Mechanisms Underlying Metabolic and Neural Defects in Zebrafish and Human Multiple Acyl-CoA Dehydrogenase Deficiency (MADD)

In humans, mutations in electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETFDH) lead to MADD/glutaric aciduria type II, an autosomal recessively inherited disorder characterized by a broad spectrum of devastating neurological, systemic and metabolic symptoms. We show that a zebrafish mutant in ETFDH, xavier, and fibroblast cells from MADD patients demonstrate similar mitochondrial and metabolic abnormalities, including reduced oxidative phosphorylation, increased aerobic glycolysis, and upregulation of the PPARG-ERK pathway. This metabolic dysfunction is associated with aberrant neural proliferation in xav, in addition to other neural phenotypes and paralysis. Strikingly, a PPARG antagonist attenuates aberrant neural proliferation and alleviates paralysis in xav, while PPARG agonists increase neural proliferation in wild type embryos. These results show that mitochondrial dysfunction, leading to an increase in aerobic glycolysis, affects neurogenesis through the PPARG-ERK pathway, a potential target for therapeutic intervention

Primary Coenzyme Q Deficiency in Pdss2 Mutant Mice Causes Isolated Renal Disease

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Eltrombopag Does Not Significantly Affect Mitochondria Function and ATP Production.

<p>A) Graph shows results of TMRE staining as a measurement of the mitochondria membrane potential in MOLM14 cells after exposure to E at various time points. CCCP, a known mitochondrial membrane potential disrupter was used as a control for the assay. Bars represent percent of control (untreated cells) mean fluorescence intensity (MFI) with SEM. B) Effect of E on mitochondrial membrane potential in AML cell lines (left panel) and primary AML cells (right panel) measured by a TMRE staining. C) Measurements of oxygen consumption rate in control and cells treated with E (5 μM) for 24 hours. The oxygen consumption rates are expressed as nanomoles O₂ consumed/ min/ 10⁶ cells. The differences in maximal and spare respiratory capacity (SRC) between control and E treated cells are statistically significant with p value from 3 independent experiments equal 0.04 and 0.01 respectively. D) Graph represents an ATP production in MOLM14 cells cultured with or without (CTR) E for 20 hours, then exposed either to IAA, AA+O or remained untreated. Dark gray bars (untreated) represent total ATP, black bars (IAA) represent ATP produced by mitochondria and light gray bars (AA+O) represent ATP produced during glycolysis.</p

Modification of intracellular ROS level abrogates eltrombopag’s effect on AML cells.

<p>A) E blocks completely increase of ROS caused by BSO and rescues cells from BSO induced death. Left panel—ROS level in MOLM14 cells untreated (CTR) or pre-treated with BSO for 72 hours and then exposed to E or left untreated. Right panel—proliferation assay—results are presented as an average and SEM of a total number of cells from 3 independent experiments. At day 0 cells cultured in the presence of BSO (50 or 100 μM) for 72hours or in plain medium (CTR) were plated at concentration 5x10^4 per 250 μL of culture medium and exposed to E or left untreated. B) Pre-loading with ferric ammonium citrate (FAC) rescues MOLM14 cells from E cytotoxic effect by inducing ROS level. Levels of H₂O₂ measured using carboxy- H₂DCFDA in MOLM14 cells treated as described above (left panel). Proliferation of MOLM14 cells un-manipulated or pre-loaded with 500 μg/mL of FAC for 24 h and then exposed to E or left untreated (CTR) (right panel).</p

Correlation between eltrombopag’s cytotoxic effect and ROS level at various drug doses and serum concentrations.

<p>A) Additive effect of E combined with antioxidant (NAC) on ROS decrease and survival of MOLM14 cells. Left panel—levels of H₂O₂ measured using carboxy- H₂DCFDA in MOLM14 cells exposed to E and NAC for 1 hour. Right panel—Proliferation of MOLM14 cells exposed to E (2–5 μM) alone NAC (15 mM) alone or combination. Results are presented as an average of total number of cells counted at days: 1 and 2 and SEM from 2 independent experiments. B) Increasing serum concentration decreases effect of E on ROS level (top panels) and cell survival (bottom panels) in AML cell lines. Results are presented as an average of percent of control of total number of cells counted at day 2 and SEM from 2 independent experiments.</p

Effectiveness of E cytotoxicity presented as a half maximal growth inhibitory concentration (CC₅₀).

<p>The CC₅₀ was calculated for primary AML samples (top) and AML cell lines (bottom) at days: 4 and 2 respectively, when cultured at 2% serum.</p><p>Effectiveness of E cytotoxicity presented as a half maximal growth inhibitory concentration (CC₅₀).</p