Dark-adapted versus bleached state in fluorescence lifetime imaging ophthalmoscopy

Purpose: The (early) detection of diseases based on metabolic changes in the retina is the goal of the novel autofluorescence lifetime ophthalmoscopy (FLIO) technique. These metabolic changes can be detected as alterations in the fundus autofluorescence (FAF) lifetimes. The influences of the photopigment bleaching and photobleaching on the FAF lifetimes are unknown. Thus, we performed a volunteer study to investigate these influences. Methods: In 21 healthy volunteers (23.6±3.8 years) time-resolved FAF was measured with a FLIO device (30° of fundus, excitation at 473nm, detection in two spectral channels: 500-560nm (ch1) and 560-720nm (ch2), time-correlated single photon counting method). All subjects had a crystalline lens. The pupil was dilated with 0.5% Tropicamid. After volunteers adapted to the dark using a custom-made lightproof eyewear over a period of 30 min, the first FLIO measurement was recorded (dark-adapted state). Subsequently, one eye was bleached for 1 min using a luminance of 3200cd/m2, followed by a FLIO measurement (bleached state). The fluorescence lifetimes were estimated from the FAF decays, based on three exponential functions, using the software FLIMX (www.flimx.de). Average values from the central region, and the inner and outer rings of the ETDRS grid were utilized to compare both bleaching states using analysis of variance, Friedman, and post hoc tests. Results : Only ch2 yielded significant changes (p<0.05) for the fluorescence lifetime τ2 from all ETDRS regions (+19-28ps), for the fluorescence lifetime τ1 (+6ps) and the mean fluorescence lifetime (+6ps) in the central area that were less than 10% in magnitude. Additionally, the acquisition time in the bleached state was significantly reduced by approximately 20% on average, compared to the dark-adapted state. The fluorescence lifetime differences caused by bleaching were much smaller than pathological states known from literature. Conclusions: We conclude that bleaching is not relevant for current clinical FLIO applications because of the small magnitude of the elicited fluorescence lifetime changes. Thus, it is advisable to instruct patients to wait in a bright room before FLIO measurements. If the expected changes in the fluorescence lifetime in a specific experimental paradigm are small, FLIO users should follow a strict acquisition protocol in terms of the photopigment bleaching state of the patients to obtain the most reliable results

Bleaching effects and fluorescence lifetime imaging ophthalmoscopy

This study investigates the influence of photopigment bleaching on autofluorescence lifetimes in the fundus in 21 young healthy volunteers. Three measurements of 30° retinal fields in two spectral channels (SSC: 498–560 nm, LSC: 560–720 nm) were obtained for each volunteer using fluorescence lifetime imaging ophthalmoscopy (FLIO). After dark-adaptation by wearing a custom-made lightproof mask for 30 minutes, the first FLIO-measurement was recorded (dark-adapted state). Subsequently, the eye was bleached for 1 minute (luminance: 3200 cd/m2), followed by a second FLIO-measurement (bleached state). Following an additional 10 minute dark adaptation using the mask, a final FLIO-measurement was recorded (recovered state). Average values of the fluorescence lifetimes were calculated from within different areas of a standardized early treatment diabetic retinopathy study (ETDRS) grid (central area, inner and outer rings). The acquisition time in the bleached state was significantly shortened by approximately 20%. The SSC did not show any significant changes in fluorescence lifetimes with photopigment bleaching, only the LSC showed small but significant bleaching-related changes in the fluorescence lifetimes τ1 and τ2 from all regions, as well as the mean fluorescence lifetime in the central area. The fluorescence lifetime differences caused by bleaching were by far less significant than pathological changes caused by eye diseases. The magnitudes of fluorescence lifetime changes are <10% and do not interfere with healthy or disease related FLIO patterns. Thus, we conclude that bleaching is not a relevant confounder in current clinical applications of FLIO

Spatial alanine metabolism determines local growth dynamics of textitEscherichia coli colonies

Bacteria commonly live in spatially structured biofilm assemblages, which are encased by an extracellular matrix. Metabolic activity of the cells inside biofilms causes gradients in local environmental conditions, which leads to the emergence of physiologically differentiated subpopulations. Information about the properties and spatial arrangement of such metabolic subpopulations, as well as their interaction strength and interaction length scales are lacking, even for model systems like textitEscherichia coli colony biofilms grown on agar-solidified media. Here, we use an unbiased approach, based on temporal and spatial transcriptome and metabolome data acquired during textitE. coli colony biofilm growth, to study the spatial organization of metabolism. We discovered that alanine displays a unique pattern among amino acids and that alanine metabolism is spatially and temporally heterogeneous. At the anoxic base of the colony, where carbon and nitrogen sources are abundant, cells secrete alanine textitvia the transporter AlaE. In contrast, cells utilize alanine as a carbon and nitrogen source in the oxic nutrient-deprived region at the colony mid-height, textitvia the enzymes DadA and DadX. This spatially structured alanine cross-feeding influences cellular viability and growth in the cross-feeding-dependent region, which shapes the overall colony morphology. More generally, our results on this precisely controllable biofilm model system demonstrate a remarkable spatiotemporal complexity of metabolism in biofilms. A better characterization of the spatiotemporal metabolic heterogeneities and dependencies is essential for understanding the physiology, architecture, and function of biofilms

Volunteering in the care of people with severe mental illness: a systematic review

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Real-life assessment of standardized contrast-enhanced ultrasound (CEUS) and CEUS algorithms (CEUS LI-RADS®/ESCULAP) in hepatic nodules in cirrhotic patients—a prospective multicenter study

Objectives Hepatocellular carcinoma (HCC) can be diagnosed non-invasively with contrast-enhanced ultrasound (CEUS) in cirrhosis if the characteristic pattern of arterial phase hyperenhancement followed by hypoenhancement is present. Recent studies suggest that diagnosis based on this “hyper-hypo” pattern needs further refinement. This study compares the diagnostic accuracies of standardized CEUS for HCC according to the current guideline definition and following the newly developed CEUS algorithms (CEUS LI-RADS®, ESCULAP) in a prospective multicenter real-life setting. Methods Cirrhotic patients with liver lesions on B-mode ultrasound were recruited prospectively from 04/2018 to 04/2019, and clinical and imaging data were collected. The CEUS standard included an additional examination point after 4–6 min in case of no washout after 3 min. The diagnostic accuracies of CEUS following the guidelines (“hyper-hypo” pattern), based on the examiner’s subjective interpretation (“CEUS subjective”), and based on the CEUS algorithms ESCULAP and CEUS LI-RADS® were compared. Results In total, 470 cirrhotic patients were recruited in 43 centers. The final diagnosis was HCC in 378 cases (80.4%) according to the reference standard (histology 77.4%, MRI 16.4%, CT 6.2%). The “hyper-hypo” pattern yielded 74.3% sensitivity and 63% specificity. “CEUS subjective” showed a higher diagnostic accuracy (sensitivity, 91.5%; specificity, 67.4%; positive predictive value, 92%; negative predictive value, 66%). Sensitivity was higher for ESCULAP (95%) and “CEUS subjective” (91.5%) versus CEUS LI-RADS® (65.2%; p < 0.001). Specificity was highest for CEUS LI-RADS® (78.6%; p < 0.001). Conclusions CEUS has an excellent diagnostic accuracy for the non-invasive diagnosis of HCC in cirrhosis. CEUS algorithms may be a helpful refinement of the “hyper-hypo” pattern defined by current HCC guidelines. Key Points • Contrast-enhanced ultrasound (CEUS) has a high diagnostic accuracy for the non-invasive diagnosis of hepatocellular carcinoma (HCC) in cirrhosis. • The CEUS algorithm ESCULAP (Erlanger Synopsis for Contrast-enhanced Ultrasound for Liver lesion Assessment in Patients at risk) showed the highest sensitivity, whereas the CEUS LI-RADS® (Contrast-Enhanced UltraSound Liver Imaging Reporting and Data System) algorithm yielded the highest specificity. • A standardized CEUS examination procedure with an additional examination point in the late phase, after 4–6 min in lesions with no washout after 3 min, is vital

High-throughput screening for industrial enzyme production hosts by droplet microfluidics

A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its a-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in a-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains

250 labels used to stigmatise people with mental illness

<p>Abstract</p> <p>Background</p> <p>The stigma against people with mental illness is a major barrier to help-seeking in young people for mental health problems. The objective of this study was to investigate the extent of stigma in relation to treatment avoidance in 14 year-old school students in England in relation to how they refer to people with mental illness.</p> <p>Methods</p> <p>This is a qualitative, cross-sectional study. The data were gathered as part of the baseline assessment for an intervention study intended to reduce stigma among 14 year old school students. The participating schools were two grammar (selective) schools and three comprehensive (non-selective) schools. At the start of the lesson, the students were asked 'What sorts of words or phrases might you use to describe someone who experiences mental health problems?' Words and terms used to refer to mental illness were enumerated. Using the grounded theory approach, words and terms were grouped in terms of their denotative and connotative meanings. Labels were then derived to capture the key themes attached by the students to the concepts of mental illness. The frequencies of occurrence for each word were also tabulated.</p> <p>Results</p> <p>400 of the 472 participating students (85%) provided 250 words and terms to describe a person with mental illness. Five themes were identified from the data. The first theme called 'popular derogatory terms' (116 items) accounted for nearly half of the words examined. The second theme occurred less often and was described as 'negative emotional state' (61 items). The third theme demonstrated the confusion of young people between physical disabilities, learning difficulties and mental health problems (38 items). The use of psychiatric diagnoses (15 items) and terms related to violence (9 items) were unexpectedly uncommon.</p> <p>Conclusion</p> <p>Our findings suggest the hypothesis that help-seeking by mentally ill young people may be improved by interventions that address both their lack of factual information about mental illness, and those which reduce their strong negative emotional reactions towards people with mental illness.</p