Comparison of Immersed Boundary Simulations of Heart Valve Hemodynamics Against In Vitro 4D Flow MRI Data.

The immersed boundary (IB) method is a mathematical framework for fluid-structure interaction problems (FSI) that was originally developed to simulate flows around heart valves. Direct comparison of FSI simulations around heart valves against experimental data is challenging, however, due to the difficulty of performing robust and effective simulations, the complications of modeling a specific physical experiment, and the need to acquire experimental data that is directly comparable to simulation data. Such comparators are a necessary precursor for further formal validation studies of FSI simulations involving heart valves. In this work, we performed physical experiments of flow through a pulmonary valve in an in vitro pulse duplicator, and measured the corresponding velocity field using 4D flow MRI (4-dimensional flow magnetic resonance imaging). We constructed a computer model of this pulmonary artery setup, including modeling valve geometry and material properties via a technique called design-based elasticity, and simulated flow through it with the IB method. The simulated flow fields showed excellent qualitative agreement with experiments, excellent agreement on integral metrics, and reasonable relative error in the entire flow domain and on slices of interest. These results illustrate how to construct a computational model of a physical experiment for use as a comparator.

Controlled Comparison of Simulated Hemodynamics Across Tricuspid and Bicuspid Aortic Valves.

Bicuspid aortic valve is the most common congenital heart defect, affecting 1-2% of the global population. Patients with bicuspid valves frequently develop dilation and aneurysms of the ascending aorta. Both hemodynamic and genetic factors are believed to contribute to dilation, yet the precise mechanism underlying this progression remains under debate. Controlled comparisons of hemodynamics in patients with different forms of bicuspid valve disease are challenging because of confounding factors, and simulations offer the opportunity for direct and systematic comparisons. Using fluid-structure interaction simulations, we simulate flows through multiple aortic valve models in a patient-specific geometry. The aortic geometry is based on a healthy patient with no known aortic or valvular disease, which allows us to isolate the hemodynamic consequences of changes to the valve alone. Four fully-passive, elastic model valves are studied: a tricuspid valve and bicuspid valves with fusion of the left- and right-, right- and non-, and non- and left-coronary cusps. The resulting tricuspid flow is relatively uniform, with little secondary or reverse flow, and little to no pressure gradient across the valve. The bicuspid cases show localized jets of forward flow, excess streamwise momentum, elevated secondary and reverse flow, and clinically significant levels of stenosis. Localized high flow rates correspond to locations of dilation observed in patients, with the location related to which valve cusps are fused. Thus, the simulations support the hypothesis that chronic exposure to high local flow contributes to localized dilation and aneurysm formation.

A Mechanistic Lumped Parameter Model of the Berlin Heart EXCOR to Analyze Device Performance and Physiologic Interactions.

PURPOSE: The Berlin Heart EXCOR (BH) is the only FDA-approved, extracorporeal pulsatile ventricular assist device (VAD) for infants and children with heart failure. Clinicians control four settings on the device-systolic and diastolic drive pressures, device pump rate, and systolic time as a percentage of the pump cycle. However, interactions between BH pneumatics and the native circulation remain poorly understood. Thus, establishing appropriate device size and settings can be challenging on a patient-to-patient basis. METHODS: In this study we develop a novel lumped parameter network based on simplified device mechanics. We perform parametric studies to characterize device behavior, study interactions between the left ventricle (LV) and BH across different device settings, and develop patient-specific simulations. We then simulate the impact of changing device parameters for each of three patients. RESULTS: Increasing systolic pressure and systolic time increased device output. We identified previously unobserved cycle-to-cycle variations in LV-BH interactions that may impact patient health. Patient-specific simulations demonstrated the model's ability to replicate BH performance, captured trends in LV behavior after device implantation, and emphasized the importance of device rate and volume in optimizing BH efficiency. CONCLUSION: We present a novel, mechanistic model that can be readily adjusted to study a wide range of device settings and clinical scenarios. Physiologic interactions between the BH and the native LV produced large variability in cardiac loading. Our findings showed that operating the BH at a device rate greater than the patient's native heart decreases variability in physiological interactions between the BH and LV, increasing cardiac offloading while maintaining cardiac output. Device rates that are close to the resting heart rate may result in unfavorable cardiac loading conditions. Our work demonstrates the utility of our model to investigate BH performance for patient-specific physiologies.

In Vitro Assessment of Right Ventricular Outflow Tract Anatomy and Valve Orientation Effects on Bioprosthetic Pulmonary Valve Hemodynamics.

