Targeted delivery of lysosomal enzymes to the endocytic compartment in human cells using engineered extracellular vesicles.

Targeted delivery of lysosomal enzymes to the endocytic compartment of human cells represents a transformative technology for treating a large family of lysosomal storage diseases (LSDs). Gaucher disease is one of the most common types of LSDs caused by mutations to the lysosomal β-glucocerebrosidase (GBA). Here, we describe a genetic strategy to produce engineered exosomes loaded with GBA in two different spatial configurations for targeted delivery to the endocytic compartment of recipient cells. By fusing human GBA to an exosome-anchoring protein: vesicular stomatitis virus glycoprotein (VSVG), we demonstrate that the chimeric proteins were successfully integrated into exosomes which were secreted as extracellular vesicles (EVs) by producer cells. Isolation and molecular characterization of EVs confirmed that the fusion proteins were loaded onto exosomes without altering their surface markers, particle size or distribution. Further, enzyme-loaded exosomes/EVs added to cultured medium were taken up by recipient cells. Further, the endocytosed exosomes/EVs targeted to endocytic compartments exhibited a significant increase in GBA activity. Together, we have developed a novel method for targeting and delivery of lysosomal enzymes to their natural location: the endocytic compartment of recipient cells. Since exosomes/EVs have an intrinsic ability to cross the blood-brain-barrier, our technology may provide a new approach to treat severe types of LSDs, including Gaucher disease with neurological complications

Towards Inclusion in Museums: Multisensory and Cross-Modal Translations/Interpretations of Visual Artworks

Access to art and cultural works is a fundamental human right, irrespective of abilities and human differences. However, traditional museum experiences heavily rely on visual perception, which creates barriers for visitors—especially for those who are unable to access art through sight. How can visual art be “translated” into other modalities, and what might be their affordances, limitations, and impact? This qualitative investigation focused on a graduate course on multisensory museum experiences embedded within a unique partnership between the Art Gallery of Ontario and OCAD University. Observations and interviews with students, instructors, museum visitors, and stakeholders (including community members with vision impairments and museum professionals) revealed: a range of translation/interpretation strategies, from “literal” (mapping visually perceived spatial properties of artworks to non-visual perceptual modalities) to “constructivist” (non-literal mappings that aim to engender audience memories that are akin to what might have inspired the original artwork); transformative student journeys, such as building meaningful connections with art; and significant impact on diverse audiences and students. This study revealed promising directions for inclusive museums, a preliminary technical language to support the design of translations/ interpretations, and a need for theoretically informed and tested standards to guide these designs and practices

Sorting Nexin 1 Down-Regulation Promotes Colon Tumorigenesis

PURPOSE: Colon cancer is one of the most common human malignancies, yet studies have only begun to identify the multiple mechanisms that underlie the development of this tumor. In this study, we have identified a novel mechanism, dysregulation of endocytic sorting, which promotes colon cancer development. EXPERIMENTAL DESIGN: Immunohistochemical and microarray analyses were done on human colon cancer tissue specimens to determine the levels of one endocytic protein, sorting nexin 1 (SNX1). SW480 cells, a human colon cancer cell line that retains a relatively high level of SNX1 expression, were used to assess the effects of down-regulating this protein by small hairpin RNA. Activation of signal transduction cascades was evaluated in these cells using Western blotting, and multiple functional assays were done. RESULTS: We determined by immunohistochemistry that the level of SNX1 was significantly down-regulated in 75% of human colon cancers. In corroborative studies using microarray analysis, SNX1 message was significantly decreased (log(2) ratio less than -1) for 8 of 19 colon carcinomas. Cell lines with reduced SNX1 levels showed increased proliferation, decreased apoptosis, and decreased susceptibility to anoikis. They also showed increased activation of epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 in response to epidermal growth factor. This increased activation was abolished by inhibition of endocytosis. CONCLUSIONS: These data suggest that loss of SNX1 may play a significant role in the development and aggressiveness of human colon cancer, at least partially through the mechanism of increased signaling from endosomes. Further, these findings suggest that dysregulation of endocytic proteins may represent a new paradigm in the process of carcinogenesis.Fil: Nguyen, Lananh N.. University of Washington; Estados UnidosFil: Holdren, Matthew S.. University of Washington; Estados UnidosFil: Nguyen, Anthony P.. Baylor College of Medicine; Estados UnidosFil: Furuya, Momoko H.. University of Washington; Estados UnidosFil: Bianchini, Michele. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Levy, Estrella Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Mordoh, Jose. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Liu, Annie. University of Washington; Estados UnidosFil: Guncay, Gabriela D.. University of Washington; Estados UnidosFil: Campbell, Jean S.. University of Washington; Estados UnidosFil: Parks, W. Tony. University of Washington; Estados Unido

