Activation of Type 1 Cannabinoid Receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca2+](i)) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.Fundacao para a Ciencia e a Tecnologia - Portugal [POCTI/SAU-NEU/68465/2006, PTDC/SAU-NEU/104415/2008, PTDC/SAU-NEU/101783/2008, POCTI/SAU-NEU/110838/2009]; Fundacao Calouste Gulbenkian [96542]; Fundacao para a Ciencia e Tecnologiainfo:eu-repo/semantics/publishedVersio

HumMeth27QCReport: an R package for quality control and primary analysis of Illumina Infinium methylation data

<p>Abstract</p> <p>Background</p> <p>The study of the human DNA methylome has gained particular interest in the last few years. Researchers can nowadays investigate the potential role of DNA methylation in common disorders by taking advantage of new high-throughput technologies. Among these, Illumina Infinium assays can interrogate the methylation levels of hundreds of thousands of CpG sites, offering an ideal solution for genome-wide methylation profiling. However, like for other high-throughput technologies, the main bottleneck remains at the stage of data analysis rather than data production.</p> <p>Findings</p> <p>We have developed <it>HumMeth27QCReport</it>, an R package devoted to researchers wanting to quickly analyse their Illumina Infinium methylation arrays. This package automates quality control steps by generating a report including sample-independent and sample-dependent quality plots, and performs primary analysis of raw methylation calls by computing data normalization, statistics, and sample similarities. This package is available at CRAN repository, and can be integrated in any Galaxy instance through the implementation of ad-hoc scripts accessible at Galaxy Tool Shed.</p> <p>Conclusions</p> <p>Our package provides users of the Illumina Infinium Methylation assays with a simplified, automated, open-source quality control and primary analysis of their methylation data. Moreover, to enhance its use by experimental researchers, the tool is being distributed along with the scripts necessary for its implementation in the Galaxy workbench. Finally, although it was originally developed for HumanMethylation27, we proved its compatibility with data generated with the HumanMethylation450 Bead Chip.</p

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form

Development of a Ki-67-based clinical trial assay for neoadjuvant endocrine therapy response monitoring in breast cancer.

Purpose The recent publication of the ACOSOG Z1031 trial results demonstrated that Ki-67 proliferation marker-based neoadjuvant endocrine therapy response monitoring could be used for tailoring the use of adjuvant chemotherapy in ER+HER2-negative breast cancer patients. In this paper, we describe the development of the Ki-67 clinical trial assay used for this study.Methods Ki-67 assay assessment focused on reproducing a 2.7% Ki-67 cut-point (CP) required for calculating the Preoperative Endocrine Prognostic Index and a 10% CP for poor endocrine therapy response identification within the first month of neoadjuvant endocrine treatment. Image analysis was assessed to increase the efficiency of the scoring process. Clinical outcome concordance for two independent Ki-67 scores was the primary performance metric.Results Discordant scores led to a triage approach where cases with complex histological features that software algorithms could not resolve were flagged for visual point counting (17%). The final Ki-67 scoring approach was run on T1/2 N0 cases from the P024 and POL trials (N = 58). The percent positive agreement for the 2.7% CP was 87.5% (95% CI 61.7-98.5%); percent negative agreement 88.9% (95% CI: 65.3-98.6%). Minor discordance did not affect the ability to predict similar relapse-free outcomes (Log-Rank P = 0.044 and P = 0.055). The data for the 10% early triage CP in the POL trial were similar (N = 66), the percentage positive agreement was 100%, and percent negative agreement 93.55% (95% CI: 78.58-99.21%). The independent survival predictions were concordant (Log-rank P = 0.0001 and P = 0.01).Conclusions We have developed an efficient and reproducible Ki-67 scoring system that was approved by the Clinical Trials Evaluation Program for NCI-supported neoadjuvant endocrine therapy trials. Using the methodology described here, investigators are able to identify a subgroup of patients with ER+HER2-negative breast cancer that can be safely managed without the need of adjuvant chemotherapy