PURPOSE: The congenital heart defect Tetralogy of Fallot (ToF) affects 1 in 2500 newborns annually in the US and typically requires surgical repair of the right ventricular outflow tract (RVOT) early in life, with variations in surgical technique leading to large disparities in RVOT anatomy among patients. Subsequently, often in adolescence or early adulthood, patients usually require surgical placement of a xenograft or allograft pulmonary valve prosthesis. Valve longevity is highly variable for reasons that remain poorly understood. METHODS: This work aims to assess the performance of bioprosthetic pulmonary valves in vitro using two 3D printed geometries: an idealized case based on healthy subjects aged 11 to 13 years and a diseased case with a 150% dilation in vessel diameter downstream of the valve. Each geometry was studied with two valve orientations: one with a valve leaflet opening posterior, which is the native pulmonary valve position, and one with a valve leaflet opening anterior. RESULTS: Full three-dimensional, three-component, phase-averaged velocity fields were obtained in the physiological models using 4D flow MRI. Flow features, particularly vortex formation and reversed flow regions, differed significantly between the RVOT geometries and valve orientations. Pronounced asymmetry in streamwise velocity was present in all cases, while the diseased geometry produced additional asymmetry in radial flows. Quantitative integral metrics demonstrated increased secondary flow strength and recirculation in the rotated orientation for the diseased geometry. CONCLUSIONS: The compound effects of geometry and orientation on bioprosthetic valve hemodynamics illustrated in this study could have a crucial impact on long-term valve performance.

Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography.

Supercritical fluid chromatography (SFC) has become the fastest growing analytical tool for chiral and achiral small-molecule pharmaceutical separations. The benefits from savings in cost (as a result of lower solvent and energy consumption), and time have made SFC a proven effective tool for solving many analytical problems for small-molecules over the years. There is, however, a gap in the application of SFC for larger biomolecules, proteins and peptides. There has been a notable increase of protein- and peptide-based drug therapies that contain a higher-order structure important to their efficacy. These studies leverage the use of size exclusion chromatography coupled with hydrogen-deuterium exchange (SEC-HDX) methodology and circular dichroism (CD) spectroscopy to probe global conformational structures of model peptides and proteins following purification by preparative SFC. It was demonstrated that bradykinin and insulin can be used in SFC purification, and moreover, insulin was able to recover its original higher-order structure when compared to pre-purification insulin by three orthogonal techniques: 1) calculated percent alpha-helicity based on CD spectra, 2) alpha-helix - temperature hysteresis analysis by CD and 3) SEC-HDX-MS at different temperatures. However, it was shown that the higher order structures of the other three model proteins used in the study (ubiquitin, cytochrome C, and apomyoglobin) were significantly modified during SFC purification and were unable to re-fold to their original conformations. The present workflow was applied successfully to several peptide therapeutic programs at our comp any and in addition can be applied for small proteins.

Combination of circular dichroism spectroscopy and size-exclusion chromatography coupled with HDX-MS for studying global conformational structures of peptides in solution.

Misfolding of therapeutic peptides and proteins can lead to numerous issues, ranging in severity, including loss of function, aggregation, immunogenicity, and cytotoxicity. A primary component of protein folding is secondary structure, including α-helices and ß-sheets. Many native peptides and proteins are predominately α-helical therefore, it is of critical importance to develop robust and reliable analytical tools to investigate protein higher order structure, including the percentage of α-helix under various conditions, to evaluate protein folding and prevent the negative effects of misfolding. However, given the complexity of protein folding and higher order structure, it is unlikely that one technique will provide a comprehensive analysis. To bridge this gap, this study presents the combination of two orthogonal techniques - circular dichroism (CD) and size-exclusion chromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS) to investigate global peptide and protein conformations. Also, the incorporation of trifluoroethanol (TFE), a known stabilizer of α-helical structures, into the analyses, aims to enhance the discrimination power of these two techniques by increasing the alpha helical stability range of study. CD data was used to estimate the percent of α-helix content and its thermal stability while online SEC-HDX-MS screening compared global conformational changes of each peptide based on a difference in the number of deuterons exchanged to protons, ΔHDX. The workflow described in this report can be very beneficial in pharmaceutical development. The model peptides were chosen to demonstrate the workflow with commercially available compounds. The goal of this study was to show a proof-of-concept for direct correlation of these methodologies and to estimate the percentage of α-helix content at a particular ΔHDX, which is indicative of the state of protein folding.

Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances.

Nucleotides are among the most frequently used chemical building blocks in the research, development and manufacture of drug substances. They are composed of three highly polar subunit molecules (a nucleobase, a sugar, and at least one phosphate group), which makes their separation and analysis very challenging by conventional liquid chromatography techniques. Herein, we describe a simple, efficient, and cost-effective ion-exchange chromatography (IEC) method for the separation and purification of over 20 nucleotides. This method combines the use of a Tosoh TSKgel SuperQ-5P W resin in conjunction with a fully aqueous eluent profile (ammonium bicarbonate-based) that allows for a straightforward scale-up transition and convenient drying process with minimal environmental impact. This generic method was optimized using chromatography simulation software (ACD Labs/LC Simulator) and successfully applied to the preparative purification of multicomponent nucleotide mixtures using readily available Fast Protein Liquid Chromatography (FPLC) instrumentation. These IEC method conditions can be effectively applied as the starting point for method development and isolation of other highly polar nucleotide species beyond those investigated in this study.

