In this work, we present the synthesis and application of fluorescent rhodamine B hydrazide for the derivatization of simple oligosaccharides and complex glycans using a hydrazone formation chemistry approach. The labeling conditions and the experimental setup of CE/LIF were optimized by analyzing oligosaccharide standards. The CE/LIF separations were performed in polybrene-coated capillaries eliminating the need for the purification step after derivatization. The addition of methanol to the background electrolyte significantly increased the LIF detection sensitivity reaching the limits of detection in the attomole range. The resolution of carbohydrate samples was improved by using long (98 cm) capillaries and polymer additives (polybrene). The developed method was applied for CE/LIF and CE-MS analysis of N-linked glycans released from bovine ribonuclease B and the therapeutic monoclonal antibody of trastuzumab.
- Klíčová slova
- Capillary electrophoresis, Fluorescence, Glycan, Labeling, Mass spectrometry, Rhodamine B hydrazide,
- MeSH
- elektroforéza kapilární * metody MeSH
- fluorescenční barviva * chemie MeSH
- fluorescenční spektrometrie metody MeSH
- hmotnostní spektrometrie metody MeSH
- lasery MeSH
- oligosacharidy * chemie analýza MeSH
- polysacharidy * analýza chemie MeSH
- rhodaminy * chemie MeSH
- skot MeSH
- trastuzumab chemie analýza MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fluorescenční barviva * MeSH
- oligosacharidy * MeSH
- polysacharidy * MeSH
- rhodamine B MeSH Prohlížeč
- rhodaminy * MeSH
- trastuzumab MeSH
The placenta plays a critical role in maternal-fetal nutrient transport and fetal protection against drugs. Creating physiological in vitro models to study these processes is crucial, but technically challenging. This study introduces an efficient cell model that mimics the human placental barrier using co-cultures of primary trophoblasts and primary human umbilical vein endothelial cells (HUVEC) on a Transwell®-based system. Monolayer formation was examined over 7 days by determining transepithelial electrical resistance (TEER), permeability of Lucifer yellow (LY) and inulin, localization of transport proteins at the trophoblast membrane (immunofluorescence), and syncytialization markers (RT-qPCR/ELISA). We analysed diffusion-based (caffeine/antipyrine) and transport-based (leucine/Rhodamine-123) processes to study the transfer of physiologically relevant compounds. The latter relies on the adequate localization and function of the amino-acid transporter LAT1 and the drug transporter P-glycoprotein (P-gp) which were studied by immunofluorescence microscopy and application of respective inhibitors (2-Amino-2-norbornanecarboxylic acid (BCH) for LAT1; cyclosporine-A for P-gp). The formation of functional monolayer(s) was confirmed by increasing TEER values, low LY transfer rates, minimal inulin leakage, and appropriate expression/release of syncytialization markers. These results were supported by microscopic monitoring of monolayer formation. LAT1 was identified on the apical and basal sides of the trophoblast monolayer, while P-gp was apically localized. Transport assays confirmed the inhibition of LAT1 by BCH, reducing both intracellular leucine levels and leucine transport to the basal compartment. Inhibiting P-gp with cyclosporine-A increased intracellular Rhodamine-123 concentrations. Our in vitro model mimics key aspects of the human placental barrier. It represents a powerful tool to study nutrient and drug transport mechanisms across the placenta, assisting in evaluating safer pregnancy therapies.
