Daniel J. Geiszler
Dec 1, 2020; 0:TIR120.002216v1-mcp.TIR120.002216
Technological Innovation and Resources

Johannes Griss
Dec 1, 2020; 19:2115-2124
Technological Innovation and Resources

Diana Samodova
Dec 1, 2020; 19:2139-2156
Technological Innovation and Resources

Post-transcriptional modifications of pre-mRNAs expand the diversity of proteomes in higher eukaryotes. In the brain, these modifications diversify the functional output of many critical neuronal signal molecules. In this study, we identified a brain-specific A-to-I RNA editing that changed glutamine to arginine (Q/R) at exon 20 and an alternative splicing of exon 4 in Tmem63b, which encodes a ubiquitously expressed osmosensitive cation channel. The channel isoforms lacking exon 4 occurred in ∼80% of Tmem63b mRNAs in the brain but were not detected in other tissues, suggesting a brain-specific splicing. We found that the Q/R editing was catalyzed by Adar2 (Adarb1) and required an editing site complementary sequence located in the proximal 5' end of intron 20. Moreover, the Q/R editing was almost exclusively identified in the splicing isoform lacking exon 4, indicating a coupling between the editing and the splicing. Elimination of the Q/R editing in brain-specific Adar2 knockout mice did not affect the splicing efficiency of exon 4. Furthermore, transfection with the splicing isoform containing exon 4 suppressed the Q/R editing in primary cultured cerebellar granule neurons. Thus, our study revealed a coupling between an RNA editing and a distant alternative splicing in the Tmem63b pre-mRNA, in which the splicing plays a dominant role. Finally, physiological analysis showed that the splicing and the editing coordinately regulate Ca2+ permeability and osmosensitivity of channel proteins, which may contribute to their functions in the brain.

Montgomery Blencowe
Dec 23, 2020; 0:jlr.RA120000713v1-jlr.RA120000713
Research Articles

Nicholas D. LeBlond
Dec 1, 2020; 61:1697-1706
Research Articles

Auguste Dargent
Dec 1, 2020; 61:1776-1783
Research Articles

Thibaut Bourgeois
Dec 11, 2020; 0:jlr.RA120000737v1-jlr.RA120000737
Research Articles

Amanda D.V. MacCannell
Nov 18, 2020; 0:jlr.ILR120001204v1-jlr.ILR120001204
Images in Lipid Research

If the response variable is in the CLASS statement variable list before the class variables that also appear in the MODEL statement, and an OM-data-set is used, least squares means results for several of the statistical procedures are incorrect.

Recent studies have highlighted an important role for lysophosphatidylcholine acyltransferase 3 (LPCAT3) in controlling the PUFA composition of cell membranes in the liver and intestine. In these organs, LPCAT3 critically supports cell membrane-associated processes such as lipid absorption or lipoprotein secretion. However, the role of LPCAT3 in macrophages remains controversial. Here, we investigated LPCAT3’s role in macrophages both in vitro and in vivo in mice with atherosclerosis and obesity. To accomplish this, we used the LysMCre strategy to develop a mouse model with conditional Lpcat3 deficiency in myeloid cells (Lpcat3KOMac). We observed that partial Lpcat3 deficiency (approx. 75% reduction) in macrophages alters the PUFA composition of all phospholipid (PL) subclasses, including phosphatidylinositols and phosphatidylserines. A reduced incorporation of C20 PUFAs (mainly arachidonic acid [AA]) into PLs was associated with a redistribution of these FAs toward other cellular lipids such as cholesteryl esters. Lpcat3 deficiency had no obvious impact on macrophage inflammatory response or endoplasmic reticulum (ER) stress; however, Lpcat3KOMac macrophages exhibited a reduction in cholesterol efflux in vitro. In vivo, myeloid Lpcat3 deficiency did not affect atherosclerosis development in LDL receptor deficient mouse (Ldlr-/-) mice. Lpcat3KOMac mice on a high-fat diet displayed a mild increase in hepatic steatosis associated with alterations in several liver metabolic pathways and in liver eicosanoid composition. We conclude that alterations in AA metabolism along with myeloid Lpcat3 deficiency may secondarily affect AA homeostasis in the whole liver, leading to metabolic disorders and triglyceride accumulation.

