scholarly journals Properties of Fluorescent Far-Red Anti-TNF Nanobodies

Antibodies ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 43 ◽  
Author(s):  
Ekaterina Gorshkova ◽  
Grigory Efimov ◽  
Ksenia Ermakova ◽  
Ekaterina Vasilenko ◽  
Diana Yuzhakova ◽  
...  
Keyword(s):  
Autoimmune Diseases ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Humanized Mice ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Theranostic Agent ◽  
Variable Heavy

Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

Cellular Uptake of Carbon Nanotubes Conjugated to Semiconductor Quantum Dots for Breast Cancer Imaging and Treatment Applications

2010 ◽  
Author(s):  
Kristen A. Zimmermann ◽  
Jianfei Zhang ◽  
Harry Dorn ◽  
Christopher Rylander ◽  
Marissa Nichole Rylander
Keyword(s):  
Carbon Nanotubes ◽  
Quantum Dots ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Properties ◽  
Breast Cancer Imaging ◽  
Fluorescent Markers ◽  
Green Fluorescent

Carbon nanotubes (CNTs) are attractive materials for early detection, treatment, and imaging of cancer malignancies; however, they are limited by their inability to be monitored in vitro and in vivo [1]. Unlabeled CNTs are difficult to distinguish using elemental analysis because they are composed entirely of carbon, which is also characteristic of cellular membranes. Although some single walled nanotubes (SWNT) have been found to exhibit fluorescent properties, not all particles in a single batch fluoresce [2]. Additionally, these emissions may be too weak to be detected using conventional imaging modalities [3]. Incorporating fluorescent markers, such as fluorescent proteins or quantum dots, allows the non-fluorescent particles to be visualized. Previously, fluorophores, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), have been used to visualize and track cells or other particles in biological environments, but their low quantum yield and tendency to photobleach generate limitations for their use in such applications.

scholarly journals Whole-Body Imaging of Sequestration of Plasmodium falciparum in the Rat

2005 ◽  
Vol 73 (11) ◽  
pp. 7736-7746 ◽  
Author(s):  
Fredrik Pettersson ◽  
Anna M. Vogt ◽  
Cathrine Jonsson ◽  
Bobo W. Mok ◽  
Alireza Shamaei-Tousi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasmodium Falciparum ◽  
Severe Malaria ◽  
Whole Body ◽  
Surgical Removal ◽  
Whole Body Imaging ◽  
Rat Lung ◽  
Body Imaging

ABSTRACT The occlusion of vessels by packed Plasmodium falciparum-infected (iRBC) and uninfected erythrocytes is a characteristic postmortem finding in the microvasculature of patients with severe malaria. Here we have employed immunocompetent Sprague-Dawley rats to establish sequestration in vivo. Human iRBC cultivated in vitro and purified in a single step over a magnet were labeled with 99mtechnetium, injected into the tail vein of the rat, and monitored dynamically for adhesion in the microvasculature using whole-body imaging or imaging of the lungs subsequent to surgical removal. iRBC of different lines and clones sequester avidly in vivo while uninfected erythrocytes did not. Histological examination revealed that a multiadhesive parasite adhered in the larger microvasculature, inducing extensive intravascular changes while CD36- and chondroitin sulfate A-specific parasites predominantly sequester in capillaries, inducing no or minor pathology. Removal of the adhesive ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), preincubation of the iRBC with sera to PfEMP1 or preincubation with soluble PfEMP1-receptors prior to injection significantly reduced the sequestration. The specificity of iRBC binding to the heterologous murine receptors was confirmed in vitro, using primary rat lung endothelial cells and rat lung cryosections. In offering flow dynamics, nonmanipulated endothelial cells, and an intact immune system, we believe this syngeneic animal model to be an important complement to existing in vitro systems for the screening of vaccines and adjunct therapies aiming at the prevention and treatment of severe malaria.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals Supramolecular Encapsulation of Small-Ultra Red Fluorescent Proteins in Virus-Like Nanoparticles for Non-Invasive In Vivo Imaging Agents

2020 ◽  
Author(s):  
Fabian C. Herbert ◽  
Olivia Brohlin ◽  
Tyler Galbraith ◽  
Candace Benjamin ◽  
Cesar A. Reyes ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ex Vivo ◽  
Imaging Agents ◽  
Non Invasive ◽  
Red Fluorescent Proteins ◽  
Ex Vivo Imaging

<div> <div> <div> <p>Icosahedral virus-like particles (VLPs) derived from bacteriophages Qβ and PP7 encapsulating small-ultra red fluorescent protein (smURFP) were produced using a versatile supramolecualr capsid dissassemble-reassemble approach. The generated fluorescent VLPs display identical structural properties to their non-fluorescent analogs. Encapsulated smURFP shows indistinguishable photochemical properties to its unencapsulated counterpart, exhibits outstanding stability towards pH, and produces bright in vitro images following phagocytosis by macrophages. In vivo imaging allows biodistribution to be imaged at different time points. Ex vivo imaging of intravenously administered encapsulated smURFP reveleas localization in the liver and </p> </div> </div> <div> <div> <p>kidneys after 2 h blood circulation and substantial elimination constructs as non-invasive in vivo imaging agents. </p> </div> </div> </div>

scholarly journals Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

2020 ◽  
Author(s):  
Husam Sabah Auhim ◽  
Bella L. Grigorenko ◽  
Tessa Harris ◽  
Igor V. Polyakov ◽  
Colin Berry ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Life Sciences ◽  
Intermediate Stage ◽  
Open Conformation ◽  
Oxidation Step ◽  
Canonical Amino Acid

AbstractFluorescent proteins (FPs) have revolutionised the life sciences but the mechanism of chromophore maturation is still not fully understood. Incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; the crystal structure reveals the presence of O2 located above a dehydrated enolate imidazolone (I) ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an “open” conformation, potentially allowing O2 access to the chromophore. Absorption spectroscopy supported by QM/MM simulations suggest that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H2O2 upon irradiation of this intermediate, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation, and the mechanistic insights opens up new approaches for engineering optically controlled fluorescent proteins.

scholarly journals Fluorescence Phenomena in Amyloid and Amyloidogenic Bionanostructures

Crystals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 668 ◽  
Author(s):  
B. Apter ◽  
N. Lapshina ◽  
H. Barhom ◽  
B. Fainberg ◽  
A. Handelman ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Structures ◽  
Visible Spectrum ◽  
Biological Tissues ◽  
New Generation ◽  
Β Sheet

Nanoscale optical labeling is an advanced bioimaging tool. It is mostly based on fluorescence (FL) phenomena and enables the visualization of single biocells, bacteria, viruses, and biological tissues, providing monitoring of functional biosystems in vitro and in vivo, and the imaging-guided transportation of drug molecules. There is a variety of FL biolabels such as organic molecular dyes, genetically encoded fluorescent proteins (green fluorescent protein and homologs), semiconductor quantum dots, carbon dots, plasmonic metal gold-based nanostructures and more. In this review, a new generation of FL biolabels based on the recently found biophotonic effects of visible FL are described. This intrinsic FL phenomenon is observed in any peptide/protein materials folded into β-sheet secondary structures, irrespective of their composition, complexity, and origin. The FL effect has been observed both in natural amyloid fibrils, associated with neurodegenerative diseases (Alzheimer’s, Parkinson’s, and more), and diverse synthetic peptide/protein structures subjected to thermally induced biological refolding helix-like→β-sheet. This approach allowed us to develop a new generation of FL peptide/protein bionanodots radiating multicolor, tunable, visible FL, covering the entire visible spectrum in the range of 400–700 nm. Newly developed biocompatible nanoscale biomarkers are considered as a promising tool for emerging precise biomedicine and advanced medical nanotechnologies (high-resolution bioimaging, light diagnostics, therapy, optogenetics, and health monitoring).

scholarly journals Far-red fluorescent tags for protein imaging in living tissues

2009 ◽  
Vol 418 (3) ◽  
pp. 567-574 ◽  
Author(s):  
Dmitry Shcherbo ◽  
Christopher S. Murphy ◽  
Galina V. Ermakova ◽  
Elena A. Solovieva ◽  
Tatiana V. Chepurnykh ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Imaging Techniques ◽  
Fluorescent Imaging ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Fluorescent Tags ◽  
Living Tissues ◽  
Fluorescent Tag

A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.

Multipotent Adult Progenitor Cells (MAPCs) Improve Cardiac Function after Ischemic Injury.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1701-1701
Author(s):  
Jakub Tolar ◽  
Xiahong Wang ◽  
Scott Bell ◽  
Yasuhiro Nakamura ◽  
Ron T. McElmurry ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Fluorescent Protein ◽  
Sleeping Beauty ◽  
Left Ventricular ◽  
Whole Body ◽  
Myocardial Repair ◽  
Surgical Suture ◽  
Injured Myocardium

Abstract MAPCs are pluripotent cells derived from mesenchymal stromal cells (MSCs) in adult bone marrow. In contrast to MSCs, MAPCs differentiate into various lineages of mesodermal, ectodermal and endodermal origin, and contribute to numerous terminally differentiated tissues in the recipients. This capacity is enhanced in the setting of injury, suggesting a possible role of MAPCs in repair and regeneration in disease states. We aimed to investigate the capacity of MAPCs to aid in myocardial repair in hearts with postinfarction remodeling. We reasoned that as MAPCs differentiate to both endothelium and cardiomyocytes in vitro and as engraftment of delivered cells depends on establishing adequate blood flow in the ischemic region, MAPCs may represent the optimal cell type to contribute to both angiogenesis and the parenchymal tissue to regenerate function of injured myocardium. To study engraftment and survival of MAPCs, we labeled adult murine C57BL/6 MAPCs with firefly luciferase and DsRed2 fluorescent protein using non-viral Sleeping Beauty transposons, and injected them into myocardium of C57BL/6 adult mice with acute myocardial infarction (AMI). Mice were anesthetized, intubated and mechanically ventilated using a small-animal respirator. Under a stereomicroscope the heart was accessed via left thoracotomy. The left anterior descending coronary artery was ligated at mid-level between apex and base with a 9-0 surgical suture to produce AMI. Twenty minutes later, intramyocardial injections of labeled MAPCs in saline, or saline alone were administered at five distinct injection sites at boarder zone of AMI (total MAPC dose = 106/mouse). Chest was closed in layers and animals were allowed to recover. Mice were followed with echocardiography and in vivo whole body bioluminescent imaging. Seventy days after AMI, MAPCs recipients (N=6) had significantly less severe left ventricular (LV) dilatation evidenced by a smaller LV end-diastolic and LV end-systolic dimensions when compared to control mice infused with saline (N=4) (average±standard deviation, 4.7±0.2 mm versus 5.3±0.5 mm, p=0.05; and 3.7±0.2 mm versus 4.4±0.6 mm, p=0.03, respectively). In addition, ejection and shortening fractions were significantly higher in MAPC recipients (36±2% versus 30±3%, p=0.004; and 20±1% versus 16±2%, p=0.004, respectively). Luciferase signals emitted from donor MAPCs were easily detectable in MAPC recipients 100 days after MAPC infusion, at which point the animals were harvested. Analyses are ongoing to determine whether MAPCs and their progeny contributed to expansion of coronary vasculature (capillary density), or formed or modified injured myocardial tissue. Alternatively, both populations of repair cells could have been derived from donor (DsRed2+) MAPCs, or donor MAPCs could have provided permissive local environment to recruit recipient cells and enhance endogeneous regeneration. In summary, these findings provide evidence that MAPCs persist long term in injured myocardium and document the potential of MAPCs for improvement of cardiac function after ischemic myocardial injury. Jakub Tolar and Xiaohong Wang contributed equally to this study.

scholarly journals Magnetically Assisted Control of Stem Cells Applied in 2D, 3D and In Situ Models of Cell Migration

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1563
Author(s):  
Richard Harrison ◽  
Jeni Luckett ◽  
Sarah Marsh ◽  
Hilda Anaid Lugo Leija ◽  
Shelanah Salih ◽  
...  
Keyword(s):  
Stem Cells ◽  
Magnetic Particles ◽  
Control Cell ◽  
3D Models ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Iron Oxide Particles ◽  
Magnetic Forces

The success of cell therapy approaches is greatly dependent on the ability to precisely deliver and monitor transplanted stem cell grafts at treated sites. Iron oxide particles, traditionally used in vivo for magnetic resonance imaging (MRI), have been shown to also represent a safe and efficient in vitro labelling agent for mesenchymal stem cells (MSCs). Here, stem cells were labelled with magnetic particles, and their resulting response to magnetic forces was studied using 2D and 3D models. Labelled cells exhibited magnetic responsiveness, which promoted localised retention and patterned cell seeding when exposed to magnet arrangements in vitro. Directed migration was observed in 2D culture when adherent cells were exposed to a magnetic field, and also when cells were seeded into a 3D gel. Finally, a model of cell injection into the rodent leg was used to test the enhanced localised retention of labelled stem cells when applying magnetic forces, using whole body imaging to confirm the potential use of magnetic particles in strategies seeking to better control cell distribution for in vivo cell delivery.

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