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The endogenous repair mechanisms play an important role in the recovery of nerve function after stroke, such as gliosis, synaptic plasticity, remyelination and nerve regeneration. Iron is the most abundant trace metal element in the brain and plays a crucial role in the maintenance of normal cerebral function. It is an important coenzyme factor in the process of cell metabolism, DNA synthesis, purine catabolism and neurotransmitter synthesis and decomposition. However, it is unclear what role iron plays in the long-term recovery of neurological function after stroke. In this study, we first observed that changes in iron metabolism occurred during neurological function recovery in the mice with distal middle cerebral artery occlusion (dMCAO). Our data showed that plasticity changes due to endogenous repair mechanisms resulted in improvements in cerebral cortex function. These changes involved gliosis, synaptic function reconstruction, remyelination, and activation of neural stem cells. In order to examine the potential role of iron, we synthesized liposomal-encapsulated deferoxamine (DFO) nanoparticles to further explore the effect and the mechanism of iron on the recovery of neurological function in dMCAO mice. Our results showed that liposome-DFO decreased iron deposition and reversed plasticity changes in cerebral cortex function after stroke, which delayed neurological function recovery. This experiment shows that the increasing iron level promotes endogenous repair in ischemic stroke. Our finding reveals the change regularity of iron and emphasizes the beneficial role of iron in the recovery process of neurological function, which provides an important basis for the prevention and/or treatment of ischemia-reperfusion and recovery after stroke.

Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH–induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.

Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.

We describe herein a method to enable high throughput (HTP) screening of libraries of soluble proteins such as phage-derived clones of IgG, scFv-Fc, or other Fc-fusion proteins expressed in mammalian cells via adenovirus transduction. DNA fragments of antibody single chains (scFvs) and fragment antigen-binding (Fabs) from the positive clones of the third round of bacteriophage panning against a target antigen were batch reformatted into scFv-Fc or IgG in an oriP bearing entry vector and then recombined to an adenovirus vector through Gateway technology. The resulting antibody gene-containing adenovirus libraries were added to 96-well plates seeded with mammalian cells at a ratio of 0.7 infectious viral particles per well to establish clonality. Protocol optimization improved the expression of scFv-Fc and IgGs up to 100μg/mL in 96-well plates, which is sufficient for most antibody characterizations. In addition, 78% of the wells that were positive for protein expression contain only one sequence, indicating successful establishment of clonality in a majority of wells. We have established and optimized a mammalian expression system that produces soluble protein variants in a HTP manner. The system will facilitate developing multiple downstream screening methodologies.

Introduction A novel alpha-melanocyte-stimulating hormone peptide analog CHX-A″-Re(Arg 11 )CCMSH, which targeted the melanocortin-1 receptor (MC1-R) overexpressed on melanoma cells, was investigated for its biodistribution and tumor imaging properties. Methods The metal bifunctional chelator CHX-A″ was conjugated to the melanoma targeting peptide (Arg 11 )CCMSH and cyclized by Re incorporation to yield CHX-A″-Re(Arg 11 )CCMSH. CHX-A″-Re(Arg 11 )CCMSH was labeled with 111 In, 86 Y and 68 Ga, and the radiolabeled peptides were examined in B16/F1 melanoma-bearing mice for their pharmacokinetic as well as their tumor targeting properties using small animal SPECT and PET. Results The radiolabeling efficiencies of the 111 In-, 86 Y- and 68 Ga-labeled CHX-A″-Re(Arg 11 )CCMSH peptides were >95%, resulting in specific activities of 4.44, 3.7 and 1.85 MBq/μg, respectively. Tumor uptake of the 111 In-, 86 Y- and 68 Ga-labeled peptides was rapid with 4.17±0.94, 4.68±1.02 and 2.68±0.69 %ID/g present in the tumors 2 h postinjection, respectively. Disappearance of radioactivity from the normal organs and tissues was rapid with the exception of the kidneys. Melanoma tumors were imaged with all three radiolabeled peptides 2 h postinjection. MC1-R-specific uptake was confirmed by competitive receptor blocking studies. Conclusions Melanoma tumor uptake and imaging was exhibited by the 111 In-, 86 Y- and 68 Ga-labeled Re(Arg 11 )CCMSH peptides, although the tumor uptake was moderated by low specific activity. The facile radiolabeling properties of CHX-A″-Re(Arg 11 )CCMSH allow it to be employed as a melanoma imaging agent with little or no purification after 111 In, 86 Y and 68 Ga labeling.