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Richard, B. C., Bayer, T. A., Lind, S., Shevchenko, G. & Bergquist, J. (2019). A simplified and sensitive immunoprecipitation mass spectrometry protocol for the analysis of amyloid-beta peptides in brain tissue. CLINICAL MASS SPECTROMETRY, 14, 83-88
Open this publication in new window or tab >>A simplified and sensitive immunoprecipitation mass spectrometry protocol for the analysis of amyloid-beta peptides in brain tissue
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2019 (English)In: CLINICAL MASS SPECTROMETRY, ISSN 2376-9998, Vol. 14, p. 83-88Article in journal (Refereed) Published
Abstract [en]

In the field of Alzheimer's disease, there is an urgent need for novel analytical tools to identify disease-specific biomarkers and to evaluate therapeutics. Preclinical trials commonly employ amyloid beta (A beta) peptide signatures as a read-out. In this paper, we report a simplified and detailed protocol for robust immunoprecipitation of A beta in brain tissue prior to mass spectrometric detection exemplified by a study using transgenic mice. The established method employed murine monoclonal and rabbit polyclonal antibodies and was capable of yielding well-reproducible peaks of high intensity with low background signal intensities corresponding to various A beta forms.

Place, publisher, year, edition, pages
ELSEVIER, 2019
Keywords
MALDI-TOF MS, Amyloid beta peptides, Brain, Immunoprecipitation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-398015 (URN)10.1016/j.clinms.2019.07.001 (DOI)000496422100004 ()
Funder
Swedish Foundation for Strategic Research , SB16-0039Swedish Research Council, 621-2011-4423Swedish Research Council, 2015-4870Magnus Bergvall Foundation, 2018-01726
Available from: 2019-12-04 Created: 2019-12-04 Last updated: 2019-12-04Bibliographically approved
Asser, A., Koks, S., Soomets, U., Terasmaa, A., Sauk, M., Eltermaa, M., . . . Taba, P. (2019). Acute effects of methcathinone and manganese in mice: A dose response study. HELIYON, 5(9), Article ID e02475.
Open this publication in new window or tab >>Acute effects of methcathinone and manganese in mice: A dose response study
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2019 (English)In: HELIYON, ISSN 2405-8440, Vol. 5, no 9, article id e02475Article in journal (Refereed) Published
Abstract [en]

An intravenously injectable illicit drug made by mixing pseudoephedrine, potassium permanganate, vinegar and water, yielding methcathinone (Mcat) and manganese (Mn), induces an extrapyramidal syndrome with parkinsonism, dystonia, gait and balance disorders similar to manganism. Although the cause of the syndrome is largely attributed to Mn, the interaction of the drug's individual components is not known and the role of Mcat is possibly underestimated. Aim of the present study was to analyze dose-dependent behavioral effects of the mixture and its two main active components Mcat and Mn in an acute setting and determine the lethal doses of each substance. Three groups of C57BL/6 mice were injected intraperitoneally with (1) the drug mixture containing 10, 25, 50, 100 or 150 mg of Mcat and respectively 1.6, 3.8, 6.9, 17.1 and 22.6 mg of Mn per kilogram of body weight; (2) 10, 25, 50, 100, 150, 200 or 300 mg of racemic Mcat/kg of body weight; (3) MnCl2 10, 25 or 50 mg/kg of body weight. Locomotor activity of the animals, various signs and time of death were recorded. Lower doses (10 and 25 mg/kg) of Mcat had a clear motor activity stimulating effect and this was clearly dose-dependent. High doses of Mcat produced epileptic seizures in 74% of the animals and became lethal with the highest doses. Similarly, the mixture had a clear dose-dependent stimulating effect and the higher doses became lethal. The LD50 of the pseudoephedrine mixture was 110.2 mg of Mcat/kg and for pure Mcat 201.7 mg/kg. Mn did not prove to be lethal in doses up to 50 mg/kg, but had a strong dose dependent inhibitory effect on the animals' behavior. Our data reveal that both Mn and Mcat have a significant role in the toxicity of the mixture.