Scholarly Responses to ‘Students’ experiences of Open Distance Learning: A Samoan case study’

Scholarly Responses to ‘Students’ experiences of Open Distance Learning: A Samoan case study

Three patients with homozygous familial hypercholesterolemia: Genomic sequencing and kindred analysis.

BackgroundHomozygous Familial Hypercholesterolemia (HoFH) is an inherited recessive condition associated with extremely high levels of low-density lipoprotein (LDL) cholesterol in affected individuals. It is usually caused by homozygous or compound heterozygous functional mutations in the LDL receptor (LDLR). A number of mutations causing FH have been reported in literature and such genetic heterogeneity presents great challenges for disease diagnosis.ObjectiveWe aim to determine the likely genetic defects responsible for three cases of pediatric HoFH in two kindreds.MethodsWe applied whole exome sequencing (WES) on the two probands to determine the likely functional variants among candidate FH genes. We additionally applied 10x Genomics (10xG) Linked-Reads whole genome sequencing (WGS) on one of the kindreds to identify potentially deleterious structural variants (SVs) underlying HoFH. A PCR-based screening assay was also established to detect the LDLR structural variant in a cohort of 641 patients with elevated LDL.ResultsIn the Caucasian kindred, the FH homozygosity can be attributed to two compound heterozygous LDLR damaging variants, an exon 12 p.G592E missense mutation and a novel 3kb exon 1 deletion. By analyzing the 10xG phased data, we ascertained that this deletion allele was most likely to have originated from a Russian ancestor. In the Mexican kindred, the strikingly elevated LDL cholesterol level can be attributed to a homozygous frameshift LDLR variant p.E113fs.ConclusionsWhile the application of WES can provide a cost-effective way of identifying the genetic causes of FH, it often lacks sensitivity for detecting structural variants. Our finding of the LDLR exon 1 deletion highlights the broader utility of Linked-Read WGS in detecting SVs in the clinical setting, especially when HoFH patients remain undiagnosed after WES

A data-driven modeling method to analyze cardiomyocyte impedance data

Présentation PosterInternational audienceOne goal of the Comprehensive in vitro ProArrhythmia Assay initiative is to predict more accurately potentially torsadogenic compounds in an earlier stage of drug development. To that aim one of the CiPA component is to assess capabilities of label-free in vitro assays (impedance and extracellular field potential signals) applied to human stem cell-derived cardiomyocytes

Orchestrating Extracellular Vesicle With Dual Reporters for Imaging and Capturing in Mammalian Cell Culture

Background: Recent technological advancements have enabled live-cell imaging of intracellular organelles to monitor their biogenesis in mammalian cells. However, applying this method to gain insight into extracellular organelles, such as extracellular vesicles (EVs), presents unique challenges that require special considerations in design and engineering.Results: We have developed a dual-reporter system that combines genetic fusion, fluorescence microcopy and magnetic beads capture of EVs to study the biogenesis of EVs in mammalian cell cultures. First, we genetically produced a series of reporters by fusing a green fluorescent protein (GFP) and an affinity peptide (6xHis), with either the endogenous transmembrane protein, CD63, or EVs targeting vesicular stomatitis viral glycoprotein (VSVG). Transfection of these reporters into human 293T cells resulted in expression and integration of these reporters into pre-exosome compartments, which were subsequently released into the culture medium. Confocal imaging and nano-particle tracking analysis demonstrated that EVs were appropriately labeled and exhibited a single dominant peak in the 80–110 nm size range, indicating that isolated EVs were comprised of micro-vesicles and/or exosome subpopulations. Incubation of isolated EVs with nickel-coated magnetic beads resulted in successful capture of GFP-positive EVs. Finally, addition of EVs into culture medium was able to reveal the cellular uptake of GFP-labeled EVs by recipient cells. Taken together, our dual-reporter system provides a powerful method for both monitoring and capturing of EVs in mammalian cell culture systems.Conclusion: A dual-reporter system provides a robust tool to study the life cycle of EVs in mammalian cells from biogenesis and excretion to cellular uptake

Energy density and weight change in a long-term weight-loss trial

<p>Abstract</p> <p>Background</p> <p>Health risks linked to obesity and the difficulty most have in achieving weight loss underscore the importance of identifying dietary factors that contribute to successful weight loss.</p> <p>Methods</p> <p>This study examined the association between change in dietary energy density and weight loss over time. Subjects were 213 men and women with BMI of 30–39 kg/m²and without chronic illness enrolled in 2004 in a randomized trial evaluating behavioral treatments for long-term weight loss. Subjects completed a 62-item food frequency questionnaire at baseline and at 6, 12, and 18 months.</p> <p>Results</p> <p>Pearson correlations between BMI and energy density (kcals/g of solid food) at baseline were not significantly different from zero (r = -0.02, p = 0.84). In a longitudinal analysis, change in energy density was strongly related to change in BMI. The estimated β for change in BMI (kg/m²) of those in the quartile representing greatest decrease in energy density at 18 months compared to those in the quartile with the least was -1.95 (p = 0.006). The association was especially strong in the first six months (estimated β = -1.43), the period with greatest weight loss (mean change in BMI = -2.50 kg/m²from 0–6 months <it>vs. </it>0.23 kg/m²from 12–18 months) and the greatest contrast with respect to change in energy density.</p> <p>Conclusion</p> <p>Decreased energy density predicted weight loss in this 18 month weight loss study. These findings may have important implications for individual dietary advice and public health policies targeting weight control in the general population</p

Parcours-travail et cancers professionnels. Recherche-action en Seine Saint Denis (France)

