Étude des mécanismes moléculaires induits par Sonic hedgehog lors du guidage axonal des neurones commissuraux de la moelle épinière

Le morphogène Sonic hedgehog (Shh) est requis pour le guidage axonal des neurones commissuraux lors du développement de la moelle épinière, phénomène impliquant des événements de réorganisation du cytosquelette d’actine. Bien qu’il soit généralement admis que le cytosquelette d’actine soit régulé via les petites GTPases de la famille Rho, un effet de Shh sur ces protéines n’a jamais été observé dans aucun contexte physiologique. Nous démontrons que Shh active les petites GTPases Rac1 et Cdc42 et que cette activation est rapide et donc, compatible avec les effets de guidage induits par Shh sur les neurones commissuraux. En parallèle, nous avons étudié l’activation de la protéine Boc, qui est un récepteur de Shh requis pour le guidage axonal des neurones commissuraux. Ces résultats contribuent à raffiner notre compréhension de la transduction cellulaire induite par Shh lors du guidage axonal des neurones commissuraux.Sonic hedgehog (Shh) is required for axon guidance of commissural neurons during spinal cord development, which involves reorganization of the actin cytoskeleton. Even if it is known that this process is regulated by small Rho GTPases, an effect of Shh on these proteins has not been clearly demonstrated. In this study, we show that Shh activates the small GTPases Rac1 and Cdc42. This activation occurs rapidly, which is compatible with the guidance effects of Shh on commissural neurons. In parallel, we characterized the Shh-dependent activation of Boc, which is a Shh receptor required for commissural axon guidance. Taken together, these results help refine our understanding of the signal transduction mediated by Shh during axon guidance of commissural neurons

People living without health insurance in Durham County, North Carolina : an action-oriented community assessment

This document details the methods and findings from an Action-Oriented Community Assessment (AOCA) of people living without health insurance in Durham County, North Carolina. A group of four graduate students from the University of North Carolina School of Public Health conducted this assessment from September 2006 to April 2007 under the direction of preceptors Sarah Covington, the Coordinator of the Partnership for a Healthy Durham, and Tekola Fisseha, Director of Health Education at the Durham County Health Department (DCHD). The overarching goals of this AOCA were to: 1) inform the DCHD’s countywide health assessment with findings on the strengths and needs among people living without health insurance, in general, and those served by Lincoln Community Health Center (LCHC), in particular; and 2) engage the Lincoln community and service providers in discussing AOCA findings to determine concrete action steps for initiating change. We began the process by conducting participant observations of local neighborhoods and community events to define and gain entry into our community of interest. Next, we collected and reviewed secondary data sources for background information and also to inform our interview guides. We spoke with 47 community members and service providers to the uninsured, including 27 individual interviews and two focus groups. Our data analysis revealed nine themes on needs. Of these, the forum planning committee prioritized four as the most important and changeable for discussion at the community forum. The forum was attended by approximately 50 participants and was held at the Lyon Park Community Center in Durham on Thursday, April 19th, from 6:00 – 8:00 p.m. The purpose was to engage community members and service providers in discussing our findings and generating their own action steps. A description of these themes, as well as key action steps that were generated during the forum, are as follows: Access to Healthcare. Many people in Durham are uninsured or underinsured. Because Lincoln is the only healthcare option for many, the extent of healthcare is limited by what Lincoln is able to provide. Action Steps: 1. Begin research and planning within health department for creation of a bumper sticker that has a main telephone number for all Durham services. 2. Plan and host a health fair at a participant’s church. 3. Increase participation in the Access to Healthcare Committee of the Partnership for a Healthy Durham. Crime and Safety. Most people agree that crime and safety are important issues within the community. Although steps have been taken to address these issues, most people feel that something more can be done. Action Steps: 1. Put pressure on church congregations to take a role in making neighborhoods safer. 2. Parents will make efforts to stress the importance of obeying the law to their children. 3. Make a connection with the kids in your neighborhood and community. Education. Community members agree that limited education hinders their ability to obtain adequate employment, health insurance, and a living wage. Action Steps: 1. Increase awareness of educational opportunities available in Durham. 2. Develop programs for parents promoting the importance of education for their children. 3. Increase the variety of educational opportunities in Durham, such as more certificate programs. Race Relations. Durham is rich with diversity. The increasing diversity of the community sometimes results in misunderstandings and conflict between different racial and ethnic groups. These tensions intensify pre-existing racism, which negatively affects daily interactions within the community. Action Steps: 1. Advocate for cultural competency at workplaces. 2. Speak to youth groups about race relations in Durham. 3. Step out of one’s comfort zone. Other domains and themes found, but were not discussed at the community forum were: Transportation: Access to, safety of, and other limitations of available transportation affect people’s ability to access healthcare settings, employment opportunities, and other local resources. Employment: Limited options for employment in the community combined with limited work experience and educational attainment increase the difficulty to obtain adequate employment. Housing: The ability to obtain quality and affordable housing is limited and is a fundamental issue in the community which affects all aspects of life (i.e. employment, transportation, healthcare, etc). Health & Healthy Living: Despite a variety of community resources that promote healthy living, there are also numerous barriers (i.e. limited time and money for proper diet and exercise). Healthy living is often not prioritized by individuals because of these and other more immediate concerns.Master of Public Healt

