A proof-of-concept assay for quantitative and optical assessment of drug-induced toxicity in renal organoids.

Kidneys are complex organs, and reproducing their function and physiology in a laboratory setting remains difficult. During drug development, potential compounds may exhibit unexpected nephrotoxic effects, which imposes a significant financial burden on pharmaceutical companies. As a result, there is an ongoing need for more accurate model systems. The use of renal organoids to simulate responses to nephrotoxic insults has the potential to bridge the gap between preclinical drug efficacy studies in cell cultures and animal models, and the stages of clinical trials in humans. Here we established an accessible fluorescent whole-mount approach for nuclear and membrane staining to first provide an overview of the organoid histology. Furthermore, we investigated the potential of renal organoids to model responses to drug toxicity. For this purpose, organoids were treated with the chemotherapeutic agent doxorubicin for 48 h. When cell viability was assessed biochemically, the organoids demonstrated a significant, dose-dependent decline in response to the treatment. Confocal microscopy revealed visible tubular disintegration and a loss of cellular boundaries at high drug concentrations. This observation was further reinforced by a dose-dependent decrease of the nuclear area in the analyzed images. In contrast to other approaches, in this study, we provide a straightforward experimental framework for drug toxicity assessment in renal organoids that may be used in early research stages to assist screen for potential adverse effects of compounds.

Real-time monitoring of immediate drug response and adaptation upon repeated treatment in a microfluidic chip system.

Microfluidic tissue culture and organ-on-a-chip models provide efficient tools for drug testing in vivo and are considered to become the basis of in vitro test systems to analyze drug response, drug interactions and toxicity to complement and reduce animal testing. A major limitation is the efficient recording of drug action. Here we present an efficient experimental setup that allows long-term cultivation of cells in a microfluidic system in combination with continuous recording of luciferase reporter gene expression. The system combines a sensitive cooled luminescence camera system in combination with a custom build miniaturized incubation chamber. The setup allows to monitor time-dependent activation, but also the end of drug response. Repeated activation and recovery as well as varying durations of drug treatment periods can be monitored, and different modes of drug activity can be visualized.

pVHL-mediated SMAD3 degradation suppresses TGF-ß signaling.

Transforming growth factor ß (TGF-ß) signaling plays a fundamental role in metazoan development and tissue homeostasis. However, the molecular mechanisms concerning the ubiquitin-related dynamic regulation of TGF-ß signaling are not thoroughly understood. Using a combination of proteomics and an siRNA screen, we identify pVHL as an E3 ligase for SMAD3 ubiquitination. We show that pVHL directly interacts with conserved lysine and proline residues in the MH2 domain of SMAD3, triggering degradation. As a result, the level of pVHL expression negatively correlates with the expression and activity of SMAD3 in cells, Drosophila wing, and patient tissues. In Drosophila, loss of pVHL leads to the up-regulation of TGF-ß targets visible in a downward wing blade phenotype, which is rescued by inhibition of SMAD activity. Drosophila pVHL expression exhibited ectopic veinlets and reduced wing growth in a similar manner as upon loss of TGF-ß/SMAD signaling. Thus, our study demonstrates a conserved role of pVHL in the regulation of TGF-ß/SMAD3 signaling in human cells and Drosophila wing development.

Preselector.uni-jena.de: optimize your cloning-a resource for identifying restriction enzymes for preselection reactions.

Preselection digests are a common strategy to reduce the background in the ligation step of molecular cloning. However, choosing fitting restriction enzymes by hand is not trivial and may lead to errors, potentially costing a lot of time and work. We therefore created preselector.uni-jena.de (https://preselector.uni-jena.de/), a free online tool to find such restriction enzymes. The tool uses regular expressions to find recognition sites of restriction enzymes in the DNA sequences provided by the user. This new tool compares the sets of restriction sites and reports the enzymes that cut one sequence but not the other sequences to the user. These enzymes are then the ones suitable for a preselection digest. Thus, preselector.uni-jena.de is a fast, reliable, and free-to-use tool to help researchers designing preselection digestion strategies for their cloning.

The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes.

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.

Decipher the complexity of cis-regulatory regions by a modified Cas9.

