Semaphorin 3A Binds to the Perineuronal Nets via Chondroitin Sulfate Type E Motifs in Rodent Brains

Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186–200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity.     

Recycling antimalarial leads for cancer: Antiproliferative properties of N-cinnamoyl chloroquine analogues

Cinnamic acids and quinolines are known as useful scaffolds in the discovery of antitumor agents. Therefore, N-cinnamoylated analogues of chloroquine, recently reported as potent dual-action antimalarials, were evaluated against three different cancer cell lines: MKN-28, Caco-2, and MCF-7. All compounds display anti-proliferative activity in the micromolar range against the three cell lines tested, and most of them were more active than their parent drug, chloroquine, against all cell lines tested. Hence, N-cinnamoyl-chloroquine analogues are a good start towards development of affordable antitumor leads.     

Polyethylenimine of various molecular weights as adjuvant for transfection mediated by cationic liposomes

Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.     

Alarm scent-marking during predatory attempts in the Cabrera vole (Microtus cabrerae Thomas, 1906)

The alarm pheromones often released by animals under stressful situations seem to elicit behavioral changes in conspecifics, which in the appropriate context can be viewed as anti-predatory responses. However, the releasing of alarm pheromones associated with predatory events has not been demonstrated in mammals. In the current study with wild-caught Cabrera voles, we carried out experiments in the laboratory and in the field to assess the release of alarm pheromones in scent-marks during simulated predatory events and disclose their effects on conspecifics. We first conducted an assay wherein voles where let to scent-mark a clean substrate in the absence of disturbance (control) and under the simulation of predatory events. Contrarily to the control, no fecal boli were released and the area marked with urine was significantly larger during the predatory simulation. In a subsequent assay, we assessed the voles’ preference between urine-marks released under predatory simulation and in control conditions. Voles showed a significant preference by control substrates. Finally, a third assay was carried out in the vole’s habitat wherein the individual activity was monitored by radio-tracking before and after placement of urine-marks obtained during the conditions described above. The vole’s activity was only reduced near the urine-marks released during the simulated predatory events. The results suggest that: (1) during predatory attempts, Cabrera voles release an alarm pheromone in their urine-marks; (2) the putative alarm pheromone reduces the voles’ activity in the surroundings of the marked area; (3) the putative alarm pheromone persists in the field affecting conspecifics’ activity for several days.     

Ocellatin‐PT antimicrobial peptides: High‐resolution microscopy studies in antileishmania models and interactions with mimetic membrane systems

Although the mechanism of action of antimicrobial peptides (AMPs) is not clear, they can interact electrostatically with the cell membranes of microorganisms. New ocellatin‐PT peptides were recently isolated from the skin secretion of Leptodactylus pustulatus. The secondary structure of these AMPs and their effect on Leishmania infantum cells, and on different lipid surface models was characterized in this work. The results showed that all ocellatin‐PT peptides have an α‐helix structure and five of them (PT3, PT4, PT6 to PT8) have leishmanicidal activity; PT1 and PT2 affected the cellular morphology of the parasites and showed greater affinity for leishmania and bacteria‐mimicking lipid membranes than for those of mammals. The results show selectivity of ocellatin‐PTs to the membranes of microorganisms and the applicability of biophysical methods to clarify the interaction of AMPs with cell membranes.     

Population estimates of the breeding birds of the Tinhosas islands (Gulf of Guinea), the only major seabird colony of the eastern tropical Atlantic

The Tinhosas islands, in São Tomé e Príncipe, host the most important seabird breeding colony in the Gulf of Guinea, but information on its conservation status was hitherto unpublished or anecdotal, the last assessment having been performed in 1997. A two-day expedition to the Tinhosas islands was undertaken to estimate the status of breeding seabirds in 2013. Four of the five seabird species known to breed in São Tomé e Príncipe, namely Brown Booby Sula leucogaster, Sooty Tern Onychoprion fuscatus, Brown Noddy Anous stolidus and Black Noddy Anous minutus, occur on the Tinhosas. A decrease of 80% in Brown Booby numbers, possibly due to occasional exploitation, and a 30% increase in Sooty Tern and Black Noddy numbers, were found compared to 1997 data although survey methods differed. Breeding of Brown Noddy and Madeiran Storm-petrel Hydrobates castro remains unconfirmed. Our estimates confirmed that BirdLife International Important Bird and Biodiversity Area criteria are met for at least one species, the Sooty Tern. The islands are not legally protected, nonetheless, apart from moderate levels of disturbance by fishermen who land on Tinhosa Grande, no alien species were seen, and no immediate threats to the Tinhosas colony were detected. Multiple visits within and between years are recommended, to census breeders, monitor threats and establish breeding phenologies.     

Potential applications of stress solutes from extremophiles in protein folding diseases and healthcare

Protein misfolding, aggregation and deposition in the brain, in the form of amyloid, are implicated in the etiology of several neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and prion diseases. Drugs available on the market reduce the symptoms, but they are not a cure. Therefore, it is urgent to identify promising targets and develop effective drugs. Preservation of protein native conformation and/or inhibition of protein aggregation seem pertinent targets for drug development. Several studies have shown that organic solutes, produced by extremophilic microorganisms in response to osmotic and/or heat stress, prevent denaturation and aggregation of model proteins. Among these stress solutes, mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, diglycerol phosphate and ectoine are effective in preventing amyloid formation by Alzheimer’s Aβ peptide and/or α-synuclein in vitro. Moreover, mannosylglycerate is a potent inhibitor of Aβ and α-synuclein aggregation in living cells, and mannosylglyceramide and ectoine inhibit aggregation and reduce prion peptide-induced toxicity in human cells. This review focuses on the efficacy of stress solutes from hyper/thermophiles and ectoines to prevent amyloid formation in vitro and in vivo and their potential application in drug development against protein misfolding diseases. Current and envisaged applications of these extremolytes in neurodegenerative diseases and healthcare will also be addressed.