Chronic Adaptions in Quadriceps Fascicle Mechanics Are Related to Altered Knee Biomechanics After Anterior Cruciate Ligament Reconstruction.

Following anterior cruciate ligament reconstruction (ACLR), patients exhibit abnormal walking mechanics and quadriceps dysfunction. Quadriceps dysfunction has been largely attributed to muscle atrophy and weakness. While important, these factors do not capture intrinsic properties of muscle that govern its ability to generate force and withstand load. While fascicle abnormalities after ACLR have been documented in early stages of recovery (<12 mo), long-term effects of ACLR on fascicle mechanics remain unexplored. We evaluated quadriceps fascicle mechanics during walking 3 years post-ACLR and examined the relationship with knee mechanics. Participants included 24 individuals with ACLR and 24 Controls. Linear mixed models compared the ACLR, Contralateral, and Controls limbs for (1) quadriceps strength, (2) fascicle architecture and mechanics, and (3) knee mechanics. No difference in strength or overall fascicle length excursions was found between limbs. The ACLR limb exhibited longer fascicles at heel strike and peak knee extension moment (P < .001-.004), and smaller fascicle angles at heel strike, peak knee extension moment, and overall suppressed fascicle angle excursions (P < .001-.049) relative to the Contralateral and/or Control limb. This indicates an abnormality in fascicle architecture and mechanics following ACLR and suggests abnormalities in contractile function that cannot be explained by muscle weakness and may contribute to long-term gait irregularities.

Effect of Sampling Rate, Filtering, and Torque Onset Detection on Quadriceps Rate of Torque Development and Torque Steadiness.

Quadriceps rate of torque development (RTD) and torque steadiness are valuable metrics for assessing explosive strength and the ability to control force over a sustained period of time, which can inform clinical assessments of knee function. Despite their widespread use, there is a significant gap in standardized methodology for measuring these metrics, which limits their utility in comparing outcomes across different studies and populations. To address these gaps, we evaluated the influence of sampling rates, signal filtering, and torque onset detection on RTD and torque steadiness. Twenty-seven participants with a history of a primary anterior cruciate ligament reconstruction (N = 27 (11 male/16 female), age = 23 ± 8 years, body mass index = 26 ± 4 kg/m2) and thirty-two control participants (N = 32 (13 male/19 female), age = 23 ± 7 years, body mass index = 23 ± 3 kg/m2) underwent isometric quadriceps strength testing, with data collected at 2222 Hz on an isokinetic dynamometer. The torque-time signal was downsampled to approximately 100 and 1000 Hz and processed using a low-pass, zero-lag Butterworth filter with a range of cutoff frequencies spanning 10-200 Hz. The thresholds used to detect torque onset were defined as 0.1 Nm, 1 Nm, and 5 Nm. RTD between 0 and 100 ms, 0 and 200 ms, and 40-160 ms was computed, as well as absolute and relative torque steadiness. Relative differences were computed by comparing all outcomes to the "gold standard" values computed, with a sampling rate of 2222 Hz, a cutoff frequency in the low-pass filter of 150 Hz, and torque onset of 1 Nm, and compared utilizing linear mixed models. While all combinations of signal collection and processing parameters reached statistical significance (p < 0.05), these differences were consistent between injured and control limbs. Additionally, clinically relevant differences (+/-10%) were primarily observed through torque onset detection methods and primarily affected RTD between 0 and 100 ms. Although measurements of RTD and torque steadiness were generally robust against diverse signal collection and processing parameters, the selection of torque onset should be carefully considered, especially in early RTD assessments that have shorter time epochs.

Beyond weakness: Exploring intramuscular fat and quadriceps atrophy in ACLR recovery.

