Search results for: confocal fluorescence microscopy

Authors: Andrey V. Khromov, Antonida V. Makhotenko, Ekaterina A. Snigir, Svetlana S. Makarova, Natalia O. Kalinina, Valentin V. Makarov, Mikhail E. Taliansky

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Due to its simplicity, CRISPR/Cas9 has become widely used and capable of inducing mutations in the genes of organisms of various kingdoms. The aim of this work was to develop applications for the efficient modification of DNA coding sequences of phytoene desaturase (PDS), coilin and vacuolar invertase (Solanum tuberosum) genes, and to develop a new nanoparticles carrier efficient technology to deliver the CRISPR/Cas9 system for editing the plant genome. For each of the genes - coilin, PDS and vacuolar invertase, five single RNA guide (sgRNAs) were synthesized. To determine the most suitable nanoplatform, two types of NP platforms were used: magnetic NPs (MNPS) and gold NPs (AuNPs). To test the penetration efficiency, they were functionalized with fluorescent agents - BSA * FITS and GFP, as well as labeled Cy3 small-sized RNA. To measure the efficiency, a fluorescence and confocal microscopy were used. It was shown that the best of these options were AuNP - both in the case of proteins and in the case of RNA. The next step was to check the possibility of delivering components of the CRISPR/Cas9 system to plant cells for editing target genes. AuNPs were functionalized with a ribonucleoprotein complex consisting of Cas9 and corresponding to target genes sgRNAs, and they were biolistically bombarded to axillary buds and apical meristems of potato plants. After the treatment by the best NP carrier, potato meristems were grown to adult plants. DNA isolated from this plants was sent to a preliminary fragment of the analysis to screen out the non-transformed samples, and then to the NGS. The present work was carried out with the financial support from the Russian Science Foundation (grant No. 16-16-04019).

Keywords: biobombardment, coilin, CRISPR/Cas9, nanoparticles, NPs, PDS, sgRNA, vacuolar invertase

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Authors: Waroton Paisuwan, Mongkol Sukwattanasinitt, Mamoru Tobisu, Anawat Ajavakom

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Novel dihydroquinoline derivatives (DHP and DHP-OH) were synthesized in one pot via a tandem trimerization-cyclization of methylpropiolate. DHP and DHP-OH possess strong blue fluorescence with high quantum efficiencies over 0.70 in aqueous media. DHP-OH displays a remarkable fluorescence quenching selectively to the presence of Au3+ through the oxidation of dihydropyridine to pyridinium ion as confirmed by NMR and HRMS. DHP-OH was used to demonstrate the quantitative analysis of Au3+ in water samples with the limit of detection of 33 ppb and excellent recovery (>95%). This fluorescent probe was also applied for the determination of Au3+ residue in the gold nanoparticle solution and a paper-based sensing strip for the on-site detection of Au3+.

Keywords: Gold(III) ion detection, Fluorescent sensor, Fluorescence quenching, Dihydropyridine, Gold nanoparticles (AuNPs)

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Authors: Omar Dib, Rita Yaacoub, Luc Eveleigh, Nathalie Locquet, Hussein Dib, Ali Bassal, Christophe B. Y. Cordella

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The potential of front-face fluorescence coupled with chemometric techniques, namely parallel factor analysis (PARAFAC) and multiple linear regression (MLR) as a rapid analysis tool to characterize Lebanese virgin olive oils was investigated. Fluorescence fingerprints were acquired directly on 102 Lebanese virgin olive oil samples in the range of 280-540 nm in excitation and 280-700 nm in emission. A PARAFAC model with seven components was considered optimal with a residual of 99.64% and core consistency value of 78.65. The model revealed seven main fluorescence profiles in olive oil and was mainly associated with tocopherols, polyphenols, chlorophyllic compounds and oxidation/hydrolysis products. 23 MLR regression models based on PARAFAC scores were generated, the majority of which showed a good correlation coefficient (R > 0.7 for 12 predicted variables), thus satisfactory prediction performances. Acid values, peroxide values, and Delta K had the models with the highest predictions, with R values of 0.89, 0.84 and 0.81 respectively. Among fatty acids, linoleic and oleic acids were also highly predicted with R values of 0.8 and 0.76, respectively. Factors contributing to the model's construction were related to common fluorophores found in olive oil, mainly chlorophyll, polyphenols, and oxidation products. This study demonstrates the interest of front-face fluorescence as a promising tool for quality control of Lebanese virgin olive oils.

Keywords: front-face fluorescence, Lebanese virgin olive oils, multiple Linear regressions, PARAFAC analysis

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Authors: Amanda M. Tadini, Houssam Hajjoul, Gustavo Nicolodelli, Stéphane Mounier, Célia R. Montes, Débora M. B. P. Milori

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Soil organic matter (SOM) plays an important role in maintaining soil productivity and accounting for the promotion of biological diversity. The main components of the SOM are the humic substances which can be fractionated according to its solubility in humic acid (HA), fulvic acids (FA) and humin (HU). The determination of the chemical properties of organic matter as well as its interaction with metallic species is an important tool for understanding the structure of the humic fractions. Fluorescence spectroscopy has been studied as a source of information about what is happening at the molecular level in these compounds. Specially, soils of Amazon region are an important ecosystem of the planet. The aim of this study is to understand the molecular and structural composition of HA samples from Spodosol of Amazonia using the fluorescence Emission-Excitation Matrix (EEM) and Time Resolved Fluorescence Spectroscopy (TRFS). The results showed that the samples of HA showed two fluorescent components; one has a more complex structure and the other one has a simpler structure, which was also seen in TRFS through the evaluation of each sample lifetime. Thus, studies of this nature become important because it aims to evaluate the molecular and structural characteristics of the humic fractions in the region that is considered as one of the most important regions in the world, the Amazon.

