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Telomerase-negative tumors can maintain telomere length by alternative lengthening of telomeres (ALT) but the mechanism behind ALT is poorly understood. Aggressive Neuroblastoma (NB), in particular, relapsed tumors are positive for ALT (ALT+) which suggests that better dissection of the ALT mechanism could provide novel therapeutic opportunities. TERRA long non-coding RNA (lncRNA) which is derived from the telomere ends is localized to telomeres in R-loop dependent manner and is essential for telomere maintenance. In the present study, we provide evidence that RNA modification at theN6position of internal adenosine (m6A) in TERRA RNA by methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells and that loss of TERRA m6A/METTL3 leads to telomere damage. We observed that R-loop enriched TERRA is abundantly m6A modified and m6A mediated recruitment of hnRNPA2B1 to TERRA RNA is essential for R-loop formation. Our data suggest that m6A drives telomere targeting of TERRA via R-loop and this m6A mediated R-loop formation could be a widespread mechanism utilized by other chromatin-interacting lncRNAs. Furthermore, treating ALT+ NB cells with METTL3 inhibitor leads to compromised telomere targeting of TERRA and accumulation of DNA damage over telomere, suggesting METTL3 inhibition could be a therapeutic opportunity for ALT+ NB.

Objective Incretins are known to influence lipid metabolism in the intestine when administered as pharmacologic agents. The aggregate influence of endogenous incretins on chylomicron production and clearance is less clear, particularly in light of opposing effects of co-secreted hormones. Here, we tested the hypothesis that physiological levels of incretins may impact on production or clearances rates of chylomicrons and VLDL. Design and methods A group of 22 overweight/obese men was studied to determine associations between plasma levels of glucagon-like peptides 1 and 2 (GLP-1 and GLP-2) and glucose-dependent insulinotropic polypeptide (GIP) after a fat-rich meal and the production and clearance rates of apoB48- and apoB100-containing triglyceride-rich lipoproteins. Subjects were stratified by above- and below-median incretin response (area under the curve). Results Stratification yielded subgroups that differed about two-fold in incretin response. There were neither differences in apoB48 production rates in chylomicrons or VLDL fractions nor in apoB100 or triglyceride kinetics in VLDL between men with above- vs below-median incretin responses. The men with above-median GLP-1 and GLP-2 responses exhibited higher postprandial plasma and chylomicron triglyceride levels, but this could not be related to altered kinetic parameters. No differences were found between incretin response subgroups and particle clearance rates. Conclusion We found no evidence for a regulatory effect of endogenous incretins on contemporaneous chylomicron or VLDL metabolism following a standardised fat-rich meal. The actions of incretins at pharmacological doses may not be reflected at physiological levels of these hormones.

Resident tissue macrophages (RTMs) are organ-specialized phagocytes responsible for the maintenance and protection of tissue homeostasis. It is well established that tissue diversity is reflected by the heterogeneity of RTMs origin and phenotype. However, much less is known about tissue-specific phagocytic and proteolytic macrophage functions. Here, using quantitative proteomics approach, we identify cathepsins as key determinants of phagosome maturation in primary peritoneal, lung and brain resident macrophages. The data further uncover cathepsin K (CtsK) as a molecular marker for lung phagosomes required for intracellular protein and collagen degradation. Pharmacological blockade of CtsK activity diminished phagosomal proteolysis and collagenolysis in lung resident macrophages. Furthermore, pro-fibrotic TGF-β negatively regulated CtsK-mediated phagosomal collagen degradation independently from classical endocytic proteolytic pathways. In humans, phagosomal CtsK activity was reduced in COPD lung macrophages and non-COPD lung macrophages exposed to cigarette smoke extract. Taken together, this study provides a comprehensive map of how peritoneal, lung and brain tissue environment shapes phagosomal composition, revealing CtsK as a key molecular determinant of lung phagosomes contributing to phagocytic collagen clearance in lungs.

