scholarly journals Unraveling mitotic protein networks by 3D multiplexed epitope drug screening

2017 ◽  
Author(s):  
Lorenz Maier ◽  
Stefan Kallenberger ◽  
Katharina Jechow ◽  
Marcel Waschow ◽  
Roland Eils ◽  
...  
Keyword(s):  
Drug Targets ◽  
Network Effects ◽  
Protein Localization ◽  
Three Dimensional ◽  
Immunofluorescent Staining ◽  
Functional Protein ◽  
Spatially Resolved ◽  
Antibody Staining ◽  
Drug Assays ◽  
Automated Technology

Three-dimensional protein localization intricately determines the functional coordination of cellular processes. The complex spatial context of protein landscape has been assessed by multiplexed immunofluorescent staining1–3or mass spectrometry4, applied to 2D cell culture with limited physiological relevance5or tissue sections. Here, we present3D SPECS, an automated technology for3DSpatial characterization ofProteinExpressionChanges by microscopic Screening. This workflow encompasses iterative antibody staining of proteins, high-content imaging, and machine learning based classification of mitotic states. This is followed by mapping of spatial protein localization into a spherical, cellular coordinate system, the basis used for model-based prediction of spatially resolved affinities of various mitotic proteins. As a proof-of-concept, we mapped twelve epitopes in 3D cultured epithelial breast spheroids and investigated the network effects of mitotic cancer drugs with known limited success in clinical trials6–8. Our approach reveals novel insights into spindle fragility and global chromatin stress, and predicts unknown interactions between proteins in specific mitotic pathways.3D SPECS’sability to map potential drug targets by multiplexed immunofluorescence in 3D cell cultured models combined with our automized high content assay will inspire future functional protein expression and drug assays.

scholarly journals Detection of functional protein domains by unbiased genome-wide forward genetic screening

2017 ◽  
Author(s):  
Mareike Herzog ◽  
Fabio Puddu ◽  
Julia Coates ◽  
Nicola Geisler ◽  
Josep V Forment ◽  
...  
Keyword(s):  
Drug Targets ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Gene Knockout ◽  
Mammalian Cell Culture ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Protein Domains ◽  
Functional Protein ◽  
Suppressor Mutations

ABSTRACTGenetic and chemo-genetic interactions have played key roles in elucidating the molecular mechanisms by which certain chemicals perturb cellular functions. Many studies have employed gene knockout collections or gene disruption/depletion strategies to identify routes for evolving resistance to chemical agents. By contrast, searching for point-mutational genetic suppressors that can identify separation- or gain-of-function mutations, has been limited even in simpler, genetically amenable organisms such as yeast, and has not until recently been possible in mammalian cell culture systems. Here, by demonstrating its utility in identifying suppressors of cellular sensitivity to the drugs camptothecin or olaparib, we describe an approach allowing systematic, large-scale detection of spontaneous or chemically-induced suppressor mutations in yeast and in haploid mouse embryonic stem cells in a short timeframe, and with potential applications in essentially any other haploid system. In addition to its utility for molecular biology research, this protocol can be used to identify drug targets and to predict mechanisms leading to drug resistance. Mapping suppressor mutations on the primary sequence or three-dimensional structures of protein suppressor hits provides insights into functionally relevant protein domains, advancing our molecular understanding of protein functions, and potentially helping to improve drug design and applicability.

scholarly journals Designed and biologically active protein lattices

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shih-Ting Wang ◽  
Brian Minevich ◽  
Jianfang Liu ◽  
Honghu Zhang ◽  
Dmytro Nykypanchuk ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Biologically Active ◽  
Double Layers ◽  
Active Protein ◽  
Functional Protein ◽  
Protein Arrays ◽  
Iron Storage ◽  
Nanoscale Systems ◽  
Framework Design ◽  
2D And 3D

AbstractVersatile methods to organize proteins in space are required to enable complex biomaterials, engineered biomolecular scaffolds, cell-free biology, and hybrid nanoscale systems. Here, we demonstrate how the tailored encapsulation of proteins in DNA-based voxels can be combined with programmable assembly that directs these voxels into biologically functional protein arrays with prescribed and ordered two-dimensional (2D) and three-dimensional (3D) organizations. We apply the presented concept to ferritin, an iron storage protein, and its iron-free analog, apoferritin, in order to form single-layers, double-layers, as well as several types of 3D protein lattices. Our study demonstrates that internal voxel design and inter-voxel encoding can be effectively employed to create protein lattices with designed organization, as confirmed by in situ X-ray scattering and cryo-electron microscopy 3D imaging. The assembled protein arrays maintain structural stability and biological activity in environments relevant for protein functionality. The framework design of the arrays then allows small molecules to access the ferritins and their iron cores and convert them into apoferritin arrays through the release of iron ions. The presented study introduces a platform approach for creating bio-active protein-containing ordered nanomaterials with desired 2D and 3D organizations.

scholarly journals PhytoSFDM version 1.0.0: Phytoplankton Size and Functional Diversity Model

2016 ◽  
Vol 9 (11) ◽  
pp. 4071-4085 ◽  
Author(s):  
Esteban Acevedo-Trejos ◽  
Gunnar Brandt ◽  
S. Lan Smith ◽  
Agostino Merico
Keyword(s):  
Open Source ◽  
Open Source Software ◽  
Marine Ecosystem ◽  
Three Dimensional ◽  
Functional Trait ◽  
Global Ocean ◽  
Total Biomass ◽  
Spatially Resolved ◽  
Phytoplankton Communities ◽  
Natural Complexity

Abstract. Biodiversity is one of the key mechanisms that facilitate the adaptive response of planktonic communities to a fluctuating environment. How to allow for such a flexible response in marine ecosystem models is, however, not entirely clear. One particular way is to resolve the natural complexity of phytoplankton communities by explicitly incorporating a large number of species or plankton functional types. Alternatively, models of aggregate community properties focus on macroecological quantities such as total biomass, mean trait, and trait variance (or functional trait diversity), thus reducing the observed natural complexity to a few mathematical expressions. We developed the PhytoSFDM modelling tool, which can resolve species discretely and can capture aggregate community properties. The tool also provides a set of methods for treating diversity under realistic oceanographic settings. This model is coded in Python and is distributed as open-source software. PhytoSFDM is implemented in a zero-dimensional physical scheme and can be applied to any location of the global ocean. We show that aggregate community models reduce computational complexity while preserving relevant macroecological features of phytoplankton communities. Compared to species-explicit models, aggregate models are more manageable in terms of number of equations and have faster computational times. Further developments of this tool should address the caveats associated with the assumptions of aggregate community models and about implementations into spatially resolved physical settings (one-dimensional and three-dimensional). With PhytoSFDM we embrace the idea of promoting open-source software and encourage scientists to build on this modelling tool to further improve our understanding of the role that biodiversity plays in shaping marine ecosystems.

Growth Evolution of (Zn,Cd)Se Quantum Dots Deduced from Spatially Resolved Structural and Optical Characterization

1999 ◽  
Vol 571 ◽  
Author(s):  
K. Leonard ◽  
D. Hommel ◽  
A. Stockmann ◽  
H. Selke ◽  
J. Seufert ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Quantum Wells ◽  
Three Dimensional ◽  
High Energy ◽  
Growth Direction ◽  
Growth Mode ◽  
Gradual Transition ◽  
Mode Transition ◽  
Spatially Resolved ◽  
Composition Fluctuations

