Accumulation and toxicity of biologically produced gold nanoparticles in different types of specialized mammalian cells.

The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15 nm in size with a zeta potential of -35.8 mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F-actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell-specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F-actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells.

Total and Unilateral Peak Nasal Inspiratory Flow and Unilateral Visual Analogue Scale: Rationale in Indication and Outcomes of Septoplasty?

Septoplasty is one of the most frequently performed procedures in otorhinolaryngology. Proper selection of patients for septoplasty as well as examination is essential for good outcome of surgery. The aim of this study was to evaluate the suitability of combination of 2 simple, cheap, and easily performed examinations for indication and outcomes of septoplasty to evaluate the effectiveness in this type of surgery. Seventy patients were enrolled from 2 tertiary centers from the period of April 2017 to March 2018, who all underwent septoplasty for nasal septal deviation. Total and unilateral peak nasal inspiratory flow (PNIF), unilateral visual analogue scale, and nasal endoscopy for indication and outcomes of septoplasty were performed. Furthermore, assessment of possible correlation between subjective and objective data before and 3 months after septoplasty was done. Statistical analysis was carried out on the obtained data. Postoperatively the total and both unilateral PNIF measurements increased significantly compared with corresponding preoperative values and similarly means of visual analogue scale scores on both sides decreased 3 months after the procedure. No significant correlation was seen between objective and subjective outcomes neither before the surgery, nor postoperatively. The study confirmed the suitability of using PNIF as a simple and inexpensive tool that can be a reasonable alternative to rhinomanometry and which is able to reflect changes after septoplasty. Simultaneously, along with the expected improvement in the total PNIF and unilateral PNIF on deviated side, a slight but significant improvement in PNIF values on the nondeviated side was also demonstrated.

Spatially and Temporally Resolved Detection of Arsenic in a Capillary Dielectric Barrier Discharge by Hydride Generation High-Resolved Optical Emission Spectrometry.

A new method for arsenic detection by optical emission spectrometry (OES) is presented. Arsine (AsH3) is generated from liquid solutions by means of hydride generation (HG) and introduced into a capillary dielectric barrier discharge (DBD) where it is atomized and excited. A great challenge in OES is the reduction of the recorded background signal, because it negatively affects the limit of detection (LOD). In conventional DBD/OES methods, the signal intensity of the line of interest, in this case arsenic, is integrated over a long time scale. However, due to the pulsed character of the plasma, the plasma on-time is only a small fraction of the integration time. Therefore, a high amount of noise is added to the actual signal in each discharge cycle. To circumvent this, in the present study the emitted light from the DBD is collected by a fast gated iCCD camera, which is mounted on a modified monochromator. The experimental arrangement enables the recording of the emission signal of arsenic in the form of a monochromatic 2D-resolved picture. The temporal resolution of the iCCD camera in the nanosecond range provides the information at which point in time and how long arsenic is excited in the discharge. With use of this knowledge, it is possible to integrate only the arsenic emission by temporally isolating the signal from the background. With the presented method, the LOD for arsenic could be determined to 93 pg mL-1 with a calibration curve linear over 4 orders of magnitude. As a consequence, the developed experimental approach has a potential for both mechanistic studies of arsine atomization and excitation in DBD plasmas as well as routine applications, in which arsenic determination at ultratrace levels is required.

Atomization of Bismuthane in a Dielectric Barrier Discharge: A Mechanistic Study.