In X-linked thrombocytopenia with thalassemia (XLTT; OMIM 314050), caused by the mutation p.R216Q in exon 4 of the GATA1 gene, male hemizygous patients display macrothrombocytopenia, bleeding diathesis and a b-thalassemia trait. Herein, we describe findings in two unrelated Swedish XLTT families with a bleeding tendency exceeding what is expected from the thrombocytopenia. Blood tests revealed low P-PAI-1 and P-factor 5, and elevated S-thrombopoietin levels. Transmission electron microscopy showed diminished numbers of platelet a- and dense granules. The proteomes of isolated blood platelets from five male XLTT patients, compared to five sex- and agematched controls, were explored. Quantitative mass spectrometry showed alterations of 83 proteins (fold change ≥±1.2, q

Introduction Human ovulation is a biologically complex process that involves several biochemical factors, promoting follicular rupture and release of a fertilizable oocyte. Proteins which are present in follicular fluid at high concentrations during ovulation are likely to be active participants in the biochemical pathways of ovulation. The aim of the study was to identify, by use of a modern proteomic technique, proteins of human follicular fluid which are differentially regulated during ovulation of the natural menstrual cycle. Material and methods This prospective experimental study over 3 years included women planned for laparoscopic sterilization. During surgery, retrieval of the dominant follicle was performed either at the preovulatory stage or during ovulation. Four women of preovulatory phase and four women of ovulatory phase met the predetermined criteria of hormone levels for respective phases, and samples of these were finally included out of the 15 women operated. Follicular fluid was aspirated from the excised follicle and subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ) technology for isobaric tagging of peptides. This enables simultaneous identification and quantification of proteins. The protein profiles of the follicular fluid of the preovulatory phase and the ovulatory phase were analyzed, and proteins that were present were identified. Results A total of 502 proteins were identified, several of which previously have not been identified in human follicular fluid. Of the 115 proteins that were found in all samples, 20 proteins were at higher levels during ovulation. These were inflammatory-related proteins, coagulation factors, proteins in lipid metabolism, complement factors and antioxidants. Five proteins were present in lower levels during ovulation, with three being enzymes and the other two proteins of lipid metabolism and iron transport. Conclusions Twenty-five follicular fluid proteins, with differential regulation during ovulation, were identified in human follicular fluid of the natural menstrual cycle. These proteins may have essential roles in the ovulatory cascade.

Cells maintain reserves in their metabolic and translational capacities as a strategy to quickly respond to changing environments. Here we quantify these reserves by stepwise reducing nitrogen availability in yeast steady-state chemostat cultures, imposing severe restrictions on total cellular protein and transcript content. Combining multi-omics analysis with metabolic modeling, we find that seven metabolic superpathways maintain >50% metabolic capacity in reserve, with glucose metabolism maintaining >80% reserve capacity. Cells maintain >50% reserve in translational capacity for 2490 out of 3361 expressed genes (74%), with a disproportionately large reserve dedicated to translating metabolic proteins. Finally, ribosome reserves contain up to 30% sub-stoichiometric ribosomal proteins, with activation of reserve translational capacity associated with selective upregulation of 17 ribosomal proteins. Together, our dataset provides a quantitative link between yeast physiology and cellular economics, which could be leveraged in future cell engineering through targeted proteome streamlining., Cells maintain reserves in their metabolic and translational capacities enabling fast response to changing environments. Here, the authors quantify reserves in yeast by stepwise reduction in nitrogen availability and a combination of multi-omic analysis and metabolic modelling.

A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories., Graphical Abstract Highlights A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb. 103 reports were received from 76 diverse laboratories worldwide. Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method. Consensus values were derived and similar performance across all experimental methods was noted., Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

Cardiac amyloidosis is a severe condition leading to restrictive cardiomyopathy and heart failure. Mass spectrometry-based methods for cardiac amyloid subtyping have become important diagnostic tools but are currently used only in a few reference laboratories. Such methods include laser-capture microdissection to ensure the specific analysis of amyloid deposits. Here we introduce a direct proteomics-based method for subtyping of cardiac amyloidosis. Endomyocardial biopsies were retrospectively analysed from fresh frozen material of 78 patients with cardiac amyloidosis and from 12 biopsies of unused donor heart explants. Cryostat sections were digested with trypsin and analysed with liquid chromatography - mass spectrometry, and data were evaluated by proteomic software. With a diagnostic threshold set to 70% for each of the four most common amyloid proteins affecting the heart (LC κ, LC λ, TTR and SAA), 65 of the cases (87%) could be diagnosed, and of these, 61 cases (94%) were in concordance with the original diagnoses. The specimens were also analysed for the summed intensities of the amyloid signature proteins (ApoE, ApoA-IV and SAP). The intensities were significantly higher (p < 0.001) for all assigned cases compared with controls. Cardiac amyloidosis can be successfully subtyped without the prior enrichment of amyloid deposits with laser microdissection.

