4D in vivo imaging of glomerular barrier function in a zebrafish podocyte injury model

AimZebrafish larvae with their simplified pronephros are an ideal model to study glomerular physiology. Although several groups use zebrafish larvae to assess glomerular barrier function, temporary or slight changes are still difficult to measure. The aim of this study was to investigate the potential of in vivo two‐photon microscopy (2‐PM) for long‐term imaging of glomerular barrier function in zebrafish larvae.MethodsAs a proof of principle, we adapted the nitroreductase/metronidazole model of targeted podocyte ablation for 2‐PM. Combination with a strain, which expresses eGFP‐vitamin D‐binding protein in the blood plasma, led to a strain that allowed induction of podocyte injury with parallel assessment of glomerular barrier function. We used four‐dimensional (4D) 2‐PM to assess eGFP fluorescence over 26 h in the vasculature and in tubules of multiple zebrafish larvae (5 days post‐fertilization) simultaneously.ResultsBy 4D 2‐PM, we observed that, under physiological conditions, eGFP fluorescence was retained in the vasculature and rarely detected in proximal tubule cells. Application of metronidazole induced podocyte injury and cell death as shown by TUNEL staining. Induction of podocyte injury resulted in a dramatic decrease of eGFP fluorescence in the vasculature over time (about 50% and 90% after 2 and 12 h respectively). Loss of vascular eGFP fluorescence was paralleled by an endocytosis‐mediated accumulation of eGFP fluorescence in proximal tubule cells, indicating proteinuria.ConclusionWe established a microscopy‐based method to monitor the dynamics of glomerular barrier function during induction of podocyte injury in multiple zebrafish larvae simultaneously over 26 h.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136708/1/apha12754.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136708/2/apha12754_am.pd

Evaluation of endogenous miRNA reference genes across different zebrafish strains, developmental stages and kidney disease models

The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo. Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments. Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences. All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies

Prolonged podocyte depletion in larval zebrafish resembles mammalian focal and segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is a histological pattern frequently found in patients with nephrotic syndrome that often progress to end-stage kidney disease. The initial step in development of this histologically defined entity is injury and ultimately depletion of podocytes, highly arborized interdigitating cells on the glomerular capillaries with important function for the glomerular filtration barrier. Since there are still no causal therapeutic options, animal models are needed to develop new treatment strategies. Here, we present an FSGS-like model in zebrafish larvae, an eligible vertebrate model for kidney research. In a transgenic zebrafish strain, podocytes were depleted, and the glomerular response was investigated by histological and morphometrical analysis combined with immunofluorescence staining and ultrastructural analysis by transmission electron microscopy. By intravenous injection of fluorescent high-molecular weight dextran, we confirmed leakage of the size selective filtration barrier. Additionally, we observed severe podocyte foot process effacement of remaining podocytes, activation of proximal tubule-like parietal epithelial cells identified by ultrastructural cytomorphology, and expression of proximal tubule markers. These activated cells deposited extracellular matrix on the glomerular tuft which are all hallmarks of FSGS. Our findings indicate that glomerular response to podocyte depletion in larval zebrafish resembles human FSGS in several important characteristics. Therefore, this model will help to investigate the disease development and the effects of potential drugs in a living organism

Chemical composition and radiative properties of nascent particulate matter emitted by an aircraft turbofan burning conventional and alternative fuels

Aircraft engines are a unique source of carbonaceous aerosols in the upper troposphere. There, these particles can more efficiently interact with solar radiation than at ground. Due to the lack of measurement data, the radiative forcing from aircraft exhaust aerosol remains uncertain. To better estimate the global radiative effects of aircraft exhaust aerosol, its optical properties need to be comprehensively characterized. In this work we present the link between the chemical composition and the optical properties of the particulate matter (PM) measured at the engine exit plane of a CFM56-7B turbofan. The measurements covered a wide range of power settings (thrust), ranging from ground idle to take-off, using four different fuel blends of conventional Jet A-1 and hydro-processed ester and fatty acids (HEFA) biofuel. At the two measurement wavelengths (532 and 870&thinsp;nm) and for all tested fuels, the absorption and scattering coefficients increased with thrust, as did the PM mass. The analysis of elemental carbon (EC) and organic carbon (OC) revealed a significant mass fraction of OC (up to 90&thinsp;%) at low thrust levels, while EC mass dominated at medium and high thrust. The use of HEFA blends induced a significant decrease in the PM mass and the optical coefficients at all thrust levels. The HEFA effect was highest at low thrust levels, where the EC mass was reduced by up to 50&thinsp;%–60&thinsp;%. The variability in the chemical composition of the particles was the main reason for the strong thrust dependency of the single scattering albedo (SSA), which followed the same trend as the fraction of OC to total carbon (TC). Mass absorption coefficients (MACs) were determined from the correlations between aerosol light absorption and EC mass concentration. The obtained MAC values (MAC532=7.5±0.3&thinsp;m2&thinsp;g−1 and MAC870=5.2±0.9&thinsp;m2&thinsp;g−1) are in excellent agreement with previous literature values of absorption cross section for freshly generated soot. While the MAC values were found to be independent of the thrust level and fuel type, the mass scattering coefficients (MSCs) significantly varied with thrust. For cruise conditions we obtained MSC532=4.5±0.4&thinsp;m2&thinsp;g−1 and MSC870=0.54±0.04&thinsp;m2&thinsp;g−1, which fall within the higher end of MSCs measured for fresh biomass smoke. However, the latter comparison is limited by the strong dependency of MSC on the particles' size, morphology and chemical composition. The use of the HEFA fuel blends significantly decreased PM emissions, but no changes were observed in terms of EC∕OC composition and radiative properties.</p

