Uptake and recycling of pro-BDNF for transmitter-induced secretion by cortical astrocytes

Activity-dependent secretion of brain-derived neurotrophic factor (BDNF) is thought to enhance synaptic plasticity, but the mechanisms controlling extracellular availability and clearance of secreted BDNF are poorly understood. We show that BDNF is secreted in its precursor form (pro-BDNF) and is then cleared from the extracellular space through rapid uptake by nearby astrocytes after θ-burst stimulation in layer II/III of cortical slices, a paradigm resulting in long-term potentiation of synaptic transmission. Internalization of pro-BDNF occurs via the formation of a complex with the pan-neurotrophin receptor p75 and subsequent clathrin-dependent endocytosis. Fluorescence-tagged pro-BDNF and real-time total internal reflection fluorescence microscopy in cultured astrocytes is used to monitor single endocytic vesicles in response to the neurotransmitter glutamate. We find that endocytosed pro-BDNF is routed into a fast recycling pathway for subsequent soluble NSF attachment protein receptor–dependent secretion. Thus, astrocytes contain an endocytic compartment competent for pro-BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia.     

Inter-individual variability and conspecific densities: consequences for population regulation and range expansion

The presence of conspecifics can strongly modulate the quality of a breeding site. Both positive and negative effects of conspecifics can act on the same individuals, with the final balance between its costs and benefits depending on individual characteristics. A particular case of inter-individual variation found in many avian species is chromatic variability. Among birds, plumage coloration can co-vary with morphology, physiology and behavior as well as with age. These relationships suggest that cost-benefit balances of conspecific presence may be different for individuals with different colorations. We investigated whether inter-individual variability affects population regulation and expansion processes by analyzing potential differences in density-dependent productivity and settlement patterns in relation to plumage coloration in a population of a long-lived avian species recently undergoing a notable increase in numbers and distribution range. Our results show strong variation in the effect of density on productivity of breeding pairs depending on plumage coloration of their members. Productivity of dark birds decreased along the breeding density gradient while that of lighter breeders remained unchanged with conspecific density. In a similar way, our results showed an uneven occupation of localities by individuals with different plumage coloration in relation to local densities, with the breeding of lighter harriers more aggregated than that of dark-brown ones. At a population scale, darker birds had higher probability of colonization of the most isolated, empty sites. Explanations for species range expansion and population regulation usually make the inferred assumption that species traits are similar among individuals. However, in most species, there could be individual variation in niche requirements or dispersal propensities among individuals with different traits. Our results contribute to the growing appreciation that the individual traits, but not the average trait at the level of species, are important during population regulation and expansion processes.     

Covalent adducts between tRNA (m5U54)-methyltransferase and RNA substrates.

The interaction of tRNA (m5U54)-methyltransferase (RUMT) with in vitro synthesized unmodified tRNA and a 17-base oligoribonucleotide analog of the T-arm of tRNA in the absence of AdoMet has been investigated. Binary complexes are formed which are isolable on nitrocellulose filters and are composed of noncovalent and covalent complexes in nearly equal amounts. The covalent RUMT-RNA complexes are stable to SDS-PAGE and migrate slower than free enzyme or RNA. Kinetic and thermodynamic constants involved in formation and disruption of noncovalent and covalent binary complexes have been determined and interpreted in the context of steady-state kinetic parameters of the enzyme-catalyzed methylation and 5-H exchange of substrate. The results show that the isolable covalent complex is kinetically incompetent as an intermediate for methylation. Isotope trapping experiments show that when AdoMet is added to preformed binary complex, all bound tRNA is converted to methylated product; thus, the covalent complexes are chemically competent to form products. We have concluded that, after a reversible binary complex is formed, the catalytic thiol adds to the 6-carbon of the U54 of tRNA. The initial adduct leaves the reaction pathway to protonation at carbon 5; the latter can deprotonate and re-enter the pathway to form methylated product. It is speculated that covalent binary RUMT-RNA adducts may serve as depots of enzyme-tRNA complexes primed for methylation, or in unknown roles with RNAs other than tRNA.     

PRMT5 silencing selectively affects MTAP-deleted mesothelioma: In vitro evidence of a novel promising approach

Malignant mesothelioma (MM) is an aggressive asbestos-related cancer of the serous membranes. Despite intensive treatment regimens, MM is still a fatal disease, mainly due to the intrinsic resistance to current therapies and the lack of predictive markers and new valuable molecular targets. Protein arginine methyltransferase 5 (PRMT5) inhibition has recently emerged as a potential therapy against methylthioadenosine phosphorylase (MTAP)-deficient cancers, in which the accumulation of the substrate 5'-methylthioadenosine (MTA) inhibits PRMT5 activity, thus sensitizing the cells to further PRMT5 inhibition. Considering that the MTAP gene is frequently codeleted with the adjacent cyclin-dependent kinase inhibitor 2A (CDKN2A) locus in MM, we assessed whether PRMT5 could represent a therapeutic target also for this cancer type. We evaluated PRMT5 expression, the MTAP status and MTA content in normal mesothelial and MM cell lines. We found that both administration of exogenous MTA and stable PRMT5 knock-down, by short hairpin RNAs (shRNAs), selectively reduced the growth of MTAP-deleted MM cells. We also observed that PRMT5 knock-down in MTAP-deficient MM cells reduced the expression of E2F1 target genes involved in cell cycle progression and of factors implicated in epithelial-to-mesenchymal transition. Therefore, PRMT5 targeting could represent a promising new therapeutic strategy against MTAP-deleted MMs.     

Revisiting Street Intersections Using Slot-Based Systems

Since their appearance at the end of the 19th century, traffic lights have been the primary mode of granting access to road intersections. Today, this centuries-old technology is challenged by advances in intelligent transportation, which are opening the way to new solutions built upon slot-based systems similar to those commonly used in aerial traffic: what we call Slot-based Intersections (SIs). Despite simulation-based evidence of the potential benefits of SIs, a comprehensive, analytical framework to compare their relative performance with traffic lights is still lacking. Here, we develop such a framework. We approach the problem in a novel way, by generalizing classical queuing theory. Having defined safety conditions, we characterize capacity and delay of SIs. In the 2-road crossing configuration, we provide a capacity-optimal SI management system. For arbitrary intersection configurations, near-optimal solutions are developed. Results theoretically show that transitioning from a traffic light system to SI has the potential of doubling capacity and significantly reducing delays. This suggests a reduction of non-linear dynamics induced by intersection bottlenecks, with positive impact on the road network. Such findings can provide transportation engineers and planners with crucial insights as they prepare to manage the transition towards a more intelligent transportation infrastructure in cities.     

Studies on DNA damage: Discordant responses of rate of DNA disentanglement (viscosimetrically evaluated) and alkaline elution rate,obtained for several compounds

Alkaline elution is a well-known method for detecting DNA damage. Recently we have developed a viscosimetric method that is even more sensitive than alkaline elution. Here we report that the two methods, although apparently both revealing alkaline DNA fragmentation, can give dramatically different results for a significant series of compounds. We suspect that alkaline elution might reveal not only DNA fragmentation but also the extent of disentanglement of chromatin structure, whereas this DNA disentanglement rate, when evaluated viscosimetrically, is more strictly correlated with the initiation of DNA unwinding.     

In vitro chloroquine resistance modulation study on fresh isolates of Brazilian Plasmodium falciparum: intrinsic antimalarial activity of phenothiazine drugs

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination.