Amplification-Free Strategy for miRNA Quantification in Human Serum Using Single Particle ICP-MS and Gold Nanoparticles as Labels.

MicroRNAs (miRNAs), which are short single-stranded RNA sequences between 18 and 24 nucleotides, are known to play a crucial role in gene expression. Changes in their expression are not only involved in many diseases but also as a response to physiological changes, such as physical exercise. In this work, a new analytical strategy for the sensitive and specific analysis of miRNA sequences in human plasma is presented. The developed strategy does not depend on any nucleic acid amplification process and can be obtained in direct correlation to the number of events obtained by using single-particle ICP-MS measurements. The high selectivity of the assay (up to single nucleotide polymorphisms) can be achieved by a double hybridization process of the target miRNA with a complementary capture oligonucleotide that is conjugated to a magnetic microparticle and simultaneously with a complementary reporter oligonucleotide conjugated to a gold nanoparticle. Thanks to the novel approach followed in this method, the stoichiometry of the oligonucleotide-nanoparticle conjugates does not need to be addressed for the quantification of the target miRNA, which also represents a big advantage over other similar methods. The optimized method is applied to the determination of a miRNA as a biomarker of physical exercise in non-spiked human serum samples, and the results are validated against rt-qPCR. The achieved sensitivity permits the direct differentiation among sedentary and sportive subjects. This general platform can be easily applied to any other sequence by only modifying the capture and reporter oligonucleotides, paving the way for multiple clinically interesting applications.

Determination of selenium-containing species, including nanoparticles, in selenium-enriched Lingzhi mushrooms.

Mushrooms are considered a valuable food source due to their high protein and fibre and low fat content, among the other health benefits of their consumption. Selenium is an essential nutrient and is renowned for its chemo-preventative properties. In this study, batches of selenium-enriched Lingzhi mushrooms were prepared by growing mycelium and fruit in substrates containing various concentrations of sodium selenite. The mushroom fruit accumulated low levels of selenium with selenomethionine being the most abundant form in all enriched samples. Conversely, the mycelium showed significant selenium accumulation but relatively low proportions of selenomethionine. The red colour of the selenium-enriched mycelia indicated the probable presence of selenium nanoparticles, which was confirmed by single-particle inductively coupled plasma-mass spectrometry. Mean particle diameters of 90-120 nm were observed, with size distributions of 60-250 nm. Additional analysis with transmission electron microscopy confirmed this size distribution and showed that the biogenic selenium nanoparticles were roughly spherical in shape and contained elemental selenium.

Single-Cell ICP-MS in Combination with Fluorescence-Activated Cell Sorting for Investigating the Effects of Nanotransported Cisplatin(IV) Prodrugs.

The combined use of fluorescence-activated cell sorting (FACS) and single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) is reported, for the first time, in this work. It is applied to evaluate the differences between the cellular uptake of ultrasmall iron oxide nanoparticles (FeNPs) loaded with cisplatin(IV) prodrug (FeNPs-Pt(IV)) and cisplatin regarding cell viability. For this aim, FACS is applied to separate viable, apoptotic, and necrotic A2780 ovarian cancer cells after exposing them to the nanotransported prodrug and cisplatin, respectively. The different sorted cell populations are individually analyzed using quantitative SC-ICP-MS to address the intracellular amount of Pt. The highest Pt intracellular content occurs in the apoptotic cell population (about 2.1 fg Pt/cell) with a narrow intercellular distribution when using FeNPs-Pt(IV) nanoprodrug and containing the largest number of cells (75% of the total). In the case of the cisplatin-treated cells, the highest Pt content (about 1.6 fg Pt/cell) could be determined in the viable sorted cell population. The combined methodology, never explored before, permits a more accurate picture of the effect of the intracellular drug content together with the cell death mechanisms associated with the free drug and the nanotransported prodrug, respectively, and opens the door to many possible single-cell experiments in sorted cell populations.

Elucidating the Therapeutic Potential of Bis(Maltolato)OxoVanadium(IV): The Protective Role of Copper in Cellular Metabolism.

