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Eleonora Veglia, Alessandro Pini, Aldo Moggio, Cristina Grange, Federica Premoselli, Gianluca Miglio, Katerina Tiligada, Roberto Fantozzi, Paul L Chazot, Arianna Carolina Rosa
Histamine has been reported to decrease the ultrafiltration coefficient, which inversely correlates with glomerular permselectivity, however the mechanism(s) underling this effect have never been investigated. This study aimed to assess whether histamine could exert a direct detrimental effect on podocyte permeability and the possible involvement of two key proteins for the glomerular slit diaphragm (SD) integrity, zonula occludens-1 (ZO-1) and P-cadherin. The effect of histamine (100 pM-1000nM) on coloured podocytes junctional integrity was evaluated functionally by a transwell assay of monolayer permeability and morphologically by electron microscopy...
October 14, 2016: Pharmacological Research: the Official Journal of the Italian Pharmacological Society
Avik Roy, Madhuchhanda Kundu, Malabendu Jana, Rama K Mishra, Yeni Yung, Chi-Hao Luan, Frank J Gonzalez, Kalipada Pahan
Peroxisome proliferator-activated receptor-α (PPARα) regulates hepatic fatty acid catabolism and mediates the metabolic response to starvation. Recently we found that PPARα is constitutively activated in nuclei of hippocampal neurons and controls plasticity via direct transcriptional activation of CREB. Here we report the discovery of three endogenous PPARα ligands-3-hydroxy-(2,2)-dimethyl butyrate, hexadecanamide, and 9-octadecenamide-in mouse brain hippocampus. Mass spectrometric detection of these compounds in mouse hippocampal nuclear extracts, in silico interaction studies, time-resolved FRET analyses, and thermal shift assay results clearly indicated that these three compounds served as ligands of PPARα...
October 17, 2016: Nature Chemical Biology
Gnanasambandan Ramanathan, Santu Ghosh, Ramprasad Elumalai, Soundararajan Periyasamy, Bhaskar V K S Lakkakula
BACKGROUND & OBJECTIVES: Autosomal dominant polycystic kidney disease (ADPKD) is an inherited systemic disorder, characterized by the fluid filled cysts in the kidneys leading to end stage renal failure in later years of life. Hypertension is one of the major factors independently contributing to the chronic kidney disease (CKD) progression. The renin-angiotensin aldosterone system (RAAS) genes have been extensively studied as hypertension candidate genes. The aim of the present study was to investigate the role of angiotensin converting enzyme tagging - single nucleotide polymorphisms (ACE tag-SNPs) in progression of CKD in patients with ADPKD...
June 2016: Indian Journal of Medical Research
Liqin Wang, Miao Xu, Noel Southall, Wei Zheng, Shuishu Wang
Tuberculosis (TB) still kills over 1.5 million people per year despite available anti-TB drugs. The emergence of drug-resistant TB poses a major threat to public health and prompts for an urgent need for new and more effective drugs. The long duration needed to treat TB by the current TB drugs, which target the essential cellular activities, inevitably leads to the emergence of drug-resistance. PhoP of Mycobacterium tuberculosis (MTB), an essential virulence factor, is a novel target for drug development. Guided by the crystal structure of the PhoP-DNA complex, we designed and developed a robust high-throughput screening assay for identification of PhoP inhibitors that disrupt the PhoP-DNA binding...
October 10, 2016: Combinatorial Chemistry & High Throughput Screening
Yanjun Wang, Luzhu Yang, Baoxin Li, Yan Jin
As a universal biomarker, telomerase is one of the promising targets for cancer diagnosis and therapy. Therefore, it is meaningful to develop facile, robust and sensitive methods for evaluation of telomerase activity. Herein, combined with fluorescence resonance energy transfer (FRET), we creatively designed a gold nanorod (GNR)-based FRET method to detect telomerase activity from cell extracts. As the signal probe, carboxyfluorescein-modified DNA probes (F-DNA) hold negative electricity. The electrostatic interaction between F-DNA and positively charged GNRs makes F-DNA close to GNRs, which leads to weak FRET between the F-DNA and GNRs...
October 17, 2016: Analyst
Tapas Paul, Subhas Chandra Bera, Nidhi Agnihotri, Padmaja P Mishra
A remarkable observation about adsorption and desorption mechanism of single-stranded oligo nucleotides and hybridization of double stranded DNA (ds-DNA) on graphene oxide (GO) surface has been monitored using ensemble and single molecule fluorescence methods. The probe and target DNA, both labeled individually with fluorescence resonance energy transfer (FRET) pair and having similar adsorption affinity towards the GO surface are used to provide the detailed insight into the hybridization mechanism. The single molecule-FRET (sm-FRET) results reveals the DNA hybridization mechanism to happen "in situ", i...