Altered Neurocircuitry in the Dopamine Transporter Knockout Mouse Brain

The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn^(2+) into the prefrontal cortex indicated that DAT KO mice have a truncated Mn^(2+) distribution within this circuitry with little accumulation beyond the thalamus or contralateral to the injection site. By contrast, WT littermates exhibit Mn^(2+) transport into more posterior midbrain nuclei and contralateral mesolimbic structures at 26 hr post-injection. Thus, DAT KO mice appear, at this level of anatomic resolution, to have preserved cortico-striatal-thalamic connectivity but diminished robustness of reward-modulating circuitry distal to the thalamus. This is in contradistinction to the state of this circuitry in serotonin transporter KO mice where we observed more robust connectivity in more posterior brain regions using methods identical to those employed here

Size limits the formation of liquid jets during bubble bursting

A bubble reaching an air–liquid interface usually bursts and forms a liquid jet. Jetting is relevant to climate and health as it is a source of aerosol droplets from breaking waves. Jetting has been observed for large bubbles with radii of R≫100 μm. However, few studies have been devoted to small bubbles (R<100 μm) despite the entrainment of a large number of such bubbles in sea water. Here we show that jet formation is inhibited by bubble size; a jet is not formed during bursting for bubbles smaller than a critical size. Using ultrafast X-ray and optical imaging methods, we build a phase diagram for jetting and the absence of jetting. Our results demonstrate that jetting in bubble bursting is analogous to pinching-off in liquid coalescence. The coalescence mechanism for bubble bursting may be useful in preventing jet formation in industry and improving climate models concerning aerosol production

Impaired Nuclear Nrf2 Translocation Undermines the Oxidative Stress Response in Friedreich Ataxia

BACKGROUND: Friedreich ataxia originates from a decrease in mitochondrial frataxin, which causes the death of a subset of neurons. The biochemical hallmarks of the disease include low activity of the iron sulfur cluster-containing proteins (ISP) and impairment of antioxidant defense mechanisms that may play a major role in disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We thus investigated signaling pathways involved in antioxidant defense mechanisms. We showed that cultured fibroblasts from patients with Friedreich ataxia exhibited hypersensitivity to oxidative insults because of an impairment in the Nrf2 signaling pathway, which led to faulty induction of antioxidant enzymes. This impairment originated from previously reported actin remodeling by hydrogen peroxide. CONCLUSIONS/SIGNIFICANCE: Thus, the defective machinery for ISP synthesis by causing mitochondrial iron dysmetabolism increases hydrogen peroxide production that accounts for the increased susceptibility to oxidative stress

Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach

Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase

Evaluation of Group Genetic Ancestry of Populations from Philadelphia and Dakar in the Context of Sex-Biased Admixture in the Americas

Population history can be reflected in group genetic ancestry, where genomic variation captured by the mitochondrial DNA (mtDNA) and non-recombining portion of the Y chromosome (NRY) can separate female- and male-specific admixture processes. Genetic ancestry may influence genetic association studies due to differences in individual admixture within recently admixed populations like African Americans.We evaluated the genetic ancestry of Senegalese as well as European Americans and African Americans from Philadelphia. Senegalese mtDNA consisted of approximately 12% U haplotypes (U6 and U5b1b haplotypes, common in North Africa) while the NRY haplotypes belonged solely to haplogroup E. In Philadelphia, we observed varying degrees of admixture. While African Americans have 9-10% mtDNAs and approximately 31% NRYs of European origin, these results are not mirrored in the mtDNA/NRY pools of European Americans: they have less than 7% mtDNAs and less than 2% NRYs from non-European sources. Additionally, there is <2% Native American contribution to Philadelphian African American ancestry and the admixture from combined mtDNA/NRY estimates is consistent with the admixture derived from autosomal genetic data. To further dissect these estimates, we have analyzed our samples in the context of different demographic groups in the Americas.We found that sex-biased admixture in African-derived populations is present throughout the Americas, with continual influence of European males, while Native American females contribute mainly to populations of the Caribbean and South America. The high non-European female contribution to the pool of European-derived populations is consistently characteristic of Iberian colonization. These data suggest that genomic data correlate well with historical records of colonization in the Americas