Preferential uptake of antibody targeted calcium phosphosilicate nanoparticles by metastatic triple negative breast cancer cells in co-cultures of human metastatic breast cancer cells plus bone osteoblasts.

Calcium phosphosilicate nanoparticles (CPSNPs) are bioresorbable nanoparticles that can be bioconjugated with targeting molecules and encapsulate active agents and deliver them to tumor cells without causing damage to adjacent healthy tissue. Data obtained in this study demonstrated that an anti-CD71 antibody on CPSNPs targets these nanoparticles and enhances their internalization by triple negative breast cancer cells in-vitro. Caspase 3,7 activation, DNA damage, and fluorescent microscopy confirmed the apoptotic breast cancer response caused by targeted anti-CD71-CPSNPs encapsulated with gemcitabine monophosphate, the active metabolite of the chemotherapeutic gemcitabine used to treat cancers including breast and ovarian. Targeted anti-CD71-CPSNPs encapsulated with the fluorophore, Rhodamine WT, were preferentially internalized by breast cancer cells in co-cultures with osteoblasts. While osteoblasts partially internalized anti-CD71-GemMP-CPSNPs, their cell growth was not affected. These results suggest that CPSNPs may be used as imaging tools and selective drug delivery systems for breast cancer that has metastasized to bone.

Constructing Electron Levers in Perovskite Nanocrystals to Regulate the Local Electron Density for Intensive Chemodynamic Therapy.

The local electron density of an atom is one key factor that determines its chemical properties. Regulating electron density can promote the atom's reactivity and so reduce the reaction activation energy, which is highly desired in many chemical applications. Herein, we report an intra-crystalline electron lever strategy, which can regulate the electron density of reaction centre atoms via manipulating ambient lattice states, for Fenton activity improvement. Typically, with the assistance of ultrasound, the Mn4+ -O-Fe3+ bond in BiFe0.97 Mn0.03 O3 perovskite nanocrystals can drive valence electrons and free electrons to accumulate on Fe atoms by a polarization electric field originated from the designed lattice strain. The increase of electron density significantly improves the catalytic activity of Fe, decreasing the activation energy of BiFe0.97 Mn0.03 O3 -mediated Fenton reaction by 52.55 %, and increasing the . OH yield by 9.21-fold. This study provides a new way to understand the sono-Fenton chemistry, and the increased . OH production enables a highly effective chemodynamic therapy.

Characterization and photoprotective potentiality of lime dwelling Pseudomonas mediated melanin as sunscreen agent against UV-B radiations.

Prolonged exposure to Ultra Violet Radiation (UVR) adversely alters the functions of many skin cell types causing skin cancer and photoaging, which had led to increase in demand for more safe and natural sunscreens against UVR. The present study focuses on production, structural characterization and evaluation of photoprotective nature of melanin pigment derived from lime dwelling Pseudomonas sp. Melanin was characterized by solubility, UV-Vis, FT-IR, 13C-CPMAS, ESI-MS spectroscopy, including particle size, melting point and elemental analyses. In vitro cytotoxicity and photo-protective effect of Pseudomonas derived melanin (Mel-P) against UV-B (Broad Band-BB) radiations were assessed on mouse fibroblasts NIH 3 T3 cell lines. Reactive Oxygen Species (ROS) generated in NIH 3 T3 cells upon UV-B (BB) exposure was determined and quantified by Fluorescent microscopic and Flow cytometric analyses. A natural melanin obtained from Pseudomonas sp. contains 5,6- dihydroxy indole 2-carboxyic acid (DHICA) as its basic constituent and possess typical properties of eumelanin as revealed by the characterization studies. Mel-P has shown cell viability of 61.33 ± 6.58% at the concentration of 500 µg/mL proving its non-cytotoxic effect. Owing to its anti-oxidant property, melanin efficiently protected the mouse fibroblast cells from UV-B (BB) irradiation in a dose dependant manner demonstrating its potential as an active photoprotective agent.

In vitro comparisons of microscale and nanoscale calcium silicate particles.

