Assembly Mechanism of Mucin and von Willebrand Factor Polymers.

The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.

The coding capacity of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic1. To understand the pathogenicity and antigenic potential of SARS-CoV-2 and to develop therapeutic tools, it is essential to profile the full repertoire of its expressed proteins. The current map of SARS-CoV-2 coding capacity is based on computational predictions and relies on homology with other coronaviruses. As the protein complement varies among coronaviruses, especially in regard to the variety of accessory proteins, it is crucial to characterize the specific range of SARS-CoV-2 proteins in an unbiased and open-ended manner. Here, using a suite of ribosome-profiling techniques2-4, we present a high-resolution map of coding regions in the SARS-CoV-2 genome, which enables us to accurately quantify the expression of canonical viral open reading frames (ORFs) and to identify 23 unannotated viral ORFs. These ORFs include upstream ORFs that are likely to have a regulatory role, several in-frame internal ORFs within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; instead, virus translation dominates host translation because of the high levels of viral transcripts. Our work provides a resource that will form the basis of future functional studies.

Using Constellation Pharmacology to Characterize a Novel α-Conotoxin from Conus ateralbus.

The venom of cone snails has been proven to be a rich source of bioactive peptides that target a variety of ion channels and receptors. α-Conotoxins (αCtx) interact with nicotinic acetylcholine receptors (nAChRs) and are powerful tools for investigating the structure and function of the various nAChR subtypes. By studying how conotoxins interact with nAChRs, we can improve our understanding of these receptors, leading to new insights into neurological diseases associated with nAChRs. Here, we describe the discovery and characterization of a novel conotoxin from Conus ateralbus, αCtx-AtIA, which has an amino acid sequence homologous to the well-described αCtx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic αCtx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that αCtx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an α7 positive allosteric modulator, PNU-120596 (PNU). However, αCtx-AtIA did not display any activity in the absence of PNU. These findings were further validated using two-electrode voltage clamp electrophysiology performed on oocytes overexpressing mouse α3ß4, α6/α3ß4 and α7 nAChRs subtypes. We observed that αCtx-AtIA displayed no or low potency in blocking α3ß4 and α6/α3ß4 receptors, respectively, but improved potency and selectivity to block α7 nAChRs when compared with αCtx-PeIA. Through the synthesis of two additional analogs of αCtx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide.

Cross-cultural adaptation, validation and psychometric evaluation of the International Hip Outcome Tool 12 (iHOT12) to Hebrew.

BACKGROUND: The "International Hip Outcome Tool 12" (iHOT12) is a self-administered patient-reported outcome tool for measuring health-related quality of life and physical functioning in young and active patients with hip pathology. Since the iHOT12 has become widely used, we sought to translate and validate it for Hebrew-speaking populations. The aims of this study were: (1) To translate and culturally adapt the iHOT12 into Hebrew using established guidelines. (2) To test the new Hebrew version for validity, and (3) reliability. METHODS: The iHOT12 was translated and culturally adapted from English to Hebrew (iHOT12-H) according to the COSAMIN guidelines. For validity, the iHOT12-H and Western Ontario and McMaster universities osteoarthritis index (WOMAC) were completed by 200 patients with hip pathology. Exploratory factor analysis was used to assess structural validity. Subsequently, 51 patients repeated the iHOT12-H within a 2-week interval. Intraclass Correlation Coefficient (ICC), Cronbach alpha, and Standard Error of Measurement (SEM) were calculated to assess reliability. RESULTS: Construct validity: iHOT12-H correlated strongly to the WOMAC scores (r = -0.82, P < 0.001, Spearman). Factor analysis revealed a two-factor structure. Cronbach's alpha was 0.953 confirming internal consistency to be highly satisfactory. Test-retest correlation of the iHOT12-H was excellent with an ICC = 0.956 (95% CI 0.924-0.974). There was no floor or ceiling effect. CONCLUSION: The iHOT12 Hebrew version has excellent reliability, good construct validity and can be used as a measurement tool for physical functioning and quality of life in young, physically active patients with hip pathology. This study will serve Israeli researchers in evaluating treatment effectiveness for these patients. Moreover, it will also enable multinational cooperation in the study of hip pathology.

