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Incidence and reporting of sharps injuries amongst ENT surgeons.
J Laryngol Otol. 2016 Feb 29;:1-6
Authors: Vijendren A, Sanchez J, Yung M
Abstract
BACKGROUND: Sharps injuries are a common occupational hazard amongst surgeons. Limited work has been conducted on their effects within the ENT community.
METHODS: A literature review was performed and a survey on sharps injuries was distributed to the entire membership of ENT-UK electronically.
RESULTS: The literature review revealed 3 studies, with 2 of them performed more than 20 years ago. A total of 323 completed questionnaires were returned (24 per cent response rate). Of the respondents, 26.6 per cent reported having experienced sharps injuries. There was no statistical difference between the occurrence of sharps injuries and the grade, length of time spent in the specialty or subspecialty of respondents. Only 33.7 per cent of afflicted clinicians reported all their injuries as per local institutional policies. No seroconversions were reported.
CONCLUSION: The study found poor evidence on sharps injuries amongst ENT surgeons, and low reporting rates that were comparable to other studies conducted in the UK. This highlights the need for further research and increasing awareness on sharps injuries regulations within the specialty.
PMID: 26924350 [PubMed - as supplied by publisher]
CRISPR/Cas9 treatment for Duchenne muscular dystrophy
Cell Research advance online publication, March 1 2016. doi:10.1038/cr.2016.28
Authors: Jerry R Mendell & Louise R Rodino-Klapac
Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de-ubiquitinase OTUB1 as a negative regulator of RAS mono- and di-ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild-type RAS cells. An increase of OTUB1 expression is commonly observed in non-small-cell lung carcinomas harboring wild-type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer development.
Mitochondrial dysfunction and altered proteostasis are central features of neurodegenerative diseases. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests oligopeptides, including the mitochondrial targeting sequences that are cleaved from proteins imported across the inner mitochondrial membrane and the mitochondrial fraction of amyloid beta (Aβ). We identified two siblings carrying a homozygous PITRM1 missense mutation (c.548G>A, p.Arg183Gln) associated with an autosomal recessive, slowly progressive syndrome characterised by mental retardation, spinocerebellar ataxia, cognitive decline and psychosis. The pathogenicity of the mutation was tested in vitro, in mutant fibroblasts and skeletal muscle, and in a yeast model. A Pitrm1+/– heterozygous mouse showed progressive ataxia associated with brain degenerative lesions, including accumulation of Aβ-positive amyloid deposits. Our results show that PITRM1 is responsible for significant Aβ degradation and that impairment of its activity results in Aβ accumulation, thus providing a mechanistic demonstration of the mitochondrial involvement in amyloidotic neurodegeneration.
Excess cellular iron increases reactive oxygen species (ROS) production and causes cellular damage. Mitochondria are the major site of iron metabolism and ROS production; however, few studies have investigated the role of mitochondrial iron in the development of cardiac disorders, such as ischemic heart disease or cardiomyopathy (CM). We observe increased mitochondrial iron in mice after ischemia/reperfusion (I/R) and in human hearts with ischemic CM, and hypothesize that decreasing mitochondrial iron protects against I/R damage and the development of CM. Reducing mitochondrial iron genetically through cardiac-specific overexpression of a mitochondrial iron export protein or pharmacologically using a mitochondria-permeable iron chelator protects mice against I/R injury. Furthermore, decreasing mitochondrial iron protects the murine hearts in a model of spontaneous CM with mitochondrial iron accumulation. Reduced mitochondrial ROS that is independent of alterations in the electron transport chain's ROS producing capacity contributes to the protective effects. Overall, our findings suggest that mitochondrial iron contributes to cardiac ischemic damage, and may be a novel therapeutic target against ischemic heart disease.
WBP2 encodes the WW domain-binding protein 2 that acts as a transcriptional coactivator for estrogen receptor α (ESR1) and progesterone receptor (PGR). We reported that the loss of Wbp2 expression leads to progressive high-frequency hearing loss in mouse, as well as in two deaf children, each carrying two different variants in the WBP2 gene. The earliest abnormality we detect in Wbp2-deficient mice is a primary defect at inner hair cell afferent synapses. This study defines a new gene involved in the molecular pathway linking hearing impairment to hormonal signalling and provides new therapeutic targets.
