Augmentation of VAMP-catalytic activity of botulinum neurotoxin serotype B does not result in increased potency in physiological systems.

Botulinum neurotoxins (BoNTs) are used extensively as therapeutic agents. Serotypes A and B are available as marketed products. Higher doses of BoNT/B are required to reach an efficacy similar to that of products containing BoNT/A. Advances in our understanding of BoNT/B mechanism of action have afforded the opportunity to make rational modifications to the toxin aimed at increasing its activity. Recently, a mutation in the light chain of BoNT/B (S201P) was described that increases the catalytic activity of the isolated BoNT/B light chain in biochemical assays. In this study, we have produced two full-length recombinant BoNT/B toxins in E.coli-one wild type (rBoNT/B1) and one incorporating the S201P mutation (rBoNT/B1(S201P)). We have compared the activity of these two molecules along with a native BoNT/B1 in biochemical cell-free assays and in several biological systems. In the cell-free assay, which measured light-chain activity alone, rBoNT/B1(S201P) cleaved VAMP-2 and VAMP-1 substrate with an activity 3-4-fold higher than rBoNT/B1. However, despite the enhanced catalytic activity of rBoNT/B1(S201P), there was no significant difference in potency between the two molecules in any of the in vitro cell-based assays, using either rodent spinal cord neurons or cortical neurons. Similarly in ex vivo tissue preparations rBoNT/B1(S201P) was not significantly more potent than rBoNT/B1 at inhibiting either diaphragm or detrusor (bladder) muscle activity in C57BL/6N and CD1 mice. Finally, no differences between rBoNT/B1 and rBoNT/B1(S201P) were observed in an in vivo digit abduction score (DAS) assay in C57BL/6N mice, either in efficacy or safety parameters. The lack of translation from the enhanced BoNT/B1(S201P) catalytic activity to potency in complex biological systems suggests that the catalytic step is not the rate-limiting factor for BoNT/B to reach maximum efficacy. In order to augment the efficacy of BoNT/B in humans, strategies other than enhancing light chain activity may need to be considered.

Group I mGluR Induced LTD of NMDAR-synaptic Transmission at the Schaffer Collateral but not Temperoammonic Input to CA1.

NMDA receptors are composed of multiple subunits and are crucial in the induction of synaptic plasticity and learning and memory. In this study, application of the group I mGlu receptor agonist, DHPG, caused LTD of NMDA-EPSCs (DHPG-LTDNMDA) of the Schaffer collateral, but not of NMDA-EPSCs of the temperoammonic pathway onto CA1 neurons of the hippocampus. DHPGLTDNMDA did not alter the sensitivity of NMDA-EPSC to the GluN2B-antagonist, Ro25-6981, indicating that the postsynaptic NMDA receptor subunit composition remained unchanged following DHPG-LTDNMDA. Furthermore, blockade of GluN2B receptors did not affect the induction of DHPG-LTDNMDA. These results demonstrate a difference in the plasticity of NMDA receptors between two synapses onto the same CA1 neuron, but indicate that the subunit composition of NMDA receptors does not account for this difference.

Professional medical writing support and the quality of randomised controlled trial reporting: a cross-sectional study.

OBJECTIVES: Authors may choose to work with professional medical writers when writing up their research for publication. We examined the relationship between medical writing support and the quality and timeliness of reporting of the results of randomised controlled trials (RCTs). DESIGN: Cross-sectional study. STUDY SAMPLE: Primary reports of RCTs published in BioMed Central journals from 2000 to 16 July 2014, subdivided into those with medical writing support (n=110) and those without medical writing support (n=123). MAIN OUTCOME MEASURES: Proportion of items that were completely reported from a predefined subset of the Consolidated Standards of Reporting Trials (CONSORT) checklist (12 items known to be commonly poorly reported), overall acceptance time (from manuscript submission to editorial acceptance) and quality of written English as assessed by peer reviewers. The effect of funding source and publication year was examined. RESULTS: The number of articles that completely reported at least 50% of the CONSORT items assessed was higher for those with declared medical writing support (39.1% (43/110 articles); 95% CI 29.9% to 48.9%) than for those without (21.1% (26/123 articles); 95% CI 14.3% to 29.4%). Articles with declared medical writing support were more likely than articles without such support to have acceptable written English (81.1% (43/53 articles); 95% CI 67.6% to 90.1% vs 47.9% (23/48 articles); 95% CI 33.5% to 62.7%). The median time of overall acceptance was longer for articles with declared medical writing support than for those without (167 days (IQR 114.5-231 days) vs 136 days (IQR 77-193 days)). CONCLUSIONS: In this sample of open-access journals, declared professional medical writing support was associated with more complete reporting of clinical trial results and higher quality of written English. Medical writing support may play an important role in raising the quality of clinical trial reporting.

Auxiliary subunits of the CKAMP family differentially modulate AMPA receptor properties.

AMPA receptor (AMPAR) function is modulated by auxiliary subunits. Here, we report on three AMPAR interacting proteins-namely CKAMP39, CKAMP52 and CKAMP59-that, together with the previously characterized CKAMP44, constitute a novel family of auxiliary subunits distinct from other families of AMPAR interacting proteins. The new members of the CKAMP family display distinct regional and developmental expression profiles in the mouse brain. Notably, despite their structural similarities they exert diverse modulation on AMPAR gating by influencing deactivation, desensitization and recovery from desensitization, as well as glutamate and cyclothiazide potency to AMPARs. This study indicates that AMPAR function is very precisely controlled by the cell-type specific expression of the CKAMP family members.

mGlu1 receptor-induced LTD of NMDA receptor transmission selectively at Schaffer collateral-CA1 synapses mediates metaplasticity.

Hippocampal CA1 pyramidal neurons receive inputs from entorhinal cortex directly via the temporoammonic (TA) pathway and indirectly via the Schaffer collateral (SC) pathway from CA3. NMDARs at synapses of both pathways are critical for the induction of synaptic plasticity, information processing, and learning and memory. We now demonstrate that, in the rat hippocampus, activity-dependent mGlu1 receptor-mediated LTD (mGlu1-LTD) of NMDAR-mediated transmission (EPSC(NMDA)) at the SC-CA1 input prevents subsequent LTP of AMPAR-mediated transmission. In contrast, there was no activity-dependent mGlu1-LTD of EPSC(NMDA) at the TA-CA1 pathway, or effects on subsequent plasticity of AMPAR-mediated transmission. Therefore, the two major pathways delivering information to CA1 pyramidal neurons are subject to very different plasticity rules.

Coexpressed auxiliary subunits exhibit distinct modulatory profiles on AMPA receptor function.

Gating properties and surface trafficking of AMPA receptors (AMPARs) are modulated by auxiliary subunits. Here we studied the function of coexpressed auxiliary subunits belonging to two different classes. We focused on TARP γ-8 and CKAMP44 in dentate gyrus (DG) granule cells, since both subunits are highly expressed in this cell type. TARP γ-8 and CKAMP44 decrease the rate of deactivation but have an opposing influence on receptor desensitization, which accounts for their differential modulation of synaptic short-term plasticity. Furthermore, long-term plasticity (LTP) requires TARP γ-8 but not CKAMP44. The coexpression of both auxiliary subunits is necessary for the efficient targeting of AMPARs to the cell surface of DG granule cells. Finally, electrophysiological and biochemical evidence support the notion that CKAMP44 and TARP γ-8 can be contained in the same AMPAR complex.

Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons.

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague-Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.

Emerging developments in RNA therapeutics.

Exciting developments have recently emerged in the field of RNA therapeutics, with potential applications in the treatment of human diseases. The second International Conference on RNA in drug development was held to highlight several novel RNA-based technologies, including different approaches to silence gene expression, the broad range of diagnostic and therapeutic applications for aptamers, and the targeting of RNA with small molecules. Highlights of the meeting included the utilisation of RNA interference to silence genes, with applications for the treatment of both cancer and viral infections, and for systemic silencing of gene expression. Novel approaches to safer drug design using aptamers were presented, which would enable control of their therapeutic activity to be achieved with antidote oligonucleotides. Updates were also presented on the clinical and preclinical development of ribozymes and aptamers, including good progress in increasing the half-life of these molecules in serum.