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Background and Purpose Posterior cerebral artery occlusion (PCAo) can cause long-term disability, yet randomized controlled trials to guide optimal reperfusion strategy are lacking. We compared the outcomes of PCAo patients treated with endovascular thrombectomy (EVT) with or without intravenous thrombolysis (IVT) to patients treated with IVT alone. Methods From the multicenter retrospective Posterior cerebraL ArTery Occlusion (PLATO) registry, we included patients with isolated PCAo treated with reperfusion therapy within 24 hours of onset between January 2015 and August 2022. The primary outcome was the distribution of the modified Rankin Scale (mRS) at 3 months. Other outcomes comprised 3-month excellent (mRS 0–1) and independent outcome (mRS 0–2), early neurological improvement (ENI), mortality, and symptomatic intracranial hemorrhage (sICH). The treatments were compared using inverse probability weighted regression adjustment. Results Among 724 patients, 400 received EVT+/-IVT and 324 IVT alone (median age 74 years, 57.7% men). The median National Institutes of Health Stroke Scale score on admission was 7, and the occluded segment was P1 (43.9%), P2 (48.3%), P3–P4 (6.1%), bilateral (1.0%), or fetal posterior cerebral artery (0.7%). Compared to IVT alone, EVT+/-IVT was not associated with improved functional outcome (adjusted common odds ratio [OR] 1.07, 95% confidence interval [CI] 0.79–1.43). EVT increased the odds for ENI (adjusted OR [aOR] 1.49, 95% CI 1.05–2.12), sICH (aOR 2.87, 95% CI 1.23–6.72), and mortality (aOR 1.77, 95% CI 1.07–2.95). Conclusion Despite higher odds for early improvement, EVT+/-IVT did not affect functional outcome compared to IVT alone after PCAo. This may be driven by the increased risk of sICH and mortality after EVT.

A common genetic risk factor for bipolar disorder is CACNA1C, a gene that is also critical for cardiac rhythm. The impact of CACNA1C mutations on bipolar patient cardiac rhythm is unknown. Here, we report the cardiac electrophysiological implications of a bipolar disorder–associated genetic risk factor in CACNA1C using patient induced pluripotent stem cell-derived cardiomyocytes. Results indicate that the CACNA1C bipolar disorder–related mutation causes cardiac electrical impulse conduction slowing mediated by impaired intercellular coupling via connexin 43 gap junctions. In vitro gene therapy to restore connexin 43 expression increased cardiac electrical impulse conduction velocity and protected against thioridazine-induced QT prolongation. Patients positive for bipolar disorder CACNA1C genetic risk factors may have elevated proarrhythmic risk for adverse events in response to psychiatric medications that slow conduction or prolong the QT interval. This in vitro diagnostic tool enables cardiac testing specific to patients with psychiatric disorders to determine their sensitivity to off-target effects of psychiatric medications.

Introduction Vertebral artery stenting represents a viable option in treating symptomatic vertebral artery atherosclerotic stenosis. However, its safety and efficacy in improving neurological status and reducing recurrent strokes remain to be established. Methods We systematically searched PubMed, Embase, and Cochrane databases using keywords with Boolean operators to increase search sensitivity and specificity (‘extracranial’; ‘stenting’; ‘vertebral artery’; ‘stenosis’). We included articles reporting patients > 18 years old with symptomatic extracranial vertebral artery stenoses due to atherosclerosis treated with stenting (with or without angioplasty). Those reporting patients with vertebral dissections were excluded. Patients’ characteristics, procedural details, complications, and outcomes were extracted. Estimated rates weighted for sample size were generated for each variable using fixed and random effects models. Results From 89 unique studies identified, 21 met our inclusion criteria and were included in the analysis, comprising 1499 patients with 1570 lesions. The mean age was 65 years, and 74.4% were men. The mean baseline NIHSS was 4.6. The most frequent stenosis location was the V1 segment of the vertebral artery (88.4%). Device‐related issues occurred in 1.8%. Periprocedural ischemic and hemorrhagic complications occurred in 2.1% and 1.7%, respectively. Neurological improvement post‐procedure was observed in 84.2%, and the in‐hospital mortality rate was 2.1%. During follow‐up, in‐stent stenosis >50% was observed in 12.8%, and retreatment with either re‐stenting or angioplasty was performed in 10.8%. Ischemic complications during follow‐up occurred in 5%, and the overall mortality rate was 5%. Conclusion Stenting for the treatment of atherosclerotic vertebral artery stenosis is technically feasible, associated with a high rate of neurological improvement and low short and long‐term complication rates.