Place, publisher, year, edition, pages
ELSEVIER SCI LTD, 2019
Keywords
Neuroscience, Toxicology, Behavioral neuroscience, Dose-response relationship, Nervous system, Neurotoxicology, Ephedrone, Toxic parkinsonism, Manganese, Methcathinone
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-396626 (URN)10.1016/j.heliyon.2019.e02475 (DOI)000488879100006 ()
Available from: 2019-11-13 Created: 2019-11-13 Last updated: 2019-11-13Bibliographically approved
Källsten, M., Pijnappel, M., Hartmann, R., Lehmann, F., Kovac, L., Lind, S. & Bergquist, J. (2019). Application of triple quadrupole mass spectrometry for the characterization of antibody-drug conjugates. Analytical and Bioanalytical Chemistry, 411(12), 2569-2576
Open this publication in new window or tab >>Application of triple quadrupole mass spectrometry for the characterization of antibody-drug conjugates
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2019 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 12, p. 2569-2576Article in journal (Refereed) Published
Abstract [en]

Antibody-drug conjugates (ADCs) are an inherently heterogeneous class of biotherapeutics, the development of which requires extensive characterization throughout. During the earliest phases of preclinical development, when synthetic routes towards the desired conjugate are being assessed, the main interest lies in the determination of the average drug-to-antibody ratio (DAR) of a given batch as well as information about different conjugation species. There has been a trend in mass spectrometry (MS)-based characterization of ADCs towards the use of high-resolving mass spectrometry for many of these analyses. Considering the high cost for such an instrument, the evaluation of cheaper and more accessible alternatives is highly motivated. We have therefore tested the applicability of a quadrupole mass analyzer for the aforementioned characterizations. Eight ADCs consisting of trastuzumab and varying stoichiometries of Mc-Val-Cit-PABC-monomethyl auristatin E conjugated to native cysteines were synthesized and served as test analytes. The average DAR value and molecular weights (Mw) of all detected chains from the quadrupole mass analyzer showed surprisingly high agreement with results obtained from a time-of-flight (TOF) mass analyzer and hydrophobic interaction chromatography (HIC)-derived values for all investigated ADC batches. Acquired Mw were within 80ppm of TOF-derived values, and DAR was on average within 0.32 DAR units of HIC-derived values. Quadrupole mass spectrometers therefore represent a viable alternative for the characterization of ADC in early-stage development.

Place, publisher, year, edition, pages
SPRINGER HEIDELBERG, 2019
Keywords
Reversed-phase liquid chromatography, Antibody-drug conjugates, Drug-to-antibody ratio, Triple quadrupole mass spectrometry
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-383155 (URN)10.1007/s00216-019-01699-0 (DOI)000464948100011 ()30848315 (PubMedID)
Funder
Swedish Foundation for Strategic Research , ID14-0081Swedish Foundation for Strategic Research , SB16-0039Swedish Research Council, 2015-4870
Available from: 2019-05-10 Created: 2019-05-10 Last updated: 2020-01-31Bibliographically approved
Hawkes, J. A., Sjöberg, P. J. R., Bergquist, J. & Tranvik, L. (2019). Complexity of dissolved organic matter in the molecular size dimension: insights from coupled size exclusion chromatography electrospray ionisation mass spectrometry. Paper presented at Conference on Challenges in Analysis of Complex Natural Mixtures, Univ Edinburgh, Edinburgh, Scotland, May 13-15, 2019. Faraday discussions (Online), 218, 52-71
Open this publication in new window or tab >>Complexity of dissolved organic matter in the molecular size dimension: insights from coupled size exclusion chromatography electrospray ionisation mass spectrometry
2019 (English)In: Faraday discussions (Online), ISSN 1359-6640, E-ISSN 1364-5498, Vol. 218, p. 52-71Article in journal (Refereed) Published
Abstract [en]

