Tuula A. Nyman

• Matikainen S, Nyman TA, Cypryk W. Function and regulation of non-canonical caspase-4/5/11 inflammasome. J Immunol. 2020 Jun 15;204(12):3063-3069.
• Cypryk W, Nyman TA, Matikainen S. From Inflammasome to Exosome-Does Extracellular Vesicle Secretion Constitute an Inflammasome-Dependent Immune Response? Front Immunol. 2018 Sep 25;9:2188.
• Nyman TA, Lorey MB, Cypryk W, Matikainen S. Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome, global protein secretion, and T cells. Expert Rev Proteomics. 2017 May;14(5):395-407
• Lorey MB, Rossi K, Eklund KK, Nyman TA*, Matikainen S*. Global characterization of protein secretion from human macrophages following non-canonical caspase-4/5 inflammasome activation. Mol Cell Proteomics. 2017 Apr;16(4 suppl 1):S187-S199
• Cypryk W, Lorey M, Puustinen A, Nyman TA, Matikainen S. Proteomic and Bioinformatic Characterization of Extracellular Vesicles Released from Human Macrophages upon Influenza A Virus Infection. J Proteome Res. 2017;16:217-227
• Söderholm S, Fu Y, Gaelings L, Belanov S, Yetukuri L, Berlinkov M, Cheltsov AV, Anders S, Aittokallio T, Nyman TA, Matikainen S, Kainov DE. Multi-Omics Studies towards Novel Modulators of Influenza A Virus-Host Interaction.Viruses. 2016 Sep 29;8(10). pii: E269. Review.
• Välimäki E, Cypryk W, Virkanen J, Nurmi K, Turunen PM, Eklund KK, Åkerman KE, Nyman TA, Matikainen S. Calpain Activity Is Essential for ATP-Driven Unconventional Vesicle-Mediated Protein Secretion and Inflammasome Activation in Human Macrophages. J Immunol. 2016;197:3315-3325
• Söderholm S, Kainov DE, Öhman T, Denisova OV, Schepens B, Kulesskiy E, Imanishi SY, Corthals G, Hintsanen P, Aittokallio T, Saelens X, Matikainen S*, Nyman TA*. Phosphoproteomics to characterize host response during influenza A virus infection of human macrophages. Mol Cell Proteomics. 2016;15:3203-3219
• Christensen MH, Jensen SB, Miettinen JJ, Luecke S, Prabakaran T, Reinert LS, Mettenleiter T, Chen ZJ, Knipe DM, Sandri-Goldin RM, Enquist LW, Hartmann R, Mogensen TH, Rice SA, Nyman TA, Matikainen S, Paludan SR. HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression. EMBO J. 2016;35:1385-99.
• Öhman T, Söderholm S, Paidikondala M, Lietzén N, Matikainen S, Nyman TA. Phosphoproteome characterization reveals that Sendai-virus infection activates mTOR signaling in human epithelial cells. Proteomics. 2015;15:2087-97.
• Öhman T, Söderholm S, Hintsanen P, Välimäki E, Lietzén N, MacKintosh C, Aittokallio T, Matikainen S, Nyman TA. Phosphoproteomics Combined with Quantitative 14-3-3-affinity Capture Identifies SIRT1 and RAI as Novel Regulators of Cytosolic dsRNA Recognition Pathway. Mol Cell Proteomics. 2014;13,2604-17.
• Cypryk W, Öhman T, Eskelinen E-L, Matikainen S, Nyman TA. Quantitative proteomics of extracellular vesicles released from human monocyte-derived macrophages upon β-glucan stimulation. J Proteome Res. 2014;13:2468-77.
• Öhman T, Teirilä L, Lahesmaa AM, Kankkunen P, Veckman V, Saijo S, Wolff H, Hautaniemi S, Nyman TA*, Matikainen S*. Dectin-1 pathway activates robust autophagy-dependent unconventional protein secretion in human macrophages, J Immunol. 2014;192:5952-62.
• Välimäki E, Miettinen J, Lietzén N, Matikainen S*, Nyman TA*. Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages. Mol Cell Proteomics. 2013;12:749-63.
• Miettinen JJ, Matikainen S, Nyman TA. Global secretome characterisation of Herpes simplex virus 1 infected human primary macrophages, J Virol. 2012;86:12770-8.
• Lietzén N, Öhman T*, Rintahaka J*, Julkunen I, Aittokallio T, Matikainen S*, Nyman TA*. Quantitative subcellular proteome and secretome profiling of Influenza A virus-infected human primary macrophages. PLoS Pathogens 2011 May;7(5):e1001340.

*equal contribution

• Rønneberg, Jørgen Dybevik; Hjeltnes, Martine; Krieger, Josephine; Elias, Joshua E.; Stensland, Maria & Holm, Kristian [Show all 15 contributors for this article] (2026). An epithelial GPR35 isoform supports tumor-associated transcriptional and metabolic phenotypes. FEBS Letters. ISSN 0014-5793. p. 1–23. doi: 10.1002/1873-3468.70423.
• Niemi, Essi Maria; Nyman, Tuula Anneli; Attramadal, Håvard; Hoel, Henrik; Rosales, Antonio & Hisdal, Jonny [Show all 7 contributors for this article] (2026). Proteomic evaluation and comparison of native and decellularized human saphenous vein extracellular matrices. Frontiers in Bioengineering and Biotechnology. 14. doi: 10.3389/fbioe.2026.1773372. Full text in Research Archive
• Canavati, Christina; Oppebøen, Mari; Verma, Radha; Misceo, Doriana; Frengen, Eirik & Salvador, Cathrin Lytomt [Show all 29 contributors for this article] (2026). Biallelic SUPT4H1 variants cause a multisystem neurodevelopmental disorder associated with disrupted transcription. Genetics in Medicine. ISSN 1098-3600. 28(5). doi: 10.1016/j.gim.2026.102553. Full text in Research Archive Show summary

  Purpose We aimed to define the clinical and genetic basis of an autosomal recessive neurodevelopmental disorder identified in 3 unrelated families with an overlapping multisystem phenotype. Methods Exome or genome sequencing was performed in 6 affected individuals from 3 families, revealing biallelic variants in SUPT4H1. Functional effects were evaluated using CRISPR and RNAi knockdown in C. elegans. Transcriptomic and proteomic profiling were conducted on patient-derived fibroblasts. Phylogenetic profiling assessed evolutionary conservation and coevolution. Dopamine response was evaluated in 2 affected siblings. Results All individuals carried biallelic SUPT4H1 variants—1 frameshift and 2 missense variants. Shared features included intellectual disability, dystonia, speech impairment, craniofacial dysmorphism, skeletal anomalies, and enamel hypoplasia. Functional modeling in C. elegans confirmed pathogenicity through neuromotor deficits. Multiomics analyses revealed dysregulation of developmental gene networks and the transcriptional machinery with disrupted RNA polymerase complexes and cell-cycle regulators. Dopamine treatment improved motor symptoms and normalized cerebrospinal neurotransmitter levels in 2 siblings. Conclusion Biallelic variants in SUPT4H1 cause a previously unrecognized multisystem neurodevelopmental disorder. These findings underscore the importance of disrupted transcription in human disease and demonstrate how integrated multiomics and cross-species modeling can reveal underlying mechanisms and provide a foundation for future studies of transcriptional regulation.

• Gjerde, Christiane Helgestad; Kleinmanns, Katrin; Langer, Anika; Garibay, Gorka Ruiz de; Rozmus, Ezekiel & Stangeland, Gina Nyhus [Show all 22 contributors for this article] (2026). A 3D ovarian cancer metastasis model using a decellularised peritoneal matrix to study therapy response. EBioMedicine. 124. doi: 10.1016/j.ebiom.2026.106135. Full text in Research Archive Show summary

  Background: High-grade serous ovarian carcinoma (HGSOC) presents a significant therapeutic challenge. Late-stage disease is frequently associated with peritoneal carcinomatosis. The peritoneal metastases exhibit a unique tumour microenvironment (TME) distinct from the primary tumours and other metastatic sites. Understanding the critical influence of the extracellular matrix (ECM) in shaping the tumour phenotype is essential for the development of effective new therapies. Methods: This study introduces a three-dimensional (3D) model of HGSOC peritoneal metastases using a porcine decellularised peritoneal-derived ECM scaffold, referred to as peritoneal matrix (PerMa). Findings: We show that the decellularisation maintains the structural integrity and composition of ECM molecules. Comparative analysis reveals structural, compositional, and mechanical similarities between porcine and human peritoneal matrices, underscoring the porcine model's translational relevance for modelling human peritoneum physiology. The PerMa supports the 3D growth of HGSOC cell lines. The model enables the assessment of sensitivity to traditional chemotherapy and novel cell-based immunotherapy through confocal imaging and quantification of cell volume. Interpretation: Our model offers a valuable platform for investigating peritoneal carcinomatosis in HGSOC, with the potential to contribute significantly to developing novel therapeutic approaches.

• Monteuuis, Geoffray; Awadhpersad, Ryan; Kolk, Daan van der; Singh, Sachin; Nyman, Tuula A. & Malyutina, Alina [Show all 13 contributors for this article] (2025). Vacuolar-type H+-ATPase-mediated extra-organellar buffering resolves mitochondrial dysfunction. Nature Communications. 17(1). doi: 10.1038/s41467-025-66656-1. Full text in Research Archive Show summary

  Abstract Mitochondrial dysfunction underlies a wide range of human diseases, including primary mitochondrial disorders, neurodegeneration, cancer, and ageing. To preserve cellular homeostasis, organisms have evolved adaptive mechanisms that coordinate nuclear and mitochondrial gene expression. Here, we use genome-wide CRISPR knockout screening to identify cell fitness pathways that support survival under impaired mitochondrial protein synthesis. The strongest suppressor of aberrant mitochondrial translation defects – besides a compendium of known mitochondrial translation quality control factors – is the loss of the vacuolar-type H + -ATPase (v-ATPase), a key regulator of intracellular acidification, nutrient sensing, and growth signaling. We show that partial v-ATPase loss reciprocally modulates mitochondrial membrane potential (ΔΨ m ) and cristae structure in both cancer cell lines and mitochondrial disease patient-derived models. Our findings uncover an extra-organellar buffering mechanism whereby partial v-ATPase inhibition mitigates mitochondrial dysfunction by altering pH homeostasis and driving metabolic rewiring as a protective response that promotes cell fitness.

• Saber, Saber H.; Yak, Nyakuoy; Yong, Xuan Ling Hilary; Bong, Yih Tyng; Leeson, Hannah & Dai, Chuan-Yang [Show all 27 contributors for this article] (2025). DDHD2 provides a flux of saturated fatty acids for neuronal energy and function. Nature Metabolism. 7(10), p. 2117–2141. doi: 10.1038/s42255-025-01367-x. Full text in Research Archive Show summary

  Although fatty acids support mitochondrial ATP production in most tissues, neurons are believed to rely exclusively on glucose for energy. Here we show that genetic ablation of the triglyceride and phospholipid lipase Ddhd2 impairs mitochondrial respiration and ATP synthesis in cultured neurons, despite increased glycolysis. This defect arises from reduced levels of long-chain saturated free fatty acids, particularly myristic, palmitic and stearic acids, normally released in an activity-dependent manner by Ddhd2. Inhibition of mitochondrial fatty acid import in wild-type neurons similarly reduced mitochondrial respiration and ATP production. Saturated fatty acyl-coenzyme A treatment restored mitochondrial energy production in Ddhd2 knockout neurons. When provided in combination, these activated fatty acyl-CoA supplements also rescued defects in membrane trafficking, synaptic function and protein homeostasis. These findings uncover that neurons perform β-oxidation of endogenous long-chain free fatty acids to meet ATP demands and reveal a potential therapeutic strategy for hereditary spastic paraplegia 54 caused by DDHD2 mutations.

• Kangas, Petra; Nyman, Tuula Anneli; Metsähonkala, Liisa; Junnila, Jouni; Karttunen, Jenni & Jokinen, Tarja S. (2025). Proteome profiling of cerebrospinal fluid-derived extracellular vesicles reveals potential biomarkers for drug-resistant epilepsy. Clinical Proteomics. ISSN 1542-6416. 22(1). doi: 10.1186/s12014-025-09569-x. Full text in Research Archive Show summary

  Background Epilepsy is one of the most common neurological disorders in humans and in dogs. Treatment currently focuses on alleviating symptoms, and a wide range of anti-seizure medications (ASMs) is available. Still, over one-third of patients have an inadequate response to ASM. The proteome of cerebrospinal fluid (CSF)-derived extracellular vesicles (EVs) offers a potential source of biomarkers for drug-resistant epilepsy (DRE). Methods We utilised a spontaneous canine epilepsy model to study the proteomic content of CSF-derived EVs as a source of biomarkers for DRE. We included 37 drug-naïve dogs with recent onset epilepsy and confirmed diagnosis of idiopathic epilepsy. CSF samples were collected at the onset of epilepsy. After the first visit, ASM treatment was started in all dogs and they were followed up for at least 12 months. After the follow-up period, based on their response to ASM treatment, dogs were grouped as either drug-responsive or drug-resistant. We isolated CSF-derived EVs with ultrafiltration combined with size-exclusion chromatography and then performed proteomic analysis with liquid chromatography-tandem mass spectrometry. A comparison between the drug-responsive and drug-resistant dogs was conducted regarding clinical factors and CSF-derived EV proteomic data. Results Younger age at seizure onset and occurrence of cluster seizures were identified as risk factors for drug-resistance. The proteomic analysis of normalised data identified five proteins with differential abundance between the two groups: KRT4, an uncharacterised immunoglobulin-like domain-containing protein (IgDCPa), F2, DSC1b, and LOC607874. A receiver operating characteristic analysis was performed, revealing a predictive value of ≥ 0.90 for two combinations of three proteins (KRT4, IgDCPa, and F2 (area under curve (AUC) = 0.91, confidence interval (CI) = 0.78-1.00); DSC1b, F2, and IgDCPa (AUC = 0.90, CI = 0.78-1.00)). Conclusions Proteins with differential abundance studied here are associated with epilepsy due to their potential involvement in critical processes such as neuroprotection, inflammation, cell integrity, and immune response. The observed reduction in the abundance of these proteins in drug-resistant dogs suggests that disruptions in these processes may contribute to the severity of the condition and its resistance to treatment. Results from this pilot study warrant further study in a larger cohort

• Dalmao-Fernandez, Andrea; Katare, Parmeshwar Bajirao; Bakke, Hege Gilbø; Hamarsland, Håvard; Ellefsen, Stian & Singh, Sachin [Show all 10 contributors for this article] (2025). The use of three-dimensional primary human myospheres to explore skeletal muscle effects of in vivo krill oil supplementation. In vitro models. ISSN 2731-3433. 4(2), p. 145–155. doi: 10.1007/s44164-025-00087-6. Full text in Research Archive Show summary

  Supplementation with krill oil has shown effects on whole-body lipid and glucose metabolism, as well as on skeletal muscle strength and function. We previously showed that krill oil intervention in vivo promoted fatty acid metabolism and protein synthesis in cultured human myotubes in a two-dimensional (2D) model. The aim of this study was to explore the effects of krill oil supplementation in vivo in a 3D myosphere model, and to compare a the human skeletal muscle 3D cell model to a 2D model. Myospheres were formed from myoblasts obtained before and after 7 weeks of in vivo krill oil intervention. Glucose and oleic acid metabolism were assessed, and transcriptomic and proteomic analyses were performed. In vivo intervention with krill oil increased glucose metabolism in myospheres, while no effect was observed on fatty acid metabolism. Transcriptomic analyses of myospheres after krill oil intervention showed increased expression of genes involved in pathways like motor proteins and hypertrophy, as well as in calcium signaling, of which motor proteins and hypertrophy pathways have not been described in 2D myotube cultures. Proteomic analyses after krill oil intervention showed increased expression of proteins in glycolysis/gluconeogenesis and fatty acid degradation. Comparison of proteins expressed in the 3D myosphere model and the 2D myotube model at the basal level showed that in myospheres, mitochondrial gene expression and translation dominated, while in 2D cultures, mitochondrial organization and response to oxidative stress were more important. These findings suggest that in vivo krill oil intervention induces different metabolic effects when comparing 3D and 2D cultures. In contrast to the 2D model, data obtained with the 3D model showed gene expression changes that are more compatible with previously observed results in vivo concerning skeletal muscle motoric function. Hence, the 3D cell model might better reflect krill oil-induced modifications in skeletal muscle performance in vivo than the 2D model.

