The empowerment of translational research: lessons from laminopathies

The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.     

Synthesis and proteomic activity evaluation of a new isotope-coded affinity tagging (ICAT) reagent

During recent years, quantitative proteome profiling has taken advantage of incorporating the traditional stable isotope dilution analysis into global scale or discovery-based proteomic experiments that use mass spectrometers as detectors to allow the pairwise study of differently expressed proteins. Quantitative protein analysis by means of the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS) enables the pairwise comparison of protein expression levels in biological samples. Herein, a modified ICAT reagent, named BAA-ICAT (beta-alanine-arm-ICAT) in which the polyether linker is replaced by a more water-soluble polyamide one, was investigated.     

Systematic screens for human disease genes, from yeast to human and back

Systematic screens for human disease genes have emerged in recent years, due to the wealth of information provided by genome sequences and large scale datasets. Here we review how integration of genomic data in yeast and human is helping to elucidate the genetic basis of mitochondrial diseases. The identification of nearly all yeast mitochondrial proteins and many of their functional interactions provides insight into the role of mitochondria in cellular processes. This information enables prioritization of the candidate genes underlying mitochondrial disorders. In an iterative fashion, the link between predicted human candidate genes and their disease phenotypes can be experimentally tested back in yeast.     

The impact of mitochondrial DNA on human lifespan: a view from studies on centenarians

The role of inherited and somatic mutations of mitochondrial DNA (mtDNA) in aging and longevity is complex and highly controversial, owing to its peculiar genetics, including the phenomenon of heteroplasmy. Most of the data on mtDNA and longevity have been obtained on humans and particularly on centenarians, i. e., people who escaped or delayed the major age-related pathologies and reached the extreme limit of human lifespan. In this review we summarize the most recent advances in this field that suggest a consistent role in human longevity of both germ-line inherited and somatically acquired mutations. The particular case of the association with longevity of the somatic C150T mutation is extensively discussed, challenging the tenet that mtDNA mutations are basically detrimental. We also stress several limitations of our present knowledge, regarding the difficulty in extrapolating to humans the results obtained in animal models, owing to a variety of biological differences, including the very limited genetic variability of mtDNA in the strains used in laboratory experiments. The use of high-throughput technologies and the extensive analysis, possibly at the single cell level, of different tissues and cell types derived from the same individual will help in disentangling the complexity of mtDNA in aging and longevity.     

An experimental biomimetic platform for artificial olfaction

Artificial olfactory systems have been studied for the last two decades mainly from the point of view of the features of olfactory neuron receptor fields. Other fundamental olfaction properties have only been episodically considered in artificial systems. As a result, current artificial olfactory systems are mostly intended as instruments and are of poor benefit for biologists who may need tools to model and test olfactory models. Herewith, we show how a simple experimental approach can be used to account for several phenomena observed in olfaction. An artificial epithelium is formed as a disordered distributed layer of broadly selective color indicators dispersed in a transparent polymer layer. The whole epithelium is probed with colored light, imaged with a digital camera and the olfactory response upon exposure to an odor is the change of the multispectral image. The pixels are treated as olfactory receptor neurons, whose optical properties are used to build a convergence classifier into a number of mathematically defined artificial glomeruli. A non-homogenous exposure of the test structure to the odours gives rise to a time and spatial dependence of the response of the different glomeruli strikingly similar to patterns observed in the olfactory bulb. The model seems to mimic both the formation of glomeruli, the zonal nature of olfactory epithelium, and the spatio-temporal signal patterns at the glomeruli level. This platform is able to provide a readily available test vehicle for chemists developing optical indicators for chemical sensing purposes and for biologists to test models of olfactory system organization.     

Dispensing an enzyme-conjugated solution into an ELISA plate by adapting ink-jet printers

The rapid and precise delivery of small volumes of bio-fluids (from picoliters to nanoliters) is a key feature of modern bioanalytical assays. Commercial ink-jet printers are low-cost systems which enable the dispensing of tiny droplets at a rate which may exceed 10⁴ Hz per nozzle. Currently, the main ejection technologies are piezoelectric and bubble-jet. We adapted two commercial printers, respectively a piezoelectric and a bubble-jet one, for the deposition of immunoglobulins into an ELISA plate. The objective was to perform a comparative evaluation of the two classes of ink-jet technologies in terms of required hardware modifications and possible damage on the dispensed molecules. The hardware of the two printers was modified to dispense an enzyme conjugate solution, containing polyclonal rabbit anti-human IgG labelled with HRP in 7 wells of an ELISA plate. Moreover, the ELISA assay was used to assess the functional activity of the biomolecules after ejection. ELISA is a common and well-assessed technique to detect the presence of particular antigens or antibodies in a sample. We employed an ELISA diagnostic kit for the qualitative screening of anti-ENA antibodies to verify the ability of the dispensed immunoglobulins to bind the primary antibodies in the wells. Experimental tests showed that the dispensing of immunoglobulins using the piezoelectric printer does not cause any detectable difference on the outcome of the ELISA test if compared to manual dispensing using micropipettes. On the contrary, the thermal printhead was not able to reliably dispense the bio-fluid, which may mean that a surfactant is required to modify the wetting properties of the liquid.     

Purification and characterization of the enantioselective esterase from Kluyveromyces marxianus CBS 1553

An intracellular esterase from the yeast Kluyveromyces marxianus CBS 1553 with interesting enantioselective hydrolytic activity towards racemic esters of 1,2-O-isopropylidene glycerol (IPG) was purified and characterized. Optimal culture conditions for the obtainment of the enantioselective esterase on a 5 l-fermentation scale were investigated. Two esterase activities (EST1 and EST2) in the crude cell extract were identified by native PAGE with specific activity staining and separated from each other by anion-exchange chromatography. EST1 showed higher activity and enantioselectivity than EST2 in the resolution of racemic IPG acetate and was further purified by hydrophobic interaction chromatography and preparative electrophoresis (final specific activity approximately = 300 U mg(-1), showing a main protein band with a molecular mass of 29 kDa. EST1 showed optimal activity between pH 8.0 and 10.0 and was stable in the pH range 7.0-10.0. Moreover, it was rather thermostable and active up to 80 degrees C, and retained most of its activity in the presence of 15% (v/v) of various organic solvents. The enzyme showed similar Vmax in the hydrolysis of the acetate esters of IPG, whereas the Km value towards (S)-IPG acetate was significantly lower than the one towards the (R)-enantiomer (5.3 and 70 microM, respectively). Finally, comparison of EST1 activity in the presence of different glycerol esters and synthetic substrates with different chain lengths showed a strong preference of this biocatalyst for short-chain substrates.     

Human fetal aorta-derived vascular progenitor cells: identification and potential application in ischemic diseases

Vasculogenesis, the formation of blood vessels in embryonic or fetal tissue mediated by immature vascular cells (i.e., angioblasts), is poorly understood. Here we report a summary of our recent studies on the identification of a population of vascular progenitor cells (VPCs) in human fetal aorta. These undifferentiated mesenchymal cells co-express endothelial and myogenic markers (CD133+, CD34+, KDR+, desmin+) and are localized in outer layer of the aortic stroma of 11–12 weeks old human fetuses. Under stimulation with VEGF-A or PDGF-BB, VPCs give origin to a mixed population of mature endothelial and mural cells, respectively. When embedded in a three-dimensional collagen gel, VPCs organize into cohesive cellular cords that resembled mature vascular structures. The therapeutic efficacy of a small number of VPCs transplanted into ischemic limb muscle was demonstrated in immunodeficient mice. Investigation of the effect of VPCs on experimental heart ischemia and on diabetic ischemic ulcers in mice is in progress and seems to confirm their efficacy. On the whole, fetal aorta represents an important source for the investigation of phenotypic and functional features of human vascular progenitor cells.