Cell fate determinant Llgl1 is required for propagation of acute myeloid leukemia

Scribble complex proteins can influence cell fate decisions and self-renewal capacity of hematopoietic cells. While specific cellular functions of Scribble complex members are conserved in mammalian hematopoiesis, they appear to be highly context dependent. Using CRISPR/Cas9-based genetic screening, we have identified Scribble complex-related liabilities in AML including LLGL1. Despite its reported suppressive function in HSC self-renewal, inactivation of LLGL1 in AML confirms its relevant role for proliferative capacity and development of AML. Its function was conserved in human and murine models of AML and across various genetic backgrounds. Inactivation of LLGL1 results in loss of stemness-associated gene-expression including HoxA-genes and induces a GMP-like phenotype in the leukemia stem cell compartment. Re-expression of HoxA9 facilitates functional and phenotypic rescue. Collectively, these data establish LLGL1 as a specific dependency and putative target in AML and emphasizes its cell-type specific functions

Recent Progress on the Marylie/Impact Beam Dynamics Code

MARYLIE/IMPACT (ML/I) is a hybrid code that combines the beam optics capabilities of MARYLIE with the parallel Particle-In-Cell capabilities of IMPACT. In addition to combining the capabilities of these codes, ML/I has a number of powerful features, including a choice of Poisson solvers, a fifth-order rf cavity model, multiple reference particles for rf cavities, a library of soft-edge magnet models, representation of magnet systems in terms of coil stacks with possibly overlapping fields, and wakefield effects. The code allows for map production, map analysis, particle tracking, and 3D envelope tracking, all within a single, coherent user environment. ML/I has a front end that can read both MARYLIE input and MAD lattice descriptions. The code can model beams with or without acceleration, and with or without space charge. Developed under a US DOE Scientific Discovery through Advanced Computing (SciDAC) project, ML/I is well suited to large-scale modeling, simulations having been performed with up to 100M macroparticles. The code inherits the powerful fitting and optimizing capabilities of MARYLIE augmented for the new features of ML/I. The combination of soft-edge magnet models, high-order capability, space charge effects, and fitting/optimization capabilities, make ML/I a powerful code for a wide range of beam optics design problems. This paper provides a description of the code and its unique capabilities

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns(-/-) zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS-/- kidney cells and injection into ctns(-/-) zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns(-/-) zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns(-/-) larvae, and restoration of the zebrafish pronephros function.Immunopathology of vascular and renal diseases and of organ and celltransplantationIP

Diverse uncultivated ultra-small bacterial cells in groundwater.

Bacteria from phyla lacking cultivated representatives are widespread in natural systems and some have very small genomes. Here we test the hypothesis that these cells are small and thus might be enriched by filtration for coupled genomic and ultrastructural characterization. Metagenomic analysis of groundwater that passed through a ~0.2-μm filter reveals a wide diversity of bacteria from the WWE3, OP11 and OD1 candidate phyla. Cryogenic transmission electron microscopy demonstrates that, despite morphological variation, cells consistently have small cell size (0.009±0.002 μm(3)). Ultrastructural features potentially related to cell and genome size minimization include tightly packed spirals inferred to be DNA, few densely packed ribosomes and a variety of pili-like structures that might enable inter-organism interactions that compensate for biosynthetic capacities inferred to be missing from genomic data. The results suggest that extremely small cell size is associated with these relatively common, yet little known organisms

Diverse uncultivated ultra-small bacterial cells in groundwater.

Bacteria from phyla lacking cultivated representatives are widespread in natural systems and some have very small genomes. Here we test the hypothesis that these cells are small and thus might be enriched by filtration for coupled genomic and ultrastructural characterization. Metagenomic analysis of groundwater that passed through a ~0.2-μm filter reveals a wide diversity of bacteria from the WWE3, OP11 and OD1 candidate phyla. Cryogenic transmission electron microscopy demonstrates that, despite morphological variation, cells consistently have small cell size (0.009±0.002 μm(3)). Ultrastructural features potentially related to cell and genome size minimization include tightly packed spirals inferred to be DNA, few densely packed ribosomes and a variety of pili-like structures that might enable inter-organism interactions that compensate for biosynthetic capacities inferred to be missing from genomic data. The results suggest that extremely small cell size is associated with these relatively common, yet little known organisms