Rapid activation of FAK/mTOR/p70S6K/PAK1-signaling controls the early testosterone-induced actin reorganization in colon cancer cells

Actin cytoskeleton reorganization initiated by testosterone conjugates through activation of membrane androgen receptors (mAR) has recently been reported in colon tumor cells. This mAR-induced actin reorganization was recognized as a critical initial event, controlling apoptosis and inhibiting cell migration. The present study addressed the molecular signaling regulating the rapid actin remodeling initiated upon testosterone-induced mAR activation in Caco2 colon tumor cells. We report early phosphorylation of the Focal Adhesion Kinase (FAK), followed by substantial early phosphorylation of mammalian target of rapamycin (mTOR), S6 kinase (p70S6K) and the actin regulating p21-activated kinase (PAK1). Pharmacological inhibition of FAK-sensitive phosphatidylinositide-3-kinase (PI-3K), a known element of mAR-signaling, fully abrogated the testosterone-induced actin reorganization and the activation of mTOR, p70S6K and PAK1. Similarly, inhibition of mTOR blocked p70S6K and PAK1 phosphorylation and actin remodeling. Pretreatment of the cells with the intracellular androgen receptor (iAR) antagonist flutamide or silencing iAR through siRNA did not influence mTOR phosphorylation and actin reorganization, indicating specific mAR-induced testosterone effects that are independent of iAR signaling. In conclusion, we demonstrate for the first time a new mAR-governed pathway involving FAK/PI-3K and mTOR/p70S6K/PAK1-cascade that regulates early actin reorganization in colon cancer cells.     

Incidence and Prevalence of Major Central Nervous System Involvement in Systemic Lupus Erythematosus: A 3-Year Prospective Study of 370 Patients

Background

The incidence and prevalence of CNS involvement in SLE remains unclear owing to conflicting results in the published studies. The aim of the study was to evaluate the incidence and prevalence of major definite CNS events in SLE patients.

Methods

370 SLE patients with no previous history of CNS involvement were prospectively evaluated in a tertiary hospital referral center for 3 years. Major CNS manifestations were codified according to ACR definitions, including chorea, aseptic meningitis, psychosis, seizures, myelopathy, demyelinating syndrome, acute confusional state and strokes. Minor CNS events were excluded. ECLAM and SLEDAI-SELENA Modification scores were used to evaluate disease activity and SLICC/ACR Damage Index was used to assess accumulated damage.

Results

16/370 (4.3%) patients presented with a total of 23 major CNS events. These included seizures (35%), strokes (26%), myelopathy (22%), optic neuritis (8.7%), aseptic meningitis (4.3%) and acute psychosis (4.3%). Incidence was 7.8/100 person years. Among hospitalizations for SLE, 13% were due to CNS manifestations. Epileptic seizures were associated with high disease activity, while myelopathy correlated with lower disease activity and NMO-IgG antibodies (P≤0.05). Stroke incidence correlated with APS coexistence (P = 0.06). Overall, CNS involvement correlated with high ECLAM and SLEDAI scores (P<0.001).

Conclusions

Clinically severe CNS involvement is rare in SLE patients, accounting for 7.8/100 person years. CNS involvement correlates with high disease activity and coexistence of specific features that define the respective CNS syndromes.     

Comparative study of flow in right-sided and left-sided aortas: numerical simulations in patient-based models

A right-sided aorta is a rare malformation which may be associated with other various types of congenital heart disease. We utilised haemodynamic, echocardiographic measurements, computerised tomography and image reconstruction software packages that were integrated in a computational fluid dynamics model to determine blood flow patterns in patient-based aortas. In the left-sided aorta, a systolic clockwise rotational component was present, while helical flow was depicted in the aortic arch that was converted in the descending aorta as counter-rotating vortices with accompanying retrograde flow. The right-sided configuration has not altered the orientation of the three-dimensional vortex, but intensification of polymorphic flow patterns, alterations in wall shear stress distribution and development of a lateral pressure gradient at the area of an aneurysmal anomaly was observed. Moreover, increments of Reynolds, Womersley and Dean numbers were evident. These phenomena along with the formation of the aneurysm might influence cardiovascular risk in patients with right-sided aortas.     

The Impact of Biofumigation and Chemical Fumigation Methods on the Structure and Function of the Soil Microbial Community