• Chernigovskaya, Maria; Quy, Khang Le; Stensland, Maria; Singh, Sachin; Nelson, Rowan & Yilmaz, Melih [Show all 19 contributors for this article] (2025). Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases. Cell Systems. ISSN 2405-4712. 16(11). doi: 10.1016/j.cels.2025.101449. Full text in Research Archive Show summary

  The circulating antibody (Ab) repertoire is crucial for immune protection, holding significant immunological and biotechnological value. While bottom-up mass spectrometry (MS) is widely used for profiling the sequence diversity of circulating Abs (Ab repertoire sequencing [Ab-seq]), it has not been thoroughly benchmarked. We quantified the replicability and robustness of Ab-seq using six monoclonal Ab spike-ins in 70 combinations of concentration and oligoclonality, with and without polyclonal serum immunoglobulin G (IgG) background. Each combination underwent four protease treatments and was analyzed across four experimental and three technical replicates, totaling 3,360 liquid chromatography-tandem MS (LC-MS/MS) runs. We quantified the dependence of Ab-seq identification on Ab sequence, concentration, protease, presence of background IgGs, and bioinformatics methods. Integrating the data from experimental replicates, proteases, and bioinformatics tools enhanced Ab identification. De novo sequencing performed similarly to database-dependent methods at higher Ab concentrations, but de novo Ab reconstruction remains challenging. Our work provides a foundational resource for the field of MS-based Ab profiling. A record of this paper’s transparent peer review process is included in the supplemental information

• Rydland, Anne; Heinicke, Fatima; Nyman, Tuula Anneli; Siebke, Anne-Marie; Flåm, Siri Tennebø & Stensland, Maria [Show all 11 contributors for this article] (2025). Newly-diagnosed rheumatoid arthritis patients have elevated levels of plasma extracellular vesicles with protein cargo altered towards inflammatory processes. Scientific Reports. 15(1). doi: 10.1038/s41598-025-96325-8. Full text in Research Archive Show summary

  Extracellular vesicles (EVs) are implicated in rheumatoid arthritis (RA) but have mainly been assessed in RA patients taking disease modifying anti-rheumatic drugs. EVs are nanoparticles important in cell-cell communication and their molecular cargo are biomarker candidates. We characterized the protein profiles of EVs from blood plasma from newly diagnosed, treatment naïve RA patients (N = 32) and compared them to healthy controls (N = 20), by size exclusion chromatography-based EV enrichment coupled with high-resolution quantitative proteomics. The RA patients had higher EV concentration and larger EVs than controls. A total of 682 EV proteins were reliably quantified, and the overall profiles were distinctly different between patients and controls. Specifically, 26 proteins were significantly upregulated and 31 downregulated in RA patients, with several proteins acting in inflammatory networks and with immunologically important upstream regulators. The RA associated EVs appear, based on the tissue expression of their cargo proteins, to originate mainly from hepatocytes or immune cells, like neutrophils. Interestingly, the strongest RA associated EV proteins were inflammatory molecules, like SAA1 and S100A9, already suggested as biomarkers in RA. Furthermore, the RA associated EV proteins were generally not correlated with total serum protein levels, stressing the importance of EV transport of inflammatory proteins in RA pathogenesis.

• Tang, Jia Xin; Cabrera-Orefice, Alfredo; Meisterknecht, Jana; Taylor, Lucie S.; Monteuuis, Geoffray & Stensland, Maria [Show all 17 contributors for this article] (2025). COA5 has an essential role in the early stage of mitochondrial complex IV assembly. Life Science Alliance (LSA). 8(3). doi: 10.26508/lsa.202403013. Full text in Research Archive Show summary

  Pathogenic variants in cytochrome c oxidase assembly factor 5 (COA5), a proposed complex IV (CIV) assembly factor, have been shown to cause clinical mitochondrial disease with two siblings affected by neonatal hypertrophic cardiomyopathy manifesting a rare, homozygous COA5 missense variant (NM_001008215.3: c.157G>C, p.Ala53Pro). The most striking observation in the affected individuals was an isolated impairment in the early stage of mitochondrial CIV assembly. In this study, we report an unrelated family in whom we have identified the same COA5 variant with patient-derived fibroblasts and skeletal muscle biopsies replicating an isolated CIV deficiency. A CRISPR/Cas9-edited homozygous COA5 knockout U2OS cell line with a similar biochemical profile was generated to interrogate the functional role of the human COA5 protein. Mitochondrial complexome profiling pinpointed a role of COA5 in early CIV assembly, more specifically, its involvement in the stage between MTCO1 maturation and the incorporation of MTCO2. We therefore propose that the COA5 protein plays an essential role in the biogenesis of MTCO2 and its integration into the early CIV assembly intermediate for downstream assembly of the functional holocomplex.

• Carrasco-Rojas, Javiera; Zavala, Gabriela; Contreras-Lopez, Rafael; Olivares, Belén; Larsen, Miriam Aarsund & Inngjerdingen, Marit [Show all 13 contributors for this article] (2025). Artificial cell-derived vesicles by extrusion, a novel docetaxel drug delivery system for lung cancer. Journal of Drug Delivery Science and Technology. ISSN 1773-2247. 106. doi: 10.1016/j.jddst.2025.106693. Full text in Research Archive
• Courtade, Sandra Monica Bach de; Eikenes, Marte; Sheng, Ying; Nyman, Tuula Anneli; Bliksrud, Yngve Thomas & Scheffler, Katja [Show all 7 contributors for this article] (2025). Identification of determinants for variability in mitochondrial biochemical complex activities. Biochimica et Biophysica Acta - Bioenergetics. ISSN 0005-2728. 1866(2). doi: 10.1016/j.bbabio.2025.149553. Full text in Research Archive Show summary

  Diagnostics of mitochondrial disease requires a combination of clinical evaluations and biochemical characterization. However, the large normal variation in mitochondrial complex activity limits the precision of biochemical diagnostics. Thus, identifying factors that contribute to such variations could enhance diagnostic accuracy. In comparison, inbred mice demonstrate much less variations in brain mitochondrial activity, but a clear reduction with age. Interestingly, pretreatment of mouse brain mitochondria with the detergent dodecyl maltoside abolishes the reduction. We therefore postulated that DDM pretreatment could be valuable tool for distinguishing between variations caused by posttranslational modifications and those caused by genetic heterogeneity. In this study, we evaluated the effects of age, DDM sensitivity, oxidative damage and single nucleotide polymorphism on biochemical complex activity and the proteome of human muscle mitochondria, which serve as reference standards for mitochondrial diagnostics. Our results indicate that mtDNA variants are the primary contributors to the diversity in biochemical activity in human muscle mitochondria from healthy individuals.

• Wu, Jingyun; Sanz, Irene Santos Garcia; Delgado, Iván Eiriz; Brüning, Thomas; Kvasnička, Aleš & Friedecký, David [Show all 8 contributors for this article] (2025). Sex-dependent efficacy of sphingosine-1-phosphate receptor agonist FTY720 in mitigating Huntington's disease. Pharmacological Research. ISSN 1043-6618. 211. doi: 10.1016/j.phrs.2024.107557. Full text in Research Archive Show summary

  Huntington's disease (HD) is a debilitating neurodegenerative disorder characterized by severe motor deficits, cognitive decline and psychiatric disturbances. An early and significant morphological hallmark of HD is the activation of astrocytes triggered by mutant huntingtin, leading to the release of inflammatory mediators. Fingolimod (FTY), an FDA-approved sphingosine-1-phosphate (S1P) receptor agonist is used to treat multiple sclerosis (MS), a neuroinflammatory disease, and has shown therapeutic promise in other neurological conditions. Our study aimed to investigate the therapeutic potential of FTY for treating HD by utilizing a well-characterized mouse model of HD (zQ175dn) and wild-type littermates. The study design included a crossover, long-term oral treatment with 1 mg/kg to 2 mg/kg FTY from the age of 15–46 weeks (n = 128). Different motor behavior and physiological parameters were assessed throughout the study. The findings revealed that FTY rescued disease-related body weight loss in a sex-dependent manner, indicating its potential to regulate metabolic disturbances and to counteract neurodegenerative processes in HD. FTY intervention also rescued testicular atrophy, restored testis tissue structure in male mice suggesting a broader impact on peripheral tissues affected by huntingtin pathology. Histological analyses of the brain revealed delayed accumulation of activated astrocytes contributing to the preservation of the neural microenvironment by reducing neuroinflammation. The extent of FTY-related disease improvement was sex-dependent. Motor functions and body weight improved mostly in female mice with sustained estrogen levels, whereas males had to compensate for the ongoing, disease-related testis atrophy and the loss of androgen production. Our study underscores the beneficial therapeutic effects of FTY on HD involving endogenous steroid hormones and their important anabolic effects. It positions FTY as a promising candidate for therapeutic interventions targeting various aspects of HD pathology. Further studies are needed to fully evaluate its therapeutic potential in patients.

• Skagen, Christine; Stevanovic, Stanislava; Bakke, Hege Gilbø; Nyman, Tuula Anneli; Stensland, Maria & Kase, Eili Tranheim [Show all 9 contributors for this article] (2025). Reduced lipid and glucose oxidation and reduced lipid synthesis in AMPKα2/ myotubes. Archives of Physiology and Biochemistry. ISSN 1381-3455. 131(3), p. 483–492. doi: 10.1080/13813455.2024.2449409. Full text in Research Archive Show summary

  Adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) plays a crucial role in regulation ofmetabolic homeostasis. To understand the role of the catalytic α2 subunit of AMPK in skeletal muscleenergy metabolism, myotube cultures were established from AMPKα2+/+ and AMPKα2-/- mice. Myotubesfrom AMPKα2-/- mice had lower basal oleic acid and glucose oxidation compared to myotubes fromAMPKα2+/+ mice. However, the relative response to mitochondrial uncoupling was increased for oleicacid oxidation. Incorporation of acetate into lipids was also lower in myotubes from AMPKα2-/- mice.Proteomics analysis revealed that AMPKα2-/- myotubes had upregulated pathways related to mitochondrialfunction and fatty acid oxidation, and decreased pathways related to fatty acid biosynthesis. Inconclusion, ablation of AMPKα2 catalytic subunit in skeletal muscle cells resulted in reduced basaloxidation of glucose and fatty acids, however upregulated pathways related to mitochondrial functionand fatty acid oxidation and reduced lipid formation

• Montico, Barbara; Giurato, Giorgio; Guerrieri, Roberto; Colizzi, Francesca; Salvati, Annamaria & Nassa, Giovanni [Show all 24 contributors for this article] (2025). Suppression of Spry1 reduces HIF1α-dependent glycolysis and impairs angiogenesis in BRAF-mutant cutaneous melanoma. Journal of Experimental & Clinical Cancer Research. 44(1). doi: 10.1186/s13046-025-03289-8. Full text in Research Archive Show summary

  Background About 50% of cutaneous melanoma (CM) harbors the activating BRAFV600 mutation which exerts most of the oncogenic effects through the MAPK signaling pathway. In the last years, a number of MAPK modulators have been identified, including Spry1. In this context, we have recently demonstrated that knockout of Spry1 (Spry1KO) in BRAFV600-mutant CM led to cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and reduced tumor growth in vivo. Despite these findings, however, the precise molecular mechanism linking Spry1 to BRAFV600-mutant CM remains to be elucidated. Materials and methods Immunoprecipitation coupled to mass spectrometry was employed to gain insight into Spry1 interactome. Spry1 gene was knocked-out using the CRISPR strategy in the BRAF-mutant cell lines. Transmission electron microscopy was used to assess the relationship between Spry1 expression and mitochondrial morphology. By using in vitro and in vivo models, the effects of Spry1KO were investigated through RNA-sequencing, quantitative real-time PCR, Western blot, and immunofluorescence analyses. The Seahorse XF24 assay allowed real-time measurement of cellular metabolism in our model. Angiogenic potential was assessed through in vitro tube formation assays and in vivo CD31 staining. Results Spry1 was mainly located in mitochondria in BRAFV600-mutant CM cells where it interacted with key molecules involved in mitochondrial homeostasis. Spry1 loss resulted in mitochondrial shape alterations and dysfunction, which associated with increased reactive oxygen species production. In agreement, we found that nuclear hypoxia-inducible factor-1 alpha (HIF1α) protein levels were reduced in Spry1KO clones both in vitro and in vivo along with the expression of its glycolysis related genes. Accordingly, Ingenuity Pathway Analysis identified “HIF1α Signaling” as the most significant molecular and cellular function affected by Spry1 silencing, whereas the glycolytic function was significantly impaired in Spry1 depleted BRAFV600-mutant CM cells. In addition, our results indicated that the expression of the vascular endothelial growth factor A was down-regulated following Spry1KO, possibly as a result of mitochondrial dysfunction. Consistently, we observed a substantial impairment of angiogenesis, as assessed by the tube formation assay in vitro and the immunofluorescence staining of CD31 in vivo. Conclusions Altogether, these findings identify Spry1 as a potential regulator of mitochondrial homeostasis, and uncover a previously unrecognized role for Spry1 in regulating nuclear HIF1α expression and angiogenesis in BRAFV600-mutant CM. Significance Spry1KO profoundly impacts on mitochondria homeostasis, while concomitantly impairing HIF1α-dependent glycolysis and reducing angiogenesis in BRAF-mutant CM cells, thus providing a potential therapeutic target to improve BRAFV600-mutant CM treatment.

• Abusdal, Merisa; Markussen, Kjersti Normann; Nyman, Tuula A; Øystese, Kristin Astrid Berland; Sundaram, Arvind & Dahlberg, Daniel [Show all 10 contributors for this article] (2024). PCSK1N as a Tumor Size Marker and an ER Stress Response Protein in Corticotroph Pituitary Adenomas. Journal of Clinical Endocrinology and Metabolism (JCEM). ISSN 0021-972X. 110(4), p. 1065–1075. doi: 10.1210/clinem/dgae643. Full text in Research Archive Show summary

  Abstract Context Silent corticotroph adenoma (SCA) exhibits more tumor aggressiveness features than functioning adenomas (FCAs). Objective We aimed to investigate proprotein convertase subtilisin/kexin type 1 inhibitor (PCSK1N) expression in CA and examine if endoplasmic reticulum (ER) stress-induced responses affect cell survival in a corticotroph tumor cell model. Methods Clinical and imaging characteristics were recorded in 33 patients with FCA (20 women, 11 macroadenomas) and 18 SCAs (8 women, all macroadenomas). Gene expression of pro-opiomelanocortin (POMC), T-box transcription factor 19(TBX19)/TPIT, proprotein convertase subtilisin/kexin type 1 (PCSK1)/PC1/3, and its inhibitor PCSK1N, was measured by reverse transcription–quantitative polymerase chain reaction in adenoma tissue. Mouse pituitary corticotroph tumor (AtT-20) cells were treated with tanespimycin (17-AAG), an HSP90 chaperone inhibitor, to induce ER stress, followed by gene and protein analyses. Results POMC, TPIT, and PCSK1 expression were higher, whereas PCSK1N was lower in FCA compared to SCA. PCSK1N correlated with POMC (rs = −0.514; P < .001), TPIT (rs = −0.386; P = .005), PCSK1 (rs = −0.3691; P = .008), and tumor largest diameter (rs = 0.645; P < .001), in all CA. Induction of ER stress by 17-AAG in AtT-20 cells led to a decrease of Pomc and an increase of Pcsk1n gene expression at 24 hours. Moreover, a downregulation of cell cycle, apoptosis, and senescence pathways, and alterations in cell adhesion and cytoskeleton, were observed at the protein level. Conclusion PCSK1N is higher in SCA compared with FCA, and associated with corticotroph cell markers and tumor size. PCSK1N is likely to be part of the adaptive response to ER stress, potentially conferring a survival advantage to the corticotroph tumor cell in conjunction with other proteins.

• Gorska, Anna Maria; Sanz, Irene Santos Garcia; Eiriz, Iván; Brüning, Thomas; Nyman, Tuula Anneli & Pahnke, Jens (2024). Evaluation of cerebrospinal fluid (CSF) and interstitial fluid (ISF) mouse proteomes for the validation and description of Alzheimer's disease biomarkers. Journal of Neuroscience Methods. ISSN 0165-0270. 411:110239, p. 1–16. doi: 10.1016/j.jneumeth.2024.110239. Full text in Research Archive Show summary

  Background Mass spectrometry (MS)-based cerebrospinal fluid (CSF) proteomics is an important method for discovering biomarkers of neurodegenerative diseases. CSF serves as a reservoir for interstitial fluid (ISF), and extensive communication between the two fluid compartments helps to remove waste products from the brain. New method We performed proteomic analyses of both CSF and ISF fluid compartments using intracerebral microdialysis to validate and detect novel biomarkers of Alzheimer's disease (AD) in APPtg and C57Bl/6J control mice. Results We identified up to 625 proteins in ISF and 4483 proteins in CSF samples. By comparing the biofluid profiles of APPtg and C57Bl/6J mice, we detected 37 and 108 significantly up- and downregulated candidates, respectively. In ISF, 7 highly regulated proteins, such as Gfap, Aldh1l1, Gstm1, and Txn, have already been implicated in AD progression, whereas in CSF, 9 out of 14 highly regulated proteins, such as Apba2, Syt12, Pgs1 and Vsnl1, have also been validated to be involved in AD pathogenesis. In addition, we also detected new interesting regulated proteins related to the control of synapses and neurotransmission (Kcna2, Cacng3, and Clcn6) whose roles as AD biomarkers should be further investigated. Comparison with existing methods This newly established combined protocol provides better insight into the mutual communication between ISF and CSF as an analysis of tissue or CSF compartments alone. Conclusions The use of multiple fluid compartments, ISF and CSF, for the detection of their biological communication enables better detection of new promising AD biomarkers.

