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.

Co-fractionation MS (CF-MS) is a technique with potential to characterize endogenous and unmanipulated protein complexes on an unprecedented scale. However this potential has been offset by a lack of guidelines for best-practice CF-MS data collection and analysis. To obtain such guidelines, this study thoroughly evaluates novel and published Saccharomyces cerevisiae CF-MS data sets using very high proteome coverage libraries of yeast gold standard complexes. A new method for identifying gold standard complexes in CF-MS data, Reference Complex Profiling, and the Extending 'Guilt-by-Association' by Degree (EGAD) R package are used for these evaluations, which are verified with concurrent analyses of published human data. By evaluating data collection designs, which involve fractionation of cell lysates, it is found that near-maximum recall of complexes can be achieved with fewer samples than published studies. Distributing sample collection across orthogonal fractionation methods, rather than a single high resolution data set, leads to particularly efficient recall. By evaluating 17 different similarity scoring metrics, which are central to CF-MS data analysis, it is found that two metrics rarely used in past CF-MS studies – Spearman and Kendall correlations – and the recently introduced Co-apex metric frequently maximize recall, whereas a popular metric—Euclidean distance—delivers poor recall. The common practice of integrating external genomic data into CF-MS data analysis is also evaluated, revealing that this practice may improve the precision and recall of known complexes but is generally unsuitable for predicting novel complexes in model organisms. If studying nonmodel organisms using orthologous genomic data, it is found that particular subsets of fractionation profiles (e.g. the lowest abundance quartile) should be excluded to minimize false discovery. These assessments are summarized in a series of universally applicable guidelines for precise, sensitive and efficient CF-MS studies of known complexes, and effective predictions of novel complexes for orthogonal experimental validation.

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem MS coupled with liquid chromatography (LC–MS/MS), we found 142 nonreductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2), a TCA cycle enzyme, was the only protein to show an increase in Sia content during capacitation. Interestingly, although the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A₁ activity, a decrease in the activity of ACO2 (i.e. stereospecific isomerization of citrate to isocitrate) occurred when sialylation increased (N612). The latter was confirmed by N612D recombinant protein tagged with both His and GFP. The replacement of Sia for the negatively charged Aspartic acid in the N612D mutant caused complete loss of aconitase activity compared with the WT. Computer modeling show that N612 sits atop the catalytic site of ACO2. The introduction of Sia causes a large conformational change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

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.

Coronavirus disease 2019 (COVID-19) is a highly contagious infection and threating the human lives in the world. The elevation of cytokines in blood is crucial to induce cytokine storm and immunosuppression in the transition of severity in COVID-19 patients. However, the comprehensive changes of serum proteins in COVID-19 patients throughout the SARS-CoV-2 infection is unknown. In this work, we developed a high-density antibody microarray and performed an in-depth proteomics analysis of serum samples collected from early COVID-19 (n = 15) and influenza (n = 13) patients. We identified a large set of differentially expressed proteins (n = 132) that participate in a landscape of inflammation and immune signaling related to the SARS-CoV-2 infection. Furthermore, the significant correlations of neutrophil and lymphocyte with the CCL2 and CXCL10 mediated cytokine signaling pathways was identified. These information are valuable for the understanding of COVID-19 pathogenesis, identification of biomarkers and development of the optimal anti-inflammation therapy.

MS-based proteome profiling has become increasingly comprehensive and quantitative, yet a persistent shortcoming has been the relatively large samples required to achieve an in-depth measurement. Such bulk samples, typically comprising thousands of cells or more, provide a population average and obscure important cellular heterogeneity. Single-cell proteomics capabilities have the potential to transform biomedical research and enable understanding of biological systems with a new level of granularity. Recent advances in sample processing, separations and MS instrumentation now make it possible to quantify >1000 proteins from individual mammalian cells, a level of coverage that required an input of thousands of cells just a few years ago. This review discusses important factors and parameters that should be optimized across the workflow for single-cell and other low-input measurements. It also highlights recent developments that have advanced the field and opportunities for further development.

