Search results for: evogliptin tartrate

Authors: Rajeshkumar Kanubhai Patel, Neha Sudhirkumar Mochi

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A simple and accurate stability-indicating, reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous quantitation of Evogliptin tartrate and Metformin HCl in pharmaceutical dosage forms, following ICH guidelines. Forced degradation was performed under various stress conditions including acid, base, oxidation, thermal, and photodegradation. The method utilized an Eclipse C18 column (250 mm × 4.6 mm, 5 µm) with a mobile phase of 5 mM 1-hexane sulfonic acid sodium salt in water and 0.2% v/v TEA (45:55 %v/v), adjusted to pH 3.0 with OPA, at a flow rate of 1.0 mL/min. Detection at 254.4 nm using a PDA detector showed good resolution of degradation products and both drugs. Linearity was observed within 1-5 µg/mL for Evogliptin tartrate and 100-500 µg/mL for Metformin HCl, with % recovery between 99-100% and precision within acceptable limits (%RSD < 2%). The method proved to be specific, precise, accurate, and robust for routine analysis of these drugs.

Keywords: stability indicating RP-HPLC, evogliptin tartrate, metformin HCl, validation

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Authors: Indranil Chakraborty, Kalyan Mandal

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Herein, we synthesize MnFe₂O₄ nano−hollow spheres (NHSs) of average diameter 100 nm through a facile template free solvothermal process and carry out a time dependent morphological study to investigate their process of core excavation. Further, a surface engineering of as−synthesized MnFe₂O₄ NHSs has been executed with organic disodium tartrate dihydrate ligand and interestingly, the surface modified MnFe₂O₄ NHSs are found to capable of emerging multicolor fluorescence starting from blue, green to red. The magnetic measurements through vibrating sample magnetometer demonstrate that room temperature superparamagnetic nature of MnFe₂O₄ NHSs remains unaltered after surface modification. Moreover, functionalized MnFe₂O₄ NHSs are found to exhibit excellent reusable photocatalytic efficiency in the degradation of cationic dye, methylene blue with rate constant of 2.64×10−2 min.

Keywords: nano hollow sphere, tartrate modification, multiple fluorescence, catalytic property

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Authors: Amanda Cristina de Oliveira, Elias de Souza Monteiro Filho, Roberta Ceriani

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Liquid-liquid extraction in aqueous two-phase systems (ATPSs) constitutes a powerful tool for purifying bio-materials, such as cells, organelles, proteins, among others. In this work, the extraction of the bovine serum albumin (BSA) has been studied in systems formed by polyethylene glycol (PEG) (1500, 4000, and 6000 g.mol⁻¹) + potassium sodium tartrate + water at 303.15°K. Phase diagrams were obtained by turbidimetry and Merchuk’s method (1998). The experimental tie-lines were described using the Othmer-Tobias and Bancroft correlations. ATPSs were correlated with the nonrandom two-liquid (NRTL) model. The results were considered excellent according to global root-mean-square deviations found which were between 0,72 and 1,13%. The concentrations of the proteins in each phase were determined by spectrophotometry at 280 nm, finding partition efficiencies greater than 71%.

Keywords: aqueous two phases systems, bovine serum albumin , liquid-liquid extraction, polyethylene glycol

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Authors: B. M. Patil, S. R. Dharwadkar

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Zirconolite (CaZrTi2O7) is one of the three major phases in the synthetic ceramic 'SYNROC' which is used for immobilization of high-level nuclear waste and also acts as photocatalytic and photophysical properties. In the present work the nanocrystalline CaZrTi2O7 was synthesized from Calcium Zirconyl Titanate tartrate precursor (CZTT) employing two different heating techniques such as Conventional heating (Muffle furnace) and Microwave heating (Microwave Oven). Thermal decomposition of the CZTT precursors in air yielded nanocrystalline CaZrTi2O7 powder as the end product. The products obtained by annealing the CZTT precursor using both heating method were characterized using simultaneous TG-DTA, FTIR, XRD, SEM, TEM, NTA and thermodilatometric study. The physical characteristics such as crystallinity, morphology and particle size of the product obtained by heating the CZTT precursor at the different temperatures in a Muffle furnace and Microwave oven were found to be significantly different. The microwave heating technique considerably lowered the synthesis temperature of CaZrTi2O7. The influence of microwave heating was more pronounced as compared to Muffle furnace heating. The details of the synthesis of CaZrTi2O7 from CZTT precursor are discussed.

Keywords: CZTT, CaZrTi2O7, microwave, SYNROC, zirconolite

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Authors: Monalisa Pal, Kalyan Mandal

Abstract:

Herein we report the molecular functionalization of promising transition metal oxide nanostructures, such as Co3O4 nanocubes, using nontoxic and biocompati-ble organic ligand sodium tartrate. The electronic structural modification of the nanocubes imparted through functionalization and subsequent water solubilization reveals multiple absorption bands in the UV-vis region. Further surface modification of the solubilized nanocubes, leads to the emergence of intrinsic multi-color fluorescence (from blue, cyan, green to red region of the spectrum), upon excitation at proper wavelengths, where the respective excitation wavelengths have a direct correlation with the observed UV-vis absorption bands. Using a multitude of spectroscopic tools we have investigated the mechanistic insight behind the origin of different UV-vis absorption bands and emergence of multicolor photoluminescence from the functionalized nanocubes. Our detailed study shows that ligand to metal charge transfer (LMCT) from tartrate ligand to Co2+/Co3+ ions and d-d transitions involving Co2+/Co3+ ions are responsible for generation of this novel optical properties. Magnetic study reveals that, antiferromagnetic nature of Co3O4 nanocubes changes to ferromagnetic behavior upon functionalization, however, the overall magnetic response was very weak. To combine strong magnetism with this novel optical property, we followed the same surface modification strategy in case of CoFe2O4 nanoparticles, which reveals that irrespective of size and shape, all Co-based oxides can develop intrinsic multi-color fluorescence upon facile functionalization with sodium tartrate ligands and the magnetic response was significantly higher. Surface modified Co-based oxide nanostructures also show excellent catalytic activity in degradation of biologically and environmentally harmful dyes. We hope that, our developed facile functionalization strategy of Co-based oxides will open up new opportunities in the field of biomedical applications such as bio-imaging and targeted drug delivery.

Keywords: co-based oxide nanostructures, functionalization, multi-color fluorescence, catalysis

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Authors: Hye Kyung Kim, Myung-Gyou Kim, Kang-Hyun Leem

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The deer velvet antler is well known for its traditional medicinal value and is widely used in the clinic. It has been considered to possess bone-strengthening activity. The goal of this study was to investigate the anti-osteoporotic effect of deer antler velvet on ovariectomized rats (OVX), and their possible mechanism of the action. In the first step, the in vitro effects of DAE on bone loss were determined. The proliferation, collagen content and alkaline phosphatase (ALP) activity of human osteoblastic MG-63 cells and osteoclastogenesis from bone marrow-derived precursor cells were measured. The in vivo experiment confirmed the positive effect of DAE on bone tissue. 3-month old female Sparague-Dawley rats were either sham operated or OVX, and administered DAE (20 and 100 mg/kg) for 4 weeks. DAE increased MG-63 cell proliferation and ALP activity in a dose-dependent manner. Collagen content was also increased by DAE treatment. However, the effect of DAE on bone resorption was not observed. OVX rats supplemented with DAE showed osteoprotective effects as the bone ALP level was increased and c-terminal telopeptide level was decreased by 100 mg/kg DAE treatment compared with OVX controls. Moreover, the tartrate-resistant acid phosphatase-5b level was also decreased by DAE treatment. The present study suggests that DAE is effective in preventing bone loss in OVX rats, and may be potential therapeutic agents for the treatment of postmenopausal osteoporosis.

Keywords: bone ALP, c-terminal telopeptide, deer antler, osteoporosis, ovariectomy, tartrate-resistant acid phosphatase-5b

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Authors: Indranil Chakraborty, Kalyan Mandal

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Three new α-hydroxy carboxylate group functionalized MnFe₂O₄ nanoparticles (NPs) have been developed to explore the microscopic origin of ligand modified fluorescence and magnetic properties of nearly monodispersed MnFe₂O₄ NPs. The surface functionalization has been carried out with three small organic ligands (tartrate, malate, and citrate) having different number of α-hydroxy carboxylate functional group along with steric effect. Detailed study unveils that α-hydroxy carboxylate moiety of the ligands plays key role to generate intrinsic fluorescence in functionalized MnFe₂O₄ NPs through the activation of ligand to metal charge transfer transitions, associated with ligand-Mn²⁺/Fe³⁺ interactions along with d-d transition corresponding to d-orbital energy level splitting of Fe³⁺ ions on NP surface. Further, MnFe₂O₄ NPs show a maximum 140.88% increase in coercivity and 97.95% decrease in magnetization compared to its bare one upon functionalization. The ligands that induce smallest crystal field splitting of d-orbital energy level of transition metal ions are found to result in strongest ferromagnetic activation of the NPs. Finally, our developed tartrate functionalized MnFe₂O₄ (T-MnFe₂O₄) NPs have been utilized for studying DNA binding interaction and nuclease activity for stimulating their beneficial activities toward diverse biomedical applications. The spectroscopic measurements indicate that T-MnFe₂O₄ NPs bind calf thymus DNA by intercalative mode. The ability of T-MnFe₂O₄ NPs to induce DNA cleavage was studied by gel electrophoresis technique where the complex is found to promote the cleavage of pBR322 plasmid DNA from the super coiled form I to linear coiled form II and nicked coiled form III with good efficiency. This may be taken into account for designing new biomolecular detection agents and anti-cancer drug which can open up a new door toward diverse non-invasive biomedical applications.

Keywords: MnFe₂O₄ nanoparticle, α-hydroxy carboxylic acid, comparative fluorescence, magnetism study, DNA interaction, nuclease activity

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Authors: Wendong Yang, Changhai Wang, Valeria Arrighi

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Low ink sintering temperature is desired for flexible electronics, as it would widen the application of the ink on temperature-sensitive substrates where the selection of silver precursor is very critical. In this paper, four types of organic silver precursors, silver carbonate, silver oxalate, silver tartrate and silver itaconate, were synthesized using an ion exchange method, firstly. Various characterization methods were employed to investigate their physical phase, chemical composition, morphologies and thermal decomposition behavior. It was found that silver oxalate had the ideal thermal property and showed the lowest decomposition temperature. An ink was then formulated by complexing the as-prepared silver oxalate with ethylenediamine in organic solvents. Results show that a favorable conductive film with a uniform surface structure consisting of silver nanoparticles and few voids could be produced from the ink at a sintering temperature of 150 °C.

Keywords: conductive ink, electrical property, film, organic silver

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Authors: Chih-Chang Yeh, Min-Fang Yang, Hsin-I Chang

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Chitosan is a cationic polysaccharide derived from the partial deacetylation of chitin. Hyaluronic acid (HA), chondroitin sulfate (CS) and heparin (HP) are anionic glycosaminoglycans (GCGs) which can regulate osteogenic activity. In this study, chitosan membranes were prepared by glutaraldehyde crosslinking reaction and then complexed with three different types of GCGs. 7F2 osteoblasts-like cells and macrophages Raw264.7 were used as models to study the influence of chitosan membranes on osteometabolism. Although chitosan membranes are highly hydrophilic, the membranes associated with GCG-chitosan complexes showed about 60-70% cell attachment. Furthermore, the membranes associated with HP-chitosan complexes could increase ALP activity in comparison with chitosan films only. Three types of the membranes associated with GCG-chitosan complexes could significantly inhibit LPS induced-nitric oxide expression. In addition, chitosan membranes associated with HP and HA can down-regulate tartrate-resistant acid phosphatase (TRAP) activity but not CS-chitosan complexes. Based on these results, we conclude that chitosan membranes associated with HP can increase ALP activity in osteoblasts and chitosan membranes associated with HP and HA reduce TRAP activity in osteoclasts.

Keywords: osteoblast, osteoclast, chitosan, glycosaminoglycan

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Authors: M. Heysem Arslan, Ikilem Gocek, U. Kivanc Sahin

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Natural dyestuff, extracted from Juglans Regia L., a kind of walnut, was used to dye 100% cotton gabardine fabric. The main goal of this study was to enhance dyeing process of cotton fabric with Juglans Regia L. dyestuff in terms of color fastness values by designing and developing a mordant application process. Within the context of this study, different mordants such as tannic acid, gallic acid, ascorbic acid, potassium sodium tartrate tetrahydrate, calcium carbonate, iron (II) sulphate heptahydrate, aluminum potassium sulphate dodecahydrate and their combinations were applied in the mordanting processes. Spectrophotometric analysis, color fastness to washing and color fastness to light tests were carried out on the fabric samples. In this study, it was shown that by using the right combination of mordants with a proper application process, it is possible to improve color fastness values of cotton fabric samples dyed with natural dyestuff.

Keywords: extraction, Juglans Regia L., mordanting process, natural dyestuff

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Authors: Swarali Hingse, Shraddha Digole, Uday Annapure

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The present study investigates the biotransformation of ferulic acid to vanillin by Pycnoporus cinnabarinus and its optimization using one-factor-at-a-time method as well as statistical approach. Effect of various physicochemical parameters and medium components was studied using one-factor-at-a-time method. Screening of the significant factors was carried out using L25 Taguchi orthogonal array and then these selected significant factors were further optimized using response surface methodology (RSM). Significant media components obtained using Taguchi L25 orthogonal array were glucose, KH2PO4 and yeast extract. Further, a Box Behnken design was used to investigate the interactive effects of the three most significant media components. The final medium obtained after optimization using RSM containing glucose (34.89 g/L), diammonium tartrate (1 g/L), yeast extract (1.47 g/L), MgSO4•7H2O (0.5 g/L), KH2PO4 (0.15 g/L), and CaCl2•2H2O (20 mg/L) resulted in amplification of vanillin production from 30.88 mg/L to 187.63 mg/L.

Keywords: ferulic acid, pycnoporus cinnabarinus, response surface methodology, vanillin

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Authors: Boonyalitpun P., Pruckpattranon P., Thonghom A., Rotpenpian N.

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Osteoarthritis is a musculoskeletal and neuromuscular abnormality, masticatory muscle, and other tissue that causes pain and breaks down the articular surface of the temporomandibular joint (TMJ). The aim of this study is to investigate the change in the mandibular condyle, in terms of thickness and porosity, and osteoclast marker in the mandibular condyle of TMJ induced osteoarthritis mice (TMJ-OA mice). We investigated the bony changes in the TMJ structure of a complete Freund adjuvant (CFA)-injected TMJ in a mice model over 28 days. On day 28, we observed any change in the TMJ by a micro computed tomography scan (micro-CT scan) in the parameters of trabecular microarchitecture. Then we studied the thickness of the condyles by hematoxylin and eosin staining. Moreover, we calculated the area around the TMJ’s condylar head containing the osteoclast expression by TRAP (Tartrate-resistant acid phosphatase) immunohistochemistry staining. The result found that the parameter of a micro-CT scan was no different from microarchitecture in the TMJ compared with the control group; however, mandibular condyles of the TMJ-OA group was significantly thinner than the control groups, and the osteoclast expression significantly increased in the TMJ-OA group. Therefore, our findings suggest that CFA-induced TMJ-OA represents an expression of osteoclast mandibular condyle of the TMJ, which is the proposed mechanism for a TMJ-OA model.

