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This manuscript has been withdrawn by the Author.

This article has been withdrawn by the authors as part of this review overlapped with the contents of Pietrangelo A and Ridgway ND. 2018. Cellular and Molecular Life Sciences. 75; 3079-98.

Adiponectin, an adipocyte-derived protein, has anti-atherogenic and anti-diabetic effects, but how it confers the anti-atherogenic effects is not well understood. To study the anti-atherogenic mechanisms of adiponectin, we examined whether it interacts with atherogenic low-density lipoprotein (LDL) to attenuate LDL’s atherogenicity. L5, the most electronegative subfraction of LDL, induces atherogenic responses similarly to copper-oxidized LDL (oxLDL). Unlike native LDL endocytosed via the LDL receptor, L5 and oxLDL are internalized by cells via the lectin-like oxidized LDL receptor-1 (LOX-1). Using enzyme-linked immunosorbent assays (ELISAs), we showed that adiponectin preferentially bound oxLDL but not native LDL. In Chinese hamster ovary (CHO) cells transfected with LOX-1 or LDL receptor, adiponectin selectively inhibited the uptake of oxLDL but not of native LDL, respectively. Furthermore, adiponectin suppressed the internalization of oxLDL in human coronary artery endothelial cells (HCAECs) and THP-1–derived macrophages. Western blot analysis of human plasma showed that adiponectin was abundant in L5 but not in L1, the least electronegative subfraction of LDL. Sandwich ELISAs with anti-adiponectin and anti–apolipoprotein B antibodies confirmed the binding of adiponectin to L5 and oxLDL. In LOX-1–expressing CHO cells, adiponectin inhibited cellular responses to oxLDL and L5, including nuclear factor-B activation and ERK phosphorylation. In HCAECs, adiponectin inhibited oxLDL-induced endothelin-1 secretion and ERK phosphorylation. Conversely, oxLDL suppressed the adiponectin-induced activation of adenosine monophosphate–activated protein kinase in COS-7 cells expressing adiponectin receptor AdipoR1. Our findings suggest that adiponectin binds and inactivates atherogenic LDL, providing novel insight into the anti-atherogenic mechanisms of adiponectin.

Apolipoproteins C-I, C-II and C-III interact with ApoE to regulate lipoprotein metabolism and contribute to Alzheimer’s disease pathophysiology. In plasma, apoC-I and C-II exist as truncated isoforms, while apoC-III exhibits multiple glycoforms. This study aimed to 1. delineate apoC-I, C-II and C-III isoform profiles in CSF and plasma in a cohort of non-demented older individuals (n = 61), and 2. examine the effect of APOE4 on these isoforms and their correlation with CSF Aβ42, a surrogate of brain amyloid accumulation. The isoforms of the apoCs were immunoaffinity enriched and measured with MALDI-TOF mass spectrometry, revealing a significantly higher percentage of truncated apoC-I and apoC-II in CSF compared to matched plasma, with positive correlation between CSF and plasma. A greater percentage of monosialylated and disialylated apoC-III isoforms was detected in CSF, accompanied by a lower percentage of the two non-sialylated apoC-III isoforms, with significant linear correlations between CSF and plasma. Furthermore, a greater percentage of truncated apoC-I in CSF, and apoC-II in plasma and CSF, was observed in individuals carrying at least one apoE E4 allele. Increased apoC-I and apoC-II truncations were  associated with lower CSF Aβ42. Finally, monosialylated apoC-III was lower, and disialylated apoC-III greater in the CSF of E4 carriers. Together, these results reveal distinct patterns of the apoCs isoforms in CSF, implying CSF-specific apoCs processing. These patterns were accentuated in APOE E4 allele carriers, suggesting an association between APOE4 genotype and Alzheimer’s disease pathology with apoCs processing and function in the brain.

Microtubules are polymers composed of αβ-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human iPS cell-derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in C. reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry-mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms (CRPs) in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that CRPs mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function. 

TG-rich lipoprotein (TRL)-related biomarkers, including TRL-cholesterol (TRL-C), remnant-like lipoprotein particle-cholesterol (RLP-C), and apoC-III have been associated with atherosclerosis. However, their prognostic values have not been fully determined, especially in patients with previous CAD. This study aimed to examine the associations of TRL-C, RLP-C, and apoC-III with incident cardiovascular events (CVEs) in the setting of secondary prevention of CAD. Plasma TRL-C, RLP-C, and total apoC-III were directly measured. A total of 4,355 participants with angiographically confirmed CAD were followed up for the occurrence of CVEs. During a median follow-up period of 5.1 years (interquartile range: 3.9–6.4 years), 543 (12.5%) events occurred. Patients with incident CVEs had significantly higher levels of TRL-C, RLP-C, and apoC-III than those without events. Multivariable Cox analysis indicated that a log unit increase in TRL-C, RLP-C, and apoC-III increased the risk of CVEs by 49% (95% CI: 1.16–1.93), 21% (95% CI: 1.09–1.35), and 40% (95% CI: 1.11–1.77), respectively. High TRL-C, RLP-C, and apoC-III were also independent predictors of CVEs in individuals with LDL-C levels ≤1.8 mmol/l (n = 1,068). The addition of RLP-C level to a prediction model resulted in a significant increase in discrimination, and all three TRL biomarkers improved risk reclassification. Thus, TRL-C, RLP-C, and apoC-III levels were independently associated with incident CVEs in Chinese CAD patients undergoing statin therapy.

