Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction.

The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.

Mechanistic safety assessment via multi-omic characterisation of systemic pathway perturbations following in vivo MAT2A inhibition.

The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.

Predicting Knee Joint Kinematics from Wearable Sensor Data in People with Knee Osteoarthritis and Clinical Considerations for Future Machine Learning Models.

Deep learning models developed to predict knee joint kinematics are usually trained on inertial measurement unit (IMU) data from healthy people and only for the activity of walking. Yet, people with knee osteoarthritis have difficulties with other activities and there are a lack of studies using IMU training data from this population. Our objective was to conduct a proof-of-concept study to determine the feasibility of using IMU training data from people with knee osteoarthritis performing multiple clinically important activities to predict knee joint sagittal plane kinematics using a deep learning approach. We trained a bidirectional long short-term memory model on IMU data from 17 participants with knee osteoarthritis to estimate knee joint flexion kinematics for phases of walking, transitioning to and from a chair, and negotiating stairs. We tested two models, a double-leg model (four IMUs) and a single-leg model (two IMUs). The single-leg model demonstrated less prediction error compared to the double-leg model. Across the different activity phases, RMSE (SD) ranged from 7.04° (2.6) to 11.78° (6.04), MAE (SD) from 5.99° (2.34) to 10.37° (5.44), and Pearson's R from 0.85 to 0.99 using leave-one-subject-out cross-validation. This study demonstrates the feasibility of using IMU training data from people who have knee osteoarthritis for the prediction of kinematics for multiple clinically relevant activities.

Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines.

Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.

Human Activity Recognition for People with Knee Osteoarthritis-A Proof-of-Concept.

Clinicians lack objective means for monitoring if their knee osteoarthritis patients are improving outside of the clinic (e.g., at home). Previous human activity recognition (HAR) models using wearable sensor data have only used data from healthy people and such models are typically imprecise for people who have medical conditions affecting movement. HAR models designed for people with knee osteoarthritis have classified rehabilitation exercises but not the clinically relevant activities of transitioning from a chair, negotiating stairs and walking, which are commonly monitored for improvement during therapy for this condition. Therefore, it is unknown if a HAR model trained on data from people who have knee osteoarthritis can be accurate in classifying these three clinically relevant activities. Therefore, we collected inertial measurement unit (IMU) data from 18 participants with knee osteoarthritis and trained convolutional neural network models to identify chair, stairs and walking activities, and phases. The model accuracy was 85% at the first level of classification (activity), 89-97% at the second (direction of movement) and 60-67% at the third level (phase). This study is the first proof-of-concept that an accurate HAR system can be developed using IMU data from people with knee osteoarthritis to classify activities and phases of activities.

Development of a Machine Learning Model for the Estimation of Hip and Lumbar Angles in Ballet Dancers.

OBJECTIVE: Accurate field-based assessment of dance kinematics is important to understand the etiology, and thus prevention and management, of hip and back pain. The study objective was to develop a machine learning model to estimate thigh elevation and lumbar sagittal plane angles during ballet leg lifting tasks, using wearable sensor data. METHODS: Female dancers (n=30) performed ballet-specific leg lifting tasks to the front, side, and behind the body. Dancers wore six wearable sensors (100 Hz). Data were simultaneously collected using an 18-camera motion analysis system (250 Hz). Due to synchronization and hardware malfunction issues, only 23 dancers had usable data. Using leave-one-out cross-validation, machine learning models were compared with the optic motion capture system using root mean square error (RMSE) in degrees and correlation coefficients (r) over the complete movement profile of each leg lift and mean absolute error (MAE) and Bland Altman plots for peak angle accuracy. RESULTS: The average RMSE for model estimation was 6.8° for thigh elevation angle and 5.6° for lumbar spine sagittal plane angle, with respective MAE of 6.3°and 5.7°. There was a strong correlation between the machine learning model and optic motion capture for peak angle values (thigh r=0.86, lumbar r=0.96). CONCLUSION: The models developed demonstrated an acceptable degree of accuracy for the estimation of thigh elevation angle and lumbar spine sagittal plane angle during dance-specific leg lifting tasks. This provides potential for a near-real-time, field-based measurement system.

