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  • 501.
    Qundos, Ulrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hong, Mun-Gwan
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Tybring, Gunnel
    Divers, Mark
    Odeberg, Jacob
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Profiling post-centrifugation delay of serum and plasma with antibody bead arrays2013Ingår i: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 95, nr SI, s. 46-54Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several biobanking initiatives have emerged to create extensive collections of specimen for biomedical studies and various analytical platforms. An affinity proteomic analysis with antibody suspension bead arrays was conducted to investigate the influence of the pre-analytical time and temperature conditions on blood derived samples. Serum and EDTA plasma prepared from 16 individuals was centrifuged and aliquots were kept either at 4. °C or in ambient temperature for 1. h and up to 36. h prior to first storage. Multiplexed protein profiles of post-centrifugation delay were generated in 384 biotinylated samples using 373 antibodies that targeted 343 unique proteins. Very few profiles were observed as significantly altered by the studied temperature and time intervals. Single binder and sandwich assays revealed decreasing levels of caldesmon 1 (CALD1) related to EDTA standard tubes and prolonged post-centrifugation delay of 36. h. Indications from changes in CALD1 levels require further confirmation in independent material, but the current data suggests that samples should preferentially be frozen during the day of collection when to be profiled with antibody arrays selected for this study. Biological significance: Affinity-based profiling of serum and plasma by microarray assays can provide unique opportunities for the discovery of biomarkers. It is though often not known how differences in sample handling after collection influence the downstream analysis. By profiling three types of blood preparations for alterations in protein profiles with respect to time and temperature post centrifugation, we addressed an important component in the analysis and of such specimen. We believe that this analysis adds valuable information to be considered when biobanking blood derived samples.This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.

  • 502.
    Qundos, Ulrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johannesson, H.
    Fredolini, Claudia
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    O'Hurley, G.
    Branca, R.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Wiklund, F.
    Bjartell, A.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Analysis of plasma from prostate cancer patients links decreased carnosine dipeptidase 1 levels to lymph node metastasis2014Ingår i: Translational Proteomics, ISSN 2212-9634, Vol. 2, nr 1, s. 14-24Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is a need for a better differentiation of aggressive tumors in prostate cancer to design a tailored treatment for each patient, preferably by a minimally invasive analysis of blood samples. In a previous study, we discovered a decrease of plasma levels of carnosine dipeptidase 1 (CNDP1) in association with aggressive prostate cancer. Now this relation has been investigated and characterized further by generating several new antibodies for extended analysis of CNDP1 in plasma. Multi-antibody sandwich assays were developed and applied to 1214 samples from two Swedish cohorts that confirmed decreased levels of CNDP1 in plasma from patients with advanced disease. Therein, data from CNDP1 assays allowed a better differentiation between tumor N stages than clinical tPSA, but did not when classifying T or M stages. Further investigations can now elucidate mechanisms behind decreasing levels of CNDP1 in plasma and primary in regards to lymph node metastasis.

  • 503.
    Qundos, Ulrika
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johannesson, Henrik
    Fredolini, Claudia
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    O´Hurley, Gillian
    Branca, Rui
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Wiklund, Fredrik
    Bjartell, Anders
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Plasma levels of carnosine dipeptidase 1 decrease in prostate cancer patients with lymph node metastasisManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    There is a need for a better differentiation of aggressive tumors in prostate cancer to design a tailored treatment for each patient, preferably by a minimally invasive analysis of blood samples. In a previous study, we discovered a decrease of plasma levels of carnosine dipeptidase 1 (CNDP1) in association with aggressive prostate cancer. Now this relation has been investigated and characterized further by generating several new antibodies for extended analysis of CNDP1 in plasma. Multi-antibody sandwich assays were developed and applied to 1,214 samples from two Swedish cohorts that confirmed decreased levels of CNDP1 in plasma for patients with advanced disease. Therein, CNDP1 assays revealed superior differentiation for tumor N stages than clinical tPSA. Further investigations can now elucidate mechanisms behind decreasing levels of CNDP1 in plasma and primary in regards to lymph node metastasis.

  • 504. Regev, A
    et al.
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Danish Tech Univ, Denmark.
    Yosef, Nir
    et al.,
    The Human Cell Atlas2017Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 6, artikel-id e27041Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

  • 505.
    Remnestål, Julia
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Bergström, Sofia
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Olofsson, Jennie
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Sjöstedt, Evelina
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Blennow, Kaj
    Zetterberg, Henrik
    Zettergren, Anna
    Kern, Silke
    Skoog, Ingmar
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Månberg, Anna
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Association of CSF proteins with tau and amyloid beta levels in asymptomatic 70-year-olds2021Ingår i: Alzheimer's Research & Therapy, E-ISSN 1758-9193, Vol. 13, nr 1, artikel-id 54Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background Increased knowledge of the evolution of molecular changes in neurodegenerative disorders such as Alzheimer's disease (AD) is important for the understanding of disease pathophysiology and also crucial to be able to identify and validate disease biomarkers. While several biological changes that occur early in the disease development have already been recognized, the need for further characterization of the pathophysiological mechanisms behind AD still remains. Methods In this study, we investigated cerebrospinal fluid (CSF) levels of 104 proteins in 307 asymptomatic 70-year-olds from the H70 Gothenburg Birth Cohort Studies using a multiplexed antibody- and bead-based technology. Results The protein levels were first correlated with the core AD CSF biomarker concentrations of total tau, phospho-tau and amyloid beta (A beta 42) in all individuals. Sixty-three proteins showed significant correlations to either total tau, phospho-tau or A beta 42. Thereafter, individuals were divided based on CSF A beta 42/A beta 40 ratio and Clinical Dementia Rating (CDR) score to determine if early changes in pathology and cognition had an effect on the correlations. We compared the associations of the analysed proteins with CSF markers between groups and found 33 proteins displaying significantly different associations for amyloid-positive individuals and amyloid-negative individuals, as defined by the CSF A beta 42/A beta 40 ratio. No differences in the associations could be seen for individuals divided by CDR score. Conclusions We identified a series of transmembrane proteins, proteins associated with or anchored to the plasma membrane, and proteins involved in or connected to synaptic vesicle transport to be associated with CSF biomarkers of amyloid and tau pathology in AD. Further studies are needed to explore these proteins' role in AD pathophysiology.

  • 506.
    Remnestål, Julia
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Bergström, Sofia
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Olofsson, Jennie
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Sjöstedt, Evelina
    Karolinska Institutet.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Blennow, Kaj
    The Sahlgrenska Academy, University of Gothenburg.
    Zetterberg, Henrik
    The Sahlgrenska Academy, University of Gothenburg; UCL Institute of Neurology, London; UK Dementia Research Institute at UCL, London.
    Zettergren, Anna
    The Sahlgrenska Academy, University of Gothenburg.
    Kern, Silke
    The Sahlgrenska Academy, University of Gothenburg.
    Skoog, Ingmar
    The Sahlgrenska Academy, University of Gothenburg.
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Månberg, Anna
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Novel CSF protein pattern correlates with biomarker evidence of tau and amyloid β pathology in asymptomatic 70-year-oldsManuskript (preprint) (Övrigt vetenskapligt)
  • 507.
    Remnestål, Julia
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Just, David
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Mitsios, Nicholas
    Fredolini, Claudia
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Mulder, Jan
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kultima, Kim
    Ingelsson, Martin
    Kilander, Lena
    Lannfelt, Lars
    Svenningsson, Per
    Nellgard, Bengt
    Zetterberg, Henrik
    Blennow, Kaj
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Häggmark-Månberg, Anna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    CSF profiling of the human brain enriched proteome reveals associations of neuromodulin and neurogranin to Alzheimer's disease2016Ingår i: PROTEOMICS - Clinical Applications, ISSN 1862-8346, E-ISSN 1862-8354, Vol. 10, nr 12, s. 1242-1253Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Purpose: This study is part of a larger effort aiming to expand the knowledge of brain-enriched proteins in human cerebrospinal fluid (CSF) and to provide novel insight into the relation between such proteins and different neurodegenerative diseases. Experimental design: Here 280 brain-enriched proteins in CSF from patients with Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are profiled. In total, 441 human samples of ventricular CSF collected post mortem and lumbar CSF collected ante mortem are analyzed using 376 antibodies in a suspension bead array setup, utilizing a direct labelling approach. Results: Among several proteins displaying differentiated profiles between sample groups, we focus here on two synaptic proteins, neuromodulin (GAP43) and neurogranin (NRGN). They are both found at elevated levels in CSF from AD patients in two independent cohorts, providing disease-associated profiles in addition to verifying and strengthening previously observed patterns. Increased levels are also observed for patients for whom the AD diagnosis was not established at the time of sampling. Conclusions and clinical relevance: These findings indicate that analyzing the brain-enriched proteins in CSF is of particular interest to increase the understanding of the CSF proteome and its relation to neurodegenerative disorders. In addition, this study lends support to the notion that measurements of these synaptic proteins could potentially be of great relevance in future diagnostic tests for AD.

  • 508.
    Remnestål, Julia
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab. Swedish FTD Initiat, Stockholm, Sweden..
    Oijerstedt, Linn
    Swedish FTD Initiat, Stockholm, Sweden.;Karolinska Inst, Div Neurogeriatr, Ctr Alzheimer Res, Dept Neurobiol Care Sci & Soc, S-17164 Solna, Sweden.;Karolinska Univ Hosp, Unit Hereditary Dementias, Theme Aging, Stockholm, Sweden..
    Ullgren, Abbe
    Swedish FTD Initiat, Stockholm, Sweden.;Karolinska Inst, Div Neurogeriatr, Ctr Alzheimer Res, Dept Neurobiol Care Sci & Soc, S-17164 Solna, Sweden..
    Olofsson, Jennie
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. Swedish FTD Initiat, Stockholm, Sweden..
    Bergström, Sofia
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. Swedish FTD Initiat, Stockholm, Sweden..
    Kultima, Kim
    Uppsala Univ, Dept Med Sci, Clin Chem, Uppsala, Sweden..
    Ingelsson, Martin
    Uppsala Univ, Dept Publ Hlth & Caring Sci, Geriatr, Uppsala, Sweden..
    Kilander, Lena
    Uppsala Univ, Dept Publ Hlth & Caring Sci, Geriatr, Uppsala, Sweden..
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. Karolinska Inst, Dept Neurosci, Solna, Sweden..
    Månberg, Anna
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. Swedish FTD Initiat, Stockholm, Sweden..
    Graff, Caroline
    Swedish FTD Initiat, Stockholm, Sweden.;Karolinska Inst, Div Neurogeriatr, Ctr Alzheimer Res, Dept Neurobiol Care Sci & Soc, S-17164 Solna, Sweden.;Karolinska Univ Hosp, Unit Hereditary Dementias, Theme Aging, Stockholm, Sweden..
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. Swedish FTD Initiat, Stockholm, Sweden..
    Altered levels of CSF proteins in patients with FTD, presymptomatic mutation carriers and non-carriers2020Ingår i: Translational Neurodegeneration, ISSN 2047-9158, Vol. 9, nr 1, artikel-id 27Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The clinical presentations of frontotemporal dementia (FTD) are diverse and overlap with other neurological disorders. There are, as of today, no biomarkers in clinical practice for diagnosing the disorders. Here, we aimed to find protein markers in cerebrospinal fluid (CSF) from patients with FTD, presymptomatic mutation carriers and non-carriers. Methods: Antibody suspension bead arrays were used to analyse 328 proteins in CSF from patients with behavioural variant FTD (bvFTD, n = 16) and progressive primary aphasia (PPA, n = 13), as well as presymptomatic mutation carriers (PMC, n = 16) and non-carriers (NC, n = 8). A total of 492 antibodies were used to measure protein levels by direct labelling of the CSF samples. The findings were further examined in an independent cohort including 13 FTD patients, 79 patients with Alzheimer's disease and 18 healthy controls. Results: We found significantly altered protein levels in CSF from FTD patients compared to unaffected individuals (PMC and NC) for 26 proteins. The analysis show patterns of separation between unaffected individuals and FTD patients, especially for those with a clinical diagnosis of bvFTD. The most statistically significant differences in protein levels were found for VGF, TN-R, NPTXR, TMEM132D, PDYN and NF-M. Patients with FTD were found to have higher levels of TN-R and NF-M, and lower levels of VGF, NPTXR, TMEM132D and PDYN, compared to unaffected individuals. The main findings were reproduced in the independent cohort. Conclusion: In this pilot study, we show a separation of FTD patients from unaffected individuals based on protein levels in CSF. Further investigation is required to explore the CSF profiles in larger cohorts, but the results presented here has the potential to enable future clinical utilization of these potential biomarkers within FTD.

  • 509.
    Remnestål, Julia
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Öijerstedt, Linn
    Karolinska Institutet.
    Ullgren, Abbe
    Karolinska Institutet.
    Olofsson, Jennie
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Bergström, Sofia
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Kultima, Kim
    Uppsala Universitet.
    Ingelsson, Martin
    Uppsala Universitet.
    Kilander, Lena
    Uppsala Universitet.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Månberg, Anna
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Graff, Caroline
    Karolinska Institutet.
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Altered levels of CSF proteins in patients with FTD, presymptomatic mutation carriers and non-carriersIngår i: Translational Neurodegeneration, ISSN 2047-9158Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background. The clinical presentations of frontotemporal dementia (FTD) are diverse and overlap with other neurological disorders. There are, as of today, no biomarkers in clinical practice for diagnosing the disorders. Here, we aimed to find protein markers in cerebrospinal fluid (CSF) from patients with FTD, presymptomatic mutation carriers and non-carriers.

    Methods. Antibody suspension bead arrays were used to analyse 328 proteins in CSF from patients with behavioural variant FTD (bvFTD, n=16) and progressive primary aphasia (PPA, n=13), as well as presymptomatic mutation carriers (PMC, n=16) and non-carriers (NC, n=8). A total of 492 antibodies were used to measure protein levels by direct labelling of the CSF samples. The findings were further examined in an independent cohort including 13 FTD patients, 79 patients with Alzheimer’s disease and 18 healthy controls.

    Results. We found significantly altered protein levels in CSF from FTD patients compared to unaffected individuals (PMC and NC) for 26 proteins. The analysis show patterns of separation between unaffected individuals and FTD patients, especially for those with a clinical diagnosis of bvFTD. The most statistically significant differences in protein levels were found for VGF, TN-R, NPTXR, TMEM132D, PDYN and NF-M. Patients with FTD were found to have higher levels of TN-R and NF-M, and lower levels of VGF, NPTXR, TMEM132D and PDYN, compared to unaffected individuals. The main findings were reproduced in the independent cohort.

    Conclusion. In this pilot study, we show a separation of FTD patients from unaffected individuals based on protein levels in CSF. Further investigation is required to explore the CSF profiles in larger cohorts, but the results presented here has the potential to enable future clinical utilization of these potential biomarkers within FTD.

  • 510.
    Renberg, Björn
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Nordin, Jon
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Merca, Anna
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Feldwisch, Joachim
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Eriksson Karlström, Amelie
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Affibody molecules in protein capture microarrays: Evaluation of multidomain ligands and different detection formats2007Ingår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, nr 1, s. 171-179Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The importance of the ligand presentation format for the production of protein capture microarrays was evaluated using different Affibody molecules, produced either as single 6 kDa monomers or genetically linked head-to-tail multimers containing up to four domains. The performances in terms of selectivity and sensitivity of the monomeric and the multidomain Affibody molecules were compared by immobilization of the ligands on microarray slides, followed by incubation with fluorescent-labeled target protein. An increase in signal intensities for the multimers was demonstrated, with the most pronounced difference observed between monomers and dimers. A protein microarray containing six different dimeric Affibody ligands with specificity for IgA, IgE, IgG, TNF-alpha, insulin, or Taq DNA polymerase was characterized for direct detection of fluorescent-labeled analytes. No cross-reactivity was observed and the limits of detection were 600 fM for IgA, 20 pM for IgE, 70 fM for IgG, 20 pM for TNF-alpha, 60 pM for insulin, and 10 pM for Taq DNA polymerase. Also, different sandwich formats for detection of unlabeled protein were evaluated and used for selective detection of IgA or TNF-alpha in human serum or plasma samples, respectively. Finally, the presence of IgA was determined using detection of directly Cy5-labeled normal or IgA-deficient serum samples.

