Circadian Rhythm Disruption in Hepatocellular Carcinoma Investigated by Integrated Analysis of Bulk and Single-Cell RNA Sequencing Data.

Circadian rhythms are essential regulators of a multitude of physiological and behavioral processes, such as the metabolism and function of the liver. Circadian rhythms are crucial to liver homeostasis, as the liver is a key metabolic organ accountable for the systemic equilibrium of the body. Circadian rhythm disruption alone is sufficient to cause liver cancer through the maintenance of hepatic metabolic disorder. Although there is evidence linking CRD to hepatocarcinogenesis, the precise cellular and molecular mechanisms that underlie the circadian crosstalk that leads to hepatocellular carcinoma remain unknown. The expression of CRD-related genes in HCC was investigated in this study via bulk RNA transcriptomic analysis and single-cell sequencing. Dysregulated CRD-related genes are predominantly found in hepatocytes and fibroblasts, according to the findings. By using a combination of single-cell RNA sequencing and bulk RNA sequencing analyses, the dysregulated CRD-related genes ADAMTS13, BIRC5, IGFBP3, MARCO, MT2A, NNMT, and PGLYRP2 were identified. The survival analysis using the Kaplan-Meier method revealed a significant correlation between the expression levels of BIRC5 and IGFBP3 and the survival of patients diagnosed with HCC.

Circulating Exosomes from Septic Mice Activate NF-κB/MIR17HG Pathway in Macrophages.

Circulating exosomes derived from polymicrobial sepsis contain various non-coding RNAs and proteins. Isobaric tags for a relative or absolute quantitation proteomic analysis of the exosomal content revealed 70 dysregulated proteins in the circulating exosomes from septic mice. Next-generation sequencing was used to profile the long non-coding RNA expression in primary cultured macrophages treated with exosomes obtained from the blood of septic C57BL/6 mice, and it was discovered that the nuclear factor-kappa B (NF-κB)/miR-17-92a-1 cluster host gene (MIR17HG) pathways were activated in the macrophages. The inhibition of MIR17HG expression by RNA interference resulted in significantly decreased cell viability. RNA pull-down assays of MIR17HG revealed that ten protein targets bind to MIR17HG. Interaction networks of proteins pulled down by MIR17HG were constructed using GeneMANIA, and their functions were mainly involved in ribonucleoprotein granules, type I interferons, the regulation of organelle assembly, the biosynthesis of acetyl coenzyme A, as a signal transducer and activator of transcription (STAT) protein phosphorylation, and mRNA splicing. Furthermore, RNA interference inhibited MIR17HG expression, resulting in significantly decreased cell survival. In conclusion, this work discovered considerable MIR17HG overexpression in macrophages treated with circulating exosomes from sepsis-affected animals. This study's findings assist us in comprehending the role of exosomes in modulating inflammatory responses and mediating pathogenic pathways in macrophages during sepsis.

Cathelicidin Antimicrobial Peptide Acts as a Tumor Suppressor in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is associated with high rates of metastasis and recurrence, and is one of the most common causes of cancer-associated death worldwide. This study examined the protein changes within circulating exosomes in patients with HCC against those in healthy people using isobaric tags for a relative or absolute quantitation (iTRAQ)-based quantitative proteomics analysis. The protein levels of von Willebrand factor (VWF), cathelicidin antimicrobial peptide (CAMP), and proteasome subunit beta type-2 (PSMB2) were altered in HCC. The increased levels of VWF and PSMB2 but decreased CAMP levels in the serum of patients with HCC were validated by enzyme-linked immunosorbent assays. The level of CAMP (the only cathelicidin found in humans) also decreased in the circulating exosomes and buffy coat of the HCC patients. The serum with reduced levels of CAMP protein in the HCC patients increased the cell proliferation of Huh-7 cells; this effect was reduced following the addition of CAMP protein. The depletion of CAMP proteins in the serum of healthy people enhances the cell proliferation of Huh-7 cells. In addition, supplementation with synthetic CAMP reduces cell proliferation in a dose-dependent manner and significantly delays G1-S transition in Huh-7 cells. This implies that CAMP may act as a tumor suppressor in HCC.

