The 3D muscle morphology and intramuscular innervation of the digital bellies of flexor digitorum profundus: Clinical implications for botulinum toxin injection sites.

Spasticity of flexor digitorum profundus is frequently managed with botulinum toxin injections. Knowledge of the 3D morphology and intramuscular innervation of the digital bellies of flexor digitorum profundus is necessary to optimize the injections. The purpose of this study was to digitize and model in 3D the contractile and connective tissue elements of flexor digitorum profundus to determine muscle morphology, model and map the intramuscular innervation and propose sites for botulinum toxin injection. Fiber bundles (FBs)/aponeuroses and intramuscular nerve branches were dissected and digitized in 12 formalin embalmed cadaveric specimens. Cartesian coordinate data were reconstructed into 3D models as in situ to visualize and compare the muscle morphology and intramuscular innervation patterns of the bellies of flexor digitorum profundus. The 3rd, 4th and 5th digital bellies were superficial to the 2nd digital belly and located adjacent to each other in all specimens. Each digital belly had distinct intramuscular innervation patterns. The 2nd digital belly received intramuscular branches from the anterior interosseus nerve (AIN). The superior half of the 3rd digital belly was innervated intramuscularly by the ulnar nerve (n = 4) or by both the anterior interosseus and ulnar nerves (n = 1). The inferior half of the belly received dual innervation from the anterior interosseus and ulnar nerves in 2 specimens, or exclusively from the AIN (n = 2) or the ulnar nerve (n = 1). The 4th digital belly was innervated by intramuscular branches of the ulnar nerve. One main branch, after coursing through the 4th digital belly, entered the lateral aspect of the 5th digital belly and arborized intramuscularly. The morphology of the FBs, aponeuroses and intramuscular innervation of the digital bellies of FDP were mapped and modelled volumetrically in 3D as in situ. Previous studies were not volumetric nor identified the course of the intramuscular nerve branches within each digital belly. Based on the intramuscular innervation of each of the digital bellies, one possible optimized botulinum toxin injection location was proposed. This injection location, at the junction of the superior and middle thirds of the forearm, would be located in dense nerve terminal zones of the anterior interosseus and ulnar nerves. Future anatomical and clinical investigations are necessary to evaluate the efficacy of these anatomical findings in the management of spasticity.

Quantification of needle angles for lumbar medial branch denervation targeting the posterior half of the superior articular process: an osteological study.

BACKGROUND: Lumbar medial branch radiofrequency ablation (RFA) is a common intervention to manage chronic axial low back pain originating from the facet joints. A more parasagittal approach targeting the posterior half of the lateral neck of superior articular process (SAP) was previously proposed. However, specific needle angles to achieve parallel placement at this target site have not been investigated. OBJECTIVE: To quantify and compare the needle angles, on posterior and lateral views, to achieve parallel placement of electrodes along the medial branch at the posterior half of the lateral neck of SAP at each lumbar vertebral level (L1-L5) and sacrum. DESIGN: Osteological Study. METHODS: Twelve disarticulated lumbosacral spines (n = 72 individual bones) were used in this study. Needles were placed along the periosteum of the posterior half of the lateral neck of SAP, bilaterally and photographed. Mean needle angles for each vertebral level (L1-L5) and sacrum were quantified, and statistical differences were analyzed. RESULTS: The posterior view provided the degrees of lateral displacement from the parasagittal plane (abduction angle), while the lateral view provided the degrees of declination (cranial-to-caudal angle) of the needle. Mean needle angles at each level varied, ranging from 5.63 ± 5.76° to 14.50 ± 14.24° (abduction angle, posterior view) and 40.17 ± 7.32° to 64.10 ± 9.73° (cranial-to-caudal angle, lateral view). In posterior view, a < 10-degree needle angle interval was most frequently identified (57.0% of needle placements). In lateral view, the 40-50-degree (L1-L2), 50-60-degree (L3-L5), and 60-70-degree (sacrum) needle angle intervals occurred most frequently (54.2%, 50.0%, and 41.7% of needle placements, respectively). CONCLUSIONS: Targeting the posterior half of the lateral neck of SAP required <10-degree angulation from parasagittal plane in majority of cases. However, variability of needle angles suggests a standard "one-size-fits-all" approach may not be the optimal technique.

Translatability of In Vitro Stress for Predicting Deamidation and Oxidation Biotransformation on Biotherapeutics.