Vanadium (V) is a trace mineral whose biological activity, role as a micronutrient, and pharmacotherapeutic applications remain unknown. Over the last years, interest in V has increased due to its potential use as an antidiabetic agent mediated by its ability to improve glycemic metabolism. However, some toxicological aspects limit its potential therapeutic application. The present study aims to evaluate the effect of the co-treatment with copper (Cu) and bis(maltolato)oxovanadium(IV) (BMOV) as a possible strategy to reduce the toxicity of BMOV. Treating hepatic cells with BMOV reduced cell viability under the present conditions, but cell viability was corrected when cells were co-incubated with BMOV and Cu. Additionally, the effect of these two minerals on nuclear and mitochondrial DNA was evaluated. Co-treatment with both metals reduced the nuclear damage caused by BMOV. Moreover, treatment with these two metals simultaneously tended to reduce the ND1/ND4 deletion of the mitochondrial DNA produced with the treatment using BMOV alone. In conclusion, these results showed that combining Cu and V could effectively reduce the toxicity associated with V and enhance its potential therapeutic applications.

Intracellular Biotransformation of Ultrasmall Iron Oxide Nanoparticles and Their Effect in Cultured Human Cells and in Drosophila Larvae In Vivo.

A systematic investigation on the cellular uptake, intracellular dissolution, and in vitro biological effects of ultra-small (<10 nm) iron hydroxide adipate/tartrate coated nanoparticles (FeAT-NPs) was carried out in intestinal Caco-2, hepatic HepG2 and ovarian A2780 cells, and the nucleotide excision repair (NER) deficient GM04312 fibroblasts. Quantitative evaluation of the nanoparticles uptake, as well as their transformation within the cell cytosol, was performed by inductively coupled plasma mass spectrometry (ICP-MS), alone or in combination with high performance liquid chromatography (HPLC). The obtained results revealed that FeAT-NPs are effectively taken up in a cell type-dependent manner with a minimum dissolution after 3 h. These results correlated with no effects on cell proliferation and minor effects on cell viability and reactive oxygen species (ROS) production for all the cell lines under study. Moreover, the comet assay results revealed significant DNA damage only in GM04312 cells. In vivo genotoxicity was further studied in larvae from Drosophila melanogaster, using the eye-SMART test. The obtained results showed that FeAT-NPs were genotoxic only with the two highest tested concentrations (2 and 5 mmol·L−1 of Fe) in surface treatments. These data altogether show that these nanoparticles represent a safe alternative for anemia management, with high uptake level and controlled iron release.

A drought-responsive rice amidohydrolase is the elusive plant guanine deaminase with the potential to modulate the epigenome.

Drought stress in plants causes differential expression of numerous genes. One of these differentially expressed genes in rice is a specific amidohydrolase. We characterized this amidohydrolase gene on the rice chromosome 12 as the first plant guanine deaminase (OsGDA1). The biochemical activity of GDA is known from tea and coffee plants where its catalytic product, xanthine, is the precursor for theine and caffeine. However, no plant gene that is coding for GDA is known so far. Recombinant OsGDA1 converted guanine to xanthine in vitro. Measurement of guanine and xanthine contents in the OsGDA1 knockout (KO) line and in the wild type Tainung 67 rice plants also suggested GDA activity in vivo. The content of cellular xanthine is important because of its catabolic products allantoin, ureides, and urea which play roles in water and nitrogen stress tolerance among others. The identification of OsGDA1 fills a critical gap in the S-adenosyl-methionine (SAM) to xanthine pathway. SAM is converted to S-adenosyl-homocysteine (SAH) and finally to xanthine. SAH is a potent inhibitor of DNA methyltransferases, the reduction of which leads to increased DNA methylation and gene silencing in Arabidopsis. We report that the OsGDA1 KO line exhibited a decrease in SAM, SAH and adenosine and an increase in rice genome methylation. The OsGDA1 protein phylogeny combined with mutational protein destabilization analysis suggested artificial selection for null mutants, which could affect genome methylation as in the KO line. Limited information on genes that may affect epigenetics indirectly requires deeper insights into such a role and effect of purine catabolism and related genetic networks.

The Modulation of SCO2730/31 Copper Chaperone/Transporter Orthologue Expression Enhances Secondary Metabolism in Streptomycetes.