• Le, Quy Khang; Chernigovskaia, Mariia; Stensland, Maria; Singh, Sachin; Leem, Jinwoo & Revale, Santiago [Show all 14 contributors for this article] (2024). Benchmarking and integrating human B-cell receptor genomic and antibody proteomic profiling. npj Systems Biology and Applications. 10. doi: 10.1038/s41540-024-00402-z. Full text in Research Archive Show summary

  Immunoglobulins (Ig), which exist either as B-cell receptors (BCR) on the surface of B cells or as antibodies when secreted, play a key role in the recognition and response to antigenic threats. The capability to jointly characterize the BCR and antibody repertoire is crucial for understanding human adaptive immunity. From peripheral blood, bulk BCR sequencing (bulkBCR-seq) currently provides the highest sampling depth, single-cell BCR sequencing (scBCR-seq) allows for paired chain characterization, and antibody peptide sequencing by tandem mass spectrometry (Ab-seq) provides information on the composition of secreted antibodies in the serum. Yet, it has not been benchmarked to what extent the datasets generated by these three technologies overlap and complement each other. To address this question, we isolated peripheral blood B cells from healthy human donors and sequenced BCRs at bulk and single-cell levels, in addition to utilizing publicly available sequencing data. Integrated analysis was performed on these datasets, resolved by replicates and across individuals. Simultaneously, serum antibodies were isolated, digested with multiple proteases, and analyzed with Ab-seq. Systems immunology analysis showed high concordance in repertoire features between bulk and scBCR-seq within individuals, especially when replicates were utilized. In addition, Ab-seq identified clonotype-specific peptides using both bulk and scBCR-seq library references, demonstrating the feasibility of combining scBCR-seq and Ab-seq for reconstructing paired-chain Ig sequences from the serum antibody repertoire. Collectively, our work serves as a proof-of-principle for combining bulk sequencing, single-cell sequencing, and mass spectrometry as complementary methods towards capturing humoral immunity in its entirety.

• Hilander, Taru; Awadhpersad, Ryan; Monteuuis, Geoffray; Broda, Krystyna L.; Pohjanpelto, Max & Pyman, Elizabeth [Show all 15 contributors for this article] (2024). Supernumerary proteins of the human mitochondrial ribosomal small subunit are integral for assembly and translation. iScience. 27:110185(7), p. 1–20. doi: 10.1016/j.isci.2024.110185. Full text in Research Archive Show summary

  Mitochondrial ribosomes (mitoribosomes) have undergone substantial evolutionary structural remodeling accompanied by loss of ribosomal RNA, while acquiring unique protein subunits located on the periphery. We generated CRISPR-mediated knockouts of all 14 unique (mitochondria-specific/supernumerary) human mitoribosomal proteins (snMRPs) in the small subunit to study the effect on mitoribosome assembly and protein synthesis, each leading to a unique mitoribosome assembly defect with variable impact on mitochondrial protein synthesis. Surprisingly, the stability of mS37 was reduced in all our snMRP knockouts of the small and large ribosomal subunits and patient-derived lines with mitoribosome assembly defects. A redox-regulated CX9C motif in mS37 was essential for protein stability, suggesting a potential mechanism to regulate mitochondrial protein synthesis. Together, our findings support a modular assembly of the human mitochondrial small ribosomal subunit mediated by essential supernumerary subunits and identify a redox regulatory role involving mS37 in mitochondrial protein synthesis in health and disease.

• Kang, Yilin; Hepojoki, Jussi; Maldonado, Rocio Sartori; Mito, Takayuki; Terzioglu, Mügen & Manninen, Tuula [Show all 19 contributors for this article] (2024). Ancestral allele of DNA polymerase gamma modifies antiviral tolerance. Nature. ISSN 0028-0836. 628, p. 844–853. doi: 10.1038/s41586-024-07260-z. Full text in Research Archive Show summary

  Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1,2,3,4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

• Katare, Parmeshwar Bajirao; Dalmao-Fernandez, Andrea; Mengeste, Abel Mulu; Navabakbar, Farnaz; Hamarsland, Håvard & Ellefsen, Stian [Show all 12 contributors for this article] (2024). Krill oil supplementation in vivo promotes increased fuel metabolism and protein synthesis in cultured human skeletal muscle cells. Frontiers in Nutrition. 11:1452768, p. 1–14. doi: 10.3389/fnut.2024.1452768. Full text in Research Archive Show summary

  Introduction: Krill oil is a dietary supplement derived from Antarctic krill; a small crustacean found in the ocean. Krill oil is a rich source of omega-3 fatty acids, specifically eicosapentaenoic acid and docosahexaenoic acid, as well as the antioxidant astaxanthin. The aim of this study was to investigate the effects of krill oil supplementation, compared to placebo oil (high oleic sunflower oil added astaxanthin), in vivo on energy metabolism and substrate turnover in human skeletal muscle cells. Methods: Skeletal muscle cells (myotubes) were obtained before and after a 7-week krill oil or placebo oil intervention, and glucose and oleic acid metabolism and leucine accumulation, as well as effects of different stimuli in vitro, were studied in the myotubes. The functional data were combined with proteomic and transcriptomic analyses. Results: In vivo intervention with krill oil increased oleic acid oxidation and leucine accumulation in skeletal muscle cells, however no effects were observed on glucose metabolism. The krill oil-intervention-induced increase in oleic acid oxidation correlated negatively with changes in serum low-density lipoprotein (LDL) concentration. In addition, myotubes were also exposed to krill oil in vitro. The in vitro study revealed that 24 h of krill oil treatment increased both glucose and oleic acid metabolism in myotubes, enhancing energy substrate utilization. Transcriptomic analysis comparing myotubes obtained before and after krill oil supplementation identified differentially expressed genes associated with e.g., glycolysis/gluconeogenesis, metabolic pathways and calcium signaling pathway, while proteomic analysis demonstrated upregulation of e.g., LDL-receptor in myotubes obtained after the krill oil intervention. Conclusion: These findings suggest that krill oil intervention promotes increased fuel metabolism and protein synthesis in human skeletal muscle cells, with potential implications for metabolic health.

• Loivamaa, Iida; Sillanpää, Annika; Deptula, Paulina; Chamlagain, Bhawani; Edelmann, Minnamari & Auvinen, Petri [Show all 10 contributors for this article] (2024). Aerobic adaptation and metabolic dynamics of Propionibacterium freudenreichii DSM 20271: insights from comparative transcriptomics and surfaceome analysis. mSystems. 9:e00615-24(10), p. 1–26. doi: 10.1128/msystems.00615-24. Full text in Research Archive Show summary

  Propionibacterium freudenreichii (PFR) DSM 20271T is a bacterium known for its ability to thrive in diverse environments and to produce vitamin B12. Despite its anaerobic preference, recent studies have elucidated its ability to prosper in the presence of oxygen, prompting a deeper exploration of its physiology under aerobic conditions. Here, we investigated the response of DSM 20271T to aerobic growth by employing comparative transcriptomic and surfaceome analyses alongside metabolite profiling. Cultivation under controlled partial pressure of oxygen (pO2) conditions revealed significant increases in biomass formation and altered metabolite production, notably of vitamin B12, pseudovitamin-B12, propionate, and acetate, under aerobic conditions. Transcriptomic analysis identified differential expression of genes involved in lactate metabolism, tricarboxylic acid cycle, and electron transport chain, suggesting metabolic adjustments to aerobic environments. Moreover, surfaceome analysis unveiled growth environment-dependent changes in surface protein abundance, with implications for adaptation to atmospheric conditions. Supplementation experiments with key compounds highlighted the potential for enhancing aerobic growth, emphasizing the importance of iron and α-ketoglutarate availability. Furthermore, in liquid culture, FeSO4 supplementation led to increased heme production and reduced vitamin B12 production, highlighting the impact of oxygen and iron availability on the metabolic pathways. These findings deepen our understanding of PFR’s physiological responses to oxygen availability and offer insights for optimizing its growth in industrial applications.

• Stevanovic, Stanislava; Dalmao-Fernandez, Andrea; Mohamed, Derya; Nyman, Tuula Anneli; Kostovski, Emil & Iversen, Per Ole [Show all 11 contributors for this article] (2024). Time-dependent reduction in oxidative capacity among cultured myotubes from spinal cord injured individuals. Acta Physiologica. ISSN 1748-1708. 240:e14156(7), p. 1–15. doi: 10.1111/apha.14156. Full text in Research Archive Show summary

  Background Skeletal muscle adapts in reaction to contractile activity to efficiently utilize energy substrates, primarily glucose and free fatty acids (FA). Inactivity leads to atrophy and a change in energy utilization in individuals with spinal cord injury (SCI). The present study aimed to characterize possible inactivity-related differences in the energy metabolism between skeletal muscle cells cultured from satellite cells isolated 1- and 12-months post-SCI. Methods To characterize inactivity-related disturbances in spinal cord injury, we studied skeletal muscle cells isolated from SCI subjects. Cell cultures were established from biopsy samples from musculus vastus lateralis from subjects with SCI 1 and 12 months after the injury. The myoblasts were proliferated and differentiated into myotubes before fatty acid and glucose metabolism were assessed and gene and protein expressions were measured. Results The results showed that glucose uptake was increased, while oleic acid oxidation was reduced at 12 months compared to 1 month. mRNA expressions of PPARGC1α, the master regulator of mitochondrial biogenesis, and MYH2, a determinant of muscle fiber type, were significantly reduced at 12 months. Proteomic analysis showed reduced expression of several mitochondrial proteins. Conclusion In conclusion, skeletal muscle cells isolated from immobilized subjects 12 months compared to 1 month after SCI showed reduced fatty acid metabolism and reduced expression of mitochondrial proteins, indicating an increased loss of oxidative capacity with time after injury.

• Saidu, Nathaniel Edward Bennett; Larsen, Miriam Aarsund; Sørensen, Eva; Stensland, Maria; Nyman, Tuula Anneli & ULVMOEN, AINA [Show all 8 contributors for this article] (2023). Identifying a core protein signature of small extracellular vesicles derived from B-cell precursor acute lymphoblastic leukaemia. Scandinavian Journal of Immunology. ISSN 0300-9475. 99(3), p. 1–10. doi: 10.1111/sji.13341. Full text in Research Archive Show summary

  Abstract Acute paediatric leukaemia is diagnosed and monitored via bone marrow aspirate assessment of blasts as a measure of minimal residual disease. Liquid biopsies in the form of blood samples could greatly reduce the need for invasive bone marrow aspirations, but there are currently no blood markers that match the sensitivity of bone marrow diagnostics. Circulating extracellular vesicles (EVs) represent candidate biomarkers that may reflect the blast burden in bone marrow, and several studies have reported on the utility of EVs as biomarkers for adult haematological malignancies. Increased levels of EVs have been reported for several haematological malignancies, and we similarly report here elevated EV concentrations in plasma from paediatric BCP‐ALL patients. Plasma EVs are very heterogeneous in terms of their cellular origin, thus identifying a cancer selective EV‐marker is challenging. Here, we undertook a reductionistic approach to identify protein markers selectively associated to plasma EVs derived from BCP‐ALL patients. The EV proteome of primary BCP‐ALL cell‐derived EVs were compared against EVs from healthy donor B cells and the BCP‐ALL cell line REH, and further against EVs isolated from plasma of healthy paediatric donors and paediatric BCP‐ALL patients. With this approach, we identified a signature of 6 proteins (CD317, CD38, IGF2BP1, PCNA, CSDE1, and GPR116) that were specifically identified in BCP‐ALL derived EVs only and not in healthy control EVs, and that could be exploited as diagnostic biomarkers.

• Misceo, Doriana; Lirussi, Lisa; Strømme, Petter; Sumathipala, Dulika Sanjeewani; Guerin, Andrea & Wolf, Nicole I [Show all 14 contributors for this article] (2023). A homozygous POLR1A variant causes leukodystrophy and affects protein homeostasis. Brain. ISSN 0006-8950. 146(8), p. 3513–3527. doi: 10.1093/brain/awad086. Full text in Research Archive Show summary

  Abstract RNA polymerase I transcribes ribosomal DNA to produce precursor 47S rRNA. Post-transcriptional processing of this rRNA generates mature 28S, 18S and 5.8S rRNAs, which form the ribosomes, together with 5S rRNA, assembly factors and ribosomal proteins. We previously reported a homozygous variant in the catalytic subunit of RNA polymerase I, POLR1A, in two brothers with leukodystrophy and progressive course. However, the disease mechanism remained unknown. In this report, we describe another missense variant POLR1A NM_015425.3:c.1925C>A; p.(Thr642Asn) in homozygosity in two unrelated patients. Patient 1 was a 16-year-old male and Patient 2 was a 2-year-old female. Both patients manifested neurological deficits, with brain MRIs showing hypomyelinating leukodystrophy and cerebellar atrophy; and in Patient 1 additionally with hypointensity of globi pallidi and small volume of the basal ganglia. Patient 1 had progressive disease course, leading to death at the age of 16.5 years. Extensive in vitro experiments in fibroblasts from Patient 1 documented that the mutated POLR1A led to aberrant rRNA processing and degradation, and abnormal nucleolar homeostasis. Proteomics data analyses and further in vitro experiments documented abnormal protein homeostasis, and endoplasmic reticulum stress responses. We confirm that POLR1A biallelic variants cause neurodegenerative disease, expand the knowledge of the clinical phenotype of the disorder, and provide evidence for possible pathological mechanisms leading to POLR1A-related leukodystrophy.

• Frerker, Nadine; Karlsen, Tommy Aleksander; Stensland, Maria; Nyman, Tuula Anneli; Rayner, Simon & Brinchmann, Jan Engelsen (2023). Comparison between articular chondrocytes and mesenchymal stromal cells for the production of articular cartilage implants. Frontiers in Bioengineering and Biotechnology. 11. doi: 10.3389/fbioe.2023.1116513. Full text in Research Archive Show summary

  Focal lesions of articular cartilage give rise to pain and reduced joint function and may, if left untreated, lead to osteoarthritis. Implantation of in vitro generated, scaffold-free autologous cartilage discs may represent the best treatment option. Here we compare articular chondrocytes (ACs) and bone marrow-derived mesenchymal stromal cells (MSCs) for their ability to make scaffold-free cartilage discs. Articular chondrocytes produced more extracellular matrix per seeded cell than mesenchymal stromal cells. Quantitative proteomics analysis showed that articular chondrocyte discs contained more articular cartilage proteins, while mesenchymal stromal cell discs had more proteins associated with cartilage hypertrophy and bone formation. Sequencing analysis revealed more microRNAs associated with normal cartilage in articular chondrocyte discs, and large-scale target predictions, performed for the first time for in vitro chondrogenesis, suggested that differential expression of microRNAs in the two disc types were important mechanisms behind differential synthesis of proteins. We conclude that articular chondrocytes should be preferred over mesenchymal stromal cells for tissue engineering of articular cartilage.

• Gorski, Katarin; Jackson, Christopher B.; Nyman, Tuula Anneli; Rezov, Veronika; Battersby, Brendan J. & Lehesjoki, Anna-Elina (2023). Progressive mitochondrial dysfunction in cerebellar synaptosomes of cystatin B-deficient mice. Frontiers in Molecular Neuroscience. 16:1175851, p. 1–12. doi: 10.3389/fnmol.2023.1175851. Full text in Research Archive Show summary

  The involvement of mitochondrial dysfunction in cystatin B (CSTB) deficiency has been suggested, but its role in the onset of neurodegeneration, myoclonus, and ataxia in the CSTB-deficient mouse model ( Cstb −/− ) is yet unknown. CSTB is an inhibitor of lysosomal and nuclear cysteine cathepsins. In humans, partial loss-of-function mutations cause the progressive myoclonus epilepsy neurodegenerative disorder, EPM1. Here we applied proteome analysis and respirometry on cerebellar synaptosomes from early symptomatic ( Cstb −/− ) mice to identify the molecular mechanisms involved in the onset of CSTB-deficiency associated neural pathogenesis. Proteome analysis showed that CSTB deficiency is associated with differential expression of mitochondrial and synaptic proteins, and respirometry revealed a progressive impairment in mitochondrial function coinciding with the onset of myoclonus and neurodegeneration in ( Cstb −/− ) mice. This mitochondrial dysfunction was not associated with alterations in mitochondrial DNA copy number or membrane ultrastructure. Collectively, our results show that CSTB deficiency generates a defect in synaptic mitochondrial bioenergetics that coincides with the onset and progression of the clinical phenotypes, and thus is likely a contributor to the pathogenesis of EPM1.

• Hammarén, Milka Marjut; Luukinen, Hanna; Sillanpää, Alina; Remans, Kim; Lapouge, Karine & Custódio, Tânia [Show all 14 contributors for this article] (2023). In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies. mSystems. 8:e01073-22(3), p. 1–27. doi: 10.1128/msystems.01073-22. Full text in Research Archive Show summary

  The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface–binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection.