Biotransformation leading to single residue modifications (e.g., deamidation, oxidation) can contribute to decreased efficacy/potency, poor pharmacokinetics, and/or toxicity/immunogenicity for protein therapeutics. Identifying and characterizing such liabilities in vivo are emerging needs for biologics drug discovery. In vitro stress assays involving PBS for deamidation or AAPH for oxidation are commonly used for predicting liabilities in manufacturing and storage and are sometimes considered a predictive tool for in vivo liabilities. However, reports discussing their in vivo translatability are limited. Herein, we introduce a mass spectrometry workflow that characterizes in vivo oxidation and deamidation in pharmacokinetically relevant compartments for diverse protein therapeutic modalities. The workflow has low bias of <10% in quantitating degradation in the relevant pharmacokinetic concentration range for monkey and rabbit serum/plasma (1-100 µg/mL) and allows for high sequence coverage (∼85%) for discovery/monitoring of amino acid modifications. For oxidation and deamidation, the assay was precise, with percent coefficient of variation of <8% at 1-100 µg/mL and ≤6% method-induced artifacts. A high degree of in vitro and in vivo correlation was observed for deamidation on the six diverse protein therapeutics (seven liability sites) tested. In vivo translatability for oxidation liabilities were not observed for the 11 molecules tested using in vitro AAPH stress. One of the molecules dosed in eyes resulted in a false positive and a false negative prediction for in vivo oxidation following AAPH stress. Finally, peroxide stress was also tested but resulted in limited success (1 out of 4 molecules) in predicting oxidation liabilities.

High-Throughput Analyses of Therapeutic Antibodies Using High-Field Asymmetric Waveform Ion Mobility Spectrometry Combined with SampleStream and Intact Protein Mass Spectrometry.

Intact protein mass spectrometry (MS) coupled with liquid chromatography was applied to characterize the pharmacokinetics and stability profiles of therapeutic proteins. However, limitations from chromatography, including throughput and carryover, result in challenges with handling large sample numbers. Here, we combined intact protein MS with multiple front-end separations, including affinity capture, SampleStream, and high-field asymmetric waveform ion mobility spectrometry (FAIMS), to perform high-throughput and specific mass measurements of a multivalent antibody with one antigen-binding fragment (Fab) fused to an immunoglobulin G1 (IgG1) antibody. Generic affinity capture ensures the retention of both intact species 1Fab-IgG1 and the tentative degradation product IgG1. Subsequently, the analytes were directly loaded into SampleStream, where each injection occurs within ∼30 s. By separating ions prior to MS detection, FAIMS further offered improvement in signal-overnoise by ∼30% for denatured protein MS via employing compensation voltages that were optimized for different antibody species. When enhanced FAIMS transmission of 1Fab-IgG1 was employed, a qualified assay was established for spiked-in serum samples between 0.1 and 25 µg/mL, resulting in ∼10% accuracy bias and precision coefficient of variation. Selective FAIMS transmission of IgG1 as the degradation surrogate product enabled more sensitive detection of clipped species for intact 1Fab-IgG1 at 5 µg/mL in serum, generating an assay to measure 1Fab-IgG1 truncation between 2.5 and 50% with accuracy and precision below 20% bias and coefficient of variation. Our results revealed that the SampleStream-FAIMS-MS platform affords high throughput, selectivity, and sensitivity for characterizing therapeutic antibodies from complex biomatrices qualitatively and quantitatively.

Quantifying KRAS G12C Covalent Drug Inhibitor Activity in Mouse Tumors Using Mass Spectrometry.

The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.

Intramuscular aponeuroses and fiber bundle morphology of the five bellies of flexor digitorum superficialis: A three-dimensional modeling study.

Restoring balanced function of the five bellies of flexor digitorum superficialis (FDS) following injury requires knowledge of the muscle architecture and the arrangement of the contractile and connective tissue elements. No three-dimensional (3D) studies of FDS architecture were found in the literature. The purpose was to (1) digitize/model in 3D the contractile/connective tissue elements of FDS, (2) quantify/compare architectural parameters of the bellies and (3) assess functional implications. The fiber bundles (FBs)/aponeuroses of the bellies of FDS were dissected and digitized (MicroScribe® Digitizer) in 10 embalmed specimens. Data were used to construct 3D models of FDS to determine/compare the morphology of each digital belly and quantify architectural parameters to assess functional implications. FDS consists of five morphologically and architecturally distinct bellies, a proximal belly, and four digital bellies. FBs of each belly have unique attachment sites to one or more of the three aponeuroses (proximal/distal/median). The proximal belly is connected through the median aponeurosis to the bellies of the second and fifth digits. The third belly exhibited the longest mean FB length (72.84 ± 16.26 mm) and the proximal belly the shortest (30.49 ± 6.45 mm). The third belly also had the greatest mean physiological cross-sectional area, followed by proximal/second/fourth/fifth. Each belly was found to have distinct excursion and force-generating capabilities based on their 3D morphology and architectural parameters. Results of this study provide the basis for the development of in vivo ultrasound protocols to study activation patterns of FDS during functional activities in normal and pathologic states.

Quantification of Needle Angles for Traditional Lumbar Medial Branch Radiofrequency Ablation: An Osteological Study.

BACKGROUND: Clinical outcomes following lumbar medial branch radiofrequency ablation (RFA) have been inconsistent. One possible reason is less-than-optimal placement of the electrode along the medial branch at the lateral neck of superior articular process (SAP). Needle angles that define optimal placement (i.e., parallel to the medial branch) may be helpful for consistent technical performance of RFA. Despite its importance, there is a lack of anatomical studies that quantify RFA needle placement angles. OBJECTIVE: To quantify and compare needle angles to achieve parallel placement along the medial branch as it courses on the middle two-quarters of the lateral neck of the SAP at the L1-L5 vertebrae. DESIGN: Osteological Study. METHODS: Ten lumbar vertebral columns were used in this study. Needles were placed along the periosteum of the middle two-quarters of the lateral neck of SAP. Mean needle angles for L1-L5 were quantified and compared using posterior (n = 100) and lateral (n = 100) photographs. RESULTS: Mean needle angles varied ranging from 29.29 ± 17.82° to 47.22 ± 16.27° lateral to the parasagittal plane (posterior view) and 33.53 ± 10.23° to 49.19 ± 10.69° caudal to the superior vertebral endplate (lateral view). Significant differences in mean angles were found between: L1/L3 (P = .008), L1/L4 (P = .003), and L1/L5 (P = .040) in the posterior view and L1/L3 (P = .042), L1/L4 (P < .001), L1/L5 (P < .001), L2/L4 (P = .004), and L2/L5 (P = .004) in lateral view. CONCLUSIONS: Variability of needle angles suggest a standard "one-size-fits-all" approach may not be the optimal technique. Future research is necessary to determine optimal patient-specific needle angles from a more detailed and granular analysis of fluoroscopic landmarks.

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry.

Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFire-time-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach.

KRAS is one of the most frequently mutated oncogenes, with KRAS G12C recently becoming an actionable target for small molecule intervention. GDC-6036 is an investigational KRAS G12C inhibitor that acts by irreversibly binding to the switch II pocket of KRAS G12C when in the inactive GDP-bound state, thereby blocking GTP binding and activation. Assessing target engagement is an essential component of clinical drug development, helping to demonstrate mechanistic activity, guide dose selection, understand pharmacodynamics as it relates to clinical response, and explore resistance. Here, we report the development of an ultra-sensitive approach for assessing KRAS G12C engagement. Immunoaffinity enrichment with a commercially available anti-RAS antibody was combined with a targeted 2D-LC-MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. A KRAS G12C-positive non-small cell lung cancer xenograft model was dosed with GDC-6036 to assess the feasibility of this assay for analyzing small core needle biopsies. As predicted, dose-dependent KRAS G12C engagement was observed. To date, a sensitivity of 0.08 fmol/µg of total protein has been achieved for both free and GDC-6036-bound KRAS G12C with as little as 4 µg of total protein extracted from human tumor samples. This sub-fmol/µg level of sensitivity provides a powerful potential approach to assess covalent inhibitor target engagement at the site of action using core needle tumor biopsies from clinical studies.

A Cross Company Perspective on the Assessment of Therapeutic Protein Biotransformation.

Unlike with new chemical entities, the biotransformation of therapeutic proteins (TPs) has not been routinely investigated or included in regulatory filings. Nevertheless, there is an expanding pool of evidence suggesting that a more in-depth understanding of biotransformation could better aid the discovery and development of increasingly diverse modalities. For instance, such biotransformation analysis of TPs affords important information on molecular stability, which in turn may shed light on any potential impact on binding affinity, potency, pharmacokinetics, efficacy, safety, or bioanalysis. This perspective summarizes the current practices in studying biotransformation of TPs and related findings in the biopharmaceutical industry. Various TP case studies are discussed, and a fit-for-purpose approach is recommended when investigating their biotransformation. In addition, we provide a decision tree to guide the biotransformation characterization for selected modalities. By raising the awareness of this important topic, which remains relatively underexplored in the development of TPs (Bolleddula et al., 2022), we hope that current and developing practices can pave the way for establishing a consensus on the biotransformation assessment of TPs. SIGNIFICANCE STATEMENT: This article provides a comprehensive perspective of the current practices for exploring the biotransformation of therapeutic proteins across the drug development industry. We, the participants of the Innovation and Quality therapeutic protein absorption distribution metabolism excretion working group, recommend and summarize appropriate approaches for conducting biotransformation studies to support internal decision making based on the data generated in discovery and development.

Using a peptide-based mass spectrometry approach to quantitate proteolysis of an intact heterogeneous procollagen substrate by BMP1 for antagonistic antibody screening.

Proteases are critical proteins involved in cleaving substrates that may impact biological pathways, cellular processes, or disease progression. In the biopharmaceutical industry, modulating the levels of protease activity is an important strategy for mitigating many types of diseases. While a variety of analytical tools exist for characterizing substrate cleavages, in vitro functional screening for antibody inhibitors of protease activity using physiologically relevant intact protein substrates remains challenging. In addition, detecting such large protein substrates with high heterogeneity using high-throughput mass spectrometry screening has rarely been reported in the literature with concerns for assay robustness and sensitivity. In this study, we established a peptide-based in vitro functional screening assay for antibody inhibitors of mouse bone morphogenic protein 1 (mBMP1) metalloprotease using a heterogeneous recombinant 66-kDa mouse Procollagen I alpha 1 chain (mProcollagen) substrate. We compared several analytical tools including capillary gel electrophoresis Western blot (CE-Western blot), as well as both intact protein and peptide-based mass spectrometry (MS) to quantitate the mBMP1 proteolytic activity and its inhibition by antibodies using this heterogeneous mProcollagen substrate. We concluded that the peptide-based mass spectrometry screening assay was the most suitable approach in terms of throughput, sensitivity, and assay robustness. We then optimized our mBMP1 proteolysis reaction after characterizing the enzyme kinetics using the peptide-based MS assay. This assay resulted in Z' values ranging from 0.6 to 0.8 from the screening campaign. Among over 1200 antibodies screened, IC50 characterization was performed on the top candidate hits, which showed partial or complete inhibitory activities against mBMP1.

Ultrasound-Guided Gluteal Fascial Plane Block for the Treatment of Chronic Refractory Greater Trochanteric Pain Syndrome - Technique Description and Anatomical Correlation Study.

INTRODUCTION: Greater trochanteric pain syndrome may often mimic pain generated from other sources. However, it is most commonly caused by gluteus medius and gluteus minimus tendinopathy or tear. The purpose of this technical report was to: 1) describe the ultrasound-guided fascial plane block technique targeting the superior gluteal nerve in the plane between gluteus medius/gluteus minimus to treat moderate-to-severe, chronic, refractory greater trochanteric pain syndrome; 2) anatomically correlate the procedure with cadaveric dissections demonstrating the structures being imaged and the tissues along the needle trajectory; 3) demonstrate the feasibility of the technique with serial dissection of one cadaveric specimen following injection with color dye. TECHNIQUE DESCRIPTION: The ultrasound-guided fascial plane block targeting the superior gluteal nerve to treat moderate-to-severe, chronic, refractory greater trochanteric pain syndrome has been outlined with supporting ultrasound scans and anatomical dissections. The cadaveric dissections are correlated to the ultrasound scans of a healthy volunteer and provide visualization of the tissues in the needle trajectory. The feasibility study in a cadaveric specimen showed adequate stain of the superior gluteal nerve without spread to the piriformis muscle belly, the sciatic nerve, or the inferior gluteal nerve. CONCLUSIONS: This ultrasound-guided fascial plane block is a feasible option for blocking the superior gluteal nerve without inadvertent involvement of the sciatic and inferior gluteal nerves. Further randomized controlled clinical trials are necessary to assess the clinical efficacy of the gluteus medius/gluteus minimus fascial plane block to treat moderate-to-severe, chronic, refractory greater trochanteric pain syndrome.