Keywords: Amazonian soil, characterization, fluorescence, humic acid, lifetime

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Authors: Eva Filová, Jana Daňková, Věra Sovková, Matej Daniel

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Titanium alloys are biocompatible metals that are widely used in clinical practice as load bearing implants. The chemical modification may influence cell adhesion, proliferation, and differentiation as well as stiffness of the material. The aim of the study was to evaluate the adhesion, growth and differentiation of pig mesenchymal stem cells on the novel beta titanium alloy Ti36Nb6Ta compared to standard medical titanium alloy Ti6Al4V. Discs of Ti36Nb6Ta and Ti6Al4V alloy were sterilized by ethanol, put in 48-well plates, and seeded by pig mesenchymal stem cells at the density of 60×103/cm2 and cultured in Minimum essential medium (Sigma) supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cell viability was evaluated using MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay;Promega), cell proliferation using Quant-iT™ ds DNA Assay Kit (Life Technologies). Cells were stained immunohistochemically using monoclonal antibody beta-actin, and secondary antibody conjugated with AlexaFluor®488 and subsequently the spread area of cells was measured. Cell differentiation was evaluated by alkaline phosphatase assay using p-nitrophenyl phosphate (pNPP) as a substrate; the reaction was stopped by NaOH, and the absorbance was measured at 405 nm. Osteocalcin, specific bone marker was stained immunohistochemically and subsequently visualized using confocal microscopy; the fluorescence intensity was analyzed and quantified. Moreover, gene expression of osteogenic markers osteocalcin and type I collagen was evaluated by real-time reverse transcription-PCR (qRT-PCR). For statistical evaluation, One-way ANOVA followed by Student-Newman-Keuls Method was used. For qRT-PCR, the nonparametric Kruskal-Wallis Test and Dunn's Multiple Comparison Test were used. The absorbance in MTS assay was significantly higher on titanium alloy Ti6Al4V compared to beta titanium alloy Ti36Nb6Ta on days 7 and 14. Mesenchymal stem cells were well spread on both alloys, but no difference in spread area was found. No differences in alkaline phosphatase assay, fluorescence intensity of osteocalcin as well as the expression of type I collagen, and osteocalcin genes were observed. Higher expression of type I collagen compared to osteocalcin was observed for cells on both alloys. Both beta titanium alloy Ti36Nb6Ta and titanium alloy Ti6Al4V Ti36Nb6Ta supported mesenchymal stem cellsˈ adhesion, proliferation and osteogenic differentiation. Novel beta titanium alloys Ti36Nb6Ta is a promising material for bone implantation. The project was supported by the Czech Science Foundation: grant No. 16-14758S, the Grant Agency of the Charles University, grant No. 1246314 and by the Ministry of Education, Youth and Sports NPU I: LO1309.

Keywords: beta titanium, cell growth, mesenchymal stem cells, titanium alloy, implant

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Authors: Mona Hassan Aburahma, Alaa Hamed Salama

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Ufasomes “unsaturated fatty acids liposomes” are unique nano-sized self-assembled bilayered vesicles that can be easily created from the readily available unsaturated fatty acid. Ufasomes are formed due to weak associative interaction of the fully ionized and unionized fatty acids into bilayers structures. In the ufasomes constructs, the fatty acid molecules are oriented with their hydrocarbon tails directed toward the membrane interior and the carboxyl groups are in contact with water. Although ufasomes can be employed as a safe vesicular carrier for drugs, the extreme instability of their aqueous dispersions hinders their effective use in drug delivery field. Accordingly, in our study, lyophilized gels containing ufasomes were prepared using a simple assembling technique form the readily available oleic acid to overcome the colloidal instability of the ufasomes dispersions and convert them into accurate unit dosage forms. The influence of changing cholesterol percentage relative to oleic acid on the ufasomes vesicles were investigated using factorial design. The optimized oleic acid ufasomes comprised nanoscaled spherical vesicles. Scanning electron micrographs of the lyophilized gels revealed that the included ufasomes were intact, non-aggregating, and preserved their spherical morphology. Rheological characterization (viscosity and shear stress versus shear rate) of reconstituted ufasomal lyophilized gel ensured the ease of application. The capability of the ufasomes, included in the gel, to penetrate deep through the mucosa layers was illustrated using ex-vivo confocal laser imaging, thereby, highlighting the feasibility of stabilizing ufasomes using lyophilized gel platforms.