• Krapf, Solveig; Lund, Jenny; Bakke, Hege Gilbø; Nyman, Tuula Anneli; Bartesaghi, Stefano & Peng, Xiao-Rong [Show all 9 contributors for this article] (2023). SENP2 knockdown in human adipocytes reduces glucose metabolism and lipid accumulation, while increases lipid oxidation. Metabolism Open. 18. doi: 10.1016/j.metop.2023.100234. Full text in Research Archive Show summary

  Adipose tissue is one of the main regulative sites for energy metabolism. Excess lipid storage and expansion of white adipose tissue (WAT) is the primary contributor to obesity, a strong predisposing factor for development of insulin resistance. Sentrin-specific protease (SENP) 2 has been shown to play a role in metabolism in murine fat and skeletal muscle cells, and we have previously demonstrated its role in energy metabolism of human skeletal muscle cells. In the present work, we have investigated the impact of SENP2 on fatty acid and glucose metabolism in primary human fat cells by using cultured primary human adipocytes to knock down the SENP2 gene. Glucose uptake and oxidation, as well as accumulation and distribution of oleic acid into complex lipids were decreased, while oleic acid oxidation was increased in SENP2-knockdown cells compared to control adipocytes. Furthermore, lipogenesis was reduced by SENP2-knockdown in adipocytes. Although TAG accumulation relative to total uptake was unchanged, there was increased mRNA expression of metabolically relevant genes such as UCP1 and PPARGC1A and mRNA and proteomic data revealed increased levels of mRNA and proteins related to mitochondrial function by SENP2-knockdown. In conclusion, SENP2 is an important regulator of energy metabolism in primary human adipocytes and its knockdown reduce glucose metabolism and lipid accumulation, while increasing lipid oxidation in human adipocytes.

• Lunde, Ngoc Nguyen; Osoble, Nimo Mukhtar Mohamud; Dalmao-Fernandez, Andrea; Lukacs, Alfreda Serwah; Jafari, Abbas & Singh, Sachin [Show all 10 contributors for this article] (2023). Interplay between Cultured Human Osteoblastic and Skeletal Muscle Cells: Effects of Conditioned Media on Glucose and Fatty Acid Metabolism. Biomedicines. 11:2908(11), p. 1–20. doi: 10.3390/biomedicines11112908. Full text in Research Archive Show summary

  The interplay between skeletal muscle and bone is primarily mechanical; however, biochemical crosstalk by secreted mediators has recently gained increased attention. The aim of this study was to investigate metabolic effects of conditioned medium from osteoblasts (OB-CM) on myotubes and vice versa. Human skeletal muscle cells incubated with OB-CM showed increased glucose uptake and oxidation, and mRNA expression of the glucose transporter (GLUT) 1, while fatty acid uptake and oxidation, and mRNA expression of the fatty acid transporter CD36 were decreased. This was supported by proteomic analysis, where expression of proteins involved in glucose uptake, glycolytic pathways, and the TCA cycle were enhanced, and expression of several proteins involved in fatty acid metabolism were reduced. Similar effects on energy metabolism were observed in human bone marrow stromal cells differentiated to osteoblastic cells incubated with conditioned medium from myotubes (SKM-CM), with increased glucose uptake and reduced oleic acid uptake. Proteomic analyses of the two conditioned media revealed many common proteins. Thus, our data may indicate a shift in fuel preference from fatty acid to glucose metabolism in both cell types, induced by conditioned media from the opposite cell type, possibly indicating a more general pattern in communication between these tissues.

• Harrison, Sean Philip; Siller, Richard; Tanaka, Yoshiaki; Dugarte, Maria Eugenia Chollet; Morena-Barrio, María Eugenia de la & Xiang, Yangfei [Show all 36 contributors for this article] (2023). Scalable production of tissue-like vascularized liver organoids from human PSCs. Experimental and Molecular Medicine. ISSN 1226-3613. 55, p. 2005–2024. doi: 10.1038/s12276-023-01074-1. Full text in Research Archive Show summary

  Abstract The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.

• Kangas, Petra; Nyman, Tuula Anneli; Metsähonkala, Liisa; Burns, Cameron; Tempest, Robert & Williams, Tim [Show all 8 contributors for this article] (2023). Towards optimised extracellular vesicle proteomics from cerebrospinal fluid. Scientific Reports. 13:9564, p. 1–13. doi: 10.1038/s41598-023-36706-z. Full text in Research Archive Show summary

  Abstract The proteomic profile of extracellular vesicles (EVs) from cerebrospinal fluid (CSF) can reveal novel biomarkers for diseases of the brain. Here, we validate an ultrafiltration combined with size-exclusion chromatography (UF-SEC) method for isolation of EVs from canine CSF and probe the effect of starting volume on the EV proteomics profile. First, we performed a literature review of CSF EV articles to define the current state of art, discovering a need for basic characterisation of CSF EVs. Secondly, we isolated EVs from CSF by UF-SEC and characterised the SEC fractions by protein amount, particle count, transmission electron microscopy, and immunoblotting. Data are presented as mean ± standard deviation. Using proteomics, SEC fractions 3–5 were compared and enrichment of EV markers in fraction 3 was detected, whereas fractions 4–5 contained more apolipoproteins. Lastly, we compared starting volumes of pooled CSF (6 ml, 3 ml, 1 ml, and 0.5 ml) to evaluate the effect on the proteomic profile. Even with a 0.5 ml starting volume, 743 ± 77 or 345 ± 88 proteins were identified depending on whether ‘matches between runs’ was active in MaxQuant. The results confirm that UF-SEC effectively isolates CSF EVs and that EV proteomic analysis can be performed from 0.5 ml of canine CSF.

• Cypryk, Wojciech; Czernek, Liliana; Horodecka, Katarzyna; Chrzanowski, Jędrzej; Stanczak, Marcin & Nurmi, Katariina [Show all 14 contributors for this article] (2023). Lipopolysaccharide Primes Human Macrophages for Noncanonical Inflammasome-Induced Extracellular Vesicle Secretion. Journal of Immunology. ISSN 0022-1767. 210(3), p. 322–334. doi: 10.4049/jimmunol.2200444. Full text in Research Archive
• Aas, Vigdis; Øvstebø, Reidun; Brusletto, Berit; Aspelin, Trude; Trøseid, Anne-Marie Siebke & Qureshi, Saba [Show all 10 contributors for this article] (2023). Distinct microRNA and protein profiles of extracellular vesicles secreted from myotubes from morbidly obese donors with type 2 diabetes in response to electrical pulse stimulation. Frontiers in Physiology. 14. doi: 10.3389/fphys.2023.1143966. Full text in Research Archive Show summary

  Lifestyle disorders like obesity, type 2 diabetes (T2D), and cardiovascular diseases can be prevented and treated by regular physical activity. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from six morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter. Size and concentration of EV subtypes were characterized by nanoparticle tracking analysis, surface markers were examined by flow cytometry and Western blotting, and morphology was confirmed by transmission electron microscopy. Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray, and selected microRNAs (miRs) validated by real time RT-qPCR. The size and concentration of exosomes and MV were unaffected by EPS. Of the 400 miRs identified in the EVs, EPS significantly changed the level of 15 exosome miRs, of which miR-1233-5p showed the highest fold change. The miR pattern of MV was unaffected by EPS. Totally, about 1000 proteins were identified in exosomes and 2000 in MV. EPS changed the content of 73 proteins in exosomes, 97 in MVs, and of these four were changed in both exosomes and MV (GANAB, HSPA9, CNDP2, and ATP5B). By matching the EPS-changed miRs and proteins in exosomes, 31 targets were identified, and among these several promising signaling factors. Of particular interest were CNDP2, an enzyme that generates the appetite regulatory metabolite Lac-Phe, and miR-4433b-3p, which targets CNDP2. Several of the regulated miRs, such as miR-92b-5p, miR-320b, and miR-1233-5p might also mediate interesting signaling functions. In conclusion, we have used a combined transcriptome-proteome approach to describe how EPS affected the cargo of EVs derived from myotubes from morbidly obese patients with T2D, and revealed several new factors, both miRs and proteins, that might act as exercise factors.

• Høyer, Helle; Busk, Øyvind Løvold; Esbensen, Qin Ying; Røsby, Oddveig; Hilmarsen, Hilde Tveitan & Russell, Michael Bjørn [Show all 9 contributors for this article] (2022). Clinical characteristics and proteome modifications in two Charcot-Marie-Tooth families with the AARS1 Arg326Trp mutation. BMC Neurology. 22(1). doi: 10.1186/s12883-022-02828-6. Full text in Research Archive Show summary

  Background: Aminoacyl tRNA-synthetases are ubiquitously-expressed enzymes that attach amino acids to their cognate tRNA molecules. Mutations in several genes encoding aminoacyl tRNA-synthetases, have been associated with peripheral neuropathy, i.e. AARS1, GARS1, HARS1, YARS1 and WARS1. The pathogenic mechanism underlying AARS1-related neuropathy is not known. Methods: From 2012 onward, all probands presenting at Telemark Hospital (Skien, Norway) with peripheral neuropathy were screened for variants in AARS1 using an "in-house" next-generation sequencing panel. DNA from patient's family members was examined by Sanger sequencing. Blood from affected family members and healthy controls were used for quantification of AARS1 mRNA and alanine. Proteomic analyses were conducted in peripheral blood mononuclear cells (PBMC) from four affected family members and five healthy controls. Results: Seventeen individuals in two Norwegian families affected by Charcot-Marie-Tooth disease (CMT) were characterized in this study. The heterozygous NM_001605.2:c.976C > T p.(Arg326Trp) AARS1 mutation was identified in ten affected family members. All living carriers had a mild to severe length-dependent sensorimotor neuropathy. Three deceased obligate carriers aged 74-98 were reported to be unaffected, but were not examined in the clinic. Proteomic studies in PBMC from four affected individuals suggest an effect on the immune system mediated by components of a systemic response to chronic injury and inflammation. Furthermore, altered expression of proteins linked to mitochondrial function/dysfunction was observed. Proteomic data are available via ProteomeXchange using identifier PXD023842. Conclusion: This study describes clinical and neurophysiological features linked to the p.(Arg326Trp) variant of AARS1 in CMT-affected members of two Norwegian families. Proteomic analyses based on of PBMC from four CMT-affected individuals suggest that involvement of inflammation and mitochondrial dysfunction might contribute to AARS1 variant-associated peripheral neuropathy.

• Salvati, Annamaria; Melone, Viola; Sellitto, Assunta; Rizzo, Francesca; Tarallo, Roberta & Nyman, Tuula Anneli [Show all 9 contributors for this article] (2022). Combinatorial targeting of a chromatin complex comprising Dot1L, menin and the tyrosine kinase BAZ1B reveals a new therapeutic vulnerability of endocrine therapy-resistant breast cancer. Breast Cancer Research. ISSN 1465-5411. 24:52(1), p. 1–23. doi: 10.1186/s13058-022-01547-7. Full text in Research Archive Show summary

  Background Targeting vulnerabilities of cancer cells by inhibiting key regulators of cell proliferation or survival represents a promising way to overcome resistance to current therapies. In breast cancer (BC), resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor alpha (ERα) signaling to the genome. Targeting components of the ERα pathway in these tumors represents, therefore, a rational way toward effective new treatments. Interaction proteomics identified several proteins associated with ERα in BC cells, including epigenetic complexes controlling gene transcription comprising the scaffold protein menin and the histone methyltransferase Dot1L. Methods We combined chromatin immunoprecipitation, transcriptome sequencing, siRNA-mediated gene knockdown (kd), pharmacological inhibition coupled to cellular and functional assays and interaction proteomics in antiestrogen (AE)-sensitive and AE-resistant human BC cell models to: map menin and Dot1L chromatin localization, search for their common and specific target genes, measure the effects of single or combinatorial knockdown or pharmacological inhibition of these proteins on cell proliferation and survival, and characterize their nuclear interactomes. Results Dot1L and menin associate in MCF-7 cells chromatin, where they co-localize in a significant fraction of sites, resulting in co-regulation of genes involved, among others, in estrogen, p53, HIF1α and death receptor signaling, regulation of cell cycle and epithelial-to-mesenchymal transition. Specific inhibitors of the two factors synergize with each other for inhibition of cell proliferation of AE (tamoxifen or fulvestrant)-sensitive and AE-resistant BC cells. Menin and Dot1L interactomes share a sizeable fraction of their nuclear partners, the majority being known BC fitness genes. Interestingly, these include B-WICH and WINAC complexes that share BAZ1B, a bromodomain protein comprising a tyrosine–protein kinase domain playing a central role in chromatin remodeling and transcriptional regulation. BAZ1B kd caused significant inhibition of ERα expression, proliferation and transcriptome changes resulting in inhibition of estrogen, myc, mTOR, PI3K and AKT signaling and metabolic pathways in AE-sensitive and AE-resistant BC cells. Conclusions Identification of a functional interplay between ERα, Dot1L, menin and BAZ1B and the significant effects of their co-inhibition on cell proliferation and survival in cell models of endocrine therapy-resistant BC reveal a new therapeutic vulnerability of these aggressive diseases.

• Larsen, Miriam Aarsund; Nyman, Tuula Anneli; Stensland, Maria Ekman; Wu, Yunjie & Inngjerdingen, Marit (2022). Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins. Frontiers in Immunology. 13:977353, p. 1–12. doi: 10.3389/fimmu.2022.977353. Full text in Research Archive Show summary

  NK cells can broadly target and kill malignant cells via release of cytolytic proteins. NK cells also release extracellular vesicles (EVs) that contain cytolytic proteins, previously shown to induce apoptosis of a variety of cancer cells in vitro and in vivo . The EVs released by NK cells are likely very heterogeneous, as vesicles can be released from the plasma membrane or from different intracellular compartments. In this study, we undertook a fractionation scheme to enrich for cytolytic NK-EVs. NK-EVs were harvested from culture medium from the human NK-92 cell line or primary human NK cells grown in serum-free conditions. By combining ultracentrifugation with downstream density-gradient ultracentrifugation or size-exclusion chromatography, distinct EV populations were identified. Density-gradient ultracentrifugation led to separation of three subpopulations of EVs. The different EV isolates were characterized by label-free quantitative mass spectrometry and western blotting, and we found that one subpopulation was primarily enriched for plasma membrane proteins and tetraspanins CD37, CD82, and CD151, and likely represents microvesicles. The other major subpopulation was enriched in intracellularly derived markers with high expression of the endosomal tetraspanin CD63 and markers for intracellular organelles. The intracellularly derived EVs were highly enriched in cytolytic proteins, and possessed high apoptotic activity against HCT-116 colon cancer spheroids. To further enrich for cytolytic EVs, immunoaffinity pulldowns led to the isolation of a subset of EVs containing the cytolytic granule marker NKG7 and the majority of vesicular granzyme B content. We therefore propose that EVs containing cytolytic proteins may primarily be released via cytolytic granules.

• Mengeste, Abel Mulu; Nikolic, Natasa; Fernandez, Andrea Dalmao; Feng, Yuan Zeng; Nyman, Tuula Anneli & Kersten, Sander [Show all 11 contributors for this article] (2022). Insight Into the Metabolic Adaptations of Electrically Pulse-Stimulated Human Myotubes Using Global Analysis of the Transcriptome and Proteome. Frontiers in Physiology. 13. doi: 10.3389/fphys.2022.928195. Full text in Research Archive Show summary

  Electrical pulse stimulation (EPS) has proven to be a useful tool to interrogate cell-specific responses to muscle contraction. In the present study, we aimed to uncover networks of signaling pathways and regulatory molecules responsible for the metabolic effects of exercise in human skeletal muscle cells exposed to chronic EPS. Differentiated myotubes from young male subjects were exposed to EPS protocol 1 (i.e. 2 ms, 10 V, and 0.1 Hz for 24 h), whereas myotubes from middle-aged women and men were exposed to protocol 2 (i.e. 2 ms, 30 V, and 1 Hz for 48 h). Fuel handling as well as the transcriptome, cellular proteome, and secreted proteins of EPS-treated myotubes from young male subjects were analyzed using a combination of high-throughput RNA sequencing, high-resolution liquid chromatography-tandem mass spectrometry, oxidation assay, and immunoblotting. The data showed that oxidative metabolism was enhanced in EPS-exposed myotubes from young male subjects. Moreover, a total of 81 differentially regulated proteins and 952 differentially expressed genes (DEGs) were observed in these cells after EPS protocol 1. We also found 61 overlapping genes while comparing the DEGs to mRNA expression in myotubes from the middle-aged group exposed to protocol 2, assessed by microarray. Gene ontology (GO) analysis indicated that significantly regulated proteins and genes were enriched in biological processes related to glycolytic pathways, positive regulation of fatty acid oxidation, and oxidative phosphorylation, as well as muscle contraction, autophagy/mitophagy, and oxidative stress. Additionally, proteomic identification of secreted proteins revealed extracellular levels of 137 proteins were changed in myotubes from young male subjects exposed to EPS protocol 1. Selected putative myokines were measured using ELISA or multiplex assay to validate the results. Collectively, our data provides new insight into the transcriptome, proteome and secreted proteins alterations following in vitro exercise and is a valuable resource for understanding the molecular mechanisms and regulatory molecules mediating the beneficial metabolic effects of exercise.

