Generation of miniaturized planar ecombinant antibody arrays using a microcantilever-based printer.

Miniaturized (Ø 10 µm), multiplexed (>5-plex), and high-density (>100 000 spots cm(-2)) antibody arrays will play a key role in generating protein expression profiles in health and disease. However, producing such antibody arrays is challenging, and it is the type and range of available spotters which set the stage. This pilot study explored the use of a novel microspotting tool, Bioplume(TM)-consisting of an array of micromachined silicon cantilevers with integrated microfluidic channels-to produce miniaturized, multiplexed, and high-density planar recombinant antibody arrays for protein expression profiling which targets crude, directly labelled serum. The results demonstrated that 16-plex recombinant antibody arrays could be produced-based on miniaturized spot features (78.5 um(2), Ø 10 µm) at a 7-125-times increased spot density (250 000 spots cm(-2)), interfaced with a fluorescent-based read-out. This prototype platform was found to display adequate reproducibility (spot-to-spot) and an assay sensitivity in the pM range. The feasibility of the array platform for serum protein profiling was outlined.

Functionalization of optical nanotip arrays with an electrochemical microcantilever for multiplexed DNA detection.

Optical nanotip arrays fabricated on etched fiber bundles were functionalized with DNA spots. Such unconventional substrates (3D and non-planar) are difficult to pattern with standard microfabrication techniques but, using an electrochemical cantilever, up to 400 spots were electrodeposited on the nanostructured optical surface in 5 min. This approach allows each spot to be addressed individually and multiplexed fluorescence detection is demonstrated. Finally, remote fluorescence detection was performed by imaging through the optical fiber bundle itself after hybridisation with the complementary sequence.

Expression of insulin-responsive glucose transporter GLUT4 mRNA in the rat brain and spinal cord: an in situ hybridization study.

Following a previous immunocytochemical study of GLUT4 in the rat brain and spinal cord (J. Comp. Neurol. 399 (1998) 492), we now report the distribution and cellular expression of GLUT4 mRNA in the CNS using reverse transcription-polymerase chain reaction and non-radioactive in situ hybridization (ISH). The former technique demonstrated the expression of GLUT4 in the different regions examined while ISH with a specific riboprobe allowed the anatomical localization of GLUT4 mRNA. A strong hybridization signal was detected in the piriform and entorhinal cortices and in the pyramidal cell layer of the hippocampal CA1-CA3 areas. Numerous moderately labeled cells were additionally observed in the dentate gyrus granular layer, subiculum and most neocortical areas, as well as in different nuclei of the limbic and motor systems. In contrast, positive cell groups were scarce in the hypothalamus. In the hindbrain, a strong expression of GLUT4 mRNA was observed in the large cell bodies of the red nucleus and cerebellar Purkinje cell layer. Moreover, different groups of moderately labeled cells were found in the deep cerebellar and medullary motor nuclei, in various reticular fields and in the ventral horn of the spinal cord. The present results of ISH mostly agree with the immunocytochemical data reported by our group, although the immunoreactive cells were generally less numerous. However, the fact that a high expression of GLUT4 mRNA was observed in cell bodies of the piriform lobe, hippocampus and substantia nigra, whereas the immunoreactivity for GLUT4 was low in these regions, suggests the existence of post-transcriptional regulation of GLUT4 expression which may depend on the physiological conditions of the animals.

Motoneuron morphological alterations before and after the onset of the disease in the wobbler mouse.

The wobbler mutant mouse displays a recessively inherited neurological disease with degeneration of motoneurons and is considered to be an animal model for human motoneuron diseases. Mutant mice can be clinically recognised at about 3-4 weeks of age but a polymorphic marker close to the wobbler gene offers the opportunity of a preclinical diagnosis. Using this polymorphic marker we performed morphometric (cell size) analysis of spinal cord motoneurons from 10 to 40 days post natal (PN). We observed at day 16 PN a transient appearance of swollen motoneurons, probably those that present vacuolar degeneration a little later and possibly die. One week later, from 21 days onwards, we found that the subpopulation of large motoneurons was depleted in the mutant mice. The absence of large motoneurons may have important physiological consequences and the loss or absence of differentiation of this particular subpopulation of motoneurons may be a key event in the course of the disease.