Bone tissue remodels throughout life in response to mechanical loads. Impaired activities of bone cells (osteocytes, osteoblasts and osteoclasts) result in a disruption of the bone remodelling cycle, which eventually leads to bone disorders such as osteoporosis. To develop efficient therapeutic strategies against bone disorders, new tools are needed to unravel the bone remodelling cycle at the molecular level. Here, we developed a microfluidic platform, which should allow understanding the bone remodelling cycle in much more detail and ultimately be used to discover new therapeutic compounds. We focused specifically on studying cell-cell communication between osteocytes and osteoblasts cells via connexin 43-gap junctions. Therefore, a new cell printing method was developed to create living cellular bone cell arrays in a microfluidic channel. Several cell printing designs where osteocytes and osteoblasts heterotypically interacted at localized interfaces were evaluated. Physical contacts between the bone cells were characterised at high resolution by correlative atomic force microscopy (AFM) - fluorescence microscopy. We demonstrated that the platform is compatible with single-cell mechanostimulation by AFM nanoindentation and subsequent fluorescent analysis of the mechanoresponse. As a proof of concept, we showed the functionality of the platform by analysing the induced in vivo-like Ca++ wave in the printed osteocyte-osteoblast network upon mechanical stimulation by fluid flow shear stress.
Campylobacteriosis is a widespread infectious disease, leading to a major health and economic burden. Chickens are considered as the most common infection source for humans. Campylobacter mainly multiplies in the mucus layer of their caeca. No effective control measures are currently available, but passive immunisation of chickens with pathogen-specific maternal IgY antibodies, present in egg yolk of immunised chickens, reduces Campylobacter colonisation. To explore this strategy further, anti-Campylobacter nanobodies, directed against the flagella and major outer membrane proteins, were fused to the constant domains of chicken IgA and IgY, combining the benefits of nanobodies and the effector functions of the Fc-domains. The designer chimeric antibodies were effectively produced in leaves of Nicotiana benthamiana and seeds of Arabidopsis thaliana. Stable expression of the chimeric antibodies in seeds resulted in production levels between 1% and 8% of the total soluble protein. These in planta produced antibodies do not only bind to their purified antigens but also to Campylobacter bacterial cells. In addition, the anti-flagellin chimeric antibodies are reducing the motility of Campylobacter bacteria. These antibody-containing Arabidopsis seeds can be tested for oral passive immunisation of chickens and, if effective, the chimeric antibodies can be produced in crop seeds.
The fast emergence of multi-resistant pathogenic yeasts is caused by the extensive—and sometimes unnecessary—use of broad-spectrum antimicrobial drugs. To rationalise the use of broad-spectrum antifungals, it is essential to have a rapid and sensitive system to identify the most appropriate drug. Here, we developed a microfluidic chip to apply the recently developed optical nanomotion detection (ONMD) method as a rapid antifungal susceptibility test. The microfluidic chip contains no-flow yeast imaging chambers in which the growth medium can be replaced by an antifungal solution without disturbing the nanomotion of the cells in the imaging chamber. This allows for recording the cellular nanomotion of the same cells at regular time intervals of a few minutes before and throughout the treatment with an antifungal. Hence, the real-time response of individual cells to a killing compound can be quantified. In this way, this killing rate provides a new measure to rapidly assess the susceptibility of a specific antifungal. It also permits the determination of the ratio of antifungal resistant versus sensitive cells in a population.
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