Anticancer dinuclear Ir(III) complex activates Nrf2 and interferes with NAD(H) in cancer cells.

Dinuclear complex [Ir2(µ-L1)(η5-Cp*)2Cl2](PF6)2 (1) exhibits low micromolar cytotoxic activity in vitro in various human cancer cells (GI50 = 1.7-3.0 µM) and outperformed its mononuclear analogue [Ir(η5-Cp*)Cl(L2)]PF6 (2; GI50 > 40.0 µM); Cp* = pentamethylcyclopentadienyl, L1 = 4-chloro-2,6-bis[5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl]pyridine, L2 = 5-(pyridin-2-yl)-1,3,4-thiadiazol-2-amine. Compound 1 upregulated the Keap1/Nrf2 oxidative stress-protective pathway in the treated MV4-11 acute myeloid leukemia cells. In connection with the redox-mediated mode of action of 1, its NADH-oxidizing activity was detected in solution (1H NMR), while NAD+ remained intact (with formate as a hydride source). Surprisingly, only negligible NADH oxidation was detected in the presence of the reduced glutathione and ascorbate. Following the results of in-solution experiments, NAD(H) concentration was assessed in 1-treated MV4-11 cancer cells. Besides the intracellular NADH oxidation in the presence of 1, the induced oxidative stress also led to a decrease of NAD+, resulting in depletion of both NAD+/NADH coenzymes. The discussed findings provide new insight into the biochemical effects of catalytic anticancer compounds that induce cell death via a redox-mediated mode of action.

In vivo assessment of TiO2 based wear nanoparticles in periprosthetic tissues.

A multimodal approach combining inductively coupled plasma mass spectrometry (ICP-MS), single-particle ICP-MS (spICP-MS), scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS) and Raman spectroscopy enabled a deeper insight into the balance between total titanium (Ti), the soluble titanium fraction and titanium dioxide based particle fraction levels in periprosthetic tissues collected from patients undergoing revision surgery. Hydrofluoric acid usage in the sample digestion allowed for complete digestion of TiO2 particles, thus enabling accurate estimation of total Ti levels. The TiO2 fraction represents 38-94% of the titanium load in the six samples where particles were detected, and the fraction is present mainly in samples from patients with aseptically loosened total hip arthroplasty. Further attention was given to this fraction determining the elemental composition, particle count, particle size and modification of TiO2. The spICP-MS analysis confirmed the presence of the TiO2-derived (nano)particles (NPs) with a 39- to 187-nm median size and particle count up to 2.3 × 1011 particles per gram of tissue. On top of that, the SEM-EDS confirmed the presence of the TiO2 nanoparticles with 230-nm median size and an anatase crystal phase was determined by Raman spectroscopy. This study presents a novel multimodal approach for TiO2 particle determination and characterization in tissue samples and is the first in vivo study of this character.

Direct elemental analysis of plant oils by inductively coupled plasma mass spectrometry: Simple sample dilution combined with oxygen introduction into the plasma.

Assessment of trace metal concentrations in plant oils has been considered a crucial quality control marker for potential health risks, oil flavour, and oxidative stability. A straightforward inductively coupled plasma mass spectrometry (ICP-MS) methodology was developed and validated through introduction of argon:oxygen gas mixture into plasma, allowing for a direct elemental analysis of organic matrices. This approach offers the advantage of a simple one-step preparation of plant oil samples with negligible contamination risks. The complete solubilization of the oil matrix enables the determination of total metal content from a single test tube with low dilution factor of 5. The modified plasma conditions resulted in the development of a robust and accurate ICP-MS method providing limits of detection at sub ng·g-1 levels. The ICP-MS method allowed the determination of trace levels of Ba, Cd, Cu, Fe, Mn, Pb, Sn, V, and Zn in olive, sunflower and rapeseed oils.

Impact of the central atom and halido ligand on the structure, antiproliferative activity and selectivity of half-sandwich Ru(II) and Ir(III) complexes with a 1,3,4-thiadiazole-based ligand.