This paper investigates the relationship between apparent size distribution and molecular complexity of dissolved organic matter from the natural environment. We used a high pressure size exclusion chromatography (HPSEC) method coupled to UV-Vis diode array detection (UV-DAD) and electrospray ionisation mass spectrometry (ESI-MS) in order to compare the apparent size of natural organic matter, determined by HPSEC-UV and the molecular mass determined online by ESI-MS. We found that there was a clear discrepancy between the two methods, and found evidence for an important pool of organic matter that has a strong UV absorbance and no ESI-MS signal. Contrary to some previous research, we found no evidence that apparently high molecular weight organic matter is constituted by aggregates of low molecular weight (<1000 Da) material. Furthermore, our results suggest that the majority of apparent size variability within the ESI ionisable pool of organic matter is due to secondary interaction and exclusion effects on the HPSEC column, and not true differences in hydrodynamic size or intermolecular aggregation.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-384038 (URN)10.1039/c8fd00222c (DOI)000481497900003 ()31120465 (PubMedID)
Conference
Conference on Challenges in Analysis of Complex Natural Mixtures, Univ Edinburgh, Edinburgh, Scotland, May 13-15, 2019
Available from: 2019-06-04 Created: 2019-06-04 Last updated: 2019-10-03Bibliographically approved
Karunasekera, H., Pettersson, J., Mi, J., Bergquist, J. & Daniel, G. (2019). Copper tolerance of the soft-rot fungus Phialophora malorum grown in-vitro revealed by microscopy and global protein expression. International Biodeterioration & Biodegradation, 137, 147-152
Open this publication in new window or tab >>Copper tolerance of the soft-rot fungus Phialophora malorum grown in-vitro revealed by microscopy and global protein expression
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2019 (English)In: International Biodeterioration & Biodegradation, ISSN 0964-8305, E-ISSN 1879-0208, Vol. 137, p. 147-152Article in journal (Refereed) Published
Abstract [en]

In this study, we used proteomics in conjunction with microscopy to study differences in the proteome and hyphal morphology of the copper tolerant soft rot fungus Phialophora malorum grown in media containing 0.064, 0.64% Cu as CuSO4. Unique proteins were found in the control and the copper-treated (0.064% CuSO4) samples. Of five unique proteins found in the 0.064% CuSO4 treated cultures, ATP synthase subunit alpha is considered to play an important role in copper tolerance as it is involved in the biosynthesis of fatty acids and steroids and may relate to morphological changes associated with hyphal cell walls of the fungus when grown in the presence of copper. ICP-AES analyses showed total mycelial Cu to increase with media Cu with 5246- and 16535 mu g Cu/g dry wt mycelia respectively found in 0.064 and 0.64% Cu-cultures after 6 weeks growth. Rubeanic acid staining of 0.064% mycelia showed Cu bound in intracellular bodies while most Cu was found as extracellular precipitates on the surfaces of hyphae in 0.64% Cu. SEM showed hyphal surfaces enrobed in fibrillar polysaccharides to which Cu was bound.

Keywords
Copper tolerance, ICP-AES, LC-MS/MS, Phialophora malorum, Proteomics, Soft rot fungi
National Category
Wood Science
Identifiers
urn:nbn:se:uu:diva-378196 (URN)10.1016/j.ibiod.2018.12.001 (DOI)000458470800017 ()
Funder
Swedish Research Council Formas, 2011-416Swedish Research Council Formas, 2011-6383-19675Swedish Research Council Formas, 621-2015-4870
Available from: 2019-03-07 Created: 2019-03-07 Last updated: 2019-03-07Bibliographically approved
Leito, I., Teearu, A., Bobacka, J., Randon, J. & Bergquist, J. (2019). EACH (Excellence in Analytical Chemistry), an Erasmus Mundus Joint Programme: progress and success. Analytical and Bioanalytical Chemistry, 411(23), 5913-5921
Open this publication in new window or tab >>EACH (Excellence in Analytical Chemistry), an Erasmus Mundus Joint Programme: progress and success
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2019 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 23, p. 5913-5921Article in journal, Editorial material (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER HEIDELBERG, 2019
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-394143 (URN)10.1007/s00216-019-01988-8 (DOI)000482448100002 ()31392438 (PubMedID)
Available from: 2019-10-04 Created: 2019-10-04 Last updated: 2019-10-04Bibliographically approved
Zayny, A., Almokhtar, M., Wikvall, K., Ljunggren, Ö., Ubhayasekera, K., Bergquist, J., . . . Norlin, M. (2019). Effects of glucocorticoids on vitamin D3-metabolizing 24-hydroxylase (CYP24A1) in Saos-2 cells and primary human osteoblasts. Molecular and Cellular Endocrinology, 496, Article ID 110525.
Open this publication in new window or tab >>Effects of glucocorticoids on vitamin D3-metabolizing 24-hydroxylase (CYP24A1) in Saos-2 cells and primary human osteoblasts
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2019 (English)In: Molecular and Cellular Endocrinology, ISSN 0303-7207, E-ISSN 1872-8057, Vol. 496, article id 110525Article in journal (Refereed) Published
Abstract [en]

