Acetaminophen is both bronchodilatory and bronchoprotective in human precision cut lung slice airways.

Epidemiologic studies have demonstrated an association between acetaminophen (APAP) use and the development of asthma symptoms. However, few studies have examined relationships between APAP-induced signaling pathways associated with the development of asthma symptoms. We tested the hypothesis that acute APAP exposure causes airway hyper-responsiveness (AHR) in human airways. Precision cut lung slice (PCLS) airways from humans and mice were used to determine the effects of APAP on airway bronchoconstriction and bronchodilation and to assess APAP metabolism in lungs. APAP did not promote AHR in normal or asthmatic human airways ex vivo. Rather, high concentrations mildly bronchodilated airways pre-constricted with carbachol (CCh), histamine (His), or immunoglobulin E (IgE) cross-linking. Further, the addition of APAP prior to bronchoconstrictors protected the airways from constriction. Similarly, in vivo treatment of mice with APAP (200 mg/kg IP) resulted in reduced bronchoconstrictor responses in PCLS airways ex vivo. Finally, in both mouse and human PCLS airways, exposure to APAP generated only low amounts of APAP-protein adducts, indicating minimal drug metabolic activity in the tissues. These findings indicate that acute exposure to APAP does not initiate AHR, that high-dose APAP is protective against bronchoconstriction, and that APAP is a mild bronchodilator.

Multiple microRNAs function as self-protective modules in acetaminophen-induced hepatotoxicity in humans.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure. Yet the mechanisms underlying adaptive tolerance toward APAP-induced liver injury are not fully understood. To better understand molecular mechanisms contributing to adaptive tolerance to APAP is an underpinning foundation for APAP-related precision medicine. In the current study, the mRNA and microRNA (miRNA) expression profiles derived from next generation sequencing data for APAP-treated (5 and 10 mM) HepaRG cells and controls were analyzed systematically. Putative miRNAs targeting key dysregulated genes involved in APAP hepatotoxicity were selected using in silico prediction algorithms, un-biased gene ontology, and network analyses. Luciferase reporter assays, RNA electrophoresis mobility shift assays, and miRNA pull-down assays were performed to investigate the role of miRNAs affecting the expression of dysregulated genes. Levels of selected miRNAs were measured in serum samples obtained from children with APAP overdose (58.6-559.4 mg/kg) and from healthy controls. As results, 2758 differentially expressed genes and 47 miRNAs were identified. Four of these miRNAs (hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p) suppressed drug metabolizing enzyme (DME) levels involved in APAP-induced liver injury by downregulating HNF1A, HNF4A and NR1I2 expression. Exogenous transfection of these miRNAs into HepaRG cells effectively rescued them from APAP toxicity, as indicated by decreased alanine aminotransferase levels. Importantly, hsa-miR-320a and hsa-miR-877-5p levels were significantly elevated in serum samples obtained from children with APAP overdose compared to health controls. Collectively, these data indicate that hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p suppress DME expression involved in APAP-induced hepatotoxicity and they contribute to an adaptive response in hepatocytes.

Potential of extracellular microRNAs as biomarkers of acetaminophen toxicity in children.

UNLABELLED: Developing biomarkers for detecting acetaminophen (APAP) toxicity has been widely investigated. Recent studies of adults with APAP-induced liver injury have reported human serum microRNA-122 (miR-122) as a novel biomarker of APAP-induced liver injury. The goal of this study was to examine extracellular microRNAs (miRNAs) as potential biomarkers for APAP liver injury in children. Global levels of serum and urine miRNAs were examined in three pediatric subgroups: 1) healthy children (n=10), 2) hospitalized children receiving therapeutic doses of APAP (n=10) and 3) children hospitalized for APAP overdose (n=8). Out of 147 miRNAs detected in the APAP overdose group, eight showed significantly increased median levels in serum (miR-122, -375, -423-5p, -30d-5p, -125b-5p, -4732-5p, -204-5p, and -574-3p), compared to the other groups. Analysis of urine samples from the same patients had significantly increased median levels of four miRNAs (miR-375, -940, -9-3p and -302a) compared to the other groups. Importantly, correlation of peak serum APAP protein adduct levels (an indicator of the oxidation of APAP to the reactive metabolite N-acetyl-para-quinone imine) with peak miRNA levels showed that the highest correlation was observed for serum miR-122 (R=0.94; p<0.01) followed by miR-375 (R=0.70; p=0.05). CONCLUSION: Our findings demonstrate that miRNAs are increased in children with APAP toxicity and correlate with APAP protein adducts, suggesting a potential role as biomarkers of APAP toxicity.