Optimized Glycopeptide Enrichment Method-It Is All About the Sauce.

Protein glycosylation is a family of posttranslational modifications that play a crucial role in many biological pathways and diseases. The enrichment and analysis of such a diverse family of modifications are very challenging because of the number of possible glycan-peptide combinations. Among the methods used for the enrichment of glycopeptides, boronic acid never lived up to its promise. While most studies focused on improving the affinity of the boronic acids to the sugars, we discovered that the buffer choice is just as important for successful enrichment if not more so. We show that an amine-less buffer allows for the best glycoproteomic coverage, in human plasma and brain specimens, improving total quantified glycopeptides by over 10-fold, and reaching 1598 N-linked glycopeptides in the brain and 737 in nondepleted plasma. We speculate that amines compete with the glycans for boronic acid binding, and therefore the elimination of them improved the method significantly.

RawBeans: A Simple, Vendor-Independent, Raw-Data Quality-Control Tool.

Every laboratory performing mass-spectrometry-based proteomics strives to generate high-quality data. Among the many factors that impact the outcome of any experiment in proteomics is the LC-MS system performance, which should be monitored within each specific experiment and also long term. This process is termed quality control (QC). We present an easy-to-use tool that rapidly produces a visual, HTML-based report that includes the key parameters needed to monitor the LC-MS system performance, with a focus on monitoring the performance within an experiment. The tool, named RawBeans, generates a report for individual files or for a set of samples from a whole experiment. We anticipate that it will help proteomics users and experts evaluate raw data quality independent of data processing. The tool is available at https://bitbucket.org/incpm/prot-qc/downloads. The mass-spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD022816.

Phosphoproteomics of Fibroblast Growth Factor 1 (FGF1) Signaling in Chondrocytes: Identifying the Signature of Inhibitory Response.

Fibroblast growth factor (FGF) signaling is vital for many biological processes, beginning with development. The importance of FGF signaling for skeleton formation was first discovered by the analysis of genetic FGFR mutations which cause several bone morphogenetic disorders, including achondroplasia, the most common form of human dwarfism. The formation of the long bones is mediated through proliferation and differentiation of highly specialized cells - chondrocytes.Chondrocytes respond to FGF with growth inhibition, a unique response which differs from the proliferative response of the majority of cell types; however, its molecular determinants are still unclear. Quantitative phosphoproteomic analysis was utilized to catalogue the proteins whose phosphorylation status is changed upon FGF1 treatment. The generated dataset consists of 756 proteins. We could localize the divergence between proliferative (canonical) and inhibitory (chondrocyte specific) FGF transduction pathways immediately upstream of AKT kinase. Gene Ontology (GO) analysis of the FGF1 regulated peptides revealed that many of the identified phosphorylated proteins are assigned to negative regulation clusters, in accordance with the observed inhibitory growth response. This is the first time a comprehensive subset of proteins involved in FGF inhibitory response is defined. We were able to identify a number of targets and specifically discover glycogen synthase kinase3ß (GSK3ß) as a novel key mediator of FGF inhibitory response in chondrocytes.

Characterization of the First Conotoxin from Conus ateralbus, a Vermivorous Cone Snail from the Cabo Verde Archipelago.

Conus ateralbus is a cone snail endemic to the west side of the island of Sal, in the Cabo Verde Archipelago off West Africa. We describe the isolation and characterization of the first bioactive peptide from the venom of this species. This 30AA venom peptide is named conotoxin AtVIA (δ-conotoxin-like). An excitatory activity was manifested by the peptide on a majority of mouse lumbar dorsal root ganglion neurons. An analog of AtVIA with conservative changes on three amino acid residues at the C-terminal region was synthesized and this analog produced an identical effect on the mouse neurons. AtVIA has homology with δ-conotoxins from other worm-hunters, which include conserved sequence elements that are shared with δ-conotoxins from fish-hunting Conus. In contrast, there is no comparable sequence similarity with δ-conotoxins from the venoms of molluscivorous Conus species. A rationale for the potential presence of δ-conotoxins, that are potent in vertebrate systems in two different lineages of worm-hunting cone snails, is discussed.