Calcium silicate (CaSi) materials have been used for bone repair and generation due to their osteogenic properties. Tailoring the surface chemistry and structure of CaSi can enhance its clinical performance. There is no direct comparison between microscale and nanoscale CaSi particles. Therefore, this article aimed to compare and evaluate the surface chemistry, structure, and in vitro properties of microscale CaSi (µCaSi) and nanoscale CaSi (nCaSi) particles synthesized by the sol-gel method and precipitation method, respectively. As a result, the semi-crystalline µCaSi powders were assemblies of irregular microparticles containing a major ß-dicalcium silicate phase, while the amorphous nCaSi powders consisted of spherical particles with a size of 100 nm. After soaking in a Tris-HCl solution, the amount of Si ions released from nCaSi was higher than that released from µCaSi, but there was no significant difference in Ca ion release between the two CaSi particles. Compared to microscale CaSi (µCaSi), nanoscale CaSi (nCaSi) significantly enhanced the growth and differentiation of human mesenchymal stem cells (hMSC) and inhibited the function of RAW 264.7 macrophages. In the case of antibacterial activity against Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus), nanoscale nCaSi displayed a higher bacteriostatic ratio, a greater growth inhibition zone and more reactive oxygen species (ROS) production than microscale µCaSi. The conclusion is that nanoscale CaSi had greater antibacterial and osteogenic activity compared to microscale CaSi. Next generation CaSi-based materials with unique properties are emerging to meet specific clinical needs.

Investigation into Enhancing Capecitabine Efficacy in Colorectal Cancer by Inhibiting Focal Adhesion Kinase Signaling.

BACKGROUND/AIM: Capecitabine is a pro-drug of 5-fluorouracil (5-FU), and is an orally available chemotherapeutic used to treat colorectal cancer (CRC). Recently, research has focused on improving its efficacy at lower doses in order to minimize its well-known toxicities. In this study, we investigated the possibility of improving the antitumor effect of capecitabine against CRC by destabilizing focal adhesion kinase (FAK) signaling. MATERIALS AND METHODS: Optimal dosages for capecitabine and lactate calcium salt (LCS) were determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide MTT assay. The viability of CRC cells was investigated by MTT and clonogenic assays after single or combination treatment with capecitabine and LCS. Western blot analyses were used to determine changes in the expression of components of the FAK and AKT signaling cascade, and this information was used to elucidate the underlying mechanism. A xenograft model was established to evaluate the antitumor efficacy of the combination treatment, as well as its necrotic effect and organ toxicity. RESULTS: The addition of LCS to capecitabine treatment led to an increase in the proteolysis of the FAK signaling cascade components, including SRC proto-oncogene, non-receptor tyrosine kinase; AKT serine/threonine kinase 1; and nuclear factor-kappa B, resulting in a decrease in the viability and clonogenic ability of CRC cells. In vivo antitumor efficacy, including tumor necrosis, was significantly increased with the combination treatment relative to both single treatments, and no organ toxicity was found in any experimental group. CONCLUSION: The addition of LCS increased the anticancer efficacy of capecitabine at a lower dose than is currently used in human patients.

Biocompatible silk/calcium silicate/sodium alginate composite scaffolds for bone tissue engineering.

Scaffolds are crucial for bone tissue engineering since their compositions and properties could significantly affect the seeded cells' behavior. In this study, we developed an interpenetrating network hydrogel by utilizing Ca2+ from calcium silicate (CS) to simultaneously crosslink silk fibroin (SF) and sodium alginate (SA). Afterwards, the hydrogels were lyophilized to obtain scaffolds and systematically evaluated by physical characterizations, in vitro cytocompatibility and alkaline phosphatase (ALP) assay. We found that CS inside the porous structure of SF/CS/SA scaffolds could remarkably enhance hydrophilicity, degradation, compression resistance, bioactivity and pH of SF/CS/SA scaffolds. Scaffolds with CS concentrations of 25% and 12% (25/CS and 12/CS) could dominantly stimulate proliferation of bone marrow stromal cells (BMSCs). Besides, BMSCs cultured with 25/CS and 12/CS scaffolds showed high ALP activity, respectively. Consequently, this study suggested SF/CS/SA scaffolds possess potential in non-loading bone tissue engineering application.

Nanoscale synchrotron X-ray speciation of iron and calcium compounds in amyloid plaque cores from Alzheimer's disease subjects.