Vitamin D is essential for bone function and deficiency in active vitamin D hormone can lead to bone disorders. Long-term treatment with glucocorticoids results in osteoporosis and increased risk of fractures. Much remains unclear regarding the effects of these compounds in bone cells. In the current study, human osteosarcoma Saos-2 cells and primary human osteoblasts were found to express mRNA for the vitamin D receptor as well as activating and deactivating enzymes in vitamin D-3 metabolism. These bone cells exhibited CYP24A1-mediated 24-hydroxylation which is essential for deactivation of the active vitamin form. However, bioactivating vitamin D-3 hydroxylase activities could not be detected in either of these cells. Several glucocorticoids, including prednisolone, down regulated CYP24A1 mRNA and CYP24A1-mediated 24-hydroxylase activity in both Saos-2 and primary human osteoblasts. Also, prednisolone significantly suppressed a human CYP24A1 promoter-luciferase reporter gene in Saos-2 cells co-transfected with the glucocorticoid receptor. Thus, the results of the present study show suppression by glucocorticoids on CYP24A1 mRNA, CYP24A1-mediated metabolism and CYP24A1 promoter activity in human osteoblast-like cells. As part of this study we examined if glucocorticoids are formed locally in Saos-2 cells. The experiments indicate formation of 11-deoxycortisol, a steroid with glucocorticoid activity, which can bind the glucocorticoid receptor. Our data showing suppression by glucocorticoids on CYP24A1 expression in human osteoblasts suggest a previously unknown mechanism for effects of glucocorticoids in human bone, where these compounds may interfere with regulation of active vitamin D levels.

Keywords
Vitamin D, Bone, Metabolism, Osteoblast, Osteosarcoma, Steroid
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-395784 (URN)10.1016/j.mce.2019.110525 (DOI)000487328200005 ()31352041 (PubMedID)
Funder
Swedish Research Council, 2015-4870
Available from: 2019-10-28 Created: 2019-10-28 Last updated: 2019-10-28Bibliographically approved
Manell, H., Kristinsson, H., Kullberg, J., Ubhayasekera, S. J., Mörwald, K., Staaf, J., . . . Bergsten, P. (2019). Hyperglucagonemia in youth is associated with high plasma free fatty acids, visceral adiposity and impaired glucose tolerance. Pediatric Diabetes, 20(7), 880-891
Open this publication in new window or tab >>Hyperglucagonemia in youth is associated with high plasma free fatty acids, visceral adiposity and impaired glucose tolerance
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2019 (English)In: Pediatric Diabetes, ISSN 1399-543X, E-ISSN 1399-5448, Vol. 20, no 7, p. 880-891Article in journal (Refereed) Published
Abstract [en]

Objective: To delineate mechanisms for fasting hyperglucagonemia in childhood obesity bystudying the associations between fasting plasma glucagon concentrations and plasmalipid parameters and fat compartments.

Methods: Cross-sectional study of children and adolescents with obesity (n=147) and leancontrols (n=43). Differences in free fatty acids (FFA), triglycerides, insulin and fatcompartments (quantified by magnetic resonance imaging) across quartiles of fastingplasma glucagon concentration were analysed. Differences in OGTT glucagonresponse was tested in high vs low FFAs, triglycerides and insulin. Human islets ofLangerhans were cultured at 5.5 mmol/l glucose and in the absence or presence of aFFA mixture with total FFA concentration of 0.5 mmol/l and glucagon secretionquantified.

Results: In children with obesity, the quartile with the highest fasting glucagon had higherinsulin (201±174 vs 83±39 pmol/l, p<0.01), FFAs (383±52 vs 338±109 μmol/l,p=0.02), triglycerides (1.5±0.9 vs 1.0±0.7 mmol/l, p<0.01), visceral adipose tissuevolume (1.9±0.8 vs 1.2±0.3 dm3, p<0.001) and a higher prevalence of impairedglucose tolerance (41% vs 8%, p=0.01) than the lowest quartile. During OGTT,children with obesity and high insulin had a worse suppression of glucagon during thefirst 10 minutes after glucose intake. Glucagon secretion was 2.6-fold higher in isletstreated with FFAs than in those not treated with FFAs.4

Conclusion: Hyperglucagonemia in childhood obesity is associated with hyperinsulinemia, highplasma FFAs, high plasma triglycerides, visceral adiposity and impaired glucosetolerance. The glucagonotropic effect of FFAs on isolated human islets provides apotential mechanism linking high fasting plasma FFAs and glucagon levels.

