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Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications., Competing Interests: AB, LS, LD, AP, FS, EH, CE, HJ, HY, GF, DC No competing interests declared, EG Now works for Boehringer Ingelheim Pharma GmbH & Co KG, MP Board member of Tartan Cell Technologies Ltd, JS Board member of Tartan Cell Technologies Ltd and Glencoe Software Ltd, AL Board member of Tartan Cell Technologies Ltd and Platinum Informatics Ltd, (© 2024, Brenes et al.)

Male contraceptive options and infertility treatments are limited, and almost all innovation has been limited to updates to medically assisted reproduction protocols and methods. To accelerate the development of drugs that can either improve or inhibit fertility, we established a small molecule library as a toolbox for assay development and screening campaigns using human spermatozoa. We have profiled all compounds in the Sperm Toolbox in several automated high-throughput assays that measure stimulation or inhibition of sperm motility or the acrosome reaction. We have assayed motility under non-capacitating and capacitating conditions to distinguish between pathways operating under these different physiological states. We also assayed cell viability to ensure any effects on sperm function are specific. A key advantage of our studies is that all compounds are assayed together in the same experimental conditions, which allows quantitative comparisons of their effects in complementary functional assays. We have combined the resulting datasets to generate fingerprints of the Sperm Toolbox compounds on sperm function. The data are included in an on-line R-based app for convenient querying., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gruber et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A growing community is constructing a next-generation file format (NGFF) for bioimaging to overcome problems of scalability and heterogeneity. Organized by the Open Microscopy Environment (OME), individuals and institutes across diverse modalities facing these problems have designed a format specification process (OME-NGFF) to address these needs. This paper brings together a wide range of those community members to describe the cloud-optimized format itself-OME-Zarr-along with tools and data resources available today to increase FAIR access and remove barriers in the scientific process. The current momentum offers an opportunity to unify a key component of the bioimaging domain-the file format that underlies so many personal, institutional, and global data management and analysis tasks., (© 2023. The Author(s).)

Open and practical exchange, dissemination, and reuse of specimens and data have become a fundamental requirement for life sciences research. The quality of the data obtained and thus the findings and knowledge derived is thus significantly influenced by the quality of the samples, the experimental methods, and the data analysis. Therefore, a comprehensive and precise documentation of the pre-analytical conditions, the analytical procedures, and the data processing are essential to be able to assess the validity of the research results. With the increasing importance of the exchange, reuse, and sharing of data and samples, procedures are required that enable cross-organizational documentation, traceability, and non-repudiation. At present, this information on the provenance of samples and data is mostly either sparse, incomplete, or incoherent. Since there is no uniform framework, this information is usually only provided within the organization and not interoperably. At the same time, the collection and sharing of biological and environmental specimens increasingly require definition and documentation of benefit sharing and compliance to regulatory requirements rather than consideration of pure scientific needs. In this publication, we present an ongoing standardization effort to provide trustworthy machine-actionable documentation of the data lineage and specimens. We would like to invite experts from the biotechnology and biomedical fields to further contribute to the standard., Competing Interests: The authors report that they have no conflicts of interest., (© 2023 The Authors. Learning Health Systems published by Wiley Periodicals LLC on behalf of University of Michigan.)

There is a need for non-hormonal contraceptives. One area that needs further investigation is the development of male contraceptives. Comparatively little is understood about potential drug targets in men to achieve a reversible contraceptive effect. In this article, we review the need for male contraceptives and some thoughts around the characteristics of a male contraceptive and the potential development pathway. We then discuss different potential approaches to discovering male contraceptives and then highlight potential targets that have been discussed in the literature., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Masson SAS.)

Recent advances in fluorescence microscopy techniques and tissue clearing, labeling, and staining provide unprecedented opportunities to investigate brain structure and function. These experiments' images make it possible to catalog brain cell types and define their location, morphology, and connectivity in a native context, leading to a better understanding of normal development and disease etiology. Consistent annotation of metadata is needed to provide the context necessary to understand, reuse, and integrate these data. This report describes an effort to establish metadata standards for three-dimensional (3D) microscopy datasets for use by the Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative and the neuroscience research community. These standards were built on existing efforts and developed with input from the brain microscopy community to promote adoption. The resulting 3D Microscopy Metadata Standards (3D-MMS) includes 91 fields organized into seven categories: Contributors, Funders, Publication, Instrument, Dataset, Specimen, and Image. Adoption of these metadata standards will ensure that investigators receive credit for their work, promote data reuse, facilitate downstream analysis of shared data, and encourage collaboration., (© 2022. The Author(s).)