Translational biomarkers of acetaminophen-induced acute liver injury.

Acetaminophen (APAP) is a commonly used analgesic drug that can cause liver injury, liver necrosis and liver failure. APAP-induced liver injury is associated with glutathione depletion, the formation of APAP protein adducts, the generation of reactive oxygen and nitrogen species and mitochondrial injury. The systems biology omics technologies (transcriptomics, proteomics and metabolomics) have been used to discover potential translational biomarkers of liver injury. The following review provides a summary of the systems biology discovery process, analytical validation of biomarkers and translation of omics biomarkers from the nonclinical to clinical setting in APAP-induced liver injury.

Identification of DCAF1 by Clinical Exome Sequencing and Methylation Analysis as a Candidate Gene for Autism and Intellectual Disability: A Case Report.

Autism spectrum disorder (ASD) comprises a heterogeneous group of neurodevelopmental disorders and occurs in all racial, ethnic, and socioeconomic groups. Cutting-edge technologies are contributing to understanding genetic underpinnings in ASD. The reported patient is a 32-year-old male and as an infant was noted to have microcephaly, hypospadias, pulmonary vascular anomaly, and small stature. He was diagnosed with Cornelia De Lange Syndrome (CDLS) at that time based on the clinical features. As a child, he had autistic features and intellectual disabilities and as diagnoses with autism and intellectual disability. He was referred as an adult to our neurodiversity clinic and a full exome trio sequencing with reflex to mitochondrial genes identified a de novo variant of uncertain significance in a candidate gene, DCAF1. The specific variant was c.137 C > T (p.Thr46Ile) in exon 4 in the DCAF1 gene. In silico analysis supports a deleterious effect on protein structure/function. DCAF1 participates with DDB1 and CUL4 as a part of the E3 ubiquitin ligase complex. The E3 ligase complex has been associated with a syndromic form of X-linked intellectual disability. The DDB1/CUL4 E3 ubiquitination complex plays a role in methylation-dependent ubiquitination. Next, a methylation study identified a signature similar to the methylation pattern found in X- linked intellectual disability type 93. This is associated with variants of the BRWD3 gene, which is linked with the functioning of the DDB1/CUL4 E3 ubiquitination complex. Taken together, this suggests that the de novo DCAF1 variant may be a newly identified molecular cause of autism and intellectual disability.

Integrated microRNA-mRNA Expression Profiling Identifies Novel Targets and Networks Associated with Autism.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder, with mutations in hundreds of genes contributing to its risk. Herein, we studied lymphoblastoid cell lines (LCLs) from children diagnosed with autistic disorder (n = 10) and controls (n = 7) using RNA and miRNA sequencing profiles. The sequencing analysis identified 1700 genes and 102 miRNAs differentially expressed between the ASD and control LCLs (p ≤ 0.05). The top upregulated genes were GABRA4, AUTS2, and IL27, and the top upregulated miRNAs were hsa-miR-6813-3p, hsa-miR-221-5p, and hsa-miR-21-5p. The RT-qPCR analysis confirmed the sequencing results for randomly selected candidates: AUTS2, FMR1, PTEN, hsa-miR-15a-5p, hsa-miR-92a-3p, and hsa-miR-125b-5p. The functional enrichment analysis showed pathways involved in ASD control proliferation of neuronal cells, cell death of immune cells, epilepsy or neurodevelopmental disorders, WNT and PTEN signaling, apoptosis, and cancer. The integration of mRNA and miRNA sequencing profiles by miRWalk2.0 identified correlated changes in miRNAs and their targets' expression. The integration analysis found significantly dysregulated miRNA-gene pairs in ASD. Overall, these findings suggest that mRNA and miRNA expression profiles in ASD are greatly altered in LCLs and reveal numerous miRNA-gene interactions that regulate critical pathways involved in the proliferation of neuronal cells, cell death of immune cells, and neuronal development.

