Urease-powered nanobots for radionuclide bladder cancer therapy.

Bladder cancer treatment via intravesical drug administration achieves reasonable survival rates but suffers from low therapeutic efficacy. To address the latter, self-propelled nanoparticles or nanobots have been proposed, taking advantage of their enhanced diffusion and mixing capabilities in urine when compared with conventional drugs or passive nanoparticles. However, the translational capabilities of nanobots in treating bladder cancer are underexplored. Here, we tested radiolabelled mesoporous silica-based urease-powered nanobots in an orthotopic mouse model of bladder cancer. In vivo and ex vivo results demonstrated enhanced nanobot accumulation at the tumour site, with an eightfold increase revealed by positron emission tomography in vivo. Label-free optical contrast based on polarization-dependent scattered light-sheet microscopy of cleared bladders confirmed tumour penetration by nanobots ex vivo. Treating tumour-bearing mice with intravesically administered radio-iodinated nanobots for radionuclide therapy resulted in a tumour size reduction of about 90%, positioning nanobots as efficient delivery nanosystems for bladder cancer therapy.

Neurogenesis redirects ß-catenin from adherens junctions to the nucleus to promote axonal growth.

Here, we show that, in the developing spinal cord, after the early Wnt-mediated Tcf transcription activation that confers dorsal identity to neural stem cells, neurogenesis redirects ß-catenin from the adherens junctions to the nucleus to stimulate Tcf-dependent transcription in a Wnt-independent manner. This new ß-catenin activity regulates genes implicated in several aspects of contralateral axon growth, including axon guidance and adhesion. Using live imaging of ex-vivo chick neural tube, we showed that the nuclear accumulation of ß-catenin and the rise in Tcf-dependent transcription both initiate before the dismantling of the adherens junctions and remain during the axon elongation process. Notably, we demonstrated that ß-catenin activity in post-mitotic cells depends on TCF7L2 and is central to spinal commissural axon growth. Together, our results reveal Wnt-independent Tcf/ß-catenin regulation of genes that control the growth and guidance of commissural axons in chick spinal cord.

The P4-ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast.

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.

Metastatic recurrence in colorectal cancer arises from residual EMP1+ cells.

Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.

The LEGO® brick road to open science and biotechnology.

LEGO® is a brand of toys that have entertained generations of children. Beyond amusement, LEGO® bricks also constitute a building ecosystem of their own that creators from the general public, as well as scientists and engineers, can use to design and assemble devices for all purposes, including scientific research and biotechnology. We describe several of these constructions to highlight the construction properties of LEGO® and their advantages, caveats, and impact in biotechnology. We also discuss how this emerging trend in LEGO® building pairs with a growing interest in open-access and frugal science which aims to provide access to technology to all scientists regardless of financial wealth and technological prowess.

Highlights from the 2016-2020 NEUBIAS training schools for Bioimage Analysts: a success story and key asset for analysts and life scientists.

NEUBIAS, the European Network of Bioimage Analysts, was created in 2016 with the goal of improving the communication and the knowledge transfer among the various stakeholders involved in the acquisition, processing and analysis of biological image data, and to promote the establishment and recognition of the profession of Bioimage Analyst. One of the most successful initiatives of the NEUBIAS programme was its series of 15 training schools, which trained over 400 new Bioimage Analysts, coming from over 40 countries. Here we outline the rationale behind the innovative three-level program of the schools, the curriculum, the trainer recruitment and turnover strategy, the outcomes for the community and the career path of analysts, including some success stories. We discuss the future of the materials created during this programme and some of the new initiatives emanating from the community of NEUBIAS-trained analysts, such as the NEUBIAS Academy. Overall, we elaborate on how this training programme played a key role in collectively leveraging Bioimaging and Life Science research by bringing the latest innovations into structured, frequent and intensive training activities, and on why we believe this should become a model to further develop in Life Sciences.

Bioimage analysis workflows: community resources to navigate through a complex ecosystem.

Workflows are the keystone of bioimage analysis, and the NEUBIAS (Network of European BioImage AnalystS) community is trying to gather the actors of this field and organize the information around them.  One of its most recent outputs is the opening of the F1000Research NEUBIAS gateway, whose main objective is to offer a channel of publication for bioimage analysis workflows and associated resources. In this paper we want to express some personal opinions and recommendations related to finding, handling and developing bioimage analysis workflows.  The emergence of "big data" in bioimaging and resource-intensive analysis algorithms make local data storage and computing solutions a limiting factor. At the same time, the need for data sharing with collaborators and a general shift towards remote work, have created new challenges and avenues for the execution and sharing of bioimage analysis workflows. These challenges are to reproducibly run workflows in remote environments, in particular when their components come from different software packages, but also to document them and link their parameters and results by following the FAIR principles (Findable, Accessible, Interoperable, Reusable) to foster open and reproducible science. In this opinion paper, we focus on giving some directions to the reader to tackle these challenges and navigate through this complex ecosystem, in order to find and use workflows, and to compare workflows addressing the same problem. We also discuss tools to run workflows in the cloud and on High Performance Computing resources, and suggest ways to make these workflows FAIR.

AutoScanJ: A Suite of ImageJ Scripts for Intelligent Microscopy.

We developed AutoscanJ, a suite of ImageJ scripts enabling to image targets of interest by automatically driving a motorized microscope at the corresponding locations. For live samples, our software can sequentially detect biological events from their onset and further image them at high resolution, an action that would be impractical by user operation. For fixed samples, the software can dramatically reduce the amount of data acquired and the acquisition duration in situations where statistically few targets of interest are observed per field of view. AutoScanJ is compatible with motorized fluorescence microscopes controlled by Leica LAS AF/X or Micro-Manager. The software is straightforward to set up and new custom image analysis workflows to detect targets of interest can be simply implemented and shared with minimal efforts as independent ImageJ macro functions. We illustrate five different application scenarios with the system ranging from samples fixed on micropatterned surfaces to live cells undergoing several rounds of division. The target detection functions for these applications are provided and can be used as a starting point and a source of inspiration for new applications. Overall, AutoScanJ helps to optimize microscope usage by autonomous operation, and it opens up new experimental avenues by enabling the real-time detection and selective imaging of transient events in live microscopy.

The NEUBIAS Gateway: a hub for bioimage analysis methods and materials.

We introduce the NEUBIAS Gateway, a new platform for publishing materials related to bioimage analysis, an interdisciplinary field bridging computer science and life sciences. This emerging field has been lacking a central place to share the efforts of the growing group of scientists addressing biological questions using image data. The Gateway welcomes a wide range of publication formats including articles, reviews, reports and training materials. We hope the Gateway further supports this important field to grow and helps more biologists and computational scientists learn about and contribute to these efforts.

From whole-organ imaging to in-silico blood flow modeling: A new multi-scale network analysis for revisiting tissue functional anatomy.

We present a multi-disciplinary image-based blood flow perfusion modeling of a whole organ vascular network for analyzing both its structural and functional properties. We show how the use of Light-Sheet Fluorescence Microscopy (LSFM) permits whole-organ micro-vascular imaging, analysis and modelling. By using adapted image post-treatment workflow, we could segment, vectorize and reconstruct the entire micro-vascular network composed of 1.7 million vessels, from the tissue-scale, inside a ∼ 25 × 5 × 1 = 125mm3 volume of the mouse fat pad, hundreds of times larger than previous studies, down to the cellular scale at micron resolution, with the entire blood perfusion modeled. Adapted network analysis revealed the structural and functional organization of meso-scale tissue as strongly connected communities of vessels. These communities share a distinct heterogeneous core region and a more homogeneous peripheral region, consistently with known biological functions of fat tissue. Graph clustering analysis also revealed two distinct robust meso-scale typical sizes (from 10 to several hundred times the cellular size), revealing, for the first time, strongly connected functional vascular communities. These community networks support heterogeneous micro-environments. This work provides the proof of concept that in-silico all-tissue perfusion modeling can reveal new structural and functional exchanges between micro-regions in tissues, found from community clusters in the vascular graph.