Despite the crucial function of the small intestine in nutrient uptake our understanding of the molecular events underlying the digestive function is still rudimentary. Recent studies demonstrated that enterocytes do not direct the entire dietary triacylglycerol toward immediate chylomicron synthesis. Especially after high-fat challenges, parts of the resynthesized triacylglycerol are packaged into cytosolic lipid droplets for transient storage in the endothelial layer of the small intestine. The reason for this temporary storage of triacylglycerol is not completely understood. To utilize lipids from cytosolic lipid droplets for chylomicron synthesis in the endoplasmic reticulum, stored triacylglycerol has to be hydrolyzed either by cytosolic lipolysis or lipophagy. Interestingly, triacylglycerol storage and chylomicron secretion rates are unevenly distributed along the small intestine, with the proximal jejunum exhibiting the highest intermittent storage capacity. We hypothesize that correlating hydrolytic enzyme activities with the reported distribution of triacylglycerol storage and chylomicron secretion in different sections of the small intestine is a promising strategy to determine key enzymes in triacylglycerol remobilization. We employed a serine hydrolase specific activity-based labeling approach in combination with quantitative proteomics to identify and rank hydrolases based on their relative activity in 11 sections of the small intestine. Moreover, we identified several clusters of enzymes showing similar activity distribution along the small intestine. Merging our activity-based results with substrate specificity and subcellular localization known from previous studies, carboxylesterase 2e and arylacetamide deacetylase emerge as promising candidates for triacylglycerol mobilization from cytosolic lipid droplets in enterocytes.

Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer to spermatozoa key proteins that are essential in promoting motility and subsequent fertilization success. Using the domestic cat model, the objectives were to (1) characterize and compare protein content of EVs between segments of the epididymis, and (2) compare EV protein compositions between normo- and teratospermic individuals (producing >60% of abnormal spermatozoa). Epididymal EVs from adult cats were isolated and assessed via liquid chromatography tandem MS. Both male types shared 3008 proteins in total, with 98 and 20 EV proteins unique to normospermic and teratospermic males, respectively. Expression levels of several proteins changed between epididymal segments in both male types. Several proteins in both groups were related to sperm motility (e.g. hexokinase 1, adenylate kinase isoenzyme) and zona pellucida or oolemma binding (e.g. disintegrin and metalloproteinase domain proteins, zona binding proteins 1 and 2). Interestingly, seven cauda-derived EV proteins trended downward in teratospermic compared with normospermic males, which may relate to poor sperm quality. Collective results revealed, for the first time, EV proteins related to sequential sperm maturation with differences observed between normospermic and teratospermic individuals.

Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-B activation. However, the precise mechanism that links protein aggregation to NF-B-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IBα-loss with consequent NF-B activation. Four known mechanisms of IBα-loss i.e. the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IBα-loss was due to its sequestration along with IBβ into insoluble aggregates, thereby releasing NF-B. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-B subunit p65, which stably interacts with IBα under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IBα under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IBα after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IBα-nuclear import. The concurrent aggregation of IBα, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IBα and its consequent binding and termination of NF-B activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.

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.

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Cover art by Julie Newdoll for MCP April issue.

After successful completion of the Human Genome Project (HGP), HUPO has recently officially launched a global Human Proteome Project (HPP) which is designed to map the entire human protein set. Given the presence of about 30% undisclosed proteins out of 20,300 protein gene products, a systematic global effort is necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP groups employ the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge base. The HPP participants will take advantage of the output and cross-analyses from the ongoing HUPO initiatives and a chromosome-based protein mapping strategy, termed C-HPP with many national teams currently engaged. In addition, numerous biologically-driven projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents and tools for protein studies and analyses, and a stronger basis for personalized medicine. HUPO urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

This article provides an introduction to Fourier transform-based mass spectrometry (FTMS). The key performance characteristics of FTMS, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of FTMS technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for his/her application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook.

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The histidine kinase Hik33 plays important roles in mediating cyanobacterial response to divergent types of abiotic stresses including cold, salt, high light (HL), and osmotic stresses. However, how these functions are regulated by Hik33 remains to be addressed. Using a hik33-deficient strain (hik33) of Synechocystis sp. PCC 6803 (Synechocystis) and quantitative proteomics, we found that Hik33 depletion induces differential protein expression highly similar to that induced by divergent types of stresses. This typically includes downregulation of proteins in photosynthesis and carbon assimilation that are necessary for cell propagation, and upregulation of heat shock proteins, chaperons, and proteases that are important for cell survival. This observation indicates that depletion of Hik33 alone mimics divergent types of abiotic stresses, and that Hik33 could be important for preventing abnormal stress response in the normal condition. Moreover, we found the majority of proteins of plasmid origin were significantly upregulated in hik33, though their biological significance remains to be addressed. Together, the systematically characterized Hik33-regulated cyanobacterial proteome, which is largely involved in stress responses, builds the molecular basis for Hik33 as a general regulator of stress responses.

