491 research outputs found

    Function2Gene: A gene selection tool to increase the power of genetic association studies by utilizing public databases and expert knowledge

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    <p>Abstract</p> <p>Background</p> <p>Many common disorders have multiple genetic components which convey increased susceptibility. SNPs have been used to identify genetic components which are associated with a disease. Unfortunately, many studies using these methods suffer from low reproducibility due to lack of power.</p> <p>Results</p> <p>We present a set of programs which implement a novel method for searching for disease-associated genes using prior information to select and order genes from publicly available databases by their prior likelihood of association with the disease. These programs were used in a published study of childhood-onset SLE which yielded novel associations with modest sample size.</p> <p>Conclusion</p> <p>Using prior information to decrease the size of the problem space to an amount commensurate with available samples and resources while maintaining appropriate power enables researchers to increase their likelihood of discovering reproducible associations.</p

    Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

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    Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike most GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility

    Does Preexisting Antiplatelet Treatment Influence Postthrombolysis Intracranial Hemorrhage in Community‚Äźtreated Ischemic Stroke Patients? An Observational Study

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    Objectives Intracranial hemorrhage ( ICH ) after acute stroke thrombolysis is associated with poor outcomes. Previous investigations of the relationship between preexisting antiplatelet use and the safety of intravenous ( IV ) thrombolysis have been limited by low event rates. The objective of this study was to determine whether preexisting antiplatelet therapy increased the risk of ICH following acute stroke thrombolysis. The primary hypothesis was that antiplatelet use would not be associated with radiographic evidence of ICH after controlling for relevant confounders. Methods Consecutive cases of thrombolysis patients treated in the emergency department (ED) were identified using multiple methods. Retrospective data were collected from four hospitals from 1996 to 2004 and 24 other hospitals from 2007 to 2010 as part of a cluster‚Äźrandomized trial. The same chart abstraction tool was used during both time periods, and data were subjected to numerous quality control checks. Hemorrhages were classified using a prespecified methodology: ICH was defined as presence of hemorrhage in radiographic interpretations of follow‚Äźup imaging (primary outcome). Symptomatic ICH ( sICH ) was defined as radiographic ICH with associated clinical worsening. A multivariable logistic regression model was constructed to adjust for clinical factors previously identified to be related to postthrombolysis ICH. Sensitivity analyses were conducted where the unadjusted and adjusted results from this study were combined with those of previously published external studies on this topic via meta‚Äźanalytic techniques. Results There were 830 patients included, with 47% having documented preexisting antiplatelet treatment. The mean (¬Ī standard deviation [SD]) age was 69¬†(¬Ī15)¬†years, and the cohort was 53% male. The unadjusted proportion of patients with any ICH was 15.1% without antiplatelet use and 19.3% with antiplatelet use (absolute risk difference¬†=¬†4.2%, 95% confidence interval [CI]¬†=¬†‚ąí1.2% to 9.6%); for sICH this was 6.1% without antiplatelet use and 9% with antiplatelet use (absolute risk difference¬†=¬†3.1%, 95% CI¬†=¬†‚ąí1% to 6.7%). After adjusting for confounders, antiplatelet use was not significantly associated with radiographic ICH (odds ratio [OR]¬†=¬†1.1, 95% CI¬†=¬†0.8 to 1.7) or sICH (OR¬†=¬†1.3, 95% CI¬†=¬†0.7 to 2.2). In patients 81¬†years and older, there was a higher risk of radiographic ICH (absolute risk difference¬†=¬†11.9%, 95% CI¬†=¬†0.1% to 23.6%). The meta‚Äźanalyses combined the findings of this investigation with previous similar work and found increased unadjusted risks of radiographic ICH (absolute risk difference¬†=¬†4.9%, 95% CI¬†=¬†0.7% to 9%) and sICH (absolute risk difference¬†=¬†4%, 95% CI¬†=¬†2.3% to 5.6%). The meta‚Äźanalytic adjusted OR of sICH for antiplatelet use was 1.6 (95% CI¬†=¬†1.1 to 2.4). Conclusions The authors did not find that preexisting antiplatelet use was associated with postthrombolysis ICH or sICH in this cohort of community treated patients. Preexisting tobacco use, younger age, and lower severity were associated with lower odds of sICH . The meta‚Äźanalyses demonstrated small, but statistically significant increases in the absolute risk of radiographic ICH and sICH , along with increased odds of sICH in patients with preexisting antiplatelet use. Resumen ¬ŅInfluye el Tratamiento Antiagregante Previo en la Hemorragia Intracraneal tras la Trombolisis en los Pacientes con Ictus Isqu√©micos Tratados en la Comunidad? Un Estudio Observacional Objetivos La hemorragia intracraneal ( HIC ) tras la trombolisis de un ictus agudo se asocia con malos resultados. Los estudios previos de la relaci√≥n entre el uso de antiagregantes y la seguridad de la trombolisis intravenosa ( IV ) han estado limitados por los porcentajes bajos de sucesos. El objetivo de este estudio fue determinar si el tratamiento antiagregante previo est√° asociado con la evidencia radiol√≥gica de HIC tras el control por los factores de confusi√≥n relevantes. Metodolog√≠a Se identificaron los casos consecutivos de pacientes tratados con trombolisis en el SU de m√ļltiples formas. Se recogieron los datos de forma retrospectiva de cuatro hospitales de 1996 a 2004 y de 24 hospitales distintos de 2007 a 2010 como parte de un ensayo cl√≠nico aleatorizado en racimos. Se utiliz√≥ la misma tabla resumen de historia cl√≠nica durante ambos periodos de tiempo y los datos fueron sometidos a numerosos controles de calidad. Las hemorragias se clasificaron siguiendo una metodolog√≠a preestablecida: la HIC se defini√≥ como la presencia de hemorragia en las interpretaciones radiol√≥gicas de las im√°genes de seguimiento (resultado primario); y la HIC sintom√°tica ( HIC s) se defini√≥ como la HIC radiol√≥gica asociada con un empeoramiento cl√≠nico. Se construy√≥ un modelo multivariable de regresi√≥n log√≠stica para ajustar los factores cl√≠nicos previamente identificados como relacionados con un la HIC tras la trombolisis. Los an√°lisis de sensibilidad se realizaron mediante t√©cnicas de metan√°lisis y se combinaron los resultados ajustados y no ajustados de esta investigaci√≥n con los estudios externos previamente publicados en este tema. Resultados Se incluyeron 830 pacientes, de los cuales el 47% ten√≠a documentado tratamiento antiagregante previo. La media de edad fue de 69 a√Īos, y el 53% eran varones. La proporci√≥n no ajustada de pacientes con cualquier tipo de HIC fue del 15,1% sin toma de antiagregante y del 19,3% con la toma de antiagregante (diferencia del riesgo absoluto 4,2%, IC 95%¬†=¬†‚ąí1,2% a 9,6%); y para las HIC s fue del 6,1% sin toma de antiagregantes y del 9% con la toma de antiagregantes (diferencia absoluta del riesgo 3,1%, IC 95%¬†=¬†‚ąí1% a 6,7%). Tras ajustar por los factores de confusi√≥n, la toma de antiagregantes no se asoci√≥ de forma significativa con la HIC radiol√≥gica ( OR 1,1, IC 95%¬†=¬†0,8 a 1,7]) o HIC s ( OR 1,3, IC 95%¬†=¬†0,7 a 2,2). En los pacientes de 81 a√Īos o m√°s, hubo mayor riesgo de HIC radiol√≥gica (diferencia de riesgo absoluta 11,9%, IC 95%¬†=¬†0,1% a 23,6%). El metan√°lisis que combin√≥ los hallazgos de esta investigaci√≥n con los trabajos similares previos encontr√≥ un riesgo no ajustado incrementado para la HIC radiol√≥gica (diferencia absoluta del riesgo 4,9%, IC 95%¬†=¬†0,7% a 9%) y de HIC s (diferencia absoluta del riesgo 4%, IC 95%¬†=¬†2,3% a 5,6%). La odds ratio ajustada del metan√°lisis de HIC s para los pacientes con tratamiento de antigregantes fue de 1,6 ( IC 95%¬†=¬†1,1 a 2,4). Conclusiones Los autores no encontraron que la toma previa de antigregantes se asocie con la HIC o la HIC s tras la trombolisis en esta cohorte de pacientes tratados en la comunidad. El consumo previo de tabaco, la edad m√°s joven y la menor gravedad se asociaron con odds ratio menores de HIC s. El metan√°lisis demostr√≥ un incremento bajo, aunque estad√≠sticamente significativo, de riesgo absoluto de HIC radiol√≥gica o de HIC s, con una odds ratio aumentada de HIC s en los pacientes con toma previa de antiagregantes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96759/1/acem12077.pd

    Plasma membrane cholesterol as a regulator of human and rodent P2X7 receptor activation and sensitization.

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    P2X7 receptors are nonselective cation channels gated by high extracellular ATP, but with sustained activation, receptor sensitization occurs, whereby the intrinsic pore dilates, making the cell permeable to large organic cations, which eventually leads to cell death. P2X7 receptors associate with cholesterol-rich lipid rafts, but it is unclear how this affects the properties of the receptor channel. Here we show that pore-forming properties of human and rodent P2X7 receptors are sensitive to perturbations of cholesterol levels. Acute depletion of cholesterol with 5 mm methyl-ő≤-cyclodextrin (MCD) caused a substantial increase in the rate of agonist-evoked pore formation, as measured by the uptake of ethidium dye, whereas cholesterol loading inhibited this process. Patch clamp analysis of P2X7 receptor currents carried by Na(+) and N-methyl-D-glucamine (NMDG(+)) showed enhanced activation and current facilitation following cholesterol depletion. This contrasts with the inhibitory effect of methyl-ő≤-cyclodextrin reported for other P2X subtypes. Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating. These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.This work was supported by the Biotechnology and Biological Sciences Research Council (BB/F001320/1), the David James Studentship, Department of Pharmacology, University of Cambridge and the Marshall Scholarship.This paper was originally published in The Journal of Biological Chemistry (Robinson LE, Shridar M, Smith P, Murrell-Lagnado RD, The Journal of Biological Chemistry 2014, 289, 46, 31983‚Äď31994, doi:10.1074/jbc.M114.574699

    Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells

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    Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement ‚Äď a stage between initial cellular internalization and final gene silencing of siRNA delivery systems

    Methyl-ő≤-Cyclodextrins Preferentially Remove Cholesterol from the Liquid Disordered Phase in Giant Unilamellar Vesicles

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    Methyl-ő≤-cyclodextrins (Mő≤CDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by Mő≤CD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of Mő≤CD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of Mő≤CD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27¬įC. Under these conditions, individual GUVs present Chol distributed into lo and ld phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that Mő≤CD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the Mő≤CD concentration. At high concentrations, Mő≤CD also removes phospholipids

    LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery

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    Neurotransmitter receptor trafficking during synaptic plasticity requires the concerted action of multiple signaling pathways and the protein transport machinery. However, little is known about the contribution of lipid metabolism during these processes. In this paper, we addressed the question of the role of cholesterol in synaptic changes during long-term potentiation (LTP). We found that N-methyl-d-aspartate-type glutamate receptor (NMDAR) activation during LTP induction leads to a rapid and sustained loss or redistribution of intracellular cholesterol in the neuron. A reduction in cholesterol, in turn, leads to the activation of Cdc42 and the mobilization of GluA1-containing őĪ-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) from Rab11-recycling endosomes into the synaptic membrane, leading to synaptic potentiation. This process is accompanied by an increase of NMDAR function and an enhancement of LTP. These results imply that cholesterol acts as a sensor of NMDAR activation and as a trigger of downstream signaling to engage small GTPase (guanosine triphosphatase) activation and AMPAR synaptic delivery during LTP.Peer Reviewe
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