A Photoalkylative Fluorogenic Probe of Guttiferone A for Live Cell Imaging and Proteome Labeling in Plasmodium falciparum

Guttiferone A (GA) 1, a polycyclic polyprenylated acylphloroglucinol (PPAP) isolated from the plant Symphonia globulifera (Clusiaceae), constitutes a novel hit in antimalarial drug discovery. PPAPs do not possess identified biochemical targets in malarial parasites up to now. Towards this aim, we designed and evaluated a natural product-derived photoactivatable probe AZC-GA 5, embedding a photoalkylative fluorogenic motif of the 7-azidocoumarin (AZC) type, devoted to studying the affinity proteins interacting with GA in Plasmodium falciparum. Probe 5 manifested a number of positive functional and biological features, such as (i) inhibitory activity in vitro against P. falciparum blood-stages that was superimposable to that of GA 1, dose–response photoalkylative fluorogenic properties (ii) in model conditions using bovine serum albumin (BSA) as an affinity protein surrogate, (iii) in live P. falciparum-infected erythrocytes, and (iv) in fresh P. falciparum cell lysate. Fluorogenic signals by photoactivated AZC-GA 5 in biological settings were markedly abolished in the presence of excess GA 1 as a competitor, indicating significant pharmacological specificity of the designed molecular probe relative to the native PPAP. These results open the way to identify the detected plasmodial proteins as putative drug targets for the natural product 1 by means of proteomic analysis.

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Genotypes and phenotypes of resistance in Ecuadorian Plasmodium falciparum

Malaria Journal ◽

10.1186/s12936-019-3044-z ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 1

Author(s):

Gabriela Valenzuela ◽

L. Enrique Castro ◽

Julio Valencia-Zamora ◽

Claudia A. Vera-Arias ◽

Petra Rohrbach ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Live Cell Imaging ◽

Cell Imaging ◽

Chloroquine Resistance ◽

In Vitro Assays ◽

Wild Type ◽

The Status ◽

Transport Assay ◽

Resistance Markers

Abstract Background Malaria continues to be endemic in the coast and Amazon regions of Ecuador. Clarifying current Plasmodium falciparum resistance in the country will support malaria elimination efforts. In this study, Ecuadorian P. falciparum parasites were analysed to determine their drug resistance genotypes and phenotypes. Methods Molecular analyses were performed to search for mutations in known resistance markers (Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, k13). Pfmdr1 copy number was determined by qPCR. PFMDR1 transporter activity was characterized in live parasites using live cell imaging in combination with the Fluo-4 transport assay. Chloroquine, quinine, lumefantrine, mefloquine, dihydroartemisinin, and artemether sensitivities were measured by in vitro assays. Results The majority of samples from this study presented the CVMNT genotype for Pfcrt (72–26), NEDF SDFD mutations in Pfmdr1 and wild type genotypes for Pfdhfr, Pfdhps and k13. The Ecuadorian P. falciparum strain ESM-2013 showed in vitro resistance to chloroquine, but sensitivity to quinine, lumefantrine, mefloquine, dihydroartemisinin and artemether. In addition, transport of the fluorochrome Fluo-4 from the cytosol into the digestive vacuole (DV) of the ESM-2013 strain was minimally detected in the DV. All analysed samples revealed one copy of Pfmdr1. Conclusion This study indicates that Ecuadorian parasites presented the genotype and phenotype for chloroquine resistance and were found to be sensitive to SP, artemether-lumefantrine, quinine, mefloquine, and dihydroartemisinin. The results suggest that the current malaria treatment employed in the country remains effective. This study clarifies the status of anti-malarial resistance in Ecuador and informs the P. falciparum elimination campaigns in the country.

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A new visible-light-excitable ICT-CHEF-mediated fluorescence ‘turn-on’ probe for the selective detection of Cd2+ in a mixed aqueous system with live-cell imaging

Dalton Transactions ◽

10.1039/c4dt02463j ◽

2015 ◽

Vol 44 (12) ◽

pp. 5763-5770 ◽

Cited By ~ 58

Author(s):

Shyamaprosad Goswami ◽

Krishnendu Aich ◽

Sangita Das ◽

Chitrangada Das Mukhopadhyay ◽

Deblina Sarkar ◽

...

Keyword(s):

Visible Light ◽

Live Cell Imaging ◽

Cell Imaging ◽

Aqueous System ◽

Live Cell ◽

Selective Detection ◽

Turn On ◽

Fluorescence Turn On

A new quinoline based sensor was developed and applied for the selective detection of Cd2+ both in vitro and in vivo.

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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BMP4-induced symmetry breaking in a 3D model of the human epiblast

10.21203/rs.2.9730/v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Mijo Simunovic ◽

Ali H. Brivanlou ◽

Eric D. Siggia

Keyword(s):

Live Cell Imaging ◽

3D Model ◽

Epithelial To Mesenchymal Transition ◽

Cell Imaging ◽

Embryonic Stem ◽

Primitive Streak ◽

Mesenchymal Transition ◽

Pluripotency Markers ◽

Anterior Posterior

Abstract We describe the protocol of generating a 3D stem-cell-based model of the human pre-gastrulation epiblast by culturing human embryonic stem cells in a mix of hydrogel and Matrigel. Much like the epiblast of an in vitro attached day-10 human embryo, this model is an epithelial sphere with a cavity at its center, it is expressing key pluripotency markers, and it displays apico-basal polarity. The 3D colonies can further be differentiated with morphogens and in the case of intermediate concentrations of BMP4, they break the anterior-posterior symmetry characterized by an asymmetric expression of a primitive streak marker and showing signs of epithelial to mesenchymal transition. The protocol described here is suitable for immunofluorescence staining and for live-cell imaging.

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A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03342-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 356-364 ◽

Cited By ~ 52

Author(s):

Wesley Wu ◽

Zachary Herrera ◽

Danny Ebert ◽

Katie Baska ◽

Seok H. Cho ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Antimalarial Drug ◽

Drug Targets ◽

Specific Activity ◽

Drug Candidate ◽

Content Type ◽

Chemical Rescue ◽

Growth Inhibitory ◽

Parasite Populations

ABSTRACTThe apicoplast is an essential plastid organelle found inPlasmodiumparasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the “Malaria Box” library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stagePlasmodium falciparumgrowth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations inP. falciparumIspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activityin vitrowith a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action.

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