An acute pulmonary embolism (aPE) is characterised by occlusion of one or more pulmonary arteries. Physiological disturbance may be minimal, but often cardiac output decreases as the right ventricle ...attempts to overcome increased afterload. Additionally, ventilation‐perfusion mismatches can develop in affected vascular beds, reducing systemic oxygenation. Incidence is reported at 50–75 per 100 000 in Australia and New Zealand, with 30‐day mortality rates ranging from 0.5% to over 20%. Incidence is likely to increase with the ageing population, increased survival of patients with comorbidities that are considered risk factors and improving sensitivity of imaging techniques. Use of clinical prediction scores, such as the Wells score, has assisted in clinical decision‐making and decreased unnecessary radiological investigations. However, imaging (i.e. computed tomography pulmonary angiography or ventilation‐perfusion scans) is still necessary for objective diagnosis. Anti‐coagulation remains the foundation of PE management. Haemodynamically unstable patients require thrombolysis unless absolutely contraindicated, while stable patients with right ventricular dysfunction or ischaemia should be aggressively anti‐coagulated. Stable patients with no right ventricular dysfunction can be discharged home early with anti‐coagulation and review. However, treatment should be case dependent with full consideration of the patient’s clinical state. Direct oral anti‐coagulants have become an alternative to vitamin K antagonists and are facilitating shorter hospital admissions. Additionally, duration of anti‐coagulation must be decided by considering any provoking factors, bleeding risk and comorbid state. Patients with truly unprovoked or idiopathic PE often require indefinite treatment, while in provoked cases it is typically 3 months with some patients requiring longer periods of 6–12 months.
Background
There is increasing evidence supporting early discharge of patients with acute pulmonary embolism (aPE) deemed ‘low prognostic risk’ as a safe and viable alternative to admission if ...identified correctly by guideline algorithms.
Aim
To determine if risk stratification guidelines were followed accurately in an Australian tertiary hospital.
Methods
Patients admitted to the emergency department with a diagnosis of PE were included from December 2012 to December 2017. The 272 patients were retrospectively assessed for prognostic risk prior to and after release of the 2014 European Society of Cardiology (ESC) guidelines. This included the simplified Pulmonary Embolism Severity Index (sPESI), and evidence of right heart dysfunction. Thereafter, patients were dichotomised into low (i.e. sPESI = 0) and non‐low (i.e. sPESI ≥1 with or without the evidence of right heart dysfunction) prognostic risk groups.
Results
Prior to ESC guideline release, 52 (65%) of the 80 patients diagnosed with PE were non‐low risk and 12 (23%) of these were discharged home; 11 (91.7%) of the 12 discharges had unrecognised sPESI medical history components. After ESC guideline release, 122 (63.5%) of the 192 patients were non‐low risk and 20 (16.4%) of these were discharged home; 18 (90%) of the 20 discharges had unrecognised sPESI medical history components.
Conclusion
We found that the sPESI score is not adequately applied in determining prognostic risk for acute PE. In cases of non‐low‐risk discharge, both prior to and after ESC guideline release, the medical history components of the sPESI score are under‐recognised as a marker of increased prognostic risk.
Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in Neisseria meningitidis and other Gram-negative ...pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Mass Spec Studio package was designed to support the extraction of hydrogen-deuterium exchange and covalent labeling data for a range of mass spectrometry (MS)-based workflows, to integrate with ...restraint-driven protein modeling activities. In this report, we present an extension of the underlying Studio framework and provide a plug-in for crosslink (XL) detection. To accommodate flexibility in XL methods and applications, while maintaining efficient data processing, the plug-in employs a peptide library reduction strategy via a presearch of the tandem-MS data. We demonstrate that prescoring linear unmodified peptide tags using a probabilistic approach substantially reduces search space by requiring both crosslinked peptides to generate sparse data attributable to their linear forms. The method demonstrates highly sensitive crosslink peptide identification with a low false positive rate. Integration with a Haddock plug-in provides a resource that can combine multiple sources of data for protein modeling activities. We generated a structural model of porcine transferrin bound to TbpB, a membrane-bound receptor essential for iron acquisition in Actinobacillus pleuropneumoniae. Using mutational data and crosslinking restraints, we confirm the mechanism by which TbpB recognizes the iron-loaded form of transferrin, and note the requirement for disparate sources of restraint data for accurate model construction. The software plugin is freely available at www.msstudio.ca.
The data analysis practices associated with hydrogen–deuterium exchange mass spectrometry (HX-MS) lag far behind that of most other MS-based protein analysis tools. A reliance on external tools from ...other fields and a persistent need for manual data validation restrict this powerful technology to the expert user. Here, we provide an extensive upgrade to the HX data analysis suite available in the Mass Spec Studio in the form of two new apps (HX-PIPE and HX-DEAL), completing a workflow that provides an HX-tailored peptide identification capability, accelerated validation routines, automated spectral deconvolution strategies, and a rich set of exportable graphics and statistical reports. With these new tools, we demonstrate that the peptide identifications obtained from undeuterated samples generated at the start of a project contain information that helps predict and control the extent of manual validation required. We also uncover a large fraction of HX-usable peptides that remains unidentified in most experiments. We show that automated spectral deconvolution routines can identify exchange regimes in a project-wide manner, although they remain difficult to accurately assign in all scenarios. Taken together, these new tools provide a robust and complete solution suitable for the analysis of high-complexity HX-MS data.
In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There ...is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry identifies an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, where the synaptic complex supports hierarchical processing of the ends and scaffold assembly, requiring displacement of the catalytic subunit and tension release through kinase activity.
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•The synaptic complex formed by DNA-PK adopts a staggered “head-to-head” arrangement•A plug domain functions to block the end of the double-strand DNA•When bound to DNA and primed with nucleotide, the holoenzyme adopts a tensed state•The geometry supports the assembly of scaffolding factor to manage repair transitions
Hepburn et al. used an integrative approach relying on mass spectrometry to demonstrate that the massive trimeric complex DNA-PK caps DNA double-strand breaks with a strongly protective plug-like domain in a large, unstable synaptic structure that holds the two ends far apart.
Proteomics methodology has expanded to include protein structural analysis, primarily through cross-linking mass spectrometry (XL-MS) and hydrogen–deuterium exchange mass spectrometry (HX-MS). ...However, while the structural proteomics community has effective tools for primary data analysis, there is a need for structure modeling pipelines that are accessible to the proteomics specialist. Integrative structural biology requires the aggregation of multiple distinct types of data to generate models that satisfy all inputs. Here, we describe IMProv, an app in the Mass Spec Studio that combines XL-MS data with other structural data, such as cryo-EM densities and crystallographic structures, for integrative structure modeling on high-performance computing platforms. The resource provides an easily deployed bundle that includes the open-source Integrative Modeling Platform program (IMP) and its dependencies. IMProv also provides functionality to adjust cross-link distance restraints according to the underlying dynamics of cross-linked sites, as characterized by HX-MS. A dynamics-driven conditioning of restraint values can improve structure modeling precision, as illustrated by an integrative structure of the five-membered Polycomb Repressive Complex 2. IMProv is extensible to additional types of data.
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•A resource for streamlining integrative structural modeling using XL-MS.•A method for contextualizing crosslink restraints with HX-MS data.•Improved model precision arises from dynamics-controlled cross-link restraints.•A complete structural model of the PRC2 complex.
Proteomics has much to contribute to the structural analysis of cells, particularly considering the new developments in computational structural prediction. Cross-linking MS data can use structural building blocks (from empirical or modeled sources) to assemble models of higher-order complexes involving many proteins. In this work, we present a resource (IMProv) that reduces the barrier to integrative structural modeling and improves the precision of modeling exercises by incorporating dynamics (in the form of hydrogen/deuterium exchange data) to condition the cross-linking restraints.
Hydrogen-deuterium exchange mass spectrometry (HX-MS) has made important contributions to the study of protein structure and function. Unfortunately, it is not known for low limits of detection, when ...compared with other forms of peptide-based or bottom-up protein MS methods. Systems perform poorly on sub-pmol quantities of protein states with greater than 300 kDa of unique sequences. The HX-MS analysis of complex protein states would be possible if proteomics-grade configurations could be used reliably, but temperature and temporal constraints have proven to be significant design challenges. Here, we describe an integrated HX-MS ion source operating on a vented-column geometry, which brings regulated column cooling right to the spray tip. The design offers chromatographic peak widths of 2-6 s (FWHM). It provides stable operation at 500 nL min
, while retaining deuteration levels comparable to conventional geometries. We demonstrate at least a 50-fold improvement in protein consumption levels, and illustrate robustness by measuring peptide-averaged protection factors for 90% of DNA-PKcs, a 469 kDa protein, from 0.5 pmol injections.
Hydrogen-deuterium exchange mass spectrometry (HX-MS) has made important contributions to the study of protein structure and function. Unfortunately, it is not known for low limits of detection, when ...compared with other forms of peptide-based or bottom-up protein MS methods. Systems perform poorly on sub-pmol quantities of protein states with greater than 300 kDa of unique sequences. The HX-MS analysis of complex protein states would be possible if proteomics-grade configurations could be used reliably, but temperature and temporal constraints have proven to be significant design challenges. Here, we describe an integrated HX-MS ion source operating on a vented-column geometry, which brings regulated column cooling right to the spray tip. The design offers chromatographic peak widths of 2-6 s (FWHM). It provides stable operation at 500 nL min
−1
, while retaining deuteration levels comparable to conventional geometries. We demonstrate at least a 50-fold improvement in protein consumption levels, and illustrate robustness by measuring peptide-averaged protection factors for 90% of DNA-PKcs, a 469 kDa protein, from 0.5 pmol injections.
An in-source column chiller supports nanoHX-MS workflows for analyzing proteins from cellular extracts.
Structural Mass Spectrometry (SMS) provides a comprehensive toolbox for the analysis of protein structure and function. It offers multiple sources of structural information that are increasingly ...useful for integrative structural modeling of complex protein systems. As MS-based structural workflows scale to larger systems, consistent and coherent data interpretation resources are needed to better support modeling. Unlike the proteomics community, practitioners of SMS lack adequate computational tools. Here, we review new developments in the Mass Spec Studio: an expandable ecosystem of workflows for the analysis of complementary SMS techniques with linkages to modeling. Current functionality in the Studio (version 2) supports three major SMS workflows (crosslinking, hydrogen/deuterium exchange and covalent labelling) and two pipelines for structural modeling, with a special focus on data integration. The Mass Spec Studio is an architecture focused on rapid and robust extension of functionality by a community of developers.
This review surveys the new data analysis capabilities within the Mass Spec Studio, a rich framework for rapid software development specifically targeting the community of structural proteomics and structural mass spectrometry. Updates to crosslinking, hydrogen/deuterium-exchange and covalent labeling apps are provided as well as a utility for translating such analyses into restraints that support integrative structural modeling. These new capabilities, together with the underlying design tools and content, provide the community with a wealth of resources to tackle complex structural problem and design new approaches to data analysis.
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•Mass spectrometry offers a diverse set of techniques for protein structure analysis.•Compared to proteomics, structural MS techniques are not well supported in software.•Accurate structural modeling requires the integration of complementary data types.•The Studio provides data analysis pipelines and integration for structural modeling.•The Studio enables rapid and robust development of new analytical workflows.
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