Keywords: ufasomes, lyophilized gel, confocal scanning microscopy, rheological characterization, oleic acid

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Authors: Przemyslaw J. Jodlowski, Roman J. Jedrzejczyk, Damian K. Chlebda, Anna Dziedzicka, Lukasz Kuterasinski, Anna Gancarczyk, Maciej Sitarz

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The aim of this study was to obtain highly active catalysts based on non-noble metal oxides supported on zirconia prepared via a sonochemical method. In this study, the influence of the stabilizers addition during the preparation step was checked. The final catalysts were characterized by using such characterization methods as X-ray Diffraction (XRD), nitrogen adsorption, X-ray fluorescence (XRF), scanning electron microscopy (SEM) equipped with energy dispersive X-ray spectrometer (EDS), transmission electron microscopy (TEM) and µRaman. The proposed preparation method allowed to obtain uniformly dispersed metal-oxide nanoparticles at the support’s surface. The catalytic activity of prepared catalyst samples was measured in a methane combustion reaction. The activity of the catalysts prepared by the sonochemical method was considerably higher than their counterparts prepared by the incipient wetness method.

Keywords: methane catalytic combustion, nanoparticles, non-noble metals, sonochemistry

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Authors: A. G. Pershina, P. S. Postnikov, M. E. Trusova, D. O. Burlakova, A. E. Sazonov

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Invention of magnetic-fluorescent nanocomposites is a rapidly developing area of research. The magnetic-fluorescent nanocomposite attractiveness is connected with the ability of simultaneous management and control of such nanocomposites by two independent methods based on different physical principles. These nanocomposites are applied for the solution of various essential scientific and experimental biomedical problems. The aim of this research is development of principle approach to nanobiohybrid structures with magnetic and fluorescent properties design. The surface of carbon-coated iron nanoparticles (Fe@C) were covalently modified by 4-carboxy benzenediazonium tosylate. Recombinant fluorescent protein TagGFP2 (Eurogen) was obtained in E. coli (Rosetta DE3) by standard laboratory techniques. Immobilization of TagGFP2 on the nanoparticles surface was provided by the carbodiimide activation. The amount of COOH-groups on the nanoparticle surface was estimated by elemental analysis (Elementar Vario Macro) and TGA-analysis (SDT Q600, TA Instruments. Obtained nanocomposites were analyzed by FTIR spectroscopy (Nicolet Thermo 5700) and fluorescence microscopy (AxioImager M1, Carl Zeiss). Amount of the protein immobilized on the modified nanoparticle surface was determined by fluorimetry (Cary Eclipse) and spectrophotometry (Unico 2800) with the help of preliminary obtained calibration plots. In the FTIR spectra of modified nanoparticles the adsorption band of –COOH group around 1700 cm-1 and bands in the region of 450-850 cm-1 caused by bending vibrations of benzene ring were observed. The calculated quantity of active groups on the surface was equal to 0,1 mmol/g of material. The carbodiimide activation of COOH-groups on nanoparticles surface results to covalent immobilization of TagGFP2 fluorescent protein (0.2 nmol/mg). The success of immobilization was proved by FTIR spectroscopy. Protein characteristic adsorption bands in the region of 1500-1600 cm-1 (amide I) were presented in the FTIR spectrum of nanocomposite. The fluorescence microscopy analysis shows that Fe@C-TagGFP2 nanocomposite possesses fluorescence properties. This fact confirms that TagGFP2 protein retains its conformation due to immobilization on nanoparticles surface. Magnetic-fluorescent nanocomposite was obtained as a result of unique design solution implementation – the fluorescent protein molecules were fixed to the surface of superparamagnetic carbon-coated iron nanoparticles using original diazonium salts.

Keywords: carbon-coated iron nanoparticles, diazonium salts, fluorescent protein, immobilization

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Authors: Stephanie Hernández-Carreón, Judith Virginia Ríos-Arana

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Microplastics (MPs) are plastic particles smaller than 5 mm that has been detected in soil, air, organisms, and mostly water around the world. Most studies have focused on MPs detection in marine waters, and less so in freshwater, such is the case of Mexico, where studies about MPs in freshwaters are limited. One of the most important rivers in the country is The Rio Grande/Río Bravo, a natural border between Mexico and the United States. Its waters serve different purposes, such as fishing, habitat to endemic species, electricity generation, agriculture, and drinking water sources, among others. Despite its importance, the river’s waters have not been analyzed to determine the presence of MPs; therefore, the purpose of this research is to determine if the Rio Bravo/Rio Grande is polluted with microplastics. For doing so, three sites (Borderland, Casa de Adobe, and Guadalupe) along the El Paso-Juárez metroplex have been sampled: 30 L of water were filtered through a plankton net (64 µm) in each site and sediments-composed samples were collected. Water samples and sediments were 1) digested with a hydrogen peroxide solution (30%), 2) resuspended in a calcium chloride solution (1.5 g/cm3) to separate MPs, and 3) filtered through a 0.45 µm nitrocellulose membrane. Processed water samples were dyed with Nile Red (1 mg/ml ethanol) and analyzed by fluorescence microscopy. Two water samples have been analyzed until January 2023: Casa de Adobe and Borderland finding a concentration of 5.67 particles/L and 5.93 particles/L, respectively. Three types of particles were observed: fibers, fragments, and films, fibers being the most abundant. These data, as well as the data obtained from the rest of the samples, will be analyzed by an ANOVA (α=0.05). The concentrations and types of particles found in the Río Bravo correspond with other studies on rivers associated with urban environments and agricultural activities in China, where a range of 3.67—10.7 particles/L was reported in the Wei River. Even though we are in the early stages of the study, and three new sites will be sampled and analyzed in 2023 to provide more data about this issue in the river, this presents the first evidence of microplastic pollution in the Rio Grande.

Keywords: microplastics, fresh water, Rio Bravo, fluorescence microscopy

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Authors: Francisco Pedroza-Montero, Jesús Naín Pedroza-Montero, Diego Soto-Puebla, Osiris Alvarez-Bajo, Beatriz Castaneda, Sofía Navarro-Espinoza, Martín Pedroza-Montero

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Nanodiamonds have been widely studied for their physical properties, including chemical inertness, biocompatibility, optical transparency from the ultraviolet to the infrared region, high thermal conductivity, and mechanical strength. In this work, we studied how the fluorescence spectrum of nanodiamonds quenches concerning the concentration in aqueous solutions systematically ranging from 0.1 to 10 mg/mL. Our results demonstrated a non-linear fluorescence quenching as the concentration increases for both of the NV zero-phonon lines; the 5 mg/mL concentration shows the maximum fluorescence emission. Furthermore, this behaviour is theoretically explained as an electronic recombination process that modulates the intensity in the NV centres. Finally, to gain more insight, the FRET methodology is used to determine the fluorescence efficiency in terms of the fluorophores' separation distance. Thus, the concentration level is simulated as follows, a small distance between nanodiamonds would be considered a highly concentrated system, whereas a large distance would mean a low concentrated one. Although the 5 mg/mL concentration shows the maximum intensity, our main interest is focused on the concentration of 0.5 mg/mL, which our studies demonstrate the optimal human cell viability (99%). In this respect, this concentration has the feature of being as biocompatible as water giving the possibility to internalize it in cells without harming the living media. To this end, not only can we track nanodiamonds on the surface or inside the cell with excellent precision due to their fluorescent intensity, but also, we can perform thermometry tests transforming a fluorescence contrast image into a temperature contrast image.

Keywords: nanodiamonds, fluorescence spectroscopy, concentration, bioimaging, thermometry

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Authors: Darshna Sharma, Suban K. Sahoo

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The detection of intracellular fluoride in human cancer cell HeLa was achieved by chemosensors derived from vitamin B6 cofactors using fluorescence imaging technique. These sensors were first synthesized by condensation of pyridoxal/pyridoxal phosphate with 2-amino(thio)phenol. The anion recognition ability was explored by experimental (UV-VIS, fluorescence and 1H NMR) and theoretical DFT [(B3LYP/6-31G(d,p)] methods in DMSO and mixed DMSO-H2O system. All the developed sensors showed both naked-eye detectable color change and remarkable fluorescence enhancement in the presence of F- and AcO-. The anion recognition was occurred through the formation of hydrogen bonded complexes between these anions and sensor, followed by the partial deprotonation of sensor. The detection limit of these sensors were down to micro(nano) molar level of F- and AcO-.

Keywords: chemosensors, fluoride, acetate, turn-on, live cells imaging, DFT

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Authors: Nioushasadat Haji Seyed Javadi, Ailar Hajimohammadi, Nasser Khalili

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The common method to improve the performance of asphalt binders is through modification. The utilization of recycled plastics for asphalt modification has been the subject of research studies due to their environmental and economic benefits over using commercial polymers. Polypropylene (PP) is one of the most available recycled plastics in Australia. Unlike other plastics, its contamination with other plastics during the recycling process is negligible. Therefore, the quality of recycled plastic is high, which makes it a good candidate for road construction applications. To assess its effectiveness for bitumen modification, three different grades of PP were selected. The PP grades were compared for blendability with bitumen, and the best suitable grade was chosen for further studies. The PP-modified bitumen and the base bitumen were then compared through physical and rheological properties. The stability of the PP-modified bitumen at elevated temperatures was measured, and the morphology of the samples before and after the storage stability was characterized by fluorescent microscopy. The results showed that PP had a significant influence on reducing the penetration and increasing the viscosity and the rutting resistance of the virgin bitumen. Storage stability test results indicated that the difference between the softening point of the top and bottom section of the tube sample is below the defined limit, which means the PP-modified bitumen is storage stable. However, the fluorescence microscopy results showed that the distribution of the PP particles in the bitumen matrix in the top and bottom sections of the tube are significantly different, which is an indicator of poor storage stability.

Keywords: polypropylene, waste plastic, bitumen, road pavements, storage stability, fluorescent microscopy, morphology

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Authors: Neha Singh

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Background: Nowadays, the nanoparticle is gaining unexceptional attention in targeted drug delivery. But before proceeding to this episode of accomplishment, the journey and closure of these nanoparticles inside the cells should be disentangle. Being foreign for the cells, nanoparticles will easily getcleared off without any effective outcome. As the cancer cells withhold these nanoparticles for a longer period of time, more will be the drug’s effect. Chlorpromazine is a cationic amphiphilic drug which is believed to inhibit clathrin-coated pit formation by a reversible translocation of clathrin and its adapter proteins from the plasma membrane to intracellular vesicles. Chlorpromazine has a role in increasing the retention of nanoparticles in cancer cells. The mechanism of action how this small molecule increases the retention of nanoparticles is still uncovered. Method: Polymeric nanoparticle (PLGA) with Cyanine3.5 dye were synthesized by solvent evaporation method and characterized for size and zeta potential. FTIR was also done. Pulse and chase studies with and without inhibitor were done to check the retention of nanoparticle using fluorescence microscopy. Mean fluorescence intensity was measured by ImageJ software. Results: Increased retention of nanoparticle with inhibitor was observed in both pulse and chase studies. Conclusion: Our results demonstrate that by repurposing these small molecule inhibitor, we can increase the retention of nanoparticle at the targeted site.

Keywords: nanoparticle, endocytosis, clathrin inhibitor, cancer cell

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Authors: T. Ferreira, A. Kulkarni, C. Bretscher, K. Richter, M. Ehrlich, A. Marchini

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H-1 protoparvovirus (H-1PV) is a virus with inherent oncolytic and oncosuppressive activities while remaining non-pathogenic in humans. H-1PV was the first oncolytic parvovirus to undergo clinical testing. Results from trials in patients with glioblastoma or pancreatic carcinoma showed an excellent safety profile and first signs of efficacy. H-1PV infection is vastly dependent on cellular factors, from cell attachment and entry to viral replication and egress. Hence, we believe that the characterisation of the parvovirus life cycle would ultimately help further improve H-1PV clinical outcome. In the present study, we explored the entry pathway of H-1PV in cervical HeLa and glioma NCH125 cancer cell lines. Electron and confocal microscopy showed viral particles associated with clathrin-coated pits and vesicles, providing the first evidence that H-1PV cell entry occurs through clathrin-mediated endocytosis. Accordingly, we observed that by blocking clathrin-mediated endocytosis with hypertonic sucrose, chlorpromazine, or pitstop 2, H-1PV transduction was markedly decreased. Accordingly, siRNA-mediated knockdown of AP2M1, which retains a crucial role in clathrin-mediated endocytosis, verified the reliance of H-1PV on this route to enter HeLa and NCH125 cancer cells. By contrast, we found no evidence of viral entry through caveolae-mediated endocytosis. Indeed, pre-treatment of cells with nystatin or methyl-β-cyclodextrin, both inhibitors of caveolae-mediated endocytosis, did not affect viral transduction levels. Unexpectedly, siRNA-mediated knockdown of caveolin-1, the main driver of caveolae-mediated endocytosis, increased H-1PV transduction, suggesting caveolin-1 is a negative modulator of H-1PV infection. We also show that H-1PV entry is dependent on dynamin, a protein responsible for mediating the scission of vesicle neck and promoting further internalisation. Furthermore, since dynamin inhibition almost completely abolished H-1PV infection, makes it unlikely that H-1PV uses macropinocytosis as an alternative pathway to enter cells. After viral internalisation, H-1PV passes through early to late endosomes as observed by confocal microscopy. Inside these endocytic compartments, the acidic environment proved to be crucial for a productive infection. Inhibition of acidification of pH dramatically reduced H-1PV transduction. Besides, a fraction of H-1PV particles was observed inside LAMP1-positive lysosomes, most likely following a non-infectious route. To the author's best knowledge, this is the first study to characterise the cell entry pathways of H-1PV. Along these lines, this work will further contribute to understand H-1PV oncolytic properties as well as to improve its clinical potential in cancer virotherapy.

Keywords: clathrin-mediated endocytosis, H-1 parvovirus, oncolytic virus, virus entry

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Authors: Zhengyu Yan, Yan Yu, Jianqiu Chen

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A novel sensing system has been designed for naphthalene detection based on the quenched fluorescence signal of CdS quantum dots. The fluorescence intensity of the system reduced significantly after adding CdS quantum dots to the water pollution model because of the fluorescent static quenching f mechanism. Herein, we have demonstrated the facile methodology can offer a convenient and low analysis cost with the recovery rate as 97.43%-103.2%, which has potential application prospect.

Keywords: CdS quantum dots, modification, detection, naphthalene

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Authors: O. S. Adeyemi, C. G. Whiteley

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The present study determined the toxic potential of metal nanoparticles in cell culture system. Silver and gold nanoparticles were synthesized and characterized following established "green" protocols. The synthesized nanoparticles, in varying concentrations ranging from 0.1–100 µM were evaluated for toxicity in metastatic MDA-MB-231 cells. The nanoparticles promoted a generation of reactive oxygen species and reduced cell viability to less than 50% in the demonstration of cellular toxicity. The nanoparticles; gold and the silver-gold mixture had IC50 values of 56.65 and 18.44 µM respectively. The IC50 concentration for silver nanoparticles could not be determined. Furthermore, the probe of the cell death using flow cytometry and confocal microscopy revealed the partial involvement of apoptosis as well as necrosis. Our results revealed cellular toxicity caused by the nanoparticles but the mechanism remains yet undefined.

Keywords: cell death, nanomedicine, nanotoxicology, toxicity

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Authors: Neeraj Neeraj, Soumen Basu, Banibrata Maity

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Glutathione (GSH) is one of the most important biomolecules having small molecular weight, which helps in various cellular functions like regulation of gene, xenobiotic metabolism, preservation of intracellular redox activities, signal transduction, etc. Therefore, the detection of GSH requires huge attention by using extremely selective and sensitive techniques. Herein, a rapid fluorometric nanosensor is designed by combining carbon dots (Cdots) and MnO₂ nanoparticles of different sizes for the detection of GSH. The bottom-up approach, i.e., microwave method, was used for the preparation of the water soluble and greatly fluorescent Cdots by using ascorbic acid as a precursor. MnO₂ nanospheres of different sizes (large, medium, and small) were prepared by varying the ratio of concentration of methionine and KMnO₄ at room temperature, which was confirmed by HRTEM analysis. The successive addition of MnO₂ nanospheres in Cdots results fluorescence quenching. From the fluorescence intensity data, Stern-Volmer quenching constant values (KS-V) were evaluated. From the fluorescence intensity and lifetime analysis, it was found that the degree of fluorescence quenching of Cdots followed the order: large > medium > small. Moreover, fluorescence recovery studies were also performed in the presence of GSH. Fluorescence restoration studies also show the order of turn on follows the same order, i.e., large > medium > small, which was also confirmed by quantum yield and lifetime studies. The limits of detection (LOD) of GSH in presence of Cdots@different sized MnO₂ nanospheres were also evaluated. It was observed thatLOD values were in μM region and lowest in case of large MnO₂ nanospheres. The separation distance (d) between Cdots and the surface of different MnO₂ nanospheres was determined. The d values increase with increase in the size of the MnO₂ nanospheres. In summary, the synthesized Cdots@MnO₂ nanocomposites acted as a rapid, simple, economical as well as environmental-friendly nanosensor for the detection of GSH.

Keywords: carbon dots, fluorescence, glutathione, MnO₂ nanospheres, turn off-on

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Authors: Guohua Cao, Xu Dong

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X-ray Fluorescence Molecular Imaging (XFMI) holds great promise as a low-cost molecular imaging modality for biomedical applications with high chemical sensitivity. However, for in vivo biomedical applications, a key technical bottleneck is the relatively low chemical sensitivity of XFMI, especially at a reasonably low radiation dose. In laboratory x-ray source based XFMI, one of the main factors that limits the chemical sensitivity of XFMI is the scattered x-rays. We will present our latest findings on improving the chemical sensitivity of XFMI using excitation beam spectrum optimization. XFMI imaging experiments on two mouse-sized phantoms were conducted at three different excitation beam spectra. Our results show that the minimum detectable concentration (MDC) of iodine can be readily increased by five times via excitation spectrum optimization. Findings from this investigation could find use for in vivo pre-clinical small-animal XFMI in the future.

Keywords: molecular imaging, X-ray fluorescence, chemical sensitivity, X-ray scattering

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Authors: Murtada Ahmed Oshi, Jin-Wook Yoo

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Layer-by-layer (LBL) coating has gained popularity for drug delivery of therapeutic drugs. Herein we described a novel approach for enhancing the therapeutic efficiency of the locally administered dexamethasone (Dex) for inflammatory bowel disease (IBD). We utilized a LBL-coating technique on Dex microcrystals (DexMCs) with multiple layers of polyelectrolytes composed of poly (allylamine hydrochloride) (PAH), poly (sodium 4-styrene sulfonate) (PSS) and Eudragit® S100 (ES). The successful deposition of the layers onto DexMCs surfaces were confirmed through zeta potential measurement and confocal laser scanning microscopy. The surface morphology was investigated through scanning electron microscopy. The drug encapsulation efficiency was 95% with a mean particle size of 2 µm and negative surface charge (-40 mV). Moreover, in vitro drug release study showed a minimum release of the drug ( 15%) at an acidic condition during initial first 5 h, followed by sustained-release at an alkaline condition. For in vivo study, LBL-DxMCs were administered orally to ICR mice suffering from dextran sulfate sodium-induced colitis. LBL-DxMCs substantially enhanced anti-IBD activities as compared to DxMCs. Macroscopic, histological and biochemical (tumor necrosis factor-α, interleukin-6 and myeloperoxidase) examinations revealed marked improvements of colitis signs in the mice treated with LBL-DxMCs compared with those treated with DxMCs. Overall, LBL-DxMCs could be a suitable candidate for the treatment of IBD.

Keywords: dexamethasone, inflammatory bowel disease, LBL-coating, polyelectrolytes

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Authors: Rejane Maria P. da Silva, Mariana X. Milagre, João Victor de S. Araujo, Leandro A. de Oliveira, Renato A. Antunes, Isolda Costa

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The Al-Cu-Li alloys are advanced materials for aerospace application because of their interesting mechanical properties and low density when compared with conventional Al-alloys. However, Al-Cu-Li alloys are susceptible to localized corrosion. The near-surface deformed layer (NSDL) induced by the rolling process during the production of the alloy and its removal by polishing can influence on the corrosion susceptibility of these alloys. In this work, the influence of surface preparation effects on the electrochemical activity of AA2098-T351 (Al–Cu–Li alloy) was investigated using a correlation between surface chemistry, microstructure, and electrochemical activity. Two conditions were investigated, polished and as-received surfaces of the alloy. The morphology of the two types of surfaces was investigated using confocal laser scanning microscopy (CLSM) and optical microscopy. The surface chemistry was analyzed by X-ray Photoelectron Spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDS). Global electrochemical techniques (potentiodynamic polarization and EIS technique) and a local electrochemical technique (Localized Electrochemical Impedance Spectroscopy-LEIS) were used to examine the electrochemical activity of the surfaces. The results obtained in this study showed that in the as-received surface, the near-surface deformed layer (NSDL), which is composed of Mg-rich bands, influenced the electrochemical behavior of the alloy. The results showed higher electrochemical activity to the polished surface condition compared to the as-received one.

Keywords: Al-Cu-Li alloys, surface preparation effects, electrochemical techniques, localized corrosion

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Authors: Alaa Hamed Salama, Rehab Nabil Shamma

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Introduction: Polymeric micelles that can deliver drug to intended sites of the eye have attracted much scientific attention recently. The aim of this study was to review the aqueous-based formulation of drug-loaded polymeric micelles that hold significant promise for ophthalmic drug delivery. This study investigated the synergistic performance of mixed polymeric micelles made of linear and branched poly (ethylene oxide)-poly (propylene oxide) for the more effective encapsulation of Lornoxicam (LX) as a hydrophobic model drug. Methods: The co-micellization process of 10% binary systems combining different weight ratios of the highly hydrophilic poloxamers; Synperonic® PE/P84, and Synperonic® PE/F127 and the hydrophobic poloxamine counterpart (Tetronic® T701) was investigated by means of photon correlation spectroscopy and cloud point. The drug-loaded micelles were tested for their solubilizing capacity towards LX. Results: Results showed a sharp solubility increase from 0.46 mg/ml up to more than 4.34 mg/ml, representing about 136-fold increase. Optimized formulation was selected to achieve maximum drug solubilizing power and clarity with lowest possible particle size. The optimized formulation was characterized by 1HNMR analysis which revealed complete encapsulation of the drug within the micelles. Further investigations by histopathological and confocal laser studies revealed the non-irritant nature and good corneal penetrating power of the proposed nano-formulation. Conclusion: LX-loaded polymeric nanomicellar formulation was fabricated allowing easy application of the drug in the form of clear eye drops that do not cause blurred vision or discomfort, thus achieving high patient compliance.

Keywords: confocal laser scanning microscopy, Histopathological studies, Lornoxicam, micellar solubilization

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Authors: Omar Drissi, Nadia El Harfaoui, Khalid Nouneh, Rachid Hsissou, Badre Daoudi

Abstract:

The pulp endures of having a lower importance, incompletely because of the way that it has been less studied, and it has been recognized as a pivotal product got from biomass that can be utilized in different fields. The current research focuses on pulp of Argania spinosa (L). To this end, the aim is to study the characteristics and properties of Argan pulp, such as shape, chemical and macromineral composition. As a result, X-Ray Fluorescence (XRF), Fourier transform infrared spectroscopy (FTIR), and Scanning Electron Microscopy (SEM) were used in the research.

Keywords: argania spinose, argan pulp, argan bio-waste, green synthesis, silver nanoparticles, valorization

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Authors: Tej Varma Y, Debi D. Pant

Abstract:

Photophysics and rotational dynamics of the fluorescent probe, 6-methoxyquinoline (6MQ) with cationic surfactant, alkyltrimethylammonium bromide (nTAB) micelle solutions have been investigated (n = 12, 14 and 16). Absorption and emission peaks of the dye have been observed to shift at concentrations around critical micellar concentration (cmc) of nTAB compared to that of bulk solutions suggesting probe is in a lower polar environment. The probe senses changes in polarity (ET (30)) brought about by variation of surfactant chain length concentration and is invariably solubilized in the aqueous interface or palisade layer. The order of change in polarity observed was DTAB > CTAB > TTAB. The binding constant study shows that the probe binds strongest with TTAB (is of the order TTAB > CTAB > DTAB) due to deeper penetration into the micelle. The anisotropy decay for the probe in all the nTAB micelles studied have been rationalized based on a two-step model consisting of fast-restricted rotation of the probe and slow lateral diffusion of the probe in the micelle that is coupled to the overall rotation of the micelle. Fluorescence lifetime measurements of probe in the cationic micelles demonstrate the close proximity of the 6MQ to the Br - counterions. The fluorescence lifetimes of TTAB and DTAB are much shorter than in CTAB. These results indicate that 6MQ resides to a substantial degree in the head group region of the micelles. All the changes observed in the steady state fluorescence, microenvironment, fluorescence lifetimes, fluorescence anisotropy, and other calculations are in agreement with each other suggesting binding of the cationic surfactant with the neutral dye molecule.

Keywords: photophysics, chain length, ntaB, micelles

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Authors: Bokolombe Pitchou Ngoy, John Mack, Tebello Nyokong

Abstract:

Synthesis and photochemical studies of BODIPY dyes have been investigated in this work in order to have a broad benchmark of this functionalized photosensitizer for biological applications such as photodynamic therapy or antimicrobial activity. The common acid catalyzed synthetic method was used, and BODIPY dyes were obtained in quite a good yield (25 %) followed by bromination and Knoevenagel condensation to afford the BODIPY dyes conjugated with maximum absorbance in the near-infrared region of the electromagnetic spectrum. The fluorescence lifetimes, fluorescence quantum yield, and Singlet oxygen quantum yield of the conjugated BODIPY dyes were determined in different solvents by using Time Correlation Single Photon Counting (TCSPC), fluorimeter, and Laser Flash Photolysis respectively. It was clearly shown that the singlet oxygen quantum yield was higher in THF followed by DMSO compared to another solvent. The same trend was observed for the fluorescence lifetimes.

Keywords: BODIPY, photodynamic therapy, photosensitizer, singlet oxygen

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Authors: K. A. Laptinskiy, S. A. Burikov, A. M. Vervald, S. A. Dolenko, T. A. Dolenko

Abstract:

The article presents the results of the application of artificial neural networks to separate the fluorescent contribution of nanodiamonds used as biomarkers, adsorbents and carriers of drugs in biomedicine, from a fluorescent background of own biological fluorophores. The principal possibility of solving this problem is shown. Use of neural network architecture let to detect fluorescence of nanodiamonds against the background autofluorescence of egg white with high accuracy - better than 3 ug/ml.