Caregiver Strategies to Enhance Participation in Children With Autism Spectrum Disorder.

Participation is necessary for childhood development, however, children with disabilities participate in fewer activities than their nondisabled peers. This study identified strategies caregivers use to increase participation in home- and community-based activities for children with autism spectrum disorder. Survey responses of 44 caregivers were analyzed through open and axial coding to develop a central theme and five broad themes. Caregiver strategies which produce a participation outcome fell into five themes: (a) adapters/facilitators, (b) pragmatic considerations, (c) social reframing, (d) sensory adjustments, and (e) desperate measures. Nonproductive strategies were identified where the child did not participate in the activity. Adaptations/facilitators were more likely used in the home, whereas community-based strategies were more often pragmatic considerations. Nonproductive strategies occurred more frequently in the community. When evaluating a child's participation, occupational therapy (OT) practitioners should take into consideration the context of the activity to identify appropriate and helpful strategies.

Capillary electrophoresis coupled to negative mode electrospray ionization-mass spectrometry using an electrokinetically-pumped nanospray interface with primary amines grafted to the interior of a glass emitter.

We demonstrate an electrokinetically pumped sheath flow nanospray interface for capillary electrophoresis coupled to negative mode electrospray mass spectrometry. In this interface, application of an electric field generates electro-osmotic flow at the interior of a glass emitter that is pulled to a 10-20µm inner diameter orifice. Electro-osmotic flow pumps liquid around the distal tip of the separation capillary, ensheathing analyte into the electrospray electrolyte. In negative ion mode, negative potential applied to an untreated glass emitter drives sheath flow away from the emitter orifice, decreasing the stability and efficiency of the spray. In this manuscript, we treat a portion of the interior of the electrospray emitter with 3-aminopropyltrimethoxysilane, which grafts primary amines to the interior. The amines take on a positive charge, which reverses electro-osmosis and generates stable sheath flow to the emitter orifice under negative potential. Negative mode operation is demonstrated by analyzing a metabolite extract from stage 1 Xenopus laevis embryos. Production of the treated emitters was quite reproducible. We evaluated the performance of three emitters using a set of amino acids; the relative standard deviation in peak intensity was 7% for the most intense component.

High speed capillary zone electrophoresis-mass spectrometry via an electrokinetically pumped sheath flow interface for rapid analysis of amino acids and a protein digest.

While capillary zone electrophoresis (CZE) has been used to produce very rapid and efficient separations, coupling these high-speed separations with mass spectrometry (MS) has been challenging. Now, with much faster and sensitive mass spectrometers, it is possible to take full advantage of the CZE speed and reconstruct the fast migrating peaks. Here are three high-speed CZE-MS analyses via an electrokinetically pumped sheath-flow interface. The first separation demonstrates CZE-ESI-MS of an amino acid mixture with a 2-min separation, >50,000 theoretical plates, low micromolar concentration detection limits, and subfemtomole mass detection limits (LTQ XL mass spectrometer). The second separation with our recently improved third-generation CE-MS interface illustrates a 20 amino acid separation in ∼7min with an average over 200,000 plate counts, and results in almost-baseline resolution of structural isomers, leucine and isoleucine. The third separation is of a BSA digest with a reproducible CZE separation and mass spectrometry detection in 2min. CZE-MS/MS analysis of the BSA digest identified 31 peptides, produced 52% sequence coverage, and generated a peak capacity of ∼40 across the 1-min separation window (Q-Exactive mass spectrometer).

Online SERS detection and characterization of eight biologically-active peptides separated by capillary zone electrophoresis.

There is a need for low cost, sensitive and chemical specific detectors for routine characterization of biomolecules. In this study, we utilize sheath-flow surface-enhanced Raman scattering (SERS) to analyze a mixture of eight biologically-active peptides separated by capillary zone electrophoresis (CZE). Analysis of the SERS electropherogram resulting from online detection resolves the characteristic Raman bands attributed to the amino acid constituents of each peptide, which enables identification. The detection limit by SERS was found to be 10(-8) M. Our results suggest that the structural information obtained from the detected vibrational modes provides complementary characterization to other chemically specific detectors like mass spectrometry and improved chemical identification over other commonly used optical-based post-chromatographic detection methods. In addition, the sheath-flow SERS detection results in band narrowing in the observed electropherogram that enables distinction of closely migrating species. The results presented here indicate that this platform can provide fast, robust, reproducible, and chemical specific detection to facilitate the characterization of peptides.