Lipoprotein (a) [Lp(a)] is a well-known risk factor for cardiovascular disease, but analysis on Lp(a) and renal dysfunction is scarce. We aimed to investigate prospectively the association of serum Lp(a) with the risk of reduced renal function, and further investigated whether diabetic or hypertensive status modified such association. Six thousand two hundred and fifty-seven Chinese adults aged ≤40 years and free of reduced renal function at baseline were included in the study. Reduced renal function was defined as estimated glomerular filtration rate <60 ml/min/1.73 m². During a mean follow-up of 4.4 years, 158 participants developed reduced renal function. Each one-unit increase in log₁₀-Lp(a) (milligrams per deciliter) was associated with a 1.99-fold (95% CI 1.15–3.43) increased risk of incident reduced renal function; the multivariable-adjusted odds ratio (OR) for the highest tertile of Lp(a) was 1.61 (95% CI 1.03–2.52) compared with the lowest tertile (P for trend = 0.03). The stratified analysis showed the association of serum Lp(a) and incident reduced renal function was more prominent in participants with prevalent diabetes [OR 4.04, 95% CI (1.42–11.54)] or hypertension [OR 2.18, 95% CI (1.22–3.89)]. A stronger association was observed in the group with diabetes and high Lp(a) (>25 mg/dl), indicating a combined effect of diabetes and high Lp(a) on the reduced renal function risk. An elevated Lp(a) level was independently associated with risk of incident reduced renal function, especially in diabetic or hypertensive patients.

Ion mobility brings an additional dimension of separation to LC–MS, improving identification of peptides and proteins in complex mixtures. A recently introduced timsTOF mass spectrometer (Bruker) couples trapped ion mobility separation to TOF mass analysis. With the parallel accumulation serial fragmentation (PASEF) method, the timsTOF platform achieves promising results, yet analysis of the data generated on this platform represents a major bottleneck. Currently, MaxQuant and PEAKS are most used to analyze these data. However, because of the high complexity of timsTOF PASEF data, both require substantial time to perform even standard tryptic searches. Advanced searches (e.g. with many variable modifications, semi- or non-enzymatic searches, or open searches for post-translational modification discovery) are practically impossible. We have extended our fast peptide identification tool MSFragger to support timsTOF PASEF data, and developed a label-free quantification tool, IonQuant, for fast and accurate 4-D feature extraction and quantification. Using a HeLa data set published by Meier et al. (2018), we demonstrate that MSFragger identifies significantly (~30%) more unique peptides than MaxQuant (1.6.10.43), and performs comparably or better than PEAKS X+ (~10% more peptides). IonQuant outperforms both in terms of number of quantified proteins while maintaining good quantification precision and accuracy. Runtime tests show that MSFragger and IonQuant can fully process a typical two-hour PASEF run in under 70 min on a typical desktop (6 CPU cores, 32 GB RAM), significantly faster than other tools. Finally, through semi-enzymatic searching, we significantly increase the number of identified peptides. Within these semi-tryptic identifications, we report evidence of gas-phase fragmentation before MS/MS analysis.

Mass spectrometry has become an indispensable tool for the characterization of glycosylation across biological systems. Our ability to generate rich fragmentation of glycopeptides has dramatically improved over the last decade yet our informatic approaches still lag behind. Although glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using N-linked glycopeptides of Campylobacter fetus subsp. fetus as well as O-linked glycopeptides of Acinetobacter baumannii and Burkholderia cenocepacia revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses before database searching. Using this approach, we demonstrate how wide tolerance searching can be used to compare glycan use across bacterial species by examining the glycoproteomes of eight Burkholderia species (B. pseudomallei; B. multivorans; B. dolosa; B. humptydooensis; B. ubonensis, B. anthina; B. diffusa; B. pseudomultivorans). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes.

