| 1. |
- Gennebäck, Nina, 1982-, et al.
(författare)
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Growth factor stimulation of cardiomyocytes induces changes in the transcriptional contents of secreted exosomes
- 2013
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Ingår i: Journal of Extracellular Vesicles. - : Co-Action Publishing. - 2001-3078.
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Tidskriftsartikel (refereegranskat)abstract
- Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in targets cells. We recently reported that cultured cardiomyocytes are able to release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the transcriptional content of the released exosomes.Exosomes were isolated from media collected from cultured cardiomyocytes (HL-1) with or without growth factor treatment (TGF-β2 and PDGF-BB), with a series of differential centrifugations, including preparative ultracentrifugation and separation with a sucrose gradient. The exosomes were characterized with dynamic light scattering (DLS), electron microscopy (EM) and Western blot and analyzed with Illumina whole genome microarray gene expression.The exosomes were rounded in shape and had an average size of 50–90 nm in diameter with no difference between treatment groups. Analysis of the mRNA content in repeated experiments conclusively revealed 505 transcripts in the control group, 562 in the TGF-β2-treated group and 300 in the PDGF-BB-treated group. Common transcripts (217) were found in all 3 groups.We show that the mode of stimulation of parental cells affects the characteristics of exosomes released. Hence, there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated, or not stimulated, with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system.
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| 2. |
- Malm, Linus, 1980-, et al.
(författare)
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Metabolomic Quality Assessment of EDTA Plasma and Serum Samples
- 2016
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Ingår i: Biopreservation and Biobanking. - : Mary Ann Liebert Inc. - 1947-5535 .- 1947-5543. ; 14:5, s. 416-423
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Tidskriftsartikel (refereegranskat)abstract
- Handling and processing of blood can significantly alter the molecular composition and consistency of biobank samples and can have a major impact on the identification of biomarkers. It is thus crucial to identify tools to determine the quality of samples to be used in biomarker discovery studies. In this study, a non-targeted gas chromatography/time-of-flight mass spectrometry (GC-TOFMS) metabolomic strategy was used with the aim of identifying quality markers for serum and plasma biobank collections lacking proper documentation of preanalytical handling. The effect of postcentrifugation delay was examined in serum stored in tubes with gel separation plugs and ethylenediaminetetraacetic acid (EDTA) plasma in tubes with or without gel separation plugs. The change in metabolic pattern was negligible in all sample types processed within 3 hours after centrifugation regardless of whether the samples were kept at 4 degrees C or 22 degrees C. After 8 and 24 hours postcentrifugation delay before aliquoting, there was a pronounced increase in the number of affected metabolites, as well as in the magnitude of the observed changes. No protective effect on the metabolites was observed in gel-separated EDTA plasma samples. In a separate series of experiments, lactate and glucose levels were determined in plasma to estimate the effect of precentrifugation delay. This separate experiment indicates that the lactate to glucose ratio may serve as a marker to identify samples with delayed time to centrifugation. Although our data from the untargeted GC-TOFMS analysis did not identify any specific markers, we conclude that plasma and serum metabolic profiles remain quite stable when plasma and serum are centrifuged and separated from the blood cells within 3 hours.
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| 3. |
- Malm, Linus, 1980-, et al.
(författare)
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Predicting amyloid aggregation rates of proteins using multivariate analysis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Several diseases have been linked to the presence of extracellular protein deposits of β-rich aggregates, known as amyloid fibrils. The formation of these fibrils and their precursors has been identified as key players in the development of these diseases. It is therefore desirable to gain a deeper understanding of the mechanism of amyloid aggregation.In this study we have used multivariate analysis to elucidate the most important physicochemical and structural factors of amino acids that are important for the amyloid aggregation. We used a combination of principal component analysis, orthogonal partial least squares and auto- and cross-covariance to investigate a database consisting of amyloid aggregation rate measurements of 77 AcP mutants.Our results show that changes in hydrophobic patterns, charge and β-sheet propensity is common for mutants with the largest changes in amyloid propensity. In addition, we can also, with reasonable accuracy, predict the amyloid aggregation rate of a test set of AcP mutants that were not used to create the initial aggregation model. Thus, the multivariate approach used in this study is shown to be powerful tools to extract important factors of protein amyloid aggregation that are hidden in the growing pool of available experimental data of amyloid aggregation.