BACKGROUND: Understanding complex mechanisms of human transcriptional regulation remains a major challenge. Classical reporter studies already enabled the discovery of cis-regulatory elements within the non-coding DNA; however, the influence of genomic context and potential interactions are still largely unknown. Using a modified Cas9 activation complex we explore the complexity of renin transcription in its native genomic context. METHODS: With the help of genomic editing, we stably tagged the native renin on chromosome 1 with the firefly luciferase and stably integrated a programmable modified Cas9 based trans-activation complex (SAM-complex) by lentiviral transduction into human cells. By delivering five specific guide-RNA homologous to specific promoter regions of renin we were able to guide this SAM-complex to these regions of interest. We measured gene expression and generated and compared computational models. RESULTS: SAM complexes induced activation of renin in our cells after renin specific guide-RNA had been provided. All possible combinations of the five guides were subjected to model analysis in linear models. Quantifying the prediction error and the calculation of an estimator of the relative quality of the statistical models for our given set of data revealed that a model incorporating interactions in the proximal promoter is the superior model for explanation of the data. CONCLUSION: By applying our combined experimental and modelling approach we can show that interactions occur within the selected sequences of the proximal renin promoter region. This combined approach might potentially be useful to investigate other genomic regions. Our findings may help to better understand the transcriptional regulation of human renin.

Imidazopyridines as Potent KDM5 Demethylase Inhibitors Promoting Reprogramming Efficiency of Human iPSCs.

Pioneering human induced pluripotent stem cell (iPSC)-based pre-clinical studies have raised safety concerns and pinpointed the need for safer and more efficient approaches to generate and maintain patient-specific iPSCs. One approach is searching for compounds that influence pluripotent stem cell reprogramming using functional screens of known drugs. Our high-throughput screening of drug-like hits showed that imidazopyridines-analogs of zolpidem, a sedative-hypnotic drug-are able to improve reprogramming efficiency and facilitate reprogramming of resistant human primary fibroblasts. The lead compound (O4I3) showed a remarkable OCT4 induction, which at least in part is due to the inhibition of H3K4 demethylase (KDM5, also known as JARID1). Experiments demonstrated that KDM5A, but not its homolog KDM5B, serves as a reprogramming barrier by interfering with the enrichment of H3K4Me3 at the OCT4 promoter. Thus our results introduce a new class of KDM5 chemical inhibitors and provide further insight into the pluripotency-related properties of KDM5 family members.

Liver-Kidney-on-Chip To Study Toxicity of Drug Metabolites.

Advances in organ-on-chip technologies for the application in in vitro drug development provide an attractive alternative approach to replace ethically controversial animal testing and to establish a basis for accelerated drug development. In recent years, various chip-based tissue culture systems have been developed, which are mostly optimized for cultivation of one single cell type or organoid structure and lack the representation of multi organ interactions. Here we present an optimized microfluidic chip design consisting of interconnected compartments, which provides the possibility to mimic the exchange between different organ specific cell types and enables to study interdependent cellular responses between organs and demonstrate that such tandem system can greatly improve the reproducibility and efficiency of toxicity studies. In a simplified liver-kidney-on-chip model, we showed that hepatic cells that grow in microfluidic conditions abundantly and stably expressed metabolism-related biomarkers. Moreover, we applied this system for investigating the biotransformation and toxicity of Aflatoxin B1 (AFB1) and Benzoalphapyrene (BαP), as well as the interaction with other chemicals. The results clearly demonstrate that the toxicity and metabolic response to drugs can be evaluated in a flow-dependent manner within our system, supporting the importance of advanced interconnected multiorgans in microfluidic devices for application in in vitro toxicity testing and as optimized tissue culture systems for in vitro drug screening.

Human microRNA-299-3p decreases invasive behavior of cancer cells by downregulation of Oct4 expression and causes apoptosis.

PURPOSE: Oct4 was reported to be one of the most important pluripotency transcription factors in the biology of stem cells including cancer stem cells, and progressed malignant cells. Here we report the investigation of gene expression control of Oct4 by selected human microRNAs and the physiological effect of Oct4 silencing in invasive cancer cells. METHODS AND RESULTS: High throughput luciferase activity assay revealed the microRNA-299-3p to be the most effective in reducing gene expression of Oct4, which was confirmed by Western blot analysis and Oct4 promoter activity in a target luciferase assay. Furthermore, it could be demonstrated that downregulation of Oct4 by microRNAs-299-3p in breast cancer and fibrosarcoma cells lead to a decreased invasiveness in a microfluidic chip assay. Additionally, microRNA-299-3p causes apoptosis in cancer cells. Comparison with Oct4 specific siRNA transfection confirmed that this effect is primary due to the blockade of Oct4 expression. CONCLUSION: The results suggest that microRNA-299-3p is an interesting target for potential clinical use. It may be able to decrease invasive behaviour of carcinoma cells; or even kill these cells by causing apoptosis.

