Targeted volume correlative light and electron microscopy of an environmental marine microorganism.

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

MicroRNAs Influence the Migratory Ability of Human Umbilical Vein Endothelial Cells.

To identify miRNAs that are involved in cell migration in human umbilical vein endothelial cells (HUVECs), we employed RNA sequencing under high glucose incubation and text mining within the databases miRWalk and TargetScanHuman using 83 genes that regulate HUVECs migration. From both databases, 307 predicted miRNAs were retrieved. Differentially expressed miRNAs were determined by exposing HUVECs to high glucose stimulation, which significantly inhibited the migratory ability of HUVECs as compared to cells cultured in normal glucose. A total of 35 miRNAs were found as differently expressed miRNAs in miRNA sequencing, and 4 miRNAs, namely miR-21-3p, miR-107, miR-143-3p, and miR-106b-5p, were identified as overlapping hits. These were subjected to hub gene analysis and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG), identifing 71 pathways which were influenced by all four miRNAs. The influence of all four miRNAs on HUVEC migration was phenomorphologically confirmed. miR21 and miR107 promoted migration in HUVECs while miR106b and miR143 inhibited migration. Pathway analysis also revealed eight shared pathways between the four miRNAs. Protein-protein interaction (PPI) network analysis was then performed to predict the functionality of interacting genes or proteins. This revealed six hub genes which could firstly be predicted to be related to HUVEC migration.

PITX1 Is a Regulator of TERT Expression in Prostate Cancer with Prognostic Power.

The current risk stratification in prostate cancer (PCa) is frequently insufficient to adequately predict disease development and outcome. One hallmark of cancer is telomere maintenance. For telomere maintenance, PCa cells exclusively employ telomerase, making it essential for this cancer entity. However, TERT, the catalytic protein component of the reverse transcriptase telomerase, itself does not suit as a prognostic marker for prostate cancer as it is rather low expressed. We investigated if, instead of TERT, transcription factors regulating TERT may suit as prognostic markers. To identify transcription factors regulating TERT, we developed and applied a new gene regulatory modeling strategy to a comprehensive transcriptome dataset of 445 primary PCa. Six transcription factors were predicted as TERT regulators, and most prominently, the developmental morphogenic factor PITX1. PITX1 expression positively correlated with telomere staining intensity in PCa tumor samples. Functional assays and chromatin immune-precipitation showed that PITX1 activates TERT expression in PCa cells. Clinically, we observed that PITX1 is an excellent prognostic marker, as concluded from an analysis of more than 15,000 PCa samples. PITX1 expression in tumor samples associated with (i) increased Ki67 expression indicating increased tumor growth, (ii) a worse prognosis, and (iii) correlated with telomere length.

High-content analysis of microRNAs involved in the phenotype regulation of vascular smooth muscle cells.

In response to vascular injury vascular smooth muscle cells (VSMCs) alternate between a differentiated (contractile) and a dedifferentiated (synthetic) state or phenotype. Although parts of the signaling cascade regulating the phenotypic switch have been described, the role of miRNAs is still incompletely understood. To systematically address this issue, we have established a microscopy-based quantitative assay and identified 23 miRNAs that induced contractile phenotypes when over-expressed. These were then correlated to miRNAs identified from RNA-sequencing when comparing cells in the contractile and synthetic states. Using both approaches, six miRNAs (miR-132-3p, miR-138-5p, miR-141-3p, miR-145-5p, miR-150-5p, and miR-22-3p) were filtered as candidates that induce the phenotypic switch from synthetic to contractile. To identify potentially common regulatory mechanisms of these six miRNAs, their predicted targets were compared with five miRNAs sharing ZBTB20, ZNF704, and EIF4EBP2 as common potential targets and four miRNAs sharing 16 common potential targets. The interaction network consisting of these 19 targets and additional 18 hub targets were created to facilitate validation of miRNA-mRNA interactions by suggesting the most plausible pairs. Furthermore, the information on drug candidates was integrated into the network to predict novel combinatorial therapies that encompass the complexity of miRNAs-mediated regulation. This is the first study that combines a phenotypic screening approach with RNA sequencing and bioinformatics to systematically identify miRNA-mediated pathways and to detect potential drug candidates to positively influence the phenotypic switch of VSMCs.

An RNA Interference/Adeno-Associated Virus Vector-Based Combinatorial Gene Therapy Approach Against Hepatitis E Virus.