Growing implications of glycosylation in physiological occurrences and human disease have prompted intensive focus on revealing glycomic perturbations through absolute and relative quantification. Empowered by seminal methodologies and increasing capacity for detection, identification, and characterization, the past decade has provided a significant increase in the number of suitable strategies for glycan and glycopeptide quantification. Mass spectrometry-based strategies for glycomic quantitation have grown to include metabolic incorporation of stable isotopes, deposition of mass difference and mass defect isotopic labels, and isobaric chemical labeling, providing researchers with ample tools for accurate and robust quantitation. Beyond this, workflows have been designed to harness instrument capability for label-free quantification and numerous software packages have been developed to facilitate reliable spectrum scoring. In this review, we present and highlight the most recent advances in chemical labeling and associated techniques for glycan and glycopeptide quantification.

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.

Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as MS instrumentation and software improve, so this review aims to help equip researchers with necessary information to choose appropriate enrichment strategies that best complement these efforts.

Glycosylation is a highly diverse set of co- and post-translational modification of proteins. For mammalian glycoproteins, glycosylation is often site-, tissue- and species-specific, and diversified by microheterogeneity. Multitudinous biochemical, cellular, physiological and organismic effects of their glycans have been revealed, either intrinsic to the carrier proteins or mediated by endogenous reader proteins with carbohydrate recognition domains. Furthermore, glycans frequently form the first line of access by or defense from foreign invaders, and new roles for nucleocytoplasmic glycosylation are blossoming. We now know enough to conclude that the same general principles apply in invertebrate animals and unicellular eukaryotes – different branches of which spawned the plants or fungi and animals. The two major driving forces for exploring the glycomes of invertebrates and protists are (i) to understand the biochemical basis of glycan-driven biology in these organisms, especially of pathogens, and (ii) to uncover the evolutionary relationships between glycans, their biosynthetic enzyme genes, and biological functions for new glycobiological insights. With an emphasis on emerging areas of protist glycobiology, here we offer an overview of glycan diversity and evolution, to promote future access to this treasure trove of glycobiological processes.

Regulation of gene expression is essential for the functioning of all eukaryotic organisms. Understanding gene expression regulation requires determining which proteins interact with regulatory elements in chromatin. Mass spectrometry-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation, as it allows studying protein function and protein complex formation in their in vivo chromatin-bound context. Total chromatin isolated from cells can be directly analysed using mass spectrometry or further fractionated into transcriptionally active and inactive chromatin prior to MS-based analysis. Newly formed chromatin that is assembled during DNA replication can also be specifically isolated and analysed. Furthermore, capturing specific chromatin domains facilitates the identification of previously unknown transcription factors interacting with these domains. Finally, in recent years, advances have been made towards identifying proteins that interact with a single genomic locus of interest. In this review, we highlight the power of chromatin proteomics approaches and how these provide complementary alternatives compared to conventional affinity purification methods. Furthermore, we discuss the biochemical challenges that should be addressed to consolidate and expand the role of chromatin proteomics as a key technology in the context of gene expression regulation and epigenetics research in health and disease.

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.

Histone post-translational modifications (PTMs) are one of the main mechanisms of epigenetic regulation. Dysregulation of histone PTMs leads to many human diseases, such as cancer. Due to its high-throughput, accuracy, and flexibility, mass spectrometry (MS) has emerged as a powerful tool in the epigenetic histone modification field, allowing the comprehensive and unbiased analysis of histone PTMs and chromatin-associated factors. Coupled with various techniques from molecular biology, biochemistry, chemical biology and biophysics, MS has been employed to characterize distinct aspects of histone PTMs in the epigenetic regulation of chromatin functions. In this review we will describe advancements in the field of MS that have facilitated the analysis of histone PTMs and chromatin biology.  

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.

In all cells, proteins are continuously synthesized and degraded in order to maintain protein homeostasis and modify gene expression levels in response to stimuli. Collectively, the processes of protein synthesis and degradation are referred to as protein turnover. At steady state, protein turnover is constant to maintain protein homeostasis, but in dynamic responses, proteins change their rates of synthesis and degradation in order to adjust their proteomes to internal or external stimuli. Thus, probing the kinetics and dynamics of protein turnover lends insight into how cells regulate essential processes such as growth, differentiation, and stress response. Here we outline historical and current approaches to measuring the kinetics of protein turnover on a proteome-wide scale in both steady-state and dynamic systems, with an emphasis on metabolic tracing using stable-isotope-labeled amino acids. We highlight important considerations for designing proteome turnover experiments, key biological findings regarding the conserved principles of proteome turnover regulation, and future perspectives for both technological and biological investigation.