Despite the huge impact of data resources in genomics and structural biology, until now there has been no central archive for biological data for all imaging modalities. The BioImage Archive is a new data resource at the European Bioinformatics Institute (EMBL-EBI) designed to fill this gap. In its initial development BioImage Archive accepts bioimaging data associated with publications, in any format, from any imaging modality from the molecular to the organism scale, excluding medical imaging. The BioImage Archive will ensure reproducibility of published studies that derive results from image data and reduce duplication of effort. Most importantly, the BioImage Archive will help scientists to generate new insights through reuse of existing data to answer new biological questions, and provision of training, testing and benchmarking data for development of tools for image analysis. The archive is available at https://www.ebi.ac.uk/bioimage-archive/., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Study Question: Can a high-throughput screening (HTS) platform facilitate male fertility drug discovery?, Summary Answer: An HTS platform identified a large number of compounds that enhanced sperm motility., What Is Known Already: Several efforts to find small molecules modulating sperm function have been performed but none have used high-throughput technology., Study Design, Size, Duration: Healthy donor semen samples were used and samples were pooled (3-5 donors per pool). Primary screening was performed singly; dose-response screening was performed in duplicate (using independent donor pools)., Participants/materials, Setting, Methods: Spermatozoa isolated from healthy donors were prepared by density gradient centrifugation and incubated in 384-well plates with compounds (6.25 μM) to identify those compounds with enhancing effects on motility. Approximately 17 000 compounds from the libraries, ReFRAME, Prestwick, Tocris, LOPAC, CLOUD and MMV Pathogen Box, were screened. Dose-response experiments of screening hits were performed to confirm the enhancing effect on sperm motility. Experiments were performed in a university setting., Main Results and the Role of Chance: From our primary single concentration screening, 105 compounds elicited an enhancing effect on sperm motility compared to dimethylsulphoxide-treated wells. Confirmed enhancing compounds were grouped based on their annotated targets/target classes. A major target class, phosphodiesterase inhibitors, were identified, in particular PDE10A inhibitors as well as number of compounds not previously known to enhance human sperm motility, such as those related to GABA signalling., Large Scale Data: N/A., Limitations, Reasons for Caution: Although this approach provides data about the activity of the compound, it is only a starting point. For example, further substantive experiments are necessary to provide a more comprehensive picture of each compound's activity, the effect on the kinetics of the cell populations and subpopulations, and their potential mechanisms of action. Compounds have been tested with prepared donor spermatozoa, incubated under non-capacitating conditions, and only incubated with compounds for a relatively short period of time. Therefore, the effect of compounds under different conditions, for example in whole semen, for longer incubation times, or using samples from patient groups, may be different and require further study. All experiments were performed in vitro., Wider Implications of the Findings: This phenotypic screening assay identified a large number of compounds that increased sperm motility. In addition to furthering our understanding of human sperm function, for example identifying new avenues for discovery, we highlight potential compounds as promising start-point for a medicinal chemistry programme for potential enhancement of male fertility. Moreover, with disclosure of the results of screening, we present a substantial resource to inform further work in the field., Study Funding/competing Interest(s): This study was supported by the Bill and Melinda Gates Foundation, Scottish Funding Council and Scottish Universities Life Science Alliance. C.L.R.B. is Editor for RBMO. C.L.R.B. receives funding from Chief Scientists Office (Scotland), ESHRE and Genus PLC, consulting fees from Exscientia and lecture fees from Cooper Surgical and Ferring. S.M.d.S. is an Associate Editor of Human Reproduction, and an Associate Editor of Reproduction and Fertility. S.M.d.S. receives funding from Cooper Surgical and British Dietetic Society. No other authors declared a COI., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

The rapid pace of innovation in biological imaging and the diversity of its applications have prevented the establishment of a community-agreed standardized data format. We propose that complementing established open formats such as OME-TIFF and HDF5 with a next-generation file format such as Zarr will satisfy the majority of use cases in bioimaging. Critically, a common metadata format used in all these vessels can deliver truly findable, accessible, interoperable and reusable bioimaging data., (© 2021. The Author(s).)