Exploring the Regulatory Role of XIST-microRNAs/mRNA Network in Circulating CD4+ T Cells of Hepatocellular Carcinoma Patients.

Hepatocellular carcinoma (HCC) is one of the most common cancers and the main cause of cancer-related death globally. Immune dysregulation of CD4+ T cells has been identified to play a role in the development of HCC. Nevertheless, the underlying molecular pathways of CD4+ T cells in HCC are not completely known. Thus, a better understanding of the dysregulation of the lncRNA-miRNA/mRNA network may yield novel insights into the etiology or progression of HCC. In this study, circulating CD4+ T cells were isolated from the whole blood of 10 healthy controls and 10 HCC patients for the next-generation sequencing of the expression of lncRNAs, miRNAs, and mRNAs. Our data showed that there were different expressions of 34 transcripts (2 lncRNAs, XISTs, and MIR222HGs; 29 mRNAs; and 3 other types of RNA) and 13 miRNAs in the circulating CD4+ T cells of HCC patients. The expression of lncRNA-XIST-related miRNAs and their target mRNAs was confirmed using real-time quantitative polymerase chain reaction (qPCR) on samples from 100 healthy controls and 60 HCC patients. The lncRNA-miRNA/mRNA regulation network was created using interaction data generated from ENCORI and revealed there are positive correlations in the infiltration of total CD4+ T cells, particularly resting memory CD4+ T cells, and negative correlations in the infiltration of Th1 CD4+ T cells.

iTRAQ Proteomics Identified the Potential Biomarkers of Coronary Artery Lesion in Kawasaki Disease and In Vitro Studies Demonstrated That S100A4 Treatment Made HCAECs More Susceptible to Neutrophil Infiltration.

Coronary artery lesions (CAL) are a major complication of Kawasaki disease (KD). The early prediction of CAL enables the medical personnel to apply adequate medical intervention. We collected the serum samples from the KD patients with CAL (n = 32) and those without CAL (n = 31), followed by a global screening with isobaric tagging for relative and absolute quantification (iTRAQ) technology and specific validation with an enzyme-linked immunosorbent assay (ELISA). iTRAQ identified 846 proteins in total in the serum samples, and four candidate proteins related to CAL were selected for ELISA validation as follows: Protein S100-A4 (S100A4), Catalase (CAT), Folate receptor gamma (FOLR3), and Galectin 10 (CLC). ELISA validation showed that the S100A4 level was significantly higher in KD patients with CAL than in those without CAL (225.2 ± 209.5 vs. 143.3 ± 83 pg/mL, p < 0.05). In addition, KD patients with CAL had a significantly lower CAT level than those without CAL (1.6 ± 1.5 vs. 2.7 ± 2.3 ng/mL, p < 0.05). Next, we found that S100A4 treatment on human coronary artery endothelial cells (HCAECs) reduced the abundance of cell junction proteins, which promoted the migration of HCAECs. Further assays also demonstrated that S100A4 treatment enhanced the permeability of the endothelial layer. These results concluded that S100A4 treatment resulted in an incompact endothelial layer and made HCAECs more susceptible to in vitro neutrophil infiltration. In addition, both upregulated S100A4 and downregulated CAT increased the risk of CAL in KD. Further in vitro study implied that S100A4 could be a potential therapeutic target for CAL in KD.

A new technique for separating platelet-rich plasma by a copolymer device － without a centrifugation process.

