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Post-translational modification (PTM) of antigens provides an additional source of specificities targeted by immune responses to tumors or pathogens, but identifying antigen PTMs and assessing their role in shaping the immunopeptidome is challenging. Here we describe the Protein Modification Integrated Search Engine (PROMISE), an antigen discovery pipeline that enables the analysis of 29 different PTM combinations from multiple clinical cohorts and cell lines. We expanded the antigen landscape, uncovering human leukocyte antigen class I binding motifs defined by specific PTMs with haplotype-specific binding preferences and revealing disease-specific modified targets, including thousands of new cancer-specific antigens that can be shared between patients and across cancer types. Furthermore, we uncovered a subset of modified peptides that are specific to cancer tissue and driven by post-translational changes that occurred in the tumor proteome. Our findings highlight principles of PTM-driven antigenicity, which may have broad implications for T cell-mediated therapies in cancer and beyond.

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.

S1: Study population characteristics of CT Study; S2: Sensitivities and specificities of the 5-marker blood test obtained in the CTStudy (Training Study).

INTRODUCTION: Molecular mechanisms underlying the well-known association between Type 2 diabetes (T2D) and late-life cognitive impairment are unknown. Alterations in glyco post-translational modifications (PTM) of serum proteins reflect altered cellular physiology and are associated with T2D and impaired cognition. This novel large-scale mass spectrometry based glycoproteomics study aimed to identify therapeutic PTM targets that may link T2D with poor cognition. METHODS: We performed LC-MS/MS–based novel glycoproteomic profiling of sera samples from 23 cognitively normal older adults with T2D. At baseline all people had normal cognition, but during 36-months, 8 participants developed cognitive impairment while 15 maintained normal cognition RESULTS: We identified and quantified a total of 1,866 unique glycoproteoforms (i.e., peptides with a post-transcription sugar modification) that matched 201 annotated proteins. Group differences were observed for 72 glycoproteoforms at baseline. At the time of cognitive testing three years later, there were group differences for 137 glycoproteoforms. Group differences were observed for 14 glycoproteoforms both at baseline and at 3-year follow-up. DISCUSSION: Glycoproteoforms may contribute to cognitive impairment in T2D, may identify adults with T2D at risk for cognitive impairment and are high value therapeutic targets for new therapies to mitigate the negative cognitive effects of T2D in old age.

Invadopodia are adhesive, actin-rich protrusions formed by metastatic cancer cells that degrade the extracellular matrix and facilitate invasion. They support the metastatic cascade by a spatially and temporally coordinated process whereby invading cells bind to the matrix, degrade it by specific metalloproteinases, and mechanically penetrate diverse tissue barriers by forming actin-rich extensions. However, despite the apparent involvement of invadopodia in the metastatic process, the molecular mechanisms that regulate invadopodia formation and function are still largely unclear. In this study, we have explored the involvement of the key Hippo pathway co-regulators, namely YAP, and TAZ, in invadopodia formation and matrix degradation. Towards that goal, we tested the effect of depletion of YAP, TAZ, or both on invadopodia formation and activity in multiple human cancer cell lines. We report that knockdown of YAP and TAZ or their inhibition by verteporfin induces a significant elevation in matrix degradation and invadopodia formation in several cancer cell lines. Conversely, overexpression of these proteins strongly suppresses invadopodia formation and matrix degradation. Proteomic and transcriptomic profiling of MDA-MB-231 cells, following co-knockdown of YAP and TAZ, revealed a significant change in the levels of key invadopodia-associated proteins, including the crucial proteins Tks5 and MT1-MMP (MMP14). Collectively, our findings show that YAP and TAZ act as negative regulators of invadopodia formation in diverse cancer lines, most likely by reducing the levels of essential invadopodia components. Dissecting the molecular mechanisms of invadopodia formation in cancer invasion may eventually reveal novel targets for therapeutic applications against invasive cancer.

