Making a case for outpatient parenteral antimicrobial therapy (OPAT).

In the wake of the COVID-19 pandemic, and its negative impact on both acute and elective care and decline in available inpatient resources, there is an imperative to maximize safe and effective alternatives to inpatient hospital care. Properly governed outpatient parenteral antimicrobial therapy (OPAT) services embed the principles of antimicrobial stewardship (AMS) (including use of early oral therapy) and support admission avoidance and early discharge for a growing range of patient groups with complex infections through well-organized multidisciplinary team working. Expansion of OPAT aligns with the UK's national strategy to deliver care closer to home and cost-effectively maximize use of inpatient resources. OPAT serves as an exemplar to other ambulatory services and presents opportunities for developing and assuring AMS strategies within the rapidly developing hospital-at-home and virtual ward environments.

Supported Biomembrane Systems Incorporating Multiarm Polymers and Bioorthogonal Tethering.

To functionalize interfaces with supported biomembranes and membrane proteins, the challenge is to build stabilized and supported systems that mimic the native lipid microenvironment. Our objective is to control substrate-to-biomembrane spacing and the tethering chemistry so proteoliposomes can be fused and conjugated without perturbation of membrane protein function. Furthermore, the substrates need to exhibit low protein and antibody nonspecific binding to use these systems in assays. We have employed protein orthogonal coupling schemes in concert with multiarm poly(ethylene glycol) (PEG) technology to build supported biomembranes on microspheres. The lipid bilayer structures and tailored substrates of the microsphere-supported biomembranes were analyzed via flow cytometry, confocal fluorescence, and super-resolution imaging microscopy, and the lateral fluidity was quantified using fluorescence recovery after photobleaching (FRAP) techniques. Under these conditions, the 4-arm-PEG20,000-NH2 based configuration gave the most desirable tethering system based on lateral diffusivity and coverage.

Fluorescent azobenzene-confined coiled-coil mesofibers.

Fluorescent protein biomaterials have important applications such as bioimaging in pharmacological studies. Self-assembly of proteins, especially into fibrils, is known to produce fluorescence in the blue band. Capable of self-assembly into nanofibers, we have shown we can modulate its aggregation into mesofibers by encapsulation of a small hydrophobic molecule. Conversely, azobenzenes are hydrophobic small molecules that are virtually non-fluorescent in solution due to their highly efficient photoisomerization. However, they demonstrate fluorogenic properties upon confinement in nanoscale assemblies by reducing the non-radiative photoisomerization. Here, we report the fluorescence of a hybrid protein-small molecule system in which azobenzene is confined in our protein assembly leading to fiber thickening and increased fluorescence. We show our engineered protein Q encapsulates AzoCholine, bearing a photoswitchable azobenzene moiety, in the hydrophobic pore to produce fluorescent mesofibers. This study further investigates the photocontrol of protein conformation as well as fluorescence of an azobenze-containing biomaterial.

Apoptosis detection via automated algorithms to analyze biomarker translocation in reporter cells.

Rapid, efficient, and robust quantitative analyses of dynamic apoptotic events are essential in a high-throughput screening workflow. Currently used methods have several bottlenecks, specifically, limitations in available fluorophores for downstream assays and misinterpretation of statistical image data analysis. In this study, we developed cytochrome-C (Cyt-C) and caspase-3/-8 reporter cell lines using lung (PC9) and breast (T47D) cancer cells, and characterized them from the response to apoptotic stimuli. In these two reporter cell lines, the spatial fluorescent signal translocation patterns served as reporters of activations of apoptotic events, such as Cyt-C release and caspase-3/-8 activation. We also developed a vision-based, tunable, automated algorithm in MATLAB to implement the robust and accurate analysis of signal translocation in single or multiple cells. Construction of the reporter cell lines allows live monitoring of apoptotic events without the need for any other dyes or fixatives. Our algorithmic implementation forgoes the use of simple image statistics for more robust analytics. Our optimized algorithm can achieve a precision greater than 90% and a sensitivity higher than 85%. Combining our automated algorithm with reporter cells bearing a single-color dye/fluorophore, we expect our approach to become an integral component in the high-throughput drug screening workflow.

