scholarly journals A sensitive and specific genetically encodable biosensor for potassium ions

2021 ◽  
Author(s):  
Sheng-Yi Wu ◽  
Yurong Wen ◽  
Nelson Bernard Calixte Serre ◽  
Cathrine Charlotte Heiede Laursen ◽  
Andrea Grostøl Dietz ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Directed Evolution ◽  
Biological Systems ◽  
Critical Role ◽  
Potassium Ions ◽  
In Vivo Detection ◽  
Highly Sensitive ◽  
Multiple Species

Potassium ions (K+) play a critical role as an essential electrolyte in all biological systems. Here we report the crystal structure-guided optimization and directed evolution of an improved genetically encoded fluorescent K+ biosensor, GINKO2. GINKO2 is highly sensitive and specific for K+ and enables in vivo detection of K+ dynamics in multiple species.

scholarly journals Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis

2020 ◽  
Author(s):  
Ayushi Agrawal ◽  
Chandra Kanth Bandi ◽  
Tucker Burgin ◽  
Youngwoo Woo ◽  
Heather B. Mayes ◽  
...  
Keyword(s):  
Single Cell ◽  
Click Chemistry ◽  
Directed Evolution ◽  
Cell Sorting ◽  
Screening Methods ◽  
Carbohydrate Active Enzymes ◽  
E Coli ◽  
Highly Sensitive ◽  
Increased Activity

AbstractEngineering of carbohydrate-active enzymes like glycosynthases for chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable directed evolution based protein engineering methods. Currently there are no ultrahigh-throughput screening methods available for rapid and highly sensitive single cell-based screening of evolved glycosynthase enzymes employing azido sugars as substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides (versus free inorganic azides) that facilitated ultrahigh-throughput in-vivo single cell-based assay of glycosynthase activity. This discovery has led to the development of a directed evolution methodology for screening and sorting glycosynthase mutants for synthesis of desired fucosylated oligosaccharides. Our screening technique facilitated rapid fluorescence activated cell sorting of a large library of glycosynthase variants (>106 mutants) expressed in E. coli to identify several novel mutants with increased activity for β-fucosyl-azide activated donor sugars towards desired acceptor sugars, demonstrating the broader applicability of this methodology.

scholarly journals Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex

2006 ◽  
Vol 398 (3) ◽  
pp. 353-360 ◽  
Author(s):  
Jingzhi Li ◽  
Yunkun Wu ◽  
Xinguo Qian ◽  
Bingdong Sha
Keyword(s):  
Crystal Structure ◽  
Close Contact ◽  
Critical Role ◽  
Peptide Binding ◽  
Structural Basis ◽  
Cellular Processes ◽  
Terminal Form ◽  
Charged Residues ◽  
Domain I

Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40–Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a β-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge–charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.

scholarly journals Optical imaging-guided cancer therapy with fluorescent nanoparticles

2009 ◽  
Vol 7 (42) ◽  
pp. 3-18 ◽  
Author(s):  
Shan Jiang ◽  
Muthu Kumara Gnanasammandhan ◽  
Yong Zhang
Keyword(s):  
Cancer Therapy ◽  
Optical Imaging ◽  
Critical Role ◽  
Therapeutic Agents ◽  
Fluorescent Nanoparticles ◽  
Highly Sensitive ◽  
Imaging Probes ◽  
Recent Developments

The diagnosis and treatment of cancer have been greatly improved with the recent developments in nanotechnology. One of the promising nanoscale tools for cancer diagnosis is fluorescent nanoparticles (NPs), such as organic dye-doped NPs, quantum dots and upconversion NPs that enable highly sensitive optical imaging of cancer at cellular and animal level. Furthermore, the emerging development of novel multi-functional NPs, which can be conjugated with several functional molecules simultaneously including targeting moieties, therapeutic agents and imaging probes, provides new potentials for clinical therapies and diagnostics and undoubtedly will play a critical role in cancer therapy. In this article, we review the types and characteristics of fluorescent NPs, in vitro and in vivo imaging of cancer using fluorescent NPs and multi-functional NPs for imaging-guided cancer therapy.

