Hereditary hearing loss: a 96 gene targeted sequencing protocol reveals novel alleles in a series of Italian and Qatari patients

Deafness is a really common disorder in humans. It can begin at any age with any degree of severity. Hereditary hearing loss is characterized by a vast genetic heterogeneity with more than 140 loci described in humans but only 65 genes so far identified. Families affected by hearing impairment would have real advantages from an early molecular diagnosis that is of primary relevance in genetic counseling. In this perspective, here we report a family-based approach employing Ion Torrent DNA sequencing technology to analyze coding and UTR regions of 96 genes related to hearing function and loss in a first series of 12 families coming from Italy and Qatar. Using this approach we were able to find the causative gene in 4 out of these 12 families (33%). In particular 5 novel alleles were identified in the following genes LOXHD1, TMPRSS3, TECTA and MYO15A already associated with hearing impairment. Our study confirms the usefulness of a targeted sequencing approach despite larger numbers are required for further validation and for defining a molecular epidemiology picture of hearing loss in these two countries.     

In Silico Models for Dynamic Connected Cell Cultures Mimicking Hepatocyte-Endothelial Cell-Adipocyte Interaction Circle

The biochemistry of a system made up of three kinds of cell is virtually impossible to work out without the use of in silico models. Here, we deal with homeostatic balance phenomena from a metabolic point of view and we present a new computational model merging three single-cell models, already available from our research group: the first model reproduced the metabolic behaviour of a hepatocyte, the second one represented an endothelial cell, and the third one described an adipocyte. Multiple interconnections were created among these three models in order to mimic the main physiological interactions that are known for the examined cell phenotypes. The ultimate aim was to recreate the accomplishment of the homeostatic balance as it was observed for an in vitro connected three-culture system concerning glucose and lipid metabolism in the presence of the medium flow. The whole model was based on a modular approach and on a set of nonlinear differential equations implemented in Simulink, applying Michaelis-Menten kinetic laws and some energy balance considerations to the studied metabolic pathways. Our in silico model was then validated against experimental datasets coming from literature about the cited in vitro model. The agreement between simulated and experimental results was good and the behaviour of the connected culture system was reproduced through an adequate parameter evaluation. The developed model may help other researchers to investigate further about integrated metabolism and the regulation mechanisms underlying the physiological homeostasis.     

Sensing scaffolds to monitor cellular activity using impedance measurements

Scaffolds are cell adhesive matrices for the realisation of tissue constructs. Here we describe how scaffolds for tissue engineering can also be used as sensors for monitoring cellular activity such as adhesion and spreading. Carbon nanotube polymer composites were fabricated into membranes and scaffolds with electro-conductive properties. Impedance techniques were used to measure the effects of media and cell cultures on composite membranes and the results were analysed using lumped parameter models. We show that protein adhesion can be distinguished from cell adhesion as the impedance changes are much smaller for the latter (5%). In the presence of cells, impedance changes are of the order of 40% and can be correlated with adhesion, spreading and changes in cell density.     

Membrane lipids and ethanol tolerance in the mouse. The influence of dietary fatty acid composition

Mice of the TO Swiss strain received diets containing different amounts of saturated or unsaturated fat throughout life. These diets produced characteristic changes in cardiac phospholipid fatty acid composition, but produced no significant differences in fatty acid composition of phospholipids from a crude membrane fraction of brain. When littermates of these animals were exposed to ethanol vapour in an inhalation chamber it was observed that mice which had received a diet high in saturated fat lost the righting reflex at an estimated concentration of ethanol in blood higher than that required for mice receiving a control diet, or a diet rich in polyunsaturated fat. Analysis of the brain membrane fraction from those animals which had received ethanol revealed that mice receiving the highly saturated fat diet now had a significantly greater proportion of saturated fatty acids in brain membrane phospholipids. These results are discussed in relation to the hypothesis that brain membrane lipid composition may influence the behavioural response to ethanol.     

Expression of C-type natriuretic peptide and its receptor NPR-B in cardiomyocytes

C-type natriuretic peptide (CNP) was recently found in myocardium at the mRNA and protein levels, but it is not known whether cardiomyocytes are able to produce CNP. The aim of this study was to determine the expression of CNP and its specific receptor NPR-B in cardiac cells, both in vitro and ex vivo. CNP, brain natriuretic peptide (BNP) and natriuretic peptide receptor (NPR)-B mRNA expression were examined by RT-PCR in the H9c2 rat cardiac myoblast cell line, in neonatal rat primary cardiomyocytes and in human umbilical vein endothelial cells (HUVECs) as control. CNP protein expression was probed in cardiac tissue sections obtained from adult male minipigs by immunohistochemistry, and in H9c2 cells both by immunocytochemistry and by specific radioimmunoassay. The results showed that cardiac cells as well as endothelial cells were able to produce CNP. Unlike cardiomyocytes, as expected, in endothelial cells expression of BNP was not detected. NPR-B mRNA expression was found in both cell types. Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination (H9c2: CNP = 0.86 ± 0.083 pg/mg) and by immunocytochemistry studies. By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes. Expression of CNP in cardiac cells at gene and protein levels suggests that the heart is actively involved in the production of CNP.