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Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the gene SACS, encoding the 520 kDa protein sacsin. Although sacsin's physiological role is largely unknown, its sequence domains suggest a molecular chaperone or protein quality control function. Consequences of its loss include neurofilament network abnormalities, specifically accumulation and bundling of perikaryal and dendritic neurofilaments. To investigate if loss of sacsin affects intermediate filaments more generally, the distribution of vimentin was analysed in ARSACS patient fibroblasts and in cells where sacsin expression was reduced. Abnormal perinuclear accumulation of vimentin filaments, which sometimes had a cage-like appearance, occurred in sacsin-deficient cells. Mitochondria and other organelles were displaced to the periphery of vimentin accumulations. Reorganization of the vimentin network occurs in vitro under stress conditions, including when misfolded proteins accumulate. In ARSACS patient fibroblasts HSP70, ubiquitin and the autophagy-lysosome pathway proteins Lamp2 and p62 relocalized to the area of the vimentin accumulation. There was no overall increase in ubiquitinated proteins, suggesting the ubiquitin-proteasome system was not impaired. There was evidence for alterations in the autophagy-lysosome pathway. Specifically, in ARSACS HDFs cellular levels of Lamp2 were elevated while levels of p62, which is degraded in autophagy, were decreased. Moreover, autophagic flux was increased in ARSACS HDFs under starvation conditions. These data show that loss of sacsin effects the organization of intermediate filaments in multiple cell types, which impacts the cellular distribution of other organelles and influences autophagic activity., (© The Author 2017. Published by Oxford University Press.)

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 enables us to generate targeted sequence changes in the genomes of cells and organisms. However, off-target effects have been a persistent problem hampering the development of therapeutics based on CRISPR/Cas9 and potentially confounding research results. Efforts to improve Cas9 specificity, like the development of RNA-guided FokI-nucleases (RFNs), usually come at the cost of editing efficiency and/or genome targetability. To overcome these limitations, we engineered improved chimeras of RFNs that enable higher cleavage efficiency and provide broader genome targetability, while retaining high fidelity for genome editing in human cells. Furthermore, we developed a new RFN ortholog derived from Staphylococcus aureus Cas9 and characterize its utility for efficient genome engineering. Finally, we demonstrate the feasibility of RFN orthologs to functionally hetero-dimerize to modify endogenous genes, unveiling a new dimension of RFN target design opportunities., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Breastfeeding has been shown to enhance global measures of intelligence in children. However, few studies have examined associations between breastfeeding and specific cognitive task performance in the first 2 y of life, particularly in an Asian population., Objective: We assessed associations between early infant feeding and detailed measures of cognitive development in the first 2 y of life in healthy Asian children born at term., Design: In a prospective cohort study, neurocognitive testing was performed in 408 healthy children (aged 6, 18, and 24 mo) from uncomplicated pregnancies (i.e., birth weight >2500 and <4000 g, gestational age ≥37 wk, and 5-min Apgar score ≥9). Tests included memory (deferred imitation, relational binding, habituation) and attention tasks (visual expectation, auditory oddball) as well as the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III). Children were stratified into 3 groups (low, intermediate, and high) on the basis of breastfeeding duration and exclusivity., Results: After potential confounding variables were controlled for, significant associations and dose-response relations were observed for 4 of the 15 tests. Higher breastfeeding exposure was associated with better memory at 6 mo, demonstrated by greater preferential looking toward correctly matched items during early portions of a relational memory task (i.e., relational binding task: P-trend = 0.015 and 0.050 for the first two 1000-ms time bins, respectively). No effects of breastfeeding were observed at 18 mo. At 24 mo, breastfed children were more likely to display sequential memory during a deferred imitation memory task (P-trend = 0.048), and toddlers with more exposure to breastfeeding scored higher in receptive language [+0.93 (0.23, 1.63) and +1.08 (0.10, 2.07) for intermediate- and high-breastfeeding groups, respectively, compared with the low-breastfeeding group], as well as expressive language [+0.58 (-0.06, 1.23) and +1.22 (0.32, 2.12) for intermediate- and high-breastfeeding groups, respectively] assessed via the BSID-III., Conclusions: Our findings suggest small but significant benefits of breastfeeding for some aspects of memory and language development in the first 2 y of life, with significant improvements in only 4 of 15 indicators. Whether the implicated processes confer developmental advantages is unknown and represents an important area for future research. This trial was registered at www.clinicaltrials.gov as NCT01174875., (© 2015 American Society for Nutrition.)