• Hautala, Katja; Pursiainen, Jami; Näreaho, Anu; Nyman, Tuula Anneli; Varmanen, Pekka & Sukura, Antti [Show all 8 contributors for this article] (2022). Label-free quantitative proteomics and immunoblotting identifies immunoreactive and other excretory-secretory (E/S) proteins of Anoplocephala perfoliata. Frontiers in Immunology. 13:1045468, p. 1–14. doi: 10.3389/fimmu.2022.1045468. Full text in Research Archive Show summary

  Anoplocephala perfoliata is a common tapeworm in horses causing colic and even mortalities. Current diagnostic tests to detect A. perfoliata infections have their limitations and an improved method is needed. Immunoreactive excretory/secretory proteins (E/S proteome) of this parasite can provide promising candidates for diagnostic tests. We compared E/S proteins produced by small (length < 20 mm, width < 5 mm) and large (length 20 to 40 mm, width 5 to 10 mm) A. perfoliata worms in vitro by label-free quantitative proteomics using a database composed of related Hymenolepis diminuta, Echinococcus multilocularis/granulosus and Taenia aseatica proteins for protein identifications. Altogether, 509 E/S proteins were identified after incubating the worms in vitro for three and eight hours. The greatest E/S proteome changes suggested both worm size- and time-dependent changes in cytoskeleton remodeling, apoptosis, and production of antigens/immunogens. The E/S proteins collected at the three-hour time point represented the natural conditions better than those collected at the eight-hour time point, and thereby contained the most relevant diagnostic targets. Immunoblotting using antibodies from horses tested positive/negative for A. perfoliata indicated strongest antigenicity/immunogenicity with 13-, 30- and 100-kDa proteins, involving a thioredoxin, heat-shock chaperone 90 (Hsp90), dynein light chain component (DYNLL), tubulin-specific chaperone A (TBCA) and signaling pathway modulators (14-3-3 and Sj-Ts4). This is among the first studies identifying new diagnostic targets and A. perfoliata antigens eliciting a IgG-response in horses.

• Reigada, Ines; San-Martin-Gadolino, Paola; Gilbert-Girard, Shella; Chiaro, Jacopo; Cerullo, Vincenzo & Savijoki, Kirsi [Show all 9 contributors for this article] (2021). Surfaceome and Exoproteome Dynamics in Dual-Species Pseudomonas aeruginosa and Staphylococcus aureus Biofilms. Frontiers in Microbiology. 12:672975, p. 1–23. doi: 10.3389/fmicb.2021.672975. Full text in Research Archive Show summary

  Bacterial biofilms are an important underlying cause for chronic infections. By switching into the biofilm state, bacteria can evade host defenses and withstand antibiotic chemotherapy. Despite the fact that biofilms at clinical and environmental settings are mostly composed of multiple microbial species, biofilm research has largely been focused on single-species biofilms. In this study, we investigated the interaction between two clinically relevant bacterial pathogens ( Staphylococcus aureus and Pseudomonas aeruginosa ) by label-free quantitative proteomics focusing on proteins associated with the bacterial cell surfaces (surfaceome) and proteins exported/released to the extracellular space (exoproteome). The changes observed in the surfaceome and exoproteome of P. aeruginosa pointed toward higher motility and lower pigment production when co-cultured with S. aureus . In S. aureus , lower abundances of proteins related to cell wall biosynthesis and cell division, suggesting increased persistence, were observed in the dual-species biofilm. Complementary phenotypic analyses confirmed the higher motility and the lower pigment production in P. aeruginosa when co-cultured with S. aureus. Higher antimicrobial tolerance associated with the co-culture setting was additionally observed in both species. To the best of our knowledge, this study is among the first systematic explorations providing insights into the dynamics of both the surfaceome and exoproteome of S. aureus and P. aeruginosa dual-species biofilms.

• Savijoki, Kirsi; Myllymäki, Henna; Luukinen, Hanna; Paulamäki, Lauri; Vanha-aho, Leena-Maija & Svorjova, Aleksandra [Show all 12 contributors for this article] (2021). Surface-Shaving Proteomics of Mycobacterium marinum Identifies Biofilm Subtype-Specific Changes Affecting Virulence, Tolerance, and Persistence. mSystems. 6:e00500(3), p. 1–23. doi: 10.1128/mSystems.00500-21. Full text in Research Archive Show summary

  The complex cell wall and biofilm matrix (ECM) act as key barriers to antibiotics in mycobacteria. Here, the ECM and envelope proteins of Mycobacterium marinum ATCC 927, a nontuberculous mycobacterial model, were monitored over 3 months by label-free proteomics and compared with cell surface proteins on planktonic cells to uncover pathways leading to virulence, tolerance, and persistence. We show that ATCC 927 forms pellicle-type and submerged-type biofilms (PBFs and SBFs, respectively) after 2 weeks and 2 days of growth, respectively, and that the increased CelA1 synthesis in this strain prevents biofilm formation and leads to reduced rifampicin tolerance. The proteomic data suggest that specific changes in mycolic acid synthesis (cord factor), Esx1 secretion, and cell wall adhesins explain the appearance of PBFs as ribbon-like cords and SBFs as lichen-like structures. A subpopulation of cells resisting 64× MIC rifampicin (persisters) was detected in both biofilm subtypes and already in 1-week-old SBFs. The key forces boosting their development could include subtype-dependent changes in asymmetric cell division, cell wall biogenesis, tricarboxylic acid/glyoxylate cycle activities, and energy/redox/iron metabolisms. The effect of various ambient oxygen tensions on each cell type and nonclassical protein secretion are likely factors explaining the majority of the subtype-specific changes. The proteomic findings also imply that Esx1-type protein secretion is more efficient in planktonic (PL) and PBF cells, while SBF may prefer both the Esx5 and nonclassical pathways to control virulence and prolonged viability/persistence. In conclusion, this study reports the first proteomic insight into aging mycobacterial biofilm ECMs and indicates biofilm subtype-dependent mechanisms conferring increased adaptive potential and virulence of nontuberculous mycobacteria.

• Røberg-Larsen, Hanne; Lundanes, Elsa; Nyman, Tuula Anneli; Berven, Frode Steingrimsen & Wilson, Steven Ray Haakon (2021). Liquid chromatography, a key tool for the advancement of single-cell omics analysis. Analytica Chimica Acta. ISSN 0003-2670. 1178:338551, p. 1–9. doi: 10.1016/j.aca.2021.338551. Full text in Research Archive
• Snapkov, Igor; Chernigovskaia, Maria; Sinitcyn, Pavel; Le, Quy Khang; Nyman, Tuula Anneli & Greiff, Victor (2021). Progress and challenges in mass spectrometry-based analysis of antibody repertoires. Trends in Biotechnology. ISSN 0167-7799. 00, p. 1–19. doi: 10.1016/j.tibtech.2021.08.006. Full text in Research Archive Show summary

  Humoral immunity is divided into the cellular B cell and protein-level antibody responses. High-throughput sequencing has advanced our understanding of both these fundamental aspects of B cell immunology as well as aspects pertaining to vaccine and therapeutics biotechnology. Although the protein-level serum and mucosal antibody repertoire make major contributions to humoral protection, the sequence composition and dynamics of antibody repertoires remain underexplored. This limits insight into important immunological and biotechnological parameters such as the number of antigen-specific antibodies, which are for example, relevant for pathogen neutralization, microbiota regulation, severity of autoimmunity, and therapeutic efficacy. High-resolution mass spectrometry (MS) has allowed initial insights into the antibody repertoire. We outline current challenges in MS-based sequence analysis of antibody repertoires and propose strategies for their resolution.

• Kaivola, Juha; Nyman, Tuula Anneli & Matikainen, Sampsa (2021). Inflammasomes and sars-cov-2 infection. Viruses. 13:2513(12), p. 1–11. doi: 10.3390/v13122513. Full text in Research Archive Show summary

  SARS-CoV-2 is a new type of coronavirus that has caused worldwide pandemic. The disease induced by SARS-CoV-2 is called COVID-19. A majority of people with COVID-19 have relatively mild respiratory symptoms. However, a small percentage of COVID-19 patients develop a severe disease where multiple organs are affected. These severe forms of SARS-CoV-2 infections are associated with excessive production of pro-inflammatory cytokines, so called “cytokine storm”. Inflammasomes, which are protein complexes of the innate immune system orchestrate development of local and systemic inflammation during virus infection. Recent data suggest involvement of inflammasomes in severe COVID-19. Activation of inflammasome exerts two major effects: it activates caspase-1-mediated processing and secretion of pro-inflammatory cytokines IL-1β and IL-18, and induces inflammatory cell death, pyroptosis, via protein called gasdermin D. Here, we provide comprehensive review of current understanding of the activation and possible functions of different inflammasome structures during SARS-CoV-2 infection and compare that to response caused by influenza A virus. We also discuss how novel SARS-CoV-2 mRNA vaccines activate innate immune response, which is a prerequisite for the activation of protective adaptive immune response.

• Wanichawan, Pimthanya; Skogestad, Jonas; Lunde, Marianne; Støle, Thea Parsberg; Stensland, Maria & Nyman, Tuula Anneli [Show all 10 contributors for this article] (2021). Design of a proteolytically stable sodium-calcium exchanger 1 activator peptide for in vivo studies. Frontiers in Pharmacology. 12(638646). doi: 10.3389/fphar.2021.638646. Full text in Research Archive Show summary

  The cardiac sodium–calcium exchanger (NCX1) is important for normal Na + - and Ca 2+ -homeostasis and cardiomyocyte relaxation and contraction. It has been suggested that NCX1 activity is reduced by phosphorylated phospholemman (pSer68-PLM); however its direct interaction with PLM is debated. Disruption of the potentially inhibitory pSer68-PLM-NCX1 interaction might be a therapeutic strategy to increase NCX1 activity in cardiac disease. In the present study, we aimed to analyze the binding affinities and kinetics of the PLM-NCX1 and pSer68-PLM-NCX1 interactions by surface plasmon resonance (SPR) and to develop a proteolytically stable NCX1 activator peptide for future in vivo studies. The cytoplasmic parts of PLM (PLM cyt ) and pSer68-PLM (pSer68-PLM cyt ) were found to bind strongly to the intracellular loop of NCX1 (NCX1 cyt ) with similar K D values of 4.1 ± 1.0 nM and 4.3 ± 1.9 nM, but the PLM cyt -NCX1 cyt interaction showed higher on/off rates. To develop a proteolytically stable NCX1 activator, we took advantage of a previously designed, high-affinity PLM binding peptide (OPT) that was derived from the PLM binding region in NCX1 and that reverses the inhibitory PLM (S68D)-NCX1 interaction in HEK293. We performed N- and C-terminal truncations of OPT and identified PYKEIEQLIELANYQV as the minimum sequence required for pSer68-PLM binding. To increase peptide stability in human serum, we replaced the proline with an N-methyl-proline (NOPT) after identification of N-terminus as substitution tolerant by two-dimensional peptide array analysis. Mass spectrometry analysis revealed that the half-life of NOPT was increased 17-fold from that of OPT. NOPT pulled down endogenous PLM from rat left ventricle lysate and exhibited direct pSer68-PLM binding in an ELISA-based assay and bound to pSer68-PLM cyt with a K D of 129 nM. Excess NOPT also reduced the PLM cyt -NCX1 cyt interaction in an ELISA-based competition assay, but in line with that NCX1 and PLM form oligomers, NOPT was not able to outcompete the physical interaction between endogenous full length proteins. Importantly, cell-permeable NOPT-TAT increased NCX1 activity in cardiomyocytes isolated from both SHAM-operated and aorta banded heart failure (HF) mice, indicating that NOPT disrupted the inhibitory pSer68-PLM-NCX1 interaction. In conclusion, we have developed a proteolytically stable NCX1-derived PLM binding peptide that upregulates NCX1 activity in SHAM and HF cardiomyocytes.

• Stamnæs, Jorunn; Stray, Daniel; Stensland, Maria; Sarna, Vikas Kumar; Nyman, Tuula Anneli & Lundin, Knut Erik Aslaksen [Show all 7 contributors for this article] (2021). In well‐treated celiac patients low‐level mucosal inflammation predicts response to 14‐day gluten challenge. Advanced Science. 8:2003526(4), p. 1–7. doi: 10.1002/advs.202003526. Full text in Research Archive Show summary

  In celiac disease (CeD), gluten activates adaptive immune cells that cause damage to the small intestinal mucosa. Histological evaluation of intestinal biopsies allows for grading of disease severity. CeD can effectively be treated with a life-long gluten-free diet. Gluten challenge of treated CeD patients is used to confirm diagnosis and to test drug efficacy in clinical trials, but patients respond with different magnitudes to the same gluten challenge. In this study of 19 well-treated CeD patients, proteome analysis of total tissue or isolated epithelial cell compartment from formalin-fixed paraffin embedded biopsies collected before and after 14-day gluten challenge demonstrates that patients with strong mucosal response to challenge have signs of ongoing tissue inflammation already before challenge. This low-level tissue inflammation at baseline is paralleled by increased gluten specific CD4+ T-cell frequencies in the gut and presence of a low-level blood inflammatory profile. Thus, apparently well-treated CeD is frequently not entirely quiescent, with presence of low-grade inflammation and antigluten immunity in the gut mucosa. Histology assessment alone appears insufficient to judge full recovery and gut mucosal healing of CeD patients. The findings raise a concern whether a seemingly proper gluten-free diet is able to curb gut inflammation in all CeD patients.

• San-Martin-Galindo, Paola; Rosqvist, Emil; Tolvanen, Stiina; Miettinen, Ilkka; Savijoki, Kirsi & Nyman, Tuula Anneli [Show all 8 contributors for this article] (2021). Modulation of virulence factors of Staphylococcus aureus by nanostructured surfaces. Materials & design. ISSN 0264-1275. 208:109879, p. 1–19. doi: 10.1016/j.matdes.2021.109879. Full text in Research Archive Show summary

  Investigating and understanding the response of microbes to various surfaces requires a versatile parametrisation of the surface, and multiple assays that captures the complexity of the biofilm structures. Here, Staphylococcus aureus biofilm viability, polysaccharide poly-N-acetylglucosamine, and proteins on the cell surface were analysed with agar plate- and well plate-based biofilm formation assays. Biofilms were grown on a set of nanostructured polymeric surfaces, which were thoroughly characterised for their surface chemistry and topography. Surface hydrophobicity, summit density as well as peak and valley structure were found to influence the microbial viability and exopolysaccharide abundance level in the agar plate assay. In the well plate assay, surface chemical parameters had a lesser influence on the viability, but roughness caused by valley structures increased the viability and decreased the exopolysaccharide expression. Surface proteins relating to pathogenicity were affected by the biofilm formation assay. The abundance profile of these proteins correlated clearly with several roughness parameters, especially fine structure parameters in the agar plate assay and lateral roughness in the well plate assay. These results highlight the necessity of describing the material surfaces with a versatile set of different roughness parameters to completely understand what specific features of a surface drive a certain bacterial response.

• Quiles-Jiménez, Ana M T; Gregersen, Ida; Segers, Filip; Skarpengland, Tonje; Kroustallaki, Pinelopi & Yang, Kuan [Show all 30 contributors for this article] (2021). DNA glycosylase Neil3 regulates vascular smooth muscle cell biology during atherosclerosis development. Atherosclerosis. ISSN 0021-9150. 324, p. 123–132. doi: 10.1016/j.atherosclerosis.2021.02.023. Full text in Research Archive Show summary

  Background and aims Atherogenesis involves a complex interaction between immune cells and lipids, processes greatly influenced by the vascular smooth muscle cell (VSMC) phenotype. The DNA glycosylase NEIL3 has previously been shown to have a role in atherogenesis, though whether this is due to its ability to repair DNA damage or to other non-canonical functions is not yet clear. Hereby, we investigate the role of NEIL3 in atherogenesis, specifically in VSMC phenotypic modulation, which is critical in plaque formation and stability. Methods Chow diet-fed atherosclerosis-prone Apoe−/− mice deficient in Neil3, and NEIL3-abrogated human primary aortic VSMCs were characterized by qPCR, and immunohistochemical and enzymatic-based assays; moreover, single-cell RNA sequencing, mRNA sequencing, and proteomics were used to map the molecular effects of Neil3/NEIL3 deficiency in the aortic VSMC phenotype. Furthermore, BrdU-based proliferation assays and Western blot were performed to elucidate the involvement of the Akt signaling pathway in the transdifferentiation of aortic VSMCs lacking Neil3/NEIL3. Results We show that Neil3 deficiency increases atherosclerotic plaque development without affecting systemic lipids. This observation was associated with a shift in VSMC phenotype towards a proliferating, lipid-accumulating and secretory macrophage-like cell phenotype, without changes in DNA damage. VSMC transdifferentiation in Neil3-deficient mice encompassed increased activity of the Akt signaling pathway, supported by cell experiments showing Akt-dependent proliferation in NEIL3-abrogated human primary aortic VSMCs. Conclusions Our findings show that Neil3 deficiency promotes atherosclerosis development through non-canonical mechanisms affecting VSMC phenotype involving activation of the Akt signaling pathway.

• Borowicz, Pawel; Gjerstad, Vibeke Sundvold; Chan, Hanna; Abrahamsen, Greger; Kjelstrup, Hanna & Nyman, Tuula Anneli [Show all 7 contributors for this article] (2021). Tyr192 Regulates Lymphocyte-Specific Tyrosine Kinase Activity in T Cells. Journal of Immunology. ISSN 0022-1767. 207(4), p. 1128–1137. doi: 10.4049/jimmunol.2001105. Full text in Research Archive Show summary

  TCR signaling critically depends on the tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase). Two phosphotyrosines, the activating pTyr394 and the inhibitory pTyr505, control Lck activity. Recently, pTyr192 in the Lck SH2 domain emerged as a third regulator. How pTyr192 may affect Lck function remains unclear. In this study, we explored the role of Lck Tyr192 using CRISPR/Cas9-targeted knock-in mutations in the human Jurkat T cell line. Our data reveal that both Lck pTyr394 and pTyr505 are controlled by Lck Tyr192. Lck with a nonphosphorylated SH2 domain (Lck Phe192) displayed hyperactivity, possibly by promoting Lck Tyr394 transphosphorylation. Lck Glu192 mimicking stable Lck pTyr192 was inhibited by Tyr505 hyperphosphorylation. To overcome this effect, we further mutated Tyr505. The resulting Lck Glu192/Phe505 displayed strongly increased amounts of pTyr394 both in resting and activated T cells. Our results suggest that a fundamental role of Lck pTyr192 may be to protect Lck pTyr394 and/or pTyr505 to maintain a pool of already active Lck in resting T cells. This provides an additional mechanism for fine-tuning of Lck as well as T cell activity.

• Skagen, Christine; Nyman, Tuula Anneli; Xiao-Rong, Peng; O'Mahony, Sven; Kase, Eili Tranheim & Rustan, Arild Christian [Show all 7 contributors for this article] (2021). Chronic treatment with terbutaline increases glucose and oleic acid oxidation and protein synthesis in cultured human myotubes. Current Research in Pharmacology and Drug Discovery (CRPHAR). 2, p. 1–11. doi: 10.1016/j.crphar.2021.100039. Full text in Research Archive
• Vestad, Beate; Nyman, Tuula A.; Hove-Skovsgaard, Malene; Stensland, Maria; Hoel, Hedda Benedicte & Siebke, Anne-Marie [Show all 13 contributors for this article] (2021). Plasma extracellular vesicles in people living with HIV and type 2 diabetes are related to microbial translocation and cardiovascular risk. Scientific Reports. 11(1). doi: 10.1038/s41598-021-01334-y. Full text in Research Archive Show summary

  Abstract HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotoxemia and increased cardiovascular risk. We investigated the potential role of plasma extracellular vesicles (EVs) in relation to these processes. Plasma EVs were isolated by size exclusion chromatography in fasting individuals with HIV and T2D (n = 16), T2D only (n = 14), HIV only (n = 20) or healthy controls (n = 19), and characterized by transmission electron microscopy, western blot, nanoparticle tracking analysis and quantitative proteomics. The findings were compared to gut microbiota alterations, lipopolysaccharide levels and cardiovascular risk profile. Individuals with concomitant HIV and T2D had higher plasma EV concentration, which correlated closely with plasma lipopolysaccharides, triglycerides and Framingham score, but not with gut microbiota alterations. Proteomic analyses identified 558 human proteins, largely related to cardiometabolic disease genes and upstream regulation of inflammatory pathways, including IL-6 and IL-1β, as well as 30 bacterial proteins, mostly from lipopolysaccharide-producing Proteobacteria. Our study supports that EVs are related to microbial translocation processes in individuals with HIV and T2D. Their proteomic content suggests a contributing role in low-grade inflammation and cardiovascular risk development. The present approach for exploring gut-host crosstalk can potentially identify novel diagnostic biomarkers and therapeutic targets.

• Holm, Sverre; Kared, Hassen; Michelsen, Annika Elisabet; Kong, Xiang Yi; Dahl, Tuva Børresdatter & Schultz, Nina Haagenrud [Show all 21 contributors for this article] (2021). Immune complexes, innate immunity, and NETosis in ChAdOx1 vaccine-induced thrombocytopenia. European Heart Journal. ISSN 0195-668X. 42(39), p. 4064–4072. doi: 10.1093/eurheartj/ehab506. Full text in Research Archive Show summary

  Aims - We recently reported five cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) 7–10 days after receiving the first dose of the ChAdOx1 nCoV-19 adenoviral vector vaccine against corona virus disease 2019 (COVID-19). We aimed to investigate the pathogenic immunological responses operating in these patients.<p> <p>Methods and results - We assessed circulating inflammatory markers by immune assays and immune cell phenotyping by flow cytometry analyses and performed immunoprecipitation with anti-platelet factor (PF)4 antibody in plasma samples followed by mass spectrometry from all five patients. A thrombus was retrieved from the sinus sagittal superior of one patient and analysed by immunohistochemistry and flow cytometry. Precipitated immune complexes revealed multiple innate immune pathway triggers for platelet and leucocyte activation. Plasma contained increased levels of innate immune response cytokines and markers of systemic inflammation, extensive degranulation of neutrophils, and tissue and endothelial damage. Blood analyses showed activation of neutrophils and increased levels of circulating H3Cit, dsDNA, and myeloperoxidase–DNA complex. The thrombus had extensive infiltration of neutrophils, formation of neutrophil extracellular traps (NETs), and IgG deposits.<p> <p>Conclusions - The results show that anti-PF4/polyanion IgG-mediated thrombus formation in VITT patients is accompanied by a massive innate immune activation and particularly the fulminant activation of neutrophils including NETosis. These results provide novel data on the immune response in this rare adenoviral vector-induced VITT.

• Mirlashari, Mohammad Reza; Vetlesen, Annette; Nissen-Meyer, Lise Sofie Haug; Stensland, Maria Ekman; Singh, Sachin Kumar & Nyman, Tuula Anneli [Show all 7 contributors for this article] (2021). Proteomic study of apheresis platelets made HLA class I deficient for transfusion of refractory patients. PROTEOMICS - Clinical Applications. ISSN 1862-8346. 15:e2100022(6), p. 1–8. doi: 10.1002/prca.202100022. Full text in Research Archive Show summary

  Purpose Refractoriness can occur after repeated platelet (PLT) transfusions because of alloimmunization to HLA class I antigens on transfused PLTs and generation of anti-HLA antibodies that bind to the foreign PLTs and initiate their destruction. Such refractoriness can be overcome by provision of HLA-matched PLTs from HLA typed donors. However, since the procedure is both expensive and time-consuming, an alternative approach is to deplete PLTs of HLA class I molecules by a brief treatment with citric acid, on ice. This is shown to be feasible without damaging PLT function. We used label free quantitative mass spectrometry (MS)-based proteomics to investigate the effect of acid treatment on apheresis PLTs for combatting immunologic PLT refractoriness. Experimental Design Proteomic analyses are undertaken using PLTs from seven apheresis concentrates, which were split in two with or without acid treatment. Results In total 1717 proteins in apheresis PLTs were quantified using proteomics. Data are available via ProteomeXchange with identifier PXD027893 . Of these, the amount of 80 proteins changed significantly after acid treatment, but overall there were not any major differences in proteomes between samples with and without acid treatment. Conclusions and Clinical Relevance In general, the changes of PLT proteins after treatment with citric acid were quite small and functionally safe. Hence, this result taken together with our previously published data indicates that acid treated PLTs can be used for treatment of patients with PLT refractoriness and opens up for a clinical trial.

• Nyman, Tuula Anneli; Gigantino, Valerio; Salvati, Annamaria; Giurato, G; Palumbo, D & Strianese, O [Show all 10 contributors for this article] (2020). Identification of Antiestrogen-Bound Estrogen Receptor α Interactomes in Hormone-Responsive Human Breast Cancer Cell Nuclei. Proteomics. ISSN 1615-9853. 20(19-20). doi: 10.1002/pmic.202000135. Full text in Research Archive
• Savijoki, Kirsi; Miettinen, Ilkka; Nyman, Tuula Anneli; Kortesoja, Maarit; Hanski, Leena & Varmanen, Pekka [Show all 7 contributors for this article] (2020). Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus. Microorganisms. 8:106(1), p. 1–24. doi: 10.3390/microorganisms8010106. Full text in Research Archive Show summary

  The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus

• Krapf, Solveig Astrid; Lund, Jenny; Lundkvist, Malin; Dale, Marianne Grebstad; Nyman, Tuula Anneli & Thoresen, G. Hege [Show all 7 contributors for this article] (2020). Pancreatic cancer cells show lower oleic acid oxidation and their conditioned medium inhibits oleic acid oxidation in human myotubes. Pancreatology (Print). ISSN 1424-3903. 20(4), p. 676–682. doi: 10.1016/j.pan.2020.04.014. Full text in Research Archive
• Harrison, Sean; Siller, Richard; Tanaka, Yoshiaki; Xiang, Yangfei; Patterson, Benjamin & Kempf, Henning [Show all 29 contributors for this article] (2020). Scalable production of tissue-like vascularised liver organoids from human PSCs. BioRxiv. ISSN 0362-4331. doi: 10.1101/2020.12.02.406835. Full text in Research Archive
• Eieland, Alexander K.; Normann, Kjersti R.; Sundaram, Arvind Y. M.; Nyman, Tuula A.; Øystese, Kristin A. B. & Lekva, Tove [Show all 9 contributors for this article] (2020). Distinct pattern of endoplasmic reticulum protein processing and extracellular matrix proteins in functioning and silent corticotroph pituitary adenomas. Cancers. 12(10), p. 1–20. doi: 10.3390/cancers12102980. Full text in Research Archive Show summary

  Functioning (FCA) and silent corticotroph (SCA) pituitary adenomas act differently from a clinical perspective, despite both subtypes showing positive TBX19 (TPIT) and/or adrenocorticotropic hormone (ACTH) staining by immunohistochemistry. They are challenging to treat, the former due to functional ACTH production and consequently hypercortisolemia, and the latter due to invasive and recurrent behavior. Moreover, the molecular mechanisms behind their distinct behavior are not clear. We investigated global transcriptome and proteome changes in order to identify signaling pathways that can explain FCA and SCA differences (e.g., hormone production vs. aggressive growth). In the transcriptomic study, cluster analyses of differentially expressed genes revealed two distinct groups in accordance with clinical and histological classification. However, in the proteomic study, a greater degree of heterogeneity within the SCA group was found. Genes and proteins related to protein synthesis and vesicular transport were expressed by both adenoma groups, although different types and a distinct pattern of collagen/extracellular matrix proteins were presented by each group. Moreover, several genes related to endoplasmic reticulum protein processing were overexpressed in the FCA group. Together, our findings shed light on the different repertoires of activated signaling pathways in corticotroph adenomas, namely, the increased protein processing capacity of FCA and a specific pattern of adhesion molecules that may play a role in the aggressiveness of SCA.

• Szibor, Marten; Schreckenberg, Rolf; Gizatullina, Zemfira; Dufour, Eric; Wiesnet, Marion & Dhandapani, Praveen K. [Show all 20 contributors for this article] (2020). Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia. Journal of Cellular and Molecular Medicine. ISSN 1582-1838. 24(6), p. 3534–3548. doi: 10.1111/jcmm.15043. Full text in Research Archive Show summary

  Cardiac ischaemia‐reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re‐perfused post‐ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post‐anoxic heart mitochondria. However, post‐ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up‐regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro‐fibrotic and pro‐apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label‐free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC‐derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.

• Louwe, Maria Cornelia; Olsen, Maria Belland; Kaasbøll, Ole Jørgen; Yang, Kuan; Fosshaug, Linn Elisabeth Lillerud & Alfsnes, Katrine [Show all 25 contributors for this article] (2020). Absence of NLRP3 Inflammasome in Hematopoietic Cells Reduces Adverse Remodeling After Experimental Myocardial Infarction. JACC: Basic to Translational Science. ISSN 2452-302X. 5(12), p. 1210–1224. doi: 10.1016/j.jacbts.2020.09.013. Full text in Research Archive Show summary

  An inflammatory response is required for tissue healing after a myocardial infarction (MI), but the process must be balanced to prevent maladaptive remodeling. This study shows that improved survival and cardiac function following MI, in mice deficient for the NLRP3 inflammasome, can be recapitulated in wild-type mice receiving bone marrow from Nlrp3−/− mice. This suggests that NLRP3 activation in hematopoietic cells infiltrating in the myocardium increases mortality and late ventricular remodeling. Our data should encourage performing clinical trials directly targeting NLRP3 inflammasome and their inflammatory cytokines (interleukin-1β and -18) in MI patients.

• Mathiesen, Sabrina Bech; Lunde, Marianne; Stensland, Maria; Martinsen, Marita; Nyman, Tuula Anneli & Christensen, Geir Arve [Show all 7 contributors for this article] (2020). The cardiac syndecan-2 interactome. Frontiers in Cell and Developmental Biology. 8:792, p. 1–18. doi: 10.3389/fcell.2020.00792. Full text in Research Archive Show summary

  The extracellular matrix (ECM) is important in cardiac remodeling and syndecans have gained increased interest in this process due to their ability to convert changes in the ECM to cell signaling. In particular, syndecan-4 has been shown to be important for cardiac remodeling, whereas the role of its close relative syndecan-2 is largely unknown in the heart. To get more insight into the role of syndecan-2, we here sought to identify interaction partners of syndecan-2 in rat left ventricle. By using three different affinity purification methods combined with mass spectrometry (MS) analysis, we identified 30 novel partners and 9 partners previously described in the literature, which together make up the first cardiac syndecan-2 interactome. Eleven of the novel partners were also verified in HEK293 cells (i.e., AP2A2, CAVIN2, DDX19A, EIF4E, JPH2, MYL12A, NSF, PFDN2, PSMC5, PSMD11, and RRAD). The cardiac syndecan-2 interactome partners formed connections to each other and grouped into clusters mainly involved in cytoskeletal remodeling and protein metabolism, but also into a cluster consisting of a family of novel syndecan-2 interaction partners, the CAVINs. MS analyses revealed that although syndecan-2 was significantly enriched in fibroblast fractions, most of its partners were present in both cardiomyocytes and fibroblasts. Finally, a comparison of the cardiac syndecan-2 and -4 interactomes revealed surprisingly few protein partners in common.

• Matikainen, Sampsa; Nyman, Tuula Anneli & Cypryk, Wojciech (2020). Function and regulation of noncanonical caspase-4/5/11 inflammasome. Journal of Immunology. ISSN 0022-1767. 204(12), p. 3063–3069. doi: 10.4049/jimmunol.2000373. Full text in Research Archive
• Gorski, Katarin; Spoljaric, Albert; Nyman, Tuula Anneli; Kaila, Kai; Battersby, Brendan J. & Lehesjoki, Anna-Elina (2020). Quantitative Changes in the Mitochondrial Proteome of Cerebellar Synaptosomes From Preclinical Cystatin B-Deficient Mice. Frontiers in Molecular Neuroscience. 13:570640, p. 1–13. doi: 10.3389/fnmol.2020.570640. Full text in Research Archive Show summary

  Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is a neurodegenerative disorder caused by loss-of-function mutations in the cystatin B (CSTB) gene. Progression of the clinical symptoms in EPM1 patients, including stimulus-sensitive myoclonus, tonic-clonic seizures, and ataxia, are well described. However, the cellular dysfunction during the presymptomatic phase that precedes the disease onset is not understood. CSTB deficiency leads to alterations in GABAergic signaling, and causes early neuroinflammation followed by progressive neurodegeneration in brains of a mouse model, manifesting as progressive myoclonus and ataxia. Here, we report the first proteome atlas from cerebellar synaptosomes of presymptomatic Cstb-deficient mice, and propose that early mitochondrial dysfunction is important to the pathogenesis of altered synaptic function in EPM1. A decreased sodium- and chloride dependent GABA transporter 1 (GAT-1) abundance was noted in synaptosomes with CSTB deficiency, but no functional difference was seen between the two genotypes in electrophysiological experiments with pharmacological block of GAT-1. Collectively, our findings provide novel insights into the early onset and pathogenesis of CSTB deficiency, and reveal greater complexity to the molecular pathogenesis of EPM1.

• Matikainen, Sampsa; Nyman, Tuula A. & Cypryk, Wojciech (2020). Inflammasomes: Exosomal miRNAs loaded for action. Journal of Cell Biology. ISSN 0021-9525. 219:e202008130(10), p. 1–2. doi: 10.1083/jcb.202008130. Full text in Research Archive Show summary

  Inflammasomes are multiprotein complexes of the innate immune system. Their activation leads to robust secretion of exosomes. In this issue, Wozniak et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.201912074) reveal a connection between inflammasome-mediated cleavage of Rab-interacting lysosomal protein (RILP) and sequence-specific loading of miRNAs into exosomes.

• Nassa, Giovanni; Giurato, Giorgio; Salvati, Annamaria; Gigantino, Valerio; Pecoraro, Giovanni & Lamberti, Jessica [Show all 10 contributors for this article] (2019). The RNA-mediated estrogen receptor α interactome of hormone-dependent human breast cancer cell nuclei. Scientific Data. 6(1), p. 173–173. doi: 10.1038/s41597-019-0179-2. Full text in Research Archive Show summary

  Estrogen Receptor alpha (ERα) is a ligand-inducible transcription factor that mediates estrogen signaling in hormone-responsive cells, where it controls key cellular functions by assembling in gene-regulatory multiprotein complexes. For this reason, interaction proteomics has been shown to represent a useful tool to investigate the molecular mechanisms underlying ERα action in target cells. RNAs have emerged as bridging molecules, involved in both assembly and activity of transcription regulatory protein complexes. By applying Tandem Affinity Purification (TAP) coupled to mass spectrometry (MS) before and after RNase digestion in vitro, we generated a dataset of nuclear ERα molecular partners whose association with the receptor involves RNAs. These data provide a useful resource to elucidate the combined role of nuclear RNAs and the proteins identified here in ERα signaling to the genome in breast cancer and other cell types.

• Alexandrova, Elena; Giurato, Giorgio; Saggese, Pasquale; Pecoraro, Giovanni; Lamberti, Jessica & Ravo, Maria [Show all 16 contributors for this article] (2019). Interaction proteomics identifies estrogen receptor beta association with multiple chromatin repressive complexes to inhibit cholesterol biosynthesis and exert an oncosuppressive role in triple-negative breast cancer. Molecular & Cellular Proteomics. ISSN 1535-9476. doi: 10.1074/mcp.RA119.001817. Full text in Research Archive Show summary

  Triple-negative breast cancer (TNBC) is characterized by poor response to therapy and low overall patient survival. Recently, Estrogen Receptor beta (ERβ) has been found to be expressed in a fraction of TNBCs where, due to its oncosuppressive actions on the genome, it represents a potential therapeutic target, provided a better understanding of its actions in these tumors becomes available. To this end, the cell lines Hs 578T, MDA-MB-468 and HCC1806, representing the claudin-low, basal-like 1 and 2 TNBC molecular subtypes respectively, were engineered to express ERβ under the control of a Tetracycline-inducible promoter and used to investigate the effects of this transcription factor on gene activity. The antiproliferative effects of ERβ in these cells were confirmed by multiple functional approaches, including transcriptome profiling and global mapping of receptor binding sites in the genome, that revealed direct negative regulation by ERβ of genes, encoding for key components of cellular pathways associated to TNBC aggressiveness representing novel therapeutic targets such as angiogenesis, invasion, metastasis and cholesterol biosynthesis. Supporting these results, interaction proteomics by immunoprecipitation coupled to nano LC-MS/MS mass spectrometry revealed ERβ association with several potential nuclear protein partners, including key components of regulatory complexes known to control chromatin remodelling, transcriptional and post-transcriptional gene regulation and RNA splicing. Among these, ERβ association with the Polycomb Repressor Complexes 1 and 2 (PRC1/2), known for their central role in gene regulation in cancer cells, was confirmed in all three TNBC subtypes investigated, suggesting its occurrence independently from the cellular context. These results demonstrate a significant impact of ERβ in TNBC genome activity mediated by its cooperation with regulatory multiprotein chromatin remodelling complexes, providing novel ground to devise new strategies for the treatment of these diseases based on ligands affecting the activity of this nuclear receptor or some of its protein partners

• Hiltunen, Anna K.; Savijoki, Kirsi; Nyman, Tuula Anneli; Miettinen, Ilkka; Ihalainen, Petri & Peltonen, Jouko [Show all 7 contributors for this article] (2019). Structural and functional dynamics of staphylococcus aureus biofilms and biofilm matrix proteins on different clinical materials. Microorganisms. 7(12). doi: 10.3390/microorganisms7120584. Full text in Research Archive Show summary

  Medical device-associated staphylococcal infections are a common and challenging problem. However, detailed knowledge of staphylococcal biofilm dynamics on clinically relevant surfaces is still limited. In the present study, biofilm formation of the Staphylococcus aureus ATCC 25923 strain was studied on clinically relevant materials-borosilicate glass, plexiglass, hydroxyapatite, titanium and polystyrene-at 18, 42 and 66 h. Materials with the highest surface roughness and porosity (hydroxyapatite and plexiglass) did not promote biofilm formation as efficiently as some other selected materials. Matrix-associated poly-N-acetyl-β-(1-6)-glucosamine (PNAG) was considered important in young (18 h) biofilms, whereas proteins appeared to play a more important role at later stages of biofilm development. A total of 460 proteins were identified from biofilm matrices formed on the indicated materials and time points-from which, 66 proteins were proposed to form the core surfaceome. At 18 h, the appearance of several r-proteins and glycolytic adhesive moonlighters, possibly via an autolysin (AtlA)-mediated release, was demonstrated in all materials, whereas classical surface adhesins, resistance- and virulence-associated proteins displayed greater variation in their abundances depending on the used material. Hydroxyapatite-associated biofilms were more susceptible to antibiotics than biofilms formed on titanium, but no clear correlation between the tolerance and biofilm age was observed. Thus, other factors, possibly the adhesive moonlighters, could have contributed to the observed chemotolerant phenotype. In addition, a protein-dependent matrix network was observed to be already well-established at the 18 h time point. To the best of our knowledge, this is among the first studies shedding light into matrix-associated surfaceomes of S. aureus biofilms grown on different clinically relevant materials and at different time points

• Lunde, Ngoc Dieu Nguyen; Gregersen, Ida; Ueland, Thor; Shetelig, Christian; Holm, Sverre & Kong, Xiang Yi [Show all 31 contributors for this article] (2019). Legumain is upregulated in acute cardiovascular events and associated with improved outcome – potentially related to anti-inflammatory effects on macrophages. Atherosclerosis. ISSN 0021-9150. p. 1–9. doi: 10.1016/j.atherosclerosis.2019.12.008. Full text in Research Archive Show summary

  Background and aims We have previously found increased levels of the cysteine protease legumain in plasma and plaques from patients with carotid atherosclerosis. This study further investigated legumain during acute cardiovascular events. Methods Circulating levels of legumain from patients and legumain released from platelets were assessed by enzyme-linked-immunosorbent assay. Quantitative PCR and immunoblotting were used to study expression, while localization was visualized by immunohistochemistry. Results In the SUMMIT Malmö cohort (n = 339 with or without type 2 diabetes and/or cardiovascular disease [CVD], and 64 healthy controls), the levels of circulating legumain were associated with the presence of CVD in non-diabetics, with no relation to outcome. In symptomatic carotid plaques and in samples from both coronary and intracerebral thrombi obtained during acute cardiovascular events, legumain was co-localized with macrophages in the same regions as platelets. In vitro, legumain was shown to be present in and released from platelets upon activation. In addition, THP-1 macrophages exposed to releasate from activated platelets showed increased legumain expression. Interestingly, primary peripheral blood mononuclear cells stimulated with recombinant legumain promoted anti-inflammatory responses. Finally, in a STEMI population (POSTEMI; n = 272), patients had significantly higher circulating legumain before and immediately after percutaneous coronary intervention compared with healthy controls (n = 67), and high levels were associated with improved outcome. Conclusions Our data demonstrate for the first time that legumain is upregulated during acute cardiovascular events and is associated with improved outcome.

• Yang, Kuan; Lauritzen, Knut Husø; Olsen, Maria Belland; Dahl, Tuva Børresdatter; Ranheim, Trine & Ahmed, Muhammad Shakil [Show all 12 contributors for this article] (2019). Low cellular NAD+ compromises lipopolysaccharide-induced inflammatory responses via inhibiting TLR4 signal transduction in human monocytes. Journal of Immunology. ISSN 0022-1767. 203(6), p. 1598–1608. doi: 10.4049/jimmunol.1801382. Full text in Research Archive
• Savijoki, Kirsi; Nyman, Tuula Anneli; Kainulainen, Veera; Miettinen, Ilkka; Siljamaki, Pia & Fallarero, Adyary [Show all 9 contributors for this article] (2019). Growth mode and carbon source impact the surfaceome dynamics of lactobacillus rhamnosus GG. Frontiers in Microbiology. 10. doi: 10.3389/fmicb.2019.01272. Full text in Research Archive Show summary

  Bacterial biofilms have clear implications in disease and in food applications involving probiotics. Here, we show that switching the carbohydrate source from glucose to fructose increased the biofilm formation and the total surface-antigenicity of a well-known probiotic, Lactobacillus rhamnosus GG. Surfaceomes (all cell surface-associated proteins) of GG cells grown with glucose and fructose in planktonic and biofilm cultures were identified and compared, which indicated carbohydrate source-dependent variations, especially during biofilm growth. The most distinctive differences under these conditions were detected with several surface adhesins (e.g., MBF, SpaC pilus protein and penicillin-binding proteins), enzymes (glycoside hydrolases, PrsA, PrtP, PrtR, and HtrA) and moonlighting proteins (glycolytic, transcription/translation and stress-associated proteins, r-proteins, tRNA synthetases, Clp family proteins, PepC, PepN, and PepA). The abundance of several known adhesins and candidate moonlighters, including enzymes acting on casein-derived peptides (ClpP, PepC, and PepN), increased in the biofilm cells grown on fructose, from which the surface-associated aminopeptidase activity mediated by PepC and PepN was further confirmed by an enzymatic assay. The mucus binding factor (MBF) was found most abundant in fructose grown biofilm cells whereas SpaC adhesin was identified specifically from planktonic cells growing on fructose. An additional indirect ELISA indicated both growth mode- and carbohydrate-dependent differences in abundance of SpaC, whereas the overall adherence of GG assessed with porcine mucus indicated that the carbon source and the growth mode affected mucus adhesion. The adherence of GG cells to mucus was almost completely inhibited by anti-SpaC antibodies regardless of growth mode and/or carbohydrate source, indicating the key role of the SpaCBA pilus in adherence under the tested conditions. Altogether, our results suggest that carbon source and growth mode coordinate mechanisms shaping the proteinaceous composition of GG cell surface, which potentially contributes to resistance, nutrient acquisition and cell-cell interactions under different conditions. In conclusion, the present study shows that different growth regimes and conditions can have a profound impact on the adherent and antigenic features of GG, thereby providing new information on how to gain additional benefits from this probiotic.

• Nassa, Giovanni; Salvati, Annamaria; Tarallo, Roberta; Gigantino, Valerio; Alexandrova, Elena & Memoli, Domenico [Show all 18 contributors for this article] (2019). Inhibition of histone methyltransferase DOT1L silences ER gene and blocks proliferation of antiestrogen-resistant breast cancer cells. Indian Journal of Pure & Applied Physics. ISSN 0019-5596. 5(2), p. 1–14. doi: 10.1126/sciadv.aav5590. Full text in Research Archive Show summary

  Breast cancer (BC) resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor α (ERα) signaling, and ways to block ERα pathway in these tumors are sought after. We identified the H3K79 methyltransferase DOT1L as a novel cofactor of ERα in BC cell chromatin, where the two proteins colocalize to regulate estrogen target gene transcription. DOT1L blockade reduces proliferation of hormone-responsive BC cells in vivo and in vitro, consequent to cell cycle arrest and apoptotic cell death, with widespread effects on ER-dependent gene transcription, including ERα and FOXA1 gene silencing. Antiestrogen-resistant BC cells respond to DOT1L inhibition also in mouse xenografts, with reduction in ERα levels, H3K79 methylation, and tumor growth. These results indicate that DOT1L is an exploitable epigenetic target for treatment of endocrine therapy-resistant ERα-positive BCs

• Richter, Uwe; Ng, Kah Ying; Suomi, Fumi; Marttinen, Paula; Turunen, Taina & Jackson, Christopher [Show all 18 contributors for this article] (2019). Mitochondrial stress response triggered by defects in protein synthesis quality control. Life Science Alliance (LSA). 2:e201800219(1), p. 1–17. doi: 10.26508/lsa.201800219. Full text in Research Archive Show summary

  Mitochondria have a compartmentalized gene expression system dedicated to the synthesis of membrane proteins essential for oxidative phosphorylation. Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. Pathogenic mutations that impede the function of the mitochondrial matrix quality control protease complex composed of AFG3L2 and paraplegin cause a multifaceted clinical syndrome. At the cell and molecular level, defects to this quality control complex are defined by impairment to mitochondrial form and function. Here, we establish the etiology of these phenotypes. We show how disruptions to the quality control of mitochondrial protein synthesis trigger a sequential stress response characterized first by OMA1 activation followed by loss of mitochondrial ribosomes and by remodelling of mitochondrial inner membrane ultrastructure. Inhibiting mitochondrial protein synthesis with chloramphenicol completely blocks this stress response. Together, our data establish a mechanism linking major cell biological phenotypes of AFG3L2 pathogenesis and show how modulation of mitochondrial protein synthesis can exert a beneficial effect on organelle homeostasis.

• Mathiesen, Sabrina Bech; Lunde, Marianne; Aronsen, Jan Magnus; Romaine, Andreas; Kaupang, Anita & Martinsen, Marita [Show all 11 contributors for this article] (2019). The cardiac syndecan-4 interactome reveals a role for syndecan-4 in nuclear translocation of muscle LIM protein (MLP). Journal of Biological Chemistry. ISSN 0021-9258. 294(22), p. 8717–8731. doi: 10.1074/jbc.RA118.006423. Full text in Research Archive Show summary

  Costameres are signaling hubs at the sarcolemma and important contact points between the extracellular matrix and cell interior, sensing and transducing biomechanical signals into a cellular response. The transmembrane proteoglycan syndecan-4 localizes to these attachment points and has been shown to be important in the initial stages of cardiac remodeling, but its mechanistic function in the heart remains insufficiently understood. Here, we sought to map the cardiac interactome of syndecan-4 to better understand its function and downstream signaling mechanisms. By combining two different affinity purification methods with MS analysis, we found that the cardiac syndecan-4 interactome consists of 21 novel and 29 previously described interaction partners. Nine of the novel partners were further validated to bind syndecan-4 in HEK293 cells (i.e. CAVIN1/PTRF, CCT5, CDK9, EIF2S1, EIF4B, MPP7, PARVB, PFKM, and RASIP). We also found that 19 of the 50 interactome partners bind differently to syndecan-4 in the left ventricle lysate from aortic-banded heart failure (ABHF) rats compared with SHAM-operated animals. One of these partners was the well-known mechanotransducer muscle LIM protein (MLP), which showed direct and increased binding to syndecan-4 in ABHF. Nuclear translocation is important in MLP-mediated signaling, and we found less MLP in the nuclear-enriched fractions from syndecan-4-/- mouse left ventricles but increased nuclear MLP when syndecan-4 was overexpressed in a cardiomyocyte cell line. In the presence of a cell-permeable syndecan-4-MLP disruptor peptide, the nuclear MLP level was reduced. These findings suggest that syndecan-4 mediates nuclear translocation of MLP in the heart

• Giurato, Giorgio; Nassa, Giovanni; Salvati, Annamaria; Alexandrova, Elena; Rizzo, Francesca & Nyman, Tuula Anneli [Show all 8 contributors for this article] (2018). Quantitative mapping of RNA-mediated nuclear estrogen receptor β interactome in human breast cancer cells. Scientific Data. 5:180031, p. 1–9. doi: 10.1038/sdata.2018.31. Full text in Research Archive Show summary

  The nuclear receptor estrogen receptor 2 (ESR2, ERβ) modulates cancer cell proliferation and tumor growth, exerting an oncosuppressive role in breast cancer (BC). Interaction proteomics by tandem affinity purification coupled to mass spectrometry was previously applied in BC cells to identify proteins acting in concert with ERβ to control key cellular functions, including gene transcription, RNA splicing and post-transcriptional mRNA regulation. These studies revealed an involvement of RNA in ERβ interactome assembly and functions. By applying native protein complex purification followed by nano LC-MS/MS before and after in vitro RNA removal, we generated a large dataset of newly identified nuclear ERβ interactors, including a subset associating with the receptor via RNA bridging. These datasets will be useful to investigate further the role of ERβ, nuclear RNAs and the other proteins identified here in BC and other cell types.

• Nassa, Giovanni; Giurato, Giorgio; Cimmino, Giovanni; Rizzo, Francesca; Ravo, Maria & Salvati, Annamaria [Show all 13 contributors for this article] (2018). Splicing of platelet resident pre-mRNAs upon activation by physiological stimuli results in functionally relevant proteome modifications. Scientific Reports. 8:498, p. 1–12. doi: 10.1038/s41598-017-18985-5. Full text in Research Archive Show summary

  Platelet activation triggers thrombus formation in physiological and pathological conditions, such as acute coronary syndromes. Current therapies still fail to prevent thrombotic events in numerous patients, indicating that the mechanisms modulating platelet response during activation need to be clarified. The evidence that platelets are capable of de novo protein synthesis in response to stimuli raised the issue of how megakaryocyte-derived mRNAs are regulated in these anucleate cell fragments. Proteogenomics was applied here to investigate this phenomeon in platelets activated in vitro with Collagen or Thrombin Receptor Activating Peptide. Combining proteomics and transcriptomics allowed in depth platelet proteome characterization, revealing a significant effect of either stimulus on proteome composition. In silico analysis revealed the presence of resident immature RNAs in resting platelets, characterized by retained introns, while unbiased proteogenomics correlated intron removal by RNA splicing with changes on proteome composition upon activation. This allowed identification of a set of transcripts undergoing maturation by intron removal during activation and resulting in accumulation of the corresponding peptides at exon-exon junctions. These results indicate that RNA splicing events occur in platelets during activation and that maturation of specific pre-mRNAs is part of the activation cascade, contributing to a dynamic fine-tuning of the transcriptome

• Frohnmeyer, Esther; Deptula, Paulina; Nyman, Tuula Anneli; Laine, Pia K.S.; Vihinen, Helena & Paulin, Lars [Show all 11 contributors for this article] (2018). Secretome profiling of Propionibacterium freudenreichii reveals highly variable responses even among the closely related strains. Microbial Biotechnology. ISSN 1751-7907. 11(3), p. 510–526. doi: 10.1111/1751-7915.13254. Full text in Research Archive Show summary

  This study compared the secretomes (proteins exported out of the cell) of Propionibacterium freudenreichii of different origin to identify plausible adaptation factors. Phylosecretomics indicated strain-specific variation in secretion of adhesins/invasins (SlpA, InlA), cell-wall hydrolysing (NlpC60 peptidase, transglycosylase), protective (RpfB) and moonlighting (DnaK, GroEL, GaPDH, IDH, ENO, ClpB) enzymes and/or proteins. Detailed secretome comparison suggested that one of the cereal strains (JS14) released a tip fimbrillin (FimB) in to the extracellular milieu, which was in line with the electron microscopy and genomic analyses, indicating the lack of surface-associated fimbrial-like structures, predicting a mutated type-2 fimbrial gene cluster (fimB-fimA-srtC2) and production of anchorless FimB. Instead, the cereal strain produced high amounts of SlpB that tentatively mediated adherent growth on hydrophilic surface and adherence to hydrophobic material. One of the dairy strains (JS22), producing non-covalently bound surface-proteins (LspA, ClpB, AraI) and releasing SlpA and InlA into the culture medium, was found to form clumps under physiological conditions. The JS22 strain lacked SlpB and displayed a non-clumping and biofilm-forming phenotype only under conditions of increased ionic strength (300 mM NaCl). However, this strain cultured under the same conditions was not adherent to hydrophobic support, which supports the contributory role of SlpB in mediating hydrophobic interactions. Thus, this study reports significant secretome variation in P. freudenreichii and suggests that strain-specific differences in protein export, modification and protein-protein interactions have been the driving forces behind the adaptation of this bacterial species.