Half-sandwich complexes [Ru(η6-pcym)(L1)X]PF6 (1, 3) and [Ir(η5-Cp*)(L1)X]PF6 (2, 4) featuring a thiadiazole-based ligand L1 (2-(furan-2-yl)-5-(pyridin-2-yl)-1,3,4-thiadiazole) were synthesized and characterized by varied analytical methods, including single-crystal X-ray diffraction (X = Cl or I, pcym = p-cymene, Cp* = pentamethylcyclopentadienyl). The structures of the molecules were analysed and interpreted using computational methods such as Density Functional Theory (DFT) and Quantum Theory of Atoms in Molecules (QT-AIM). A 1H NMR spectroscopy study showed that complexes 1-3 exhibited hydrolytic stability while 4 underwent partial iodido/chlorido ligand exchange in phosphate-buffered saline. Moreover, 1-4 demonstrated the ability to oxidize NADH (reduced nicotinamide adenine dinucleotide) to NAD+ with Ir(III) complexes 2 and 4 displaying higher catalytic activity compared to their Ru(II) analogues. None of the complexes interacted with reduced glutathione (GSH). Additionally, 1-4 exhibited greater lipophilicity than cisplatin. In vitro biological analyses were performed in healthy cell lines (CCD-18Co colon and CCD-1072Sk foreskin fibroblasts) as well as in cisplatin-sensitive (A2780) and -resistant (A2780cis) ovarian cancer cell lines. The results indicated that Ir(III) complexes 2 and 4 had no effect on human fibroblasts, demonstrating their selectivity. In contrast, complexes 1 and 4 exhibited moderate inhibitory effects on the metabolic and proliferation activities of the cancer cells tested (selectivity index SI > 3.4 for 4 and 2.6 for cisplatin; SI = IC50(A2780)/IC50(CCD-18Co)), including the cisplatin-resistant cancer cell line. Based on these findings, it is possible to emphasize that mainly complex 4 could represent a further step in the development of selective and highly effective anticancer agents, particularly against resistant tumour types.

Detailed insight into chromium species released from failed CoCrMo implants: Ex vivo periprosthetic tissues study.

This unique study provides information on Cr species and their distribution in periprosthetic tissues of patients with metal-on-polyethylene joint implants. Co-Cr-Mo alloy has been widely used in joint replacement and represents a source of metal derived species. In the case of chromium, previous studies on periprosthetic tissues revealed mainly Cr(III) distribution, whereas the potential release of carcinogenic Cr(VI) species has been still a subject of debate. Here, an analytical approach utilizing speciation and fractionation was developed to analyze periprosthetic tissue samples collected from wide range of patients with failed total hip or knee replacements. The results reveal that Cr(III) is mainly released in the form of insoluble CrPO4 and Cr2 O3 particles. The highest Cr contents were found in periprosthetic tissues of patients suffering from aseptic loosening and having more Cr-based implants in the body. Cr species penetrated tissue layers, but their levels decreased with the distance from an implant. The detailed speciation/fractionation study carried out using the set of consecutive periprosthetic tissues of a patient with extensive metallosis showed the presence of trace amounts of free Cr(III), nanoparticles, and metal-protein complexes, but the majority of Cr still occurred in CrPO4 form. Carcinogenic Cr(VI) species were not detected. Up to date, there is no published human tissue study focused on the detailed speciation of both soluble and insoluble Cr-based species in the context of failing total hip and knee replacements.

Bottom-up uncertainty evaluation of complex measurements from correlated performance data: Determination of total Cr in yeast by ICP-MS after acid digestion.

The objective interpretation of a measurement result requires knowing the associated uncertainty. The cost-effective collection of measurement performance data on the same day produces correlated values that can affect measurement uncertainty evaluation. This work describes a novel methodology for the bottom-up evaluation of measurements based on complex sample pretreatment and the instrumental quantification of the prepared sample applicable to correlated inputs. The numerical Kragten method is used to combine the uncertainty components shared in various analyte recovery determinations. The developed methodology was applied to the determination of total chromium in yeast samples by ICP-MS after microwave-assisted acid digestion. The developed analysis of yeast samples is fit for monitoring the contamination of this product since it is associated with a relative expanded uncertainty, U', lower than 20%, ranging from 8.4% to 10.0% in determinations of Cr between 0.125 mg/kg and 305.5 mg/kg. Duplicate analyses are adequate for reference materials production (U' < 7%).

Stability of a half-sandwich Os(II) complex with indomethacin-functionalized ligand in the presence of carboxypeptidase A.

In the presence of carboxypeptidase, the hydrolytically stable complex [Os(η6-pcym)(L2)Cl]PF6 (2) partially released the bioactive substituent indomethacin, bound through the amide bond to the chelating 2-(1,3,4-thiadiazol-2-yl)pyridine-based moiety of L2. Stability in the presence of other relevant biomolecules (GSH, NADH, GMP) and cancer cell viability were also studied.