BACKGROUND: Platelet-rich plasma (PRP) is beneficial for clinical applications in various medical fields. Although commercial PRP preparation kits are already available in the market, most of these kits employ centrifugation. METHODS: We used a new cationic copolymer coating on a polyurethane (PU) sponge to promote platelet separation from the blood. This copolymer showed no cytotoxicity against cell viability or hemolysis. We further evaluated the efficiency of the new PRP preparation device by comparing it with that of a commercially available kit (RegenKit-THT). RESULTS: We demonstrated that PRP obtained using copolymer device contains high concentrations of platelets and angiogenic growth factors (epidermal growth factor, vascular endothelial growth factor-A, growth differentiation factor 2, and interleukin-8). The separated PRP also displayed beneficial effects on cell migration, angiogenesis, and matrix metalloproteinase gene expression. CONCLUSION: Based on these results, we developed a cationic copolymer-coated PU sponge as a PRP preparation device without the need for any centrifugation.

MicroRNAs serve as prediction and treatment-response biomarkers of attention-deficit/hyperactivity disorder and promote the differentiation of neuronal cells by repressing the apoptosis pathway.

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.

Gut microbiota and plasma cytokine levels in patients with attention-deficit/hyperactivity disorder.

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-1ß, 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 ß-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.

Recovery of Dysregulated Genes in Cancer-Related Lower Limb Lymphedema After Supermicrosurgical Lymphaticovenous Anastomosis - A Prospective Longitudinal Cohort Study.

PURPOSE: This study aims at profiling the expression of dysregulated genes in circulating monocytes of patients with cancer-related lower limb lymphedema before and after treatment with supermicrosurgical lymphaticovenous anastomosis (LVA). MATERIALS AND METHODS: This prospective longitudinal cohort study enrolled 51 women with post-treatment gynecological cancer, including those with unilateral lymphedema (study group, n = 25) and those without (control group, n = 26). Venous blood samples obtained from the study group before and after LVA and those from the controls were sent for next-generation sequencing, which was validated by real-time PCR. Dysregulated gene expression in the study group, relative to expression in the controls, was recorded before LVA. After one month, postoperative changes in the expression of the identified genes were evaluated. Protein-protein interaction (PPI) was used to investigate dysregulated genes whose expression returned to baseline levels after LVA. RESULTS: Of the 148 preoperative dysregulated genes, which comprised 108 up- and 40 down-regulated genes, 78 genes, consisting of 69 up- and 9 down-regulated genes, showed post-LVA recovery to baseline levels. Through PPI analysis, five functional modules involving immunity, lipid metabolism, oxidative stress, transcriptional regulators, and tumor suppression, as well as six hub genes (CCL2, LPL, PDK4, FOXO3, EGR1, and DUSP5), were identified. Cross-linking and co-regulated genes between modules were also identified. CONCLUSION: Localized lymphedema leads to dysregulated gene expression in circulating monocytes. The current study is the first to identify the hub genes related to lymphedema and demonstrate the recovery of some dysregulated genes after LVA.

BST-1 as a serum protein biomarker involved in neutrophil infiltration in schizophrenia.

OBJECTIVES: Schizophrenia is a serious mental illness. The serum protein biomarkers of schizophrenia were explored using isobaric tags for relative and absolute quantitation (iTRAQ) technology. The underlying function of the identified protein biomarker was also investigated. METHODS: We first collected serum samples from 12 schizophrenia patients and 12 healthy control (HC) subjects, followed by global screening with iTRAQ and tandem mass spectrometry (LC-MS/MS). In total, 691 serum proteins were detected and eight proteins, including ZYX, OSCAR, TPM4, SDPR, BST1, ARGHDB, ITIH5 and SH3BGRL3, were selected for further specific validation with enzyme-linked immunosorbent assay (ELISA) on the serum samples from 52 schizophrenia patients and 50 HC subjects. RESULTS: Schizophrenia patients had significantly lower serum level of BST1 and higher ITIH5 level than the HC subjects did. Using the levels of BST1, ITIH5 and OSCAR combined with machine learning algorithm, we developed a prediction model of schizophrenia with an auROC value 0.78. Moreover, in vitro cell assay confirmed that BST1 significantly repressed neutrophil infiltration through endothelial layer, highlighted the anti-inflammation nature of BST1. CONCLUSIONS: Four novel protein markers (BST1, ITIH5, SDPR, and OSCAR) of schizophrenia were identified, and BST-1 could serve as a serum protein biomarker involved in neutrophil infiltration in schizophrenia.

Exosomes Secreted by Adipose-Derived Stem Cells Following FK506 Stimulation Reduce Autophagy of Macrophages in Spine after Nerve Crush Injury.

Macrophages emerge in the milieu around innervated neurons after nerve injuries. Following nerve injury, autophagy is induced in macrophages and affects the regulation of inflammatory responses. It is closely linked to neuroinflammation, while the immunosuppressive drug tacrolimus (FK506) enhances nerve regeneration following nerve crush injury and nerve allotransplantation with additional neuroprotective and neurotrophic functions. The combined use of FK506 and adipose-derived stem cells (ADSCs) was employed in cell therapy for organ transplantation and vascularized composite allotransplantation. This study aimed to investigate the topical application of exosomes secreted by ADSCs following FK506 treatment (ADSC-F-exo) to the injured nerve in a mouse model of sciatic nerve crush injury. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile the potential exosomal proteins involved in autophagy. Immunohistochemical analysis revealed that nerve crush injuries significantly induced autophagy in the dorsal root ganglia and dorsal horn of the spinal segments. Locally applied ADSC-F-exo significantly reduced autophagy of macrophages in the spinal segments after nerve crush injury. Proteomic analysis showed that of the 22 abundant exosomal proteins detected in ADSC-F-exo, heat shock protein family A member 8 (HSPA8) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) are involved in exosome-mediated autophagy reduction.

Disparity of Hepatocellular Carcinoma in Tumor Microenvironment-Related Genes and Infiltrating Immune Cells between Asian and Non-Asian Populations.

Hepatocellular carcinoma (HCC) is the most common cause of primary liver cancer deaths worldwide. The major risk factors for liver cancer development are cirrhosis, hepatitis B virus (HBV), hepatitis C virus (HCV) infection, and chronic alcohol abuse. HCC displays heterogeneity in terms of biology, etiology, and epidemiology. In Southeast Asia and Africa, chronic HBV infection is a major risk factor for HCC, whereas chronic HCV infection is a risk factor for HCC in western countries and Japan. Environmental and genetic conditions also play a role in the regional and temporal variations in the incidence of HCC. In this study, we used the ESTIMATE (ESTIMATE, Estimation of stromal and immune cells in malignant tumor tissues using expression data) algorithm and the CIBERSOFT tool to analyze gene expression profiles and infiltrating immune cells in HCC between Asian and non-Asian patients. The results showed that stromal and immune scores were dependent on overall survival (OS) in non-Asian patients but not in Asian patients. Kaplan-Meier survival analysis revealed four differentially expressed genes (DEGs) that were significantly associated with OS in non-Asian patients only. CIBERSORT (CIBERSORT, Cell type identification by estimating relative subsets of known RNA transcripts) analysis indicated that the composition of infiltrating immune cells was significantly different between Asian and non-Asian patients. By parsing the subclasses of HCC, the ability to predict prognosis and guide therapeutic targets for potentially actionable HCC may be improved.

Enhanced Nerve Regeneration by Exosomes Secreted by Adipose-Derived Stem Cells with or without FK506 Stimulation.

Exosomes secreted by adipose-derived stem cells (ADSC-exo) reportedly improve nerve regeneration after peripheral nerve injury. Herein, we investigated whether pretreatment of ADSCs with FK506, an immunosuppressive drug that enhances nerve regeneration, could secret exosomes (ADSC-F-exo) that further augment nerve regeneration. Designed exosomes were topically applied to injured nerve in a mouse model of sciatic nerve crush injury to assess the nerve regeneration efficacy. Outcomes were determined by histomorphometric analysis of semi-thin nerve sections stained with toluidine blue, mouse neurogenesis PCR array, and neurotrophin expression in distal nerve segments. Isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile potential exosomal proteins facilitating nerve regeneration. We observed that locally applied ADSC-exo and ADSC-F-exo significantly enhanced nerve regeneration after nerve crush injury. Pretreatment of ADSCs with FK506 failed to produce exosomes possessing more potent molecules for enhanced nerve regeneration. Proteomic analysis revealed that of 192 exosomal proteins detected in both ADSC-exo and ADSC-F-exo, histone deacetylases (HDACs), amyloid-beta A4 protein (APP), and integrin beta-1 (ITGB1) might be involved in enhancing nerve regeneration.

Increased Angiogenesis by Exosomes Secreted by Adipose-Derived Stem Cells upon Lipopolysaccharide Stimulation.

Exosomes secreted by adipose-derived stem cells (ADSCs) enhance angiogenesis and wound healing. However, in clinical settings, wounds may be infected by various bacteria or pathogens. We investigated whether human ADSCs stimulated with lipopolysaccharide (LPS) secrete exosomes (ADSC-LPS-exo) that augment the angiogenesis of human umbilical vein endothelial cells (HUVECs). ExoQuick-TC exosome precipitation solution was used to purify exosomes from human ADSC culture media in the presence or absence of 1 µg/mL LPS treatment for 24 h. The uptake of ADSC-LPS-exo significantly induced the activation of cAMP response element binding protein (CREB), activating protein 1 (AP-1), and nuclear factor-κB (NF-κB) signaling pathways and increased the migration of and tube formation in HUVECs. RNA interference with CREB, AP-1, or NF-κB1 significantly reduced the migration of and tube formation in HUVECs treated with ADSC-LPS-exo. An experiment with an antibody array for 25 angiogenesis-related proteins revealed that only interleukin-8 expression was significantly upregulated in HUVECs treated with ADSC-LPS-exo. In addition, proteomic analysis revealed that eukaryotic translation initiation factor 4E, amyloid beta A4 protein, integrin beta-1, and ras-related C3 botulinum toxin substrate 1 may be potential candidates involved in ADSC-LPS-exo-mediated enhanced angiogenesis.

Identification and characterization of hADSC-derived exosome proteins from different isolation methods.

Exosomes are secreted into the extracellular space by most cell types and contain various molecular constituents, which play roles in many biological processes. Adipose-derived mesenchymal stem cells (ADSCs) can differentiate into a variety of cell types and secrete a series of paracrine factors through exosomes. ADSC-derived exosomes have shown diagnostic and therapeutic potential in many clinical diseases. The molecular components are critical for their mechanisms. Several methods have been developed for exosome purification, including ultracentrifugation, ultrafiltration, density gradient purification, size-based isolation, polymer precipitation and immuno-affinity purification. Thus, we employed four methods to isolate exosomes from the hADSC culture medium, including ultracentrifugation, size exclusion chromatography, ExoQuick-TC precipitation and ExoQuick-TC ULTRA isolation. Following exosome isolation, we performed quantitative proteomic analysis of the exosome proteins using isobaric tags for relative and absolute quantification (iTRAQ) labelling, combined with 2D-LC-MS/MS. There were 599 universal and 138 stably expressed proteins in hADSC-derived exosomes. We proved that these proteins were potential hADSC-derived exosomes markers, including CD109, CD166, HSPA4, TRAP1, RAB2A, RAB11B and RAB14. From the quantitative proteomic analysis, we demonstrated that hADSC-derived exosome protein expression varied, with lipopolysaccharide (LPS) treatment, in the different isolation methods. Pathway analysis and proliferation, migration and endothelial tube formation assays showed varying effects in cells stimulated with hADSC-derived exosomes from different isolation methods. Our study revealed that different isolation methods might introduce variations in the protein composition in exosomes, which reflects their effects on biological function. The pros and cons of these methods are important points to consider for downstream research applications.

