Transparency of reporting in molecular diagnostics

The major advances made over the past few years in molecular and cell biology are providing a progressively more detailed understanding of the molecular pathways that control normal processes and become dysregulated in disease. This has resulted in the documentation of numerous genetic, epigenetic, transcriptomic, proteomic and metabolomic biomarkers that promise earlier disease detection, more accurate patient stratification and better prognosis. Furthermore, molecular fingerprinting of diseases can be predictive of drug response and so assist with specific targeting of drugs against disease-associated molecules and function

Alkyl radical geometry controls geminate cage recombination in alkylcobalamins

Abstract: The radical pair that results from photolysis of adenosylcob(II1)alamin (AdoCbl"') undergoes primary geminate recombination with a first-order rate constant of 1 x lo9 s-l. In contrast, methylcob(II1)alamin (CH3Cbl"') and aristeromicylcob(II1)alamin (AriCblII', the carbocyclic analogue of AdoCbl"' in which the ribofuranose ring oxygen has been replaced with a methylene group) does not undergo primary geminate recombination. The ribofwanose group enables a high rate of geminate recombination in the [Ado' Cbl"'] radical pair. This may be due to a stereoelectronic (p-anomeric) effect that maintains a pyramidal geometry at the 5'-carbon of the 5'-deoxyadenosyl radical, or it may be due to hindered rotation about the C4t-C5, bond such that /?-elimination to the olefin is prevented. Recombination in the geminate singlet radical pair is in competition with diffusive escape to form a solvent-separated radical pair. Hyperfine coupling from Co" promotes intersystem crossing to the triplet radical pair (Chagovetz, A. M.; Grissom, C. B. J. Am. Chem. SOC. 1993, 115, 12152). Recombination of the [CH3' Cbl"] radical pair is not prevented by a lack of intersystem crossing, as neither unlabeled or I3C-labeled CH3Cbl"' undergoes geminate recombination. There is only a small difference in the rate of diffusive recombination in the solvent cage for AdoCbl"', AriCbl"', and CH3Cbl"' following photolysis: 2.01 x 10" s-l, 2.20 x lo4 s-l, and 1.16 x lo4 s-l. The rate of diffusive recombination is limited by productive collisions and not by radical geometry or intersystem crossing. The CF3' radical that results from photolysis of (trifluoromethyl)cob(III)alamin (CF3Cbl"') maintains its pyramidal geometry and undergoes faster diffusive recombination in the solvent cage at 51 x lo4 s-l. The C-Co bond dissociation enthalpy in AriCbl"' is 37 f 1.4 kcaymol. The profound difference in geminate recombination rates for AdoCbl"' and CH3Cbl"' is consistent with their different biological roles as enzymatic cofactors: AdoCbl"' is an initiator of radical chain chemistry in the active site, whereas CH3Cbl"' is a methyl group donor in an S~2-type process

Kinetics of Multiplex Hybridization: Mechanisms and Implications☆

Quantitative analysis of DNA microarray data is complicated by uncertainties inherent to the experimental setup. Using computer simulations and real-time experimental results, we have previously demonstrated effects of multiplex reactions on a single sensing zone of an array, which may be a leading factor in erroneous interpretation of experimental data. We suggest here that a simplified three-component kinetic model may present a sufficient approximation to describe the general case of DNA sensing in a complex sample milieu. We show that, by analyzing the real-time hybridization kinetics of a nontarget species, we can perform quantitative analysis of unlabeled targets of interest within a broad dynamic range of concentrations

Competitive Displacement of DNA during Surface Hybridization

Using real-time dual-color fluorescence detection, we have experimentally tracked individual target species during competitive DNA surface hybridization in a two-component sample. Our experimental results demonstrate displacement of the lower affinity species by the higher affinity species and corroborate recent theoretical models describing competitive DNA surface hybridization. Competition at probe sites complementary to one of the two DNA species was monitored in separate experiments for two different target pairs. Each pair differs in sequence by a single nucleotide polymorphism, and one pair includes a folding target. We propose a mechanistic interpretation of the differences between hybridization curves of targets in multi-component and single-component experiments

Neurodegeneration severity can be predicted from early microglia alterations monitored in vivo in a mouse model of chronic glaucoma

Microglia serve key homeostatic roles, and respond to neuronal perturbation and decline with a high spatiotemporal resolution. The course of all chronic CNS pathologies is thus paralleled by local microgliosis and microglia activation, which begin at early stages of the disease. However, the possibility of using live monitoring of microglia during early disease progression to predict the severity of neurodegeneration has not been explored. Because the retina allows live tracking of fluorescent microglia in their intact niche, here we investigated their early changes in relation to later optic nerve neurodegeneration. To achieve this, we used the DBA/2J mouse model of inherited glaucoma, which develops progressive retinal ganglion cell degeneration of variable severity during aging, and represents a useful model to study pathogenic mechanisms of retinal ganglion cell decline that are similar to those in human glaucoma. We imaged CX3CR1+/GFP microglial cells in vivo at ages ranging from 1 to 5 months by confocal scanning laser ophthalmoscopy (cSLO) and quantified cell density and morphological activation. We detected early microgliosis at the optic nerve head (ONH), where axonopathy first manifests, and could track attenuation of this microgliosis induced by minocycline. We also observed heterogeneous and dynamic patterns of early microglia activation in the retina. When the same animals were aged and analyzed for the severity of optic nerve pathology at 10 months of age, we found a strong correlation with the levels of ONH microgliosis at 3 to 4 months. Our findings indicate that live imaging and monitoring the time course and levels of early retinal microgliosis and microglia activation in glaucoma could serve as indicators of future neurodegeneration severity

Deconvolution analysis for classifying gastric adenocarcinoma patients based on differential scanning calorimetry serum thermograms

Recently, differential scanning calorimetry (DSC) has been acknowledged as a novel tool for diagnosing and monitoring several diseases. This highly sensitive technique has been traditionally used to study thermally induced protein folding/unfolding transitions. In previous research papers, DSC profiles from blood samples of patients were analyzed and they exhibited marked differences in the thermal denaturation profile. Thus, we investigated the use of this novel technology in blood serum samples from 25 healthy subjects and 30 patients with gastric adenocarcinoma (GAC) at different stages of tumor development with a new multiparametric approach. The analysis of the calorimetric profiles of blood serum from GAC patients allowed us to discriminate three stages of cancer development (I to III) from those of healthy individuals. After a multiparametric analysis, a classification of blood serum DSC parameters from patients with GAC is proposed. Certain parameters exhibited significant differences (P < 0.05) and allowed the discrimination of healthy subjects/patients from patients at different tumor stages. The results of this work validate DSC as a novel technique for GAC patient classification and staging, and offer new graphical tools and value ranges for the acquired parameters in order to discriminate healthy from diseased subjects with increased disease burden.Peer Reviewe