Messenger RNA-based vaccines against COVID-19 induce a robust anti-SARS-CoV-2 antibody response with potent viral neutralization activity. Antibody effector functions are determined by their constant region subclasses and by their glycosylation patterns, but their role in vaccine efficacy is unclear. Moreover, whether vaccination induces antibodies similar to those in patients with COVID-19 remains unknown. We analyze BNT162b2 vaccine-induced IgG subclass distribution and Fc glycosylation patterns and their potential to drive effector function via Fcγ receptors and complement pathways. We identify unique and dynamic pro-inflammatory Fc compositions that are distinct from those in patients with COVID-19 and convalescents. Vaccine-induced anti-Spike IgG is characterized by distinct Fab- and Fc-mediated functions between different age groups and in comparison to antibodies generated during natural viral infection. These data highlight the heterogeneity of Fc responses to SARS-CoV-2 infection and vaccination and suggest that they support long-lasting protection differently., Graphical abstract, The Fc structures of IgGs produced during infection and vaccination have important roles in shaping the immune response. Farkash et al. show that vaccine- and infection-induced anti-SARS-CoV-2 IgGs differ in their Fc regions and in the engagement of complement and Fc receptors, implying distinct effector functions and immunity.

Many integral membrane proteins are produced by translocon-associated ribosomes. The assembly of ribosomes translating membrane proteins on the translocons is mediated by a conserved system, composed of the signal recognition particle and its receptor (FtsY in Escherichia coli). FtsY is a peripheral membrane protein, and its role late during membrane protein targeting involves interactions with the translocon. However, earlier stages in the pathway have remained obscure, namely, how FtsY targets the membrane in vivo and where it initially docks. Our previous studies have demonstrated co-translational membrane-targeting of FtsY translation intermediates and identified a nascent FtsY targeting-peptide. Here, in a set of in vivo experiments, we utilized tightly stalled FtsY translation intermediates, pull-down assays and site-directed cross-linking, which revealed FtsY-nascent chain-associated proteins in the cytosol and on the membrane. Our results demonstrate interactions between the FtsY-translating ribosomes and cytosolic chaperones, which are followed by directly docking on the translocon. In support of this conclusion, we show that translocon over-expression increases dramatically the amount of membrane associated FtsY-translating ribosomes. The co-translational contacts of the FtsY nascent chains with the translocon differ from its post-translational contacts, suggesting a major structural maturation process. The identified interactions led us to propose a model for how FtsY may target the membrane co-translationally. On top of our past observations, the current results may add another tier to the hypothesis that FtsY acts stoichiometrically in targeting ribosomes to the membrane in a constitutive manner.

Early Stage; Hepatocellular Carcinoma Etapa temprana; Carcinoma hepatocelular Etapa primera; Carcinoma hepatocel·lular Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide, has an incidence rate equal to mortality. Over 80% of HCC cases occur within a high-risk population, mainly patients with both cirrhosis and chronic hepatitis B or C. With a 5-year survival rate ranging from 90% for early stage HCC, there is a high medical need for the early detection of HCC. In this study, we systematically evaluated biomarkers mentioned in international guidelines and peer-reviewed literature for HCC surveillance and diagnosis with the aim of identifying combinations that display high sensitivity and specificity for early stage HCC. Fifty biomarkers were measured in the first sample panel, panel A (n = 110), and subjected to univariate analysis. Of these, 35 biomarkers (38 assays) from panel A and an additional 13 biomarkers from the literature were prioritized for subsequent multivariate evaluation with lasso regression and exhaustive search of two- to four-biomarker combinations (panel B). Panel B included 1,081 samples from patients with HCC (n = 308) or with chronic liver diseases (n = 740). Among all patients, 61.0% had hepatitis B, 32.9% had hepatitis C, and 60.5% had cirrhosis; 40.6% of patients with HCC had early stage cancer. Protein induced by vitamin K absence-II (PIVKA-II; also known as des-gamma-carboxy prothrombin [DCP]) and alpha-fetoprotein (AFP) demonstrated the best clinical performance, both individually and in combination, and the addition of a third biomarker (Lens culinaris agglutinin-reactive fraction of AFP [AFP-L3], cartilage oligomeric matrix protein [COMP], insulin-like growth factor-binding protein 3 [IGFBP3], or matrix metalloproteinase 3 [MMP3]) further increased sensitivity for the detection of both early stage and all-stage HCC. The addition of age and sex to the three-biomarker panel resulted in an improved diagnostic performance. Conclusion: The combination of AFP and PIVKA-II, with either IGFBP3, COMP or MMP3, plus age and sex, demonstrated the best performance for the detection of early- and all-stage HCC. These novel panels performed similar to that of the GALAD score (sex [gender], age, plus serum levels of AFP, AFP-L3 and DCP [PIVKA-II]), a promising screening tool developed for HCC detection. Supported by Roche Diagnostics GmbH.