Renal nitrate clearance in chronic kidney disease.

BACKGROUND: Nitric oxide (NO) is rapidly oxidised in humans to nitrite and nitrate, with nitrate being present in much greater abundance. These oxidation products can be recycled back into nitric oxide via a complex entero-salivary pathway, thus preserving NO activity. Approximately 65% of circulating nitrate is excreted in the urine in 48 h, with the excretory pathway of the remainder unknown. The effect of declining renal function on nitrate clearance is unknown METHODS: Forty five subjects, 21 M, 24F, median age 69 (range 27-75 years) with renal function assessed by CKD-EPI eGFR between 9 and 89 ml/min/1.73 m2 completed the study. Following a 24 h low nitrate diet a microplate spectrophotometric method was employed to measure plasma nitrate concentration and 24 h urinary nitrate excretion were measured to determine renal nitrate clearance. RESULTS: There was a strong positive correlation between urinary nitrate clearance and eGFR, (Spearman R = 0.7665, p < 0.0001) with a moderate negative correlation between plasma nitrate concentration and CKD-EPI eGFR, (Spearman's R = -0.37, p = 0.012). There was a trend between fractional excretion of nitrate and CKD-EPI eGFR (ml/min/1.73 m2) Spearman's R 0.27, p = 0.07 though this did not reach statistical significance. Plasma nitrate concentration and serum creatinine concentration were positively correlated, Spearman's R = 0.39, p = 0.008. CONCLUSIONS: We have observed a strong positive association between renal nitrate clearance and renal function such that plasma nitrate rises as renal function falls. Fractional excretion of nitrate appears to decline as renal function falls. As such, urinary nitrate excretion is unlikely to be a reliable marker of endogenous NO synthesis in settings where renal function is altered.

γ-Secretase Partitioning into Lipid Bilayers Remodels Membrane Microdomains after Direct Insertion.

γ-Secretase is a multisubunit complex that catalyzes intramembranous cleavage of transmembrane proteins. The lipid environment forms membrane microdomains that serve as spatio-temporal platforms for proteins to function properly. Despite substantial advances in the regulation of γ-secretase, the effect of the local membrane lipid microenvironment on the regulation of γ-secretase is poorly understood. Here, we characterized and quantified the partitioning of γ-secretase and its substrates, the amyloid precursor protein (APP) and Notch, into lipid bilayers using solid-supported model membranes. Notch substrate is preferentially localized in the liquid-disordered (Ld) lipid domains, whereas APP and γ-secretase partition as single or higher complex in both phases but highly favor the ordered phase, especially after recruiting lipids from the ordered phase, indicating that the activity and specificity of γ-secretase against these two substrates are modulated by membrane lateral organization. Moreover, time-elapse measurements reveal that γ-secretase can recruit specific membrane components from the cholesterol-rich Lo phase and thus creates a favorable lipid environment for substrate recognition and therefore activity. This work offers insight into how γ-secretase and lipid modulate each other and control its activity and specificity.

Prediction of improved therapeutics for fabry disease patients generated by mutagenesis of the α-galactosidase A active site, dimer interface, and glycosylation region.

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme, α-galactosidase A that induces the accumulation of the substrate globotriaosylceramide. Currently approved enzyme replacement therapy using recombinant human α-galactosidase A improves patient symptoms but a majority of patients experience adverse events due to the multiple infusions required for full therapeutic efficacy. Our approach is to use medicinal chemistry and phylogenic comparisons to introduce mutations into the human enzyme to increase catalytic activity and/or stability to generate an improved therapeutic enzyme that may require fewer infusions. We designed mutations at three regions of the human α-galactosidase A: the active site, the dimer interface, and a site for glycosylation. The M208E mutation, adjacent to the Y207 active site residue, increased enzyme activity 3.01-fold. This mutation introduced a charged Glu residue that is adjacent to the Y207 active site residue and close to a site of N-glycosylation. The W277C mutation, designed to promote dimer stability, introduced a strong thiol-aromatic interaction (Cys-Phe) at the dimer interface and increased activity 2.31-fold. The W277C and M208E mutations modify the structure of the enzyme into forms with enhanced thermal stability 3.7- and 3.9-fold, respectively and positive cooperativity resulting in increased Hill coefficient from 1.0 to 4.60 and 3.47, respectively. Enhanced thermal stability and positive cooperativity predict improved in vivo activity and superior therapeutic properties. Our results demonstrate the value of in vitro mutagenesis for α-galactosidase A and support future perspectives to validate these results in Fabry disease patients.

