The ferrozine (FZ) assay is a vital oxidation state-specific colorimetric assay for the quantification of Fe2+ ions in environmental samples due to its sharp increase in absorbance at 562 nm upon addition of Fe2+. However, it has yet to be applied to corresponding fluoresence assays which typically offer higher sensitivites and lower detection limits. In this article we present for the first time its pairing with upconverting luminescent nanomaterials to enable detection of Fe2+via the inner filter effect using a low-power continuous wave diode laser (45 mW). Upon near infra-red excitation at 980 nm, the overlap of the upconversion emission of Er3+ at approximately 545 nm and the absorbance of the FZ:Fe2+ complex at 562 nm enabled measurement in the change of UCNP emission response as a function of Fe2+ concentration in a ratiometric manner. We first applied large, ultra-bright poly(acrylic acid) (PAA)-capped Gd2O2S:Yb3+,Er3+ UCNPs upconverting nanoparticles (UCNPs) for the detection of Fe2+ using FZ as the acceptor. The probe displayed good selectivity and sensitivity for Fe2+, with a low limit of detection (LoD) of 2.74 μM. Analogous results employing smaller (31 nm) PAA-capped hexagonal-phase NaYF4:Yb3+,Er3+ UCNPs synthesised in our lab were achieved, with a lower LoD towards Fe2+ of 1.43 μM. These results illustrate how the ratiometric nature of the system means it is applicable over a range of particle sizes, brightnesses and nanoparticle host matrices. Preliminary investigations also found the probes capable of detecting micromolar concentrations of Fe2+ in turbid solutions.

A novel integrated analyzer was developed for the in situ determination of two-dimensional (2D) dissolved Fe(II) distributions in sediment pore water. The analyzer utilized gel enrichment and optical imaging techniques. An image probe mainly consisting of a gel holder and portable document scanner was designed to be inserted into sediment. The gel holder exposed to the sediment was made to hold a polyacrylamide gel strip (diffusive gel) and polyacrylamide gel strip impregnated with C18 and coated with ferrozine (concentrating gel). The concentrating gel strip could accumulate the dissolved Fe(II) in pore water and produce a magenta-colored Fe(II)-ferrozine compound on the gel strip in two dimensions. The portable document scanner sealed in a transparent box and stuck onto the back of the gel holder could record gel images from the back of the concentrating gel strip. Gel images with grayscale intensities were acquired and analyzed using ImageJ software, and Fe(II) concentration was determined based on a deployment time related calibration curve established in the laboratory. The measurement accuracy and precision were investigated. The quantitative range reached up to 200 μmol L-1. The method and analyzer exhibit distinct characteristics of in situ enrichment and measurement; they were successfully applied to determine the 2D Fe(II) distribution in lake and marine sediment pore waters.

An efficient microwave-assisted extraction (MAE) combined with in-capillary [Fe(ferrozine)3]2+-capillary electrophoresis-Diode Array Detector (in-capillary [Fe(ferrozine)3]2+-CE-DAD) was developed to screen active components with the ability to chelate ferrous ions and determine the total antioxidant activity. The MAE conditions, including methanol concentration, extraction power, extraction time, and the ratio of material to liquid, were optimized by an L9(34) orthogonal experiment. Background buffer, voltage, and cartridge temperature that affect the separation of six compounds were optimized. It was found that rutin and quercetin were the main components chelating ferrous ions in Flos Sophorae Immaturus (Flos Sophorae) by the in-capillary [Fe(ferrozine)3]2+-CE-DAD. The recoveries were ranged from 95.2% to 104%. It was concluded that the MAE combined with in-capillary [Fe(ferrozine)3]2+-CE-DAD method was a simple, reliable, and efficient tool for screening active components from the complex traditional Chinese medicine samples and evaluating their ability to chelate ferrous ions.