Multidisciplinary Consulting Team for Complicated Cases of Neurodevelopmental and Neurobehavioral Disorders: Assessing the Opportunities and Challenges of Integrating Pharmacogenomics into a Team Setting.

Neurodevelopmental disorders have steadily increased in incidence in the United States. Over the past decade, there have been significant changes in clinical diagnoses and treatments some of which are due to the increasing adoption of pharmacogenomics (PGx) by clinicians. In this pilot study, a multidisciplinary team at the Arkansas Children's Hospital North West consulted on 27 patients referred for difficult-to-manage neurodevelopmental and/or neurobehavioral disorders. The 27 patients were evaluated by the team using records review, team discussion, and pharmacogenetic testing. OneOme RightMed® (Minneapolis, MN, USA) and the Arkansas Children's Hospital comprehensive PGx test were used for drug prescribing guidance. Of the 27 patients' predicted phenotypes, the normal metabolizer was 11 (40.8%) for CYP2C19 and 16 (59.3%) for CYP2D6. For the neurodevelopmental disorders, the most common comorbid conditions included attention-deficit hyperactivity disorder (66.7%), anxiety disorder (59.3%), and autism (40.7%). Following the team assessment and PGx testing, 66.7% of the patients had actionable medication recommendations. This included continuing current therapy, suggesting an appropriate alternative medication, starting a new therapy, or adding adjunct therapy (based on their current medication use). Moreover, 25.9% of patients phenoconverted to a CYP2D6 poor metabolizer. This retrospective chart review pilot study highlights the value of a multidisciplinary treatment approach to deliver precision healthcare by improving physician clinical decisions and potentially impacting patient outcomes. It also shows the feasibility to implement PGx testing in neurodevelopmental/neurobehavioral disorders.

Impaired sodium excretion and increased blood pressure in mice with targeted deletion of renal epithelial insulin receptor.

Renal tubule epithelial cells express the insulin receptor (IR); however, their value has not been firmly established. We generated mice with renal epithelial cell-specific knockout of the IR by Cre-recombinase-loxP recombination using a kidney-specific (Ksp) cadherin promoter. KO mice expressed significantly lower levels of IR mRNA and protein in kidney cortex (49-56% of the WT) and medulla (32-47%) homogenates. Immunofluorescence showed the greatest relative reduction in the thick ascending limb and collecting duct cell types. Body weight, kidney weight, and food and water intakes were not different from WT littermates. However, KO mice had significantly increased basal systolic blood pressure (BP, 15 mm Hg higher) as measured by radiotelemetry. In response to a volume load by gavage (20 ml/kg of body weight, 0.9% NaCl, 15% dextrose), KO mice had impaired natriuresis (37 +/- 10 versus 99 +/- 9 mmol of Na(+) per 2 h in WT). Furthermore, volume load led to a sustained increase in BP in KO mice only. In contrast, insulin administration i.p. (0.5 units/kg of body weight) resulted in a significant fall in BP in WT, but not in KO mice. To test the role of reduced renal nitric oxide (NO) production in these responses, basal urinary nitrates plus nitrites excretion (UNOx) was measured and found to be 61% lower in KO vs. WT mice. Furthermore, acute insulin increased UNOx by 202% in the WT, relative to a significantly blunted rise (67%) in KO animals. These results illuminate a previously uncharacterized role for renal IR to reduce BP and facilitate sodium and water excretion, possibly via NO production.

