Engineering Self-Calibrating Nanoprobes with Two-Photon-Activated Fluorescence Resonance Energy Transfer for Ratiometric Imaging of Biological Selenocysteine.

Selenocysteine (Sec) has proven to be the dominant active site of diverse selenoproteins that are directly linked with human health and disease. Thus, understanding the critical functions and dynamics of endogenous Sec at cellular and tissue levels is highly demanded. However, no method has been reported that is capable of providing reliable quantitative imaging analysis of Sec in living systems, especially in deep tissues, with low background signal and high sensitivity and imaging resolution simultaneously. To address this challenge, we herein report a novel class of engineered Sec-responsive fluorescent nanoprobes that combines two-photon excitation with Förster resonance energy transfer (FRET) mechanisms for direct, yet selective, sensing and imaging of biological Sec over abundant competing biothiols. Specifically, the two-photon excitation at the near-infrared window can minimize light scattering and background signals in tissues, thus offering improved spatial and temporal imaging of deep living tissues with reduced background interference. Moreover, a reasonable FRET donor-acceptor pair has further been designed and verified by theoretical calculation. The acceptor undergoes intramolecular rearrangement specifically in response to the nucleophilic attack of Sec, hence triggering remarkable FRET-mediated ratiometric fluorescence enhancement for sensitive and reliable quantification of Sec through self-calibration of two emission channels. These striking properties, along with good water solubility and biocompatibility, suggest that this strategy may serve as a valuable imaging tool for studying various Sec-related biological events in complex biological systems.

Investigation of free seleno-amino acids in extra-virgin olive oil by mixed mode solid phase extraction cleanup and enantioselective hydrophilic interaction liquid chromatography-tandem mass spectrometry.

An analytical method for determining seleno-methionine (SeMet), methyl-seleno-cysteine and seleno-cystine in extra-virgin olive oil (EVOO) was developed and validated. EVOO sample (15 g) was diluted with hexane, extracted with methanol/water 80:20 (v/v), and cleaned up by a reversed phase/strong cation exchange solid phase extraction. Analysis was performed by chiral hydrophilic interaction liquid chromatography-tandem mass spectrometry. Process efficiency ranged between 49 and 97% and trueness between 87 and 126%, with intermediate precision, expressed as standard deviation, lower than 10%. Method detection limits (MDLs) and method quantification limits (MQLs) were lower than 1 µg kg-1. Thirty-two EVOO samples from different Italian regions were analyzed for both total Se and single seleno-amino acids determination. Only l-SeMet was found at level MQL (0.2 µg kg-1)-1.42 µg kg-1 in ten samples, while total Se was in the range of MDL-9.1 µg kg-1. Concentration of l-SeMet (5-6% of total Se) and total Se correlated very well to each other (R2 = 0.995).

Selective Cytotoxic Activity of Se-Methyl-Seleno-L-Cysteine- and Se-Polysaccharide-Containing Extracts from Shiitake Medicinal Mushroom, Lentinus edodes (Agaricomycetes).

Numerous formulations derived from the shiitake medicinal mushroom, Lentinus edodes, demonstrate anticancer activities. We hypothesized that isolates from selenium (Se)-enriched mycelia of L. edodes would possess stronger cancer-preventive properties than current preparations. The aim of this study was to investigate whether the presence of Se-methyl-seleno-L-cysteine in mycelial extracts of L. edodes affects their cytotoxic activity (makes them stronger) or whether they are as effective as Se-containing polysaccharides. Extracts were prepared from Se-containing mycelia under various conditions and assayed for cytotoxic activity in cancer (PC3 and HeLa) and normal (HMEC-1) cell lines. The chemical composition of the extracts was examined; specifically, the amounts of potentially cytotoxic Se compounds (methylselenocysteine, selenomethionine, and Se-containing polysaccharides) were measured. The relationship between extract composition and biological activity was characterized. Mycelial cultures were cultivated in a 10-L bioreactor in medium enriched with sodium selenite. Mycelial extracts were prepared either at 100°C or at 4°C in acidic solution. Total Se content was determined using the atomic absorption spectrometry method, and methylselenocysteine and selenomethionine contents were measured using reverse-phase high-performance liquid chromatography. Protein, carbohydrate, and polyphenolic contents were determined with spectrophotometric methods, and Se-containing polysaccharides were measured with the use of precipitation. Anticancer activity of mycelial extracts was examined using the MTT cell viability assay. Extracts containing Se-methyl-seleno-L-cysteine or Se-polysaccharides prepared at 4°C and 100°C, respectively, display moderate, time-dependent, specific cytotoxic activity in HeLa and PC3 cell lines. The effect in HeLa cells is more pronounced in the extract prepared at 4°C than at 100°C. The effect is almost equal for the PC3 cell line. However, both extracts have no effect or only slightly stimulate normal (HMEC-1) cell viability. The selective cytotoxic activity of L. edodes extracts in cancer (PC3 and HeLa) cells is due to the presence of both Se-methyl-seleno-L-cysteine and selenated polysaccharides, perhaps in combination with other active ingredients.

The response of broccoli (Brassica oleracea convar. italica) varieties on foliar application of selenium: uptake, translocation, and speciation.

A model small-scale field experiment was set up to investigate selenium (Se) uptake by four different varieties of broccoli plants, as well as the effect of Se foliar application on the uptake of essential elements for plants calcium (Ca), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), phosphorus (P), sulfur (S), and zinc (Zn). Foliar application of sodium selenate (Na2SeO4) was carried out at two rates (25 and 50 g Se/ha), and an untreated control variant was included. Analyses of individual parts of broccoli were performed, whereby it was found that Se in the plant accumulates mainly in the flower heads and slightly less in the leaves, stems, and roots, regardless of the Se rate and broccoli variety. In most cases, there was a statistically significant increase of Se content in all parts of the plant, while there was no confirmed systematic influence of the addition of Se on the changing intake of other monitored elements. Selenization of broccoli leads to an effective increase in the Se content at a rate of 25 g/ha, whereas the higher rate did not result in a substantial increase of Se content compared to the lower rate in all varieties. Therefore, the rate of 25 g/ha can be recommended as effective to produce broccoli with an increased Se content suitable for consumption. Moreover, Se application resulted in an adequate increase of the main organic compounds of Se, such as selenocystine (SeCys2), selenomethionine (SeMet), and Se-methylselenocysteine (Se-MeSeCys).

Evaluation of selenium species in selenium-enriched pakchoi (Brassica chinensis Jusl var parachinensis (Bailey) Tsen & Lee) using mixed ion-pair reversed phase HPLC-ICP-MS.

HPLC-ICP-MS based on ion-paired reversed phase chromatography for the selenium speciation using the mixture of 1-butanesulfonic acid (BA) and trifluoroacetic acid (TFA) as the mixed ion-pairing reagents was developed and applied to selenium-enriched pakchoi (Brassica chinensis Jusl var parachinensis (Bailey) Tsen & Lee). Several conditions of ion-paired reversed phase HPLC-ICP-MS, such as pH of the mobile phase, concentration of ion pairing reagents, types and length of analytical column, and flow rate of the mobile phase, were optimised for five selenium species; selenate (Se(VI)), Selenite (se(IV)), selenocysteine (SeC), Se-methylselenocysteine (SeMC) and selenomethionine (SeM). The results showed that the optimum conditions for pH, BA and TFA condition, type of separating column and flow rate, were 4.5, 8mM, 4mM, C18 (250 mm length × 4.6mm I.D) and 1.2 mL min(-1), respectively. These conditions archived separation of the organic selenium species. The limits of detection (LOD) and quantitation (LOQ) of each selenium species were lower than 5 and 16 ng Se mL(-1), respectively. Furthermore, the recoveries of most selenium species were good, except for SeC. In this research, selenium-enriched pakchoi was cultivated by supplementing inorganic selenium from selenate into sand. The result showed that inorganic selenium, SeMC, SeM and several unknown species were found in selenium-enriched pakchoi sprouts by using the proposed method. Thereby, the biotransformation of selenate in pakchoi was similar to other Brassicaceae plants such as kale and broccoli.

Determination of selenomethionine and seleno-methyl-selenocysteine in biota by ultrasonic-assisted enzymatic digestion and multi-shot stir bar sorptive extraction-thermal desorption-gas chromatography-mass spectrometry.

