One-Year Injury History and Safe Dance Practices Among Female Highland Dancers in Canada.

AIMS: Over 8,650 Highland dancers registered to compete in Royal Scottish Official Board of Highland Dancing events worldwide in 2019. While the burden of dance-related injuries is high among dancers, there are few studies examining Highland dance. The purpose of this study was to describe the prevalence of self-reported 1-year injury history and safe dance practices among female Highland dancers. METHODS: Sixty-five female Canadian Highland dancers (median age 18; range 14-47) completed an anonymous online survey at the beginning of the 2019 championship season. Demographics (i.e., age, body mass index), exposure (e.g., months/year dancing), safe dance practices (e.g., environmental, physical, psychological), and 1-year injury history (i.e., yes/no) were self-reported. Three definitions of dance-related injury were used: 1) time-loss (missed ≥1 class, practice, performance, and/or competition); 2) medical attention (requiring professional medical care); and 3) any physical complaint that affected full participation. RESULTS: Most participants were training at the elite standard/premier level (86%, 95%CI 75-93) and for ≥8 months/year (83%, 95%CI 75-93). The proportion of dancers reporting at least one physical complaint in the previous 1 year was 71% (95%CI 58-81). Sixty percent (95%CI 47-71) of dancers reported ≥1 medical attention and/or time-loss injury. All participants reported warming up regularly, with 59% (95%CI 46-70) participating in regular cool-downs. CONCLUSION: The prevalence of 1-year injury history among female Highland dancers is high. Education on the benefits of safe dance practice for Highland dancers may be useful. Prospective cohort studies are needed to understand the dynamic nature of dance injuries across a full competitive season.

Instructor Social Support in the Group Physical Activity Context: Older Participants' Perspectives.

Instructors in organized physical activity classes can be a source of social support through their relationships with participants, influence on participants' interactions with each other, and design of activities. Grounded in interpretive description, the objective of this study was to examine older adults' experiences of and their perspectives on group physical activity instructors' supportive behaviors. Observations of 16 group physical activity classes (N = 295) and focus groups or interviews with N = 38 class participants aged ≥ 55 (n = 29 women) were conducted at four municipal recreation facilities in a Canadian city. Five themes shed light on how instructors provided social support: (a) supporting autonomous engagement, (b) developing caring connections, (c) fostering trust through expert instruction, (d) managing conflict directly and effectively, and (e) creating a climate where people want to go. Instructor training should consider older adults' social support needs and help instructors embody behaviors that support continued physical activity participation, thereby contributing to healthy aging.

Social Supports and Barriers for Older Adults Not Participating in Group Physical Activity.

Group physical activity can provide physical and social benefits; however, social barriers or a lack of social support may affect participation. This study examined social-support needs and barriers among older adults who were not participating in group physical activities. Using interpretive description, semistructured interviews were conducted with 38 older adults (M = 70.9 years; 81.6% women). Themes were grouped into two categories. Category 1, expectations and initial impressions, consisted of the following: (a) Groups cannot meet everyone's expectations or interests, (b) groups are intimidating to join, and (c) the need for inclusive programming. Category 2, social processes within group physical activity, consisted of (a) modeling physical activity behaviors, (b) sharing information and suggestions about physical activity opportunities, and (c) encouragement and genuine interest. Outreach to this population should aim to address these barriers and utilize these supportive behaviors to reduce feelings of intimidation and promote participation among older adults.

Ultraviolet Photodissociation Mass Spectrometry for Analysis of Biological Molecules.

The development of new ion-activation/dissociation methods continues to be one of the most active areas of mass spectrometry owing to the broad applications of tandem mass spectrometry in the identification and structural characterization of molecules. This Review will showcase the impact of ultraviolet photodissociation (UVPD) as a frontier strategy for generating informative fragmentation patterns of ions, especially for biological molecules whose complicated structures, subtle modifications, and large sizes often impede molecular characterization. UVPD energizes ions via absorption of high-energy photons, which allows access to new dissociation pathways relative to more conventional ion-activation methods. Applications of UVPD for the analysis of peptides, proteins, lipids, and other classes of biologically relevant molecules are emphasized in this Review.

N- and O-Glycosylation of the SARS-CoV-2 Spike Protein.

Covid-19 pandemic outbreak is the reason of the current world health crisis. The development of effective antiviral compounds and vaccines requires detailed descriptive studies of SARS-CoV-2 proteins. The SARS-CoV-2 spike (S) protein mediates virion binding to the human cells through its interaction with the ACE2 cell surface receptor and is one of the prime immunization targets. A functional virion is composed of three S1 and three S2 subunits created by furin cleavage of the spike protein at R682, a polybasic cleavage site that differs from the SARS-CoV spike protein of 2002. By analysis of the protein produced in HEK293 cells, we observe that the spike is O-glycosylated on a threonine (T678) near the furin cleavage site occupied by core-1 and core-2 structures. In addition, we have identified eight additional O-glycopeptides on the spike glycoprotein and confirmed that the spike protein is heavily N-glycosylated. Our recently developed liquid chromatography-mass spectrometry methodology allowed us to identify LacdiNAc structural motifs on all occupied N-glycopeptides and polyLacNAc structures on six glycopeptides of the spike protein. In conclusion, our study substantially expands the current knowledge of the spike protein's glycosylation and enables the investigation of the influence of O-glycosylation on its proteolytic activation.

Long-term outcomes of left atrial appendage occlusion in high-risk atrial fibrillation patients: 4-year follow up data.

Long-term follow-up data of left atrial appendage (LAA) occlusion in patients with atrial fibrillation (AF) are sparse. To address these data gaps, we analysed the 4-year outcomes of AF patients following LAA occlusion. The was a retrospective cohort study of high-risk patients with AF who underwent successful implantation of the Amulet device at our center between 2014 and 2017. Study endpoints were the rate of stroke, major bleeding and all-cause mortality. We included 71 patients (35.2% females) with a median age of 78 (IQR 73-82) years. Over a median follow-up period of 46 (IQR 19-56) months, the annual rate of ischemic stroke was 1.06 events/100 patient-years (95% CI 0-2.35), hemorrhagic stroke was 1.06 events/100 patient-years (95% CI 0-2.35) and major extracranial bleeding that required unplanned hospital admission was 1.84 events/100 patient-years (95% CI 0.25-3.43). A total of 28 (39.4%) patients died during this period with an annual mortality rate of 10.29 events/100 patient-years (95% CI 7.25-13.32). Our experience suggests that LAA occlusion using the Amulet device appears to be associated with a low risk of ischemic stroke in high-risk AF patients who are deemed unsuitable for oral anticoagulation; however, these patients have a high rate of mortality over the medium to long-term follow-up, and an ongoing significant risk of bleeding and thrombotic events.

Top-Down Characterization of Denatured Proteins and Native Protein Complexes Using Electron Capture Dissociation Implemented within a Modified Ion Mobility-Mass Spectrometer.

Electron-based fragmentation methods have revolutionized biomolecular mass spectrometry, in particular native and top-down protein analysis. Here, we report the use of a new electromagnetostatic cell to perform electron capture dissociation (ECD) within a quadrupole/ion mobility/time-of-flight mass spectrometer. This cell was installed between the ion mobility and time-of-flight regions of the instrument, and fragmentation was fast enough to be compatible with mobility separation. The instrument was already fitted with electron transfer dissociation (ETD) between the quadrupole and mobility regions prior to modification. We show excellent fragmentation efficiency for denatured peptides and proteins without the need to trap ions in the gas phase. Additionally, we demonstrate native top-down backbone fragmentation of noncovalent protein complexes, leading to comparable sequence coverage to what was achieved using the instrument's existing ETD capabilities. Limited collisional ion activation of the hemoglobin tetramer before ECD was reflected in the observed fragmentation pattern, and complementary ion mobility measurements prior to ECD provided orthogonal evidence of monomer unfolding within this complex. The approach demonstrated here provides a powerful platform for both top-down proteomics and mass spectrometry-based structural biology studies.

Towards mapping electrostatic interactions between Kdo2-lipid A and cationic antimicrobial peptides via ultraviolet photodissociation mass spectrometry.

Cationic antimicrobial peptides (CAMPs) have been known to act as multi-modal weapons against Gram-negative bacteria. As a new approach to investigate the nature of the interactions between CAMPs and the surfaces of bacteria, native mass spectrometry and two MS/MS strategies (ultraviolet photodissociation (UVPD) and higher energy collisional activation (HCD)) are used to examine formation and disassembly of saccharolipid·peptide complexes. Kdo2-lipid A (KLA) is used as a model saccharolipid to evaluate complexation with a series of cationic peptides (melittin and three analogs). Collisional activation of the KLA·peptide complexes results in the disruption of electrostatic interactions, resulting in apo-sequence ions with shifts in the distribution of ions compared to the fragmentation patterns of the apo-peptides. UVPD of the KLA·peptide complexes results in both apo- and holo-sequence ions of the peptides, the latter in which the KLA remains bound to the truncated peptide fragment despite cleavage of a covalent bond of the peptide backbone. Mapping both the N- and C-terminal holo-product ions gives insight into the peptide motifs (specifically an electropositive KRKR segment and a proline residue) that are responsible for mediating the electrostatic interactions between the cationic peptides and saccharolipid.

Percutaneous Device Closure of Paravalvular Leak: Combined Experience From the United Kingdom and Ireland.

BACKGROUND: Paravalvular leak (PVL) occurs in 5% to 17% of patients following surgical valve replacement. Percutaneous device closure represents an alternative to repeat surgery. METHODS: All UK and Ireland centers undertaking percutaneous PVL closure submitted data to the UK PVL Registry. Data were analyzed for association with death and major adverse cardiovascular events (MACE) at follow-up. RESULTS: Three hundred eight PVL closure procedures were attempted in 259 patients in 20 centers (2004-2015). Patient age was 67±13 years; 28% were female. The main indications for closure were heart failure (80%) and hemolysis (16%). Devices were successfully implanted in 91% of patients, via radial (7%), femoral arterial (52%), femoral venous (33%), and apical (7%) approaches. Nineteen percent of patients required repeat procedures. The target valve was mitral (44%), aortic (48%), both (2%), pulmonic (0.4%), or transcatheter aortic valve replacement (5%). Preprocedural leak was severe (61%), moderate (34%), or mild (5.7%) and was multiple in 37%. PVL improved postprocedure (P<0.001) and was none (33.3%), mild (41.4%), moderate (18.6%), or severe (6.7%) at last follow-up. Mean New York Heart Association class improved from 2.7±0.8 preprocedure to 1.6±0.8 (P<0.001) after a median follow-up of 110 (7-452) days. Hospital mortality was 2.9% (elective), 6.8% (in-hospital urgent), and 50% (emergency) (P<0.001). MACE during follow-up included death (16%), valve surgery (6%), late device embolization (0.4%), and new hemolysis requiring transfusion (1.6%). Mitral PVL was associated with higher MACE (hazard ratio [HR], 1.83; P=0.011). Factors independently associated with death were the degree of persisting leak (HR, 2.87; P=0.037), New York Heart Association class (HR, 2.00; P=0.015) at follow-up and baseline creatinine (HR, 8.19; P=0.001). The only factor independently associated with MACE was the degree of persisting leak at follow-up (HR, 3.01; P=0.002). CONCLUSION: Percutaneous closure of PVL is an effective procedure that improves PVL severity and symptoms. Severity of persisting leak at follow-up is independently associated with both MACE and death. Percutaneous closure should be considered as an alternative to repeat surgery.

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.

Characterization of hydrogen bonding motifs in proteins: hydrogen elimination monitoring by ultraviolet photodissociation mass spectrometry.

Determination of structure and folding of certain classes of proteins remains intractable by conventional structural characterization strategies and has spurred the development of alternative methodologies. Mass spectrometry-based approaches have a unique capacity to differentiate protein heterogeneity due to the ability to discriminate populations, whether minor or major, featuring modifications or complexation with non-covalent ligands on the basis of m/z. Cleavage of the peptide backbone can be further utilized to obtain residue-specific structural information. Here, hydrogen elimination monitoring (HEM) upon ultraviolet photodissociation (UVPD) of proteins transferred to the gas phase via nativespray ionization is introduced as an innovative approach to deduce backbone hydrogen bonding patterns. Using well-characterized peptides and a series of proteins, prediction of the engagement of the amide carbonyl oxygen of the protein backbone in hydrogen bonding using UVPD-HEM is demonstrated to show significant agreement with the hydrogen-bonding motifs derived from molecular dynamics simulations and X-ray crystal structures.

193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology.

Protein-protein interfaces and architecture are critical to the function of multiprotein complexes. Mass spectrometry-based techniques have emerged as powerful strategies for characterization of protein complexes, particularly for heterogeneous mixtures of structures. In the present study, activation and dissociation of three tetrameric protein complexes (streptavidin, transthyretin, and hemoglobin) in the gas phase was undertaken by 193 nm ultraviolet photodissociation (UVPD) for the characterization of higher order structure. High pulse energy UVPD resulted in the production of dimers and low charged monomers exhibiting symmetrical charge partitioning among the subunits (the so-called symmetrical dissociation pathways), consistent with the subunit organization of the complexes. In addition, UVPD promoted backbone cleavages of the monomeric subunits, the abundances of which corresponded to the more flexible loop regions of the proteins.

UVliPiD: A UVPD-Based Hierarchical Approach for De Novo Characterization of Lipid A Structures.