Activation of non-shivering thermogenesis (NST) in brown adipose tissue (BAT) has been proposed as an anti-obesity treatment. Moreover, cold-induced glucose uptake could normalize blood glucose levels in insulin-resistant patients. It is therefore important to identify novel regulators of NST and cold-induced glucose uptake. Mammalian target of rapamycin complex 2 (mTORC2) mediates insulin-stimulated glucose uptake in metabolic tissues, but its role in NST is unknown. We show that mTORC2 is activated in brown adipocytes upon β-adrenergic stimulation. Furthermore, mice lacking mTORC2 specifically in adipose tissue (AdRiKO mice) are hypothermic, display increased sensitivity to cold, and show impaired cold-induced glucose uptake and glycolysis. Restoration of glucose uptake in BAT by overexpression of hexokinase II or activated Akt2 was sufficient to increase body temperature and improve cold tolerance in AdRiKO mice. Thus, mTORC2 in BAT mediates temperature homeostasis via regulation of cold-induced glucose uptake. Our findings demonstrate the importance of glucose metabolism in temperature regulation.
There is increasing evidence that common molecular pathways in neurons are closely linked with mitochondrial function and that mitochondrial dysfunction is connected to various forms of neurodegenerative diseases. For instance, mitochondria are involved in amyloid-β (Aβ) deposition in Alzheimer's disease, although the exact molecular pathways remain largely unknown. Brunetti et al (2015) in this issue of EMBO Molecular Medicine provide a novel link between Aβ accumulation and mitochondria. A pathogenic mutation in a Norwegian family in the mitochondrial metallopeptidase PITRM1 is found to underlie a novel mitochondrial neurodegenerative phenotype associated with Aβ accumulation.
MicroRNA-10b (miR-10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes, while absent in normal neuroglial cells of the brain. miR-10b inhibition strongly impairs proliferation and survival of cultured glioma cells, including glioma-initiating stem-like cells (GSC). Although several miR-10b targets have been identified previously, the common mechanism conferring the miR-10b-sustained viability of GSC is unknown. Here, we demonstrate that in heterogeneous GSC, miR-10b regulates cell cycle and alternative splicing, often through the non-canonical targeting via 5'UTRs of its target genes, including MBNL1-3, SART3, and RSRC1. We have further assessed the inhibition of miR-10b in intracranial human GSC-derived xenograft and murine GL261 allograft models in athymic and immunocompetent mice. Three delivery routes for the miR-10b antisense oligonucleotide inhibitors (ASO), direct intratumoral injections, continuous osmotic delivery, and systemic intravenous injections, have been explored. In all cases, the treatment with miR-10b ASO led to targets’ derepression, and attenuated growth and progression of established intracranial GBM. No significant systemic toxicity was observed upon ASO administration by local or systemic routes. Our results indicate that miR-10b is a promising candidate for the development of targeted therapies against all GBM subtypes.
While increased VEGF-A has been associated with neovascular age-related macular degeneration (AMD), it is not known whether VEGF-A may also promote other age-related eye diseases. Here, we show that an increase in VEGF-A is sufficient to cause multiple distinct common aging diseases of the eye, including cataracts and both neovascular and non-exudative AMD-like pathologies. In the lens, increased VEGF-A induces age-related opacifications that are associated with ERK hyperactivation, increased oxidative damage, and higher expression of the NLRP3 inflammasome effector cytokine IL-1β. Similarly, increased VEGF-A induces oxidative stress and IL-1β expression also in the retinal pigment epithelium (RPE). Targeting NLRP3 inflammasome components or Il1r1 strongly inhibited not only VEGF-A-induced cataract formation, but also both neovascular and non-exudative AMD-like pathologies. Moreover, increased VEGF-A expression specifically in the RPE was sufficient to cause choroidal neovascularization (CNV) as in neovascular AMD, which could be inhibited by RPE-specific inactivation of Flk1, while Tlr2 inactivation strongly reduced CNV. These findings suggest a shared pathogenic role of VEGF-A-induced and NLRP3 inflammasome-mediated IL-1β activation for multiple distinct ocular aging diseases.
Design Features for Linguistically-Mediated Meaning Construction: The Relative Roles of the Linguistic and Conceptual Systems in Subserving the Ideational Function of Language.