Muscle weakness following anterior cruciate ligament reconstruction (ACLR) increases the risk of posttraumatic osteoarthritis (OA). However, focusing solely on muscle weakness overlooks other aspects like muscle composition, which could hinder strength recovery. Intramuscular fat is a non-contractile element linked to joint degeneration in idiopathic OA, but its role post-ACLR has not been thoroughly investigated. To bridge this gap, we aimed to characterize quadriceps volume and intramuscular fat in participants with ACLR (male/female = 15/9, age = 22.8 ± 3.6 years, body mass index [BMI] = 23.2 ± 1.9, time since surgery = 3.3 ± 0.9 years) and in controls (male/female = 14/10, age = 22.0 ± 3.1 years, BMI = 23.3 ± 2.6) while also exploring the associations between intramuscular fat and muscle volume with isometric strength. Linear mixed effects models assessed (I) muscle volume, (II) intramuscular fat, and (III) strength between limbs (ACLR vs. contralateral vs. control). Regression analyses were run to determine if intramuscular fat or volume were associated with quadriceps strength. The ACLR limb was 8%-11% smaller than the contralateral limb (p < 0.05). No between-limb differences in intramuscular fat were observed (p 0.091-0.997). Muscle volume but not intramuscular fat was associated with strength in the ACLR and control limbs (p < 0.001-0.002). We demonstrate that intramuscular fat does not appear to be an additional source of quadriceps dysfunction following ACLR and that muscle size only explains some of the variance in muscle strength.

Associations between Body Mass Index, Gait Mechanics and Trochlear Cartilage Thickness in those with ACL Reconstruction.

PURPOSE: High body mass index (BMI) is a strong predictor of post-traumatic osteoarthritis (OA) after anterior cruciate ligament reconstruction (ACLR). Altered gait mechanics are independently affected by BMI and ACLR, and influence OA risk. Yet, evidence directly assessing the impact of high BMI on gait or cartilage characteristics after ACLR are limited. Here, we evaluated if high BMI moderates associations between gait and trochlear cartilage structure in individuals with ACLR. METHODS: Treadmill walking biomechanics were evaluated in forty normal BMI and twenty-four high BMI participants with ACLR at self-selected speeds. Normalized and absolute peak and cumulative loads (i.e., impulse) were extracted for peak knee flexion and adduction moments (KFM, KAM) and vertical ground reaction force (GRF). Medial and lateral femoral cartilage thickness and medial: lateral thickness ratios were assessed via ultrasound. RESULTS: Those with ACLR and high BMI walked with reduced normalized peak vertical GRFs, and greater absolute peak and cumulative loads compared to normal BMI individuals with ACLR. Those with ACLR and high BMI also exhibited thinner cartilage and greater medial: lateral ratios in ACLR limbs compared to contralateral limbs whereas normal BMI individuals with ACLR exhibited thicker ACLR limb cartilage. Lastly, greater peak KAM and KAM cumulative load were associated with thicker lateral cartilage and lesser medial: lateral thickness ratios, but only in the high BMI group. CONCLUSIONS: We observed those with high BMI after ACLR exhibited trochlear cartilage structural alterations not observed in normal BMI patients, while differential associations between loading outcomes and cartilage thickness in ACLR knees were observed between groups. Those with high BMI after ACLR may require different therapeutic strategies to optimize joint health in this subset of patients.

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing.

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to measure EMG signals in preclinical models. Although classical resources exist describing the principles of epimysial electrode fabrication, there is a sparsity of illustrative information translating electrode theory to practice. To remedy this, we provide an updated, easy-to-follow guide on fabricating and testing a low-cost epimysial electrode. Electrodes were made by folding and inserting two platinum-iridium foils into a precut silicone base to form the contact surfaces. Next, coated stainless steel wires were welded to each contact surface to form the electrode leads. Lastly, a silicone mixture was used to seal the electrode. Ex vivo testing was conducted to compare our custom-fabricated electrode to an industry standard electrode in a saline bath, where high levels of signal agreement (sine [intraclass correlation - ICC= 0.993], square [ICC = 0.995], triangle [ICC = 0.958]), and temporal-synchrony (sine [r = 0.987], square [r = 0.990], triangle [r= 0.931]) were found across all waveforms. Low levels of electrode impedance were also quantified via electrochemical impedance spectroscopy. An in vivo performance assessment was also conducted where the vastus lateralis muscle of a rat was surgically instrumented with the custom-fabricated electrode and signaling was acquired during uphill and downhill walking. As expected, peak EMG activity was significantly lower during downhill walking (0.008 ± 0.005 mV) than uphill (0.031 ± 0.180 mV, p = 0.005), supporting the validity of the device. The reliability and biocompatibility of the device were also supported by consistent signaling during level walking at 14 days and 56 days post implantation (0.01 ± 0.007 mV, 0.012 ± 0.007 mV respectively; p > 0.05) and the absence of histological inflammation. Collectively, we provide an updated workflow for the fabrication and testing of low-cost epimysial electrodes.