October 17, 2016: Journal of Physical Chemistry. B
Ana V Ferreira, Ilana Perelshtein, Nina Perkas, Aharon Gedanken, Joana Cunha, Artur Cavaco-Paulo
Chronic wound fluids have elevated concentration of human neutrophil elastase (HNE) which can be used as inflammation/infection marker. Our goal is to develop functional materials for fast diagnosis of wound inflammation/infection by using HNE as a specific marker. For that, fluorogenic peptides with a HNE-specific cleavage sequence were incorporated into traditional textile dressings, to allow real-time detection of the wound status. Two different fluorogenic approaches were studied in terms of intensity of the signal generated upon HNE addition: a fluorophore 7-amino-4-trifluormethylcoumarin (AFC) conjugated to a HNE-specific peptide and two fluorophore/quencher pairs (FAM/Dabcyl and EDANS/Dabcyl) coupled to a similar peptide as a Förster resonance energy transfer (FRET) strategy...
October 15, 2016: Applied Microbiology and Biotechnology
I Velázquez-López, E León-Cruz, J P Pardo, A Sosa-Peinado, M González-Andrade
Eight new fluorescent biosensors of human calmodulin (hCaM) using Alexa Fluor(®) 350, 488, 532, and 555 dyes were constructed. These biosensors are thermodynamically stable, functional, and highly sensitive to ligands of the CaM. They resolve the problem of CaM ligands with similar spectroscopic properties to the intrinsic and extrinsic fluorophores of other biosensors previously reported. Additionally, they can be used in studies of protein-protein interaction through Förster resonance energy transfer (FRET)...
October 12, 2016: Analytical Biochemistry
Oliver G Weingart, Martin J Loessner
Botulinum neurotoxins (BoNT) are the most toxic substances known, and their neurotoxic properties and paralysing effects are exploited for medical treatment of a wide spectrum of disorders. To accurately quantify the potency of a pharmaceutical BoNT preparation, its physiological key activities (binding to membrane receptor, translocation, and proteolytic degradation of SNARE proteins) need to be determined. To date, this was only possible using animal models, or, to a limited extent, cell-based assays. We here report a novel in vitro system for BoNT/B analysis, based on nerve-cell mimicking liposomes presenting motoneuronal membrane receptors required for BoNT binding...
October 12, 2016: Toxicology and Applied Pharmacology
Chan-I Chung, Ryoji Makino, Yuki Ohmuro-Matsuyama, Hiroshi Ueda
Fluorescence-based biosensor probes, especially those based on Förster resonance energy transfer (FRET) between fluorescent protein (FP) variants, are widely used to monitor various biological phenomena, often detecting ligand-induced association of the receptor domains. While antibodies are fertile sources of specific receptors for various biomolecules, their potential has not been fully exploited. In this study, we used a fluorescent probe comprising FP-fused antibody variable region fragments to detect a bone metabolism biomarker, osteocalcin (BGP), by using fluorescence spectrometry/microscopy...
October 12, 2016: Journal of Bioscience and Bioengineering
Elizaveta A Kovrigina, Brian Pattengale, Chuanwu Xia, Azamat R Galiakhmetov, Jier Huang, Jung-Ja P Kim, Evgenii L Kovrigin
NADPH-cytochrome P450 oxidoreductase (CYPOR) was demonstrated to undergo large conformational rearrangements in its functional cycle. Using a new FRET approach based on femtosecond transient absorption spectroscopy (TA), we determined the donor-acceptor distance distribution in the reduced and oxidized states of CYPOR. Unmatched time resolution of TA enabled the quantitative assessment of the donor-acceptor FRET indicating that CYPOR assumes a closed conformation in both reduced and oxidized states in the absence of the redox partner...
October 14, 2016: Biochemistry
Luke J Higgins, Cristian A Marocico, Vasilios D Karanikolas, Alan P Bell, John J Gough, Graham P Murphy, Peter J Parbrook, A Louise Bradley
A range of seven different Ag plasmonic arrays formed using nanostructures of varying shape, size and gap were fabricated using helium-ion lithography (HIL) on an InGaN/GaN quantum well (QW) substrate. The influence of the array geometry on plasmon-enhanced Förster resonance energy transfer (FRET) from a single InGaN QW to a ∼80 nm layer of CdSe/ZnS quantum dots (QDs) embedded in a poly(methyl methacrylate) (PMMA) matrix is investigated. It is shown that the energy transfer efficiency is strongly dependent on the array properties and an efficiency of ∼51% is observed for a nanoring array...
