High-throughput sequencing of hematologic malignancies and other cancers has revealed recurrent mis-sense mutations of genes encoding pre-mRNA splicing factors. The essential splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired U2AF2 mutations, namely N196K or G301D amino acid substitutions associated with leukemia or solid tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2 proteins, each bound to a prototypical AdML polypyrimidine tract, at 1.5, 1.4, or 1.7 Å resolutions. The N196K residue appears to stabilize the open conformation of U2AF2 with an inter-RNA recognition motif hydrogen bond, in agreement with an increased apparent RNA-binding affinity of the N196K-substituted protein. The G301D residue remains in a similar position as the WT residue, where unfavorable proximity to the RNA phosphodiester could explain the decreased RNA-binding affinity of the G301D-substituted protein. We found that expression of the G301D-substituted U2AF2 protein reduces splicing of a minigene transcript carrying prototypical splice sites. We further show that expression of either N196K- or G301D-substituted U2AF2 can subtly alter splicing of representative endogenous transcripts, despite the presence of endogenous, WT U2AF2 such as would be present in cancer cells. Altogether, our results demonstrate that acquired U2AF2 mutations such as N196K and G301D are capable of dysregulating gene expression for neoplastic transformation.

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.

1 July 2020

23 July 2020

26 August 2020

Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week–old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.

Silencing the Guns in Africa by 2030: Lessons from Mozambique 17 February 2023 — 7:00AM TO 9:00AM Anonymous (not verified) 7 February 2023 Addis Ababa and online

A hybrid event in Addis Ababa reflecting on Mozambique’s 2019 peace agreement and the lessons it offers for the African Union’s ‘Silencing the Guns’ agenda by 2030.

This event will explore opportunities for furthering the AU’s Silencing the Guns agenda by 2030 to assist Africa’s transformative development, highlighting lessons learnt from Mozambique’s experience.

The ‘Silencing the Guns in Africa’ agenda, a flagship initiative of the African Union’s (AU) Agenda 2063, aspires to end all wars and conflict, prevent genocide, and stop gender-based violence.

The 2019 peace agreement in Mozambique and the subsequent disarmament, demobilization and reintegration process supported by the United Nations (UN) but implemented by Mozambique’s government and institutions, provides experience and learning for other continental conflicts that have recently ended or resumed.

Mozambique is seeking to break from the cyclical ‘conflict trap’ where once a country experiences one civil war, it is significantly more likely to experience additional episodes of violence.

Since the end of Mozambique’s civil war in 1992, targeted armed conflict by RENAMO resumed in 2013 and ended through the new agreement in August 2019. The final reintegration into civilian life of former Mozambican combatants of opposition RENAMO will be completed in 2023.

Mozambique and Switzerland – a key supporter of successive Mozambican peace processes – have become non-permanent members of the UN Security Council for the first time in their respective histories.

At a moment when old vulnerabilities and new threats are apparent on the African continent, this seminar, held by Chatham House in partnership with the United Nations Development Programme (UNDP), explores opportunities to furthering the AU’s Silencing the Guns agenda by 2030 to assist Africa’s transformative development, as outlined by the UNDP in a report published in February 2022.

This hybrid event is held in partnership with the African Union Commission and the United Nations Development Programme (UNDP).

This event will also be broadcast live via the Africa Programme Facebook page.

Visual Abstract

Madison T Wright
Nov 18, 2020; 0:RA120.002168v1-mcp.RA120.002168
Research

Qin Zhang
Nov 17, 2020; 0:RA120.002325v1-mcp.RA120.002325
Research

Leandro Xavier Neves
Nov 11, 2020; 0:RA120.002227v1-mcp.RA120.002227
Research

Gle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where, and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel 10-amino acid aggregation-prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation-prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and nonstressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis.

Genetic mutations related to ALS, a progressive neurological disease, have been discovered in the gene encoding σ-1 receptor (σ1R). We previously reported that σ1RE102Q elicits toxicity in cells. The σ1R forms oligomeric states that are regulated by ligands. Nevertheless, little is known about the effect of ALS-related mutations on oligomer formation. Here, we transfected NSC-34 cells, a motor neuronal cell line, and HEK293T cells with σ1R-mCherry (mCh), σ1RE102Q-mCh, or nontagged forms to investigate detergent solubility and subcellular distribution using immunocytochemistry and fluorescence recovery after photobleaching. The oligomeric state was determined using crosslinking procedure. σ1Rs were soluble to detergents, whereas the mutants accumulated in the insoluble fraction. Within the soluble fraction, peak distribution of mutants appeared in higher sucrose density fractions. Mutants formed intracellular aggregates that were co-stained with p62, ubiquitin, and phosphorylated pancreatic eukaryotic translation initiation factor-2-α kinase in NSC-34 cells but not in HEK293T cells. The aggregates had significantly lower recovery in fluorescence recovery after photobleaching. Acute treatment with σ1R agonist SA4503 failed to improve recovery, whereas prolonged treatment for 48 h significantly decreased σ1RE102Q-mCh insolubility and inhibited apoptosis. Whereas σ1R-mCh formed monomers and dimers, σ1RE102Q-mCh also formed trimers and tetramers. SA4503 reduced accumulation of the four types in the insoluble fraction and increased monomers in the soluble fraction. The σ1RE102Q insolubility was diminished by σ1R-mCh co-expression. These results suggest that the agonist and WT σ1R modify the detergent insolubility, toxicity, and oligomeric state of σ1RE102Q, which may lead to promising new treatments for σ1R-related ALS.

Type I and III interferons induce expression of the “myxovirus resistance proteins” MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles (“biomolecular condensates”). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40–50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20–30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.

Priyanka Tripathi
Dec 30, 2020; 0:jlr.RA120001190v1-jlr.RA120001190
Research Articles

Abudukadier Abulizi
Dec 1, 2020; 61:1565-1576
Research Articles

Yueming Hu
Dec 11, 2020; 0:jlr.RA120000919v1-jlr.RA120000919
Research Articles

Roux-en-Y gastric bypass (RYGB) is one of the most commonly performed weight-loss procedures, but how severe obesity and RYGB affects circulating HDL-associated microRNAs (miRNAs) remains unclear. Here, we aim to investigate how HDL-associated miRNAs are regulated in severe obesity and how weight loss after RYGB surgery affects HDL-miRNAs. Plasma HDL were isolated from patients with severe obesity (n=53) before, 6 and 12 months after RYGB by immunoprecipitation using goat anti-human apoA-I microbeads. HDL were also isolated from 18 healthy participants. miRNAs were extracted from isolated HDL and levels of miR-24, miR-126, miR-222 and miR-223 were determined by TaqMan miRNA assays. We found that HDL-associated miR-126, miR-222 and miR-223 levels, but not miR-24 levels, were significantly higher in patients with severe obesity when compared with healthy controls. There were significant increases in HDL-associated miR-24, miR-222 and miR-223 at 12 months after RYGB. Additionally, cholesterol efflux capacity and paraoxonase (PON1) activity were increased and intracellular adhesion molecule-1 (ICAM-1) levels decreased. The increases in HDL-associated miR-24 and miR-223 were positively correlated with increase in cholesterol efflux capacity (r=0.326, P=0.027 and r=0.349, P=0.017 respectively). An inverse correlation was observed between HDL-associated miR-223 and ICAM-1 at baseline. Together, these findings show that HDL-associated miRNAs are differentially regulated in healthy versus patients with severe obesity and are altered after RYGB. These findings provide insights into how miRNAs are regulated in obesity before and after weight reduction, and may lead to the development of novel treatment strategies for obesity and related metabolic disorders.

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. 

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.