Metallotriazine complexes possess interesting biological and medicinal properties, and the present study focuses on the synthesis, characterization, and antimicrobial activity of four novel copper-triazine derivatives in search of potent antibacterial and antifungal drug leads. In this study, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4,4'-disulfonic acid monosodium salt (L1, ferrozine) and 3-(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5,5'-disulfonic acid disodium salt (L2, ferene) have been used as ligands to study the complexation towards copper(II). The synthesized complexes, [CuCl2(ferrozine)]·7H2O·MeOH (1), [CuCl2(ferrozine)2]·5H2O·MeOH (2), [CuCl2(ferene)]·H2O·MeOH (3), and [CuCl2(ferene)2]·H2O·MeOH (4), have been characterized spectroscopically, and preliminary bioassays have been carried out. FTIR spectroscopic data have shown that N=N and C=N stretching frequencies of complexes have been shifted towards lower frequencies in comparison with that of the ligands, confirming new bond formation between Cu and N, which in turn lowers the strength of N=N and C=N bonds. In addition, a bathochromic shift has been observed for UV-visible spectra of complexes (1), (2), (3), and (4). Furthermore, elemental analysis data have been useful to obtain empirical formulas of these complexes and to establish the purity of each complex. Complexes (1) and (2) have shown antibacterial activity for both S. aureus (ATCC® 25923) and E. coli (ATCC® 25922) at 1 mg/disc concentration, and ferrozine has shown a larger inhibition zone against the clinical sample of C. albicans at 1 mg/disc concentration in comparison with the positive control, fluconazole.

Aldehyde:ferredoxin oxidoreductases (AORs) have been isolated and biochemically-characterized from a handful of anaerobic or facultative aerobic archaea and bacteria. They catalyze the ferredoxin (Fd)-dependent oxidation of aldehydes to acids. Recently, the involvement of AOR in the reduction of organic acids to alcohols with electrons derived from sugar or synthesis gas was demonstrated, with alcohol dehydrogenases (ADHs) carrying out the reduction of the aldehyde to the alcohol (AOR-ADH pathway). Here, we describe the biochemical characterization of an AOR of the thermophilic fermentative bacterium Thermoanaerobacter sp. strain X514 (AORX514). The putative aor gene (Teth514_1380) including a 6x-His-tag was introduced into the genome of the genetically-accessible, related species Thermoanaerobacter kivui. The protein was purified to apparent homogeneity, and indeed revealed AOR activity, as measured by acetaldehyde-dependent ferredoxin reduction. AORX514 was active over a wide temperature (10 to 95 °C) and pH (5.5 to 11.5) range, utilized a wide variety of aldehydes (short and branched-chained, aliphatic, aromatic) and resembles archaeal sensu stricto AORs, as the protein is active in a homodimeric form. The successful, recombinant production of AORX514 in a related, well-characterized and likewise strict anaerobe paves the road towards structure-function analyses of this enzyme and possibly similar oxygen-sensitive or W/Mo-dependent proteins in the future.

In this study, the effects of salinity on growth, fatty acid, essential oil, and phenolic composition of cumin (Cuminum cyminum L.) seeds as well as the antioxidant activities of their extracts were investigated. Plants were treated with different concentrations of NaCl treatment: 0, 50, 75, and 125 mmoL. Plant growth was significantly reduced with the severity of saline treatment. This also caused important reductions in the seed yield and yield components. Besides, NaCl treatments affected fatty acid composition. Petroselinic and linoleic acids proportions diminished consistently with the increase in NaCl concentration, whereas palmitic acid proportion increased. Furthermore, NaCl enhanced essential oil production in C. cyminum seeds and induced marked changes on the essential oil quality. Essential oil chemotype was modified from γ-terpinene/1-phenyl-1,2 ethanediol in control to γ-terpinene/β-pinene in salt stressed plants. Total polyphenol content was higher in treated seeds, and salinity improved the amount of individual phenolic compounds. Moreover, antioxidant activities of the extracts were determined by four different test systems, namely 2,2-diphenyl-1-picrylhydrazyl, β-carotene/linoleic acid chelating, and reducing power assays. The highest antioxidant activities were reveled in severe stressed plants. In this case, cumin seeds produced under saline conditions may function as a potential source of essential oil and antioxidant compounds, which could support the utilization of this plant in a large field of applications such as food industry.