Front Psychol. 2016;7:156
Authors: Evans V
Abstract
Recent research in language and cognitive science proposes that the linguistic system evolved to provide an "executive" control system on the evolutionarily more ancient conceptual system (e.g., Barsalou et al., 2008; Evans, 2009, 2015a,b; Bergen, 2012). In short, the claim is that embodied representations in the linguistic system interface with non-linguistic representations in the conceptual system, facilitating rich meanings, or simulations, enabling linguistically mediated communication. In this paper I build on these proposals by examining the nature of what I identify as design features for this control system. In particular, I address how the ideational function of language-our ability to deploy linguistic symbols to convey meanings of great complexity-is facilitated. The central proposal of this paper is as follows. The linguistic system of any given language user, of any given linguistic system-spoken or signed-facilitates access to knowledge representation-concepts-in the conceptual system, which subserves this ideational function. In the most general terms, the human meaning-making capacity is underpinned by two distinct, although tightly coupled representational systems: the conceptual system and the linguistic system. Each system contributes to meaning construction in qualitatively distinct ways. This leads to the first design feature: given that the two systems are representational-they are populated by semantic representations-the nature and function of the representations are qualitatively different. This proposed design feature I term the bifurcation in semantic representation. After all, it stands to reason that if a linguistic system has a different function, vis-à-vis the conceptual system, which is of far greater evolutionary antiquity, then the semantic representations will be complementary, and as such, qualitatively different, reflecting the functional distinctions of the two systems, in collectively giving rise to meaning. I consider the nature of these qualitatively distinct representations. And second, language itself is adapted to the conceptual system-the semantic potential-that it marshals in the meaning construction process. Hence, a linguistic system itself exhibits a bifurcation, in terms of the symbolic resources at its disposal. This design feature I dub the birfucation in linguistic organization. As I shall argue, this relates to two distinct reference strategies available for symbolic encoding in language: what I dub words-to-world reference and words-to-words reference. In slightly different terms, this design feature of language amounts to a distinction between a lexical subsystem, and a grammatical subsystem.
PMID: 26925000 [PubMed - as supplied by publisher]
Two cardiovascular outcome trials established niacin 3 g daily prevents hard cardiac events. However, as detailed in part I of this series, an extended-release (ER) alternative at only 2 g nightly demonstrated no comparable benefits in two outcome trials, implying the alternative is not equivalent to the established cardioprotective regimen. Since statins leave a significant treatment gap, this presents a major opportunity for developers. Importantly, the established regimen is cardioprotective, so the pathway is likely beneficial. Moreover, though effective, the established cardioprotective regimen is cumbersome, limiting clinical use. At the same time, the ER alternative has been thoroughly discredited as a viable substitute for the established cardioprotective regimen. Therefore, by exploiting the pathway and skillfully avoiding the problems with the established cardioprotective regimen and the ER alternative, developers could validate cardioprotective variations facing little meaningful competition from their predecessors. Thus, shrewd developers could effectively tap into a gold mine at the grave of the ER alternative. The GPR109A receptor was discovered a decade ago, leading to a large body of evidence commending the niacin pathway to a lower cardiovascular risk beyond statins. While mediating niacin’s most prominent adverse effects, GPR109A also seems to mediate anti-lipolytic, anti-inflammatory, and anti-atherogenic effects of niacin. Several developers are investing heavily in novel strategies to exploit niacin’s therapeutic pathways. These include selective GPR109A receptor agonists, niacin prodrugs, and a niacin metabolite, with encouraging early phase human data. In part II of this review, we summarize the accumulated results of these early phase studies of emerging niacin mimetics.
As testosterone replacement therapy (TRT) has emerged as a commonly prescribed therapy for symptomatic low testosterone, conflicting data have been reported in terms of both its efficacy and potential adverse outcomes. One of the most controversial associations has been that of TRT and cardiovascular morbidity and mortality. This review briefly provides background on the history of TRT, the indications for TRT, and the data behind TRT for symptomatic low testosterone. It then specifically delves into the rather limited data for cardiovascular outcomes of those with low endogenous testosterone and those who receive TRT. The available body of literature strongly suggests that more work, by way of clinical trials, needs to be done to better understand the impact of testosterone and TRT on the cardiovascular system.
Gemfibrozil is a lipid-modifying agent that belongs to the fibric acid derivative class. Fibric acid derivatives activate peroxisome proliferator activated receptor α (PPAR-α). The primary role of these agents in clinical practice is for the management of hypertriglyceridemia. Triglycerides may be reduced by as much as 74 % in some patients. In addition to lowering triglycerides, these agents can also decrease very low-density lipoprotein cholesterol (VLDL-C) and low-density lipoprotein cholesterol (LDL-C) as well as raise high-density lipoprotein cholesterol (HDL-C). Based on the 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults and the National Lipid Association, pharmacologic therapy to reduce triglycerides should be considered when triglyceride levels are ≥500 mg/dL. While the use of gemfibrozil has decreased over the years for a variety of reasons, muscle-associated adverse effects is the predominant reason and the one that is most clinically relevant. However, despite these concerns, there are situations in which the use of gemfibrozil in combination with a statin may be necessary. Understanding the metabolism of gemfibrozil and the degree of interaction with the various statins will assist health-care providers to optimize safety when this combination is clinically indicated.
Inflammatory markers are important prognostic factors in various cancers. This study investigated whether inflammatory markers of the Glasgow prognostic score (GPS) predicted progression-free survival (PFS) and overall survival (OS) for patients with all cases of epithelial ovarian cancer (OC). Pretreatment GPS was examined for the correlations with PFS and OS in 216 patients in all stages of epithelial OC. Statistical analyses were performed using the Mann–Whitney U-test. PFS and OS were analyzed using the Kaplan–Meier method. Cox's proportional hazard regression was used for univariate and multivariate analyses. For all patients, the median PFS was 35.1 months, and median OS was 46.7 months; follow-up range was 1–162 months. Kaplan–Meier analysis revealed that patients with high GPS (GPS 2) at pretreatment had a shorter PFS and OS than did patients with lower GPS (GPS 0 + 1) in for early, advanced, and all-stages of OC (PFS: P < 0.001 for early-, advanced- and all-stages; OS; P < 0.001 for early- and all-stage, P = 0.015 for advanced-stage). GPS (GPS 2) was also found to be an independent predictor of both recurrence (P = 0.002) and survival (P = 0.001) of all cases of epithelial OC by a multivariate analysis. GPS can serve as an indicator of poor prognosis in patients with all stages of epithelial OC, including early-stage disease and regardless of histology.
This study investigated whether inflammatory markers of the Glasgow prognostic score (GPS) predicted progression-free survival (PFS) and overall survival (OS) for patients with all cases of epithelial ovarian cancer (OC). GPS can serve as an indicator of poor prognosis in patients with all stages of OC cancer, including early-stage disease and regardless of histology.
This study aimed to compare the oncological results between unplanned excision (UE) and planned excision (PE) of malignant soft tissue tumor and to examine the impact of residual tumor (ReT) after UE. Nonmetastatic soft tissue sarcomas surgically treated in 1996–2012 were included in this study. Disease-specific survival (DSS), metastasis-free survival (MFS), and local-recurrence-free survival (LRFS) were stratified according to the tumor location and American Joint Committee on Cancer Classification 7th edition stage. Independent prognostic parameters were identified by Cox proportional hazard models. Two-hundred and ninety PEs and 161 UEs were identified. Significant difference in oncological outcome was observed only for LRFS probability of retroperitoneal sarcomas (5-year LRFS: 33.0% [UE] vs. 71.0% [PE], P = 0.018). Among the 142 UEs of extremity and trunk, ReT in re-excision specimen were found in 75 cases (53%). UEs with ReT had significantly lower survival probabilities and a higher amputation rate than UEs without ReT (5-year DSS: 68.8% vs. 92%, P < 0.001; MFS: 56.1% vs. 90.9%, P < 0.001; LRFS: 75.8% vs. 98.4%, P = <0.001; amputation rate 18.5% vs. 1.8%, P = 0.003). The presence of ReT was an independent poor prognostic predictor for DSS, MFS, and LRFS with hazard ratios of 2.02 (95% confidence interval (CI), 1.25–3.26), 1.62 (95% CI, 1.05–2.51) and 1.94 (95% CI, 1.05–3.59), respectively. Soft tissue sarcomas should be treated in specialized centers and UE should be avoided because of its detrimental effect especially when ReT remains after UE.
Residual disease in unplanned excision of soft tissue sarcoma was a poor prognostic predictor for all oncological outcomes. Soft tissue sarcomas should be treated in specialized centers and unplanned excision should be avoided because of its detrimental effect especially when residual disease remains after unplanned excision.
Posttranslational modifications of proteins play an important role in biology. For example, phosphorylation is a key component in signal transduction in all three domains of life, and histones can be modified in such a variety of ways that a histone code for gene regulation has been proposed. Shotgun proteomics is commonly used to identify posttranslational modifications as well as chemical modifications from sample processing. However, it favors the detection of abundant peptides over the repertoire presented, and the data analysis usually requires advance specification of modification masses and target amino acids, their number constrained by available computational resources. Recent advances in data independent acquisition mass spectrometry technologies such as SWATH-MS enable a deeper recording of the peptide contents of samples, including peptides with modifications. Here, we present a novel approach that applies the power of SWATH-MS analysis to the automated pursuit of modified peptides. With the new SWATHProphetPTM functionality added to the open source SWATHProphet software, precursor ions consistent with a modification are identified along with the mass and localization of the modification in the peptide sequence in a sensitive and unrestricted manner without the need to anticipate the modifications in advance. Using this method, we demonstrate the detection of a wide assortment of modified peptides, many unanticipated, in samples containing unpurified synthetic peptides and human urine, as well as in phospho-enriched human tissue culture cell samples.
Sjögren's syndrome is a chronic autoimmune disorder characterized by lymphocytic infiltration and hypofunction of salivary and lacrimal glands. This loss of salivary function leads to oral dryness, impaired swallowing and speech, and increased infection and is associated with other autoimmune diseases and an increased risk of certain cancers. Despite the implications of this prevalent disease, diagnosis currently takes years, partly due to the diversity in patient presentation. Saliva is a complicated biological fluid with major constituents, including heavily glycosylated mucins MUC5B and MUC7, important for its viscoelastic and hydrating and lubricating properties. This study investigated Sjögren's patient's perception of dryness (bother index questionnaires) along with the rheological, protein composition, and glycan analysis of whole mouth saliva and the saliva on the mucosal surface (residual mucosal saliva) to understand the properties that most affect patient wellbeing. Sjögren's patients exhibited a statistically significant reduction in residual mucosal saliva, salivary flow rate, and extensional rheology, spinnbarkeit (stringiness). Although the concentration of mucins MUC5B and MUC7 were similar between patients and controls, a comparison of protein Western blotting and glycan staining identified a reduction in mucin glycosylation in Sjögren's, particularly on MUC7. LC-MS/MS analysis of O-glycans released from MUC7 by β-elimination revealed that although patients had an increase in core 1 sulfation, the even larger reduction in sialylation resulted in a global decline of charged glycans. This was primarily due to the loss of the extended core 2 disialylated structure, with and without fucosylation. A decrease in the extended, fucosylated core 2 disialylated structure on MUC7, residual mucosal wetness, and whole mouth saliva flow rate appeared to have a negative and cumulative effect on the perception of oral dryness. The observed changes in MUC7 glycosylation could be a potential diagnostic tool for saliva quality and taken into consideration for future therapies for this multifactorial syndrome.
Covalent post-translational modifications (PTMs) of proteins can regulate the structural and functional state of a protein in the absence of primary changes in the underlying sequence. Common PTMs include phosphorylation, acetylation, and methylation. Histone proteins are critical regulators of the genome and are subject to a highly abundant and diverse array of PTMs. To highlight the functional complexity added to the proteome by lysine methylation signaling, here we will focus on lysine methylation of histone proteins, an important modification in the regulation of chromatin and epigenetic processes. We review the signaling pathways and functions associated with a single residue, H4K20, as a model chromatin and clinically important mark that regulates biological processes ranging from the DNA damage response and DNA replication to gene expression and silencing.
All large scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here, we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropyl alcohol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through 13CD3-methionine labeling (heavy-methyl SILAC) of Saccharomyces cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared with target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methyl-PSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-β-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together, these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl-PSMs in large scale LC-MS/MS methylation site discovery experiments and make recommendations on how to reduce methyl-PSM FDRs in samples not amenable to heavy isotope labeling. Data are available via ProteomeXchange with the data identifier PXD002857.
Normal cell growth is characterized by a regulated epigenetic program that drives cellular activities such as gene transcription, DNA replication, and DNA damage repair. Perturbation of this epigenetic program can lead to events such as mis-regulation of gene transcription and diseases such as cancer. To begin to understand the epigenetic program correlated to the development of melanoma, we performed a novel quantitative mass spectrometric analysis of histone post-translational modifications mis-regulated in melanoma cell culture as well as patient tumors. Aggressive melanoma cell lines as well as metastatic melanoma were found to have elevated histone H3 Lys27 trimethylation (H3K27me3) accompanied by overexpressed methyltransferase EZH2 that adds the specific modification. The altered epigenetic program that led to elevated H3K27me3 in melanoma cell culture was found to directly silence transcription of the tumor suppressor genes RUNX3 and E-cadherin. The EZH2-mediated silencing of RUNX3 and E-cadherin transcription was also validated in advanced stage human melanoma tissues. This is the first study focusing on the detailed epigenetic mechanisms leading to EZH2-mediated silencing of RUNX3 and E-cadherin tumor suppressors in melanoma. This study underscores the utility of using high resolution mass spectrometry to identify mis-regulated epigenetic programs in diseases such as cancer, which could ultimately lead to the identification of biological markers for diagnostic and prognostic applications.
Cross-linking/mass spectrometry resolves protein–protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers.
Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such "trivalent hypermethylation" (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can now decode crosstalk involving up to three modified sites.
The highly conserved yeast R2TP complex, consisting of Rvb1, Rvb2, Pih1, and Tah1, participates in diverse cellular processes ranging from assembly of protein complexes to apoptosis. Rvb1 and Rvb2 are closely related proteins belonging to the AAA+ superfamily and are essential for cell survival. Although Rvbs have been shown to be associated with various protein complexes including the Ino80 and Swr1chromatin remodeling complexes, we performed a systematic quantitative proteomic analysis of their associated proteins and identified two additional complexes that associate with Rvb1 and Rvb2: the chaperonin-containing T-complex and the 19S regulatory particle of the proteasome complex. We also analyzed Rvb1 and Rvb2 purified from yeast strains devoid of PIH1 and TAH1. These analyses revealed that both Rvb1 and Rvb2 still associated with Hsp90 and were highly enriched with RNA polymerase II complex components. Our analyses also revealed that both Rvb1 and Rvb2 were recruited to the Ino80 and Swr1 chromatin remodeling complexes even in the absence of Pih1 and Tah1 proteins. Using further biochemical analysis, we showed that Rvb1 and Rvb2 directly interacted with Hsp90 as well as with the RNA polymerase II complex. RNA-Seq analysis of the deletion strains compared with the wild-type strains revealed an up-regulation of ribosome biogenesis and ribonucleoprotein complex biogenesis genes, down-regulation of response to abiotic stimulus genes, and down-regulation of response to temperature stimulus genes. A Gene Ontology analysis of the 80 proteins whose protein associations were altered in the PIH1 or TAH1 deletion strains found ribonucleoprotein complex proteins to be the most enriched category. This suggests an important function of the R2TP complex in ribonucleoprotein complex biogenesis at both the proteomic and genomic levels. Finally, these results demonstrate that deletion network analyses can provide novel insights into cellular systems.
Deleted in breast cancer 1 (DBC1) has emerged as an important regulator of multiple cellular processes, ranging from gene expression to cell cycle progression. DBC1 has been linked to tumorigenesis both as an inhibitor of histone deacetylases, HDAC3 and sirtuin 1, and as a transcriptional cofactor for nuclear hormone receptors. However, despite mounting interest in DBC1, relatively little is known about the range of its interacting partners and the scope of its functions. Here, we carried out a functional proteomics-based investigation of DBC1 interactions in two relevant cell types, T cells and kidney cells. Microscopy, molecular biology, biochemistry, and mass spectrometry studies allowed us to assess DBC1 mRNA and protein levels, localization, phosphorylation status, and protein interaction networks. The comparison of DBC1 interactions in these cell types revealed conserved regulatory roles for DBC1 in gene expression, chromatin organization and modification, and cell cycle progression. Interestingly, we observe previously unrecognized DBC1 interactions with proteins encoded by cancer-associated genes. Among these interactions are five components of the SWI/SNF complex, the most frequently mutated chromatin remodeling complex in human cancers. Additionally, we identified a DBC1 interaction with TBL1XR1, a component of the NCoR complex, which we validated by reciprocal isolation. Strikingly, we discovered that DBC1 associates with proteins that regulate the circadian cycle, including DDX5, DHX9, and SFPQ. We validated this interaction by colocalization and reciprocal isolation. Functional assessment of this association demonstrated that DBC1 protein levels are important for regulating CLOCK and BMAL1 protein oscillations in synchronized T cells. Our results suggest that DBC1 is integral to the maintenance of the circadian molecular clock. Furthermore, the identified interactions provide a valuable resource for the exploration of pathways involved in DBC1-associated tumorigenesis.
Glioblastoma is one of the most malignant brain tumors with poor prognosis and their development and progression are known to be driven by glioblastoma stem cells. Although glioblastoma stem cells lose their cancer stem cell properties during cultivation in serum-containing medium, little is known about the molecular mechanisms regulating signaling alteration in relation to reduction of stem cell-like characteristics. To elucidate the global phosphorylation-related signaling events, we performed a SILAC-based quantitative phosphoproteome analysis of serum-induced dynamics in glioblastoma stem cells established from the tumor tissues of the patient. Among a total of 2876 phosphorylation sites on 1584 proteins identified in our analysis, 732 phosphorylation sites on 419 proteins were regulated through the alteration of stem cell-like characteristics. The integrative computational analyses based on the quantified phosphoproteome data revealed the relevant changes of phosphorylation levels regarding the proteins associated with cytoskeleton reorganization such as Rho family GTPase and Intermediate filament signaling, in addition to transforming growth factor-β receptor type-2 (TGFBR2) as a prominent upstream regulator involved in the serum-induced phosphoproteome regulation. The functional association of transforming growth factor-β receptor type-2 with stem cell-like properties was experimentally validated through signaling perturbation using the corresponding inhibitors, which indicated that transforming growth factor-β receptor type-2 could play an important role as a novel cell fate determinant in glioblastoma stem cell regulation.
Defective copper excretion from hepatocytes in Wilson's disease causes accumulation of copper ions with increased generation of reactive oxygen species via the Fenton-type reaction. Here we developed a nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry coupled with the isotope-dilution method for the simultaneous quantification of oxidatively induced DNA modifications. This method enabled measurement, in microgram quantities of DNA, of four oxidative stress-induced lesions, including direct ROS-induced purine cyclonucleosides (cPus) and two exocyclic adducts induced by byproducts of lipid peroxidation, i.e. 1,N6-etheno-2'-deoxyadenosine (dA) and 1,N2-etheno-2'-deoxyguanosine (dG). Analysis of liver tissues of Long-Evans Cinnamon rats, which constitute an animal model of human Wilson's disease, and their healthy counterparts [i.e. Long-Evans Agouti rats] showed significantly higher levels of all four DNA lesions in Long-Evans Cinnamon than Long-Evans Agouti rats. Moreover, cPus were present at much higher levels than dA and dG lesions. In contrast, the level of 5-hydroxymethyl-2'-deoxycytidine (5-HmdC), an oxidation product of 5-methyl-2'-deoxycytidine (5-mdC), was markedly lower in the liver tissues of Long-Evans Cinnamon than Long-Evans Agouti rats, though no differences were observed for the levels of 5-mdC. In vitro biochemical assay showed that Cu2+ ions could directly inhibit the activity of Tet enzymes. Together, these results suggest that aberrant copper accumulation may perturb genomic stability by elevating oxidatively induced DNA lesions, and by altering epigenetic pathways of gene regulation.
Cholangiocellular carcinoma (CCC) and pancreatic ductal adenocarcinoma (PDAC) are two highly aggressive cancer types that arise from epithelial cells of the pancreatobiliary system. Owing to their histological and morphological similarity, differential diagnosis between CCC and metastasis of PDAC located in the liver frequently proves an unsolvable issue for pathologists. The detection of biomarkers with high specificity and sensitivity for the differentiation of these tumor types would therefore be a valuable tool. Here, we address this problem by comparing microdissected CCC and PDAC tumor cells from nine and eleven cancer patients, respectively, in a label-free proteomics approach. The novel biomarker candidates were subsequently verified by immunohistochemical staining of 73 CCC, 78 primary, and 18 metastatic PDAC tissue sections. In the proteome analysis, we found 180 proteins with a significantly differential expression between CCC and PDAC cells (p value < 0.05, absolute fold change > 2). Nine candidate proteins were chosen for an immunohistochemical verification out of which three showed very promising results. These were the annexins ANXA1, ANXA10, and ANXA13. For the correct classification of PDAC, ANXA1 showed a sensitivity of 84% and a specificity of 85% and ANXA10 a sensitivity of 90% at a specificity of 66%. ANXA13 was higher abundant in CCC. It presented a sensitivity of 84% at a specificity of 55%. In metastatic PDAC tissue ANXA1 and ANXA10 showed similar staining behavior as in the primary PDAC tumors (13/18 and 17/18 positive, respectively). ANXA13, however, presented positive staining in eight out of eighteen secondary PDAC tumors and was therefore not suitable for the differentiation of these from CCC. We conclude that ANXA1 and ANXA10 are promising biomarker candidates with high diagnostic values for the differential diagnosis of intrahepatic CCC and metastatic liver tumors deriving from PDAC.
Breast cancer was the second leading cause of cancer related mortality for females in 2014. Recent studies suggest histone H1 phosphorylation may be useful as a clinical biomarker of breast and other cancers because of its ability to recognize proliferative cell populations. Although monitoring a single phosphorylated H1 residue is adequate to stratify high-grade breast tumors, expanding our knowledge of how H1 is phosphorylated through the cell cycle is paramount to understanding its role in carcinogenesis. H1 analysis by bottom-up MS is challenging because of the presence of highly homologous sequence variants expressed by most cells. These highly basic proteins are difficult to analyze by LC-MS/MS because of the small, hydrophilic nature of peptides produced by tryptic digestion. Although bottom-up methods permit identification of several H1 phosphorylation events, these peptides are not useful for observing the combinatorial post-translational modification (PTM) patterns on the protein of interest. To complement the information provided by bottom-up MS, we utilized a top-down MS/MS workflow to permit identification and quantitation of H1 proteoforms related to the progression of breast cells through the cell cycle. Histones H1.2 and H1.4 were observed in MDA-MB-231 metastatic breast cells, whereas an additional histone variant, histone H1.3, was identified only in nonneoplastic MCF-10A cells. Progressive phosphorylation of histone H1.4 was identified in both cell lines at mitosis (M phase). Phosphorylation occurred first at S172 followed successively by S187, T18, T146, and T154. Notably, phosphorylation at S173 of histone H1.2 and S172, S187, T18, T146, and T154 of H1.4 significantly increases during M phase relative to S phase, suggesting that these events are cell cycle-dependent and may serve as markers for proliferation. Finally, we report the observation of the H1.2 SNP variant A18V in MCF-10A cells.