Supervised machine learning for the prediction of infection on admission to hospital: a prospective observational cohort study.

BACKGROUND: Infection diagnosis can be challenging, relying on clinical judgement and non-specific markers of infection. We evaluated a supervised machine learning (SML) algorithm for diagnosing bacterial infection using routinely available blood parameters on presentation to hospital. METHODS: An SML algorithm was developed to classify cases into infection versus no infection using microbiology records and six available blood parameters (C-reactive protein, white cell count, bilirubin, creatinine, ALT and alkaline phosphatase) from 160203 individuals. A cohort of patients admitted to hospital over a 6 month period had their admission blood parameters prospectively inputted into the SML algorithm. They were prospectively followed up from admission to classify those who fulfilled clinical case criteria for a community-acquired bacterial infection within 72 h of admission using a pre-determined definition. Predictive ability was assessed using receiver operating characteristics (ROC) with cut-off values for optimal sensitivity and specificity explored. RESULTS: One hundred and four individuals were included prospectively. The median (range) cohort age was 65 (21-98) years. The majority were female (56/104; 54%). Thirty-six (35%) were diagnosed with infection in the first 72 h of admission. Overall, 44/104 (42%) individuals had microbiological investigations performed. Treatment was prescribed for 33/36 (92%) of infected individuals and 4/68 (6%) of those with no identifiable bacterial infection. Mean (SD) likelihood estimates for those with and without infection were significantly different. The infection group had a likelihood of 0.80 (0.09) and the non-infection group 0.50 (0.29) (P < 0.01; 95% CI: 0.20-0.40). ROC AUC was 0.84 (95% CI: 0.76-0.91). CONCLUSIONS: An SML algorithm was able to diagnose infection in individuals presenting to hospital using routinely available blood parameters.

Effect of adding a mobile health intervention to a multimodal antimicrobial stewardship programme across three teaching hospitals: an interrupted time series study.

Objectives: To evaluate the impact of adding a mobile health (mHealth) decision support system for antibiotic prescribing to an established antimicrobial stewardship programme (ASP). Methods: In August 2011, the antimicrobial prescribing policy was converted into a mobile application (app). A segmented regression analysis of interrupted time series was used to assess the impact of the app on prescribing indicators, using data (2008-14) from a biannual point prevalence survey of medical and surgical wards. There were six data points pre-implementation and six data points post-implementation. Results: There was an increase in compliance with policy (e.g. compliance with empirical therapy or expert advice) in the two specialties of medicine (6.48%, 95% CI = -1.25 to 14.20) and surgery (6.63%, 95% CI = 0.15-13.10) in the implementation period, with a significant sudden change in level in surgery ( P < 0.05). There was an increase, though not significant, in medicine (15.20%, 95% CI = -17.81 to 48.22) and surgery (35.97%, 95% CI = -3.72 to 75.66) in the percentage of prescriptions that had a stop/review date documented. The documentation of indication decreased in both medicine (-16.25%, 95% CI = -42.52 to 10.01) and surgery (-14.62%, 95% CI = -42.88 to 13.63). Conclusions: Introducing the app into an existing ASP had a significant impact on the compliance with policy in surgery, and a positive, but not significant, effect on documentation of stop/review date in both specialties. The negative effect on the third indicator may reflect a high level of compliance pre-intervention, due to existing ASP efforts. The broader value of providing an antimicrobial policy on a digital platform, e.g. the reach and access to the policy, should be measured using indicators more sensitive to mHealth interventions.