Molecular Dysregulation in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) comprises a heterogeneous group of neurodevelopmental disorders with a strong heritable genetic component. At present, ASD is diagnosed solely by behavioral criteria. Advances in genomic analysis have contributed to numerous candidate genes for the risk of ASD, where rare mutations and s common variants contribute to its susceptibility. Moreover, studies show rare de novo variants, copy number variation and single nucleotide polymorphisms (SNPs) also impact neurodevelopment signaling. Exploration of rare and common variants involved in common dysregulated pathways can provide new diagnostic and therapeutic strategies for ASD. Contributions of current innovative molecular strategies to understand etiology of ASD will be explored which are focused on whole exome sequencing (WES), whole genome sequencing (WGS), microRNA, long non-coding RNAs and CRISPR/Cas9 models. Some promising areas of pharmacogenomic and endophenotype directed therapies as novel personalized treatment and prevention will be discussed.

MicroRNA Expression Profiles in Autism Spectrum Disorder: Role for miR-181 in Immunomodulation.

BACKGROUND: MicroRNAs (miRNAs) are important regulators of molecular pathways in psychiatric disease. Here, we examine differential miRNAs expression in lymphoblastoid cell lines (LCLs) derived from 10 individuals with autism spectrum disorder (ASD) and compare them to seven typically developing unrelated age- and gender-matched controls and 10 typically developing siblings. Small RNAseq analysis identified miRNAs, and selected miRNAs were validated using quantitative real-time polymerase reaction (qRT-PCR). KEGG analysis identified target pathways, and selected predicted mRNAs were validated using qRT-PCR. RESULTS: Small RNAseq analysis identified that multiple miRNAs differentiated ASD from unrelated controls and ASD from typically developing siblings, with only one, hsa-miR-451a_R-1, being in common. Verification with qRT-PCR showed that miR-320a differentiated ASD from both sibling and unrelated controls and that several members of the miR-181 family differentiated ASD from unrelated controls. Differential expression of AKT2, AKT3, TNF α and CamKinase II predicted by KEGG analysis was verified by qRT-PCR. Expression of CamKinase II ßwas found to be correlated with the severity of stereotyped behavior of the ASD participants. CONCLUSIONS: This study provides insight into the mechanisms regulating molecular pathways in individuals with ASD and identifies differentiated regulated genes involved in both the central nervous system and the immune system.

Implementing Pharmacogenomics Testing: Single Center Experience at Arkansas Children's Hospital.

Pharmacogenomics (PGx) is a growing field within precision medicine. Testing can help predict adverse events and sub-therapeutic response risks of certain medications. To date, the US FDA lists over 280 drugs which provide biomarker-based dosing guidance for adults and children. At Arkansas Children's Hospital (ACH), a clinical PGx laboratory-based test was developed and implemented to provide guidance on 66 pediatric medications for genotype-guided dosing. This PGx test consists of 174 single nucleotide polymorphisms (SNPs) targeting 23 clinically actionable PGx genes or gene variants. Individual genotypes are processed to provide per-gene discrete results in star-allele and phenotype format. These results are then integrated into EPIC- EHR. Genomic indicators built into EPIC-EHR provide the source for clinical decision support (CDS) for clinicians, providing genotype-guided dosing.

Granzyme B and miR-378a Interaction in Acetaminophen Toxicity in Children.

