Co-localization of acinar markers and insulin in pancreatic cells of subjects with type 2 diabetes

To search for clues suggesting that beta cells may generate by transdifferentiation in humans, we assessed the presence of cells double positive for exocrine (amylase, carboxypeptidase A) and endocrine (insulin) markers in the pancreas of non-diabetic individuals (ND) and patients with type 2 diabetes (T2D). Samples from twelve ND and twelve matched T2D multiorgan donors were studied by electron microscopy, including amylase and insulin immunogold labeling; carboxypeptidase A immunofluorescence light microscopy assessment was also performed. In the pancreas from four T2D donors, cells containing both zymogen-like and insulin-like granules were observed, scattered in the exocrine compartment. Nature of granules was confirmed by immunogold labeling for amylase and insulin. Double positive cells ranged from 0.82 to 1.74 per mm2, corresponding to 0.26±0.045% of the counted exocrine cells. Intriguingly, cells of the innate immune systems (mast cells and/or macrophages) were adjacent to 33.3±13.6% of these hybrid cells. No cells showing co-localization of amylase and insulin were found in ND samples by electron microscopy. Similarly, cells containing both carboxypeptidase A and insulin were more frequently observed in the diabetic pancreata. These results demonstrate more abundant presence of cells containing both acinar markers and insulin in the pancreas of T2D subjects, which suggests possible conversion from one cellular type to the other and specific association with the diseased condition

Microcontact printing trapping air : a versatile tool for protein microarray fabrication

The present work introduces a new method for the fabrication of protein micro-patterns, microcontact printing trapping air. The method is based on microcontact printing, a well-established soft-lithographic technique for printing bioactive protein patterns. Usually, the stamp used is made of poly(dimethylsiloxane) obtained by replicating a lithographically microfabricated silicon master. In microcontact printing, the dimensions of the features in the stamp are critical, since the high compressibility of poly(dimethylsiloxane) causes high aspect ratio features to collapse, leading to the printing of undesired areas. In most cases, this is an unwanted effect, which interferes with the printing quality. In this work we used a poly(dimethylsiloxane) stamp bearing an array of micro-posts which, when placed over a flat surface, collapses with consequent formation of an air gap around the entire array. This effect is linked to the distance between the posts that form the array and can be exploited for the fabrication of protein microarrays having a remarkably low background noise for fluorescence detection

Biomolecules and cells on surfaces : fundamental concepts

Not available

Protein patterning by microcontact printing using pyramidal PDMS stamps

Micro-contact printing, μCP, is a well-established soft-lithography technique for printing biomolecules. μCP uses stamps made of Poly(dimethylsiloxane), PDMS, made by replicating a microstructured silicon master fabricated by semiconductor manufacturing processes. One of the problems of the μCP is the difficult control of the printing process, which, because of the high compressibility of PDMS, is very sensitive to minute changes in the applied pressure. This over-sensitive response leads to frequent and/or uncontrollable collapse of the stamps with high aspect ratios, thus decreasing the printing accuracy and reproducibility. Here we present a straightforward methodology of designing and fabricating PDMS structures with an architecture which uses the collapse of the stamp to reduce, rather than enlarge the variability of the printing. The PDMS stamp, organized as an array of pyramidal micro-posts, whose ceiling collapses when pressed on a flat surface, replicates the structure of the silicon master fabricated by anisotropic wet etching. Upon application of pressure, depending on the size of, and the pitch between, the PDMS pyramids, an air gap is formed surrounding either the entire array, or individual posts. The printing technology, which also exhibits a remarkably low background noise for fluorescence detection, may find applications when the clear demarcation of the shapes of protein patterns and the distance between them are critical, such as microarrays and studies of cell patterning

Protein interaction with combinatorial surfaces

No abstract available

Microbeads on microposts: an inverted architecture for bead microarrays

The rapid development of genomics and proteomics requires accelerated improvement of the microarrays density, multiplexing, readout capabilities and cost-effectiveness. The bead arrays are increasingly attractive because of their self-assembly-based fabrication, which alleviates many problems of top-down microfabrication. Here we present a simple, reliable, robust and modular technique for the fabrication of bead microarrays, which combines the directed assembling of beads in microstructures and PDMS-based replica molding. The beads are first self-assembled in pyramidal microwells fabricated by anisotropic etching of silicon substrates, then transferred on the apex of PDMS pyramids that replicate the silicon microstructures. The arrays are chemically and biochemically robust; they are spatially addressable and have the potential for being informationally addressable; and they appear to offer better readout capabilities than the classical microarrays

Fungal growth in confined microfabricated networks

The understanding and control of cell growth in confined microenvironments has application to a variety of fields including cell biosensor development, medical device fabrication, and pathogen control. While the majority of work in these areas has focused on mammalian and bacterial cell growth, this study reports on the growth behavior of fungal cells in three-dimensionally confined PDMS microenvironments of a scale similar to that of individual hyphae. The general responses of hyphae to physical confinement included continued apical extension against barriers, resultant filament bending and increased rates of subapical branching with apparent directionality towards structure openings. Overall, these responses promoted continued extension of hyphae through the confined areas and away from the distal regions of the fungal colony. The induction of branching by apical obstruction provides a means of controlling the growth and branching of fungal hyphae through purposefully designed microstructures

Negotiation of obstacles by fungi in micro-fabricated structures : to turn or not to turn?

Polymer microstructures were used to examine the manner in which fungal filaments negotiate obstacles in confined environments. When faced with an obstacle requiring a right-angle turn, two different responses were observed. In 21% of cases, hyphae turned around the corner and continued growth, while in the remaining 79% of cases, filaments continued apical growth into the corner, resulting in bending of the distal portions of the filament. The different reactions could not be linked to physical constraints (e.g., filament flexibility) since the filament deflection required to negotiate the obstacle was the same in all cases. Instead, the response appeared to be related to the original direction of growth at the time of filament formation (branching), with filaments turning only if the resultant growth vector was no more than 90/spl deg/ from their original branching vector. The results suggest that filaments are somehow able to retain a memory of their original branching direction, consistent with an overall survival strategy based on continued growth away from the colony center and into the surrounding environment

Amplification of protein adsorption on micro/nanostructures for microarray applications

The fabrication and operation of biodevices require the accurate control of the concentration of the bioactive molecules on the surface, as well as the preservation of their bioactivity, in particular proteins that have a significant propensity for surface-induced denaturation. A method that allows the adsorption of proteins on ‘combinatorial’ micro/nano-surfaces fabricated via laser ablation of a thin metal layer deposited on a polymer has been recently proposed1. The present study investigates the relationship between the amplification of the protein adsorption and their molecular characteristics (total molecular surface; and charge- and hydrophobicity-specific surface). The adsorption of five proteins with very different molecular characteristics, i.e. alpha-chymotrypsin, human serum albumin, human immunoglobulin, lysozyme, and myoglobin, has been characterized using quantitative fluorescence measurements and atomic force microscopy. It has been found that the ‘combinatorial’ nature of the micro/nano-channels surface allows for the increased adsorption of molecularly different proteins, comparing with the adsorption on flat surfaces. This amplification increases for proteins with lower molecular surface which can capitalize better on the newly created surface and nano-environments. Importantly, the adsorption on micro/nano-fabricated structures appears to be less dependent on the local molecular descriptors, i.e. hydrophobicity and charges, due to the combinatorialization of the nano-areas presented to the proteins. The amplification of adsorption is important, ranging from 3- to 10-fold, with a higher amplification for smaller, globular proteins