Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16,485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the substrates of greatest affinity.
Mutations of FBXL4, which encodes an orphan mitochondrial F-box protein, are a recently identified cause of encephalomyopathic mtDNA depletion. Here, we describe the detailed clinical and biochemical phenotype of a neonate presenting with hyperlactatemia, leukoencephalopathy, arrhythmias, pulmonary hypertension, dysmorphic features, and lymphopenia. Next-generation sequencing in the proband identified a homozygous frameshift, c.1641_1642delTG, in FBXL4, with a surrounding block of SNP marker homozygosity identified by microarray. Muscle biopsy showed a paucity of mitochondria with ultrastructural abnormalities, mitochondrial DNA depletion, and profound deficiency of all respiratory chain complexes. Cell-based mitochondrial phenotyping in fibroblasts showed mitochondrial fragmentation, decreased basal and maximal respiration, absence of ATP-linked respiratory and leak capacity, impaired survival under obligate aerobic respiration, and reduced mitochondrial inner membrane potential, with relative sparing of mitochondrial mass. Cultured fibroblasts from the patient exhibited a more oxidized glutathione ratio, consistent with altered cellular redox poise. High-resolution respirometry of permeabilized muscle fibers showed marked deficiency of oxidative phosphorylation using a variety of mitochondrial energy substrates and inhibitors. This constitutes the fourth and most detailed report of FBXL4 deficiency to date. In light of our patient's clinical findings and genotype (homozygous frameshift), this phenotype likely represents the severe end of the FBXL4 clinical spectrum.
BackgroundClinical research has recently demonstrated that vitamin D deficiency (VDD) is highly prevalent in the pediatric intensive care unit (PICU) and associated with worse clinical course. Multiple adult ICU trials have suggested that optimization of vitamin D status through high-dose supplementation may reduce mortality and improve other clinically relevant outcomes; however, there have been no trials of rapid normalization in the PICU setting. The objective of this study is to evaluate the safety and efficacy of an enteral weight-based cholecalciferol loading dose regimen in critically ill children with VDD.Methods/designThe VITdAL-PICU pilot study is designed as a multicenter placebo-controlled phase II dose evaluation pilot randomized controlled trial. We aim to randomize 67 VDD critically ill children using a 2:1 randomization schema to receive loading dose enteral cholecalciferol (10,000 IU/kg, maximum of 400,000 IU) or a placebo solution. Participants, caregivers and outcome assessors will be blinded to allocation. Eligibility criteria include ICU patient, aged 37 weeks to 18 years, expected ICU length of stay more than 48 h, anticipated access to bloodwork at 7 days, and VDD (blood total 25 hydroxyvitamin D < 50 nmol/L). The primary objective is to determine whether the dosing protocol normalizes vitamin D status, defined as a blood total 25(OH)D concentration above 75 nmol/L. Secondary objectives include an examination of the safety of the dosing regimen (e.g. hypercalcemia, hypercalciuria, nephrocalcinosis), measures of vitamin D axis function (e.g. calcitriol levels, immune function), and protocol feasibility (eligibility criteria, protocol deviations, blinding).DiscussionDespite significant observational literature suggesting VDD to be a modifiable risk factor in the PICU setting, there is no robust clinical trial evidence evaluating the benefits of rapid normalization. This phase II clinical trial will evaluate an innovative weight-based dosing regimen intended to rapidly and safely normalize vitamin D levels in critically ill children. Study findings will be used to inform the design of a multicenter phase III trial evaluating the clinical and economic benefits to rapid normalization. Recruitment for this trial was initiated in January 2016 and is expected to continue until November 30, 2017.Trial registrationClinicaltrials.gov NCT02452762 Electronic supplementary materialThe online version of this article (10.1186/s40814-017-0214-z) contains supplementary material, which is available to authorized users.
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