Vitamin D3 Partly Antagonizes Advanced-Glycation Endproducts-Induced NFκB Activation in Mouse Podocytes.

BACKGROUND/AIMS: We have previously shown that advanced glycation-endproducts (AGEs) induced NFκB activation in differentiated mouse podocytes. This NFκB activation may contribute to the progression of renal disease and mediation of fibrosis by various mechanisms. This study was undertaken to test whether this detrimental response may be reversed by vitamin D3 or its analogue paricalcitol. METHODS: Differentiated mouse podocytes were challenged with glycated bovine serum albumin (AGE-BSA), or non-glycated control BSA (in the presence or absence of various concentrations of vitamin D3 (decostriol, 1α,25-dihydroxyvitamin D3)) or its active analog paricalcitol. Quantitative mRNA expressions were measured by real-time PCR, whereas protein expressions were determined by Western blotting followed by densitometry. Cytoplasmic and nuclear protein expression of the NFκB subunit p65 (Rel A) were determined by Western blotting. Furthermore, the ratio of phosphorylated to non-phosphorylated IκB-α was measured using specific antibodies. Electrophoretic mobility shift assays and a capture ELISA assay were used to assess NFκB transactivation in vitro. In addition, NFκB transactivation was also monitored in HEK-NFκBIA reporter cells using live cell luminometry. RESULTS: Podocytes expressed the receptor for vitamin D. The vitamins did not suppress receptor for AGEs (RAGE) expression; instead, they rather upregulated RAGE. Although vitamin D3 and paricalcitol partly and differentially modified some of the studied parameters, both hormones inhibited AGE-BSA-induced NFκB transactivation, presumably by various mechanisms including the upregulation of IκB-α protein, keeping NFκB sequestered in an inactive state in the cytoplasm. CONCLUSION: Vitamin D3 or its analog paricalcitol partly prevented AGE-mediated NFκB activation, an important feature of diabetic nephropathy (DN). Whether this in vitro finding is of clinical relevance to prevent/treat DN requires further studies.

Ethyl 2-((4-Chlorophenyl)amino)thiazole-4-carboxylate and Derivatives Are Potent Inducers of Oct3/4.

The octamer-binding transcription factor 4 (Oct3/4) is a master gene in the transcriptional regulatory network of pluripotent cells. Repression of Oct3/4 in embryonic stem cells (ESCs) is associated with cell differentiation and loss of pluripotency, whereas forced overexpression in cooperation with other transcriptional factors, such as Nanog, Sox2, and Lin28, can reprogram somatic cells back into pluripotent cells, termed induced pluripotent stem cells (iPSCs). However, random integration and potential tumorigenic transformation caused by viral transduction limit the clinical application of iPSCs. By performing a cell-based high throughput screening (HTS) campaign, we identified several potential small molecules as inducers of Oct3/4 expression. Here we report a lead structure ethyl 2-((4-chlorophenyl)amino)-thiazole-4-carboxylate, termed O4I2, showing high activity in enforcing Oct3/4 expression. On the basis of chemical expansion, we further identified derivatives having increased activities toward Oct3/4 induction. Thus, O4I2 and its derivatives should provide a new class of small molecules suitable for iPSC generation.

Identification of 2-[4-[(4-Methoxyphenyl)methoxy]-phenyl]acetonitrile and Derivatives as Potent Oct3/4 Inducers.

Reprogramming somatic cells into induced-pluripotent cells (iPSCs) provides new access to all somatic cell types for clinical application without any ethical controversy arising from the use of embryonic stem cells (ESCs). Established protocols for iPSCs generation based on viral transduction with defined factors are limited by low efficiency and the risk of genetic abnormality. Several small molecules have been reported as replacements for defined transcriptional factors, but a chemical able to replace Oct3/4 allowing the generation of human iPSCs is still unavailable. Using a cell-based High Throughput Screening (HTS) campaign, we identified that 2-[4-[(4-methoxyphenyl)methoxy]phenyl]acetonitrile (1), termed O4I1, enhanced Oct3/4 expression. Structural verification and modification by chemical synthesis showed that O4I1 and its derivatives not only promoted expression and stabilization of Oct3/4 but also enhanced its transcriptional activity in diverse human somatic cells, implying the possible benefit from using this class of compounds in regenerative medicine.

The major autoantibody epitope on factor H in atypical hemolytic uremic syndrome is structurally different from its homologous site in factor H-related protein 1, supporting a novel model for induction of autoimmunity in this disease.

