This article has been withdrawn by the authors. This article did not comply with the editorial guidelines of MCP. Specifically, single peptide based protein identifications of 9-19% were included in the analysis and discussed in the results and conclusions. We wish to withdraw this article and resubmit a clarified, corrected manuscript for review.

Intact glycopeptide identification has long been known as a key and challenging barrier to the comprehensive and accurate understanding the role of glycosylation in an organism. Intact glycopeptide analysis is a blossoming field that has received increasing attention in recent years. Mass spectrometry (MS)-based strategies and relative software tools are major drivers that have greatly facilitated the analysis of intact glycopeptides, particularly intact N-glycopeptides. This manuscript provides a systematic review of the intact glycopeptide identification process using mass spectrometry data generated in shotgun proteomic experiments, which typically focus on N-glycopeptide analysis. Particular attention is paid to the software tools that have been recently developed in the last decade for the interpretation and quality control of glycopeptide spectra acquired using different MS strategies. The review also provides information about the characteristics and applications of these software tools, discusses their advantages and disadvantages, and concludes with a discussion of outstanding tools.

Mass spectrometry-based glycoproteomics has gone through some incredible developments over the last few years. Technological advances in glycopeptide enrichment, fragmentation methods, and data analysis workflows have enabled the transition of glycoproteomics from a niche application, mainly focused on the characterization of isolated glycoproteins, to a mature technology capable of profiling thousands of intact glycopeptides at once. In addition to numerous biological discoveries catalyzed by the technology, we are also observing an increase in studies focusing on global protein glycosylation and the relationship between multiple glycosylation sites on the same protein. It has become apparent that just describing protein glycosylation in terms of micro- and macro-heterogeneity, respectively the variation and occupancy of glycans at a given site, is not sufficient to describe the observed interactions between sites. In this perspective we propose a new term, meta-heterogeneity, to describe a higher level of glycan regulation: the variation in glycosylation across multiple sites of a given protein. We provide literature examples of extensive meta-heterogeneity on relevant proteins such as antibodies, erythropoietin, myeloperoxidase and a number of serum and plasma proteins. Furthermore, we postulate on the possible biological reasons and causes behind the intriguing meta-heterogeneity observed in glycoproteins.

Complex protein glycosylation occurs through biosynthetic steps in the secretory pathway that create macro- and microheterogeneity of structure and function.  Required for all life forms, glycosylation diversifies and adapts protein interactions with binding partners that underpin interactions at cell surfaces and pericellular and extracellular environments. Because these biological effects arise from heterogeneity of structure and function, it is necessary to measure their changes as part of the quest to understand nature.  Quite often, however, the assumption behind proteomics that post-translational modifications are discrete additions that can be modeled using the genome as a template does not apply to protein glycosylation.  Rather, it is necessary to quantify the glycosylation distribution at each glycosite and to aggregate this information into a population of mature glycoproteins that exist in a given biological system.  To date, mass spectrometric methods for assigning singly glycosylated peptides are well-established.  But it is necessary to quantify glycosylation heterogeneity accurately in order to gauge the alterations that occur during biological processes.  The task is to quantify the glycosylated peptide forms as accurately as possible and then apply appropriate bioinformatics algorithms to the calculation of micro- and macro-similarities.  In this review, we summarize current approaches for protein quantification as they apply to this glycoprotein similarity problem.

This review covers recent developments in glycosaminoglycan (GAG) analysis via mass spectrometry (MS). GAGs participate in a variety of biological functions, including cellular communication, wound healing, and anticoagulation, and are important targets for structural characterization. GAGs exhibit a diverse range of structural features due to the variety of O- and N-sulfation modifications and uronic acid C-5 epimerization that can occur, making their analysis a challenging target. Mass spectrometry approaches to the structure assignment of GAGs have been widely investigated, and new methodologies remain the subject of development. Advances in sample preparation, tandem MS techniques (MS/MS), on-line separations and automated analysis software have advanced the field of GAG analysis. These recent developments have led to remarkable improvements in the precision and time efficiency for the structural characterization of GAGs.

O-GlcNAcylation, the addition of a single N-acetylglucosamine residue to serine and threonine residues of cytoplasmic, nuclear, or mitochondrial proteins, is a widespread regulatory post-translational modification. It is involved in response to nutritional status and stress and its dysregulation is associated with diseases ranging from Alzheimer’s to diabetes.  While the modification was first detected over thirty-five years ago, research into the function of O-GlcNAcylation has accelerated dramatically in the last ten years due to the development of new enrichment and mass spectrometry techniques that facilitate its analysis.  This article summarizes methods for O-GlcNAc enrichment, key mass spectrometry instrumentation advancements, particularly those that allow modification site localization, and software tools that allow analysis of data from O-GlcNAc modified peptides.

Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analysed the glyco/proteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyse Sequential Window Acquisition of all THeoretical mass spectra (SWATH) Data Independent Acquisition (DIA) mass spectrometry data based on glycopeptides identified by Byonic. We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the Liqueur de Tirage for secondary fermentation. We found that aging a cuvée on lees for 24 months compared to 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. This work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic SWATH/DIA workflow that is broadly applicable to other sample types.

Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins most notably the up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the up-regulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.

The glycoprotein spike (S) on the surface of SARS-CoV-2 is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunit. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, completely of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells, regardless of the glycosites’ location. Moreover, the N-glycan compositions were conserved among different sizes of subunits. Our study indicate that the S protein N-glycosylation occurs regularly at each site, albeit the occupied N-glycans were diverse and heterogenous. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.

Esophageal squamous cell cancer (ESCC) is an aggressive malignancy with poor therapeutic outcomes. However, the alterations in proteins and post-translational modifications (PTMs) leading to the pathogenesis of ESCC remains unclear. Here, we provide the comprehensive characterization of the proteome, phosphorylome, lysine acetylome and succinylome for ESCC and matched control cells using quantitative proteomic approach. We identify abnormal protein and post-translational modification (PTM) pathways, including significantly downregulated lysine succinylation sites in cancer cells. Focusing on hyposuccinylation, we reveal that this altered PTM was enriched on enzymes of metabolic pathways inextricably linked with cancer metabolism. Importantly, ESCC malignant behaviors such as cell migration are inhibited once the level of succinylation was restored in vitro or in vivo. This effect was further verified by mutations to disrupt succinylation sites in candidate proteins. Meanwhile, we found that succinylation has a negative regulatory effect on histone methylation to promote cancer migration. Finally, hyposuccinylation is confirmed in primary ESCC specimens. Our findings together demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing new insights into the functional significance of PTM in cancer biology.

