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Investigates whether cultured bovine oviduct epithelial cells and fibroblasts synthesize leukemia inhibitory factor (LIF). Measurement of the LIF production in the conditioned medium of oviduct epithelial cells and fibroblasts using LIF enzyme-linked immunosorbent assay; LIF reverse transcriptase-polymerase chain reaction in extracts of RNA from oviduct epithelial/fibroblast cells.

Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7× increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca 2+ responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca 2+ concentration using fluorescence lifetime imaging microscopy (FLIM)., (© 2024. The Author(s).)

Recurrent somatic mutations in the BRG1/BRM-associated factor (BAF) chromatin remodeling complex subunit ARID1A occur frequently in advanced urothelial, endometrial, and ovarian clear cell carcinomas, creating an alternative chromatin state that may be exploited therapeutically. The histone methyltransferase EZH2 has been previously identified as targetable vulnerability in the context of ARID1A mutations. In this study, we describe the discovery of tulmimetostat, an orally available, clinical stage EZH2 inhibitor, and it elucidates the aspects of its application potential in ARID1A mutant tumors. Tulmimetostat administration achieved efficacy in multiple ARID1A mutant bladder, ovarian, and endometrial tumor models and improved cisplatin response in chemotherapy-resistant models. Consistent with its comprehensive and durable level of target coverage, tulmimetostat demonstrated greater efficacy than other PRC2-targeted inhibitors at comparable or lower exposures in a bladder cancer xenograft mouse model. Tulmimetostat mediated extensive changes in gene expression, in addition to a profound reduction in global H3K27me3 levels in tumors. Phase I clinical pharmacokinetic and pharmacodynamic data indicated that tulmimetostat exhibits durable exposure and profound target engagement. Importantly, a tulmimetostat controlled gene expression signature identified in whole blood from a cohort of 32 patients with cancer correlated with tulmimetostat exposure, representing a pharmacodynamic marker for the assessment of target coverage for PRC2-targeted agents in the clinic. Collectively, these data suggest that tulmimetostat has the potential to achieve clinical benefit in solid tumors as a monotherapy but also in combination with chemotherapeutic agents, and may be beneficial in various indications with recurrent ARID1A mutations. Significance: The EZH2 inhibitor tulmimetostat achieves comprehensive target inhibition in ARID1A mutant solid tumor models and cancer patients that can be assessed with a pharmacodynamic gene signature in peripheral blood., (©2024 The Authors; Published by the American Association for Cancer Research.)

Genetically encoded fluorescent calcium indicators have revolutionized neuroscience and other biological fields by allowing cellular-resolution recording of physiology during behavior. However, we currently lack bright, genetically targetable indicators in the near infrared that can be used in animals. Here, we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains that can be genetically targeted to specific cell populations. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several dye-ligands that efficiently label the central nervous system in animals. When bound to a near-infrared dye-ligand, WHaloCaMP1a is more than twice as bright as jGCaMP8s, and shows a 7× increase in fluorescence intensity and a 2.1 ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a with near-infrared fluorescence emission to image Ca 2+ responses in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae, and to quantitate calcium concentration using fluorescence lifetime imaging microscopy (FLIM)., Competing Interests: Competing interests H. F. and E. R. S. have filed patent applications on tryptophan-containing chemigenetic fluorescent indicators. L. D. L. and J. B. G have filed patents and patent applications on fluorinated and azetidine-containing rhodamines.

We present a method to automatically identify and track nuclei in time-lapse microscopy recordings of entire developing embryos. The method combines deep learning and global optimization. On a mouse dataset, it reconstructs 75.8% of cell lineages spanning 1 h, as compared to 31.8% for the competing method. Our approach improves understanding of where and when cell fate decisions are made in developing embryos, tissues, and organs., (© 2022. The Author(s).)

Background: Transcranial Doppler ultrasound (TCD) is a frequently used method to monitor brain perfusion during and following carotid endarterectomy (CEA). Our aim was to define the normally occurring changes of intracranial hemodynamics in patients undergoing CEA measuring recently developed TCD parameters., Methods: A retrospective, single-center cohort study was performed. Patients undergoing CEA were evaluated pre- and postoperatively from day 0 to day 3 measuring middle cerebral artery flow velocity (MCAFV). The following parameters were analyzed: the first systolic peak (Sys1), the second systolic peak (Sys2) and diastolic flow velocity at a fixed time after heartbeat onset (Dias@560). These parameters linearly decrease with age and were, therefore, transformed to Z-scores., Results: Three hundred eighteen patients were included with a mean age of 70.8 years. Most patients were male (71%). Compared to preoperatively, the Z-scores of Sys1 and Sys2 were larger on postoperative day 3: +1.12 standard deviation (SD) or 16.0 cm/s (CI: 0.93 to 1.32; P<0.001) and +0.55 SD or 7.8 cm/s (CI: 0.35 to 0.74; P<0.001), respectively. The Z-score for Dias@560 was smaller than preoperatively: -0.23 SD or -1.9 cm/s (CI: -0.41 to -0.05, P=0.015)., Conclusions: Under normal circumstances Sys1 profits more from CEA than Sys2, whilst diastolic flow velocity decreases. This indicates a return to normal arteriolar vascular resistance. Carefully describing normal changes in MCAFV, may in future enable discrimination of abnormalities, such as hyperperfusion syndrome.

Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1 μM to 10 mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed ∼3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals., Competing Interests: Declaration of interests L.L.L., J.S.M., and R.T.L. are holders of US Patent US9939437B2, which covers iGlucoSnFR. The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mammalian heart is derived from multiple cell lineages; however, our understanding of when and how the diverse cardiac cell types arise is limited. We mapped the origin of the embryonic mouse heart at single-cell resolution using a combination of transcriptomic, imaging, and genetic lineage labeling approaches. This mapping provided a transcriptional and anatomic definition of cardiac progenitor types. Furthermore, it revealed a cardiac progenitor pool that is anatomically and transcriptionally distinct from currently known cardiac progenitors. Besides contributing to cardiomyocytes, these cells also represent the earliest progenitor of the epicardium, a source of trophic factors and cells during cardiac development and injury. This study provides detailed insights into the formation of early cardiac cell types, with particular relevance to the development of cell-based cardiac regenerative therapies., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

An important question in early neural development is the origin of stochastic nuclear movement between apical and basal surfaces of neuroepithelia during interkinetic nuclear migration. Tracking of nuclear subpopulations has shown evidence of diffusion - mean squared displacements growing linearly in time - and suggested crowding from cell division at the apical surface drives basalward motion. Yet, this hypothesis has not yet been tested, and the forces involved not quantified. We employ long-term, rapid light-sheet and two-photon imaging of early zebrafish retinogenesis to track entire populations of nuclei within the tissue. The time-varying concentration profiles show clear evidence of crowding as nuclei reach close-packing and are quantitatively described by a nonlinear diffusion model. Considerations of nuclear motion constrained inside the enveloping cell membrane show that concentration-dependent stochastic forces inside cells, compatible in magnitude to those found in cytoskeletal transport, can explain the observed magnitude of the diffusion constant., Competing Interests: AA, AH, YW, SB, PK, WH No competing interests declared, RG Reviewing editor, eLife, (© 2020, Azizi et al.)

Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system., Competing Interests: Declaration of Interests H.R.U. is a co-inventor on patent applications covering the CUBIC reagents and a cofounder of CUBICStars Inc. P.J.K. is a co-inventor on patents and patent applications covering multi-view and adaptive light-sheet microscopy. P.O. is a cofounder of Certerra Inc. and Certego Therapeutics Inc., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.

The ability to visualize and quantitatively measure dynamic biological processes in vivo and at high spatiotemporal resolution is of fundamental importance to experimental investigations in developmental biology. Light-sheet microscopy is particularly well suited to providing such data, since it offers exceptionally high imaging speed and good spatial resolution while minimizing light-induced damage to the specimen. We review core principles and recent advances in light-sheet microscopy, with a focus on concepts and implementations relevant for applications in developmental biology. We discuss how light-sheet microcopy has helped advance our understanding of developmental processes from single-molecule to whole-organism studies, assess the potential for synergies with other state-of-the-art technologies, and introduce methods for computational image and data analysis. Finally, we explore the future trajectory of light-sheet microscopy, discuss key efforts to disseminate new light-sheet technology, and identify exciting opportunities for further advances.

