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

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

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

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

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.

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

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

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.

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

Cullin RING E3 Ligases (CRLs) ubiquitylate hundreds of important cellular substrates. Here we have assembled and purified the Ankyrin repeat and SOCS Box protein 9 CUL5 RBX2 Ligase (ASB9-CRL) in vitro and show how it ubiquitylates one of its substrates, CKB. CRLs occasionally collaborate with RING between RING E3 ligases (RBRLs) and indeed, mass spectrometry analysis showed that CKB is specifically ubiquitylated by the ASB9-CRL-ARIH2-UBE2L3 complex. Addition of other E2s such as UBE2R1 or UBE2D2 contribute to polyubiquitylation but do not alter the sites of CKB ubiquitylation. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis revealed that CUL5 neddylation allosterically exposes its ARIH2 binding site, promoting high affinity binding, and it also sequesters the NEDD8 E2 (UBE2F) binding site on RBX2. Once bound, ARIH2 helices near the Ariadne domain active site are exposed, presumably relieving its autoinhibition. These results allow us to propose a model of how neddylation activates ASB-CRLs to ubiquitylate their substrates.

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

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

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

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

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

Deep proteome coverage in bottom-up proteomics requires peptide-level fractionation to simplify the complex peptide mixture before analysis by tandem mass spectrometry. By decreasing the number of co-eluting precursor peptide ions, fractionation effectively reduces the complexity of the sample leading to higher sample coverage and reduced bias towards high abundance precursors that are preferentially identified in data-dependent acquisition strategies. To achieve this goal, we report a bead-based off-line peptide fractionation method termed CIF or Carboxylate modified magnetic bead-based isopropanol gradient peptide fractionation. CIF is an extension of the SP3 (single-pot solid-phase-enhanced sample preparation) strategy and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange (SCX) and reversed phase (RP)-based chromatography. We demonstrate that CIF is an effective offline separation strategy capable of increasing the depth of peptide analyte coverage both when used alone or as a second dimension of peptide fractionation in conjunction with high pH RP. These features make it ideally suited for a wide range of proteomic applications including the affinity purification of low abundance bait proteins.

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

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

Prices, Products and Priorities: Meeting Refugees’ Energy Needs in Burkina Faso and Kenya Other resource sysadmin 24 January 2018

As the number of displaced people increases, and aid budgets come under further pressure, the imperative to identify cost-effective and sustainable solutions for delivering energy to refugees is more pressing than ever.

This paper examines the issue of energy and displacement in detail, using insights from refugees in camps in Burkina Faso and Kenya. It seeks to promote a better understanding of their energy needs, priorities and preferences, and explores how increased access to energy might help to achieve lasting impact in the two camps surveyed. The paper is based on primary research from the Goudoubo camp in Burkina Faso and the Kakuma I camp in Kenya, but the analysis and conclusions are pertinent in the wider context of camps for forcibly displaced people.

