Disparities in Screening for Substance Use Among Injured Adolescents.

This cohort study compares rates of biochemical substance use screening among injured adolescents by sociodemographic characteristics including sex, race, ethnicity, and insurance status.

Characteristics of overnight video-EEG monitoring in infantile epileptic spasms syndrome at 2-week follow-up.

OBJECTIVE: To determine the optimal duration of electroencephalography (EEG) recording to detect epileptic spasms (ES) based on inpatient overnight video-EEG monitoring in patients with infantile epileptic spasms syndrome (IESS) at the 2-week follow-up. METHODS: Patients with IESS and overnight EEG monitoring between January 2020 and June 2022 were retrospectively reviewed. Time-to-ES, time-to-sleep and time-to-epileptic encephalopathy (EE) per the Burden of Amplitudes and Epileptiform Discharges (BASED 2021) score. BASED 2021 score were reported. ES and sleep detection sensitivity were calculated with respect to monitoring time. Etiology, treatment, and EEG features were assessed for strength of association with continued ES. Time-to-event analysis was performed with the first ES as the event of interest. RESULTS: Of 90 patients, 39 (43%) continued to have ES; 78.6% with EE continued to have ES, whereas only 27.4% without EE had ES (odds ratio [OR] 12.05). Structural etiologies were also associated with continued ES (OR 5.24). ES detection was 35.9%, 76.9%, and 84.6% at 1, 4, and 6 h, respectively, with corresponding negative likelihood ratios (NLRs) of .64, .23, and .15. ES detection reached >90% and >95% at 14 and 19 h, respectively. Sleep detection was 52.2%, 84.4%, and 95.6% at 1, 4, and 6 h, respectively, and captured in all patients by 11 h. EE was observed by 6 h for all associated patients. SIGNIFICANCE: Typical routine EEG durations (<1 h) were not sufficient to detect ES, EE, or sleep in patients with IESS at the 2-week follow-up. Four hour outpatient EEG will capture ES in 77% and sleep in 84% of the patients. EE, if present, was shortly after sleep onset. Additional monitoring of up to 19 h was needed to capture >95% of patients with ES. Although EE was strongly associated with continued ES, 27.4% of patients without EE demonstrated ES. This study will help guide adequate duration of EEG monitoring at the 2-week follow-up for patients with IESS.

POLCAM: instant molecular orientation microscopy for the life sciences.

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, which can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field-of-view errors, used simulations to optimize experimental design and developed a fast algorithm based on Stokes parameter estimation that can operate over 1,000-fold faster than the state of the art, enabling near-instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study α-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.

Single-molecule digital sizing of proteins in solution.

The physical characterization of proteins in terms of their sizes, interactions, and assembly states is key to understanding their biological function and dysfunction. However, this has remained a difficult task because proteins are often highly polydisperse and present as multicomponent mixtures. Here, we address this challenge by introducing single-molecule microfluidic diffusional sizing (smMDS). This approach measures the hydrodynamic radius of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. smMDS allows for ultrasensitive sizing of proteins down to femtomolar concentrations and enables affinity profiling of protein interactions at the single-molecule level. We show that smMDS is effective in resolving the assembly states of protein oligomers and in characterizing the size of protein species within complex mixtures, including fibrillar protein aggregates and nanoscale condensate clusters. Overall, smMDS is a highly sensitive method for the analysis of proteins in solution, with wide-ranging applications in drug discovery, diagnostics, and nanobiotechnology.

Non-Operative Management of Uncomplicated Appendicitis in Children: Can Patients Be Discharged From the Emergency Department?

INTRODUCTION: Non-operative management of uncomplicated appendicitis in children is a safe alternative to laparoscopic appendectomy. The purpose of this study is to determine the feasibility of nonoperative outpatient management of uncomplicated appendicitis in pediatric patients. METHODS: A standardized pathway for non-operative outpatient management of uncomplicated appendicitis in children (NOMA-C) was implemented in a university pediatric surgery practice starting October 2021. Those who met criteria would be considered for discharge from the emergency department (ED) with oral antibiotics and close followup over the following year. A quality improvement project started concurrently to monitor patients for recurrent symptoms of appendicitis. RESULTS: A total of 121 patients were diagnosed with acute appendicitis during the study period (October 2021 to April 2023). Forty-five children (38%) met criteria for the NOMA-C protocol, and 11 patients/families chose appendectomy. Of the 34 patients who pursued nonoperative management, 14 patients were admitted to the hospital and 20 patients were discharged from the ED. Total time in the ED/hospital was significantly shorter for patients discharged from the ED (7 hours versus 23 hours; p<0.001). At one year follow up only 4 (12%) had undergone appendectomy. There were no adverse events for patients who underwent nonoperative management. CONCLUSION: A protocol offering non-operative management of appendicitis with an option for outpatient management was feasible and safe. Outpatient management was associated with shorter ED/hospital stays than those admitted. Future studies should evaluate whether this protocol can be adopted by EDs without pediatric surgery services to avoid the need for transfer.