Metabolic flux can be regulated by a variety of different mechanisms, but the organization of these mechanisms within the metabolic network has remained unknown. Here we test the hypothesis that flux control mechanisms are not distributed randomly in the metabolic network, but rather organized according to pathway. Combining proteomics, phosphoproteomics, and metabolic modeling, we report the largest collection of flux-enzyme-phosphoenzyme relationships to date in Saccharomyces cerevisiae. In support of the hypothesis, we show that (i) amino acid metabolic pathways are predominantly regulated by enzyme abundance stemming from transcriptional regulation; (ii) upper glycolysis and associated pathways, by inactivating enzyme phosphorylation; (iii) lower glycolysis and associated pathways, by activating enzyme phosphorylation; and (iv) glycolipid/glycophospholipid pathways, by a combination of enzyme phosphorylation and metabolic compartmentalization. We delineate the evolutionary history for the observed organization of flux control mechanisms in yeast central metabolic pathways, furthering our understanding of the regulation of metabolism and its evolution.

Like most other cell surface proteins, αHere, we have established the first comprehensive site-specific glycan map of αWe expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control αGlycosylation of α

Molecular biomarkers of ionizing radiation (IR) exposure are a promising new tool in various disciplines: they can give necessary information for adaptive treatment planning in cancer radiotherapy, enable risk projection for radiation-induced survivorship diseases, or facilitate triage and intervention in radiation hazard events. However, radiation biomarker discovery has not yet resolved the most basic features of personalized medicine: age and sex. To overcome this critical bias in biomarker identification, we quantitated age and sex effects and assessed their relevance in the radiation response across the blood proteome. We used high-throughput mass spectrometry on blood plasma collected 24 hours after 0.5 Gy total body irradiation (15 MV nominal photon energy) from male and female C57BL/6N mice at juvenile (7-weeks-old) or adult (18-weeks-old) age. We also assessed sex and strain effects using juvenile male and female BALB/c nude mice. We showed that age and sex created significant effects in the proteomic response regarding both extent and functional quality of IR-induced responses. Furthermore, we found that age and sex effects appeared non-linear and were often end-point specific. Overall, age contributed more to differences in the proteomic response than sex, most notably in immune responses, oxidative stress, and apoptotic cell death. Interestingly, sex effects were pronounced for DNA damage & repair pathways and associated cellular outcome (pro-survival vs. pro-apoptotic). Only one protein (AHSP) was identified as a potential general biomarker candidate across age and sex, while GMNN, REG3B, and SNCA indicated some response similarity across age. This low yield advocated that unisex or uniage biomarker screening approaches are not feasible. In conclusion, age- and sex-specific screening approaches should be implemented as standard protocol to ensure robustness and diagnostic power of biomarker candidates. Bias-free molecular biomarkers are a necessary progression towards personalized medicine and integral for advanced adaptive cancer radiotherapy and risk assessment.

Molecular biomarkers of ionizing radiation (IR) exposure are a promising new tool in various disciplines: they can give necessary information for adaptive treatment planning in cancer radiotherapy, enable risk projection for radiation-induced survivorship diseases, or facilitate triage and intervention in radiation hazard events. However, radiation biomarker discovery has not yet resolved the most basic features of personalized medicine: age and sex. To overcome this critical bias in biomarker identification, we quantitated age and sex effects and assessed their relevance in the radiation response across the blood proteome. We used high-throughput mass spectrometry on blood plasma collected 24 h after 0.5 Gy total body irradiation (15 MV nominal photon energy) from male and female C57BL/6 N mice at juvenile (7-weeks-old) or adult (18-weeks-old) age. We also assessed sex and strain effects using juvenile male and female BALB/c nude mice. We showed that age and sex created significant effects in the proteomic response regarding both extent and functional quality of IR-induced responses. Furthermore, we found that age and sex effects appeared non-linear and were often end-point specific. Overall, age contributed more to differences in the proteomic response than sex, most notably in immune responses, oxidative stress, and apoptotic cell death. Interestingly, sex effects were pronounced for DNA damage and repair pathways and associated cellular outcome (pro-survival vs. pro-apoptotic). Only one protein (AHSP) was identified as a potential general biomarker candidate across age and sex, while GMNN, REG3B, and SNCA indicated some response similarity across age. This low yield advocated that unisex or uniage biomarker screening approaches are not feasible. In conclusion, age- and sex-specific screening approaches should be implemented as standard protocol to ensure robustness and diagnostic power of biomarker candidates. Bias-free molecular biomarkers are a necessary progression towards personalized medicine and integral for advanced adaptive cancer radiotherapy and risk assessment.