Streptomycetes are important biotechnological bacteria that produce several clinically bioactive compounds. They have a complex development, including hyphae differentiation and sporulation. Cytosolic copper is a well-known modulator of differentiation and secondary metabolism. The interruption of the Streptomyces coelicolor SCO2730 (copper chaperone, SCO2730::Tn5062 mutant) blocks SCO2730 and reduces SCO2731 (P-type ATPase copper export) expressions, decreasing copper export and increasing cytosolic copper. This mutation triggers the expression of 13 secondary metabolite clusters, including cryptic pathways, during the whole developmental cycle, skipping the vegetative, non-productive stage. As a proof of concept, here, we tested whether the knockdown of the SCO2730/31 orthologue expression can enhance secondary metabolism in streptomycetes. We created a SCO2730/31 consensus antisense mRNA from the sequences of seven key streptomycetes, which helped to increase the cytosolic copper in S. coelicolor, albeit to a lower level than in the SCO2730::Tn5062 mutant. This antisense mRNA affected the production of at least six secondary metabolites (CDA, 2-methylisoborneol, undecylprodigiosin, tetrahydroxynaphtalene, α-actinorhodin, ε-actinorhodin) in the S. coelicolor, and five (phenanthroviridin, alkylresorcinol, chloramphenicol, pikromycin, jadomycin G) in the S. venezuelae; it also helped to alter the S. albus metabolome. The SCO2730/31 consensus antisense mRNA designed here constitutes a tool for the knockdown of SCO2730/31 expression and for the enhancement of Streptomyces' secondary metabolism.

Fragmentation of Proteins in the Corona of Gold Nanoparticles As Observed in Live Cell Surface-Enhanced Raman Scattering.

Surface-enhanced Raman scattering (SERS) can provide information on the structure, composition, and interaction of molecules in the proximity of gold nanoparticles, thereby enabling studies of adsorbed biomolecules in vivo. Here, the processing of the protein corona and the corresponding protein-nanoparticle interactions in live J774 cells incubated with gold nanoparticles was characterized by SERS. Samples of isolated cytoplasm, devoid of active processing, of the same cell line were used as references. The occurrence of the most important SERS signals was compared in both types of samples. The comparison of signal abundances, supported by multivariate assessment, suggests a decreased nanoparticle-peptide backbone interaction and an increased contribution of denatured proteins in endolysosomal compartments, indicating an interaction of protein fragments with the gold nanoparticles in the endolysosome of the living cells. To study the protein fragmentation in a model and to confirm the assignment of specific spectral signatures in the live cell spectra, SERS data were collected from a solution of bovine serum albumin (BSA) digested by trypsin as an enzymatic model and from solutions of intact BSA and trypsin. The spectra from the enzymatic model confirm the strong interaction of protein fragments with the gold nanoparticles in the endolysosomal compartments. By proteomic analysis, using combined sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry of the extracted hard corona, we directly identified protein fragments, some originating from the culture medium. The results illustrate the use of appropriate models for the validation of SERS spectra and have potential implications for further developments of SERS as an in vivo analytical and biomedical tool.

Analytical strategies to study the formation and drug delivery capabilities of ferritin-encapsulated cisplatin in sensitive and resistant cell models.

One of the limitations in the use of cisplatin is its low penetration into cells. In addition, some cells develop the so called resistance, a multifactorial event that decreases significantly the intracellular cisplatin concentration. To circumvent these limitations, recent studies are focused on the use of nanocarriers that permit, among others, to achieve higher drug uptake. In this work, ferritin is evaluated as a nanostructured cisplatin-delivery system in cell models of ovarian cancer. One of the key aspects is the characterization of the encapsulated product, and for this aim, a battery of analytical techniques, including size exclusion chromatography (SEC) coupled to UV detection and to inductively coupled plasma mass spectrometry (ICP-MS) together with transmission electron microscopy (TEM), is conducted. Higher level of incorporation occurs when using initial concentrations of the Fe-containing form of the protein at 10 mg/mL and 1 mg/mL cisplatin solution. The incorporation of the free and encapsulated cisplatin is addressed in A2780 and A2780CIS, sensitive and cisplatin-resistant cell lines, respectively, showing a significantly higher uptake of the encapsulated form. These values ranged from 5- to 9-fold in the sensitive line and 2-4 in the resistant model, being always more pronounced at the lower doses. Functionality of the drug after encapsulation is addressed by monitoring the presence of Pt in DNA and normalizing DNA concentration through simultaneous P and Pt measurements by ICP-MS. Time elapsed between exposure and Pt detection in DNA proved to be critical in the encapsulated model, showing the slower drug release mechanism from the ferritin nanocage that could be advantageously used for a controlled therapy. Graphical abstract.