Quiescin sulfhydryl oxidase 1 (QSOX1) glycosite mutation perturbs secretion but not Golgi localization.

Quiescin sulfhydryl oxidase 1 (QSOX1) catalyzes the formation of disulfide bonds in protein substrates. Unlike other enzymes with related activities, which are commonly found in the endoplasmic reticulum, QSOX1 is localized to the Golgi apparatus or secreted. QSOX1 is upregulated in quiescent fibroblast cells and secreted into the extracellular environment, where it contributes to extracellular matrix assembly. QSOX1 is also upregulated in adenocarcinomas, though the extent to which it is secreted in this context is currently unknown. To achieve a better understanding of factors that dictate QSOX1 localization and function, we aimed to determine how post-translational modifications affect QSOX1 trafficking and activity. We found a highly conserved N-linked glycosylation site to be required for QSOX1 secretion from fibroblasts and other cell types. Notably, QSOX1 lacking a glycan at this site arrives at the Golgi, suggesting that it passes endoplasmic reticulum quality control but is not further transported to the cell surface for secretion. The QSOX1 transmembrane segment is dispensable for Golgi localization and secretion, as fully luminal and transmembrane variants displayed the same trafficking behavior. This study provides a key example of the effect of glycosylation on Golgi exit and contributes to an understanding of late secretory sorting and quality control.

Observation versus treatment among men with favorable risk prostate cancer in a community-based integrated health care system: a retrospective cohort study.

BACKGROUND: The objective of this study was to describe overall survival and the management of men with favorable risk prostate cancer (PCa) within a large community-based health care system in the United States. METHODS: A retrospective cohort study was conducted using linked electronic health records from men aged ≥40 years with favorable risk PCa (T1 or 2, PSA ≤15, Gleason ≤7 [3 + 4]) diagnosed between January 2005 and October 2013. Cohorts were defined as receiving any treatment (IMT) or no treatment (OBS) within 6 months after index PCa diagnosis. Cohorts' characteristics were compared between OBS and IMT; monitoring patterns were reported for OBS within the first 18 and 24 months. Cox Proportional Hazards models were used for multivariate analysis of overall survival. RESULTS: A total of 1425 men met the inclusion criteria (OBS 362; IMT 1063). The proportion of men managed with OBS increased from 20% (2005) to 35% (2013). The OBS group was older (65.6 vs 62.8 years, p < 0.01), had higher Charlson comorbidity index scores (CCI ≥2, 21.5% vs 12.2%, p < 0.01), and had a higher proportion of low-risk PCa (65.2% vs 55.0%, p < 0.01). For the OBS cohort, 181 of the men (50%) eventually received treatment. Among those remaining on OBS for ≥24 months (N = 166), 88.6% had ≥1 follow-up PSA test and 26.5% received ≥1 follow-up biopsy within the 24 months. The unadjusted mortality rate was higher for OBS compared with IMT (2.7 vs 1.3/100 person-years [py]; p < 0.001). After multivariate adjustment, there was no significant difference in all-cause mortality between OBS and IMT groups (HR 0.73, p = 0.138). CONCLUSIONS: Use of OBS management increased over the 10-year study period. Men in the OBS cohort had a higher proportion of low-risk PCa. No differences were observed in overall survival between the two groups after adjustment of covariates. These data provide insights into how favorable risk PCa was managed in a community setting.

Triple-Stage Mass Spectrometry Unravels the Heterogeneity of an Endogenous Protein Complex.

Protein complexes often represent an ensemble of different assemblies with distinct functions and regulation. This increased complexity is enabled by the variety of protein diversification mechanisms that exist at every step of the protein biosynthesis pathway, such as alternative splicing and post transcriptional and translational modifications. The resulting variation in subunits can generate compositionally distinct protein assemblies. These different forms of a single protein complex may comprise functional variances that enable response and adaptation to varying cellular conditions. Despite the biological importance of this layer of complexity, relatively little is known about the compositional heterogeneity of protein complexes, mostly due to technical barriers of studying such closely related species. Here, we show that native mass spectrometry (MS) offers a way to unravel this inherent heterogeneity of protein assemblies. Our approach relies on the advanced Orbitrap mass spectrometer capable of multistage MS analysis across all levels of protein organization. Specifically, we have implemented a two-step fragmentation process in the inject flatapole device, which was converted to a linear ion trap, and can now probe the intact protein complex assembly, through its constituent subunits, to the primary sequence of each protein. We demonstrate our approach on the yeast homotetrameric FBP1 complex, the rate-limiting enzyme in gluconeogenesis. We show that the complex responds differently to changes in growth conditions by tuning phosphorylation dynamics. Our methodology deciphers, on a single instrument and in a single measurement, the stoichiometry, kinetics, and exact position of modifications, contributing to the exposure of the multilevel diversity of protein complexes.

Regulation of AP-1 by MAPK Signaling in Metal-Stressed Sea Anemone.

BACKGROUND/AIMS: AP-1 transcription factor plays a conserved role in the immediate response to stress. Activation of AP-1 members jun and fos is mediated by complex signaling cascades to control cell proliferation and survival. To understand the evolution of this broadly-shared pathway, we studied AP-1 regulation by MAPK signaling in a basal metazoan. METHODS: Metal- stressed cnidarian Nematostella vectensis anemones were tested with kinase inhibitors and analyzed for gene expression levels and protein phosphorylation. RESULTS: We show that in cnidarian, AP-1 is regulated differently than in bilaterian models. ERK2 and ERK5, the main MAPK drivers of AP-1 activation in Bilateria, down-regulated fos1 and jun1 transcription in anemones exposed to metal stress, whereas p38 MAPK, triggered transcription of jun1 but not fos1. Furthermore, our results reveal that GSK3-ß is the main driver of the immediate stress response in Nematostella. GSK3-ß triggered transcription of AP-1 and two other stress-related genes, egr1 and hsp70. Finally, phylogenetic analysis and protein characterization show that while MAPKs and GSK3-ß are evolutionarily conserved, Fos and Jun proteins in Nematostella and other cnidarians lack important regulatory and phosphorylation sites found in Bilateria. CONCLUSION: These findings reveal alternative network interactions of conserved signaling kinases, providing insight into the evolutionary plasticity of immediate stress response mechanisms.

The dynamically evolving nematocyst content of an anthozoan, a scyphozoan, and a hydrozoan.

Nematocytes, the stinging cells of cnidarians, are the most evolutionarily ancient venom apparatus. These nanosyringe-like weaponry systems reach pressures of approximately 150 atmospheres before discharging and punching through the outer layer of the prey or predator at accelerations of more than 5 million g, making them one of the fastest biomechanical events known. To gain better understanding of the function of the complex, phylum-specific nematocyst organelle, and its venom payload, we compared the soluble nematocyst's proteome from the sea anemone Anemonia viridis, the jellyfish Aurelia aurita, and the hydrozoan Hydra magnipapillata, each belonging to one of the three basal cnidarian lineages which diverged over 600 Ma. Although the basic morphological and functional characteristics of the nematocysts of the three organisms are similar, out of hundreds of proteins identified in each organism, only six are shared. These include structural proteins, a chaperone which may help maintain venon activity over extended periods, and dickkopf, an enigmatic Wnt ligand which may also serve as a toxin. Nevertheless, many protein domains are shared between the three organisms' nematocyst content suggesting common proteome functionalities. The venoms of Hydra and Aurelia appear to be functionally similar and composed mainly of cytotoxins and enzymes, whereas the venom of the Anemonia is markedly unique and based on peptide neurotoxins. Cnidarian venoms show evidence for functional recruitment, yet evidence for diversification through positive selection, common to other venoms, is lacking. The final injected nematocyst payload comprises a mixture of dynamically evolving proteins involved in the development, maturation, maintenance, and discharge of the nematocysts, which is unique to each organism and potentially to each nematocyst type.

