Elevated incubation temperature improves later-life swimming endurance in juvenile Chinook salmon, Oncorhynchus tshawytscha.

The effect of incubation and rearing temperature on muscle development and swimming endurance under a high-intensity swimming test was investigated in juvenile Chinook salmon (Oncorhynchus tshawytscha) in a hatchery experiment. After controlling for the effects of fork length (LF ) and parental identity, times to fatigue of fish were higher when fish were incubated or reared at warmer temperatures. Significant differences among combinations of pre- and post-emergence temperatures conformed to 15-15°C > 15-9°C > 9-9°C > 7-9°C > 7-7°C in 2011 when swimming tests were conducted at 300 accumulated temperature units post-emergence and 15-9°C > (7-9°C = 7-7°C) in 2012 when swimming tests were conducted at an LF of c. 40 mm. The combination of pre- and post-emergence temperatures also affected the number and size of muscle fibres, with differences among temperature treatments in mean fibre cross-sectional area persisting after controlling for LF and parental effects. Nonetheless, neither fibre number nor fibre size accounted for significant variation in swimming endurance. Thus, thermal carryover effects on swimming endurance were not mediated by thermal imprinting of muscle structure. This is the first study to test how temperature, body size and muscle structure interact to affect swimming endurance during early development in salmon.

Evaluation of a Secure Laptop-Based Testing Program in an Undergraduate Nursing Program: Students' Perspective.

Recently, the many robust learning management systems, and the availability of affordable laptops, have made secure laptop-based testing a reality on many campuses. The undergraduate nursing program at the authors' university began to implement a secure laptop-based testing program in 2009, which allowed students to use their newly purchased laptops to take quizzes and tests securely in classrooms. After nearly 5 years' secure laptop-based testing program implementation, a formative evaluation, using a mixed method that has both descriptive and correlational data elements, was conducted to seek constructive feedback from students to improve the program. Evaluation data show that, overall, students (n = 166) believed the secure laptop-based testing program helps them get hands-on experience of taking examinations on the computer and gets them prepared for their computerized NCLEX-RN. Students, however, had a lot of concerns about laptop glitches and campus wireless network glitches they experienced during testing. At the same time, NCLEX-RN first-time passing rate data were analyzed using the χ2 test, and revealed no significant association between the two testing methods (paper-and-pencil testing and the secure laptop-based testing) and students' first-time NCLEX-RN passing rate. Based on the odds ratio, however, the odds of students passing NCLEX-RN the first time was 1.37 times higher if they were taught with the secure laptop-based testing method than if taught with the traditional paper-and-pencil testing method in nursing school. It was recommended to the institution that better quality of laptops needs to be provided to future students, measures needed to be taken to further stabilize the campus wireless Internet network, and there was a need to reevaluate the Laptop Initiative Program.

Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high Plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in HeLa cells.

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.

Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.

Polo-like kinase-1 (PLK1) is an essential mitotic kinase regulating multiple aspects of the cell division process. Activation of PLK1 requires phosphorylation of a conserved threonine residue (Thr 210) in the T-loop of the PLK1 kinase domain, but the kinase responsible for this has not yet been affirmatively identified. Here we show that in human cells PLK1 activation occurs several hours before entry into mitosis, and requires aurora A (AURKA, also known as STK6)-dependent phosphorylation of Thr 210. We find that aurora A can directly phosphorylate PLK1 on Thr 210, and that activity of aurora A towards PLK1 is greatly enhanced by Bora (also known as C13orf34 and FLJ22624), a known cofactor for aurora A (ref. 7). We show that Bora/aurora-A-dependent phosphorylation is a prerequisite for PLK1 to promote mitotic entry after a checkpoint-dependent arrest. Importantly, expression of a PLK1-T210D phospho-mimicking mutant partially overcomes the requirement for aurora A in checkpoint recovery. Taken together, these data demonstrate that the initial activation of PLK1 is a primary function of aurora A.

Serendipitous alkylation of a Plk1 ligand uncovers a new binding channel.

