Intravital Microscopy With an Airy Beam Light Sheet Microscope Improves Temporal Resolution and Reduces Surgical Trauma.

Intravital microscopy has emerged as a powerful imaging tool, which allows the visualization and precise understanding of rapid physiological processes at sites of inflammation in vivo, such as vascular permeability and leukocyte migration. Leukocyte interactions with the vascular endothelium can be characterized in the living organism in the murine cremaster muscle. Here, we present a microscopy technique using an Airy Beam Light Sheet microscope that has significant advantages over our previously used confocal microscopy systems. In comparison, the light sheet microscope offers near isotropic optical resolution and faster acquisition speed, while imaging a larger field of view. With less invasive surgery we can significantly reduce side effects such as bleeding, muscle twitching, and surgical inflammation. However, the increased acquisition speed requires exceptional tissue stability to avoid imaging artefacts. Since respiratory motion is transmitted to the tissue under investigation, we have developed a relocation algorithm that removes motion artefacts from our intravital microscopy images. Using these techniques, we are now able to obtain more detailed 3D time-lapse images of the cremaster vascular microcirculation, which allow us to observe the process of leukocyte emigration into the surrounding tissue with increased temporal resolution in comparison to our previous confocal approach.

Targeted protein degradation using chimeric human E2 ubiquitin-conjugating enzymes.

Proteins can be targeted for degradation by engineering biomolecules that direct them to the eukaryotic ubiquitination machinery. For instance, the fusion of an E3 ubiquitin ligase to a suitable target binding domain creates a 'biological Proteolysis-Targeting Chimera' (bioPROTAC). Here we employ an analogous approach where the target protein is recruited directly to a human E2 ubiquitin-conjugating enzyme via an attached target binding domain. Through rational design and screening we develop E2 bioPROTACs that induce the degradation of the human intracellular proteins SHP2 and KRAS. Using global proteomics, we characterise the target-specific and wider effects of E2 vs. VHL-based fusions. Taking SHP2 as a model target, we also employ a route to bioPROTAC discovery based on protein display libraries, yielding a degrader with comparatively weak affinity capable of suppressing SHP2-mediated signalling.

An Artificial Intelligence Generated Automated Algorithm to Measure Total Kidney Volume in ADPKD.

Introduction: Accurate tools to inform individual prognosis in patients with autosomal dominant polycystic kidney disease (ADPKD) are lacking. Here, we report an artificial intelligence (AI)-generated method for routinely measuring total kidney volume (TKV). Methods: An ensemble U-net algorithm was created using the nnUNet approach. The training and internal cross-validation cohort consisted of all 1.5T magnetic resonance imaging (MRI) data acquired using 5 different MRI scanners (454 kidneys, 227 scans) in the CYSTic consortium, which was first manually segmented by a single human operator. As an independent validation cohort, we utilized 48 sequential clinical MRI scans with reference results of manual segmentation acquired by 6 individual analysts at a single center. The tool was then implemented for clinical use and its performance analyzed. Results: The training or internal validation cohort was younger (mean age 44.0 vs. 51.5 years) and the female-to-male ratio higher (1.2 vs. 0.94) compared to the clinical validation cohort. The majority of CYSTic patients had PKD1 mutations (79%) and typical disease (Mayo Imaging class 1, 86%). The median DICE score on the clinical validation data set between the algorithm and human analysts was 0.96 for left and right kidneys with a median TKV error of -1.8%. The time taken to manually segment kidneys in the CYSTic data set was 56 (±28) minutes, whereas manual corrections of the algorithm output took 8.5 (±9.2) minutes per scan. Conclusion: Our AI-based algorithm demonstrates performance comparable to manual segmentation. Its rapidity and precision in real-world clinical cases demonstrate its suitability for clinical application.

A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation.

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

Evaluating the performance of artificial intelligence software for lung nodule detection on chest radiographs in a retrospective real-world UK population.

OBJECTIVES: Early identification of lung cancer on chest radiographs improves patient outcomes. Artificial intelligence (AI) tools may increase diagnostic accuracy and streamline this pathway. This study evaluated the performance of commercially available AI-based software trained to identify cancerous lung nodules on chest radiographs. DESIGN: This retrospective study included primary care chest radiographs acquired in a UK centre. The software evaluated each radiograph independently and outputs were compared with two reference standards: (1) the radiologist report and (2) the diagnosis of cancer by multidisciplinary team decision. Failure analysis was performed by interrogating the software marker locations on radiographs. PARTICIPANTS: 5722 consecutive chest radiographs were included from 5592 patients (median age 59 years, 53.8% women, 1.6% prevalence of cancer). RESULTS: Compared with radiologist reports for nodule detection, the software demonstrated sensitivity 54.5% (95% CI 44.2% to 64.4%), specificity 83.2% (82.2% to 84.1%), positive predictive value (PPV) 5.5% (4.6% to 6.6%) and negative predictive value (NPV) 99.0% (98.8% to 99.2%). Compared with cancer diagnosis, the software demonstrated sensitivity 60.9% (50.1% to 70.9%), specificity 83.3% (82.3% to 84.2%), PPV 5.6% (4.8% to 6.6%) and NPV 99.2% (99.0% to 99.4%). Normal or variant anatomy was misidentified as an abnormality in 69.9% of the 943 false positive cases. CONCLUSIONS: The software demonstrated considerable underperformance in this real-world patient cohort. Failure analysis suggested a lack of generalisability in the training and testing datasets as a potential factor. The low PPV carries the risk of over-investigation and limits the translation of the software to clinical practice. Our findings highlight the importance of training and testing software in representative datasets, with broader implications for the implementation of AI tools in imaging.

Characterization of the Native Disulfide Isomers of the Novel χ-Conotoxin PnID: Implications for Further Increasing Conotoxin Diversity.

χ-Conotoxins are known for their ability to selectively inhibit norepinephrine transporters, an ability that makes them potential leads for treating various neurological disorders, including neuropathic pain. PnID, a peptide isolated from the venom of Conus pennaceus, shares high sequence homology with previously characterized χ-conotoxins. Whereas previously reported χ-conotoxins seem to only have a single native disulfide bonding pattern, PnID has three native isomers due to the formation of different disulfide bond patterns during its maturation in the venom duct. In this study, the disulfide connectivity and three-dimensional structure of these disulfide isomers were explored using regioselective synthesis, chromatographic coelution, and solution-state nuclear magnetic resonance spectroscopy. Of the native isomers, only the isomer with a ribbon disulfide configuration showed pharmacological activity similar to other χ-conotoxins. This isomer inhibited the rat norepinephrine transporter (IC50 = 10 ± 2 µM) and has the most structural similarity to previously characterized χ-conotoxins. In contrast, the globular isoform of PnID showed more than ten times less activity against this transporter and the beaded isoform did not display any measurable biological activity. This study is the first report of the pharmacological and structural characterization of an χ-conotoxin from a species other than Conus marmoreus and is the first report of the existence of natively-formed conotoxin isomers.

Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration.

Cardiac injury leads to the loss of cardiomyocytes, which are rapidly replaced by the proliferation of the surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronized live imaging, RNA sequencing, and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterize macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium, which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signaling. The identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair.

Reconstructing codependent cellular cross-talk in lung adenocarcinoma using REMI.

Cellular cross-talk in tissue microenvironments is fundamental to normal and pathological biological processes. Global assessment of cell-cell interactions (CCIs) is not yet technically feasible, but computational efforts to reconstruct these interactions have been proposed. Current computational approaches that identify CCI often make the simplifying assumption that pairwise interactions are independent of one another, which can lead to reduced accuracy. We present REMI (REgularized Microenvironment Interactome), a graph-based algorithm that predicts ligand-receptor (LR) interactions by accounting for LR dependencies on high-dimensional, small-sample size datasets. We apply REMI to reconstruct the human lung adenocarcinoma (LUAD) interactome from a bulk flow-sorted RNA sequencing dataset, then leverage single-cell transcriptomics data to increase the cell type resolution and identify LR prognostic signatures among tumor-stroma-immune subpopulations. We experimentally confirmed colocalization of CTGF:LRP6 among malignant cell subtypes as an interaction predicted to be associated with LUAD progression. Our work presents a computational approach to reconstruct interactomes and identify clinically relevant CCIs.

Military deployment's impact on the surgeon's practice.

