Évaluation de différentes composantes chromatographiques d'un système nano-LC-MS pour des applications protéomiques

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Proteomics-based confirmation of protein expression and correction of annotation errors in the Brucella abortus genome

<p>Abstract</p> <p>Background</p> <p>Brucellosis is a major bacterial zoonosis affecting domestic livestock and wild mammals, as well as humans around the globe. While conducting proteomics studies to better understand <it>Brucella abortus </it>virulence, we consolidated the proteomic data collected and compared it to publically available genomic data.</p> <p>Results</p> <p>The proteomic data was compiled from several independent comparative studies of <it>Brucella abortus </it>that used either outer membrane blebs, cytosols, or whole bacteria grown in media, as well as intracellular bacteria recovered at different times following macrophage infection. We identified a total of 621 bacterial proteins that were differentially expressed in a condition-specific manner. For 305 of these proteins we provide the first experimental evidence of their expression. Using a custom-built protein sequence database, we uncovered 7 annotation errors. We provide experimental evidence of expression of 5 genes that were originally annotated as non-expressed pseudogenes, as well as start site annotation errors for 2 other genes.</p> <p>Conclusions</p> <p>An essential element for ensuring correct functional studies is the correspondence between reported genome sequences and subsequent proteomics studies. In this study, we have used proteomics evidence to confirm expression of multiple proteins previously considered to be putative, as well as correct annotation errors in the genome of <it>Brucella abortus </it>strain 2308.</p

Identification of Circulating Endocan-1 and Ether Phospholipids as Biomarkers for Complications in Thalassemia Patients

Despite advances in our knowledge and attempts to improve therapies, β-thalassemia remains a prevalent disorder with increased risk for the development of cardiomyopathy. Using an untargeted discovery-based lipidomic workflow, we uncovered that transfusion-dependent thalassemia (TDT) patients had a unique circulating lipidomic signature consisting of 387 lipid features, allowing their significant discrimination from healthy controls (Q-value &lt; 0.01). In particular, TDT patients had elevated triacylglycerols and long-chain acylcarnitines, albeit lower ether phospholipids or plasmalogens, sphingomyelins, and cholesterol esters, reminiscent of that previously characterized in cardiometabolic diseases resulting from mitochondrial and peroxisomal dysfunction. Discriminating lipid (sub)classes correlated differentially with clinical parameters, reflecting blood (ether phospholipids) and iron (cholesterol ester) status or heart function (triacylglycerols). We also tested 15 potential serum biomarkers related to cardiometabolic disease and found that both lipocalin-2 and, for the first time, endocan-1 levels were significantly elevated in TDT patients and showed a strong correlation with blood parameters and three ether diacylglycerophosphatidylcholine species. In conclusion, this study identifies new characteristics of TDT patients which may have relevance in developing biomarkers and therapeutics

Lipidomics unveils lipid dyshomeostasis and low circulating plasmalogens as biomarkers in a monogenic mitochondrial disorder

Mitochondrial dysfunction characterizes many rare and common age-associated diseases. The biochemical consequences, underlying clinical manifestations, and potential therapeutic targets, remain to be better understood. We tested the hypothesis that lipid dyshomeostasis in mitochondrial disorders goes beyond mitochondrial fatty acid β-oxidation, particularly in liver. This was achieved using comprehensive untargeted and targeted lipidomics in a case-control cohort of patients with Leigh syndrome French-Canadian variant (LSFC), a mitochondrial disease caused by mutations in LRPPRC, and in mice harboring liver-specific inactivation of Lrpprc (H-Lrpprc–/–). We discovered a plasma lipid signature discriminating LSFC patients from controls encompassing lower levels of plasmalogens and conjugated bile acids, which suggest perturbations in peroxisomal lipid metabolism. This premise was reinforced in H-Lrpprc–/– mice, which compared with littermates recapitulated a similar, albeit stronger peroxisomal metabolic signature in plasma and liver including elevated levels of very-long-chain acylcarnitines. These mice also presented higher transcript levels for hepatic markers of peroxisome proliferation in addition to lipid remodeling reminiscent of nonalcoholic fatty liver diseases. Our study underscores the value of lipidomics to unveil unexpected mechanisms underlying lipid dyshomeostasis ensuing from mitochondrial dysfunction herein implying peroxisomes and liver, which likely contribute to the pathophysiology of LSFC, but also other rare and common mitochondrial diseases

SLC25A46 is required for mitochondrial lipid homeostasis and cristae maintenance and is responsible for Leigh syndrome

