The Belgian Association for Study of the Liver Guidance Document on the Management of Adult and Paediatric Non-Alcoholic Fatty Liver Disease.

Non-Alcoholic Fatty Liver Disease (NAFLD) is highly prevalent and associated with considerable liver-related and non-liverrelated morbidity and mortality. There is, however, a lot of uncertainty on how to handle NAFLD in clinical practice. The current guidance document, compiled under the aegis of the Belgian Association for the Study of the Liver by a panel of experts in NAFLD, from a broad range of different specialties, covers many questions encountered in daily clinical practice regarding diagnosis, screening, therapy and follow-up in adult and paediatric patients. Guidance statements in this document are based on the available evidence whenever possible. In case of absence of evidence or inconsistency of the data, guidance statements were formulated based on consensus of the expert panel. This guidance document is intended as a help for clinicians (general practitioners and all involved specialties) to implement the most recent evidence and insights in the field of NAFLD within a Belgian perspective.

Genetics of sudden cardiac death in the young.

Sudden cardiac death (SCD) has an enormous impact on those who are left behind, evoking strong feelings of anxiety and incomprehension because such a dramatic event was not anticipated. Moreover, over the last decade a prominent genetic contribution to the pathogenesis of SCD has been unveiled. As many inherited cardiac diseases show an autosomal dominant pattern of inheritance, the risk of carrying the same inherited predisposition is a real concern for the relatives. In this article, we discuss the major causes of primary electrical disorders, cardiomyopathies and thoracic aortic dissection and address issues in genotype-phenotype correlation, personalized management and cardiogenetic counselling.

Prediction of HIV-1 drug susceptibility phenotype from the viral genotype using linear regression modeling.

Linear regression modeling on a database of HIV-1 genotypes and phenotypes was applied to predict the HIV-1 resistance phenotype from the viral genotype. In this approach, the phenotypic measurement is estimated as the weighted sum of the effects of individual mutations. Higher order interaction terms (mutation pairs) were included to account for synergistic and antagonistic effects between mutations. The most significant mutations and interactions identified by the linear regression models for 17 approved antiretroviral drugs are reported. Although linear regression modeling is a statistical data-driven technique focused on obtaining the best possible prediction, many of these mutations are also known resistance-associated mutations, indicating that the statistical models largely reflect well characterized biological phenomena. The performance of the models in predicting in vitro susceptibility phenotype and virologic response in treated patients is described. In addition to a high concordance with in vitro measured fold change, which was the primary aim of model design, the models per drug show good predictivity of therapy response for regimens including that drug, even in the absence of other clinically relevant factors such as background regimen.

Heuristic statistical analysis of fluorescence fluctuation data with bright spikes: application to ligand binding to the human serotonin receptor expressed in Escherichia coli cells.

A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore-coated beads that mimic the behaviour of multi-liganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.

Fluorescence fluctuation analysis for the study of interactions between oligonucleotides and polycationic polymers.

The interactions between a cationic polymer, poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), and negatively charged rhodamine-labeled 25-mer phosphodiester oligonucleotides (Rh-ONs) were studied by fluorescence fluctuation spectroscopy and other techniques. The composition of the pDMAEMA/Rh-ON complexes was investigated as a function of the charge ratio (+/-) by increasing the pDMAEMA concentration and keeping the Rh-ON concentration constant. We applied two different methods for analyzing the fluorescence fluctuation profiles of the pDMAEMA/Rh-ON complexes, which depended on their composition. First, we analyzed the data with the photon counting histogram (PCH) technique, which determines the molecular brightness and the concentration of fluorophores (Chen et al, 1999). A particular challenge for the data analysis is the occurrence of sudden fluorescence bursts in the fluorescence fluctuation profiles, which are linked to the appearance of multimolecular complexes (i. e. when several Rh-ONs were present in one complex). A quantitative interpretation of the analysis for the complexes remains challenging and is connected to the rarity of the fluorescent bursts, which do not provide sufficient data statistics. To specifically address the problem of the fluorescent bursts we employed a method described by Van Craenenbroeck et al. (1999). This method, applicable only when data were integrated over much longer time bins, allowed us to estimate the number of fluorescence bursts which could be considered as a relative measure of the amount of multimolecular complexes present. When monomolecular complexes were formed, i. e. at high values of the charge ratio, highly intense fluorescence peaks were not present and the interpretation of the PCH analysis was more straightforward. The molecular brightness of the species (epsilon), as revealed from PCH analysis, was greater than epsilon for the free Rh-ONs, indicating that the Rh-ONs were attached to pDMAEMA chains.

Fluorescence correlation spectroscopy: molecular recognition at the single molecule level.

Fluorescence correlation spectroscopy (FCS) is a fluorescence microscopy technique that allows the study of molecular interactions in extremely low volumes, at nanomolar concentrations, even when binding is not accompanied by a fluorescence change. It can be applied directly in living cells. FCS clearly considerably extends the possibilities of the classical techniques used in molecular recognition studies and can be considered to belong to a growing group of techniques that allow detection at the single molecule level. In this review, several applications of FCS, both in vitro and in vivo, will be discussed.

Quantitative characterization of the binding of fluorescently labeled colchicine to tubulin in vitro using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a new technique that allows the determination of the diffusion constant of a fluorescent molecule in solution. Also, the binding of the fluorescent molecule to a target can be analyzed, if the difference in the diffusion coefficients of the free and bound ligand is sufficiently large. With FCS, the interaction between fluorescein-colchicine (FC) and tubulin has been studied in vitro. A fast and reversible binding is observed with an association constant at room temperature of (3.9 +/- 0.1) x 10(4) M-1. No competition with colchicine is seen, indicating that FCS reveals the existence of a new binding site on tubulin. FCS is not able to show the binding of FC to the original colchicine binding site, even though it exists, because the fluorescence of FC is strongly quenched upon binding to this site. This quenching is evident in spectrofluorometry experiments, revealing a slow binding of FC to tubulin that is subject to competition with colchicine. FCS allows the determination of the diffusion coefficients of both free and bound fluorescent colchicine which were found to be (2.6 +/- 0.2) x 10(-)10 and (2.0 +/- 0.2) x 10(-)11 m2 s-1, respectively. It can be concluded that fluorescent labeling, especially of small molecules, can interfere considerably with the binding behavior that is being studied. Although general qualitative effects in vivo are similar for colchicine and its fluorescein derivative, this quantitative study of the binding to tubulin presents a nuanced view, and the existence of a second binding site for FC can even explain some conflicting indications in the literature.