LOBSTER: an environment to design bioimage analysis workflows for large and complex fluorescence microscopy data.

SUMMARY: Open source software such as ImageJ and CellProfiler greatly simplified the quantitative analysis of microscopy images but their applicability is limited by the size, dimensionality and complexity of the images under study. In contrast, software optimized for the needs of specific research projects can overcome these limitations, but they may be harder to find, set up and customize to different needs. Overall, the analysis of large, complex, microscopy images is hence still a critical bottleneck for many Life Scientists. We introduce LOBSTER (Little Objects Segmentation and Tracking Environment), an environment designed to help scientists design and customize image analysis workflows to accurately characterize biological objects from a broad range of fluorescence microscopy images, including large images exceeding workstation main memory. LOBSTER comes with a starting set of over 75 sample image analysis workflows and associated images stemming from state-of-the-art image-based research projects. AVAILABILITY AND IMPLEMENTATION: LOBSTER requires MATLAB (version ≥ 2015a), MATLAB Image processing toolbox, and MATLAB statistics and machine learning toolbox. Code source, online tutorials, video demonstrations, documentation and sample images are freely available from: https://sebastients.github.io. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MosaicExplorerJ: Interactive stitching of terabyte-size tiled datasets from lightsheet microscopy.

We introduce MosaicExplorerJ, an ImageJ macro to stitch 3D tiles from terabyte-size microscopy datasets. As opposed to existing software, stitching does not require any prior information on the actual positions of the tiles, sample fiducials, or conversion of raw TIFF images, and the stitched images can be explored instantly. MosaicExplorerJ was specifically designed to process lightsheet microscopy datasets from optically cleared samples. It can handle multiple fluorescence channels, dual-side lightsheet illumination and dual-side camera detection.

Sidekick Is a Key Component of Tricellular Adherens Junctions that Acts to Resolve Cell Rearrangements.

Tricellular adherens junctions are points of high tension that are central to the rearrangement of epithelial cells. However, the molecular composition of these junctions is unknown, making it difficult to assess their role in morphogenesis. Here, we show that Sidekick, an immunoglobulin family cell adhesion protein, is highly enriched at tricellular adherens junctions in Drosophila. This localization is modulated by tension, and Sidekick is itself necessary to maintain normal levels of cell bond tension. Loss of Sidekick causes defects in cell and junctional rearrangements in actively remodeling epithelial tissues like the retina and tracheal system. The adaptor proteins Polychaetoid and Canoe are enriched at tricellular adherens junctions in a Sidekick-dependent manner; Sidekick functionally interacts with both proteins and directly binds to Polychaetoid. We suggest that Polychaetoid and Canoe link Sidekick to the actin cytoskeleton to enable tricellular adherens junctions to maintain or transmit cell bond tension during epithelial cell rearrangements.

Reversible silencing of endogenous receptors in intact brain tissue using 2-photon pharmacology.

The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast, and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and 2-photon excitation using pulsed near-infrared lasers to reversibly silence metabotropic glutamate 5 (mGlu5) receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 µm. Thus, 2-photon pharmacology using alloswitch allows investigating mGlu5-dependent processes in wild-type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles.

Regional vulnerability and spreading of hyperphosphorylated tau in seeded mouse brain.

We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool.

Adherens Junction Length during Tissue Contraction Is Controlled by the Mechanosensitive Activity of Actomyosin and Junctional Recycling.

During epithelial contraction, cells generate forces to constrict their surface and, concurrently, fine-tune the length of their adherens junctions to ensure force transmission. While many studies have focused on understanding force generation, little is known on how junctional length is controlled. Here, we show that, during amnioserosa contraction in Drosophila dorsal closure, adherens junctions reduce their length in coordination with the shrinkage of apical cell area, maintaining a nearly constant junctional straightness. We reveal that junctional straightness and integrity depend on the endocytic machinery and on the mechanosensitive activity of the actomyosin cytoskeleton. On one hand, upon junctional stretch and decrease in E-cadherin density, actomyosin relocalizes from the medial area to the junctions, thus maintaining junctional integrity. On the other hand, when junctions have excess material and ruffles, junction removal is enhanced, and high junctional straightness and tension are restored. These two mechanisms control junctional length and integrity during morphogenesis.

