Jerneren F.,University of Oxford |
Soderquist M.,Denator Inc. |
Karlsson O.,Karolinska Institutet
Journal of Pharmacological and Toxicological Methods | Year: 2015
Introduction: The field of lipid research has made progress and it is now possible to study the lipidome of cells and organelles. A basic requirement of a successful lipid study is adequate pre-analytical sample handling, as some lipids can be unstable and postmortem changes can cause substantial accumulation of free fatty acids (FFAs). Methods: The aim of the present study was to investigate the effects of conductive heat stabilization and euthanasia methods on FFA levels in the rat brain and liver using liquid chromatography tandem mass spectrometry. Results: The analysis of brain homogenates clearly demonstrated phospholipase activity and time-dependent post-sampling changes in the lipid pool of snap frozen non-stabilized tissue. There was a significant increase in FFAs already at 2min, which continued over time. Heat stabilization was shown to be an efficient method to reduce phospholipase activity and ex vivo lipolysis. Post-sampling effects due to tissue thawing and sample preparation induced a massive release of FFAs (up to 3700%) from non-stabilized liver and brain tissues compared to heat stabilized tissue. Furthermore, the choice of euthanasia method significantly influenced the levels of FFAs in the brain. The FFAs were decreased by 15-44% in the group of animals euthanized by pentobarbital injection compared with CO2 inhalation or decapitation. Discussion: Our results highlight the importance of considering euthanasia methods and pre-analytical treatment in lipid analysis, factors which may otherwise interfere with the outcome of the experiments. © 2014 Elsevier Inc.
Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2012.2.1.1-3 | Award Amount: 3.53M | Year: 2012
Since biology is by and large a 3-dimensional phenomenon, 3D imaging has a significant impact on many challenges in life sciences. 3D matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an emerging label-free 3D imaging technology with high potential in proteomics and metabolomics. 3D MALDI-IMS is based on 2D MALDI-IMS, which in the last years has proven its value in metabolomics, glycomics, lipidomics, peptidomics, and proteomics. However, 3D MALDI-IMS cannot tap its full potential due to the lack of computational methods for processing large and complex 3D IMS data. Our main goal is to realise 3D label-free proteomics and metabolomics by providing new statistical methods for the reproducible collection of 3D MALDI-IMS data, and for un- and supervised statistical analysis and interpretation of this data. We will validate our methods in diabetes research, surgical metabolomics, and natural products research. To reach our global goal, we will develop methodologies for reproducible collection of 3D MALDI-IMS data, develop a statistical simulator of MALDI-IMS data and evaluation strategies for statistical methods, develop preprocessing, unsupervised, and supervised methods of statistical analysis of 3D MALDI-IMS data, evaluate the developed methods on simulated and real-life proteomics and metabolomics data, and implement them using efficient graphic processing unit (GPU) architecture. This project is of high importance for research-intensive SME partners, Denator and SagivTech, since it would create a new market segment for them. Expertise of partners in biomedicine and their strong connections to clinical partners ensure adoption of the new methods into clinical practice. The developed methods and protocols can be potentially used in any biomedical application where 3D MALDI-IMS is advantageous such as cancer and diabetes diagnostics, disease pathway elucidation, histopathology, and discovery of biomarkers or antibiotics.
Boren M.,Denator Inc.
Methods in Molecular Biology | Year: 2011
The ability to adequately measure the phosphorylation state of a protein has major biological as well as clinical relevance. Due to its variable nature, reversible protein phosphorylations are sensitive to changes in the tissue environment. Stabilizor T1 is a system for rapid inactivation of enzymatic activity in biological samples. Enzyme inactivation is accomplished using thermal denaturation in a rapid, homogeneous, and reproducible fashion without the need for added inhibitors. Using pCREB(Ser133) as a model system, the applicability of the Stabilizor system to preserve a rapidly lost phosphorylation is shown. © 2011 Springer Science+Business Media, LLC.
