Ribosome profiling reveals novel regulation of C9ORF72 GGGGCC repeat-containing RNA translation.

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5' cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

Dysregulated molecular pathways in amyotrophic lateral sclerosis-frontotemporal dementia spectrum disorder.

Frontotemporal dementia (FTD), the second most common form of dementia in people under 65 years of age, is characterized by progressive atrophy of the frontal and/or temporal lobes. FTD overlaps extensively with the motor neuron disease amyotrophic lateral sclerosis (ALS), especially at the genetic level. Both FTD and ALS can be caused by many mutations in the same set of genes; the most prevalent of these mutations is a GGGGCC repeat expansion in the first intron of C9ORF72 As shown by recent intensive studies, some key cellular pathways are dysregulated in the ALS-FTD spectrum disorder, including autophagy, nucleocytoplasmic transport, DNA damage repair, pre-mRNA splicing, stress granule dynamics, and others. These exciting advances reveal the complexity of the pathogenic mechanisms of FTD and ALS and suggest promising molecular targets for future therapeutic interventions in these devastating disorders.

GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport.

The GGGGCC (G4C2) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia. The basis for pathogenesis is unknown. To elucidate the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2-repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. We show that these transgenic animals display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.

ALS-linked protein disulfide isomerase variants cause motor dysfunction.

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease.

Optical parameters and hardness of two maxillofacial elastomers after immersion in different solutions of Brazilian green propolis extract.

STATEMENT OF PROBLEM: Maxillofacial elastomers undergo physical and mechanical degradation with disinfecting solutions. Solutions of Brazilian green propolis extract may be suitable alternatives for infection control of maxillofacial prostheses. However, their effects on the properties of the material are unknown. PURPOSE: The purpose of this in vitro study was to evaluate the effect of disinfection with solutions of Brazilian green propolis extract on the transmittance, translucency parameter, contrast ratio, and hardness of 2 maxillofacial elastomers (MDX4-4210 and MED-4014). MATERIAL AND METHODS: Fifty disk-shaped specimens (3×10 mm) of each elastomer were randomly and equally divided into 4 groups of disinfectant agents and 1 control group: 3 separate groups of 11% green propolis extracts including aqueous (PAQ), glycolic (PGL), and alcoholic (PAL), a 2% chlorhexidine gluconate (CHX) group, and the control group of distilled water. Specimens were subjected to disinfection by immersion 3 times a week for 60 days. Color differences (ΔE values) were calculated with CIELab and CIEDE2000 formulas. Optical parameters and Shore A hardness were determined at 2 time points: at baseline and after the period of specimen disinfection. Data were analyzed by parametric and nonparametric analysis of variance and by multiple-comparison tests (α=.05). RESULTS: The ΔE values of specimens immersed in 11% PAL were not clinically acceptable for either elastomer. Regarding translucency parameter and contrast ratio, the immersion in 11% PAL and 11% PGL resulted in greater opacity and lower translucency of the material. Mean Shore A hardness values were not statistically significantly different at baseline or after 60 days of immersion in the solutions. CONCLUSIONS: The solution of Brazilian green propolis extract tested showed changes in optical parameters. Elastomers immersed in 11% alcoholic green propolis extract showed clinically unacceptable color and translucency changes. All hardness values of the tested elastomers were clinically acceptable after immersion in all tested disinfectant groups.

How long is the ovary relevant for synthesis of steroids after menopause?

This study aimed to determine whether the ovaries synthesize estradiol (E2), testosterone (T), and androstenedione (A) after menopause. The first group (30 patients) underwent surgical menopause (SM) - their ovaries were removed due to a benign condition around the time of menopause. The second group (30 patients) consisted of patients with natural menopause (NM). The E2 median level was 10.0 pg/ml (CI ± 2.18) and 9.5 pg/ml (CI ± 1.63) in the NM and SM groups (p = .69), respectively. The median level of total T was 0.12 ng/ml (CI ± 0.01) and 0.11 ng/ml (CI ± 0.03) in NM and SM, respectively (p = .96). The median level of A was 783.85 pg/ml (CI ± 154.39) and 883.48 pg/dl (CI ± 201.03) in NM and SM, respectively (p = .57). The FAI (free androgen index) was 1.06 (CI ± 0.24) and 1.35 (CI ± 0.68) for NM and SM, respectively (p = .98). We concluded that 5-10 years after menopause the ovaries are no longer relevant for sex steroid synthesis.

Targeted manipulation of the sortilin-progranulin axis rescues progranulin haploinsufficiency.

