Expression and characterization of recombinant rattusin, an α-defensin-related peptide with a homodimeric scaffold formed by intermolecular disulfide exchanges.

Rattusin is an α-defensin-related peptide isolated from the small intestine of rats. The primary sequence of linear rattusin is composed of 31 amino acids containing five cysteines with a unique spacing pattern. It forms a homodimeric scaffold in which the primary structure occurs in an antiparallel fashion formed by five intermolecular disulfide (SS) bonds. Rattusin is a highly potent antibiotic, which not only exhibits broad-spectrum antimicrobial activity, but also maintains its antimicrobial activity at physiological salt concentrations. Therefore, to develop new antibiotics based on rattusin, structural and functional studies of rattusin should be performed. For this purpose, large amounts of linear rattusin precursor must be obtained through appropriate preparation methods. Therefore, we established a mass production technique for linear rattusin by using recombinant protein expression and purification procedures. We verified that structure and activity of the recombinant rattusin are identical to the chemically synthesized rattusin. The described method for producing recombinant rattusin provides a high yield of rattusin, which can be used to study the biochemical and functional properties of rattusin and for the development of rattusin-based peptide antibiotics.

A Novel Dimeric Short Peptide Derived from α-Defensin-Related Rattusin with Improved Antimicrobial and DNA-Binding Activities.

Rattusin, an α-defensin-related antimicrobial peptide isolated from the small intestine of rats, has been previously characterized through NMR spectroscopy to elucidate its three-dimensional structure, revealing a C2 homodimeric scaffold stabilized by five disulfide bonds. This study aimed to identify the functional region of rattusin by designing and synthesizing various short analogs, subsequently leading to the development of novel peptide-based antibiotics. The analogs, designated as F1, F2, F3, and F4, were constructed based on the three-dimensional configuration of rattusin, among which F2 is the shortest peptide and exhibited superior antimicrobial efficacy compared to the wild-type peptide. The central cysteine residue of F2 prompted an investigation into its potential to form a dimer at neutral pH, which is critical for its antimicrobial function. This activity was abolished upon the substitution of the cysteine residue with serine, indicating the necessity of dimerization for antimicrobial action. Further, we synthesized ß-hairpin-like analogs, both parallel and antiparallel, based on the dimeric structure of F2, which maintained comparable antimicrobial potency. In contrast to rattusin, which acts by disrupting bacterial membranes, the F2 dimer binds directly to DNA, as evidenced by fluorescence assays and DNA retardation experiments. Importantly, F2 exhibited negligible cytotoxicity up to 515 µg/mL, assessed via hemolysis and MTT assays, underscoring its potential as a lead compound for novel peptide-based antibiotic development.

Development of a Low-Power Instrumented Mouthpiece for Directly Measuring Head Acceleration in American Football.

Instrumented mouthpieces (IM) offer a means of measuring head impacts that occur in sport. Direct measurement of angular head kinematics is preferential for accuracy; however, existing IMs measure angular velocity and differentiate the measurement to calculate angular acceleration, which can limit bandwidth and consume more power. This study presents the development and validation of an IM that uses new, low-power accelerometers for direct measurement of linear and angular acceleration over a broad range of head impact conditions in American football. IM sensor accuracy for measuring six-degree-of-freedom head kinematics was assessed using two helmeted headforms instrumented with a custom-fit IM and reference sensor instrumentation. Head impacts were performed at 10 locations and 6 speeds representative of the on-field conditions associated with injurious and non-injurious impacts in American football. Sensor measurements from the IM were highly correlated with those from the reference instrumentation located at the maxilla and skull center of gravity. Based on pooled data across headform and impact location, R2 ≥ 0.94, mean absolute error (AE) ≤ 7%, and mean relative impact angle ≤ 11° for peak linear and angular acceleration and angular velocity while R2 ≥ 0.90 and mean AE ≤ 7% for kinematic-based injury metrics used in helmet tests.

Surface Contact Features, Impact Obliquity, and Preimpact Rotational Motion in Concussive Helmet-to-Ground Impacts: Assessment via a New Impact Test Device.

This paper reports the development of a test device for replicating unique features of concussion-causing helmet-to-ground impacts. Helmet-to-ground impacts are characterized by an oblique impact velocity vector, preimpact rotational motion of the helmeted head, and an impact into a compliant frictional surface of unknown effective mass. No helmet assessment testing program replicates these impact characteristics, yet they influence brain injury risk and therefore may influence helmet design priorities. To replicate these mechanics, the carriage of a drop tower was modified by the addition of a curvilinear bearing track and a hinged torso-neck fixture to which a helmeted head of a Hybrid III anthropomorphic test device was mounted. Preimpact rotational motion of the head was imparted by forcing a link arm to follow the curvilinear path as the carriage fell under gravity. At impact, the rotating helmeted head struck a vertically mounted surface. The ground impact features of head kinematics are illustrated by comparing rear impacts into a rigid, low-friction surface against those into a compliant frictional surface simulating turf. With the rigid, low-friction surface, the head experienced a change in rotational rate of approximately 40 rad/s, which corresponded to a peak rotational acceleration of approximately αy = - 4000 rad/s2. In contrast, peak rotational acceleration with the compliant frictional surface was approximately αy = - 1000 rad/s2 while the helmet was in contact with the surface. Neck loads were significantly greater with the compliant frictional surface. Translational head acceleration was less sensitive to the surface characteristics, with the peak of the anterior-posterior component essentially unchanged.

Finite Element Model of a Deformable American Football Helmet Under Impact.

Despite the use of helmets in American football, brain injuries are still prevalent. To reduce the burden of these injuries, novel impact mitigation systems are needed. The Vicis Zero1 (VZ1) American football helmet is unique in its use of multi-directional buckling structures sandwiched between a deformable outer shell and a stiff inner shell. The objective of this study was to develop a model of the VZ1 and to assess this unique characteristic for its role in mitigating head kinematics. The VZ1 model was developed using a bottom-up framework that emphasized material testing, constitutive model calibration, and component-level validation. Over 50 experimental tests were simulated to validate the VZ1 model. CORrelation and Analysis (CORA) was used to quantify the similarity between experimental and model head kinematics, neck forces, and impactor accelerations and forces. The VZ1 model demonstrated good correlation with an overall mean CORA score of 0.86. A parametric analysis on helmet compliance revealed that the outer shell and column stiffness influenced translational head kinematics more than rotational. For the material parameters investigated, head linear acceleration ranged from 80 to 220 g, whereas angular velocity ranged from 37 to 40 rad/s. This helmet model is open-source and serves as an in silico design platform for helmet innovation.

Development and Evaluation of a Test Method for Assessing the Performance of American Football Helmets.

As more is learned about injury mechanisms of concussion and scenarios under which injuries are sustained in football games, methods used to evaluate protective equipment must adapt. A combination of video review, videogrammetry, and laboratory reconstructions was used to characterize concussive impacts from National Football League games during the 2015-2017 seasons. Test conditions were generated based upon impact locations and speeds from this data set, and a method for scoring overall helmet performance was created. Head kinematics generated using a linear impactor and sliding table fixture were comparable to those from laboratory reconstructions of concussive impacts at similar impact conditions. Impact tests were performed on 36 football helmet models at two laboratories to evaluate the reproducibility of results from the resulting test protocol. Head acceleration response metric, a head impact severity metric, varied 2.9-5.6% for helmet impacts in the same lab, and 3.8-6.0% for tests performed in a separate lab when averaged by location for the models tested. Overall inter-lab helmet performance varied by 1.1 ± 0.9%, while the standard deviation in helmet performance score was 7.0%. The worst helmet performance score was 33% greater than the score of the best-performing helmet evaluated by this study.

Development of Open-Source Dummy and Impactor Models for the Assessment of American Football Helmet Finite Element Models.

The objective of this study was to develop and validate a set of Hybrid-III head and neck (HIII-HN) and impactor models that can be used to assess American football design modifications with established dummy-based injury metrics. The model was validated in two bare-head impact test configurations used by the National Football League and research groups to rank and assess helmet performance. The first configuration was a rigid pendulum impact to three locations on the HIII head (front, rear, side) at 3 m/s. The second configuration was a set of eight 5.5 m/s impacts to the HIII head at different locations using a linear impactor with a compliant end cap. The ISO/TS 18571 objective rating metric was used to quantify the correlation between the experimental and model head kinematics (linear and rotational velocity and acceleration) and neck kinetics (neck force and moment). The HIII-HN model demonstrated good correlation with overall mean ISO scores of 0.69-0.78 in the pendulum impacts and 0.65-0.81 in the linear impacts. These publically available models will serve as an in silico design platform that will be useful for investigating novel football helmet designs and studying human head impact biomechanics, in general.