- Klíčová slova
- LAT1, P‐gp, co‐culture, endothelial cell, placental barrier, polarized monolayer, primary trophoblast, transport,
- MeSH
- biologické modely MeSH
- biologický transport MeSH
- endoteliální buňky pupečníkové žíly (lidské) * metabolismus MeSH
- inulin metabolismus MeSH
- isochinoliny MeSH
- kokultivační techniky MeSH
- leucin metabolismus MeSH
- lidé MeSH
- maternofetální výměna látek * MeSH
- P-glykoprotein metabolismus MeSH
- placenta * metabolismus MeSH
- rhodamin 123 metabolismus MeSH
- těhotenství MeSH
- trofoblasty * metabolismus MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- inulin MeSH
- isochinoliny MeSH
- leucin MeSH
- lucifer yellow MeSH Prohlížeč
- P-glykoprotein MeSH
- rhodamin 123 MeSH
Polymersomes are synthetic vesicles with potential use in healthcare, chemical transformations in confined environment (nanofactories), and in the construction of artificial cells and organelles. In this framework, one of the most important features of such supramolecular structures is the permeability behavior allowing for selective control of mass exchange between the inner and outer compartments. The use of biological and synthetic nanopores in this regard is the most common strategy to impart permeability nevertheless, this typically requires fairly complex strategies to enable porosity. Yet, investigations concerning the permeability of polymer vesicles to different analytes still requires further exploration and, taking these considerations into account, we have detailed investigated the permeability behavior of a variety of polymersomes with regard to different analytes (water, protons, and rhodamine B) which were selected as models for solvents, ions, and small molecules. Polymersomes based on hydrophilic blocks of poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) or PEO (poly(ethylene oxide)) linked to the non-responsive blocks poly[N-(4-isopropylphenylacetamide)ethyl methacrylate] (PPPhA) or poly(methyl methacrylate) (PMMA), or to the stimuli pH-responsive block poly[2-(diisopropylamino)ethyl methacrylate] (PDPA) have been investigated. Interestingly, the produced PEO-based vesicles are notably larger than the ones produced using PHPMA-containing block copolymers. The experimental results reveal that all the vesicles are inherently permeable to some extent with permeability behavior following exponential profiles. Nevertheless, polymersomes based on PMMA as the hydrophobic component were demonstrated to be the least permeable to the small molecule rhodamine B as well as to water. The synthetic vesicles based on the pH-responsive PDPA block exhibited restrictive and notably slow proton permeability as attributed to partial chain protonation upon acidification of the medium. The dye permeability was evidenced to be much slower than ion or solvent diffusion, and in the case of pH-responsive assemblies, it was demonstrated to also depend on the ionic strength of the environment. These findings are understood to be highly relevant towards polymer selection for the production of synthetic vesicles with selective and time-dependent permeability, and it may thus contribute in advancing biomimicry and nanomedicine.
- Klíčová slova
- Block copolymers, Permeability, Polymersomes, Self-assembly,
- MeSH
- hydrofobní a hydrofilní interakce MeSH
- koncentrace vodíkových iontů MeSH
- permeabilita * MeSH
- polymery * chemie MeSH
- povrchové vlastnosti MeSH
- rhodaminy * chemie MeSH
- umělé buňky chemie MeSH
- velikost částic MeSH
- voda chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- polymery * MeSH
- rhodamine B MeSH Prohlížeč
- rhodaminy * MeSH
- voda MeSH
An in-depth investigation was conducted on a promising composite material (BiVO4/TiO2), focusing on its potential toxicity, photoinduced catalytic properties, as well as its antibiofilm and antimicrobial functionalities. The preparation process involved the synthesis of 2D TiO2 using the lyophilization method, which was subsequently functionalized with sphere-like BiVO4 through wet impregnation. Finally, we developed BiVO4/TiO2 S-scheme heterojunctions which can greatly promote the separation of electron-hole pairs to achieve high photocatalytic performance. The evaluation of concentration- and time-dependent viability inhibition was performed on human lung carcinoma epithelial A549 cells. This assessment included the estimation of glutathione levels and mitochondrial dehydrogenase activity. Significantly, the BiVO4/TiO2 composite demonstrated minimal toxicity towards A549 cells. Impressively, the BiVO4/TiO2 composite exhibited notable photocatalytic performance in the degradation of rhodamine B (k=0.135 min-1) and phenol (k=0.016 min-1). In terms of photoinduced antimicrobial performance, the composite effectively inactivated both gram-negative E. coli and gram-positive E. faecalis bacteria upon 60 minutes of UV-A light exposure, resulting in a significant log 6 (log 10 CFU/mL) reduction in bacterial count. In addition, a 49 % reduction of E. faecalis biofilm was observed. These promising results can be attributed to the unique 2D morphology of TiO2 modified by sphere-like BiVO4, leading to an increased generation of (intracellular) hydroxyl radicals, which plays a crucial role in the treatments of both organic pollutants and bacteria. This research has significant potential for various applications, particularly in addressing environmental contamination and microbial infections.