The impact of cardiovascular co-morbidities and duration of diabetes on the association between microvascular function and glycaemic control.

BACKGROUND: Good glycaemic control in type 2 diabetes (T2DM) protects the microcirculation. Current guidelines suggest glycaemic targets be relaxed in advanced diabetes. We explored whether disease duration or pre-existing macrovascular complications attenuated the association between hyperglycaemia and microvascular function. METHODS: 743 participants with T2DM (n = 222), cardiovascular disease (CVD = 183), both (n = 177) or neither (controls = 161) from two centres in the UK, underwent standard clinical measures and endothelial dependent (ACh) and independent (SNP) microvascular function assessment using laser Doppler imaging. RESULTS: People with T2DM and CVD had attenuated ACh and SNP responses compared to controls. This was additive in those with both (ANOVA p < 0.001). In regression models, cardiovascular risk factors accounted for attenuated ACh and SNP responses in CVD, whereas HbA1c accounted for the effects of T2DM. HbA1c was associated with ACh and SNP response after adjustment for cardiovascular risk factors (adjusted standardised beta (ß) -0.096, p = <0.008 and -0.135, p < 0.001, respectively). Pre-existing CVD did not modify this association (ß -0.099; p = 0.006 and -0.138; p < 0.001, respectively). Duration of diabetes accounted for the association between HbA1c and ACh (ß -0.043; p = 0.3), but not between HbA1c and SNP (ß -0.105; p = 0.02). CONCLUSIONS: In those with T2DM and CVD, good glycaemic control is still associated with better microvascular function, whereas in those with prolonged disease this association is lost. This suggests duration of diabetes may be a better surrogate for "advanced disease" than concomitant CVD, although this requires prospective validation.

Phase Composition Control in Microsphere-Supported Biomembrane Systems.

The popularization of studies in membrane protein lipid phase coexistence has prompted the development of new techniques to construct and study biomimetic systems with cholesterol-rich lipid microdomains. Here, microsphere-supported biomembranes with integrated α-helical peptides, referred to as proteolipobeads (PLBs), were used to model peptide/protein partitioning within DOPC/DPPC/cholesterol phase-separated membranes. Due to the appearance of compositional heterogeneity and impurities in the formation of model PLB assemblies, fluorescence-activated cell sorting (FACS) was used to characterize and sort PLB populations on the basis of disordered phase (Ld) content. In addition, spectral imaging was used to assess the partitioning of FITC-labeled α-helical peptide between fluorescently labeled Ld phase and unlabeled ordered phase (Lo) phase lipid microdomains. The apparent peptide partition coefficient, Kp,app, was measured to be 0.89 ± 0.06, indicating a slight preference of the peptide for the Lo phase. A biomimetic motif of the Lo phase concentration enhancement of the biotinyl-peptide ligand display in proteolipobeads was also observed. Finally, peptide mobility was measured by FRAP separately in each lipid phase, yielding diffusivities of 0.036 ± 0.005 and 0.014 ± 0.003 µm2/s in the Ld and Lo phases, respectively.

Monodisperse polystyrene foams via polymerization of foamed emulsions: structure and mechanical properties.

Foamed styrene-in-water emulsions can serve as templates for solid polystyrene foams as the pore size dpore in the solid polystyrene foam matches the bubble size dbubble of the liquid foam template. By producing monodisperse foamed emulsions with a microfluidic device it is possible to adjust the pore size, the connectivity of the pores, as well as the density of the solid polystyrene foams. The pore size can be tuned either by varying the gas pressure used to form the emulsion or by varying the chip dimension. Using three different chip dimensions we are able to produce monodisperse polystyrene foams with pore sizes ranging from 115 µm up to 588 µm. The relative density can be varied easily in a range from 0.10 to 0.30. Increasing the liquid fraction leads initially to smaller interconnections and ultimately to a mainly closed cell foam. It is practically impossible to produce a fully closed cell foam since, even at high liquid fractions, two adjacent bubbles eventually touch and form a film that will rupture during polymerization. By closely investigating the structure of the polystyrene foams we noticed an additional porosity in the pore walls which matches the water content of the styrene-in-water emulsion. During polymerization, the styrene droplets in the aqueous matrix fuse and build up a continuous but porous structure which makes up the pore walls of the macropores. This additionally porosity also leads to lower Young's and shear moduli than expected, as predicted by Gibson and Ashby's model. The relationship between relative density and moduli is in good agreement with the model.

Imaging and Functional Analysis of γ-Secretase and Substrate in a Proteolipobead System with an Activity-Based Probe.

Investigation of intramembranal protease catalysis demands the generation of intact biomembrane assemblies with structural integrity and lateral mobility. Here, we report the development of a microsphere supported-biomembrane platform enabling characterization of γ-secretase and substrate within proteolipobead assemblies via microscopy and flow cytometry. The active enzyme loading levels were tracked using an activity-based probe, with the biomembranes delineated by carbocyanine lipid reporters. Proteolipobeads formed from HeLa proteoliposomes gave rise to homogeneous distributions of active γ-secretase within supported biomembranes with native-like fluidity. The substrate loading into supported biomembranes was detergent-dependent, as evidenced by even colocalization of substrate and lipid tracers in confocal 3D imaging of individual proteolipobeads. Moreover, the loading level was tunable with bulk substrate concentration. γ-Secretase substrate cleavage and its inhibition within γ-secretase proteolipobeads were observed. This platform offers a means to visualize enzyme and substrate loading, activity, and inhibition in a controllable biomembrane microenvironment.

Biodegradable, Tethered Lipid Bilayer-Microsphere Systems with Membrane-Integrated α-Helical Peptide Anchors.

Supported lipid bilayers (SLBs) are ideally suited for the study of biomembrane-biomembrane interactions and for the biomimicry of cell-to-cell communication, allowing for surface ligand displays that contain laterally mobile elements. However, the SLB paradigm does not include three-dimensionality and biocompatibility. As a way to bypass these limitations, we have developed a biodegradable form of microsphere SLBs, also known as proteolipobeads (PLBs), using PLGA microspheres. Microspheres were synthesized using solvent evaporation and size selected with fluorescence activated cell sorting (FACS). Biomembranes were covalently tethered upon fusion to microsphere supports via short-chain PEG spacers connecting membrane-integrated α-helical peptides and the microsphere surface, affecting membrane diffusivity and mobility as indicated by confocal FRAP analysis. Membrane heterogeneities, which are attributed to PLGA hydrophobicity and rough surface topography, are curtailed by the addition of PEG tethers. This method allows for the presentation of tethered, laterally mobile biomembranes in three dimensions with functionally embedded attachment peptides for mobile ligand displays.

Outpatient parenteral antimicrobial therapy and antimicrobial stewardship: challenges and checklists.

Outpatient parenteral antimicrobial therapy (OPAT) has become, for many countries, an established form of healthcare delivery. At the same time, there have been calls to ensure the prudent use of the existing antimicrobial armamentarium. For OPAT, this presents a dilemma. On one hand, stewardship principles look for the most effective agent with minimal collateral effects. In OPAT, whilst the aims of the service are similar, convenience of dosing to optimize early hospital discharge or admission avoidance may take precedence over an agent's spectrum of activity. This brief article aims to highlight the importance and explore the challenges of antimicrobial stewardship in the context of OPAT. Within the UK, the safe and effective use of antimicrobials is modelled around the IDSA/Society for Healthcare Epidemiology of America stewardship practice guidelines with local customization where appropriate. Current UK stewardship practice principles were compared with published good practice recommendations for OPAT to identify how OPAT could support the broader antimicrobial stewardship agenda. It is essential that antimicrobial stewardship teams should understand the challenges faced in the non-inpatient setting and the potential benefits/lower risks associated with avoided admission or shortened hospital stay in this population. Within its limitations, OPAT should practise stewardship principles, including optimization of intravenous to oral switch and the reporting of outcomes, healthcare-associated infections and re-admission rates. OPAT should report to the antimicrobial stewardship team. Ideally the OPAT team should be formally represented within the stewardship framework. A checklist has been proposed to aid OPAT services in ensuring they meet their stewardship agenda.