Anatomical Study of the Innervation of Triangular Fibrocartilage Complex and Distal Radioulnar and Radiocarpal Joints: Implications for Denervation.

PURPOSE: Open and percutaneous denervation is an emerging technique for joint pain. This study investigated the course and distribution of the articular branches innervating the triangular fibrocartilage complex (TFCC), distal radioulnar joint (DRUJ), and radiocarpal joint (RCJ) relative to bony and soft tissue landmarks to guide wrist denervation procedures. METHODS: Fourteen formalin-embalmed specimens were serially dissected to expose the origin, course, and distribution of articular branches innervating the TFCC, DRUJ, and RCJ. Bony and soft tissue landmarks to localize each articular branch were documented and visualized on a 3-dimensional reconstruction of the bones of the distal forearm and hand. RESULTS: The TFCC was innervated by articular branches from the posterior interosseus nerve (10 of 14 specimens), dorsal cutaneous branch of the ulnar nerve (14 of 14 specimens), palmar cutaneous branch of the ulnar nerve (12 of 14 specimens), and medial antebrachial cutaneous nerve (9 of 14 specimens). The DRUJ was innervated by the posterior interosseus nerve (9 of 14 specimens) and anterior interosseus nerve (14 of 14 specimens). The RCJ was innervated by the posterior interosseus nerve (14 of 14 specimens), superficial branch of the radial nerve (5 of 14 specimens), lateral antebrachial cutaneous nerve (14 of 14 specimens), and palmar cutaneous branch of the median nerve (10 of 14 specimens). CONCLUSIONS: Multiple nerves were found to innervate the TFCC, DRUJ, and RCJ. The relationship of anatomical landmarks to specific articular branches supplying the TFCC, DRUJ, and RCJ can inform selective denervation procedures based on the structural origin of pain. CLINICAL RELEVANCE: The detailed documentation of the spatial relationship of the nerve supply to the wrist provides clinicians with the anatomical basis to optimize current, and develop new denervation protocols to manage chronic wrist pain.

Parametric Multi-Scale Modeling of the Zygomaticus Major and Minor: Implications for Facial Reanimation.

ABSTRACT: Facial paralysis can severely impact functionality and mental health. Facial reanimation surgery can improve facial symmetry and movement. Zygomaticus minor (Zmin) and zygomaticus major (Zmaj) are 2 important perioral muscles, that function to elevate the upper lip, contributing to the formation of a smile. The objective of this study was to analyze the morphology in three-dimensional (3D) and quantify architectural parameters of Zmin and Zmaj. In ten formalin-embalmed specimens, Zmin and Zmaj were serially dissected and digitized at the fiber bundle level. The 2 muscles were modeled in 3D to construct high fidelity models. The 3D models were used to assess muscle morphology and quantify architectural parameters including mean fiber bundle length, physiological cross-sectional area, and line of action. Zygomaticus minor fiber bundles were oriented horizontally or slightly obliquely and had a muscular attachment to the medial modiolus. Zygomaticus minor was found to either have no partitions or medial and lateral partitions. Specimens with partitions were divided into type 1 and type 2. Type 1 consisted of a medial partition with fiber bundles attaching to the zygomatic bone at the inferior margin of the orbit. The type 2 medial partition attached to the lateral margin of the orbit to attach to the zygomatic bone. Zygomaticus major had obliquely oriented fiber bundles with most specimens having inferior and superior partitions attaching to the inferior aspect of the zygomatic bone. Zygomaticus major was found to have a greater mean fiber bundle length and physiological cross-sectional area than Zmin. The direction of the line of action of Zmin and Zmaj was closely related to fiber bundle arrangement. Detailed 3D anatomical understanding of Zmin and Zmaj, at the fiber bundle level, is critical for reconstructive surgeons performing dynamic facial reanimation. This data can be used to assist with selecting the ideal donor site for reconstruction.

Characterizing and Quantitating Therapeutic Tethered Multimeric Antibody Degradation Using Affinity Capture Mass Spectrometry.