BACKGROUND AND AIM: Hepatic phase I drug-metabolizing enzymes CYP2E1, CYP1A2 and CYP3A4 catalyze the biotransformation of Acetaminophen (APAP) and are important in the mediation of toxicity. The potential role of other hepatic and non-hepatic Phase I enzymes in APAP toxicity has not been established. METHODS: PCR array containing 84 genes involved in phase I drug metabolism was examined in subgroups of hospitalized children for APAP overdose, categorized as no toxicity (ALT ≤ 45 IU/L, n=5) and moderate toxicity (ALT ≥ 500 IU/L, n=5). RESULTS: Significant downregulation was observed for ALDH6A1, CYP4F12 and GZMB in the no toxicity subgroup and ALDH1A1, CYP27A1 and GZMB in the moderate toxicity subgroup. qRTPCR confirmed significant downregulation for ALDH1A1, CYP4F12, and GZMB. In-silico analysis identified GZMB 3'UTR to be a target of miR-378a-5p. Overexpression of miR-378a-5p reduced the luciferase activity of GZMB 3'UTR reporter plasmid reportedly by 50%. NK-92 cells transfected with the miR-378a-5p mimic extended the effect of APAP on GZMB protein expression compared to mimic controls. In addition, miR-378a-5p was significantly upregulated in blood samples of children with APAP overdose undergoing NAC treatment. CONCLUSION: Overall, our study suggests the presence of a novel signaling pathway, whereby miR- 378a-5p inhibits GZMB expression in children with APAP overdose.

High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity.

BACKGROUND: Commercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity. RESULTS: The human Genome-Wide SpliceArray(TM) (GWSA), a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC) Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform. CONCLUSION: Significant changes were detected independent of transcriptional activity, indicating that the controls for transcript generation and transcription are distinct, and require novel tools in order to detect changes in specific transcript quantity. Our results demonstrate that the SpliceArray(TM) design will provide researchers with a robust platform to detect and quantify specific changes not only in overall gene expression, but also at the individual transcript level.

Isoform-specific regulation by N(G),N(G)-dimethylarginine dimethylaminohydrolase of rat serum asymmetric dimethylarginine and vascular endothelium-derived relaxing factor/NO.

Asymmetric dimethylarginine (ADMA), which inhibits NO synthase, is inactivated by N(G),N(G)-dimethylarginine dimethylaminohydrolase (DDAH). We tested whether DDAH-1 or -2 regulates serum ADMA (S(ADMA)) and/or endothelium-derived relaxing factor (EDRF)/NO. Small inhibitory (si)RNAs targeting DDAH-1 or -2, or an siRNA control were given intravenously to rats. After 72 hours, EDRF/NO was assessed from acetylcholine-induced, NO synthase-dependent relaxation and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate for NO activity in isolated mesenteric resistance vessels (MRVs). Expression of mRNA for DDAH-1 versus -2 was 2- and 7-fold higher in the kidney cortex and liver, respectively, whereas expression of DDAH-2 versus -1 was 5-fold higher in MRVs. The proteins and mRNAs for DDAH-1 or -2 were reduced selectively by 35% to 85% in the kidney cortex, liver, and MRVs 72 hours following the corresponding siRNA. S(ADMA) was increased only after siDDAH-1 (266+/-25 versus 342+/-39 [mean+/-SD] nmol x L(-1); P<0.005), whereas EDRF/NO responses and NO activity were not changed consistently by siDDAH-1 but were greatly reduced after siDDAH-2. Mean arterial pressure was not changed significantly by any siRNA. In conclusion, S(ADMA) is regulated by DDAH-1, which is expressed at sites of ADMA metabolism in the kidney cortex and liver, whereas EDRF/NO is regulated primarily by DDAH-2, which is expressed strongly in blood vessels. This implies specific functions of DDAH isoforms.

Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention.

In vitro studies suggest that collecting duct-derived (CD-derived) endothelin-1 (ET-1) can regulate renal Na reabsorption; however, the physiologic role of CD-derived ET-1 is unknown. Consequently, the physiologic effect of selective disruption of the ET-1 gene in the CD of mice was determined. Mice heterozygous for aquaporin2 promoter Cre recombinase and homozygous for loxP-flanked exon 2 of the ET-1 gene (called CD-specific KO of ET-1 [CD ET-1 KO] mice) were generated. These animals had no CD ET-1 mRNA and had reduced urinary ET-1 excretion. CD ET-1 KO mice on a normal Na diet were hypertensive, while body weight, Na excretion, urinary aldosterone excretion, and plasma renin activity were unchanged. CD ET-1 KO mice on a high-Na diet had worsened hypertension, reduced urinary Na excretion, and excessive weight gain, but showed no differences between aldosterone excretion and plasma renin activity. Amiloride or furosemide reduced BP in CD ET-1 KO mice on a normal or high-Na diet and prevented excessive Na retention in salt-loaded CD ET-1 KO mice. These studies indicate that CD-derived ET-1 is an important physiologic regulator of renal Na excretion and systemic BP.