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics., (© 2021 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.)

Cell migration research has become a high-content field. However, the quantitative information encapsulated in these complex and high-dimensional datasets is not fully exploited owing to the diversity of experimental protocols and non-standardized output formats. In addition, typically the datasets are not open for reuse. Making the data open and Findable, Accessible, Interoperable, and Reusable (FAIR) will enable meta-analysis, data integration, and data mining. Standardized data formats and controlled vocabularies are essential for building a suitable infrastructure for that purpose but are not available in the cell migration domain. We here present standardization efforts by the Cell Migration Standardisation Organisation (CMSO), an open community-driven organization to facilitate the development of standards for cell migration data. This work will foster the development of improved algorithms and tools and enable secondary analysis of public datasets, ultimately unlocking new knowledge of the complex biological process of cell migration., (© The Author(s) 2020. Published by Oxford University Press.)

In stimulated emission depletion (STED) nanoscopy, the major origin of decreased signal-to-noise ratio within images can be attributed to sample photobleaching and strong optical aberrations. This is due to STED utilizing a high-power depletion laser (increasing the risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here, we demonstrate a custom-built STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating tissue. We introduce and investigate a state of the art image denoising method by block-matching and collaborative 3D filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise and further enhance the image quality. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well established Fourier ring correlation metric. Results achieved through the combination of aberration correction and tailored image processing are experimentally validated through super-resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under an 80 μm thick layer of tissue with lateral and axial resolution of 204 and 310 nm, respectively.

Faced with the need to support a growing number of whole slide imaging (WSI) file formats, our team has extended a long-standing community file format (OME-TIFF) for use in digital pathology. The format makes use of the core TIFF specification to store multi-resolution (or "pyramidal") representations of a single slide in a flexible, performant manner. Here we describe the structure of this format, its performance characteristics, as well as an open-source library support for reading and writing pyramidal OME-TIFFs.

Stimulated emission depletion (STED) nanoscopy is one of a suite of modern optical microscopy techniques capable of bypassing the conventional diffraction limit in fluorescent imaging. STED makes use of a spiral phase mask to enable 2D super-resolution imaging whereas to achieve full volumetric 3D super-resolution an additional bottle-beam phase mask must be applied. The resolution achieved in biological samples 10 µm or thicker is limited by aberrations induced mainly by scattering due to refractive index heterogeneity in the sample. These aberrations impact the fidelity of both types of phase mask, and have limited the application of STED to thicker biological systems. Here we apply an automated adaptive optics solution to correct the performance of both STED masks, enhancing robustness and expanding the capabilities of this nanoscopic technique. Corroboration in terms of successful high-quality imaging of the full volume of a 15µm mitotic spindle with resolution of 50nm x 50nm x 150nm achieved in all three dimensions is presented., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

The Type VI secretion system (T6SS) is widely used by bacterial pathogens as an effective weapon against bacterial competitors and is also deployed against host eukaryotic cells in some cases. It is a contractile nanomachine which delivers toxic effector proteins directly into target cells by dynamic cycles of assembly and firing. Bacterial cells adopt distinct post-translational regulatory strategies for deployment of the T6SS. 'Defensive' T6SSs assemble and fire in response to incoming attacks from aggressive neighbouring cells, and can utilise the Threonine Protein Phosphorylation (TPP) regulatory pathway to achieve this control. However, many T6SSs are 'offensive', firing at all-comers without the need for incoming attack or other cell contact-dependent signal. Post-translational control of the offensive mode has been less well defined but can utilise components of the same TPP pathway. Here, we used the anti-bacterial T6SS of Serratia marcescens to elucidate post-translational regulation of offensive T6SS deployment, using single-cell microscopy and genetic analyses. We show that the integration of the TPP pathway with the negative regulator TagF to control core T6SS machine assembly is conserved between offensive and defensive T6SSs. Signal-dependent PpkA-mediated phosphorylation of Fha is required to overcome inhibition of membrane complex assembly by TagF, whilst PppA-mediated dephosphorylation promotes spatial reorientation and efficient killing. In contrast, the upstream input of the TPP pathway defines regulatory strategy, with a new periplasmic regulator, RtkS, shown to interact with the PpkA kinase in S. marcescens. We propose a model whereby the opposing actions of the TPP pathway and TagF impose a delay on T6SS re-assembly after firing, providing an opportunity for spatial re-orientation of the T6SS in order to maximise the efficiency of competitor cell targeting. Our findings provide a better understanding of how bacterial cells deploy competitive weapons effectively, with implications for the structure and dynamics of varied polymicrobial communities., Competing Interests: The authors have declared that no competing interests exist.