Altered metabolism of biometals in the brain is a key feature of Alzheimer's disease, and biometal interactions with amyloid-ß are linked to amyloid plaque formation. Iron-rich aggregates, including evidence for the mixed-valence iron oxide magnetite, are associated with amyloid plaques. To test the hypothesis that increased chemical reduction of iron, as observed in vitro in the presence of aggregating amyloid-ß, may occur at sites of amyloid plaque formation in the human brain, the nanoscale distribution and physicochemical states of biometals, particularly iron, were characterised in isolated amyloid plaque cores from human Alzheimer's disease cases using synchrotron X-ray spectromicroscopy. In situ X-ray magnetic circular dichroism revealed the presence of magnetite: a finding supported by ptychographic observation of an iron oxide crystal with the morphology of biogenic magnetite. The exceptional sensitivity and specificity of X-ray spectromicroscopy, combining chemical and magnetic probes, allowed enhanced differentiation of the iron oxides phases present. This facilitated the discovery and speciation of ferrous-rich phases and lower oxidation state phases resembling zero-valent iron as well as magnetite. Sequestered calcium was discovered in two distinct mineral forms suggesting a dynamic process of amyloid plaque calcification in vivo. The range of iron oxidation states present and the direct observation of biogenic magnetite provide unparalleled support for the hypothesis that chemical reduction of iron arises in conjunction with the formation of amyloid plaques. These new findings raise challenging questions about the relative impacts of amyloid-ß aggregation, plaque formation, and disrupted metal homeostasis on the oxidative burden observed in Alzheimer's disease.

Variation in Extracellular Detoxification Is a Link to Different Carcinogenicity among Chromates in Rodent and Human Lungs.

Inhalation of soluble chromium(VI) is firmly linked with higher risks of lung cancer in humans. However, comparative studies in rats have found a high lung tumorigenicity for moderately soluble chromates but no tumors for highly soluble chromates. These major species differences remain unexplained. We investigated the impact of extracellular reducers on responses of human and rat lung epithelial cells to different Cr(VI) forms. Extracellular reduction of Cr(VI) is a detoxification process, and rat and human lung lining fluids contain different concentrations of ascorbate and glutathione. We found that reduction of chromate anions in simulated lung fluids was principally driven by ascorbate with only minimal contribution from glutathione. The addition of 500 µM ascorbate (∼rat lung fluid concentration) to culture media strongly inhibited cellular uptake of chromate anions and completely prevented their cytotoxicity even at otherwise lethal doses. While proportionally less effective, 50 µM extracellular ascorbate (∼human lung fluid concentration) also decreased uptake of chromate anions and their cytotoxicity. In comparison to chromate anions, uptake and cytotoxicity of respirable particles of moderately soluble CaCrO4 and SrCrO4 were much less sensitive to suppression by extracellular ascorbate, especially during early exposure times and in primary bronchial cells. In the absence of extracellular ascorbate, chromate anions and CaCrO4/SrCrO4 particles produced overall similar levels of DNA double-stranded breaks, with less soluble particles exhibiting a slower rate of breakage. Our results indicate that a gradual extracellular dissolution and a rapid internalization of calcium chromate and strontium chromate particles makes them resistant to detoxification outside the cells, which is extremely effective for chromate anions in the rat lung fluid. The detoxification potential of the human lung fluid is significant but much lower and insufficient to provide a threshold-type dose dependence for soluble chromates.

Boosting the Peroxidase-Like Activity of Nanostructured Nickel by Inducing Its 3+ Oxidation State in LaNiO3 Perovskite and Its Application for Biomedical Assays.

Catalytic nanomaterials with intrinsic enzyme-like activities, called nanozymes, have recently attracted significant research interest due to their unique advantages relative to natural enzymes and conventional artificial enzymes. Among the nanozymes developed, particular interests have been devoted to nanozymes with peroxidase mimicking activities because of their promising applications in biosensing, bioimaging, biomedicine, etc. Till now, lots of functional nanomaterials have been used to mimic peroxidase. However, few studies have focused on the Ni-based nanomaterials for peroxidase mimics. In this work, we obtained the porous LaNiO3 nanocubes with high peroxidase-like activity by inducing its 3+ oxidation state in LaNiO3 perovskite and optimizing the morphology of LaNiO3 perovskite. The peroxidase mimicking activity of the porous LaNiO3 nanocubes with Ni3+ was about 58~fold and 22~fold higher than that of NiO with Ni2+ and Ni nanoparticles with Ni0. More, the porous LaNiO3 nanocubes exhibited about 2-fold higher activity when compared with LaNiO3 nanoparticles. Based on the superior peroxidase-like activity of porous LaNiO3 nanocubes, facile colorimetric assays for H2O2, glucose, and sarcosine detection were developed. Our present work not only demonstrates a useful strategy for modulating nanozymes' activities but also provides promising bioassays for clinical diagnostics.