Fe3+-TiO2 (Fe-TiO2) thin films were successfully prepared using a "sandwich" approach. TiO2 NPs were doped with different Fe3+ content (0.05%, 0.1%, 0.2% molar ratio), and the modified TiO2 NPs were deposited on glass flat support by dip coating.Structural, morphological, optical, and photocatalytic properties of Fe-TiO2 thin films were studied. XPS spectra confirm the presence of Ti, Fe, O, and defective -OH groups at the material surface. The Fe 2p spectrum demonstrates the existence of Fe3+. SEM images indicate that the incorporation of Fe3+ deforms in some degree the homogeneity of the TiO2 system. Additionally, incorporation of Fe3+ ions to the network creates an impurity band near the VB due to the oxygen vacancies, resulting in the reduction of the effective optical band gap. Photocatalytic activity of fabricated thin films in the elimination of sulfamethoxazole (SMT) follows pseudo first-order kinetics. The highest SMT removal yields were achieved using the sample with 0.05%Fe. Additionally, the use of greater thicknesses improves the removal performance. However, material detachment limits the maximum usable value around 6 µm.Finally, stability and reusability of catalysts were confirmed studying the photocatalytic activity over three cycles and evaluating that no Fe3+ leaching occurred.

Background: Diabetes mellitus, a metabolic disease, is a major health concern today throughout the world. Callistemon lanceolatus (Myrtaceae), commonly known as bottlebrush, has been used by Indian tribal communities for the treatment of many diseases. The purpose of this study was to explore antioxidant and antihyperglycemic potential of methanolic and aqueous extracts of the stem of C. lanceolatus in vitro and in vivo. Methods: Phytoconstituents of C. lanceolatus stem were extracted in methanol and water sequentially followed by phytochemical analysis. The in vitro antioxidant potential of aqueous and methanolic extracts was assessed by metal ion chelating, free radical scavenging, and reducing power assays. The in vivo antihyperglycemic activity of the oral methanolic extract was studied in alloxan-induced diabetic rats. Bodyweight and blood glucose were monitored regularly. After the treatment period, serum was examined for total cholesterol, triglycerides, high-density lipoprotein (HDL), bilirubin, creatinine, urea, glutamate pyruvate transaminase (SGPT), glutamate oxaloacetate transaminase (SGOT), and alkaline phosphatase (ALP). Results: Methanolic extract exhibited superior antioxidant activity to aqueous extract. A marked increase in levels of serum markers, viz., glucose, triglycerides, total cholesterol, bilirubin, urea, creatinine, SGOT, SGPT, and ALP along with a reduction in HDL was observed in diabetic rats. Methanol extract treatment for 28 days accounted for a decrease in blood glucose and other metabolic markers accompanied by an improvement in body weight and HDL level in hyperglycemic rats. Conclusions: The present study suggests that C. lanceolatus methanolic stem extract possesses antioxidant and antihyperglycemic activities and has potential as a therapeutic agent in diabetes.

Low molecular mass iron (LMrFe) can appear in plasma when the transferrin becomes fully iron loaded. Such iron poses a risk factor for oxidative damage, and for microbial virulence. A previous novel approach to the detection and measurement of LMrFe in plasma was the use of the iron-binding properties of the glycopeptide antitumour antibiotic bleomycin and its ability to degrade DNA in the presence of oxygen, bound iron, and an iron reducing agent. Since bleomycin is a non-physiological ligand with iron-binding and redox cycling properties, it has been suggested that it may not be a valid biological model for detecting and measuring LMrFe. To address these concerns we have developed a biological approach to the detection and measurement of LMrFe based on the activation of iron-requiring aconitase. Parallel measurements, in a variety of clinical conditions in which there was a complete saturation of the plasma transferrin, showed that the bleomycin assay and the aconitase assay can give similar results for LMrFe.

