Mouse Aurora A: expression in Escherichia coli and purification.

Aurora kinases have recently become some of the most intensely pursued oncology targets for the design of small-molecule inhibitors. Most of the active Aurora-A protein variants are currently being expressed from baculoviruses in insect cells, while catalytically impaired proteins can also be generated in and purified from Escherichia coli. In this study we present a method of expressing large quantities of active mouse Aurora-A kinase domain as an N-terminal glutathione-S-transferase fusion protein in bacteria and outline a simple purification method that produces greater than 99% pure protein samples suitable for enzymatic assays and X-ray crystallography. The methods described in this report simplify mouse Aurora-A expression and purification, and may aid in the production of other difficult kinases in prokaryotes.

Structural white matter abnormalities in patients with idiopathic dystonia.

We investigated whether structural white matter abnormalities, in the form of disruption of axonal coherence and integrity as measured with diffusion tensor imaging (DTI), constitute an underlying pathological mechanism of idiopathic dystonia (ID), independent of genotype status. We studied seven subjects with ID: all had cervical dystonia as their main symptom (one patient also had spasmodic dysphonia and two patients had concurrent generalized dystonia, both DYT1-negative). We compared DTI MR images of patients with 10 controls, evaluating differences in mean diffusivity (MD) and fractional anisotropy (FA). ID was associated with increased FA values in the thalamus and adjacent white matter, and in the white matter underlying the middle frontal gyrus. ID was also associated with increase in MD in adjacent white matter to the pallidum and putamen bilaterally, left caudate, and in subcortical hemispheric regions, including the postcentral gyrus. Abnormal FA and MD in patients with ID indicate that abnormal axonal coherence and integrity contribute to the pathophysiology of dystonia. These findings suggest that ID is not only a functional disorder, but also associated with structural brain changes. Impaired connectivity and disrupted flow of information may contribute to the impairment of motor planning and regulation in dystonia.

The range of motor activation in the normal human cortex using bold FMRI.

Understanding and documenting the nature of normal human brain functional motor activation using functional Magnetic Resonance Imaging (fMRI) is necessary, if valid statements are to be made about normal and disease functional states using fMRI activation maps. The present study examines activation maps in "normal" adults. Six healthy adult volunteers performed three motor tasks isolating the tongue, non-dominant foot, and non-dominant thumb during a single magnetic resonance imaging (MRI)/(fMRI) scanning session. Group maps demonstrated discrete areas of activation that were task dependent. The degree of variability between the anatomical central location of global maximum intensity for each individual may mean extra care should be applied when using the global maximum to define the area of activation. These differences may represent anatomical variability among individuals, task complexity, paradigm design, data analysis techniques or a combination thereof, which form the basis of our ongoing research endeavors. Standard notions of strongly associated functions as related to anatomic foci may need to be revised.

Functional Magnetic Resonance Imaging (fMRI): A Brief Exercise for an Undergraduate Laboratory Course.

Functional neuroimaging represents an important technique for the study of the brain. However, the skills necessary for collecting, processing, and analyzing functional magnetic resonance imaging (fMRI) data sets are complex and relatively few undergraduate programs offer students an opportunity to acquire these skills or to observe functional neuroimaging. We report here on our experiences working with functional neuroimaging in an undergraduate laboratory course and suggest resources for the implementation of a similar exercise in a comparable setting. This exercise is structured so that four class meetings are devoted to functional neuroimaging. During these sessions, we discuss the basics of fMRI, study design, the advantages and disadvantages of this technique for the study of brain function as well as a general overview of data processing and analysis. Due to the college's proximity to a medical school, we are able to offer students an opportunity to observe functional neuroimaging sessions (however, this component is not critical for the completion of this exercise). Two final class sessions are devoted to data processing and presentation as well as writing up the experimental results. The exercise culminates in a paper based on the American Psychological Association format for a small number of subjects. At the conclusion of the exercise, students were surveyed to assess their impressions of the lab sessions. The results from these surveys indicate that students found this portion of the laboratory course to be a very positive experience. While this lab exercise does require some initial set up, we believe it stimulates the development of critical thinking skills with a technique that is used increasingly in neuroscience research. Both print and online resources are suggested to assist faculty in setting up a similar exercise.