Lymphaticovenous Anastomosis Supermicrosurgery Decreases Oxidative Stress and Increases Antioxidant Capacity in the Serum of Lymphedema Patients.

BACKGROUND: Excess lymphedematous tissue causes excessive oxidative stress in lymphedema. Lymphaticovenous anastomosis (LVA) supermicrosurgery is currently emerging as the first-line surgical intervention for lymphedema. No data are available regarding the changes in serum proteins correlating to oxidative stress and antioxidant capacity before and after LVA. METHODS: A total of 26 patients with unilateral lower limb lymphedema confirmed by lymphoscintigraphy were recruited, and venous serum samples were collected before (pre-LVA) and after LVA (post-LVA). In 16 patients, the serum proteins were identified by isobaric tags for relative and absolute quantitation-based quantitative proteomic analysis with subsequent validation of protein expression by enzyme-linked immunosorbent assay. An Oxidative Stress Panel Kit was used on an additional 10 patients. Magnetic resonance (MR) volumetry was used to measure t limb volume six months after LVA. RESULTS: This study identified that catalase (CAT) was significantly downregulated after LVA (pre-LVA vs. post-LVA, 2651 ± 2101 vs. 1448 ± 593 ng/mL, respectively, p = 0.033). There were significantly higher levels of post-LVA serum total antioxidant capacity (pre-LVA vs. post-LVA, 441 ± 81 vs. 488 ± 59 µmole/L, respectively, p = 0.031) and glutathione peroxidase (pre-LVA vs. post-LVA, 73 ± 20 vs. 92 ± 29 U/g, respectively, p = 0.018) than pre-LVA serum. In addition, after LVA, there were significantly more differences between post-LVA and pre-LVA serum levels of CAT (good outcome vs. fair outcome, -2593 ± 2363 vs. 178 ± 603 ng/mL, respectively, p = 0.021) and peroxiredoxin-2 (PRDX2) (good outcome vs. fair outcome, -7782 ± 7347 vs. -397 ± 1235 pg/mL, respectively, p = 0.037) in those patients with good outcomes (≥40% volume reduction in MR volumetry) than those with fair outcomes (<40% volume reduction in MR volumetry). CONCLUSIONS: The study revealed that following LVA, differences in some specific oxidative stress markers and antioxidant capacity can be found in the serum of patients with lymphedema.

ArfGAP1 acts as a GTPase-activating protein for human ADP-ribosylation factor-like 1 protein.

ADP-ribosylation factors (Arfs) and Arf-like (Arl) GTPases are key regulators of intracellular vesicle trafficking and Golgi structure. Both Arf and Arl proteins cycle between active GTP-bound and inactive GDP-bound forms, where guanine nucleotide exchange factors (GEFs) regulate the exchange of GDP for GTP, whereas GTPase-activating proteins (GAPs) promote the hydrolysis of bound GTP. Human Arl1 is located at the trans-Golgi network (TGN) and regulates the function and structure of the Golgi complex. However, neither GEFs nor GAPs for human Arl1 have been identified. Here, we report that ArfGAP1, an Arf1 GAP, can promote GTP hydrolysis of Arl1. We show that ArfGAP1 directly interacts with GTP-bound Arl1 and exhibits GAP activity toward Arl1 in vitro. Exogenous expression of ArfGAP1, but not ArfGAP2 and ArfGAP3, causes dissociation of endogenous Arl1 from the TGN. In addition, GAP activity-deficient ArfGAP1 fails to regulate the Golgi localization of Arl1. Using an activity pull-down assay, we demonstrated that ArfGAP1 regulates the levels of Arl1-GTP in cells expressing ArfGAP1-myc or with ArfGAP1 knockdown. Finally, we observed that, similar to expression of putative active Arl1 (Arl1QL), ArfGAP1 knockdown impairs endosome-to-TGN retrograde transport of the Shiga toxin B-subunit. Thus, our findings support the idea that ArfGAP1 acts as an Arl1 GAP to regulate the function of Arl1 in vesicle trafficking at the TGN.