Toward quantitative three-dimensional microvascular networks segmentation with multiview light-sheet fluorescence microscopy.

Three-dimensional (3-D) large-scale imaging of microvascular networks is of interest in various areas of biology and medicine related to structural, functional, developmental, and pathological issues. Light-sheet fluorescence microscopy (LSFM) techniques are rapidly spreading and are now on the way to offer operational solutions for large-scale tissue imaging. This contribution describes how reliable vessel segmentation can be handled from LSFM data in very large tissue volumes using a suitable image analysis workflow. Since capillaries are tubular objects of a few microns scale radius, they represent challenging structures to reliably reconstruct without distortion and artifacts. We provide a systematic analysis of multiview deconvolution image processing workflow to control and evaluate the accuracy of the reconstructed vascular network using various low to high level, metrics. We show that even if low-level structural metrics are sensitive to isotropic imaging enhancement provided by a larger number of views, functional high-level metrics, including perfusion permeability, are less sensitive. Hence, combining deconvolution and registration onto a few number of views appears sufficient for a reliable quantitative 3-D vessel segmentation for their possible use for perfusion modeling.

Differential requirements for Tousled-like kinases 1 and 2 in mammalian development.

The regulation of chromatin structure is critical for a wide range of essential cellular processes. The Tousled-like kinases, TLK1 and TLK2, regulate ASF1, a histone H3/H4 chaperone, and likely other substrates, and their activity has been implicated in transcription, DNA replication, DNA repair, RNA interference, cell cycle progression, viral latency, chromosome segregation and mitosis. However, little is known about the functions of TLK activity in vivo or the relative functions of the highly similar TLK1 and TLK2 in any cell type. To begin to address this, we have generated Tlk1- and Tlk2-deficient mice. We found that while TLK1 was dispensable for murine viability, TLK2 loss led to late embryonic lethality because of placental failure. TLK2 was required for normal trophoblast differentiation and the phosphorylation of ASF1 was reduced in placentas lacking TLK2. Conditional bypass of the placental phenotype allowed the generation of apparently healthy Tlk2-deficient mice, while only the depletion of both TLK1 and TLK2 led to extensive genomic instability, indicating that both activities contribute to genome maintenance. Our data identifies a specific role for TLK2 in placental function during mammalian development and suggests that TLK1 and TLK2 have largely redundant roles in genome maintenance.

Tetraspanin 6: a pivotal protein of the multiple vesicular body determining exosome release and lysosomal degradation of amyloid precursor protein fragments.

BACKGROUND: The mechanisms behind Aß-peptide accumulation in non-familial Alzheimer's disease (AD) remain elusive. Proteins of the tetraspanin family modulate Aß production by interacting to γ-secretase. METHODS: We searched for tetraspanins with altered expression in AD brains. The function of the selected tetraspanin was studied in vitro and the physiological relevance of our findings was confirmed in vivo. RESULTS: Tetraspanin-6 (TSPAN6) is increased in AD brains and overexpression in cells exerts paradoxical effects on Amyloid Precursor Protein (APP) metabolism, increasing APP-C-terminal fragments (APP-CTF) and Aß levels at the same time. TSPAN6 affects autophagosome-lysosomal fusion slowing down the degradation of APP-CTF. TSPAN6 recruits also the cytosolic, exosome-forming adaptor syntenin which increases secretion of exosomes that contain APP-CTF. CONCLUSIONS: TSPAN6 is a key player in the bifurcation between lysosomal-dependent degradation and exosome mediated secretion of APP-CTF. This corroborates the central role of the autophagosomal/lysosomal pathway in APP metabolism and shows that TSPAN6 is a crucial player in APP-CTF turnover.