Biofumigation (BIOF) is carried out mainly by the incorporation of brassica plant parts into the soil, and this fumigation activity has been linked to their high glucosinolate (GSL) content. GSLs are hydrolyzed by the endogenous enzyme myrosinase to release isothiocyanates (ITCs). A microcosm study was conducted to investigate the effects induced on the soil microbial community by the incorporation of broccoli residues into soil either with (BM) or without (B) added myrosinase and of chemical fumigation, either as soil application of 2-phenylethyl ITC (PITC) or metham sodium (MS). Soil microbial activity was evaluated by measuring fluorescein diacetate hydrolysis and soil respiration. Effects on the structure of the total microbial community were assessed by phospholipid fatty acid analysis, while the impact on important fungal (ascomycetes (ASC)) and bacterial (ammonia-oxidizing bacteria (AOB)) guilds was evaluated by denaturating gradient gel electrophoresis (DGGE). Overall, B, and to a lesser extent BM, stimulated microbial activity and biomass. The diminished effect of BM compared to B was particularly evident in fungi and Gram-negative bacteria and was attributed to rapid ITC release following the myrosinase treatment. PITC did not have a significant effect, whereas an inhibitory effect was observed in the MS-treated soil. DGGE analysis showed that the ASC community was temporarily altered by BIOF treatments and more persistently by the MS treatment, while the structure of the AOB community was not affected by the treatments. Cloning of the ASC community showed that MS application had a deleterious effect on potential plant pathogens like Fusarium, Nectria, and Cladosporium compared to BIOF treatments which did not appear to inhibit them. Our findings indicate that BIOF induces changes on the structure and function of the soil microbial community that are mostly related to microbial substrate availability changes derived from the soil amendment with fresh organic materials.     

Passive smoking reduces and vitamin C increases exercise-induced oxidative stress: Does this make passive smoking an anti-oxidant and vitamin C a pro-oxidant stimulus?

The current interpretative framework states that, for a certain experimental treatment (usually a chemical substance) to be classified as “anti-oxidant”, it must possess the property of reducing (or even nullifying) exercise-induced oxidative stress. The aim of the study was to compare side by side, in the same experimental setup, redox biomarkers responses to an identical acute eccentric exercise session, before and after chronic passive smoking (considered a pro-oxidant stimulus) or vitamin C supplementation (considered an anti-oxidant stimulus). Twenty men were randomly assigned into either passive smoking or vitamin C group. All participants performed two acute eccentric exercise sessions, one before and one after either exposure to passive smoking or vitamin C supplementation for 12 days. Vitamin C, oxidant biomarkers (F₂-isoprostanes and protein carbonyls) and the non-enzymatic antioxidant (glutathione) were measured, before and after passive smoking, vitamin C supplementation or exercise. It was found that chronic exposure to passive smoking increased the level of F₂-isoprostanes and decreased the level of glutathione at rest, resulting in minimal increase or absence of oxidative stress after exercise. Conversely, chronic supplementation with vitamin C decreased the level of F₂-isoprostanes and increased the level of glutathione at rest, resulting in marked exercise-induced oxidative stress. Contrary to the current scientific consensus, our results show that, when a pro-oxidant stimulus is chronically delivered, it is more likely that oxidative stress induced by subsequent exercise is decreased and not increased. Reversely, it is more likely to find greater exercise-induced oxidative stress after previous exposure to an anti-oxidant stimulus. We believe that the proposed framework will be a useful tool to reach more pragmatic explanations of redox biology phenomena.     

Towards analyzing the potential of exosomes to deliver microRNA therapeutics

Exosome selectivity mechanisms underlying exosome–target cell interactions and the specific traits affecting their capability to communicate still remain unclear. Moreover, the capacity of exosomes to efficiently deliver their molecular cargos intracellularly needs precise investigation towards establishing functional exosome‐based delivery platforms exploitable in the clinical practice. The current study focuses on: (a) exosome production from normal MRC‐5 and Vero cells growing in culture, (b) physicochemical characterization by dynamic light scattering (DLS) and cryo‐transmission electron microscopy; (c) cellular uptake studies of rhodamine‐labeled exosomes in normal and cancer cells, providing to exosomes either “autologous” or “heterologous” cellular delivery environments; and (d) loading exogenous Alexa Fluor 488‐labeled siRNA into exosomes for the assessment of their delivering capacity by immunofluorescence in a panel of recipient cells. The data obtained thus far indicate that MRC‐5 and Vero exosomes, indeed exhibit an interesting delivering profile, as promising “bio‐shuttles,” being pharmacologically exploitable in the context of theranostic applications.     

Clustering by common friends finds locally significant proteins mediating modules

MOTIVATION: Much research has been dedicated to large-scale protein interaction networks including the analysis of scale-free topologies, network modules and the relation of domain-domain to protein-protein interaction networks. Identifying locally significant proteins that mediate the function of modules is still an open problem. Method: We use a layered clustering algorithm for interaction networks, which groups proteins by the similarity of their direct neighborhoods. We identify locally significant proteins, called mediators, which link different clusters. We apply the algorithm to a yeast network. RESULTS: Clusters and mediators are organized in hierarchies, where clusters are mediated by and act as mediators for other clusters. We compare the clusters and mediators to known yeast complexes and find agreement with precision of 71% and recall of 61%. We analyzed the functions, processes and locations of mediators and clusters. We found that 55% of mediators to a cluster are enriched with a set of diverse processes and locations, often related to translocation of biomolecules. Additionally, 82% of clusters are enriched with one or more functions. The important role of mediators is further corroborated by a comparatively higher degree of conservation across genomes. We illustrate the above findings with an example of membrane protein translocation from the cytoplasm to the inner nuclear membrane. AVAILABILITY: All software is freely available under Supplementary information.