Many experimental studies concerning hypoxia or ischemia have reported a decrease in intra/extracellular pH and massive dopamine (DA) release in the striatum. The present work investigated whether the increase in striatal extracellular DA is related to acidification or to lactate production. Striatal perfusion of lactic acid (pH 5.5) by microdialysis in conscious freely-moving rats induced an increase in extracellular concentrations of DA and catabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), as a probable result of acidification. Perfusion with sodium lactate (pH 7.4) failed to modify DA and catabolite release, whereas orthophosphoric acid produced the same effect as lactic acid. As lactic acidosis is known to induce a displacement of iron from its uptake sites, the possible role of this metal in response to acidosis was studied by perfusing ferrozine, an iron complexing agent, at the same time as lactic acid. The results showed that ferrous ions are involved in the process and suggested that oxygen free radicals play a role in the extracellular release of DA. Thus, lactic acid perfusion in rat striatum would appear to be a useful model for in vivo studies of the mechanisms responsible for increases in extracellular DA during hypoxia and ischemia.

Previous studies have suggested that sugars enhance iron bioavailability, possibly through either chelation or altering the oxidation state of the metal, however, results have been inconclusive. Sugar intake in the last 20 years has increased dramatically, and iron status disorders are significant public health problems worldwide; therefore understanding the nutritional implications of iron-sugar interactions is particularly relevant. In this study we measured the effects of sugars on non-heme iron bioavailability in human intestinal Caco-2 cells and HepG2 hepatoma cells using ferritin formation as a surrogate marker for iron uptake. The effect of sugars on iron oxidation state was examined by measuring ferrous iron formation in different sugar-iron solutions with a ferrozine-based assay. Fructose significantly increased iron-induced ferritin formation in both Caco-2 and HepG2 cells. In addition, high-fructose corn syrup (HFCS-55) increased Caco-2 cell iron-induced ferritin; these effects were negated by the addition of either tannic acid or phytic acid. Fructose combined with FeCl3 increased ferrozine-chelatable ferrous iron levels by approximately 300%. In conclusion, fructose increases iron bioavailability in human intestinal Caco-2 and HepG2 cells. Given the large amount of simple and rapidly digestible sugars in the modern diet their effects on iron bioavailability may have important patho-physiological consequences. Further studies are warranted to characterize these interactions.

Pyrococcus furiosus rubredoxin (PfRd), a small, monomeric, 53 residues-long, iron-containing, electron-transfer protein of known structure is sometimes referred to as being the most structurally-stable protein known to man. Here, using a combination of mutational and spectroscopic (CD, fluorescence, and NMR) studies of differently made holo- and apo-forms of PfRd, we demonstrate that it is not the presence of iron, or even the folding of the PfRd chain into a compact well-folded structure that causes holo-PfRd to display its extraordinary thermal stability, but rather the correct iron binding-guided packing of certain residues (specifically, Trp3, Phe29, Trp36, and also Tyr10) within a tight aromatic cluster of six residues in PfRd's hydrophobic core. Binding of the iron atom appears to play a remarkable role in determining subtle details of residue packing, forcing the chain to form a hyper-thermally stable native structure which is kinetically stable enough to survive (subsequent) removal of iron. On the other hand, failure to bind iron causes the same chain to adopt an equally well-folded native-like structure which, however, has a differently-packed aromatic cluster in its core, causing it to be only as stable as any other ordinary mesophile-derived rubredoxin. Our studies demonstrate, perhaps for the very first time ever that hyperthermal stability in proteins can owe to subtle differences in residue packing vis a vis mesostable proteins, without there being any underlying differences in either amino acid sequence, or bound ligand status.

N-[(2-Hydroxy-3-Trimethylammonium) Propyl] Chitosan Chloride Derivatives (HTCC), based on low molecular weight crab chitosan, were synthesized by the alkylation reaction with a degree of substitution of 10-98%. The chemical structure was confirmed by 1H NMR and IR-spectra. Physical and chemical characteristics and a number of properties were defined. All HTCC derivatives were soluble at pH 7.4. HTCCs have an inhibitory capacity on the growth of the studied microorganisms. The introduction of quaternary ammonium groups into chitosan molecule contributed to the increase of antibacterial activity of derivatives. HTCC53 showed antifungal activity and at a concentration of 500 mg/ml completely inhibited the growth of mycelial fungi F. oxysporum, A. alternata and C. herbarum. When studying the ability of HTCCs to absorb DPPH radicals, it was found that samples of HTCC10 and HTCC40 showed high inhibitory capacity at a concentration of >15 mg/ml. It was shown that the chelating ability of HTCCs decreased by reducing the number of free amino groups. HTCC10-HTCC53 demonstrated the maximum values of chelating ability at a concentration of 4-10 mg/ml. Due to the solubility at neutral pH values and the properties shown, obtained chitosan derivatives can be used in clinical practice, pharmaceutical and food industries in the future.