October 14, 2016: Nanoscale
Myriam A Badr, Jose R Pinto, Michael W Davidson, P Bryant Chase
Cardiac troponin C (cTnC) is a key effector in cardiac muscle excitation-contraction coupling as the Ca2+ sensing subunit responsible for controlling contraction. In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP) and an acceptor fluorescent protein (YFP). The sensors report Ca2+ and Mg2+ binding, and relay global structural information about the structural relationship between cTnC's N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca2+ binding in the presence or absence of Mg2+...
2016: PloS One
Thomas E Kraft, Monique R Heitmeier, Marina Putanko, Rachel L Edwards, Ma Xenia G Ilagan, Maria A Payne, Joseph M Autry, David D Thomas, Audrey R Odom, Paul W Hruz
The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose-sensor...
October 10, 2016: Antimicrobial Agents and Chemotherapy
Jiang Zhang, Fangrui Lin, Liuying Chai, Lichun Wei, Tongsheng Chen
No abstract text is available yet for this article.
October 1, 2016: Journal of Biomedical Optics
Yuanwei Wang, Yihua Zhu, Jianfei Huang, Jin Cai, Jingrun Zhu, Xiaoling Yang, Jianhua Shen, Hao Jiang, Chunzhong Li
Perovskite quantum dots with superior optical performances are supposed to be desirable for fluorescence resonance energy transfer (FRET) based fluorescence detection, which is popular for its accuracy and simplicity. In this work, a monolithic superhydrophobic polystyrene fiber membrane encapsulated with CsPbBr3 perovskite quantum dots (CPBQDs/PS FM) was prepared via electrospinning. Combining the outstanding optical properties of CPBQDs and stability of PS, this CPBQDs/PS FM exhibits high quantum yields (~91%) and narrow half-peak width (~16 nm), nearly 100% fluorescence retention after being immersed in water for 10 days, 79...
October 13, 2016: Journal of Physical Chemistry Letters
Pingsheng Huang, Huijuan Song, Yumin Zhang, Jinjian Liu, Ju Zhang, Weiwei Wang, Jianfeng Liu, Chen Li, Deling Kong
The objective of this study was to investigate the spatiotemporal delivery of nanomedicines by an injectable, thermosensitive and nanoparticle self-aggregated hydrogel for peritumoral chemotherapy. Doxorubicin (Dox) was taken as the model medicine, which was encapsulated into poly(ε-caprolactone-co- 1,4,8-trioxa[4.6]spiro-9-undecanone)-poly(ethylene glycol)-poly(ε-caprolactone-co -1,4,8-trioxa[4.6]spiro-9-undecanone) (PECT) nanoparticles (PECT/Dox NPs). Macroscale hydrogel was formed by thermosensitive self-aggregation of PECT/Dox NPs in aqueous solution...
October 12, 2016: ACS Applied Materials & Interfaces
Yitao Feng, Lu Zhang, Shaowen Wu, Zhijun Liu, Xin Gao, Xu Zhang, Maili Liu, Jianwei Liu, Xuhui Huang, Wenning Wang
The glutamine binding protein (GlnBP) binds l-glutamine and cooperates with its cognate transporters during glutamine uptake. Crystal structure analysis has revealed an open and a closed conformation for apo- and holo-GlnBP, respectively. However, the detailed conformational dynamics have remained unclear. Herein, we combined NMR spectroscopy, MD simulations, and single-molecule FRET techniques to decipher the conformational dynamics of apo-GlnBP. The NMR residual dipolar couplings of apo-GlnBP were in good agreement with a MD-derived structure ensemble consisting of four metastable states...
October 12, 2016: Angewandte Chemie
Joachim Behar, Ambhighainath Ganesan, Jin Zhang, Yael Yaniv
Sinoatrial nodal cells (SANCs) generate spontaneous action potentials (APs) that control the cardiac rate. The brain modulates SANC automaticity, via the autonomic nervous system, by stimulating membrane receptors that activate (adrenergic) or inactivate (cholinergic) adenylyl cyclase (AC). However, these opposing afferents are not simply additive. We showed that activation of adrenergic signaling increases AC-cAMP/PKA signaling, which mediates the increase in the SANC AP firing rate (i.e., positive chronotropic modulation)...
2016: Frontiers in Physiology
Aruna K Mora, Sushant Murudkar, A Alamelu, Prabhat K Singh, Subrata Chattopadhyay, Sukhendu Nath
Early detection of amyloid fibrils is very important for the timely diagnosis of several neurological diseases. Thioflavin-T (ThT) is a gold standard fluorescent probe for amyloid fibrils and has been used for the last few decades. However, due to its positive charge, ThT is incapable of crossing the blood-brain barrier and cannot be used for in vivo imaging of fibrils. In the present work, we synthesized a neutral ThT derivative, 2-[2'-Me,4'-(dimethylamino)phenyl]benzothiazole (2Me-DABT), which showed a strong affinity towards the amyloid fibrils...
October 11, 2016: Chemistry: a European Journal
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