Inhibition of NaV1.6 sodium channel currents by a novel series of 1,4-disubstituted-triazole derivatives obtained via copper-catalyzed click chemistry

We have synthesized and evaluated a series of 1,4-disubstituted-triazole derivatives for inhibition of the rat Na_V1.6 sodium channel isoform, an isoform thought to play an important role in controlling neuronal firing. Starting from a series of 2,4(1H)-diarylimidazoles previously published, we decided to extend the SAR study by replacing the imidazole with a different heterocyclic scaffold and by varying the aryl substituents on the central aromatic ring. The 1,4-disubstituted 1,2,3-triazoles were prepared employing the copper-catalyzed azide–alkyne cycloaddition (CuAAC). Many of the new molecules were able to block the rNa_v1.6 currents at 10 μM by over 20%, displaying IC₅₀ values ranging in the low micromolar, thus indicating that triazole can efficiently replace the central heterocyclic core. Moreover, the introduction of a long chain at C4 of the central triazole seems beneficial for increased rNa_v1.6 current block, whereas the length of N1 substituent seems less crucial for inhibition, as long as a phenyl ring is not direcly connected to the triazole. These results provide additional information on the structural features necessary for block of the voltage-gated sodium channels. These new data will be exploited in the preparation of new compounds and could result in potentially useful AEDs.     

ARE PREGNANT WOMEN FETAL CONTAINERS?

Purdy explores the argument that women's rights to control their bodies should be subordinated to the welfare of their fetuses. She gives examples ofinstances where women's decisions about pregnancy and childbirth have been overridden by physicians and judges. She examines the nature of the mother fetus relationship, the social context of the current conflict between maternal and fetal rights, and the extent of a woman's legally enforceable duty to her fetus. Purdy acknowledges that women may owe a reasonable duty to their fetuses to try to prevent disease or handicap. However, given the uncertainties of modern medicine, the value-laden nature of many physicians' decisions, and the lack of adequate adequate prenatal care, women should not be held responsible for situations that are due largely to society's shortcomings. Purdy concludes that it is unjust to use the law to coerce women into accepting medical advice.     

Integrating multiple analytical approaches to spatially delineate and characterize genetic population structure: an application to boreal caribou (<Emphasis Type="Italic">Rangifer tarandus caribou</Emphasis>) in central Canada

Individual-based clustering (IBC) methods have become increasingly popular for the characterization and delineation of genetic population units for numerous species. These methods delineate populations based on the genetic assumptions of a breeding unit which may provide a better representation of the behaviour of the species. The increasing use of IBC has resulted in the development of several analytical models all of which vary in their theoretical assumptions to infer genetic population structure. In this paper, we report a comparative strategy utilizing three IBC methods to characterize the spatial genetic structure of the boreal population of woodland caribou (Rangifer tarandus caribou) in central Canada. In addition, we implement both tests for isolation-by-distance (IBD) and frequency-based assignment tests to validate the consensus genetic clusters as defined by IBC. We also compare indirect metrics of genetic diversity and gene flow using both a priori defined herds and the IBC defined populations. Although our results show some concordance between both pre-defined herds and IBC derived genetic clusters, the IBC analyses identified a cluster that was cryptic to observation-based caribou herds and found no difference between several adjacent herds. By comparing multiple IBC methods and integrating both IBD and indirect genetic diversity metrics a posteriori, our strategy provides an effective means to delineate wildlife population structure and accurately assess genetic diversity and connectivity.     

Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics

Background

Amyotrophic lateral sclerosis (ALS)-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. Since only the mutants, but not the endogenous wild-type FUS, are associated with stress granules under most of the stress conditions reported to date, the relationship between FUS and stress granules represents a mutant-specific phenotype and thus may be of significance in mutant-induced pathogenesis. While the association of mutant-FUS with stress granules is well established, the effect of the mutant protein on stress granules has not been examined. Here we investigated the effect of mutant-FUS on stress granule formation and dynamics under conditions of oxidative stress.

Results

We found that expression of mutant-FUS delays the assembly of stress granules. However, once stress granules containing mutant-FUS are formed, they are more dynamic, larger and more abundant compared to stress granules lacking FUS. Once stress is removed, stress granules disassemble more rapidly in cells expressing mutant-FUS. These effects directly correlate with the degree of mutant-FUS cytoplasmic localization, which is induced by mutations in the nuclear localization signal of the protein. We also determine that the RGG domains within FUS play a key role in its association to stress granules. While there has been speculation that arginine methylation within these RGG domains modulates the incorporation of FUS into stress granules, our results demonstrate that this post-translational modification is not involved.

Conclusions

Our results indicate that mutant-FUS alters the dynamic properties of stress granules, which is consistent with a gain-of-toxic mechanism for mutant-FUS in stress granule assembly and cellular stress response.

     

The interaction of cationic antimicrobial peptides with vesicles containing synthetic glycolipids as models of the outer membrane of gram-negative bacteria.

Two simple lipid A analogues methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside (GL1) and methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside 4-O-phosphate (GL2) were synthesized and used for preparing mixed phosphocholine vesicles as models of the outer membrane of gram-negative bacteria. The interaction of these model membranes with magainin 2, a representative of the alpha-helical membrane active peptides, and apidaecin Ib and drosocin, two insect Pro-rich peptides which do not act at the level of the cellular membrane, were studied by CD and dye-releasing experiments. The CD spectra of apidaecin Ib and drosocin in the presence of GL1- or GL2-containing vesicles were consistent with largely unordered structures, whereas, according to the CD spectra, magainin 2 adopted an amphipathic alpha-helical conformation, particularly in the presence of negatively charged bilayers. The ability of the peptides to fold into amphipathic conformations was strictly correlated to their ability to bind and to permeabilize phospholipid as well as glycolipid membranes. Apidaecin Ib and drosocin, which are unable to adopt an amphipathic structure, showed negligible dye-leakage activity even in the presence of GL2-containing vesicles. It is reasonable to suppose that, as for the killing mechanism, the two classes of antimicrobial peptides follow different patterns to cross the bacterial outer membrane.     

Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30-60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 × 10⁶ sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 × 10⁶ sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 × 10⁶ sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.     

Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.