No abstract

Deep Neural Networks (DNN) are widely used to carry out segmentation tasks in biomedical images. Most DNNs developed for this purpose are based on some variation of the encoderdecoder U-Net architecture. Here we show that Res-CR-Net, a new type of fully convolutional neural network, which was originally developed for the semantic segmentation of microscopy images, and which does not adopt a U-Net architecture, is very effective at segmenting the lung fields in chest X-rays from either healthy patients or patients with a variety of lung pathologies.

Supramolecular cup-shaped lipoprotein structures called porosomes embedded in the cell plasma membrane mediate fractional release of intra-vesicular contents from cells during secretion. The presence of porosomes, have been documented in many cell types including neurons, acinar cells of the exocrine pancreas, growth hormone secreting cells of the pituitary, and mouse insulin-secreting beta cell insulinomas called Min6. Electron micrographs of the nerve terminal in rat brain and of Min6 cells, suggest that besides the docking, fusion and content release of neurotransmitter containing synaptic vesicles and insulin containing granules, multi-vesicular bodies and exosome-like vesicles are released at the porosome complex. To test this hypothesis, the porosome-associated calcium transporting ATPase 1 [SPCA1] encoded by the ATP2C1 gene, was knocked-out in Min6 cells using CRISPR, to study glucose-stimulated insulin and exosome release. In agreement with electron micrographs, results from the study demonstrate a loss of both glucose-stimulated insulin and exosome release in the ATP2C1 knockout Min6 cells. These results further confirm the role of the porosome complex in insulin secretion and establishes a new paradigm in porosome-mediated exosome release in beta cells of the endocrine pancreas.

Expansion microscopy (ExM) involves the use of hydration-competent polymers to physically expand biological specimens approximately 4-fold linear increase to achieve 70 nanometer resolution using an ordinary diffraction limited optical microscope. Optimal conditions however for antigen retention during the expansion process and the relative expansion between organelles within cells has remained unclear. It is reported that different tissues expand to different extents, suggesting that although isotropic expansion is believed to occur, different subcellular compartments with different composition would undergo anisotropic or differential expansion (DiEx). Consequently, there would be distortion of the native shape and size of subcellular compartments upon expansion, parameters which are critical in assessing cellular states in health and disease. Here we report optimal fixation and expansion conditions that retain structural integrity of cells while exhibiting up to 8-fold linear and therefore 512-fold volumetric expansion. Anisotropic expansion is observed not just between tissues, but between different subcellular compartments and even within subcellular compartments themselves.Combining image analysis and machine learning, we provide an approach for the rapid and precise measurement of cellular and subcellular structures in expanded tissue. Using both manual and computation assessment of morphometric parameters, we demonstrate expansion to be anisotropic and therefore refer to this method as differential expansion microscopy (DiExM).

Swelling of secretory vesicles is critical for the regulated expulsion of intravesicular contents from cells during secretion. At the secretory vesicle membrane of the exocrine pancreas and neurons, GTP-binding G proteins, vH + -ATPase, potassium channels and AQP water channels, are among the players implicated in vesicle volume regulation. Here we report in insulin secreting MIN6 cells, the requirement of vH + -ATPase-mediated intracellular acidification on glucosestimulated insulin release. MIN6 cells exposed to the vH + -ATPase inhibitor Bafilomycin A show decreased acidification of the cytosolic compartment that include insulin-carrying granules. Additionally, a loss of insulin granule association with the cell plasma membrane is demonstrated following Bafilomycin A treatment and results in a decrease in glucose-stimulated insulin secretion and accumulation of intracellular insulin. These results suggest that vH + -ATPase-mediated intracellular acidification is required both at the level of secretory vesicles and the cell plasma membrane for cell secretion.