Bicyclic ß-Sheet Mimetics that Target the Transcriptional Coactivator ß-Catenin and Inhibit Wnt Signaling.

Protein complexes are defined by the three-dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been applied for example, as inhibitors of protein-protein interactions (PPIs). Even though ß-sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure-based design of ß-sheet mimetics targeting the intracellular protein ß-catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of ß-catenin, a macrocyclic peptide was designed and its crystal structure in complex with ß-catenin obtained. Using this structure, we designed a library of bicyclic ß-sheet mimetics employing a late-stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to ß-catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other ß-sheet-mediated PPIs.

Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5.

Protein arginine methyltransferases (PRMTs) are of great interest for the development of therapeutics due to their involvement in a number of malignancies, such as lung and colon cancer. PRMT5 catalyzes the formation of symmetrical dimethylarginine of a wide variety of substrates and is responsible for the majority of this mark within cells. To gain insight into the mechanism of PRMT5 inhibition, we co-expressed the human PRMT5:MEP50 complex (hPRMT5:MEP50) in insect cells for a detailed mechanistic study. In this report, we carry out steady state, product, and dead-end inhibitor studies that show hPRMT5:MEP50 uses a rapid equilibrium random order mechanism with EAP and EBQ dead-end complexes. We also provide evidence of ternary complex formation in solution using hydrogen/deuterium exchange mass spectrometry. Isotope exchange and intact protein mass spectrometry further rule out ping-pong as a potential enzyme mechanism, and finally, we show that PRMT5 exhibits a pre-steady state burst that corresponds to an initial slow turnover with all four active sites of the hetero-octamer being catalytically active.

Lipidated Stapled Peptides Targeting the Acyl Binding Protein UNC119.

The acyl-binding UNC119 proteins mediate the activation and transport of various N-myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α-helical peptides that target UNC119a. By using the hydrocarbon peptide-stapling approach, cell-permeable binders of UNC119a were generated that induced the accumulation of cytokinetic and binucleated cells; this suggests UNC119a as a potential target for the inhibition of cytokinesis.

Running quietly reduces ground reaction force and vertical loading rate and alters foot strike technique.

This study aimed to determine if a quantifiable relationship exists between the peak sound amplitude and peak vertical ground reaction force (vGRF) and vertical loading rate during running. It also investigated whether differences in peak sound amplitude, contact time, lower limb kinematics, kinetics and foot strike technique existed when participants were verbally instructed to run quietly compared to their normal running. A total of 26 males completed running trials for two sound conditions: normal running and quiet running. Simple linear regressions revealed no significant relationships between impact sound and peak vGRF in the normal and quiet conditions and vertical loading rate in the normal condition. t-Tests revealed significant within-subject decreases in peak sound, peak vGRF and vertical loading rate during the quiet compared to the normal running condition. During the normal running condition, 15.4% of participants utilised a non-rearfoot strike technique compared to 76.9% in the quiet condition, which was corroborated by an increased ankle plantarflexion angle at initial contact. This study demonstrated that quieter impact sound is not directly associated with a lower peak vGRF or vertical loading rate. However, given the instructions to run quietly, participants effectively reduced peak impact sound, peak vGRF and vertical loading rate.

The Acute Effects of Ball Pressure on Anticipation Timing Following a Series of Purposeful Headers in Adult Football (Soccer) Players.