Atomization of bismuthane in a planar dielectric barrier discharge (DBD) atomizer was investigated using a variety of probes, including atomic absorption spectrometry (AAS) to monitor distribution of free atoms along the optical path and direct analysis in real time (DART) coupled to an Orbitrap mass spectrometer to identify the structure of the species arising from the hydride generator as well as the atomizer. Results obtained with the DBD were compared to those from a conventional externally heated quartz tube atomizer (QTA). Free Bi atoms were essentially absent outside the central part of the DBD atomizer, suggesting their high reactivity. The gas phase analyte fraction transported beyond the confines of the DBD or QTA atomizers, quantified by inductively coupled plasma mass spectrometry (ICP-MS), was less than 10%. The amount of Bi found in acidic leachates of the interiors of both atomizers, representing the fraction retained on their surfaces, was ca. 90%. These complementary experiments comprising the determination of recovered Bi in the nitric acid leachates from deposition in the atomizer on the one hand and quantification of the Bi fraction transportable outside the atomizer on the other, were in excellent agreement, providing 100% mass balance of detected analyte. The high fraction of Bi deposited in the atomizers indicates significant reactivity of free Bi atoms, which is in accord with the fact that almost no free Bi atoms exist beyond the physical boundaries of the DBD. The extent of interference from other hydride forming elements (As, Sb, Se) on Bi response by AAS using DBD and QTA atomizers was investigated, with the former atomizer providing superior performance. Compared to QTA, DBD provided 2 orders of magnitude and 1 order of magnitude, respectively, better resistance to interference from Se and Sb.

Fate of the capping agent of biologically produced gold nanoparticles and adsorption of enzymes onto their surface.

Enzymotherapy based on DNase I or RNase A has often been suggested as an optional strategy for cancer treatment. The efficacy of such procedures is limited e.g. by a short half-time of the enzymes or a low rate of their internalization. The use of nanoparticles, such as gold nanoparticles (AuNPs), helps to overcome these limits. Specifically, biologically produced AuNPs represent an interesting variant here due to naturally occurring capping agents (CA) on their surface. The composition of the CA depends on the producing microorganism. CAs are responsible for the stabilization of the nanoparticles, and promote the direct linking of targeting and therapeutic molecules. This study provided proof of enzyme adsorption onto gold nanoparticles and digestion efficacy of AuNPs-adsorbed enzymes. We employed Fusarium oxysporum extract to produce AuNPs. These nanoparticles were round or polygonal with a size of about 5 nm, negative surface charge of about - 33 mV, and maximum absorption peak at 530 nm. After the adsorption of DNAse I, RNase A, or Proteinase K onto the AuNPs surface, the nanoparticles exhibited shifts in surface charge (values between - 22 and - 13 mV) and maximum absorption peak (values between 513 and 534 nm). The ability of AuNP-enzyme complexes to digest different targets was compared to enzymes alone. We found a remarkable degradation of ssDNA, and dsDNA by AuNP-DNAse I, and a modest degradation of ssRNA by AuNP-RNase A. The presence of particular enzymes on the AuNP surface was proved by liquid chromatography-mass spectrometry (LC-MS). Using SDS-PAGE electrophoresis, we detected a remarkable digestion of collagen type I and fibrinogen by AuNP-proteinase K complexes. We concluded that the biologically produced AuNPs directly bound DNase I, RNase A, and proteinase K while preserving their ability to digest specific targets. Therefore, according to our results, AuNPs can be used as effective enzyme carriers and the AuNP-enzyme conjugates can be effective tools for enzymotherapy.

Effect of Cu Alloying on Mechanical Properties of Medium-C Steel after Long-Time Tempering at 500 °C.

This research studies the influence of the copper alloying of medium-carbon steel on mechanical properties after quenching and tempering at 500 °C. The microstructure was characterised using SEM, EBSD, TEM, and XRD analysis. The mechanical properties were comprehensively investigated using hardness measurements, tensile and Charpy impact tests and solid solution, grain boundary, dislocation, and precipitation strengthening contributions were estimated. Higher yield strength for Cu-alloyed steel was confirmed at about 35-73 MPa. The precipitation strengthening contribution from Cu precipitates in the range of 11-49 MPa was calculated. The interaction between Cu precipitates and dislocations retards the decrease in dislocation density. Similar values of effective grain size of martensite crystals were measured for Cu-alloyed and Cu-free steel as well. Copper alloyed steel exhibited significantly deteriorated impact toughness, total plastic elongation, and reduction of area. The size of Cu precipitates ranged from 8.3 nm after tempering at 500 °C for 6 h to 13.9 nm after tempering for 48 h.

Direct analysis of methylated trivalent arsenicals in mouse liver by hydride generation-cryotrapping-atomic absorption spectrometry.