Open-sourced semi-automatic program for ultrasound assessments of femoral trochlea cartilage health.

We developed an open-sourced program that accounts for the current limitations of B-mode ultrasound and extends the clinical utility of ultrasound for assessing femoral trochlear cartilage thickness. Images were collected on 18 patients with a history of knee surgery. By failing to account for ultrasound acoustics and beam refraction, cartilage thickness was underestimated by 26% and overestimated by 0-4%, respectively (p < 0.001). Comprehensive thickness measurements achieved by measuring the Euclidean distance between every point were significantly different than traditional single-location measurements or by using the area/length (p = 0.004-0.006). Sub-regions were significantly different than all whole regions of interest (p = 0.001-0.012).

Anxiety symptoms and theory of mind in older and younger adults: curvilinearity moderated by age group.

OBJECTIVES: Theory of mind (ToM), the ability to reason about mental states, declines in later life. While anxiety symptoms may predict ToM abilities, the nature of associations requires more elucidation. Further, it is unknown whether age group moderates associational patterns. We examined associations between anxiety symptoms (linear and curvilinear) and cognitive ToM (C-ToM) and affective ToM (A-ToM); and moderation by age group (older vs. younger adults). METHODS: In a sample of healthy younger (n = 90, Mage = 20.17 years) and older adults (n = 87, Mage = 71.52), we used hierarchical regressions with polynomial and interaction terms to assess the association between anxiety symptoms and ToM in younger and older adults. RESULTS: Anxiety symptoms were associated with C-ToM but not A-ToM. Age group interacted linearly with anxiety (ß = -1.64, p = .02), and with anxiety's quadratic polynomial (ß = .84, p = .04). The inverted-U shaped association in younger adults (highest C-ToM at moderate anxiety) contrasted with older adults' linear decline in C-ToM with increasing anxiety. CONCLUSION: We highlight the importance of anxiety symptoms for predicting ToM, and clarify that associations vary by age. Elucidating associations may be critical to developing interventions that improve social wellbeing.

Scoping review of rational polytherapy in patients with drug-resistant epilepsy.

There is a paucity of literature regarding the optimal selection of combination antiseizure medications (ASMs) for drug-resistant epilepsy (DRE). The aim of this scoping review is to evaluate current evidence related to "rational polytherapy" among adults with DRE. Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Scoping Reviews (PRISMA-SCr) guidelines, PubMed, ProQuest, CINAHL, and Cochrane databases were searched using DRE- and polytherapy-related keywords. The exclusion criteria applied included: non-English; non-human studies; non-research studies; participants less than 18 years; status epilepticus; ASM monotherapy; and certain ASMs. In Covidence, two researchers independently reviewed articles for inclusion at each phase, with a third resolving conflicts. Data were extracted, with quality appraisal using the Mixed Methods Appraisal Tool (MMAT). Of the 6477 studies imported for screening, 33 studies were included. Clinical, humanistic, and economic outcomes were reported by 26, 12, and one study, respectively. Common efficacy-related clinical outcomes included ≥50% reduction in seizure frequency (n = 14), seizure freedom (n = 14), and percent reduction in seizure frequency (n = 8). Common humanistic outcomes included quality of life (n = 4), medication adherence (n = 2), sleep-related outcomes (n = 2), and physician and patient global assessments (n = 2). The economic study reported quality-adjusted life years. The median MMAT score was 80 (range: 60-100). Two studies referenced the standard definition of DRE, whereas five studies did not specifically define DRE. Gaps in the literature include limited generalizability, minimal reports in pregnancy, and lack of optimal ASM combinations, among others. Strengths of the evidence include addressing a variety of outcomes. Inconsistent definitions of DRE, small sample sizes, and heterogeneity among studies limit the ability to draw meaningful conclusions. Optimal combinations of ASMs for rational polytherapy for DRE is unclear.