Animal survival requires a functioning nervous system to develop during embryogenesis. Newborn neurons must assemble into circuits producing activity patterns capable of instructing behaviors. Elucidating how this process is coordinated requires new methods that follow maturation and activity of all cells across a developing circuit. We present an imaging method for comprehensively tracking neuron lineages, movements, molecular identities, and activity in the entire developing zebrafish spinal cord, from neurogenesis until the emergence of patterned activity instructing the earliest spontaneous motor behavior. We found that motoneurons are active first and form local patterned ensembles with neighboring neurons. These ensembles merge, synchronize globally after reaching a threshold size, and finally recruit commissural interneurons to orchestrate the left-right alternating patterns important for locomotion in vertebrates. Individual neurons undergo functional maturation stereotypically based on their birth time and anatomical origin. Our study provides a general strategy for reconstructing how functioning circuits emerge during embryogenesis. VIDEO ABSTRACT., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Light-sheet imaging of cleared and expanded samples creates terabyte-sized datasets that consist of many unaligned three-dimensional image tiles, which must be reconstructed before analysis. We developed the BigStitcher software to address this challenge. BigStitcher enables interactive visualization, fast and precise alignment, spatially resolved quality estimation, real-time fusion and deconvolution of dual-illumination, multitile, multiview datasets. The software also compensates for optical effects, thereby improving accuracy and enabling subsequent biological analysis.

The thirteen nuclear cleavages that give rise to the Drosophila blastoderm are some of the fastest known cell cycles [1]. Surprisingly, the fertilized egg is provided with at most one-third of the dNTPs needed to complete the thirteen rounds of DNA replication [2]. The rest must be synthesized by the embryo, concurrent with cleavage divisions. What is the reason for the limited supply of DNA building blocks? We propose that frugal control of dNTP synthesis contributes to the well-characterized deceleration of the cleavage cycles and is needed for robust accumulation of zygotic gene products. In support of this model, we demonstrate that when the levels of dNTPs are abnormally high, nuclear cleavages fail to sufficiently decelerate, the levels of zygotic transcription are dramatically reduced, and the embryo catastrophically fails early in gastrulation. Our work reveals a direct connection between metabolism, the cell cycle, and zygotic transcription., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Whole-brain imaging allows for comprehensive functional mapping of distributed neural pathways, but neuronal perturbation experiments are usually limited to targeting predefined regions or genetically identifiable cell types. To complement whole-brain measures of activity with brain-wide manipulations for testing causal interactions, we introduce a system that uses measured activity patterns to guide optical perturbations of any subset of neurons in the same fictively behaving larval zebrafish. First, a light-sheet microscope collects whole-brain data that are rapidly analyzed by a distributed computing system to generate functional brain maps. On the basis of these maps, the experimenter can then optically ablate neurons and image activity changes across the brain. We applied this method to characterize contributions of behaviorally tuned populations to the optomotor response. We extended the system to optogenetically stimulate arbitrary subsets of neurons during whole-brain imaging. These open-source methods enable delineating the contributions of neurons to brain-wide circuit dynamics and behavior in individual animals.

We describe the implementation and use of an adaptive imaging framework for optimizing spatial resolution and signal strength in a light-sheet microscope. The framework, termed AutoPilot, comprises hardware and software modules for automatically measuring and compensating for mismatches between light-sheet and detection focal planes in living specimens. Our protocol enables researchers to introduce adaptive imaging capabilities in an existing light-sheet microscope or use our SiMView microscope blueprint to set up a new adaptive multiview light-sheet microscope. The protocol describes (i) the mechano-optical implementation of the adaptive imaging hardware, including technical drawings for all custom microscope components; (ii) the algorithms and software library for automated adaptive imaging, including the pseudocode and annotated source code for all software modules; and (iii) the execution of adaptive imaging experiments, as well as the configuration and practical use of the AutoPilot framework. Setup of the adaptive imaging hardware and software takes 1-2 weeks each. Previous experience with light-sheet microscopy and some familiarity with software engineering and building of optical instruments are recommended. Successful implementation of the protocol recovers near diffraction-limited performance in many parts of typical multicellular organisms studied with light-sheet microscopy, such as fruit fly and zebrafish embryos, for which resolution and signal strength are improved two- to fivefold.

The mouse embryo has long been central to the study of mammalian development; however, elucidating the cell behaviors governing gastrulation and the formation of tissues and organs remains a fundamental challenge. A major obstacle is the lack of live imaging and image analysis technologies capable of systematically following cellular dynamics across the developing embryo. We developed a light-sheet microscope that adapts itself to the dramatic changes in size, shape, and optical properties of the post-implantation mouse embryo and captures its development from gastrulation to early organogenesis at the cellular level. We furthermore developed a computational framework for reconstructing long-term cell tracks, cell divisions, dynamic fate maps, and maps of tissue morphogenesis across the entire embryo. By jointly analyzing cellular dynamics in multiple embryos registered in space and time, we built a dynamic atlas of post-implantation mouse development that, together with our microscopy and computational methods, is provided as a resource. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustacean Parhyale hawaiensis with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT). In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic., Competing Interests: CW, JT, TP, ES, BH, LG, SP, SS, PK, PT, AP No competing interests declared, (© 2018, Wolff et al.)

Mechanics plays a key role in the development of higher organisms. However, understanding this relationship is complicated by the difficulty of modeling the link between local forces generated at the subcellular level and deformations observed at the tissue and whole-embryo levels. Here we propose an approach first developed for lipid bilayers and cell membranes, in which force-generation by cytoskeletal elements enters a continuum mechanics formulation for the full system in the form of local changes in preferred curvature. This allows us to express and solve the system using only tissue strains. Locations of preferred curvature are simply related to products of gene expression. A solution, in that context, means relaxing the system's mechanical energy to yield global morphogenetic predictions that accommodate a tendency toward the local preferred curvature, without a need to explicitly model force-generation mechanisms at the molecular level. Our computational framework, which we call SPHARM-MECH, extends a 3D spherical harmonics parameterization known as SPHARM to combine this level of abstraction with a sparse shape representation. The integration of these two principles allows computer simulations to be performed in three dimensions on highly complex shapes, gene expression patterns, and mechanical constraints. We demonstrate our approach by modeling mesoderm invagination in the fruit-fly embryo, where local forces generated by the acto-myosin meshwork in the region of the future mesoderm lead to formation of a ventral tissue fold. The process is accompanied by substantial changes in cell shape and long-range cell movements. Applying SPHARM-MECH to whole-embryo live imaging data acquired with light-sheet microscopy reveals significant correlation between calculated and observed tissue movements. Our analysis predicts the observed cell shape anisotropy on the ventral side of the embryo and suggests an active mechanical role of mesoderm invagination in supporting the onset of germ-band extension., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision. This strategy allowed us to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. Our results demonstrate the versatility of these new dyes in cells, tissues and animals.

Organogenesis and tissue development occur through sequential stepwise processes leading to increased lineage restriction and loss of pluripotency. An exception to this appears in the adult human breast, where rare variant epithelial cells exhibit pluripotency and multilineage differentiation potential when removed from the signals of their native microenvironment. This phenomenon provides a unique opportunity to study mechanisms that lead to cellular reprogramming and lineage plasticity in real time. Here, we show that primary human mammary epithelial cells (HMECs) lose expression of differentiated mammary epithelial markers in a manner dependent on paracrine factors and epigenetic regulation. Furthermore, we demonstrate that HMEC reprogramming is dependent on gene silencing by the DNA methyltransferase DNMT3A and loss of histone transcriptional marks following downregulation of the methyltransferase DOT1L. These results demonstrate that lineage commitment in adult tissues is context dependent and highlight the plasticity of somatic cells when removed from their native tissue microenvironment., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Optimal image quality in light-sheet microscopy requires a perfect overlap between the illuminating light sheet and the focal plane of the detection objective. However, mismatches between the light-sheet and detection planes are common owing to the spatiotemporally varying optical properties of living specimens. Here we present the AutoPilot framework, an automated method for spatiotemporally adaptive imaging that integrates (i) a multi-view light-sheet microscope capable of digitally translating and rotating light-sheet and detection planes in three dimensions and (ii) a computational method that continuously optimizes spatial resolution across the specimen volume in real time. We demonstrate long-term adaptive imaging of entire developing zebrafish (Danio rerio) and Drosophila melanogaster embryos and perform adaptive whole-brain functional imaging in larval zebrafish. Our method improves spatial resolution and signal strength two to five-fold, recovers cellular and sub-cellular structures in many regions that are not resolved by non-adaptive imaging, adapts to spatiotemporal dynamics of genetically encoded fluorescent markers and robustly optimizes imaging performance during large-scale morphogenetic changes in living organisms.