Dans le domaine des cancers d’origine professionnelle, la toxicologie et l’épidémiologie ont apporté depuis longtemps des connaissances permettant d’identifier des relations causales ou des corrélations statistiques entre des substances toxiques et certains cancers. Mais la réalité sociale du travail dans laquelle s’inscrit l’exposition à des cancérogènes professionnels reste inconnue. La démarche de recherche-action pluridisciplinaire qui concerne la surveillance des cancers professionnels en Seine Saint Denis vise à améliorer la connaissance, la reconnaissance et la prévention des cancers d’origine professionnelle dans ce département. Une phase exploratoire réalisée en 2001 dans deux services du centre hospitalier universitaire Avicenne de Bobigny a permis la mise au point d’outils méthodologiques pour cette démarche. L’objectif est de construire une connaissance qualitative des expositions professionnelles à des cancérogènes à partir de la reconstitution des parcours-travail de patients atteints de cancer résidant en Seine Saint Denis.Nous présentons dans cet article la problématique de cette recherche et ses premiers résultats. L’élaboration d’une typologie des parcours-travail de ces patients vise à inscrire l’exposition à des cancérogènes professionnels en référence à l’histoire combinée de l’emploi, de la qualification et de l’activité de travail de ces patients. Le classement des malades de l’étude selon des types de parcours différents permet de dégager une différenciation des histoires d’exposition aux cancérogènes. L’approche par la typologie des parcours-travail permet de surmonter la difficulté d’analyse des expositions cumulées tout au long de la vie professionnelle et de qualifier ces expositions en référence aux objectifs de prévention et de réparation. Il s’agit d’inscrire ces expositions dans un contexte et une histoire individuels et collectifs dans l’espace et dans le temps et d’analyser les conditions de la reconnaissance en maladie professionnelle.In the field of occupational cancer, toxicology and epidemiology have produced a substantial amount of data that have been used to identify the causal relationship and statistical correlation between toxic substances and some types of cancer. However, little information is available on the social reality of work and occupational exposure to carcinogenic agents. A multidisciplinary and proactive scientific study, based on interviews with incident cases, was undertaken in a highly industrialized suburb of Paris. The preliminary study, carried out in the Avicenne hospital (attached to the University of Paris13, Bobigny), allowed us to build methodological tools. Our goal was to collect qualitative data on occupational exposure to carcinogenic agents in the job histories of new patients living in Seine Saint Denis.In this paper, we present out theoretical and methodological approach, along with some results. Exposure to carcinogens was very common in our study group. We identified different types of job histories involving a combination of blue-collar work and occupational exposure to carcinogens. It was then possible to differentiate between cases due to past occupational exposure to carcinogenic agents and those involving workers who are still exposed in their current positions. Where exposure occurs, there is clearly an urgent need for preventive strategies to avoid future cases of cancer. Through the study, we were able to register cumulated and multiple exposure throughout a worker’s job history and to qualify that exposure. The job history study is not necessarily the only way of identifying whether or not patients have been exposed to carcinogenic agents, but it is a way of developing two other kinds of knowledge, namely how the exposure histories fit into an individual and collective history, both spatially and temporally, and how to recognize and compensate for occupational disease procedures - in other words, to see how the compensation system rules are applied and how they can be changed to suit the specific situation of occupational cancer victims.En el ámbito de los cánceres de origen profesional, la toxicología y la epidemiología han proporcionado, desde hace mucho tiempo, conocimientos que permiten identificar las relaciones causales o las correlaciones estadísticas entre las sustancias tóxicas y ciertos cánceres. Pero la realidad social del trabajo en la cual se desarrolla la exposición a carcinógenos profesionales permanece desconocida. El enfoque de investigación proactiva multidisciplinaria a propósito del seguimiento de los cánceres profesionales en Seine Saint Denis tiene por objeto de mejorar el conocimiento, el reconocimiento y la prevención de los cánceres de origen profesional en este distrito de las afueras de París. Una fase exploratoria realizada en 2001 en dos servicios del centro hospitalario universitario Avicenne de Bobigny ha permitido la elaboración de instrumentos metodológicos para este enfoque. El objetivo es construir un conocimiento cualitativo de las exposiciones profesionales a carcinógenos a partir de la reconstitución de los recorridos laborales de pacientes viviendo en Seine Saint Denis que padecen de cáncer.En este artículo, presentamos la problemática de esta investigación y sus primeros resultados. La elaboración de una tipología de los recorridos laborales de estos pacientes tiene por objetivo subrayar la exposición a carcinógenos profesionales en referencia a la historia combinada de su empleo, su calificación y de su actividad de trabajo. La clasificación de los enfermos según los diferentes recorridos permite destacar una diferenciación de las historias de exposición a carcinógenos. El enfoque por tipología de los recorridos laborales permite superar la dificuldad de análisis de las exposiciones cumuladas a lo largo de la vida profesional y de calificar estas exposiciones en relación con los objetivos de prevención y de reparación. Se trata de clasificar estas exposiciones en un contexto y una historia individuales y colectivos en el espacio y en el tiempo y analizar las condiciones de su reconocimiento como enfermedad profesional