Hepatitis E virus (HEV) is a major public health problem with limited therapeutic options. Here, we engineered adeno-associated viral vectors of serotype 6 (AAV6) to express short hairpin RNAs (shRNAs) against HEV transcripts with the prospect of down-regulating HEV replication in vivo. We designed 20 different shRNAs, targeting the genome of the HEV genotype 3 (GT3) Kernow-C1 p6 strain, for delivery upon AAV6 transduction. Using an original selectable HEV GT3 reporter replicon, we identified three shRNAs that efficiently down-regulated HEV replication. We further confirmed their inhibitory potency with full-length HEV infection. Seventy-two hours following transduction, HEV replication in both systems decreased by up to 95%. The three most potent inhibitory shRNAs identified were directed against the methyltransferase domain, the junction region between the open reading frames (ORFs), and the 3´ end of ORF2. Targeting all three regions by multiplexing the shRNAs further enhanced their inhibitory potency over a prolonged period of up to 21 days following transduction. Conclusion: Combining RNA interference and AAV vector-based gene therapy has great potential for suppressing HEV replication. Our strategy to target the viral RNA with multiplexed shRNAs should help to counteract viral escape through mutations. Considering the widely documented safety of AAV vector-based gene therapies, our approach is, in principle, amenable to clinical translation.

Hyperparameter optimization for image analysis: application to prostate tissue images and live cell data of virus-infected cells.

PURPOSE: Automated analysis of microscopy image data typically requires complex pipelines that involve multiple methods for different image analysis tasks. To achieve best results of the analysis pipelines, method-dependent hyperparameters need to be optimized. However, complex pipelines often suffer from the fact that calculation of the gradient of the loss function is analytically or computationally infeasible. Therefore, first- or higher-order optimization methods cannot be applied. METHODS: We developed a new framework for zero-order black-box hyperparameter optimization called HyperHyper, which has a modular architecture that separates hyperparameter sampling and optimization. We also developed a visualization of the loss function based on infimum projection to obtain further insights into the optimization problem. RESULTS: We applied HyperHyper in three different experiments with different imaging modalities, and evaluated in total more than 400.000 hyperparameter combinations. HyperHyper was used for optimizing two pipelines for cell nuclei segmentation in prostate tissue microscopy images and two pipelines for detection of hepatitis C virus proteins in live cell microscopy data. We evaluated the impact of separating the sampling and optimization strategy using different optimizers and employed an infimum projection for visualizing the hyperparameter space. CONCLUSIONS: The separation of sampling and optimization strategy of the proposed HyperHyper optimization framework improves the result of the investigated image analysis pipelines. Visualization of the loss function based on infimum projection enables gaining further insights on the optimization process.

High-Density Cell Arrays for Genome-Scale Phenotypic Screening.

Due to high associated costs and considerable time investments of cell-based screening, there is a strong demand for new technologies that enable preclinical development and tests of diverse biologicals in a cost-saving and time-efficient manner. For those reasons we developed the high-density cell array (HD-CA) platform, which miniaturizes cell-based screening in the form of preprinted and ready-to-run screening arrays. With the HD-CA technology, up to 24,576 samples can be tested in a single experiment, thereby saving costs and time for microscopy-based screening by 75%. Experiments on the scale of the entire human genome can be addressed in a real parallel manner, with screening campaigns becoming more comfortable and devoid of robotics infrastructure on the user side. The high degree of miniaturization enables working with expensive reagents and rare and difficult-to-obtain cell lines. We have also optimized an automated imaging procedure for HD-CA and demonstrate the applicability of HD-CA to CRISPR-Cas9- and RNAi-mediated phenotypic assessment of the gene function.

Impact of the Assembly-Activating Protein on Molecular Evolution of Synthetic Adeno-Associated Virus Capsids.

Over the last decade, the role of the assembly-activating protein (AAP) has begun to be dissected for the formation of adeno-associated virus (AAV) capsids based on different viral serotypes. Recently, the authors' group has specifically studied AAP's relevance during production of AAV gene therapy vectors in mammalian or insect cells, and AAP was found to be essential for capsid protein stabilization and generation of functional vector particles. Here, the lingering question is additionally addressed of whether molecular AAV evolution via DNA family shuffling of viral capsid genes would perturb AAP functionality due to concurrent and inadvertent recombination of the AAP open reading frame. To this end, a battery of complementary experiments was conducted in which: (1) the ability of chimeric AAP from AAVDJ, a hybrid of serotypes 2, 8, and 9, was tested to rescue AAP knockouts in the three parental serotypes; (2) the functionality of 60 chimeric AAPs extracted from five shuffled, unselected capsid libraries was measured; (3) whether production of different shuffled libraries, 10 wild-type serotypes or 25 individual chimeric capsids, can be enhanced by overexpression of AAP cocktails was assessed; and (4) the activity of 12 chimeric AAPs isolated from a shuffled library that was iteratively selected in vivo in mouse livers was studied. Collectively, the data demonstrate a remarkable tolerance of AAP for recombination via DNA family shuffling, evidenced by the findings that (1) all chimeric AAPs studied here retained at least partial activity, even in cases where the cognate hybrid capsid may be non-functional, and that (2) ectopic AAP overexpression did not enhance production of shuffled AAV chimeras or libraries, implying that the inherently encoded hybrid AAP variants are sufficiently active. Together, this work provides compelling evidence that AAP is not rate limiting during AAV capsid shuffling and thereby relieves a major concern in the field of AAV vector evolution.

Comparison of Cell Arrays and Multi-Well Plates in Microscopy-Based Screening.

Multi-well plates and cell arrays enable microscopy-based screening assays in which many samples can be analysed in parallel. Each of the formats possesses its own strengths and weaknesses, but reference comparisons between these platforms and their application rationale is lacking. We aim to fill this gap by comparing two RNA interference (RNAi)-mediated fluorescence microscopy-based assays, namely epidermal growth factor (EGF) internalization and cell cycle progression, on both platforms. Quantitative analysis revealed that both platforms enabled the generation of data with the appearance of the expected phenotypes significantly distinct from the negative controls. The measurements of cell cycle progression were less variable in multi-well plates. The result can largely be attributed to higher cell numbers resulting in less data variability when dealing with the assay generating phenotypic cell subpopulations. The EGF internalization assay with a uniform phenotype over nearly the whole cell population performed better on cell arrays than in multi-well plates. The result was achieved by scoring five times less cells on cell arrays than in multi-well plates, indicating the efficiency of the cell array format. Our data indicate that the choice of the screening platform primarily depends on the type of the cellular assay to achieve a maximum data quality and screen efficiency.

Super-Resolution Microscopy Techniques and Their Potential for Applications in Radiation Biophysics.

Fluorescence microscopy is an essential tool for imaging tagged biological structures. Due to the wave nature of light, the resolution of a conventional fluorescence microscope is limited laterally to about 200 nm and axially to about 600 nm, which is often referred to as the Abbe limit. This hampers the observation of important biological structures and dynamics in the nano-scaled range ~10 nm to ~100 nm. Consequentially, various methods have been developed circumventing this limit of resolution. Super-resolution microscopy comprises several of those methods employing physical and/or chemical properties, such as optical/instrumental modifications and specific labeling of samples. In this article, we will give a brief insight into a variety of selected optical microscopy methods reaching super-resolution beyond the Abbe limit. We will survey three different concepts in connection to biological applications in radiation research without making a claim to be complete.

Fully Automated Targeted Confocal and Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) enables imaging of biological structures in the nanometre range. Long measurement times are the consequence of this kind of microscopy due to the need of acquiring thousands of images. We built a setup that automatically detects target structures using confocal microscopy and images them with SMLM. Utilizing the Konstanz Information Miner (KNIME), we were able to connect a confocal microscope with an SMLM unit for targeted screening. In this process, we developed KNIME plugins to communicate with the microscope components and combined them to a workflow. Thus, measuring biological nanometre-sized structures in a sufficient number to get statistical significance becomes feasible. For proof of principle HIV-1 assembly complexes in HeLa cells derived from transfection of replication deficient viral construct were imaged by a fully automated screen.

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

Workflows for microscopy image analysis and cellular phenotyping.

In large scale biological experiments, like high-throughput or high-content cellular screening, the amount and the complexity of images to be analyzed are steadily increasing. To handle and process these images, well defined image processing and analysis steps need to be performed by applying dedicated workflows. Multiple software tools have emerged with the aim to facilitate creation of such workflows by integrating existing methods, tools, and routines, and by adapting them to different applications and questions, as well as making them reusable and interchangeable. In this review, we describe workflow systems for the integration of microscopy image analysis techniques with focus on KNIME and Galaxy.

The long non-coding RNA LINC00152 is essential for cell cycle progression through mitosis in HeLa cells.

In recent years, long non-coding RNA (lncRNA) research has identified essential roles of these transcripts in virtually all physiological cellular processes including tumorigenesis, but their functions and molecular mechanisms are poorly understood. In this study, we performed a high-throughput siRNA screen targeting 638 lncRNAs deregulated in cancer entities to analyse their impact on cell division by using time-lapse microscopy. We identified 26 lncRNAs affecting cell morphology and cell cycle including LINC00152. This transcript was ubiquitously expressed in many human cell lines and its RNA levels were significantly upregulated in lung, liver and breast cancer tissues. A comprehensive sequence analysis of LINC00152 revealed a highly similar paralog annotated as MIR4435-2HG and several splice variants of both transcripts. The shortest and most abundant isoform preferentially localized to the cytoplasm. Cells depleted of LINC00152 arrested in prometaphase of mitosis and showed reduced cell viability. In RNA affinity purification (RAP) studies, LINC00152 interacted with a network of proteins that were associated with M phase of the cell cycle. In summary, we provide new insights into the properties and biological function of LINC00152 suggesting that this transcript is crucial for cell cycle progression through mitosis and thus, could act as a non-coding oncogene.