Genome-wide association studies (GWAS) have implicated ~380 genetic loci for plasma lipid regulation. However, these loci only explain 17-27% of the trait variance and a comprehensive understanding of the molecular mechanisms has not been achieved. In this study, we utilized an integrative genomics approach leveraging diverse genomic data from human populations to investigate whether genetic variants associated with various plasma lipid traits, namely total cholesterol (TC), high and low density lipoprotein cholesterol (HDL and LDL), and triglycerides (TG), from GWAS were concentrated on specific parts of tissue-specific gene regulatory networks. In addition to the expected lipid metabolism pathways, gene subnetworks involved in ‘interferon signaling’, ‘autoimmune/immune activation’, ‘visual transduction’, and ‘protein catabolism’ were significantly associated with all lipid traits. Additionally, we detected trait-specific subnetworks, including cadherin-associated subnetworks for LDL, glutathione metabolism for HDL, valine, leucine and isoleucine biosynthesis for TC, and insulin signaling and complement pathways for TG. Finally, utilizing gene-gene relations revealed by tissue-specific gene regulatory networks, we detected both known (e.g. APOH, APOA4, and ABCA1) and novel (e.g. F2 in adipose tissue) key regulator genes in these lipid-associated subnetworks. Knockdown of the F2 gene (Coagulation Factor II, Thrombin) in 3T3-L1 and C3H10T1/2 adipocytes reduced gene expression of Abcb11, Apoa5, Apof, Fabp1, Lipc, and Cd36, reduced intracellular adipocyte lipid content, and increased extracellular lipid content, supporting a link between adipose thrombin and lipid regulation. Our results shed light on the complex mechanisms underlying lipid metabolism and highlight potential novel targets for lipid regulation and lipid-associated diseases.

Bile acids (BAs) have been established as ubiquitous regulatory molecules implicated in a large variety of healthy and pathological processes. However, the scope of BA heterogeneity is often underrepresented in current literature. This is due in part to inadequate detection methods, which fail to distinguish the individual constituents of the BA pool. Thus, the primary aim of this study was to develop a method that would allow the simultaneous analysis of specific C24 BA species, and to apply that method to biological systems of interest. Herein, we describe the generation and validation of an LC-MS/MS assay for quantification of numerous BAs in a variety of cell systems and relevant biofluids and tissue. These studies included the first baseline level assessment for planar BAs, including allocholic acid, in cell lines, biofluids, and tissue in a nonhuman primate (NHP) laboratory animal, Macaca mulatta, in healthy conditions. These results indicate that immortalized cell lines make poor models for the study of BA synthesis and metabolism, whereas human primary hepatocytes represent a promising alternative model system. We also characterized the BA pool of M. mulatta in detail. Our results support the use of NHP models for the study of BA metabolism and pathology in lieu of murine models. Moreover, the method developed here can be applied to the study of common and planar C24 BA species in other systems.

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

Lipopolysaccharide (LPS) is a key player for innate immunity activation. It is therefore a prime target for sepsis treatment, as antibiotics are not sufficient to improve outcome during septic shock. An extracorporeal removal method by polymyxin (PMX) B direct hemoperfusion (PMX-DHP) is used in Japan, but recent trials failed to show a significant lowering of circulating LPS levels after PMX-DHP therapy. PMX-DHP has a direct effect on LPS molecules. However, LPS is not present in a free form in the circulation, as it is mainly carried by lipoproteins, including LDLs. Lipoproteins are critical for physiological LPS clearance, as LPSs are carried by LDLs to the liver for elimination. We hypothesized that LDL apheresis could be an alternate method for LPS removal. First, we demonstrated in vitro that LDL apheresis microbeads are almost as efficient as PMX beads to reduce LPS concentration in LPS-spiked human plasma, whereas it is not active in PBS. We found that PMX was also adsorbing lipoproteins, although less specifically. Then, we found that endogenous LPS of patients treated by LDL apheresis for familial hypercholesterolemia is also removed during their LDL apheresis sessions, with both electrostatic-based devices and filtration devices. Finally, LPS circulating in the plasma of septic shock and severe sepsis patients with gram-negative bacteremia was also removed in vitro by LDL adsorption. Overall, these results underline the importance of lipoproteins for LPS clearance, making them a prime target to study and treat endotoxemia-related conditions.

The dysregulation of myeloid-derived cell metabolism can drive atherosclerosis. AMP-activated protein kinase (AMPK) controls various aspects of macrophage dynamics and lipid homeostasis, which are important during atherogenesis. Using LysM-Cre to drive the deletion of both the α1 and α2 catalytic subunits (MacKO), we aimed to clarify the role of myeloid-specific AMPK signaling in male and female mice made acutely atherosclerotic by injection of AAV vector encoding a gain-of-function mutant PCSK9 (PCSK9-AAV) and WD feeding. After 6 weeks of WD feeding, mice received a daily injection of either the AMPK activator A-769662 or a vehicle control for an additional 6 weeks. Following this (12 weeks total), we assessed myeloid cell populations and differences between genotype or sex were not observed. Similarly, aortic sinus plaque size, lipid staining, and necrotic area did not differ in male and female MacKO mice compared with their littermate floxed controls. Moreover, therapeutic intervention with A-769662 showed no treatment effect. There were also no observable differences in the amount of circulating total cholesterol or triglyceride, and only minor differences in the levels of inflammatory cytokines between groups. Finally, CD68+ area and markers of autophagy showed no effect of either lacking AMPK signaling or AMPK activation. Our data suggest that while defined roles for each catalytic AMPK subunit have been identified, complete deletion of myeloid AMPK signaling does not significantly impact atherosclerosis. Additionally, these findings suggest that intervention with the first-generation AMPK activator A-769662 is not able to stem the progression of atherosclerosis.