Effects of Coprecipitation Conditions on the Electrochemical Properties of Cobalt-Free LiNi0.9Mn0.1-xAlxO2 Cathodes.

Commercializing high-nickel, cobalt-free cathodes, such as LiNi0.9Mn0.1-xAlxO2 (NMA-90), hinges on effectively incorporating Al3+ during the hydroxide coprecipitation reaction. However, Al3+ coprecipitation is nontrivial as Al3+ possesses unique precipitation properties compared to Ni2+ and Mn2+, which impact the final precursor morphology and consequently the cathode properties. In this study, the nuance of Al3+ coprecipitation and its influence on the cycling stability of NMA with increasing Al3+ content is elucidated. While low reaction pH and ammonia concentration are suitable for producing Al-free LiNi0.9Mn0.1O2 (NM-90) effectively, the same coprecipitation environment leads to porous precursor morphology and poor cycle life in Al-containing LiNi0.9Mn0.08Al0.02O2 (NMA-900802) and LiNi0.9Mn0.05Al0.05O2 (NMA-900505). By systematically increasing the reaction pH and ammonia concentration for the Al-containing compositions, the precursor morphology becomes denser and the cathode cycling stability is greatly improved. It is hypothesized that the improvement in cycling stability stems from the reduction in Al(OH)3 nucleation, which promotes hydroxide particle growth with optimal Al3+ incorporation into the cathode lattice.

Introduction to Entrustable Professional Activities for Pediatric Surgery.

BACKGROUND: Entrustable Professional Activities (EPAs) have emerged as a valuable tool in medical education, enabling the assessment of trainee competence in a real-world context. Despite its growing popularity in other medical specialties, the use of EPAs in pediatric surgery is still relatively new. METHODS: This article provides an overview of the development and application of EPAs in pediatric surgery. The key concepts and principles of EPAs, including the historical development and current implementation in other medical specialties, will also be reviewed. Definition of pediatric surgery EPAs, advantages, and challenges of implementing EPAs in pediatric surgery, and the evidence supporting the use of EPAs in pediatric surgical training will also be examined. RESULTS: The use of EPAs in pediatric surgery has shown promise in providing a comprehensive and standardized approach to assessing trainee competence. By focusing on the essential tasks and responsibilities of a pediatric surgeon, EPAs can provide a framework for measuring trainee progress and ensuring that they possess the necessary skills to perform their duties independently. Challenges in implementing EPAs in pediatric surgery include the need for consensus on the definition and scope of EPAs, as well as the establishment of appropriate assessment and evaluation tools. CONCLUSION: The use of EPAs in pediatric surgery education and training has the potential to enhance the training and ultimately the quality of care delivered to pediatric surgical patients. The development of appropriate assessment and evaluation tools and frameworks will be crucial in implementing EPAs effectively in this field. TYPE OF STUDY: Review.

Six Years of Quality Improvement in Pectus Excavatum Repair: Implementation of Intercostal Nerve Cryoablation and ERAS Protocols for Patients Undergoing Nuss Procedure.

BACKGROUND: The Nuss procedure for pectus excavatum is associated with prolonged hospitalizations due to pain. We evaluated implementation of intercostal nerve cryoablation and enhanced recovery after surgery (ERAS) protocols on outcomes of Nuss procedures performed over six years at a single institution. METHODS: This retrospective cohort study included patients who underwent Nuss procedure from 10/2017 to 09/2023. Patients received epidurals prior to 06/2019, cryoablation from 06/2019 to 07/2021, and ERAS with cryoablation and intraoperative methadone administration after 07/2021. We used multivariable linear regression to evaluate length of stay (LOS), inpatient morphine milligram equivalents (MMEs), and discharge opioids. We assessed the balancing measures of operative time, postoperative pain scores, and complications. RESULTS: We identified 62 patients; 15 who received epidurals, 18 cryoablation, and 29 cryoablation with ERAS. Cryoablation was associated with a 62.3% (p < 0.001) decrease in length of stay, an 86.6% (p < 0.001) decrease in inpatient MMEs, and a 72.9% (p < 0.001) decrease in discharge opioids. Cryoablation was additionally associated with 24.5% (p = 0.02) longer operative times and 46.4% (p = 0.04) higher postoperative day one pain scores. Subsequent implementation of an ERAS protocol was associated with a further 82.8% (p = 0.04) decrease in discharge opioids and a 25.0% (p = 0.04) decrease in postoperative day one pain scores. CONCLUSIONS: Over six years of quality improvement efforts, we found the implementation of cryoablation and ERAS protocols to be associated with a significant decrease in length of stay and opioid exposures. Protocolized pain management and cryoablation may work synergistically to improve outcomes without compromising patient experience. LEVEL OF EVIDENCE: Level III - Retrospective comparative study.

