Fluorescent nanosensors for intracellular measurements: synthesis, characterization, calibration, and measurement

Measurement of intracellular acidification is important for understanding fundamental biological pathways as well as developing effective therapeutic strategies. Fluorescent pH nanosensors are an enabling technology for real-time monitoring of intracellular acidification. The physicochemical characteristics of nanosensors can be engineered to target specific cellular compartments and respond to external stimuli. Therefore, nanosensors represent a versatile approach for probing biological pathways inside cells. The fundamental components of nanosensors comprise a pH-sensitive fluorophore (signal transducer) and a pH-insensitive reference fluorophore (internal standard) immobilized in an inert non-toxic matrix. The inert matrix prevents interference of cellular components with the sensing elements as well as minimizing potentially harmful effects of some fluorophores on cell function. Fluorescent nanosensors are synthesized using standard laboratory equipment and are detectable by non-invasive widely accessible imaging techniques. The outcomes of studies employing this technology are dependent on reliable methodology for performing measurements. In particular, special consideration must be given to conditions for sensor calibration, uptake conditions and parameters for image analysis. We describe procedures for: (1) synthesis and characterization of polyacrylamide and silica based nanosensors, (2) nanosensor calibration and (3) performing measurements using fluorescence microscopy

B-cell dysregulation in Crohn's disease is partially restored with infliximab therapy

Background: B-cell depletion can improve a variety of chronic inflammatory diseases, but does not appear beneficial for patients with Crohn's disease. Objective: To elucidate the involvement of B cells in Crohn's disease, we here performed an 'in depth' analysis of intestinal and blood B-cells in this chronic inflammatory disease. Methods: Patients with Crohn's disease were recruited to study B-cell infiltrates in intestinal biopsies (n = 5), serum immunoglobulin levels and the phenotype and molecular characteristics of blood B-cell subsets (n = 21). The effects of infliximab treatment were studied in 9 patients. Results: Granulomatous tissue showed infiltrates of B lymphocytes rather than Ig-secreting plasma cells. Circulating transitional B cells and CD21low B cells were elevated. IgM memory B cells were reduced and natural effector cells showed decreased replication histories and somatic hypermutation (SHM) levels. In contrast, IgG and IgA memory B cells were normally present and their Ig gene transcripts carried increased SHM levels. The numbers of transitional and natural effector cells were normal in patients who responded clinically well to infliximab. Conclusions: B cells in patients with Crohn's disease showed signs of chronic stimulation with localization to granulomatous tissue and increased molecular maturation of IgA and IgG. Therapy with TNFα-blockers restored the defect in IgM memory B-cell generation and normalized transitional B-cell levels, making these subsets candidate markers for treatment monitoring. Together, these results suggest a chronic, aberrant B-cell response in patients with Crohn's disease, which could be targeted with new therapeutics that specifically regulate B-cell function

Matterwave interferometric velocimetry of cold Rb atoms

We consider the matterwave interferometric measurement of atomic velocities, which forms a building block for all matterwave inertial measurements. A theoretical analysis, addressing both the laboratory and atomic frames and accounting for residual Doppler sensitivity in the beamsplitter and recombiner pulses, is followed by an experimental demonstration, with measurements of the velocity distribution within a 20 μK cloud of rubidium atoms. Our experiments use Raman transitions between the long-lived ground hyperfine states, and allow quadrature measurements that yield the full complex interferometer signal and hence discriminate between positive and negative velocities. The technique is most suitable for measurement of colder samples

Organizational and Leadership Implications for Transformational Development

Transformational development is a concept of change that originated in the Christian context but has now become generally used in the work of both secular and faith-based organizations. The growing use of the concept by organizations that are fundamentally different has naturally led to some confusion about what the concept means and what it takes to effectively implement it. In this article, we describe the key features of the concept and how they are important in determining the organizational requirements for its effective implementation. Drawing on a few cases, the paper highlights the centrality of faith in transformational development work

Attribution of global foodborne disease to specific foods: Findings from a World Health Organization structured expert elicitation

Background Recently the World Health Organization, Foodborne Disease Burden Epidemiology Reference Group (FERG) estimated that 31 foodborne diseases (FBDs) resulted in over 600 million illnesses and 420,000 deaths worldwide in 2010. Knowing the relative role importance of different foods as exposure routes for key hazards is critical to preventing illness. This study reports the findings of a structured expert elicitation providing globally comparable food source attribution estimates for 11 major FBDs in each of 14 world subregions. Methods and findings We used Cooke’s Classical Model to elicit and aggregate judgments of 73 international experts. Judgments were elicited from each expert individually and aggregated using both equal and performance weights. Performance weighted results are reported as they increased the informativeness of estimates, while retaining accuracy. We report measures of central tendency and uncertainty bounds on food source attribution estimate. For some pathogens we see relatively consistent food source attribution estimates across subregions of the world; for others there is substantial regional variation. For example, for non-typhoidal salmonellosis, pork was of minor importance compared to eggs and poultry meat in the American and African subregions, whereas in the European and Western Pacific subregions the importance of these three food sources were quite similar. Our regional results broadly agree with estimates from earlier European and North American food source attribution research. As in prior food source attribution research, we find relatively wide uncertainty bounds around our median estimates. Conclusions We present the first worldwide estimates of the proportion of specific foodborne diseases attributable to specific food exposure routes. While we find substantial uncertainty around central tendency estimates, we believe these estimates provide the best currently available basis on which to link FBDs and specific foods in many parts of the world, providing guidance for policy actions to control FBDs.This study was commissioned and paid for by the World Health Organization (WHO). Aspinall & Associates and Gibb Epidemiology Consulting LLC provided support in the form of salaries for authors [WA, HJG], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ sectio

Wetlands and low-gradient topography are associated with longer hydrologic transit times in Precambrian Shield headwater catchments

The estimation of hydrologic transit times in a catchment provides insights into the integrated effects of water storage, mixing dynamics, and runoff generation processes. There has been limited effort to estimate transit times in southern boreal Precambrian Shield landscapes, which are characteristically heterogeneous with surface cover including till, thin soils, bedrock outcrops, and depressional wetland features that play contrasting hydrologic roles. This study presents approximately 3.5 years of precipitation and streamflow water isotope data and estimates mean transit times (MTTs) and the young water fraction (py) across six small catchments in the Muskoka-Haliburton region of south-central Ontario. The main objectives were to define a typical range of MTTs for headwater catchments in this region and to identify landscape variables that best explain differences in MTTs/py using airborne light detection and ranging and digital terrain analysis. Of the transit time distributions, the two parallel linear reservoir and gamma distributions best describe the hydrology of these catchments, particularly because of their ability to capture more extreme changes related to events such as snowmelt. The estimated MTTs, regardless of the modelling approach or distribution used, are positively associated with the percent wetland area and negatively with mean slope in the catchments. In this landscape, low-gradient features such as wetlands increase catchment scale water storage when antecedent conditions are dryer and decrease transit times when there is a moisture surplus, which plausibly explains the increases in MTTs and mean annual runoff from catchments with significant coverage of these landscape features

Nonlinear dynamics of single freely-floating microbubbles under prolonged insonation

The acoustic nonlinear responses of ultrasound contrast agent microbubbles (MBs) are of great interest for both diagnostic and therapeutic applications. Previously, optical and acoustical methods were developed to characterize single bubbles floating against a rigid wall. However, there is a need to develop an efficient approach for statistical measurement of single freely-floating MBs. Here we combine simultaneous optical sizing and sensitive acoustical characterization measurement to study quantitatively the nonlinear dynamics of single freely-floating bubbles under prolonged ultrasound exposure. The nonlinearity (ε2f, ε3f) and asymmetry (i.e., compression-dominant behavior) of bubble vibrations were found to increase with increasing oscillation amplitude, and reach the maximum for bubbles at resonance. Moreover, with the same fundamental response (εf), the second harmonic response (ε2f) of bubbles smaller than the resonance size is 150% stronger than bubbles bigger than the resonance size. The data showed agreement with numerical simulations based on the shell-buckling model by Marmottant et al. The new system shows its great potential for in vitro characterization of contrast agent MB populations