The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site–associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.

Tandem mass tag (TMT) is a multiplexing technology widely-used in proteomic research. It enables relative quantification of proteins from multiple biological samples in a single MS run with high efficiency and high throughput. However, experiments often require more biological replicates or conditions than can be accommodated by a single run, and involve multiple TMT mixtures and multiple runs. Such larger-scale experiments combine sources of biological and technical variation in patterns that are complex, unique to TMT-based workflows, and challenging for the downstream statistical analysis. These patterns cannot be adequately characterized by statistical methods designed for other technologies, such as label-free proteomics or transcriptomics. This manuscript proposes a general statistical approach for relative protein quantification in MS- based experiments with TMT labeling. It is applicable to experiments with multiple conditions, multiple biological replicate runs and multiple technical replicate runs, and unbalanced designs. It is based on a flexible family of linear mixed-effects models that handle complex patterns of technical artifacts and missing values. The approach is implemented in MSstatsTMT, a freely available open-source R/Bioconductor package compatible with data processing tools such as Proteome Discoverer, MaxQuant, OpenMS, and SpectroMine. Evaluation on a controlled mixture, simulated datasets, and three biological investigations with diverse designs demonstrated that MSstatsTMT balanced the sensitivity and the specificity of detecting differentially abundant proteins, in large-scale experiments with multiple biological mixtures.

Glutathionylation is an important posttranslational modification that protects proteins from further oxidative damage as well as influencing protein structure and activity. In the present study, we demonstrate that the cysteine-42 residue in protein arginine N-methyltransferase 5 (PRMT5) is glutathionylated in aged mice or in cells that have been exposed to oxidative stress. Deglutathionylation of this protein is catalyzed by glutaredoxin-1 (Grx1). Using mutagenesis and subsequent biochemical analyses, we show that glutathionylation decreased the binding affinity of PRMT5 with methylosome protein-50 (MEP50) and reduced the methyltransferase activity of PRMT5. Furthermore, overexpression of PRMT5-C42A mutant caused a significant increase in histone methylation in HEK293T and A549 cells and promoted cell growth, whereas overexpression of the PRMT5-C42D mutant, a mimic of glutathionylated PRMT5, inhibited cell proliferation. Taken together, our results demonstrate a new mechanism of regulation of PRMT5 methyltransferases activity and suggest that PRMT5 glutathionylation is partly responsible for reactive oxygen species-mediated cell growth inhibition.

Renal Cell Carcinoma (RCC) is one of the most commonly diagnosed cancers worldwide with research efforts dramatically improving understanding of the biology of the disease. To investigate the role of the immune system in treatment-naïve clear cell Renal Cell Carcinoma (ccRCC), we interrogated the immune infiltrate in patient-matched ccRCC tumor samples, benign normal adjacent tissue (NAT) and peripheral blood mononuclear cells (PBMCs isolated from whole blood, focusing our attention on the myeloid cell infiltrate. Using flow cytometric, MS, and ExCYT analysis, we discovered unique myeloid populations in PBMCs across patient samples. Furthermore, normal adjacent tissues and ccRCC tissues contained numerous myeloid populations with a unique signature for both tissues. Enrichment of the immune cell (CD45⁺) fraction and subsequent gene expression analysis revealed a number of myeloid-related genes that were differentially expressed. These data provide evidence, for the first time, of an immunosuppressive and pro-tumorigenic role of myeloid cells in early, clinically localized ccRCC. The identification of a number of immune proteins for therapeutic targeting provides a rationale for investigation into the potential efficacy of earlier intervention with single-agent or combination immunotherapy for ccRCC.