Prevalence of Vancomycin-Resistant Enterococci and Antimicrobial Residues in Wastewater and Surface Water.

Due to the extensive use of antimicrobial agents in human and veterinary medicine, residues of various antimicrobials get into wastewater and, subsequently, surface water. On the one hand, a combination of processes in wastewater treatment plants aims to eliminate chemical and biological pollutants; on the other hand, this environment may create conditions suitable for the horizontal transfer of resistance genes and potential selection of antibiotic-resistant bacteria. Wastewater and surface water samples (Morava River) were analyzed to determine the concentrations of 10 antibiotics and identify those exceeding so-called predicted no-effect environmental concentrations (PNECs). This study revealed that residues of five of the tested antimicrobials, namely ampicillin, clindamycin, tetracycline, tigecycline and vancomycin, in wastewater samples exceeded the PNEC. Vancomycin concentrations were analyzed with respect to the detected strains of vancomycin-resistant enterococci (VRE), in which the presence of resistance genes, virulence factors and potential relationship were analyzed. VRE were detected in 16 wastewater samples (11%) and two surface water samples (6%). The PNEC of vancomycin was exceed in 16% of the samples. Since the detected VRE did not correlate with the vancomycin concentrations, no direct relationship was confirmed between the residues of this antimicrobials and the presence of the resistant strains.

Tutorial and spreadsheet for the evaluation of instrumental quantification uncertainty by the linear weighted regression model: Determination of elemental impurities in a nasal spray by ICP-MS.

A tutorial and spreadsheet for the validation and bottom-up uncertainty evaluation of quantifications performed by instrumental methods of analysis based on linear weighted calibrations is presented. The developed tool automatically assesses if calibrator values uncertainty is negligible given instrumental signal precision, assesses signal homoscedasticity by the Levene's test, guides the selection of weighting factors and evaluates the fitness of the regression model to define the calibration curve. The spreadsheet allows the use of the linear weighted regression model without the need for collecting many replicate signals of calibrators and sample by taking previously developed detailed models of signal precision variation in the calibration interval after adjustments to the daily precision conditions. This tool was successfully applied to the determination of the mass concentration of Cd, Pb, As, Hg, Co, V and Ni in a nasal spray by ICP-MS after samples dilution and acidification. The developed uncertainty models were checked through the analysis of nasal sprays after spiking with known analyte concentration levels. The metrological compatibility between estimated and reference analyte levels for 95% or 99% confidence level supports uncertainty model adequacy. The spiked samples were quantified from many replicate signals but uncertainty evaluation from duplicate calibrator and sample signals was assessed by randomly selecting calibrators and sample signals and by numerically defining a minimum acceptable success rate of the compatibility tests. The developed model was proven adequate to quantify the uncertainty of the studied measurements.

The number of lymphocytes increases in the periprosthetic tissues with increasing time of implant service in non-metal-on-metal total joint arthroplasties: A role of metallic particles?

BACKGROUND: The objective of the study was to determine the association between periprosthetic concentrations of selected metals and changes induced in periprosthetic tissues (PT). METHODS: PT from 24 patients with metal-on-polyethylene or ceramic-on-polyethylene total joint replacements (TJRs) were examined. Samples underwent histological examination including quantification of cellular populations. Determination of metals was performed according to the published methodology. Results were processed using correlation analysis and Principal Component Analysis (PCA), respectively. RESULTS: Growing concentration of metals in the PT was found as a function of length of exposure (LoE). Differences in Ti, Co, Cr and V concentrations (per α = 0.05) depended on the type of alloy the implants were made from. On the contrary, the implant composition did not reflect in the different numbers of immune cells per 1 high power field, not even in distribution of the membrane type according to the Krenn classification. PCA revealed several clusters in dependence on the LoE, type of the membrane and presence of immune cells. High representation of lymphocytes in the PT was typical for clusters with the longest LoE while a higher representation of neutrophils was typical for a shorter time to reoperation. CONCLUSIONS: Correlation between the LoE and concentrations of metals in its surroundings was demonstrated. However, the tissue image analysis cannot differentiate finer, potentially metal-induced tissue changes. Importantly, the tissues become more similar with an increasing LoE. We draw a conclusion about predominantly non-specific stimulation of the PT jointly by metal and polyethylene particles in non-metal-on-metal TJRs.

Measurement uncertainty evaluation from correlated validation data: Determination of elemental impurities in pharmaceutical products by ICP-MS.