Growing evidence suggest that the methylated trivalent metabolites of inorganic arsenic (iAs), methylarsonite (MAs(III)) and dimethylarsinite (DMAs(III)), contribute to adverse effects of iAs exposure. However, the lack of suitable methods has hindered the quantitative analysis of MAs(III) and DMAs(III) in complex biological matrices. Here, we show that hydride generation-cryotrapping-atomic absorption spectrometry can quantify both MAs(III) and DMAs(III) in livers of mice exposed to iAs. No sample extraction is required, thus limiting MAs(III) or DMAs(III) oxidation prior to analysis. The limits of detection are below 6 ng As/g of tissue, making this method suitable even for studies examining low exposures to iAs.

Effect of additives on cadmium chemical vapor generation and reliable quantification of generation efficiency.

Chemical vapor generation (CVG) of cadmium was optimized based on response from atomic absorption spectrometry (AAS) with a heated quartz tube atomizer (QTA). Effect of several modifiers on analytical performance was studied. These additives were: inorganic salts of Cr3+, Ti4+ and Co2+ and their on-line synthesized complexes with KCN and thiourea, respectively. The use of these additives resulted in sensitivity enhancement, better repeatability and correspondingly in improvement of overall CVG efficiency. The latter was quantified by two independent approaches: a) by means of 115mCd radioactive indicator, b) from comparison of sensitivities obtained with conventional solution nebulization and with CVG, both coupled simultaneously to inductively coupled plasma mass spectrometry. Both approaches provided comparable results. The highest efficiency, between 60 and 70%, was reached in the presence of Cr3+/KCN and Ti4+/KCN while 19% was achieved in Co2+/ascorbic acid/thiourea environment. Highly irreproducible results with low CVG efficiency ranging from 2.5 to 15% were reached in the absence of any additives. The generated cadmium species were identified to be mostly free atoms regardless of the additives presence or their absence. Cr3+/KCN environment was selected as the most robust for CVG of Cd reaching sensitivity of 6.6 s ng-1 Cd and limit of detection of 60 pg mL-1 Cd (9 pg Cd absolute) with detection by QTA-AAS.

Oxidation State Specific Generation of Arsines from Methylated Arsenicals Based on L- Cysteine Treatment in Buffered Media for Speciation Analysis by Hydride Generation - Automated Cryotrapping - Gas Chromatography-Atomic Absorption Spectrometry with the Multiatomizer.

An automated system for hydride generation - cryotrapping- gas chromatography - atomic absorption spectrometry with the multiatomizer is described. Arsines are preconcentrated and separated in a Chromosorb filled U-tube. An automated cryotrapping unit, employing nitrogen gas formed upon heating in the detection phase for the displacement of the cooling liquid nitrogen, has been developed. The conditions for separation of arsines in a Chromosorb filled U-tube have been optimized. A complete separation of signals from arsine, methylarsine, dimethylarsine, and trimethylarsine has been achieved within a 60 s reading window. The limits of detection for methylated arsenicals tested were 4 ng l(-1). Selective hydride generation is applied for the oxidation state specific speciation analysis of inorganic and methylated arsenicals. The arsines are generated either exclusively from trivalent or from both tri- and pentavalent inorganic and methylated arsenicals depending on the presence of L-cysteine as a prereductant and/or reaction modifier. A TRIS buffer reaction medium is proposed to overcome narrow optimum concentration range observed for the L-cysteine modified reaction in HCl medium. The system provides uniform peak area sensitivity for all As species. Consequently, the calibration with a single form of As is possible. This method permits a high-throughput speciation analysis of metabolites of inorganic arsenic in relatively complex biological matrices such as cell culture systems without sample pretreatment, thus preserving the distribution of tri- and pentavalent species.

Behavior of selenium hydride in heated quartz tube and dielectric barrier discharge atomizers.

Atomization of SeH2 in an externally heated multiple microflame quartz tube atomizer (MMQTA) as well as planar dielectric barrier discharge (DBD) atomizer was investigated using a variety of probes. Deposits of Se on inner surfaces of the atomizers were quantified and their distribution visualized by autoradiography with 75Se radiotracer. The gas phase fraction of Se transported beyond the confines of the atomizers was also determined. In the MMQTA, a 15% mass fraction of Se was deposited in a narrow zone at both colder ends of the optical arm (100-400 °C). By contrast, a 25-40% mass fraction of Se was deposited homogeneously along the entire length of the optical arm of the DBD, depending on detection technique employed. The fraction of Se transported outside the MMQTA approached 90%, whereas it was 50-70% in the DBD. The presence of H2 was essential for atomization of selenium hydride in both atomizers. The gaseous effluent arising from the hydride generator as well as the atomizers was investigated by direct analysis in real time (DART) coupled to an Orbitrap-mass spectrometer, enabling identification of major gas phase species of Se.

Sample preparation for arsenic speciation analysis in baby food by generation of substituted arsines with atomic absorption spectrometry detection.

A slurry sampling procedure for arsenic speciation analysis in baby food by arsane generation, cryogenic trapping and detection with atomic absorption spectrometry is presented. Several procedures were tested for slurry preparation, including different reagents (HNO3, HCl and tetramethylammonium hydroxide - TMAH) and their concentrations, water bath heating and ultrasound-assisted agitation. The best results for inorganic arsenic (iAs) and dimethylarsinate (DMA) were reached when using 3molL-1 HCl under heating and ultrasound-assisted agitation. The developed method was applied for the analysis of five porridge powder and six baby meal samples. The trueness of the method was checked with a certified reference material (CRM) of total arsenic (tAs), iAs and DMA in rice (ERM-BC211). Arsenic recoveries (mass balance) for all samples and CRM were performed by the determination of the tAs by inductively coupled plasma mass spectrometry (ICP-MS) after microwave-assisted digestion and its comparison against the sum of the results from the speciation analysis. The relative limits of detection were 0.44, 0.24 and 0.16µgkg-1 for iAs, methylarsonate and DMA, respectively. The concentrations of the most toxic arsenic species (iAs) in the analyzed baby food samples ranged between 4.2 and 99µgkg-1 which were below the limits of 300, 200 and 100µgkg-1 set by the Brazilian, Chinese and European legislation, respectively.

Direct analysis and stability of methylated trivalent arsenic metabolites in cells and tissues.

Chronic ingestion of water containing inorganic arsenic (iAs) has been linked to a variety of adverse health effects, including cancer, hypertension and diabetes. Current evidence suggests that the toxic methylated trivalent metabolites of iAs, methylarsonous acid (MAs(III)) and dimethylarsinous acid (DMAs(III)) play a key role in the etiology of these diseases. Both MAs(III) and DMAs(III) have been detected in urine of subjects exposed to iAs. However, the rapid oxidation of DMAs(III) and, to a lesser extent, MAs(III) in oxygen-rich environments leads to difficulties in the analysis of these metabolites in samples of urine collected in population studies. Results of our previous work indicate that MAs(III) and DMAs(III) are relatively stable in a reducing cellular environment and can be quantified in cells and tissues. In the present study, we used the oxidation state-specific hydride generation-cryotrapping-atomic absorption spectroscopy (HG-CT-AAS) to examine the presence and stability of these trivalent metabolites in the liver of mice and in UROtsa/F35 cells exposed to iAs. Tri- and pentavalent metabolites of iAs were analyzed directly (without chemical extraction or digestion). Liver homogenates prepared in cold deionized water and cell culture medium and lysates were stored at either 0 °C or -80 °C for up to 22 days. Both MAs(III) and DMAs(III) were stable in homogenates stored at -80 °C. In contrast, DMAs(III) in homogenates stored at 0 °C began to oxidize to its pentavalent counterpart after 1 day; MAs(III) remained stable for at least 3 weeks under these conditions. MAs(III) and DMAs(III) generated in UROtsa/F35 cultures were stable for 3 weeks when culture media and cell lysates were stored at -80 °C. These results suggest that samples of cells and tissues represent suitable material for the quantitative, oxidation state-specific analysis of As in laboratory and population studies examining the metabolism or toxic effects of this metalloid.