Propranolol treatment of infantile hemangioma endothelial cells: A molecular analysis

Infantile hemangiomas (IHs) are non-malignant, largely cutaneous vascular tumors affecting approximately 5–10% of children to varying degrees. During the first year of life, these tumors are strongly proliferative, reaching an average size ranging from 2 to 20 cm. These lesions subsequently stabilize, undergo a spontaneous slow involution and are fully regressed by 5 to 10 years of age. Systemic treatment of infants with the non-selective β-adrenergic receptor blocker, propranolol, has demonstrated remarkable efficacy in reducing the size and appearance of IHs. However, the mechanism by which this occurs is largely unknown. In this study, we sought to understand the molecular mechanisms underlying the effectiveness of β blocker treatment in IHs. Our data reveal that propranolol treatment of IH endothelial cells, as well as a panel of normal primary endothelial cells, blocks endothelial cell proliferation, migration, and formation of the actin cytoskeleton coincident with alterations in vascular endothelial growth factor receptor-2 (VEGFR-2), p38 and cofilin signaling. Moreover, propranolol induces major alterations in the protein levels of key cyclins and cyclin-dependent kinase inhibitors, and modulates global gene expression patterns with a particular affect on genes involved in lipid/sterol metabolism, cell cycle regulation, angiogenesis and ubiquitination. Interestingly, the effects of propranolol were endothelial cell-type independent, affecting the properties of IH endothelial cells at similar levels to that observed in neonatal dermal microvascular and coronary artery endothelial cells. This data suggests that while propranolol markedly inhibits hemangioma and normal endothelial cell function, its lack of endothelial cell specificity hints that the efficacy of this drug in the treatment of IHs may be more complex than simply blockage of endothelial function as previously believed

Rapid model-guided design of organ-scale synthetic vasculature for biomanufacturing

Our ability to produce human-scale bio-manufactured organs is critically limited by the need for vascularization and perfusion. For tissues of variable size and shape, including arbitrarily complex geometries, designing and printing vasculature capable of adequate perfusion has posed a major hurdle. Here, we introduce a model-driven design pipeline combining accelerated optimization methods for fast synthetic vascular tree generation and computational hemodynamics models. We demonstrate rapid generation, simulation, and 3D printing of synthetic vasculature in complex geometries, from small tissue constructs to organ scale networks. We introduce key algorithmic advances that all together accelerate synthetic vascular generation by more than 230-fold compared to standard methods and enable their use in arbitrarily complex shapes through localized implicit functions. Furthermore, we provide techniques for joining vascular trees into watertight networks suitable for hemodynamic CFD and 3D fabrication. We demonstrate that organ-scale vascular network models can be generated in silico within minutes and can be used to perfuse engineered and anatomic models including a bioreactor, annulus, bi-ventricular heart, and gyrus. We further show that this flexible pipeline can be applied to two common modes of bioprinting with free-form reversible embedding of suspended hydrogels and writing into soft matter. Our synthetic vascular tree generation pipeline enables rapid, scalable vascular model generation and fluid analysis for bio-manufactured tissues necessary for future scale up and production.Comment: 58 pages (19 main and 39 supplement pages), 4 main figures, 9 supplement figure

New Retinal Pigment Epithelial Cell Model to Unravel Neuroprotection Sensors of Neurodegeneration in Retinal Disease

Retinal pigment epithelial (RPE) cells sustain photoreceptor integrity, and when this function is disrupted, retinal degenerations ensue. Herein, we characterize a new cell line from human RPE that we termed ABC. These cells remarkably recapitulate human eye native cells. Distinctive from other epithelia, RPE cells originate from the neural crest and follow a neural development but are terminally differentiated into “epithelial” type, thus sharing characteristics with their neuronal lineages counterparts. Additionally, they form microvilli, tight junctions, and honeycomb packing and express distinctive markers. In these cells, outer segment phagocytosis, phagolysosome fate, phospholipid metabolism, and lipid mediator release can be studied. ABC cells display higher resistance to oxidative stress and are protected from senescence through mTOR inhibition, making them more stable in culture. The cells are responsive to Neuroprotectin D1 (NPD1), which downregulates inflammasomes and upregulates antioxidant and anti-inflammatory genes. ABC gene expression profile displays close proximity to native RPE lineage, making them a reliable cell system to unravel signaling in uncompensated oxidative stress (UOS) and retinal degenerative disease to define neuroprotection sites.Fil: Asatryan, Aram. State University of Louisiana; Estados UnidosFil: Calandria, Jorgelina M.. State University of Louisiana; Estados UnidosFil: Kautzmann, Marie-Audrey. State University of Louisiana; Estados UnidosFil: Jun, Bokkyoo. State University of Louisiana; Estados UnidosFil: Gordon, William C.. State University of Louisiana; Estados UnidosFil: Do, Khanh V. State University of Louisiana; Estados UnidosFil: Bhattacharjee, S.. State University of Louisiana; Estados UnidosFil: Pham, Thang L.. State University of Louisiana; Estados UnidosFil: Bermúdez, Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Mateos, Melina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Heap, Jessica. State University of Louisiana; Estados UnidosFil: Bazan, Nicolás G.. State University of Louisiana; Estados Unido

Aberrant GlyRS-HDAC6 interaction linked to axonal transport deficits in Charcot-Marie-Tooth neuropathy.