Summary points

• There is a low level of energy access in the refugee camps of Kakuma I and Goudoubo, which contributes to poverty and hampers relief and development efforts. Trying to meet basic cooking, lighting and phone-charging needs is costly for refugees, consuming a significant share of stretched monthly budgets.
• The predominant cooking solution consists of basic improved cookstoves burning wood and charcoal. The ‘three-stone fire’ method also remains commonplace. Three out of five families in Kakuma I report health problems due to smoke from cookstoves.
• Street lighting is a high priority for residents, due to concerns about security and safety in camps. In Goudoubo, 86 per cent of survey respondents said that more household members would go out after dark if there were better public lighting.
• A significant proportion of refugees would pay for cleaner and more efficient energy technologies, but many lack the financial resources required, and the development of markets for such products remains partially contingent on sustained financial support.
• There is a need for a diversity of energy technologies that give varying levels and qualities of service; a ‘one size fits all’ approach is inappropriate if universal access to sustainable energy is to be achieved.
• Clean cookstoves and fuels (LPG, ethanol, biogas, etc.) are in high demand, but require much greater investment if they are to be introduced at scale. Solid biomass and improved cookstoves will continue to be important cooking solutions in Kakuma I and Goudoubo, as well as in other refugee camps. A shift to more efficient cooking can be achieved at little or no extra cost for the significant proportion of people who still cook on three-stone fires.
• Users of quality-verified household solar products spend dramatically less on light and power than do people using inferior technologies. Strong brand recognition and a high willingness to pay indicate a large market and a significant opportunity for the solar private sector.
• Centralized electricity supply solutions – mini-grids or grid connections – are more economic than multiple standalone diesel generators. The current piecemeal and ad hoc approach, with each facility managing its own power supply, is inherently wasteful. Greater coordination among humanitarian clusters is required so that centralized solutions can be assessed, designed, financed and implemented.
• Collecting data on energy expenditure and use, as well as quantification of the wide ranging impacts of improved technologies, is necessary to build a compelling case for investment in electricity infrastructure. In addition, engaging refugees on their needs, preferences and willingness to pay can improve the sustainability and impact of energy interventions.
• Private-sector and market development approaches offer long-term, cost-effective solutions for refugees and can also benefit host communities. As the number of displaced people in the world increases, and as aid budgets come under further pressure, the imperative to identify cost-effective and sustainable solutions is more pressing than ever.

Chatham House is a part of the Moving Energy Initiative, a consortium working towards clean energy for refugees. For more information visit movingenergy.earth

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

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

Summary

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

Cooking in Displacement Settings: Engaging the Private Sector in Non-wood-based Fuel Supply Research paper sysadmin 22 January 2019

In displacement settings, providing cooking solutions that reduce negative impacts on the environment and health remains a challenge for local governments, humanitarian agencies, businesses and refugees.

Summary

• Providing adequate cooking fuel and clean-burning, fuel-efficient stoves in displacement settings has long been a major challenge for local authorities, humanitarian agencies, non-governmental organizations, local communities and refugees themselves. Refugees generally have limited access to modern cooking solutions. Most either depend on insufficient humanitarian agency handouts of ‘in-kind’ firewood or have to travel long distances to collect firewood.
• There is significant potential for private-sector engagement in this context – which, though largely overlooked to date, could result in win-win scenarios for all stakeholders. Refugee camps and other displacement settings present opportunities for private-sector cooking fuel companies to expand their customer bases, with the added advantage for vendors of offering concentrated demand and scope for economies of scale.
• For the Kakuma refugee camp complex in Kenya, the Moving Energy Initiative (MEI) decided to engage with the private sector directly. The MEI requested expressions of interest from local private-sector companies for expanding sales and distribution of fuels in the complex through the concession. The winning company – National Oil Corporation of Kenya – is to receive a prize of $50,000 for its proposed concession to supply liquefied petroleum gas both to refugees in the Kakuma complex and to the surrounding host community.
• The MEI also conducted interviews with various stakeholders in other contexts and countries who are engaged in efforts to develop market-based approaches to providing clean, fuel-efficient cooking solutions to refugees.
• Based on the interviews and the concession process, the MEI recommends greater donor investment and longer-term guaranteed funding for cooking interventions. This is needed to allow sufficient time to build sustainable markets and secure the requisite engagement and investments from the private sector.
• Larger, longer-term investments by the private sector – supported through partnerships with donors and humanitarian agencies – in infrastructure and demand creation (both in and outside the refugee community) can reduce the price of alternative solutions and support a gradual transition away from subsidies.

Adopting a Market-based Approach to Boost Energy Access in Displaced Contexts Research paper sysadmin 25 March 2019

This paper evaluates the market-based approaches adopted in the MEI projects in Kenya and Burkina Faso. It articulates how such commercial strategies can be applied to the delivery of energy in displacement settings and compares this to real world examples.