Diagnostic Performance of Risk of Ovarian Malignancy Algorithm Against CA125 and HE4 in Connection With Ovarian Cancer: A Meta-analysis.

OBJECTIVES: The aim of this study was to determine whether the Risk of Ovarian Malignancy Algorithm (ROMA) is more accurate than the human epididymis 4 (HE4) or carbohydrate antigen 125 (CA125) biomarkers with respect to the differential diagnosis of women with a pelvic mass. The secondary objective is to assess the performance of ROMA in early-stage ovarian cancer (OC) and late-stage OC, as well as premenopausal and postmenopausal patient populations. METHODS/MATERIALS: The PubMed and Google Scholar databases were searched for relevant clinical studies. Eligibility criteria included comparison of ROMA with both HE4 and CA125 levels in OC (unspecified, epithelial, and borderline ovarian tumors), use of only validated ROMA assays, presentation of area under the curve and sensitivity/specificity data, and results from early-stage OC, late-stage OC and premenopausal and postmenopausal women. Area under the curve (AUC), sensitivity/specificity, and the diagnostic odds ratio (DOR) results were summarized. RESULTS: Five studies were selected comprising 1975 patients (premenopausal, n = 1033; postmenopausal, n = 925; benign, n = 1387; early stage, n = 192; and late stage, n = 313). On the basis of the AUC (95% confidence interval) data for all patients, ROMA (0.921 [0.855-0.960]) had a numerically greater diagnostic performance than CA125 (0.883 [0.771-0.950]) and HE4 (0.899 [0.835-0.943]). This was also observed in each of the subgroup populations, in particular, the postmenopausal patients and patients with early OC. The sensitivity and specificity (95% confidence interval) results showed ROMA (sensitivity, 0.873 [0.752-0.940]; specificity, 0.855 [0.719-0.932]) to be numerically superior to CA125 (sensitivity, 0.796 [0.663-0.885]; specificity, 0.825 [0.662-0.919]) and HE4 (sensitivity, 0.817 [0.683-0.902]; specificity, 0.851 [0.716-0.928]) in all patients and for the early- and late-stage OC subgroups. Finally, the ROMA log DOR results were better than HE4 and CA125 log DOR results especially for the early-stage patient group. CONCLUSIONS: The results presented support the use of ROMA to improve clinical decision making, most notably in patients with early OC.

Squeezers and leaf-cutters: differential diversification and degeneration of the venom system in toxicoferan reptiles.

Although it has been established that all toxicoferan squamates share a common venomous ancestor, it has remained unclear whether the maxillary and mandibular venom glands are evolving on separate gene expression trajectories or if they remain under shared genetic control. We show that identical transcripts are simultaneously expressed not only in the mandibular and maxillary glands, but also in the enigmatic snake rictal gland. Toxin molecular frameworks recovered in this study were three-finger toxin (3FTx), CRiSP, crotamine (beta-defensin), cobra venom factor, cystatin, epididymal secretory protein, kunitz, L-amino acid oxidase, lectin, renin aspartate protease, veficolin, and vespryn. We also discovered a novel low-molecular weight disulfide bridged peptide class in pythonid snake glands. In the iguanian lizards, the most highly expressed are potentially antimicrobial in nature (crotamine (beta-defensin) and cystatin), with crotamine (beta-defensin) also the most diverse. However, a number of proteins characterized from anguimorph lizards and caenophidian snakes with hemotoxic or neurotoxic activities were recruited in the common toxicoferan ancestor and remain expressed, albeit in low levels, even in the iguanian lizards. In contrast, the henophidian snakes express 3FTx and lectin toxins as the dominant transcripts. Even in the constricting pythonid and boid snakes, where the glands are predominantly mucous-secreting, low-levels of toxin transcripts can be detected. Venom thus appears to play little role in feeding behavior of most iguanian lizards or the powerful constricting snakes, and the low levels of expression argue against a defensive role. However, clearly the incipient or secondarily atrophied venom systems of these taxa may be a source of novel compounds useful in drug design and discovery.