Keywords
Childhood obesity, glucagon, free fatty acids, insulin, visceral adiposity, impaired glucose tolerance, type 2 diabetes
National Category
Pediatrics Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-380313 (URN)10.1111/pedi.12890 (DOI)000476081000001 ()31271247 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 279153EXODIAB - Excellence of Diabetes Research in SwedenErnfors FoundationErik, Karin och Gösta Selanders FoundationSwedish Research Council, 2015-4870Swedish Diabetes Association
Available from: 2019-03-26 Created: 2019-03-26 Last updated: 2019-12-06Bibliographically approved
Zhu, Y., Qi, X., Yu, C., Yu, S., Zhang, C., Zhang, Y., . . . Mi, J. (2019). Identification of prothymosin alpha (PTMA) as a biomarker for esophageal squamous cell carcinoma (ESCC) by label-free quantitative proteomics and Quantitative Dot Blot (QDB). Clinical Proteomics, 16, Article ID 12.
Open this publication in new window or tab >>Identification of prothymosin alpha (PTMA) as a biomarker for esophageal squamous cell carcinoma (ESCC) by label-free quantitative proteomics and Quantitative Dot Blot (QDB)
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2019 (English)In: Clinical Proteomics, ISSN 1542-6416, E-ISSN 1559-0275, Vol. 16, article id 12Article in journal (Refereed) Published
Abstract [en]

Background: Esophageal cancer (EC) is one of the malignant tumors with a poor prognosis. The early stage of EC is asymptomatic, so identification of cancer biomarkers is important for early detection and clinical practice.

Methods: In this study, we compared the protein expression profiles in esophageal squamous cell carcinoma (ESCC) tissues and adjacent normal esophageal tissues from five patients through high-resolution label-free mass spectrometry. Through bioinformatics analysis, we found the differentially expressed proteins of ESCC. To perform the rapid identification of biomarkers, we adopted a high-throughput protein identification technique of Quantitative Dot Blot (QDB). Meanwhile, the QDB results were verified by classical immunohistochemistry.

Results: In total 2297 proteins were identified, out of which 308 proteins were differentially expressed between ESCC tissues and normal tissues. By bioinformatics analysis, the four up-regulated proteins (PTMA, PAK2, PPP1CA, HMGB2) and the five down-regulated proteins (Caveolin, Integrin beta-1, Collagen alpha-2(VI), Leiomodin-1 and Vinculin) were selected and validated in ESCC by Western Blot. Furthermore, we performed the QDB and IHC analysis in 64 patients and 117 patients, respectively. The PTMA expression was up-regulated gradually along the progression of ESCC, and the PTMA expression ratio between tumor and adjacent normal tissue was significantly increased along with the progression. Therefore, we suggest that PTMA might be a potential candidate biomarker for ESCC.

Conclusion: In this study, label-free quantitative proteomics combined with QDB revealed that PTMA expression was up-regulated in ESCC tissues, and PTMA might be a potential candidate for ESCC. Since Western Blot cannot achieve rapid and high-throughput screening of mass spectrometry results, the emergence of QDB meets this demand and provides an effective method for the identification of biomarkers.

Place, publisher, year, edition, pages
BMC, 2019
Keywords
Esophageal squamous cell carcinoma (ESCC), Label-free quantitative proteomics, Prothymosin alpha (PTMA), Quantitative Dot Blot (QDB)
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-382384 (URN)10.1186/s12014-019-9232-6 (DOI)000463784200001 ()30988666 (PubMedID)
Available from: 2019-04-26 Created: 2019-04-26 Last updated: 2019-04-26Bibliographically approved
Bergquist, J. (2019). Leveraging the power of mass spectrometry to unravel complex brain pathologies. CLINICAL MASS SPECTROMETRY, 14, 63-65
Open this publication in new window or tab >>Leveraging the power of mass spectrometry to unravel complex brain pathologies
2019 (English)In: CLINICAL MASS SPECTROMETRY, ISSN 2376-9998, Vol. 14, p. 63-65Article in journal, Editorial material (Other academic) Published
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-398130 (URN)10.1016/j.clinms.2019.08.002 (DOI)000496422100001 ()
Available from: 2019-12-04 Created: 2019-12-04 Last updated: 2019-12-04Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-4597-041x

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