  • 511.
    Reuterswärd, Philippa
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Bergström, Sofia
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Orikiiriza, Judy
    Makerere Univ, Coll Hlth Sci, Infect Dis Inst, Kampala, Uganda..
    Lindquist, Elisabeth
    Umeå Univ, Dept Mol Biol, Umeå, Sweden..
    Bergström, Sven
    Umeå Univ, Dept Mol Biol, Umeå, Sweden..
    Svahn Andersson, Helene
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ayoglu, Burcu
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Wahlgren, Mats
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Normark, Johan
    Umeå Univ, Dept Mol Biol, Umeå, Sweden..
    Ribacke, Ulf
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Levels of human proteins in plasma associated with acute paediatric malaria2018Ingår i: Malaria Journal, ISSN 1475-2875, E-ISSN 1475-2875, Vol. 17, artikel-id 426Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The intimate interaction between the pathophysiology of the human host and the biology of the Plasmodium falciparum parasite results in a wide spectrum of disease outcomes in malaria. Development of severe disease is associated with a progressively augmented imbalance in pro- and anti-inflammatory responses to high parasite loads and sequestration of parasitized erythrocytes. Although these phenomena collectively constitute common denominators for the wide variety of discrete severe malaria manifestations, the mechanistic rationales behind discrepancies in outcome are poorly understood. Exploration of the human pathophysiological response by variations in protein profiles in plasma presents an excellent opportunity to increase the understanding. This is ultimately required for better prediction, prevention and treatment of malaria, which is essential for ongoing elimination and eradication efforts. Results: An affinity proteomics approach was used to analyse 541 paediatric plasma samples collected from community controls and patients with mild or severe malaria in Rwanda. Protein profiles were generated with an antibody-based suspension bead array containing 255 antibodies targetting 115 human proteins. Here, 57 proteins were identified with significantly altered levels (adjusted p-values<0.001) in patients with malaria compared to controls. From these, the 27 most significant proteins (adjusted p-values<10(-14)) were selected for a stringent analysis approach. Here, 24 proteins showed elevated levels in malaria patients and included proteins involved in acute inflammatory response as well as cell adhesion. The remaining three proteins, also implicated in immune regulation and cellular adhesivity, displayed lower abundance in malaria patients. In addition, 37 proteins (adjusted p-values<0.05) were identified with increased levels in patients with severe compared to mild malaria. This set includes, proteins involved in tissue remodelling and erythrocyte membrane proteins. Collectively, this approach has been successfully used to identify proteins both with known and unknown association with different stages of malaria. Conclusion: In this study, a high-throughput affinity proteomics approach was used to find protein profiles in plasma linked to P. falciparum infection and malaria disease progression. The proteins presented herein are mainly involved in inflammatory response, cellular adhesion and as constituents of erythrocyte membrane. These findings have a great potential to provide increased conceptual understanding of host-parasite interaction and malaria pathogenesis.

  • 512.
    Reuterswärd, Philippa
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Sofia, Bergström
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Orikiiriza, Judy
    Lindquist, Elisabeth
    Bergström, Sven
    Andersson Svahn, Helene
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Ayoglu, Burcu
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Wahlgren, Mats
    Normark, Johan
    Ribacke, Ulf
    Nilsson, Peter
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Levels of human proteins in plasma as indicators for acute severe pediatric malariaManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Background

    Existing low resource diagnostics for malaria infection suffer from sensitivity and specificity issues while lacking sufficient prognostic value. Identifying human host proteins could improve the possibilities to predict the risk of development of acute severe malaria. This will possible enable improved treatment and thereby lead to a decrease in mortality of malaria infected children. Furthermore, discovering host proteins with altered levels during active infection could generate leads to better understand host-parasite interaction.

    Results

    Here, we have analyzed a total of 541 pediatric plasma samples that were collected from community controls and individuals with mild or severe malaria in Rwanda. Protein profiles of these plasma samples were generated with an antibody-based suspension bead array containing 255 antibodies targeting 115 human proteins. We present 22 proteins with a strong discriminatory capacity (adjusted p-values below 10-19) for separating malaria cases from community controls. This panel of proteins contains among others acute phase proteins and proteins connected to cell adhesion and migration. Among these, three proteins showed lower plasma levels in the group of malaria-infected individuals compared to the control group. One of these proteins is the anti-adhesive secreted protein acidic and cysteine rich (SPARC) with possible connections to parasite cytoadhesion. A multi-protein panel of six proteins, including SPARC, could differentiate between controls and malaria cases with an AUC of 0.98. Furthermore, a panel of 37 proteins, including proteins associated to erythrocyte membranes, was identified as candidates for separation of mild and severe malaria patients (adjusted pvalues below 0.05).

    Conclusion

    The herein identified set of human proteins has a significant discriminatory capacity between community controls and malaria cases. We also present proteins offering the possibility to enable stratification and risk prediction for the development of severe malaria. This constitutes an important set that could enable enhanced understanding and thereby also possibilities for better treatment of acute severe pediatric malaria.

     

  • 513. Rexhepaj, Elton
    et al.
    Agnarsdottir, Margret
    Bergman, Julia
    Edqvist, Per-Henrik
    Bergqvist, Michael
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Gallagher, William M.
    Lundberg, Emma
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ponten, Fredrik
    A Texture Based Pattern Recognition Approach to Distinguish Melanoma from Non-Melanoma Cells in Histopathological Tissue Microarray Sections2013Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 8, nr 5, s. e62070-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aims: Immunohistochemistry is a routine practice in clinical cancer diagnostics and also an established technology for tissue-based research regarding biomarker discovery efforts. Tedious manual assessment of immunohistochemically stained tissue needs to be fully automated to take full advantage of the potential for high throughput analyses enabled by tissue microarrays and digital pathology. Such automated tools also need to be reproducible for different experimental conditions and biomarker targets. In this study we present a novel supervised melanoma specific pattern recognition approach that is fully automated and quantitative. Methods and Results: Melanoma samples were immunostained for the melanocyte specific target, Melan-A. Images representing immunostained melanoma tissue were then digitally processed to segment regions of interest, highlighting Melan-A positive and negative areas. Color deconvolution was applied to each region of interest to separate the channel containing the immunohistochemistry signal from the hematoxylin counterstaining channel. A support vector machine melanoma classification model was learned from a discovery melanoma patient cohort (n = 264) and subsequently validated on an independent cohort of melanoma patient tissue sample images (n = 157). Conclusion: Here we propose a novel method that takes advantage of utilizing an immuhistochemical marker highlighting melanocytes to fully automate the learning of a general melanoma cell classification model. The presented method can be applied on any protein of interest and thus provides a tool for quantification of immunohistochemistry-based protein expression in melanoma.

  • 514.
    Rimini, Rebecca
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sundberg, Marten
    Sjöberg, Ronald
    Klevebring, Daniel
    Gry, Marcus
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Validation of serum protein profiles by a dual antibody array approach2009Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In recent years, affinity-based technologies have become important tools for serum profiling to uncover protein expression patterns linked to disease state or therapeutic effects. In this study, we describe a path towards the production of an antibody microarray to allow protein profiling of biotinylated human serum samples with reproducible sensitivity in the picomolar range. With the availability of growing numbers of affinity reagents, protein profiles are to be validated in efficient manners and we describe a cross-platform strategy based on data concordance with a suspension bead array to interrogate the identical set of antibodies with the same cohort of serum samples. Comparative analysis enabled to screen for high-performing antibodies, which were displaying consistent results across the two platforms and targeting known serum components. Moreover, data processing methods such as sample referencing and normalization were evaluated for their effects on inter-platform agreement. Our work suggests that mutual validation of protein expression profiles using alternative microarray platforms holds great potential in becoming an important and valuable component in affinity-based high-throughput proteomic screenings as it allows to narrow down the number of discovered targets prior to orthogonal, uniplexed validation approaches.

  • 515.
    Robinson, Jonathan L.
    et al.
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, Gothenburg, Sweden.;Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, Gothenburg, Sweden..
    Feizi, Amir
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, Gothenburg, Sweden.;Novo Nordisk Res Ctr Oxford, Old Campus Rd, Oxford, England..
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nielsen, Jens
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, Gothenburg, Sweden ; Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, Gothenburg, Sweden ; Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, DK-2800 Lyngby, Denmark.
    A Systematic Investigation of the Malignant Functions and Diagnostic Potential of the Cancer Secretome2019Ingår i: Cell Reports, E-ISSN 2211-1247, Vol. 26, nr 10, s. 2622-+Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The collection of proteins secreted from a cell-the secretome-is of particular interest in cancer pathophysiology due to its diagnostic potential and role in tumorigenesis. However, cancer secretome studies are often limited to one tissue or cancer type or focus on biomarker prediction without exploring the associated functions. We therefore conducted a pan-cancer analysis of secretome gene expression changes to identify candidate diagnostic biomarkers and to investigate the underlying biological function of these changes. Using transcriptomic data spanning 32 cancer types and 30 healthy tissues, we quantified the relative diagnostic potential of secretome proteins for each cancer. Furthermore, we offer a potential mechanism by which cancer cells relieve secretory pathway stress by decreasing the expression of tissue-specific genes, thereby facilitating the secretion of proteins promoting invasion and proliferation. These results provide a more systematic understanding of the cancer secretome, facilitating its use in diagnostics and its targeting for therapeutic development.