We obtained unanticipated synthetic byproducts from alkylation of the δ(1) nitrogen (N3) of the histidine imidazole ring of the polo-like kinase-1 (Plk1) polo-box domain (PBD)-binding peptide PLHSpT. For the highest-affinity byproduct, bearing a C(6)H(5)(CH(2))(8)- group, a Plk1 PBD cocrystal structure revealed a new binding channel that had previously been occluded. An N-terminal PEGylated version of this peptide containing a hydrolytically stable phosphothreonyl residue (pT) bound the Plk1 PBD with affinity equal to that of the non-PEGylated parent but showed markedly less interaction with the PBDs of the two closely related proteins Plk2 and Plk3. Treatment of cultured cells with this PEGylated peptide resulted in delocalization of Plk1 from centrosomes and kinetochores and in chromosome misalignment that effectively induced mitotic block and apoptotic cell death. This work provides insights that might advance efforts to develop Plk1 PBD-binding inhibitors as potential Plk1-specific anticancer agents.

Development of cyclic peptomer inhibitors targeting the polo-box domain of polo-like kinase 1.

The polo-box domain (PBD) of polo-like kinase 1 (Plk1) is essentially required for the function of Plk1 in cell proliferation. The availability of the phosphopeptide-binding pocket on PBD provides a unique opportunity to develop novel protein-protein interaction inhibitors. Recent identification of a minimal 5-residue-long phosphopeptide, PLHSpT, as a Plk1 PBD-specific ligand has led to the development of several peptide-based inhibitors, but none of them is cyclic peptide. Through the combination of single-peptoid mimics and thio-ether bridged cyclization, we successfully demonstrated for the first time two cyclic peptomers, PL-116 and PL-120, dramatically improved the binding affinity without losing mono-specificity against Plk1 PBD in comparison with the linear parental peptide, PLHSpT. These cyclic peptomers could serve as promising templates for future drug designs to inhibit Plk1 PBD.

Peptoid-Peptide hybrid ligands targeting the polo box domain of polo-like kinase 1.

We replaced the amino terminal Pro residue of the Plk1 polo-box-domain-binding pentapeptide (PLHSpT) with a library of N-alkyl-Gly "peptoids", and identified long-chain tethered phenyl moieties giving greater than two-orders-of-magnitude affinity enhancement. Further simplification by replacing the peptoid residue with appropriate amides gave low-nanomolar affinity N-acylated tetrapeptides. Binding of the N-terminal long-chain phenyl extension was demonstrated by X-ray co-crystal data.

Tranexamic acid mediates proinflammatory and anti-inflammatory signaling via complement C5a regulation in a plasminogen activator-dependent manner.

BACKGROUND: Both tissue plasminogen activator (tPA) in the circulation and urokinase (uPA) in tissues cleave plasminogen (PLG) to plasmin to promote clot lysis. Tranexamic acid (TXA) blocks both the tPA-dependent generation of plasmin on blood clots as well as active plasmin binding to polymerized fibrin, and is commonly administered for bleeding in trauma to limit fibrinolysis. In addition to lysing clots, however, active plasmin also cleaves complement proteins, potentially enhancing inflammation. Because TXA does not block uPA-dependent plasmin generation from PLG and instead augments it, we hypothesized that administration of TXA could enhance or inhibit proinflammatory C5a formation in a PLG activator-dependent manner. METHODS: Citrate platelet-poor plasma (PPP) and PPP depleted of complement protein C3 or PLG were obtained from healthy donors and commercial sources. Platelet-poor plasma was treated ex vivo with or without TXA and either with or without tPA or with or without uPA. Clotting was then induced by calcium and thrombin in clotted PPP experiments, while unclotted PPP experiments were treated with vehicle controls. C5a levels were measured via enzyme-linked immunosorbent assay. Data were expressed as mean ± SEM. RESULTS: Plasmin-mediated fibrinolysis by tPA in clotted PPP led to an approximately threefold increase in C5a production (p < 0.0001), which was significantly inhibited by TXA (p < 0.001). Paradoxically, when fibrinolysis was induced by uPA, TXA treatment led to further increases in C5a production beyond uPA alone (p < 0.0001). Furthermore, clotting was not required for C5a generation from uPA + TXA. C3 depletion had no effect on C5a production, while depletion of PLG eliminated it. CONCLUSIONS: Tranexamic acid administration can have proinflammatory or anti-inflammatory effects through regulating C5a generation by plasmin, depending on the predominating PLG activator. Tranexamic acid may cause significant inflammatory C5a elevations in injured tissues by augmenting uPA-mediated plasmin generation in a fibrin-independent manner. In contrast, TXA reduces C5a generation during tPA-mediated fibrinolysis that may reduce inflammatory responses. In vivo validation of these novel ex vivo findings is warranted and may have important clinical consequences.

