Search

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Katare, Dalmao-Fernandez, Mengeste, Navabakbar, Hamarsland, Ellefsen, Berge, Bakke, Nyman, Kase, Rustan and Thoresen.)

Propionibacterium freudenreichii ( PFR ) DSM 20271 T 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 20271 T to aerobic growth by employing comparative transcriptomic and surfaceome analyses alongside metabolite profiling. Cultivation under controlled partial pressure of oxygen (pO 2 ) 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, FeSO 4 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., Importance: The study of the response of Propionibacterium freudenreichii to aerobic growth is crucial for understanding how this bacterium adapts to different environments and produces essential compounds like vitamin B12. By investigating its physiological changes under aerobic conditions, we can gain insights into its metabolic adjustments and potential for enhanced growth. These findings not only deepen our understanding of P. freudenreichii' s responses to oxygen availability but also offer valuable information for optimizing its growth in industrial applications. This research sheds light on the adaptive mechanisms of this bacterium, providing a foundation for further exploration and potential applications in various fields., Competing Interests: The authors declare no conflict of interest.

Purpose: Silent corticotroph adenoma (SCA) exhibits more tumor aggressiveness features than functioning adenomas (FCA). We aimed to investigate PCSK1N expression in CA and examine if 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 SCA (8 women, all macroadenomas). Gene expression of proopiomelanocortin (POMC), T-box transcription factor 19(TBX19)/TPIT, proprotein convertase subtilisin/kexin type 1(PCSK1)/PC1/3, and its inhibitor PCSK1N, was measured by RT-qPCR in adenoma tissue.Mouse pituitary corticotroph tumor (AtT-20) cells were treated with tanespimycin (17-AAG), a 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 <0.001), TPIT (rs= -0.386, p = 0.005), PCSK1 (rs= -0.3691, p = 0.008), and tumor largest diameter (rs= 0.645, p <0.001), 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 24h. Moreover, a downregulation of cell cycle, apoptosis, and senescence pathways, and alterations in cell adhesion and cytoskeleton were observed at the protein level., Conclusions: 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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

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., (© 2024. The Author(s).)

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., (© 2024 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

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 CX 9 C 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., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

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) response 1-4 . 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 symptoms 5 , 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 individuals 6 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., (© 2024. The Author(s).)

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., (© 2023 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology.)

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.

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., (© 2023. The Author(s).)

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&#95;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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

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. IMPORTANCE With the currently available antibiotics, the treatment of TB takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms., Competing Interests: The authors declare no conflict of interest.

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., (© 2023. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gorski, Jackson, Nyman, Rezov, Battersby and Lehesjoki.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Aas, Øvstebø, Brusletto, Aspelin, Trøseid, Qureshi, Eid, Olstad, Nyman and Haug.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Frerker, Karlsen, Stensland, Nyman, Rayner and Brinchmann.)

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., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stefano Bartesaghi and Xiao-Rong Peng are employees at AstraZeneca. The remaining authors have no competing interests to declare., (© 2023 The Authors.)

Human macrophages secrete extracellular vesicles (EVs) loaded with numerous immunoregulatory proteins. Vesicle-mediated protein secretion in macrophages is regulated by poorly characterized mechanisms; however, it is now known that inflammatory conditions significantly alter both the quantities and protein composition of secreted vesicles. In this study, we employed high-throughput quantitative proteomics to characterize the modulation of EV-mediated protein secretion during noncanonical caspase-4/5 inflammasome activation via LPS transfection. We show that human macrophages activate robust caspase-4-dependent EV secretion upon transfection of LPS, and this process is also partially dependent on NLRP3 and caspase-5. A similar effect occurs with delivery of the LPS with Escherichia coli-derived outer membrane vesicles. Moreover, sensitization of the macrophages through TLR4 by LPS priming prior to LPS transfection dramatically augments the EV-mediated protein secretion. Our data demonstrate that this process differs significantly from canonical inflammasome activator ATP-induced vesiculation, and it is dependent on the autocrine IFN signal associated with TLR4 activation. LPS priming preceding the noncanonical inflammasome activation significantly enhances vesicle-mediated secretion of inflammasome components caspase-1, ASC, and lytic cell death effectors GSDMD, MLKL, and NINJ1, suggesting that inflammatory EV transfer may exert paracrine effects in recipient cells. Moreover, using bioinformatics methods, we identify 15-deoxy-Δ12,14-PGJ2 and parthenolide as inhibitors of caspase-4-mediated inflammation and vesicle secretion, indicating new therapeutic potential of these anti-inflammatory drugs., (Copyright © 2023 by The American Association of Immunologists, Inc.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aarsund, Nyman, Stensland, Wu and Inngjerdingen.)

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&#95;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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mengeste, Nikolić, Dalmao Fernandez, Feng, Nyman, Kersten, Haugen, Kase, Aas, Rustan and Thoresen.)