Introduction Previous studies did not show a difference in terms of safety and efficacy among first‐line stent retriever (SR), contact aspiration (CA), or combined techniques for proximal large vessel occlusion strokes. However, the optimal reperfusion therapy in patients with primary distal medium vessel occlusion (DMVO) strokes is uncertain. We aimed to compare the clinical and procedural outcomes among first‐line SR, CA and combined techniques in patients with primary DMVO. Methods This is a retrospective analysis of a prospectively maintained database from 14 comprehensive stroke centers in the US and Europe. Patients were included if they had a primary DMVO stroke due to MCA‐M3/M4, ACA‐A1/A2‐3, or PCA‐P1/P2‐3 and underwent mechanical thrombectomy with first‐line SR, CA, or combined technique. The primary outcome was FPE defined as eTICI 2c/3 on the first pass. Secondary outcomes included mFPE defined as eTICI 2b/3 on the first pass, successful reperfusion defined as eTICI 2b/3 at the end of the procedure. Clinical outcomes included 90‐day mRS0‐1 and 90‐day mRS0‐2. Safety measures included procedural complications, symptomatic intracranial hemorrhage (sICH), and 90‐day mortality. The secondary analysis aimed to identify the procedural and clinical outcomes in 3 matched cohorts: (SR vs. CA), (SR vs. combined technique), and (CA vs. combined technique). Results A total of 365 patients were eligible for analysis; 38.1% were female, with a mean age of 69.3 years and a median NIHSS score of 11 [7‐18]. The first‐line SR group consisted of n=74 (20.3%), CA group n=142 (38.9%), and combined technique group n=149 (40.8%). Patients with first‐line SR or combined technique had a lower median NIHSS score, less frequent MCA‐M3 segment occlusion, and less usage of the rescue strategy compared to those with first‐line CA, P

Summary: hiPSC-CMs are being considered by the Food and Drug Administration and other regulatory agencies for in vitro cardiotoxicity screening to provide human-relevant safety data. Widespread adoption of hiPSC-CMs in regulatory and academic science is limited by the immature, fetal-like phenotype of the cells. Here, to advance the maturation state of hiPSC-CMs, we developed and validated a human perinatal stem cell-derived extracellular matrix coating applied to high-throughput cell culture plates. We also present and validate a cardiac optical mapping device designed for high-throughput functional assessment of mature hiPSC-CM action potentials using voltage-sensitive dye and calcium transients using calcium-sensitive dyes or genetically encoded calcium indicators (GECI, GCaMP6). We utilize the optical mapping device to provide new biological insight into mature chamber-specific hiPSC-CMs, responsiveness to cardioactive drugs, the effect of GCaMP6 genetic variants on electrophysiological function, and the effect of daily β-receptor stimulation on hiPSC-CM monolayer function and SERCA2a expression.

Background: Infarct volume measured from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices is critical to in vivo stroke models. In this study, we developed an interactive, tunable, software that automatically computes whole-brain infarct metrics from serial TTC-stained brain sections. Methods: Three rat ischemic stroke cohorts were used in this study (Total n = 91 rats; Cohort 1 n = 21, Cohort 2 n = 40, Cohort 3 n = 30). For each, brains were serially-sliced, stained with TTC and scanned on both anterior and posterior sides. Ground truth annotation and infarct morphometric analysis (e.g., brain-Vbrain, infarct-Vinfarct, and non-infarct-Vnon-infarct volumes) were completed by domain experts. We used Cohort 1 for brain and infarct segmentation model development (n = 3 training cases with 36 slices [18 anterior and posterior faces], n = 18 testing cases with 218 slices [109 anterior and posterior faces]), as well as infarct morphometrics automation. The infarct quantification pipeline and pre-trained model were packaged as a standalone software and applied to Cohort 2, an internal validation dataset. Finally, software and model trainability were tested as a use-case with Cohort 3, a dataset from a separate institute. Results: Both high segmentation and statistically significant quantification performance (correlation between manual and software) were observed across all datasets. Segmentation performance: Cohort 1 brain accuracy = 0.95/f1-score = 0.90, infarct accuracy = 0.96/f1-score = 0.89; Cohort 2 brain accuracy = 0.97/f1-score = 0.90, infarct accuracy = 0.97/f1-score = 0.80; Cohort 3 brain accuracy = 0.96/f1-score = 0.92, infarct accuracy = 0.95/f1-score = 0.82. Infarct quantification (cohort average): Vbrain (ρ = 0.87, p

Background Toward development of diagnostics for cryptogenic stroke, we hypothesize that histomic features of stroke blood clots retrieved by mechanical thrombectomy could be used to delineate stroke etiology. Methods Clots were retrieved from patients undergoing thrombectomy, and etiology was determined by the trial of TOAST (Trial of Org 10172 in Acute Stroke Treatment) score. After sectioning and hematoxylin and eosin staining, clot components (red blood cells [RBCs], fibrin–platelet aggregates [FPs], and white blood cells [WBCs]) were segmented on whole slide images. Histomic features were engineered to capture structural distribution of RBC/FP regions, including radiomics, radial composition, and RBC/FP object features. To locally characterize WBCs, textural features derived from nuclear and extranuclear regions were computed from each WBC to define classes, which we summarized into class frequency distributions. Univariate and multivariate statistics were used to identify significant differences in engineered features between large artery atherosclerosis (LAA) and cardioembolic cases. The top 3 significant RBC/FP and WBC features were used to train a complement Naïve Bayes model, which was then used to predict the etiology of cryptogenic cases. Results In our data (n=53), 31 clots were cardioembolic, 8 were LAA, 4 were of strokes of other determined etiology, and 10 were cryptogenic. We identified 17 significant RBC/FP features and 3 significant WBC class frequency distributions that were different between cardioembolic and LAA. A complement Naïve Bayes model accurately classified cardioembolic versus LAA with a validation area under the receiver operating characteristic curve of 0.87±0.03, a performance substantially higher to using clot component percent composition (area under the receiver operating characteristic curve=0.69±0.16) that is the current state‐of‐the‐art. Further, cryptogenic cases were reliably classified as cardioembolic or LAA in cross‐validation analysis. Conclusion We present a first‐of‐its‐kind histomics pipeline to robustly quantify the complex structure and WBC heterogeneity in acute ischemic stroke clots and classify cryptogenic cases. We hope this work begins to pave the way for histopathology biomarkers for stroke etiology diagnosis.

Cloutier et al. discover that human embryonic stem cells (hESCs) cultured with media containing inhibitors of GSK3 proteins undergo erosion of X-chromosome inactivation, which equalizes X-linked gene expression between females and males. The findings inform the faithful culture of hESCs.

OBJECTIVES/GOALS: Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy (PL), we initiated whole-exome sequencing for our patients with PL syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel truncating EBF2 variant. METHODS/STUDY POPULATION: Using 3T3-L1 and human primary subcutaneous preadipocytes, we performed loss-of-function and gene rescue experiments. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen) at 5% CO2. After lentivirus transfection, cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone (0.25µM), insulin (1µg/ml) and isobutyl methylxanthine (0.5 mM). Total RNA was extracted using RNeasy Mini Kit (Qiagen) and cDNA was synthesized using IScript (Bio-Rad). Real-time qPCR was performed using TaqMan probes for Pparg and Fabp4, two key adipogenesis markers. RESULTS/ANTICIPATED RESULTS: Patient was found to carry a heterozygous nonsense mutation in exon 6 of EBF2, causing the premature termination of the protein at amino acid position 165. Adipogenesis was significantly suppressed in 3T3L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed marked reduction of intracellular lipid content and Pparg and Fabp4 expression (>80% reduction). With lentiviral gene transfer, EBF2 fully rescued adipogenic potential, whereas the truncated variant EBF2 did not. Of note, 3T3-L1 cells transfected with the EBF2 variant displayed impaired adipogenesis, suggesting a dominant-negative effect of the EBF2 variant on adipogenesis. We confirmed the dominant effect of the EBF2 variant in human adipocyte differentiation. DISCUSSION/SIGNIFICANCE: Our data suggest that EBF2 is indispensable for adipogenesis. The loss of function and dominant-negative effect of the truncating variant of EBF2 likely plays a pathogenic role in PL. Whole exome sequencing of PL patients and ex-vivo functional analysis help identify novel gene variants and better understand the molecular pathogenesis of PL.

Background Recent widespread adoption of a transradial approach in adults has encouraged its use and expansion in children; however, the safety and feasibility of the transradial approach in the latter has not been established. We evaluated these characteristics in our pediatric case series and compared our results with those in the literature. Methods Our prospectively maintained database was retrospectively searched for consecutive patients ≤18 years of age who underwent diagnostic and interventional neuroangiography through the transradial approach. Patient demographics, indications for the procedure, use of ultrasound guidance, arterial size at the access site, intra‐ and postprocedure complications, and outcomes were recorded. For the literature review, systematic searches of PubMed, MEDLINE, and Embase databases were conducted using keywords with Boolean operators (“radial artery” AND “pediatric”) for studies published in English between January 2000 and September 2021. Continuous variables were reported as means or medians and respective standard deviations and interquartile ranges according to data normality. Categorical variables were reported as frequencies. Results Twenty‐one patients were included in our series (mean age, 16.6±2.23 years, range 9–17 years; male sex, 11 [52.4%]). The transradial approach was used for diagnostic angiography in 15 cases (71.4%) and intervention in 6 (28.6%). Ultrasound guidance was used and a “radial cocktail” (verapamil‐heparin‐nitroglycerin) was given in all cases. Mean radial artery access‐site diameter was 2.2±0.46 mm. Two cases (9.5%) required conversion to femoral access. Two patients (9.5%) suffered reversible vasospasm. No radial artery occlusion or permanent neurologic deficits were recorded. The systematic review showed results similar to ours for vasospasm rates (6.3% and 9.5%, respectively) and procedural outcomes (92% and 91%, respectively). [Correction added on November 8, 2022, after first online publication: In the preceding sentence, the value 7% was changed to 6.3%, and the value 93% was changed to 92%.] Conclusion Our results and the literature review demonstrate that the transradial approach is a safe and feasible option for pediatric patients. Routine use of ultrasound guidance, selection of appropriately sized catheters, and prophylactic use of vasodilators and antispasmodics can help ensure the success of the procedure and limit common access‐site complications.

Background Time to reperfusion is believed to influence clinical outcomes following thrombectomy mainly through reduction of infarct growth. In this study, we aim to understand whether clinical outcomes can be influenced by ischemia duration (penumbral time) independent of postintervention infarct volume by comparing outcomes following thrombectomy in patients with similar (and small) follow‐up infarct volumes. Methods We performed a retrospective analysis of a prospectively maintained large‐vessel occlusion stroke thrombectomy database across 3 US centers. Demographic, clinical, radiological, and outcomes data of patients with anterior circulation large‐vessel occlusion (internal carotid or middle cerebral artery M1) stroke who had a witnessed‐onset stroke, had substantial penumbral volumes, achieved successful reperfusion (modified thrombolysis in cerebral infarction 2b‐3), and had a follow‐up infarct volume of

Abstract Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting “undruggable” regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand–membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

Background Patients presenting with large vessel occlusion and a large ischemic core (>50 mL) have been consistently undersampled in the major endovascular thrombectomy (ET) trials. As such, equipoise exists as to whether ET is associated with improved outcomes over medical therapy alone in this population. Methods Prospectively collected databases from 4 US centers were reviewed to identify patients with baseline ischemic cores >50 mL based on computed tomography perfusion imaging using RAPID software (iSchemaView), who were treated with ET or medical therapy alone between January 2014 and October 2019. Baseline characteristics, procedural information, and clinical follow‐up data were collected. A matched‐control comparison of these patients was performed. Results A total of 167 patients were included, of whom 92 received medical therapy alone and 75 underwent ET. Seventy‐five pairs were obtained after matching for baseline ischemic core volume, National Institutes of Health Stroke Scale score, and age. Rate of 90‐day good outcome was significantly higher in the ET arm (28.4% versus 4.9%, P=0.002). In univariate analysis of ET patients, the rate of good outcome was significantly higher among patients treated within

Background: Studying the relationship between hemodynamics and local intracranial aneurysm (IA) pathobiology can help us understand the natural history of IA. We characterized the relationship between the IA wall appearance, using intraoperative imaging, and the hemodynamics from CFD simulations. Methods: Three-dimensional geometries of 15 IAs were constructed and used for CFD. Two-dimensional intraoperative images were subjected to wall classification using a machine learning approach, after which the wall type was mapped onto the 3D surface. IA wall regions included thick (white), normal (purple-crimson), and thin/translucent (red) regions. IA-wide and local statistical analyses were performed to assess the relationship between hemodynamics and wall type. Results: Thin regions of the IA sac had significantly higher WSS, Normalized WSS, WSS Divergence and Transverse WSS, compared to both normal and thick regions. Thicker regions tended to co-locate with significantly higher RRT than thin regions. These trends were observed on a local scale as well. Regression analysis showed a significant positive correlation between WSS and thin regions and a significant negative correlation between WSSD and thick regions. Conclusion: Hemodynamic simulation results were associated with the intraoperatively observed IA wall type. We consistently found that elevated WSS and WSSNorm were associated with thin regions of the IA wall rather than thick and normal regions.

Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) have been used extensively to model inherited heart diseases, but hiPSC-CM models of ischemic heart disease are lacking. Here, our objective was to generate an hiPSC-CM model of ischemic heart disease. To this end, hiPSCs were differentiated into functional hiPSC-CMs and then purified using either a simulated ischemia media or by using magnetic antibody-based purification targeting the nonmyocyte population for depletion from the cell population. Flow cytometry analysis confirmed that each purification approach generated hiPSC-CM cultures that had more than 94% cTnT+ cells. After purification, hiPSC-CMs were replated as confluent syncytial monolayers for electrophysiological phenotype analysis and protein expression by Western blotting. The phenotype of metabolic stress–selected hiPSC-CM monolayers recapitulated many of the functional and structural hallmarks of ischemic CMs, including elevated diastolic calcium, diminished calcium transient amplitude, prolonged action potential duration, depolarized resting membrane potential, hypersensitivity to chemotherapy-induced cardiotoxicity, depolarized mitochondrial membrane potential, depressed SERCA2a expression, reduced maximal oxygen consumption rate, and abnormal response to β1-adrenergic receptor stimulation. These findings indicate that metabolic selection of hiPSC-CMs generated cell populations with phenotype similar to what is well known to occur in the setting of ischemic heart failure and thus provide a opportunity for study of human ischemic heart disease.

Introduction Rheumatic heart disease (RHD) causes significant morbidity and mortality in young people from disadvantaged populations. Early detection through echocardiography screening can facilitate early access to treatment. Large-scale implementation of screening could be feasible with the combination of inexpensive standalone ultrasound transducers and upskilling non-expert practitioners to perform abbreviated echocardiography.Methods and analysis A prospective cross-sectional study will evaluate an abbreviated echocardiography screening protocol for the detection of latent (asymptomatic) RHD in high-risk populations. The study will evaluate the diagnostic accuracy of health worker conducted single parasternal long axis view with a sweep using handheld devices (SPLASH) (Philips Lumify S4-1 phased array transducer). Each participant will have at least one reference test performed on the same day by an expert echocardiographer. Diagnosis of RHD will be determined by a panel of three experts, using 2012 World Heart Federation criteria. Sensitivity and specificity of the index test will be calculated with 95% CIs, to determine diagnostic accuracy of a screen-and-refer approach to echocardiography screening for RHD. Remote review of SPLASH images obtained by health workers will facilitate evaluation of the sensitivity and specificity of an alternative approach, using external review of health worker obtained SPLASH images to decide onward referral.Ethics and dissemination Ethics approval was obtained from the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research, for the project to be carried out in Timor-Leste (HREC 2019-3399), and in Australia, following review by the Aboriginal Ethics subcommittee (HREC 2019-334). Ethical and technical approval was granted in Timor-Leste, by the Institute National of Health Research Ethics and Technical Committee (1073-MS-INS/GDE/VII/2019). Study results will be disseminated in the communities involved in the study, and through peer-reviewed publications and conference abstracts.Trial registration number The Australia New Zealand Clinical Trials Registry (ACTRN12620000122954).

BackgroundRecently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress.MethodsWe performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling.ResultsWe first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction.ConclusionsCollectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

Background: VWE in contrast-enhanced magnetic resonance imaging (MRI) is a potential biomarker for the evaluation of IA. The common practice to identify IAs with VWE is mainly based on a visual inspection of MR images, which is subject to errors and inconsistencies. Here, we develop and validate a tool for the visualization, quantification and objective identification of regions with VWE. Methods: N = 41 3D T1-MRI and 3D TOF-MRA IA images from 38 patients were obtained and co-registered. A contrast-enhanced MRI was normalized by the enhancement intensity of the pituitary stalk and signal intensities were mapped onto the surface of IA models generated from segmented MRA. N = 30 IAs were used to identify the optimal signal intensity value to distinguish the enhancing and non-enhancing regions (marked by an experienced neuroradiologist). The remaining IAs (n = 11) were used to validate the threshold. We tested if the enhancement area ratio (EAR—ratio of the enhancing area to the IA surface-area) could identify high risk aneurysms as identified by the ISUIA clinical score. Results: A normalized intensity of 0.276 was the optimal threshold to delineate enhancing regions, with a validation accuracy of 81.7%. In comparing the overlap between the identified enhancement regions against those marked by the neuroradiologist, our method had a dice coefficient of 71.1%. An EAR of 23% was able to discriminate high-risk cases with an AUC of 0.7. Conclusions: We developed and validated a pipeline for the visualization and objective identification of VWE regions that could potentially help evaluation of IAs become more reliable and consistent.

Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5′ untranslated region (UTR) of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP’s pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the Clustered Regularly Interspaced Palindromic Repeats/deficient CRISPR associated protein 9 (CRISPR/dCas9) system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCas9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 messenger RNA (mRNA) expression despite promoter and repeat methylation. However, these changes at the transcript level were not coupled with a significant elevation in FMRP protein expression in FXS cells. These studies demonstrate that directing a transcriptional activator to CGG repeats is sufficient to selectively reactivate FMR1 mRNA expression in Fragile X patient stem cells.