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of protein phosphatase 2A containing the B56 regulatory subunit (PP2A-B56). This phosphatase controls the amount of phosphorylation of several kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. Like PP2A-B56, Bod1 directly localizes to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localization of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1's binding to and inhibition of PP2A-B56, peaks in prometaphase when PP2A-B56 localization to kinetochores is highest. We demonstrate here that kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Crucially, Bod1 depletion functionally affects Ndc80 phosphorylation at the N-terminal serine 55 (S55), as well as a number of other phosphorylation sites within the outer kinetochore, including Knl1 at serine 24 and 60 (S24, S60), and threonine T943 and T1155 (T943, T1155). Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80, and Knl1 phosphorylation, including sites that mediate the attachment of microtubules to kinetochores., (© 2017 The Authors.)

Access to primary research data is vital for the advancement of science. To extend the data types supported by community repositories, we built a prototype Image Data Resource (IDR) that collects and integrates imaging data acquired across many different imaging modalities. IDR links data from several imaging modalities, including high-content screening, super-resolution and time-lapse microscopy, digital pathology, public genetic or chemical databases, and cell and tissue phenotypes expressed using controlled ontologies. Using this integration, IDR facilitates the analysis of gene networks and reveals functional interactions that are inaccessible to individual studies. To enable re-analysis, we also established a computational resource based on Jupyter notebooks that allows remote access to the entire IDR. IDR is also an open source platform that others can use to publish their own image data. Thus IDR provides both a novel on-line resource and a software infrastructure that promotes and extends publication and re-analysis of scientific image data., Competing Interests: Competing Financial Interests J. R. S is affiliated with Glencoe Software, Inc., an open-source US-based commercial company that provides commercial licenses for OME software.

Here we report a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family, where individuals that are homozygous for the mutation have no detectable BOD1 mRNA or protein. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 substrates by modulating Protein Phosphatase 2A (PP2A) activity during mitosis. We report that fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. However, in contrast to the mitotic arrest observed in BOD1-siRNA treated HeLa cells, patient-derived cells progressed through mitosis with no apparent segregation defects but at an accelerated rate compared to controls. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, we found that in normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and provide evidence for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, we established two Drosophila models, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together our results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila we provide evidence for a conserved role of BOD1 in the development and maintenance of cognitive features.

High content screening (HCS) experiments create a classic data management challenge-multiple, large sets of heterogeneous structured and unstructured data, that must be integrated and linked to produce a set of "final" results. These different data include images, reagents, protocols, analytic output, and phenotypes, all of which must be stored, linked and made accessible for users, scientists, collaborators and where appropriate the wider community. The OME Consortium has built several open source tools for managing, linking and sharing these different types of data. The OME Data Model is a metadata specification that supports the image data and metadata recorded in HCS experiments. Bio-Formats is a Java library that reads recorded image data and metadata and includes support for several HCS screening systems. OMERO is an enterprise data management application that integrates image data, experimental and analytic metadata and makes them accessible for visualization, mining, sharing and downstream analysis. We discuss how Bio-Formats and OMERO handle these different data types, and how they can be used to integrate, link and share HCS experiments in facilities and public data repositories. OME specifications and software are open source and are available at https://www.openmicroscopy.org., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitosis ensures equal segregation of the genome and is controlled by a variety of ubiquitylation signals on substrate proteins. However, it remains unexplored how the versatile ubiquitin code is read out during mitotic progression. Here, we identify the ubiquitin receptor protein UBASH3B as an important regulator of mitosis. UBASH3B interacts with ubiquitylated Aurora B, one of the main kinases regulating chromosome segregation, and controls its subcellular localization but not protein levels. UBASH3B is a limiting factor in this pathway and is sufficient to localize Aurora B to microtubules prior to anaphase. Importantly, targeting Aurora B to microtubules by UBASH3B is necessary for the timing and fidelity of chromosome segregation in human cells. Our findings uncover an important mechanism defining how ubiquitin attachment to a substrate protein is decoded during mitosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which cell cycle mediators, such as CDKs, temporally control the activity of PHD1, directly altering the regulation of HIF1α and Cep192., (© 2016. Published by The Company of Biologists Ltd.)

