Quantitative measures of motor development in Angelman syndrome.

Angelman Syndrome is a rare neurodevelopmental disorder characterized by developmental delay, lack of speech, seizures, intellectual disability, characteristic behavior, and movement disorders. Clinical gait analysis provides the opportunity for movement quantification to investigate an observed maladaptive change in gait pattern and offers an objective outcome of change. Pressure-sensor-based technology, inertial and activity monitoring, and instrumented gait analysis (IGA) were employed to define motor abnormalities in Angelman syndrome. Temporal-spatial gait parameters of persons with Angelman Syndrome (pwAS) show deficiencies in gait performance through walking speed, step length, step width, and walk ratio. pwAS walk with reduced step lengths, increased step width, and greater variability. Three-dimensional motion kinematics showed increased anterior pelvic tilt, hip flexion, and knee flexion. PwAS have a walk ratio more than two standard deviations below controls. Dynamic electromyography showed prolonged activation of knee extensors, which was associated with a decreased range of motion and the presence of hip flexion contractures. Use of multiple gait tracking modalities revealed that pwAS exhibit a change in gait pattern to a flexed knee gait pattern.  Cross-sectional studies of individuals with AS show a regression toward this maladaptive gait pattern over development in pwAS ages 4-11. PwAS unexpectedly did not have spasticity associated with change in gait pattern. Multiple quantitative measures of motor patterning may offer early biomarkers of gait decline consistent with critical periods of intervention, insight into appropriate management strategies, objective primary outcomes, and early indicators of adverse events.

Creation of an advancing adult and pediatric neurology resident EEG curriculum.

BACKGROUND: The Accreditation Council for Graduate Medical Education (ACGME) milestones state that neurology residents should be able to "interpret common EEG abnormalities, recognize normal EEG variants, and create a report." Yet, recent studies have shown that only 43% of neurology residents express confidence in interpreting EEG without supervision and can recognize less than half of normal and abnormal EEG patterns. Our objective was to create a curriculum to improve both confidence and competence in reading EEGs. METHODS: At Vanderbilt University Medical Center (VUMC), adult and pediatric neurology residents have required EEG rotations in their first and second years of neurology residency and can choose an EEG elective in their third year. A curriculum consisting of specific learning objectives, self-directed modules, EEG lectures, epilepsy-related conferences, supplemental educational material, and tests was created for each of the three years of training. RESULTS: Since the implementation of an EEG curriculum at VUMC from September 2019 until November 2022, 12 adult and 21 pediatric neurology residents completed pre- and post-rotation tests. Among the 33 residents, there was a statistically significant improvement in post-rotation test scores, with a mean score improvement of 17% (60.0 ± 12.9 to 77.9 ± 11.8, n = 33, p < 0.0001). When differentiated by training, the mean improvement of 18.8% in the adult cohort was slightly higher than in the pediatric cohort, 17.3%, though it was not significantly different. Overall improvement was significantly increased in the junior resident cohort with a 22.6% improvement in contrast to 11.5% in the senior resident cohort (p = 0.0097 by Student's t-test, n = 14 junior residents and 15 senior residents). DISCUSSION: With the creation of an EEG curriculum specific to each year of neurology residency, adult and pediatric neurology residents demonstrated a statistically significant mean improvement between pre- and post-rotation test scores. The improvement was significantly higher in junior residents in contrast to senior residents. Our structured and comprehensive EEG curriculum objectively improved EEG knowledge in all neurology residents at our institution. The findings may suggest a model which other neurology training programs may consider for the implementation of a similar curriculum to both standardize and address gaps in resident EEG education.

Prevention of premature death and seizures in a Depdc5 mouse epilepsy model through inhibition of mTORC1.

Mutations in DEP domain containing 5 (DEPDC5) are increasingly appreciated as one of the most common causes of inherited focal epilepsy. Epilepsies due to DEPDC5 mutations are often associated with brain malformations, tend to be drug-resistant, and have been linked to an increased risk of sudden unexplained death in epilepsy (SUDEP). Generation of epilepsy models to define mechanisms of epileptogenesis remains vital for future therapies. Here, we describe a novel mouse model of Depdc5 deficiency with a severe epilepsy phenotype, generated by conditional deletion of Depdc5 in dorsal telencephalic neuroprogenitor cells. In contrast to control and heterozygous mice, Depdc5-Emx1-Cre conditional knockout (CKO) mice demonstrated macrocephaly, spontaneous seizures and premature death. Consistent with increased mTORC1 activation, targeted neurons were enlarged and both neurons and astrocytes demonstrated increased S6 phosphorylation. Electrophysiologic characterization of miniature inhibitory post-synaptic currents in excitatory neurons was consistent with impaired post-synaptic response to GABAergic input, suggesting a potential mechanism for neuronal hyperexcitability. mTORC1 inhibition with rapamycin significantly improved survival of CKO animals and prevented observed seizures, including for up to 40 days following rapamycin withdrawal. These data not only support a primary role for mTORC1 hyperactivation in epilepsy following homozygous loss of Depdc5, but also suggest a developmental window for treatment which may have a durable benefit for some time even after withdrawal.

Electroencephalogram background and head ultrasound together stratify seizure risk in neonates undergoing hypothermia.

The benefits of continuous electroencephalography (cEEG) monitoring in the intensive care unit (ICU) are increasingly appreciated, though expanding indications for cEEG may strain resources. The current standard of care in babies with hypoxic-ischemic encephalopathy (HIE) undergoing therapeutic hypothermia (TH) includes cEEG monitoring throughout the entire TH and rewarming process (at least 72 h). Recent cEEG data demonstrate that most seizures occur within the first 24 h of monitoring. We hypothesized that abnormal head imaging and EEG background could stratify seizure risk in babies with HIE undergoing TH to identify candidates for early cEEG discontinuation. In this retrospective review of 126 neonates undergoing TH and cEEG, we identified seizures in 38 (30%) neonates, 33 (87%) of whom seized within the first 24 h of cEEG monitoring. EEG background was graded and demonstrated that 90% of neonates with seizures had a moderately/markedly abnormal background versus 33% of neonates who did not seize (p < 0.0001). Additionally, while head ultrasound (HUS) obtained before EEG did not stratify seizure risk alone, no neonates with both a normal/mildly abnormal EEG background and a normal HUS (0/25) experienced seizures in contrast to 60% (24/40) neonates with both an abnormal EEG background and an abnormal HUS (p < 0.0001). Our data suggest that neonates with abnormal EEG backgrounds and abnormal HUS should be monitored for seizures throughout TH and rewarming, while neonates with normal/mildly abnormal EEG backgrounds and normal HUS are at low risk of seizures after 24 h of monitoring, and thus would be candidates for early cEEG discontinuation.

Nutritional Formulation for Patients with Angelman Syndrome: A Randomized, Double-Blind, Placebo-Controlled Study of Exogenous Ketones.

BACKGROUND: Angelman syndrome (AS) patients often respond to low glycemic index therapy to manage refractory seizures. These diets significantly affect quality of life and are challenging to implement. These formulations may have benefits in AS even in the absence of biomarkers suggesting ketosis. OBJECTIVES: We aimed to compare an exogenous medical food ketone formulation (KF) with placebo for the dietary management of AS. METHODS: This randomized, double-blind, placebo-controlled, crossover clinical trial was conducted in an academic center from 15 November, 2018 to 6 January, 2020. Thirteen participants with molecularly confirmed AS aged 4-11 y met the criteria and completed the 16-wk study. The study consisted of four 4-wk phases: a baseline phase, a blinded KF or placebo phase, a washout phase, and the crossover phase with alternate blinded KF or placebo. Primary outcomes were safety and tolerability rated by retention in the study and adherence to the formulation. Additional secondary outcomes of safety in this nonverbal population included blood chemistry, gastrointestinal health, seizure burden, cortical irritability, cognition, mobility, sleep, and developmental staging. RESULTS: Data were compared between the baseline, KF, and placebo epochs. One participant exited the trial owing to difficulty consuming the formulation. Adverse events included an increase in cholesterol in 1 subject when consuming KF and a decrease in albumin in 1 subject when consuming placebo. Stool consistency improved with KF consumption, from 6.04 ± 1.61 at baseline and 6.35 ± 1.55 during placebo to 4.54 ± 1.19 during KF (P = 0.0027). Electroencephalograph trends showed a decrease in Δ frequency power during the KF arm and event-related potentials suggested a change in the frontal memory response. Vineland-3 showed improved fine motor skills in the KF arm. CONCLUSIONS: The exogenous KF appears safe. More data are needed to determine the utility of exogenous ketones as a nutritional approach in children with AS.This trial was registered at clinicaltrials.gov as NCT03644693.

DEPDC5 haploinsufficiency drives increased mTORC1 signaling and abnormal morphology in human iPSC-derived cortical neurons.

Mutations in the DEPDC5 gene can cause epilepsy, including forms with and without brain malformations. The goal of this study was to investigate the contribution of DEPDC5 gene dosage to the underlying neuropathology of DEPDC5-related epilepsies. We generated induced pluripotent stem cells (iPSCs) from epilepsy patients harboring heterozygous loss of function mutations in DEPDC5. Patient iPSCs displayed increases in both phosphorylation of ribosomal protein S6 and proliferation rate, consistent with elevated mTORC1 activation. In line with these findings, we observed increased soma size in patient iPSC-derived cortical neurons that was rescued with rapamycin treatment. These data indicate that human cells heterozygous for DEPDC5 loss-of-function mutations are haploinsufficient for control of mTORC1 signaling. Our findings suggest that human pathology differs from mouse models of DEPDC5-related epilepsies, which do not show consistent phenotypic differences in heterozygous neurons, and support the need for human-based models to affirm and augment the findings from animal models of DEPDC5-related epilepsy.

Spectrum of KV 2.1 Dysfunction in KCNB1-Associated Neurodevelopmental Disorders.

OBJECTIVE: Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel KV 2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. METHODS: We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant KV 2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. RESULTS: Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild-type KV 2.1. Quantification of protein expression also identified variants with reduced total KV 2.1 expression or deficient cell-surface expression. INTERPRETATION: Our study establishes a platform for rapid screening of KV 2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype. ANN NEUROL 2019;86:899-912.

Cerebral aquaporin-4 expression is independent of seizures in tuberous sclerosis complex.

Astrocytes serve many functions in the human brain, many of which focus on maintenance of homeostasis. Astrocyte dysfunction in Tuberous Sclerosis Complex (TSC) has long been appreciated with activation of the mTORC1 signaling pathway resulting in gliosis and possibly contributing to the very frequent phenotype of epilepsy. We hypothesized that aberrant expression of the astrocyte protein aquaporin-4 (AQP4) may be present in TSC and contribute to disease pathology. Characterization of AQP4 expression in epileptic cortex from TSC patients demonstrated a diffuse increase in AQP4. To determine if this was due to exposure to seizures, we examined Aqp4 expression in mouse models of TSC in which Tsc1 or Tsc2 inactivation was targeted to astrocytes or glial progenitors, respectively. Loss of either Tsc1 or Tsc2 from astrocytes resulted in a marked increase in Aqp4 expression which was sensitive to mTORC1 inhibition with rapamycin. Our findings in both TSC epileptogenic cortex and in a variety of astrocyte culture models demonstrate for the first time that AQP4 expression is dysregulated in TSC. The extent to which AQP4 contributes to epilepsy in TSC is not known, though the similarities in AQP4 expression between TSC and temporal lobe epilepsy supports further studies targeting AQP4 in TSC.

Pilot study on renal magnetic resonance diffusion tensor imaging: are quantitative diffusion tensor imaging values useful in the evaluation of children with ureteropelvic junction obstruction?

BACKGROUND: Ureteropelvic junction (UPJ) obstruction is a common cause of renal injury in children. Indications for surgery are still controversial. Currently, there is no threshold to differentiate patients with suspected UPJ obstruction requiring surgery from the ones that do not, or to predict renal outcome after surgery. Several studies have demonstrated that diffusion tensor imaging (DTI) results may correlate with microstructural changes in the kidneys. OBJECTIVE: To evaluate the feasibility of using DTI to identify UPJ obstruction kidneys. MATERIALS AND METHODS: We analyzed functional MR urography (fMRU) with renal DTI (b=0 and b=400, 20 directions, 1.5 Tesla, no respiratory triggering) in 26 kidneys of 19 children (mean age: 6.15 years) by comparing 13 kidneys with UPJ obstruction configuration that underwent pyeloplasty following the fMRU, and 13 anatomically normal age- and gender-matched kidneys. DTI tractography was reconstructed using a fractional anisotropy threshold of 0.10 and an angle threshold of 55°. User-defined regions of interest (ROIs) of the renal parenchyma (excluding collecting system) were drawn to quantify DTI parameters: fractional anisotropy, apparent diffusion coefficient (ADC), track length and track volume. The failure rate was evaluated. RESULTS: All DTI parameters changed with age; fractional anisotropy decreased (P<0.032). Track volume and track length increased (P<0.05). ADC increased with age in normal kidneys (P<0.001) but not in UPJ obstruction kidneys (P=0.11). After controlling for age, the fractional anisotropy (UPJ obstruction mean: 0.18, normal kidney mean: 0.21; P=0.001) and track length (UPJ obstruction mean: 11.9 mm, normal kidney mean: 15.4 mm; P<0.001) were lower in UPJ obstruction vs. normal kidneys. There was a trend toward a higher ADC in UPJ obstruction kidneys vs. normal kidneys (P=0.062). The failure rate in UPJ obstruction kidneys due to technical limitations of DTI was 13/26 (50%). CONCLUSION: We demonstrated that fractional anisotropy is lower in UPJ obstruction than in normal kidneys. It is necessary to improve this technique to increase the success rate and to perform more studies to evaluate if a decrease in fractional anisotropy can differentiate UPJ obstruction kidneys from hydronephrotic kidneys without UPJ obstruction.

Relationship of renal apparent diffusion coefficient and functional MR urography in children with pelvicalyceal dilation.

OBJECTIVE: The aim is to evaluate the age-related changes and relationship of renal apparent diffusion coefficient (ADC) against the morphological and functional changes detected by functional magnetic resonance urography (fMRU) in children with pelvicalyceal dilation, with suspected or known ureteropelvic junction obstruction. MATERIALS AND METHODS: We retrospectively analyzed fMRUs with diffusion-weighted imaging (DWI) of the kidney in 35 subjects (25 males; median age: 7.1 years, range: 0.3-22.7 years) with 70 kidneys (40 with pelvicalyceal dilation and 30 with no pelvicalyceal dilation). Inclusion criteria were pelvicalyceal dilation, the absence of duplex kidneys and no ureteric dilation. DWI was performed with 3 diffusion gradient directions (b values = 0, 200, 500, 800 and 1,000 s/mm2). Metrics for fMRU included calyceal and renal transit times (CTT, RTT), time-to-peak (TTP), differential renal function based on volume (vDRF), Patlak number (pDRF) and combined volume and Patlak number (vpDRF). The grades of pelvicalyceal dilation, cortical thinning and corticomedullary differentiation were evaluated. The relationship between ADC values and the fMRU parameters was analyzed. RESULTS: ADC increases with age in kidneys without pelvicalyceal dilation (R2=0.37, P<0.001). Renal ADC does not correlate with any of the morphological or fMRU parameters (P>0.07). The median ADC of kidneys without pelvicalyceal dilation was 3.73×10-3 mm2/s (range: 2.78-5.37×0-3 mm2/s) and the median ADC of kidneys with pelvicalyceal dilation was 3.82×10-3 mm2/s (range: 2.70-5.70×10-3 mm2/s). There was no correlation between ADC and the absolute differences of vDRF or pDRF (P>0.33). CONCLUSION: Renal ADC does not correlate with morphological and functional results of fMRU changes in children with pelvicalyceal dilation due to suspected or known ureteropelvic junction obstruction.

Myelin volume fraction imaging with MRI.

MRI is a valuable tool to assess myelin during development and demyelinating disease processes. While multiexponential T2 and quantitative magnetization transfer measures correlate with myelin content, neither provides the total myelin volume fraction. In many cases correlative measures are adequate; but to assess microstructure of myelin, (e.g. calculate the g-ratio using MRI), an accurate measure of myelin volume fraction is imperative. Using a volumetric model of white matter, we relate MRI measures of myelin to absolute measures of myelin volume fraction and compare them to quantitative histology. We assess our approach in control mice along with two models of hypomyelination and one model of hypermyelination and find strong agreement between MRI and histology amongst models. This work investigates the sensitivities of MRI myelin measures to changes in axon geometry and displays promise for estimating g-ratio from MRI.

Experimental studies of g-ratio MRI in ex vivo mouse brain.

This study aimed to experimentally evaluate a previously proposed MRI method for mapping axonal g-ratio (ratio of axon diameters, measured to the inner and outer boundary of myelin). MRI and electron microscopy were used to study excised and fixed brains of control mice and three mouse models of abnormal white matter. The results showed that g-ratio measured with MRI correlated with histological measures of myelinated axon g-ratio, but with a bias that is likely due to the presence of non-myelinated axons. The results also pointed to cases where the MRI g-ratio model simplifies to be primarily a function of total myelin content.

Efficacy of artisanal preparations of cannabidiol for the treatment of epilepsy: Practical experiences in a tertiary medical center.

Medically refractory epilepsy continues to be a challenge worldwide, and despite an increasing number of medical therapies, approximately 1 in 3 patients continues to have seizures. Cannabidiol (CBD), one of many constituents of the Cannabis sativa or marijuana plant, has received renewed interest in the treatment of epilepsy. While highly purified CBD awaits Food and Drug Administration (FDA) approval, artisanal formulations of CBD are readily available and are seeing increased use in our patient population. Although randomized controlled trials of CBD are ongoing and promising, data regarding artisanal formulations of CBD are minimal and largely anecdotal. Here, we report a retrospective study to define the efficacy of artisanal CBD preparations in children with epilepsy. Given the known interaction between CBD and clobazam, we also conducted a subgroup comparison to determine if clobazam use was related to any beneficial effects of CBD. Additionally, we compared response rates with CBD and with clobazam alone within an overlapping patient cohort. A pediatric cohort with epilepsy of 108 patients was identified through a medical record search for patients using CBD oil. The addition of CBD resulted in 39% of patients having a >50% reduction in seizures, with 10% becoming seizure-free. The responder rate for clobazam was similar. No patients achieved CBD monotherapy, although the weaning of other antiepileptic drugs (AEDs) became possible in 22% of patients. A comparable proportion had AED additions during CBD therapy. With concomitant use of clobazam, 44% of patients had a 50% reduction in seizures upon addition of CBD compared with 33% in the population not taking clobazam; this difference was not statistically significant. The most common reported side effect of CBD was sedation in less than 4% of patients, all of whom were also taking clobazam. Increased alertness and improved verbal interactions were reported in 14% of patients in the CBD group and 8% of patients in the CBD and clobazam group. Benefits were more marked in the CBD alone group, in contrast to the CBD and clobazam group, but this difference was not statistically significant. In summary, these findings support efficacy of artisanal CBD preparations in seizure reduction with few significant side effects. The response to CBD was independent of concurrent clobazam use, although clobazam may contribute to the sedation seen with concurrent CBD use.

Multi-compartment microscopic diffusion imaging.

This paper introduces a multi-compartment model for microscopic diffusion anisotropy imaging. The aim is to estimate microscopic features specific to the intra- and extra-neurite compartments in nervous tissue unconfounded by the effects of fibre crossings and orientation dispersion, which are ubiquitous in the brain. The proposed MRI method is based on the Spherical Mean Technique (SMT), which factors out the neurite orientation distribution and thus provides direct estimates of the microscopic tissue structure. This technique can be immediately used in the clinic for the assessment of various neurological conditions, as it requires only a widely available off-the-shelf sequence with two b-shells and high-angular gradient resolution achievable within clinically feasible scan times. To demonstrate the developed method, we use high-quality diffusion data acquired with a bespoke scanner system from the Human Connectome Project. This study establishes the normative values of the new biomarkers for a large cohort of healthy young adults, which may then support clinical diagnostics in patients. Moreover, we show that the microscopic diffusion indices offer direct sensitivity to pathological tissue alterations, exemplified in a preclinical animal model of Tuberous Sclerosis Complex (TSC), a genetic multi-organ disorder which impacts brain microstructure and hence may lead to neurological manifestations such as autism, epilepsy and developmental delay.

A revised model for estimating g-ratio from MRI.

A key measure of white matter health is the g-ratio, which is defined as the ratio between the inner axon radius and the outer, myelinated, axon radius. Recent methods have been proposed to measure the g-ratio non-invasively using the relationship between two magnetic resonance imaging (MRI) measures. While this relationship is intuitive, it predicates on the simplifying assumption that g-ratio is constant across axons. Here, we extend the model to account for a distribution of g-ratio values within an imaging voxel, and evaluate this model with quantitative histology from normal and hypomyelinated mouse brains.

Evaluation of diffusion kurtosis imaging in ex vivo hypomyelinated mouse brains.

Diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), and DKI-derived white matter tract integrity metrics (WMTI) were experimentally evaluated ex vivo through comparisons to histological measurements and established magnetic resonance imaging (MRI) measures of myelin in two knockout mouse models with varying degrees of hypomyelination. DKI metrics of mean and radial kurtosis were found to be better indicators of myelin content than conventional DTI metrics. The biophysical WMTI model based on the DKI framework reported on axon water fraction with good accuracy in cases with near normal axon density, but did not provide additional specificity to myelination. Overall, DKI provided additional information regarding white matter microstructure compared with DTI, making it an attractive method for future assessments of white matter development and pathology.