The approach of peptide-based anti-cancer vaccination has proven the ability to induce cancer-specific immune responses in multiple studies for various cancer entities. However, clinical responses remain so far limited to single patients and broad clinical applicability was not achieved. Therefore, further efforts are required to improve peptide vaccination in order to integrate this low side effect therapy into the clinical routine of cancer therapy. To design clinically effective peptide vaccines in the future, different issues have to be addressed and optimized comprising antigen target selection as well as choice of optimal adjuvants and vaccination schedules. Furthermore, the combination of peptide-based vaccines with other immuno- and molecular targeted therapies as well as the development of predictive biomarkers could further improve efficacy. In this review, current approaches in the development of peptide-based vaccines and critical implications for optimal vaccine design are discussed.

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.

Gender-smart Procurement: Policies for Driving Change Research paper sysadmin 14 December 2017

Governments should use public procurement policy as a strategic lever to accelerate gender-inclusive economic growth through the application of state spending power.

• Governments need to rethink public procurement policy. They need to use it as a strategic lever to accelerate gender-inclusive economic growth through the application of state spending power, while maintaining rigorous governance standards. Reform of public procurement to make it more gender-inclusive could create a ‘diversity dividend’ through increased job creation and economic growth. Gender-smart procurement policies could also mitigate economic and business risk by rendering supply chains more diverse.
• In 2014, G20 members agreed to reduce the gender gap in the labour market by 25 per cent by 2025. Procurement policy is one of the most powerful tools governments have to achieve this goal. All G20 members, regardless of the differences in their legal frameworks, can implement measures that will increase the ability of women to benefit from procurement policy.
• Public procurement accounts for around one-fifth of global gross domestic product (GDP). It is estimated that women entrepreneurs supply only 1 per cent of this market. Women’s businesses face considerable barriers to accessing procurement tenders and winning procurement contracts. The inadequate design of many procurement processes prevents more inclusive gender outcomes for citizens. Governments should redefine procurement policies to make explicit the requirement that increasing women’s workforce participation, through greater use of female suppliers, is a key objective when selecting bids for procurement contracts.
• Through the use of policy and spending levers, governments can play four primary roles in encouraging procurement from enterprises owned by women, or from businesses committed to promoting female labour participation. These roles are:
  - To direct reforms of government procurement – reviewing procurement policies and practices to ensure sustainable and inclusive procurement;
  - To reduce barriers to women’s participation in the economy – creating the support mechanisms to ensure an environment in which businesses owned by women can flourish;
  - To help scale up gender-smart procurement in the private sector – expanding government’s role in encouraging private companies to spend more of their procurement budgets with women’s businesses; and
  - To encourage increased transparency on the issues – creating and sharing procurement databases and lessons learned, especially at the regional level.
• Companies can also benefit from having more diverse supply chains and volunteering for accreditation schemes. They can start conversations with government about national regulation that encourages diversity in procurement, leading by example.
• The G20 should set measurable and time-bound targets in the area of gender-smart procurement, to build on the momentum of UN reforms and incorporate good practice in supply chain management.

Annual Review 2017-18 Other resource sysadmin 18 July 2018

Explore the institute’s output, activities and achievements from the past year examining how to make the world more secure in uncertain times; new thinking on how societies can flourish and be prosperous; and how to contribute to a more just society.

Director’s statement

I wrote a year ago that the world has entered one of the most politically turbulent periods in the modern era. This rings even more true now, as relations have seriously deteriorated between the US administration and its European allies, while instability persists across the Middle East, and China and Russia increase their strategic influence.

Over the past year, Chatham House has assessed these changes and proposed ways to manage the associated risks, while seeking out opportunities to expand prosperity and security across the world. Adam Ward, our deputy director, led the publication of the first Chatham House Expert Perspectives report on risks and opportunities in international affairs, to coincide with our fifth annual London Conference on 21–22 June 2018. The conference was attended by over 450 participants from 71 countries.