Bioinformatics and functional magnetic resonance imaging in clinical populations: practical aspects of data collection, analysis, interpretation, and management.

In this paper the authors review the issues associated with bioinformatics and functional magnetic resonance (fMR) imaging in the context of neurosurgery. They discuss the practical aspects of data collection, analysis, interpretation, and the management of large data sets, and they consider the challenges involved in the adoption of fMR imaging into clinical neurosurgical practice. Their goal is to provide neurosurgeons and other clinicians with a better understanding of some of the current issues associated with bioinformatics or neuroinformatics and fMR imaging. Thousands to tens of thousands of images are typically acquired during an fMR imaging session. It is essential to follow an activation task paradigm exactly to obtain an accurate representation of cortical activation. These images are then interactively postprocessed offline to produce an activation map, or in some cases a series of maps. The maps may then be viewed and interpreted in consultation with a neurosurgeon and/or other clinicians. After this consultation, long-term archiving of the processed fMR activation maps along with the standard structural MR images is a complex but necessary final step in this process. The fMR modality represents a valuable tool in the neurosurgical planning process that is still in the developmental stages for routine clinical use, but holds exceptional promise for patient care.

Anticoagulants (thrombin inhibitors) and aspirin synergize with P2Y12 receptor antagonism in thrombosis.

BACKGROUND: This study was designed to determine whether (1) P2Y12 antagonism synergizes with other antithrombotics and (2) anticoagulants (thrombin inhibitors) affect the antithrombotic activity elicited by P2Y12 antagonism. METHODS AND RESULTS: Thrombosis was achieved by perfusion of human and murine blood through type III collagen-coated capillaries at arterial shear rate. CT50547, a direct-acting P2Y12 antagonist, inhibited thrombosis in PPACK- but not heparin-anticoagulated human blood. In contrast, CT50547 inhibited thrombosis in aspirin-treated individuals independently of the anticoagulant. Thrombin and TXA2 also synergized with P2Y12 in the absence of anticoagulation, because combined treatment of aspirin or C921-78 (a factor Xa inhibitor) with CT50547 or 2-MeSAMP (a P2Y12 antagonist) inhibited the thrombotic process, whereas all treatments failed to inhibit thrombosis when used individually. Synergism was also observed ex vivo when P2Y12-deficient (P2Y12-/-) mice were administered aspirin or coagulation inhibitors (C921-78 and bivalirudin). Finally, using intravital microscopy, we found that both C921-78 and bivalirudin abrogated the thrombotic process in P2Y12+/- mice, whereas each showed only partial efficacy in P2Y12+/+ animals. CONCLUSIONS: Our study indicates that (1) thrombin inhibitors and aspirin have a demonstrable synergy of antithrombotic activity with P2Y12 antagonism and (2) the in vitro analysis of the antithrombotic activity of P2Y12 antagonists is affected by the anticoagulant used for blood collection. This suggests that the antithrombotic potential of P2Y12 antagonists in vitro may be overestimated in anticoagulated samples of blood and best achieved in vivo by the inclusion of aspirin and/or a thrombin inhibitor.

P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries.

The critical role for ADP in arterial thrombogenesis was established by the clinical success of P2Y12 antagonists, currently used at doses that block 40-50% of the P2Y12 on platelets. This study was designed to determine the role of P2Y12 in platelet thrombosis and how its complete absence affects the thrombotic process. P2Y12-null mice were generated by a gene-targeting strategy. Using an in vivo mesenteric artery injury model and real-time continuous analysis of the thrombotic process, we observed that the time for appearance of first thrombus was delayed and that only small, unstable thrombi formed in P2Y12-/- mice without reaching occlusive size, in the absence of aspirin. Platelet adhesion to vWF was impaired in P2Y12-/- platelets. While adhesion to fibrinogen and collagen appeared normal, the platelets in thrombi from P2Y12-/- mice on collagen were less dense and less activated than their WT counterparts. P2Y12-/- platelet activation was also reduced in response to ADP or a PAR-4-activating peptide. Thus, P2Y12 is involved in several key steps of thrombosis: platelet adhesion/activation, thrombus growth, and stability. The data suggest that more aggressive strategies of P2Y12 antagonism will be antithrombotic without the requirement of aspirin cotherapy and may provide benefits even to the aspirin-nonresponder population.