How is income generated by outpatient parenteral antibiotic treatment (OPAT) in the UK? Analysis of payment tariffs for cellulitis.

OBJECTIVES: We determined the available mechanisms to generate income from outpatient parenteral antimicrobial therapy (OPAT) in the UK and calculated the revenue generated from treatment of an episode of cellulitis. METHODS: Revenue was calculated for patients receiving treatment for cellulitis as an inpatient and for patients receiving OPAT by a series of different payment pathways. Selected established OPAT services in Northern Ireland, Scotland and Wales, where Payment-by-Results (PbR) does not operate, were contacted to determine individual national funding arrangements. RESULTS: In England, a traditional inpatient episode for uncomplicated cellulitis requiring 7 days of treatment generated £1361 of revenue, while OPAT generated revenue ranging from £773 to £2084 for the same length of treatment depending on the payment pathway used. Treatment using OPAT to avoid admission entirely generated £2084, inpatient admission followed by transfer to a virtual OPAT ward at day 2 generated £1361 and inpatient admission followed by discharge from hospital to OPAT at day 2 generated £773. In Northern Ireland, Scotland and Wales block contracts were used and no income was calculable for an individual episode of cellulitis. CONCLUSIONS: No single funding mechanism supports OPAT across the UK. In England, revenue generated by OPAT providers from treatment of cellulitis varied with the OPAT payment pathway used, but equalled or exceeded the income generated from equivalent inpatient care. Cost savings for OPAT and reuse of released inpatient beds will increase revenue further. A single OPAT tariff is proposed.

Ageing modifies the effects of beetroot juice supplementation on 24-hour blood pressure variability: An individual participant meta-analysis.

OBJECTIVES: Abnormal circadian oscillations of blood pressure (BP) and nocturnal-diurnal BP differences (i.e., dipping) increase cardiovascular risk. Whether inorganic nitrate supplementation influences 24-hr BP variability is currently unknown. We studied the effects of high-nitrate beetroot juice supplementation on BP variability measured by 24-hr ambulatory BP monitoring (24-hr ABPM) in older subjects. METHODS: Data from four independent randomised clinical trials were collated. Eighty-five older participants (age range: 55-76 years) were included in the final database. Two trials had an open-label, parallel design and two trials had a cross-over, double-blind design. Participants were randomised to either beetroot juice or placebo. Changes in 24-hr ABPM (daily, diurnal, nocturnal), variability (weighted-SDs), night-dipping, morning surge for systolic and diastolic BP were measured. Meta-analysis was conducted to obtain pooled estimates of the effect size for each BP outcome. Sub-group analyses were conducted to evaluate the influence of age, BMI, gender, BP status and changes in nitrite concentrations on the effect size. RESULTS: The pooled effect of beetroot juice on all BP outcomes was not significant. Beetroot juice ingestion determined a significant decrease in nocturnal systolic BP variability in subjects aged less than 65 y (2.8 mmHg, -4.5 -1.0, p = 0.002) compared to the older group (≥ 65 y; 1.0 mmHg, -2.2 4.2, p = 0.54). A greater change in NO2(-) concentrations after beetroot supplementation was associated with significant differences for nocturnal mean (-3.4 mmHg, -0.6 -2.4, p = 0.02) and variability (-0.8 mmHg, -1.5 -0.06, p = 0.03) of systolic BP. CONCLUSIONS: The vascular responsiveness to inorganic nitrate may be modified by mechanisms of vascular ageing influencing the reducing capacity to convert inorganic nitrate into nitrite and tissue-specific responses to dietary nitrate supplementation.

Engineered coiled-coil protein microfibers.

