Mutations in the Mitochondrial Methionyl-tRNA Synthetase Cause a Neurodegenerative Phenotype in Flies and a Recessive Ataxia (ARSAL) in Humans

The study of Drosophila neurodegenerative mutants combined with genetic and biochemical analyses lead to the identification of multiple complex mutations in 60 patients with a novel form of ataxia/leukoencephalopathy

The role of oleic acid and cholesterol in neonatal diet

Oleic acid (18:1) and cholesterol are not considered to be essential dietary nutrients for adults or infants. Human milk provides a significant amount of each of these nutrients. In contrast, most infant formulas contain relatively low 18:1 and cholesterol. The overall objective of this thesis was to determine the importance of 18:1 and cholesterol in natural milk to infant nutrition. Recent studies found reduced 18:1 in brain total lipid of piglets fed formula with 17% 18:1 rather than sow milk providing 37% 18:1. Oleic acid is a major fatty acid in brain myelin lipid and is rapidly deposited during myelination. It is important, therefore, to know if reduced 18:1 in brain total lipid reflects deposition of myelin lipid with reduced 18:1 and/or delayed myelination, or is related to changes in other brain membranes. The first part of this thesis determined the fatty acid composition of myelin total lipid, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and of plasma and liver phospholipid (PL) from piglets fed from birth to 15 d with formula containing low (17%) or high (38%) 18:1, or sow milk. Brain 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP'ase) activity and cerebrosides concentration were also determined and used as indicators of myelination. Piglets fed the low 18:1 formula had lower 18:1 in their plasma and liver PL than sow milk-fed piglets. Formula providing a similar level of 18:1 to sow milk resulted in higher 18:1 in piglet plasma and liver PL than in sow milk-fed piglets. Brain cerebrosides and CNP'ase activity and myelin 18:1 were similar in sow milk- and formula-fed piglets, irrespective of the formula 18:1 content. These studies suggest that supplying 18:1 in formula in a similar quantity to natural milk is not essential to normal accretion of 18:1 in brain myelin. Several studies have reported that plasma cholesterol and PL levels of 20:4n-6 are lower and PL levels of 18:2n-6 are higher in infants fed formula than in infants fed human milk. Plasma cholesterol and possibly the dietary intake of cholesterol, could be related to plasma PL n-6 fatty acid metabolism because plasma PL 18:2n-6 is usually used for esterification of plasma free cholesterol. Whether the low cholesterol content of infant formula compared to human milk is related to the difference in plasma n-6 fatty acids between infants fed human milk and formula is not known. The second part of this thesis determined the effect of feeding a formula containing low (0.05 mmol/L) or high (1.09 mmol/L) cholesterol content, or sow milk, on plasma, liver and bile lipid fatty acids and liver LDL receptor mass in piglets fed from birth to 18 days. Liver microsomal HMG CA reductase activity and plasma lathosterol were assayed as indices of liver and body cholesterol synthesis, respectively. Providing cholesterol in the formula did not correct the significantly lower plasma cholesterol, or plasma and liver PL 20:4n-6 associated with formula feeding. The liver total cholesterol and cholesterol esters, biliary bile acids and PL concentration were significantly higher and the liver HMG CoA reductase activity and plasma lathosterol:cholesterol ratio were significantly lower in piglets fed the formula with cholesterol than in piglets fed the formula without cholesterol. No evidence of lower hepatic LDL receptor mass was found in piglets fed sow milk compared to piglets fed formula. The results show marked differences in hepatic and bile n-6 fatty acid concentration between artificially and naturally fed piglets which do not seem to be explained by the difference in dietary cholesterol intake. Whether or not cholesterol and 18:1 should be added to infant formula in concentrations similar to that of natural milk is still unknown. Results from this thesis do not provide evidence that they should be added.Land and Food Systems, Faculty ofGraduat

Identification and preliminary characterization of a 75-kDa hemin- and hemoglobin-binding outer membrane protein of Actinobacillus pleuropneumoniae serotype 1