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., Competing Interests: Declaration of interests V.G. declares advisory board positions in aiNET GmbH and Enpicom BV. V.G. is a consultant for Roche/Genentech. The remaining authors have no interests to declare., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

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., (© 2021. The Author(s).)

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., (© 2021 Wiley-VCH GmbH.)

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., 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., 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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Single-cell analysis can allow for an in-depth understanding of diseases, diagnostics, and aid the development of therapeutics. However, single-cell analysis is challenging, as samples are both extremely limited in size and complex. But the concept is gaining promise, much due to novel sample preparation approaches and the ever-improving field of mass spectrometry. The mass spectrometer's output is often linked to the preceding compound separation step, typically being liquid chromatography (LC). In this review, we focus on LC's role in single-cell omics. Particle-packed nano LC columns (typically 50-100 μm inner diameter) have traditionally been the tool of choice for limited samples, and are also used for single cells. Several commercial products and systems are emerging with single cells in mind, featuring particle-packed columns or miniaturized pillar array systems. In addition, columns with inner diameters as narrow as 2 μm are being explored to maximize sensitivity. Hence, LC column down-scaling is a key focus in single-cell analysis. But narrow columns are associated with considerable technical challenges, while single cell analysis may be expected to become a "routine" service, requiring higher degrees of robustness and throughput. These challenges and expectations will increase the need and attention for the development (and even the reinvention) of alternative nano LC column formats. Therefore, monolith columns and even open tubular columns may finally find their "killer-application" in single cell analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

TCR signaling critically depends on the tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase). Two phosphotyrosines, the activating pTyr 394 and the inhibitory pTyr 505 , control Lck activity. Recently, pTyr 192 in the Lck SH2 domain emerged as a third regulator. How pTyr 192 may affect Lck function remains unclear. In this study, we explored the role of Lck Tyr 192 using CRISPR/Cas9-targeted knock-in mutations in the human Jurkat T cell line. Our data reveal that both Lck pTyr 394 and pTyr 505 are controlled by Lck Tyr 192 Lck with a nonphosphorylated SH2 domain (Lck Phe 192 ) displayed hyperactivity, possibly by promoting Lck Tyr 394 transphosphorylation. Lck Glu 192 mimicking stable Lck pTyr 192 was inhibited by Tyr 505 hyperphosphorylation. To overcome this effect, we further mutated Tyr 505 The resulting Lck Glu 192 /Phe 505 displayed strongly increased amounts of pTyr 394 both in resting and activated T cells. Our results suggest that a fundamental role of Lck pTyr 192 may be to protect Lck pTyr 394 and/or pTyr 505 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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

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. IMPORTANCE Mycobacteria are naturally resilient, and mycobacterial infections are notoriously difficult to treat with antibiotics, with biofilm formation being the main factor complicating the successful treatment of tuberculosis (TB). The present study shows that nontuberculous Mycobacterium marinum ATCC 927 forms submerged- and pellicle-type biofilms with lichen- and ribbon-like structures, respectively, as well as persister cells under the same conditions. We show that both biofilm subtypes differ in terms of virulence-, tolerance-, and persistence-conferring activities, highlighting the fact that both subtypes should be targeted to maximize the power of antimycobacterial treatment therapies.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Reigada, San-Martin-Galindo, Gilbert-Girard, Chiaro, Cerullo, Savijoki, Nyman, Fallarero and Miettinen.)

Objective: In vivo studies have reported several beneficial metabolic effects of β-adrenergic receptor agonist administration in skeletal muscle, including increased glucose uptake, fatty acid metabolism, lipolysis and mitochondrial biogenesis. Although these effects have been widely studied in vivo , the in vitro data are limited to mouse and rat cell lines. Therefore, we sought to discover the effects of the β 2 -adrenergic receptor agonist terbutaline on metabolism and protein synthesis in human primary skeletal muscle cells., Methods: Human cultured myotubes were exposed to terbutaline in various concentrations (0.01-30 ​μM) for 4 or 96 ​h. Thereafter uptake of [ 14 C]deoxy-D-glucose, oxydation of [ 14 C]glucose and [ 14 C]oleic acid were measured. Incorporation of [ 14 C]leucine, gene expression by qPCR and proteomics analyses by mass spectrometry by the STAGE-TIP method were performed after 96 ​h exposure to 1 and 10 ​μM of terbutaline., Results: The results showed that 4 ​h treatment with terbutaline in concentrations up to 1 ​μM increased glucose uptake in human myotubes, but also decreased both glucose and oleic acid oxidation along with oleic acid uptake in concentrations of 10-30 ​μM. Moreover, administration of terbutaline for 96 ​h increased glucose uptake (in terbutaline concentrations up to 1 ​μM) and oxidation (1 ​μM), as well as oleic acid oxidation (0.1-30 ​μM), leucine incorporation into cellular protein (1-10 ​μM) and upregulated several pathways related to mitochondrial metabolism (1 ​μM). Data are available via ProteomeXchange with identifier PXD024063., Conclusion: These results suggest that β 2 -adrenergic receptor have direct effects in human skeletal muscle affecting fuel metabolism and net protein synthesis, effects that might be favourable for both type 2 diabetes and muscle wasting disorders., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiao-Rong Peng and Gavin O'Mahony are employees of AstraZeneca and may own stock or stock options., (© 2021 The Authors.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wanichawan, Skogestad, Lunde, Støle, Stensland, Nyman, Sjaastad, Sejersted, Aronsen and Carlson.)