Imaging data are used in the life and biomedical sciences to measure the molecular and structural composition and dynamics of cells, tissues, and organisms. Datasets range in size from megabytes to terabytes and usually contain a combination of binary pixel data and metadata that describe the acquisition process and any derived results. The OMERO image data management platform allows users to securely share image datasets according to specific permissions levels: data can be held privately, shared with a set of colleagues, or made available via a public URL. Users control access by assigning data to specific Groups with defined membership and access rights. OMERO's Permission system supports simple data sharing in a lab, collaborative data analysis, and even teaching environments. OMERO software is open source and released by the OME Consortium at www.openmicroscopy.org.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.  Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

Cell differentiation is ubiquitous and facilitates division of labor and development. Bacteria are capable of multicellular behaviors that benefit the bacterial community as a whole. A striking example of bacterial differentiation occurs throughout the formation of a biofilm. During Bacillus subtilis biofilm formation, a subpopulation of cells differentiates into a specialized population that synthesizes the exopolysaccharide and the TasA amyloid components of the extracellular matrix. The differentiation process is indirectly controlled by the transcription factor Spo0A that facilitates transcription of the eps and tapA (tasA) operons. DegU is a transcription factor involved in regulating biofilm formation. Here, using a combination of genetics and live single-cell cytological techniques, we define the mechanism of biofilm inhibition at high levels of phosphorylated DegU (DegU∼P) by showing that transcription from the eps and tapA promoter regions is inhibited. Data demonstrating that this is not a direct regulatory event are presented. We demonstrate that DegU∼P controls the frequency with which cells activate transcription from the operons needed for matrix biosynthesis in favor of an off state. Subsequent experimental analysis led us to conclude that DegU∼P functions to increase the level of Spo0A∼P, driving cell fate differentiation toward the terminal developmental process of sporulation.

The Protein Data Bank in Europe (PDBe) has developed web-based tools for the visualisation and analysis of 3D electron microscopy (3DEM) structures in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB). The tools include: (1) a volume viewer for 3D visualisation of maps, tomograms and models, (2) a slice viewer for inspecting 2D slices of tomographic reconstructions, and (3) visual analysis pages to facilitate analysis and validation of maps, tomograms and models. These tools were designed to help non-experts and experts alike to get some insight into the content and assess the quality of 3DEM structures in EMDB and PDB without the need to install specialised software or to download large amounts of data from these archives. The technical challenges encountered in developing these tools, as well as the more general considerations when making archived data available to the user community through a web interface, are discussed., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Proper chromosome segregation depends on correct attachments between microtubules and kinetochores. Budding yeast have been thought to achieve these attachments with different kinetics than other eukaryotes. Now, deploying specialized data processing techniques to achieve super-resolution images, Marco et al. demonstrate that this tractable cell-cycle model system shares more similarities with plants and animals than previously thought., (Copyright © 2013 Elsevier Inc. All rights reserved.)

PHD1 belongs to the family of prolyl-4-hydroxylases (PHDs) that is responsible for posttranslational modification of prolines on specific target proteins. Because PHD activity is sensitive to oxygen levels and certain byproducts of the tricarboxylic acid cycle, PHDs act as sensors of the cell's metabolic state. Here, we identify PHD1 as a critical molecular link between oxygen sensing and cell-cycle control. We show that PHD1 function is required for centrosome duplication and maturation through modification of the critical centrosome component Cep192. Importantly, PHD1 is also required for primary cilia formation. Cep192 is hydroxylated by PHD1 on proline residue 1717. This hydroxylation is required for binding of the E3 ubiquitin ligase SCF(Skp2), which ubiquitinates Cep192, targeting it for proteasomal degradation. By modulating Cep192 levels, PHD1 thereby affects the processes of centriole duplication and centrosome maturation and contributes to the regulation of cell-cycle progression., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Polo-like kinase 1 (PLK1) critically regulates mitosis through its dynamic localization to kinetochores, centrosomes and the midzone. The polo-box domain (PBD) and activity of PLK1 mediate its recruitment to mitotic structures, but the mechanisms regulating PLK1 dynamics remain poorly understood. Here, we identify PLK1 as a target of the cullin 3 (CUL3)-based E3 ubiquitin ligase, containing the BTB adaptor KLHL22, which regulates chromosome alignment and PLK1 kinetochore localization but not PLK1 stability. In the absence of KLHL22, PLK1 accumulates on kinetochores, resulting in activation of the spindle assembly checkpoint (SAC). CUL3-KLHL22 ubiquitylates Lys 492, located within the PBD, leading to PLK1 dissociation from kinetochore phosphoreceptors. Expression of a non-ubiquitylatable PLK1-K492R mutant phenocopies inactivation of CUL3-KLHL22. KLHL22 associates with the mitotic spindle and its interaction with PLK1 increases on chromosome bi-orientation. Our data suggest that CUL3-KLHL22-mediated ubiquitylation signals degradation-independent removal of PLK1 from kinetochores and SAC satisfaction, which are required for faithful mitosis.