Injury risk functions based on population-based finite element model responses: Application to femurs under dynamic three-point bending.

OBJECTIVE: The goal of this study was to explore a framework for developing injury risk functions (IRFs) in a bottom-up approach based on responses of parametrically variable finite element (FE) models representing exemplar populations. METHODS: First, a parametric femur modeling tool was developed and validated using a subject-specific (SS)-FE modeling approach. Second, principal component analysis and regression were used to identify parametric geometric descriptors of the human femur and the distribution of those factors for 3 target occupant sizes (5th, 50th, and 95th percentile males). Third, distributions of material parameters of cortical bone were obtained from the literature for 3 target occupant ages (25, 50, and 75 years) using regression analysis. A Monte Carlo method was then implemented to generate populations of FE models of the femur for target occupants, using a parametric femur modeling tool. Simulations were conducted with each of these models under 3-point dynamic bending. Finally, model-based IRFs were developed using logistic regression analysis, based on the moment at fracture observed in the FE simulation. In total, 100 femur FE models incorporating the variation in the population of interest were generated, and 500,000 moments at fracture were observed (applying 5,000 ultimate strains for each synthesized 100 femur FE models) for each target occupant characteristics. RESULTS: Using the proposed framework on this study, the model-based IRFs for 3 target male occupant sizes (5th, 50th, and 95th percentiles) and ages (25, 50, and 75 years) were developed. The model-based IRF was located in the 95% confidence interval of the test-based IRF for the range of 15 to 70% injury risks. The 95% confidence interval of the developed IRF was almost in line with the mean curve due to a large number of data points. CONCLUSIONS: The framework proposed in this study would be beneficial for developing the IRFs in a bottom-up manner, whose range of variabilities is informed by the population-based FE model responses. Specifically, this method mitigates the uncertainties in applying empirical scaling and may improve IRF fidelity when a limited number of experimental specimens are available.

Prediction of the structural response of the femoral shaft under dynamic loading using subject-specific finite element models.

The goal of this study was to predict the structural response of the femoral shaft under dynamic loading conditions using subject-specific finite element (SS-FE) models and to evaluate the prediction accuracy of the models in relation to the model complexity. In total, SS-FE models of 31 femur specimens were developed. Using those models, dynamic three-point bending and combined loading tests (bending with four different levels of axial compression) of bare femurs were simulated, and the prediction capabilities of five different levels of model complexity were evaluated based on the impact force time histories: baseline, mass-based scaled, structure-based scaled, geometric SS-FE, and heterogenized SS-FE models. Among the five levels of model complexity, the geometric SS-FE and the heterogenized SS-FE models showed statistically significant improvement on response prediction capability compared to the other model formulations whereas the difference between two SS-FE models was negligible. This result indicated the geometric SS-FE models, containing detailed geometric information from CT images with homogeneous linear isotropic elastic material properties, would be an optimal model complexity for prediction of structural response of the femoral shafts under the dynamic loading conditions. The average and the standard deviation of the RMS errors of the geometric SS-FE models for all the 31 cases was 0.46 kN and 0.66 kN, respectively. This study highlights the contribution of geometric variability on the structural response variation of the femoral shafts subjected to dynamic loading condition and the potential of geometric SS-FE models to capture the structural response variation of the femoral shafts.

Validation of Shoulder Response of Human Body Finite-Element Model (GHBMC) Under Whole Body Lateral Impact Condition.

In previous shoulder impact studies, the 50th-percentile male GHBMC human body finite-element model was shown to have good biofidelity regarding impact force, but under-predicted shoulder deflection by 80% compared to those observed in the experiment. The goal of this study was to validate the response of the GHBMC M50 model by focusing on three-dimensional shoulder kinematics under a whole-body lateral impact condition. Five modifications, focused on material properties and modeling techniques, were introduced into the model and a supplementary sensitivity analysis was done to determine the influence of each modification to the biomechanical response of the body. The modified model predicted substantially improved shoulder response and peak shoulder deflection within 10% of the observed experimental data, and showed good correlation in the scapula kinematics on sagittal and transverse planes. The improvement in the biofidelity of the shoulder region was mainly due to the modifications of material properties of muscle, the acromioclavicular joint, and the attachment region between the pectoralis major and ribs. Predictions of rib fracture and chest deflection were also improved because of these modifications.

Biofidelity evaluation of WorldSID and ES-2re under side impact conditions with and without airbag.