• Sikorski, Krzysztof; Mehta, Adi; Inngjerdingen, Marit; Thakor, Flourina Kumar; Kling, Simon & Kalina, Tomas [Show all 14 contributors for this article] (2018). A high-throughput pipeline for validation of antibodies. Nature Methods. ISSN 1548-7091. 15(11), p. 909–912. doi: 10.1038/s41592-018-0179-8. Full text in Research Archive Show summary

  Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).

• Cypryk, Wojciech; Nyman, Tuula Anneli & Matikainen, Sampsa (2018). From inflammasome to exosome-does extracellular vesicle secretion constitute an inflammasome-dependent immune response? Frontiers in Immunology. 9:2188, p. 1–8. doi: 10.3389/fimmu.2018.02188. Full text in Research Archive Show summary

  Inflammasomes are intracellular protein complexes of pattern recognition receptors and caspase-1, with essential functions in regulating inflammatory responses of macrophages and dendritic cells. The primary role of inflammasomes is to catalyze processing and secretion of pro-inflammatory cytokines IL-1β and IL-18. Recently, intracellular non-canonical inflammasome activation by caspases-4/5, which are also regulators of pyroptosis via processing gasdermin D, has been elucidated. Caspase-1, the effector protease of inflammasome complex, is also known to modulate secretion of large number of other proteins. Thereby, besides its known role in processing pro-inflammatory cytokines, the inflammasome turns into a universal regulator of protein secretion, which allows the danger-exposed cells to release various proteins in order to alert and guide neighboring cells. Majority of these proteins are not secreted through the conventional ER-Golgi secretory pathway. Instead, they are segregated in membrane-enclosed compartment and secreted in nanosized extracellular vesicles, which protect their cargo and guide it for delivery. Growing evidence indicates that inflammasome activity correlates with enhanced secretion of extracellular vesicles and modulation of their protein cargo. This inflammasome-driven unconventional, vesicle-mediated secretion of multitude of immunoregulatory proteins may constitute a novel paradigm in inflammatory responses. In this mini review we discuss the current knowledge and highlight unsolved questions about metabolic processes, signals, and mechanisms linking inflammasome activity with regulated extracellular vesicle secretion of proteins. Further investigations on this relationship may in the future help understanding the significance of extracellular vesicle secretion in inflammatory diseases such as atherosclerosis, gouty arthritis, asthma, Alzheimer's and many others.

• Aittomäki, Saara; Valanne, Susanna; Lehtinen, Tapio; Matikainen, Sampsa; Nyman, Tuula Anneli & Rämet, Mika [Show all 7 contributors for this article] (2017). Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense. The FASEB Journal. ISSN 0892-6638. 31(11), p. 4770–4782. doi: 10.1096/fj.201700296R. Full text in Research Archive
• Lorey, Martina B.; Rossi, Katriina; Eklund, Kari K.; Nyman, Tuula Anneli & Matikainen, Sampsa (2017). Global characterization of protein secretion from human macrophages following non-canonical caspase-4/5 inflammasome activation. Molecular & Cellular Proteomics. ISSN 1535-9476. 16(4), p. S187–S199. doi: 10.1074/mcp.M116.064840. Full text in Research Archive
• Deptula, Paulina; Chamlagain, Bhawani; Edelmann, Minnamari; Sangsuwan, Panchanit; Nyman, Tuula Anneli & Savijoki, Kirsi [Show all 8 contributors for this article] (2017). Food-like growth conditions support production of active vitamin B12 by Propionibacterium freudenreichii 2067 without DMBI, the lower ligand base, or cobalt supplementation. Frontiers in Microbiology. 8. doi: 10.3389/fmicb.2017.00368. Full text in Research Archive
• Nyman, Tuula Anneli; Cypryk, Wojciech; Lorey, Martina B.; Puustinen, Anne & Matikainen, Sampsa (2017). Proteomic and bioinformatic characterization of extracellular vesicles released from human macrophages upon influenza A virus infection. Journal of Proteome Research. ISSN 1535-3893. doi: 10.1021/acs.jproteome.6b00596. Full text in Research Archive Show summary

  Influenza A viruses (IAVs) are aggressive pathogens that cause acute respiratory diseases and annual epidemics in humans. Host defense against IAV infection is initiated by macrophages, which are the principal effector cells of the innate immune system. We have previously shown that IAV infection of human macrophages is associated with robust secretion of proteins via conventional and unconventional protein release pathways. Here we have characterized unconventional, extracellular vesicle (EV)-mediated protein secretion in human macrophages during IAV infection using proteomics, bioinformatics, and functional studies. We demonstrate that at 9 h postinfection a robust EV-mediated protein secretion takes place. We identified 2359 human proteins from EVs of IAV-infected macrophages compared with 1448 proteins identified from EVs of control cells. Bioinformatic analysis shows that many proteins involved in translation, like components of spliceosome machinery and the ribosome, are secreted in EVs in response to IAV infection. Our data also shows that EVs derived from IAV-infected macrophages contain fatty acid-binding proteins, antiviral cytokines, copper metabolism Murr-1 domain proteins, and autophagy-related proteins. In addition, our data suggest that secretory autophagy plays a role in activating EV-mediated protein secretion during IAV infection.

• Dumont, Vincent; Tolvanen, Tuomas A.; Kuusela, Sara; Wang, Hong; Nyman, Tuula Anneli & Lindfors, Sonja [Show all 12 contributors for this article] (2017). PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease. The FASEB Journal. ISSN 0892-6638. 31(9), p. 3978–3990. doi: 10.1096/fj.201601265R. Full text in Research Archive
• Nyman, Tuula Anneli; Nassa, Giovanni; Tarallo, Roberta; Giurato, Giorgio; Bruno, G & Ravo, Maria [Show all 18 contributors for this article] (2017). The nuclear receptor ERβ engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading. Genome Biology. ISSN 1465-6906. doi: 10.1186/s13059-017-1321-0. Full text in Research Archive Show summary

  BACKGROUND: The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. RESULTS: Applying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ + vs ERβ - cells, and before and after AGO2 knock-down in ERβ + cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability. CONCLUSIONS: These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions.

• Nyman, Tuula Anneli; Matikainen, Sampsa; Lorey, Martina B.; Rossi, M & Eklund, Kari K. (2017). Global characterization of protein secretion from human macrophages following non-canonical caspase-4/5 inflammasome activation. Molecular & Cellular Proteomics. ISSN 1535-9476. doi: 10.1074/mcp.M116.064840. Full text in Research Archive Show summary

  Gram-negative bacteria are associated with a wide spectrum of infectious diseases in humans. Inflammasomes are cytosolic protein complexes that are assembled when the cell encounters pathogens or other harmful agents. The non-canonical caspase-4/5 inflammasome is activated by Gram-negative bacteria-derived lipopolysaccharide (LPS) and by endogenous oxidized phospholipids. Protein secretion is a critical component of the innate immune response. Here, we have used label-free quantitative proteomics to characterize global protein secretion in response to non-canonical inflammasome activation upon intracellular LPS recognition in human primary macrophages. Before proteomics, the total secretome was separated into two fractions, enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation. We identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction. From these, 640 were identified from both fractions suggesting that the non-canonical inflammasome activates multiple, partly overlapping protein secretion pathways. We identified several secreted proteins that have a critical role in host response against severe Gram-negative bacterial infection. The soluble secretome (RS fraction) was highly enriched with inflammation-associated proteins upon intracellular LPS recognition. Several ribosomal proteins were highly abundant in the EV fraction upon infection, and our data strongly suggest that secretion of translational machinery and concomitant inhibition of translation are important parts of host response against Gram-negative bacteria sensing caspase-4/5 inflammasome. Intracellular recognition of LPS resulted in the secretion of two metalloproteinases, adisintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and MMP14, in the enriched EV fraction. ADAM10 release was associated with the secretion of TNF, a key inflammatory cytokine, and M-CSF, an important growth factor for myeloid cells probably through ADAM10-dependent membrane shedding of these cytokines. Caspase-4/5 inflammasome activation also resulted in secretion of danger-associated molecules S100A8 and prothymosin-α in the enriched EV fraction. Both S100A8 and prothymosin-α are ligands for toll-like receptor 4 recognizing extracellular LPS, and they may contribute to endotoxic shock during non-canonical inflammasome activation.

• Nyman, Tuula Anneli; Matikainen, Sampsa; Cypryk, Wojciech & Lorey, Martina B. (2017). Mass spectrometry-based proteomic exploration of the human immune system -focus on the inflammasome, global protein secretion, and T cells. Expert Review of Proteomics. ISSN 1478-9450. doi: 10.1080/14789450.2017.1319768. Full text in Research Archive Show summary

  The immune system is our defense system against microbial infections and tissue injury, and understanding how it works in detail is essential for developing drugs for different diseases. Mass spectrometry-based proteomics can provide in-depth information on the molecular mechanisms involved in immune responses. Areas covered: Summarized are the key immunology findings obtained with MS-based proteomics in the past five years, with a focus on inflammasome activation, global protein secretion, mucosal immunology, immunopeptidome and T cells. Special focus is on extracellular vesicle-mediated protein secretion and its role in immune responses. Expert commentary: Proteomics is an essential part of modern omics-scale immunology research. To date, MS-based proteomics has been used in immunology to study protein expression levels, their subcellular localization, secretion, post-translational modifications, and interactions in immune cells upon activation by different stimuli. These studies have made major contributions to understanding the molecular mechanisms involved in innate and adaptive immune responses. New developments in proteomics offer constantly novel possibilities for exploring the immune system. Examples of these techniques include mass cytometry and different MS-based imaging approaches which can be widely used in immunology.

• Bulanova, Daria; Ianevski, Aleksandr; Bugai, Andrii; Akimov, Yevhen; Kuivanen, Suvi & Paavilainen, Henrik [Show all 43 contributors for this article] (2017). Antiviral properties of chemical inhibitors of cellular anti-apoptotic Bcl-2 proteins. Viruses. 9(10). doi: 10.3390/v9100271. Full text in Research Archive Show summary

  Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.

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• Chan, Hanna; Borowicz, Pawel; Gilmour, Brian Christopher; Stensland, Maria; Garcia-Loza, Ivan & Phuyal, Santosh [Show all 10 contributors for this article] (2026). The TSAd-DOK2 axis regulates CD8+ T cell effector function. Full text in Research Archive
• Chan, Hanna; Borowicz, Pawel; Gilmour, Brian Christopher; Stensland, Maria; Loza, Ivan Garcia & Phuyal, Santosh [Show all 10 contributors for this article] (2024). Identification of novel interactors of T cell Specific Adaptor protein TSAd - An adaptor molecule with elusive function. Full text in Research Archive
• Chan, Hanna; Borowicz, Pawel; Gilmour, Brian Christopher; Stensland, Maria; Loza, Ivan Garcia & Phuyal, Santosh [Show all 10 contributors for this article] (2024). Identification of novel interactors of T cell specific adaptor protein TSAd - an adaptor molecule with elusive function. Full text in Research Archive
• Ueland, Thor; Äikäs, Lauri A.O.; Dahl, Tuva Børresdatter; Gregersen, Ida; Olsen, Maria Belland & Michelsen, Annika Elisabet [Show all 20 contributors for this article] (2023). Low-density lipoprotein particles carrying proinflammatory proteins with altered aggregation pattern detected in COVID-19 patients 3 months after hospitalization. Journal of Infection. ISSN 0163-4453. 86(5), p. 489–492. doi: 10.1016/j.jinf.2023.02.024. Full text in Research Archive
• Chan, Hanna; Borowicz, Pawel; Kjelstrup, Hanna; Gilmour, Brian Christopher; Loza, Ivan Garcia & Stensland, Maria [Show all 8 contributors for this article] (2022). Novel interaction partners of the SH2 domain in T cell specific adaptor protein (TSAd). Full text in Research Archive
• Foss, Kari Bente; Aas, Vigdis; Hussein, Abdille; Hussain, Misbah; Brusletto, Berit Sletbakk & Olstad, Ole Kristoffer [Show all 10 contributors for this article] (2021). Extracellular vesicles from skeletal muscle cells isolated from extremely obese patients with and without type 2-diabetes show different protein- and miRNA-pattern. Full text in Research Archive
• Bakketeig, Eris Aas; Olsen, Christine; Berg, Henriette Engen; Nyman, Tuula Anneli; Thiede, Bernd & Lundanes, Elsa [Show all 8 contributors for this article] (2021). Optimization of concatenation of fractions in comprehensive two dimensional liquid chromatography bottom-up proteomics. Full text in Research Archive Show summary

  Two dimensional liquid chromatography (2D-LC) has long been a commonly used technique in bottom-up proteomics, and is as relevant today as ever [1, 2]. To maximize the number of protein identifications, analytical chemists have strived to obtain 2D-LC systems with a high degree of orthogonality, which is needed to reach the required high peak capacities. A system utilizing reversed-phase chromatography (RP) in both column dimensions with a large difference in pH has proven to yield a higher peak capacity for peptides, even with a lower degree of orthogonality compared to other combinations [3]. The present study is based on Reubsaet, et al. [4]. The study is a comprehensive offline RPRP 2D-LC system with high pH (>9) in the first dimension and low pH (3) in the second dimension. The method’s purpose is to maximise protein identifications from one trypsinated protein sample. To achieve maximum identifications, Reubsaet incorporated the concept of concatenation of fractions. In practice, concatenation is applied through collecting the column output from the first dimension in eight numbered vials, where the 1st, 9th, 17th, … fractions are collected in the first vial, the 2nd, 10th, 18th, … are collected in the second, etc. [4]. In the present study, the fractionation scheme is examined by changing the total number of vials in which samples are concatenated, the number of fractions in each vial, and the collection time for each fraction. As the method demands high precision and is very timeconsuming, automatization is practically essential. Automatization is achieved with the spider fractionation scheme, proposed and shown by Kulak, et al.[5]. Here, a 10-port injector connects the first dimension column to each sample vial, and switches between them automatically, reducing the strain on the operator drastically, while removing human errors. The optimization of the concatenation will lead to higher numbers of identified proteins in proteomics.

• Borowicz, Pawel; Sundvold-Gjerstad, Vibeke; Chan, Hanna; Abrahamsen, Greger; Kjelstrup, Hanna & Medina, Daniel [Show all 8 contributors for this article] (2019). Tyr192 in the Lck SH2 domain affects the regulation of Lck activity. Full text in Research Archive
• Borowicz, Pawel; Sundvold-Gjerstad, Vibeke; Abrahamsen, Greger; Kjelstrup, Hanna; Nyman, Tuula Anneli & Spurkland, Anne (2018). Lck’s interactome is influenced by phosphorylation of Tyr192 in the Lck SH2 domain. Full text in Research Archive
• Borowicz, Pawel; Sundvold-Gjerstad, Vibeke; Abrahamsen, Greger; Kjelstrup, Hanna; Nyman, Tuula Anneli & Spurkland, Anne (2018). Lck’s interactome is influenced by phosphorylation of Tyr192 in the Lck SH2 domain. Full text in Research Archive
• Borowicz, Pawel; Sundvold-Gjerstad, Vibeke; Abrahamsen, Greger; Kjelstrup, Hanna; Nyman, Tuula Anneli & Spurkland, Anne (2018). Lck’s interactome is influenced by phosphorylation of Tyr192 in the Lck SH2 domain. Full text in Research Archive
• Bakketeig, Eris Aas; Wilson, Steven Ray Haakon; Lundanes, Elsa; Berg, Henriette Engen; Olsen, Christine & Reubsaet, Leon [Show all 8 contributors for this article] (2021). The setup and optimization of a complete global proteomics two-dimensional liquid chromatography system, using Spider Fractionation. Universitetet i Oslo. Full text in Research Archive

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