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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: KEAL has privately or via his employer been a consultant during the two last years for Chugai Pharmaceutical, Bioniz Therapeutics, Interexon Actobiotics, Amyra Biotech AG and Dr. Falk Pharma GMBH. LMS has privately or via his employer been a consultant during the two last years for Amagma Therapeutics, Chugai Pharmaceutical, Bioniz Therapeutics, Interexon Actobiotics, UCB Biopharma, Merck and GSK. The other authors declare no conflicts of interest., (© 2021 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

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., Competing Interests: This work was supported by grants from the K.G. Jebsen Inflammation Research Centre, South-Eastern Norway Regional Health Authority, and Norwegian Research Council. Dr. Yndestad is currently an employee of Pfizer Norway; the current work was conducted when he was employed by Oslo University Hospital. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2020 The Authors.)

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., (Copyright © 2020 Gorski, Spoljaric, Nyman, Kaila, Battersby and Lehesjoki.)

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.

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., (© 2020 Matikainen et al.)

Estrogen receptor alpha (ERα) is a ligand-inducible transcription factor which mediates estrogen actions in hormone-responsive tumors and is targeted by effective anticancer therapies based on the ERα antagonist ligands, selective estrogen receptor modulators (such as Tamoxifen/TAM) or disruptors (such as Fulvestrant/ICI). Despite its importance for cancer therapy, including acquired resistance to endocrine therapy, the molecular basis of ERα response to different ligands is not fully known to date. Interaction proteomics shows great potential to identify and characterize molecular mechanisms of disease based on physical and functional protein-protein interaction networks. Tandem affinity purification coupled to mass spectrometry is applied here for mapping in hormone-responsive breast cancer cells nuclei, the ERα interactomes, induced by each of the two classes of antiestrogens. The results provide new insights on the molecular bases for antiestrogen-mediated control of ERα function and reveal new potential ways to overcome endocrine therapy resistance in cancer., (© 2020 Wiley-VCH GmbH.)

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., (Copyright © 2020 Mathiesen, Lunde, Stensland, Martinsen, Nyman, Christensen and Carlson.)

Inflammasomes are multiprotein complexes of the innate immune system that orchestrate development of inflammation by activating the secretion of proinflammatory cytokines, IL-1β and IL-18. The LPS of Gram-negative bacteria have been shown to activate a novel, noncanonical inflammasome by directly binding in the cytosol to human caspase-4 and mouse caspase-11. Activation of noncanonical inflammasome exerts two major effects: it activates the NLRP3-caspase-1-mediated processing and secretion of IL-1β and IL-18 and induces the inflammatory cell death, pyroptosis, via gasdermin D. This previously unexpected cytosolic LPS sensing of the innate immune system provides critical hints for host response to Gram-negative bacterial infections and development of different inflammatory diseases. However, many of its molecular regulatory mechanisms are yet to be discovered. In this review, we provide comprehensive analysis of current understanding of intracellular LPS detection and pyroptosis via noncanonical inflammasome and discuss the recently proposed mechanisms of its function and regulation., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Background: /Objectives: We aimed to metabolically compare healthy primary human pancreatic epithelial cells (hPEC) to a pancreatic cancer cell line (PANC-1) and explore the effect on energy metabolism of exposing primary human myotubes to conditioned medium from hPEC and PANC-1 cells., Methods: Differences in metabolism were examined with radiolabeled glucose, oleic acid and lactic acid, and by qPCR. Mass spectrometry-based proteomics was used to study global protein secretion from the two cell types. Pathway analyses were performed., Results: PANC-1 cells tended to have higher glucose uptake, production of lactic acid, and glucose oxidation compared to hPEC cells. PANC-1 cells had higher uptake but lower oxidation of oleic acid, and mitochondrial reserve capacity from oleic acid was lower in PANC-1 cells. These differences in energy metabolism were reflected by differences in gene expressions and pathway analyses of the secretome. Conditioned medium from PANC-1 cells attenuated oleic acid oxidation in primary human myotubes., Conclusions: Metabolic characterization of the PANC-1 cells revealed a glycolytic phenotype since they had an active glucose oxidation. Furthermore, PANC-1 cells showed a lower oleic acid oxidation and secreted a high amount of proteins into conditioned medium that also induced a reduced oleic acid oxidation in myotubes., Competing Interests: Declaration of competing interest None., (Copyright © 2020 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)