Treatment of BRAF‐mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient ...benefit. We use live‐cell imaging, single‐cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug. Drug‐adapted cells up‐regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly. This phenotype is transiently stable, reverting to the drug‐naïve state within 9 days of drug withdrawal. Transcriptional profiling of cell lines and human tumors implicates a c‐Jun/ECM/FAK/Src cascade in de‐differentiation in about one‐third of cell lines studied; drug‐induced changes in c‐Jun and NGFR levels are also observed in xenograft and human tumors. Drugs targeting the c‐Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (Emax) of RAF/MEK kinase inhibitors by promoting cell killing. Thus, analysis of reversible drug resistance at a single‐cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.
Synopsis
Responses of BRAFV600E melanoma cells to vemurafenib were studied at the single‐cell level using live‐cell imaging and by transcriptional and biochemical profiling to uncover a slowly dividing, de‐differentiated cell state associated with drug resistance but inhibitable by drug combinations.
Cell‐to‐cell variability in BRAFV600E melanomas generates drug‐tolerant subpopulations.
The drug‐tolerant, slowly dividing NFGRHigh state is transiently heritable.
Drugs against a proposed c‐Jun/ECM/FAK/Src cascade block acquisition of this phenotype.
The NGFRHigh drug‐tolerant state is also blocked by BET inhibitors in vitro and in vivo.
Drugs that block adaptation by cell subpopulations increase cell killing by RAF/MEK inhibitors.
LINCS‐compliant data and methods are freely available to enhance reproducibility.
Responses of BRAFV600E melanoma cells to vemurafenib were studied at the single‐cell level using live‐cell imaging and by transcriptional and biochemical profiling to uncover a slowly dividing, de‐differentiated cell state associated with drug resistance but inhibitable by drug combinations.
Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to ...correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications.
Pyramid Stereo Matching Network Chang, Jia-Ren; Chen, Yong-Sheng
2018 IEEE/CVF Conference on Computer Vision and Pattern Recognition
Conference Proceeding
Odprti dostop
Recent work has shown that depth estimation from a stereo pair of images can be formulated as a supervised learning task to be resolved with convolutional neural networks (CNNs). However, current ...architectures rely on patch-based Siamese networks, lacking the means to exploit context information for finding correspondence in ill-posed regions. To tackle this problem, we propose PSMNet, a pyramid stereo matching network consisting of two main modules: spatial pyramid pooling and 3D CNN. The spatial pyramid pooling module takes advantage of the capacity of global context information by aggregating context in different scales and locations to form a cost volume. The 3D CNN learns to regularize cost volume using stacked multiple hourglass networks in conjunction with intermediate supervision. The proposed approach was evaluated on several benchmark datasets. Our method ranked first in the KITTI 2012 and 2015 leaderboards before March 18, 2018. The codes of PSMNet are available at: https://github.com/JiaRenChang/PSMNet.
Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to ...formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.
Here, a completely new readout technique, so-called “Click” SERS, has been developed based on Raman scattered light splice derived from nanoparticle (NP) assemblies. The single and narrow (1–2 nm) ...emission originating from triple bond-containing reporters undergoes dynamic combinatorial output, by means of controllable splice of SERS-active NPs analogous to small molecule units in click chemistry. Entirely different to conventional “sole code related to sole target” readout protocol, the intuitional, predictable and uniquely identifiable “Click” SERS is relies on the number rather than the intensity of combinatorial emissions. By this technique, 10-plex synchronous biomarkers detection under a single scan, and accurate cellular imaging under double exposure have been achieved. “Click” SERS demonstrated multiple single band Raman scattering could be an authentic optical analysis method in biomedicine.
The physiochemical determinants of drug-target interactions in the microenvironment of the cell are complex and generally not defined by simple diffusion and intrinsic chemical reactivity. ...Non-specific interactions of drugs and macromolecules in cells are rarely considered formally in assessing pharmacodynamics. Here, we demonstrate that non-specific interactions lead to very slow incorporation kinetics of DNA binding drugs. We observe a rate of drug incorporation in cell nuclei three orders of magnitude slower than in vitro due to anomalous drug diffusion within cells. This slow diffusion, however, has an advantageous consequence: it leads to virtually irreversible binding of the drug to specific DNA targets in cells. We show that non-specific interactions drive slow drug diffusion manifesting as slow reaction front propagation. We study the effect of non-specific interactions in different cellular compartments by permeabilization of plasma and nuclear membranes in order to pinpoint differential compartment effects on variability in intracellular drug kinetics. These results provide the basis for a comprehensive model of the determinants of intracellular diffusion of small-molecule drugs, their target-seeking trajectories, and the consequences of these processes on the apparent kinetics of drug-target interactions.