Atypical hemolytic uremic syndrome (aHUS) is characterized by complement attack against host cells due to mutations in complement proteins or autoantibodies against complement factor H (CFH). It is unknown why nearly all patients with autoimmune aHUS lack CFHR1 (CFH-related protein-1). These patients have autoantibodies against CFH domains 19 and 20 (CFH19-20), which are nearly identical to CFHR1 domains 4 and 5 (CFHR14-5). Here, binding site mapping of autoantibodies from 17 patients using mutant CFH19-20 constructs revealed an autoantibody epitope cluster within a loop on domain 20, next to the two buried residues that are different in CFH19-20 and CFHR14-5. The crystal structure of CFHR14-5 revealed a difference in conformation of the autoantigenic loop in the C-terminal domains of CFH and CFHR1, explaining the variation in binding of autoantibodies from some aHUS patients to CFH19-20 and CFHR14-5. The autoantigenic loop on CFH seems to be generally flexible, as its conformation in previously published structures of CFH19-20 bound to the microbial protein OspE and a sialic acid glycan is somewhat altered. Cumulatively, our data suggest that association of CFHR1 deficiency with autoimmune aHUS could be due to the structural difference between CFHR1 and the autoantigenic CFH epitope, suggesting a novel explanation for CFHR1 deficiency in the pathogenesis of autoimmune aHUS.

Similarity in targets with REST points to neural and glioblastoma related miRNAs.

There are groups of genes that need coordinated repression in multiple contexts, for example if they code for proteins that work together in a pathway or in a protein complex. Redundancy of biological regulatory networks implies that such coordinated repression might occur at both the pre- and post-transcriptional level, though not necessarily simultaneously or under the same conditions. Here, we propose that such redundancy in the global regulatory network can be detected by the overlap between the putative targets of a transcriptional repressor, as identified by a ChIP-seq experiment, and predicted targets of a microRNA (miRNA). To test this hypothesis, we used publicly available ChIP-seq data of the neural transcriptional repressor RE1 silencing transcription factor (REST) from 15 different cell samples. We found 20 miRNAs, each of which shares a significant amount of predicted targets with REST. The set of predicted associations between these 20 miRNAs and the overlapping REST targets is enriched in known miRNA targets. Many of the detected miRNAs have functions related to neural identity and glioblastoma, which could be expected from their overlap in targets with REST. We propose that the integration of experimentally determined transcription factor binding sites with miRNA-target predictions provides functional information on miRNAs.

Autoantibodies to complement components in C3 glomerulopathy and atypical hemolytic uremic syndrome.

The alternative pathway of complement is implicated in the pathogenesis of several renal diseases, such as atypical hemolytic uremic syndrome, dense deposit disease and other forms of C3 glomerulopathy. The underlying complement defects include genetic and/or acquired factors, the latter in the form of autoantibodies. Because the autoimmune forms require a specific treatment, in part different from that of the genetic forms, it is important to detect the autoantibodies as soon as possible and understand their characteristics. In this overview, we summarize the types of anti-complement autoantibodies detected in such diseases, i.e. autoantibodies to factor H, factor I, C3b, factor B and those against the C3 convertases (C3 nephritic factor and C4 nephritic factor). We draw attention to newly described autoantibodies and their characteristics, and highlight similarities and differences in the autoimmune forms of these diseases.

An engineered construct combining complement regulatory and surface-recognition domains represents a minimal-size functional factor H.

Complement is an essential humoral component of innate immunity; however, its inappropriate activation leads to pathology. Polymorphisms, mutations, and autoantibodies affecting factor H (FH), a major regulator of the alternative complement pathway, are associated with various diseases, including age-related macular degeneration, atypical hemolytic uremic syndrome, and C3 glomerulopathies. Restoring FH function could be a treatment option for such pathologies. In this article, we report on an engineered FH construct that directly combines the two major functional regions of FH: the N-terminal complement regulatory domains and the C-terminal surface-recognition domains. This minimal-size FH (mini-FH) binds C3b and has complement regulatory functions similar to those of the full-length protein. In addition, we demonstrate that mini-FH binds to the FH ligands C-reactive protein, pentraxin 3, and malondialdehyde epitopes. Mini-FH was functionally active when bound to the extracellular matrix and endothelial cells in vitro, and it inhibited C3 deposition on the cells. Furthermore, mini-FH efficiently inhibited complement-mediated lysis of host-like cells caused by a disease-associated FH mutation or by anti-FH autoantibodies. Therefore, mini-FH could potentially be used as a complement inhibitor targeting host surfaces, as well as to replace compromised FH in diseases associated with FH dysfunction.