• Development of long-term energy solutions in displacement settings tends to be perceived as investment that falls outside the remit of emergency responses. In addition, when emergency energy supply measures are implemented they often result in expensive, unreliable and unhealthy energy provision for those in protracted or recurrent crises.
• There is widespread agreement among humanitarian and development experts that an effective refugee response should include long-term development solutions as well as emergency relief.
• The energy access imperative is more pronounced when considering the need for effective energy distribution in practically all camp activities and basic necessities: pumping and treatment of clean water; heating and cooling for food storage and cooking; energy for livelihood activities; and provision of light for schooling, hospitals and the prevention of violence against women and children.
• Minor shifts in household energy use to basic solar lighting options and non-wood fuels would save $303 million annually on refugee fuel costs.
• Within refugee contexts in Kenya and Burkina Faso, the MEI sought to examine opportunities to use market interventions, rather than in-kind distributions, to improve clean energy access over the long-term and test the delivery of market-based approaches.

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

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

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

The Chatham House Sustainable Laboratories Initiative: Prior Assessment Tool Other resource sysadmin 14 June 2019

Laboratories are critical for supporting effective infectious disease surveillance and outbreak response. This tool is meant to help structure a conversation between funding partners and recipient countries on how to most effectively establish or repurpose laboratories in low-resource environments.

Introduction

Laboratories are critical for supporting effective infectious disease surveillance and outbreak response, and lack of adequate laboratory capacity is a global challenge. As part of global health security initiatives, cooperative threat reduction efforts and international development programmes, sophisticated laboratories have been provided to mitigate biological threats and bolster a country’s capacity for detection, diagnosis and storage of high-consequence pathogens. Very often, these use the assumptions, standards and templates applied in high-income countries. However, it can be difficult or even impossible to sustain these facilities in low-resource environments. There can sometimes be limited local technical capacity and capability, which can result in a high reliance on imported expertise, skills, equipment and other resources. Sustainability can therefore be hard to achieve. In addition, when a funding partner withdraws, the laboratories can become disused, foundering without the trained personnel and financial resources to sustain them.

To help address this situation, a proposal gaining increasing support internationally is to adopt an approach based on a local risk assessment, whereby laboratories are appropriately and optimally tailored to the local risks and to the resources available, both in the short and longer term, without compromising biosafety and biosecurity.

A Chatham House workshop was convened in Abuja, Nigeria, in 2018 to explore what West African countries would find most appropriate in terms of building laboratory capacity, what the main challenges have been so far, and what needs to be done to improve the sustainability of laboratories in the region. It emerged that there was a need for a more structured conversation between the funding partner and recipient country early in the process – prior to embarking on the detailed planning phase for the establishment or repurposing of a laboratory. This should involve careful consideration and an assessment of existing and planned capacity, needs and contextual issues, together with proposals for how to address the issues revealed, so that any ensuing laboratory demonstrably supports the national strategy and therefore flourishes.

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

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli. MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication.

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

Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex–forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.

All 30 Major League teams will wear special "MLB 150" patches on their uniforms for the entire 2019 season in honor of the 150th anniversary of the 1869 Cincinnati Red Stockings, the first openly all-salaried professional baseball team.

We're reluctant to finalize our list of offseason winners because, as you may have heard, there are some prominent unsigned free agents. Not just Bryce Harper and Manny Machado, either.

On this opening week of Spring Training, all 30 Major League teams have one thing in common: optimism. Here's an optimism cheat sheet for each of them.

With less than a week left until Angels pitchers and catchers report to Spring Training, it's time for the final Angels Inbox of the offseason.

All 30 Major League teams will wear special "MLB 150" patches on their uniforms for the entire 2019 season in honor of the 150th anniversary of the 1869 Cincinnati Red Stockings, the first openly all-salaried professional baseball team.

Brad Ausmus held his first media session of the spring on the day pitchers and catchers officially reported on Tuesday, giving updates on rehabbing players such as Shohei Ohtani, Albert Pujols and Zack Cozart.

Spring Training is officially here for the Angels, with pitchers and catchers reporting to Tempe Diablo Stadium on Tuesday. And there's a new face at manager, with Brad Ausmus replacing longtime skipper Mike Scioscia this offseason.

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

On this opening week of Spring Training, all 30 Major League teams have one thing in common: optimism. Here's an optimism cheat sheet for each of them.