Progranulin (GRN) mutations causing haploinsufficiency are a major cause of frontotemporal lobar degeneration (FTLD-TDP). Recent discoveries demonstrating sortilin (SORT1) is a neuronal receptor for PGRN endocytosis and a determinant of plasma PGRN levels portend the development of enhancers targeting the SORT1-PGRN axis. We demonstrate the preclinical efficacy of several approaches through which impairing PGRN's interaction with SORT1 restores extracellular PGRN levels. Our report is the first to demonstrate the efficacy of enhancing PGRN levels in iPSC neurons derived from frontotemporal dementia (FTD) patients with PGRN deficiency. We validate a small molecule preferentially increases extracellular PGRN by reducing SORT1 levels in various mammalian cell lines and patient-derived iPSC neurons and lymphocytes. We further demonstrate that SORT1 antagonists and a small-molecule binder of PGRN588₋593, residues critical for PGRN-SORT1 binding, inhibit SORT1-mediated PGRN endocytosis. Collectively, our data demonstrate that the SORT1-PGRN axis is a viable target for PGRN-based therapy, particularly in FTD-GRN patients.

AIDS and jail: social representations of women in freedom deprivation situations.

OBJECTIVE: To graspthe AIDS social representations built by freedom-deprived women. METHOD: Descriptive study with a quali-quantitative approach that involved 174 convicted women in a women's prison in a capital city of the Brazilian northeastern region. Aword-association test was applied in October and November 2014, using AIDS as a stimulus. The corpuswas processed usingIramuteq software. Descending Hierarchical Classification and Correspondence Factor Analysis were applied. RESULTS: The content that comprises the social representation of AIDS was influenced by the prison context, which was pervaded by a lack of assistance, lack of knowledge, discrimination, and suffering that disclosed vulnerability to HIV/AIDS factors such as unprotected sex and object sharing. This underlines the stigma and fear of the illness, in addition to favoring and supporting negative feelings and a sense of rejection. CONCLUSION: To consider the use of this representational amalgam to ensure a comprehensive, contextualized care can help redirect practices, motivate self-care practices, and reduce prejudiced attitudes. OBJETIVO: Apreenderas representações sociais sobre a aids construídas por mulheres privadas de liberdade. MÉTODO: Estudo descritivo, com abordagem quali-quantitativa que envolveu 174 apenadas de Presídio Feminino situado em capital do nordeste brasileiro. Aplicou-se o Teste de Associação Livre de Palavras, em outubro e novembro de 2014, utilizando-se do estímulo aids. O corpus foi processado pelo software Iramuteq, sendo efetuadas a Classificação Hierárquica Descendente e Análise Fatorial de Correspondência. RESULTADOS: Os conteúdos que compõem a representação social sobre aids são influenciados pelo contexto prisional, permeado dedesassistência, desconhecimento, discriminação e condições de sofrimento, revelando fatores de vulnerabilidade ao HIV/Aids como atividade sexual desprotegida e compartilhamento de objetos; reiterando o estigma e o temor à doença; e favorecendo e sustentando sentimentos negativos e de rejeição. CONCLUSÃO:: Considerar este amálgama representacional na garantia de um cuidado integral e contextualizado pode contribuir para redirecionar práticas, motivar condutas de autocuidado e reduzir atitudes preconceituosas.

FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions.

C9ORF72 repeat expansion is the most common genetic mutation in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Abnormal dipeptide repeat proteins (DPRs) generated from repeat-associated non-AUG (RAN) translation of repeat-containing RNAs are thought to be pathogenic; however, the mechanisms are unknown. Here we report that (GR)80 and (PR)80 are toxic in neuronal and non-neuronal cells in Drosophila. In contrast to reported shorter poly(GR) forms, (GR)80 is mostly localized throughout the cytosol without detectable accumulation in the nucleolus, accompanied by suppression of Notch signaling and cell loss in the wing. Some Notch target genes are also downregulated in brains and iPSC-derived cortical neurons of C9ORF72 patients. Increased Notch expression largely suppressed (GR)80-induced cell loss in the wing. When co-expressed in Drosophila, HeLa cells, or human neurons, (GA)80 recruited (GR)80 into cytoplasmic inclusions, partially decreasing the toxicity of (GR)80 and restoring Notch signaling in Drosophila. Thus, different DPRs have opposing roles in cell loss and we identify the Notch pathway as one of the receptor signaling pathways that might be compromised in C9ORF72 FTD/ALS.

Postpartum Treatment With Immunoglobulin Does Not Prevent Relapses of Multiple Sclerosis in the Mother.

Multiple sclerosis (MS) is a chronic, neurological, immune-mediated disease that can worsen in the postpartum period. There is no consensus on the use of immunoglobulin for prevention of disease relapses after delivery. We have shown that the controversial beneficial effect of immunoglobulin given immediately after birth could not be observed in patients with MS.

[Preventive practices in the elderly and vulnerability to HIV]. / Práticas preventivas de idosos e a vulnerabilidade ao HIV.

OBJECTIVE: To know the vulnerability of the elderly to the HIV infection in the context of preventive practices. METHOD: Exploratory qualitative study, lead from December 2012 to May 2013, with 37 nursing Coexistence Groups in João Pessoa--Paraiba. The Focus Group was elected as the research technique, and the empirical material obtained was subjected to a Content Analysis Technique, thematic modality. RESULTS: The elderly recognize the importance of preventive practices, but they face difficulties in its use when their emotional relationships with their partners do not favor preventive behavior, resulting in vulnerability. The elderly showed the population groups most vulnerable to HIV and do not recognize themselves as such. CONCLUSION: The complexity of the various contexts experienced by the elderlies of this study indicate the need for more research that allows advances in the understanding of subjectivity imposed in relations that underlie the aging process and the experience of sexuality in this age group.

