Search results for "Dye"

2016

RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from Bacteria, Archaea and Eukarya. RNAs bearing 2'-O-methylations show increased resistance to degradation and enhanced stability in helices. While the exact role of each 2'-O-Me residue remained elusive, the catalytic protein Fibrillarin (Nop1 in yeast) responsible for 2'-O-methylation in eukaryotes, is associated with human pathologies. Therefore, there is an urgent need to precisely map and quantify hundreds of 2'-O-Me residues in RNA using high-throughput technologies. Here, we develop a reliable protocol using alkaline fragmentation of total RNA coupled to a commonly used ligation approach, …

In vivo fluorescent cercariae reveal the entry portals of Cardiocephaloides longicollis (Rudolphi, 1819) Dubois, 1982 (Strigeidae) into the gilthead …

2019

Background Despite their complex life-cycles involving various types of hosts and free-living stages, digenean trematodes are becoming recurrent model systems. The infection and penetration strategy of the larval stages, i.e. cercariae, into the fish host is poorly understood and information regarding their entry portals is not well-known for most species. Cardiocephaloides longicollis (Rudolphi, 1819) Dubois, 1982 (Digenea, Strigeidae) uses the gilthead seabream (Sparus aurata L.), an important marine fish in Mediterranean aquaculture, as a second intermediate host, where they encyst in the brain as metacercariae. Labelling the cercariae with in vivo fluorescent dyes helped us to track the…

Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe.

2016

Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density,…

Double-exponential kinetics of binding and redistribution of the fluorescent dyes in cell membranes witness for the existence of lipid microdomains.

2018

Abstract New technique of detecting lateral heterogeneity of the plasma membrane of living cells by means of membrane-binding fluorescent dyes is proposed. The kinetics of dye incorporation into the membrane or its lateral diffusion inside the membrane is measured and decomposed into exponential components by means of the Maximum Entropy Method. Two distinct exponential components are obtained consistently in all cases for several fluorescent dyes, two different cell lines and in different types of experiments including spectroscopy, flow cytometry and fluorescence recovery after photobleaching. These components are attributed to the liquid-ordered and disordered phases in the plasma membra…

Single Particle Plasmon Sensors as Label-Free Technique To Monitor MinDE Protein Wave Propagation on Membranes.

2016

We use individual gold nanorods as pointlike detectors for the intrinsic dynamics of an oscillating biological system. We chose the pattern forming MinDE protein system from Escherichia coli (E. coli), a prominent example for self-organized chemical oscillations of membrane-associated proteins that are involved in the bacterial cell division process. Similar to surface plasmon resonance (SPR), the gold nanorods report changes in their protein surface coverage without the need for fluorescence labeling, a technique we refer to as NanoSPR. Comparing the dynamics for fluorescence labeled and unlabeled proteins, we find a reduction of the oscillation period by about 20%. The absence of photoble…

FRET-based method for evaluation of the efficiency of reversible and irreversible sonoporation.

2017

It is widely known that not all of the treated cells survive after introduction of exogenous molecules via any physical method. Therefore, it is important to develop methods that would allow simultaneous evaluation of both molecular delivery efficiency and cell viability. This study presents Forster resonance energy transfer (FRET)-based method that allows molecular transfer and cell viability evaluation in a single measurement by employing two common fluorescent dyes, namely, ethidium bromide and trypan blue. The method has been validated using cell sonoporation. The FRET-based method allows the efficiency evaluation of both reversible and irreversible sonoporation in a single experiment. …

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

2018

The hepatitis E virus (HEV) is an emerging pathogen showing a considerable increase in the number of reported cases in Europe mainly related to the ingestion of contaminated food. As with other relevant viral foodborne pathogens, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for HEV detection in clinical, food, and environmental samples, but these procedures cannot discriminate between inactivated and potentially infectious viruses. Thus, the aim of this study was to develop a viability PCR method to discriminate between native, heat-, and high-pressure processing (HPP)-treated HEV using the hepatitis A virus (HAV) as a cultivable surrogate. To thi…

Molecular docking-based design and development of a highly selective probe substrate for UDP-glucuronosyltransferase 1A10

2018

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescen…

Identification of accessory olfactory system and medial amygdala in the zebrafish

2017

AbstractZebrafish larvae imprint on visual and olfactory cues of their kin on day 5 and 6 postfertilization, respectively. Only imprinted (but not non-imprinted) larvae show strongly activated crypt (and some microvillous) cells demonstrated by pERK levels after subsequent exposure to kin odor. Here, we investigate the olfactory bulb of zebrafish larvae for activated neurons located at the sole glomerulus mdG2 which receives crypt cell input. Imprinted larvae show a significantly increased activation of olfactory bulb cells compared to non-imprinted larvae after exposure to kin odor. Surprisingly, pERK activated Orthopedia-positive cell numbers in the intermediate ventral telencephalic nucl…

Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples

2018

Shellfish contamination by human noroviruses (HuNoVs) is a serious health and economic problem. Recently an ISO procedure based on RT-qPCR for the quantitative detection of HuNoVs in shellfish has been issued, but these procedures cannot discriminate between inactivated and potentially infectious viruses. The aim of the present study was to optimize a pretreatment using PMAxx to better discriminate between intact and heat-treated HuNoVs in shellfish and sewage. To this end, the optimal conditions (30 min incubation with 100 μM of PMAxx and 0.5% of Triton, and double photoactivation) were applied to mussels, oysters and cockles artificially inoculated with thermally-inactivated (99 °C for 5 …