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Autofluorescence of Breast Cancer Proteins
摘要： Background: The intensity of the blood as well as tissue autofluorescence (fluorescence of endogenous fluorophores) shows current structure of protein mixture, its current conformation (native or denatured state), concentration or activity depending on the external and internal conditions. Nicotine amide adenine dinucleotide (NAD+) is a cofactor in redox reactions of glycolysis and the citric acid cycle in eukaryotic cells and it can be a marker of the intensity of mitochondria metabolism as well as the presence of oxygen in the cells. The concentration of reduced nicotinamide adenine dinucleotide (NADH) varies during normoxia, hypoxia and hyperoxia cells. Cancer cells increase the concentration of NADH during hypoxia and anoxia. The reason of NADH cumulation is microenvironment with low or no oxygen supply which induces glycolysis as a preferential source of energy. Glycolysis is faster, does not need oxygen but is less effective than oxidative phosphorylation. Focus: The aim of this work was to measure the structure of blood plasma and mammary gland homogenates of patients at three stages of breast cancer in comparison to healthy subjects using fluorescence analysis and atomic force microscopy. The blood plasma of patients with breast cancer had a different structure of proteins in comparison to healthy subjects. The blood plasma and homogenate of patients with breast cancer showed significant increase in autofluorescence intensity, which represents mixture of various endogenous fluorophores in particular porphyrins, collagen, NAD and flavins. Prospect: The information about complex summary of fluorescence intensity of all mixtures of endogenous fluorophores may in the future serve as rapid preliminary markers of cancer. The fluorescence analysis might be a non-traditional methodology for an early rapid diagnosis of breast cancer in the next clinical practice.
关键词： breast cancer proteins,blood plasma,Nicotine amide adenine dinucleotide,breast cancer homogenate,fluorophores,autofluorescence
更新于2025-09-04 15:30:14
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Simulation of Spectra of Red Fluorescent Protein Mutants
摘要： This paper presents the results of describing red fluorescent proteins using the combined quantum mechanics/molecular mechanics approach and describing the quantum-mechanical subsystem by the density functional tight-binding (DFTB) method. Based on the calculated vertical electronic transition energies, it is concluded that this method is suitable for estimating equilibrium geometry configurations, but it cannot be used for the subsequent estimation of vertical electronic transition energies.
关键词： QM/MM,DFTB,fluorescent proteins
更新于2025-09-04 15:30:14
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Choosing the right label for single-molecule tracking in live bacteria: side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes
摘要： Visualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins (PA-FPs). An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and results from single-molecule localization and tracking experiments. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.
关键词： Halo tag,single-molecule tracking,photoactivatable fluorescent protein,Escherichia coli,fluorophores,super-resolution microscopy,DNA-binding proteins
更新于2025-09-04 15:30:14
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Characterization of a DNA Aptamer for Ovarian Cancer Clinical Tissue Recognition and in Vivo Imaging
摘要： Backgrounds/Aims: Ovarian cancer is the most lethal gynaecologic malignancy and is difficult to detect early. The inefficient early diagnosis of ovarian cancer is the main contributor to its high mortality rate. Aptamers, as chemical antibodies, are single-stranded DNA or RNA oligonucleotides that target cells or molecules with high affinity. Methods: Binding ability of R13 was measured by flow cytometry analysis. Stability of R13 was tested in blood serum of an ovarian cancer patient. Internalization of R13 was verified by confocal microscope imaging. 80 cases ovarian cancer tissues, 10 cases normal ovary tissues in a microarray and 6 fallopian tube tissues were prepared for this study. R13’s target ability was further confirmed in vivo tumor models in NOD/SCID mice. Results: In this study, we found aptamer R13 bound to ovarian cancer cells with dissociation constants in the nanomolar range. Moreover, these results were further confirmed by tissue imaging. Next we demonstrated that the targets of R13 are membrane proteins and that its internalization occurs in a caveolae-mediated and clathrin-mediated manner. The target function of R13 was determined by imaging A2780 tumours in mouse models. Conclusion: These findings suggest that R13 is a promising novel tool to diagnose and deliver drugs to treat ovarian cancer.
关键词： Endocytosis,DNA aptamer,Membrane proteins,Ovarian cancer,Nude mouse,Tissue microarray
更新于2025-09-04 15:30:14
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RNA-protein UV-crosslinking Assay
摘要： RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria et al., 2016). Here we have described the protocol which (1) covalently links transiently interacting RNA-protein complexes by UV crosslinking, (2) removes the unprotected RNA by RNase digestion and (3) detects the RNA-protein complexes by SDS-PAGE analysis. This protocol provides a rapid and reliable means to directly assay RNA-protein interactions and their kinetics using purified proteins and also help in identifying novel RNA-protein interactions
关键词： UV-crosslinking,RNA-protein interaction,RNA-binding proteins
更新于2025-09-04 15:30:14