Quantitative Analysis of Transferrin Receptor 1 (TfR1) in Individual Breast Cancer Cells by Means of Labeled Antibodies and Elemental (ICP-MS) Detection.

Cells are able to precisely control the amount of iron they acquire in the form of transferrin (TF)-bound iron by modulating the synthesis of transferrin receptor 1 (TfR1). In tumor cells, elevated TfR1 seems to be related to poorer outcome for patients. Thus, the direct measurement of this biomarker in breast cancer tissues and cells might serve as a prognosis biomarker. In this work, we have used Nd-labeled antibodies to tag the TfR1 present on the cell surface of two cell models of breast cancer with different malignancy (MCF7 and MDA-MB 231). For this aim, monoclonal antibody anti-TfR1 is first labeled with a polymeric chelator (MAXPAR) with the subsequent incorporation of several isotopic 143Nd atoms. The characterization of the labeled antibody revealed a stoichiometry of 21 Nd atoms per antibody molecule that can be used for further quantification experiments. This antibody is used for cell tagging followed by single-cell analysis using inductively coupled plasma mass spectrometry (ICP-MS) detection. In this case, cell introduction is conducted using a high-efficiency nebulizer and spray chamber to achieve transport efficiencies of up to 55% for cells. Quantitative results revealed a number of receptors per cell significantly higher in the case of the most malignant phenotype (MDA-MB-231). Absolute and relative TfR1 concentration values are obtained in individual cells for the first time using the proposed system.

Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples.

Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40 µg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).

Gold nanoparticles: Distribution, bioaccumulation and toxicity. In vitro and in vivo studies.

Concerns about the bioaccumulation and toxicity of gold nanoparticles inside humans have recently risen. HT-29 and HepG2 cell lines and Wistar rats were exposed to 10, 30 or 60 nm gold nanoparticles to determine their tissue distribution, subcellular location and deleterious effects. Cell viability, ROS production and DNA damage were evaluated in vitro. Lipid peroxidation and protein carbonylation were determined in liver. ICP-MS measurements showed the presence of gold in intestine, kidney, liver, spleen, feces and urine. Subcellular locations of gold nanoparticles were observed in colon cells and liver samples by transmission electron microscopy. Inflammatory markers in liver and biochemical parameters in plasma were measured to assess the inflammatory status and presence of tissue damage. The size of the nanoparticles determined differences in the biodistribution and the excretion route. The smallest nanoparticles showed more deleterious effects, confirmed by their location inside the cell nucleus and the higher DNA damage.

The Use of Stable Isotopic Tracers in Metallomics Studies.

Mass spectrometry represents an essential technique in Metallomics studies. It permits the identification of metal-containing molecules as part of the metallome as well as their quantification at low concentration levels. The technique becomes even more powerful in combination with the use of isotopically enriched species. Provided that they are stable, these isotopically labelled species can be easily distinguished from their natural counterparts by mass spectrometric techniques. This capability permits that these species are used for accurate and precise quantitative experiments and/or metabolic studies applying inductively coupled plasma as ionization source. In this chapter, we present the different concepts of using stable isotope tracers and isotope dilution analysis as quantification strategy. Besides some fundamental aspects, various examples from Metallomics studies, for instance, on the preparation of isotopically enriched metalloproteins and determination by isotope dilution analysis or the exploration of the biological pathways of Se species, are shown in order to demonstrate the usefulness of isotopes.

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR.

Hepcidin-25 was identified as the main iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry (MS/MS), high-resolution mass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D) model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.

Elemental labelling and mass spectrometry for the specific detection of sulfenic acid groups in model peptides: a proof of concept.