The lipid A domain of the endotoxic lipopolysaccharide layer of Gram-negative bacteria is comprised of a diglucosamine backbone to which a variable number of variable length fatty acyl chains are anchored. Traditional characterization of these tails and their linkages by nuclear magnetic resonance (NMR) or mass spectrometry is time-consuming and necessitates databases of pre-existing structures for structural assignment. Here, we introduce an automated de novo approach for characterization of lipid A structures that is completely database-independent. A hierarchical decision-tree MS(n) method is used in conjunction with a hybrid activation technique, UVPDCID, to acquire characteristic fragmentation patterns of lipid A variants from a number of Gram-negative bacteria. Structural assignments are derived from integration of key features from three to five spectra and automated interpretation is achieved in minutes without the need for pre-existing information or candidate structures. The utility of this strategy is demonstrated for a mixture of lipid A structures from an enzymatically modified E. coli lipid A variant. A total of 27 lipid A structures were discovered, many of which were isomeric, showcasing the need for a rapid de novo approach to lipid A characterization.

Charge site assignment in native proteins by ultraviolet photodissociation (UVPD) mass spectrometry.

Characterization of all gas-phase charge sites of natively sprayed proteins and peptides is demonstrated using 193 nm UVPD. The high sequence coverage offered by UVPD is exploited for the accurate determination of charge sites in protein systems up to 18 kDa, allowing charge site to be studied as a function of protein conformation and the presence of disulfide bonds. Charging protons are found on both basic sidechains and on the amide backbone of less basic amino acids such as serine, glutamine, and proline. UVPD analysis was performed on the 3+ charge state of melittin, the 5+ to 8+ charge states of ubiquitin, and the 8+ charge state of reduced and oxidized ß-lactoglobulin. The location of charges in gas-phase proteins is known to impact structure; molecular modeling of different charge site motifs of 3+ melittin demonstrates how placement of protons in simulations can dramatically impact the predicted structure of the molecule. The location of positive charge sites in ubiquitin and ß-lactoglobulin are additionally found to depend on the presence or absence of salt-bridges, columbic repulsion across the length of the peptide, and protein conformation. Charge site isomers are demonstrated for ubiquitin and ß-lactoglobulin but found to be much less numerous than previously predicted.

Percutaneous closure of postinfarction ventricular septal defect: in-hospital outcomes and long-term follow-up of UK experience.

BACKGROUND: Postinfarction ventricular septal defect carries a grim prognosis. Surgical repair offers reasonable outcomes in patients who survive a healing phase. Percutaneous device implantation represents a potentially attractive early alternative. METHODS AND RESULTS: Postinfarction ventricular septal defect closure was attempted in 53 patients from 11 centers (1997-2012; aged 72±11 years; 42% female). Nineteen percent had previous surgical closure. Myocardial infarction was anterior (66%) or inferior (34%). Time from myocardial infarction to closure procedure was 13 (first and third quartiles, 5-54) days. Devices were successfully implanted in 89% of patients. Major immediate complications included procedural death (3.8%) and emergency cardiac surgery (7.5%). Immediate shunt reduction was graded as complete (23%), partial (62%), or none (15%). Median length of stay after the procedure was 5.0 (2.0-9.0) days. Fifty-eight percent survived to discharge and were followed up for 395 (63-1522) days, during which time 4 additional patients died (7.5%). Factors associated with death after postinfarction ventricular septal defect closure included the following: age (hazard ratio [HR]=1.04; P=0.039), female sex (HR=2.33; P=0.043), New York Heart Association class IV (HR=4.42; P=0.002), cardiogenic shock (HR=3.75; P=0.003), creatinine (HR=1.007; P=0.003), defect size (HR=1.09; P=0.026), inotropes (HR=4.18; P=0.005), and absence of revascularization therapy for presenting myocardial infarction (HR=3.28; P=0.009). Prior surgical closure (HR=0.12; P=0.040) and immediate shunt reduction (HR=0.49; P=0.037) were associated with survival. CONCLUSIONS: Percutaneous closure of postinfarction ventricular septal defect is a reasonably effective treatment for these extremely high-risk patients. Mortality remains high, but patients who survive to discharge do well in the longer term.

Resolution of Stepwise Cooperativities of Copper Binding by the Homotetrameric Copper-Sensitive Operon Repressor (CsoR): Impact on Structure and Stability.

The cooperativity of ligand binding is central to biological regulation and new approaches are needed to quantify these allosteric relationships. Herein, we exploit a suite of mass spectrometry (MS) experiments to provide novel insights into homotropic Cu-binding cooperativity, gas-phase stabilities and conformational ensembles of the D2 -symmetric, homotetrameric copper-sensitive operon repressor (CsoR) as a function of Cu(I) ligation state. Cu(I) binding is overall positively cooperative, but is characterized by distinct ligation state-specific cooperativities. Structural transitions occur upon binding the first and fourth Cu(I) , with the latter occurring with significantly higher cooperativity than previous steps; this results in the formation of a holo-tetramer that is markedly more resistant than apo-, and partially ligated CsoR tetramers toward surface-induced dissociation (SID).

Gas-phase helical peptides mimic solution-phase behavior.

In solution, α-helices are stabilized at the termini by a variety of different capping interactions. Study of these interactions in the gas phase provides a unique means to explore the intrinsic properties that cause this stabilization. Evidence of helical and globular conformations is presented here for gas-phase, doubly charged peptides of sequence XAnK, wherein X is D, N, Q, or L. The relative abundance of the helical conformation is found to vary as a function of peptide length and the identity of the first amino acid, consistent with solution phase studies that have looked at the identity of the first amino acid. The N-terminal, b ion fragments of the doubly charged precursor peptides are shown to form helical and globular conformations. The stability of the helical fragments is examined as a function of fragment length, N-terminal amino acid, precursor conformation, and the activation energy used to generate the fragment. At lower collision energies, helical b ions preferentially form, particularly from helical precursors. The abundance of the helical b ion population is observed to dramatically decrease for NAn and DAn b ions smaller than the b10; simulations suggest this feature is due to the b10 having two complete turns of the helix, while the b9 and smaller ions have only a partial second turn, suggesting the b10 is the lower limit for stable helical conformations in b ions. Use of higher collision energies promotes the formation of globular structures in the b ions. This characteristic is attributed to increased conformational dynamics and subsequently improved proton transfer kinetics from the b ion's C-terminal oxazolone ring to the N-terminus.

Bringing Dance to Older Adults: Program Experts' Perspectives on the Role of Community Dance Classes to Support Older Adults.

BACKGROUND: Dancing offers several health and wellness benefits for older adults: it may promote physical literacy (PL) and positively influence the aging process. Yet, limited research considers the perspectives of those with experience working with older adults and in community dance programming. OBJECTIVE: The purpose of this study was to understand program experts' perspectives on how older adult community dance can promote PL and contribute to age-friendly cities and community initiatives. METHODS AND FINDINGS: Four themes were identified from semi-structured interviews with five program experts: (1) expert instructors tailor classes to participants' needs and interests; (2) the heart of what draws us to dancing: authentic experience and social connection; (3) elitist, ableist, and gendered assumptions of dance prevent social inclusion of older adults in dancing spaces; and (4) collaboration across sectors is needed to offer accessible, sustainable, and valued dance programming. DISCUSSION: Recommendations for developing and implementing older adult community dance programming are described.

The influence glutamic acid in protonated b3 → b2 formation from VGEIG and related analogs.

A direct pathway for the fragmentation of peptide b3 fragment ions to b2 ions has, until now, not been identified. Experimental evidence for the formation of a b3 anhydride structure and isomerization to an extended macrocycle is demonstrated here by comparison of the completely different fragmentation patterns of the b3 ions generated from protonated VGEIG and its methyl ester. In particular, the absence of a b2 ion in the fragmentation spectrum of the methyl ester b3 indicates that facile fragmentation of an anhydride-type b3 is responsible for virtually all b2 ions formed. The stability of this b3 structure and the ease with which it fragments to the b2 may be responsible for the relatively high abundance of the b3 and b2 ions. IRMPD action spectroscopy measurements indicate the presence of a ring protonated oxazolone in the b2 population. VGEIG and three related analogs, VALEIG, VADEIG, and V(Aib)EIG were studied by QCID-HDX-SORI experiments in an FT-ICR instrument, and provide significant evidence for extensive alpha proton scrambling in an ion-molecule complex formed between the b2 and neutral loss fragment following formation of the b2. MS3 and HDX of VG(2,2-d2)EIG indicate that the scrambled b2 ions have the same structure as the unscrambled b2. Based on these data and with the support of molecular modeling, we propose a new mechanism for this scrambling, in which the alpha protons are transferred in a multistep pathway during an ion-molecule complex formed between the b2 and amino-terminated anhydride ring neutral loss component.

Antimicrobial Resistance.

Antimicrobial resistance is developing rapidly and threatens to outstrip the rate at which new antimicrobials are introduced. Genetic recombination allows bacteria to rapidly disseminate genes encoding for antimicrobial resistance within and across species. Antimicrobial use creates a selective evolutionary pressure, which leads to further resistance. Antimicrobial stewardship, best use, and infection prevention are the most effective ways to slow the spread and development of antimicrobial resistance.