Furthermore, using our new simulation centre on the Stavros Niarchos Foundation (SNF) Floor, our research teams are ‘stress-testing’ their ideas in exercises involving both practitioners and experts. For example, in June 2017 the Centre on Global Health Security and the Africa Programme hosted our first scenario exercise, which explored how to respond to a humanitarian emergency that required landmine clearance. Since then, we have hosted other simulation and scenario planning exercises, including on cyberattacks, Brexit ‘futures’ and the rise of the populist parties in Europe.

We have also used the new facilities to host events supported by modern audiovisual systems; to stress-test our own communications strategy; and even to conduct a round of scenario-based interactive job interviews. The new media studio and editing facilities have led us to create more multimedia content, which has, in turn, enabled us to reach expanded and more diverse audiences for our work. The new SNF Floor has also provided staff with an open, multifunctional meeting area and has alleviated some of the space pressures which had been created by our growing staff numbers.

As you will read in the following pages, our research is currently centred around three themes: making the world more secure in uncertain times; offering new ideas on how societies can flourish and be prosperous; and contributing to a more just society. A growing number of cross-cutting projects enable us to address topics within each of these themes, including cyberthreats, transatlantic relations, the future of the EU–China economic relationship, regulating data, vulnerabilities in global food trade and implementing universal health coverage.

Our ability to do so has been enhanced by the appointment of several new senior research staff: Hans Kundnani as senior fellow in the Europe Programme; Champa Patel as head of the Asia- Pacific Programme; and Leslie Vinjamuri as head of the US and the Americas Programme and dean of the Queen Elizabeth II Academy for Leadership in International Affairs. In addition, Bernice Lee will take over as the new research director of our Global Economy and Finance Department, alongside her continuing role as executive director of the Hoffmann Centre for Sustainable Resource Economy.

During the coming months, we will lay the foundations to mark the institute’s centenary in the summer of 2020. In doing so, we will draw on our archives to acknowledge the institute’s unique achievements over the past 100 years, recognize those many individuals who have supported us along the way, and set out our objectives for the future. In preparation, we have already launched a series of members’ events with a historical focus, examining how the lessons from the past can inform international affairs and policy thinking today.

In closing, I would like to pay tribute and offer my sincere thanks to Stuart Popham, who steps down this year after six years as chair of the institute and 13 years between 2005 and 2018 as a member of Council. Stuart has been a tremendous support and guide to me, and to the institute as a whole, during this period. His measured advice to management and collegiate leadership of Council have been invaluable during a period in which the institute has more than doubled in size, and in which it has thrived despite the increasingly competitive environment for policy convening, analysis and ideas. We look forward to welcoming Stuart back to Chatham House as a highly engaged individual member, and wish him all the best for the future.

Robin Niblett CMG

Exploring Public International Law and the Rights of Individuals with Chinese Scholars - Part One Other resource sysadmin 29 October 2018

As part of a roundtable series, Chatham House and China University of Political Science and Law (CUPL) jointly organized this four-day meeting at Chatham House for international lawyers to discuss a wide range of issues related to public international law and the rights of individuals.

The specific objectives were to:

• create a platform for Chinese international law academics working on international human rights law issues to present their thinking and exchange ideas with counterparts from outside China;
• build stronger understanding within the wider international law community of intellectual debates taking place in China about the international human rights system and China’s role within it;
• support networking between Chinese and non-Chinese academics working on international human rights and related areas of international law.

The roundtable forms part of a wider Chatham House project exploring China’s impact on the international human rights system and was inspired by early discussions with a burgeoning community of Chinese academics thinking, writing (mainly in Chinese) and teaching about international human rights law.

For China University of Political Science and Law, one of the largest and most prestigious law schools in China and perhaps the only university in the world with an entire faculty of international law, the initiative is part of a drive to forge partnerships beyond China in the international law field.

The roundtable had a total of 22 participants, 10 Chinese (from universities and other academic institutions in Beijing and Shanghai) and 12 non-Chinese (from Australia, Germany, the Netherlands, Switzerland, the United Kingdom and the United States).

All discussions were held in English under the Chatham House Rule.

Exploring Public International Law and the Rights of Individuals with Chinese Scholars - Part Two Other resource sysadmin 30 October 2018

As part of a roundtable series, Chatham House and China University of Political Science and Law (CUPL) held a two-day roundtable meeting in Beijing on public international law and the rights of individuals.

The specific objectives were to:

• create a platform for Chinese international law academics working on international human rights law issues to present their thinking and exchange ideas with counterparts from outside China;
• build stronger understanding within the wider international law community of intellectual debates taking place in China about the international human rights system and China’s role within it;
• support networking between Chinese and non-Chinese academics working on international human rights and related areas of international law.

