Wnt-5a Promotes Neural Development and Differentiation by Regulating CDK5 via Ca2+/Calpain Pathway

Background/Aims: The Wnt signaling pathway has essential functions in the central nervous system, where it regulates the major physiological functions of neurons, including development, differentiation, and plasticity. Wnt signaling controls these cellular events; however, how Wnt pathways integrate into a coherent developmental program remains unclear. Methods: The expression and secretion of different WNT ligands (Wnt-1, Wnt-3a, Wnt-4, Wnt-5a, Wnt-11), and the levels and activities of cyclin-dependent kinases (CDK2, CDK4, CDK6/cyclin D, cyclin E) or CDK5 (CDK5/p35 and p25) were measured in Rat cortex at different embryonic stages, and in RA/BDNF-induced differentiated SH-SY5Y cell model, by Quantitative real-time PCR (qPCR), western blotting, ELISA, and in vitro CDK5 kinase assays. MAP2-BrdU double staining was used to assess cell differentiation and cell cycle exit in an RA/BDNF-induced differentiated SH-SY5Y cell model. The effects of CDK5 and Ca2+/calpain signaling were assessed using specific chemical inhibitors. Results: We found that Wnt-1 was unchanged and Wnt-3a was attenuated, whereas Wnt-4, Wnt-5a, and Wnt-11 were markedly up-regulated, during the development of neurons and differentiated SH-SY5Y cells. Simultaneously, the activity of CDK5 was elevated. Furthermore, we describe crosstalk between non-canonical Wnt signaling and CDK5 in the development of neurons and differentiated SH-SY5Y cells. Wnt-5a, a non-canonical Wnt ligand, regulated CDK5 via Ca2+/calpain signaling in both neuronal development and differentiation. Inhibition of Wnt-5a diminished CDK5 kinase activity via the Ca2+/calpain pathway, thereby attenuating RA-BDNF induced SH-SY5Y cell differentiation. Conclusion: Wnt-5a signaling is a significant regulator of neuronal development and differentiation and upregulates CDK5 kinase activity via Ca2+/calpain signaling

Whole-genome sequencing of the snub-nosed monkey provides insights into folivory and evolutionary history

Colobines are a unique group of Old World monkeys that principally eat leaves and seeds rather than fruits and insects. We report the sequencing at 146× coverage, de novo assembly and analyses of the genome of a male golden snub-nosed monkey (Rhinopithecus roxellana) and resequencing at 30× coverage of three related species (Rhinopithecus bieti, Rhinopithecus brelichi and Rhinopithecus strykeri). Comparative analyses showed that Asian colobines have an enhanced ability to derive energy from fatty acids and to degrade xenobiotics. We found evidence for functional evolution in the colobine RNASE1 gene, encoding a key secretory RNase that digests the high concentrations of bacterial RNA derived from symbiotic microflora. Demographic reconstructions indicated that the profile of ancient effective population sizes for R. roxellana more closely resembles that of giant panda rather than its congeners. These findings offer new insights into the dietary adaptations and evolutionary history of colobine primates

Mapping Paratope on Antithrombotic Antibody 6B4 to Epitope on Platelet Glycoprotein Ibalpha via Molecular Dynamic Simulations

<div><p>Binding of platelet receptor glycoprotein Ibα (GPIbα) to the A1 domain of von Willebrand factor (vWF) is a critical step in both physiologic hemostasis and pathologic thrombosis, for initiating platelet adhesion to subendothelium of blood vessels at sites of vascular injury. Gain-of-function mutations in GPIbα contribute to an abnormally high-affinity binding of platelets to vWF and can lead to thrombosis, an accurate complication causing heart attack and stroke. Of various antithrombotic monoclonal antibodies (mAbs) targeting human GPIbα, 6B4 is a potent one to inhibit the interaction between GPIbα and vWF-A1 under static and flow conditions. Mapping paratope to epitope with mutagenesis experiments, a traditional route in researches of these antithrombotic mAbs, is usually expensive and time-consuming. Here, we suggested a novel computational procedure, which combines with homology modeling, rigid body docking, free and steered molecular dynamics (MD) simulations, to identify key paratope residues on 6B4 and their partners on GPIbα, with hypothesis that the stable hydrogen bonds and salt bridges are the important linkers between paratope and epitope residues. Based on a best constructed model of 6B4 bound with GPIbα, the survival ratios and rupture times of all detected hydrogen bonds and salt bridges in binding site were examined via free and steered MD simulations and regarded as indices of thermal and mechanical stabilizations of the bonds, respectively. Five principal paratope residues with their partners were predicted with their high survival ratios and/or long rupture times of involved hydrogen bonds, or with their hydrogen bond stabilization indices ranked in top 5. Exciting, the present results were in good agreement with previous mutagenesis experiment data, meaning a wide application prospect of our novel computational procedure on researches of molecular of basis of ligand-receptor interactions, various antithrombotic mAbs and other antibodies as well as theoretically design of biomolecular drugs.</p> </div

Variation of interatomic distance versus steered simulation time.

<p>The interatomic distances of the six representative bonds under stretching were plotted against simulation time, where all descriptions for line types, bonds and their lengths are same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042263#pone-0042263-g004" target="_blank">Figure 4</a>. These time courses of interatomic distances showed that, the 5^th and 16^th bonds were very quickly ruptured (A and D), in comparison with others, in which the 9^th and 1^st bonds would maintain more long time (C and F) than 4^th and 10^th bonds (B and E).</p

Hydrogen bonds and salt bridges with higher stabilization in Top 8.

*<p>HBSI expresses the index of hydrogen bond stabilization (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042263#s2" target="_blank">Materials and Methods</a>).</p

The false discovery rate, sensitivity and specificity of three different positive criterions.

<p>The false discovery rate was evaluated by Eq. 1, and Eq. 3 was used to estimated the sensitivity and specificity, with use of data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042263#pone.0042263.s006" target="_blank">Table S2</a> in Suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042263#s2" target="_blank">Materials</a>.</p

The CDRs and identified paratope residues of 6B4 [16].

*<p>The positions of residues in 6B4-ScFv were expressed with serial numbering from N-terminal of heavy chain to C-terminal of light chain.</p>†<p>The serial numbers, 166, 167, 168, 233 and 106, are corresponding to those in Kabat numbering, such as 27D, 27E, 28, 93 and 100C, respectively. ^†The residue sequences, contributed to their respective CDRs (CDR H1, H2, H3, L1, L2 and L3), follow the serial numbering too.</p