Role of biochar amendment in mitigation of nitrogen loss and greenhouse gas emission during sewage sludge composting.

The objective of the present study was to mitigate the greenhouse gas (GHG) emissions during composting of dewatered fresh sewage sludge (DFSS) employing biochar combined with zeolite (B+Z) and low dosage of lime (B+L). The 12% biochar was mixed at a 10%, 15% and 30% of zeolite and 1% lime, while without any additives was used as control. The results indicated that the combine use of B+Z was significantly increased the enzymatic activities and reduced the ammonia loss 58.03-65.17% as compare to B+L amended treatment, while CH4 92.85-95.34% and N2O 95.14-97.28% decreased than control. The B+L1% amendment significantly increased the organic matter degradation but the reduction was lower than B+Z and that could reduce the CH4 and N2O emission by 55.17-63.08% and 62.24-65.53% as compare to control, respectively. Overall our results demonstrated that 12%B+Z10% addition into DFSS can be potentially used to improve the DFSS composting by mitigation of GHG emission and nitrogen loss.

Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Mechanism of Four-Coordinate Co(II)Cbl Formation.

EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosyltransferases (ACATs), implicated in the biosynthesis of adenosylcobalamin (AdoCbl). In the presence of cosubstrate ATP, ACATs raise the Co(II)/Co(I) reduction potential of their cob(II)alamin [Co(II)Cbl] substrate by >250 mV via the formation of a unique four-coordinate (4c) Co(II)Cbl species, thereby facilitating the formation of a "supernucleophilic" cob(I)alamin intermediate required for the formation of the AdoCbl product. Previous kinetic studies of EutT revealed the importance of a HX11CCX2C(83) motif for catalytic activity and have led to the proposal that residues in this motif serve as the binding site for a divalent transition metal cofactor [e.g., Fe(II) or Zn(II)]. This motif is absent in other ACAT families, suggesting that EutT employs a distinct mechanism for AdoCbl formation. To assess how metal ion binding to the HX11CCX2C(83) motif affects the relative yield of 4c Co(II)Cbl generated in the EutT active site, we have characterized several enzyme variants by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our results indicate that Fe(II) or Zn(II) binding to the HX11CCX2C(83) motif of EutT is required for promoting the formation of 4c Co(II)Cbl. Intriguingly, our spectroscopic data also reveal the presence of an equilibrium between five-coordinate "base-on" and "base-off" Co(II)Cbl species bound to the EutT active site at low ATP concentrations, which shifts in favor of "base-off" Co(II)Cbl in the presence of excess ATP, suggesting that the base-off species serves as a precursor to 4c Co(II)Cbl.

Regulation of 2,4-epibrassinolide on mineral nutrient uptake and ion distribution in Ca(NO3)2 stressed cucumber plants.

2,4-Epibrassinolide (EBL) is a plant hormone that plays a pivotal role in regulation of plants growth and development processes under abiotic stress. The investigation was carried out to study the effect of EBL on mineral nutrients uptake and distribution with ion element analysis and X-ray microanalysis in cucumber seedlings (Cucumis sativus L. cv. Jinyou No.4) under 80 mM Ca(NO3)2 stress. Our study found EBL significantly alleviated the inhibitory of P, K, Na, Mg, Fe, Mn, or Cl uptake in shoot or root by Ca(NO3)2 stress. Under Ca(NO3)2 stress, X-ray microanalysis showed that high levels of Ca by EBL treatment accumulated in the epidermal cells of root and gradually decreased from epidermal cells to stellar parenchyma. K(+) levels were restored in different cross section of roots and high K(+) level mostly accumulated in stellar parenchyma. The results of Ca(2+) ultra-structural localization showed Ca(2+) particles of antimonite precipitate by EBL were partly decreased in mesophyll and root cells, and Ca(2+) precipitate distributed in intercellular spaces again. Increased Ca(2+)-ATPase activity and ATP content by EBL were also contributed to extrude excess Ca(2+) from the cytoplasm. These results suggested that EBL could alleviate the ion damage from excess Ca(2+) through regulating mineral nutrients uptake and distribution.

