Online immobilized metal affinity chromatography/mass spectrometric analysis of changes elicited by cCMP in the murine brain phosphoproteome

An automated online immobilized metal affinity chromatography/high‐performance liquid chromatography mass spectrometric (IMAC‐HPLC/MS/MS) method was developed to study cytidine 3′,5′‐cyclic monophosphate (cCMP)‐specific protein phosphorylation, analogous to a previously successful offline IMAC method using microvolume IMAC pipette tips. The optimized method identified murine brain phosphoproteins selectively modified by challenge with cCMP, using manual interpretation of the results to confirm both phosphorylation and selectivity of response to cCMP. A number of proteins identified by this strategy have potential roles in hyperproliferation, a previously reported response to elevated levels of cCMP. Copyright © 2008 John Wiley & Sons, Ltd.     

Solution of sparse quasi-square rectangular systems by Gaussian elimination

We present a general method for the linear least-squares solutionof overdetermined and underdetermined systems. The method isparticularly efficient when the coefficient matrix is quasi-square,that is when the number of rows and number of columns is almostthe same. The numerical methods for linear least-squares problemsand minimum-norm solutions do not generally take account ofthis special characteristic. The proposed method is based onLU factorization of the original quasi-square matrix A, assumingthat A has full rank. In the overdetermined case, the LU factorsare used to compute a basis for the null space of A^T. The right-handside vector b is then projected onto this subspace and the least-squaressolution is obtained from the solution of this reduced problem.In the case of underdetermined systems, the desired solutionis again obtained through the solution of a reduced system.The use of this method may lead to important savings in computationaltime for both dense and sparse matrices. It is also shown inthe paper that, even in cases where the matrices are quite small,sparse solvers perform better than dense solvers. Some practicalexamples that illustrate the use of the method are included.     

Uniform and universal Glivenko-Cantelli classes

A class of measurable functions on a probability space is called a Glivenko-Cantelli class if the empirical measuresP _n converge to the trueP uniformly over almost surely. is a universal Glivenko-Cantelli class if it is a Glivenko-Cantelli Cantelli class for all lawsP on a measurable space, and a uniform Glivenko-Cantelli class if the convergence is also uniform inP. We give general sufficient conditions for the Glivenko-Cantelli and universal Glivenko-Cantelli properties and examples to show that some stronger conditions are not necessary. The uniform Glivenko-Cantelli property is characterized, under measurability assumptions, by an entropy condition.     

Analysis of urinary nucleosides. II. Comparison of mass spectrometric methods for the analysis of urinary nucleosides

Qualitative and quantitative analyses of urinary nucleosides have diagnostic potential as tumour markers. We have developed separation techniques linked to mass spectrometric detection in order to overcome the problems associated with past identification and quantitation methods. The three methods of analysis utilised were: gas chromatography/mass spectrometry (GC/MS), high-performance liquid chromatography/ion-trap mass spectrometry (HPLC/ITMS) and capillary liquid chromatography/triple quadrupole mass spectrometry (CapLC/TQMS). Here we compare the relative effectiveness of each of the techniques for subsequent application in the systematic study of urinary nucleoside profiles in cancer patients. All three methods proved to be valuable techniques for such urinary nucleoside analyses, and a combination rather than one single choice is concluded as the ideal.     

Spatial Taming and Trapping of Molecules

Molecular beam techniques for study of collisional and spectroscopic processes have recently been enhanced by use of static electric or magnetic fields to orient or align molecules with permanent dipole moments. A more general method is now in prospect, applicable both to alignment and to spatial trapping of molecules. This exploits the anisotropic interaction of the electric field vector of intense laser radiation with the dipole moment induced in a polarizable molecule by the laser field. The interaction creates directional superpositions of field-free states that correspond to oblate spheroidal wavefunctions, with eigenenergies that decrease with increasing field strength. We suggest that this polarizability interaction produces the marked alignment found in laser-induced dissociative ionization of CO by the Saclay group. We also present calculations illustrating the feasibility of spattal trapping. In combination with supermirror focussing and buffer-gas cooling, an intense infrared laser can typically confine molecules for long-times (-hours) within a small (-picoliter) and cold (?1°K) “pocket of light.”     

Mechanical Stimulation of Growth Plate Chondrocytes: Previous Approaches and Future Directions

Growth plate cartilage resides near the ends of long bones and is the primary driver of skeletal growth. During growth, both intrinsically and extrinsically generated mechanical stresses act on chondrocytes in the growth plate. Although the role of mechanical stresses in promoting tissue growth and homeostasis has been strongly demonstrated in articular cartilage of the major skeletal joints, effects of stresses on growth plate cartilage and bone growth are not well established. Here, we review the literature on mechanobiology in growth plate cartilage at macroscopic and microscopic scales, with particular emphasis on comparison of results obtained using different methodological approaches, as well as from whole animal and in vitro experiments. To answer these questions, macroscopic mechanical stimulators have been developed and applied to study mechanobiology of growth plate cartilage and chondrocytes. However, the previous approaches have tested a limited number of stress conditions, and the mechanobiology of a single chondrocyte has not been well studied due to limitations of the macroscopic mechanical stimulators. We explore how microfluidics devices can overcome these limitations and improve current understanding of growth plate chondrocyte mechanobiology. In particular, microfluidic devices can generate multiple stress conditions in a single platform and enable real-time monitoring of metabolism and cellular behavior using optical microscopy. Systematic characterization of the chondrocytes using microfluidics will enhance our understanding of how to use mechanical stresses to control the bone growth and the properties of tissue-engineered growth plate cartilage.