Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers and known for its extensive genetic heterogeneity, high therapeutic resistance, and strong variation in intrinsic radiosensitivity. To understand the molecular mechanisms underlying radioresistance, we screened the phenotypic response of 38 PDAC cell lines to ionizing radiation. Subsequent phosphoproteomic analysis of two representative sensitive and resistant lines led to the reproducible identification of 7,800 proteins and 13,000 phosphorylation sites (p-sites). Approximately 700 p-sites on 400 proteins showed abundance changes after radiation in all cell lines regardless of their phenotypic sensitivity. Apart from recapitulating known radiation response phosphorylation markers such as on proteins involved in DNA damage repair, the analysis uncovered many novel members of a radiation-responsive signaling network that was apparent only at the level of protein phosphorylation. These regulated p-sites were enriched in potential ATM substrates and in vitro kinase assays corroborated 10 of these. Comparing the proteomes and phosphoproteomes of radiosensitive and -resistant cells pointed to additional tractable radioresistance mechanisms involving apoptotic proteins. For instance, elevated NADPH quinine oxidoreductase 1 (NQO1) expression in radioresistant cells may aid in clearing harmful reactive oxygen species. Resistant cells also showed elevated phosphorylation levels of proteins involved in cytoskeleton organization including actin dynamics and focal adhesion kinase (FAK) activity and one resistant cell line showed a strong migration phenotype. Pharmacological inhibition of the kinases FAK by Defactinib and of CHEK1 by Rabusertib showed a statistically significant sensitization to radiation in radioresistant PDAC cells. Together, the presented data map a comprehensive molecular network of radiation-induced signaling, improves the understanding of radioresistance and provides avenues for developing radiotherapeutic strategies.

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.

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.

Amino acid hydroxylation is a common post-translational modification, which generally regulates protein interactions or adds a functional group that can be further modified. Such hydroxylation is currently considered irreversible, necessitating the degradation and re-synthesis of the entire protein to reset the modification. Here we present evidence that the cellular machinery can reverse FIH-mediated asparagine hydroxylation on intact proteins. These data suggest that asparagine hydroxylation is a flexible and dynamic post-translational modification akin to modifications involved in regulating signaling networks, such as phosphorylation, methylation and ubiquitylation.

High-speed analysis of large (prote)omics sample sets at the rate of thousands or millions of samples per day on a single platform has been a challenge since the beginning of proteomics. For many years, ESI-based MS methods have dominated proteomics because of their high sensitivity and great depth in analyzing complex proteomes. However, despite improvements in speed, ESI-based MS methods are fundamentally limited by their sample introduction, which excludes off-line sample preparation/fractionation because of the time required to switch between individual samples/sample fractions, and therefore being dependent on the speed of on-line sample preparation methods such as liquid chromatography. Laser-based ionization methods have the advantage of moving from one sample to the next without these limitations, being mainly restricted by the speed of modern sample stages, i.e. 10 ms or less between samples. This speed matches the data acquisition speed of modern high-performing mass spectrometers whereas the pulse repetition rate of the lasers (>1 kHz) provides a sufficient number of desorption/ionization events for successful ion signal detection from each sample at the above speed of the sample stages. Other advantages of laser-based ionization methods include the generally higher tolerance to sample additives and contamination compared with ESI MS, and the contact-less and pulsed nature of the laser used for desorption, reducing the risk of cross-contamination. Furthermore, new developments in MALDI have expanded its analytical capabilities, now being able to fully exploit high-performing hybrid mass analyzers and their strengths in sensitivity and MS/MS analysis by generating an ESI-like stable yield of multiply charged analyte ions. Thus, these new developments and the intrinsically high speed of laser-based methods now provide a good basis for tackling extreme sample analysis speed in the omics.

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.

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.

Specific E3 ligases target tumor suppressors for degradation. Inhibition of such E3 ligases may be an important approach to cancer treatment. RNF146 is a RING domain and PARylation-dependent E3 ligase that functions as an activator of the β-catenin/Wnt and YAP/Hippo pathways by targeting the degradation of several tumor suppressors. Tankyrases 1 and 2 (TNKS1/2) are the only known poly-ADP-ribosyltransferases that require RNF146 to degrade their substrates. However, systematic identification of RNF146 substrates have not yet been performed. To uncover substrates of RNF146 that are targeted for degradation, we generated RNF146 knockout cells and TNKS1/2-double knockout cells and performed proteome profiling with label-free quantification as well as transcriptome analysis. We identified 160 potential substrates of RNF146, which included many known substrates of RNF146 and TNKS1/2 and 122 potential TNKS-independent substrates of RNF146. In addition, we validated OTU domain-containing protein 5 and Protein mono-ADP-ribosyltransferase PARP10 as TNKS1/2-independent substrates of RNF146 and SARDH as a novel substrate of TNKS1/2 and RNF146. Our study is the first proteome-wide analysis of potential RNF146 substrates. Together, these findings not only demonstrate that proteome profiling can be a useful general approach for the systemic identification of substrates of E3 ligases but also reveal new substrates of RNF146, which provides a resource for further functional studies.