The fabrication of de novo proteins able to self-assemble on the nano- to meso-length scales is critical in the development of protein-based biomaterials in nanotechnology and medicine. Here we report the design and characterization of a protein engineered coiled-coil that not only assembles into microfibers, but also can bind hydrophobic small molecules. Under ambient conditions, the protein forms fibers with nanoscale structure possessing large aspect ratios formed by bundles of α-helical homopentameric assemblies, which further assemble into mesoscale fibers in the presence of curcumin through aggregation. Surprisingly, these biosynthesized fibers are able to form in conditions of remarkably low concentrations. Unlike previously designed coiled-coil fibers, these engineered protein microfibers can bind the small molecule curcumin throughout the assembly, serving as a depot for encapsulation and delivery of other chemical agents within protein-based 3D microenvironments.

Investigation of Head Kinematics and Brain Strain Response During Soccer Heading Using a Custom-Fit Instrumented Mouthguard.

Association football, also known as soccer in some regions, is unique in encouraging its participants to intentionally use their head to gain a competitive advantage, including scoring a goal. Repetitive head impacts are now being increasingly linked to an inflated risk of developing long-term neurodegenerative disease. This study investigated the effect of heading passes from different distances, using head acceleration data and finite element modelling to estimate brain injury risk. Seven university-level participants wore a custom-fitted instrumented mouthguard to capture linear and angular acceleration-time data. They performed 10 headers within a laboratory environment, from a combination of short, medium, and long passes. Kinematic data was then used to calculate peak linear acceleration, peak angular velocity, and peak angular acceleration as well as two brain injury metrics: head injury criterion and rotational injury criterion. Six degrees of freedom acceleration-time data were also inputted into a widely accepted finite element brain model to estimate strain-response using mean peak strain and cumulative strain damage measure values. Five headers were considered to have a 25% concussion risk. Mean peak linear acceleration equalled 26 ± 7.9 g, mean peak angular velocity 7.20 ± 2.18 rad/s, mean peak angular acceleration 1730 ± 611 rad/s2, and 95th percentile mean peak strain 0.0962 ± 0.252. Some of these data were similar to brain injury metrics reported from American football, which supports the need for further investigation into soccer heading.

Tether-supported biomembranes with α-helical peptide-based anchoring constructs.

The strict requirement of constructing a native lipid environment to preserve the structure and functionality of membrane proteins is the starting constraint when building biomaterials and sensor systems from these biomolecules. To enhance the viability of supported biomembranes systems and build new ligand display interfaces, we apply rationally designed peptides partitioned into the lipid bilayer interface. Peptides designed to form membrane-spanning α-helical anchoring domains are synthesized using solid-phase peptide synthesis. K(3)A(4)L(2)A(7)L(2)A(3)K(2)-FITC is synthesized on the 100 mg scale for use as a biomembrane anchoring molecule, where orthogonal side-chain modifications allow us to introduce probes enabling peptide localization within supported bilayers. The peptides are found to form α-helical domains within liposomes as assessed with circular dichroism spectroscopy. These peptides are designed to be incorporated into lipid bilayers supported by microspheres and serve as biomembrane anchoring moieties to amino-terminated surfaces. Here, the silica bead surface (4.7 µm diameter) is activated with homobifunctional NHS-PEG(3000)-NHS as "polymer cushion" spacers. This tethering to a subset of the K(3)A(4)L(2)A(7)L(2)A(3)K(2)-FITC molecules present in the bilayer is achieved by the fusion of liposomes followed by coupling of the peptide amino groups to the NHS presented from the silica microsphere surfaces. The biomembrane distributions of tethered and untethered K(3)A(4)L(2)A(7)L(2)A(3)K(2)-FITC are probed with confocal microscopy and are found to give 3D reconstructions consistent with largely homogeneous supported biomembranes. The fluidity of the untethered fraction of peptides within supported membranes is quantified using the fluorescence recovery after photobleaching (FRAP) technique. The presence of the PEG(3000) polymer cushion facilitated a 28.9% increase in peptide diffusivity over untethered bilayers at the lowest peptide to lipid ratio we examined. We show that rationally designed peptide-based anchors can be used to tether lipid bilayers, creating a polymer-cushioned lipid microenvironment on surfaces with high lateral mobility and facilitating the development of a new platform for ligand displays.