Keywords: artificial neural networks, fluorescence, data aggregation, biomarkers

Procedia PDF Downloads 679

Authors: Sarim Ahmad

Abstract:

The Single Cell Gel Electrophoresis Assay (SCGE, known as comet assay) is a potential, uncomplicated, sensitive and state-of-the-art technique for quantitating DNA damage at individual cell level and repair from in vivo and in vitro samples of eukaryotic cells and some prokaryotic cells, being popular in its widespread use in various areas including human biomonitoring, genotoxicology, ecological monitoring and as a tool for research into DNA damage or repair in different cell types in response to a range of DNA damaging agents, cancer risk and therapy. The method involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH > 13) conditions, and electrophoresis of the suspended lysed cells, resulting in structures resembling comets as observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend towards the anode. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by imaging software. The assay can, therefore, predict an individual’s tumor sensitivity to radiation and various chemotherapeutic drugs and further assess the oxidative stress within tumors and to detect the extent of DNA damage in various cancerous and precancerous lesions of oral cavity.

Keywords: comet assay, single cell gel electrophoresis, DNA damage, early detection test

Procedia PDF Downloads 268

Authors: M. A. Elbagermia, A. I. Alajtala, M. Alkerzab

Abstract:

This study compares the quality of different brands of Portland Cement (PC) available in Libyan market. The amounts of chemical constituents like SiO2, Al2O3, Fe2O3, CaO, MgO, SO3, and Lime Saturation Factor (LSF) were determined in accordance with Libyan (L.S.S) and Amrican (A.S.S) Standard Specifications. All the cement studies were found to be good for concrete work especially where no special property is required. The chemical and mineralogical analyses for studied clinker samples show that the dominant phases composition are C3S and C2S while the C3A and C4AF are less abundant.

Keywords: Portland cement, chemical composition, Libyan market, X-Ray fluorescence

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Authors: Sang Hun Park, Chul Gyu Song

Abstract:

Artery and vein occlusion changes observed in patients and experimental animals are unexplainable symptoms. As the fat accumulated in cardiovascular ruptures, it causes vascular blocking. Likewise, early detection of cardiovascular disease can be useful for treatment. In this study, we used the mouse femoral occlusion model to observe the arterial and venous occlusion changes without darkroom. We observed the femoral arterial flow pattern changes by proposed fluorescent imaging system using an animal model of thrombosis. We adjusted the near-infrared light source current in order to control the intensity of the fluorescent substance light. We got the clear fluorescent images and femoral artery flow pattern were measured by a 5-minute interval. The result showed that the fluorescent substance flowing in the femoral arteries were accumulated in thrombus as time passed, and the fluorescence of other vessels gradually decreased.

Keywords: thrombus, fluorescence, femoral, arteries

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Authors: Sungroul Kim, Yeonjin Kim

Abstract:

This study was conducted by comparing the concentrations of heavy metals analyzed from the same samples with three X-Ray fluorescence (XRF) spectrometer in three different global research institutions, including PAN (A Branch of Malvern Panalytical, Seoul, South Korea), RTI (Research Triangle Institute, NC, U.S.A), and aerosol laboratory in Harvard University, Boston, U.S.A. To achieve our research objectives, the indoor air filter samples were collected at homes (n=24) of adults or child asthmatics then analyzed in PAN followed by Harvard University and RTI consecutively. Descriptive statistics were conducted for data comparison as well as correlation and simple regression analysis using R version 4.0.3. As a result, detection rates of most heavy metals analyzed in three institutions were about 90%. Of the 25 elements commonly analyzed among those institutions, 16 elements showed an R² (coefficient of determination) of 0.7 or higher (10 components were 0.9 or higher). The findings of this study demonstrated that XRF was a useful device ensuring reproducibility and compatibility for measuring heavy metals in PM2.5 collected from indoor air of asthmatics’ home.

Keywords: heavy metals, indoor air quality, PM2.5, X-ray fluorescence

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Authors: Mehdi Rahimi-Nasrabadi, Seied Mahdi Pourmortazavi

Abstract:

Taguchi robust design as a statistical method was applied for optimization of the process parameters in order to tunable, simple and fast synthesis of europium (III) tungstate nanoparticles. Europium (III) tungstate nanoparticles were synthesized by a chemical precipitation reaction involving direct addition of europium ion aqueous solution to the tungstate reagent solved in aqueous media. Effects of some synthesis procedure variables i.e., europium and tungstate concentrations, flow rate of cation reagent addition, and temperature of reaction reactor on the particle size of europium (III) tungstate nanoparticles were studied experimentally in order to tune particle size of europium (III) tungstate. Analysis of variance shows the importance of controlling tungstate concentration, cation feeding flow rate and temperature for preparation of europium (III) tungstate nanoparticles by the proposed chemical precipitation reaction. Finally, europium (III) tungstate nanoparticles were synthesized at the optimum conditions of the proposed method and the morphology and chemical composition of the prepared nano-material were characterized by means of X-Ray diffraction, scanning electron microscopy, transmission electron microscopy, FT-IR spectroscopy, and fluorescence.

Keywords: europium (III) tungstate, nano-material, particle size control, procedure optimization

Procedia PDF Downloads 362