The Convergent Validity of the SWAY Balance Application to Assess Postural Stability in Military Cadets Recovering from Concussion.

Background: Concussions are often accompanied by balance disturbances. Clinically accurate evaluation systems are often expensive, large, and inaccessible to most clinicians. The Sway Balance Mobile Application (SWAY) is an accessible method to quantify balance changes. Purpose: To determine the known groups and convergent validity of the SWAY to assess balance after a concussion. Study Design: Case-Control Study. Methods: Twenty participants with acute concussion and twenty controls were recruited. At initial, one-week, and final return to activity (RTA) evaluations, all participants completed the Sports Concussion Assessment Tool (SCAT-5), and balance control measured by SWAY mBESS and NeuroCom Balance Master Sensory Organization Test (SOT). Mixed model ANOVAs were used to detect differences in SWAY mBESS and NeuroCom SOT scores with time (initial, one-week, final RTA) as the within-subjects factor and group (concussed, healthy) as the between-subjects factor. Spearman's Rho correlations explored the associations between NeuroCom SOT scores, SWAY scores, SCAT-5 symptom scores, and time in days to final RTA. Results: The sampled population was predominantly male and age (20 ± 1), and BMI differences were insignificant between groups. The SWAY did not detect differences between healthy and concussed participants and did not detect change over time [F(2,40) = .114, p = 0.89; F(2,40)= .276, p =0.60]. When assessing the relationship between the SWAY and the SOT, no correlation was found at any time point (r = -0.317 to -0.062, p > 0.05). Time to RTA demonstrated a moderate correlation with both SCAT-5 symptom severity score (r = .693, p < 0.01) and SCAT-5 total symptom score (r = .611, p < 0.01) at the one-week follow-up. Conclusion: The SWAY mBESS does not appear to be a valid balance assessment for the concussed patient. The SWAY mBESS in patients with concussion failed to demonstrate convergent validity and did not demonstrate an ability to validate known groups. When assessing the time to final RTA, the one-week post-initial assessment SCAT-5 symptom severity and total scores may help determine the length of recovery in this population. Level of Evidence: Level 3.

Host lipids regulate multicellular behavior of a predator of a human pathogen.

As symbionts of animals, microbial eukaryotes benefit and harm their hosts in myriad ways. A model microeukaryote (Capsaspora owczarzaki) is a symbiont of Biomphalaria glabrata snails and may prevent transmission of parasitic schistosomes from snails to humans. However, it is unclear which host factors determine Capsaspora's ability to colonize snails. Here, we discovered that Capsaspora forms multicellular aggregates when exposed to snail hemolymph. We identified a molecular cue for aggregation: a hemolymph-derived phosphatidylcholine, which becomes elevated in schistosome-infected snails. Therefore, Capsaspora aggregation may be a response to the physiological state of its host, and it may determine its ability to colonize snails and exclude parasitic schistosomes. Furthermore, Capsaspora is an evolutionary model organism whose aggregation may be ancestral to animals. This discovery, that a prevalent lipid induces Capsaspora multicellularity, suggests that this aggregation phenotype may be ancient. Additionally, the specific lipid will be a useful tool for further aggregation studies.

Apolipoprotein E-containing HDL decreases caspase-dependent apoptosis of memory regulatory T lymphocytes.

Plasma levels of HDL cholesterol are inversely associated with CVD progression. It is becoming increasingly clear that HDL plays important roles in immunity that go beyond its traditionally understood roles in lipid transport. We previously reported that HDL interaction with regulatory T cells (Treg) protected them from apoptosis, which could be a mechanism underlying the broad anti-inflammatory effect of HDL. Herein, we extend our work to show that HDL interacts mainly with memory Treg, particularly with the highly suppressive effector memory Treg, by limiting caspase-dependent apoptosis in an Akt-dependent manner. Reconstitution experiments identified the protein component of HDL as the primary driver of the effect, though the most abundant HDL protein, apolipoprotein A-I (APOA1), was inactive. In contrast, APOE-depleted HDL failed to rescue effector memory Treg, suggesting the critical role of APOE proteins. HDL particles reconstituted with APOE, and synthetic phospholipids blunted Treg apoptosis at physiological concentrations. The APOE3 and APOE4 isoforms were the most efficient. Similar results were obtained when lipid-free recombinant APOEs were tested. Binding experiments showed that lipid-free APOE3 bound to memory Treg but not to naive Treg. Overall, our results show that APOE interaction with Treg results in blunted caspase-dependent apoptosis and increased survival. As dysregulation of HDL-APOE levels has been reported in CVD and obesity, our data bring new insight on how this defect may contribute to these diseases.

Diagnostic Imaging for Distal Extremity Injuries in Direct Access Physical Therapy: An Observational Study.

Background: Military physical therapists practicing direct-access routinely utilize diagnostic imaging and numerous published case reports demonstrate the ability of physical therapists to diagnose and appropriately disposition patients with foot/ankle and wrist/hand fractures. However, no larger cohort studies have explored the utilization of diagnostic imaging by physical therapists to detect fractures. Hypothesis/Purpose: To describe the utilization of diagnostic imaging in foot/ankle and wrist/hand injuries by physical therapists in a direct-access sports physical therapy clinic. Study Design: Retrospective cohort study. Methods: The Agfa Impax Client 6 image viewing software (IMPAX) was searched from 2014 to 2018 for patients with diagnostic imaging ordered for foot/ankle and wrist/hand injuries. The Armed Forces Health Longitudinal Technology Application (AHLTA) electronic medical record was independently reviewed by the principal and co-investigator physical therapists. Data extracted were demographics and elements from the patient history and physical examination. Results: In foot/ankle injuries, physical therapists diagnosed a fracture in 16% of the 177 cases and waited for an average of 3.9 days and 1.3 visits before ordering imaging. In wrist/hand injuries, physical therapists diagnosed a fracture in 24% of the 178 cases and waited for an average of 3.7 days and 1.2 visits before ordering imaging. The time to definitive care from the initial physical therapy evaluation was significantly different (p = 0.04) for foot/ankle fractures (0.6 days) compared to wrist/hand fractures (5.0 days). The Ottawa Ankle Rules demonstrated a negative likelihood ratio (-LR) of 0.11 (0.02, 0.72) and a positive likelihood ratio (+LR) of 1.99 (1.62, 2.44) for the diagnosis of foot/ankle fracture. Conclusions: Physical therapists utilizing diagnostic imaging in a direct-access sports physical therapy clinic diagnosed fractures in similar proportions for foot/ankle and wrist/hand injuries and quickly dispositioned patients to definitive care for those fractures. The diagnostic accuracy of the Ottawa Ankle Rules was similar to previously reported values. Level of Evidence: Level 3.

Impaired response of memory Treg to high density lipoproteins is associated with intermediate/high cardiovascular disease risk in persons with HIV.

Cardiovascular disease (CVD) is a leading cause of enhanced morbidity and mortality in persons with HIV (PWH) in the era of highly active antiretroviral therapy (AART). However, the underlying mechanisms are not fully understood. Regulatory T cells (Treg), notably the highly suppressive memory subset, have been shown to limit CVD. Importantly, memory Treg cell numbers remain low in many treated PWH. High density lipoproteins (HDL) also protect from CVD, and we previously found that Treg-HDL interactions reduce oxidative stress in these cells. Here, we evaluated Treg-HDL interactions in PWH and whether they were operative in those higher CVD risk. To do that, we recruited a cohort of PWH with intermediate/high CVD risk (median ASCVD risk score of 13.2%, n=15) or low/borderline risk (median ASCVD risk score of 3.6%, n=14), as well as a group of statins treated PWH with intermediate/high CVD risk (median ASCVD risk score of 12.7%, n=14). We evaluated Treg frequency, phenotype and response to HDL. PWH with Int/High CVD risk had a significantly lower number of memory Treg, but memory Treg were more activated and displayed an inflammatory phenotype, versus those with Low/BL CVD risk. In untreated patients, Treg absolute numbers were negatively correlated with ASCVD score. Although HDL decreased oxidative stress in memory Treg in all subjects, memory Treg from PWH with Int/High CVD risk were significantly less responsive to HDL than those from PWH with Low/BL CVD risk. The level of oxidative stress in memory Treg positively correlated with ASCVD scores. In contrast, plasma HDL from PWH, regardless of CVD risk, retained their anti-oxidative properties, suggesting that the defect in memory Treg response to HDL is intrinsic. Statin treatment partially ameliorated the memory Treg defect. In conclusion, the defective HDL-Treg interactions may contribute to the inflammation-induced increased CVD risk observed in many AART-treated PWH.

Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux.

Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1's ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the "double super helix." Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.

Shoulder Pain of Spinal Source in the Military: A Case Series.

Musculoskeletal injury (MSI) presents the greatest threat to military mission readiness. Atraumatic shoulder pain is a common military MSI that often results in persistent functional limitations. Shoulder orthopedic evaluation presents many diagnostic challenges, due in part to the possibility of a spinal source of symptoms. This case series outlines the use of mechanical diagnosis and therapy to screen the cervical and thoracic spine in active duty (AD) service members (SMs) with a chief complaint of unchanging or worsening shoulder pain. All three SMs previously received shoulder-specific diagnoses from experienced clinicians, yet repeated movements revealed a possible spinal nociceptive driver that guided targeted intervention. Treatment directed only at the cervical spine resulted in a clinically important improvement within an average of 10 days from the initial evaluation, return to duty (RTD) within an average of 32 days, and continued resolution at 3 months. SMs can independently complete the screening process with guidance from healthcare providers, ultimately shaping the treatment strategy and possibly facilitating self-management of future recurrence. This case series demonstrates that identification of shoulder pain of spinal source in the military population may be an important step in facilitating timely RTD. These cases also highlight the use of a standardized, systematic method to screen the cervical and thoracic spine that concurrently reveals the indicated treatment. Further research to determine the prevalence of shoulder pain of spinal source in the AD population and its impact on RTD rates has the potential to reduce the substantial burden of MSI in the military.

Apolipoprotein E content of VLDL limits LPL-mediated triglyceride hydrolysis.

High levels of circulating triglycerides (TGs), or hypertriglyceridemia, are key components of metabolic diseases, such as type 2 diabetes, metabolic syndrome, and CVD. As TGs are carried by lipoproteins in plasma, hypertriglyceridemia can result from overproduction or lack of clearance of TG-rich lipoproteins (TRLs) such as VLDLs. The primary driver of TRL clearance is TG hydrolysis mediated by LPL. LPL is regulated by numerous TRL protein components, including the cofactor apolipoprotein C-II, but it is not clear how their effects combine to impact TRL hydrolysis across individuals. Using a novel assay designed to mimic human plasma conditions in vitro, we tested the ability of VLDL from 15 normolipidemic donors to act as substrates for human LPL. We found a striking 10-fold difference in hydrolysis rates across individuals when the particles were compared on a protein or a TG basis. While VLDL TG contents moderately correlated with hydrolysis rate, we noticed substantial variations in non-apoB proteins within these particles by MS. The ability of LPL to hydrolyze VLDL TGs did not correlate with apolipoprotein C-II content, but it was strongly inversely correlated with apolipoprotein E (APOE) and, to a lesser extent, apolipoprotein A-II. Addition of exogenous APOE inhibited LPL lipolysis in a dose-dependent manner. The APOE3 and (particularly) APOE4 isoforms were effective at limiting LPL hydrolysis, whereas APOE2 was not. We conclude that APOE on VLDL modulates LPL activity and could be a relevant factor in the pathogenesis of metabolic disease.

The Effect of Blood Flow Restriction Therapy on Shoulder Function Following Shoulder Stabilization Surgery: A Case Series.

Background: Traumatic shoulder instability is a common injury in athletes and military personnel. Surgical stabilization reduces recurrence, but athletes often return to sport before recovering upper extremity rotational strength and sport-specific abilities. Blood flow restriction (BFR) may stimulate muscle growth without the need for heavy resistance training post-surgically. Hypothesis/Purpose: To observe changes in shoulder strength, self-reported function, upper extremity performance, and range of motion (ROM) in military cadets recovering from shoulder stabilization surgery who completed a standard rehabilitation program with six weeks of BFR training. Study Design: Prospective case series. Methods: Military cadets who underwent shoulder stabilization surgery completed six weeks of upper extremity BFR training, beginning post-op week six. Primary outcomes were shoulder isometric strength and patient-reported function assessed at 6-weeks, 12-weeks, and 6-months postoperatively. Secondary outcomes included shoulder ROM assessed at each timepoint and the Closed Kinetic Chain Upper Extremity Stability Test (CKCUEST), the Upper Extremity Y-Balance Test (UQYBT), and the Unilateral Seated Shotput Test (USPT) assessed at the six-month follow-up. Results: Twenty cadets performed an average 10.9 BFR training sessions over six weeks. Statistically significant and clinically meaningful increases in surgical extremity external rotation strength (p < 0.001; mean difference, .049; 95% CI: .021, .077), abduction strength (p < 0.001; mean difference, .079; 95% CI: .050, .108), and internal rotation strength (p < 0.001; mean difference, .060; CI: .028, .093) occurred from six to 12 weeks postoperatively. Statistically significant and clinically meaningful improvements were reported on the Single Assessment Numeric Evaluation (p < 0.001; mean difference, 17.7; CI: 9.4, 25.9) and Shoulder Pain and Disability Index (p < 0.001; mean difference, -31.1; CI: -44.2, -18.0) from six to 12 weeks postoperatively. Additionally, over 70 percent of participants met reference values on two to three performance tests at 6-months. Conclusion: While the degree of improvement attributable to the addition of BFR is unknown, the clinically meaningful improvements in shoulder strength, self-reported function, and upper extremity performance warrant further exploration of BFR during upper extremity rehabilitation. Level of Evidence: 4, Case Series.

Pregnancy is accompanied by larger high density lipoprotein particles and compositionally distinct subspecies.

Pregnancy is accompanied by significant physiological changes, which can impact the health and development of the fetus and mother. Pregnancy-induced changes in plasma lipoproteins are well documented, with modest to no impact observed on the generic measure of high density lipoprotein (HDL) cholesterol. However, the impact of pregnancy on the concentration and composition of HDL subspecies has not been examined in depth. In this prospective study, we collected plasma from 24 nonpregnant and 19 pregnant women in their second trimester. Using nuclear magnetic resonance (NMR), we quantified 11 different lipoprotein subspecies from plasma by size, including three in the HDL class. We observed an increase in the number of larger HDL particles in pregnant women, which were confirmed by tracking phospholipids across lipoproteins using high-resolution gel-filtration chromatography. Using liquid chromatography-mass spectrometry (LC-MS), we identified 87 lipid-associated proteins across size-speciated fractions. We report drastic shifts in multiple protein clusters across different HDL size fractions in pregnant females compared with nonpregnant controls that have major implications on HDL function. These findings significantly elevate our understanding of how changes in lipoprotein metabolism during pregnancy could impact the health of both the fetus and the mother.

Gait Retraining Improves Running Impact Loading and Function in Previously Injured U.S. Military Cadets: A Pilot Study.

INTRODUCTION: Running-related musculoskeletal injury (RRI) among U.S. military service members continues to negatively impact force readiness. There is a paucity of evidence supporting the use of RRI interventions, such as gait retraining, in military populations. Gait retraining has demonstrated effectiveness in altering running biomechanics and reducing running load. The purpose of this pilot study was to investigate the clinical effect of a gait retraining intervention on a military cadet population recovering from a lower-extremity RRI. MATERIALS AND METHODS: The study design is a pilot study. Before study initiation, institutional approval was granted by the Keller Army Community Hospital Office of Human Research Protections. Nine rearfoot strike (RFS) runners recovering from a lower-extremity RRI at the U.S. Military Academy were prospectively enrolled and completed a gait retraining intervention. Participants followed-up with their assigned medical provider 6 times over 10 weeks for a clinical evaluation and running gait retraining. Gait retraining was provided utilizing verbal, visual, and audio feedback to facilitate a change in running foot strike pattern from RFS to non-rearfoot strike (NRFS) and increase preferred running step rate. At pre-intervention and post-intervention running ground reaction forces (GRF) [average vertical loading rate (AVLR), peak vertical GRF], kinematic (foot strike pattern) and temporospatial (step rate, contact time) data were collected. Participants self-reported their level of function via the Single Assessment Numeric Evaluation, Patient-Specific Functional Scale, and total weekly running minutes. Paired samples t-tests and Wilcoxon signed rank tests were used to compare pre- and post-intervention measures of interest. Values of P < .05 were considered statistically significant. RESULTS: Nine patients completed the 10-week intervention (age, 20.3 ± 2.2 years; height, 170.7 ± 13.8 cm; mass, 71.7 ± 14.9 kg; duration of injury symptoms, 192.4 ± 345.5 days; running speed, 2.8 ± 0.38 m/s). All nine runners (100%) transitioned from RFS to NRFS. Left AVLR significantly decreased from 60.3 ± 17.0 bodyweight per second (BW/s) before intervention to 25.9 ± 9.1 BW/s after intervention (P = 0.008; effect size (d) = 2.5). Right AVLR significantly decreased from 60.5 ± 15.7 BW/s to 32.3 ± 12.5 BW/s (P < .001; d = 2.0). Similarly, step rate increased from 169.9 ± 10.0 steps per minute (steps/min) before intervention to 180.5 ± 6.5 steps/min following intervention (P = .005; d = 1.3). Single Assessment Numeric Evaluation scores improved significantly from 75 ± 23 to 100 ± 8 (P = .008; d = 1.5) and Patient-Specific Functional Scale values significantly improved from 6 ± 2.3 to 9.5 ± 1.6 (P = .007; d = 1.8) after intervention. Peak vertical GRF (left, P = .127, d = 0.42; right, P = .052, d = 0.53), contact time (left, P = 0.127, d = 0.42; right, P = 0.052, d = 0.53), and total weekly continuous running minutes (P = 0.095, d = 0.80) remained unchanged at post-intervention. All 9 patients remained injury free upon a 6-month medical record review. CONCLUSIONS: In 9 military service members with a RRI, a 10-week NRFS gait retraining intervention was effective in improving running mechanics and measures of function. Patients remained injury-free 6 months following enrollment. The outcomes of this pilot study suggest that individuals recovering from certain lower-extremity RRIs may benefit from transitioning to an NRFS running pattern.

IS STEP RATE ASSOCIATED WITH RUNNING INJURY INCIDENCE? AN OBSERVATIONAL STUDY WITH 9- MONTH FOLLOW UP.

BACKGROUND: Several strategies have been proposed to reduce loading of the lower extremity while running including step rate manipulation. It is unclear however, whether step rate influences the incidence of lower extremity injuries. PURPOSE: To examine the association between step rate and risk of injury in an adult recreational runner population. STUDY DESIGN: Prospective Cohort. METHODS: A total of 381 runners were prospectively followed for an average of nine months. Two-dimensional video was used to assess preferred step rate during a timed two-mile run or a 5K race. Injury surveillance to record sub-clinical injuries (those for which medical treatment was not sought) was performed via semi-monthly email surveys over the course of one year. Injury surveillance for clinical injuries (those for which medical treatment was sought) was performed via a full medical record review using the Armed Forces Health Longitudinal Technology Application. Clinical, sub-clinical and combined clinical and sub-clinical injury incidence were assessed in separate analyses. Injury was operationally defined as seven or more days of reduced activity due to pain. To assess the predictive validity of running step rate, the step rate of participants who did not develop a musculoskeletal injury during the observation period were compared with the running step rate of participants who did develop an injury during the observation period. RESULTS: Out of 381 runners, 16 sustained a clinical overuse injury for which medical treatment was sought. Mean step rate for clinically un-injured runners was 172 steps/min and mean step rate for clinically injured runners was 173 steps/min which was not statistically significantly different (p = 0.77.) Out of 381 runners, 95 completed all four sub-clinical injury surveys (95/381 = 25%). Out of those 95 runners, 19 sustained a clinical (n=4) or sub-clinical injury (n=15). The step rate of sub-clinically injured and non-injured runners in this sub-sample was also not statistically significantly different (p = 0.08), with a mean of 174 steps/min for the uninjured group and a mean step rate of 170 steps/min for those in the sub-clinical injured group. CONCLUSION: Preferred step rate was not associated with lower extremity injury rates in this sample of DoD runners. Additional research is needed to justify preferred step rate manipulation as a means to reduce lower extremity injury risk. LEVEL OF EVIDENCE: Level 3.

Academic pursuits in board-certified reproductive endocrinologists.

OBJECTIVE: To determine research interests of reproductive endocrinology and infertility (REI) physicians and assess their academic productivity. DESIGN: A questionnaire composed by the Society for REI (SREI) board members was e-mailed to members. PubMed was queried to quantify peer-reviewed publications. SETTING: An internal SREI questionnaire to members and online publication search. PATIENT(S): Not applicable. INTERVENTION(S): Questions involving research being performed, funding, relevance to fellow thesis, and important areas of future research. Publications were ascertained in the past 3 years, past 10 years, and total publications for SREI members. MAIN OUTCOME MEASURE(S): Question responses and number of peer-reviewed publications. RESULT(S): Most respondents currently conduct research, which was predominantly clinical. One-third have current research funding and two-thirds were ever funded. One-third had a National Institutes of Health grant and about half were principal investigators. Two-thirds had a basic science fellow thesis and 44% of respondents perform research related to their fellowship thesis. Important research areas included infertility outcomes, implantation, preimplantation genetic testing, and genetics. In the past 3 years, SREI members published 3,408 peer-reviewed articles (mean ± standard deviation [SD], 4.4 ± 9.0). In the past 10 years, SREI members had 10,162 peer-reviewed publications (mean±SD, 13.0 ± 24.3). When all publications were considered, SREI members published 24,088 peer-reviewed articles (mean±SD, 30.9 ± 53.0). CONCLUSION(S): The REI fellows have learned to construct scientific articles, which will help them to better interpret the literature in the care of patients. The SREI members continue to pursue scientific investigation, commonly related to their fellowship thesis. Respondents support SREI funding research; the success of which should be judged by publications. Overall, SREI members have demonstrated significant academic productivity and published about 1,000 articles/year for the past 10 years, affirming the importance of research training.

Modified sites and functional consequences of 4-oxo-2-nonenal adducts in HDL that are elevated in familial hypercholesterolemia.

The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein crosslinker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation product-mediated crosslinking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well-studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ± 33.8 pmol/mg) than healthy controls (15.3 ± 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq), which was 100-fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from ∼46.5 to ∼18.4% (p < 0.001). Surprisingly, ONE modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC-MS/MS analysis revealed that Lys-12, Lys-23, Lys-96, and Lys-226 in apoA-I are modified by ONE ketoamide adducts. Compared with other dicarbonyl scavengers, pentylpyridoxamine (PPM) most efficaciously blocked ONE-induced protein crosslinking in HDL and also prevented HDL dysfunction in an in vitro model of inflammation. Our findings show that ONE-HDL adducts cause HDL dysfunction and are elevated in individuals with FH who have severe hypercholesterolemia.

A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL.

APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity (P < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.

Characterization of homodimer interfaces with cross-linking mass spectrometry and isotopically labeled proteins.

Cross-linking coupled with mass spectrometry (XL-MS) has emerged as a powerful strategy for the identification of protein-protein interactions, characterization of interaction regions, and obtainment of structural information on proteins and protein complexes. In XL-MS, proteins or complexes are covalently stabilized with cross-linkers and digested, followed by identification of the cross-linked peptides by tandem mass spectrometry (MS/MS). This provides spatial constraints that enable modeling of protein (complex) structures and regions of interaction. However, most XL-MS approaches are not capable of differentiating intramolecular from intermolecular links in multimeric complexes, and therefore they cannot be used to study homodimer interfaces. We have recently developed an approach that overcomes this limitation by stable isotope-labeling of one of the two monomers, thereby creating a homodimer with one 'light' and one 'heavy' monomer. Here, we describe a step-by-step protocol for stable isotope-labeling, followed by controlled denaturation and refolding in the presence of the wild-type protein. The resulting light-heavy dimers are cross-linked, digested, and analyzed by mass spectrometry. We show how to quantitatively analyze the corresponding data with SIM-XL, an XL-MS software with a module tailored toward the MS/MS data from homodimers. In addition, we provide a video tutorial of the data analysis with this protocol. This protocol can be performed in ∼14 d, and requires basic biochemical and mass spectrometry skills.

INTER-RATER RELIABILITY OF THE SELECTIVE FUNCTIONAL MOVEMENT ASSESSMENT (SFMA) BY SFMA CERTIFIED PHYSICAL THERAPISTS WITH SIMILAR CLINICAL AND RATING EXPERIENCE.

BACKGROUND: The Selective Functional Movement Assessment (SFMA) assesses posture, muscle balance, and movement patterns in order to identify relevant musculoskeletal dysfunction in a clinical population. PURPOSE: The purposes of this study were to: (1) determine if raters with similar clinical experience and rating experience exhibit adequate agreement of the scoring for the SFMA during clinical use; (2) determine the reliability of the categorical scoring of the SFMA in a clinical population; (3) determine the reliability of the criterion checklist scoring of the SFMA in a clinical population; (4) compare the reliability of real-time assessment to recorded assessment. DESIGN: Inter-rater reliability study. METHODS: 49 clinical subjects (20.7 years ± 1.6) were simultaneously assessed in real-time by two physical therapists and were recorded with digital video cameras in the sagittal and frontal view while they performed the fifteen component movement patterns that comprise the top-tier SFMA. The third physical therapist assessed the patterns from the video. Subjects were assessed using the SFMA categorical scoring and criterion checklist scoring tools. RESULTS: The two live clinical raters demonstrated the greatest Cohen's Kappa scores (10 of 15) with moderate or better inter-rater agreement (Kappa > 0.40) using the categorical scoring tool. The overall ICC [2,1] score indicated fair to moderate agreement between all raters for the criterion checklist scoring (ICC, SEM, p-value) (0.61, 8.23, p < 0.001). Real time clinical use was the most reliable method for using the criterion checklist scoring tool (0.72, 1.95, p=0.43). CONCLUSIONS: Using the categorical and criterion checklist tools in a clinical population to score the fifteen component fundamental movements of the SFMA demonstrated moderate or better reliability when performed clinically by certified SFMA raters. LEVEL OF EVIDENCE: Reliability, Level 2.

A consensus model of human apolipoprotein A-I in its monomeric and lipid-free state.

Apolipoprotein (apo)A-I is an organizing scaffold protein that is critical to high-density lipoprotein (HDL) structure and metabolism, probably mediating many of its cardioprotective properties. However, HDL biogenesis is poorly understood, as lipid-free apoA-I has been notoriously resistant to high-resolution structural study. Published models from low-resolution techniques share certain features but vary considerably in shape and secondary structure. To tackle this central issue in lipoprotein biology, we assembled a team of structural biologists specializing in apolipoproteins and set out to build a consensus model of monomeric lipid-free human apoA-I. Combining novel and published cross-link constraints, small-angle X-ray scattering (SAXS), hydrogen-deuterium exchange (HDX) and crystallography data, we propose a time-averaged model consistent with much of the experimental data published over the last 40 years. The model provides a long-sought platform for understanding and testing details of HDL biogenesis, structure and function.

THE EFFECTIVENESS OF DRY NEEDLING AND STRETCHING VS. STRETCHING ALONE ON HAMSTRING FLEXIBILITY IN PATIENTS WITH KNEE PAIN: A RANDOMIZED CONTROLLED TRIAL.

BACKGROUND: Recently, dry needling has emerged as a popular treatment for muscular pain and impairments. While there are numerous studies detailing the benefits of dry needling for pain, few studies exist examining the effects on soft tissue mobility. PURPOSE: The purpose of this study was to determine if the addition of hamstring dry needling to a standard stretching program results in greater improvements in hamstring flexibility compared to sham dry needling and stretching in subjects with atraumatic knee pain. Additionally, squat range of motion, knee pain, and the Lower Extremity Functional Scale were compared between the two groups. STUDY DESIGN: Double blinded randomized controlled trial. METHODS: Thirty-nine subjects were randomized to receive either dry needling (n = 20) or sham (n = 19) dry needling in addition to hamstring stretching, to all detected hamstring trigger points on two visits. All dependent variables were measured at baseline, immediately post intervention, and 1, 3, and 7 days after the initial treatment. Each subject also performed hamstring stretching three times daily for one week. RESULTS: Significant improvements in hamstring range of motion and all other dependent variables were observed across time regardless of treatment group. However, the lack of significant time by group interactions indicated the improvements were not different between dry needling and sham dry needling groups. CONCLUSIONS: The results of the current randomized controlled trial suggest that two sessions of dry needling did not improve hamstring range of motion or other knee pain-related impairments more than sham dry needling in a young active population with atraumatic knee pain. LEVEL OF EVIDENCE: Therapy, Level 2.

An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding.

Apolipoprotein (apo) A-I mediates many of the anti-atherogenic functions attributed to high density lipoprotein. Unfortunately, efforts toward a high resolution structure of full-length apoA-I have not been fruitful, although there have been successes with deletion mutants. Recently, a C-terminal truncation (apoA-I(Δ185-243)) was crystallized as a dimer. The structure showed two helical bundles connected by a long, curved pair of swapped helical domains. To compare this structure to that existing under solution conditions, we applied small angle x-ray scattering and isotope-assisted chemical cross-linking to apoA-I(Δ185-243) in its dimeric and monomeric forms. For the dimer, we found evidence for the shared domains and aspects of the N-terminal bundles, but not the molecular curvature seen in the crystal. We also found that the N-terminal bundles equilibrate between open and closed states. Interestingly, this movement is one of the transitions proposed during lipid binding. The monomer was consistent with a model in which the long shared helix doubles back onto the helical bundle. Combined with the crystal structure, these data offer an important starting point to understand the molecular details of high density lipoprotein biogenesis.