- Klíčová slova
- 2D TiO2, antibiofilm, antimicrobial, cytotoxicity, photocatalysis, sphere-like BiVO4,
- MeSH
- antibakteriální látky * chemie farmakologie MeSH
- biofilmy účinky léků MeSH
- bismut * chemie MeSH
- buňky A549 MeSH
- Enterococcus faecalis účinky léků MeSH
- Escherichia coli * účinky léků MeSH
- fotochemické procesy * MeSH
- fotolýza MeSH
- katalýza MeSH
- lidé MeSH
- nanostruktury chemie MeSH
- rhodaminy chemie MeSH
- titan * chemie MeSH
- vanadáty * chemie farmakologie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky * MeSH
- bismut * MeSH
- bismuth vanadium tetraoxide MeSH Prohlížeč
- rhodamine B MeSH Prohlížeč
- rhodaminy MeSH
- titan * MeSH
- titanium dioxide MeSH Prohlížeč
- vanadáty * MeSH
Physiology-based pharmacokinetic modeling suggests that rifabutin can out-balance P-glycoprotein (P-gp) induction by concurrent P-gp inhibition. However, clinical or experimental evidence for this Janus-faced rifabutin effect is missing. Consequently, LS180 cells were exposed to a moderately (2 µM) and strongly (10 µM) P-gp-inducing concentration of rifampicin or rifabutin for 6 days. Cellular accumulation of the fluorescent P-gp substrate rhodamine 123 was evaluated using flow cytometry, either without (induction only) or with adding rifamycin drug to the cells during the rhodamine 123 efflux phase (induction + potential inhibition). Rhodamine 123 accumulation was decreased similarly by both drugs after 6-day exposure (2 µM: 55% residual fluorescence compared to non-induced cells, P < 0.01; 10 µM: 30% residual fluorescence compared to non-induced cells, P < 0.001), indicating P-gp induction. Rhodamine 123 influx transporters mRNA expressions were not affected, excluding off-target effects. Acute re-exposure to rifabutin, however, considerably re-increased rhodamine 123 accumulation (2 µM induction: re-increase by 55%, P < 0.01; 10 µM induction: 49% re-increase, P < 0.001), suggesting P-gp inhibition. In contrast, rifampicin only had weak effects (2 µM induction: no re-increase; 10 µM induction: 16% re-increase; P < 0.05). Molecular docking analysis eventually revealed that rifabutin has a higher binding affinity to the inhibitor binding site of P-gp than rifampicin (ΔG (kcal/mol) = -11.5 vs -5.3). Together, this study demonstrates that rifabutin can at least partly mask P-gp induction by P-gp inhibition, mediated by high affinity binding to the inhibitory site of P-gp.
- Klíčová slova
- Induction, Inhibition, P-glycoprotein, Rifabutin, Rifampicin,
- MeSH
- P-glykoprotein metabolismus MeSH
- rhodamin 123 metabolismus MeSH
- rifabutin * farmakologie MeSH
- rifampin * farmakologie MeSH
- simulace molekulového dockingu MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- P-glykoprotein MeSH
- rhodamin 123 MeSH
- rifabutin * MeSH
- rifampin * MeSH
The present study describes the enhanced photodegradation of organic pollutant dyes, congo red (CR) and rhodamine B (RhB) dyes under visible light irradiation. AgIO4 nanorods decorated on Bi2S3 nanoflakes in various proportions were synthesized via sono-chemical route wherein the deposition of varying amounts of AgIO4 on Bi2S3 plays a pivotal role in improving the photodegradation ability. The characterization of the as-synthesized nanohybrids was assessed by XRD, UV-vis DRS, PL, EIS, ESR, FT-IR, XPS, HR-TEM, FE-SEM, N2 adsorption and desorption techniques. The effect of initial CR and RhB dye concentration, reaction pH and usage of nanohybrid concentration were investigated where 30%-AgIO4/Bi2S3 exhibited excellent visible light photodegradation of 95.58% for CR and 96.11% for RhB dyes at 140 min and 100 min respectively. The superoxide (•O2-) and hydroxyl radicals (•OH) played predominant role in the photodegradation of CR and RhB which is experimentally confirmed by radical trapping experiments. Also, the photocatalysts exhibited good photo stability and excellent reusability. The TOC analysis confirmed the complete mineralization of CR and RhB dyes by the nanohybrid and the formation of possible intermediate and degradation pathway was delineated based on GC/MS analysis. The outstanding photodegradation performance were ascribed to the Z-scheme charge transfer path, which effectively promotes the separation and transfer of e-/h+ pairs, resulting in a strong redox activity of the accumulated charge to decompose organic dyes during the degradation reaction. The study suggested that the nanohybrid can be utilized for the removal of organic pollutants from the contaminated water bodies.
- Klíčová slova
- Bismuth sulphide, Congo red, Photodegradation, Rhodamine B, Silver iodate, Z-scheme,
- MeSH
- fotolýza MeSH
- katalýza MeSH
- Kongo červeň * MeSH
- nanotrubičky * MeSH
- rhodaminy MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- světlo MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- Kongo červeň * MeSH
- rhodamine B MeSH Prohlížeč
- rhodaminy MeSH
A new design of permeation module based on 3D printing was developed to monitor the interaction of exogenic compounds with cell membrane transporters in real-time. The fluorescent marker Rhodamine 123 (Rho123) was applied as a substrate to study the activity of the P-glycoprotein membrane transporter using the MDCKII-MDR1 genetically modified cell line. In addition, the inhibitory effect of verapamil (Ver), a prototype P-glycoprotein inhibitor, was examined in the module, demonstrating an enhanced Rho123 transfer and accumulation into cells as well as the applicability of the module for P-glycoprotein inhibitor testing. Inhibition was demonstrated for different ratios of Rho123 and Ver, and their competition in terms of interaction with the P-glycoprotein transporter was monitored in real-time. Employing the 3D-printed module, permeation testing was shortened from 8 h in the conventional module to 2 h and evaluation based on kinetic profiles in every 10 min was possible in both donor and acceptor compartments. We also show that monitoring Rho123 levels in both compartments enables calculate the amount of Rho123 accumulated inside cells without the need of cell lysis.
- Klíčová slova
- 3D printing, Cell membrane transporter, P-glycoprotein, Permeation, Real-time monitoring, Sequential injection analysis,
- MeSH
- 3D tisk MeSH
- buňky MDCK MeSH
- membránové transportní proteiny * MeSH
- P-glykoprotein * MeSH
- psi MeSH
- rhodamin 123 MeSH
- verapamil MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- membránové transportní proteiny * MeSH
- P-glykoprotein * MeSH
- rhodamin 123 MeSH
- verapamil MeSH
Herein, we introduce versatile molecular tools that enable specific delivery and visualization of photoswitchable lipids at cellular membranes, namely at the plasma membrane and internal membranes. These molecules were prepared by tethering ortho-nitrobenzyl-based fluorescent cages with a signaling lipid bearing an azobenzene photoswitch. They permit two sequential photocontrolled reactions, which are uncaging of a lipid analogue and then its repeated activation and deactivation. We used these molecules to activate GPR40 receptor transiently expressed in HeLa cells and demonstrated downstream modulation of intracellular Ca2+ levels.