A method based on stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) has been optimized for the determination of seleno-methyl-selenocysteine (SeMetSeCys) and selenomethionine (SeMet) in biota samples. Aliquots of freeze-dried tissue, a mixture of protease XIV-lipase and water were sonicated for 2min. After extraction, the extract was separated by centrifugation and subjected to derivatization and SBSE-TD-GC-MS. The parameters affecting derivatization, absorption and desorption steps were investigated. The optimized conditions consist of a derivatization with 40µL of ethyl chloroformate (ECF) in 400µL of a water:ethanol:pyridine (60:32:8) mixture, followed by dilution to 1.5mL of 70g NaClL(-1) in water at neutral pH and an extraction step using 10mm×1mm PDMS stir bar, stirring at 800rpm for 20min at room temperature (23±1°C). Three stir bars were used for the extraction of three different aliquots of the same sample and then placed in a single glass desorption liner and simultaneously desorbed for GC-MS analysis. The desorption step required the following conditions: 300°C (desorption temperature), 6min (desorption time), 50mLmin(-1) (vent flow) and -5°C (cryotrapping temperature). The method provided precise (8.1%) and accurate results in the mgSekg(-1) range (using the selected-ion monitoring-SIM mode) against certified reference material SELM-1 yeast, with recoveries higher than 80% for spiked algae and clams samples.

Hyphenated techniques for speciation of Se in in vitro gastrointestinal digests of Saccharomyces cerevisiae.

A method was developed allowing the separation, detection and identification of Se species extracted from yeast supplements during simulated digestion processes. The in vitro gastric and intestinal digests were studied for their Se compounds by successive high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES-MS-MS) analyses. The conditions for the separation were chosen as to be compatible with both ICP-MS and ES-MS-MS detection. HPLC-ICP-MS was used to screen the extracts for their Se content. By means of HPLC-ES-MS-MS, the compounds extracted were identified on-line according to their retention time, m/ z of the molecular ion and the presence of typical product ions. From these results, it was clear that the main compound extracted by both gastric and intestinal fluid was Se-methionine, which was also the main Se compound extracted by proteolytic digestion from the yeast supplements. Two other minor compounds could be identified as Se-cystine and Se(O)-methionine, a degradation product of Se-methionine.

Exploiting the 21st amino acid-purifying and labeling proteins by selenolate targeting.

Selenium is essential to human life and occurs in selenoproteins as selenocysteine (Sec), the 21st amino acid. The selenium atom endows selenocysteine with unique biochemical properties, including a low pK(a) and a high reactivity with many electrophilic agents. Here we describe the introduction of selenocysteine into recombinant non-selenoproteins produced in Escherichia coli, as part of a small tetrapeptide motif at the C terminus. This selenocysteine-containing motif could subsequently be used as a protein tag for purification of the recombinant protein, selenolate-targeted labeling with fluorescent compounds or radiolabeling with either gamma-emitting (75)Se or short-lived positron emitters such as (11)C. The results presented here thus show how a wide range of biotechnological applications can be developed starting from the insertion of selenocysteine into proteins.

Modern trends in the speciation of selenium by hyphenated techniques.

The complexity of selenium speciation in the environment and in living organisms results in broad analytical challenges. The importance of the selective determination of the particular species of this element, to understand its metabolism and biological significance in clinical chemistry, biology, toxicology, and nutrition, calls for state-of-the-art analytical techniques. In this paper hyphenated techniques are evaluated with particular emphasis on interfaced separation with element-selective detection and identification of the selenium compounds detected.

Direct determination of seleno-amino acids in biological tissues by anion-exchange separation and electrochemical detection.

Several studies have described the determination of selenium in protein extracts from tissues of marine or terrestrial animals, but have not identified the different chemical forms of selenium that are present. Selenium may be present as seleno-amino acids. Selenocysteine, for example, is a normal component of glutathione peroxidase, an antioxidant enzyme which may behave like other antioxidants, such as vitamin E, protecting tissues against methylmercury toxicity. The present study illustrates a method for the characterization of seleno-amino acids, such as selenocysteine and selenomethionine, in proteins extracted from the liver of marine mammals. The mechanism of detoxification of methylmercury, which involves seleno-compounds, is identified. The analytical determination was carried out using high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection (HPAEC-IPAD). This method allows the direct determination of underivatized amino acids, eliminating the procedure of pre- or postcolumn derivatization. The chromatographic separation was carried out on an anion-exchange column using a quaternary gradient elution. In order to optimize this method, interferences of amino acids and the influence of pH and ionic strength on the separation and electrochemical detection were studied. The IPAD response for the direct detection of amino acids is optimum at pH > 11. The detection limit (S/N = 3) for selenocysteine was found to be 450 micrograms/l. The application of this method for the identification of seleno-amino acids in protein hydrolysates is also shown.