  • 516.
    Robinson, Jonathan L.
    et al.
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden.;Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Kocabas, Pinar
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden.;Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Wang, Hao
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden.;Univ Gothenburg, Wallenberg Ctr Mol & Translat Med, Kemivagen 10, SE-41258 Gothenburg, Sweden.;Chalmers Univ Technol, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Cholley, Pierre-Etienne
    Chalmers Univ Technol, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Cook, Daniel
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Nilsson, Avlant
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Anton, Mihail
    Chalmers Univ Technol, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Ferreira, Raphael
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Domenzain, Ivan
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden.;Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Billa, Virinchi
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Limeta, Angelo
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Hedin, Alex
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Gustafsson, Johan
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden.;Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Kerkhoven, Eduard J.
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Svensson, L. Thomas
    Chalmers Univ Technol, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden..
    Palsson, Bernhard O.
    Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, DK-2800 Lyngby, Denmark.;Univ Calif San Diego, Dept Bioengn, La Jolla, CA 92093 USA.;Univ Calif San Diego, Dept Pediat, La Jolla, CA 92093 USA..
    Mardinoglu, Adil
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. Kings Coll London, Fac Dent Oral & Craniofacial Sci, Ctr Host Microbiome Interact, London WC2R 2LS, England..
    Hansson, Lena
    Chalmers Univ Technol, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden.;Novo Nordisk Res Ctr Oxford, Oxford OX3 7FZ, England..
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Centra, Wallenberg Center for Protein Research. Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, DK-2800 Lyngby, Denmark..
    Nielsen, Jens
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, SE-41258 Gothenburg, Sweden.;Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, SE-41258 Gothenburg, Sweden.;Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, DK-2800 Lyngby, Denmark.;BioInnovat Inst, Ole Maaloes Vej 3, DK-2200 Copenhagen, Denmark..
    An atlas of human metabolism2020Ingår i: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 13, nr 624, artikel-id eaaz1482Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Genome-scale metabolic models (GEMs) are valuable tools to study metabolism and provide a scaffold for the integrative analysis of omics data. Researchers have developed increasingly comprehensive human GEMs, but the disconnect among different model sources and versions impedes further progress. We therefore integrated and extensively curated the most recent human metabolic models to construct a consensus GEM, Human1. We demonstrated the versatility of Human1 through the generation and analysis of cell- and tissue-specific models using transcriptomic, proteomic, and kinetic data. We also present an accompanying web portal, Metabolic Atlas (https://www.metabolicatlas.org/), which facilitates further exploration and visualization of Human1 content. Human1 was created using a version-controlled, open-source model development framework to enable community-driven curation and refinement. This framework allows Human1 to be an evolving shared resource for future studies of human health and disease.

  • 517.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Epitope mapping of antibodies using bacterial surface display2008Ingår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 5, nr 12, s. 1039-1045Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.

  • 518.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Epitope mapping using gram-positive surface display2010Ingår i: Current Protocols in Immunology, ISSN 1934-3671, nr SUPPL. 90, s. 9.9.1-9.9.17Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibodys binding characteristics will still be the main features determining the assays reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths.

  • 519.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Discovery of epitopes for targeting the human epidermal growth factor receptor 2 (HER2) with antibodies2009Ingår i: Molecular Oncology, ISSN 1574-7891, Vol. 3, nr 3, s. 238-247Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antibodies have become valuable therapeutic agents for targeting of extracellular proteins in various diseases, including cancer, autoimmunity and cardiovascular disorders. For breast cancer, antibodies targeting the human HER2 have been shown to result in cell growth inhibition both in vitro and in patients with breast tumors. There is evidence to suggest that targeting multiple HER2 epitopes may result in increased growth inhibition making it interesting to find antibodies targeting new epitopes. Here, we report on a new scheme to discover antibodies directed to new epitopes using the extracellular domain of the HER2 as a model. Polyclonal antibodies were generated using recombinant protein fragments and affinity purified fractions of the antibodies were functionally characterized and precisely epitope mapped using bacterial surface display. Polyclonal antibodies towards a 127 amino acid recombinant protein fragment spanning between domains II and III of the HER2 were shown to bind to human ductal carcinoma cell line BT474 resulting in growth inhibition. Affinity purification demonstrated that antibodies to two separate regions from the N- and C-terminal end of the fragment exhibited the growth inhibition. Epitope mapping of the C-terminal antibodies revealed a 25 amino acid region (LPESFDGDPASNTAPLQPEQLQVF) with two distinct epitopes mediating efficient growth inhibition. The results suggest that antibodies directed towards this region of domain III of the HER2, distinct from the well-known monoclonal antibodies trastuzumab and pertuzumab, bind to the HER2 on living cells and exhibit growth inhibition. The work describes a new strategy to develop antibodies directed to non-overlapping epitopes and shows a path of pursuit to explore the epitope space of a target protein.

  • 520.
    Rockberg, Johan
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Szigyarto, Cristina AI-Khalili
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Antigen selection for design of protein epitope signature tags2004Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, nr 10, s. S5-S5Artikel i tidskrift (Övrigt vetenskapligt)
  • 521.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Prediction of antibody response using recombinant human protein fragments as antigen2009Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 18, nr 11, s. 2346-2355Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A great need exists for prediction of antibody response for the generation of antibodies toward protein targets. Earlier studies have suggested that prediction methods based on hydrophilicity propensity scale, in which the degree of exposure of the amino acid in an aqueous solvent is calculated, has limited value. Here, we show a comparative analysis based on 12,634 affinity-purified antibodies generated in a standardized manner against human recombinant protein fragments. The antibody response (yield) was measured and compared to theoretical predictions based on a large number (544) of published propensity scales. The results show that some of the scales have predictive power, although the overall Pearson correlation coefficient is relatively low (0.2) even for the best performing amino acid indices. Based on the current data set, a new propensity scale was calculated with a Pearson correlation coefficient of 0.25. The values correlated in some extent to earlier scales, including large penalty for hydrophobic and cysteine residues and high positive contribution from acidic residues, but with relatively low positive contribution from basic residues. The fraction of immunogens generating low antibody responses was reduced from 30% to around 10% if immunogens with a high propensity score (>0.48) were selected as compared to immunogens with lower scores (<0.29). The study demonstrates that a propensity scale might be useful for prediction of antibody response generated by immunization of recombinant protein fragments. The data set presented here can be used for further studies to design new prediction tools for the generation of antibodies to specific protein targets.

  • 522. Rodriguez, Henry
    et al.
    Snyder, Mike
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO). KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Andrews, Phil
    Beavis, Ronald
    Borchers, Christoph
    Chalkley, Robert J.
    Cho, Sang Yun
    Cottingham, Katie
    Dunn, Michael
    Dylag, Tomasz
    Edgar, Ron
    Hare, Peter
    Heck, Albert J. R.
    Hirsch, Roland F.
    Kennedy, Karen
    Kolar, Patrik
    Kraus, Hans-Joachim
    Mallick, Parag
    Nesvizhskii, Alexey
    Ping, Peipei
    Ponten, Fredrik
    Yang, Liming
    Yates, John R.
    Stein, Stephen E.
    Hermjakob, Henning
    Kinsinger, Christopher R.
    Apweiler, Rolf
    Recommendations from the 2008 International Summit on Proteomics Data Release and Sharing Policy: The Amsterdam Principles2009Ingår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 8, nr 7, s. 3689-3692Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Policies supporting the rapid and open sharing of genomic data have directly fueled the accelerated pace of discovery in large-scale genomics research. The proteomics community is starting to implement analogous policies and infrastructure for making large-scale proteomics data widely available on a precompetitive basis. On August 14, 2008, the National Cancer Institute (NCI) convened the "International Summit on Proteomics Data Release and Sharing Policy" in Amsterdam, The Netherlands, to identify and address potential roadblocks to rapid and open access to data. The six principles agreed upon by key stakeholders at the summit addressed issues surrounding (1) timing, (2) comprehensiveness, (3) format, (4) deposition to repositories, (5) quality metrics, and (6) responsibility for proteomics data release. This summit report explores various approaches to develop a framework of data release and sharing principles that will most effectively fulfill the needs of the funding agencies and the research community.

  • 523. Romanov, Roman A.
    et al.
    Alpar, Alan
    Zhang, Ming-Dong
    Zeisel, Amit
    Calas, Andre
    Landry, Marc
    Fuszard, Matthew
    Shirran, Sally L.
    Schnell, Robert
    Dobolyi, Arpad
    Olah, Mark
    Spence, Lauren
    Mulder, Jan
    Martens, Henrik
    Palkovits, Miklos
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Sitte, Harald H.
    Botting, Catherine H.
    Wagner, Ludwig
    Linnarsson, Sten
    Hökfelt, Tomas
    Harkany, Tibor
    A secretagogin locus of the mammalian hypothalamus controls stress hormone release2015Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 34, nr 1, s. 36-54Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A hierarchical hormonal cascade along the hypothalamic-pituitary-adrenal axis orchestrates bodily responses to stress. Although corticotropin-releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin's Ca2+ sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogin's loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness.

  • 524. Romanov, Roman A.
    et al.
    Zeisel, Amit
    Bakker, Joanne
    Girach, Fatima
    Hellysaz, Arash
    Tomer, Raju
    Alpar, Alan
    Mulder, Jan
    Clotman, Frederic
    Keimpema, Erik
    Hsueh, Brian
    Crow, Ailey K.
    Martens, Henrik
    Schwindling, Christian
    Calvigioni, Daniela
    Bains, Jaideep S.
    Mate, Zoltan
    Szabo, Gabor
    Yanagawa, Yuchio
    Zhang, Ming-Dong
    Rendeiro, Andre
    Farlik, Matthias
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Wulff, Peer
    Bock, Christoph
    Broberger, Christian
    Deisseroth, Karl
    Hokfelt, Tomas
    Linnarsson, Sten
    Horvath, Tamas L.
    Harkany, Tibor
    Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes2017Ingår i: Nature Neuroscience, ISSN 1097-6256, E-ISSN 1546-1726, Vol. 20, nr 2, s. 176-188Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of. hypothalamic organization and function.

  • 525.
    Ronaghi, Martin
    et al.
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Nyrén, Pål
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    A sequencing method based on real-time pyrophosphate1998Ingår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 281, nr 5375, s. 363-365Artikel i tidskrift (Refereegranskat)
  • 526. Ronnmark, J.
    et al.
    Gronlund, H.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A2002Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 269, nr 11, s. 2647-2655Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid, three-helix bundle domain derived from the IgG-binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. Characterization of selected clones by biosensor technology showed that five out of eight investigated affibody variants were capable of IgA binding, with dissociation constants (K (d) ) in the range between 0.5 and 3 mum. One variant (Z(IgA1) ) showing the strongest binding affinity was further analyzed, and showed that human IgA subclasses (IgA(1) and IgA(2) ) as well as secretory IgA were recognized with similar efficiencies. No detectable cross-reactivity towards human IgG, IgM, IgD or IgE was observed. The potential use of the Z(IgA1) affibody as a ligand in affinity chromatography applications was first demonstrated by selective recovery of IgA protein from a spiked Escherichia coli total cell lysate, using an affinity column containing a divalent head-to-tail Z(IgA1) affibody dimer construct as a ligand. In addition, efficient affinity recovery of IgA from unconditioned human plasma was also demonstrated.

  • 527. Ronnmark, J.
    et al.
    Hansson, M.
    Nguyen, T.
    Uhlén, Mathias
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Robert, A.
    Ståhl, Stefan
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Construction and characterization of affibody-Fc chimeras produced in Escherichia coli2002Ingår i: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 261, nr 02-jan, s. 199-211Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human I-G. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture, Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of artificial antibodies was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such artificial antibodies should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.

  • 528.
    Rosario, Dorines
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Benfeitas, Rui
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Bidkhori, Gholamreza
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Lab, Stockholm, Sweden..
    Zhang, Cheng
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Shoaie, Saeed
    Kings Coll London, Ctr Host Microbiome Interact, Dent Inst, London, England.;Karolinska Inst, Ctr Translat Microbiome Res, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Mardinoglu, Adil
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Chalmers Univ Technol, Dept Biol & Biol Engn, Gothenburg, Sweden..
    Understanding the Representative Gut Microbiota Dysbiosis in Metformin-Treated Type 2 Diabetes Patients Using Genome-Scale Metabolic Modeling2018Ingår i: Frontiers in Physiology, E-ISSN 1664-042X, Vol. 9, artikel-id 775Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Dysbiosis in the gut microbiome composition may be promoted by therapeutic drugs such as metformin, the world's most prescribed antidiabetic drug. Under metformin treatment, disturbances of the intestinal microbes lead to increased abundance of Escherichia spp., Akkermansia muciniphila, Subdoligranulum variabile and decreased abundance of Intestinibacter bartlettii. This alteration may potentially lead to adverse effects on the host metabolism, with the depletion of butyrate producer genus. However, an increased production of butyrate and propionate was verified in metformin-treated Type 2 diabetes (T2D) patients. The mechanisms underlying these nutritional alterations and their relation with gut microbiota dysbiosis remain unclear. Here, we used Genomescale Metabolic Models of the representative gut bacteria Escherichia spp., I. bartlettii, A. muciniphila, and S. variabile to elucidate their bacterial metabolism and its effect on intestinal nutrient pool, including macronutrients (e.g., amino acids and short chain fatty acids), minerals and chemical elements (e.g., iron and oxygen). We applied flux balance analysis (FBA) coupled with synthetic lethality analysis interactions to identify combinations of reactions and extracellular nutrients whose absence prevents growth. Our analyses suggest that Escherichia sp. is the bacteria least vulnerable to nutrient availability. We have also examined bacterial contribution to extracellular nutrients including short chain fatty acids, amino acids, and gasses. For instance, Escherichia sp. and S. variabile may contribute to the production of important short chain fatty acids (e.g., acetate and butyrate, respectively) involved in the host physiology under aerobic and anaerobic conditions. We have also identified pathway susceptibility to nutrient availability and reaction changes among the four bacteria using both FBA and flux variability analysis. For instance, lipopolysaccharide synthesis, nucleotide sugar metabolism, and amino acid metabolism are pathways susceptible to changes in Escherichia sp. and A. muciniphila. Our observations highlight important commensal and competing behavior, and their association with cellular metabolism for prevalent gut microbes. The results of our analysis have potential important implications for development of new therapeutic approaches in T2D patients through the development of prebiotics, probiotics, or postbiotics.

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  • 529. Rosario, Dorines
    et al.
    Bidkhori, Gholamreza
    Lee, Sunjae
    Bedarf, Janis
    Hildebrand, Falk
    Le Chatelier, Emmanuelle
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ehrlich, Stanislav Dusko
    Proctor, Gordon
    Wuellner, Ullrich
    Mardinoglu, Adil
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Shoaie, Saeed
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Systematic analysis of gut microbiome reveals the role of bacterial folate and homocysteine metabolism in Parkinson's disease2021Ingår i: Cell Reports, E-ISSN 2211-1247, Vol. 34, nr 9, artikel-id 108807Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Parkinson's disease (PD) is the most common progressive neurological disorder compromising motor functions. However, nonmotor symptoms, such as gastrointestinal (GI) dysfunction, precede those affecting movement. Evidence of an early involvement of the GI tract and enteric nervous system highlights the need for better understanding of the role of gut microbiota in GI complications in PD. Here, we investigate the gut microbiome of patients with PD using metagenomics and serum metabolomics. We integrate these data using metabolic modeling and construct an integrative correlation network giving insight into key microbial species linked with disease severity, GI dysfunction, and age of patients with PD. Functional analysis reveals an increased microbial capability to degrade mucin and host glycans in PD. Personalized community-level metabolic modeling reveals the microbial contribution to folate deficiency and hyperhomo-cysteinemia observed in patients with PD. The metabolic modeling approach could be applied to uncover gut microbial metabolic contributions to PD pathophysiology.

  • 530.
    Rosario, Dorines
    et al.
    Kings Coll London, Fac Dent Oral & Craniofacial Sci, Ctr Host Microbiome Interact, London, England..
    Boren, Jan
    Univ Gothenburg, Sahlgrenska Univ Hosp, Dept Mol & Clin Med, Gothenburg, Sweden..
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Proctor, Gordon
    Kings Coll London, Fac Dent Oral & Craniofacial Sci, Ctr Host Microbiome Interact, London, England..
    Aarsland, Dag
    Kings Coll London, Inst Psychiat Psychol & Neurosci, London, England..
    Mardinoglu, Adil
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Kings Coll London, Fac Dent Oral & Craniofacial Sci, Ctr Host Microbiome Interact, London, England..
    Shoaie, Saeed
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Kings Coll London, Fac Dent Oral & Craniofacial Sci, Ctr Host Microbiome Interact, London, England.
    Systems Biology Approaches to Understand the Host-Microbiome Interactions in Neurodegenerative Diseases2020Ingår i: Frontiers in Neuroscience, ISSN 1662-4548, E-ISSN 1662-453X, Vol. 14, artikel-id 716Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Neurodegenerative diseases (NDDs) comprise a broad range of progressive neurological disorders with multifactorial etiology contributing to disease pathophysiology. Evidence of the microbiome involvement in the gut-brain axis urges the interest in understanding metabolic interactions between the microbiota and host physiology in NDDs. Systems Biology offers a holistic integrative approach to study the interplay between the different biologic systems as part of a whole, and may elucidate the host-microbiome interactions in NDDs. We reviewed direct and indirect pathways through which the microbiota can modulate the bidirectional communication of the gut-brain axis, and explored the evidence of microbial dysbiosis in Alzheimer's and Parkinson's diseases. As the gut microbiota being strongly affected by diet, the potential approaches to targeting the human microbiota through diet for the stimulation of neuroprotective microbial-metabolites secretion were described. We explored the potential of Genome-scale metabolic models (GEMs) to infer microbe-microbe and host-microbe interactions and to identify the microbiome contribution to disease development or prevention. Finally, a systemic approach based on GEMs and 'omics integration, that would allow the design of sustainable personalized anti-inflammatory diets in NDDs prevention, through the modulation of gut microbiota was described.