Proteomics and deep sequencing comparison of seasonally active venom glands in the platypus reveals novel venom peptides and distinct expression profiles.

The platypus is a venomous monotreme. Male platypuses possess a spur on their hind legs that is connected to glands in the pelvic region. They produce venom only during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in- and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of the breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland, and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified included antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines, and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine, and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution.

VWD domain stabilization by autocatalytic Asp-Pro cleavage.

Domains known as von Willebrand factor type D (VWD) are found in extracellular and cell-surface proteins including von Willebrand factor, mucins, and various signaling molecules and receptors. Many VWD domains have a glycine-aspartate-proline-histidine (GDPH) amino-acid sequence motif, which is hydrolytically cleaved post-translationally between the aspartate (Asp) and proline (Pro). The Fc IgG binding protein (FCGBP), found in intestinal mucus secretions and other extracellular environments, contains 13 VWD domains, 11 of which have a GDPH cleavage site. In this study, we investigated the structural and biophysical consequences of Asp-Pro peptide cleavage in a representative FCGBP VWD domain. We found that endogenous Asp-Pro cleavage increases the resistance of the domain to exogenous proteolytic degradation. Tertiary structural interactions made by the newly generated chain termini, as revealed by a crystal structure of an FCGBP segment containing the VWD domain, may explain this observation. Notably, the Gly-Asp peptide bond, upstream of the cleavage site, assumed the cis configuration in the structure. In addition to these local features of the cleavage site, a global organizational difference was seen when comparing the FCGBP segment structure with the numerous other structures containing the same set of domains. Together, these data illuminate the outcome of GDPH cleavage and demonstrate the plasticity of proteins with VWD domains, which may contribute to their evolution for function in a dynamic extracellular environment.

A new biomarker panel for differential diagnosis of cholangiocarcinoma: Results from an exploratory analysis.

INTRODUCTION: Diagnosis of cholangiocarcinoma (CCA) can be challenging due to unclear imaging criteria and difficulty obtaining adequate tissue biopsy. Although serum cancer antigen 19-9 and carcinoembryonic antigen have been proposed as potential diagnostic aids, their use remains limited by insufficient sensitivity and specificity. This exploratory analysis aimed to identify individual- and combinations of serum biomarkers to distinguish CCA from hepatocellular carcinoma (HCC) and chronic liver disease (CLD) controls using samples from a published study. METHODS: This prospective, multicenter, case-control study included patients aged ≥18 years at high-risk of HCC. Serum and ethylene diamine tetraacetic acid-plasma samples were collected prior to any treatment and confirmed diagnosis of HCC or CCA. Fourteen biomarkers (measured by electrochemiluminescence immunoassays or enzyme-linked immunosorbent assays) were subjected to univariate analysis and 13 included in a multivariate analysis (per selected combinations and exhaustive search). RESULTS: Overall, 55 CCA, 306 HCC, and 733 CLD control samples were analyzed. For distinguishing CCA from HCC, alpha-fetoprotein and matrix metalloproteinase-2 (MMP-2) showed the best individual performance (area under the curve (AUC) 86.6% and 84.4%, respectively); tissue inhibitor of metalloproteinase-1 (TIMP-1) was most able to distinguish CCA from CLD (AUC 94.5%) and from HCC + CLD (AUC 88.6%). The combination of MMP-2 and TIMP-1 was the best-performing two-marker panel, with AUC >90% for all comparisons. CONCLUSION: MMP-2 and TIMP-1 are promising biomarkers that could support differential diagnosis of CCA. Incorporating these assays into the diagnostic algorithm could provide additional diagnostic information in a non-invasive, rapid manner, and could supplement existing diagnostic methods.

Egg multivesicular bodies elicit an LC3-associated phagocytosis-like pathway to degrade paternal mitochondria after fertilization.