The reference strains representing serotypes 1 to 12 of Actinobacillus pleuropneumoniae biotype 1 were examined for their ability to utilize porcine hemoglobin (Hb) or porcine hemin (Hm) as iron sources for growth. In a growth promotion assay, all of the reference strains were able to use porcine Hb, and all strains except 2 were able to use porcine Hm. Using a preliminary characterization procedure with Hm- or Hb-agarose, Hm- and Hb-binding outer membrane proteins (OMPs) of approximately 75 kDa were isolated from A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions. Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) analysis revealed a number of common tryptic peptides between the Hb-agarose- and Hm-agarose-purified 75 kDa OMPs, strongly suggesting that these peptides originate from the same protein. A database search of these peptide sequences revealed identities with proteins from various Gram-negative bacteria, including iron-regulated OMPs, transporter proteins, as well as TonB-dependent receptors. Taken together, our data suggest that A. pleuropneumoniae synthesizes potential Hm- and Hb-binding proteins that could be implicated in the iron uptake from porcine Hb and Hm

Les enjeux éthiques de la limite des ressources en santé

Dans ce livre, des chercheurs, des administrateurs, des décideurs et des professionnels de la santé se penchent sur les problèmes de justice distributive engendrés par le manque de ressources dans le système de santé au Québec. Listes d'attente, pénurie de personnel et coûts élevés des médicaments menacent l'équité du système et l'égalité d'accès aux soins. De grands enjeux se dessinent, dont le plus important consiste à assurer l'équilibre entre les besoins de la population, les moyens et les techniques accessibles pour y répondre et les objectifs sociaux. Devant la nécessité d'une réforme en profondeur du système public et pour en assurer la pérennité et l'efficacité, les auteurs dressent l'état des lieux et proposent des pistes de solution. Ayant pour but de favoriser l'interdisciplinarité, ce livre est une contribution importante aux débats en cours et deviendra une référence pour tous ceux qui, individus et groupes, cherchent à améliorer la situation

MARS2 mRNA expression, protein levels, mitochondrial protein translation, Complex I, aconitase activity, and cell proliferation.

<p>(A) Quantification of MARS2 mRNA expression levels was performed on six ARSAL cases and two control lymphoblast cell lines. Relative expression levels were normalized to GAPDH levels. ARSAL patients expressed up to 3× higher MARS2 mRNA levels compared to controls. (B) Mitochondrial protein synthesis was measured in lymphoblasts and fibroblasts from three controls and six ARSAL patients by pulse-labeling mitochondrial translation products with ³⁵S-methionine for 1 h in the presence of emetine, followed by electrophoresis on a 15%–20% linear-gradient polyacrylamide gel. The 13 mitochondrial products are identified at the left of the figure. A generalized mitochondrial translation deficiency is observed in three of the six ARSAL patients tested. ANOVA analysis revealed significance for three of the patient's mitochondrial translation levels: Ctrl 1-B4: **, Ctrl 1-B5: n.s., Ctrl 1-P24: n.s., Ctrl 2-B4: ***, Ctrl 2-B5: n.s., Ctrl 2-P24: *, Ctrl 3-B4: ***, Ctrl 3-B5: *, Ctrl 3-P24: ***. (C) Immunoblotting analysis was performed with antibodies against the proteins indicated at the left of the panel. MARS2 was visualized using a polyclonal antibody. For case E10 carrying the heterozygous deletion (c.681Δ268bpfx236X), the truncated product is detected at the estimated size of 24 kDa (arrow); ARSAL patients (B4, EE41, P24, B5, AA35, and E10) show decreased levels of MARS2 protein at the estimated normal size of MARS2 (67 kDa). The 130 kDa LRPPRC and the 12 kDa SLIRP were used as loading controls. (D) Each patient's MARS2 protein-level intensity from the Western Blot shown in (C) was quantified using ImageJ and divided by the protein-level intensities of LRRPRC and SLIRP. The results were then graphed for the controls and the patients, respectively. (E) Respiratory chain activity for Complex I was measured from patient fibroblast-derived disrupted mitochondria. Mutant mitochondria exhibit deficiency of complex I. Data are expressed as percentage control activity (mean ± s.e.m.). (F) Quantification of native and reactivated aconitase activity for ARSAL patient and control immortalized fibroblasts. Three controls and 6 ARSAL patients were used for the analysis. (G) Quantification of the proliferation rate for the same above-mentioned fibroblasts. (H) Graph showing the average age of onset for the three different genotypes involved.</p

Retinal degeneration and lifespan of <i>Aats-met</i> mutants.

<p>(A) TEM of a single ommatidium from a control 1-d-old fly eye, showing the characteristic seven dark rhabdomeres in the center. (B) TEM of a single ommatidium from the eye of a 1-d-old <i>HV/FB</i> escaper fly, showing no obvious defects. (C) TEM of the eye of a 1-d-old fly containing homozygous clones of a <i>PB</i> allele. (D) TEM of the eye of a 2-wk-old <i>HV/FB</i> escaper fly, showing the beginning of a neurodegenerative process, with a degenerating rhabdomere (arrowhead) and enlarged mitochondria (arrow). (E) TEM of the eye of a 3-wk-old escaper. (F) A neurodegenerative process is evident in clones of the <i>PB</i> allele in a 2-wk-old fly. Arrows indicate lipid droplets in pigment cells (arrowheads). (G) Quantification of 100 retinal photoreceptor rhabdomeres for the control, <i>HV/FB</i> escapers, and <i>PB</i> clone-containing mutants at different ages. (H) Quantification of the total mitochondrial area as a percentage of the retinal area: <i>HV/FB</i> mutants clearly have a higher mitochondrial content. (I) Quantification of average mitochondrial size, showing the mitochondrial number of the <i>HV/FB</i> mutant retinas (<i>n</i> = 50). (J) Graph showing the shortened lifespans of 100–200 <i>HV/FB</i> and <i>HV/HV</i> escapers of each gender compared to controls, with males denoted in blue and females in pink. Scale bars: 1 µm.</p

<i>Aats-met</i> mutants have reduced cell proliferation.

<p>(A–B) Brains of late 3^rd instar control and <i>HV/Df</i> larvae stained with Rhodamine-Phalloidin. (C–D) Wing discs of a late 3^rd instar control and mutant larvae stained with Rhodamine-Phalloidin. (E–F) Control and mutant pupae are shown. (G) Quantification of pupal length is shown. (H) Wing disc containing wild-type (outlined in yellow) and mutant clones (outlined in red) are seen. (I) Wild-type clones are significantly larger than mutant clones, quantified in 16 to 20 pairs of clones. (J–K) Cells in mutant clones in wing discs, stained with anti-Dlg, to mark the cell membrane, are similar in size to wild-type cells. (L) PH3-staining cells in mutant versus neighboring heterozygous tissue is quantified for five wing discs, indicating that there is less cell proliferation in mutant clones. Data are mean ± s.e.m. Scale bars for (A–D) and (H) are 100 microns, (E–F) are 0.3 mm, and (J–K) are 5 microns.</p

Additional file 1: of Diagnosis of late-onset Pompe disease and other muscle disorders by next-generation sequencing

Supplemental information. (DOCX 157 kb

Identification/mapping of the <i>Aats-met</i> gene.

<p>(A) ERG of the control (<i>y w</i>; <i>FRT82B iso</i>). The black and white arrowheads indicate the “on” and “off” transients, respectively. The double-pointed arrow indicates the amplitude. (B–C) ERGs of homozygous <i>HV</i> clone-containing flies at 1 d and 4 wk after eclosion. (D–E) ERGs of homozygous <i>FB</i> clone-containing flies at 1 d and 4 wk after eclosion. (F) ERG of a 1-d-old <i>HV/FB</i> escaper. (G) ERG of a 3-wk-old <i>HV/FB</i> escaper. (H) ERG of a 2-wk-old <i>HV/Df</i> fly rescued with <i>actin-Gal4</i> and <i>UAS-Aats-met</i>. (I) ERG of a 2-wk-old <i>HV/Df</i> fly rescued with <i>actin-Gal4</i> and <i>UAS-HMARS2</i>. (J) ERG of a 2-wk-old otherwise wild-type fly expressing HMARS2-FLAG driven by tub-Gal4. (K) Lethal stages of homozygous and transheretozygous allelic combinations reveal an allelic series: <i>Df>PB>FB>HV</i>. (L) The Aats-met protein's predicted domains are shown (drawn to scale), with position of mutations and percentage identity compared to human MARS2 shown. (M) The <i>Drosophila Aats-met</i> gene is homologous to the mitochondrial methionyl-tRNA synthetase genes of <i>S. cerevisiae</i>, <i>C. elegans</i>, <i>M. musculus</i>, and <i>H. sapiens</i>. (N) Colocalization of the Flag-tagged human MARS2 protein with Mito-GFP in the cell body of a neuron in the ventral nerve cord, driven by the D42-Gal4 driver, is shown.</p