Discrete Hamstring: Quadriceps Strength Ratios Do Not Represent Angle-Specific Ratios in Premier League Soccer Players.

ABSTRACT: Lunn, DE, Nicholson, G, Cooke, M, Crespo, R, Robinson, T, Price, RJ, and Walker, J. Discrete hamstring: quadriceps strength ratios do not represent angle-specific ratios in Premier League soccer players. J Strength Cond Res 37(12): 2417-2422, 2023-This study compared angle-specific hamstring:quadriceps (H:Q) ratios with their discrete counterparts during strength testing in professional male soccer players. Twenty-seven professional English Premier League soccer players were recruited for this study (age: 22 ± 4 years; stature: 1.81 ± 0.08 m; body mass: 74.7 ± 6.5 kg). Isokinetic testing of the knee flexors and extensors was conducted concentrically at two angular velocities (60° and 240°·s -1 ) and eccentrically (for the knee flexors only) at 30°·s -1 . Conventional H:Q ratio was calculated as the ratio between peak joint moment in the flexors and extensors at 60°·s -1 . Functional H:Q ratio was calculated as the peak joint moment in the flexors during the eccentric condition and the extensors at 240°·s -1 . Discrete conventional and functional H:Q ratios were 0.56 ± 0.06 and 1.28 ± 0.22, respectively. The residual differences between discrete values and angle-specific residual values were 13.60 ± 6.56% when normalized to the magnitude of the discrete value. For the functional ratios, the normalized residual was 21.72 ± 5.61%. Therefore, neither discrete ratio was representative of angle-specific ratios, although the conventional ratio had lower error overall. Therefore, practitioners should consider H:Q ratio throughout the full isokinetic range of motion, not just the discrete ratio calculated from peak joint moments, when designing and implementing training programs or monitoring injury risk, recovery from injury, and readiness to return to play.

Glacial lake outburst floods threaten millions globally.

Glacial lake outburst floods (GLOFs) represent a major hazard and can result in significant loss of life. Globally, since 1990, the number and size of glacial lakes has grown rapidly along with downstream population, while socio-economic vulnerability has decreased. Nevertheless, contemporary exposure and vulnerability to GLOFs at the global scale has never been quantified. Here we show that 15 million people globally are exposed to impacts from potential GLOFs. Populations in High Mountains Asia (HMA) are the most exposed and on average live closest to glacial lakes with ~1 million people living within 10 km of a glacial lake. More than half of the globally exposed population are found in just four countries: India, Pakistan, Peru, and China. While HMA has the highest potential for GLOF impacts, we highlight the Andes as a region of concern, with similar potential for GLOF impacts to HMA but comparatively few published research studies.

Stepwise remodeling and subcompartment formation in individual vesicles by three ESCRT-III proteins.

The endosomal sorting complex required for transport (ESCRT) is a multi-protein machinery involved in several membrane remodeling processes. Different approaches have been used to resolve how ESCRT proteins scission membranes. However, the underlying mechanisms generating membrane deformations are still a matter of debate. Here, giant unilamellar vesicles, microfluidic technology, and micropipette aspiration are combined to continuously follow the ESCRT-III-mediated membrane remodeling on the single-vesicle level for the first time. With this approach, we identify different mechanisms by which a minimal set of three ESCRT-III proteins from Entamoeba histolytica reshape the membrane. These proteins modulate the membrane stiffness and spontaneous curvature to regulate bud size and generate intraluminal vesicles even in the absence of ATP. We demonstrate that the bud stability depends on the protein concentration and membrane tension. The approaches introduced here should open the road to diverse applications in synthetic biology for establishing artificial cells with several membrane compartments.

Opportunities arising from the COVID-19: an international orthopaedic surgeons' perspective.

PURPOSE: The unprecedented COVID-19 experience has posed severe challenges to the health care system and several of these are documented in orthopaedic surgery; however, although the pandemic has also brought positive changes, these have not been precisely documented. The purpose of this survey is to identify positive perceptions by orthopaedic surgeons at an international level. METHODS: A cross-sectional, web-based survey inviting 120 orthopaedic surgeons was conducted in April 2020 querying about the positive lessons COVID-19 would teach us. From all responses, thematic codes were obtained and an exploratory thematic analysis was carried out to determine the prevalent themes. RESULTS: A total of 100 responses (83% response rate) from a total of seven countries were received. The variety of responses received were grouped into 13 different thematic codes. The thematic analysis generated two major themes: "Virtual reorganization" and "Wellness and sustainability". Fifty-four per cent of the participants reported positive changes in service reorganization and virtual consultation, whereas 30% replied with an increased feeling of well-being which overlapped with environmental benefits, including reduced paperwork, reduced travelling and increased quality time for family and reflection. CONCLUSIONS: Despite the negative aspects of the pandemic, responders reported several positive changes particularly relating to service reorganization and personal well-being. This study prompts further larger scale research to unravel further detail in those positive aspects and strongly enhance our future orthopaedic practice.

Biomimetic asymmetric bacterial membranes incorporating lipopolysaccharides.

Gram-negative bacteria are equipped with a cell wall that contains a complex matrix of lipids, proteins, and glycans, which form a rigid layer protecting bacteria from the environment. Major components of this outer membrane are the high-molecular weight and amphiphilic lipopolysaccharides (LPSs). They form the extracellular part of a heterobilayer with phospholipids. Understanding LPS properties within the outer membrane is therefore important to develop new antimicrobial strategies. Model systems, such as giant unilamellar vesicles (GUVs), provide a suitable platform for exploring membrane properties and interactions. However, LPS molecules contain large polysaccharide parts that confer high water solubility, which makes LPS incorporation in artificial membranes difficult; this hindrance is exacerbated for LPS with long polysaccharide chains, i.e., the smooth LPS. Here, a novel emulsification step of the inverted emulsion method is introduced to incorporate LPS in the outer or the inner leaflet of GUVs, exclusively. We developed an approach to determine the LPS content on individual GUVs and quantify membrane asymmetry. The asymmetric membranes with outer leaflet LPS show incorporations of 1-16 mol % smooth LPS (corresponding to 16-79 wt %), while vesicles with inner leaflet LPS reach coverages of 2-7 mol % smooth LPS (28-60 wt %). Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.

Effect of capillary fluid flow on single cancer cell cycle dynamics, motility, volume and morphology.

From primary tumours and disseminating to secondary organs, cancer cells experience a wide variety of fluid flow profiles when passing through blood vessels or the lymphatic system before extravasation. Sinusoidal capillaries are a common site for extravasation. Therefore, we aim to investigate how metastatic cancer cells react to a biophysical cue such as capillary fluid flow by quantifying its effect on metastatic cell cycle progression, motility, cell and nuclear volume, and morphology. We use MDA-MB-231 breast cancer cells genetically modified with fluorescent ubiquitination-based cell cycle indicator 2 (FUCCI2) as a model system. Single cells are trapped using a microfluidic device and exposed to different laminar flows. Quantitative time-lapse imaging in both 2D epifluorescence and 3D confocal microscopy is performed using in-house software FUCCItrack. In addition, 3D time-lapse with cell and nuclear segmentation is performed with a deep learning approach to streamline the image processing of big datasets. We show that at a single cell level, faster fluid flow leads to a shorter S/G2/M phase and an overall shorter cell cycle, as well as increase in cell motility irrespective of the flow direction. 3D time-lapse confocal imaging of MDA-FUCCI2 single cells reveals the evolution of cell and nuclear volume and morphology as a function of a specific cell cycle phase. Both cell and nuclear volume increase linearly over time. Cell morphology elongates more strongly during the S/G2/M phase, whereas the nuclear shape remains constant. Under the highest flow conditions, only during the S/G2/M phase can we observe a more elongated nucleus, while the cell sphericity remains similar to the control. Collectively, this data, together with the deep learning approach, provides new insights into the potential impact of fluid flow at a single cell level.

Inter-limb asymmetries and kicking limb preference in English premier league soccer players.

The aims of this study were: (1) to quantify interlimb asymmetries in EPL soccer players in the context of kicking limb preference and (2) to establish the relationship between interlimb asymmetries and measures of physical performance. Twenty-two players (age: 21.8 ± 4.4 years) from an EPL club performed a running gait assessment (20 km/h) and unilateral countermovement jumps, a CoD assessment (modified 505 test), and an isokinetic knee extension/flexion protocol using each leg. Asymmetries were quantified using the percentage difference method and Pearson's correlations were used to quantify the association between variables. Players displayed the greatest level of asymmetry in isokinetic strength measures (5.9-12.7%) and lower levels of asymmetry in gait (1.6-7.7%), jump (0.9-7.0%) and CoD (1.9-3.5%) assessments. The influence of the preferred kicking limb was most evident in the isokinetic assessment with the players showing dominance in the preferred limb for knee flexor strength and in the non-preferred limb for knee extensor strength. These manifested in the asymmetry values calculated for the hamstring:quadricep (H:Q) ratios at 60°/s (8.80 ± 7.82%) and 240°/s (11.22 ± 7.04%) and in the functional H:Q ratio (12.67 ± 8.25%). The asymmetry values for peak extensor moment at 240°/s showed a significant correlation (ρ = -0.55, p = 0.034) with 10 m time in the CoD assessment. These findings provide benchmark asymmetry data for soccer practitioners and reveal that kicking limb preferences may bring about interlimb differences in the H:Q ratio which raises important considerations in the design of testing batteries and injury reduction interventions.

