG16L1 mRNA persists low in KD patients with CAL. Our results showed the autophagy-related genes expressions in KD and their change after IVIG administration. This suggests that autophagy may have a protective effect on KD.

Cognitive impairment (CI) is not uncommon in dialysis patients. Various factors have been implicated. This study aims to examine mutual interaction of various clinical factors for CI in patients receiving hemodialysis. A total of 48 hemodialysis patients in outpatient clinic were recruited from 2015 to 2017. Demographics, circulating uremic toxin concentrations, miRNA concentrations, and nerve injury protein concentrations were collected. Clinical dementia rating (CDR) scores were used to stratify the functional scores of the patients. Receiver operating characteristic (ROC) analysis was used to evaluate diagnostic test performance for predicting dichotomous results, and cumulative ROC analysis was used to examine the combined contribution of clinical factors. CDR scale 0 included 15 patients (mean age, 59.1 years); CDR > 0.5 included 33 patients (mean age, 64.0 years). On cumulative ROC analysis, the major predictors of mild CI were hemoglobin, age, sex, homocysteine, neuron-specific enolase (NSE), and miR-486. The cumulative area under the curve (AUC) on combining hemoglobin, age, and miR-486 was the highest (0.897, 95% confidence interval 0.806–0.988). Two dichotomized variables reached 81.82% sensitivity and 86.67% specificity, with the likelihood ratio for positive and negative results being 6.14 and 0.21, respectively. In conclusion, hemoglobin, age, and miR-486 display high-degree combined effects on mild CI in patients receiving hemodialysis.

Background: Attention-deficit/hyperactivity disorder (ADHD) is a highly genetic neurodevelopmental disorder, and its dysregulation of gene expression involves microRNAs (miRNAs). The purpose of this study was to identify potential miRNAs biomarkers and then use these biomarkers to establish a diagnostic panel for ADHD.Design and methods: RNA samples from white blood cells (WBCs) of five ADHD patients and five healthy controls were combined to create one pooled patient library and one control library. We identified 20 candidate miRNAs with the next-generation sequencing (NGS) technique (Illumina). Blood samples were then collected from a Training Set (68 patients and 54 controls) and a Testing Set (20 patients and 20 controls) to identify the expression profiles of these miRNAs with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). We used receiver operating characteristic (ROC) curves and the area under the curve (AUC) to evaluate both the specificity and sensitivity of the probability score yielded by the support vector machine (SVM) model.Results: We identified 13 miRNAs as potential ADHD biomarkers. The ΔCt values of these miRNAs in the Training Set were integrated to create a biomarker model using the SVM algorithm, which demonstrated good validity in differentiating ADHD patients from control subjects (sensitivity: 86.8%, specificity: 88.9%, AUC: 0.94, p < 0.001). The results of the blind testing showed that 85% of the subjects in the Testing Set were correctly classified using the SVM model alignment (AUC: 0.91, p < 0.001). The discriminative validity is not influenced by patients' age or gender, indicating both the robustness and the reliability of the SVM classification model.Conclusion: As measured in peripheral blood, miRNA-based biomarkers can aid in the differentiation of ADHD in clinical settings. Additional studies are needed in the future to clarify the ADHD-associated gene functions and biological mechanisms modulated by miRNAs.

DNA and RNA samples from blood are the common examination target for non-invasive physical tests and/or biomedical studies. Since high-quality DNA and RNA samples guarantee the correctness of these tests and/or studies, we investigated the effects of storage temperature and storage duration of whole blood on DNA and RNA qualities. Subjects were enrolled to donate blood samples which were stored for different durations and at different temperatures, followed by the examinations on RNA quality, qPCR, DNA quality and DNA methylation. For RNA, we observed obvious quality decline with storage duration longer than 24 hours. Storage at low temperature does not keep RNA samples from degradation. And, storing whole blood samples in freezer dramatically damage RNA. For DNA, quality decline was not observed even with storage duration for 15 days. However, DNA methylation significantly altered with storage duration longer than three days. Storage duration within 24 hours is critical for collecting high-quality RNA samples for next-generation sequencing (NGS) assays (RIN≧8). If microarray assays are expected (RIN≧7), storage duration within 32 hours is acceptable. Although DNA is resistant within 15 days when kept in whole blood, DNA quantity dramatically decreases owing to WBC lysis. In addition, duration for more than three days significantly alter DNA methylation status, globally and locally. Our result provides a reference for dealing with blood samples.