The roundtable forms part of a wider Chatham House project exploring China’s impact on the international human rights system and was inspired by early discussions with a burgeoning community of Chinese academics thinking, writing (mainly in Chinese) and teaching about international human rights law.

For CUPL, one of the largest and most prestigious law schools in China and perhaps the only university in the world with an entire faculty of international law, the initiative is part of a drive to forge partnerships beyond China in the international law field.

The meeting in Beijing was hosted by CUPL and involved 20 participants, 10 Chinese (from universities and other academic institutions in Beijing) and 10 non-Chinese (from Australia, the Netherlands, South Africa, Switzerland, the United Kingdom and the United States).

To ensure continuity while also expanding the experts network being built, the second meeting included a mix of participants from the first meeting and some new participants.

All discussions were held in English under the Chatham House Rule.

Exploring Public International Law and the Rights of Individuals with Chinese Scholars - Part Three Other resource sysadmin 30 October 2018

As part of a roundtable series, Chatham House, China University of Political Science and Law (CUPL) and the Graduate Institute Geneva held a two-day roundtable meeting in Geneva on public international law and the rights of individuals.

The specific objectives were to:

• create a platform for Chinese international law academics working on international human rights law issues to present their thinking and exchange ideas with counterparts from outside China;
• build stronger understanding within the wider international law community of intellectual debates taking place in China about the international human rights system and China’s role within it;
• support networking between Chinese and non-Chinese academics working on international human rights and related areas of international law.

The roundtable forms part of a wider Chatham House project exploring China’s impact on the international human rights system and was inspired by early discussions with a burgeoning community of Chinese academics thinking, writing (mainly in Chinese) and teaching about international human rights law.

For CUPL, one of the largest and most prestigious law schools in China and perhaps the only university in the world with an entire faculty of international law, the initiative is part of a drive to forge partnerships beyond China in the international law field.

The meeting in Geneva was co-hosted by the Graduate Institute Geneva and involved 19 participants, 9 Chinese (from six research institutions in Beijing and Shanghai) and 11 non-Chinese (from eight research institutions in Australia, Germany, the Netherlands, Switzerland, the United Kingdom and the United States).

To ensure continuity while also expanding the expert network being built, the third meeting included a mix of participants from the first two meetings and some new participants

All discussions were held in English under the Chatham House Rule.

The Role of Sub-state and Non-state Actors in International Climate Processes: Civil Society Research paper sysadmin 27 November 2018

Given today’s challenging geopolitical conditions and the evolving nature of the international climate regime since Paris, civil society must now once again recalibrate its strategies to ensure continued and increasing relevance.

This is one of four background papers feeding into a synthesis paper entitled The Role of Sub-state and Non-state Actors in International Climate Processes.

Summary

• Following the failure of the 15th Conference of the Parties (COP 15) in Copenhagen in 2009, there was a step change in the sophistication and unity of civil society engagement on climate policy. This ensured that, subsequently, civil society was more effective in exercising multiple channels of influence around the negotiations for the Paris Agreement in 2015.
• Civil society proved to be particularly effective at harnessing the twin narratives of climate science and economics, and at leveraging an emerging multi-level governance architecture, to create political space for climate leadership.
• Given today’s challenging geopolitical conditions and the evolving nature of the international climate regime since Paris, civil society must now once again recalibrate its strategies to ensure continued and increasing relevance.
• In particular, the shift to a more ‘nationally grounded’ implementation regime focusing on individual states’ climate commitments will require civil society to become more effective at influencing domestic politics. At the same time, civil society will need to continue to seek strategic synergies at the international level.
• Civil society has a central role to play in ensuring that the first key test of the Paris ‘ratchet’ mechanism – revising countries’ pledged climate actions, or Nationally Determined Contributions (NDCs), by 2020 – is robust, science-informed and strongly rooted in domestic politics.

Moving Energy Initiative Learning Briefs Research paper sysadmin 29 March 2019

Drawing on experiences from Phase II of the MEI in Burkina Faso, Kenya and Jordan, these learning briefs highlight MEI’s approach to innovation, engagement with the private-sector and host communities, and gender-sensitive energy projects. The four learning papers are intended for practitioners and policymakers working in the humanitarian sector and host-country governments.

Findings from Phase I of the Moving Energy Initiative (MEI) in 2015, published in the Chatham House research paper Heat, Light and Power for Refugees: Saving Lives, Reducing Costs, highlight the negative impacts of limited sustainable energy provision on the security of displaced populations. The paper also identified some of the challenges for energy programmes in this sector, such as the lack of robust data on energy access and the priorities of refugee populations.

In Phase II of the MEI, Practical Action led detailed research into the energy needs of refugees in Burkina Faso and Kenya. Chatham House analysed data on global refugee energy use in displacement contexts and produced an interactive map. Energy 4 Impact explored sustainable funding options, private-sector contract models and non-wood cooking concessions. The market development and low-carbon energy initiatives in Burkina Faso, Jordan and Kenya were managed by Practical Action and Energy 4 Impact, with the support of local partners. These partners represented the MEI at multiple conferences and events to share findings and advocate for the inclusion of displaced people in the sustainable energy agenda.

Drawing on experiences from Phase II of the MEI in Burkina Faso, Kenya and Jordan, these learning briefs highlight MEI’s approach to innovation, engagement with the private-sector and host communities, and gender-sensitive energy projects. The four learning papers are intended for practitioners and policymakers working in the humanitarian sector and host-country governments.

Infrastructure Management Contracts: Improving Energy Asset Management in Displacement Settings Research paper sysadmin 17 April 2019

This paper highlights a number of options for managing electricity infrastructure in refugee camps and outlines the challenges, opportunities and operational implications associated with them. It takes the Kalobeyei settlement in Kenya as a case study.

• Building and maintaining electricity infrastructure to power offices, businesses, households and other operations in displacement settings is difficult. It is especially challenging for the Office of the United Nations High Commissioner for Refugees (UNHCR) and its partner agencies, because supplying electricity is not their core business.
• Private-sector companies exist that are willing and able to develop infrastructure management contracts to provide energy as a service in displacement settings. However, institutional barriers within humanitarian agencies persist, with short budgeting cycles in particular preventing humanitarian agencies from entering into the sorts of long-term service agreements required by the private sector.
• A number of options exist to leverage the expertise of the private sector through ‘public–private partnership’ (P3) structures. Such mechanisms can promote more efficient management of infrastructure by drawing on private-sector experience and expertise, incentivizing appropriate risk-sharing and providing options to leverage private capital in project development.
• Field work from the Kalobeyei settlement in Kenya suggests that a solar/diesel hybrid mini-grid solution was the most economical option to power camp services and infrastructure there. Compared to distributed diesel generation, the annual savings in operating costs were estimated at $49,880, with the additional investment paid back within 3.6 years.
• Humanitarian agencies need to be willing to change their policies to enable long-term service agreements. Alternatively (or, more likely, in conjunction with this option), financial mechanisms such as partial risk guarantees need to be developed to offset some of the risks. This change will need high-level support from donors and humanitarian agencies.
• Once the first infrastructure management contracts can be signed and tested in displacement locations (through the use of donor funding or otherwise) and associated data collected, it will ease the way for future investments in these types of projects.

Annual Review 2018-19 Other resource sysadmin 24 July 2019

Explore the institute’s output, activities and achievements from the past year examining how to make the world more stable in uncertain times, new thinking on how societies can promote prosperity, and how to contribute to a more just society.

Chair’s statement

I was delighted to be elected chair of Chatham House last year. It is an honour to lead such a remarkable institution and to have the opportunity to build on the legacy left by Stuart Popham, who stepped down last year as chair and whom I thank and pay tribute to.

My ambition is to ensure that the institute has an even better future than its illustrious past. We are living in unpredictable times, and I want us to be at the centre of the drive to guide the world to a healthier place both politically and economically.

Chatham House possesses world-leading convening power, which – when combined with our capacity to deliver leading, cross-cutting research – gives us a unique advantage in the field of international relations. I want us to harness these assets and better combine the strengths of our research teams so that we can address the big global challenges around economic growth; avoiding geopolitical tensions; and developing new governance systems (as outlined on page 7). This will enable us to improve our impact and effect more policy change.

I also want Chatham House to be an exciting place that attracts younger, more diverse, international audiences. We need to drive more engagement with the next generation of members and others to draw on their enthusiasm, energy and ideas. Our Common Futures Conversations project, for example, is engaging young people from 13 countries across Africa and Europe to identify their shared concerns, and is enabling them to work together to identify solutions via online communities.