Oxidative transformation of cysteine thiol groups into different functional groups is considered a significant posttranslational modification of great importance in pathological and physiological processes. A cysteine sulfenic acid (SA) residue is the transient state for thiol group oxidation and it can react with free thiols to form disulfide bonds or can be further oxidized with reactive oxygen/reactive nitrogen species (ROS/RNS) to form sulfinic and sulfonic acids. The increase in ROS/RNS concentrations is correlated to age-related diseases such as cancer and Alzheimer's disease. Since the formation of SA represents a transient state of oxidation of thiols, its formation can be considered a redox-sensitive sensor for the presence of ROS/RNS. Thereby, the detection of the short-lived SA will provide greater insight into the redox-mediated events that alter the structure and function of peptides and proteins. The aim of this study is to provide a new strategy for the highly sensitive and specific detection of SA in peptides as a proof of concept. For this aim, SA was firstly generated in model peptides on oxidation with H2O2 and then captured by the linear alkyne ß-ketoester (KE) previously linked to a lanthanide (Ln)-containing chelator (Ln-DOTA, where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The linking of the KE to DOTA was performed by click chemistry, resulting in a new reagent (Ln-DOTA-KE) that permits highly sensitive elemental (inductively coupled plasma) and molecular (electrospray) mass spectrometric detection. The new reagent (Ln-DOTA-KE) reacts specifically with SA, offering improved reactivity at physiological pH, facile derivatization and a cell-membrane-permeable compound that has promising future applications. Graphical Abstract A new derivatizing reagent for specific detection of sulphenic acid (SA) generated in model peptides by oxidation of cysteine groups is presented in this work.

Anion exchange chromatography for the determination of 5-methyl-2'-deoxycytidine: application to cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines.

Epigenetic alterations are increasingly implicated in the initiation and progression of cancer. Genome-wide (global) hypomethylation seems to occur in early neoplasia and is a feature of genomic DNA derived from solid tumour tissues like ovarian cancer. Thus, analytical methods that provide sensitive and quantitative information about cytosine methylation in DNA are currently required. In this work, we compare two different anion-exchange columns for the separation of methylated cytosine from the other DNA nucleotides: a silica-based (Tracer Extrasil SAX) column and a polystyrene/divinyl benzene-based (Mono-Q™) column. Under the optimised conditions, linearity range, precision and detection limits of the developed high-performance liquid chromatography (HPLC) method were evaluated and compared using conventional ultraviolet (UV) absorbance detection at 270 nm. Good separation of the five target nucleotides, including 5-methyl-2'-deoxycytidine monophosphate (5mdCMP) and 2'-deoxycytidine monophosphate (dCMP) was achieved on the Mono-Q™ column with a gradient elution of ammonium acetate buffer (1 M, pH 6.9) at a flow rate of 1 mL min(-1). The coupling of this column to inductively coupled plasma mass spectrometry (ICP-MS) permitted also phosphorous ((31)P) specific detection of the nucleotides. Both detection systems offered adequate analytical performance characteristics, with detection limits of 30 and 40 µg L(-1) for 5mdCMP by HPLC-UV and HPLC-ICP-MS, respectively. However, the latter method allowed the determination of the global DNA methylation level (%) without the need for external calibration. Different genomic DNA samples were analysed including calf thymus DNA and DNA from two human cancer cell lines (adenocarcinoma epithelial A549 and ovarian carcinoma A2780) using the proposed strategy. In the line A2780, the cisplatin-sensitive and cisplatin-resistant variants were analysed, finding no significant differences in the methylation percentage after treatment with cisplatin.

Determination of reduced homocysteine in human serum by elemental labelling and liquid chromatography with ICP-MS and ESI-MS detection.

Analytical methods allowing sensitive determination of reduced homocysteine (rHcy), one of the so-called biothiols, in human serum is a topic of growing interest due to its close relation to several human disorders, mainly cardiovascular diseases. Although most widely used analytical strategies to determine total Hcy involve derivatization by means of fluorescent labels, this work proposes the use of ebselen, a Se-containing labelling agent to derivatize the reactive sulfhydryl group of the Hcy molecule in its "free" reduced form, which is more likely to play different roles in disease pathogenesis. Optimization of the derivatization and separation conditions by high-performance liquid chromatography (HPLC) to isolate the excess of derivatizing reagent is carried out here using UV/VIS detection. Further, the study of the Se labelling reaction by electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides a stoichiometry of the derivative of 1:1. The main advantage of using ebselen as a labelling agent is the presence of the Se atom in the molecule that allows the use of inductively coupled plasma mass spectrometry (ICP-MS) as a sensitive and selective Se detector. The coupling of HPLC with ICP-MS provided excellent features for the determination of Se-derivatized rHcy (detection limit of 9.6 nM) in real samples. Quantification was accomplished by using post-column isotope dilution (ID) of Se in serum samples, after precipitation of the main serum proteins. Quantitative results for "free" rHcy turned out to be around 0.18-0.22 µM in serum samples from healthy individuals that could be directly analyzed without sample preconcentration.

Changes in iron metabolism and oxidative status in STZ-induced diabetic rats treated with bis(maltolato) oxovanadium (IV) as an antidiabetic agent.

The role of vanadium as a micronutrient and hypoglycaemic agent has yet to be fully clarified. The present study was undertaken to investigate changes in the metabolism of iron and in antioxidant defences of diabetic STZ rats following treatment with vanadium. Four groups were examined: control; diabetic; diabetic treated with 1 mgV/day; and Diabetic treated with 3 mgV/day. The vanadium was supplied in drinking water as bis(maltolato) oxovanadium (IV) (BMOV). The experiment had a duration of five weeks. Iron was measured in food, faeces, urine, serum, muscle, kidney, liver, spleen, and femur. Superoxide dismutase, catalase, NAD(P)H: quinone-oxidoreductase-1 (NQO1) activity, and protein carbonyl group levels in the liver were determined. In the diabetic rats, higher levels of Fe absorbed, Fe content in kidney, muscle, and femur, and NQO1 activity were recorded, together with decreased catalase activity, in comparison with the control rats. In the rats treated with 3 mgV/day, there was a significant decrease in fasting glycaemia, Fe content in the liver, spleen, and heart, catalase activity, and levels of protein carbonyl groups in comparison with the diabetic group. In conclusion BMOV was a dose-dependent hypoglycaemic agent. Treatment with 3 mgV/day provoked increased Fe deposits in the tissues, which promoted a protein oxidative damage in the liver.

Biomarkers in Ovarian Cancer: Towards Personalized Medicine.

Ovarian cancer is one of the deadliest cancers in women. The lack of specific symptoms, especially at the initial stages of disease development, together with the malignancy heterogeneity, lower the life expectancy of patients. Aiming to improve survival rates, diagnostic and prognostic biomarkers are increasingly employed in clinics, providing gynecologists and oncologists with new tools to guide their treatment decisions. Despite the vast number of investigations, there is still an urgent need to discover more ovarian cancer subtype-specific markers which could further improve patient classification. To this end, high-throughput screening technologies, like mass spectrometry, are applied to deepen the tumoral cellular landscape and describe the malignant phenotypes. As for disease treatment, new targeted therapies, such as those based on PARP inhibitors, have shown great efficacy in destroying the tumoral cells. Likewise, drug-nanocarrier systems targeting the tumoral cells have exhibited promising results. In this narrative review, we summarize the latest achievements in the pursuit of biomarkers for ovarian cancer and recent anti-tumoral therapies.

Bioaccessibility of trace elements and Fe and Al endogenic nanoparticles in farmed insects: Pursuing quality sustainable food.

This study investigated the in vitro bioaccessibility of aluminum, copper, iron, manganese, lead, selenium, and zinc in three important species of farmed insects: the yellow mealworm (Tenebrio molitor), the house cricket (Acheta domesticus) and the migratory locust (Locusta migratoria). Results show that all three insect species constitute excellent sources of essential elements (Fe, Cu and Zn) for the human diet, contributing to the recommended dietary allowance, i.e., 10%, 50%, and 92%, respectively. A higher accumulation of Se (≥1.4 mg Se/kg) was observed with increasing exposure concentration in A. domesticus, showing the possibility of using insects as a supplements for this element. The presence of Al and Fe nanoparticles was confirmed in all three species using single particle-inductively coupled plasma-mass spectrometry and transmission electron microscopy. The results also indicate that Fe bioaccessibility declines with increasing Fe-nanoparticle concentration. These findings contribute to increase the nutritional and toxicological insights of farmed insects.