  • 531.
    Roswall, Josefine
    et al.
    Hallands Hosp Halmstad, Dept Pediat, Halmstad, Sweden.;Univ Gothenburg, Gothenburg Pediat Growth Res Ctr, Inst Clin Sci, Dept Pediat, Gothenburg, Sweden..
    Olsson, Lisa M.
    Univ Gothenburg, Sahlgrenska Acad, Inst Med, Wallenberg Lab,Dept Mol & Clin Med, Gothenburg, Sweden..
    Kovatcheva-Datchary, Petia
    Univ Gothenburg, Sahlgrenska Acad, Inst Med, Wallenberg Lab,Dept Mol & Clin Med, Gothenburg, Sweden.;Univ Wurzburg, Inst Mol Infect Biol, Wurzburg, Germany..
    Nilsson, Staffan
    Chalmers Tekniska Hgsk, Dept Math Sci, Gothenburg, Sweden.;Univ Gothenburg, Inst Biomed, Dept Lab Med, Gothenburg, Sweden..
    Tremaroli, Valentina
    Univ Gothenburg, Sahlgrenska Acad, Inst Med, Wallenberg Lab,Dept Mol & Clin Med, Gothenburg, Sweden..
    Simon, Marie-Christine
    Univ Gothenburg, Sahlgrenska Acad, Inst Med, Wallenberg Lab,Dept Mol & Clin Med, Gothenburg, Sweden..
    Kiilerich, Pia
    Univ Copenhagen, Dept Biol, Lab Genom & Mol Biomed, Copenhagen, Denmark.;Statens Serum Inst, Artillerivej 5, DK-2300 Copenhagen S, Denmark..
    Akrami, Rozita
    Univ Gothenburg, Sahlgrenska Acad, Inst Med, Wallenberg Lab,Dept Mol & Clin Med, Gothenburg, Sweden..
    Kramer, Manuela
    Univ Gothenburg, Sahlgrenska Acad, Inst Med, Wallenberg Lab,Dept Mol & Clin Med, Gothenburg, Sweden..
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Gummesson, Anders
    Univ Gothenburg, Sahlgrenska Acad, Inst Med, Wallenberg Lab,Dept Mol & Clin Med, Gothenburg, Sweden.;Sahlgrens Univ Hosp, Dept Clin Genet & Genom, Reg Vastra Gotaland, Gothenburg, Sweden..
    Kristiansen, Karsten
    Univ Copenhagen, Dept Biol, Lab Genom & Mol Biomed, Copenhagen, Denmark.;BGI Shenzhen, Shenzhen, Peoples R China..
    Dahlgren, Jovanna
    Univ Gothenburg, Gothenburg Pediat Growth Res Ctr, Inst Clin Sci, Dept Pediat, Gothenburg, Sweden.;Sahlgrens Univ Hosp, Dept Pediat, Reg Vastra Gotaland, Gothenburg, Sweden..
    Backhed, Fredrik
    Univ Gothenburg, Sahlgrenska Acad, Inst Med, Wallenberg Lab,Dept Mol & Clin Med, Gothenburg, Sweden.;Sahlgrens Univ Hosp, Dept Clin Physiol, Reg Vastra Gotaland, Gothenburg, Sweden.;Univ Copenhagen, Novo Nordisk Fdn, Fac Hlth & Med Sci, Ctr Basic Metab Res, Copenhagen, Denmark..
    Developmental trajectory of the healthy human gut microbiota during the first 5 years of life2021Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 29, nr 5, s. 765-776.e3Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The gut is inhabited by a densely populated ecosystem, the gut microbiota, that is established at birth. However, the succession by which different bacteria are incorporated into the gut microbiota is still relatively unknown. Here, we analyze the microbiota from 471 Swedish children followed from birth to 5 years of age, collecting samples after 4 and 12 months and at 3 and 5 years of age as well as from their mothers at birth using 16S rRNA gene profiling. We also compare their microbiota to an adult Swedish population. Genera follow 4 different colonization patterns during establishment where Methanobrevibacter and Christensenellaceae colonize late and do not reached adult levels at 5 years. These late colonizers correlate with increased alpha diversity in both children and adults. By following the children through age-specific community types, we observe that children have individual dynamics in the gut microbiota development trajectory.

  • 532. Salomonsson, A.
    et al.
    Micke, P.
    Mattsson, J. S. M.
    La Fleur, L.
    Isaksson, J.
    Jönsson, M.
    Nodin, B.
    Botling, J.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi.
    Jirström, K.
    Staaf, J.
    Planck, M.
    Brunnström, H.
    Comprehensive analysis of RNA binding motif protein 3 (RBM3) in non-small cell lung cancer2020Ingår i: Cancer Medicine, E-ISSN 2045-7634, Vol. 9, nr 15, s. 5609-5619Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aims High expression of the RNA-binding motif protein 3 (RBM3) correlates with improved prognosis in several major types of cancer. The aim of the present study was to examine the prognostic value of RBM3 protein and mRNA expression in non-small cell lung cancer (NSCLC).

    Methods and results Immunohistochemical expression of RBM3 was evaluated in surgically treated NSCLC from two independent patient populations (n = 213 and n = 306). Staining patterns were correlated with clinicopathological parameters, overall survival (OS), and recurrence-free interval (RFI). Cases with high nuclear RBM3 protein expression had a prolonged 5-year OS in both cohorts when analyzing adenocarcinomas separately (P = .02 and P = .01). RBM3 remained an independent prognostic factor for OS in multivariable analysis of cohort I (HR 0.44, 95% CI 0.21-0.90) and for RFI in cohort II (HR 0.38, 95% CI 0.22-0.74). In squamous cell carcinoma, there was instead an insignificant association to poor prognosis. Also, the expression levels of RBM3 mRNA were investigated in 2087 lung adenocarcinomas and 899 squamous cell carcinomas assembled from 13 and 8 public gene expression microarray datasets, respectively. The RBM3 mRNA levels were not clearly associated with patient outcome in either adenocarcinomas or squamous cell carcinomas.

    Conclusions The results from this study support that high protein expression of RBM3 is linked to improved outcome in lung adenocarcinoma.

  • 533. Sastry, Anand
    et al.
    Monk, Jonathan
    Tegel, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. Technical University of Denmark - DTU.
    Pålsson, Bernhard O.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Brunk, Elizabeth
    Machine learning in computational biology to accelerate high-throughput protein expression2017Ingår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 33, nr 16, s. 2487-2495Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Motivation: The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Results: Combining computational biology and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide the selection of protein fragments based on these characteristics to optimize high-throughput experimentation.

  • 534.
    Savolainen, Peter
    et al.
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Rosen, B
    Holmberg, Anders
    KTH, Tidigare Institutioner (före 2005), Biokemi och biokemisk teknologi.
    Leitner, T
    Uhlen, Mathias
    Lundeberg, Joachim
    Sequence analysis of domestic dog mitochondrial DNA for forensic use1997Ingår i: Journal of Forensic Sciences, ISSN 0022-1198, E-ISSN 1556-4029, Vol. 42, nr 4, s. 593-600Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A method has been developed for the direct sequencing of hypervariable region 1 (HV1) of domestic dog (Canis familiaris) and wolf (Canis lupus) mitochondrial DNA (mtDNA) using single hairs as template. The method uses a robotic work-station and an automated sequencer to allow for robust routine analysis. A population data base was created in order to investigate the forensic and population-genetic informativeness of domestic dog HV1. Sequence variation, partitioning of dog breeds among sequence variants and phylogenetic relations between the variants were determined. Samples from 102 domestic dogs of 52 different breeds and two captive wolves were analyzed. Nineteen dog sequence variants were found and the frequencies of the variants ranged from 1 to 21%. The calculated discrimination power of the region, i.e., the exclusion capacity, implied that nine out of ten disputed individuals can be excluded by this analysis. The sequence variants were found to cluster into four phylogenetic groups.

  • 535.
    Schwenk, Jochen M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Gry, Marcus
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Rimini, Rebecca
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Antibody suspension bead arrays within serum proteomics2008Ingår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, nr 8, s. 3168-3179Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antibody microarrays offer a powerful tool to screen for target proteins in complex samples. Here, we describe an approach for systematic analysis of serum, based on antibodies and using color-coded beads for the creation of antibody arrays in suspension. This method, adapted from planar antibody arrays, offers a fast, flexible, and multiplexed procedure to screen larger numbers of serum samples, and no purification steps are required to remove excess labeling substance. The assay system detected proteins down to lower picomolar levels with dynamic ranges over 3 orders of magnitude. The feasibility of this workflow was shown in a study with more than 200 clinical serum samples tested for 20 serum proteins.

  • 536.
    Schwenk, Jochen M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Igel, Ulrika
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Kato, Bernet S.
    Nicholson, George
    Karpe, Fredrik
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Comparative protein profiling of serum and plasma using an antibody suspension bead array approach2010Ingår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, nr 3, s. 532-540Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In the pursuit towards a systematic analysis of human diseases, array-based approaches within antibody proteomics offer high-throughput strategies to discover protein biomarkers in serum and plasma. To investigate the influence of sample preparation on such discovery attempts, we report on a systematic effort to compare serum and plasma protein profiles determined with an antibody suspension bead array. The intensity levels were used to define protein profiles and no significant differences between serum and plasma were observed for 79% of the 174 antibodies (targeting 156 proteins). By excluding 36 antibodies giving rise to differential intensity levels, cluster analysis revealed donor-specific rather than preparation-dependent grouping. With a cohort from a clinically relevant medical condition, the metabolic syndrome, the influence of the sample type on a multiplexed biomarker discovery approach was further investigated. Independent comparisons of protein profiles in serum and plasma revealed an antibody targeting ADAMTSL-4, a protein that would qualify to be studied further in association with the condition. In general, the preparation type had an impact on the results of the applied antibody suspension bead array, and while the technical variability was equal, plasma offered a greater biological variability and allowed to give rise to more discoveries than serum.

  • 537.
    Schwenk, Jochen M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Igel, Ulrika
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Neiman, Maja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Langen, Hanno
    Becker, Charlotte
    Bjartell, Anders
    Ponten, Fredrik
    Wiklund, Fredrik
    Gronberg, Henrik
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays2010Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, nr 11, s. 2497-2507Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is a need for high throughput methods for screening patient samples in the quest for potential biomarkers for diagnostics and patient care. Here, we used a combination of undirected target selection, antibody suspension bead arrays, and heat-induced epitope retrieval to allow for protein profiling of human plasma in a novel and systematic manner. Several antibodies were found to reveal altered protein profiles upon epitope retrieval at elevated temperatures with limits of detection improving into lower ng/ml ranges. In a study based on prostate cancer patients, several proteins with differential profiles were discovered and subsequently validated in an independent cohort. For one of the potential biomarkers, the human carnosine dipeptidase 1 protein (CNDP1), the differences were determined to be related to the glycosylation status of the targeted protein. The study shows a path of pursuit for large scale screening of biobank repositories in a flexible and proteome-wide fashion by utilizing heat-induced epitope retrieval and using an antibody suspension bead array format. Molecular & Cellular Proteomics 9:2497-2507, 2010.

  • 538.
    Schwenk, Jochen M.
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Lindberg, Johan
    Sundberg, Mårten
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics2007Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, nr 1, s. 125-132Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    One of the major challenges of antibody-based proteomics is the quality assurance of the generated antibodies to ensure specificity to the target protein. Here we describe a single tube multiplex approach to simultaneously analyze the binding of antibodies to a large number of different antigens. This bead-based assay utilizes the full multiplexing capacity theoretically offered by the Luminex suspension array technology. A protocol for an increased coupling throughput for the immobilization of antigens was developed and used to set up complex and stabile 100-plex bead mixtures. The possibility of using a two-dimensional multiplexing, in terms of high numbers of both analytes and samples or as in this case antigens and antibodies, enables the specificity of 96 antibodies versus 100 different antigens to be determined in 2 h. This high throughput analysis will potentially have great impact on the possibility for the utilization of different antibody proteomics approaches where the quality assessment of antibodies is of the utmost importance.

  • 539.
    Seijsing, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Lindborg, Malin
    Höidén-Guthenberg, I.
    Bönisch, H.
    Gunneriusson, E.
    Frejd, F.
    Abramsén, L.
    Ekblad, C.
    Löfblom, J.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Gräslund, Torbjörn
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    An engineered affibody molecule with pHdependent binding to FcRn mediates extended circulatory half-lifeof a fusion proteinManuskript (preprint) (Övrigt vetenskapligt)
  • 540.
    Seijsing, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Lindborg, Malin
    Höidén-Guthenberg, Ingmarie
    Bönisch, Heiko
    Guneriusson, Elin
    Frejd, Fredrik Y.
    Abrahmsén, Lars
    Ekblad, Caroline
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Gräslund, Torbjörn
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    An engineered affibody molecule with pH-dependent binding to FcRn mediates extended circulatory half-life of a fusion protein2014Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, nr 48, s. 17110-17115Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased halflife, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.

  • 541.
    Seijsing, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Lindborg, Malin
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Gräslund, Torbjörn
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Robust Expression of the Human Neonatal Fc Receptor in a Truncated Soluble Form and as a Full-Length Membrane-Bound Protein in Fusion with eGFP2013Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 8, nr 11, s. e81350-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.

  • 542. Sernbo, Sandra
    et al.
    Borrebaeck, Carl A. K.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Jirström, Karin
    Ek, Sara
    Nuclear T-STAR Protein Expression Correlates with HER2 Status, Hormone Receptor Negativity and Prolonged Recurrence Free Survival in Primary Breast Cancer and Decreased Cancer Cell Growth In Vitro2013Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 8, nr 7, s. e70596-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    T-STAR (testis-signal transduction and activation of RNA) is an RNA binding protein, containing an SH3-binding domain and thus potentially playing a role in integration of cell signaling and RNA metabolism. The specific function of T-STAR is unknown and its implication in cancer is poorly characterized. Expression of T-STAR has been reported in human testis, muscle and brain tissues, and is associated with a growth-inhibitory role in immortalized fibroblasts. The aim of this paper was to investigate the functional role of T-STAR through (i) survival analysis of patients with primary invasive breast cancer and (ii) experimental evaluation of the effect of T-STAR on breast cancer cell growth. T-STAR protein expression was analysed by immunohistochemistry (IHC) in tissue microarrays with tumors from 289 patients with primary invasive breast cancer, and correlations to clinicopathological characteristics, recurrence-free and overall survival (RFS and OS) and established tumor markers such as HER2 and ER status were evaluated. In addition, the function of T-STAR was investigated using siRNA-mediated knock-down and overexpression of the gene in six breast cancer cell lines. Of the tumors analysed, 86% showed nuclear T-STAR expression, which was significantly associated with an improved RFS and strongly associated with positive HER2 status and negative hormone receptor status. Furthermore, experimental data showed that overexpression of T-STAR decreased cellular growth while knock-down increased it, as shown both by thymidine incorporation and metabolic activity. In summary, we demonstrate that T-STAR protein expression correlates with an improved RFS in primary breast cancer. This is supported by functional data, indicating that T-STAR regulation is of importance both for breast cancer biology and clinical outcome but future studies are needed to determine a potential role in patient stratification.

  • 543. Shi, Tie-Jun Sten
    et al.
    Xiang, Qiong
    Zhang, Ming-Dong
    Tortoriello, Giuseppe
    Hammarberg, Henrik
    Mulder, Jan
    Fried, Kaj
    Wagner, Ludwig
    Josephson, Anna
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Harkany, Tibor
    Hökfelt, Tomas
    Secretagogin is expressed in sensory CGRP neurons and in spinal cord of mouse and complements other calcium-binding proteins, with a note on rat and human2012Ingår i: Molecular Pain, E-ISSN 1744-8069, Vol. 8, s. 80-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Secretagogin (Scgn), a member of the EF-hand calcium-binding protein (CaBP) superfamily, has recently been found in subsets of developing and adult neurons. Here, we have analyzed the expression of Scgn in dorsal root ganglia (DRGs) and trigeminal ganglia (TGs), and in spinal cord of mouse at the mRNA and protein levels, and in comparison to the well-known CaBPs, calbindin D-28k, parvalbumin and calretinin. Rat DRGs, TGs and spinal cord, as well as human DRGs and spinal cord were used to reveal phylogenetic variations. Results: We found Scgn mRNA expressed in mouse and human DRGs and in mouse ventral spinal cord. Our immunohistochemical data showed a complementary distribution of Scgn and the three CaBPs in mouse DRG neurons and spinal cord. Scgn was expressed in similar to 7% of all mouse DRG neuron profiles, mainly small ones and almost exclusively co-localized with calcitonin gene-related peptide (CGRP). This co-localization was also seen in human, but not in rat DRGs. Scgn could be detected in the mouse sciatic nerve and accumulated proximal to its constriction. In mouse spinal cord, Scgn-positive neuronal cell bodies and fibers were found in gray matter, especially in the dorsal horn, with particularly high concentrations of fibers in the superficial laminae, as well as in cell bodies in inner lamina II and in some other laminae. A dense Scgn-positive fiber network and some small cell bodies were also found in the superficial dorsal horn of humans. In the ventral horn, a small number of neurons were Scgn-positive in mouse but not rat, confirming mRNA distribution. Both in mouse and rat, a subset of TG neurons contained Scgn. Dorsal rhizotomy strongly reduced Scgn fiber staining in the dorsal horn. Peripheral axotomy did not clearly affect Scgn expression in DRGs, dorsal horn or ventral horn neurons in mouse. Conclusions: Scgn is a CaBP expressed in a subpopulation of nociceptive DRG neurons and their processes in the dorsal horn of mouse, human and rat, the former two co-expressing CGRP, as well as in dorsal horn neurons in all three species. Functional implications of these findings include the cellular refinement of sensory information, in particular during the processing of pain.