DNA Methylation in LIME1 and SPTBN2 Genes Is Associated with Attention Deficit in Children.

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.

Serum proteins may facilitate the identification of Kawasaki disease and promote in vitro neutrophil infiltration.

Kawasaki disease (KD) usually affects the children younger than 5 years of age and subsequently causes coronary artery lesions (CALs) without timely identification and treatment. Developing a robust and fast prediction method may facilitate the timely diagnosis of KD, significantly reducing the risk of CALs in KD patients. The levels of inflammatory serum proteins dramatically vary during the onsets of many immune diseases, including in KD. However, our understanding of their pathogenic roles in KD is behind satisfaction. The purpose of this study was to evaluate candidate diagnostic serum proteins and the potential mechanism in KD using iTRAQ gel-free proteomics. We enrolled subjects and conducted iTRAQ gel-free proteomics to globally screen serum proteins followed by specific validation with ELISA. Further in vitro leukocyte trans-endothelial model was also applied to investigate the pathogenesis roles of inflammatory serum proteins. We identified six KD protein biomarkers, including Protein S100-A8 (S100A8), Protein S100-A9 (S100A9), Protein S100-A12 (S100A12), Peroxiredoxin-2 (PRDX2), Neutrophil defensin 1 (DEFA1) and Alpha-1-acid glycoprotein 1 (ORM1). They enabled us to develop a high-performance KD prediction model with an auROC value of 0.94, facilitating the timely identification of KD. Further assays concluded that recombinant S100A12 protein treatment activated neutrophil surface adhesion molecules responsible for adhesion to endothelial cells. Therefore, S100A12 promoted both freshly clinically isolated neutrophils and neutrophil-like cells to infiltrate through the endothelial layer in vitro. Finally, the antibody against S100A12 may attenuate the infiltration promoted by S100A12. Our result demonstrated that evaluating S100A8, S100A9, S100A12, PRDX2, DEFA1 and ORM1 levels may be a good diagnostic tool of KD. Further in vitro study implied that S100A12 could be a potential therapeutic target for KD.

Gut microbiota and dietary patterns in children with attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder, but the underlying pathophysiological mechanisms of ADHD remain unclear. Gut microbiota has been recognized to influence brain function and behaviors. Therefore, this study aimed to determine whether imbalanced gut microbiomes identified by a 16S rRNA sequencing approach are involved in the pathophysiology of ADHD. We recruited a total of 30 children with ADHD (mean age: 8.4 years) and a total of 30 healthy controls (mean age: 9.3 years) for this study. The dietary patterns of all participants were assessed with the food frequency questionnaire. The microbiota of fecal samples were investigated using 16S rRNA V3V4 amplicon sequencing, followed by bioinformatics and statistical analyses. We found that the gut microbiota communities in ADHD patients showed a significantly higher Shannon index and Chao index than the control subjects. Furthermore, the linear discriminant analysis effect size (LEfSe) analysis was used to identify differentially enriched bacteria between ADHD patients and healthy controls. The relative abundance of Bacteroides coprocola (B. coprocola) was decreased, while the relative abundance of Bacteroides uniformis (B. uniformis), Bacteroides ovatus (B. ovatus), and Sutterella stercoricanis (S. stercoricanis) were increased in the ADHD group. Of all participants, S. stercoricanis demonstrated a significant association with the intake of dairy, nuts/seeds/legumes, ferritin and magnesium. B. ovatus and S. stercoricanis were positively correlated to ADHD symptoms. In conclusion, we suggest that the gut microbiome community is associated with dietary patterns, and linked to the susceptibility to ADHD.