BACKGROUND: As the United States withdraws from overseas conflicts, general surgeons remain deployed in support of global operations. Surgeons and surgical teams are foundational to combat casualty care; however, currently, there are few casualty producing events. Low surgical volume and acuity can have detrimental effects on surgical readiness for those frequently deployed. The surgical team cycle of deployment involves predeployment training, drawdown of clinical practice, deployment, postdeployment reintegration, and rebuilding of a patient panel. This study aims to assess these effects on typical general surgeon practices. Quantifying the overall impact of deployment may help refine and implement measures to mitigate the effects on skill retention and patient care. METHODS: Surgeon case logs of eligible surgeons deploying between January 1, 2017, and January 1, 2020, were included from participating military treatment facilities. Eligible surgeons were surgeons whose case logs were primarily at a single military treatment facility 26 weeks before and after deployment and whose deployment duration, location, and number of deployed cases were obtainable. RESULTS: Starting 26 weeks prior to deployment, analyzing in 1-week intervals toward deployment time, case count decreased by 4.8% (p < 0.0001). With each 1-week interval, postdeployment up to the 26-week mark, case count increased by 6% (p < 0.0001). Cases volumes most prominently drop 3 weeks prior to deployment and do not reach normal levels until approximately 7 weeks postdeployment. Case volumes were similar across service branches. CONCLUSION: There is a significant decrease in the number of cases performed before deployment and increase after return regardless of military branch. The perideployment surgical volume decline should be understood and mitigated appropriately; predeployment training, surgical skill retention, and measures to safely reintegrate surgeons back into their practice should be further developed and implemented. LEVEL OF EVIDENCE: Economic/Decision, Level III.

Clinical utilization of deployed military surgeons.

BACKGROUND: Combat casualty care has been shaped by the prolonged conflicts in Southwest Asia, namely Afghanistan, Iraq, and Syria. The utilization of surgeons in austere locations outside of Southwest Asia and its implication on skill retention and value have not been examined. This study hypothesizes that surgeon utilization is low in the African theater. This lack of activity is potentially damaging to surgical skill retention and patient care. METHODS: Military case logs of surgeons deployed to Africa under command of Special Operations Command Africa between January 1, 2016, and January 1, 2020, were examined. Cases were organized based on population served, general type of procedure, current procedural terminology codes, and location. RESULTS: Twenty deployment caseloads representing 74% of the deployments during the period were analyzed. In 3,294 days, 101 operations were performed, which included 45 on combat/terrorism related injuries and 19 on US personnel. East and West African deployments, combat, and noncombat zones, respectively, were compared. East Africa averaged 4.1 ± 3.8 operations per deployment, and West Africa, 7.3 ± 8.0 (p = 0.2434). In East Africa, 56.1% of total operations were related to combat/terrorism, compared with 29.6% of total operations in West Africa (p = 0.0077). West Africa had a significantly higher proportion of elective (p = 0.0002) and humanitarian cases (p = <0.0001). CONCLUSION: Surgical cases for military surgeons were uncommon in Africa. The low volumes have implications for skill retention, morale, and sustainability of military surgical end strength. Reduction in deployment lengths, deployment location adjustments, and/or skill retention strategies are required to ensure clinical peak performance and operational readiness. Failure to implement changes to current practices to optimize surgeon experience will likely decrease surgical readiness and could contribute to decreased retention of deployable military surgeons to support global operations. LEVEL OF EVIDENCE: Economic/decision, level III.

Live Imaging of Heart Injury in Larval Zebrafish Reveals a Multi-Stage Model of Neutrophil and Macrophage Migration.

Neutrophils and macrophages are crucial effectors and modulators of repair and regeneration following myocardial infarction, but they cannot be easily observed in vivo in mammalian models. Hence many studies have utilized larval zebrafish injury models to examine neutrophils and macrophages in their tissue of interest. However, to date the migratory patterns and ontogeny of these recruited cells is unknown. In this study, we address this need by comparing our larval zebrafish model of cardiac injury to the archetypal tail fin injury model. Our in vivo imaging allowed comprehensive mapping of neutrophil and macrophage migration from primary hematopoietic sites, to the wound. Early following injury there is an acute phase of neutrophil recruitment that is followed by sustained macrophage recruitment. Both cell types are initially recruited locally and subsequently from distal sites, primarily the caudal hematopoietic tissue (CHT). Once liberated from the CHT, some neutrophils and macrophages enter circulation, but most use abluminal vascular endothelium to crawl through the larva. In both injury models the innate immune response resolves by reverse migration, with very little apoptosis or efferocytosis of neutrophils. Furthermore, our in vivo imaging led to the finding of a novel wound responsive mpeg1+ neutrophil subset, highlighting previously unrecognized heterogeneity in neutrophils. Our study provides a detailed analysis of the modes of immune cell migration in larval zebrafish, paving the way for future studies examining tissue injury and inflammation.

