Comparison between 5 extractions methods in either plasma or serum to determine the optimal extraction and matrix combination for human metabolomics.

BACKGROUND: Although metabolomics continues to expand in many domains of research, methodological issues such as sample type, extraction and analytical protocols have not been standardized, impeding proper comparison between studies and future research. METHODS: In the present study, five solvent-based and solid-phase extraction methods were investigated in both plasma and serum. All these extracts were analyzed using four liquid chromatography coupled with high resolution mass spectrometry (LC-MS) protocols, either in reversed or normal-phase and with both types of ionization. The performances of each method were compared according to putative metabolite coverage, method repeatability and also extraction parameters such as overlap, linearity and matrix effect; in both untargeted (global) and targeted approaches using fifty standard spiked analytes. RESULTS: Our results verified the broad specificity and outstanding accuracy of solvent precipitation, namely methanol and methanol/acetonitrile. We also reveal high orthogonality between methanol-based methods and SPE, providing the possibility of increased metabolome coverage, however we highlight that such potential benefits must be weighed against time constrains, sample consumption and the risk of low reproducibility of SPE method. Furthermore, we highlighted the careful consideration about matrix choice. Plasma showed the most suitable in this metabolomics approach combined with methanol-based methods. CONCLUSIONS: Our work proposes to facilitate rational design of protocols towards standardization of these approaches to improve the impact of metabolomics research.

How to improve results after DCD (donation after circulation death).

The shortage of organs for transplantation has led health professionals to look for alternative sources of donors. One of the avenues concerns donors who have died after circulatory arrest. This is a special situation because the organs from these donors are exposed to warm ischaemia-reperfusion lesions that are unavoidable during the journey of the organs from the donor to the moment of transplantation in the recipient. We will address and discuss the key issues from the perspective of team organization, legislation and its evolution, and the ethical framework. In a second part, the avenues to improve the quality of organs will be presented following the itinerary of the organs between the donor and the recipient. The important moments from the point of view of therapeutic strategy will be put into perspective. New connections between key players involved in pathophysiological mechanisms and implications for innate immunity and injury processes are among the avenues to explore. Technological developments to improve the quality of organs from these recipients will be analyzed, such as perfusion techniques with new modalities of temperatures and oxygenation. New molecules are being investigated for their potential role in protecting these organs and an analysis of potential prospects will be proposed. Finally, the important perspectives that seem to be favored will be discussed in order to reposition the use of deceased donors after circulatory arrest. The use of these organs has become a routine procedure and improving their quality and providing the means for their evaluation is absolutely inevitable.

Molecular Markers of Kidney Transplantation Outcome: Current Omics Tools and Future Developments.

PURPOSE OF REVIEW: The emerging field of molecular predictive medicine is aiming to change the traditional medical approach in renal transplantation. Many studies have explored potential biomarker molecules with predictive properties in renal transplantation, issued from omics research. Herein, we review the biomarker molecules of four technologies (i.e., Genomics, Transcriptomics, Proteomics, and Metabolomics) associated with favorable kidney transplant outcomes. RECENT FINDINGS: Several panels of molecules have been associated with the outcome that the majority of markers are related to inflammation and immune response; although. other molecular ontologies are also represented, such as proteasome, growth, regeneration, and drug metabolism. Throughout this review, we highlight the lack of properly validated statistical demonstration. Indeed, the most preeminent molecular panels either remain at the limited size study stage or are not confirmed during large-scale studies. At the core of this problem, we identify the methodological shortcomings and propose a comprehensive workflow for discovery and validation of molecular biomarkers that aims to improve the relevance of these tools in the future. SUMMARY: Overall, adopting a patient management through omics approach could bring remarkable improvement to transplantation success. An increased effort and investment between scientists, medical biologists, and clinicians seem to be the path toward a proper solution.

Preservation of Organs to Be Transplanted: An Essential Step in the Transplant Process.

Organ transplantation remains the treatment of last resort in case of failure of a vital organ (lung, liver, heart, intestine) or non-vital organ (essentially the kidney and pancreas) for which supplementary treatments exist. It remains the best alternative both in terms of quality-of-life and life expectancy for patients and of public health expenditure. Unfortunately, organ shortage remains a widespread issue, as on average only about 25% of patients waiting for an organ are transplanted each year. This situation has led to the consideration of recent donor populations (deceased by brain death with extended criteria or deceased after circulatory arrest). These organs are sensitive to the conditions of conservation during the ischemia phase, which have an impact on the graft's short- and long-term fate. This evolution necessitates a more adapted management of organ donation and the optimization of preservation conditions. In this general review, the different aspects of preservation will be considered. Initially done by hypothermia with the help of specific solutions, preservation is evolving with oxygenated perfusion, in hypothermia or normothermia, aiming at maintaining tissue metabolism. Preservation time is also becoming a unique evaluation window to predict organ quality, allowing repair and/or optimization of recipient choice.

High Throughput Proteomic Exploration of Hypothermic Preservation Reveals Active Processes within the Cell Associated with Cold Ischemia Kinetic.

The demand for organs to be transplanted increases pressure on procurement centers, to the detriment of organ quality, increasing complications. New preservation protocols are urgently needed, requiring an in-depth understanding of ischemia-reperfusion mechanisms. We performed a proteomic analysis using LC-MS/MS-TOF data analyzed through R software and Cytoscape's ClueGO application, comparing the proteome of kidney endothelial cells, key cell type, subjected to 3, 6, 12, 19, and 24 h of cold ischemia and 6 h reperfusion. Critical pathways such as energy metabolism, cytoskeleton structure/transport system, and gene transcription/translation were modulated. Important time windows were revealed: a-during the first 3 h, central proteins were upregulated within these pathways; b-the majority of these upregulations were maintained until 12 h cold ischemia time (CIT); c-after that time, the overall decrease in protein expression was observed; d-at reperfusion, proteins expressed in response to cold ischemia were all downregulated. This shows that cold ischemia is not a simple slowing down of metabolism, as deep changes take place within the proteome on major pathways. Time-sensitive expression of key protein reveals possible quality biomarkers as well as potential targets for new strategies to maintain or optimize organ quality.