Instantaneous isotropic volumetric imaging of fast biological processes.

To capture highly dynamic biological processes at cellular resolution is a recurring challenge in biology. Here we show that combining selective-volume illumination with simultaneous acquisition of orthogonal light fields yields three-dimensional images with high, isotropic spatial resolution and a significant reduction of reconstruction artefacts, thereby overcoming current limitations of light-field microscopy implementations. We demonstrate medaka heart and blood flow imaging at single-cell resolution and free of motion artefacts at volume rates of up to 200 Hz.

Inverted light-sheet microscope for imaging mouse pre-implantation development.

Despite its importance for understanding human infertility and congenital diseases, early mammalian development has remained inaccessible to in toto imaging. We developed an inverted light-sheet microscope that enabled us to image mouse embryos from zygote to blastocyst, computationally track all cells and reconstruct a complete lineage tree of mouse pre-implantation development. We used this unique data set to show that the first cell fate specification occurs at the 16-cell stage.

Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT.

We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs.

Light-sheet imaging of mammalian development.

Tackling modern cell and developmental biology questions requires fast 3D imaging with sub-cellular resolution over extended periods of time. Fluorescence microscopy has emerged as a powerful tool to image biological samples with high spatial and temporal resolution with molecular specificity. In particular, the highly efficient illumination and detection scheme of light-sheet fluorescence microscopy is starting to revolutionize the way we can monitor cellular and developmental processes in vivo. Here we summarize the state-of-the art of light-sheet imaging with a focus on mammalian development - from instrumentation, mounting and sample handling to data processing.

Evaluation of participatory planning: Lessons from Hungarian Natura 2000 management planning processes.

Stakeholder participation in nature conservation policies and especially in the management of protected areas has gained importance in the last decades. These changes are underlined by democratic principles and the perceived contribution of stakeholder involvement to the effectiveness of conservation management. Evaluating participatory processes is essential to learn about the past and thus increase the quality of future processes. The evaluation can be useful for the organisations responsible for planning and management, stakeholders and policy makers as well. The present paper shows the results of a systematic evaluation of 25 participatory processes related to the development of management plans for Natura 2000 sites in Hungary between 2007 and 2015. A conceptual framework was developed to evaluate the process and outcome of participatory management planning processes. Criteria were based on the scientific literature on public participation and tailored to conservation-related management planning and stakeholder involvement. Evaluated processes were grouped in three cases based on their time range and financial sources. Overall, the analysed processes scored at a medium level, showing better performance in the process criteria than in the outcome criteria. The best case scored significantly higher in four criteria compared to the other cases: representativeness, resource availability for facilitation, new, creative ideas and impact on the plan. The main factors behind the success were (1) embeddedness of the planning process in a larger project, where the plan was a tool for conservation, (2) carrying out only one process at a time, (3) previous experience of facilitators and planners with participatory planning and (4) the opportunity and capacity to propose a payment scheme as an incentive. But even this case received low scores in some criteria: conflict resolution, early involvement and well defined goals. Based on the results we suggest that more data is needed to evaluate the implementation of the plans and, in many cases, the impact of the process on the plan. Performance can be improved with the assistance of policy makers by further developing guidelines, harmonising payment schemes with the conservation measures of the plans and providing training on conflict resolution. The evaluation framework proved to be suitable for the assessment of a large set of conservation related management planning processes, but it also had some limitations, e.g. concerning the incorporation of stakeholders' views in the evaluation.

Learning and the transformative potential of citizen science.

The number of collaborative initiatives between scientists and volunteers (i.e., citizen science) is increasing across many research fields. The promise of societal transformation together with scientific breakthroughs contributes to the current popularity of citizen science (CS) in the policy domain. We examined the transformative capacity of citizen science in particular learning through environmental CS as conservation tool. We reviewed the CS and social-learning literature and examined 14 conservation projects across Europe that involved collaborative CS. We also developed a template that can be used to explore learning arrangements (i.e., learning events and materials) in CS projects and to explain how the desired outcomes can be achieved through CS learning. We found that recent studies aiming to define CS for analytical purposes often fail to improve the conceptual clarity of CS; CS programs may have transformative potential, especially for the development of individual skills, but such transformation is not necessarily occurring at the organizational and institutional levels; empirical evidence on simple learning outcomes, but the assertion of transformative effects of CS learning is often based on assumptions rather than empirical observation; and it is unanimous that learning in CS is considered important, but in practice it often goes unreported or unevaluated. In conclusion, we point to the need for reliable and transparent measurement of transformative effects for democratization of knowledge production.

Combined analysis of gene expression, DNA copy number, and mutation profiling data to display biological process anomalies in individual breast cancers.