- MeSH
- azosloučeniny chemie účinky záření MeSH
- fluorescenční barviva chemie účinky záření MeSH
- fluorescenční mikroskopie MeSH
- HeLa buňky MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- receptory spřažené s G-proteiny metabolismus MeSH
- rhodaminy chemie účinky záření MeSH
- ultrafialové záření MeSH
- vápník metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- azosloučeniny MeSH
- FFAR1 protein, human MeSH Prohlížeč
- fluorescenční barviva MeSH
- receptory spřažené s G-proteiny MeSH
- rhodaminy MeSH
- vápník MeSH
- zelené fluorescenční proteiny MeSH
Squalene is a naturally occurring triterpene with wide industrial applications. Due to limited natural resources, production of this valuable lipid in yeast is of high commercial relevance. Typically low levels of squalene in yeast can be significantly increased by specific cultivation conditions or genetic modifications. Under normal conditions, excess squalene is stored in lipid droplets (LD), while in a Saccharomyces cerevisiae mutant unable to form LD it is distributed to cellular membranes. We present here the evidence that squalene accumulation in this LD-less mutant treated with squalene monooxygenase inhibitor terbinafine induces growth defects and loss of viability. We show that plasma membrane malfunction is involved in squalene toxicity. We have found that subinhibitory concentrations of terbinafine increased the sensitivity of LD-less mutant to several membrane-active substances. Furthermore, squalene accumulation in terbinafine-treated LD-less cells disturbed the maintenance of membrane potential and increased plasma membrane permeability to rhodamine 6G. LD-less cells treated with terbinafine showed also high sensitivity to osmotic stress. To confirm the causal relationship between squalene accumulation, loss of viability and impaired plasma membrane functions we treated LD-less cells simultaneously with terbinafine and squalene synthase inhibitor zaragozic acid. Reduction of squalene levels by zaragozic acid improved cell growth and viability and decreased plasma membrane permeability to rhodamine 6G in terbinafine-treated LD-less cells. Our results support the hypothesis that plasma membrane malfunction is involved in the mechanisms of squalene lipotoxicity in yeast cells with defective lipid storage.
- Klíčová slova
- lipid droplet, lipotoxicity, plasma membrane, squalene, yeast,
- MeSH
- buněčná membrána patologie MeSH
- lipidová tělíska chemie MeSH
- permeabilita buněčné membrány MeSH
- rhodaminy farmakologie MeSH
- Saccharomyces cerevisiae účinky léků genetika MeSH
- skvalen toxicita MeSH
- terbinafin farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- rhodamine 6G MeSH Prohlížeč
- rhodaminy MeSH
- skvalen MeSH
- terbinafin MeSH
Humic acids are often regarded as substances with a supramolecular structure which plays an important role in Nature. Their addition into hydrogels can affect their behavior and functioning in different applications. This work is focused on the properties of widely-used hydrogel based on agarose after addition of humic acids-the protonated H-form of humic acids and humic acids with methylated carboxylic groups. Hydrogels enriched by humic acids were studied in terms of their viscoelastic and transport properties. Rotational rheometry and methods employing diffusion cells were used in order to describe the influence of humic acids on the properties and behavior of hydrogels. From the point of view of rheology the addition of humic acids mainly affected the loss modulus corresponding to the relaxation of hydrogel connected with its flow. In the case of diffusion experiments, the transport of dyes (methylene blue and rhodamine) and metal ions (copper and nickel) through the hydrogel was affected by interactions between humic acids and the diffusion probes. The time lag in the hydrogel enriched by humic acids was prolonged for copper, methylene blue and rhodamine. In contrast, the presence of humic acids in hydrogel slightly increased the mobility of nickel. The strongest influence of the methylation of humic acids on diffusion was observed for methylene blue.
- Klíčová slova
- diffusion, humic acid, hydrogel, rheology, secondary structure,
- MeSH
- huminové látky * MeSH
- hydrogely chemie MeSH
- měď chemie MeSH
- methylenová modř chemie MeSH
- nikl chemie MeSH
- reologie MeSH
- rhodaminy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- huminové látky * MeSH
- hydrogely MeSH
- měď MeSH
- methylenová modř MeSH
- nikl MeSH
- rhodaminy MeSH