Profile of a Highly Selective Quaternized Pyrrolidine Betaine αvß6 Integrin Inhibitor-(3S)-3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)-4-((1S and 1R,3R)-1-methyl-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-ium-1-yl)butanoate Synthesized by Stereoselective Methylation.

A quaternary ammonium betaine 7 is described which shows exceptional potency and selectivity (1.4 to >3 logs) for the αvß6 integrin receptor over the other αv integrins as determined in cell adhesion assays. 7 is prepared by remarkably stereoselective methylation, the origins of which are discussed. The chemical, biological, physicochemical, and pharmacokinetic properties of 7 and its docking into αvß6 are described along with related analogues.

Discovery of Potent, Efficient, and Selective Inhibitors of Phosphoinositide 3-Kinase δ through a Deconstruction and Regrowth Approach.

A deconstruction of previously reported phosphoinositide 3-kinase δ (PI3Kδ) inhibitors and subsequent regrowth led to the identification of a privileged fragment for PI3Kδ, which was exploited to deliver a potent, efficient, and selective lead series with a novel binding mode observed in the PI3Kδ crystal structure.

PRaVDA: The first solid-state system for proton computed tomography.

PURPOSE: Proton CT is widely recognised as a beneficial alternative to conventional X-ray CT for treatment planning in proton beam radiotherapy. A novel proton CT imaging system, based entirely on solid-state detector technology, is presented. Compared to conventional scintillator-based calorimeters, positional sensitive detectors allow for multiple protons to be tracked per read out cycle, leading to a potential reduction in proton CT scan time. Design and characterisation of its components are discussed. An early proton CT image obtained with a fully solid-state imaging system is shown and accuracy (as defined in Section IV) in Relative Stopping Power to water (RSP) quantified. METHOD: A solid-state imaging system for proton CT, based on silicon strip detectors, has been developed by the PRaVDA collaboration. The system comprises a tracking system that infers individual proton trajectories through an imaging phantom, and a Range Telescope (RT) which records the corresponding residual energy (range) for each proton. A back-projection-then-filtering algorithm is used for CT reconstruction of an experimentally acquired proton CT scan. RESULTS: An initial experimental result for proton CT imaging with a fully solid-state system is shown for an imaging phantom, namely a 75 mm diameter PMMA sphere containing tissue substitute inserts, imaged with a passively-scattered 125 MeV beam. Accuracy in RSP is measured to be ⩽1.6% for all the inserts shown. CONCLUSIONS: A fully solid-state imaging system for proton CT has been shown capable of imaging a phantom with protons and successfully improving RSP accuracy. These promising results, together with system the capability to cope with high proton fluences (2×108 protons/s), suggests that this research platform could improve current standards in treatment planning for proton beam radiotherapy.

Effects of Combat Deployment on Anthropometrics and Physiological Status of U.S. Army Special Operations Forces Soldiers.

INTRODUCTION: U.S. Army Special Operations Forces (SOF) soldiers deploy frequently and conduct military operations through special warfare and surgical strike capabilities. Tasks required to execute these capabilities may induce physical and mental stress and have the potential to degrade soldier physiological status. No investigations have longitudinally characterized whether combat deployment alters anthropometrics or biochemical markers of physiological status in a SOF population of frequent deployers. MATERIALS AND METHODS: Effects of modern combat deployment on longitudinal changes in anthropometrics and physiological status of elite U.S. Army SOF soldiers (n = 50) were assessed. Changes in measures of body composition, grip strength, physiological status, and health behaviors from baseline to postdeployment were determined with paired t test and McNemar's statistic. Baseline measures were obtained between 4 and 8 weeks before deployment. Deployment length was a uniform duration of time between 3 and 6 months (all soldiers completed the same length of deployment). Post hoc analyses determined change in body mass within quartiles of baseline body mass with paired t test and associations between change in sex hormone-binding globulin (SHBG) and change in body mass with correlation coefficient. The study was approved by the Human Use Review Committee at the U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts. RESULTS: In response to deployment, increases in lean mass (77.1 ± 7.6 to 77.8 ± 7.5 kg), maximum grip strength (57.9 ± 7.2 to 61.6 ± 8.8 kg), and conduct of aerobic (156 ± 106 to 250 ± 182 minutes/week) and strength training (190 ± 101 to 336 ± 251 minutes/week) exercise were observed (p < 0.05). Increases in serum SHBG (35.42 ± 10.68 to 38.77 ± 12.26 nmol/L) and decreases in serum cortisol (443.2 ± 79.3 to 381.9 ± 111.6 nmol/L) were also observed (p < 0.05). Body mass changes were dependent on baseline body mass. Soldiers in the lowest quartile of baseline body mass increased body mass (75.6 ± 2.6 vs. 76.6 ± 2.8 kg, p = 0.03), as did those in the second quartile (81.6 ± 2.0 vs. 83.7 ± 3.5 kg, p = 0.02). Those in the third quartile also tended to increase body mass (89.2 ± 2.6 vs. 90.9 ± 3.3 kg, p = 0.05), while those in the upper quartile tended to decrease body mass (98.5 ± 3.6 vs. 96.7 kg, p = 0.06). Change in SHBG was inversely correlated with change in body mass (r = -0.33, p = 0.02). There were no changes in fat mass, body fat percentage, waist circumference, neck circumference, total testosterone, calculated bioavailable or free testosterone, high-sensitivity C-reactive protein, tumor necrosis factor-α, interleukin-1ß, or interleukin-6. Inflammatory markers were skewed toward lower values. CONCLUSIONS: Overall, physiological status of elite SOF soldiers characterized by multiple prior deployments was minimally impacted by combat deployment, in the absence of major unit casualties. The majority experienced some adaptive changes, including increased lean mass, grip strength, time spent engaged in exercise, and decreased levels of the stress hormone cortisol. Mechanisms contributing to inverse correlations between change in SHBG and change in body mass may be further clarified. Future investigations may also more fully characterize the degradation and optimization of health and physiological status of SOF training and deployment cycles with in-theater data collection and repeated measures.

Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones.

Polypeptide aggregation into amyloid is linked with several debilitating human diseases. Despite the inherent risk of aggregation-induced cytotoxicity, bacteria control the export of amyloid-prone subunits and assemble adhesive amyloid fibres during biofilm formation. An Escherichia protein, CsgC potently inhibits amyloid formation of curli amyloid proteins. Here we unlock its mechanism of action, and show that CsgC strongly inhibits primary nucleation via electrostatically-guided molecular encounters, which expands the conformational distribution of disordered curli subunits. This delays the formation of higher order intermediates and maintains amyloidogenic subunits in a secretion-competent form. New structural insight also reveal that CsgC is part of diverse family of bacterial amyloid inhibitors. Curli assembly is therefore not only arrested in the periplasm, but the preservation of conformational flexibility also enables efficient secretion to the cell surface. Understanding how bacteria safely handle amyloidogenic polypeptides contribute towards efforts to control aggregation in disease-causing amyloids and amyloid-based biotechnological applications.

Non-nucleoside reverse transcriptase inhibitor (NNRTI) cross-resistance: implications for preclinical evaluation of novel NNRTIs and clinical genotypic resistance testing.

OBJECTIVES: The introduction of two new non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the past 5 years and the identification of novel NNRTI-associated mutations have made it necessary to reassess the extent of phenotypic NNRTI cross-resistance. METHODS: We analysed a dataset containing 1975, 1967, 519 and 187 genotype-phenotype correlations for nevirapine, efavirenz, etravirine and rilpivirine, respectively. We used linear regression to estimate the effects of RT mutations on susceptibility to each of these NNRTIs. RESULTS: Sixteen mutations at 10 positions were significantly associated with the greatest contribution to reduced phenotypic susceptibility (≥10-fold) to one or more NNRTIs, including: 14 mutations at six positions for nevirapine (K101P, K103N/S, V106A/M, Y181C/I/V, Y188C/L and G190A/E/Q/S); 10 mutations at six positions for efavirenz (L100I, K101P, K103N, V106M, Y188C/L and G190A/E/Q/S); 5 mutations at four positions for etravirine (K101P, Y181I/V, G190E and F227C); and 6 mutations at five positions for rilpivirine (L100I, K101P, Y181I/V, G190E and F227C). G190E, a mutation that causes high-level nevirapine and efavirenz resistance, also markedly reduced susceptibility to etravirine and rilpivirine. K101H, E138G, V179F and M230L mutations, associated with reduced susceptibility to etravirine and rilpivirine, were also associated with reduced susceptibility to nevirapine and/or efavirenz. CONCLUSIONS: The identification of novel cross-resistance patterns among approved NNRTIs illustrates the need for a systematic approach for testing novel NNRTIs against clinical virus isolates with major NNRTI-resistance mutations and for testing older NNRTIs against virus isolates with mutations identified during the evaluation of a novel NNRTI.

Laser-targeted ablation of the zebrafish embryonic ventricle: a novel model of cardiac injury and repair.

BACKGROUND: While the adult zebrafish (Danio rerio) heart demonstrates a remarkable capacity for self-renewal following apical resection little is known about the response to injury in the embryonic heart. METHODS: Injury to the beating zebrafish embryo heart was induced by laser using a transgenic zebrafish expressing cardiomyocyte specific green fluorescent protein. Changes in ejection fraction (EF), heart rate (HR), and caudal vein blood flow (CVBF) assessed by video capture techniques were assessed at 2, 24 and 48 h post-laser. Change in total and mitotic ventricular cardiomyocyte number following laser injury was also assessed by counting respectively DAPI (VCt) and Phospho-histone H3 (VCm) positive nuclei in isolated hearts using confocal microscopy. RESULTS: Laser injury to the ventricle resulted in bradycardia and mild bleeding into the pericardium. At 2 h post-laser injury, there was a significant reduction in cardiac performance in lasered-hearts compared with controls (HR 117 ± 11 vs 167 ± 9 bpm, p ≤ 0.001; EF 14.1 ± 1.8 vs 20.1 ± 1.3%, p ≤ 0.001; CVBF 103 ± 15 vs 316 ± 13 µms(-1), p ≤ 0.001, respectively). Isolated hearts showed a significant reduction in VCt at 2 h post-laser compared to controls (195 ± 15 vs 238 ± 15, p ≤ 0.05). Histology showed necrosis and apoptosis (TUNEL assay) at the site of laser injury. At 24 h post-laser cardiac performance and VCt had recovered fully to control levels. Pretreatment with the cell-cycle inhibitor, aphidicolin, significantly inhibited functional recovery of the ventricle accompanied by a significant inhibition of cardiomyocyte proliferation. CONCLUSIONS: Laser-targeted injury of the zebrafish embryonic heart is a novel and reproducible model of cardiac injury and repair suitable for pharmacological and molecular studies.

Correction of deformational plagiocephaly in early infancy using the plagio cradle orthotic.

BACKGROUND: The purpose of this study was to assess early treatment of deformational plagiocephaly using the Plagio Cradle, a modifiable cranial orthotic. METHODS: Infants were included if they had treatment of deformational plagiocephaly with the Plagio Cradle beginning at 4 months or younger. Patients were prospectively stratified by the age treatment was initiated: group 1: under 10 weeks (n = 50); group 2: 10 to 20 weeks (n = 113). Pretreatment and posttreatment calvarial asymmetry was measured using direct anthropometry and reported as a transcranial difference (TCD). The end point for therapy was a TCD of 5 mm or less, falling within 2 SDs of published normative data. RESULTS: One hundred sixty-three infants were included. Initial TCD was significantly higher for group 1 as compared with group 2 (initial TCD: 11.0 vs 9.0 mm; P < 0.05). Duration of therapy was significantly longer for group 1 as compared with group 2 (6.9 vs 5.7 week; P < 0.05). Following cradle use, group 1 infants demonstrated a significantly larger change in TCD in comparison to group 2 (change in TCD: 6.0 vs 4.0 mm; P < 0.001). At the conclusion of therapy, group 1 infants trended toward greater calvarial symmetry than group 2 patients (final TCD: 4.5 vs 5.0 mm; P = 0.06) and a higher frequency of cases with full correction of asymmetry (62.4% vs 52.2%; P = 0.16). CONCLUSIONS: The Plagio Cradle can fully correct deformational plagiocephaly early in life. Nevertheless, treatment is more effective if initiated before 10 weeks of age.