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

Colorectal cancer (CRC) arises as the consequence of progressive changes from normal epithelial cells through polyp to tumor, and thus is an useful model for studying metabolic shift. In the present study, we studied the metabolomic profiles using high analyte specific gas chromatography/mass spectrometry (GC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS) to attain a systems-level view of the shift in metabolism in cells progressing along the path to CRC. Colonic tissues including tumor, polyps and adjacent matched normal mucosa from 26 patients with sporadic CRC from freshly isolated resections were used for this study. The metabolic profiles were obtained using GC/MS and LC/MS/MS. Our data suggest there was a distinct profile change of a wide range of metabolites from mucosa to tumor tissues. Various amino acids and lipids in the polyps and tumors were elevated, suggesting higher energy needs for increased cellular proliferation. In contrast, significant depletion of glucose and inositol in polyps revealed that glycolysis may be critical in early tumorigenesis. In addition, the accumulation of hypoxanthine and xanthine, and the decrease of uric acid concentration, suggest that the purine biosynthesis pathway could have been substituted by the salvage pathway in CRC. Further, there was a step-wise reduction of deoxycholic acid concentration from mucosa to tumors. It appears that to gain a growth advantage, cancer cells may adopt alternate metabolic pathways in tumorigenesis and this flexibility allows them to adapt and thrive in harsh environment.

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.

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.

CD4+ T cell responses are crucial for inducing and maintaining effective anti-cancer immunity, and the identification of human leukocyte antigen class II (HLA-II) cancer-specific epitopes is key to the development of potent cancer immunotherapies. In many tumor types, and especially in glioblastoma (GBM), HLA-II complexes are hardly ever naturally expressed. Hence, little is known about immunogenic HLA-II epitopes in GBM. With stable expression of the class II major histocompatibility complex transactivator (CIITA) coupled to a detailed and sensitive mass spectrometry based immunopeptidomics analysis, we here uncovered a remarkable breadth of the HLA-ligandome in HROG02, HROG17 and RA GBM cell lines. The effect of CIITA expression on the induction of the HLA-II presentation machinery was striking in each of the three cell lines, and it was significantly higher compared to interferon gamma (IFN) treatment. In total, we identified 16,123 unique HLA-I peptides and 32,690 unique HLA-II peptides. In order to genuinely define the identified peptides as true HLA ligands, we carefully characterized their association with the different HLA allotypes. In addition, we identified 138 and 279 HLA-I and HLA-II ligands, respectively, most of which are novel in GBM, derived from known GBM-associated tumor-antigens that have been used as source proteins for a variety of GBM vaccines. Our data further indicate that CIITA-expressing GBM cells acquired an antigen presenting cell-like phenotype as we found that they directly present external proteins as HLA-II ligands. Not only that CIITA-expressing GBM cells are attractive models for antigen discovery endeavors, but also such engineered cells have great therapeutic potential through massive presentation of a diverse antigenic repertoire.

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.

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.

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.

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.

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.

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.  

Hirschsprung disease (HSCR) is a heterogeneous group of neurocristopathy characterized by the absence of the enteric ganglia along a variable length of the intestine. Genetic defects play a major role in the pathogenesis of HSCR while family studies of pathogenic variants in all the known genes (loci) only demonstrate incomplete penetrance and variable expressivity for unknown reasons. Here, we applied large-scale, quantitative proteomics of human colon tissues from 21 patients using iTRAQ method followed by bioinformatics analysis. Selected findings were confirmed by parallel reaction monitoring (PRM) verification. At last the interesting differentially expressed proteins were confirmed by western blot. A total of 5341 proteins in human colon tissues were identified. Among them, 664 proteins with >1.2-fold difference were identified in 6 groups: groups A1 and A2 pooled protein from the ganglionic and aganglionic colon of male, long-segment HSCR patients (L-HSCR, n=7); groups B1 and B2 pooled protein from the ganglionic and aganglionic colon of male, short-segment HSCR patients (S-HSCR, n=7); and groups C1 and C2 pooled protein from the ganglionic and aganglionic colon of female, S-HSCR patients (n=7). Based on these analyses, 49 proteins from 5 pathways were selected for PRM verification, including ribosome, endocytosis, spliceosome, oxidative phosphorylation and cell adhesion. The downregulation of three neuron projection development genes ARF4, KIF5B and RAB8A in the aganglionic part of the colon were verified in 15 paired colon samples using WB. The findings of this study will shed new light on the pathogenesis of HSCR and facilitate the development of therapeutic targets.