Application of γ-aminobutyric acid demonstrates a protective role of polyamine and GABA metabolism in muskmelon seedlings under Ca(NO3)2 stress.

The effects of exogenous γ-aminobutyric acid (GABA) application on growth, polyamine and endogenous GABA metabolism in muskmelon leaves and roots were measured. Plants were treated under control or 80 mM Ca(NO3)2 stress conditions with or without foliar spraying 50 mM GABA. Ca(NO3)2 stress significantly suppressed seedling growth and GABA transaminase activity, and enhanced glutamate decarboxylase (GAD) activity and endogenous GABA levels. Polyamine (PA) biosynthesis and degradation capacity increased in parallel with increasing GAD activity. Exogenous GABA application effectively alleviated the growth inhibition caused by Ca(NO3)2 stress, and significantly enhanced the activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), polyamine oxidase (PAO), and diamine oxidase (DAO). Exogenous GABA also significantly reduced the accumulation of free putrescine (Put) and increased the levels of free spermidine (Spd) and spermine (Spm) in leaves, which improved the capacity for polyamine biosynthesis. Application of exogenous GABA under Ca(NO3)2 stress enables the plants to maintain a higher ratio of free Spd and free Spm with respect to free Put. Our data suggest that exogenous GABA has an important role in improving muskmelon seedling tolerance to Ca(NO3)2 stress by improving biosynthesis of PAs and GABA, and by preventing PA degradation. There is a potential positive feedback mechanism that results from higher endogenous GABA content and the combined effects of Ca(NO3)2 stress and exogenous GABA, which coordinately alleviate Ca(NO3)2 stress injury by enhancing PA biosynthesis and converting free Put to an insoluble bound PA form, and reduce PA degradation in muskmelon seedlings.

Long-range interaction between the Mn4CaO5 cluster and the non-heme iron center in photosystem II as revealed by FTIR spectroelectrochemistry.

It is known that inactivation of the Mn4CaO5 cluster, the catalytic center of water oxidation in photosystem II (PSII), induces a positive shift of the redox potential (Em) of the primary quinone electron acceptor QA by ∼+150 mV, resulting in suppression of the electron transfer from QA to the secondary quinone acceptor QB. Although the relevance of this Em(QA(-)/QA) shift to the photoprotection of PSII has been debated, its molecular mechanism is still enigmatic from a structural viewpoint because QA is ∼40 Å from the Mn4CaO5 cluster. In this work, we have investigated the influence of Mn depletion on the Em of the non-heme iron, which is located between QA and QB, and its surrounding structure. Electrochemical measurements in combination with Fourier transform infrared (FTIR) spectroscopy revealed that Mn depletion shifts Em(Fe(2+)/Fe(3+)) by +18 mV, which is ∼8 times smaller than the shift of Em(QA(-)/QA). Comparison of the Fe(2+)/Fe(3+) FTIR difference spectra between intact and Mn-depleted PSII samples showed that Mn depletion altered the pKa's of a His ligand to the non-heme iron, most probably the D1-His215 interacting QB, and a carboxylate group, possibly D1-Glu244, coupled with the non-heme iron. It was further shown that Mn depletion influences the C≡N vibration of bromoxynil bound to the QB site, indicative of the modification of the QB binding site. On the basis of these results, we discuss the mechanism of a long-range interaction between the donor and acceptor sides of PSII.

Application of an early warning indicator and CaO to maximize the time-space-yield of an completely mixed waste digester using rape seed oil as co-substrate.

In order to increase the organic loading rate (OLR) and hereby the performance of biogas plants an early warning indicator (EWI-VFA/Ca) was applied in a laboratory-scale biogas digester to control process stability and to steer additive dosing. As soon as the EWI-VFA/Ca indicated the change from stable to instable process conditions, calcium oxide was charged as a countermeasure to raise the pH and to bind long-chain fatty acids (LCFAs) by formation of aggregates. An interval of eight days between two increases of the OLR, which corresponded to 38% of the hydraulic residence time (HRT), was sufficient for process adaptation. An OLR increase by a factor of three within six weeks was successfully used for biogas production. The OLR was increased to 9.5 kg volatile solids (VS) m(-3) d(-1) with up to 87% of fat. The high loading rates affected neither the microbial community negatively nor the biogas production process. Despite the increase of the organic load to high rates, methane production yielded almost its optimum, amounting to 0.9 m(3)(kg VS)(-1). Beneath several uncharacterized members of the phylum Firmicutes mostly belonging to the family Clostridiaceae, a Syntrophomonas-like organism was identified that is known to live in a syntrophic relationship to methanogenic archaea. Within the methanogenic group, microorganisms affiliated to Methanosarcina, Methanoculleus and Methanobacterium dominated the community.