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between WT IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

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

A key point in achieving accurate intact glycopeptide identification is the definition of the glycan composition file that is used to match experimental with theoretical masses by a glycoproteomics search engine. At present, these files are mainly built from searching the literature and/or querying data sources focused on posttranslational modifications. Most glycoproteomics search engines include a default composition file that is readily used when processing MS data. We introduce here a glycan composition visualizing and comparative tool associated with the GlyConnect database and called GlyConnect Compozitor. It offers a web interface through which the database can be queried to bring out contextual information relative to a set of glycan compositions. The tool takes advantage of compositions being related to one another through shared monosaccharide counts and outputs interactive graphs summarizing information searched in the database. These results provide a guide for selecting or deselecting compositions in a file in order to reflect the context of a study as closely as possible. They also confirm the consistency of a set of compositions based on the content of the GlyConnect database. As part of the tool collection of the Glycomics@ExPASy initiative, Compozitor is hosted at https://glyconnect.expasy.org/compozitor/ where it can be run as a web application. It is also directly accessible from the GlyConnect database.

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.

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.

Withdrawn by Author.

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

This article has been withdrawn by the authors. We discovered an error after this manuscript was published as a Paper in Press. Specifically, we learned that the structures of glycans presented for the PD-L1 peptide were drawn and labeled incorrectly. We wish to withdraw this article and submit a corrected version for review.

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

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.

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

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.

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.

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

Cells continually degrade and replace damaged proteins. However, the high energetic demand of protein turnover generates reactive oxygen species (ROS) that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover and energetic demand remains unclear. Here, we used a proteomic approach to measure rates of protein turnover within primary fibroblasts isolated from a number of species with diverse lifespans including the longest-lived mammal, the bowhead whale. We show that organismal lifespan is negatively correlated with turnover rates of highly abundant proteins. In comparison to mice, cells from long-lived naked mole rats have slower rates of protein turnover, lower levels of ATP production and reduced ROS levels. Despite having slower rates of protein turnover, naked mole rat cells tolerate protein misfolding stress more effectively than mouse cells. We suggest that in lieu of rapid constitutive turnover, long-lived species may have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins.

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.

Exploring Public International Law Issues with Chinese Scholars – Part Four Other resource sysadmin 30 October 2018

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

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 was co-hosted with CUPL and involved 28 participants, consisting of 19 Chinese participants (from six leading research institutions in Beijing and Shanghai) and nine nonChinese participants (from eight leading research institutions in Australia, the Netherlands, the UK, Switzerland, Canada and Singapore).

To ensure continuity while also expanding the expert network being built, the fifth meeting included a mix of participants from the previous meetings and some new participants.

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

The Phillies introduced J.T. Realmuto, their new All-Star catcher, in a news conference Tuesday at the team's Spring Training complex in Clearwater, Fla.

Jean Segura became one of the Phillies' most notable offseason acquisitions, a two-time All-Star expected to give the organization its best overall production at shortstop since Jimmy Rollins left town, in part because of a little brawl he had late last season with Mariners teammate Dee Gordon.

Bryce Harper is the final superstar standing for the Phillies.

The Angels will be without left fielder Justin Upton early in Spring Training, as he's dealing with right knee patellar tendinitis but is expected to be ready for Opening Day, manager Brad Ausmus said on Monday.

Ramaphosa Must Act Fast With New Mandate in South Africa Expert comment sysadmin 23 May 2019

In the wake of South Africa’s election, political constraints will ebb momentarily. The president should seize the opportunity to deliver meaningful change.

On 25 May, Cyril Ramaphosa will be inaugurated as president of South Africa, having dragged the African National Congress (ANC) over the line in the 8 May election. The ANC gained a 57 per cent majority, its lowest vote since 1994, its status as national liberator deeply eroded by successive corruption scandals. Only Ramaphosa’s personal popularity stopped it haemorrhaging more support.

His sustained action against corrupt public servants and promises of job-creating economic growth has attracted support from beyond the ANC’s base, including a significant minority of white voters, and generated significant international goodwill. Ramaphosa now has a short window of opportunity to reset social democracy in South Africa before the political cycle of municipal, party and national elections from 2021 to 2024 forces his attention back to party politics.

Defining ‘Ramaphosa-ism’

But personal popularity is fickle, and goodwill alone will not turn around the ailing economy. To attract investment and keep the electorate on side, Ramaphosa’s government needs to move beyond pragmatic crisis responses and articulate a clear, shared vision for how market intervention can allow the economy to grow while simultaneously delivering social transformation.

Growth will be hard to achieve in the short term. The economy is expected to grow 1.2% in 2019 and 1.5% in 2020, according to the IMF. Consumer confidence remains subdued, and a decade of declining GDP per capita and increasing inequality has put a strain on households. A ‘fiscal stimulus’ in 2018 delivered very little new government spending, and over the past 10 years, the government wage bill has increased three times higher than the rate of inflation.