In this chapter, we describe a glycoproteomic approach for the identification of novel chondroitin sulfate proteoglycans (CSPGs) using a combination of biochemical enrichments, enzymatic digestions, and nanoscale liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. The identification is achieved by trypsin digestion of CSPG-containing samples, followed by enrichment of chondroitin sulfate (CS) glycopeptides by strong anion exchange chromatography (SAX). The enriched CS glycopeptides are then digested with chondroitinase ABC to depolymerize the CS polysaccharides, generating a residual hexasaccharide structure, composed of the linkage region tetrasaccharide extended with a terminal dehydrated disaccharide, still attached to the peptide. The obtained CS glycopeptides are analyzed by nLC-MS/MS, and the generated data sets are evaluated through proteomic software with adjustment in the settings to allow for glycopeptide identification. This approach has enabled the identification of several novel core proteins in human samples and in Caenorhabditis elegans. Here we specifically describe the procedure for the enrichment and characterization of CS glycopeptides from human cerebrospinal fluid (CSF).

In this chapter, we describe a glycoproteomic approach for the identification of novel chondroitin sulfate proteoglycans (CSPGs) using a combination of biochemical enrichments, enzymatic digestions, and nanoscale liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. The identification is achieved by trypsin digestion of CSPG-containing samples, followed by enrichment of chondroitin sulfate (CS) glycopeptides by strong anion exchange chromatography (SAX). The enriched CS glycopeptides are then digested with chondroitinase ABC to depolymerize the CS polysaccharides, generating a residual hexasaccharide structure, composed of the linkage region tetrasaccharide extended with a terminal dehydrated disaccharide, still attached to the peptide. The obtained CS glycopeptides are analyzed by nLC-MS/MS, and the generated data sets are evaluated through proteomic software with adjustment in the settings to allow for glycopeptide identification. This approach has enabled the identification of several novel core proteins in human samples and in Caenorhabditis elegans. Here we specifically describe the procedure for the enrichment and characterization of CS glycopeptides from human cerebrospinal fluid (CSF).

Recent studies highlight the importance of lipotoxic damage in aortic cells as the major pathogenetic contributor to atherosclerotic disease. Since the STE20-type kinase STK25 has been shown to exacerbate ectopic lipid storage and associated cell injury in several metabolic organs, we here investigate its role in the main cell types of vasculature. We depleted STK25 by small interfering RNA in human aortic endothelial and smooth muscle cells exposed to oleic acid and oxidized LDL. In both cell types, the silencing of STK25 reduces lipid accumulation and suppresses activation of inflammatory and fibrotic pathways as well as lowering oxidative and endoplasmic reticulum stress. Notably, in smooth muscle cells, STK25 inactivation hinders the shift from a contractile to a synthetic phenotype. Together, we provide several lines of evidence that antagonizing STK25 signaling in human aortic endothelial and smooth muscle cells is atheroprotective, highlighting this kinase as a new potential therapeutic target for atherosclerotic disease.

openaire: EC/H2020/305707/EU//RESOLVE Aims: To investigate how apolipoprotein C-III (apoC-III) metabolism is altered in subjects with type 2 diabetes, whether the perturbed plasma triglyceride concentrations in this condition are determined primarily by the secretion rate or the removal rate of apoC-III, and whether improvement of glycaemic control using the glucagon-like peptide-1 analogue liraglutide for 16 weeks modifies apoC-III dynamics. Materials and Methods: Postprandial apoC-III kinetics were assessed after a bolus injection of [5,5,5- 2 H 3 ]leucine using ultrasensitive mass spectrometry techniques. We compared apoC-III kinetics in two situations: in subjects with type 2 diabetes before and after liraglutide therapy, and in type 2 diabetic subjects with matched body mass index (BMI) non-diabetic subjects. Liver fat content, subcutaneous abdominal and intra-abdominal fat were determined using proton magnetic resonance spectroscopy. Results: Improved glycaemic control by liraglutide therapy for 16 weeks significantly reduced apoC-III secretion rate (561 ± 198 vs. 652 ± 196 mg/d, P = 0.03) and apoC-III levels (10.0 ± 3.8 vs. 11.7 ± 4.3 mg/dL, P = 0.035) in subjects with type 2 diabetes. Change in apoC-III secretion rate was significantly associated with the improvement in indices of glucose control (r = 0.67; P = 0.009) and change in triglyceride area under the curve (r = 0.59; P = 0.025). In line with this, the apoC-III secretion rate was higher in subjects with type 2 diabetes compared with BMI-matched non-diabetic subjects (676 ± 208 vs. 505 ± 174 mg/d, P = 0.042). Conclusions: The results reveal that the secretion rate of apoC-III is associated with elevation of triglyceride-rich lipoproteins in subjects with type 2 diabetes, potentially through the influence of glucose homeostasis on the production of apoC-III.