Phytochemicals and antioxidant capacity of tortillas obtained after lime-cooking extrusion process of whole pigmented mexican maize.

The lime-cooking extrusion represents an alternative technology for manufacturing pre-gelatinized flours for tortillas with the advantages of saving energy and generation of null effluents. The phytochemical profiles (total phenolics, anthocyanins) and antioxidant activity of four different types of whole pigmented Mexican maize [white (WM), yellow (YM), red (RM), blue maize (BM)] processed into tortillas were studied. The lime-cooking extrusion process caused a significant decrease (p < 0.05) in total phenolics and antioxidant capacity when compared to raw kernels. Most of the total phenols assayed in raw grains (76.1-84.4 %) were bound. Tortillas from extruded maize flours retained 76.4-87.5 % of total phenolics originally found in raw grains. The BM had the highest anthocyanin content (27.52 mg cyanidin 3-glucoside/100 g DW). The WM, YM, RM and NWM contained 3.3, 3.4, 2.9, and 2.2 %, respectively, of the amount of anthocyanins found in BM. The BM lost 53.5 % of total anthocyanins when processed into extruded tortillas. Approximately 64.7 to 74.5 % of bound phytochemicals from raw kernels were the primary contributors to the ORAC values. Extruded tortillas retained amongst 87.2 to 90.7 % of total hydrophilic antioxidant activity when compared to raw kernels. Compared to the data reported by other authors using the conventional process, the lime-cooking extrusion process allowed the retention of more phenolics and antioxidant compounds in all tortillas.

Is intake of vitamin D and calcium important for cardiovascular health in elderly obese patients?

There is compelling evidence that bariatric weight loss reduces cardiovascular complications; however, these still tend to be the most common cause of late death after surgical intervention. In a prospective cohort study, correlations of dietary nutrients with indexes of vascular health were sought, with emphasis on vitamin D and calcium. Clinically stable obese outpatient subjects (>60 years old, N = 44) were interviewed about dietary macro and micronutrients. Nutritional assessment targeted anthropometric and bioimpedance analysis (BIA), hematologic counts, lipid profile, glucose homeostasis, and inflammatory markers. Carotid intima-media thickness (IMT) and brachial flow-mediated dilation (FMD), along with related vascular measurements, were documented, and results were correlated by uni- and multivariate analysis, corrected for known risk factors. IMT, FMD, and also brachial basal flow were positively influenced by vitamin D (P < 0.001). Calcium appeared beneficial for brachial basal flow only (P = 0.010). No association with IMT occurred, and a negative result for FMD was elicited. Also, vitamins A and B12 were advantageous for FMD, whereas iron was deleterious for IMT. Intake of many micronutrients including calcium and vitamin D did not meet recommendations. Vitamin D displayed a beneficial profile regarding vascular health, and more attention to this nutrient should be given, especially concerning obese patients with cardiometabolic risk. Calcium exhibited less straightforward results but deserves focus as well, along with antioxidant vitamin A as well as the B-complex which were mostly deficient in this experience.

Synthesis, characterization and evaluation of bone targeting salmon calcitonin analogs in normal and osteoporotic rats.