Eskom, the state electricity provider, has debts equating to the GDP of Latvia and is not the only state-owned enterprise (SOE) that has required bailing out by the government. There are plans to break up Eskom into three separate entities but calls for deeper reform – or even privatization – are growing.

The president’s responses to these challenges will go a long way to defining ‘Ramaphosa-ism’ and the role of government in pursuing equitable economy growth.

Economic expectations under Ramaphosa

Ramaphosa was a champion of the introduction of a minimum wage and a proponent of the National Development Plan, which relies on growth to drive job creation. His support for land reform is an individual conviction as much as it is a party line, although his views are softer than many in the party, with state-owned land being the initial target.

Investor uncertainty on land tenure and regulations in mining will need to be addressed through passing key pieces of legislation on land reform and the revised Mining and Petroleum Resources Development Act.

Where Ramaphosa differs from his predecessors is his links with business. Thabo Mbeki enjoyed a relationship of mutual respect with business; this disintegrated under Jacob Zuma. Ramaphosa, however, is part of South Africa’s business community, having founded the Shanduka Group, with investments in multiple sectors including retail, telecoms and extractives, and served as chairman of MTN and Bidvest. As president, he has surrounded himself with close economic advisers from business and banking.

In the short term, anti-corruption measures and competent appointments will ease investor woes. In the long term, there is a need to improve the ease of doing business, including labour market reforms, and to make South Africa a more competitive business environment by reducing the hold of large conglomerates on the economy. Ramaphosa may also make greater use of public-private partnerships for large projects.

Political constraints

Ramaphosa faces few immediate political challenges. The ANC is still deeply divided, but although Ramaphosa does not enjoy the ideological support of the entire party, his opponents are leaderless post-Zuma, and have been unable to offer a coherent alternative. ANC Secretary General Ace Magashule has fallen into the role of interim figurehead of this faction, and allegations of corruption would make it difficult for him to aspire to national leadership.

The need to avoid splits before the election meant Ramaphosa had to make concessions, and his first cabinet in February 2018 included opponents and those accused of corruption or incompetence, such as Malusi Gigaba and Bathabile Dlamini. Such concessions to political opponents are unlikely to continue after the election.

Meanwhile, opposition parties made some advances in the election, but where Zuma was an easy target, they are still grappling with how to confront Ramaphosa. The party with the biggest gains was the Economic Freedom Fighters, whose increase of just over 4 points from the last election gave it 11 per cent of the vote this time. They will likely continue to be an effective disruptor. Ramaphosa may also be challenged by trade unions on his reforms, notably over any break-up of SOEs.

But the biggest and most immediate external political challenge for Ramaphosa will be rebuilding trust between government and society, in a context where social protest has become an alternative form of political participation. A turnout of 65 per cent may be considered normal in Western democracies but is a notable drop for a country as politicized as South Africa, driven by frustration and a sense of exclusion as much as apathy. Turnout by young people was even lower.

Achieving the vision

South Africa has all the platforms it needs to project its renewal and attract vital external investment – it is a non-permanent member of the UN Security Council, it will take over as chair of the African Union in 2020, it is a member of BRICS and it is the only African member of the G20. But in the recent past, it has struggled to tell a coherent story about its vision for the future and offer to the world.

In the immediate wake of the election, internal and external political constraints will ebb. Ramaphosa must act fast to deliver results before the election cycle starts again. To attract much needed investment stimulus, he will not only need to articulate and market his vision for South Africa, but also outline how he plans to achieve it.

The road to COP27: In conversation with US Special Presidential Envoy for Climate John Kerry 27 October 2022 — 3:00PM TO 4:00PM Anonymous (not verified) 20 October 2022 Chatham House and Online

What will progress on climate change look like at COP27?

With global attention zeroing in on COP27, policymakers and world leaders will meet in Egypt to take the next step in the fight against the climate crisis. The planet is on course to warm well beyond 1.5°C and climate hazards are increasing our exposure to climate risk. Violent and unpredictable weather events increasingly leave devastation among communities, particularly in vulnerable countries.

At the same time, the ripple effects of the conflict in Ukraine will have wide-ranging economic, social and geopolitical consequences for years to come. Whilst some finance is being made available, more is needed to properly address the damage caused by climate change and fund the transition to net zero worldwide. These challenges have become more acute as the world grapples with a growing energy crisis, the war in Ukraine and a troubling economic outlook.

Joined by US Special Presidential Envoy for Climate John Kerry, the following questions are considered:

• Is ‘1.5 degrees’ still on track?

• How can countries better collaborate to move to net zero faster?

• How can we achieve progress on adaptation, climate finance, and loss and damage?

As with all members events, questions from the audience drive the conversation.

Read the transcript.