Well-being Content Inclusion in Pharmacy Education Across the United States and Canada.

Objective. To describe the landscape of well-being content inclusion across schools and colleges of pharmacy in the United States and Canada through identification of content implementation, incorporation, and assessment.Methods. A cross-sectional survey was distributed to all accredited schools and colleges of pharmacy in the United States (n=143) and Canada (n=10). Survey questions included curricular and cocurricular timing, frequency, assessment strategies, and support for well-being initiatives, using a framework of eight dimensions (pillars) of wellness to categorize content.Results. Descriptive data analyses were applied to 99 completed surveys (65%), 89 (62%) in the United States and 10 (100%) in Canada. Well-being content was most prevalent within the cocurricular realm and incorporated into didactic and elective more than experiential curricula. The most content came from intellectual, emotional, and physical pillars, and the least content came from financial, spiritual, and environmental pillars. Less than 50% of schools and colleges of pharmacy include well-being within their strategic plans or core values. Funding is primarily at the level of the university (59%) or the school or college of pharmacy (59%). Almost half of respondents reported inclusion of some assessment, with a need for more training, expertise, and standardization.Conclusion. Survey results revealed a wide range of implementation and assessment of well-being programs across the United States and Canada. These results provide a reference point for the state of well-being programs that can serve as a call to action and research across the Academy.

Depressive symptoms among people living with HIV in permanent supportive housing: Findings from a community-based participatory study in Vancouver, Canada.

Permanent supportive housing (PSH) is an intervention addressing housing needs among marginally housed individuals. Little is known about whether and how PSH influences depressive symptoms among people living with HIV (PLHIV). This article shares results from a community-based study that, in 2016-2017, interviewed 24 residents of a PSH facility designated for PLHIV in Vancouver, Canada. The themes of taking control; social connectedness; conviviality; and relationships and supports described how the PSH environment affected depressive symptoms among the residents.

Long-Lasting Impairments in Quadriceps Mitochondrial Health, Muscle Size, and Phenotypic Composition Are Present After Non-invasive Anterior Cruciate Ligament Injury.

INTRODUCTION: Despite rigorous rehabilitation aimed at restoring muscle health, anterior cruciate ligament (ACL) injury is often hallmarked by significant long-term quadriceps muscle weakness. Derangements in mitochondrial function are a common feature of various atrophying conditions, yet it is unclear to what extent mitochondria are involved in the detrimental sequela of quadriceps dysfunction after ACL injury. Using a preclinical, non-invasive ACL injury rodent model, our objective was to explore the direct effect of an isolated ACL injury on mitochondrial function, muscle atrophy, and muscle phenotypic transitions. METHODS: A total of 40 male and female, Long Evans rats (16-week-old) were exposed to non-invasive ACL injury, while 8 additional rats served as controls. Rats were euthanized at 3, 7, 14, 28, and 56 days after ACL injury, and vastus lateralis muscles were extracted to measure the mitochondrial respiratory control ratio (RCR; state 3 respiration/state 4 respiration), mitochondrial reactive oxygen species (ROS) production, fiber cross sectional area (CSA), and fiber phenotyping. Alterations in mitochondrial function and ROS production were detected using two-way (sex:group) analyses of variance. To determine if mitochondrial characteristics were related to fiber atrophy, individual linear mixed effect models were run by sex. RESULTS: Mitochondria-derived ROS increased from days 7 to 56 after ACL injury (30-100%, P < 0.05), concomitant with a twofold reduction in RCR (P < 0.05). Post-injury, male rats displayed decreases in fiber CSA (days 7, 14, 56; P < 0.05), loss of IIa fibers (day 7; P < 0.05), and an increase in IIb fibers (day 7; P < 0.05), while females displayed no changes in CSA or phenotyping (P > 0.05). Males displayed a positive relationship between state 3 respiration and CSA at days 14 and 56 (P < 0.05), while females only displayed a similar trend at day 14 (P = 0.05). CONCLUSION: Long-lasting impairments in quadriceps mitochondrial health are present after ACL injury and play a key role in the dysregulation of quadriceps muscle size and composition. Our preclinical data indicate that using mitoprotective therapies may be a potential therapeutic strategy to mitigate alterations in muscle size and characteristic after ACL injury.

Relationship between altered knee kinematics and subchondral bone remodeling in a clinically translational model of ACL injury.

Abnormal joint kinematics are commonly reported in the acute and chronic stages of recovery after anterior cruciate ligament (ACL) injury and have long been mechanistically implicated as a primary driver in the development of posttraumatic osteoarthritis (PTOA). Though strongly theorized, it is unclear to what extent biomechanical adaptations after ACL injury culminate in the development of PTOA, as data that directly connects these factors does not exist. Using a preclinical, noninvasive ACL injury rodent model, our objective was to explore the direct effect of an isolated ACL injury on joint kinematics and the pathogenetic mechanisms involved in the development of PTOA. A total of 32, 16-week-old Long-Evans rats were exposed to a noninvasive ACL injury. Marker-less deep learning software (DeepLabCut) was used to track animal movement for sagittal-plane kinematic analyses and micro computed tomography was used to evaluate subchondral bone architecture at days 7, 14, 28, and 56 following injury. There was a significant decrease in peak knee flexion during walking (p < .05), which had a moderate-to-strong negative correlation (r = -.59 to -.71; p < .001) with subchondral bone plate porosity in all load bearing regions of the femur and tibia. Additional comprehensive analyses of knee flexion profiles revealed dramatic alterations throughout the step cycle. This occurred alongside considerable loss of epiphyseal trabecular bone and substantial changes in anatomical orientation. Knee flexion angle and subchondral bone microarchitecture are severely impacted after ACL injury. Reductions in peak knee flexion angle after ACL injury are directly associated with subchondral bone plate remodeling.

Interrater reliability and ease of use of the High-Alert Medication Stratification Tool‒Revised Prospective (HAMST-R PRO): A prospective, multisite study.

OBJECTIVE: The High-Alert Medication Stratification Tool‒Revised (HAMST-R) was originally designed to standardize the identification of high-alert medications (HAMs) according to safety risk. The primary objective of this multisite study was to assess interrater reliability of the HAMST-R PRO, a version of the tool designed to prospectively evaluate safety risk of medications during evaluation for formulary addition. METHODS: HAMST-R was designed as an objective tool to evaluate HAMs at a single site during the HAMST-R phase I study. Phase II of the study demonstrated the validity of the tool in a multisite, national study. In this third study, 11 medication safety experts from 8 health systems across the United States and 1 in Canada facilitated evaluation of medications prospectively with the HAMST-R PRO during the formulary review process for 27 medications. At each site, at least 5 individuals were asked to review each medication. Interrater reliability was evaluated using Kendall's coefficient of concordance. Ease of use was determined by participant interviews. RESULTS: Overall interrater reliability for HAMST-R PRO was found to be 0.76 (P < 0.001) across all sites, indicating substantial agreement between users. Interrater reliability among individual sites ranged from 0.52 to 0.82 (P < 0.05 for all sites). CONCLUSION: Interrater reliability of HAMST-R PRO is substantial, indicating consistency and agreement among pharmacists utilizing this tool to evaluate safety risk of medications before their addition to a health-system formulary. This information can be used to identify potential interventions for each step of the medication-use process that institutions may implement to decrease a medication's potential safety risk.

The Utility of Functional Data Analyses to Reveal Between-Limbs Asymmetries in Those With a History of Anterior Cruciate Ligament Reconstruction.

CONTEXT: Researchers have traditionally used motion capture to quantify discrete data points (peak values) during hop testing. However, these analyses restrict the evaluation to a single time point (ie, certain percentage of stance) and provide only a narrow view of movement. Applying more comprehensive analyses may help investigators identify important characteristics that are masked by discrete analyses often used to screen patients for activity. OBJECTIVE: To examine the utility of functional data analyses to reveal asymmetries that are undetectable using discrete (ie, single time point) evaluations in participants with a history of anterior cruciate ligament reconstruction (ACLR) who achieved clinical hop symmetry. DESIGN: Cross-sectional study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: Fifteen participants with unilateral ACLR (age = 21 ± 3 years, time from surgery = 4 ± 3 years) and 15 control participants without ACLR (age = 23 ± 2 years). INTERVENTION(S): Lower extremity biomechanics during the triple-hop-for-distance task for the ACLR and contralateral limbs of patients and a representative limb of control participants were measured. MAIN OUTCOME MEASURE(S): Peak sagittal-plane joint power, joint work, and power profiles were determined. RESULTS: Using discrete analyses, we identified lower peak knee power and work in the ACLR limb compared with the contralateral and control limbs (P < .05) but were unable to demonstrate differences at the ankle or hip. Using functional data analyses, we observed asymmetries at the ankle, knee, and hip between the ACLR and contralateral or control limbs throughout stance (P < .05), and it was revealed that these asymmetries stemmed from knee power deficits that were prominent during early loading. CONCLUSIONS: Despite achieving hop-distance symmetry, the ACLR knees absorbed less power. Although this information was revealed using discrete analyses, underlying asymmetries at the ankle and hip were masked. Using functional data analyses, we found interlimb asymmetries at the ankle, knee, and hip. Importantly, we found that functional data analyses more fully elucidated the extent and source of asymmetries, which can be used by clinicians and researchers alike to aid in clinical decision making.

Morphology and Anabolic Response of Skeletal Muscles Subjected to Eccentrically or Concentrically Biased Exercise.

CONTEXT: Long-term eccentric exercise is known to promote muscle growth better than concentric exercise, but its acute effect on muscle is not well understood because of misinterpreted modeling and in situ and in vitro stretch protocols. Knowing if the initial bout of eccentric exercise promotes muscle growth and limits damage is critical to understanding the effect of this mode of exercise. OBJECTIVE: To directly evaluate the immediate effects of eccentric and concentric exercises on untrained muscle when fiber strains were physiological and exercise doses were comparable. DESIGN: Controlled laboratory study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: A total of 40 skeletally mature male Long-Evans rats (age = 16 weeks, mass = 452.1 ± 35.2 g) were randomly assigned to an eccentric exercise (downhill walking, n = 16), concentric exercise (uphill walking, n = 16), or control (no exercise, n = 8) group. INTERVENTION(S): Rats were exposed to a single 15-minute bout of eccentric or concentric exercise on a motorized treadmill and then were euthanized at 6 or 24 hours postexercise. We harvested the vastus lateralis muscle bilaterally. MAIN OUTCOME MEASURE(S): The percentage increase or decrease in protein abundance in exercised animals relative to that in unexercised control animals was evaluated as elevated phosphorylated p70S6k relative to total p70S6k. Fiber damage was quantified using immunoglobulin G permeability staining. One-way analysis of variance and post hoc Tukey tests were performed. RESULTS: Rats exposed to eccentric exercise and euthanized at 24 hours had higher percentage response protein synthesis rates than rats exposed to eccentric exercise and euthanized at 6 hours (P = .02) or to concentric exercise and euthanized at 6 (P = .03) or 24 (P = .03) hours. We assessed 9446 fibers for damage and found only 1 fiber was infiltrated (in the concentric exercise group euthanized at 6 hours). Furthermore, no between-groups differences in immunoglobulin G fluorescent intensity were detected (P = .94). CONCLUSIONS: Incorporating eccentric exercise is a simple, universally available therapeutic intervention for promoting muscle recovery. A single 15-minute dose of eccentric exercise to a novice muscle can better exert an anabolic effect than a comparable dose of concentric exercise, with very limited evidence of fiber damage.

The protease-activated receptor 4 Ala120Thr variant alters platelet responsiveness to low-dose thrombin and protease-activated receptor 4 desensitization, and is blocked by non-competitive P2Y12 inhibition.

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

5B9, a monoclonal antiplatelet factor 4/heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies.

Essentials No humanized monoclonal antibody was available to study heparin-induced thrombocytopenia (HIT). We developed the first anti-platelet factor 4 (PF4)/heparin antibody with a human Fc fragment. This antibody (5B9) fully mimics the effects of human HIT antibodies. 5B9 binds two regions within PF4 that may be critical for the pathogenicity of HIT antibodies. SUMMARY: Background The diagnosis of heparin-induced thrombocytopenia (HIT) is based on clinical and biological criteria, but a standard is lacking for laboratory assays. Moreover, no humanized HIT antibody is available for pathophysiological studies. Objective To characterise 5B9, a chimeric monoclonal antibody, which fully mimics the effects of human HIT antibodies. Methods/Results 5B9, a chimeric anti-platelet factor 4/heparin complexes IgG1 antibody, was obtained after immunizing specific transgenic mice. 5B9 induced heparin FcγRIIA-dependent platelet aggregation and tissue factor mRNA synthesis in monocytes. It also induced significant thrombocytopenia and thrombin generation in mice expressing human PF4 and FcγRIIA receptors. The binding of 5B9 to PF4/H complexes was inhibited by 15 of 25 HIT plasma samples and only three of 25 samples containing non-pathogenic anti-PF4/H antibodies. KKO, a murine IgG2b HIT antibody, also inhibited the binding of 5B9 to PF4/H, suggesting that epitopes recognized by both antibodies are close. A docking analysis based on VH and VL sequences of 5B9 showed that binding of 5B9 Fab to PF4 involved 12 and 12 residues in B and D monomers, respectively, including seven previously identified as critical to the formation of a PF4/KKO complex. Two regions (Asp-7 to Thr-15 and Ala-32 to Thr-38) therefore appeared important for the binding of 5B9 and KKO on PF4 modified by heparin. Conclusions 5B9 is the first anti-PF4/H monoclonal antibody with a human Fc fragment, which induces similar cellular activation as HIT antibodies. Moreover, 5B9 binds epitopes within PF4 that are likely to be critical for the pathogenicity of HIT antibodies.

Impacts of Contrasting Alfalfa Production Systems on the Drivers of Carabid Beetle (Coleoptera: Carabidae) Community Dynamics.

Growing concerns about the environmental consequences of chemically based pest control strategies have precipitated a call for the development of integrated, ecologically based pest management programs. Carabid or ground beetles (Coleoptera:Carabidae) are an important group of natural enemies of common agricultural pests such as aphids, slugs, and other beetles. Alfalfa (Medicago sativa L.) is one of the most common forage crop species in the semi-arid western United States. In 2011, Montana alone produced 4.0 × 10(6 )Mg of alfalfa on 8.1 × 10(5 )ha for gross revenue in excess of US$4.3 × 10(8), making it the third largest crop by revenue. We conducted our study over the 2012 and 2013 growing seasons. Each year, our study consisted of three sites each with adjacent systems of monoculture alfalfa, alfalfa nurse cropped with hay barley, and an uncultivated refuge consisting of a variety of forbs and grasses. Carabid community structure differed and strong temporal shifts were detected during both 2012 and 2013. Multivariate fuzzy set ordination suggests that variation in canopy height among the three vegetation systems was primarily responsible for the differences observed in carabid community structure. Land managers may be able to enhance carabid species richness and total abundance by creating a heterogeneous vegetation structure, and nurse cropping in particular may be effective strategy to achieve this goal.