Skold K.,Denator Inc. |
Alm H.,Uppsala University |
Scholz B.,Uppsala University
Molecular and Cellular Proteomics | Year: 2013
The separation between biological and technical variation without extensive use of technical replicates is often challenging, particularly in the context of different forms of protein and peptide modifications. Biosampling procedures in the research laboratory are easier to conduct within a shorter time frame and under controlled conditions as compared with clinical sampling, with the latter often having issues of reproducibility. But is the research laboratory biosampling really less variable? Biosampling introduces within minutes rapid tissue-specific changes in the cellular microenvironment, thus inducing a range of different pathways associated with cell survival. Biosampling involves hypoxia and, depending on the circumstances, hypothermia, circumstances for which there are evolutionarily conserved defense strategies in the range of species and also are relevant for the range of biomedical conditions. It remains unclear to what extent such adaptive processes are reflected in different biosampling procedures or how important they are for the definition of sample quality. Lately, an increasing number of comparative studies on different biosampling approaches, postmortem effects and pre-sampling biological state, have investigated such immediate early biosampling effects. Commonalities between biosampling effects and a range of ischemia/reperfusion- and hypometabolism/anoxia-associated biological phenomena indicate that even small variations in post-sampling time intervals are likely to introduce a set of nonrandom and tissue-specific effects of experimental importance (both in vivo and in vitro). This review integrates the information provided by these comparative studies and discusses how an adaptive biological perspective in biosampling procedures may be relevant for sample quality issues. © 2013 by The American Society for Biochemistry and Molecular Biology.
Kultima K.,Uppsala University |
Skold K.,Denator Inc. |
Boren M.,Denator Inc.
Journal of Proteomics | Year: 2011
This review focuses on post sampling changes and how the Stabilizor system has been used to control this natural biological process and potential implications on cancer-specific biomarkers due to post sampling changes. Tissue sampling is a major traumatic event that can have drastic effects within a very short timeframe at the molecular level  resulting in loss of sample quality due to post-mortem changes. A heat-stabilization technology, using the Stabilizor system, has been developed to quickly and permanently abolish the enzymatic activity that causes these changes post-sampling and so preserve sample quality. The Stabilizor system has been shown to give better sample quality when analyzing a variety of tissues in various proteomic workflows. In this paper we discuss the impact of using heat-stabilized tissue in different proteomic applications. Based on our observations regarding the overlap between commonly changing proteins and proteins found to change post-mortem we also highlight a group of proteins of particular interest in cancer studies. © 2011 Elsevier B.V.
Denator Inc. | Date: 2012-07-09
The present invention relates to a method for rapid stabilization of fluid biological samples, such as blood samples. More specifically, the method is based on heat stabilization of the fluid biological sample absorbed in a matrix.
Denator Inc. | Date: 2010-06-08
The present invention provides methods for stabilizing a biological sample for analysis. The invention more particularly provides methods combining heat treatment and chemical fixation of biological samples in order to maintain protein primary structure and post-translational modifications, such as protein phosphorylations.
Agency: European Commission | Branch: H2020 | Program: SME-1 | Phase: PHC-12-2015-1 | Award Amount: 71.43K | Year: 2015
The risk of epidemics due to infectious diseases caused by pathogens is an immense global challenge. The need for rapid development of vaccines, antiretroviral drugs and treatment schemes is at the top of most countries and organizations agenda. Performing biomarker research on infectious diseases is an important research area to understand and monitor disease state and progression as well as enable diagnosis and vaccine- and drug development. The project aim is to further develop and verify the Stabilizor system to enable complete inactivation of pathogens, while still preserving sensitive biological biomarkers, in a rapid and secure manner. By preserving sensitive biomarkers early on in the analytical workflow, researchers will be able to conduct more efficient and reliable biomarker research. This will enable an accelerated process of developing vaccines and antiretroviral drugs, especially in the exploratory- and pre-clinical stages where identifying and assessing disease targets and biomarker candidates is the main focus. Also, this would allow for safe transportation of infected samples from the collection site in-field or between different laboratories to facilitate proper analysis. This will have profound social- and economic impact globally. This is of particular interest for research on highly contagious pathogens where extensive biocontainment precautions are required in enclosed laboratory facilities such as BSL-3 and -4 laboratories. Denators Stabilizor system preserves the quality of biological samples from the moment of excision allowing researchers to confidently identify and verify potential biomarkers. The novelty lies in the ability to rapidly heat inactive tissue samples (within 60 seconds) to achieve both complete pathogen inactivation and ensure biomarker stability. A first customer has demonstrated complete inactivation of several infectious pathogens which is a strong proof-of-concept for the applicability of the system.
Denator Inc. | Date: 2016-06-15
Chemicals for use within biotechnological analysis. Scienctific apparatus and instruments for handling of biological samples for use within biotechnological research, industry and medical care. Medical apparatus and instruments for handling of biological samples for use within biotechnological research, industry and medical care. Performance of biotechnological analysis.