Human Amnion Epithelial Cells Induced to Express Functional Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis, an autosomal recessive disorder caused by a mutation in a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), remains a leading cause of childhood respiratory morbidity and mortality. The respiratory consequences of cystic fibrosis include the generation of thick, tenacious mucus that impairs lung clearance, predisposing the individual to repeated and persistent infections, progressive lung damage and shortened lifespan. Currently there is no cure for cystic fibrosis. With this in mind, we investigated the ability of human amnion epithelial cells (hAECs) to express functional CFTR. We found that hAECs formed 3-dimensional structures and expressed the CFTR gene and protein after culture in Small Airway Growth Medium (SAGM). We also observed a polarized CFTR distribution on the membrane of hAECs cultured in SAGM, similar to that observed in polarized airway cells in vivo. Further, hAECs induced to express CFTR possessed functional iodide/chloride (I^−/Cl⁻) ion channels that were inhibited by the CFTR-inhibitor CFTR-172, indicating the presence of functional CFTR ion channels. These data suggest that hAECs may be a promising source for the development of a cellular therapy for cystic fibrosis.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

Monitoring Migration and Measuring Biomass in Benthic Biofilms: The Effects of Dark/far-red Adaptation and Vertical Migration on Fluorescence Measurements

Pulse modulated fluorescence has increasingly been used as an ecological tool to examine changes in the vertical distribution of microphytobenthic cells within the upper layers of estuarine sediments (most often using the minimum fluorescence yield F(o)) as well as to indicate the health of the community (using the maximum PS II quantum efficiency F(v)/F(m)). However, the practicalities of in situ measurements, often dictates that short dark adaptation periods must be used ( approximately 15 min). The use of far-red light as an alternative to dark adaptation was investigated in natural migratory microphytobenthic biofilms and artificial non-migratory biofilms. Prolonged periods of darkness ( approximately 24 h) were not adequate to achieve 'true' measurements of F(o) and F(v)/F(m), which require complete oxidation of Q(A) and full reversal of non-photochemical quenching (NPQ). In some instances, stable values were only achieved using far-red light. Prolonged exposure to dark/far-red light led to a downwards migration of cells in natural assemblages, as seen by a reduction in both F(o) and the maximum fluorescence yield (F(m)). In non-migratory biofilms, F(m) increased in the dark and far-red treatments, indicating a reversal of NPQ, whereas F(o) decreased in far-red light but increased in the dark. It is suggested that far-red light and darkness differentially affected the balance between NPQ reversal and Q(A) oxidation that lead to the measured F(o) yield. The use of far-red light as an alternative to dark adaptation is discussed and the implications of short (e.g., 15 min) dark adaptation times used in situ are discussed with reference to the vertical migration of cells within sediment biofilms.     

Distributions of Virus-Like Particles and Prokaryotes within Microenvironments

Microbial interactions are important for ecosystem function, but occur at the microscale and so are difficult to observe. Previous studies in marine systems have shown significant shifts in microbial community abundance and composition over scales of micrometres to centimetres. This study investigates the microscale abundance distributions of virus-like particles (VLPs) and prokaryotes in the lower reaches of a river to determine the extent to which microscale microbial patchiness exists in freshwater systems. Here we report local hotspots surrounded by gradients that reach a maximum 80 and 107 fold change in abundance over 0.9 cm for prokaryotic and VLP subpopulations. Changes in prokaryotic and VLP hotspots were tightly coupled. There were no gradients at tens of centimetres across the boundary layers, which is consistent with strong mixing and turbulence-driven aggregation found in river systems. Quantification of the patchiness shows a marked asymmetry with patches 10 times greater than background common, but depletions being rare or absent in most samples. This consistent asymmetry suggests that coldspots depleted by grazing and lysis are rapidly mixed to background concentrations, while the prevalence of hotspots indicates persistence against disruption. The hotspot to coldspot relative abundance may be useful for understanding microbial river dynamics. The patchiness indicates that the mean- field approach of bulk phase sampling misses the microbially relevant community variation and may underestimate the concentrations of these important microbial groups.     

Predicting global change impacts on plant species’ distributions: Future challenges

Given the rate of projected environmental change for the 21st century, urgent adaptation and mitigation measures are required to slow down the on-going erosion of biodiversity. Even though increasing evidence shows that recent human-induced environmental changes have already triggered species’ range shifts, changes in phenology and species’ extinctions, accurate projections of species’ responses to future environmental changes are more difficult to ascertain. This is problematic, since there is a growing awareness of the need to adopt proactive conservation planning measures using forecasts of species’ responses to future environmental changes.

There is a substantial body of literature describing and assessing the impacts of various scenarios of climate and land-use change on species’ distributions. Model predictions include a wide range of assumptions and limitations that are widely acknowledged but compromise their use for developing reliable adaptation and mitigation strategies for biodiversity. Indeed, amongst the most used models, few, if any, explicitly deal with migration processes, the dynamics of population at the “trailing edge” of shifting populations, species’ interactions and the interaction between the effects of climate and land-use.

In this review, we propose two main avenues to progress the understanding and prediction of the different processes occurring on the leading and trailing edge of the species’ distribution in response to any global change phenomena. Deliberately focusing on plant species, we first explore the different ways to incorporate species’ migration in the existing modelling approaches, given data and knowledge limitations and the dual effects of climate and land-use factors. Secondly, we explore the mechanisms and processes happening at the trailing edge of a shifting species’ distribution and how to implement them into a modelling approach. We finally conclude this review with clear guidelines on how such modelling improvements will benefit conservation strategies in a changing world.