Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format

Truncated tau interferes with the autophagy and endolysosomal pathway and results in lipid accumulation

This is the final version. Available from Springer via the DOI in this record. Availability of data and material. The data that support the findings of this study are available from the corresponding author upon reasonable request.The autophagy-lysosomal pathway plays a critical role in the clearance of tau protein aggregates that deposit in the brain in tauopathies, and defects in this system are associated with disease pathogenesis. Here, we report that expression of Tau35, a tauopathy-associated carboxy-terminal fragment of tau, leads to lipid accumulation in cell lines and primary cortical neurons. Our findings suggest that this is likely due to a deleterious block of autophagic clearance and lysosomal degradative capacity by Tau35. Notably, upon induction of autophagy by Torin 1, Tau35 inhibited nuclear translocation of transcription factor EB (TFEB), a key regulator of lysosomal biogenesis. Both cell lines and primary cortical neurons expressing Tau35 also exhibited changes in endosomal protein expression. These findings implicate autophagic and endolysosomal dysfunction as key pathological mechanisms through which disease-associated tau fragments could lead to the development and progression of tauopathy

Aryl hydrocarbon receptor utilises cellular zinc signals to maintain the gut epithelial barrier

This is the final version. Available on open access from Nature Research via the DOI in this recordData availability: All data that supports the findings here can be found in the manuscript and Supplementary Information. Uncropped and unprocessed scans of immunoblots have been provided as Supplementary Figures in the Supplementary Information. The 16S rDNA data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) database under accession code PRJNA945597. Source data are provided with this paper.Zinc and plant-derived ligands of the aryl hydrocarbon receptor (AHR) are dietary components affecting intestinal epithelial barrier function. Here, we explore whether zinc and the AHR pathway are linked. We show that dietary supplementation with an AHR pre-ligand offers protection against inflammatory bowel disease in a mouse model while protection fails in mice lacking AHR in the intestinal epithelium. AHR agonist treatment is also ineffective in mice fed zinc depleted diet. In human ileum organoids and Caco-2 cells, AHR activation increases total cellular zinc and cytosolic free Zn2+ concentrations through transcription of genes for zinc importers. Tight junction proteins are upregulated through zinc inhibition of nuclear factor kappa-light-chain-enhancer and calpain activity. Our data show that AHR activation by plant-derived dietary ligands improves gut barrier function at least partly via zinc-dependent cellular pathways, suggesting that combined dietary supplementation with AHR ligands and zinc might be effective in preventing inflammatory gut disorders.Guts UKZinPro Performance MineralsNational Key R&D Program of ChinaNational Natural Science Foundation of China (NSFC)Program for Outstanding Medical Academic LeaderShanghai Committee of Science and TechnologyChinese Scholarship CouncilKing’s College Londo

Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation

Attitude of community pharmacists toward patients with a substance-related disorder (heroin, alcohol and tobacco), estimation of harmfulness and knowledge of these substances, and continuing education: A pilot cross-sectional study

International audienceObjectives: To investigate attitude of community pharmacists toward patients with a substance-related disorder (heroin, alcohol and tobacco).Material and methods: The attitudes were assessed thanks to the Attitude to Mental Illness Questionnaire (AMIQ) for heroin, alcohol and tobacco-related disorders in three independent groups of pharmacists. Estimation of substance-related harmfulness, knowledge of substance-related disorders and activities/needs for continuing education on substance-related disorders were also recorded.Results: Thirty-five pharmacists were included (heroin: 11, alcohol: 10 and tobacco: 14). AMIQ scores for heroin-related disorder were negative and lower than for alcohol (P<0.01) and tobacco (P<0.001). AMIQ scores for alcohol-related disorder were lower than for tobacco (P<0.05). The estimation of heroin-related harmfulness was higher than for alcohol and tobacco (P<0.001). The estimations of knowledge of substance-related disorders were lower for opioid and alcohol than for tobacco (P<0.001). AMIQ scores and the needs for continuing education on each associated addiction showed a positive relation (P<0.01).Conclusion: Pharmacists had a negative attitude toward heroin and alcohol-related disorders. A positive attitude toward patients with a substance-related disorder was associated with a need for continuing education. Efforts should be made to change attitudes and to promote continuing education on heroin and alcohol-related disorders

Development of a new pediatric formulation of hydrophilic suppositories of pentobarbital and comparison with lipophilic formulations.

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FLIM, FRET and high content analysis

Fluorescence lifetime imaging (FLIM) provides a means to contrast different molecular species and to map variations in the local fluorophore molecular environment, including to read out Förster resonant energy transfer (FRET), e.g. to assay protein interactions or genetically expressed FRET biosensors. We have implemented wide-field time-gated FLIM in a modular open automated microscopy platform for high content analysis (HCA). To demonstrate its relevance to drug discovery, we have demonstrated the capability of our openFLIM HCA platform to assay interactions of low copy number endogenous proteins in yeast cells labelled with fluorescent proteins. We have also demonstrated the capability of multiwell plate FLIM assays to provide readouts of a FRET biosensor in 2-D and 3-D cell cultures