Ectopic lipid storage in the liver is considered the main risk factor for nonalcoholic steatohepatitis (NASH). Understanding the molecular networks controlling hepatocellular lipid deposition is therefore essential for developing new strategies to effectively prevent and treat this complex disease. Here, we describe a new regulator of lipid partitioning in human hepatocytes: mammalian sterile 20-like (MST) 3. We found that MST3 protein coats lipid droplets in mouse and human liver cells. Knockdown of MST3 attenuated lipid accumulation in human hepatocytes by stimulating β-oxidation and triacylglycerol secretion while inhibiting fatty acid influx and lipid synthesis. We also observed that lipogenic gene expression and acetyl-coenzyme A carboxylase protein abundance were reduced in MST3-deficient hepatocytes, providing insight into the molecular mechanisms underlying the decreased lipid storage. Furthermore, MST3 expression was positively correlated with key features of NASH (i.e., hepatic lipid content, lobular inflammation, and hepatocellular ballooning) in human liver biopsies. In summary, our results reveal a role of MST3 in controlling the dynamic metabolic balance of liver lipid catabolism vs. lipid anabolism. Our findings highlight MST3 as a potential drug target for the prevention and treatment of NASH and related complex metabolic diseases.-Cansby, E., Kulkarni, N. M., Magnusson, E., Kurhe, Y., Amrutkar, M., Nerstedt, A., Stahlman, M., Sihlbom, C., Marschall, H.-U., Boren, J., Bluher, M., Mahlapuu, M. Protein kinase MST3 modulates lipid homeostasis in hepatocytes and correlates with nonalcoholic steatohepatitis in humans.

Maternal obesity together with androgen excess in mice negatively affects placental function and maternal and fetal liver function as demonstrated by increased triglyceride content with dysfunctional expression of enzymes and transcription factors involved in de novo lipogenesis and fat storage. To identify changes in molecular pathways that might promote diseases in adulthood, we performed a global proteomic analysis using a liquid-chromatography/mass-spectrometry system to investigate total and phosphorylated proteins in the placenta and fetal liver in a mouse model that combines maternal obesity with maternal androgen excess.After ten weeks on a control diet (CD) or high fat/high sugar-diet, dams were mated with males fed the CD. Between gestational day (GD) 16.5 and GD 18.5, mice were injected with vehicle or dihydrotestosterone (DHT) and sacrificed at GD 18.5 prior to dissection of the placentas and fetal livers. Four pools of female placentas and fetal livers were subjected to a global proteomic analysis. Total and phosphorylated proteins were filtered by ANOVA q 0.05, and this was followed by two-way ANOVA to determine the effect of maternal obesity and/or androgen exposure.In placenta, phosphorylated ATP-citrate synthase was decreased due to maternal obesity, and phosphorylated catechol-O-methyltransferase (COMT) was differentially expressed due to the interaction between maternal diet and DHT exposure. In fetal liver, five total proteins and 48 proteins phosphorylated in one or more sites, were differentially expressed due to maternal obesity or androgen excess. In fetal liver, phosphorylated COMT expression was higher in fetus exposed to maternal obesity.These results suggest a common regulatory mechanism of catecholamine metabolism in the placenta and the fetal liver as demonstrated by higher phosphorylated COMT expression in the placenta and fetal liver from animals exposed to diet-induced maternal obesity and lower expression of phosphorylated COMT in animals exposed to maternal androgen excess.

Biological scaffold or implant is a popular choice for the preparation of tissue-engineered organs and has the potential to address donor shortage in the clinics. However, biological scaffolds prepared by physical or chemical agents cause damage to the extracellular matrix by potentially inducing immune responses after implantation. The current study explores an alternative route for the preparation of acellular scaffolds and explores the fate of the prepared scaffolds in a milieu of immune cells following implantation without using immunosuppressant. Using the syngeneic (Lewis male-Lewis female) and allogeneic (Brown Norway male-Lewis female) models and different tissue routes (subcutaneous vs omentum) for transplantation, normal blood vascular scaffolds were implanted which was converted to acellular vascular scaffolds by in vivo natural decellularization at the end of 2 months of observation. We also prepared chemically decellularized acellular scaffolds from normal untreated blood vascular scaffolds using a cocktail of chemicals which was also similarly placed in subcutaneous and omentum sites. Here, we applied in-depth quantitative proteomics along with histology and image analysis to comprehensively describe and compare the proteome of the natural and chemically decellularized scaffold. Our data confirm that site-specific advantages exist in modulating the ECM and regulating the immune responses (macrophage and T cells) following implantation, which possibly led to the production of an acellular scaffold (natural decellularization) under in vivo conditions. The current approach opens up the possibility to create tailor-made acellular scaffolds to build functional blood vessels. In addition, the identification of different tissue sites facilitates differential immune response against the scaffolds. This study provides a rich resource aimed toward an enhanced mechanistic understanding to study immune responses under similar settings in the field of transplantation and regenerative medicine.Impact statementThe development of a scaffold helps in the preparation of a functional organ in the clinics. In the current study, we prepared an acellular vascular scaffold by utilizing site specific tissue changes and vis-à-vis compared with a conventionally chemically prepared biological scaffold at genomic and protein level, which helped us to identify immunological trigger following implantation. The current study which was carried out without any immunosuppressive agents could help to establish (a) alternative strategies for preparing biological scaffolds as well as (b) implantable sites as potential bioreactors to circumvent any adverse immune reactions for acceptance of the scaffold/implant post implantation.

Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further to mammals including humans. Investigating molecular virus–host interactions dictating this process and the adaptations to the new hosts that follow is vital to understand zoonotic IAV spread. Receptor incompatibility has been suggested to limit zoonotic IAV transmission from the wild bird reservoir. Other barriers to interspecies transmission, particularly within the avian system, largely remain elusive. Through assessment of infection dynamics of mallard origin IAV in two different avian hosts, coupled with studies of receptor expression and host response we aimed to reveal the host-pathogen interactions in a cross-species transmission event. We found that shedding patterns and innate immune responses were highly dependent on viral genotypes, host species and inoculation routes, but less dependent on receptor expression. Further, in contrary to the prevailing dogma we demonstrate that birds can produce a wide range of different sialylated structures also found in mammals, e.g. extended N- and O-linked Neu5Acα2,6 terminated glycans. Overall, receptor incompatibility is not the sole transmission barrier for IAV between birds and to humans, but other host-pathogen factors deserve dedicated studies to achieve proper pandemic preparedness.

Background: \ud Renewed interest in triglyceride-rich lipoproteins as causative agents in cardiovascular disease mandates further exploration of the integrated metabolism of chylomicrons and very low-density lipoproteins (VLDL).\ud \ud Methods: \ud Novel tracer techniques and an integrated multi-compartmental model were used to determine the kinetics of apoB48- and apoB100-containing particles in the chylomicron and VLDL density intervals in 15 subjects with a wide range of plasma triglyceride levels.\ud \ud Results: \ud Following a fat-rich meal, apoB48 appeared in the chylomicron, VLDL1 and VLDL2 fractions in all subjects. Chylomicrons cleared rapidly from the circulation but apoB48-containing VLDL accumulated, and over the day were 3-fold higher in those with high versus low plasma triglyceride. ApoB48-containing particles were secreted directly into both the chylomicron and VLDL fractions at rates that were similar across the plasma triglyceride range studied. During fat absorption, whilst most triglyceride entered the circulation in chylomicrons, the majority of apoB48 particles were secreted into the VLDL density range.\ud \ud Conclusion: \ud The intestine secretes apoB48-containing particles not only as chylomicrons but also directly into the VLDL1 and VLDL2 density ranges both in the basal state and during dietary lipid absorption. Over the day, apoB48-containing particles appear to comprise about 20–25% of circulating VLDL and, especially in those with elevated triglycerides, form part of a slowly cleared ‘remnant’ particle population, thereby potentially increasing CHD risk. These findings provide a metabolic understanding of the potential consequences for increased CHD risk when slowed lipolysis leads to the accumulation of remnants, especially in individuals with hypertriglyceridemia.