Disparities in outpatient rural cholecystectomy outcomes.

BACKGROUND: Previous studies showed comparable outcomes for common in-patient general surgery operations, but it is unknown if this extends to outpatient operations. Our aim was to compare outpatient cholecystectomy outcomes between rural and urban hospitals. METHODS: A retrospective cohort analysis was done using the Nationwide Ambulatory Surgery Sample for patients 20-years-and-older undergoing cholecystectomy between 2016 and 2018 â€‹at rural and urban hospitals. Survey-weighted multivariable regression analysis was performed with primary outcomes including use-of-laparoscopy, complications, and patient discharge disposition. RESULTS: The most common indication for operation was cholecystitis in both hospital settings. On multivariable analysis, rural hospitals were associated with higher transfers to short-term hospitals (adjusted odds ratio [aOR] 2.40, 95%CI 1.61-3.58, p â€‹< â€‹0.01) and complications (aOR 1.39, 95%CI 1.11-1.75, p â€‹< â€‹0.01). No difference was detected with laparoscopy (aOR 1.93, 95%CI 0.73-5.13, p â€‹= â€‹0.19), routine discharge (aOR 1.50, 95%C I0.91-2.45, p â€‹= â€‹0.11), or mortality (aOR 3.23, 95%CI 0.10-100.0, p â€‹= â€‹0.51). CONCLUSIONS: Patients cared for at rural hospitals were more likely to be transferred to short-term hospitals and have higher complications. No differences were detected in laparoscopy, routine discharge or mortality.

Disturbance amplifies sensitivity of dryland productivity to precipitation variability.

Variability of the terrestrial global carbon sink is largely determined by the response of dryland productivity to annual precipitation. Despite extensive disturbance in drylands, how disturbance alters productivity-precipitation relationships remains poorly understood. Using remote-sensing to pair more than 5600 km of natural gas pipeline corridors with neighboring undisturbed areas in North American drylands, we found that disturbance reduced average annual production 6 to 29% and caused up to a fivefold increase in the sensitivity of net primary productivity (NPP) to interannual variation in precipitation. Disturbance impacts were larger and longer-lasting at locations with higher precipitation (>450 mm mean annual precipitation). Disturbance effects on NPP dynamics were mostly explained by shifts from woody to herbaceous vegetation. Severe disturbance will amplify effects of increasing precipitation variability on NPP in drylands.

An examination of the mechanisms driving the therapeutic effects of an AAV expressing a soluble variant of VEGF receptor-1.

In previous animal model studies, we demonstrated the potential of rAAV2-sVEGFRv-1, which encodes a truncated variant of the alternatively spliced soluble version of VEGF receptor-1 (VEGFR1), as a human gene therapy for age-related macular degeneration (AMD) and diabetic retinopathy (DR). Here, we elucidate in vitro some of the mechanisms by which rAAV2-sVEGFRv-1 exerts its therapeutic effects. Human umbilical vein endothelial cells (HUVECs) were infected with rAAV2-sVEGFRv-1 or a control virus vector in the presence of members of the VEGF family to identify potential binding partners via ELISA, which showed that VEGF-A, VEGF-B, and placental growth factor (PlGF) are all ligands of its transgene product. In order to determine the effects of rAAV2-sVEGFRv-1 on cell proliferation and permeability, processes that are important to the progression AMD and DR, HUVECs were infected with the therapeutic virus vector under the stimulation of VEGF-A, the major driver of the neovascularization that characterizes the forms of these conditions most associated with vision loss. rAAV2-sVEGFRv-1 treatment, as a result, markedly reduced the extent to which these processes occurred, with the latter determined by measuring zonula occludens 1 expression. Finally, the human microglial HMC3 cell line was used to show the effects of the therapeutic virus vector upon inflammatory processes, another major contributor to angiogenic eye disease pathophysiology, with rAAV2-sVEGFRv-1 reducing therein the secretion of pro-inflammatory cytokines interleukin (IL)-1ß and IL-6. Combined with our previously published in vivo data, the in vitro activity of the expressed transgene here further demonstrates the great promise of rAAV2-sVEGFRv-1 as a potential human gene therapeutic for addressing angiogenic ocular conditions.