This chapter discusses quantitative analysis of digital microscope images and presents several exercises to provide examples to explain the concept. This chapter also presents the basic concepts in quantitative analysis for imaging, but these concepts rest on a well-established foundation of signal theory and quantitative data analysis. This chapter presents several examples for understanding the imaging process as a transformation from sample to image and the limits and considerations of quantitative analysis. This chapter introduces to the concept of digitally correcting the images and also focuses on some of the more critical types of data transformation and some of the frequently encountered issues in quantization. Image processing represents a form of data processing. There are many examples of data processing such as fitting the data to a theoretical curve. In all these cases, it is critical that care is taken during all steps of transformation, processing, and quantization., (Copyright © 2007 Elsevier Inc. All rights reserved.)

Mitotic entry and progression require the activation of several mitotic kinases and the proper regulation and localization of several phosphatases. The activity and localization of each of these enzymes is tightly controlled through a series of specific activators, inhibitors and regulatory subunits. Two proteins, Ensa and Arpp-19, were recently identified as specific inhibitors of PP2A-B55 and are critical for allowing full activity of Cdk1/cyclin B and entry into mitosis. Here we show that Bod1, a protein required for proper chromosome alignment at mitosis, shares sequence similarity with Ensa and Arpp-19 and specifically inhibits the kinetochore-associated PP2A-B56 holoenzyme. PP2A-B56 regulates the stability of kinetochore-microtubule attachments by dephosphorylating several kinetochore proteins. Loss of Bod1 changes the balance of phosphorylation at kinetochores, causing defects in kinetochore function. Bod1, Ensa and Arpp-19 define a family of specific PP2A inhibitors that regulate specific PP2A holoenzymes at distinct locations and points in the cell cycle.

Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used to remove blurred signal from an image. There are two major types of deconvolution approaches, deblurring and restoration algorithms. Deblurring algorithms remove blur, but treat a series of optical sections as individual two-dimensional entities, and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed., (Copyright © 1998 Elsevier Inc. All rights reserved.)

Few technologies are more widespread in modern biological laboratories than imaging. Recent advances in optical technologies and instrumentation are providing hitherto unimagined capabilities. Almost all these advances have required the development of software to enable the acquisition, management, analysis and visualization of the imaging data. We review each computational step that biologists encounter when dealing with digital images, the inherent challenges and the overall status of available software for bioimage informatics, focusing on open-source options.

The current revolution in biological microscopy stems from the realisation that advances in optics and computational tools and automation make the modern microscope an instrument that can access all scales relevant to modern biology - from individual molecules all the way to whole tissues and organisms and from single snapshots to time-lapse recordings sampling from milliseconds to days. As these and more new technologies appear, the challenges of delivering them to the community grows as well. I discuss some of these challenges, and the examples where openly shared technology have made an impact on the field., (Copyright © 2012 WILEY Periodicals, Inc.)

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis. In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution. This assay may be modified to image a range of embryonic tissues. In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Data-intensive research depends on tools that manage multidimensional, heterogeneous datasets. We built OME Remote Objects (OMERO), a software platform that enables access to and use of a wide range of biological data. OMERO uses a server-based middleware application to provide a unified interface for images, matrices and tables. OMERO's design and flexibility have enabled its use for light-microscopy, high-content-screening, electron-microscopy and even non-image-genotype data. OMERO is open-source software, available at http://openmicroscopy.org/.