The immunogenicity of the extracellular matrix (ECM) from genetically similar (syngeneic) and dissimilar (allogeneic and xenogeneic) species has puzzled the scientific community for many years. After implantation, the literature describes an absorption of ECM material since it is biodegradable. However, no clear insight really exists to substantiate how the underlying immune and biological responses result in absorption of ECM materials. In this context, it is important to characterize infiltrating cells and identify dominant cell populations in the infiltrate. We have studied the immune response in mice after implantation of decellularized (DC) cardiac scaffolds derived from pig and mouse. The polymorphism of the infiltrate into the implanted material signifies the importance of the adaptive immune response that is distinct for xenoimplants and alloimplants. Matrix resorption takes place mainly through phagocytic cells such as mast cells, dendritic cells, and macrophages. Histochemical observations show that innate CD8+ T cells develop immune tolerance, whereas proteomic analysis predicts the different T cell progenies for alloscaffolds and xenoscaffolds. The amalgamation of graft tolerance and involvement of both B and T cell populations in the vicinity of the graft could be decisive in wound remodeling and survival of the graft. This challenging area presents potential targets for the development of immune-privileged biomaterials, immune tolerant cells, and therapeutic agents in the future. Impact statement In this study, we have characterized the allogeneic and xenogeneic immune responses for decellularized (DC) cardiac scaffolds. We postulate that although the T cells are important players for immune tolerance of DC graft, the mechanism of their differentiation inside the host is donor specific. In this study, we have reported the distinct immune responses for syngeneic DC scaffolds than allogeneic and xenogeneic scaffolds. This distinct response provides the bases for the different immune responses reported for DC homografts in the literature. This study can provide the greater insight for modification of postimplant strategies to achieve host acceptance of donor extracellular matrix scaffolds.

Vitellogenesis (“yolking” of follicles) is a bioenergetically costly stage of reproduction requiring enlargement of the liver to produce vitellogenin (VTG) yolk precursor proteins, which are transported and deposited at the ovary. VTG may, however, serve non-nutritive antioxidant functions, a hypothesis supported by empirical work on aging and other life-history transitions in several taxa. We test this hypothesis in female painted dragon lizards (Ctenophorus pictus) by examining covariation in VTG with the ovarian cycle, and relative to reactive oxygen species (ROS) including baseline superoxide (bSO). Plasma VTG decreased prior to ovulation, when VTG is deposited into follicles. VTG, however, remained elevated post-ovulation when no longer necessary for yolk provisioning and was unrelated to reproductive investment. Instead, VTG was strongly and positively predicted by prior bSO. ROS, in turn, was negatively predicted by prior VTG, while simultaneously sampled VTG was a positive predictor. These findings are consistent with the hypothesis that VTG functions as an antioxidant to counteract oxidative stress associated with vitellogenesis. The relationship between bSO and VTG was strongest in post-ovulatory females, indicating its function may be largely antioxidant at this time. In conclusion, VTG may be under selection to offset oxidative costs of reproduction in egg-producing species.

Distinct structural changes of the α2,3/α2,6-sialic acid glycosidic linkages on glycoproteins are of importance in cancer biology, inflammatory diseases, and virus tropism. Current glycoproteomic methodologies are, however, not amenable toward high-throughput characterization of sialic acid isomers. To enable such assignments, a mass spectrometry method utilizing synthetic model glycopeptides for the analysis of oxonium ion intensity ratios was developed. This method was successfully applied in large-scale glycoproteomics, thus allowing the site-specific structural characterization of sialic acid isomers.

The structural study of glycans and glycoconjugates is essential to assign their roles in homeostasis, health, and disease. Once dominated by nuclear magnetic resonance spectroscopy, mass spectrometric methods have become the preferred toolbox for the determination of glycan structures at high sensitivity. The patterns of such structures in different cellular states now allow us to interpret the sugar codes in health and disease, based on structure-function relationships. Dr. Catherine E. Costello was the 2017 recipient of the American Society for Mass Spectrometry’s Distinguished Contribution Award. In this Perspective article, we describe her seminal work in a historical and geographical context and review the impact of her research accomplishments in the field. 8