The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic ...immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high-dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics.
Organic single‐crystalline semiconductors with long‐range periodic order have attracted much attention for potential applications in electronic and optoelectronic devices due to their high carrier ...mobility, highly thermal stability, and low impurity content. Molecular doping has been proposed as a valuable strategy for improving the performance of organic semiconductors and semiconductor‐based devices. However, a fundamental understanding of the inherent doping mechanism is still a key challenge impeding its practical application. In this study, solid evidence for the “perfect” substitutional doping mechanism of the stacking mode between the guest and host molecules in organic single‐crystalline semiconductors using polarized photoluminescence spectrum measurements and first‐principles calculations is provided. The molecular host–guest doping is further exploited for efficient color‐tunable and even white organic single‐crystal‐based light‐emitting devices by controlling the doping concentration. The clarification of the molecular doping mechanism in organic single‐crystalline semiconductor host–guest system paves the way for their practical application in high‐performance electronic and optoelectronic devices.
A fundamental understanding of the molecular doping mechanism in organic single‐crystalline semiconductors is provided using polarized PL spectrum measurement and first‐principles calculations. Color‐tunable and white single‐crystal‐based organic light‐emitting devices with high performance are realized, which have important implications for the practical application of organic single‐crystalline semiconductors.
Efficient photocatalytic conversion of CO2 into energy-rich chemicals is of great significance for both environmental conservation and alleviating the energy crisis. However, convenient synthesis of ...low-cost, durable and eco-friendly photocatalysts with a novel morphology or structure for highly selective photocatalytic CO2 reduction remains a challenge. Herein, Co3O4 hierarchical nanosheets were synthesized by calcination of novel cobalt metal–organic framework (MOF) nanosheets prepared by a facile oil bath method. In such Co MOF nanosheets, 1,4-naphthalenedicarboxylic acid was chosen as the organic linker, rather than the commonly used 2-methylimidazole for ZIF-67. After thermal treatment in air, the obtained Co3O4 inherited the 2D morphology of its MOF template and evolved into hierarchical nanosheets which were composed of small nanoparticles. Benefiting from the large surface area, abundant mesoporous structure and good capability towards the separation and transfer of photo-generated charge carriers induced by less internal oxygen vacancies, the Co3O4 hierarchical nanosheets showed a CO generation rate of 39.70 μmol h−1 in visible-light photocatalytic CO2 reduction, which was superior to that of Co3O4 nanoparticles and commercial Co3O4. What's more, a CO selectivity of 77.3% was achieved, which is among the highest of cobalt-based spinel oxide photocatalysts for CO2 conversion.
In the management of light non–aqueous phase liquid (LNAPL)‐contaminated ground, numerical simulation is widely used to analyze LNAPL flow in the unsaturated soil (vadose) zone. Porosity effects on ...the hydraulic properties of unsaturated soils are highly simplified in existing mathematical models. Some important features, such as the nonlinear relation between porosity and permeability/displacement pressure, cannot be well captured. To address this problem, a new mathematical model was developed in this study, considering porosity effects on hydraulic properties of soils, including the retention behavior and permeability function of LNAPL and water. The newly developed model was implemented in MATLAB using the finite difference method and then verified by the results of a centrifuge test. Then, parametric studies were conducted to investigate the flow of LNAPL upon an active leakage at the ground surface. Based on the computed results, the influence of several factors, such as porosity magnitude, porosity distribution, and soil layering, was revealed. In particular, an increase in the porosity leads to a significant increase in the volume of LNAPL leaked into the ground, the vertical front depth, and the area of contaminated ground. This is mainly because the porosity affects not only the intrinsic permeability but also the relative permeability because (a) the intrinsic permeability of soils is larger at a higher porosity (b) when the porosity is higher, the equilibrium water saturation at a given capillary pressure is smaller. Consequently, LNAPL can achieve a larger degree of saturation and higher relative permeability.
Core Ideas
A new theoretical model is developed, considering porosity effects on soil hydraulic properties.
The model capability is verified using the results of a centrifuge test.
The results of parametric studies have revealed the importance of porosity effects.
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