Peptide-based inhibitors of Plk1 polo-box domain containing mono-anionic phosphothreonine esters and their pivaloyloxymethyl prodrugs.

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides may be used to disrupt PBD function, the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising in part from the di-anionic nature of the phosphoryl group. We report five-mer peptides containing mono-anionic pThr phosphoryl esters that exhibit single-digit nanomolar PBD binding affinities in extracellular assays and improved antimitotic efficacies in whole cell assays. The cellular efficacies of these peptides have been further enhanced by the application of bio-reversible pivaloyloxymethyl (POM) phosphoryl protection to a pThr-containing polypeptide. Our findings may redefine structural parameters for the development of PBD-binding peptides and peptide mimetics.

Identification of high affinity polo-like kinase 1 (Plk1) polo-box domain binding peptides using oxime-based diversification.

In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein-protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues.

Polo-like kinase-1 controls Aurora A destruction by activating APC/C-Cdh1.

Polo-like kinase-1 (Plk1) is activated before mitosis by Aurora A and its cofactor Bora. In mitosis, Bora is degraded in a manner dependent on Plk1 kinase activity and the E3 ubiquitin ligase SCF-betaTrCP. Here, we show that Plk1 is also required for the timely destruction of its activator Aurora A in late anaphase. It has been shown that Aurora A destruction is controlled by the auxiliary subunit Cdh1 of the Anaphase-Promoting Complex/Cyclosome (APC/C). Remarkably, we found that Plk1-depletion prevented the efficient dephosphorylation of Cdh1 during mitotic exit. Plk1 mediated its effect on Cdh1, at least in part, through direct phosphorylation of the human phosphatase Cdc14A, controlling the phosphorylation state of Cdh1. We conclude that Plk1 facilitates efficient Aurora A degradation through APC/C-Cdh1 activation after mitosis, with a potential role for hCdc14A.

Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1.

Polo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate.

Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine-threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled-coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.

Postnatal development of radial glia and the ventricular zone (VZ): a continuum of the neural stem cell compartment.

The germinal neuroepithelium, or ventricular zone (VZ) of the developing fetal brain, was once thought to transform into the non-germinal ependymal zone of the postnatal and adult brain. Persistence of neural stem cells and neurogenesis throughout postnatal life, however, suggests a continuum between embryonic and adult germinal brain centers. Here, we suggest that developmental changes in anatomy and molecular marker expression in the ventricular walls (the principal germinal centers of the brain) may have misled us into current interpretations of VZ transformation from a germinal to a non-germinal epithelium. We review previous studies and present new data indicating that a germinal layer with characteristics similar to those of the embryonic VZ persists in lateral ventricular walls of the postnatal mouse brain, a region where the adult subventricular zone (SVZ) develops and where neurogenesis persists into adult life. The early postnatal VZ is largely composed of radial glial cell bodies that remain proliferative, display interkinetic nuclear migration and serve as progenitors of new neurons. Ependymal cells then progressively populate the walls of the lateral ventricle but a subpopulation of astrocytes, derived from radial glia, remain in contact with the ventricle lumen, into which they extend a single cilium similar to that found on neuroepithelial cells and radial cells. We propose that a VZ 'compartment' is retained postnatally and that this niche may be essential for stem cell function.