Cryogels attained from natural materials offer exceptional properties in applications such as tissue engineering. Moreover, Halloysite Nanotubes (HNT) at 1:0.5 weight ratio were embedded into CS cryogels to render additional biomedical properties. The hemolysis index of CS cryogel and CS:HNT cryogels was calculated as 0.77 ± 0.41 and 0.81 ± 0.24 and defined as non-hemolytic materials. However, the blood coagulation indices of CS cryogel and CS:HNT cryogels were determined as 76 ± 2% and 68 ± 3%, suggesting a mild blood clotting capability. The maximum% swelling capacity of CS cryogel was measured as 3587 ± 186%, 4014 ± 184%, and 3984 ± 113%, at pH 1.0, pH 7.4 and pH 9.0, respectively, which were reduced to 1961 ± 288%, 2816 ± 192, 2405 ± 73%, respectively, for CS:HNT cryogel. It was found that CS cryogels can hydrolytically be degraded 41 ± 1% (by wt) in 16-day incubation, whereas the CS:HNT cryogels degraded by 30 ± 1 wt %. There is no chelation for HNT and 67.5 ± 1% Cu(II) chelation for linear CS was measured. On the other hand, the CS cryogel and CS:HNT cryogel revealed Cu(II) chelating capabilities of 60.1 ± 12.5%, and 43.2 ± 17.5%, respectively, from 0.1 mg/mL Cu(II) ion stock solution. Additionally, at 0.5 mg/mL CS, CS:HNT, and HNT, the Fe(II) chelation capacity of 99.7 ± 0.6, 86.2 ± 4.7% and only 11.9 ± 4.5% were measured, respectively, while no Fe(II) was chelated by linear CS chelated Fe(II). As the adjustable and controllable swelling properties of cryogels are important parameters in biomedical applications, the swelling properties of CS cryogels, at different solution pHs, e.g., at the solution pHs of 1.0, 7.4 and 9.0, were measured as 3587 ± 186%, 4014 ± 184%, and 3984 ± 113%, respectively, and the maximum selling% values of CS:HNT cryogels were determined as 1961 ± 288%, 2816 ± 192, 2405 ± 73%, respectively, at the same conditions. Alpha glucosidase enzyme interactions were investigated and found that CS-based cryogels can stimulate this enzyme at any CS formulation.

The purpose of this research was to study the effect of cooking chicken breast on the production of dialyzable iron (an in vitro indicator of bioavailable iron) from added ferric iron. Chicken breast muscle was cooked by boiling, baking, sautéing, or deep-frying. Cooked samples were mixed with ferric iron and either extracted with acid or digested with pepsin and pancreatin. Total and ferrous dialyzable iron was measured after extraction or digestion and compared to raw chicken samples. For uncooked samples, dialyzable iron was significantly enhanced after both extraction and digestion. All cooking methods led to markedly reduced levels of dialyzable iron both by extraction and digestion. In most cooked, digested samples dialyzable iron was no greater than the iron-only (no sample) control. Cooked samples showed lower levels of histidine and sulfhydryls but protein digestibility was not reduced, except for the sautéed sample. The results showed that, after cooking, little if any dialyzable iron results from digestion of muscle proteins. Our research indicates that, in cooked chicken, residual acid-extractable components are the most important source of dialyzable iron.