The choice for adjuvant chemotherapy in stage II colorectal cancer (CRC) is controversial as many patients are cured by surgery alone and it is difficult to identify patients with high-risk of recurrence of the disease. There is a need for better stratification of this group of patients. Mass spectrometry imaging could identify patients at risk. We report here the N-glycosylation signatures of the different cell populations in a group of stage II CRC tissue samples. The cancer cells, compared to normal epithelial cells, have increased levels of sialylation and high-mannose glycans, as well as decreased levels of fucosylation and highly branched N-glycans. When looking at the interface between cancer and its microenvironment, it seems that the cancer N-glycosylation signature spreads into the surrounding stroma at the invasive front of the tumor. This finding was more outspoken in patients with a worse outcome within this sample group.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock which has seen a rapid rise in prevalence throughout Western Europe in recent years. Following ingestion of metacercariae (parasite cysts) by the mammalian host, newly-excysted juveniles (NEJs) emerge and invade the duodenal submucosa which causes significant pathology in heavy infections. The immature larvae then migrate upwards, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients and to avoid the host immune response. Here, transcriptome analysis of four intra-mammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic disease respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that whilst a family of cathepsins B with varying S2 sub-site residues (indicating distinct substrate specificities) are differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is up-regulated in adult worms, although they are underrepresented in the secretome. The most abundant proteins in adult fluke secretions were helminth defence molecules (HDMs) that likely establish an immune environment permissive to fluke survival and/or neutralise pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognise antigens from other helminths commonly found as co-infections with rumen fluke.

Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the Binding Capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.

Thermal proteome profiling (TPP) allows for the unbiased detection of drug – target protein engagements in vivo. Traditionally, one cell type is used for TPP studies, with the risk of missing important differentially expressed target proteins. The use of whole organisms would circumvent this problem. Zebrafish embryos are amenable to such an approach. Here, we used TPP on whole zebrafish embryo lysate to identify protein targets of napabucasin, a compound that may affect Signal transducer and activator of transcription 3 (Stat3) signaling through an ill-understood mechanism. In zebrafish embryos, napabucasin induced developmental defects consistent with inhibition of Stat3 signaling. TPP profiling showed no distinct shift in Stat3 upon napabucasin treatment, but effects were detected on the oxidoreductase, Pora, which might explain effects on Stat3 signaling. Interestingly, thermal stability of several aldehyde dehydrogenases (Aldhs) was affected. Moreover, napabucasin activated ALDH enzymatic activity in vitro. Aldhs have crucial roles in retinoic acid metabolism and functionally we validated napabucasin-mediated activation of the retinoic acid pathway in zebrafish in vivo. We conclude that TPP profiling in whole zebrafish embryo lysate is feasible and facilitates direct correlation of in vivo effects of small molecule drugs with their protein targets.

The increasing consumption of high-fat foods combined with a lack of exercise is a major contributor to the burden of obesity in humans. Aerobic exercise such as running is known to provide metabolic benefits, but how the over-consumption of a high fat diet (HFD) and exercise interact is not well characterized at the molecular level. Here, we examined the plasma proteome in mice for the effects of aerobic exercise as both a treatment and as a preventative regime for animals on either HFD or a healthy control diet. This analysis detected large changes in the plasma proteome induced by the HFD, such as increased abundance of SERPINA7, ALDOB, and down-regulation of SERPINA1E, CFD (adipsin). Some of these changes were significantly reverted using exercise as a preventative measure, but not as a treatment regime. To determine if either the intensity, or duration, of exercise influenced the outcome, we compared high-intensity interval training (HIIT) and endurance running. Endurance running slightly out-performed HIIT exercise, but overall, both provided similar reversion in abundance of plasma proteins modulated by the high-fat diet including SERPINA7, APOE, SERPINA1E, and CFD. Finally, we compared the changes induced by over-consumption of HFD to previous data from mice fed an isocaloric high saturated fat (SFA) or polyunsaturated fat (PUFA) diet. This identified several common changes including increased APOC2 and APOE, but also highlighted changes specific for either over-consumption of HFD (ALDOB, SERPINA7, CFD), SFA-based diets (SERPINA1E), or PUFA-based diets (Haptoglobin - Hp). Together, these data highlight the importance of early intervention with exercise to revert HFD-induced phenotypes and suggest some of the molecular mechanisms leading to the changes in the plasma proteome generated by high fat diet consumption. Web-based interactive visualizations are provided for this dataset (larancelab.com/hfd-exercise), which give insight into diet and exercise phenotypic interactions on the plasma proteome.

The complexity and dynamics of the immensely heterogeneous glycoproteome of the prostate cancer (PCa) tumour micro-environment remain incompletely mapped, a knowledge gap that impedes our molecular-level understanding of the disease. To this end, we have used sensitive glycomics and glycoproteomics to map the protein-, cell- and tumour grade-specific N- and O-glycosylation in surgically-removed PCa tissues spanning five histological grades (n = 10/grade) and tissues from patients with benign prostatic hyperplasia (n = 5). Quantitative glycomics revealed PCa grade-specific alterations of the oligomannosidic-, paucimannosidic- and branched sialylated complex-type N-glycans, and dynamic remodelling of the sialylated core 1- and core 2-type O-glycome. Deep quantitative glycoproteomics identified ~7,400 unique N-glycopeptides from 500 N-glycoproteins and ~500 unique O-glycopeptides from nearly 200 O-glycoproteins. With reference to a recent Tissue and Blood Atlas, our data indicate that paucimannosidic glycans of the PCa tissues arise mainly from immune cell-derived glycoproteins. Further, the grade-specific PCa glycosylation arises primarily from dynamics in the cellular makeup of the PCa tumour microenvironment across grades involving increased oligomannosylation of prostate-derived glycoproteins and decreased bisecting GlcNAcylation of N-glycans carried by the extracellular matrix proteins. Further, elevated expression of several oligosaccharyltransferase subunits and enhanced N-glycoprotein site occupancy were observed associated with PCa progression. Finally, correlations between the protein-specific glycosylation and PCa progression were observed including increased site-specific core 2-type O-glycosylation of collagen VI. In conclusion, integrated glycomics and glycoproteomics have enabled new insight into the complexity and dynamics of the tissue glycoproteome associated with PCa progression generating an important resource to explore the underpinning disease mechanisms.

Malaria elimination is still pending on the development of novel tools that rely on a deep understanding of parasite biology. Proteins of all living cells undergo a myriad number of posttranslational modifications (PTMs) that are critical to multifarious life processes. An extensive proteome-wide dissection revealed a fine PTM map of most proteins in both Plasmodium falciparum, the causative agent of severe malaria, and the infected red blood cells. More than two-thirds of proteins of the parasite and its host cell underwent extensive and dynamic modification throughout the erythrocytic developmental stage. PTMs critically modulate the virulence factors involved in the host-parasite interaction and pathogenesis. Furthermore, P. falciparum stabilized the supporting proteins of erythrocyte origin by selective de-modification. Collectively, our multiple omic analyses, apart from having furthered a deep understanding of the systems biology of P. falciparum and malaria pathogenesis, provide a valuable resource for mining new antimalarial targets.

Protease activity has been associated with pathological processes that can lead to cancer development and progression. However, understanding the pathological unbalance in proteolysis is challenging since changes can occur simultaneously at protease, their inhibitor and substrate levels. Here, we present a pipeline that combines peptidomics, proteomics and peptidase predictions for studying proteolytic events in the saliva of seventy-nine patients and their association with oral squamous cell carcinoma (OSCC) prognosis. Our findings revealed differences in the saliva peptidome of patients with (pN+) or without (pN0) lymph node metastasis and delivered a panel of ten endogenous peptides correlated with poor prognostic factors plus five molecules able to classify pN0 and pN+ patients (ROC-AUC>0.85). In addition, endo- and exopeptidases putatively implicated in the processing of differential peptides were investigated using cancer tissue gene expression data from publicly repositories reinforcing their association with poorer survival rates and prognosis in oral cancer. The dynamics of the OSCC-related proteolysis was further explored via the proteomic profiling of saliva. This revealed that peptidase/endopeptidase inhibitors exhibited reduced levels in the saliva of pN+ patients, as confirmed by SRM-MS, whilst minor changes were detected in the level of saliva proteases. Taken together, our results indicated that proteolytic activity is accentuated in the saliva of OSCC patients with lymph node metastasis and, at least in part, this is modulated by reduced levels of salivary peptidase inhibitors. Therefore, this integrated pipeline provided better comprehension and discovery of molecular features with implications in the oral cancer metastasis prognosis.

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.

The Rhizobium-legume symbiosis is a beneficial interaction in which the bacterium converts atmospheric nitrogen into ammonia and delivers it to the plant in exchange for carbon compounds. This symbiosis implies the adaptation of bacteria to live inside host plant cells. In this work we apply RP-LC-MS/MS and  iTRAQ techniques to study the proteomic profile of endosymbiotic cells (bacteroids) induced by Rhizobium leguminosarum bv viciae strain UPM791 in legume nodules. Nitrogenase subunits, tricarboxylic acid cycle enzymes, and stress response proteins are amongst the most abundant from over one thousand rhizobial proteins identified in pea (Pisum sativum) bacteroids. Comparative analysis of bacteroids induced in pea and in lentil (Lens culinaris)nodules revealed the existence of a significant host-specific differential response affecting dozens of bacterial proteins, including stress-related proteins, transcriptional regulators, and proteins involved in the carbon and nitrogen metabolisms. A mutant affected in one of these proteins, homologous to a GntR-like transcriptional regulator, showed a symbiotic performance significantly  impaired in symbiosis with pea, but not with lentil plants. Analysis of the proteomes of bacteroids isolated from both hosts also revealed the presence of different sets of plant-derived nodule-specific cysteine rich (NCR) peptides, indicating that the endosymbiotic bacteria find a host-specific cocktail of chemical stressors inside the nodule. By studying variations of the bacterial response to different plant cell environments we will be able to identify specific limitations imposed by the host that might give us clues for the improvement of rhizobial performance.

The early detection of pancreatic ductal adenocarcinoma is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers CA19-9 and sTRA are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and non-cancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry approach was utilized to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of PDAC patients represented by tissue microarrays and whole tissue sections. Orthogonally, these same tissues were characterized by multi-round immunofluorescence which defined expression of CA19-9 and sTRA as well as other lectins towards carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated bi-antennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9 expressing tissues tended to be bi-, tri- and tetra-antennary structures with both core and terminal fucose residues and bisecting N-acetylglucosamines. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored tri- and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-IHC and IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes.

The molecular mechanism associated with mammalian meiosis has yet to be fully explored, and one of the main reasons for this lack of exploration is that some meiosis-essential genes are still unknown. The profiling of gene expression during spermatogenesis has been performed in previous studies, yet few studies have aimed to find new functional genes. Since there is a huge gap between the number of genes that are able to be quantified and the number of genes that can be characterized by phenotype screening in one assay, an efficient method to rank quantified genes according to phenotypic relevance is of great importance. We proposed to rank genes by the probability of their function in mammalian meiosis based on global protein abundance using machine learning. Here, nine types of germ cells focusing on continual substages of meiosis prophase I were isolated, and the corresponding proteomes were quantified by high-resolution mass spectrometry. By combining meiotic labels annotated from the MGI mouse knockout database and the spermatogenesis proteomics dataset, a supervised machine learning package, FuncProFinder, was developed to rank meiosis-essential candidates. Of the candidates whose functions were unannotated, four of ten genes with the top prediction scores, Zcwpw1, Tesmin, 1700102P08Rik and Kctd19, were validated as meiosis-essential genes by knockout mouse models. Therefore,  mammalian meiosis-essential genes could be efficiently predicted based on the protein abundance dataset, which provides a paradigm for other functional gene mining from a related abundance dataset.

Extracellular vesicle (EV) proteins from acute myeloid leukemia (AML) cell lines were analyzed using mass spectrometry. The analyses identified 2450 proteins, including 461 differentially expressed proteins (290 upregulated and 171 downregulated). CD53 and CD47 were upregulated and were selected as candidate biomarkers. The association between survival of patients with AML and the expression levels of CD53 and CD47 at diagnosis was analyzed using mRNA expression data from The Cancer Genome Atlas database. Patients with higher expression levels showed significantly inferior survival than those with lower expression levels. Enzyme-linked immunosorbent assay results of the expression levels of CD53 and CD47 from EVs in the bone marrow of patients with AML at diagnosis and at the time of complete remission with induction chemotherapy revealed that patients with downregulated CD53 and CD47 expression appeared to relapse less frequently. Network model analysis of EV proteins revealed several upregulated kinases, including LYN, CSNK2A1, SYK, CSK, and PTK2B. The potential cytotoxicity of several clinically applicable drugs that inhibit these kinases was tested in AML cell lines. The drugs lowered the viability of AML cells. The collective data suggest that AML-derived EVs could reflect essential leukemia biology.

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.

We have previously shown that multimers of plasma pentraxin-3 (PTX3) were predictive of survival in patients with sepsis. To characterize the release kinetics and cellular source of plasma protein changes in sepsis, serial samples were obtained from healthy volunteers (n=10, 3 time-points) injected with low-dose endotoxin (LPS) and analyzed using data-independent acquisition (DIA) MS. The human plasma proteome response was compared to an LPS-induced endotoxemia model in mice. Proteomic analysis of human plasma revealed a rapid neutrophil degranulation signature, followed by a rise in acute phase proteins. Changes in circulating PTX3 correlated with increases in neutrophil-derived proteins following LPS injection. Time course analysis of the plasma proteome in mice showed a time-dependent increase in multimeric PTX3, alongside increases in neutrophil-derived myeloperoxidase (MPO) upon LPS treatment. The mechanisms of oxidation-induced multimerisation of PTX3 were explored in two genetic mouse models: MPO global knock-out mice and LysM CreNox2KO mice, in which NADPH oxidase 2 (Nox2) is only deficient in myeloid cells. Nox2 is the enzyme responsible for the oxidative burst in neutrophils. Increases in plasma multimeric PTX3 were not significantly different between wildtype and MPO or LysM CreNox2KO knock-out mice. Thus, PTX3 may already be stored and released in a multimeric form. Through in vivo neutrophil depletion and multiplexed vascular proteomics, PTX3 multimer deposition within the aorta was confirmed to be neutrophil-dependent. Proteomic analysis of aortas from LPS-injected mice returned PTX3 as the most upregulated protein, where multimeric PTX3 was deposited as early as 2 h post-LPS along with other neutrophil-derived proteins. In conclusion, the rise in multimeric PTX3 upon LPS injection correlates with neutrophil-related protein changes in plasma and in aortas. MPO and myeloid Nox2 are not required for the multimerisation of PTX3; instead, neutrophil extravasation is responsible for the LPS-induced deposition of multimeric PTX3 in the aorta.

Gonadal soma-derived factor (gsdf) has been demonstrated to be essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag "pull-down" assay coupled with shotgun LC-MS/MS to identify a group of potential interacting partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1α), and actin filaments in mature medaka testis. As for the interaction with TGFβ-dynein being critical for spermatogonial division in Drosophila melanogaster, the physical interactions of Gsdf-dynein and Gsdf-eEF1α were identified through a yeast 2-hybrid (Y2H) screening of an adult testis cDNA library using Gsdf as bait, which were verified by a paired Y2H assay. Co-immunoprecipitation of Gsdf and eEF1α was defined in adult testes as supporting the requirement of a Gsdf and eEF1α interaction in testis development. Proteomics analysis (data are available via ProteomeXchange with identifier PXD022153) and ultrastrutural observations showed that Gsdf deficiency activated eEF1α-mediated protein synthesis and ribosomal biogenesis, which in turn led to the differentiation of undifferentiated germ cells. Thus, our results provide a framework and new insight into the coordination of a Gsdf (TGFβ and eEF1α complex in the basic processes of germ cell proliferation, transcriptional and translational control of sexual RNA which may be fundamentally conserved across phyla during sexual differentiation.

The goal of clinical proteomics is to identify, quantify, and characterize proteins in body fluids or tissue to assist diagnosis, prognosis, and treatment of patients. In this way, it is similar to more mature omics technologies, such as genomics, that are increasingly applied in biomedicine. We argue that, similar to those fields, proteomics also faces ethical issues related to the kinds of information that is inherently obtained through sample measurement, although their acquisition was not the primary purpose. Specifically, we demonstrate the potential to identify individuals both by their characteristic, individual-specific protein levels and by variant peptides reporting on coding single nucleotide polymorphisms. Furthermore, it is in the nature of blood plasma proteomics profiling that it broadly reports on the health status of an individual – beyond the disease under investigation. Finally, we show that private and potentially sensitive information, such as ethnicity and pregnancy status, can increasingly be derived from proteomics data. Although this is potentially valuable not only to the individual, but also for biomedical research, it raises ethical questions similar to the incidental findings obtained through other omics technologies. We here introduce the necessity of - and argue for the desirability for - ethical and human rights-related issues to be discussed within the proteomics community. Those thoughts are more fully developed in our accompanying manuscript. Appreciation and discussion of ethical aspects of proteomic research will allow for deeper, better-informed, more diverse, and, most importantly, wiser guidelines for clinical proteomics.

To identify novel autoantibodies of Takayasu arteritis (TAK) using HuProt array-based approach. A two-phase approach was adopted. In Phase I, serum samples collected from 40 TAK patients, 15 autoimmune disease patients, and 20 healthy subjects were screened to identify TAK-specific autoantibodies using human protein (HuProt) arrays. In Phase II, the identified candidate autoantibodies were validated with TAK-focused arrays using an additional cohort comprised of 109 TAK patients, 110 autoimmune disease patients, and 96 healthy subjects. Subsequently, the TAK-specific autoantibodies validated in Phase II were further confirmed using Western blot analysis. We identified and validated eight autoantibodies as potential TAK-specific diagnostic biomarkers, including anti-SPATA7, -QDPR, -SLC25A2, -PRH2, -DIXDC1, -IL17RB, -ZFAND4, and -NOLC1 antibodies, with AUC of 0.803, 0.801, 0.780, 0.696, 0.695, 0.678, 0.635 and 0.613, respectively. SPATA7 could distinguish TAK from healthy and disease controls with 73.4% sensitivity at 85.4% specificity, while QDPR showed 71.6% sensitivity at 86.4% specificity. SLC25A22 showed the highest sensitivity of 80.7%, but at lower specificity of 67.0%. In addition, PRH2, IL17RB and NOLC1 showed good specificities of 88.3%, 85.9% and 86.9%, respectively, but at lower sensitivities (<50%). Finally, DIXDC1 and ZFAND4 showed moderate performance as compared with the other autoantibodies. Using a decision tree model, we could reach a specificity of 94.2% with AUC of 0.843, a significantly improved performance as compared to that by each individual biomarker. The performance of three autoantibodies, namely anti-SPATA7, -QDPR and -PRH2, were successfully confirmed with Western blot analysis. Using this two-phase strategy, we identified and validated eight novel autoantibodies as TAK–specific biomarker candidates, three of which could be readily adopted in a clinical setting.

Data independent acquisition (DIA) is now an emerging method in bottom-up proteomics and capable of achieving deep proteome coverage and accurate label-free quantification. However, for post-translational modifications (PTM), such as glycosylation, DIA methodology is still in the early stage of development. The full characterization of glycoproteins requires site specific glycan identification as well as subsequent quantification of glycan structures at each site. The tremendous complexity of glycosylation represents a significant analytical challenge in glycoproteomics. This review focuses on the development and perspectives of DIA methodology for N- and O- glycoproteomics and posits that DIA-based glycoproteomics could be a method of choice to address some of the challenging aspects of glycoproteomics. First, the current challenges in glycoproteomics and the basic principles of DIA is briefly introduced. DIA based glycoproteomics is then summarized and described into four aspects based on the actual samples. Lastly, we discussed the important challenges and future perspectives in the field. We believe that DIA can significantly facilitate glycoproteomic studies and contribute to the development of future advanced tools and approaches in the field of glycoproteomics.

The F-box protein MORE AXILLARY GROWTH 2 (MAX2) is a central component in the signaling cascade of strigolactones (SLs) as well as of the smoke derived karrikins (KARs) and the so far unknown endogenous KAI2 ligand (KL). The two groups of molecules are involved in overlapping and unique developmental processes, and signal-specific outcomes are attributed to perception by the paralogous α/β-hydrolases DWARF14 (D14) for SL and KARRIKIN INSENSITIVE 2/ HYPOSENSITIVE TO LIGHT (KAI2/HTL) for KAR/KL. Additionally, depending on which receptor is activated, specific members of the SUPPRESSOR OF MAX2 1 (SMAX1) – LIKE (SMXL) family control KAR/KL and SL responses. As proteins that function in the same signal transduction pathway often occur in large protein complexes, we aimed at discovering new players of the MAX2, D14 and KAI2 protein network by tandem affinity purification using Arabidopsis cell cultures. When using MAX2 as a bait, various proteins were co-purified among which general components of the Skp1-Cullin-F-box complex and members of the CONSTITUTIVE PHOTOMORPHOGENIC 9 signalosome. Here, we report the identification of a novel interactor of MAX2, a type 5 serine/threonine protein phosphatase, designated PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5 (PAPP5). Quantitative affinity purification pointed at PAPP5 as being more present in KAI2 rather than D14 protein complexes. In agreement, mutant analysis suggests that PAPP5 modulates KAR/KL-dependent seed germination in suboptimal conditions and seedling development. Additionally, a phosphopeptide enrichment experiment revealed that PAPP5 might dephosphorylate MAX2 in vivo independently of the synthetic strigolactone analog, rac-GR24. Together, by analyzing the protein complexes to which MAX2, D14 and KAI2 belong, we revealed a new MAX2 interactor, PAPP5, that might act through dephosphorylation of MAX2 to control mainly KAR/KL- related phenotypes and, hence, provide another link with the light pathway.

High performance liquid chromatography has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely useful for profiling the structural diversity of glycoprotein and glycolipid glycosylation but require the availability of glycan standards and secondary orthogonal degradation strategies to validate structural assignments. A robust method for HPLC separation of glycans as their permethylated derivatives, coupled with in-line MSn fragmentation to assign structural features independent of standards, would significantly enhance the depth of knowledge obtainable from biological samples. Here, we report an optimized workflow for LC-MS analysis of permethylated glycans that includes sample preparation, mobile phase optimization, and MSn method development to resolve structural isomers on-the-fly. We report baseline separation and MSn fragmentation of isomeric N- and O-glycan structures, aided by supplementing mobile phases with Li+, which simplifies adduct heterogeneity and facilitates cross-ring fragmentation to obtain valuable monosaccharide linkage information. Our workflow has been adapted from standard proteomics-based workflows and, therefore, provides opportunities for laboratories with expertise in proteomics to acquire glycomic data with minimal deviation from existing buffer systems, chromatography media, and instrument configurations. Furthermore, our workflow does not require a mass spectrometer with high-resolution/accurate mass capabilities. The rapidly evolving appreciation of the biological significance of glycans for human health and disease requires the implementation of high-throughput methods to identify and quantify glycans harvested from sample sets of sufficient size to achieve appropriately powered statistical significance. The LC-MSn approach we report generates glycan isomeric separations, robust structural characterization, and is amenable to auto-sampling with associated throughput enhancements.

Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases and some changes appear to be disease-specific, thus it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers and enable better understanding of AGP’s role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 μl of bloodplasma in a 96 well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography based solid-phase extraction and analyzed by RP-LC-ESI-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in 3 time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP’s second glycosylation site and lower sialylation of N-glycans on AGP’s third and AGP1’s fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.

Giardia lamblia (G. lamblia) disease is a zoonosis with a-infection rate affecting the general population of the world. Despite the constant possibility of damage due to their own metabolism, G. lamblia have survived and evolved to adapt to various environments. However, research on energy-metabolism conversion in G. lamblia is limited. This study aimed to reveal the dynamic metabolism-conversion mechanism in G. lamblia under sugar starvation by detecting global lysine acetylation and 2-hydroxyisobutyrylation sites combined with quantitative proteome analyses. A total of 2999 acetylation sites on 956 proteins and 8877 2-hydroxyisobutyryl sites on 1546 proteins were quantified under sugar starvation. Integrated Kac and Khib data revealed that modified proteins were associated with arginine biosynthesis, glycolysis/gluconeogenesis, and alanine, aspartate, and glutamate metabolism. These findings suggested that lysine acetylation and 2-hydroxyisobutyrylation were ubiquitous and provided deep insight into the metabolism-conversion mechanism in G. lamblia under sugar starvation. Overall, these results can help understand the biology of G. lamblia infections and reveal the evolution rule from prokaryote to eukaryote.

Exploring Transatlantic Responses to Far-right Populism in Europe: Simulation Exercise Research paper sysadmin 1 May 2018

A new paper summarizes the findings of a recent simulation exercise exploring how governments on both sides of the Atlantic might respond to a descent towards populist authoritarianism in an EU member state.

Summary

• To better understand how governments on both sides of the Atlantic might respond to a descent towards populist authoritarianism in an EU member state, Chatham House organized a simulation event involving a group of experts drawn from the public sector, academia and NGOs.
• Simulation exercises enable the testing and modelling of the responses of different actors when presented with specific situations; participants’ interactions in a given set of circumstances are explored, and patterns of negotiation are captured and analysed.
• In this simulation, European, US and multilateral representatives were given the task of managing relations with Baltia, a fictional Eastern European state on the verge of electing a far-right nationalist, Eurosceptic government. They were then challenged to manage their relationship with Baltia after it had elected such a government, which was pushing for a ‘leave’ vote in a planned referendum on the country’s continued EU membership.
• The simulation highlighted a number of issues:
  • Limited instruments are available to liberal democratic governments where there is cause for concern regarding the outcome of an election in an allied country. There are relatively few tools at the disposal of governments to support political allies, or to prevent outcomes that are perceived as threatening democratic norms. The simulation reinforced the view that interventionist moves, either from the European Commission or from individual national governments, would be more likely to come in response to an unfavourable development rather than pre-emptively.
  • The EU, and caucuses of European states, are the main international interlocutors in this type of political crisis involving an EU member state. The US opted to play a limited role in the negotiations; the same was largely true for NATO, aside from its action in sharing intelligence about a potential coup in Baltia. France and Germany formed a natural working partnership, taking meetings together and coordinating policies first before discussing them with a wider European circle, although their positions did not always align.
  • The UK’s capacity to shape the outcome of collective EU discussions appeared more restricted, while Brexit also seemed to shape the response of other EU states to the developing situation in Baltia. Although member states were undoubtedly reluctant to see another country go down this route, they were also resolute in demonstrating a unity of approach and limited flexibility in the face of the new populist government’s attempt to divide them.

New Frontiers in Gender-responsive Governance: Five Years of the W20 Research paper sysadmin 2 November 2018

After five years of the W20, women and gender equality remain at the margin of the G20. There is a real risk of the W20 representing a one-off territorial gain at a frontier that could easily be pushed back again.

Summary

• 2018 marks the fifth anniversary of the first grouping of the W20, the engagement group of the G20 that focuses on gender-inclusive economic growth and advocates for gender equality across the G20 agenda. Formally launched under the Turkish G20 presidency in 2015, the W20 is made up of women from business, international organizations, civil society, think-tanks and academia across the G20 member states.
• This paper takes stock of the critical steps in the development of the W20 over the last five years, examining its background, rationale and foundations, and identifying the areas of economic governance where it has so far contributed the most – and those where more action is needed. The W20 has filled a gap, it but needs to carefully assess its coherence with the UN agencies, the private sector, the G7 and other G20 engagement groups.
• The establishment of the W20 has contributed to defining new frontiers for economic governance and shifting the traditional approach from gender-neutral to gender-responsive. Whereas in 2013 gender in the G20 was considered a marginal issue better dealt with by ministers for equal opportunities, now gender equality and women’s economic empowerment are part of the mainstream economic dialogue. The next step is to ensure more structural and monitored policy reforms at the G20 level.
• Already, the W20 can count among its achievements the ‘25 by 25’ female labour force participation commitment adopted at the G20’s Brisbane summit in 2014, and the Women Entrepreneurs Finance Initiative (We-Fi) and Business Women Leaders’ Taskforce, both agreed at the Hamburg summit in 2017.
• The W20 is constrained in its policy impact by limited engagement with the finance track and a lack of consistent resourcing levels. Addressing these issues would strengthen its role as a credible player in shifting global economic governance while contributing to good gender-responsive domestic policies.
• Progress on gender equality has been too slow and too peripheral to drive change in the relatively short term – over one generation, for example. G20 governments must therefore embrace active, credible policies to bring more women into the labour market, improve access to education and finance, close the pay gap, invest in social infrastructure – especially childcare and assistance for the elderly – and support female entrepreneurs. These domestic policies need to be internationally coordinated so that action and benefits can be widespread.
• A feminist, inclusive agenda at the G20 level should highlight the current empirical evidence of women’s exclusion from the benefit of their economic activity, both in G20 members and beyond. The W20 should also focus on efforts to remedy the lack of women’s representation in G20 processes and in economic governance as a whole.

Proportionality in the Conduct of Hostilities: The Incidental Harm Side of the Assessment Research paper sysadmin 6 December 2018

Clarification of international humanitarian law is important in ensuring compliance with the rule of proportionality, but a culture of compliance within armed forces and groups is also crucial.

Summary

• Military operations are taking place with increasing frequency in densely populated areas. Such operations result in loss of life and harm to civilians, as well as damage to civilian objects, (including infrastructure providing essential services). In order to protect civilians, it is imperative that armed forces and groups comply with the rules of international humanitarian law on the conduct of hostilities, including the rule of proportionality.
• The rule of proportionality prohibits attacks which may be expected to cause incidental loss of civilian life, injury to civilians, damage to civilian objects or a combination thereof, which would be excessive in relation to the concrete and direct military advantage anticipated. This research paper analyses the key steps that belligerents must take to give effect to the rule, with a particular focus on one side of proportionality assessments – the expected incidental harm.
• Those undertaking proportionality assessments before or during an attack must consider whether the expected harm will be caused by the attack, and whether that harm could be expected (that is, was it reasonably foreseeable).
• For the purpose of proportionality assessments, injury to civilians includes disease, and there is no reason in principle to exclude mental harm, even though it is currently challenging to identify and quantify it. Damage to civilian objects includes damage to elements of the natural environment.
• Once the incidental harm to be considered has been identified, a value or weight must be assigned to it. This is then balanced against the value or weight of the military advantage anticipated from the attack to determine whether the harm would be excessive.
• In the determination of whether the expected incidental harm would be excessive compared to the anticipated military advantage, ‘excessive’ is a wide but not indeterminate standard.
• Belligerents should develop methodologies so that those planning and deciding attacks are provided with all necessary information on expected incidental harm, and to assist them in assigning weight to the incidental harm to be considered.
• If it becomes apparent that the rule of proportionality will be contravened, the attack in question must be cancelled or suspended.
• Clarification of the law is important in ensuring compliance with the rule of proportionality, but a culture of compliance within armed forces and groups, inculcated by their leaders, is also crucial.

Moscow Rules: What Drives Russia to Confront the West Book sysadmin 17 January 2019

Keir Giles surveys Russia’s history and the present day to explain why its current leadership feels it has no choice but to challenge and attack the West. Recognising and accepting that this will not change in the near future will help the West find a way of dealing with Russia without risking a deeper conflict.

This book is for anyone that cannot understand why Russia and its leaders behave as they do.

The relationship between Russia and the West is once again deep in crisis. A major reason is that Western leaders have too often believed or hoped that Russia sees the world as they do — but things look very different from Moscow. This book shows that efforts at engagement with Russia that do not take this into account are a key reason for repeated disappointment and crisis.

In confronting the West, Russia is implementing strategic and doctrinal approaches that have been consistent for centuries. The roots of current Russian behaviour and demands can be traced not just to the Soviet era, but back into Tsarist foreign and domestic policy, and further to the structure and rules of Russian society. But this also gives the US and the West pointers for how to behave — and how not to — in order to manage the challenge of Russia effectively, based on past experience of both successful and unsuccessful engagement with Moscow.

The book recognizes the reality of confrontation and provides an essential introduction to grasping why Russia sees it as inevitable. Consequently, it offers a basis for building a less crisis-prone relationship with Russia.

This book is part of the Insights series.

Praise for Moscow Rules

About the author

Keir Giles is a senior consulting fellow at Chatham House, the Royal Institute of International Affairs. He also works with the Conflict Studies Research Centre (CSRC), a group of subject matter experts in Eurasian security with a particular focus on the wide range of security challenges coming from Russia.

Purchase

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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.

Online Disinformation and Political Discourse: Applying a Human Rights Framework Research paper sysadmin 5 November 2019

Although some digital platforms now have an impact on more people’s lives than does any one state authority, the international community has been slow to hold to account these platforms’ activities by reference to human rights law.

This paper examines how human rights frameworks should guide digital technology.

Summary

• Online political campaigning techniques are distorting our democratic political processes. These techniques include the creation of disinformation and divisive content; exploiting digital platforms’ algorithms, and using bots, cyborgs and fake accounts to distribute this content; maximizing influence through harnessing emotional responses such as anger and disgust; and micro-targeting on the basis of collated personal data and sophisticated psychological profiling techniques. Some state authorities distort political debate by restricting, filtering, shutting down or censoring online networks.
• Such techniques have outpaced regulatory initiatives and, save in egregious cases such as shutdown of networks, there is no international consensus on how they should be tackled. Digital platforms, driven by their commercial impetus to encourage users to spend as long as possible on them and to attract advertisers, may provide an environment conducive to manipulative techniques.
• International human rights law, with its careful calibrations designed to protect individuals from abuse of power by authority, provides a normative framework that should underpin responses to online disinformation and distortion of political debate. Contrary to popular view, it does not entail that there should be no control of the online environment; rather, controls should balance the interests at stake appropriately.
• The rights to freedom of thought and opinion are critical to delimiting the appropriate boundary between legitimate influence and illegitimate manipulation. When digital platforms exploit decision-making biases in prioritizing bad news and divisive, emotion-arousing information, they may be breaching these rights. States and digital platforms should consider structural changes to digital platforms to ensure that methods of online political discourse respect personal agency and prevent the use of sophisticated manipulative techniques.
• The right to privacy includes a right to choose not to divulge your personal information, and a right to opt out of trading in and profiling on the basis of your personal data. Current practices in collecting, trading and using extensive personal data to ‘micro-target’ voters without their knowledge are not consistent with this right. Significant changes are needed.
• Data protection laws should be implemented robustly, and should not legitimate extensive harvesting of personal data on the basis of either notional ‘consent’ or the data handler’s commercial interests. The right to privacy should be embedded in technological design (such as by allowing the user to access all information held on them at the click of a button); and political parties should be transparent in their collection and use of personal data, and in their targeting of messages. Arguably, the value of personal data should be shared with the individuals from whom it derives.
• The rules on the boundaries of permissible content online should be set by states, and should be consistent with the right to freedom of expression. Digital platforms have had to rapidly develop policies on retention or removal of content, but those policies do not necessarily reflect the right to freedom of expression, and platforms are currently not well placed to take account of the public interest. Platforms should be far more transparent in their content regulation policies and decision-making, and should develop frameworks enabling efficient, fair, consistent internal complaints and content monitoring processes. Expertise on international human rights law should be integral to their systems.
• The right to participate in public affairs and to vote includes the right to engage in public debate. States and digital platforms should ensure an environment in which all can participate in debate online and are not discouraged from standing for election, from participating or from voting by online threats or abuse.

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.

Belarusians’ views on the political crisis - April 2021 Other resource NCapeling 11 June 2021

Results of a public opinion poll conducted between 20 and 30 April 2021.

Chatham House surveyed a total of 937 respondents between 20 and 30 April 2021. Our survey sample corresponds to the general structure of Belarus’s urban population and is corrected and weighted by gender, age, size of respondents’ town of residence and education level.

Summary

• After Lukashenka, the most known political figures in Belarus are pro-protest politicians: Tsikhanouskaya, Babaryka, Tsikhanouski, Kalesnikava, Tsapkala and Latushka. Viktar Babaryka is the most popular presidential candidate among Belarusian urban citizens, and Lukashenka comes second to him.
• State organizations and bodies are still mistrusted by more than half the population, while the army is slightly more popular than other bodies. Independent media, labour unions and human rights organizations are trusted far more than state ones.
• Russia leads when it comes to positive attitudes to foreign states: one in three Belarusians feel very positive about their neighbour and 79 per cent feel positive in general. 71 per cent Belarusians feel positive about China, which is the next highest indicator. Despite propaganda efforts, Ukraine, Lithuania, Poland and EU states in general are still perceived positively by more than 60 per cent of Belarusians, with fewer than 20 per cent feeling negative about them.
• The position on Russian interference in the Belarusian political crisis is consolidated enough: 58 per cent think that Russia should remain neutral. The rest are almost equally divided between those who think Russia should support the protest movement (19 per cent) and those who think Russia should support Lukashenka (23 per cent).
• 40 per cent have changed their attitude to the Russian government since it supported Lukashenka in the crisis, and 73 per cent are sure that Russia is his sole support.
• While 32 per cent of Belarusians see union with Russia as the most appropriate foreign policy, 46 per cent would like to be in a union with both Russia and the EU simultaneously.
• Almost half the population thinks that Belarusian foreign policy in the event of a pro-protest government coming to power would be oriented toward friendship and cooperation with both Russia and the West, which correlates with how the majority thinks it should be. Although 42 per cent expect foreign policy would lean toward the EU in that situation, which is less acceptable: only 25 per cent think it ‘should’.
• The proportion of respondents who think Belarus should remain in the Collective Security Treaty Organization is 58 per cent, while 35 per cent would like to see Belarus not belonging to any military bloc and a small share (seven per cent) would prefer Belarus to join NATO.
• Almost half of the population considers Lukashenka to be the major obstacle for political stability and economic development and is sure he does not care about, and in fact is a threat to, independence.
• In general, most Belarusians have a consolidated opinion that the authorities should start negotiating with opponents and free all political prisoners.
• More than half of Belarusians think that Lukashenka must leave immediately or before the end of 2021, while 27 per cent think he should leave when his term ends in 2025. The opinion that Lukashenka should rule for another term beginning in 2025 is marginal – only ten per cent support this.
• Expectations are not so optimistic though: only 26 per cent believe he will leave office this year. One in three thinks he will leave in 2025; a further 19 per cent say that he will stay on for further terms in office.
• If Lukashenka doesn’t leave, people expect an increase in emigration and unemployment, the continuation of repression in all areas of life, falling exchange rates and the tightening of dependency on Russia.
• Opinion on abortions is still consolidated, with only 19 per cent thinking they should be banned in most or all cases. The same applies to Lukashenka’s infamous statement that society in Belarus is not ready to have a female president: only 20 per cent agree with the opinion.
• 52 per cent don’t think there is a problem of wage inequality between men and women in Belarus, while 37 per cent do see an issue.

Download a presentation of the survey’s main findings

Diagnosing social behavioural dynamics of corruption Other resource dora.popova 8 December 2021

This interactive toolkit identifies the types of social expectations which sustain selected corrupt practices and provides behaviourally-informed recommendations for changing them.

When tackling a problem as complex as corruption, it is important to understand why and how people behave in different situations where corruption occurs. In contexts where it is easier to engage in corruption than avoid it, identifying the social expectations and informal rules which sustain corrupt practices can advance corruption prevention and deepen collective action.  

Behavioural approaches to corruption offer a better understanding of diverse social settings, group dynamics, power distribution, social motivations, and expectations that contribute to a more tolerant environment for certain forms of the phenomenon. They are also highly complementary to traditional corruption measures, which tend to focus on the enforcement of legal sanctions and deterrents. Behavioural approaches, especially those inspired by social norms theory, highlight complex social characteristics and informal rules of specific corrupt practices, and effectively support implementation of more dynamic context-specific anti-corruption interventions.

Since 2016, the Chatham House Africa programme’s Social Norms and Accountable Governance (SNAG) project has adopted a behavioural approach based on social norms methodology to investigate the social beliefs which motivate different forms of corruption. Drawing on the project’s extensive evidence-gathering and analysis, this toolkit offers users navigable behavioural mapping of contextual factors, beliefs, and expectations surrounding common corrupt practices. 

It aims to support anti-corruption actors in diagnosing informal rules and social expectations which sustain corruption in some societies. It also proposes behavioural-informed guidance for developing or adapting anti-corruption interventions and activities, so they account for informal rules of behaviour such as social norms. 
 
The toolkit supports users to:

• Identify whether and how widespread corrupt practices are motivated by social beliefs and expectations.

• Understand how society influences the types of corrupt activity individuals engage in, or avoid, and the factors informing these choices

• Integrate empirical evidence and behavioural insights into anti-corruption strategies from diagnostics to design, and eventual implementation and evaluation

The toolkit presents evidence from SNAG’s research into three key corrupt practices – bribery, embezzlement, and electoral fraud. Each was examined in the context of typical situations in which they occur, such as law enforcement, healthcare, the power sector, voting, and education while critical factors such as religion, gender, and ethnicity were considered. 

The toolkit presents an overview of specific contexts and behavioural features of the practices and provides behavioural-informed recommendations. It also contains pop-up features with definitions and explanations of key concepts. The toolkit is designed to be expandable, allowing further content and behavioural dynamics to be added.   

Belarusians’ views on the political crisis - Nov 2021 Other resource NCapeling 20 January 2022

Results of a public opinion poll conducted between 1 and 10 November 2021.

Chatham House surveyed a total of 767 respondents between 1 and 10 November 2021. Our survey sample corresponds to the general structure of Belarus’s urban population and is adjusted by gender, age, education level and the size of respondents’ place of residence.

Summary

• After Aliaksandr Lukashenka, the most well-known political figures in Belarus are the protest leaders: Sviatlana Tsikhanouskaya, Viktar Babaryka, Siarhei Tsikhanouski, Valery Tsapkala, Marya Kalesnikava and Pavel Latushka.
• Lukashenka and Babaryka have the highest popularity ratings among Belarus’s urban population. At the same time, Lukashenka has a high negative rating: half of the city residents surveyed say that they would never vote for him. Babaryka has one of the lowest negative ratings in the country.
• The popularity ratings of pro-government politicians are low, and their negative ratings remain extremely high, although the latter have decreased slightly in comparison with July 2021. The popularity ratings of protest leaders are significantly higher than those of politicians who support the current government.
• Trust in state and independent organizations is polarized along political lines. Thus, respondents classed as hardcore protesters do not trust state structures and do trust independent ones, while Lukashenka’s supporters hold the opposite positions.
• Belarusians’ geopolitical orientations remain stable. The most widely preferred option for a geopolitical union is simultaneous integration with the EU and Russia.
• Belarusians have become increasingly negative about the possibility that Russian airbases could be established on Belarusian territory (48 per cent in November 2021, compared to 39 per cent in July 2021).
• Belarusians are now feeling more strongly that the situation concerning the COVID-19 pandemic has worsened: in November, more than half were beginning to respond that Belarus is unable to cope with the spread of coronavirus.
• Sanctions against Lukashenka and those around him are supported more widely than sanctions against state enterprises.
• Belarusians feel there is acute social tension in their country: almost everyone surveyed states that social tension exists, while one in every five respondents considers the situation catastrophic. A perception that there is social tension is closely related to many factors, with the strongest of all being a sense of a lack of personal safety.
• Only one-third of Belarusians are prepared to call the state built under Lukashenka their own. Most of the survey respondents do not trust this state to some degree, and do not believe that the state protects the interests of Belarusian citizens.

Download a presentation of the survey’s main findings

• Belarusians’ views on the political crisis (PDF)

• Взгляды белорусов на политический кризис (PDF)

In nucleotide excision repair, bulky DNA lesions such as UV-induced cyclobutane pyrimidine dimers are removed from the genome by concerted dual incisions bracketing the lesion, followed by gap filling and ligation. So far, two dual-incision patterns have been discovered: the prokaryotic type, which removes the damage in 11–13-nucleotide-long oligomers, and the eukaryotic type, which removes the damage in 24–32-nucleotide-long oligomers. However, a recent study reported that the UvrC protein of Mycobacterium tuberculosis removes damage in a manner analogous to yeast and humans in a 25-mer oligonucleotide arising from incisions at 15 nt from the 3´ end and 9 nt from the 5´ end flanking the damage. To test this model, we used the in vivo excision assay and the excision repair sequencing genome-wide repair mapping method developed in our laboratory to determine the repair pattern and genome-wide repair map of Mycobacterium smegmatis. We find that M. smegmatis, which possesses homologs of the Escherichia coli uvrA, uvrB, and uvrC genes, removes cyclobutane pyrimidine dimers from the genome in a manner identical to the prokaryotic pattern by incising 7 nt 5´ and 3 or 4 nt 3´ to the photoproduct, and performs transcription-coupled repair in a manner similar to E. coli.

In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3'→5' exonuclease activity of the hPolε holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3'→5' exonuclease activity of the hPolε holoenzyme. Together, the 3'→5' exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPolε holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPolε holoenzyme an efficient and faithful replicative DNA polymerase.

Fans and analysts spend the entire offseason speculating where the top free agents could go, but sometimes an under-the-radar pickup can end up making a world of difference. As positional competitions begin to heat up at Spring Training camps this month, MLB.com's beat writers were asked to identify one potentially overlooked acquisition for each of the 30 clubs. Here's who they came up with.

Jerad Eickhoff took his latest first step on Sunday morning at Spectrum Field. He threw a bullpen session, his first since sustaining a setback last month following carpal tunnel surgery in October.

The Phillies have been the favorites in the Bryce Harper sweepstakes for months, and that speculation heated up over the weekend.

Will Rhys Hoskins be bummed if Bryce Harper signs elsewhere?

MLB.com looked at each team's two highest-ranked position players in WAR, according to the Steamer projections. Here are the top 10, but keep in mind that things could change once Manny Machado and Bryce Harper find homes.