This study evaluated the biofidelity of the WorldSID and the ES-2re under whole-body side impact conditions with and without a side airbag using the biomechanical cadaveric response data generated from 4.3m/s whole-body side impact tests. Impact forces, spinal kinematics, and chest deflections were considered in the biofidelity evaluation. Average responses and response corridors of PMHS were created using a time-alignment technique to reduce variability of the PMHS responses while maintaining the sum of the time shifts to be zero for each response. Biofidelity of the two dummies was compared using a correlation and analysis (CORA) method. The WorldSID demonstrated better biofidelity than the ES-2re in terms of CORA ratings in the conditions with airbag (0.53 vs. 0.46) and without an airbag (0.57 vs. 0.49). Lastly, the kinematic analysis of the two dummies indicated an overly compliant shoulder response of the WorldSID and excessive forward rotation of the ES-2re relative to the PMHS.

Nature and etiology of hollow-organ abdominal injuries in frontal crashes.

UNLABELLED: Injuries to the hollow organs of the lower digestive system carry substantial risk of complication due to infection and blood loss, and commonly require invasive abdominal surgery to diagnose and treat. The causes of, and risk factors for, lower abdomen injury in automobile collisions are poorly understood. The goal of this study was to investigate the risk factors and potential mechanisms of hollow-organ, lower abdomen injury in belted automobile occupants in frontal collisions. A field survey data analysis was performed to examine the relationship between various occupant and collision factors and the risk of moderate or greater severity injury (i.e., Abbreviated Injury Scale, AIS 2+) to the small intestine, large intestine, or mesentery among belted occupants involved in frontal collisions. Descriptive and comparative risk factor analyses were performed with data originating from that National Automotive Sampling System Crashworthiness Data System (NASS-CDS) over the years 2000-2011. Multivariable logistic regression models were developed to describe the effects of these factors on hollow-organ injury risk. Potential injury mechanisms were further investigated through in-depth examination of select cases exhibiting hollow-organ injuries from the Crash Investigation Research and Engineering Network (CIREN). The inclusion criteria yielded 25,407 individual cases from NASS-CDS, representing a weighted population of 11,373,358 exposed automobile occupants. Within this dataset, 143 cases (weighted frequency: 7962 occupants) exhibited AIS 2+ injury to hollow abdominal organs. Multivariable regression analysis indicated a statistically significant increased risk of moderate or greater severity injuries to the hollow organs of the abdomen with increased in ΔV (odds ratio (OR) 1.07, 95% confidence interval: 1.06-1.09) and age (OR: 1.03, 1.01-1.06). Albeit non-statistically significant, a positive association between BMI and injury risk was observed, especially among obese individuals (OR: 3.55, 0.82-15.2). No association was observed for gender or seated location within the vehicle. RESULT: from this study indicate that hollow abdominal organ injury is a universal problem in frontal collisions, not confined to a specific gender or seating location. Examination of CIREN cases suggests these types of injuries are associated with direct loading of the lower abdomen by the lap belt, either through poor initial belt positioning or through a "submarining" type of kinematic where the lap belt slips off of the pelvis and loads into the abdomen. Potential countermeasures against hollow-organ abdominal injury should include measures to improve initial lap belt fit, and to retain engagement of the lap belt on the pelvis throughout the collision event.

The Contribution of Pre-impact Spine Posture on Human Body Model Response in Whole-body Side Impact.

The objective of the study was to analyze independently the contribution of pre-impact spine posture on impact response by subjecting a finite element human body model (HBM) to whole-body, lateral impacts. Seven postured models were created from the original HBM: one matching the standard driving posture and six matching pre-impact posture measured for each of six subjects tested in previously published experiments. The same measurements as those obtained during the experiments were calculated from the simulations, and biofidelity metrics based on signals correlation were established to compare the response of HBM to that of the cadavers. HBM responses showed good correlation with the subject response for the reaction forces, the rib strain (correlation score=0.8) and the overall kinematics. The pre-impact posture was found to greatly alter the reaction forces, deflections and the strain time histories mainly in terms of time delay. By modifying only the posture of HBM, the variability in the impact response was found to be equivalent to that observed in the experiments performed with cadavers with different anthropometries. The patterns observed in the responses of the postured HBM indicate that the inclination of the spine in the frontal plane plays a major role. The postured HBM sustained from 2 to 5 bone fractures, including the scapula in some cases, confirming that the pre-impact posture influences the injury outcome predicted by the simulation.