Attribute Analytics Performance Metrics from the MAM Consortium Interlaboratory Study.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.

Interplay of surface interaction and magnetic torque in single-cell motion of magnetotactic bacteria in microfluidic confinement.

Swimming microorganisms often experience complex environments in their natural habitat. The same is true for microswimmers in envisioned biomedical applications. The simple aqueous conditions typically studied in the lab differ strongly from those found in these environments and often exclude the effects of small volume confinement or the influence that external fields have on their motion. In this work, we investigate magnetically steerable microswimmers, specifically magnetotactic bacteria, in strong spatial confinement and under the influence of an external magnetic field. We trap single cells in micrometer-sized microfluidic chambers and track and analyze their motion, which shows a variety of different trajectories, depending on the chamber size and the strength of the magnetic field. Combining these experimental observations with simulations using a variant of an active Brownian particle model, we explain the variety of trajectories by the interplay between the wall interactions and the magnetic torque. We also analyze the pronounced cell-to-cell heterogeneity, which makes single-cell tracking essential for an understanding of the motility patterns. In this way, our work establishes a basis for the analysis and prediction of microswimmer motility in more complex environments.

FUCCItrack: An all-in-one software for single cell tracking and cell cycle analysis.

Beyond the more conventional single-cell segmentation and tracking, single-cell cycle dynamics is gaining a growing interest in the field of cell biology. Thanks to sophisticated systems, such as the fluorescent ubiquitination-based cell cycle indicator (FUCCI), it is now possible to study cell proliferation, migration, changes in nuclear morphology and single cell cycle dynamics, quantitatively and in real time. In this work, we introduce FUCCItrack, an all-in-one, semi-automated software to segment, track and visualize FUCCI modified cell lines. A user-friendly complete graphical user interface is presented to record and quantitatively analyze both collective cell proliferation as well as single cell information, including migration and changes in nuclear or cell morphology as a function of cell cycle. To enable full control over the analysis, FUCCItrack also contains features for identification of errors and manual corrections.

Controlled adhesion, membrane pinning and vesicle transport by Janus particles.

The interactions between biomembranes and particles are key to many applications, but the lack of controllable model systems to study them limits the progress in their research. Here, we describe how Janus polystyrene microparticles, half coated with iron, can be partially engulfed by artificial cells, namely giant vesicles, with the goals to control and investigate their adhesion and degree of encapsulation. The interaction between the Janus particles and these model cell membrane systems is mediated by electrostatic charge, offering a further mode of modulation in addition to the iron patches. The ferromagnetic particle coatings also enable manipulation and transport of the vesicles by magnetic fields.

Cell-Free Gene Expression Dynamics in Synthetic Cell Populations.

The ability to build synthetic cellular populations from the bottom-up provides the groundwork to realize minimal living tissues comprising single cells which can communicate and bridge scales into multicellular systems. Engineered systems made of synthetic micron-sized compartments and integrated reaction networks coupled with mathematical modeling can facilitate the design and construction of complex and multiscale chemical systems from the bottom-up. Toward this goal, we generated populations of monodisperse liposomes encapsulating cell-free expression systems (CFESs) using double-emulsion microfluidics and quantified transcription and translation dynamics within individual synthetic cells of the population using a fluorescent Broccoli RNA aptamer and mCherry protein reporter. CFE dynamics in bulk reactions were used to test different coarse-grained resource-limited gene expression models using model selection to obtain transcription and translation rate parameters by likelihood-based parameter estimation. The selected model was then applied to quantify cell-free gene expression dynamics in populations of synthetic cells. In combination, our experimental and theoretical approaches provide a statistically robust analysis of CFE dynamics in bulk and monodisperse synthetic cell populations. We demonstrate that compartmentalization of CFESs leads to different transcription and translation rates compared to bulk CFE and show that this is due to the semipermeable lipid membrane that allows the exchange of materials between the synthetic cells and the external environment.

Super-Resolution Imaging of Highly Curved Membrane Structures in Giant Vesicles Encapsulating Molecular Condensates.

Molecular crowding is an inherent feature of cell interiors. Synthetic cells as provided by giant unilamellar vesicles (GUVs) encapsulating macromolecules (poly(ethylene glycol) and dextran) represent an excellent mimetic system to study membrane transformations associated with molecular crowding and protein condensation. Similarly to cells, such GUVs exhibit highly curved structures like nanotubes. Upon liquid-liquid phase separation their membrane deforms into apparent kinks at the contact line of the interface between the two aqueous phases. These structures, nanotubes, and kinks, have dimensions below optical resolution. Here, these are studied with super-resolution stimulated emission depletion (STED) microscopy facilitated by immobilization in a microfluidic device. The cylindrical nature of the nanotubes based on the superior resolution of STED and automated data analysis is demonstrated. The deduced membrane spontaneous curvature is in excellent agreement with theoretical predictions. Furthermore, the membrane kink-like structure is resolved as a smoothly curved membrane demonstrating the existence of the intrinsic contact angle, which describes the wettability contrast of the encapsulated phases to the membrane. Resolving these highly curved membrane structures with STED imaging provides important insights in the membrane properties and interactions underlying cellular activities.

Directed Signaling Cascades in Monodisperse Artificial Eukaryotic Cells.

The bottom-up assembly of multicompartment artificial cells that are able to direct biochemical reactions along a specific spatial pathway remains a considerable engineering challenge. In this work, we address this with a microfluidic platform that is able to produce monodisperse multivesicular vesicles (MVVs) to serve as synthetic eukaryotic cells. Using a two-inlet polydimethylsiloxane channel design to co-encapsulate different populations of liposomes we are able to produce lipid-based MVVs in a high-throughput manner and with three separate inner compartments, each containing a different enzyme: α-glucosidase, glucose oxidase, and horseradish peroxidase. We demonstrate the ability of these MVVs to carry out directed chemical communication between the compartments via the reconstitution of size-selective membrane pores. Therefore, the signal transduction, which is triggered externally, follows a specific spatial pathway between the compartments. We use this platform to study the effects of enzyme cascade compartmentalization by direct analytical comparison between bulk, one-, two-, and three-compartment systems. This microfluidic strategy to construct complex hierarchical structures is not only suitable to study compartmentalization effects on biochemical reactions but is also applicable for developing advanced drug delivery systems as well as minimal cells in the field of bottom-up synthetic biology.

Osmotic pressure modulates single cell cycle dynamics inducing reversible growth arrest and reactivation of human metastatic cells.

Biophysical cues such as osmotic pressure modulate proliferation and growth arrest of bacteria, yeast cells and seeds. In tissues, osmotic regulation takes place through blood and lymphatic capillaries and, at a single cell level, water and osmoregulation play a critical role. However, the effect of osmotic pressure on single cell cycle dynamics remains poorly understood. Here, we investigate the effect of osmotic pressure on single cell cycle dynamics, nuclear growth, proliferation, migration and protein expression, by quantitative time-lapse imaging of single cells genetically modified with fluorescent ubiquitination-based cell cycle indicator 2 (FUCCI2). Single cell data reveals that under hyperosmotic stress, distinct cell subpopulations emerge with impaired nuclear growth, delayed or growth arrested cell cycle and reduced migration. This state is reversible for mild hyperosmotic stress, where cells return to regular cell cycle dynamics, proliferation and migration. Thus, osmotic pressure can modulate the reversible growth arrest and reactivation of human metastatic cells.

New Peak Detection Performance Metrics from the MAM Consortium Interlaboratory Study.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.

Surfactant-free production of biomimetic giant unilamellar vesicles using PDMS-based microfluidics.

Microfluidic production of giant lipid vesicles presents a paradigm-shift in the development of artificial cells. While production is high-throughput and the lipid vesicles are mono-disperse compared to bulk methods, current technologies rely heavily on the addition of additives such as surfactants, glycerol and even ethanol. Here we present a microfluidic method for producing biomimetic surfactant-free and additive-free giant unilamellar vesicles. The versatile design allows for the production of vesicle sizes ranging anywhere from ~10 to 130 µm with either neutral or charged lipids, and in physiological buffer conditions. Purity, functionality, and stability of the membranes are validated by lipid diffusion, protein incorporation, and leakage assays. Usability as artificial cells is demonstrated by increasing their complexity, i.e., by encapsulating plasmids, smaller liposomes, mammalian cells, and microspheres. This robust method capable of creating truly biomimetic artificial cells in high-throughput will prove valuable for bottom-up synthetic biology and the understanding of membrane function.

Minimal Pathway for the Regeneration of Redox Cofactors.

Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzymatic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD+ to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equivalents in the lumen. A soluble transhydrogenase subsequently utilizes NADH for reduction of NADP+ thereby making NAD+ available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diameter (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochemical process of reduction of glutathione disulfide can be driven by the transfer of reducing equivalents from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metabolism.