Diversity of biological molecules in newborn and adult immune cells contributes to differences in cell function and atopic properties. Micro RNAs (miRNAs) are reported involve in the regulation of immune system. Therefore, determining the miRNA expression profile of leukocyte sub-populations is important for understanding immune system regulation. In order to explore the unique microRNA profiling that contribute to altered immune in neonates, we comprehensively analyzed the functional miRNA signatures of eight leukocyte subsets (polymorphonuclear cells, monocytes, CD4+ T cells, CD8+ T cells, natural killer cells, B cells, plasmacytoid dendritic cells (pDCs), and myeloid dendritic cells (mDCs)) from both neonatal and adult umbilical cord and peripheral blood samples, respectively. We observed distinct miRNA profiles between adult and neonatal blood leukocyte subsets, including unique miRNA signatures for each cell lineage. Leukocyte miRNA signatures were altered after stimulation. Adult peripheral leukocytes had higher let-7b-5p expression levels compared to neonatal cord leukocytes across multiple subsets, irrespective of stimulation. Transfecting neonatal monocytes with a let-7b-5p mimic resulted in a reduction of LPS-induced IL-6 and TNF-alpha production, while transfection of a let-7b-5p inhibitor into adult monocytes enhanced IL-6 and TNF-alpha production. With this functional approach, we provide intact differential microRNA expression profiling of specific immune cell subsets between neonates and adults. These studies serve as a basis to further understand the altered immune response observed in neonates and advance the development of therapeutic strategies.

A form of systemic vasculitis, Kawasaki disease (KD) occurs most frequently in children under the age of five years old. Previous studies have found that Prostaglandin E2 (PGE2) correlates with KD, although the related mechanisms are still unknown. CD40L may also be a marker of vasculitis in KD, so this study focuses on PGE2 and CD40L expression in KD.This study consisted of a total of 144 KD patients, whose intravenous immunoglobulin (IVIG)/coronary arterial lesion (CAL) formation resistance was evaluated. PGE2 levels were evaluated in vitro to study the effect of CD40L on CD4+ T lymphocytes.PGE2 levels significantly increased after IVIG treatment (p

Kawasaki disease (KD) or Kawasaki syndrome is known as a vasculitis of small to medium-sized vessels, and coronary arteries are predominantly involved in childhood. Generally, 20-25% of untreated with IVIG and 3-5% of treated KD patients have been developed coronary artery lesions (CALs), such as dilatation and aneurysm. Understanding how coronary artery aneurysms (CAAs) are established and maintained in KD patients is therefore of great importance. Upon our previous genotyping data of 157 valid KD subjects, a genome-wide association study (GWAS) has been conducted among 11 (7%) CAA-developed KD patients to reveal five significant genetic variants passed pre-defined thresholds and resulted in two novel susceptibility protein-coding genes, which are NEBL (rs16921209 (P = 7.44 × 10(-9); OR = 32.22) and rs7922552 (P = 8.43 × 10(-9); OR = 32.0)) and TUBA3C (rs17076896 (P = 8.04 × 10(-9); OR = 21.03)). Their known functions have been reported to associate with cardiac muscle and tubulin, respectively. As a result, this might imply their putative roles of establishing CAAs during KD progression. Additionally, various model analyses have been utilized to determine dominant and recessive inheritance patterns of identified susceptibility mutations. Finally, all susceptibility genes hit by significant genetic variants were further investigated and the top three representative gene-ontology (GO) clusters were regulation of cell projection organization, neuron recognition, and peptidyl-threonine phosphorylation. Our results help to depict the potential routes of the pathogenesis of CAAs in KD patients and will facilitate researchers to improve the diagnosis and prognosis of KD in personalized medicine.

Kawasaki disease (KD) is a systemic vasculitis primarily affecting children < 5 years old. Genes significantly associated with KD mostly involve cardiovascular, immune, and inflammatory responses. Recent studies have observed stronger associations for KD risk with multiple genes compared to individual genes. Therefore, we investigated whether gene combinations influenced KD susceptibility or coronary artery lesion (CAL) formation. We examined 384 single-nucleotide polymorphisms (SNPs) for 159 immune-related candidate genes in DNA samples from KD patients with CAL (n = 73), KD patients without CAL (n = 153), and cohort controls (n = 575). Individual SNPs were first assessed by univariate analysis (UVA) and multivariate analysis (MVA). We used multifactor dimensionality reduction (MDR) to examine individual SNPs in one-, two-, and three-locus best fit models. UVA identified 53 individual SNPs that were significantly associated with KD risk or CAL formation (p < 0.10), while 35 individual SNPs were significantly associated using MVA (p ≤ 0.05). Significant associations in MDR analysis were only observed for the two-locus models after permutation testing (p ≤ 0.05). In logistic regression, combined possession of PDE2A (rs341058) and CYFIP2 (rs767007) significantly increased KD susceptibility (OR = 3.54; p = 4.14 x 10(-7)), while combinations of LOC100133214 (rs2517892) and IL2RA (rs3118470) significantly increased the risk of CAL in KD patients (OR = 5.35; p = 7.46 x 10(-5)). Our results suggest varying gene-gene associations respectively predispose individuals to KD risk or its complications of CAL.

MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species.We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5' and/or 3' ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis.Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms.