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Damage initiates a pleiotropic cellular response aimed at cellular survival when appropriate. To identify genes required for damage survival, we used a cell-based RNAi screen against the Drosophila genome and the alkylating agent methyl methanesulphonate (MMS). Similar studies performed in other model organisms report that damage response may involve pleiotropic cellular processes other than the central DNA repair components, yet an intuitive systems level view of the cellular components required for damage survival, their interrelationship, and contextual importance has been lacking. Further, by comparing data from different model organisms, identification of conserved and presumably core survival components should be forthcoming. We identified 307 genes, representing 13 signaling, metabolic, or enzymatic pathways, affecting cellular survival of MMS-induced damage. As expected, the majority of these pathways are involved in DNA repair; however, several pathways with more diverse biological functions were also identified, including the TOR pathway, transcription, translation, proteasome, glutathione synthesis, ATP synthesis, and Notch signaling, and these were equally important in damage survival. Comparison with genomic screen data from Saccharomyces cerevisiae revealed no overlap enrichment of individual genes between the species, but a conservation of the pathways. To demonstrate the functional conservation of pathways, five were tested in Drosophila and mouse cells, with each pathway responding to alkylation damage in both species. Using the protein interactome, a significant level of connectivity was observed between Drosophila MMS survival proteins, suggesting a higher order relationship. This connectivity was dramatically improved by incorporating the components of the 13 identified pathways within the network. Grouping proteins into "pathway nodes" qualitatively improved the interactome organization, revealing a highly organized "MMS survival network." We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. A biologically intuitive, highly interconnected MMS survival network was revealed after we incorporated pathway data in our interactome analysis.

To quantify changes in segmented brain volumes over 12 months in children with mucopolysaccharidosis types IIIA and IIIB (MPS IIIA and IIIB). In order to establish suitable outcome measures for clinical trials, twenty-five children greater than 2 years of age were enrolled in a prospective natural history study of MPS IIIA and IIIB at Nationwide Children's Hospital. Data from sedated non-contrast brain 3 T MRIs and neuropsychological measures were reviewed from the baseline visit and at 12-month follow-up. No intervention beyond standard clinical care was provided. Age- and sex-matched controls were gathered from the National Institute of Mental Health Data Archive. Automated brain volume segmentation with longitudinal processing was performed using FreeSurfer. Of the 25 subjects enrolled with MPS III, 17 children (4 females, 13 males) completed at least one MRI with interpretable volumetric data. The ages ranged from 2.8 to 13.7 years old (average 7.2 years old) at enrollment, including 8 with MPS IIIA and 9 with MPS IIIB. At baseline, individuals with MPS III demonstrated reduced cerebral white matter and corpus callosum volumes, but greater volumes of the lateral ventricles, cerebellar cortex, and cerebellar white matter compared to controls. Among the 13 individuals with MPS III with two interpretable MRIs, there were annualized losses or plateaus in supratentorial brain tissue volumes (cerebral cortex −42.10 ± 18.52 cm3/year [mean ± SD], cerebral white matter −4.37 ± 11.82 cm3/year, subcortical gray matter −6.54 ± 3.63 cm3/year, corpus callosum −0.18 ± 0.62 cm3/yr) and in cerebellar cortex (−0.49 ± 12.57 cm3/year), with a compensatory increase in lateral ventricular volume (7.17 ± 6.79 cm3/year). Reductions in the cerebral cortex and subcortical gray matter were more striking in individuals younger than 8 years of age. Greater cerebral cortex volume was associated with higher fine and gross motor functioning on the Mullen Scales of Early Learning, while greater subcortical gray matter volume was associated with higher nonverbal functioning on the Leiter International Performance Scale. Larger cerebellar cortex was associated with higher receptive language performance on the Mullen, but greater cerebellar white matter correlated with worse adaptive functioning on the Vineland Adaptive Behavioral Scales and visual problem-solving on the Mullen. Loss or plateauing of supratentorial brain tissue volumes may serve as longitudinal biomarkers of MPS III age-related disease progression compared to age-related growth in typically developing controls. Abnormally increased cerebellar white matter in MPS III, and its association with worse performance on neuropsychological measures, suggest the possibility of pathophysiological mechanisms distinct from neurodegeneration-associated atrophy that warrant further investigation. [ABSTRACT FROM AUTHOR]

Rationale and Objectives: We describe our preliminary experience using a 2D turbo gradient- and spin-echo (TGSE) diffusion-weighted (DW) pulse sequence with non-Cartesian BLADE trajectory at 3 T in pediatric patients. We compared the TGSE BLADE to conventional DW spin-echo echo-planar imaging (SE-EPI) in pediatric brain imaging, assessing the presence of artifacts from signal pile-ups, geometric distortion, motion, susceptibility from air-tissue interface, shunts and orthodontia, and diagnostic image quality.Materials and Methods: Data were acquired in 53 patients (10.4 ± 7.9 years). All DW imaging data were acquired precontrast, with SE-EPI first. A four-point scale for rating was used-1 (best) and 4 (worst). A neuroradiologist scored the two sequences and further noted whether the TGSE BLADE approach or SE-EPI was preferred in each case. Apparent diffusion coefficients were compared quantitatively between the two sequences in a subset of 16 patients, in 41 separate regions of interests including caudate nucleus, putamen, globus pallidus, thalamus, and pathological areas.Results: In 43.4% of the cases, TGSE BLADE was preferred; in 49.1% of the cases, both sequences were preferred equally. Average scores for SE-EPI were 2.2 ± 0.8 versus TGSE's 1.2 ± 0.4 in assessing diagnostic quality (p < 0.05). Motion artifacts were minimal on both sequences in 92.5% of the cases. In the TGSE BLADE scores, no case received a "4" for significant artifacts with marginally acceptable image quality. Apparent diffusion coefficients values between the two sequences were statistically similar, with a linear regression slope of 0.92 (r2 = 0.97).Conclusion: TGSE BLADE DW imaging exhibited less geometric distortion in the brain and reduced signal pile-ups in areas of high susceptibility than conventional SE-EPI. [ABSTRACT FROM AUTHOR]

Background Purpose Methods Results Conclusion Radiomics refers to the extraction of quantitative information from medical images and is most commonly utilized in oncology to provide ancillary information for solid tumor diagnosis, prognosis, and treatment response. The traditional radiomic pipeline involves segmentation of volumes of interest with comparison to normal brain. In other neurologic disorders, such as epilepsy, lesion delineation may be difficult or impossible due to poor anatomic definition, small size, and multifocal or diffuse distribution. Tuberous sclerosis complex (TSC) is a rare genetic disease in which brain magnetic resonance imaging (MRI) demonstrates multifocal abnormalities with variable imaging and epileptogenic features.The purpose of this study was to develop a radiomic workflow for identification of abnormal brain regions in TSC, using a whole‐brain atlas‐based approach with generation of heatmaps based on signal deviation from normal controls.This was a retrospective pilot study utilizing high‐resolution whole‐brain 3D FLAIR MRI datasets from retrospective enrollment of tuberous sclerosis complex (TSC) patients and normal controls. Subjects underwent MRI including high‐resolution 3D FLAIR sequences. Preprocessing included skull stripping, coregistration, and intensity normalization. Using the Brainnetome and Harvard‐Oxford atlases, brain regions were parcellated into 318 discrete regions. Expert neuroradiologists spatially labeled all tubers in TSC patients using ITK‐SNAP. The pyradiomics toolbox was used to extract 88 radiomic features based on IBSI guidelines, comparing tuber‐affected and non‐tuber‐affected parenchyma in TSC patients, as well as normal brain tissue in control patients. For model training and validation, regions with tubers from 20 TSC patients and 30 normal control subjects were randomly divided into two training sets (80%) and two validation sets (20%). Additional model testing was performed on a separate group of 20 healthy controls. LASSO (least absolute shrinkage and selection operator) was used to perform variable selection and regularization to identify regions containing tubers. Relevant radiomic features selected by LASSO were combined to produce a radiomic score ω, defined as the sum of squared differences from average control group values. Region‐specific ω scores were converted to heat maps and spatially coregistered with brain MRI to reflect overall radiomic deviation from normal.The proposed radiomic workflow allows for quantification of deviation from normal in 318 regions of the brain with the use of a summative radiomic score ω. This score can be used to generate spatially registered heatmaps to identify brain regions with radiomic abnormalities. The pilot study of TSC showed radiomic scores ω that were statistically different in regions containing tubers from regions without tubers/normal brain (p < 0.0001). Our model exhibits an AUC of 0.81 (95% confidence interval: 0.78–0.84) on the testing set, and the best threshold obtained on the training set, when applied to the testing set, allows us to identify regions with tubers with a specificity of 0.91 and a sensitivity of 0.60.We describe a whole‐brain atlas‐based radiomic approach to identify abnormal brain regions in TSC patients. This approach may be helpful for identifying specific regions of interest based on relatively greater signal deviation, particularly in clinical scenarios with numerous or poorly defined anatomic lesions. [ABSTRACT FROM AUTHOR]

Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the фC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UASDHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ~6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied. [ABSTRACT FROM AUTHOR]

OBJECTIVE. Functional MRI (fMRI) is clinically used for localization of eloquent cortex before surgical intervention, most commonly motor and language function in patients with tumors or epilepsy. In the pediatric population, special considerations for fMRI relate to limited examination tolerance, small head size, developing anatomy and physiology, and diverse potential abnormalities. In this article, we will highlight pearls and pitfalls of clinical pediatric fMRI including blood oxygenation level-dependent imaging principles, patient preparation, study acquisition, data postprocessing, and examination interpretation. CONCLUSION. Clinical fMRI is indicated for presurgical localization of eloquent cortex in patients with tumors, epilepsy, or other neurologic conditions and requires a solid understanding of technical considerations and data processing. In children, special approaches are needed for patient preparation as well as study design, acquisition, and interpretation. Radiologists should be cognizant of developmental neuroanatomy, causes of neuropathology, and capacity for neuroplasticity in the pediatric population.

Purpose: The aim of the present study was to investigate the molecular basis underlying a nonsyndromic presenile autosomal dominant cataract in a three-generation pedigree. The phenotype was progressive from a peripheral ring-like opacity to a total cataract with advancing age from teenage to adulthood. The visual impairment started as problem in distant vision at the age of 16 years, to diminishing vision by the age of 24., Methods: Clinical interventions included complete ophthalmological examination, a collection of case history, and pedigree details. Blood samples were collected from available family members irrespective of their clinical status. A functional candidate gene approach was employed for PCR screening and sequencing of the exons and their flanking regions of CRYGC, CRYGD, and CRYAA genes. For structural consequences of the mutated alphaA-crystallin we used the bioinformatics tool of the ExPASy server., Results: Sequence analysis of CRYGC and CRYGD genes excluded possible causative mutations but identified known polymorphisms. Sequencing of the exons of the CRYAA gene identified a sequence variation in exon 2 (292 G->A) with a substitution of Gly to Arg at position 98. All three affected members revealed this change but it was not observed in the unaffected father or sister. The putative mutation obliterated a restriction site for the enzyme BstDSI. The same was checked in controls representing the general population of the same ethnicity (n=30) and of randomly selected DNA samples from ophthalmologically normal individuals from the population-based KORA S4 study (n=96). Moreover, the Gly at position 98 is highly conserved throughout the animal kingdom. For the mutant protein, the isoelectric point was raised from pH 5.77 to 5.96. Moreover, an extended alpha-helical structure is predicted in this region., Conclusions: The G98R mutation segregates only in affected family members and is not seen in representative controls. It represents very likely the fourth dominant cataract-causing allele in CRYAA. In all reported alleles the basic amino acid Arg is involved, suggesting the major importance of the net charge of the alphaA-crystallin for functional integrity in the lens.