DMSO increases efficiency of genome editing at two non-coding loci

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) has become the tool of choice for genome editing. Despite the fact that it has evolved as a highly efficient means to edit/replace coding sequence, CRISPR/Cas9 efficiency for “clean” editing of non-coding DNA remains low. We set out to introduce a single base-pair substitution in two intronic SNPs at the FTO locus without altering nearby non-coding sequence. Substitution efficiency increased up to 10-fold by treatment of human embryonic stem cells (ESC) with non-toxic levels of DMSO (1%) before CRISPR/Cas9 delivery. Treatment with DMSO did not result in CRISPR/Cas9 off-target effects or compromise the chromosomal stability of the ESC. Twenty-four hour treatment of human ESC with DMSO before CRISPR/Cas9 delivery may prove a simple means to increase editing efficiency of non-coding DNA without incorporation of undesirable mutations

O TRABALHO NA EDUCAÇÃO EM SAÚDE SOB A PERSPECTIVA DA ERGOLOGIA DE YVES SCHWARTZ

Este estudo é parte de uma pesquisa que objetivou compreender a percepção dos trabalhadores da saúde sobre suas práticas em educação em saúde. Compõe uma pesquisa-ação de abordagem qualitativa, realizada com 39 trabalhadores de diversas categorias atuantes em equipes da Estratégia de Saúde da Família de Blumenau/SC. Realizou-se 8 grupos focais gravados em áudio e transcritos. O conteúdo foi analisado à luz dos pressupostos da Ergologia de Yves Schwartz. Os trabalhadores demonstraram dificuldade em apontar o referencial teórico-metodológico para o trabalho com educação em saúde, enfatizaram a utilização de instrumentos funcionais às experiências. Identificaram as diferenças entre o trabalho prescrito e o trabalho real. A pesquisa se constituiu em espaço de debate de normas e práticas dos trabalhadores da saúde. Concluiu-se com a necessidade de criar espaços de debates das normas e renormalização da atividade laboral no serviço de saúde

i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs

Genetic Studies of Leptin Concentrations Implicate Leptin in the Regulation of Early Adiposity.

Leptin influences food intake by informing the brain about the status of body fat stores. Rare LEP mutations associated with congenital leptin deficiency cause severe early-onset obesity that can be mitigated by administering leptin. However, the role of genetic regulation of leptin in polygenic obesity remains poorly understood. We performed an exome-based analysis in up to 57,232 individuals of diverse ancestries to identify genetic variants that influence adiposity-adjusted leptin concentrations. We identify five novel variants, including four missense variants, in LEP, ZNF800, KLHL31, and ACTL9, and one intergenic variant near KLF14. The missense variant Val94Met (rs17151919) in LEP was common in individuals of African ancestry only, and its association with lower leptin concentrations was specific to this ancestry (P = 2 × 10-16, n = 3,901). Using in vitro analyses, we show that the Met94 allele decreases leptin secretion. We also show that the Met94 allele is associated with higher BMI in young African-ancestry children but not in adults, suggesting that leptin regulates early adiposity

Single-cell genomics reveals region-specific developmental trajectories underlying neuronal diversity in the human hypothalamus.

The development and diversity of neuronal subtypes in the human hypothalamus has been insufficiently characterized. To address this, we integrated transcriptomic data from 241,096 cells (126,840 newly generated) in the prenatal and adult human hypothalamus to reveal a temporal trajectory from proliferative stem cell populations to mature hypothalamic cell types. Iterative clustering of the adult neurons identified 108 robust transcriptionally distinct neuronal subtypes representing 10 hypothalamic nuclei. Pseudotime trajectories provided insights into the genes driving formation of these nuclei. Comparisons to single-cell transcriptomic data from the mouse hypothalamus suggested extensive conservation of neuronal subtypes despite certain differences in species-enriched gene expression. The uniqueness of hypothalamic neuronal lineages was examined developmentally by comparing excitatory lineages present in cortex and inhibitory lineages in ganglionic eminence, revealing both distinct and shared drivers of neuronal maturation across the human forebrain. These results provide a comprehensive transcriptomic view of human hypothalamus development through gestation and adulthood at cellular resolution

Inverse mismatch and lesion growth in small subcortical ischaemic stroke

OBJECTIVE: Infarction typically develops within the borders of an initial hypoperfused tissue. We prospectively investigated whether in small subcortical stroke patients infarct growth can occur beyond the margins of the affected vascular territories. METHODS: In 19 consecutive patients, stroke MRI was performed within 14 h after ictus, and at days 2 and 6 (± 1). Size of diffusion and perfusion disturbances were determined. Infarct volume measured on T2-weighted images on day 6 was considered as imaging endpoint. RESULTS: At the initial examination, the mean diffusion lesion [apparent diffusion coefficient (ADC) lesion size, 1.82 ± 1.2 ml] was larger (p = 0.0002) than the perfusion lesion [mean transit time (MTT) lesion size, 0.72 ± 0.69 ml]. Such an "inverse mismatch" (ADC lesion > MTT lesion) was present in 14/19 patients at baseline and in all patients on day 2. Final lesion volume at day 6 was 3.2 ± 1.6 ml which was larger than the initial perfusion deficit (p = 0.02). CONCLUSION: In small subcortical ischaemic stroke "inverse mismatch" is frequent and infarction develops beyond the initial perfusion disturbance. This indicates that cytotoxic processes probably triggered by the infarct core are a dominant mechanism for lesion growth. Areas with normal perfusion but which are threatened by cytotoxic damage developing over several days seem prime targets for neuroprotective therapy

Correctly targeted ESC clones are pluripotent.

<p>Immunofluorescence of correctly targeted ESC clone rs1421085 (C/C) showing pluripotency molecular markers homeobox transcription factor NANOG, octamer-binding homeodomain transcription factor 4 (OCT4), glycoprotein TRA-1-80, Stage-Specific Embryonic Antigen-4 (SSEA-4), and transcription factor SRY (sex determining region Y)-box 2 (SOX2). Pluripotency markers are shown in green, nuclei are counterstained with DAPI.</p

Quality control measures of correctly targeted clones.

<p>(A, B) Results from Sanger sequencing. The SNPs rs9940128 (near rs1421085) and rs4783819 (near rs8050136) were amplified and sequenced in the same read to control for possible long deletions in the correctly targeted ESC clones. (C) Two representative karyotypic images. All correctly targeted clones tested displayed a normal karyotype.</p

Schematic representation of the <i>FTO</i> genomic locus (chr16:53,703,963–54,121,941).

<p>(A) SNPs rs1421085 (C/T) and rs8050136 (A/C) are located in the first intron of <i>FTO</i>. (B, C) CRISPR/Cas9 technology was employed to convert ESC line H9 (heterozygous for both SNPs) to homozygosity for both alleles at rs1421085 (C/C or T/T) or rs8050136 (C/C or A/A). Positions of gRNA, PAM sequence and ssODN are indicated by thick lines in blue, purple and black, respectively. SNPs are given in green. Predicted Cas9 cut sites are indicated by red arrow heads.</p