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| 4. |
- Malm, Linus, 1980-
(författare)
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Size determination of hyaluronan and multivariate analysis of amyloid prone proteins
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background.The extracellular matrix surrounds all cells within our bodies. The glycosaminoglycan hyaluronan is a major component in the extracellular matrix. Despite its structural simplicity it has been shown to be involved in several important functions. It is a lubricant and shock absorber, as well as an important player in inflammation and tumor invasion. Many of its functions are closely related to its size and concentration in tissues. Therefore methods for measuring these properties are of great importance to properly understand the role that hyaluronan play in different events. Proteins are found both inside and outside cells, and they have a wide variety of functions. The protein structure and function is determined by the properties of their building blocks, the amino acids. Several diseases have been linked to changes in the amino acid sequence of certain proteins by mutations, causing the proteins to form extracellular deposits of structures called amyloid aggregates. The aim of this thesis is to investigate the function of hyaluronan in cell cultures, develop new methods for size determination hyaluronan and to use multivariate methods to provide prediction and better understanding of factors driving protein amyloid aggregation. Methods.Cardiomyocytes and fibroblast were cultured and stimulated by different growth factors. Hyaluronan was purified and its size and concentration were measured. Crosstalk between cardiomyocytes and fibroblast were investigated and gene expression of hyaluronan synthases was determined. A new method for size measurement of hyaluronan was developed. The amyloid aggregation rate of different mutants of acylphosphatase was predicted by multivariate analysis. Results. Cardiomyocytes stimulated by PDGF-BB produced hyaluronan. Cardiomyocytes could induce fibroblast to increase its hyaluronan production, through an unknown soluble factor. The cardiomyocyte gene expression changed when stimulated by hyaluronan. GEMMA was presented as a new method for size determination of hyaluronan. Amyloid aggregation of different acylphosphatase mutants could be predicted using a multivariate regression model of the physicochemical and structural properties of the amino acid sequence. Conclusion. It was shown that cardiomyocytes are not only able to produce hyaluronan, but also induce an increased hyaluronan production in other cells. GEMMA was proven suitable for size determination of hyaluronan at very low concentrations. Multivariate analysis showed that hydrophobic patterns and charge where the most important factors for amyloid aggregation of acylphosphatase.
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| 5. |
- Malm, Linus, 1980-, et al.
(författare)
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Size determination of hyaluronan using a gas-phase electrophoretic mobility molecular analysis
- 2012
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Ingår i: Glycobiology. - Oxford : Oxford University Press. - 0959-6658 .- 1460-2423. ; 22:1, s. 7-11
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Tidskriftsartikel (refereegranskat)abstract
- Hyaluronan (HA) is a linear non-sulfated polysaccharide mainly found in the extracellular matrix. The size of HA can vary from a fewq saccharides up to at least 25,000 units, reaching molecular weights of 10x103 kDa. HA has mainly biological functions, and both its size and tissue concentration play an important role in many physiological and phatological processes. It is relatively easy ti determine the HA concentration using enzyme-linked binding protein assays, but the molecular weight of HA has so far been shown to be a more challenging task to measure. Here, we present a method for size determination of HA using gas-phase electrophoretic mobility molecular analysis (GEMMA), which utilizes the electrophoretic mobility of molecules in air to estimate the molecular weight of the analyte. We show that this method gives reliable molecular weight estimations of HA in the range 30-2400 kDa, which covers almost its whole biological range. The average measuring time for one GEMMA spectrum is between 5 and 10 min using only 6 pg of HA. In addition the peak area in a GEMMA spectrum can be used to estimate the HA concentration in the sample. The high sensitivity and small sample volumes make GEMMA an excellent tool for both size determination and estimation of concentration of samples with low HA concentration, as is the case for HA extracted from small tissue samples.
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