Fluorescence-Based High-Throughput and Targeted Image Acquisition and Analysis for Phenotypic Screening.

Applying the right acquisition method in a fluorescence imaging-based screening context is of great importance to obtain an appropriate readout and to select the right scale of the screen. In order to save imaging time and data, we have developed routines for multiscale targeted imaging, providing both a broad overview of a sample and additional in-depth information for targets of interest identified within the screen. These objects can be identified and acquired on-the-fly by an interconnection of image acquisition and image analysis.

Localisation Microscopy of Breast Epithelial ErbB-2 Receptors and Gap Junctions: Trafficking after γ-Irradiation, Neuregulin-1ß, and Trastuzumab Application.

In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin- 1 ß application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon γ -irradiation or alternatively neuregulin- 1 ß application mobilized ErbB receptors in a nucleograde fashion-a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation.

Quantification of telomere features in tumor tissue sections by an automated 3D imaging-based workflow.

The microscopic analysis of telomere features provides a wealth of information on the mechanism by which tumor cells maintain their unlimited proliferative potential. Accordingly, the analysis of telomeres in tissue sections of patient tumor samples can be exploited to obtain diagnostic information and to define tumor subgroups. In many instances, however, analysis of the image data is conducted by manual inspection of 2D images at relatively low resolution for only a small part of the sample. As the telomere feature signal distribution is frequently heterogeneous, this approach is prone to a biased selection of the information present in the image and lacks subcellular details. Here we address these issues by using an automated high-resolution imaging and analysis workflow that quantifies individual telomere features on tissue sections for a large number of cells. The approach is particularly suited to assess telomere heterogeneity and low abundant cellular subpopulations with distinct telomere characteristics in a reproducible manner. It comprises the integration of multi-color fluorescence in situ hybridization, immunofluorescence and DNA staining with targeted automated 3D fluorescence microscopy and image analysis. We apply our method to telomeres in glioblastoma and prostate cancer samples, and describe how the imaging data can be used to derive statistically reliable information on telomere length distribution or colocalization with PML nuclear bodies. We anticipate that relating this approach to clinical outcome data will prove to be valuable for pretherapeutic patient stratification.

High-Content Analysis of the Golgi Complex by Correlative Screening Microscopy.

The Golgi complex plays a central role in a number of diverse cellular processes, and numerous regulators that control these functions and/or morphology of the Golgi complex are known by now. Many of them were identified by large-scale experiments, such as RNAi-based screening. However, high-throughput experiments frequently provide only initial information that a particular protein might play a role in regulating structure and function of the Golgi complex. Multiple follow-up experiments are necessary to functionally characterize the selected hits. In order to speed up the discovery, we have established a system for correlative screening microscopy that combines rapid data collection and high-resolution imaging in one experiment. We describe here a combination of wide-field microscopy and dual-color direct stochastical optical reconstruction microscopy (dSTORM). We apply the technique to simultaneously capture and differentiate alterations of the cis- and trans-Golgi network when depleting several proteins in a singular and combinatorial manner.

Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes.

The present work addresses the question of to what extent a geometrical support acts as a physiological determining template in the setup of artificial cardiac tissue. Surface patterns with alternating concave to convex transitions of cell size dimensions were used to organize and orientate human-induced pluripotent stem cell (hIPSC)-derived cardiac myocytes and mouse neonatal cardiac myocytes. The shape of the cells, as well as the organization of the contractile apparatus recapitulates the anisotropic line pattern geometry being derived from tissue geometry motives. The intracellular organization of the contractile apparatus and the cell coupling via gap junctions of cell assemblies growing in a random or organized pattern were examined. Cell spatial and temporal coordinated excitation and contraction has been compared on plain and patterned substrates. While the α-actinin cytoskeletal organization is comparable to terminally-developed native ventricular tissue, connexin-43 expression does not recapitulate gap junction distribution of heart muscle tissue. However, coordinated contractions could be observed. The results of tissue-like cell ensemble organization open new insights into geometry-dependent cell organization, the cultivation of artificial heart tissue from stem cells and the anisotropy-dependent activity of therapeutic compounds.

Fluorescence microscopy-based RNA interference screening.

Using RNAi interference (RNAi), it is possible to study the effect of specific gene knockdowns in mammalian cells. In this protocol we present the automated preparation of "ready to transfect" multiwell plates and cell arrays, on which cells can be grown which are then reversely transfected with one type of siRNA in every individual well or spot. Additionally, different microscope types for screening approaches are compared and considerations about the information workflow are made.