Oxytocin-Gly-Lys-Arg: A Novel Cardiomyogenic Peptide

Background: Oxytocin (OT), synthesized in the heart, has the ability to heal injured hearts and to promote cardiomyogenesis from stem cells. Recently, we reported that the OT-GKR molecule, a processing intermediate of OT, potently increased the spontaneous formation of cardiomyocytes (CM) in embryonic stem D3 cells and augmented glucose uptake in newborn rat CM above the level stimulated by OT. In the present experiments, we investigated whether OT-GKR exists in fetal and newborn rodent hearts, interacts with the OT receptors (OTR) and primes the generation of contracting cells expressing CM markers in P19 cells, a model for the study of early heart differentiation. Methodology/Principal Findings: High performance liquid chromatography of newborn rat heart extracts indicated that OT-GKR was a dominant form of OT. Immunocytochemistry of mouse embryos (embryonic day 15) showed cardiac OT-GKR accumulation and OTR expression. Computerized molecular modeling revealed OT-GKR docking to active OTR sites and to V1a receptor of vasopressin. In embryonic P19 cells, OT-GKR induced contracting cell colonies and ventricular CM markers more potently than OT, an effect being suppressed by OT antagonists and OTR-specific small interfering (si) RNA. The V1a receptor antagonist and specific si-RNA also significantly reduced OT-GKR-stimulated P19 contracting cells. In comparison to OT, OT-GKR induced in P19 cells less a-actinin, myogenin and MyoD mRNA, skeletal muscle markers. Conclusions/Significance: These results raise the possibility that C-terminally extended OT molecules stimulate C

An analysis and evaluation of the WeFold collaborative for protein structure prediction and its pipelines in CASP11 and CASP12

Every two years groups worldwide participate in the Critical Assessment of Protein Structure Prediction (CASP) experiment to blindly test the strengths and weaknesses of their computational methods. CASP has significantly advanced the field but many hurdles still remain, which may require new ideas and collaborations. In 2012 a web-based effort called WeFold, was initiated to promote collaboration within the CASP community and attract researchers from other fields to contribute new ideas to CASP. Members of the WeFold coopetition (cooperation and competition) participated in CASP as individual teams, but also shared components of their methods to create hybrid pipelines and actively contributed to this effort. We assert that the scale and diversity of integrative prediction pipelines could not have been achieved by any individual lab or even by any collaboration among a few partners. The models contributed by the participating groups and generated by the pipelines are publicly available at the WeFold website providing a wealth of data that remains to be tapped. Here, we analyze the results of the 2014 and 2016 pipelines showing improvements according to the CASP assessment as well as areas that require further adjustments and research

Detection of the OT system in the E15 mouse embryo by immunocytochemistry.

<p>Somite staining with OT-GKR antibody (<b>A</b>) and selective antibodies for OT (<b>B</b>) and OTR (<b>C</b>). TUNEL reaction displaying apoptosis (<b>D</b>)<b>.</b> Control staining of somites with OT-GKR-specific antibody reabsorbed with OT-GKR (<b>E</b>) Control staining of whole embryo with OT-GKR-specific antibody reabsorbed with OT-GKR (<b>F</b>). Immunodetection of OT-GKR in the fetal mouse heart at day E15 (arrow) (<b>G</b>). Polyclonal rabbit antibody specifically recognizing OT-GKR peptide was applied to detect OT-GKR. Staining was revealed by the biotin-streptavidin method. Higher magnification of OT-GKR staining in the fetal heart (<b>H</b>). Immunofluorescence of OT-GKR (Texas Red) in cryostat sections section of cardiac tissue (stained green by troponin Alexa Fluor 488 antibody) by (<b>I</b>)<b>.</b> Immunodetection in the heart of OTR (<b>J</b>)<b>,</b> OT (<b>K</b>), AVP (<b>L</b>), and V1R (<b>M</b>)<b>.</b> Control staining with OT-GKR-specific antibody reabsorbed with OT-GKR (<b>N</b>).</p

Schematic structure of the OT-GKR-IRES-EGFP DNA construct.

<p>Abbreviations: CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; OT, oxytocin. (<b>A</b>) pcDNA3.1/Amp-OT-GKR-IRES-EGFP transfection to EC P19 cells stimulates the expression of green fluorescence (<b>B</b>) and produces OT-GKR protein marked with VA-18 antibody in red (Texas Red) <b>(C)</b>. Blue DAPI staining of cell nuclei (<b>D</b>) and merged photo (<b>E</b>). Time course of appearance of beating cell colonies in non-induced EC P19 cells transfected with OT-GKR-IRES-EGFP and in normal EC P19 cells stimulated with OT-GKR (10⁻⁶ M), and non-induced controls (NI) (<b>F</b>).</p

Molecular docking of 3-D models of activated human OTR and V1aR with OT/OT-GKR peptides obtained by the MolDock Optimizer algorithm from Molegro Virtual Docker.

<p>(<b>A</b>) The front upright view position (side view) of the OTR-OT- GKR complex structure. (<b>A1</b>) The section (rectangle) shown in panel A1 from the top a intracellular view (i.e. rotation by 90° out of plane) of the marked section in A, demonstrate OT-GKR (green) in active conformation inside the OT binding site (the transmembrane helices in red and the cavity in violet). (<b>B</b>) side view of the V1aR-OT- GKR complex, (<b>B1</b>) V1aR top view. (<b>C1</b>) detail of docking view of OTR-OT-GKR complex, and (<b>C2</b>) detail of V1aR-OT-GKR complex. <b>D</b> displays the schematic model of human OTRs with marked amino acid residues that are putatively involved in ligand-binding. The amino acid residues in black circles have been proposed as OT docking sites, and the red bars represent docking sites of OT-GKR. (<b>E</b>) Schematic model of human vasopressin V1aR binding with OT-GKR and OT. Amino acid residues are identified by a 1-letter code in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013643#pone-0013643-t001" target="_blank">Table 1</a>.</p