A thorough understanding of the molecular details of the interactions between bacteria and host are critical to ultimately prevent disease. Recent technological advances allow simultaneous analysis of host and bacterial protein and metabolic profiles from a single small tissue sample to provide insight into pathogenesis. We used the chinchilla model of human otitis media to determine, for the first time, the most expansive delineation of global changes in protein and metabolite profiles during an experimentally induced disease. After 48 h of infection with nontypeable Haemophilus influenzae, middle ear tissue lysates were analyzed by high-resolution quantitative two-dimensional liquid chromatography-tandem mass spectrometry. Dynamic changes in 105 chinchilla proteins and 66 metabolites define the early proteomic and metabolomic signature of otitis media. Our studies indicate that establishment of disease coincides with actin morphogenesis, suppression of inflammatory mediators, and bacterial aerobic respiration. We validated the observed increase in the actin-remodeling complex, Arp2/3, and experimentally showed a role for Arp2/3 in nontypeable Haemophilus influenzae invasion. Direct inhibition of actin branch morphology altered bacterial invasion into host epithelial cells, and is supportive of our efforts to use the information gathered to modify outcomes of disease. The twenty-eight nontypeable Haemophilus influenzae proteins identified participate in carbohydrate and amino acid metabolism, redox homeostasis, and include cell wall-associated metabolic proteins. Quantitative characterization of the molecular signatures of infection will redefine our understanding of host response driven developmental changes during pathogenesis. These data represent the first comprehensive study of host protein and metabolite profiles in vivo in response to infection and show the feasibility of extensive characterization of host protein profiles during disease. Identification of novel protein targets and metabolic biomarkers will advance development of therapeutic and diagnostic options for treatment of disease.
Protein phosphorylation, one of the most common and important modifications of acute and reversible regulation of protein function, plays a dominant role in almost all cellular processes. These signaling events regulate cellular responses, including proliferation, differentiation, metabolism, survival, and apoptosis. Several studies have been successfully used to identify phosphorylated proteins and dynamic changes in phosphorylation status after stimulation. Nevertheless, it is still rather difficult to elucidate precise complex phosphorylation signaling pathways. In particular, how signal transduction pathways directly communicate from the outer cell surface through cytoplasmic space and then directly into chromatin networks to change the transcriptional and epigenetic landscape remains poorly understood. Here, we describe the optimization and comparison of methods based on thiophosphorylation affinity enrichment, which can be utilized to monitor phosphorylation signaling into chromatin by isolation of phosphoprotein containing nucleosomes, a method we term phosphorylation-specific chromatin affinity purification (PS-ChAP). We utilized this PS-ChAP1 approach in combination with quantitative proteomics to identify changes in the phosphorylation status of chromatin-bound proteins on nucleosomes following perturbation of transcriptional processes. We also demonstrate that this method can be employed to map phosphoprotein signaling into chromatin containing nucleosomes through identifying the genes those phosphorylated proteins are found on via thiophosphate PS-ChAP-qPCR. Thus, our results showed that PS-ChAP offers a new strategy for studying cellular signaling and chromatin biology, allowing us to directly and comprehensively investigate phosphorylation signaling into chromatin to investigate if these pathways are involved in altering gene expression. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD002436.
In recent years, cross-linking mass spectrometry has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis. We first benchmarked xIP-MS using the structurally well-characterized phosphoribosyl pyrophosphate synthetase complex. We then applied xIP-MS to the chromatin-associated cohesin (SMC1A/3), XRCC5/6 (Ku70/86), and MCM complexes, and we provide novel structural and biological insights into their architectures and molecular function. Of note, we use xIP-MS to perform topological studies under cell cycle perturbations, showing that the xIP-MS protocol is sufficiently straightforward and efficient to allow comparative cross-linking experiments. This work, therefore, demonstrates that xIP-MS is a robust, flexible, and widely applicable methodology for interrogating chromatin-associated protein complex architectures.
Histones represent a class of proteins ideally suited to analyses by top-down mass spectrometry due to their relatively small size, the high electron transfer dissociation-compatible charge states they exhibit, and the potential to gain valuable information concerning combinatorial post-translational modifications and variants. We recently described new methods in mass spectrometry for the acquisition of high-quality MS/MS spectra of intact proteins (Anderson, L. C., English, A. M., Wang, W., Bai, D. L., Shabanowitz, J., and Hunt, D. F. (2015) Int. J. Mass Spectrom. 377, 617–624). Here, we report an extension of these techniques. Sequential ion/ion reactions carried out in a modified Orbitrap Velos Pro/EliteTM capable of multiple fragment ion fills of the C-trap, in combination with data-dependent and targeted HPLC-MS experiments, were used to obtain high resolution MS/MS spectra of histones from butyrate-treated HeLa cells. These spectra were used to identify several unique intact histone proteoforms with up to 81% sequence coverage. We also demonstrate that parallel ion parking during ion/ion proton transfer reactions can be used to separate species of overlapping m/z that are not separated chromatographically, revealing previously indiscernible signals. Finally, we characterized several truncated forms of H2A and H2B found within the histone fractions analyzed, achieving up to 93% sequence coverage by electron transfer dissociation MS/MS. Results of follow-up in vitro experiments suggest that some of the truncated histone H2A proteoforms we observed can be generated by cathepsin L, an enzyme known to also catalyze clipping of histone H3.
