Optical Coherence Tomography for live imaging of mammalian development

Understanding the nature and mechanism of congenital defects of the different organ systems in humans has heavily relied on the analysis of the corresponding mutant phenotypes in rodent models. Optical Coherence Tomography (OCT) has recently emerged as a powerful tool to study early embryonic development. This non-invasive optical methodology does not require labeling and allows visualization of embryonic tissues with single cell resolution. Here, we will discuss how OCT can be applied for structural imaging of early mouse and rat embryos in static culture, cardiodynamic and blood flow analysis, and in utero embryonic imaging at later stages of gestation, demonstrating how OCT can be used to assess structural and functional birth defects in mammalian models.     

Correcting mortality for loss to follow-up: a nomogram applied to antiretroviral treatment programmes in sub-Saharan Africa

Background

The World Health Organization estimates that in sub-Saharan Africa about 4 million HIV-infected patients had started antiretroviral therapy (ART) by the end of 2008. Loss of patients to follow-up and care is an important problem for treatment programmes in this region. As mortality is high in these patients compared to patients remaining in care, ART programmes with high rates of loss to follow-up may substantially underestimate mortality of all patients starting ART.

Methods and Findings

We developed a nomogram to correct mortality estimates for loss to follow-up, based on the fact that mortality of all patients starting ART in a treatment programme is a weighted average of mortality among patients lost to follow-up and patients remaining in care. The nomogram gives a correction factor based on the percentage of patients lost to follow-up at a given point in time, and the estimated ratio of mortality between patients lost and not lost to follow-up. The mortality observed among patients retained in care is then multiplied by the correction factor to obtain an estimate of programme-level mortality that takes all deaths into account. A web calculator directly calculates the corrected, programme-level mortality with 95% confidence intervals (CIs). We applied the method to 11 ART programmes in sub-Saharan Africa. Patients retained in care had a mortality at 1 year of 1.4% to 12.0%; loss to follow-up ranged from 2.8% to 28.7%; and the correction factor from 1.2 to 8.0. The absolute difference between uncorrected and corrected mortality at 1 year ranged from 1.6% to 9.8%, and was above 5% in four programmes. The largest difference in mortality was in a programme with 28.7% of patients lost to follow-up at 1 year.

Conclusions

The amount of bias in mortality estimates can be large in ART programmes with substantial loss to follow-up. Programmes should routinely report mortality among patients retained in care and the proportion of patients lost. A simple nomogram can then be used to estimate mortality among all patients who started ART, for a range of plausible mortality rates among patients lost to follow-up. Please see later in the article for the Editors'' Summary     

Double Emulsions Stabilized by Food Biopolymers

Double emulsions of the water-in-oil-in-water (W/O/W) type have application in the formulation of reduced-fat food products and as vehicles for encapsulation and delivery of nutrients during food digestion. Progress in the development of stable double emulsions for food use is dependent on replacing small-molecule emulsifiers and synthetic polymeric stabilizing agents by food-grade ingredients. Of particular value for conferring the required functionality are food proteins and polysaccharides. This review describes how these biopolymers have been successfully incorporated into the internal and external aqueous phases of W/O/W emulsions to improve the stability and yield of model systems. Recent advances in the use of protein–polysaccharide conjugates and complexes for the stabilization of the outer droplets of W/O/W emulsions are highlighted.     

Factors Determining the Recruitment of Inositol Trisphosphate Receptor Channels During Calcium Puffs

Puffs are localized, transient elevations in cytosolic Ca²⁺ that serve both as the building blocks of global cellular Ca²⁺ signals and as local signals in their own right. They arise from clustered inositol 1,4,5-trisphosphate receptor/channels (IP₃Rs), whose openings are coordinated by Ca²⁺-induced Ca²⁺ release (CICR). We utilized total internal reflection fluorescence imaging of Ca²⁺ signals in neuroblastoma cells with single-channel resolution to elucidate the mechanisms determining the triggering, amplitudes, kinetics, and spatial spread of puffs. We find that any given channel in a cluster has a mean probability of ∼66% of opening following opening of an initial “trigger” channel, and the probability of puff triggering thus increases steeply with increasing number of channels in a cluster (cluster size). Mean puff amplitudes scale with cluster size, but individual amplitudes vary widely, even at sites of similar cluster size, displaying similar proportions of events involving any given number of the channels in the cluster. Stochastic variation in numbers of Ca²⁺-inhibited IP₃Rs likely contributes to the variability of amplitudes of repeated puffs at a site but the amplitudes of successive puffs were uncorrelated, even though we observed statistical correlations between interpuff intervals and puff amplitudes. Initial puffs evoked following photorelease of IP₃—which would not be subject to earlier Ca²⁺-inhibition—also showed wide variability, indicating that mechanisms such as stochastic variation in IP₃ binding and channel recruitment by CICR further determine puff amplitudes. The mean termination time of puffs lengthened with increasing puff amplitude size, consistent with independent closings of channels after a given mean open time, but we found no correlation of termination time with cluster size independent of puff amplitude. The spatial extent of puffs increased with their amplitude, and puffs of similar size were of similar width, independent of cluster size.     

Factors Determining the Recruitment of Inositol Trisphosphate Receptor Channels During Calcium Puffs

Puffs are localized, transient elevations in cytosolic Ca²⁺ that serve both as the building blocks of global cellular Ca²⁺ signals and as local signals in their own right. They arise from clustered inositol 1,4,5-trisphosphate receptor/channels (IP₃Rs), whose openings are coordinated by Ca²⁺-induced Ca²⁺ release (CICR). We utilized total internal reflection fluorescence imaging of Ca²⁺ signals in neuroblastoma cells with single-channel resolution to elucidate the mechanisms determining the triggering, amplitudes, kinetics, and spatial spread of puffs. We find that any given channel in a cluster has a mean probability of ∼66% of opening following opening of an initial “trigger” channel, and the probability of puff triggering thus increases steeply with increasing number of channels in a cluster (cluster size). Mean puff amplitudes scale with cluster size, but individual amplitudes vary widely, even at sites of similar cluster size, displaying similar proportions of events involving any given number of the channels in the cluster. Stochastic variation in numbers of Ca²⁺-inhibited IP₃Rs likely contributes to the variability of amplitudes of repeated puffs at a site but the amplitudes of successive puffs were uncorrelated, even though we observed statistical correlations between interpuff intervals and puff amplitudes. Initial puffs evoked following photorelease of IP₃—which would not be subject to earlier Ca²⁺-inhibition—also showed wide variability, indicating that mechanisms such as stochastic variation in IP₃ binding and channel recruitment by CICR further determine puff amplitudes. The mean termination time of puffs lengthened with increasing puff amplitude size, consistent with independent closings of channels after a given mean open time, but we found no correlation of termination time with cluster size independent of puff amplitude. The spatial extent of puffs increased with their amplitude, and puffs of similar size were of similar width, independent of cluster size.     

Receptor Protein-tyrosine Phosphatase α Regulates Focal Adhesion Kinase Phosphorylation and ErbB2 Oncoprotein-mediated Mammary Epithelial Cell Motility

We investigated the role of protein-tyrosine phosphatase α (PTPα) in regulating signaling by the ErbB2 oncoprotein in mammary epithelial cells. Using this model, we demonstrated that activation of ErbB2 led to the transient inactivation of PTPα, suggesting that attenuation of PTPα activity may contribute to enhanced ErbB2 signaling. Furthermore, RNAi-induced suppression of PTPα led to increased cell migration in an ErbB2-dependent manner. The ability of ErbB2 to increase cell motility in the absence of PTPα was characterized by prolonged interaction of GRB7 with ErbB2 and increased association of ErbB2 with a β1-integrin-rich complex, which depended on GRB7-SH2 domain interactions. Finally, suppression of PTPα resulted in increased phosphorylation of focal adhesion kinase on Tyr-407, which induced the recruitment of vinculin and the formation of a novel focal adhesion kinase complex in response to ErbB2 activation in mammary epithelial cells. Collectively, these results reveal a new role for PTPα in the regulation of motility of mammary epithelial cells in response to ErbB2 activation.     

Effect of ultrasound on cyprids and juvenile barnacles

Settlement inhibition of barnacle (Amphibalanus amphitrite) cypris larvae resulting from exposure to ultrasound was measured at three frequencies (23, 63, and 102 kHz), applied at three acoustic pressure levels (9, 15, and 22 kPa) for exposure times of 30, 150, and 300 s. The lowest settlement was observed for 23 kHz, which also induced the highest cyprid mortality. Cyprid settlement following exposure to 23 kHz at 22 kPa for 30 s was reduced by a factor of two. Observing surface exploration by the cyprids revealed an altered behaviour following exposure to ultrasound: step length was increased, while step duration, walking pace, and the fraction of cyprids exploring the surface were significantly reduced with respect to control cyprids. The basal area of juvenile barnacles, metamorphosed from ultrasound-treated cyprids was initially smaller than unexposed individuals, but normalised over two weeks' growth. Thus, ultrasound exposure effectively reduced cyprid settlement, yet metamorphosed barnacles grew normally.