Single-Molecule Stoichiometry of Supramolecular Complexes.

The use of single-molecule microscopy is introduced as a method to quantify the photophysical properties of supramolecular complexes rapidly at ultra low concentrations (<1 nM), previously inaccessible. Using a model supramolecular system based on the host-guest complexation of cucurbit[n]uril (CB[n]) macrocycles together with a fluorescent guest (Ant910Me), we probe fluorescent CB[n] host-guest complexes in the single molecule regime. We show quantification and differentiation of host-guest photophysics and stoichiometries, both in aqueous media and noninvasively in hydrogel, by thresholding detected photons. This methodology has wide reaching implications in aiding the design of next-generation materials with programmed and controlled properties.

Responsive neurostimulation of thalamic nuclei for regional and multifocal drug-resistant epilepsy in children and young adults.

OBJECTIVE: Responsive neurostimulation (RNS) is a US FDA-approved form of neuromodulation to treat patients with focal-onset drug-resistant epilepsy (DRE) who are ineligible for or whose condition is refractory to resection. However, the FDA approval only extends to use in patients with one or two epileptogenic foci. Recent literature has shown possible efficacy of thalamic RNS in patients with Lennox-Gastaut syndrome and multifocal epilepsy. The authors hypothesized that RNS of thalamic nuclei may be effective in seizure reduction for patients with multifocal or regionalized-onset DRE. METHODS: The authors performed a retrospective chart review of all patients who had an RNS device managed at Texas Children's Hospital between July 2016 and September 2023, with at least one active electrode in the thalamic nuclei and ≥ 12 months of postimplantation follow-up. Information conveyed by the patient or their caregiver provided data on the change in the clinical seizure frequency, quality of life (QOL), and seizure severity between the preimplantation baseline visit and the last office visit (LOV). RESULTS: Thirteen patients (ages 8-24 years) were identified with active RNS leads in thalamic nuclei (11 centromedian and 2 anterior nucleus). At LOV, 46% of patients reported 50%-100% clinical seizure reduction (classified as responders), 15% reported 25%-49% reduction, and 38% reported < 25% reduction or no change. Additionally, 42% of patients reported subjective improvement in QOL and 58% reported improved seizure severity. Patients with focal cortical dysplasia (FCD) responded strongly: 3 of 5 (60%) reported ≥ 80% reduction in seizure burden and improvement in seizure severity and QOL. Patients with multifocal epilepsy and bilateral thalamocortical leads also did well, with all 3 reporting ≥ 50% reduction in seizures. CONCLUSIONS: RNS of thalamic nuclei shows promising results in reducing seizure burden for patients with multifocal or regional-onset DRE, particularly in a bilateral thalamocortical configuration or when addressing an underlying FCD.

RASP: Optimal Single Puncta Detection in Complex Cellular Backgrounds.

Super-resolution and single-molecule microscopies have been increasingly applied to complex biological systems. A major challenge of these approaches is that fluorescent puncta must be detected in the low signal, high noise, heterogeneous background environments of cells and tissue. We present RASP, Radiality Analysis of Single Puncta, a bioimaging-segmentation method that solves this problem. RASP removes false-positive puncta that other analysis methods detect and detects features over a broad range of spatial scales: from single proteins to complex cell phenotypes. RASP outperforms the state-of-the-art methods in precision and speed using image gradients to separate Gaussian-shaped objects from the background. We demonstrate RASP's power by showing that it can extract spatial correlations between microglia, neurons, and α-synuclein oligomers in the human brain. This sensitive, computationally efficient approach enables fluorescent puncta and cellular features to be distinguished in cellular and tissue environments, with sensitivity down to the level of the single protein. Python and MATLAB codes, enabling users to perform this RASP analysis on their own data, are provided as Supporting Information and links to third-party repositories.

High-density volumetric super-resolution microscopy.

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs µm-2) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs µm-2) through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high-density volumetric imaging (0.15 locs µm-2) in dense cytosolic tubulin datasets.

Targeting the activated microenvironment with endosialin (CD248)-directed CAR-T cells ablates perivascular cells to impair tumor growth and metastasis.