The goal of this analysis was to develop a computational tool that integrates the totality of gene expression, DNA copy number, and sequence abnormalities in individual cancers in the framework of biological processes. We used the hierarchical structure of the gene ontology (GO) database to create a reference network and projected mRNA expression, DNA copy number and mutation anomalies detected in single samples into this space. We applied our method to 59 breast cancers where all three types of molecular data were available. Each cancer had a large number of disturbed biological processes. Locomotion, multicellular organismal process, and signal transduction pathways were the most commonly affected GO terms, but the individual molecular events were different from case-to-case. Estrogen receptor-positive and -negative cancers had different repertoire of anomalies. We tested the functional impact of 27 mRNAs that had overexpression in cancer with variable frequency (<2-42 %) using an siRNA screen. Each of these genes inhibited cell growth in at least some of 18 breast cancer cell lines. We developed a free, on-line software tool ( http://netgoplot.org ) to display the complex genomic abnormalities in individual cancers in the biological framework of the GO biological processes. Each cancer harbored a variable number of pathway anomalies and the individual molecular events that caused an anomaly varied from case-to-case. Our in vitro experiments indicate that rare case-specific molecular abnormalities can play a functional role and driver events may vary from case-to-case depending on the constellation of other molecular anomalies.

Snowball: a novel gene family required for developmental patterning of fruiting bodies of mushroom-forming fungi (Agaricomycetes).

The morphogenesis of sexual fruiting bodies of fungi is a complex process determined by a genetically encoded program. Fruiting bodies reached the highest complexity levels in the Agaricomycetes; yet, the underlying genetics is currently poorly known. In this work, we functionally characterized a highly conserved gene termed snb1, whose expression level increases rapidly during fruiting body initiation. According to phylogenetic analyses, orthologs of snb1 are present in almost all agaricomycetes and may represent a novel conserved gene family that plays a substantial role in fruiting body development. We disrupted snb1 using CRISPR/Cas9 in the agaricomycete model organism Coprinopsis cinerea. snb1 deletion mutants formed unique, snowball-shaped, rudimentary fruiting bodies that could not differentiate caps, stipes, and lamellae. We took advantage of this phenotype to study fruiting body differentiation using RNA-Seq analyses. This revealed differentially regulated genes and gene families that, based on wild-type RNA-Seq data, were upregulated early during development and showed tissue-specific expression, suggesting a potential role in differentiation. Taken together, the novel gene family of snb1 and the differentially expressed genes in the snb1 mutants provide valuable insights into the complex mechanisms underlying developmental patterning in the Agaricomycetes. IMPORTANCE: Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are complex multicellular structures, with a spatially and temporally integrated developmental program that is, however, currently poorly known. In this study, we present a novel, conserved gene family, Snowball (snb), termed after the unique, differentiation-less fruiting body morphology of snb1 knockout strains in the model mushroom Coprinopsis cinerea. snb is a gene of unknown function that is highly conserved among agaricomycetes and encodes a protein of unknown function. A comparative transcriptomic analysis of the early developmental stages of differentiated wild-type and non-differentiated mutant fruiting bodies revealed conserved differentially expressed genes which may be related to tissue differentiation and developmental patterning fruiting body development.

RecurrenceOnline: an online analysis tool to determine breast cancer recurrence and hormone receptor status using microarray data.

In the last decades, several gene expression-based predictors of clinical behavior were developed for breast cancer. A common feature of these is the use of multiple genes to predict hormone receptor status and the probability of tumor recurrence, survival or response to chemotherapy. We developed an online analysis tool to compute ER and HER2 status, Oncotype DX 21-gene recurrence score and an independent recurrence risk classification using gene expression data obtained by interrogation of Affymetrix microarray profiles. We implemented rigorous quality control algorithms to promptly exclude any biases related to sample processing, hybridization and scanning. After uploading the raw microarray data, the system performs the complete evaluation automatically and provides a report summarizing the results. The system is accessible online at http://www.recurrenceonline.com . We validated the system using data from 2,472 publicly available microarrays. The validation of the prediction of the 21-gene recurrence score was significant in lymph node negative patients (Cox-Mantel, P = 5.6E-16, HR = 0.4, CI = 0.32-0.5). A correct classification was obtained for 88.5% of ER- and 90.5% of ER + tumors (n = 1,894). The prediction of recurrence risk in all patients by using the mean of the independent six strongest genes (P < 1E-16, HR = 2.9, CI = 2.5-3.3), of the four strongest genes in lymph node negative ER positive patients (P < 1E-16, HR = 2.8, CI = 2.2-3.5) and of the three genes in lymph node positive patients (P = 3.2E-9, HR = 2.5, CI = 1.8-3.4) was highly significant. In summary, we integrated available knowledge in one platform to validate currently used predictors and to provide a global tool for the online determination of different prognostic parameters simultaneously using genome-wide microarrays.

Genetic deficiency of Syk protects mice from autoantibody-induced arthritis.

OBJECTIVE: The Syk tyrosine kinase plays an important role in diverse functions in hematopoietic lineage cells. Although previous in vitro and pharmacologic analyses suggested Syk to be a possible player in the development of autoimmune arthritis, no in vivo genetic studies addressing that issue have yet been reported. The aim of the present study was to test whether genetic deficiency of Syk affects autoantibody-induced experimental arthritis in the K/BxN serum-transfer model. METHODS: Syk(-/-) bone marrow chimeras carrying a Syk-deficient hematopoietic system were generated by transplanting Syk(-/-) fetal liver cells into lethally irradiated wild-type recipients. After complete repopulation of the hematopoietic compartment, autoantibody-mediated arthritis was induced by injection of arthritogenic K/BxN serum. Arthritis development was monitored by macroscopic and microscopic observation of the ankle joints, micro-computed tomography of bone morphology, as well as a joint function assay. RESULTS: Genetic deficiency of Syk in the hematopoietic compartment completely blocked the development of all macroscopic and microscopic signs of arthritis. The Syk(-/-) mutation also prevented the appearance of periarticular bone erosions. Finally, Syk(-/-) bone marrow chimeras were completely protected from arthritis-induced loss of articular function. CONCLUSION: Our results indicate that Syk is critically involved in the development of all clinically relevant aspects of autoantibody-mediated K/BxN serum-transfer arthritis in experimental mice. These results provide the first in vivo genetic evidence of the role of Syk in the development of autoimmune arthritis.

Contours of citizen science: a vignette study.

Citizen science has expanded rapidly over the past decades. Yet, defining citizen science and its boundaries remained a challenge, and this is reflected in the literature-for example in the proliferation of typologies and definitions. There is a need for identifying areas of agreement and disagreement within the citizen science practitioners community on what should be considered as citizen science activity. This paper describes the development and results of a survey that examined this issue, through the use of vignettes-short case descriptions that describe an activity, while asking the respondents to rate the activity on a scale from 'not citizen science' (0%) to 'citizen science' (100%). The survey included 50 vignettes, of which five were developed as clear cases of not-citizen science activities, five as widely accepted citizen science activities and the others addressing 10 factors and 61 sub-factors that can lead to controversy about an activity. The survey has attracted 333 respondents, who provided over 5100 ratings. The analysis demonstrates the plurality of understanding of what citizen science is and calls for an open understanding of what activities are included in the field.

The Push, Pull, and Enabling Capacities Necessary for Legume Grain Inclusion into Sustainable Agri-Food Systems and Healthy Diets.

Legume grains are traditional crops that have been modernized as processed foods and animal protein alternatives in recent years. This modernization has largely been fueled by new technological developments driven by increased consumer demands for plant protein and gluten-free options. However, consumers must be mindful that legumes have other nutritional attributes besides protein that help achieve healthier diets, and recent evidence suggests that consuming 100 g of legume grains per day would promote nutrient-dense diets and could be a target level to harmonize international strategies for recommended daily allowances. The nutritional richness of legumes has been associated with a decrease in numerous disease risk factors and, given their long shelf life, legumes are excellent choices to combat food waste and may have a role to play in countries where cold storage is often a limitation in food preservation. Besides their inclusion in global diets, legumes should be included in sustainable cropping systems. The high number of edible species and cultivars available render them excellent contributors to biodiverse food and feed systems. Legume cultivation allows reducing environmental impacts by means of the ability of legumes to fix atmospheric nitrogen via a symbiosis with nitrogen-fixing bacteria, making them natural fertilizers. Still, despite the well-known health, nutritional, and environmental benefits, legumes are underrepresented in global agri-food systems. Efforts must be made to make legumes more attractive to the farmer, the industry, and ultimately the consumer, and for this proper local, regional, national, and global policy frameworks must be in place. Here, the local scenario of legumes is showcased, and the most relevant push, pull, and enabling capacities required to achieve sustainable diversified agri-food systems with legumes are discussed.

Cell and tissue manipulation with ultrashort infrared laser pulses in light-sheet microscopy.

Three-dimensional live imaging has become an indispensable technique in the fields of cell, developmental and neural biology. Precise spatio-temporal manipulation of biological entities is often required for a deeper functional understanding of the underlying biological process. Here we present a home-built integrated framework and optical design that combines three-dimensional light-sheet imaging over time with precise spatio-temporal optical manipulations induced by short infrared laser pulses. We demonstrate their potential for sub-cellular ablation of neurons and nuclei, tissue cauterization and optogenetics by using the Drosophila melanogaster and zebrafish model systems.