Metabolomics Analysis of Urine Samples from Children after Acetaminophen Overdose.

Acetaminophen (APAP), a commonly used over-the-counter analgesic, accounts for approximately fifty percent of the cases of acute liver failure (ALF) in the United States due to overdose, with over half of those unintentional. Current clinical approaches for assessing APAP overdose rely on identifying the precise time of overdose and quantitating acetaminophen alanine aminotransferase (ALT) levels in peripheral blood. Novel specific and sensitive biomarkers may provide additional information regarding patient status post overdose. Previous non-clinical metabolomics studies identified potential urinary biomarkers of APAP-induced hepatotoxicity and metabolites involved pathways of tricarboxylic acid cycle, ketone metabolism, and tryptophan metabolism. In this study, biomarkers identified in the previous non-clinical study were evaluated in urine samples collected from healthy subjects ( N = 6, median age 14.08 years) and overdose patients ( N = 13, median age 13.91 years) as part of an IRB-approved multicenter study of APAP toxicity in children. The clinical results identified metabolites from pathways previously noted, and pathway analysis indicated analogous pathways were significantly altered in both the rats and humans after APAP overdose. The results suggest a metabolomics approach may enable the discovery of specific, translational biomarkers of drug-induced hepatotoxicity that may aid in the assessment of patients.

MicroRNA regulation of CYP 1A2, CYP3A4 and CYP2E1 expression in acetaminophen toxicity.

MicroRNAs (miRNAs) that regulate the cytochrome P-450 isoforms involved in acetaminophen (APAP) toxicity were examined in HepaRG cells treated with APAP (20 mM). In-vitro studies found that APAP protein adducts were increased at 1 h, followed by ALT increases at 12 and 24 h. CYP1A2, CYP3A4 and CYP2E1 mRNA levels were decreased, while miRNAs were increased for miR-122-5p, miR-378a-5p, miR-27b-3p at 6 h and miR-125b-5p at 12 h and miR-27b-3p at 24 h. Putative miRNA binding sites on the 3'UTRs of the CYPs were identified in-silico. Overexpression of miR-122-5p and miR-378a-5p in cells suppressed protein expression of CYP1A2, CYP3A4 and CYP2E1. Luciferase reporter assays confirmed the interaction between miR-122 and the 3'UTR of the CYP1A2 and CYP3A4. Thus, the in-vitro experiments showed that miR-122-5p and miR-378a-5p upregulation were associated with translational repression of CYPs. Serum samples of children with APAP overdose had significant elevation of miR-122-5p, miR-378a-5p, miR-125b-5p and miR-27b-3p, compared to healthy controls and receiver operator curves of the miRNAs had AUCs of 91 to 100%. Collectively, the data suggest that miRNA elevations in APAP toxicity represent a regulatory response to modify CYP1A2, CYP3A4 and CYP2E1 translation due to cellular stress and injury.

Autistic Siblings with Novel Mutations in Two Different Genes: Insight for Genetic Workups of Autistic Siblings and Connection to Mitochondrial Dysfunction.

The prevalence of autism spectrum disorder (ASD) is high, yet the etiology of this disorder is still uncertain. Advancements in genetic analysis have provided the ability to identify potential genetic changes that may contribute to ASD. Interestingly, several genetic syndromes have been linked to metabolic dysfunction, suggesting an avenue for treatment. In this case study, we report siblings with ASD who had similar initial phenotypic presentations. Whole exome sequencing (WES) revealed a novel c.795delT mutation in the WDR45 gene affecting the girl, which was consistent with her eventual progression to a Rett-like syndrome phenotype including seizures along with a stereotypical cyclic breathing pattern. Interestingly, WES identified that the brother harbored a novel heterozygous Y1546H variant in the DEP domain-containing protein 5 (DEPDC5) gene, consistent with his presentation. Both siblings underwent a metabolic workup that demonstrated different patterns of mitochondrial dysfunction. The girl demonstrated statistically significant elevations in mitochondrial activity of complex I + III in both muscle and fibroblasts and increased respiration in peripheral blood mononuclear cells (PBMCs) on Seahorse Extracellular Flux analysis. The boy demonstrates a statistically significant decrease in complex IV activity in buccal epithelium and decreased respiration in PBMCs. These cases highlight the differences in genetic abnormalities even in siblings with ASD phenotypes as well as highlights the individual role of novel mutations in the WDR45 and DEPDC5 genes. These cases demonstrate the importance of advanced genetic testing combined with metabolic evaluations in the workup of children with ASD.

NADPH oxidases in the kidney.

NADPH oxidases have a distinct cellular localization in the kidney. Reactive oxygen species (ROS) are produced in the kidney by fibroblasts, endothelial cells (EC), vascular smooth muscle cells (VSMC), mesangial cells (MCs), tubular cells, and podocyte cells. All components of the phagocytic NADPH oxidase, as well as the Nox-1 and -4, are expressed in the kidney, with a prominent expression in renal vessels, glomeruli, and podocytes, and cells of the thick ascending limb of the loop of Henle (TAL), macula densa, distal tubules, collecting ducts, and cortical interstitial fibroblasts. NADPH oxidase activity is upregulated by prolonged infusion of angiotensin II (Ang II) or a high salt diet. Since these are major factors underlying the development of hypertension, renal NADPH oxidase may have an important pathophysiological role. Indeed, recent studies with small interference RNAs (siRNAs) targeted to p22( phox ) implicate p22( phox ) in Ang II-induced activation of renal NADPH oxidase and the development of oxidative stress and hypertension, while studies with apocynin implicate activation of p47( phox ) in the development of nephropathy in a rat model of type 1 diabetes mellitus (DM). Experimental studies of the distribution, signaling, and function of NADPH oxidases in the kidney are described.

Targeted metabolomic profiling indicates structure-based perturbations in serum phospholipids in children with acetaminophen overdose.

Phospholipids are an important class of lipids that act as building blocks of biological cell membranes and participate in a variety of vital cellular functions including cell signaling. Previous studies have reported alterations in phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) metabolism in acetaminophen (APAP)-treated animals or cell cultures. However, little is known about phospholipid perturbations in humans with APAP toxicity. In the current study, targeted metabolomic analysis of 180 different metabolites including 14 lysoPCs and 73 PCs was performed in serum samples from children and adolescents hospitalized for APAP overdose. Metabolite profiles in the overdose group were compared to those of healthy controls and hospitalized children receiving low dose APAP for treatment of pain or fever (therapeutic group). PCs and lysoPCs with very long chain fatty acids (VLCFAs) were significantly decreased in the overdose group, while those with comparatively shorter chain lengths were increased in the overdose group compared to the therapeutic and control groups. All ether linked PCs were decreased in the overdose group compared to the controls. LysoPC-C26:1 was highly reduced in the overdose group and could discriminate between the overdose and control groups with 100% sensitivity and specificity. The PCs and lysoPCs with VLCFAs showed significant associations with changes in clinical indicators of drug metabolism (APAP protein adducts) and liver injury (alanine aminotransferase, or ALT). Thus, a structure-dependent reduction in PCs and lysoPCs was observed in the APAP-overdose group, which may suggest a structure-activity relationship in inhibition of enzymes involved in phospholipid metabolism in APAP toxicity.