Mechanism of Action and Epitopes of Clostridium difficile Toxin B-neutralizing Antibody Bezlotoxumab Revealed by X-ray Crystallography

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.     

Phosphoregulation of the Titin-cap Protein Telethonin in Cardiac Myocytes

Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca²⁺/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca²⁺ transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca²⁺ transients.     

Antitumor efficacy,pharmacokinetic and biodistribution studies of the anticancer peptide CIGB‐552 in mouse models

Accumulation of the COMMD1 protein as a druggable pharmacology event to target cancer cells has not been evaluated so far in cancer animal models. We have previously demonstrated that a second‐generation peptide, with cell‐penetrating capacity, termed CIGB‐552, was able to induce apoptosis mediated by stabilization of COMMD1. Here, we explore the antitumor effect by subcutaneous administration of CIGB‐552 in a therapeutic schedule. Outstandingly, a significant delay of tumor growth was observed at 0.2 and 0.7 mg/kg (p < 0.01) or 1.4 mg/kg (p < 0.001) after CIGB‐552 administration in both syngeneic murine tumors and patient‐derived xenograft models. Furthermore, we evidenced that ¹³¹I‐CIGB‐552 peptide was actually accumulated in the tumors after administration by subcutaneous route. A typical serine‐proteases degradation pattern for CIGB‐552 in BALB/c mice serum was identified. Further, biological characterization of the main metabolites of the peptide CIGB‐552 suggests that the cell‐penetrating capacity plays an important role in the cytotoxic activity. This report is the first in describing the antitumor effect induced by systemic administration of a peptide that targets COMMD1 for stabilization. Moreover, our data reinforce the perspectives of CIGB‐552 for cancer targeted therapy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Symbiotic Association Between Symbiodinium and the Gastropod Strombus gigas: Larval Acquisition of Symbionts

The importance of the dinoflagellate Symbiodinium sp. was studied in the early life stages of the gastropod Strombus gigas. This dinoflagellate was not found in the eggs or the gelatinous mass surrounding the eggs of the mollusk; therefore, Symbiodinium is not inherited directly. To determine whether the planktonic veligers can acquire these algae from the environment, they were exposed to freshly isolated Symbiodinium from adult S. gigas (homologous). The optimal stage for Symbiodinium inoculation was found at 48 h post-hatching. Survival and growth rates of veligers and juveniles were higher when inoculated with freshly isolated Symbiodinium in conjunction with daily feeding of Isochrysis spp. Veligers inoculated with Symbiodinium freshly isolated from three host species elicited distinct responses: (1) veligers did not take up Symbiodinium isolated from the hydrozoan Millepora alcicornis suggesting that there is discrimination on contact prior to ingestion, (2) veligers did take up Symbiodinium isolated from the anemone Bartholomea annulata, but the algae did not persist in the host tissue suggesting that selection against this type took place after ingestion or that the algae did not divide in the host, and (3) veligers did take up Symbiodinium isolated from Pterogorgia anceps where it persisted and was associated with metamorphosis of the larvae. In contrast, the Symbiodinium freshly isolated from S. gigas were not associated with metamorphosis and required an inducer such as the red alga Laurencia poitei. These data present a significant advancement for the establishment of a new approach in the aquaculture of this important but declining Caribbean species.     

Successful Non-Surgical Deep Uterine Transfer of Porcine Morulae after 24 Hour Culture in a Chemically Defined Medium

Excellent fertility and prolificacy have been reported after non-surgical deep uterine transfers of fresh in vivo-derived porcine embryos. Unfortunately, when this technology is used with vitrified embryos, the reproductive performance of recipients is low. For this reason and because the embryos must be stored until they are transferred to the recipient farms, we evaluated the potential application of non-surgical deep uterine transfers with in vivo-derived morulae cultured for 24 h in liquid stage. In Experiment 1, two temperatures (25°C and 37°C) and two media (one fully defined and one semi-defined) were assessed. Morulae cultured in culture medium supplemented with bovine serum albumin and fetal calf serum at 38.5°C in 5% CO₂ in air were used as controls. Irrespective of medium, the embryo viability after 24 h of culture was negatively affected (P<0.05) at 25°C but not at 37°C compared with the controls. Embryo development was delayed in all experimental groups compared with the control group (P<0.001). Most of the embryos (95.7%) cultured at 37°C achieved the full or expanded blastocyst stage, and unlike the controls, none of them hatched at the end of culture. In Experiment 2, 785 morulae were cultured in the defined medium at 37°C for 24 h, and the resulting blastocysts were transferred to the recipients (n = 24). Uncultured embryos collected at the blastocyst stage (n = 750) were directly transferred to the recipients and used as controls (n = 25). No differences in farrowing rates (91.7% and 92.0%) or litter sizes (9.0±0.6 and 9.4±0.8) were observed between the groups. This study demonstrated, for the first time, that high reproductive performance can be achieved after non-surgical deep uterine transfers with short-term cultured morulae in a defined medium, which opens new possibilities for the sanitary, safe national and international trade of porcine embryos and the commercial use of embryo transfer in pigs.     

The responsiveness of TrkB to BDNF and antidepressant drugs is differentially regulated during mouse development

Background

Previous studies suggest that the responsiveness of TrkB receptor to BDNF is developmentally regulated in rats. Antidepressant drugs (AD) have been shown to increase TrkB signalling in the adult rodent brain, and recent findings implicate a BDNF-independent mechanism behind this phenomenon. When administered during early postnatal life, ADs produce long-lasting biochemical and behavioural alterations that are observed in adult animals.

Methodology

We have here examined the responsiveness of brain TrkB receptors to BDNF and ADs during early postnatal life of mouse, measured as autophosphorylation of TrkB (pTrkB).

Principal Findings

We found that ADs fail to induce TrkB signalling before postnatal day 12 (P12) after which an adult response of TrkB to ADs was observed. Interestingly, there was a temporally inverse correlation between the appearance of the responsiveness of TrkB to systemic ADs and the marked developmental reduction of BDNF-induced TrkB in brain microslices ex vivo. Basal p-TrkB status in the brain of BDNF deficient mice was significantly reduced only during early postnatal period. Enhancing cAMP (cyclic adenosine monophosphate) signalling failed to facilitate TrkB responsiveness to BDNF. Reduced responsiveness of TrkB to BDNF was not produced by the developmental increase in the expression of dominant-negative truncated TrkB.T1 because this reduction was similarly observed in the brain microslices of trkB.T1 ^−/− mice. Moreover, postnatal AD administration produced long-lasting behavioural alterations observable in adult mice, but the responses were different when mice were treated during the time when ADs did not (P4-9) or did (P16-21) activate TrkB.

Conclusions

We have found that ADs induce the activation of TrkB only in mice older than 2 weeks and that responsiveness of brain microslices to BDNF is reduced during the same time period. Exposure to ADs before and after the age when ADs activate TrkB produces differential long-term behavioural responses in adult mice.     

Inferring the skeleton cell cycle regulatory network of malaria parasite using comparative genomic and variational Bayesian approaches

The development of new antimalarial drugs is urgently needed due to elevated drug resistance in the causative agents Plasmodium parasites. An intervention strategy based on the interruption of the parasite cell cycle could be undertaken using a systems-biology aided drug discovery approach. However, little is known about the components or the mechanism of parasite cell cycle control to date. In this proof of concept study, we attempted to infer the skeleton components using comparative genomic analysis and to uncover the genetic regulatory network (GRN) ab initio using a Variational Bayesian expectation maximization (VBEM) approach.