A custom-built objective lens called the Mesolens allows relatively large biological specimens to be imaged with cellular resolution., Competing Interests: The authors declare that no competing interests exist.

The precise positioning of organ progenitor cells constitutes an essential, yet poorly understood step during organogenesis. Using primordial germ cells that participate in gonad formation, we present the developmental mechanisms maintaining a motile progenitor cell population at the site where the organ develops. Employing high-resolution live-cell microscopy, we find that repulsive cues coupled with physical barriers confine the cells to the correct bilateral positions. This analysis revealed that cell polarity changes on interaction with the physical barrier and that the establishment of compact clusters involves increased cell-cell interaction time. Using particle-based simulations, we demonstrate the role of reflecting barriers, from which cells turn away on contact, and the importance of proper cell-cell adhesion level for maintaining the tight cell clusters and their correct positioning at the target region. The combination of these developmental and cellular mechanisms prevents organ fusion, controls organ positioning and is thus critical for its proper function.

Animal development is a complex and dynamic process orchestrated by exquisitely timed cell lineage commitment, divisions, migration, and morphological changes at the single-cell level. In the past decade, extensive genetic, stem cell, and genomic studies provided crucial insights into molecular underpinnings and the functional importance of genetic pathways governing various cellular differentiation processes. However, it is still largely unknown how the precise coordination of these pathways is achieved at the whole-organism level and how the highly regulated spatiotemporal choreography of development is established in turn. Here, we discuss the latest technological advances in imaging and single-cell genomics that hold great promise for advancing our understanding of this intricate process. We propose an integrated approach that combines such methods to quantitatively decipher in vivo cellular dynamic behaviors and their underlying molecular mechanisms at the systems level with single-cell, single-molecule resolution., (Copyright © 2016 Elsevier Inc. All rights reserved.)

CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.

We present the Real-time Accurate Cell-shape Extractor (RACE), a high-throughput image analysis framework for automated three-dimensional cell segmentation in large-scale images. RACE is 55-330 times faster and 2-5 times more accurate than state-of-the-art methods. We demonstrate the generality of RACE by extracting cell-shape information from entire Drosophila, zebrafish, and mouse embryos imaged with confocal and light-sheet microscopes. Using RACE, we automatically reconstructed cellular-resolution tissue anisotropy maps across developing Drosophila embryos and quantified differences in cell-shape dynamics in wild-type and mutant embryos. We furthermore integrated RACE with our framework for automated cell lineaging and performed joint segmentation and cell tracking in entire Drosophila embryos. RACE processed these terabyte-sized datasets on a single computer within 1.4 days. RACE is easy to use, as it requires adjustment of only three parameters, takes full advantage of state-of-the-art multi-core processors and graphics cards, and is available as open-source software for Windows, Linux, and Mac OS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. Here, we demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Selective targeting of multiple myeloma cell lines through CBP/EP300 bromodomain inhibition is the result of direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4, which is essential for the viability of myeloma cells, and the concomitant repression of the IRF4 target gene c-MYC. Ectopic expression of either IRF4 or MYC antagonizes the phenotypic and transcriptional effects of CBP/EP300 bromodomain inhibition, highlighting the IRF4/MYC axis as a key component of its mechanism of action. These findings suggest that CBP/EP300 bromodomain inhibition represents a viable therapeutic strategy for targeting multiple myeloma and other lymphoid malignancies dependent on the IRF4 network.

Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)