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp^–/–) mice and age-weight matched wild-type control mice. Young (10–12-week-old) L-Mttp^–/– mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp^–/– mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKC activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp^–/– mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKC activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp^–/– mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKC activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp^–/– mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp^–/– mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp^–/– mice.

Ventilator-associated pneumonia (VAP) is a common hospital-acquired infection, leading to high morbidity and mortality. Currently, bronchoalveolar lavage (BAL) is used in hospitals for VAP diagnosis and guiding treatment options. Although BAL collection procedures are invasive, alternatives such as endotracheal aspirates (ETA) may be of diagnostic value, however, their use has not been thoroughly explored. Longitudinal ETA and BAL were collected from 16 intubated patients up to 15 days, of which 11 developed VAP. We conducted a comprehensive LC–MS/MS based proteome and metabolome characterization of longitudinal ETA and BAL to detect host and pathogen responses to VAP infection. We discovered a diverse ETA proteome of the upper airways reflective of a rich and dynamic host-microbe interface. Prior to VAP diagnosis by microbial cultures from BAL, patient ETA presented characteristic signatures of reactive oxygen species and neutrophil degranulation, indicative of neutrophil mediated pathogen processing as a key host response to the VAP infection. Along with an increase in amino acids, this is suggestive of extracellular membrane degradation resulting from proteolytic activity of neutrophil proteases. The metaproteome approach successfully allowed simultaneous detection of pathogen peptides in patients' ETA, which may have potential use in diagnosis. Our findings suggest that ETA may facilitate early mechanistic insights into host-pathogen interactions associated with VAP infection and therefore provide its diagnosis and treatment.

Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin–proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques—including MS—have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.

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

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.

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.

The tumor marker cancer antigen 15-3 (CA 15-3) is that most commonly used to monitor metastatic breast cancer during active therapy and surveillance for disease recurrence after treatment. The association of CA 15-3 and ¹⁸F-FDG PET/CT findings can be considered complementary, since any significant rise may indicate the presence of disease and imaging is able to map the tumor sites. Although current guidelines do not recommend the routine performance of CA 15-3 in asymptomatic patients being followed up after definitive breast cancer treatment, most oncologists perform serial assessment of the tumor markers as part of routine follow-up of patients. The aim of this study was to evaluate the correlation between CA 15-3 levels and ¹⁸F-FDG PET/CT scan findings in patients with recurrent breast cancer. Methods: This was a cross-sectional study with data collected retrospectively. Patients being evaluated for breast cancer recurrence with ¹⁸F-FDG PET/CT imaging and CA 15-3 level were included. Evaluation of the association between CA 15-3 levels and ¹⁸F-FDG PET/CT scan findings was then done. Results: In total, 154 cases were included in this study; 62 patients had recurrence (positive) on the ¹⁸F-FDG PET/CT scans, whereas 92 patients had normal (negative) findings on follow-up ¹⁸F-FDG PET/CT scans. There was an association between CA 15-3 levels and the presence or absence of recurrence on ¹⁸F-FDG PET/CT scans, with 84.4% (27/32) of patients who had elevated CA 15-3 levels having disease recurrence on ¹⁸F-FDG PET/CT and 84.4% (27/32) of patients who had elevated CA 15-3 levels having disease recurrence on ¹⁸F-FDG PET/CT as well as a correlation with the burden of metastases. Most patients with disease recurrence on ¹⁸F-FDG PET/CT, however, had normal CA 15-3 levels. Conclusion: Higher CA 15-3 levels correlate with breast cancer recurrence on ¹⁸F-FDG PET/CT as well as with burden of metastasis. Notably, CA 15-3 levels within the reference range do not exclude breast cancer disease recurrence since more than half of patients with recurrence had normal CA 15-3 levels. ¹⁸F-FDG PET/CT should therefore be considered in patients with suspected breast cancer recurrence but normal CA 15-3 levels.

EU-Turkey Customs Union: Lessons for Brexit 15 March 2018 — 11:00AM TO 12:00PM Anonymous (not verified) 5 March 2018 Chatham House, London

Turkey and the EU are preparing to open negotiations to modernize their 22 year old customs union and expand its scope beyond goods to include services, public procurement and a more liberal regime for agriculture. At the same time, the UK is debating whether to seek a customs union with the EU to facilitate a frictionless flow of goods and to prevent a hard border with Ireland. The speaker will discuss Turkey’s customs union modernization agenda and share his insights on the lessons for the UK’s future relationship with the EU.

Attendance at this event is by invitation only.

EU-Turkey Customs Union: Prospects for Modernization and Lessons for Brexit 12 December 2018 — 12:30PM TO 1:30PM Anonymous (not verified) 26 November 2018 Chatham House | 10 St James's Square | London | SW1Y 4LE

Turkey and the EU have been in a customs union since 1995. Both sides recognize that the current agreement is in need of modernization and have agreed to open negotiations to expand its scope to include services, public procurement, agriculture and other elements that would help bring it into the 21st century.

At the same time, the UK Parliament is debating whether to approve the agreement on the UK’s withdrawal from the EU. It includes a backstop which – if triggered – would keep the UK and the EU in a single customs territory which would limit the disruption of withdrawal but hamper Britain’s ability to pursue an independent trade policy. The political declaration proposes building on this customs arrangement as the basis for the future relationship.

In this context, the speaker will discuss the current EU-Turkey customs union arrangement and its shortcomings, examine the prospects for its modernization and share his insights on the lessons for the UK’s future trading relationship with the EU.

The event will launch the briefing paper ‘EU-Turkey Customs Union: Prospects for Modernization and Lessons for Brexit’.

Attendance at this event is by invitation only.

McKesson Medical-Surgical Inc. entered into an agreement with the Labor Department on Monday resolving employment discrimination issues involving nearly 900 Black, Hispanic, and White applicants at a distribution center

CHANDLER, Ariz., Oct. 23, 2024 — The global edge computing market is expected to grow by more than 30 percent in the next five years, serving mission-critical applications in the […]

The post Microchip Expands 64-Bit Portfolio with High-Performance PIC64HX Microprocessors for Edge Computing appeared first on HPCwire.

Noritaka Hoshi, Senior Manager, AI Platform Division, talks about the impetus for and challenges within the development of SX-Aurora TSUBASA or a massive SIMD, created to handle enormous computing and […]

The post Advance Science, Technology and Sophistication with SX-Aurora TSUBASA or Vector Processor or Vector Engine (VE) appeared first on HPCwire.

FREMONT, Calif., Nov. 6, 2024 — AMAX, an AMD Elite Partner and a global leader in end-to-end GPU cluster solutions and advanced cooling technologies, announced its latest lineup of servers powered […]

The post AMAX Expands Server Portfolio with New AMD EPYC 9005 Series Processors for AI and HPC Workloads appeared first on HPCwire.

A B-2 Spirit stealth bomber made an emergency landing at Whiteman Air Force Base in Missouri earlier this week after the high-tech aircraft malfunctioned during a training flight.

A Missouri thrift store is unraveling the mystery of a cache of World War II love letters found in a donation bin.

Up to 50% of people admit cheating on their partner.

Chief technology officers are facing an unprecedented test of digital preparedness due to the coronavirus pandemic, struggling with shortfalls of available learning devices and huge Wi-Fi access challenges.

Chief technology officers are facing an unprecedented test of digital preparedness due to the coronavirus pandemic, struggling with shortfalls of available learning devices and huge Wi-Fi access challenges.

Marc Tucker reviews David Driscoll's new book, 'Commitment and Common Sense', and describes how the Massachusetts reforms are comparable to those in top performing education systems around the world.

Watch a discussion between three educators who ran for their state legislatures about their experiences on the campaign trail.

Students returning to schools in Virginia and New York this fall will be required to participate in mental-health education as part of their health and physical education courses.