Point Mutations in Retargeted gD Eliminate the Sensitivity of EGFR/EGFRvIII-Targeted HSV to Key Neutralizing Antibodies.

Effective oncolytic virotherapy may require systemic delivery, tumor targeting, and resistance to virus-neutralizing (VN) antibodies. Since herpes simplex virus (HSV) glycoprotein D (gD) is the viral attachment/entry protein and predominant VN target, we examined the impact of gD retargeting alone and in combination with alterations in dominant VN epitopes on virus susceptibility to VN antibodies. We compared the binding of a panel of anti-gD monoclonal antibodies (mAbs) that mimic antibody specificities in human HSV-immune sera to the purified ectodomains of wild-type and retargeted gD, revealing the retention of two prominent epitopes. Substitution of a key residue in each epitope, separately and together, revealed that both substitutions (1) blocked retargeted gD recognition by mAbs to the respective epitopes, and, in combination, caused a global reduction in mAb binding; (2) protected against fusion inhibition by VN mAbs reactive with each epitope in virus-free cell-cell fusion assays; and (3) increased the resistance of retargeted HSV-1 to these VN mAbs. Although the combined modifications of retargeted gD allowed bona fide retargeting, incorporation into virions was partially compromised. Our results indicate that stacking of epitope mutations can additively block retargeted gD recognition by VN antibodies but also that improvements in gD incorporation into virus particles may be required.

Crosstalk between neuropeptides SP and CGRP in regulation of BMP2-induced bone differentiation.

AIM OF THE STUDY: The peripheral nervous system is involved in regulation of bone metabolism via sensory and sympathetic innervation. Substance P (SP) and calcitonin gene-related peptide (CGRP) are two sensory neuropeptides that have been associated with regulation of osteogenic differentiation. However, the interaction between SP and CGRP both with each other and the bone morphogenetic protein 2 (BMP2) in regulation of osteogenic differentiation has not been studied. Therefore, the aim of this study was to investigate the interaction between SP and CGRP on BMP2-induced bone differentiation using model progenitor cells. MATERIALS AND METHODS: C2C12 myoblasts and MC3T3 pre-osteoblasts were treated with SP and CGRP, both individually and in combination, in the presence of BMP2. The effects of the neuropeptides on BMP2-induced osteogenic differentiation were assessed by measuring alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic markers. RESULTS: Both SP and CGRP enhanced BMP2 signaling, Runx2 mRNA expression, as well as mineralization in vitro. Co-stimulation with SP and CGRP resulted in down-regulation of BMP2-induced bone differentiation, suggesting potential crosstalk between the two neuropeptides in regulation of BMP2 signaling. CONCLUSIONS: Based on the results shown here, CGRP can mitigate augmenting effects of SP on BMP2 signaling and the three pathways potentially converge on Runx2 to regulate BMP2-induced bone differentiation.

Crosstalk between substance P and calcitonin gene-related peptide during heterotopic ossification in murine Achilles tendon.

Heterotopic ossification (HO) is abnormal bone formation within soft tissue, usually predisposed by neurogenic or musculoskeletal trauma. Inflammation resulting from trauma is considered to be the main trigger for HO by eliciting changes within the injury site, including elevation of bone morphogenetic proteins (BMPs). Recent research, however, has also associated changes in sensory neuropeptide expression with HO. Substance P (SP) and calcitonin gene-related peptide (CGRP) are two of those neuropeptides that have been implicated with various aspects of HO, including regulation of inflammation and BMP signaling. Despite discoveries associating SP and CGRP with soft tissue HO, it remains unclear whether SP and CGRP have a direct role in the induction of HO. Here, we investigated the effect of SP and CGRP in vivo with the aid of inkjet-based biopatterning technology to controllably deliver these neuropeptides onto a murine Achilles tendon. While we did not observe any significant effect with CGRP, SP alone promoted HO in vivo with increased expression of BMP2. Remarkably, when SP and CGRP were delivered together, CGRP counteracted the effect of SP and essentially blocked SP-induced HO. This report contributes to the understanding of the complex problem of HO pathophysiology and warrants more study to better elucidate the interplay between SP and CGRP in the induction of HO. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1444-1455, 2018.

Identification of ligand binding sites of proteins using the Gaussian Network Model.

The nonlocal nature of the protein-ligand binding problem is investigated via the Gaussian Network Model with which the residues lying along interaction pathways in a protein and the residues at the binding site are predicted. The predictions of the binding site residues are verified by using several benchmark systems where the topology of the unbound protein and the bound protein-ligand complex are known. Predictions are made on the unbound protein. Agreement of results with the bound complexes indicates that the information for binding resides in the unbound protein. Cliques that consist of three or more residues that are far apart along the primary structure but are in contact in the folded structure are shown to be important determinants of the binding problem. Comparison with known structures shows that the predictive capability of the method is significant.