This initiative, and indeed all of our activities, would not be possible without funding and support. As noted in more detail in the Honorary Treasurer’s report (page 32), 2018/19 was a challenging year financially, with income totalling £16,381,000, slightly below the level recorded in 2017/18.

Although total net assets at 31 March 2019 were 3 per cent down year on year, the balance sheet remain strong and there was an inflow of cash, with the level of forward income received and pledged increasing significantly.

In this context, I am delighted to note the award of the transformational £10 million grant to facilitate the creation of the Stavros Niarchos Foundation Wing, which will help facilitate research, host our Queen Elizabeth II Academy for Leadership in International Affairs and establish a new collaboration space – the ‘CoLab’ – for engaging our new audiences. All of Chatham House’s supporters, and not least our members, remain indispensable to our success. Without their engagement, enthusiasm and input, the institute could not fulfil its mission.

I am indebted to my colleagues on Council for their support, engagement and expertise. I can say with confidence that they are actively involved in their governance responsibilities at this time when the operational, as well as financial, pressures on all charitable institutions are more intense than ever.

I would like to pay tribute to Alistair Burnett, Martin Fraenkel and Barbara Ridpath, who step down from Council this year after a total of 15 years’ service. I would also like to thank and acknowledge Robin Niblett and his team for their dedication and hard work. Some of the outcomes of their labours are highlighted on the following pages.

Lord Jim O’Neill

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)

Annual Review 2021–22 Other resource NCapeling 11 July 2022

Explore highlights from our work over the past year, including research, events, and next generation initiatives.

Chair’s statement

The past 12 months have seen new levels of instability injected into international affairs. The consequences of the COVID-19 pandemic, the continued fracturing of the international system and Russia’s war on Ukraine, which triggered the largest movement of refugees since the Second World War, have had severe impacts on trade, prices and people around the world.

Chatham House’s Russia and Eurasia Programme and its Ukraine Forum have advised allied governments on their response to the war on Ukraine, and secured the participation of Ukrainian president Volodymyr Zelenskyy in the most watched event of the institute’s history.

The challenge now is to ensure Chatham House can bring together its diverse areas of research and offer influential ideas on how to respond to the uncertain future. Council has supported the push to define clear priorities for the institute’s work, and the launch of two new research centres covering sustainability and global governance.

Drawing out the best from the institute’s expertise in the changed geopolitical landscape will be a top priority for Bronwen Maddox when she takes over as director at the end of August. Council is delighted to have found such a worthy successor to Robin Niblett. Robin’s 15 years of inspiring leadership have seen the institute more than triple in size of staff and budget. New areas of research under his tenure include cyber policy, digital governance, global health, the US and the Americas, and the UK in the world post-Brexit.

Robin also launched the Queen Elizabeth II Academy for Leadership in International Affairs, providing the base for many initiatives to engage the next generation. He also leaves the institute with a rebuilt balance sheet after securing some major long-term gifts; expanded and renovated premises; and a strong communications and digital infrastructure that has raised the profile of Chatham House with our audiences.

Council places on record the institute’s debt to Robin and to his wife, Trisha de Borchgrave. Chatham House’s supporters around the world were delighted to see Robin awarded a knighthood in HM The Queen’s Birthday Honours 2022.

I pay tribute also to my predecessor, Jim O’Neill, and I am grateful to Simon Fraser, deputy chair, and to the other four retiring members of Council (Mimi Ajibadé, Heide Baumann, Kenneth Cukier and Tim Willasey-Wilsey) for their dedication and counsel.

Council will continue to focus on strengthening the impact of the institute’s work and on improving governance, as well as providing continuity during the leadership transition. We will act as a strategic partner for Bronwen as she takes Chatham House forward. Central to this will be implementing the institute’s plan to strengthen equality, diversity and inclusion across the board. Finally, I thank the staff for their commitment through this period of transformation.

Sir Nigel Sheinwald GCMG

The faithful segregation, or “partition,” of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg351, a residue previously predicted to participate in site-specific DNA binding. In vivo, the parAR351A allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a “super-repressor.” In vitro, ParAR351A binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.

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.

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3−/− and PMS2−/− mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3−/− and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme–induced double-strand breaks. PMS2−/− and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation–induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.

A slimmed down Maikel Franco and a bulked up Scott Kingery will compete for playing time at third base this season.

The 30 prospects below all are getting very long looks this spring with an eye toward breaking camp with the parent club. Even if they start the year in the Minors, they all should get the chance to contribute at some point in the very near future.