There are many pharmacokinetic challenges associated with administering protein therapeutics, including biotransformation via clipping, deamidation, isomerization, oxidation, etc. In the case of engineered multivalent tethered antibody formats, proteolysis or deconjugation at the fusion or conjugation site present further issues. Unlike degradations associated with antibody drug conjugates, such biotransformations of tethered antibody formats usually result in degraded products with large mass differences. These large differences can result in processing or mass spectrometry response bias among the resulting product species that can lead to inaccurate stability quantitation. Herein, we describe an assay strategy for characterizing and quantitating degradations accurately for multivalent antibodies by incorporating response bias corrections. For the multivalent tethered antibody molecules selected, an ∼30-80% difference in response, compared to the cleaved product, was observed. To correct for the response bias, selected tethered multivalent antibodies and an IgG antibody (representing the stable intact and the degraded product species, respectively) were spiked in serum at known ratios for analysis. Following affinity capture, we generated calibration curves (five-parameter logistic fit p < 0.05) by plotting the measured ratios of the MS ion responses against the known spiked-in ratios (CVs < 8% for calibration standards). The qualified calibration curve (accuracy within 8% and 2% for measuring degradations of 5% and 15% product, respectively) was then used, through interpolation, to determine stability profiles for the same multivalent tethered antibody formats from both in vitro serum and pharmacokinetic study samples.

Novel Interface for High-Throughput Analysis of Biotherapeutics by Electrospray Mass Spectrometry.

With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform. Here, we describe a new analytical platform that enables direct and high-throughput coupling to electrospray ionization mass spectrometry. The SampleStream platform is compatible with both native and denaturing electrospray, operates with a throughput of up to 15 s/sample, provides extensive concentration of dilute samples, and affords similar sensitivity to comparable liquid chromatographic methods.

Parent and Provider Perspectives on Recently Incarcerated Youths' Access to Healthcare During Community Reentry.

Incarcerated youth have numerous healthcare needs, yet access to healthcare following community reentry is limited. Healthcare and juvenile justice providers, along with parents, strongly influence access to care for youth undergoing reentry. However, their perspectives are often missing from the literature. We examined parent and provider perspectives on youths' access to healthcare during community reentry. We conducted 72 longitudinal interviews with parents of youth undergoing reentry (n= 34 parents) and cross-sectional interviews with health and juvenile justice providers (n=20 providers). We performed inductive analysis of interview transcripts to identify the major themes related to access to healthcare during reentry. Respondents identified key leverage points that influence access to healthcare along the spectrum of individual, community, and policy-level factors. Parent and provider perspectives demonstrated substantial overlap, strongly concurring on the essential role of parents in linking youth to care and the external factors that limit parents' ability to connect youth to care. However, providers discussed parents not buying-in to treatment plans as a barrier to care, and parents uniquely described feeling powerless when their children were not motivated to receive care. Parents and providers agreed on priority solutions for improving care access during reentry. Immediate solutions centered on: 1) increasing reliability and continuity of providers, 2) providing free or low-cost transportation to healthcare visits, and 3) eliminating gaps in Medicaid coverage post-incarceration. Findings also signal the broader need to pursue strategies that increase family engagement in healthcare during reentry. In doing so, health and juvenile justice providers can partner with parents to overcome barriers to healthcare for youth during reentry.

Key Population Health Outcomes for Children with Medical Complexity: A Systematic Review.

INTRODUCTION: Despite the significant healthcare policy and program implications, a summary measure of health for children with medical complexity (CMC) has not been identified. It is unclear whether existing population health approaches apply to CMC. We conducted a systematic review of the existing peer-reviewed research literature on CMC to describe the health outcomes currently measured for CMC. METHODS: We searched MEDLINE and PsycINFO by linking combinations of key words from three groups of concepts: (1) pediatric, (2) medical complexity, and (3) chronicity or severity. Study eligibility criteria were research studies including CMC with any outcome reported. Data on the outcomes were systematically extracted. Iterative content analysis organized outcomes into conceptual domains and sub-domains. RESULTS: Our search yielded 3853 articles. After exclusion criteria were applied, 517 articles remained for data extraction. Five distinct outcome domains and twenty-four sub-domains emerged. Specifically, 50% of the articles studied healthcare access and use; 43% family well-being; 39% child health and well-being; 38% healthcare quality; and 25% adaptive functioning. Notably lacking were articles examining routine child health promotion as well as child mental health and outcomes related to family functioning. CONCLUSIONS: Key health domains for CMC exist. Adaptations of existing sets of metrics and additional tools are needed to fully represent and measure population health for CMC. This approach may guide policies and programs to improve care for CMC.