Dominant mutations in glycyl-tRNA synthetase (GlyRS) cause a subtype of Charcot-Marie-Tooth neuropathy (CMT2D). Although previous studies have shown that GlyRS mutants aberrantly interact with Nrp1, giving insight into the disease\u27s specific effects on motor neurons, these cannot explain length-dependent axonal degeneration. Here, we report that GlyRS mutants interact aberrantly with HDAC6 and stimulate its deacetylase activity on α-tubulin. A decrease in α-tubulin acetylation and deficits in axonal transport are observed in mice peripheral nerves prior to disease onset. An HDAC6 inhibitor used to restore α-tubulin acetylation rescues axonal transport deficits and improves motor functions of CMT2D mice. These results link the aberrant GlyRS-HDAC6 interaction to CMT2D pathology and suggest HDAC6 as an effective therapeutic target. Moreover, the HDAC6 interaction differs from Nrp1 interaction among GlyRS mutants and correlates with divergent clinical presentations, indicating the existence of multiple and different mechanisms in CMT2D. Nat Commun 2018 Mar 8; 9(1):1007

Mucosal CD8 T Cell Responses Are Shaped by Batf3-DC After Foodborne Listeria monocytogenes Infection

While immune responses have been rigorously examined after intravenous Listeria monocytogenes (Lm) infection, less is understood about its dissemination from the intestines or the induction of adaptive immunity after more physiologic models of foodborne infection. Consequently, this study focused on early events in the intestinal mucosa and draining mesenteric lymph nodes (MLN) using foodborne infection of mice with Lm modified to invade murine intestinal epithelium (InlAM Lm). InlAM Lm trafficked intracellularly from the intestines to the MLN and were associated with Batf3-independent dendritic cells (DC) in the lymphatics. Consistent with this, InlAM Lm initially disseminated from the gut to the MLN normally in Batf3–/– mice. Activated migratory DC accumulated in the MLN by 3 days post-infection and surrounded foci of InlAM Lm. At this time Batf3–/– mice displayed reduced InlAM Lm burdens, implicating cDC1 in maximal bacterial accumulation in the MLN. Batf3–/– mice also exhibited profound defects in the induction and gut-homing of InlAM Lm-specific effector CD8 T cells. Restoration of pathogen burden did not rescue antigen-specific CD8 T cell responses in Batf3–/– mice, indicating a critical role for Batf3 in generating anti-InlAM Lm immunity following foodborne infection. Collectively, these data suggest that DC play diverse, dynamic roles in the early events following foodborne InlAM Lm infection and in driving the establishment of intestinal Lm-specific effector T cells.Fil: Imperato, Jessica Nancy. Stony Brook University Renaissance School Of Medicine; Estados UnidosFil: Xu, Daqi. Uconn Health; Estados UnidosFil: Romagnoli, Pablo Alberto. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigacion Medica Mercedes y Martin Ferreyra. Grupo Vinculado Centro de Investigacion En Medicina Traslacional Severo R. Amuchastegui - Cimetsa | Universidad Nacional de Cordoba. Instituto de Investigacion Medica Mercedes y Martin Ferreyra. Grupo Vinculado Centro de Investigacion En Medicina Traslacional Severo R. Amuchastegui - Cimetsa | Instituto de Investigacion Medica Mercedes y Martin Ferreyra. Instituto de Investigacion Medica Mercedes y Martin Ferreyra. Grupo Vinculado Centro de Investigacion En Medicina Traslacional Severo R. Amuchastegui - Cimetsa.; ArgentinaFil: Qiu, Zhijuan. Stony Brook University Renaissance School Of Medicine; Estados UnidosFil: Perez, Pedro. Stony Brook University Renaissance School Of Medicine; Estados UnidosFil: Khairallah, Camille. Stony Brook University Renaissance School Of Medicine; Estados UnidosFil: Pham, Quynh Mai. Uconn Health; Estados UnidosFil: Andrusaite, Anna. University of Glasgow; Reino UnidoFil: Bravo Blas, Alberto. The Beatson Institute For Cancer Research; Reino UnidoFil: Milling, Simon W. F.. University of Glasgow; Reino UnidoFil: Lefrancois, Leo. Uconn Health; Estados UnidosFil: Khanna, Kamal M.. University of New York; Estados UnidosFil: Puddington, Lynn. Uconn Health; Estados UnidosFil: Sheridan, Brian S.. Stony Brook University Renaissance School Of Medicine; Estados Unido