Antigen processing and presentation are critical for modulating tumor-host interactions. While post-translational modifications (PTMs) can alter the binding and recognition of antigens, their identification remains challenging. Here we uncover the role PTMs may play in antigen presentation and recognition in human cancers by profiling 29 different PTM combinations in immunopeptidomics data from multiple clinical samples and cell lines. We established and validated an antigen discovery pipeline and showed that newly identified modified antigens from a murine cancer model are cancer-specific and can elicit T cell killing. Systematic analysis of PTMs across multiple cohorts defined new haplotype preferences and binding motifs in association with specific PTM types. By expanding the antigenic landscape with modifications, we uncover disease-specific targets, including thousands of novel cancer-specific antigens and reveal insight into PTM-driven antigenicity. Collectively, our findings highlight an immunomodulatory role for modified peptides presented on HLA I, which may have broad implications for T-cell mediated therapies in cancer and beyond.SignificanceMajor efforts are underway to identify cancer-specific antigens for personalized immunotherapy. Here, we enrich the immunopeptidome landscape by uncovering thousands of novel putative antigens that are post-translationally modified. We define unique preferences for PTM-driven alterations affecting HLA binding and TCR recognition, which in turn alter tumor-immune interactions.Conflict of interest statementAuthors declare no conflicts of interest.

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.

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. A high-resolution map of coding regions in the SARS-CoV-2 genome enables the identification of 23 unannotated open reading frames and quantification of the expression of canonical viral open reading frames.

Stony corals generate their calcium carbonate exoskeleton in a highly controlled biomineralization process mediated by a variety of macromolecules including proteins. Fully identifying and classifying these proteins is crucial to understanding their role in exoskeleton formation, yet no optimal method to purify and characterize the full suite of extracted coral skeletal proteins has been established and hence their complete composition remains obscure. Here, we tested four skeletal protein purification protocols using acetone precipitation and ultrafiltration dialysis filters to present a comprehensive scleractinian coral skeletal proteome. We identified a total of 60 proteins in the coral skeleton, 44 of which were not present in previously published stony coral skeletal proteomes. Extracted protein purification protocols carried out in this study revealed that no one method captures all proteins and each protocol revealed a unique set of method-exclusive proteins. To better understand the general mechanism of skeletal protein transportation, we further examined the proteins’ gene ontology, transmembrane domains, and signal peptides. We found that transmembrane domain proteins and signal peptide secretion pathways, by themselves, could not explain the transportation of proteins to the skeleton. We therefore propose that some proteins are transported to the skeleton via non-traditional secretion pathways.

Summary 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., Graphical Abstract, Highlights • Mucin glycoprotein supramolecular assembly at Golgi pH promotes polymer formation • O-glycosylated regions help orchestrate mucin assembly • CysD domains have a novel fold, calcium binding motifs, and a role in mucin assembly • Mucins and von Willebrand factor share a mechanism to form linear polymers, Javitt et al. describe molecularly how mucin glycoproteins and von Willebrand factor—complex macromolecules responsible for mucociliary clearance in the lung, the intestinal mucosal barrier, and blood clotting—use a shared mechanism to form polymers and hydrogels that protect the respiratory tract, intestinal tract, and vasculature.

Signaling via lysine methylation by protein lysine methyltransferases (PKMTs), has been linked to diverse biological and disease processes. The mono-methyltransferase SETD6 (SET-domain-containing protein 6) is a member of the PKMT family and was previously shown to regulate essential cellular processes such as the NF-κB, WNT and the oxidative stress pathways. However, on the biochemical level, little is known about the enzymatic mode of action of SETD6. Here we provide evidence that SETD6 forms high-molecular-weight structures. Specifically, we demonstrate that SETD6 monomeric, dimeric and trimeric forms are stabilized by the methyl donor, S-adenosyl-l-methionine. We then show that SETD6 has auto-methylation activity at K39 and K179, which serves as the major auto-methylation sites with a moderate auto-methylation activity toward K372. A point mutation at K179 but not at K39 and K372, located at the SET domain of SETD6, impaired SETD6 ability to form a trimer, strongly implying a link between the auto-methylation and the oligomerization state. Finally, by radioactive in vitro methylation experiments and biochemical kinetics analysis, we show that the auto-methylation at K39 and K179 increases the catalytic rate of SETD6. Collectively, our data support a model by which SETD6 auto-methylation and self-interaction positively regulate its enzymatic activity in vitro and may suggest that other PKMTs are regulated in the same manner.