Pharmaceutical products as well as active pharmaceutical ingredients (APIs) are checked for levels of elemental contaminants to guarantee medicines administration will not involve the consumption of level of contaminants greater than their maximum admissible intake. However, the conformity decision is affected by the measurement uncertainty function of analytical steps performance, used standards quality and how measurement performance is assessed during method validation. When an ingredient is considered conform, since the measured concentration is lower than the maximum limit, the risk of a false acceptance depends on how close the measured concentration is from the limit and on the measurement uncertainty. The analytical methods used for pharmaceutical analysis should be validated by ICH and USP recommendations, in order to prove measurements are fit for purpose. The validation must also be economically feasible and have an acceptable duration. This work discusses how to evaluate the uncertainty of elemental analysis in pharmaceutical ingredients from data collected during the validation of the analytical method by following ICH guidelines and USP chapters. A top-down uncertainty evaluation based on results from the analysis of a model API intermediate, with the native analyte after spiking at three concentration levels, where analyses are performed by two analysts in two different days, is presented. The impact of the correlation of some uncertainty components of collected results on the uncertainty evaluation is discussed and considered in the calculations. The developed measurement model was checked by a cross-validation procedure where some validation data was randomly removed and used for an independent model control. The developed uncertainty evaluation methodology was successfully applied to the analysis of Pd in a model API intermediate by ICP-MS after a micro-wave assisted acid digestion, where the risk of a false acceptance of the pharmaceuticals is determined. The measurement performance data and used spreadsheet are made available as Supplementary Material.

Ion-exchange HPLC-ICP-MS: A new window to chromium speciation in biological tissues.

The presented work proposes a novel analytical ICP-MS-based approach for the accurate and precise chromium speciation in biological tissues. The determination of total Cr(VI) and soluble Cr(III) species was carried out by alkaline EDTA extraction followed by their separation using ion-exchange high-performance liquid chromatography inductively coupled plasma mass spectrometry (IE-HPLC-ICP-MS). The developed method was validated according to the procedure given in the United States Food and Drug Administration guideline on the validation of bioanalytical methods. Validation parameters included limit of detection (≤ 0.03 µg g-1), limit of quantification (≤ 0.08 µg g-1), linearity (r ≥ 0.9998), intra-day and inter-day accuracy (86-110%) and precision (≤ 10%), extraction recovery (89-110%), carry-over effect and sensitivity. In addition, special attention was paid to the study of chromium species interconversion and the elimination of spectral interferences. Moreover, the validated ICP-MS method employing microwave acid digestion was used to determine the total Cr content in collected fractions. Finally, the whole ICP-MS-based methodology was applied to the analyses of two certified reference materials of hepatopancreas tissue. Obtained results indicated that the majority of chromium in biological tissues is bound to the solid residue, Cr(VI) was determined in none of the samples investigated. This is the first study focusing on soluble Cr(III), total Cr(VI), and total bound Cr species in biological tissues. It is characterized by efficient sample preparation and fast simultaneous analysis of Cr species with parallel total Cr analysis serving for chromium balance evaluation.

Content of distinct metals in periprosthetic tissues and pseudosynovial joint fluid in patients with total joint arthroplasty.

This prospective study examined the content of metals released from total joint arthroplasty into joint fluid, whole blood and periprosthetic tissues. We determined the levels of Ti, V, Nb, Co, Cr, and Mo, using inductively coupled plasma mass spectrometry, in samples from patients who underwent reoperation of total hip or knee arthroplasty. All of the patients (n = 117) included in the study had either metal on polyethylene or ceramic on polyethylene-bearing pairs. First, our results conclusively showed that the majority of released metals were deposited in periprosthetic tissues. In this context, the bloodstream turned out to be an ineffective biomarker of the effects occurring in local tissues. Second, there was a clear time-dependent nature of metallic accumulation. Based on our extensive dataset, we found significantly elevated levels of the released metals in joint fluid and periprosthetic tissues originating from loosened implants compared to stable ones, as well as recognizable differences between the groups with stable implants and aseptic loosening. Finally, it was proved that the concentrations of metals decreased dependent on the distance of the tissue from the implant. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 454-462, 2019.

Study of chromium species release from metal implants in blood and joint effusion: Utilization of HPLC-ICP-MS.

The objective of this study was to develop and validate a novel analytical procedure for determination of total chromium and Cr(III) and Cr(VI) species released from metal implants into whole blood and joint effusion. Firstly, the ion-pair chromatographic method employing reversed-phase high-performance liquid chromatography inductively coupled plasma mass spectrometry (ICP-MS) for analysis of species was developed. Secondly, all samples, protein and low molecular fractions were analyzed for their total chromium content using ICP-MS. This new measurement procedure was validated by the following parameters: limit of detection (0.13 µg L-1 for Cr(III), 0.14 µg L-1 for Cr(VI)), linearity of calibration, trueness (recovery 84-92%), intermediate precision (RSD < 5%). We determined statistically significantly higher chromium levels in joint effusion samples obtained from patients in comparison with a control group. On the other hand, no relevant difference among the concentrations of both species and total chromium in blood was observed. Our results show that the majority of chromium is present in the trivalent form and bound to proteins. This speciation study is rare in the field of speciation analysis in clinical samples. It is characterized by very fast and simple sample preparation without any changes in distribution or stability of both Cr forms and efficient simultaneous analysis of Cr species.

Rhodamine bound maghemite as a long-term dual imaging nanoprobe of adipose tissue-derived mesenchymal stromal cells.

In the last few years, magnetically labeled cells have been intensively explored, and non-invasive cell tracking and magnetic manipulation methods have been tested in preclinical studies focused on cell transplantation. For clinical applications, it is desirable to know the intracellular pathway of nanoparticles, which can predict their biocompatibility with cells and the long-term imaging properties of labeled cells. Here, we quantified labeling efficiency, localization, and fluorescence properties of Rhodamine derivatized superparamagnetic maghemite nanoparticles (SAMN-R) in mesenchymal stromal cells (MSC). We investigated the stability of SAMN-R in the intracellular space during a long culture (20 days). Analyses were based on advanced confocal microscopy accompanied by atomic absorption spectroscopy (AAS) and magnetic resonance imaging. SAMN-R displayed excellent cellular uptake (24 h of labeling), and no toxicity of SAMN-R labeling was found. 83% of SAMN-R nanoparticles were localized in lysosomes, only 4.8% were found in mitochondria, and no particles were localized in the nucleus. On the basis of the MSC fluorescence measurement every 6 days, we also quantified the continual decrease of SAMN-R fluorescence in the average single MSC during 18 days. An additional set of analyses showed that the intracellular SAMN-R signal decrease was minimally caused by fluorophore degradation or nanoparticles extraction from the cells, main reason is a cell division. The fluorescence of SAMN-R nanoparticles within the cells was detectable minimally for 20 days. These observations indicate that SAMN-R nanoparticles have a potential for application in transplantation medicine.

Characterization of microbial siderophores by mass spectrometry.

Siderophores play important roles in microbial iron piracy, and are applied as infectious disease biomarkers and novel pharmaceutical drugs. Inductively coupled plasma and molecular mass spectrometry (ICP-MS) combined with high resolution separations allow characterization of siderophores in complex samples taking advantages of mass defect data filtering, tandem mass spectrometry, and iron-containing compound quantitation. The enrichment approaches used in siderophore analysis and current ICP-MS technologies are reviewed. The recent tools for fast dereplication of secondary metabolites and their databases are reported. This review on siderophores is concluded with their recent medical, biochemical, geochemical, and agricultural applications in mass spectrometry context.

Study of phenolic profile and antioxidant activity in selected Moravian wines during winemaking process by FT-IR spectroscopy.

Wine belongs to a family of products where the quality matters. Its quality can be in principle verified using diverse physicochemical approaches, including the determination of various chemical compounds generally accepted as chemical markers of product quality. Example of such applicable compounds is a family derived from phenols. Next to a more classical approach, infrared spectroscopy can play an important role in this game. Here we sought to develop an easy to use, ultra-fast and robust method based on FT-IR with some important advantages including lower sample and solvent consumptions. The tested and evaluated method was consequently applied in a monitoring of changes in a content of total phenolic compounds (TPC) and total antioxidant activity (TAA) during a process of wine-making. It was found out that total amount of phenolic compounds differs both for individual kind of wines, namely red, white and rose, at each processing stage of the production. The content of phenolic compounds of red and white wine increased while an opposite trend was observed in rose wine. TAA values of analysed wines showed difference between individual kind of wine and indicate the same trend like phenolic profile. Antioxidant activity values relate to changes of phenolic content during production process.

Interaction of selected platinum(II) complexes containing roscovitine-based CDK inhibitors as ligands with human liver microsomal cytochrome P450.