BACKGROUND: Targeting of solid cancers with chimeric antigen receptor (CAR)-T cells is limited by the lack of suitable tumor-specific antigens and the immunosuppressive, desmoplastic tumor microenvironment that impedes CAR-T cell infiltration, activity and persistence. We hypothesized that targeting the endosialin (CD248) receptor, strongly expressed by tumor-associated pericytes and perivascular cancer-associated fibroblasts, would circumvent these challenges and offer an exciting antigen for CAR-T cell therapy due to the close proximity of target cells to the tumor vasculature, the limited endosialin expression in normal tissues and the lack of phenotype observed in endosialin knockout mice. METHODS: We generated endosialin-directed E3K CAR-T cells from three immunocompetent mouse strains, BALB/c, FVB/N and C57BL/6. E3K CAR-T cell composition (CD4+/CD8+ ratio), activity in vitro against endosialin+ and endosialin- cells, and expansion and activity in vivo in syngeneic tumor models as well as in tumor-naive healthy and wounded mice and tumor-bearing endosialin knockout mice was assessed. RESULTS: E3K CAR-T cells were active in vitro against both mouse and human endosialin+, but not endosialin-, cells. Adoptively transferred E3K CAR-T cells exhibited no activity in endosialin knockout mice, tumor-naive endosialin wildtype mice or in wound healing models, demonstrating an absence of off-target and on-target/off-tumor activity. By contrast, adoptive transfer of E3K CAR-T cells into BALB/c, FVB/N or C57BL/6 mice bearing syngeneic breast or lung cancer lines depleted target cells in the tumor stroma resulting in increased tumor necrosis, reduced tumor growth and a substantial impairment in metastatic outgrowth. CONCLUSIONS: Together these data highlight endosialin as a viable antigen for CAR-T cell therapy and that targeting stromal cells closely associated with the tumor vasculature avoids CAR-T cells having to navigate the harsh immunosuppressive tumor microenvironment. Further, the ability of E3K CAR-T cells to recognize and target both mouse and human endosialin+ cells makes a humanized and optimized E3K CAR a promising candidate for clinical development applicable to a broad range of solid tumor types.

Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

The role of high-resolution ultrasound and MRI in the evaluation of peripheral nerves in the lower extremity.

Lower extremity peripheral neuropathy is a commonly encountered neurologic disorder, which can lead to chronic pain, functional disability, and decreased quality of life for a patient. As diagnostic imaging modalities have improved, imaging has started to play an integral role in the detection and characterization of peripheral nerve abnormalities by non-invasively and accurately identifying abnormal nerves as well as potential causes of neuropathy, which ultimately leads to precise and timely treatment. Ultrasound, which has high spatial resolution and can quickly and comfortably characterize peripheral nerves in real time along with associated denervation muscle atrophy, and magnetic resonance neurography, which provides excellent contrast resolution between nerves and other tissues and between pathologic and normal segments of peripheral nerves, in addition to assessing reversible and irreversible muscle denervation changes, are the two mainstay imaging modalities used in peripheral nerve assessment. These two modalities are complimentary, and one may be more useful than the other depending on the nerve and location of pathology. Imaging must be interpreted in the context of available clinical information and other diagnostic studies, such as electrodiagnostic tests. Here, we offer a comprehensive overview of the role of high-resolution ultrasound and magnetic resonance neurography in the evaluation of the peripheral nerves of the lower extremity and their associated neuropathies.

Role of high-resolution ultrasound and magnetic resonance neurography in the evaluation of peripheral nerves in the upper extremity.

Upper extremity entrapment neuropathies are common conditions in which peripheral nerves are prone to injury at specific anatomical locations, particularly superficial regions or within fibro-osseous tunnels, resulting in pain and potential disability. Although neuropathy is primarily diagnosed clinically by physical examination and electrophysiology, imaging evaluation with ultrasound and magnetic resonance neurography are valuable complementary non-invasive and accurate tools for evaluation and can help define the site and cause of nerve dysfunction which ultimately leads to precise and timely treatment. Ultrasound, which has higher spatial resolution, can quickly and comfortably characterize the peripheral nerves in real time and can evaluate for denervation related muscle atrophy. Magnetic resonance imaging on the other hand provides excellent contrast resolution between the nerves and adjacent tissues, also between pathologic and normal segments of peripheral nerves. It can also assess the degree of muscle denervation and atrophy. As a prerequisite for nerve imaging, radiologists and sonographers should have a thorough knowledge of anatomy of the peripheral nerves and their superficial and deep branches, including variant anatomy, and the motor and sensory territories innervated by each nerve. The purpose of this illustrative article is to review the common neuropathy and nerve entrapment syndromes in the upper extremities focusing on ultrasound and magnetic resonance neurography imaging.