The purpose of this study is to investigate the acute effects of ball pressure on anticipation timing following a series of purposeful headers in adult football (soccer) players. There is evidence to suggest acute neurophysiological changes to the brain following purposeful heading; this may lead to altered anticipation timing as a result, potentially having future safety implications for players. A repeated measures crossover design was used. Seventeen participants aged between 20 and 30 years performed (i) 20 rotational headers with a lower-pressure match ball (58.6 kPa; 8.5 psi), (ii) 20 rotational headers with a higher-pressure match ball (103.4 kPa; 15 psi), or (iii) 20 non-headers (kicks) as a control each on separate days. The effect of ball pressure on anticipation timing accuracy, measured as absolute, constant, and variable errors, was assessed before and immediately after each intervention session using an anticipation timing task. Differences between group means were compared using repeated measures ANOVA and linear mixed effects models, with p-values of <0.05 considered statistically significant. No significant differences in anticipation timing accuracy across interventions were detected between control, occluded, and non-occluded trials. This finding differs from the previous literature regarding the measurable, acute effects of purposeful heading. The anticipation timing task may lack sensitivity for detecting the effects of repeated heading on brain function.

UKPMC: a full text article resource for the life sciences.

UK PubMed Central (UKPMC) is a full-text article database that extends the functionality of the original PubMed Central (PMC) repository. The UKPMC project was launched as the first 'mirror' site to PMC, which in analogy to the International Nucleotide Sequence Database Collaboration, aims to provide international preservation of the open and free-access biomedical literature. UKPMC (http://ukpmc.ac.uk) has undergone considerable development since its inception in 2007 and now includes both a UKPMC and PubMed search, as well as access to other records such as Agricola, Patents and recent biomedical theses. UKPMC also differs from PubMed/PMC in that the full text and abstract information can be searched in an integrated manner from one input box. Furthermore, UKPMC contains 'Cited By' information as an alternative way to navigate the literature and has incorporated text-mining approaches to semantically enrich content and integrate it with related database resources. Finally, UKPMC also offers added-value services (UKPMC+) that enable grantees to deposit manuscripts, link papers to grants, publish online portfolios and view citation information on their papers. Here we describe UKPMC and clarify the relationship between PMC and UKPMC, providing historical context and future directions, 10 years on from when PMC was first launched.

The Biomechanics of Healthy Older Adults Rising from the Floor Independently.

A third of older adults will fall each year and many will not be injured. Getting up from the floor in a timely manner is important, however it is unclear what technique older adults use to get themselves up off the ground unassisted, whether there are differences between men and women in getting up and what functional joint kinematics are used to rise from the floor. This study included a convenience sample of 20 older adults (65+ years) to answer these questions. Participants completed a series of movement tasks (i.e., rising from the floor using their own technique, a specified technique, walking 10 m and five repeated sit-to-stands), with temporospatial and joint kinematic data captured using an 18-camera 3D Vicon motion analysis system. Results found three techniques preferred by participants; the sit-up (n = 12), side-sit (n = 4) and the roll over (n = 4), with no differences found between sexes. The sit-up technique requires a higher degree of hip and knee flexion to complete compared to the side-sit and roll over. It may be beneficial for health professionals to work with older adults to identify their preferred technique for rising from the floor and encourage regular practice of this skill.

A Comparison of Inertial Measurement Units and Overnight Videography to Assess Sleep Biomechanics.

PURPOSE: The assessment of sleep biomechanics (comprising movement and position during sleep) is of interest in a wide variety of clinical and research settings. However, there is no standard method by which sleep biomechanics are measured. This study aimed to (1) compare the intra- and inter-rater reliability of the current clinical standard, manually coded overnight videography, and (2) compare sleep position recorded using overnight videography to sleep position recorded using the XSENS DOT wearable sensor platform. METHODS: Ten healthy adult volunteers slept for one night with XSENS DOT units in situ (on their chest, pelvis, and left and right thighs), with three infrared video cameras recording concurrently. Ten clips per participant were edited from the video. Sleeping position in each clip was coded by six experienced allied health professionals using the novel Body Orientation During Sleep (BODS) Framework, comprising 12 sections in a 360-degree circle. Intra-rater reliability was assessed by calculating the differences between the BODS ratings from repeated clips and the percentage who were rated with a maximum of one section of the XSENS DOT value; the same methodology was used to establish the level of agreement between the XSENS DOT and allied health professional ratings of overnight videography. Bennett's S-Score was used to assess inter-rater reliability. RESULTS: High intra-rater reliability (90% of ratings with maximum difference of one section) and moderate inter-rater reliability (Bennett's S-Score 0.466 to 0.632) were demonstrated in the BODS ratings. The raters demonstrated high levels of agreement overall with the XSENS DOT platform, with 90% of ratings from allied health raters being within the range of at least 1 section of the BODS (as compared to the corresponding XSENS DOT produced rating). CONCLUSIONS: The current clinical standard for assessing sleep biomechanics, manually rated overnight videography (as rated using the BODS Framework) demonstrated acceptable intra- and inter-rater reliability. Further, the XSENS DOT platform demonstrated satisfactory levels of agreement as compared to the current clinical standard, providing confidence for its use in future studies of sleep biomechanics.