ABSTRACTThe growth mode of CdSe layers grown by migration enhanced epitaxy between ZnSe barriers has been investigated. In situ reflection high-energy electron diffraction shows a gradual transition to a three-dimensional growth mode which, however, is not accompanied by a change of the surface lattice constant. High-resolution transmission electron micrographs reveal a strong Cd diffusion, leading to ternary ZnCdSe quantum wells. Furthermore. composition fluctuations perpendicular to the growth direction on a nanometer scale are found already prior to the beginning of the growth mode transition. In the case of heterostructures containing a CdSe layer that has undergone the growth mode transition, micrographs show Cd-rich quantum dots with diameters of around 8 nm and heights of around 1.5 nm within a ternary quantum well. By spatially resolved photoluminescence the emission from single quantum dots could be observed. The polarization dependence of the emission from single dots indicates an asymmetric shape of the dots with certain preferential orientations along the [110] and [110] directions.

scholarly journals Local Li-ion conductivity changes within Al stabilized Li7La3Zr2O12 and their relationship to three-dimensional variations of the bulk composition

2019 ◽  
Vol 7 (12) ◽  
pp. 6818-6831 ◽  
Author(s):  
Stefan Smetaczek ◽  
Andreas Wachter-Welzl ◽  
Reinhard Wagner ◽  
Daniel Rettenwander ◽  
Georg Amthauer ◽  
...  
Keyword(s):  
Impedance Spectroscopy ◽  
Chemical Analysis ◽  
Bulk Composition ◽  
Three Dimensional ◽  
Ion Conductivity ◽  
Spatially Resolved ◽  
Li Ion

Investigating conductivity variations in Al stabilized LLZO by combining microelectrode impedance spectroscopy with spatially resolved chemical analysis.

scholarly journals Mapping the physical and chemical properties of the planetary nebula NGC 3242

2011 ◽  
Vol 7 (S283) ◽  
pp. 448-449
Author(s):  
Hektor Monteiro ◽  
Denise Gonçalves ◽  
Marcelo Leal-Ferreira ◽  
Romano Corradi ◽  
Sebastian Sánchez
Keyword(s):  
Planetary Nebula ◽  
Three Dimensional ◽  
Chemical Properties ◽  
Small Scale ◽  
Chemical Abundances ◽  
Electronic Density ◽  
Spatially Resolved ◽  
Integral Field Spectroscopy ◽  
Main Components ◽  
Physical And Chemical

AbstractWe present optical integral field spectroscopy analysis of the main components, with the exception of the halo, as well as of the detected small-scale structures of the planetary nebulae NGC 3242. The observations were obtained with the VIMOS instrument attached to VLT-UT3. Spatially resolved maps of the electronic density (Ne), temperatures (Te) and chemical abundances, i.e., in a pixel to pixel fashion of the small and large-scales structures of this planetary nebula are determined in this work. These diagnostic and abundance maps represent important constraints for future detailed three dimensional photoionization modeling of the nebula, as well as providing important information on biases introduced by traditional slit observations.

scholarly journals Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Mary C. Macedonia ◽  
Julia L. Drewes ◽  
Nicholas O. Markham ◽  
Alan J. Simmons ◽  
Joseph T. Roland ◽  
...  
Keyword(s):  
Host Cell ◽  
Original Method ◽  
Host Cells ◽  
Poloxamer 407 ◽  
Spatially Resolved ◽  
Antibody Staining ◽  
Biospecimen Collection ◽  
Complex Interactions ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded

Abstract Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multimodal analyses of both microbes and host cells in a spatially resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in poloxamer-fixed samples. This in turn enabled us to integrate multimodal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and cross-link at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.

Structural biology and structure–function relationships of membrane proteins

2018 ◽  
Vol 47 (1) ◽  
pp. 47-61 ◽  
Author(s):  
Rosana Reis ◽  
Isabel Moraes
Keyword(s):  
Membrane Proteins ◽  
Structure Function ◽  
Structural Biology ◽  
Drug Targets ◽  
Three Dimensional ◽  
Data Bank ◽  
Technical Advances ◽  
Medical Importance ◽  
G Protein Coupled ◽  
Important Drug

Abstract The study of structure–function relationships of membrane proteins (MPs) has been one of the major goals in the field of structural biology. Many Noble Prizes regarding remarkable accomplishments in MP structure determination and biochemistry have been awarded over the last few decades. Mutations or improper folding of these proteins are associated with numerous serious illnesses. Therefore, as important drug targets, the study of their primary sequence and three-dimensional fold, combined with cell-based assays, provides vital information about their structure–function relationships. Today, this information is vital to drug discovery and medicine. In the last two decades, many have been the technical advances and breakthroughs in the field of MP structural biology that have contributed to an exponential growth in the number of unique MP structures in the Protein Data Bank. Nevertheless, given the medical importance and many unanswered questions, it will never be an excess of MP structures, regardless of the method used. Owing to the extension of the field, in this brief review, we will only focus on structure–function relationships of the three most significant pharmaceutical classes: G protein-coupled receptors, ion channels and transporters.

scholarly journals Immunocytochemical Labeling of Rhabdomeric Proteins inDrosophilaPhotoreceptor Cells Is Compromised by a Light-dependent Technical Artifact

2019 ◽  
Vol 67 (10) ◽  
pp. 745-757 ◽  
Author(s):  
Krystina Schopf ◽  
Thomas K. Smylla ◽  
Armin Huber
Keyword(s):  
Structural Changes ◽  
Protein Transport ◽  
Light Exposure ◽  
Protein Localization ◽  
Photoreceptor Cells ◽  
Antibody Staining ◽  
Technical Artifact ◽  
Stage Process ◽  
Stage 1 ◽  
Antibody Labeling

Drosophila photoreceptor cells are employed as a model system for studying membrane protein transport. Phototransduction proteins like rhodopsin and the light-activated TRPL ion channel are transported within the photoreceptor cell, and they change their subcellular distribution in a light-dependent way. Investigating the transport mechanisms for rhodopsin and ion channels requires accurate histochemical methods for protein localization. By using immunocytochemistry the light-triggered translocation of TRPL has been described as a two-stage process. In stage 1, TRPL accumulates at the rhabdomere base and the adjacent stalk membrane a few minutes after onset of illumination and is internalized in stage 2 by endocytosis after prolonged light exposure. Here, we show that a commonly observed crescent shaped antibody labeling pattern suggesting a fast translocation of rhodopsin, TRP, and TRPL to the rhabdomere base is a light-dependent antibody staining artifact. This artifact is most probably caused by the profound structural changes in the microvillar membranes of rhabdomeres that result from activation of the signaling cascade. By using alternative labeling methods, either eGFP-tags or the self-labeling SNAP-tag, we show that light activation of TRPL transport indeed results in fast changes of the TRPL distribution in the rhabdomere but not in the way described previously.

Three-dimensional spatially resolved neutron diffraction from a disordered vortex lattice

2011 ◽  
Vol 44 (2) ◽  
pp. 414-417
Author(s):  
Xi Wang ◽  
Helen A. Hanson ◽  
Xinsheng Sean Ling ◽  
Charles F. Majkrzak ◽  
Brian B. Maranville
Keyword(s):  
Neutron Diffraction ◽  
Vortex Lattice ◽  
Three Dimensional ◽  
Vortex State ◽  
Prototype System ◽  
Vortex Matter ◽  
Atomic Lattice ◽  
Spatially Resolved ◽  
Neutron Diffraction Technique ◽  
Vortex Physics

The vortex matter in bulk type II superconductors serves as a prototype system for studying the random pinning problem in condensed matter physics. Since the vortex lattice is embedded in an atomic lattice, small-angle neutron scattering (SANS) is the only technique that allows for direct structural studies. In traditional SANS methods, the scattering intensity is a measure of the structure factor averaged over the entire sample. Recent studies in vortex physics have shown that it is highly desirable to develop a SANS technique that is capable of resolving the spatial inhomogeneities in the bulk vortex state. This article reports a novel slicing neutron diffraction technique using atypical collimation and an areal detector, which allows for observing the three-dimensional disorder of the vortex matter inside an as-grown Nb single crystal.

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