Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes

In vitro models that better reflect in vivo epithelial barrier (patho-)physiology are urgently required to predict adverse drug effects. Here we introduce extracellular matrix-supported intestinal tubules in perfused microfluidic devices, exhibiting tissue polarization and transporter expression. Forty leak-tight tubules are cultured in parallel on a single plate and their response to pharmacological stimuli is recorded over 125 h using automated imaging techniques. A study comprising 357 gut tubes is performed, of which 93% are leak tight before exposure. EC50-time curves could be extracted that provide insight into both concentration and exposure time response. Full compatibility with standard equipment and user-friendly operation make this Organ-on-a-Chip platform readily applicable in routine laboratories

Imaging of Streptomyces coelicolor A3(2) with Reduced Autofluorescence Reveals a Novel Stage of FtsZ Localization

Imaging of low abundance proteins in time and space by fluorescence microscopy is typically hampered by host-cell autofluorescence. Streptomycetes are an important model system for the study of bacterial development, and undergo multiple synchronous cell division during the sporulation stage. To analyse this phenomenon in detail, fluorescence microscopy, and in particular also the recently published novel live imaging techniques, require optimal signal to noise ratios. Here we describe the development of a novel derivative of Streptomyces coelicolor A3(2) with strongly reduced autofluorescence, allowing the imaging of fluorescently labelled proteins at significantly higher resolution. The enhanced image detail provided novel localization information for the cell division protein FtsZ, demonstrating a new developmental stage where multiple FtsZ foci accumulate at the septal plane. This suggests that multiple foci are sequentially produced, ultimately connecting to form the complete Z ring. The enhanced imaging properties are an important step forward for the confocal and live imaging of less abundant proteins and for the use of lower intensity fluorophores in streptomycetes

Unintended Consequences of Conservation Actions: Managing Disease in Complex Ecosystems

Infectious diseases are increasingly recognised to be a major threat to biodiversity. Disease management tools such as control of animal movements and vaccination can be used to mitigate the impact and spread of diseases in targeted species. They can reduce the risk of epidemics and in turn the risks of population decline and extinction. However, all species are embedded in communities and interactions between species can be complex, hence increasing the chance of survival of one species can have repercussions on the whole community structure. In this study, we use an example from the Serengeti ecosystem in Tanzania to explore how a vaccination campaign against Canine Distemper Virus (CDV) targeted at conserving the African lion (Panthera leo), could affect the viability of a coexisting threatened species, the cheetah (Acinonyx jubatus). Assuming that CDV plays a role in lion regulation, our results suggest that a vaccination programme, if successful, risks destabilising the simple two-species system considered, as simulations show that vaccination interventions could almost double the probability of extinction of an isolated cheetah population over the next 60 years. This work uses a simple example to illustrate how predictive modelling can be a useful tool in examining the consequence of vaccination interventions on non-target species. It also highlights the importance of carefully considering linkages between human-intervention, species viability and community structure when planning species-based conservation actions

Safeguarding pollinators and their values to human well-being

Wild and managed pollinators provide a wide range of benefits to society in terms of contributions to food security, farmer and beekeeper livelihoods, social and cultural values, as well as the maintenance of wider biodiversity and ecosystem stability. Pollinators face numerous threats, including changes in land-use and management intensity, climate change, pesticides and genetically modified crops, pollinator management and pathogens, and invasive alien species. There are well-documented declines in some wild and managed pollinators in several regions of the world. However, many effective policy and management responses can be implemented to safeguard pollinators and sustain pollination services

Pemphigus autoimmunity: Hypotheses and realities

The goal of contemporary research in pemphigus vulgaris and pemphigus foliaceus is to achieve and maintain clinical remission without corticosteroids. Recent advances of knowledge on pemphigus autoimmunity scrutinize old dogmas, resolve controversies, and open novel perspectives for treatment. Elucidation of intimate mechanisms of keratinocyte detachment and death in pemphigus has challenged the monopathogenic explanation of disease immunopathology. Over 50 organ-specific and non-organ-specific antigens can be targeted by pemphigus autoimmunity, including desmosomal cadherins and other adhesion molecules, PERP cholinergic and other cell membrane (CM) receptors, and mitochondrial proteins. The initial insult is sustained by the autoantibodies to the cell membrane receptor antigens triggering the intracellular signaling by Src, epidermal growth factor receptor kinase, protein kinases A and C, phospholipase C, mTOR, p38 MAPK, JNK, other tyrosine kinases, and calmodulin that cause basal cell shrinkage and ripping desmosomes off the CM. Autoantibodies synergize with effectors of apoptotic and oncotic pathways, serine proteases, and inflammatory cytokines to overcome the natural resistance and activate the cell death program in keratinocytes. The process of keratinocyte shrinkage/detachment and death via apoptosis/oncosis has been termed apoptolysis to emphasize that it is triggered by the same signal effectors and mediated by the same cell death enzymes. The natural course of pemphigus has improved due to a substantial progress in developing of the steroid-sparing therapies combining the immunosuppressive and direct anti-acantholytic effects. Further elucidation of the molecular mechanisms mediating immune dysregulation and apoptolysis in pemphigus should improve our understanding of disease pathogenesis and facilitate development of steroid-free treatment of patients

The desmosome and pemphigus

Desmosomes are patch-like intercellular adhering junctions (“maculae adherentes”), which, in concert with the related adherens junctions, provide the mechanical strength to intercellular adhesion. Therefore, it is not surprising that desmosomes are abundant in tissues subjected to significant mechanical stress such as stratified epithelia and myocardium. Desmosomal adhesion is based on the Ca2+-dependent, homo- and heterophilic transinteraction of cadherin-type adhesion molecules. Desmosomal cadherins are anchored to the intermediate filament cytoskeleton by adaptor proteins of the armadillo and plakin families. Desmosomes are dynamic structures subjected to regulation and are therefore targets of signalling pathways, which control their molecular composition and adhesive properties. Moreover, evidence is emerging that desmosomal components themselves take part in outside-in signalling under physiologic and pathologic conditions. Disturbed desmosomal adhesion contributes to the pathogenesis of a number of diseases such as pemphigus, which is caused by autoantibodies against desmosomal cadherins. Beside pemphigus, desmosome-associated diseases are caused by other mechanisms such as genetic defects or bacterial toxins. Because most of these diseases affect the skin, desmosomes are interesting not only for cell biologists who are inspired by their complex structure and molecular composition, but also for clinical physicians who are confronted with patients suffering from severe blistering skin diseases such as pemphigus. To develop disease-specific therapeutic approaches, more insights into the molecular composition and regulation of desmosomes are required

Diabetic ketoacidosis

Diabetic ketoacidosis (DKA) is the most common acute hyperglycaemic emergency in people with diabetes mellitus. A diagnosis of DKA is confirmed when all of the three criteria are present — ‘D’, either elevated blood glucose levels or a family history of diabetes mellitus; ‘K’, the presence of high urinary or blood ketoacids; and ‘A’, a high anion gap metabolic acidosis. Early diagnosis and management are paramount to improve patient outcomes. The mainstays of treatment include restoration of circulating volume, insulin therapy, electrolyte replacement and treatment of any underlying precipitating event. Without optimal treatment, DKA remains a condition with appreciable, although largely preventable, morbidity and mortality. In this Primer, we discuss the epidemiology, pathogenesis, risk factors and diagnosis of DKA and provide practical recommendations for the management of DKA in adults and children