Shrinkage estimation for functional principal component scores with application to the population kinetics of plasma folate

We present the application of a nonparametric method to performing functional principal component analysis for functional curve data that consist of measurements of a random trajectory for a sample of subjects. This design typically consists of an irregular grid of time points on which repeated measurements are taken for a number of subjects. We introduce shrinkage estimates for the functional principal component scores that serve as the random effects in the model. Scatterplot smoothing methods are used to estimate the mean function and covariance surface of this model. We propose improved estimation in the neighborhood of and at the diagonal of the covariance surface, where the measurement errors are reflected. The presence of additive measurement errors motivates shrinkage estimates for the functional principal component scores. Shrinkage estimates are developed through best linear prediction and in a generalized version, aiming at minimizing one-curve-leave-out prediction error. The estimation of individual trajectories combines data obtained from that individual as well as all other individuals. We apply our methods to new data regarding the analysis of the level of 14C-folate in plasma as a function of time since dosing of healthy adults with a small tracer dose of 14C-folic acid. A time transformation was incorporated to handle design irregularity concerning the time points on which the measurements were taken. The proposed methodology, incorporating shrinkage and data-adaptive features, is seen to be well suited for describing population kinetics of 14C-folate-specific activity and random effects, and can also be applied to other functional data analysis problems.     

Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents

In the present study a shape independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of ejaculated canine sperm cells. Volumetric shrinkage during freezing of canine sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPAs (6 different combinations of freezing media were used, ranging from a media with no CPAs, and those with 0.5%, 3%, and 6% glycerol and with 0.5% and 3% Me(2)SO). Using previously published data, the canine sperm cell was modeled as a cylinder of length 105.7mum and a radius of 0.32mum with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best fit" membrane permeability parameters at 5 and 10 degrees C/min for canine sperm cells in the absence of CPAs are: L(pg)=0.52x10(-15)m(3)/Ns (0.0029mum/min-atm) and E(Lp)=64.0kJ/mol (15.3kcal/mol) (R(2)=0.99); and the corresponding parameters in the presence of CPAs ranged from L(pg)[cpa]=0.46 to 0.53x10(-15) m(3)/Ns (0.0027-0.0031mum/min-atm) and E(Lp)[cpa]=46.4-56.0kJ/mol (11.1-13.4kcal/mol). These parameters are significantly different than previously published parameters for canine and other mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that optimal rates of freezing canine sperm cells ranges from 10 to 30 degrees C/min; these theoretical cooling rates are found to be in close conformity with previously published but empirically determined optimal cooling rates.     

Detecting correlation between characters in a comparative analysis with uncertain phylogeny

Abstract.— The importance of accommodating the phylogenetic history of a group when performing a comparative analysis is now widely recognized. The typical approaches either assume the tree is known without error, or they base inferences on a collection of well-supported trees or on a collection of trees generated under a stochastic model of cladogenesis. However, these approaches do not adequately account for the uncertainty of phylogenetic trees in a comparative analysis, especially when data relevant to the phylogeny of a group are available. Here, we develop a method for performing comparative analyses that is based on an extension of Felsenstein's independent contrasts method. Uncertainties in the phylogeny, branch lengths, and other parameters are accommodated by averaging over all possible trees, weighting each by the probability that the tree is correct. We do this in a Bayesian framework and use Markov chain Monte Carlo to perform the high-dimensional summations and integrations required by the analysis. We illustrate the method using comparative characters sampled from Anolis lizards.     

Molecular classification of cancer types from microarray data using the combination of genetic algorithms and support vector machines

Simultaneous multiclass classification of tumor types is essential for future clinical implementations of microarray-based cancer diagnosis. In this study, we have combined genetic algorithms (GAs) and all paired support vector machines (SVMs) for multiclass cancer identification. The predictive features have been selected through iterative SVMs/GAs, and recursive feature elimination post-processing steps, leading to a very compact cancer-related predictive gene set. Leave-one-out cross-validations yielded accuracies of 87.93% for the eight-class and 85.19% for the fourteen-class cancer classifications, outperforming the results derived from previously published methods.     

Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.     

Altered balance of half-reactions in p-hydroxybenzoate hydroxylase caused by substituting the 2'-carbon of FAD with fluorine

Apo-p-hydroxybenzoate hydroxylase was reconstituted using 2'-fluoro-2'-deoxy-arabino-FAD, a synthetic flavin in which the hydroxyl of the 2'-center of the ribityl chain was replaced with fluorine in an inverted configuration. The absorbance spectral changes caused by the binding of either p-hydroxybenzoate (pOHB) or 2,4-dihydroxybenzoate (2,4-diOHB) indicated that the isoalloxazine of the artificial flavin adopts the more solvent-exposed "out" conformation rather than the partially buried "in" conformation near the aromatic substrate. In contrast, the flavin of the natural enzyme adopts the in conformation when pOHB is bound. Much of the behavior of the artificial enzyme can be rationalized in light of the preference of the flavin for the out conformation, including the weaker binding of pOHB, the tighter binding of 2,4-diOHB, and the slower reactions involved in the hydroxylation of pOHB and 2,4-diOHB. Particularly noteworthy is the enhancement of the reduction of the flavin by NADPH when pOHB is bound to the active site, consistent with the recent finding that the reaction occurs when the flavin adopts the out conformation (Palfey, B. A., Moran, G. R., Entsch, B., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 1153-1158). Thus, whereas the change that induces the out conformation is detrimental to the oxidative half-reaction, it improves the reductive half-reaction, showing that the control of the flavin position in p-hydroxybenzoate hydroxylase represents a compromise between the conflicting needs of two chemically disparate half-reactions, and demonstrating that the 2'-hydroxyl of FAD can serve as a critical control element in flavoenzyme catalysis.     

Ca2+-independent protein kinase Cs mediate heterologous desensitization of leukocyte chemokine receptors by opioid receptors

Heterologous desensitization of chemokine receptors by opioids has been considered to contribute to their immunosuppressive effects. Previous studies show that Met-enkephalin, an endogenous opioid, down-regulates chemotaxis of selected chemokine receptors via phosphorylation. In the present study, we further investigated the molecular mechanism of such cross-regulation. Our data showed that preincubation with Met-enkephalin inhibited both MIP-1 alpha-mediated chemotaxis and Ca(2+) flux of monocytes in a dose-dependent manner. The inhibitory effects were maximal using nanomolar concentrations of activating chemokines, a concentration found in physiological conditions. A decrease both in chemokine receptor affinity and in coupling efficiency between receptors and G protein were observed, which directly contributed to the desensitization effects. However, comparing with chemokines such as MIP-1 alpha and MCP-1, opioids did not elicit a calcium flux, failed to induce MIP-1 alpha receptors internalization, and mediated a less potent heterologous desensitization. We hypothesized that these differences might originate from the involvement of different protein kinase C (PKC) isotypes. In our studies, opioid-mediated down-regulation of MIP-1 alpha receptors could be blocked by the general PKC inhibitor calphostin C, but not by the calcium-dependent classic PKC inhibitor Go6976. Western blotting analysis and immunofluorescent staining further showed that only calcium-independent PKCs were activated upon opioid stimulation. Thus, opioids achieve desensitization of chemokine receptors via a unique pathway, involving only calcium-independent PKC isotypes.     

Rapid tau aggregation and delayed hippocampal neuronal death induced by persistent thrombin signaling

Tau hyperphosphorylation, leading to self-aggregation, is widely held to underlie the neurofibrillary degeneration found in Alzheimer's disease (AD) and other tauopathies. However, it is unclear exactly what environmental factors may trigger this pathogenetic tau hyperphosphorylation. From several perspectives, the coagulation serine protease, thrombin, has been implicated in AD and activates several different protein kinase pathways but has not previously been shown how it may contribute to AD pathogenesis. Here we report that nanomolar thrombin induced rapid tau hyperphosphorylation and aggregation in murine hippocampal neurons via protease-activated receptors, which was followed by delayed synaptophysin reduction and apoptotic neuronal death. Mechanistic study revealed that a persistent thrombin signaling via protease-activated receptor 4 and prolonged downstream p44/42 mitogenactivated protein kinase activation are at least in part responsible. These results pathogenetically linked thrombin to subpopulations of AD and other tauopathies associated with cerebrovascular damage. Such knowledge may be instrumental in transforming therapeutic paradigms.