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.

Aspergillus flavus (A. flavus), a pathogenic fungus, can produce carcinogenic and toxic aflatoxins that are a serious agricultural and medical threat worldwide. Attempts to decipher the aflatoxin biosynthetic pathway have been hampered by the lack of a high-quality genome annotation for A. flavus. To address this gap, we performed a comprehensive proteogenomic analysis using high-accuracy mass spectrometry data for this pathogen. The resulting high-quality dataset confirmed the translation of 8,724 previously-predicted genes, and identified 732 novel proteins, 269 splice variants, 447 single amino acid variants, 188 revised genes. A subset of novel proteins was experimentally validated by RT-PCR and synthetic peptides. Further functional annotation suggested that a number of the identified novel proteins may play roles in aflatoxin biosynthesis and stress responses in A. flavus. This comprehensive strategy also identified a wide range of post-translational modifications (PTMs), including 3,461 modification sites from 1,765 proteins. Functional analysis suggested the involvement of these modified proteins in the regulation of cellular metabolic and aflatoxin biosynthetic pathways. Together, we provided a high quality annotation of A. flavus genome and revealed novel insights into the mechanisms of aflatoxin production and pathogenicity in this pathogen.

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.

Staphylococcus aureus is a major cause of infections worldwide and infection results in a variety of diseases. As of no surprise, protein phosphorylation is an important game player in signaling cascades and has been shown to be involved in S. aureus virulence. Albeit long neglected, eukaryotic-type serine/threonine kinases in S. aureus have been implicated in this complex signaling cascades. Due to the sub-stoichiometric nature of protein phosphorylation and a lack of suitable analysis tools, the knowledge of these cascades is however, to date, still limited.

Here, were apply an optimized protocol for efficient phosphopeptide enrichment via Fe3+-IMAC followed by LC-MS/MS to get a better understanding of the impact of protein phosphorylation on the complex signaling networks involved in pathogenicity. By profiling a serine/threonine kinase and phosphatase mutant from a methicillin-resistant S. aureus mutant library, we generated the most comprehensive phosphoproteome dataset of S. aureus to date, aiding a better understanding of signaling in bacteria. With the identification of 3800 class I p-sites we were able to increase the number of identifications by more than 21 times compared to recent literature. In addition, we were able to identify 74 downstream targets of the only reported eukaryotic-type Ser/Thr kinase of the S. aureus strain USA300, Stk1. This work allowed an extensive analysis of the bacterial phosphoproteome and indicates that Ser/Thr kinase signaling is far more abundant than previously anticipated in S. aureus.

Sporozoites are a motile form of malaria-causing Plasmodium falciparum parasites that migrate from the site of transmission in the dermis through the bloodstream to invade hepatocytes. Sporozoites interact with many cells within the host, but the molecular identity of these interactions and their role in the pathology of malaria is poorly understood. Parasite proteins that are secreted and embedded within membranes are known to be important for these interactions, but our understanding of how they interact with each other to form functional complexes is largely unknown. Here, we compile a library of recombinant proteins representing the repertoire of cell surface and secreted proteins from the P. falciparum sporozoite and use an assay designed to detect extracellular interactions to systematically identify complexes. We identify three protein complexes including an interaction between two components of the p24 complex that is involved in the trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins through the secretory pathway. Plasmodium parasites lacking either gene are strongly inhibited in the establishment of liver stage infections. These findings reveal an important role for the p24 complex in malaria pathogenesis and show that the library of recombinant proteins represents a valuable resource to investigate P. falciparum sporozoite biology.

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.

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.

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.

Dance of the Trillions: Developing Countries and Global Finance Book sysadmin 6 July 2018

David Lubin tells the story of what makes money flow from high-income countries to lower-income ones; what makes it flow out again; and how developing countries have sought protection against the volatility of international capital flows.

Selected by the Financial Times as one of the best economics books of 2018, Dance of the Trillions traces an arc from the 1970s, when developing countries first gained access to international financial markets, to the present day.

Underlying this story is a discussion of how the relationship between developing countries and global finance appears to be moving from one governed by the ‘Washington Consensus’ to one more likely to be shaped by Beijing.

This book is part of the Insights series.

Praise for Dance of the Trillions

About the author

David Lubin is managing director and head of emerging markets economics at Citi, an American bank, where he is responsible for a team of more than 30 economists in 15 locations globally.

Purchase

• UK (via Amazon)
• Rest of world (via Brookings Institution Press)
• Students (via Browns Books)

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

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

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

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.

Jake Arrieta on Thursday offered an unexpected explanation for his second-half struggles in 2018: he pitched much of last season with an injured left knee.

Our weekly series previewing each of baseball's six divisions begins with the American League West. Let's take a team-by-team look at the biggest questions this season.