Renal cell carcinoma (RCC), arising from the proximal tubule in the kidney, accounts for approximately 85% of kidney cancers and causes over 140,000 annual deaths worldwide. In the last decade, several new therapies have been identified for treatment of metastatic RCC. Although these therapies increase survival time compared to standard care, none of them has curative properties. The nephrotoxin orellanine specifically targets proximal tubular epithelial cells, leaving other organs unaffected. We therefore hypothesized that the selective toxicity of orellanine extends to clear cell RCC (ccRCC) cells since they emanate from proximal tubular cells. Orellanine would thus target both primary and metastatic ccRCC in vitro and in vivo. We found that orellanine induces dose-dependent cell death in proximal tubular cells and in all ccRCC cells tested, both primary and cell lines, with no toxicity detected in control cells. The toxic action of orellanine involve decreased protein synthesis, disrupted cell metabolism and induction of apoptosis. In nude rats carrying human ccRCC xenografts, brief orellanine treatment eliminated more than 90% of viable tumor mass compared to control rats. This identifies orellanine as a potential treatment concept for ccRCC patients on dialysis, due to its unique selective toxicity towards ccRCC.

Background: Clinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking. Objectives: We sought to identify and quantify a novel cartilage oligomeric matrix protein (COMP) neoepitope in the synovial fluid from the joints of healthy horses and those with different stages of OA. Study design: In vitro quantitative proteomics and assay development with application in synovial fluids samples obtained from biobanks of well-characterised horses. Methods: Articular cartilage explants were incubated with or without interleukin-1β for 25 days. Media were analysed via quantitative proteomics. Synovial fluid was obtained from either normal joints (n = 15) or joints causing lameness (n = 17) or with structural OA lesions (n = 7) and analysed for concentrations of the COMP neoepitope using a custom-developed inhibition enzyme-linked immunosorbent assay (ELISA). Explants were immunostained with polyclonal antibodies against COMP and the COMP neoepitopes. Results: Semitryptic COMP peptides were identified and quantified in cell culture media from cartilage explants. A rabbit polyclonal antibody was raised against the neoepitope of the N-terminal portion of one COMP fragment (sequence SGPTHEGVC). An inhibition ELISA was developed to quantify the COMP neoepitope in synovial fluid. The mean concentration of the COMP neoepitope significantly increased in the synovial fluid from the joints responsible for acute lameness compared with normal joints and the joints of chronically lame horses and in joints with chronic structural OA. Immunolabelling for the COMP neoepitope revealed a pericellular staining in the interleukin-1β-stimulated explants. Main limitations: The ELISA is based on polyclonal antisera rather than a monoclonal antibody. Conclusions: The increase in the COMP neoepitope in the synovial fluid from horses with acute lameness suggests that this neoepitope has the potential to be a unique candidate biomarker for the early molecular changes in articular cartilage associated with OA. (Less)

Background Takotsubo syndrome (TS) is an acute cardiac condition with a substantial mortality for which no specific treatment is available. We have previously shown that isoflurane attenuates the development of left ventricular (LV) dysfunction in an experimental TS-model. We compared the effects of equi-anaesthetic doses of isoflurane, propofol and ketamine+midazolam on haemodynamics, global and regional LV systolic function and the activation of intracellular metabolic pathways in experimental TS. We hypothesized that cardioprotection in experimental TS is specific for isoflurane. Methods Forty-five rats were randomized to isoflurane (0.6 MAC, n = 15), propofol (bolus 200 mg/kg+360 mg/kg/h, n = 15) or ketamine (100 mg/kg)+midazolam (10 mg/kg, n = 15) anaesthesia. Arterial pressure, heart rate and body temperature were continuously measured and arterial blood gas analysis was performed intermittently. TS was induced by intraperitoneal injection of isoprenaline, 50 mg/kg. LV echocardiography was performed 90 min after isoprenaline injection. Apical cardiac tissue was analysed by global discovery proteomics and pathway analysis. Results Isoprenaline-induced changes in arterial blood pressure, heart rate or body temperature did not differ between groups. LV ejection fraction was higher and extent of LV akinesia was lower with isoflurane, when compared with the propofol and the ketamine+midazolam groups. In this TS-model, the proteomic analysis revealed an up-regulation of pathways involved in inflammation, coagulation, endocytosis and lipid metabolism. This up-regulation was clearly attenuated with isoflurane compared to propofol. Conclusion In an experimental model of TS, isoflurane, but not propofol, exerts a cardioprotective effect. The proteomic analysis suggests that inflammation might be involved in pathogenesis of TS.