Mitochondria are maternally inherited, but the mechanisms underlying paternal mitochondrial elimination after fertilization are far less clear. Using Drosophila, we show that special egg-derived multivesicular body vesicles promote paternal mitochondrial elimination by activating an LC3-associated phagocytosis-like pathway, a cellular defense pathway commonly employed against invading microbes. Upon fertilization, these egg-derived vesicles form extended vesicular sheaths around the sperm flagellum, promoting degradation of the sperm mitochondrial derivative and plasma membrane. LC3-associated phagocytosis cascade of events, including recruitment of a Rubicon-based class III PI(3)K complex to the flagellum vesicular sheaths, its activation, and consequent recruitment of Atg8/LC3, are all required for paternal mitochondrial elimination. Finally, lysosomes fuse with strings of large vesicles derived from the flagellum vesicular sheaths and contain degrading fragments of the paternal mitochondrial derivative. Given reports showing that in some mammals, the paternal mitochondria are also decorated with Atg8/LC3 and surrounded by multivesicular bodies upon fertilization, our findings suggest that a similar pathway also mediates paternal mitochondrial elimination in other flagellated sperm-producing organisms.

Glycoproteome-Wide Discovery of Cortical Glycoproteins That May Provide Cognitive Resilience in Older Adults.

BACKGROUND AND OBJECTIVES: Molecular omics studies have identified proteins related to cognitive resilience but unrelated to Alzheimer disease and Alzheimer disease-related dementia (AD/ADRD) pathologies. Posttranslational modifications of proteins with glycans can modify protein function. In this study, we identified glycopeptiforms associated with cognitive resilience. METHODS: We studied brains from adults with annual cognitive testing with postmortem indices of 10 AD/ADRD pathologies and proteome-wide data from dorsal lateral prefrontal cortex (DLPFC). We quantified 11, 012 glycopeptiforms from DLPFC using liquid chromatography with tandem mass spectrometry. We used linear mixed-effects models to identify glycopeptiforms associated with cognitive decline correcting for multiple comparisons (p < 5 × 10-6). Then, we regressed out the effect of AD/ADRD pathologies to identify glycopeptiforms that may provide cognitive resilience. RESULTS: We studied 366 brains, average age at death 89 years, and 70% female with no cognitive impairment = 152, mild cognitive impairment = 93, and AD = 121 cognitive status at death. In models adjusting for age, sex and education, 11 glycopeptiforms were associated with cognitive decline. In further modeling, 8 of these glycopeptiforms remained associated with cognitive decline after adjusting for AD/ADRD pathologies: NPTX2a (Est., 0.030, SE, 0.005, p = 1 × 10-4); NPTX2b (Est.,0.019, SE, 0.005, p = 2 × 10-4) NECTIN1(Est., 0.029, SE, 0.009, p = 9 × 10-4), NPTX2c (Est., 0.015, SE, 0.004, p = 9 × 10-4), HSPB1 (Est., -0.021, SE, 0.006, p = 2 × 10-4), PLTP (Est., -0.027, SE, 0.009, p = 4.2 × 10-3), NAGK (Est., -0.027, SE, 0.008, p = 1.4 × 10-3), and VAT1 (Est., -0.020, SE, 0.006, p = 1.1 × 10-3). Higher levels of 4 resilience glycopeptiforms derived through glycosylation were associated with slower decline and higher levels of 4 derived through glycation were related to faster decline. Together, these 8 glycopeptiforms accounted for an additional 6% of cognitive decline over the 33% accounted for the 10 brain pathologies and demographics. All 8 resilience glycopeptiforms remained associated with cognitive decline after adjustments for the expression level of their corresponding protein. Exploratory gene ontology suggested that molecular mechanisms of glycopeptiforms associated with cognitive decline may involve metabolic pathways including pyruvate and NADH pathways and highlighted the importance of molecular mechanisms involved in glucose metabolism. DISCUSSION: Glycopeptiforms in aging brains may provide cognitive resilience. Targeting these glycopeptiforms may lead to therapies that maintain cognition through resilience.