β-catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer

The Wnt–β-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wnt–β-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of β-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear β-catenin content. Nuclear β-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wnt–β-catenin signaling. In the presence of high nuclear β-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the β-catenin status of patients' carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.     

IL-15 expression on RA synovial fibroblasts promotes B cell survival

Introduction

The purpose of this study was to examine the role of RA Synovial Fibroblast (RASFib) IL-15 expression on B cell survival.

Methods

Magnetically sorted peripheral blood memory B cells from 15 healthy subjects were cocultured with RASFib.

Results

RASFib constitutively expressed membrane IL-15. Survival of isolated B cells cultured for 6 days, below 5%, was extended in coculture with RASFib to 52+/−8% (p<0.001). IL-15 neutralizing agents but not isotype controls, reduced this rate to 31+/−6% (p<0.05). Interestingly, rhIL-15 had no effect on isolated B cells but significantly increased their survival in coculture with RASFib. In parallel, B cell IL-15R chains were upregulated in cocultures. BAFF and VCAM-1, that are expressed on RASFib, were tested as potential candidates involved in upregulating B cell IL-15R. Culture of B cells in the presence of rhBAFF or rhVCAM-1 resulted in significantly increased survival, together with upregulation of all three IL-15R chains; in parallel, rhIL-15 potentiated the anti-apoptotic effect of BAFF and VCAM-1. Both BAFF and VCAM-1 neutralizing agents downmodulated the effect of RASFib on B cell survival and IL-15R expression. In parallel, rhIL-15 had a lower effect on the survival of B cells cocultured with RASFib in the presence of BAFF or VCAM-1 neutralizing agents. Peripheral blood B cells from 15 early RA patients demonstrated an upregulated IL-15R and increased survival in cocultures.

Conclusion

IL-15 expression on RASFib significantly contributes to the anti-apoptotic effect of RASFib on B cells. IL-15 action is facilitated by BAFF and VCAM-1 expressed on RASFib, through an upregulation of IL-15R chains.     

Lentiviral vector delivery of short hairpin RNA to NG2 and neurotrophin-3 promotes locomotor recovery in injured rat spinal cord

Background aimsIn this study we investigated the effect of neurotrophin-3 (NT-3) and knockdown of NG2, one of the main inhibitory chondroitin sulfate proteoglycans (CSPG), in the glial scar following spinal cord injury (SCI).MethodsShort hairpin (sh) RNA were designed to target NG2 and were cloned into a lentiviral vector (LV). A LV was also constructed containing NT-3. LV expressing NT-3, shRNA to NG2 or combinations of both vectors were injected directly into contused rat spinal cords 1 week post-injury. Six weeks post-injection of LV, spinal cords were examined by histology for changes in scar size and by immunohistochemistry for changes in expression of CSPG, NT-3, astrocytes, neurons and microglia/macrophages. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale.ResultsAnimals that received the combination treatment of LV shNG2 and LV NT-3 showed reduced scar size. These animals also showed an increase in levels of neurons and NG2, a decrease in levels of astrocytes and a significant functional recovery as assessed using the BBB locomotor scale at 2 weeks post-treatment.ConclusionsThe improvement in locomotor recovery and decrease in scar size shows the potential of this gene therapy approach as a therapeutic treatment for SCI.     

New insights into social and space use behaviour of the endangered Pyrenean desman (<Emphasis Type="Italic">Galemys pyrenaicus</Emphasis>)

We describe novel aspects of the social organisation of the Pyrenean desman, Galemys pyrenaicus, by studying home range and shelter use behaviour in a local population. A total of 45 individuals were trapped of which 20 provided informative radiotracking data. In contrast to the currently accepted hypothesis [Stone RD. The social organization of the Pyrenean desman (Galemys pyrenaicus) as revealed by radiotelemetry. J Zool 212:117–129; 1987b; Stone RD, Gorman ML. Social organization of the European mole (Talpa europaea) and the Pyrenean desman (Galemys pyrenaicus). Mammal Rev 15 (1):35–42; 1985] individuals were not strictly territorial. Notably, there was no aggression between conspecifics, with simultaneous use of resting sites (shelters used for more than one hour). Resting sites were not permanent or exclusive for any individual. Individuals shared resting sites simultaneously, regardless of sex or age. Our observations recall for a new evaluation of the social structure and organisation of this species based on the new evidence that reveals higher frequency of social interactions than previously described. Resting sites may play an important role in the social organisation of the species, for instance by allowing direct and indirect communicative interactions among neighbouring individuals. This finding is of significance for the management (e.g. census and population monitoring) and conservation (e.g. habitat suitability to allow social interactions) of this endemic and seriously threatened unique mammal.     

GWA Analysis for Milk Production Traits in Dairy Sheep and Genetic Support for a QTN Influencing Milk Protein Percentage in the LALBA Gene

In this study, we used the Illumina OvineSNP50 BeadChip to conduct a genome-wide association (GWA) analysis for milk production traits in dairy sheep by analyzing a commercial population of Spanish Churra sheep. The studied population consisted of a total of 1,681 Churra ewes belonging to 16 half-sib families with available records for milk yield (MY), milk protein and fat yields (PY and FY) and milk protein and fat contents (PP and FP). The most significant association identified reached experiment-wise significance for PP and FP and was located on chromosome 3 (OAR3). These results confirm the population-level segregation of a previously reported QTL affecting PP and suggest that this QTL has a significant pleiotropic effect on FP. Further associations were detected at the chromosome-wise significance level on 14 other chromosomal regions. The marker on OAR3 showing the highest significant association was located at the third intron of the alpha-lactalbumin (LALBA) gene, which is a functional and positional candidate underlying this association. Sequencing this gene in the 16 Churra rams of the studied resource population identified additional polymorphisms. One out of the 31 polymorphisms identified was located within the coding gene sequence (LALBA_g.242T>C) and was predicted to cause an amino acid change in the protein (Val27Ala). Different approaches, including GWA analysis, a combined linkage and linkage disequilibrium study and a concordance test with the QTL segregating status of the sires, were utilized to assess the role of this mutation as a putative QTN for the genetic effects detected on OAR3. Our results strongly support the polymorphism LALBA_g.242T>C as the most likely causal mutation of the studied OAR3 QTL affecting PP and FP, although we cannot rule out the possibility that this SNP is in perfect linkage disequilibrium with the true causal polymorphism.     