Discovery of a Potent and Orally Bioavailable Zwitterionic Series of Selective Estrogen Receptor Degrader-Antagonists.

Herein, we report the optimization of a meta-substituted series of selective estrogen receptor degrader (SERD) antagonists for the treatment of ER+ breast cancer. Structure-based design together with the use of modeling and NMR to favor the bioactive conformation led to a highly potent series of basic SERDs with promising physicochemical properties. Issues with hERG activity resulted in a strategy of zwitterion formation and ultimately in the identification of 38. This compound was shown to be a highly potent SERD capable of effectively degrading ERα in both MCF-7 and CAMA-1 cell lines. The low lipophilicity and zwitterionic nature led to a SERD with a clean secondary pharmacology profile and no hERG activity. Favorable physicochemical properties resulted in good oral bioavailability in preclinical species and potent in vivo activity in a mouse xenograft model.

Can Wearable Inertial Measurement Units Be Used to Measure Sleep Biomechanics? Establishing Initial Feasibility and Validity.

Wearable motion sensors, specifically, Inertial Measurement Units, are useful tools for the assessment of orientation and movement during sleep. The DOTs platform (Xsens, Enschede, The Netherlands) has shown promise for this purpose. This pilot study aimed to assess its feasibility and validity for recording sleep biomechanics. Feasibility was assessed using four metrics: Drift, Battery Life, Reliability of Recording, and Participant Comfort. Each metric was rated as Stop (least successful), Continue But Modify Protocol, Continue But Monitor Closely, or Continue Without Modifications (most successful). A convenience sample of ten adults slept for one night with a DOT unit attached to their sternum, abdomen, and left and right legs. A survey was administered the following day to assess participant comfort wearing the DOTs. A subset of five participants underwent a single evaluation in a Vicon (Oxford Metrics, Oxford, UK) motion analysis lab to assess XSENS DOTs' validity. With the two systems recording simultaneously, participants were prompted through a series of movements intended to mimic typical sleep biomechanics (rolling over in lying), and the outputs of both systems were compared to assess the level of agreement. The DOT platform performed well on all metrics, with Drift, Battery Life, and Recording Reliability being rated as Continue Without Modifications. Participant Comfort was rated as Continue But Monitor Closely. The DOT Platform demonstrated an extremely high level of agreement with the Vicon motion analysis lab (difference of <0.025°). Using the Xsens DOT platform to assess sleep biomechanics is feasible and valid in adult populations. Future studies should further investigate the feasibility of using this data capture method for extended periods (e.g., multiple days) and in other groups (e.g., paediatric populations).

Validating an inertial measurement unit for cricket fast bowling: a first step in assessing the feasibility of diagnosing back injury risk in cricket fast bowlers during a tele-sport-and-exercise medicine consultation.