Perceptions of interprofessional teamwork in low-acuity settings: a qualitative analysis.

CONTEXT: Working effectively in interprofessional teams is a core competency for all health care professionals, yet there is a paucity of instruments with which to assess the associated skills. Published medical teamwork skills assessment tools focus primarily on high-acuity situations, such as cardiopulmonary arrests and crisis events in operating rooms, and may not generalise to non-high-acuity environments, such as in-patient wards and out-patient clinics. OBJECTIVE: We undertook the current study to explore the constructs underlying interprofessional teamwork in non-high-acuity settings and team members' perspectives of essential teamwork attributes. METHODS: We used an ethnographic approach to study four interprofessional teams in two different low-acuity settings: women's HIV (human immunodeficiency virus) clinics and in-patient paediatric wards. Over a period of 17 months, we collected qualitative data through direct observations, focus groups and individual interviews. We analysed the data using qualitative thematic analysis, following an iterative process: data from our observations (20 hours in total) informed the focus group guide and focus group data informed the interview guide. To enhance the integrity of our analysis, we triangulated data sources and verified themes through member checking. RESULTS: We conducted seven focus groups and 27 individual interviews with a total of 39 study participants representing eight professions. Participants emphasised shared leadership and collaborative decision making, mutual respect, recognition of one's own and others' limitations and strengths, and the need to nurture relationships. Team members also discussed tensions around hierarchy and questioned whether doctor leadership is appropriate for interprofessional teams. Our findings indicate that there are differences in teamwork between low-acuity and high-acuity settings, and also provide insights into potential barriers to effective interprofessional teamwork. CONCLUSIONS: Our study delineates essential elements of teamwork in low-acuity settings, including desirable attributes of team members, thus laying the foundation for the development of an individual teamwork skills assessment tool.

[Young people's conception of HIV/AIDS and the use of condoms in sexual intercourse]. / Concepção de Jovens o HIV/AIDS e o USO de preservation nas relaçôes sexuais.

The aim of this study was to gather knowledge regarding the conception of young people as for HIV/AIDS and the use of condoms in sexual intercourse. Survey conducted in May, 2012, at a public school in the city of João Pessoa, Paraíba, with eleven young people of both sexes. The chosen technique of investigation was a semi-structured interview. Empirical data were organized according to categorical content analysis, the following categories emerging: "AIDS: what young people think", "AIDS prevention methods" and one subcategory "Trust in the partner as a method for HIV/AIDS prevention". As observed, there is knowledge of condom use as a preventive method against HIV/AIDS; trust in the partner and faithfulness were also cited as preventive methods in both sexes. It is suggested that, when investing in national and regional proposals, not only social inequalities are to be considered but especially the local realities of different young people in different national scenarios.

[Matrix support work: difficulties in the scope of basic healthcare]. / Trabalho do apoiador matricial: dificuldades no âmbito da atenção básica em saúde.

Qualitative research under the analysis of contents, thematic modality, aimed to identify the difficulties lived by the matricial supporter in its practice in the Primary Health Care. The scenery of the study were six units of family health located in one of the five Sanitary Districts of João Pessoa-PB. The data collection was performed from August to September 2010, through semi directed interviews, in which ten professionals who worked as matricial supporters participated. According to the speeches the difficulties faced relate to the ignorance of some professionals of the health team toward the function of the matricial supporter in the Basic Health Attention; lack of autonomy and administrative overload. In this sense, it is suggested that a process of reflection about the work of the matricial supporter with the health team aiming to acknowledge which contribution of this professional in the reorganization of the work of the team of basic attention.

The exocyst subunit EXOC2 regulates the toxicity of expanded GGGGCC repeats in C9ORF72-ALS/FTD.

GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this genetic mutation leads to neurodegeneration remains largely unknown. Using CRISPR-Cas9 technology, we deleted EXOC2, which encodes an essential exocyst subunit, in induced pluripotent stem cells (iPSCs) derived from C9ORF72-ALS/FTD patients. These cells are viable owing to the presence of truncated EXOC2, suggesting that exocyst function is partially maintained. Several disease-relevant cellular phenotypes in C9ORF72 iPSC-derived motor neurons are rescued due to, surprisingly, the decreased levels of dipeptide repeat (DPR) proteins and expanded G4C2 repeats-containing RNA. The treatment of fully differentiated C9ORF72 neurons with EXOC2 antisense oligonucleotides also decreases expanded G4C2 repeats-containing RNA and partially rescued disease phenotypes. These results indicate that EXOC2 directly or indirectly regulates the level of G4C2 repeats-containing RNA, making it a potential therapeutic target in C9ORF72-ALS/FTD.

Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons.

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had >1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization, two iPSC lines from each subject were selected, differentiated into postmitotic neurons, and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs, iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover, repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism.