Reversal of Succinylcholine Induced Apnea with an Organophosphate Scavenging Recombinant Butyrylcholinesterase

Background

Concerns about the safety of paralytics such as succinylcholine to facilitate endotracheal intubation limit their use in prehospital and emergency department settings. The ability to rapidly reverse paralysis and restore respiratory drive would increase the safety margin of an agent, thus permitting the pursuit of alternative intubation strategies. In particular, patients who carry genetic or acquired deficiency of butyrylcholinesterase, the serum enzyme responsible for succinylcholine hydrolysis, are susceptible to succinylcholine-induced apnea, which manifests as paralysis, lasting hours beyond the normally brief half-life of succinylcholine. We hypothesized that intravenous administration of plant-derived recombinant BChE, which also prevents mortality in nerve agent poisoning, would rapidly reverse the effects of succinylcholine.

Methods

Recombinant butyrylcholinesterase was produced in transgenic plants and purified. Further analysis involved murine and guinea pig models of succinylcholine toxicity. Animals were treated with lethal and sublethal doses of succinylcholine followed by administration of butyrylcholinesterase or vehicle. In both animal models vital signs and overall survival at specified intervals post succinylcholine administration were assessed.

Results

Purified plant-derived recombinant human butyrylcholinesterase can hydrolyze succinylcholine in vitro. Challenge of mice with an LD₁₀₀ of succinylcholine followed by BChE administration resulted in complete prevention of respiratory inhibition and concomitant mortality. Furthermore, experiments in symptomatic guinea pigs demonstrated extremely rapid succinylcholine detoxification with complete amelioration of symptoms and no apparent complications.

Conclusions

Recombinant plant-derived butyrylcholinesterase was capable of counteracting and reversing apnea in two complementary models of lethal succinylcholine toxicity, completely preventing mortality. This study of a protein antidote validates the feasibility of protection and treatment of overdose from succinylcholine as well as other biologically active butyrylcholinesterase substrates.     

Intracranial Somatosensory Responses with Direct Spinal Cord Stimulation in Anesthetized Sheep

The efficacy of spinal cord stimulators is dependent on the ability of the device to functionally activate targeted structures within the spinal cord, while avoiding activation of near-by non-targeted structures. In theory, these objectives can best be achieved by delivering electrical stimuli directly to the surface of the spinal cord. The current experiments were performed to study the influence of different stimulating electrode positions on patterns of spinal cord electrophysiological activation. A custom-designed spinal cord neurostimulator was used to investigate the effects of lead position and stimulus amplitude on cortical electrophysiological responses to spinal cord stimulation. Brain recordings were obtained from subdural grids placed in four adult sheep. We systematically varied the position of the stimulating lead relative to the spinal cord and the voltage delivered by the device at each position, and then examined how these variables influenced cortical responses. A clear relationship was observed between voltage and electrode position, and the magnitude of high gamma-band oscillations. Direct stimulation of the dorsal column contralateral to the grid required the lowest voltage to evoke brain responses to spinal cord stimulation. Given the lower voltage thresholds associated with direct stimulation of the dorsal column, and its possible impact on the therapeutic window, this intradural modality may have particular clinical advantages over standard epidural techniques now in routine use.     

Plasmodium falciparum Erythrocytic Stage Parasites Require the Putative Autophagy Protein PfAtg7 for Normal Growth

Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite’s erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth.     

Floral density and co-occurring congeners alter patterns of selection in annual plant communities*

Although the evolution and diversification of flowers is often attributed to pollinator-mediated selection, interactions between co-occurring plant species can alter patterns of selection mediated by pollinators and other agents. The extent to which both floral density and congeneric species richness affect patterns of net and pollinator-mediated selection on multiple co-occurring species in a community is unknown and is likely to depend on whether co-occurring plants experience competition or facilitation for reproduction. We conducted an observational study of selection on four species of Clarkia (Onagraceae) and tested for pollinator-mediated selection on two Clarkia species in communities differing in congeneric species richness and local floral density. When selection varied with community context, selection was generally stronger in communities with fewer species, where local conspecific floral density was higher, and where local heterospecific floral density was lower. These patterns suggest that intraspecific competition at high densities and interspecific competition at low densities may affect the evolution of floral traits. However, selection on floral traits was not pollinator mediated in Clarkia cylindrica or Clarkia xantiana, despite variation in pollinator visitation and the extent of pollen limitation across communities for C. cylindrica. As such, interactions between co-occurring species may alter patterns of selection mediated by abiotic agents of selection.     

Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors

NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157–165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1_157–165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1_157–165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.     

Storage and reproductive strategy of the carpet-shell clam,Ruditapes decussatus in the Gulf of Gabès (Tunisia)

In this study, the variations of glycogen concentrations in Ruditapes decussatus from Sfax coasts (Tunisia) were described in relation to the reproductive cycle. Separate analyses were made of gonad, adductor muscle and ‘remainder’. The timing of gametogenic development and spawning was evaluated using qualitative histology associated with image analysis including (1) the estimation of the gonadal occupation index (GOI), (2) surface area occupied by reserve tissues and (3) variation in oocyte diameter. The reproductive cycle of R. decussatus exhibited partial asynchronization between sexes, the major difference being observed in the duration of the spawning period. Contrary to previous studies, we observed continuous partial spawning (e.g. 50% of ripe oocytes still subsisted at the partial spawning stage). During the gametogenic cycle, GOI varied significantly in males and in females. Most oocytes were ripe and ready to spawn when their diameter reached or exceeded 45?µm. The expansion of gonad was inversely proportional to the development of the foot tissue. Glycogen concentration showed significant temporal variations between tissues, indicating the clear differentiation in energy storage between the clam organs.     

137 DNA nanostructure serum stability: greater than the sum of their parts

DNA cages hold tremendous potential to encapsulate and selectively release therapeutic drugs, and can provide useful tools to probe the size and shape dependence of nucleic acid delivery (McLaughlin & Sleiman, H. F., 2011). These structures have been shown to site-specifically present ligands, small molecule drugs, or antisense/siRNA motifs, in order to increase their therapeutic efficiency (Li & Fan, C. 2012). One of the major barriers towards their in vivo applications is the susceptibility of their strands towards nuclease degradation. A number of chemical strategies have been used to block nuclease digestion of oligonucleotides and improve potency, such as the use of a phosphorothioate backbone, 2´-O-methyl, locked nucleic acids, and short hybrid gapmers. However, the synthesis of these oligonucleotides is often complicated and expensive, driving the need for simple modifications to enhance serum stability and address in vivo biodistribution. We show here a simple method to significantly enhance the nuclease stability of DNA strands, through introduction of commercially available, single-endmodifications (Conway & Sleiman 2013). We use these oligonucleotides to construct DNA cages in a single step and in quantitative yields. Even in single-stranded form, these cages stabilize their component strands towards nucleases, with mean lifetimes as long as 62?h in 10 % (v/v) fetal bovine serum (FBS). We examine the effect of other DNA-end modifications on nuclease susceptibility. Finally, we show the ligation of these single-stranded cages into topologically interesting catenane ‘necklaces,’ with mean lifetimes in serum of ～200?h.     

Plant community responses to long-term fertilization: changes in functional group abundance drive changes in species richness

Declines in species richness due to fertilization are typically rapid and associated with increases in aboveground production. However, in a long-term experiment examining the impacts of fertilization in an early successional community, we found it took 14 years for plant species richness to significantly decline in fertilized plots, despite fertilization causing a rapid increase in aboveground production. To determine what accounted for this lag in the species richness response, we examined several potential mechanisms. We found evidence suggesting the abundance of one functional group—tall species with long-distance (runner) clonality—drove changes in species richness, and we found little support for other mechanisms. Tall runner species initially increased in abundance due to fertilization, then declined dramatically and were not abundant again until later in the experiment, when species richness and the combined biomass of all other functional groups (non-tall runner) declined. Over 86 % of the species found throughout the course of our study are non-tall runner, and there is a strong negative relationship between non-tall runner and tall runner biomass. We therefore suggest that declines in species richness in the fertilized treatment are due to high tall runner abundance that decreases the abundance and richness of non-tall runner species. By identifying the functional group that drives declines in richness due to fertilization, our results help to elucidate how fertilization decreases plant richness and also suggest that declines in richness due to fertilization can be lessened by controlling the abundance of species with a tall runner growth form.