Deregulated Cdk5 promotes oxidative stress and mitochondrial dysfunction

Oxidative stress is one of the earliest events in Alzheimer's disease (AD). A chemical genetic screen revealed that deregulated cyclin-dependent kinase 5 (Cdk5) may cause oxidative stress by compromising the cellular anti-oxidant defense system. Using novel Cdk5 modulators, we show the mechanism by which Cdk5 can induce oxidative stress in the disease's early stage and cell death in the late stage. Cdk5 dysregulation upon neurotoxic insults results in reactive oxygen species (ROS) accumulation in neuronal cells because of the inactivation of peroxiredoxin I and II. Sole temporal activation of Cdk5 also increases ROS, suggesting its major role in this process. Cdk5 inhibition rescues mitochondrial damage upon neurotoxic insults, thereby revealing Cdk5 as an upstream regulator of mitochondrial dysfunction. As mitochondrial damage results in elevated ROS and Ca(2+) levels, both of which activate Cdk5, we propose that a feedback loop occurs in late stage of AD and leads to cell death (active Cdk5 --> ROS --> excess ROS --> mitochondrial damage --> ROS --> hyperactive Cdk5 --> severe oxidative stress and cell injury --> cell death). Cdk5 inhibition upon neurotoxic insult prevents cell death significantly, supporting this hypothesis. As oxidative stress and mitochondrial dysfunction play pivotal roles in promoting neurodegeneration, Cdk5 could be a viable therapeutic target for AD.     

Simple protocols for NMR analysis of the enantiomeric purity of chiral primary amines

A simple three-component chiral derivatization protocol for determining the enantiopurity of chiral primary amines by 1H NMR spectroscopic analysis is described here. The method involves condensation of the amines with 2-formylphenylboronic acid and enantiopure 1,1'-bi-2-naphthol. This approach affords a mixture of diastereoisomeric iminoboronate esters whose ratio can be determined by the integration of well-resolved diastereotopic resonances in their 1H NMR spectra, thus enabling the enantiopurity of the parent amine to be determined easily. The protocol, as described, takes less than 90 min to complete.     

High Diversity of vacA and cagA Helicobacter pylori Genotypes in Patients with and without Gastric Cancer

Background

Helicobacter pylori is associated with chronic gastritis, peptic ulcers, and gastric cancer. The aim of this study was to assess the topographical distribution of H. pylori in the stomach as well as the vacA and cagA genotypes in patients with and without gastric cancer.

Methodology/Principal Findings

Three gastric biopsies, from predetermined regions, were evaluated in 16 patients with gastric cancer and 14 patients with dyspeptic symptoms. From cancer patients, additional biopsy specimens were obtained from tumor centers and margins; among these samples, the presence of H. pylori vacA and cagA genotypes was evaluated. Positive H. pylori was 38% and 26% in biopsies obtained from the gastric cancer and non-cancer groups, respectively (p = 0.008), and 36% in tumor sites. In cancer patients, we found a preferential distribution of H. pylori in the fundus and corpus, whereas, in the non-cancer group, the distribution was uniform (p = 0.003). A majority of the biopsies were simultaneously cagA gene-positive and -negative. The fundus and corpus demonstrated a higher positivity rate for the cagA gene in the non-cancer group (p = 0.036). A mixture of cagA gene sizes was also significantly more frequent in this group (p = 0.003). Ninety-two percent of all the subjects showed more than one vacA gene genotype; s1b and m1 vacA genotypes were predominantly found in the gastric cancer group. The highest vacA-genotype signal-sequence diversity was found in the corpus and 5 cm from tumor margins.

Conclusion/Significance

High H. pylori colonization diversity, along with the cagA gene, was found predominantly in the fundus and corpus of patients with gastric cancer. The genotype diversity observed across systematic whole-organ and tumor sampling was remarkable. We find that there is insufficient evidence to support the association of one isolate with a specific disease, due to the multistrain nature of H. pylori infection shown in this work.     

Up-regulation of nicotinic receptors by nicotine varies with receptor subtype

Recent evidence suggests that in addition to alpha4beta2 and alpha3-containing nicotinic receptors, alpha6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that alpha6beta2, like alpha3beta2 and alpha4beta2 receptors, formed high affinity epibatidine binding complexes that are pentameric, trafficked to the cell surface, and produced acetylcholine-evoked currents. Chronic nicotine exposure up-regulated alpha6beta2 receptors with differences in up-regulation time course and concentration dependence compared with alpha4beta2 receptors, the predominant high affinity nicotine binding site in brain. The alpha6beta2 receptor up-regulation required higher nicotine concentrations than for alpha4beta2 but lower than for alpha3beta2 receptors. The alpha6beta2 up-regulation occurred 10-fold faster than for alpha4beta2 and slightly faster than for alpha3beta2. Our data suggest that nicotinic receptor up-regulation is subtype-specific such that alpha6-containing receptors up-regulate in response to transient, high nicotine exposures, whereas sustained, low nicotine exposures up-regulate alpha4beta2 receptors.