  • 544. Sistani, L.
    et al.
    Rodriguez, P. Q.
    Hultenby, K.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Betsholtz, C.
    Jalanko, H.
    Tryggvason, K.
    Wernerson, A.
    Patrakka, J.
    Neuronal proteins are novel components of podocyte major processes and their expression in glomerular crescents supports their role in crescent formation2013Ingår i: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 83, nr 1, s. 63-71Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The podocyte has a central role in the glomerular filtration barrier typified by a sophisticated morphology of highly organized primary (major) and secondary (foot) processes. The molecular makeup of foot processes is well characterized, but that of major processes is poorly known. Previously, we profiled the glomerular transcriptome through large-scale sequencing and microarray profiling. Unexpectedly, the survey found expression of three neuronal proteins (Huntingtin interacting protein 1 (Hip1), neurofascin (Nfasc), and olfactomedin-like 2a (Olfml2a)), all enriched in the glomerulus. These proteins were expressed exclusively by podocytes, wherein they localized to major processes as verified by RT-PCR, western blotting, immunofluorescence, and immunoelectron microscopy. During podocyte development, these proteins colocalized with vimentin, confirming their association with major processes. Using immunohistochemistry, we found coexpression of Hip1 and Olfml2a along with the recognized podocyte markers synaptopodin and Pdlim2 in glomerular crescents of human kidneys, indicating the presence of podocytes in these lesions. Thus, three neuronal proteins are highly expressed in podocyte major process. Using these new markers we found that podocytes contribute to the formation of glomerular crescents.

  • 545. Sistani, Laleh
    et al.
    Dunér, Fredrik
    Udumala, Sunil
    Hultenby, Kjell
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Betsholtz, Christer
    Tryggvason, Karl
    Wernerson, Annika
    Patrakka, Jaakko
    Pdlim2 is a novel actin-regulating protein of podocyte foot processes2011Ingår i: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 80, nr 10, s. 1045-1054Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The slit diaphragm and the apical and basal membrane domains of podocytes are connected to each other by an actin-based cytoskeleton critical to the maintenance of the glomerular filtration barrier. In an effort to discover novel regulatory proteins of the podocyte foot process, we identified and characterized pdlim2, a member of the actin-associated LIM protein subfamily of cytosolic proteins typified by an N-terminal PDZ domain and a C-terminal LIM domain. In the kidney, the pdlim2 protein is highly specific for the glomerulus and podocyte foot processes as shown by RT-PCR, western blotting, immunofluorescence, and immunoelectron microscopy. In cultured podocytes, pdlim2 was associated with stress fibers and cortical actin. Pdlim2 seems to regulate actin dynamics in podocytes since stress fibers were stabilized in its presence. Mechanistically, pdlim2 interacts with two actin-associated podocyte proteins, alpha-actinin-4 and angiomotin-like-1, as shown by immunoprecipitation and yeast two-hybrid analyses. By semi-quantitative immunoelectron microscopy, there was a reduced expression of pdlim2 in podocytes of patients with minimal change nephrotic syndrome and membranous nephropathy, whereas its expression was unchanged in patients with focal segmental glomerulosclerosis. Hence, pdlim2 is a novel actin-regulating protein of podocyte foot processes that may have a role in the pathogenesis of glomerular diseases.

  • 546.
    Sivertsson, Åsa
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lindström, Emil
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, S-75185 Uppsala, Sweden..
    Oksvold, Per
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Katona, Borbala
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, S-75185 Uppsala, Sweden..
    Hikmet, Feria
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, S-75185 Uppsala, Sweden..
    Vuu, Jimmy
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, S-75185 Uppsala, Sweden..
    Gustavsson, Jonas
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, S-75185 Uppsala, Sweden..
    Sjöstedt, Evelina
    Karolinska Inst, Dept Neurosci, S-17177 Stockholm, Sweden..
    von Feilitzen, Kalle
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kampf, Caroline
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, S-75185 Uppsala, Sweden.;Atlas Antibodies AB, S-16869 Bromma, Sweden..
    Schwenk, Jochen M.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Systembiologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Neurosci, S-17177 Stockholm, Sweden..
    Lindskog, Cecilia
    Uppsala Univ, Dept Immunol Genet & Pathol, Rudbeck Lab, S-75185 Uppsala, Sweden..
    Enhanced Validation of Antibodies Enables the Discovery of Missing Proteins2020Ingår i: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 19, nr 12, s. 4766-4781Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The localization of proteins at a tissue- or cell-type-specific level is tightly linked to the protein function. To better understand each protein's role in cellular systems, spatial information constitutes an important complement to quantitative data. The standard methods for determining the spatial distribution of proteins in single cells of complex tissue samples make use of antibodies. For a stringent analysis of the human proteome, we used orthogonal methods and independent antibodies to validate 5981 antibodies that show the expression of 3775 human proteins across all major human tissues. This enhanced validation uncovered 56 proteins corresponding to the group of "missing proteins" and 171 proteins of unknown function. The presented strategy will facilitate further discussions around criteria for evidence of protein existence based on immunohistochemistry and serves as a useful guide to identify candidate proteins for integrative studies with quantitative proteomics methods.

  • 547.
    Sjöberg, Ronald
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Mattsson, Cecilia
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson, Eni
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Affinitets-proteomik.
    Hellström, Cecilia
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ayoglu, Burcu
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling2016Ingår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, nr 5, s. 582-592Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt (TM) Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt (TM) Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions.

  • 548.
    Sjöberg, Ronald
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Mattsson, Cecilia
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Andersson, Eni
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hellström, Cecilia
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Zhu, Heng
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Ayoglu, Brucu
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Exploration of high-density protein microarrays for antibody validation and autoimmunity profilingManuskript (preprint) (Övrigt vetenskapligt)
  • 549.
    Sjöberg, Ronald
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sundberg, Mårten
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Gundberg, Anna
    KTH, Skolan för bioteknologi (BIO).
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO).
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Validation of affinity reagents using antigen microarrays2011Ingår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, nr 5, s. 555-563Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

  • 550. Sjöstedt, Evelina
    et al.
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Hallström, Björn M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Häggmark, Anna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Mitsios, Nicholas
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Pontén, Fredrik
    Hökfelt, Tomas
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Mulder, Jan
    Defining the Human Brain Proteome Using Transcriptomics and Antibody-Based Profiling with a Focus on the Cerebral Cortex2015Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 10, nr 6, artikel-id UNSP e0130028Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mammalian brain is a complex organ composed of many specialized cells, harboring sets of both common, widely distributed, as well as specialized and discretely localized proteins. Here we focus on the human brain, utilizing transcriptomics and public available Human Protein Atlas (HPA) data to analyze brain-enriched (frontal cortex) polyadenylated messenger RNA and long non-coding RNA and generate a genome-wide draft of global and cellular expression patterns of the brain. Based on transcriptomics analysis of altogether 27 tissues, we have estimated that approximately 3% (n=571) of all protein coding genes and 13% (n=87) of the long non-coding genes expressed in the human brain are enriched, having at least five times higher expression levels in brain as compared to any of the other analyzed peripheral tissues. Based on gene ontology analysis and detailed annotation using antibody-based tissue micro array analysis of the corresponding proteins, we found the majority of brain-enriched protein coding genes to be expressed in astrocytes, oligodendrocytes or in neurons with molecular properties linked to synaptic transmission and brain development. Detailed analysis of the transcripts and the genetic landscape of brainenriched coding and non-coding genes revealed brain-enriched splice variants. Several clusters of neighboring brain-enriched genes were also identified, suggesting regulation of gene expression on the chromatin level. This multi-angle approach uncovered the brainenriched transcriptome and linked genes to cell types and functions, providing novel insights into the molecular foundation of this highly specialized organ.

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