The EGFR tyrosine kinase inhibitor gefitinib is commonly used for lung cancer patients. However, some patients eventually become resistant to gefitinib and develop progressive disease. Here, we indicate that ecto-ATP synthase, which ectopically translocated from mitochondrial inner membrane to plasma membrane, is considered as a potential therapeutic target for drug-resistant cells. Quantitative multi-omics profiling reveals that ecto-ATP synthase inhibitor mediates CK2-dependent phosphorylation of DNA topoisomerase IIα (topo IIα) at serine 1106 and subsequently increases the expression of long noncoding RNA, GAS5. Additionally, we also determine that downstream of GAS5, p53 pathway, is activated by ecto-ATP synthase inhibitor for regulation of programed cell death. Interestingly, GAS5-proteins interactomic profiling elucidates that GAS5 associates with topo IIα and subsequently enhancing the phosphorylation level of topo IIα. Taken together, our findings suggest that ecto-ATP synthase blockade is an effective therapeutic strategy via regulation of CK2/phospho-topo IIα/GAS5 network in gefitinib-resistant lung cancer cells.

We performed an in-depth characterization and comparison of the pediatric and adult urinary glycomes using a nanoLC-MS/MS based glycomics method, which included normal healthy pediatric (1–10 years, n = 21) and adult (21–50 years, n = 22) individuals. A total of 116 N-glycan compositions were identified, and 46 of them could be reproducibly quantified. We performed quantitative comparisons of the 46 glycan compositions between different age and sex groups. The results showed significant quantitative changes between the pediatric and adult cohorts. The pediatric urinary N-glycome was found to contain a higher level of high-mannose (HM), asialylated/afucosylated glycans (excluding HM), neutral fucosylated and agalactosylated glycans, and a lower level of trisialylated glycans compared with the adult. We further analyzed gender-associated glycan changes in the pediatric and adult group, respectively. In the pediatric group, there was almost no difference of glycan levels between males and females. In adult, the majority of glycans were more abundant in males than females, except the high-mannose and tetrasialylated glycans. These findings highlight the importance to consider age-matching and adult sex-matching for urinary glycan studies. The identified normal pediatric and adult urinary glycomes can serve as a baseline reference for comparisons to other disease states affected by glycosylation.

Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in mAb. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of noncollagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a nonreduced mAb.

Pathway analyses are key methods to analyze 'omics experiments. Nevertheless, integrating data from different 'omics technologies and different species still requires considerable bioinformatics knowledge.

Here we present the novel ReactomeGSA resource for comparative pathway analyses of multi-omics datasets. ReactomeGSA can be used through Reactome's existing web interface and the novel ReactomeGSA R Bioconductor package with explicit support for scRNA-seq data. Data from different species is automatically mapped to a common pathway space. Public data from ExpressionAtlas and Single Cell ExpressionAtlas can be directly integrated in the analysis. ReactomeGSA greatly reduces the technical barrier for multi-omics, cross-species, comparative pathway analyses.

We used ReactomeGSA to characterize the role of B cells in anti-tumor immunity. We compared B cell rich and poor human cancer samples from five of the Cancer Genome Atlas (TCGA) transcriptomics and two of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteomics studies. B cell-rich lung adenocarcinoma samples lacked the otherwise present activation through NFkappaB. This may be linked to the presence of a specific subset of tumor associated IgG+ plasma cells that lack NFkappaB activation in scRNA-seq data from human melanoma. This showcases how ReactomeGSA can derive novel biomedical insights by integrating large multi-omics datasets.

Endometrial carcinoma (EC) is the most common gynecologic malignancy in the United States, with limited effective targeted therapies. Endometrial tumors exhibit frequent alterations in protein kinases, yet only a small fraction of the kinome has been therapeutically explored. To identify kinase therapeutic avenues for EC, we profiled the kinome of endometrial tumors and normal endometrial tissues using Multiplexed Inhibitor Beads and Mass Spectrometry (MIB-MS). Our proteomics analysis identified a network of kinases overexpressed in tumors, including Serine/Arginine-Rich Splicing Factor Kinase 1 (SRPK1). Immunohistochemical (IHC) analysis of endometrial tumors confirmed MIB-MS findings and showed SRPK1 protein levels were highly expressed in endometrioid and uterine serous cancer (USC) histological subtypes. Moreover, querying large-scale genomics studies of EC tumors revealed high expression of SRPK1 correlated with poor survival. Loss-of-function studies targeting SRPK1 in an established USC cell line demonstrated SRPK1 was integral for RNA splicing, as well as cell cycle progression and survival under nutrient deficient conditions. Profiling of USC cells identified a compensatory response to SRPK1 inhibition that involved EGFR and the up-regulation of IGF1R and downstream AKT signaling. Co-targeting SRPK1 and EGFR or IGF1R synergistically enhanced growth inhibition in serous and endometrioid cell lines, representing a promising combination therapy for EC.

Stroke remains a leading cause of death and disability worldwide. Despite continuous advances, the identification of key molecular signatures in the hyper-acute phase of ischemic stroke is still a primary interest for translational research on stroke diagnosis, prognosis, and treatment. Data integration from high-throughput -omics techniques has become crucial to unraveling key interactions among different molecular elements in complex biological contexts, such as ischemic stroke. Thus, we used advanced data integration methods for a multi-level joint analysis of transcriptomics and proteomics data sets obtained from mouse brains at 2 h after cerebral ischemia. By modeling net-like correlation structures, we identified an integrated network of genes and proteins that are differentially expressed at a very early stage after stroke. We validated 10 of these deregulated elements in acute stroke, and changes in their expression pattern over time after cerebral ischemia were described. Of these, CLDN20, GADD45G, RGS2, BAG5, and CTNND2 were next evaluated as blood biomarkers of cerebral ischemia in mice and human blood samples, which were obtained from stroke patients and patients presenting stroke-mimicking conditions. Our findings indicate that CTNND2 levels in blood might potentially be useful for distinguishing ischemic strokes from stroke-mimicking conditions in the hyper-acute phase of the disease. Furthermore, circulating GADD45G content within the first 6 h after stroke could also play a key role in predicting poor outcomes in stroke patients. For the first time, we have used an integrative biostatistical approach to elucidate key molecules in the initial stages of stroke pathophysiology and highlight new notable molecules that might be further considered as blood biomarkers of ischemic stroke.

Ankylosing Spondylitis (AS) is a common, inflammatory rheumatic disease, which primarily affects the axial skeleton and is associated with sacroiliitis, uveitis and enthesitis. Unlike other autoimmune rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus, autoantibodies have not yet been reported to be a feature of AS. We therefore wished to determine if plasma from patients with AS contained autoantibodies and if so, characterize and quantify this response in comparison to patients with Rheumatoid Arthritis (RA) and healthy controls. Two high-density nucleic acid programmable protein arrays expressing a total of 3498 proteins were screened with plasma from 25 patients with AS, 17 with RA and 25 healthy controls. Autoantigens identified were subjected to Ingenuity Pathway Analysis in order to determine patterns of signalling cascades or tissue origin. 44% of patients with Ankylosing Spondylitis demonstrated a broad autoantibody response, as compared to 33% of patients with RA and only 8% of healthy controls. Individuals with AS demonstrated autoantibody responses to shared autoantigens, and 60% of autoantigens identified in the AS cohort were restricted to that group. The AS patients autoantibody responses were targeted towards connective, skeletal and muscular tissue, unlike those of RA patients or healthy controls. Thus, patients with AS show evidence of systemic humoral autoimmunity and multispecific autoantibody production. Nucleic Acid Programmable Protein Arrays constitute a powerful tool to study autoimmune diseases.

In quantitative proteomics work, the differences in expression of many separate proteins are routinely examined to test for significant differences between treatments. This leads to the multiple hypothesis testing problem: when many separate tests are performed many will be significant by chance and be false positive results. Statistical methods such as the false discovery rate (FDR) method that deal with this problem have been disseminated for more than one decade. However a survey of proteomics journals shows that such tests are not widely implemented in one commonly used technique, quantitative proteomics using two-dimensional electrophoresis (2-DE). We outline a selection of multiple hypothesis testing methods, including some that are well known and some lesser known, and present a simple strategy for their use by the experimental scientist in quantitative proteomics work generally. The strategy focuses on the desirability of simultaneous use of several different methods, the choice and emphasis dependent on research priorities and the results in hand. This approach is demonstrated using case scenarios with experimental and simulated model data.

Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring (PRM)-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor (EGF)-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11-18) or acquired resistance (11-18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11-18 cell model was associated not only with previously reported upregulation of MET, but also with upregulation of FLK2 and downregulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with PRM data. Multiplexed PRM assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706.

Glycopeptides in peptide or digested protein samples pose a number of analytical and bioinformatics challenges beyond those posed by unmodified peptides or peptides with smaller posttranslational modifications. Exact structural elucidation of glycans is generally beyond the capability of a single mass spectrometry experiment, so a reasonable level of identification for tandem mass spectrometry, taken by several glycopeptide software tools, is that of peptide sequence and glycan composition, meaning the number of monosaccharides of each distinct mass, for example HexNAc(2)Hex(5) rather than man5. Even at this level, however, glycopeptide analysis poses challenges:  finding glycopeptide spectra when they are a tiny fraction of the total spectra; assigning spectra with unanticipated glycans, not in the initial glycan database; and finding, scoring, and labeling diagnostic peaks in tandem mass spectra.  Here we discuss recent improvements to Byonic, a glycoproteomics search program, that address these three issues. Byonic now supports filtering spectra by m/z peaks, so that the user can limit attention to spectra with diagnostic peaks, for example, at least two out of three of 204.087 for HexNAc, 274.092 for NeuAc (with water loss), and 366.139 for HexNAc-Hex, all within a set mass tolerance, for example, ± 0.01 Daltons. Also new is glycan "wildcard" search, which allows an unspecified mass within a user-set mass range to be applied to N- or O-linked glycans and enables assignment of spectra with unanticipated glycans. Finally the next release of Byonic supports user-specified peak annotations from user-defined posttranslational modifications. We demonstrate the utility of these new software features by finding previously unrecognized glycopeptides in publicly available data, including glycosylated neuropeptides from rat brain.

Biological functions emerge from complex and dynamic networks of protein-protein interactions. Because these protein-protein interaction networks, or interactomes, represent pairwise connections within a hierarchically organized system, it is often useful to identify higher-order associations embedded within them, such as multi-member protein complexes. Graph-based clustering techniques are widely used to accomplish this goal, and dozens of field-specific and general clustering algorithms exist. However, interactomes can be prone to errors, especially when inferred from high-throughput biochemical assays. Therefore, robustness to network-level noise is an important criterion for any clustering algorithm that aims to generate robust, reproducible clusters. Here, we tested the robustness of a range of graph-based clustering algorithms in the presence of noise, including algorithms common across domains and those specific to protein networks. Strikingly, we found that all of the clustering algorithms tested here markedly amplified noise within the underlying protein interaction network. Randomly rewiring only 1% of network edges yielded more than a 50% change in clustering results, indicating that clustering markedly amplified network-level noise. Moreover, we found the impact of network noise on individual clusters was not uniform: some clusters were consistently robust to injected noise while others were not. To assist in assessing this, we developed the clust.perturb R package and Shiny web application to measure the reproducibility of clusters by randomly perturbing the network. We show that clust.perturb results are predictive of real-world cluster stability: poorly reproducible clusters as identified by clust.perturb are significantly less likely to be reclustered across experiments. We conclude that graph-based clustering amplifies noise in protein interaction networks, but quantifying the robustness of a cluster to network noise can separate stable protein complexes from spurious associations.

Thyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for T3 and T4 hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism (CH). Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification–mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several CH-variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and engagement by targeting factors for ER-associated degradation (ERAD). Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for one Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis.

Open searching has proven to be an effective strategy for identifying both known and unknown modifications in shotgun proteomics experiments. Rather than being limited to a small set of user-specified modifications, open searches identify peptides with any mass shift that may correspond to a single modification or a combination of several modifications. Here we present PTM-Shepherd, a bioinformatics tool that automates characterization of PTM profiles detected in open searches based on attributes such as amino acid localization, fragmentation spectra similarity, retention time shifts, and relative modification rates. PTM-Shepherd can also perform multi-experiment comparisons for studying changes in modification profiles, e.g. in data generated in different laboratories or under different conditions. We demonstrate how PTM-Shepherd improves the analysis of data from formalin-fixed paraffin-embedded samples, detects extreme underalkylation of cysteine in some datasets, discovers an artefactual modification introduced during peptide synthesis, and uncovers site-specific biases in sample preparation artifacts in a multi-center proteomics profiling study.

Urinary proteomics studies have primarily focused on identifying markers of chronic kidney disease (CKD) progression. Here, we aimed to determine urinary markers of CKD renal parenchymal injury through proteomics analysis in animal kidney tissues and cells and in the urine of patients with CKD. Label-free quantitative proteomics analysis based on liquid chromatography-tandem mass spectrometry was performed on urine samples obtained from 6 normal controls and 9, 11, and 10 patients with CKD stages 1, 3, and 5, respectively, and on kidney tissue samples from a rat CKD model by 5/6 nephrectomy. Tandem mass tag-based quantitative proteomics analysis was performed for primary cultured glomerular endothelial cells (GECs) and proximal tubular epithelial cells (PTECs) before and after inducing 24-h hypoxia injury. Upon hierarchical clustering, out of 858 differentially expressed proteins (DEPs) in the urine of CKD patients, the levels of 416 decreased and 403 increased sequentially according to the disease stage, respectively. Among 2965 DEPs across 5/6 nephrectomized and sham-operated rat kidney tissues, 86 DEPs showed same expression patterns in the urine and kidney tissue. After cross-validation with two external animal proteome datasets, 38 DEPs were organized; only 10 DEPs, including serotransferrin, gelsolin, poly ADP-ribose polymerase 1, neuroblast differentiation-associated protein AHNAK, microtubule-associated protein 4, galectin-1, protein S, thymosin beta-4, myristoylated alanine-rich C-kinase substrate, and vimentin were finalized by screening human GECs and PTECs data. Among these ten potential candidates for universal CKD marker, validation analyses for protein S and galectin-1 were conducted. Galectin-1 was observed to have a significant inverse correlation with renal function as well as higher expression in glomerulus with chronic injury than protein S. This constitutes the first multi-sample proteomics study for identifying key renal-expressed proteins associated with CKD progression. The discovered proteins represent potential markers of chronic renal cell and tissue damage and candidate contributors to CKD pathophysiology.

Nuclear Disarmament and the Protection of Cultural Heritage Research paper sysadmin 6 October 2017

States possessing nuclear weapons should be called upon to consider and publish the risks posed to cultural heritage, and their mitigation strategies, in their nuclear-weapons doctrines and policies.

Summary

• Renewed risk assessments for nuclear weapons and policies are taking place around the world in light of nuclear modernization and the changing geostrategic environment that is making the use of nuclear weapons more likely. As such the humanitarian impacts of nuclear weapons and tests have received increased attention. However, the effect on cultural heritage has so far been neglected.
• The potential for armed conflict to destroy cultural heritage has been recognized in international law since 1954. There is significant evidence on the impact of nuclear weapons on cultural heritage including the consequences of their use in Hiroshima and Nagasaki and the effect of nuclear-testing programmes in places of cultural significance since 1945. States that possess nuclear weapons have increased liabilities and responsibilities to protect cultural heritage and cultural rights. The need to protect cultural heritage should strengthen the case for reducing and eliminating nuclear weapons.
• Failure to take into account the protection of heritage in the development of nuclear weapons policies – including disarmament, non-proliferation and arms-control negotiations – significantly undermines states’ existing commitments to protecting heritage threatened by conflict.
• Risk assessments of the impact of nuclear weapons on cultural heritage and important cultural artefacts – and methods of preventing such catastrophic damage – should be part of protecting cultural heritage in every country and the subject of informed public debate. A new body of knowledge on the full range of nuclear weapons impacts would introduce a fresh perspective to inform decision-makers, international organizations and the public in thinking about nuclear weapons policies and practices.
• Risk and resilience frameworks, which provide sets of solutions for risk assessments, would allow assessments of nuclear weapons threats to heritage and highlight vulnerabilities that need to be addressed. Such frameworks would provide a basis for policymakers to identify the world’s cultural heritage most at risk and help develop mitigation strategies to ensure that it is protected. In particular, states possessing nuclear weapons should be called upon to consider and publish the risks posed to cultural heritage, and their mitigation strategies, in their nuclear weapons doctrines and policies, as a contribution to transparency and confidence-building, and as a responsibility to the world’s shared heritage. International organizations, such as the UN Educational, Scientific, and Cultural Organization (UNESCO), have a role to play in bridging security perspectives with protecting cultural heritage.

Healthy Diets from Sustainable Production: Indonesia Research paper sysadmin 24 January 2019

Indonesia has an uncommon chance to bypass the negative trajectory of diets in other emerging economies and build a healthy and sustainable food system.

Summary

• Indonesia is approaching a key point on its development pathway. Rapidly declining poverty, a growing and urbanizing middle class with increased purchasing power and consumption patterns, and a diminishing contribution of agriculture to overall GDP are all set to fundamentally reorient much in society.
• Dietary change is at the heart of the public health and environmental challenges now facing Indonesia. Rates of obesity and diet-related non-communicable diseases such as type 2 diabetes are on the increase, while high levels of childhood undernutrition persist. This double burden of malnutrition presents a critical challenge for the future of Indonesian public health.
• At the same time, shifts in diet are placing increased pressure on the environment, threatening biodiversity and species loss and rapidly increasing risks for land-use change, climate change and freshwater use. In Indonesia, these environmental impacts of agriculture are driven both by domestic consumption of food and biofuels, and by a focus on export-led agricultural growth – particularly palm oil, rubber, coffee and cocoa.
• A core political focus on achieving national self-sufficiency in five strategic commodities – soy, rice, maize, sugar and beef – which has led to some price distortion, and the growing influence of modernized retail are potentially at odds with a transition to healthy diets from sustainable production.
• The components to support an ambitious national food strategy already exist, but are either underutilized or misdirected. Indonesia’s national dietary guidelines and examples of successful food-based social services, together with the country’s potential to lead the sustainable production and consumption agenda, both regionally and internationally, and its commitments under both the UN Sustainable Development Goals and the Paris Agreement on climate change, can all be harnessed to foster improved diets.
• There is a need to align high-level policy strategies across environment, public health and food issues. Mainstreaming the principles of a healthy diet within existing food policy and partnering with food providers and local pioneers to champion these efforts can help to ensure that healthy diets, produced sustainably, become the norm.
• Between now and 2020, when Indonesia embarks on the final five-year tranche of its National Long-Term Development Plan, there is an important window of opportunity to take decisive action that will influence the future trajectory of the population’s health and that of its environment, as well as contribute substantively to the global fight against climate change and biodiversity loss.
• 2019 will, meanwhile, be a critical election year in Indonesia, with both presidential and legislative elections due. Signals from Indonesian media and civil society organizations indicate that poverty reduction and social equity – including affordability of good food and healthcare – will be among the flagship issues for voters.
• The moment is thus ripe for a bold new vision for a sustainable food system that supports healthy diets for all. In choosing to act now, Indonesia could lay the foundations for a more resilient and equitable development pathway that prioritizes improved public health while at the same time safeguarding some of the world’s most important ecosystems for future generations.

Subsidies and Sustainable Agriculture: Mapping the Policy Landscape Research paper sysadmin 10 December 2019

Agricultural subsidies shape production and consumption patterns, with potentially significant effects on poverty, nutrition and other sustainability concerns. This paper maps the different types of support provided by governments to the agricultural sector, and highlights some of the complex political economy dynamics that underpin the relevant policies.

Summary

• Agricultural subsidies, a mainstay of government policy, have a large part in shaping production and consumption patterns, with potentially significant effects as regards poverty, food security, nutrition, and other sustainability concerns such as climate change, land use practices and biodiversity.
• There are multiple types of direct and indirect support provided by governments to various actors in the agricultural sector; and in terms of political economy, there are complex dynamics underpinning the policies that sustain these subsidies.
• Overall, subsidies targeting producers have the most significant effect on production, and the greater trade-distorting effect. These subsidies promote domestic production and discourage imports, leading to overproduction that is largely disposed of on the international market, with the help of export subsidies. This can tend to intensify negative environmental agricultural practices, such as cultivating marginal land, unsustainable types of intensification, or incentivizing excessive pesticide and fertilizer use.
• On the other hand, producer subsidies that are not tied to output of a specific commodity (i.e. delinked) have far fewer distorting impacts and could help to deliver sustainable outcomes. For example, this type of subsidies can require crop diversification or be linked to conservation of permanent grassland.
• Subsidies that enable transfers to consumers, for example through food stamp programmes, also serve to delink production from consumption, can foster healthier diets, can play an important role in delivering food accessibility and security among low-income groups, and can represent one of the less trade-distorting subsidies.
• If subsidies are to be reformed to help promote healthier diets and encourage more sustainable production, it is essential to understand not only the type and amount of support that key countries provide, but also the domestic dynamics that can shape such policies.
• While price support, input subsidies or investment aids remain the central pillars of programmes in large developing countries such as Brazil, China or India, other economies – notably including the EU and Japan – focus on direct payments, support for general services and set-aside schemes, as well as significant border protection. The US, for its part, has tended to focus on subsidized insurance schemes and food programmes for poorer consumers.
• If subsidies are to deliver policy objectives, their design and implementation should delink production from consumption. For example, consumer subsidies designed to deliver nutrition and food security, or payments for environmental services to enable more environmentally friendly production systems, could prove to be the most effective, least trade-distorting means of achieving more sustainable and equitable agricultural production.
• The political economy of food means that the removal of subsidies is often highly sensitive, and tends to be met with significant resistance. However, reform that delinks support from production through a gradual transition process could ultimately prove successful in delivering effective subsidy schemes.
• Effective subsidy schemes must by design be truly result- and performance-based, supported by robust and objective indicators. At the same time, engaging multiple actors along key commodity value chains – including leading importing and exporting countries, traders and transporters – could lead to the development of international, commodity-specific arrangements that are able to deliver effective nutrition and sustainability goals.

Aaron Nola established himself as one of the best pitchers in baseball in 2018. The Phillies ensured on Wednesday that he will be the ace in their rotation for possibly the next five seasons.

Cesar Hernandez scored 91 runs last season, the Phillies' highest mark since Rollins scored 102 in 2012. Now the luster is no longer there for players who score 100 runs or post 100 RBIs, but Hernandez should score at least 100 runs this season, if he stays healthy and if the Phillies' offense improves as expected.

After undergoing surgery in late June to repair a torn left labrum in his left shoulder that required nine anchors to be inserted to keep everything in place, Zack Cozart is healthy and looking for a bounce-back season with the Angels.

Angels first baseman Albert Pujols met with the media for the first time this spring on Sunday and said he's fully healthy after undergoing arthroscopic surgery on his left knee in late August.