In order to assess the therapeutic efficacy of an antiresorptive drug with imparted bone targeting potential using bisphosphonate (BP) conjugation and an improved pharmacokinetic profile using PEGylation, we synthesized, characterized and evaluated in vivo efficacy of bone-targeting PEGylated salmon calcitonin (sCT) analog (sCT-PEG-BP). sCT-PEG-BP was compared with non-PEGylated bone targeting sCT analog (sCT-BP) and unmodified, commercially available sCT. sCT-PEG-BP conjugates were characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis. The effect of PEG-BP or BP upon sCT secondary structure was examined by Circular Dichroism and sCT-PEG-BP was evaluated for in vitro bone mineral Hydroxyapatite (HA) binding ability and calcium salts specificity using a binding assay for bone HA and several calcium salts. Anti-calcitonin antibody binding ability of these analogs was determined using enzyme-linked immunosorbent assay (ELISA), by reacting bone targeting sCT analogs with calcium phosphate coated Osteologic® plates and detecting the bound sCT using anti-sCT antibody. Potential cytotoxicity of these compounds was evaluated in monocytic RAW 264.7 cells, and sCT bioactivity was evaluated using an in vitro intracellular cAMP stimulation assay in human T47D breast cancer cells. Finally, in vivo efficacy of each compound was evaluated by determining the plasma levels of calcium after s.c. administration in normal rats, and in a rat model of Osteoporosis, secondary to ovariectomy (OVX). In vivo micro-computed tomography (micro-CT) was used to temporally map and quantify alterations in bone volume and bone mineral density (BMD) in the same animals at 1, 4, 8 and 12 weeks after OVX surgery. Sixteen 6 week old virgin female rats underwent OVX surgery followed by the daily s.c. injection of 2.5IU/kg/day sCT or equivalent analogs, and compared to four sham-operated, placebo treated control rats. Our results showed the chemical coupling of PEG-BP or BP to sCT altered its secondary structure without altering its antibody binding ability. sCT analogs retained strong sCT bioactivity, were non-toxic to RAW 264.7 cells in culture and elicited a comparable hypocalcemic effect to that of unmodified sCT in normal rats. Compared to marketed unmodified sCT, sCT-PEG-BP showed significantly improved efficacy in terms of preserving bone volume, BMD and trabecular micro-architecture in osteoporotic rats at the initial dose tested. Bisphosphonate-mediated targeting of PEGylated sCT to bone represents a new class of targeted antiresorptive compounds that has not previously been attempted.

Assessment of oral bioaccessibility of arsenic in playground soil in Madrid (Spain): a three-method comparison and implications for risk assessment.

Three methodologies to assess As bioaccessibility were evaluated using playground soil collected from 16 playgrounds in Madrid, Spain: two (Simplified Bioaccessibility Extraction Test: SBET, and hydrochloric acid-extraction: HCl) assess gastric-only bioaccessibility and the third (Physiologically Based Extraction Test: PBET) evaluates mouth-gastric-intestinal bioaccessibility. Aqua regia-extractable (pseudo total) As contents, which are routinely employed in risk assessments, were used as the reference to establish the following percentages of bioaccessibility: SBET-63.1; HCl-51.8; PBET-41.6, the highest values associated with the gastric-only extractions. For Madrid playground soils--characterised by a very uniform, weakly alkaline pH, and low Fe oxide and organic matter contents--the statistical analysis of the results indicates that, in contrast with other studies, the highest percentage of As in the samples was bound to carbonates and/or present as calcium arsenate. As opposed to the As bound to Fe oxides, this As is readily released in the gastric environment as the carbonate matrix is decomposed and calcium arsenate is dissolved, but some of it is subsequently sequestered in unavailable forms as the pH is raised to 5.5 to mimic intestinal conditions. The HCl extraction can be used as a simple and reliable (i.e. low residual standard error) proxy for the more expensive, time consuming, and error-prone PBET methodology. The HCl method would essentially halve the estimate of carcinogenic risk for children playing in Madrid playground soils, providing a more representative value of associated risk than the pseudo-total concentrations used at present.

Hydrogen-related enhancement of in vivo antioxidant ability in the brain of rats fed coral calcium hydride.

This study explored the effect of coral calcium hydride (CCH) on rat intrahippocampal antioxidant ability by measuring the PCAM nitroxide radical decay ratio when CCH was (a) co-perfused into the hippocampus and (b) fed orally to the rats for 4 weeks under a freely moving state. Estimation of the in vivo antioxidant effect was obtained by administration of the blood-brain barrier-permeable PCAM nitroxide radical and the measured PCAM radical decay ratio then correlated to the amount of antioxidant in the brain using electron spin resonance (ESR) spectroscopy combined with microdialysis. The half-life periods of PCAM in rats treated with CCH in both the co-perfusion and orally fed groups were significantly shorter compared to the control group. These results clarify the mechanism that CCH may exert antioxidant activity by significantly enhancing the basal endogenous antioxidant ability in the hippocampus through a synergistic effect with α-tocopherol and ascorbic acid.