Multi-organ abnormalities and mTORC1 activation in zebrafish model of multiple acyl-CoA dehydrogenase deficiency.

Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa(vu463) ) that has an inactivating mutation in the etfa gene. dxa(vu463) recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa(vu463) zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa(vu463) zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.

Deletion of Rictor in neural progenitor cells reveals contributions of mTORC2 signaling to tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder with severe neurologic manifestations, including epilepsy, autism, anxiety and attention deficit hyperactivity disorder. TSC is caused by the loss of either the TSC1 or TSC2 genes that normally regulate the mammalian target of rapamycin (mTOR) kinase. mTOR exists within two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Loss of either TSC gene leads to increased mTORC1 but decreased mTORC2 signaling. As the contribution of decreased mTORC2 signaling to neural development and homeostasis has not been well studied, we generated a conditional knockout (CKO) of Rictor, a key component of mTORC2. mTORC2 signaling is impaired in the brain, whereas mTORC1 signaling is unchanged. Rictor CKO mice have small brains and bodies, normal lifespan and are fertile. Cortical layering is normal, but neurons are smaller than those in control brains. Seizures were not observed, although excessive slow activity was seen on electroencephalography. Rictor CKO mice are hyperactive and have reduced anxiety-like behavior. Finally, there is decreased white matter and increased levels of monoamine neurotransmitters in the cerebral cortex. Loss of mTORC2 signaling in the cortex independent of mTORC1 can disrupt normal brain development and function and may contribute to some of the neurologic manifestations seen in TSC.

Probing a 3,4'-bis-guanidinium diaryl derivative as an allosteric inhibitor of the Ras pathway.

Mutations in the Ras-pathway occur in 40-45% of colorectal cancer patients and these are refractory to treatment with anti-EGFR-targeted therapies. With this in mind, we have studied novel guanidinium-based compounds with demonstrated ability to inhibit protein kinases. We have performed docking studies with several proteins involved in the Ras-pathway and evaluated 3,4'-bis-guanidinium derivatives as inhibitors of B-Raf. Compound 3, the most potent in this series, demonstrated strong cytotoxicity in (WT)B-Raf colorectal cancer cells and also cells with (V600E)B-Raf mutations. Cell death was induced by apoptosis, detected by cleavage of PARP. Compound 3 also potently inhibited ERK1/2 signalling, inhibited EGFR activation, as well as Src, STAT3 and AKT phosphorylation. Mechanistically, compound 3 did not inhibit ATP binding to B-Raf, but direct assay of B-Raf activity was inhibited in vitro. Summarizing, we have identified a novel B-Raf type-III inhibitor that exhibits potent cellular cytotoxicity.

Optimizing functional MR urography: prime time for a 30-minutes-or-less fMRU.

BACKGROUND: Current protocols for functional MR urography (fMRU) require long scan times, limiting its widespread use. OBJECTIVE: Our goal was to use pre-defined criteria to reduce the number of sequences and thus the examination time without compromising the morphological and functional results. MATERIALS AND METHODS: The standard fMRU protocol in our department consists of eight sequences, including a 17-min dynamic post-contrast scan. Ninety-nine children and young adults (43 male, 56 female, mean age 7 years) were evaluated with this protocol. Each sequence was retrospectively analyzed for its utility and factors that affect its duration. RESULTS: Mean scan time to perform the eight sequences, without including the variable time between sequences, was 40.5 min. Five sequences were categorized as essential: (1) sagittal T2 for planning the oblique coronal plane, (2) axial T2 with fat saturation for the assessment of corticomedullary differentiation and parenchymal thickness, (3) coronal 3-D T2 with fat saturation for multiplanar and 3-D reconstructions, (4) pre-contrast coronal T1 with fat saturation to ensure an appropriate scan prior to injecting the contrast material and (5) the coronal post-contrast dynamic series. Functional information was obtained after 8 min of dynamic imaging in the majority of children. The coronal fat-saturated T2, coronal T1, and post-contrast sagittal fat-saturated T1 sequences did not provide additional information. Because of the effects of pelvicalyceal dilation and ureteropelvic angle on the renal transit time, prone position is recommended, at least in children with high-grade pelvicalyceal dilation. CONCLUSION: Comprehensive fMRU requires approximately 19 min for sequence acquisition. Allowing for time between sequences and motion correction, the total study time can be reduced to about 30 min. Four pre-contrast sequences and a shortened post-contrast dynamic scan, optimally with the child in prone position, are sufficient.