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.

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.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

scholarly journals Antimicrobial Peptides as Infection Imaging Agents: Better Than Radiolabeled Antibiotics

2012 ◽  
Vol 2012 ◽  
pp. 1-19 ◽  
Author(s):  
Muammad Saeed Akhtar ◽  
Muhammad Babar Imran ◽  
Muhammad Afzal Nadeem ◽  
Abubaker Shahid
Keyword(s):  
Nuclear Medicine ◽  
Antimicrobial Peptides ◽  
Imaging Techniques ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Nuclear Medicine Imaging ◽  
Human Immune System ◽  
Large Member ◽  
Infection And Inflammation ◽  
Radiolabeled Compounds

Nuclear medicine imaging techniques offer whole body imaging for localization of number and site of infective foci inspite of limitation of spatial resolution. The innate human immune system contains a large member of important elements including antimicrobial peptides to combat any form of infection. However, development of antibiotics against bacteria progressed rapidly and gained popularity over antimicrobial peptides but even powerful antimicrobials failed to reduce morbidity and mortality due to emergence of mutant strains of bacteria resulting in antimicrobial resistance. Differentiation between infection and inflammation using radiolabeled compounds with nuclear medicine techniques has always been a dilemma which is still to be resolved. Starting from nonspecific tracers to specific radiolabeled tracers, the question is still unanswered. Specific radiolabeled tracers included antibiotics and antimicrobial peptides which bind directly to the bacteria for efficient localization with advanced nuclear medicine equipments. However, there are merits and demerits attributed to each. In the current paper, radiolabeled antibiotics and radiolabeled peptides for infection localization have been discussed starting with the background of primitive nonspecific tracers. Radiolabeled antimicrobial peptides have certain merits compared with labeled antibiotics which make them superior agents for localization of infective focus.

scholarly journals Comparison between whole-body MRI and Fluorine-18-Fluorodeoxyglucose PET or PET/CT in oncology: a systematic review

2013 ◽  
Vol 47 (3) ◽  
pp. 206-218 ◽  
Author(s):  
Mario Ciliberto ◽  
Fabio Maggi ◽  
Giorgio Treglia ◽  
Federico Padovano ◽  
Lucio Calandriello ◽  
...  
Keyword(s):  
Systematic Review ◽  
Fdg Pet ◽  
Imaging Techniques ◽  
Diagnostic Methods ◽  
Whole Body ◽  
Published Data ◽  
Whole Body Imaging ◽  
Positron Emission ◽  
Pet Ct ◽  
Fluorine 18 Fluorodeoxyglucose

Abstract Background. The aim of the article is to systematically review published data about the comparison between positron emission tomography (PET) or PET/computed tomography (PET/CT) using Fluorine-18-Fluorodeoxyglucose (FDG) and whole-body magnetic resonance imaging (WB-MRI) in patients with different tumours. Methods. A comprehensive literature search of studies published in PubMed/MEDLINE, Scopus and Embase databases through April 2012 and regarding the comparison between FDG-PET or PET/CT and WB-MRI in patients with various tumours was carried out. Results. Forty-four articles comprising 2287 patients were retrieved in full-text version, included and discussed in this systematic review. Several articles evaluated mixed tumours with both diagnostic methods. Concerning the specific tumour types, more evidence exists for lymphomas, bone tumours, head and neck tumours and lung tumours, whereas there is less evidence for other tumour types. Conclusions. Overall, based on the literature findings, WB-MRI seems to be a valid alternative method compared to PET/CT in oncology. Further larger prospective studies and in particular cost-effectiveness analysis comparing these two whole-body imaging techniques are needed to better assess the role of WB-MRI compared to FDG-PET or PET/ CT in specific tumour types.

scholarly journals Efficient generation of endogenous protein reporters for mouse preimplantation embryos

2020 ◽  
Author(s):  
Dan O’Hagan ◽  
Amy Ralston
Keyword(s):  
High Efficiency ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Preimplantation Embryos ◽  
List Type ◽  
Gene Coding ◽  
Endogenous Protein ◽  
Mouse Lines

SummaryFluorescent proteins and epitope tags can reveal protein localization in cells and animals. However, the large size of many tags hinders efficient genome targeting. Accordingly, many studies have relied on characterizing overexpressed proteins, which might not recapitulate endogenous protein activities. We present two approaches for higher throughput production of endogenous protein reporters. Our first approach makes use of a split fluorescent protein mNeonGreen2 (mNG2). Knock-in of a small portion of the mNG2 gene, in frame with gene coding regions of interest was highly efficient in embryos, eliminating the need to establish mouse lines. When complemented by the larger portion of the mNG2 gene, fluorescence was reconstituted and endogenous protein localization faithfully reported in living embryos. However, we report a threshold of detection using this approach. By contrast, the V5 epitope enabled high efficiency and higher sensitivity protein reporting. We describe complementary advantages and prospective applications of these two approaches.HighlightsSplit fluorescent protein for in vivo protein localization in living embryosV5 tagging for in vivo localization of low abundance proteinsBypassing the need for founder mouse lines for preimplantation studiesGuidelines and strategies for implementation and prospective applications

PIT-1 Protein Localization at Different Optical Sections in a Single Living Cell Using FRET Microscopy and Green Fluorescent Proteins

1997 ◽  
Vol 3 (S2) ◽  
pp. 133-134 ◽  
Author(s):  
Ammasi Periasamy ◽  
Richard N. Day
Keyword(s):  
Transcription Factors ◽  
Living Cell ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Resonance Energy ◽  
Living Cells ◽  
Specific Gene ◽  
Green Fluorescent ◽  
Prl Gene

The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.

scholarly journals Identifying CD38+ cells in patients with multiple myeloma: first-in-human imaging using copper-64–labeled daratumumab

2020 ◽  
Vol 4 (20) ◽  
pp. 5194-5202
Author(s):  
Amrita Krishnan ◽  
Vikram Adhikarla ◽  
Erasmus K. Poku ◽  
Joycelynne Palmer ◽  
Ammar Chaudhry ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Sensitivity And Specificity ◽  
Imaging Techniques ◽  
Phase 1 ◽  
Whole Body ◽  
Human Antibody ◽  
Whole Body Imaging ◽  
Positron Emission ◽  
Pet Ct

Abstract 18F-Fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is one of the most widely used imaging techniques to detect multiple myeloma (MM). Intracellular FDG uptake depicts in vivo metabolic activity, which can be seen in both malignant and nonmalignant cells, resulting in limited sensitivity and specificity. Our group showed preclinically that tracing MM dissemination using a CD38-directed human antibody, daratumumab, that is radioconjugated with 64Cu via the chelator DOTA (64Cu-daratumumab), led to improved sensitivity and specificity over that of FDG. Here, we report the results of a phase 1 trial designed to (1) assess the safety and feasibility of 64Cu-daratumumab PET/CT and (2) preliminarily evaluate and characterize the ability of 64Cu-daratumumab to accurately detect or exclude MM lesions. A total of 12 daratumumab-naive patients were imaged. Prior to the injection of 15 mCi/5 mg of 64Cu-daratumumab, patients were treated with 0 (n = 3), 10 (n = 3), 45 (n = 3), or 95 mg (n = 3) of unlabeled daratumumab to assess its effect on image quality. No significant adverse events were observed from either unlabeled daratumumab or 64Cu-daratumumab. Of the dose levels tested, 45 mg unlabeled daratumumab was the most optimal in terms of removing background signal without saturating target sites. 64Cu-daratumumab PET/CT provided safe whole-body imaging of MM. A trial comparing the sensitivity and specificity of 64Cu-daratumumab PET/CT with that of FDG PET/CT is planned. This trial was registered at www.clinicaltrials.gov as #NCT03311828.

scholarly journals Comparative study reveals better far-red fluorescent protein for whole body imaging

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
K.E. Luker ◽  
P. Pata ◽  
I.I. Shemiakina ◽  
A. Pereverzeva ◽  
A.C. Stacer ◽  
...  
Keyword(s):  
Comparative Study ◽  
Fluorescent Protein ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Red Fluorescent Protein ◽  
Body Imaging

Bright far-red fluorescent protein for whole-body imaging

2007 ◽  
Vol 4 (9) ◽  
pp. 741-746 ◽  
Author(s):  
Dmitry Shcherbo ◽  
Ekaterina M Merzlyak ◽  
Tatiana V Chepurnykh ◽  
Arkady F Fradkov ◽  
Galina V Ermakova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Red Fluorescent Protein ◽  
Body Imaging
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