Abstract Mitochondria form a dynamic network that responds to physiological signals and metabolic stresses by altering the balance between fusion and fission. Mitochondrial fusion is orchestrated by conserved GTPases MFN1/2 and OPA1, a process coordinated in yeast by Ugo1, a mitochondrial metabolite carrier family protein. We uncovered a homozygous missense mutation in SLC25A46, the mammalian orthologue of Ugo1, in a subject with Leigh syndrome. SLC25A46 is an integral outer membrane protein that interacts with MFN2, OPA1, and the mitochondrial contact site and cristae organizing system (MICOS) complex. The subject mutation destabilizes the protein, leading to mitochondrial hyperfusion, alterations in endoplasmic reticulum (ER) morphology, impaired cellular respiration, and premature cellular senescence. The MICOS complex is disrupted in subject fibroblasts, resulting in strikingly abnormal mitochondrial architecture, with markedly shortened cristae. SLC25A46 also interacts with the ER membrane protein complex EMC, and phospholipid composition is altered in subject mitochondria. These results show that SLC25A46 plays a role in a mitochondrial/ER pathway that facilitates lipid transfer, and link altered mitochondrial dynamics to early‐onset neurodegenerative disease and cell fate decisions

Remodeling of lipid landscape in high fat fed very-long chain acyl-CoA dehydrogenase null mice favors pro-arrhythmic polyunsaturated fatty acids and their downstream metabolites

International audienceVery-long chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of mitochondrial long chain (LC) fatty acid β-oxidation (FAO). Inherited VLCAD deficiency (VLCADD) predisposes to neonatal arrhythmias whose pathophysiology is still not understood. We hypothesized that VLCADD results in global disruption of cardiac complex lipid homeostasis, which may set conditions predisposing to arrhythmia. To test this, we assessed the cardiac lipidome and related molecular markers in seven-month-old VLCAD-/- mice, which mimic to some extent the human cardiac phenotype. Mice were sacrificed in the fed or fasted state after receiving for two weeks a chow or a high-fat diet (HFD), the latter condition being known to worsen symptoms in human VLCADD. Compared to their littermate counterparts, HFD/fasted VLCAD-/- mouse hearts displayed the following lipid alterations: (1) Lower LC, but higher VLC-acylcarnitines accumulation, (2) higher levels of arachidonic acid (AA) and lower docosahexaenoic acid (DHA) contents in glycerophospholipids (GPLs), as well as (3) corresponding changes in pro-arrhythmogenic AA-derived isoprostanes and thromboxane B2 (higher), and anti-arrythmogenic DHA-derived neuroprostanes (lower). These changes were associated with remodeling in the expression of gene or protein markers of (1) GPLs remodeling: higher calcium-dependent phospholipase A2 and lysophosphatidylcholine-acyltransferase 2, (2) calcium handling perturbations, and (3) endoplasmic reticulum stress. Altogether, these results highlight global lipid dyshomeostasis beyond FAO in VLCAD-/- mouse hearts, which may set conditions predisposing the hearts to calcium mishandling and endoplasmic reticulum stress and thereby may contribute to the pathogenesis of arrhythmias in VLCADD in mice as well as in humans

Dependency on external supports:An addition to community vulnerability

In developing countries Non-governmental organizations (NGO) often get involved in development activities and provide supports for diminishing vulnerability through improving community capacities. Considering the context of involvement of NGOs, this research through studying two settlements in the Haor region of Bangladesh investigates the community resilience status, more specifically their current expectations from NGOs. Findings indicate that NGOs’ usual projects focus mainly on enhancing food security and eliminating poverty, targeting socio-economic and/or non-structural issues, whereas, communities from flood-prone Haor region prefer to have infrastructure-based development for ensuring permanent protection of their settlement from flood damages. NGOs’ regular projects or programs are small in scale and usually don’t enable communities to act independently now or in the future. Even after receiving external supports for extended periods, communities expect to receive further external supports for further development or improvement which can enhance their flood resilience level. In this way, the ‘incentive-induced development’, as an outcome of organized forms (organizational, technological and financial) of external supports, develops dependency on external supports among these communities. Identifying dependency on external supports is a new addition to community vulnerability. This research contributes to understanding risks and questions the usual practices of providing NGO supports to vulnerable communities

Intracellular adaptation of Brucella abortus

Macrophages were infected with virulent B. abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration, adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strain’s adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infectionsLos macrófagos se infectaron con la cepa 2308 de B. abortus o la cepa 19 atenuada. Las bacterias intracelulares se recuperaron en diferentes momentos después de la infección y se compararon sus proteomas. La cepa virulenta inicialmente redujo la mayoría de la biosíntesis y alteró su respiración, adaptaciones que se revirtieron más tarde en la infección. La cepa atenuada no pudo igualar la magnitud de los ajustes de la cepa virulenta. Los resultados proporcionan una visión de los mecanismos utilizados por Brucella para establecer infecciones intracelulares.Escuela de Medicina Veterinari