Highly sensitive room-temperature method of non-invasive in vivo detection of magnetic nanoparticles

2009 ◽  
Vol 321 (10) ◽  
pp. 1658-1661 ◽  
Author(s):  
Maxim P. Nikitin ◽  
Petr M. Vetoshko ◽  
Nikolai A. Brusentsov ◽  
Petr I. Nikitin
Keyword(s):  
Magnetic Nanoparticles ◽  
Room Temperature ◽  
Non Invasive ◽  
In Vivo Detection ◽  
Temperature Method ◽  
Highly Sensitive

scholarly journals In vivo detection of optically-evoked opioid peptide release

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Ream Al-Hasani ◽  
Jenny-Marie T Wong ◽  
Omar S Mabrouk ◽  
Jordan G McCall ◽  
Gavin P Schmitz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Opioid Peptide ◽  
Neuronal Circuitry ◽  
Endogenous Peptide ◽  
In Vivo Detection ◽  
Highly Sensitive ◽  
Peptide Release ◽  
Freely Moving ◽  
The Brain

Though the last decade has seen accelerated advances in techniques and technologies to perturb neuronal circuitry in the brain, we are still poorly equipped to adequately dissect endogenous peptide release in vivo. To this end we developed a system that combines in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents.

Highly Sensitive Near-Infrared Fluorophores for in Vivo Detection of Amyloid-β Plaques in Alzheimer’s Disease

2015 ◽  
Vol 58 (17) ◽  
pp. 6972-6983 ◽  
Author(s):  
Hualong Fu ◽  
Mengchao Cui ◽  
Liu Zhao ◽  
Peiyu Tu ◽  
Kaixiang Zhou ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Near Infrared ◽  
Amyloid Β ◽  
In Vivo Detection ◽  
Highly Sensitive

Radiolabelling of TiO2 Nanoparticle Libraries for Toxicological Investigations

2009 ◽  
Vol 1209 ◽  
Author(s):  
Anthony W. Musumeci ◽  
Lawrence R Gahan ◽  
Tijana Rajh ◽  
Darren J Martin ◽  
Suzanne V Smith
Keyword(s):  
Titanium Dioxide ◽  
Biological Systems ◽  
Tio2 Nanoparticle ◽  
Robust Methods ◽  
Nanoparticle Interactions ◽  
Highly Sensitive ◽  
Gamma Emitter

AbstractTo further our understanding of nanoparticle interactions with biological systems, it is important that highly sensitive, reliable and robust methods for labelling particles are established. We report here the application of a series of bi-functional cage ligands to radiolabel a range (i.e. shapes and sizes) of titanium dioxide (TiO2) particles. The cages were covalently attached to the surface of the particles via the use of a dopac derivative and then radiolabelled with a gamma emitting radioisotope. The final radiolabelled nanoparticles proved to be stable in solution and the method easy and robust. The application of a gamma emitter allows the radiolabelled particles to be tracked in vivo and in the environment.

scholarly journals In vivo detection of optically-evoked opioid peptide release

2018 ◽  
Author(s):  
Ream Al-Hasanil ◽  
Jenny-Marie T. Wong ◽  
Omar S. Mabrouk ◽  
Jordan G. McCall ◽  
Gavin P. Schmitz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Opioid Peptide ◽  
Neuronal Circuitry ◽  
Endogenous Peptide ◽  
In Vivo Detection ◽  
Highly Sensitive ◽  
Peptide Release ◽  
Freely Moving ◽  
The Brain

AbstractThough the last decade has seen accelerated advances in techniques and technologies to perturb neuronal circuitry in the brain, we are still poorly equipped to adequately dissect endogenous peptide release in vivo. To this end we developed a system that combines in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents.

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

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