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.

Myxozoa is a diverse, speciose group of microscopic parasites, recently placed within the phylum Cnidaria. Myxozoans are highly reduced in size and complexity relative to free-living cnidarians, yet they have retained specialized organelles known as polar capsules, akin to the nematocyst stinging capsules of free-living species. Whereas in free-living cnidarians the stinging capsules are used for prey capture or defense, in myxozoans they have the essential function of initiating the host infection process. To explore the evolutionary adaptation of polar capsules to parasitism, we used as a model organism Ceratonova shasta, which causes lethal disease in salmonids. Here, we report the first isolation of C. shasta myxospore polar capsules using a tailored dielectrophoresis-based microfluidic chip. Using electron microscopy and functional analysis we demonstrated that C. shasta tubules have no openings and are likely used to anchor the spore to the host. Proteomic analysis of C. shasta polar capsules suggested that they have retained typical structural and housekeeping proteins found in nematocysts of jellyfish, sea anemones and Hydra, but have lost the most important functional group in nematocysts, namely toxins. Our findings support the hypothesis that polar capsules and nematocysts are homologous organelles, which have adapted to their distinct functions.

This study explored short-term healthcare costs of men managed with observation strategies (OBS) vs immediate treatment (IMT) for favorable risk prostate cancer (PCa) from the Geisinger Health System, a single integrated health system in Pennsylvania, as evidence from the community setting is limited.A retrospective cohort study was conducted using electronic health records from men aged ≥40 years diagnosed with favorable risk PCa (T1 or 2, PSA ≤15 ng/mL, Gleason ≤7 [3 + 4]) between January 2005 and October 2013. Prostate-specific healthcare costs were compared between the OBS and IMT cohorts in men with ≥3 years of follow-up and available linked claims data. Sub-group analyses focused on those men with low-risk PCa (T1-2a, PSA ≤10 ng/mL, Gleason ≤6). Sensitivity analysis stratified the study sample in three cohorts: OBS, switched from OBS to definitive treatment (OBS switch), and IMT.A total of 352 patients were included (OBS = 70 and IMT = 282). Compared with IMT, OBS resulted in significantly lower cumulative PCa-related healthcare costs for the first 3 years ($15,785 vs $23,177; p-value.001). The main cost drivers were outpatient procedures. The OBS cohort had the lowest incremental PCa-related healthcare costs in the first 3 years (OBS: $5,011 vs OBS switch: $26,040, net cost savings = $21,029, p .001; OBS: $5,011 vs IMT: $24,064, net cost savings = $19,053, p .001).In favorable risk PCa, half of the patients who initially chose OBS eventually underwent treatment after their PCa diagnosis. As expected, OBS was associated with reduced disease management costs compared with IMT.

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 kinase3β (GSK3β) as a novel key mediator of FGF inhibitory response in chondrocytes.

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.

Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multilevel experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native MS structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution, three-dimensional structures, highly resembled that of the human complex. Using cryoelectron microscopy single-particle analysis, we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies., Mass spectrometry approaches enabled structural elucidation of unique properties of different 20S proteasomes, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

Purpose: In initial studies that included colorectal cancer patients undergoing diagnostic colonoscopy, we had identified a serum marker combination able to detect colorectal cancer with similar diagnostic performance as fecal immunochemical test (FIT). In this study, we aimed to validate the results in participants of a large colorectal cancer screening study conducted in the average-risk, asymptomatic screening population. Experimental Design: We tested serum samples from 1,200 controls, 420 advanced adenoma patients, 4 carcinoma in situ patients, and 36 colorectal cancer patients with a 5-marker blood test [carcinoembryonic antigen (CEA)+anti-p53+osteopontin+seprase+ferritin]. The diagnostic performance of individual markers and marker combinations was assessed and compared with stool test results. Results: AUCs for the detection of colorectal cancer and advanced adenomas with the 5-marker blood test were 0.78 [95% confidence interval (CI), 0.68–0.87] and 0.56 (95% CI, 0.53–0.59), respectively, which now is comparable with guaiac-based fecal occult blood test (gFOBT) but inferior to FIT. With cutoffs yielding specificities of 80%, 90%, and 95%, the sensitivities for the detection of colorectal cancer were 64%, 50%, and 42%, and early-stage cancers were detected as well as late-stage cancers. For osteopontin, seprase, and ferritin, the diagnostic performance in the screening setting was reduced compared with previous studies in diagnostic settings while CEA and anti-p53 showed similar diagnostic performance in both settings. Conclusions: Performance of the 5-marker blood test under screening conditions is inferior to FIT even though it is still comparable with the performance of gFOBT. CEA and anti-p53 could contribute to the development of a multiple marker blood-based test for early detection of colorectal cancer. Clin Cancer Res; 22(7); 1725–33. ©2015 AACR.

Animal venom is a complex cocktail of bioactive chemicals that traditionally drew interest mostly from biochemists and pharmacologists. However, in recent years the evolutionary and ecological importance of venom is realized as this trait has direct and strong influence on interactions between species. Moreover, venom content can be modulated by environmental factors. Like many other fields of biology, venom research has been revolutionized in recent years by the introduction of systems biology approaches, i.e., genomics, transcriptomics and proteomics. The employment of these methods in venom research is known as 'venomics'. In this review we describe the history and recent advancements of venomics and discuss how they are employed in studying venom in general and in particular in the context of evolutionary ecology. We also discuss the pitfalls and challenges of venomics and what the future may hold for this emerging scientific field.

Snake venom L-amino acid oxidases (svLAAOs) are an interesting class of enzymes with important biological activities. Their participation in key metabolic processes, including pathological disorders, suggest that svLAAOs are potential lead compounds in drug discovery. However, their short-term stability defies their applications. This paper describes the stability studies together with functional and structural characterization of the LAAO bordonein-L. It has 498 amino acid residues, one N-glycosylation site and two disulfide bonds, revealed by high-resolution MS/MS. Molecular modeling approach showed its monomer folds into three conserved domains: FAD, substrate and helical domains. Differential scanning fluorimetry showed the enzyme tends to destabilize from neutral to basic pHs and in presence of mono/bivalent ions and it is highly stabilized by acid pHs and its substrates. However, high concentrations of L-amino acids decrease bordonein-L enzyme activity. Dynamic light scattering revealed bordonein-L remains in the dimeric and monodisperse form, so aggregation does not cause the rapidly decrease of enzyme activity. In vitro, the enzyme exhibited cytotoxicity against fibroblast cell line and killed Leishmania amazonensis promastigotes, intensified by substrate addition. Concluding, our results provide biochemistry and biophysical insights to improve LAAOs stability and better approaches to long-term storage. Moreover, our study emphasizes the importance of proper buffers choice mainly in cell-based assays.

Animal venoms are complex chemical arsenals containing hundreds or even thousands of diverse small molecules, peptides, and proteins that affect a myriad of molecular targets. Because this biochemical arsenal is metabolically expensive, venom must be used sparingly and therefore one might predict that venom evolution would result in retention of only the most essential toxins for predation and/or defence. However, this presents a strong dichotomy in the functional requirements of venoms that are used for both predation and defence, which results in extensive functional redundancy. Here we show that spiders can overcome this potential metabolic burden by modulating the biochemical composition of their venom. We demonstrate that the venom peptidome of the lethal Australian funnel-web spider Hadronyche infensa changes both qualitatively and quantitatively throughout a series of defensive secretions — secretion of the most potent insecticidal toxins is preceded by secretion of non-insecticidal, presumably defensive, toxins. Using mass spectrometry imaging, we show that modulation of toxin secretion is facilitated by differential storage of toxins in the venom gland. We propose that the establishment of distinct venom-gland zipcodes for toxin production is an adaptation that reduces the metabolic expense of venom production and perhaps also serves to minimize effects from development of toxin resistance during predator-prey co-evolution. Our results highlight the importance of considering behavioral aspects of natural venom secretions in understanding toxin function and evolution.

Fatty acid synthase 1 (FAS I) from Mycobacterium. tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4’-Phosphopantetheine (4’-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4’-PP transferase, termed acyl carrier protein synthase (AcpS). In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid.We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structurefunction analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

Before the introduction of modern imaging techniques and the recent developments in molecular diagnosis, tumor markers (TMs) were among the few available diagnostic tools for the management of cancer patients. Easily obtained from serum or plasma samples, TMs are minimally invasive and convenient, and the associated costs are low. Single TMs were traditionally used but these have come under scrutiny due to their low sensitivity and specificity when used, for example, in a screening setting. However, recent research has shown superior performance using a combination of multiple TMs as a panel for assessment, or as part of validated algorithms that also incorporate other clinical factors. In addition, newer TMs have been discovered that have an increased sensitivity and specificity profile for defined malignancies. The aim of this review is to provide a concise overview of the appropriate uses of both traditional and newer TMs and their roles in diagnosis, prognosis, and the monitoring of patients in current clinical practice. We also look at the future direction of TMs and their integration with other diagnostic modalities and other emerging serum based biomarkers, such as circulating nucleic acids, to ultimately advance diagnostic performance and improve patient management.

© 2015 Elsevier Ltd. Summary Arthropod venoms consist primarily of peptide toxins that are injected into their prey with devastating consequences. Venom proteins are thought to be recruited from endogenous body proteins and mutated to yield neofunctionalized toxins with remarkable affinity for specific subtypes of ion channels and receptors. However, the evolutionary history of venom peptides remains poorly understood. Here we show that a neuropeptide hormone has been convergently recruited into the venom of spiders and centipedes and evolved into a highly stable toxin through divergent modification of the ancestral gene. High-resolution structures of representative hormone-derived toxins revealed they possess a unique structure and disulfide framework and that the key structural adaptation in weaponization of the ancestral hormone was loss of a C-terminal α helix, an adaptation that occurred independently in spiders and centipedes. Our results raise a new paradigm for toxin evolution and highlight the value of structural information in providing insight into protein evolution.

With increasing network bandwidths, stateful firewalls are likely to become communication bottlenecks in networks. To mitigate this problem, we propose to bypass selected traffic around firewalls using software-defined networking (SDN). We discuss various approaches and elaborate the following concept. A controller samples outgoing packets at the firewall using sFlow to detect congestion. In case of congestion, flows already admitted by the firewall are identified and offloaded at an appropriate rate by installing flow-specific bypass rules on an OpenFlow-capable switch. We suggest two different algorithms to select appropriate flows and provide a proof-of-concept implementation in a network testbed using the Ryu controller framework. Experimental results illustrate the system behavior at different load levels with and without offloading. We provide an analytical system model to predict the offloading performance for other system parameters than experimentally evaluated and validate the model with our experimental results. A parameter study suggests that the offloaded traffic rate may be a multiple of the firewall's capacity if the switch supports sufficient flow rules or is able to match for TCP flags.

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

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-β is the main driver of the immediate stress response in Nematostella. GSK3-β 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-β 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.

Purpose Vintafolide (EC145) is a folic acid–desacetylvinblastine conjugate that binds to the folate receptor (FR), which is expressed on the majority of epithelial ovarian cancers. This randomized phase II trial evaluated vintafolide combined with pegylated liposomal doxorubicin (PLD) compared with PLD alone. The utility of an FR-targeted imaging agent, 99mTc-etarfolatide (EC20), in selecting patients likely to benefit from vintafolide was also examined. Patients and Methods Women with recurrent platinum-resistant ovarian cancer who had undergone ≤ two prior cytotoxic regimens were randomly assigned at a 2:1 ratio to PLD (50 mg/m2 intravenously [IV] once every 28 days) with or without vintafolide (2.5 mg IV three times per week during weeks 1 and 3). Etarfolatide scanning was optional. The primary objective was to compare progression-free survival (PFS) between the groups. Results The intent-to-treat population comprised 149 patients. Median PFS was 5.0 and 2.7 months for the vintafolide plus PLD and PLD-alone arms, respectively (hazard ratio [HR], 0.63; 95% CI, 0.41 to 0.96; P = .031). The greatest benefit was observed in patients with 100% of lesions positive for FR, with median PFS of 5.5 compared with 1.5 months for PLD alone (HR, 0.38; 95% CI, 0.17 to 0.85; P = .013). The group of patients with FR-positive disease (10% to 90%) experienced some PFS improvement (HR, 0.873), whereas patients with disease that did not express FR experienced no PFS benefit (HR, 1.806). Conclusion Vintafolide plus PLD is the first combination to demonstrate an improvement over standard therapy in a randomized trial of patients with platinum-resistant ovarian cancer. Etarfolatide can identify patients likely to benefit from vintafolide.

ObjectivesThe 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/MaterialsThe 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.ResultsFive 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.ConclusionsThe results presented support the use of ROMA to improve clinical decision making, most notably in patients with early OC.