This study aimed to validate an array-based inertial measurement unit to measure cricket fast bowling kinematics as a first step in assessing feasibility for tele-sport-and-exercise medicine. We concurrently captured shoulder girdle relative to the pelvis, trunk lateral flexion, and knee flexion angles at front foot contact of eight cricket medium-fast bowlers using inertial measurement unit and optical motion capture. We used one sample t-tests and 95% limits of agreement (LOA) to determine the mean difference between the two systems and Smallest Worth-while Change statistic to determine whether any differences were meaningful. A statistically significant (p < 0.001) but small mean difference of -4.7° ± 8.6° (95% Confidence Interval (CI) [-3.1° to -6.4°], LOA [-22.2 to 12.7], SWC 3.9°) in shoulder girdle relative to the pelvis angle was found between the systems. There were no statistically significant differences between the two systems in trunk lateral flexion and knee flexion with the mean differences being 0.1° ± 10.8° (95% CI [-1.9° to 2.2°], LOA [-22.5 to 22.7], SWC 1.2°) and 1.6° ± 10.1° (95% CI [-0.2° to 3.3°], LOA [-19.2 to 22.3], SWC 1.9°) respectively. The inertial measurement unit-based system tested allows for accurate measurement of specific cricket fast bowling kinematics and could be used in determining injury risk in the context of tele-sport-and-exercise-medicine.

Discovery of a Potent and Selective ATAD2 Bromodomain Inhibitor with Antiproliferative Activity in Breast Cancer Models.

ATAD2 is an epigenetic bromodomain-containing target which is overexpressed in many cancers and has been suggested as a potential oncology target. While several small molecule inhibitors have been described in the literature, their cellular activity has proved to be underwhelming. In this work, we describe the identification of a novel series of ATAD2 inhibitors by high throughput screening, confirmation of the bromodomain region as the site of action, and the optimization campaign undertaken to improve the potency, selectivity, and permeability of the initial hit. The result is compound 5 (AZ13824374), a highly potent and selective ATAD2 inhibitor which shows cellular target engagement and antiproliferative activity in a range of breast cancer models.

Head Accelerations during a 1-on-1 Rugby Tackling Drill Performed by Experienced Rugby Union Players.

Rugby Union is a popular sport played by males and females worldwide, from junior to elite levels. The highly physical skill of tackling occurs every few seconds throughout a match and various injuries associated with tackling are relatively common. Of particular interest are head injuries that result in a concussion. Recently, repeated non-injurious head impacts in sport have attracted the attention of researchers interested in brain health. Therefore, this study assessed head movement during repeated rugby tackle drills among experienced Rugby Union players. Experienced male and female participants performed 15 1-on-1 tackles in a motion analysis laboratory to measure the head movements of the ball carrier and tackler during each tackle, using three-dimensional motion capture. The average peak acceleration of the head for ball carriers was 28.9 ± 24.08 g and 36.67 ± 28.91 g for the tacklers. This study found that the type of head impacts common while performing a tackle in Rugby Union are similar to those experienced by soccer players during heading, which has been found to alter brain function that lasts hours after the event. This has important implications for player health and suggests that mitigation strategies should be considered for Rugby Union.

Functional calibration does not improve the concurrent validity of magneto-inertial wearable sensor-based thorax and lumbar angle measurements when compared with retro-reflective motion capture.

 Magneto-inertial measurement unit (MIMU) systems allow calculation of simple sensor-to-sensor Euler angles, though this process does not address sensor-to-segment alignment, which is important for deriving meaningful MIMU-based kinematics. Functional sensor-to-segment calibrations have improved concurrent validity for elbow and knee angle measurements but have not yet been comprehensively investigated for trunk or sport-specific movements. This study aimed to determine the influence of MIMU functional calibration on thorax and lumbar joint angles during uni-planar and multi-planar, sport-specific tasks. It was hypothesised that functionally calibrating segment axes prior to angle decomposition would produce smaller differences than a non-functional method when both approaches were compared with concurrently collected 3D retro-reflective derived angles. Movements of 10 fast-medium cricket bowlers were simultaneously recorded by MIMUs and retro-reflective motion capture. Joint angles derived from four different segment definitions were compared, with three incorporating functionally defined axes. Statistical parametric mapping and root mean squared differences (RMSD) quantified measurement differences one-dimensionally and zero-dimensionally, respectively. Statistical parametric mapping found no significant differences between MIMU and retro-reflective data for any method across bowling and uni-planar trunk movements. The RMSDs for the functionally calibrated methods and non-functional method were not significantly different. Functional segment calibration may be unnecessary for MIMU-based measurement of thorax and lumbar joint angles.