BACKGROUND: We studied the interaction of oxaliplatin derivatives involving cytotoxic adenine-based cyclin-dependent kinase inhibitors, with human liver microsomal cytochrome P450. METHODS AND RESULTS: The activities of 9 human liver microsomal CYP forms (CYPs 1A2, 7-ethoxyresorufin O-deethylation; 2A6, coumarin 7-hydroxylation; 2B6, 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation; 2C8, luciferin-6´ methyl ether demethylation; 2C9, diclofenac 4´-hydroxylation, 6´-deoxyluciferin hydroxylation; 2C19, (S)-mephenytoin 4´-hydroxylation; 2D6, bufuralol 1´-hydroxylation, 2E1, chlorzoxazone 6-hydroxylation; 3A4, testosterone 6ß-hydroxylation, luciferin-6´ benzyl ether debenzylation) were tested using HPLC, fluorescence and luminescence product detection. At 100 µM platinum(II) oxalato complex concentration, CYP inhibition was in general 25%-50%, except for the CYP3A4 form which showed roughly twice the inhibition (72%-95%). At low complex concentration (10 µM), the difference in inhibition of CYP3A4 and other forms was even more pronounced. Dixon and Lineweaver-Burk plots indicated a partially noncompetitive mechanism of CYP3A4 inhibition. CONCLUSIONS: The tested complexes significantly inhibit human liver microsomal CYP3A4 activity even at clinically relevant concentrations. This could be a serious drawback for the use of these compounds in clinical practice.

Mesenchymal stromal cell labeling by new uncoated superparamagnetic maghemite nanoparticles in comparison with commercial Resovist--an initial in vitro study.

OBJECTIVE: Cell therapies have emerged as a promising approach in medicine. The basis of each therapy is the injection of 1-100×10(6) cells with regenerative potential into some part of the body. Mesenchymal stromal cells (MSCs) are the most used cell type in the cell therapy nowadays, but no gold standard for the labeling of the MSCs for magnetic resonance imaging (MRI) is available yet. This work evaluates our newly synthesized uncoated superparamagnetic maghemite nanoparticles (surface-active maghemite nanoparticles - SAMNs) as an MRI contrast intracellular probe usable in a clinical 1.5 T MRI system. METHODS: MSCs from rat and human donors were isolated, and then incubated at different concentrations (10-200 µg/mL) of SAMN maghemite nanoparticles for 48 hours. Viability, proliferation, and nanoparticle uptake efficiency were tested (using fluorescence microscopy, xCELLigence analysis, atomic absorption spectroscopy, and advanced microscopy techniques). Migration capacity, cluster of differentiation markers, effect of nanoparticles on long-term viability, contrast properties in MRI, and cocultivation of labeled cells with myocytes were also studied. RESULTS: SAMNs do not affect MSC viability if the concentration does not exceed 100 µg ferumoxide/mL, and this concentration does not alter their cell phenotype and long-term proliferation profile. After 48 hours of incubation, MSCs labeled with SAMNs show more than double the amount of iron per cell compared to Resovist-labeled cells, which correlates well with the better contrast properties of the SAMN cell sample in T2-weighted MRI. SAMN-labeled MSCs display strong adherence and excellent elasticity in a beating myocyte culture for a minimum of 7 days. CONCLUSION: Detailed in vitro tests and phantom tests on ex vivo tissue show that the new SAMNs are efficient MRI contrast agent probes with exclusive intracellular uptake and high biological safety.

Expression response of duplicated metallothionein 3 gene to copper stress in Silene vulgaris ecotypes.

Metallothioneins (MTs) were identified as important players in metal metabolism. MT3 gene presents a key metallothionein controlling copper homeostasis in plants. We have selected one cupricolous and one non-cupricolous ecotype to isolate and analyse the MT3 gene in Silene vulgaris. For expression data comparison, we have also included other metal-tolerant ecotypes. Based on a S. vulgaris BAC library screening, we have identified and sequenced a genomic clone containing MT3 gene (SvMT3). We found that SvMT3 gene has been locally duplicated in a tandem arrangement. Expression analysis and complementation studies using yeast mutants showed that both copies of the SvMT3 gene were functional. Moreover, we examined the expression of MT3 gene(s) in selected ecotypes under different copper treatments to show the tissue-specific expression response to copper stress. We demonstrated that higher copper concentrations specifically affected MT3 expression among ecotypes. Our analysis shows that MT3a has similar expression pattern in cupricolous ecotypes while MT3b has common expression features shared by all metallophyte S. vulgaris ecotypes. Our data indicate that down-regulation of MT3b root expression in higher copper concentrations is associated with copper stress. We propose that there might be a specific regulation of SvMT3s transcription depending on the type of heavy metal tolerance.