Selective ROCK2 inhibition in focal cerebral ischemia

Objective: Rho-associated kinase (ROCK) is a key regulator of numerous processes in multiple cell types relevant in stroke pathophysiology. ROCK inhibitors have improved outcome in experimental models of acute ischemic or hemorrhagic stroke. However, the relevant ROCK isoform (ROCK1 or ROCK2) in acute stroke is not known. Methods: We characterized the pharmacodynamic and pharmacokinetic profile, and tested the efficacy and safety of a novel selective ROCK2 inhibitor KD025 (formerly SLx-2119) in focal cerebral ischemia models in mice. Results: KD025 dose-dependently reduced infarct volume after transient middle cerebral artery occlusion. The therapeutic window was at least 3 h from stroke onset, and the efficacy was sustained for at least 4 weeks. KD025 was at least as efficacious in aged, diabetic or female mice, as in normal adult males. Concurrent treatment with atorvastatin was safe, but not additive or synergistic. KD025 was also safe in a permanent ischemia model, albeit with diminished efficacy. As one mechanism of protection, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, implicating enhanced collateral flow. Unlike isoform-nonselective ROCK inhibitors, KD025 did not cause significant hypotension, a dose-limiting side effect in acute ischemic stroke. Interpretation Altogether, these data show that KD025 is efficacious and safe in acute focal cerebral ischemia in mice, implicating ROCK2 as the relevant isoform in acute ischemic stroke. Data suggest that selective ROCK2 inhibition has a favorable safety profile to facilitate clinical translation

Inversion of calcite twin data for paleostress orientations and magnitudes: A new technique tested and calibrated on numerically-generated and natural data

The inversion of calcite twin data is a powerful tool to reconstruct paleostresses sustained by carbonate rocks during their geological history. Following Etchecopar's (1984) pioneering work, this study presents a new technique for the inversion of calcite twin data that reconstructs the 5 parameters of the deviatoric stress tensors from both monophase and polyphase twin datasets. The uncertainties in the parameters of the stress tensors reconstructed by this new technique are evaluated on numerically-generated datasets. The technique not only reliably defines the 5 parameters of the deviatoric stress tensor, but also reliably separates very close superimposed stress tensors (30° of difference in maximum principal stress orientation or switch between σ3 and σ2 axes). The technique is further shown to be robust to sampling bias and to slight variability in the critical resolved shear stress. Due to our still incomplete knowledge of the evolution of the critical resolved shear stress with grain size, our results show that it is recommended to analyze twin data subsets of homogeneous grain size to minimize possible errors, mainly those concerning differential stress values. The methodological uncertainty in principal stress orientations is about ± 10°; it is about ± 0.1 for the stress ratio. For differential stresses, the uncertainty is lower than ± 30%. Applying the technique to vein samples within Mesozoic limestones from the Monte Nero anticline (northern Apennines, Italy) demonstrates its ability to reliably detect and separate tectonically significant paleostress orientations and magnitudes from naturally deformed polyphase samples, hence to fingerprint the regional paleostresses of interest in tectonic studies

Laser Diode Thermal Desorption-positive mode Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry applied for the ultra-fast quantitative analysis of BKM120 in human plasma: method validation and its application to clinical studies

A sensitive and ultra-fast method utilizing the LDTD-APCI ion source coupled to tandem mass spectrometry was developed and validated for the quantitative analysis of BKM120 in human plasma. Samples originating from protein precipitation (PP) followed by salting-out assisted liquid-liquid extraction (SALLE) were spotted on the LazWell™ plate prior their thermal desorption and analysis by tandem mass spectrometry. Two MRM transitions (m/z 411.4367.4 and m/z 411.4307.4) were used to quantify BKM120. The present method was validated in terms of selectivity, linearity, accuracy, precision, stability, extraction recovery and matrix effect. The linearity of the method was found to be within the concentration range of 5.00 and 2000 ng mL-1. The sample analysis run time was 10 seconds as compared to 4.5 min for the current validated LC-ESI-MS/MS method. The LDTD-APCI-MS/MS method was successfully applied for the analysis of BKM120 in clinical samples obtained from 3 clinical studies. The resultant data was consistent with the results obtained with the validated LC-ESI-MS/MS assay. This work demonstrates that LDTD-APCI-MS/MS is a reliable method for the ultra-fast quantitative analysis of BKM120 and can be used to support its bioanalysis in the frame of the clinical trials

Is EBSD a more efficient tool than universal stage to collect calcite twin data as used in calcite twin inversion for stress?

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