Moringa stenopetala is a multipurpose plant having high nutritional and medicinal values. The aim of this study is to evaluate the effect of time and temperature of ultrasound-assisted extraction on bioactive compounds and antioxidant activities of M. stenopetala leaf extract. The ultrasound-assisted extraction took place at each of 30, 40 and 50 °C for 10, 20 and 30 min. The study also included the analysis of the interaction effects of time and temperature on the total phenolic content, total flavonoid content, antioxidant activity (ABTS and DPPH assay), FRAP and chelating activity. The highest total phenolic content, expressed in mg gallic acid equivalents per g dry mass, was 46.6 and total flavonoid content, expressed in mg catechin equivalents per g dry mass, was 20.4 at 40 °C for 20 min. Under the same conditions, the highest antioxidant activities evaluated by DPPH, ABTS and FRAP, expressed in mg Trolox equivalents per g dry mass, were 336.5, 581.8 and 133.3 respectively, and chelating activity, expressed in mg EDTA equivalents per g dry mass, was 28.4. The lowest amounts of bioactive compounds and antioxidant activities were observable when the extraction occurred at 50 °C for 30 min, followed by the extraction at lower temperature (30 °C) for shorter time (10 min). The morphological analysis of the residues obtained after extraction using scanning electron microscope indicated that there was a higher ultrasonic destruction of the structural components of the sample at longer extraction time. Therefore, ultrasound-assisted extraction at a temperature of 40 °C for 20 min is the best time-temperature combination to extract bioactive compounds from M. stenopetala leaves.

Oxidative stress plays an important role in development of diabetes mellitus. Satureja bachtiarica Bunge (Lamiaceae) is a rich source of bioactive compounds with antioxidant properties.

Toona sinensis (A. Juss.) Roemer is an endemic species of Toona genus native to Asia. Its crude extract exhibits an effective anti-oxidant capacity against oxidative models, but the intrinsic substances responsible for this capacity in the extract remains unclear and is yet to be studied comprehensively.

Infection processes displayed by pathogens require the acquisition of essential inorganic nutrients and trace elements from the host to survive and proliferate. Without a doubt, iron is a crucial trace metal for all living organisms and also a pivotal component in the host-parasite interactions. In particular, the host reduces the iron available to face the infectious disease, increasing iron transport proteins' expression and activating the heme synthesis and degradation pathways. Moreover, recent findings have suggested that iron metabolism modulation in fish promotes the immune response by reducing cellular iron toxicity. We hypothesized that recombinant proteins related to iron metabolism could modulate the fish's immune system through iron metabolism and iron-responsive genes. Here a chimeric iron transport protein (IPath®) was bioinformatically designed and then expressed in a recombinant bacterial system. The IPath® protein showed a significant chelating activity under in vitro conditions and biological activity. Taking this evidence, a vaccine candidate based on IPath® was evaluated in Atlantic salmon challenged with three different fish pathogens. Experimental trials were conducted using two fish groups: one immunized with IPath® and another injected with adjutant as the control group. After 400 accumulated thermal units (ATUs), two different infection trials were performed. In the first one, fish were infected with the bacterium Aeromonas salmonicida, and in a second trial, fish were exposed to the ectoparasite Caligus rogercresseyi and subsequently infected with the intracellular bacterium Piscirickettsia salmonis. Fish immunized with IPath® showed a significant delay in the mortality curve in response to A. salmonicida and P. salmonis infections. However, no significant differences between infected and control fish groups were observed at the end of the experiment. Notably, sea lice burden reduction was observed in vaccinated Atlantic salmon. Transcriptional analysis evidenced a high modulation of iron-homeostasis-related genes in fish vaccinated with IPath® compared to the control group during the infection. Moreover, increasing expression of Atlantic salmon IgT was associated with IPath® immunization. This study provides evidence that the IPath® protein could be used as an antigen or booster in commercial fish vaccines, improving the immune response against relevant pathogens for salmon aquaculture.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is known to undergo reduction mediated by ferrous iron in the presence of minerals, including magnetite. Idealized laboratory conditions may not provide representative reaction kinetics or pathways compared to field conditions. The effects of magnetite mineral morphology, the aquifer material matrix, the presence of aqueous Fe(II), and the buffer identity on RDX reduction kinetics and intermediate formation are investigated in this work. Reactions in bicarbonate buffer were substantially slower than those performed in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, and the presence of quartz and clays in magnetite-containing aquifer material resulted in slower reaction kinetics and production of additional iron oxide phases. Buffer identity also changed the rate controlling step and reaction product distribution. Conditions as close to those expected in field systems are necessary to evaluate the reaction rates and pathways of RDX in reduced groundwater systems.