Keywords: condyle, osteoarthritis, osteoclast, temporomandibular joint

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Authors: Chane-Yu Lai, Xiang-Yu Huang

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This study conducted an assessment of sampling result by using the new development rotation filtration device (RFD) filled with porous media filters integrating the method of cyclone centrifugal spins. The testing system established for the experiment used corn oil and potassium sodium tartrate tetrahydrate (PST) as challenge aerosols and were produced by using an Ultrasonic Atomizing Nozzle, a Syringe Pump, and a Collison nebulizer. The collection efficiency of RFD for oil aerosol was assessed by using an Aerodynamic Particle Sizer (APS) and a Fidas® Frog. The results of RFD for the liquid particles condition indicated the cutoff size was 1.65 µm and 1.02 µm for rotation of 0 rpm and 9000 rpm, respectively, under an 80 PPI (pores per inch）foam with a thickness of 80 mm, and sampling velocity of 13.5 cm/s. As the experiment increased the foam thickness of RFD, the cutoff size reduced from 1.62 µm to 1.02 µm. However, when increased the foam porosity of RFD, the cutoff size reduced from 1.26 µm to 0.96 µm. Moreover, as increased the sampling velocity of RFD, the cutoff size reduced from 1.02 µm to 0.76 µm. These discrepancies of above cutoff sizes of RFD all had statistical significance (P < 0.05). The cutoff size of RFD for three experimental conditions of generated liquid oil particles, solid PST particles or both liquid oil and solid PST particles was 1.03 µm, 1.02 µm, or 0.99 µm, respectively, under a 80 PPI foam with thickness of 80 mm, rotation of 9000 rpm, and sampling velocity of 13.5 cm/s. In addition, under the best condition of the experiment, two hours of sampling loading, the RFD had better collection efficiency for particle diameter greater than 0.45 µm, under a 94 PPI nickel mesh with a thickness of 68 mm, rotation of 9000 rpm, and sampling velocity of 108.3 cm/s. The experiment concluded that increased the thickness of porous media, face velocity, and porosity of porous media of RFD could increase the collection efficiency of porous media for sampling oil particles. Moreover, increased the rotation speed of RFD also increased the collection efficiency for sampling oil particles. Further investigation is required for those above operation parameters for RFD in this study in the future.

Keywords: oil aerosol, porous media filter, rotation, filtration

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Authors: Barrera C. Monserrat, Sierra-Alvarez Reyes, Pat-Espadas Aurora, Moreno Andrade Ivan

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Antimony is a toxic and carcinogenic metalloid considered a pollutant of priority interest by the United States Environmental Protection Agency. It is present in the environment in two oxidation states: antimonite (Sb (III)) and antimony (Sb (V)). Sb (III) is toxic to several aquatic organisms, but the potential inhibitory effect of Sb species for microorganisms has not been extensively evaluated. The fate and possible toxic impact of antimony on aerobic and anaerobic wastewater treatment systems are unknown. For this reason, the objective of this study was to evaluate the microbial toxicity of Sb (V) and Sb (III) in aerobic and anaerobic environments. Sb(V) and Sb(III) were used as potassium hexahydroxoantimonate (V) and potassium antimony tartrate, respectively (Sigma-Aldrich). The toxic effect of both Sb species in anaerobic environments was evaluated on methanogenic activity and the inhibition of hydrogen production of microorganisms from a wastewater treatment bioreactor. For the methanogenic activity, batch experiments were carried out in 160 mL serological bottles; each bottle contained basal mineral medium (100 mL), inoculum (1.5 g of VSS/L), acetate (2.56 g/L) as substrate, and variable concentrations of Sb (V) or Sb (III). Duplicate bioassays were incubated at 30 ± 2°C on an orbital shaker (105 rpm) in the dark. Methane production was monitored by gas chromatography. The hydrogen production inhibition tests were carried out in glass bottles with a working volume of 0.36 L. Glucose (50 g/L) was used as a substrate, pretreated inoculum (5 g VSS/L), mineral medium and varying concentrations of the two species of antimony. The bottles were kept under stirring and at a temperature of 35°C in an AMPTSII device that recorded hydrogen production. The toxicity of Sb on aerobic microorganisms (from a wastewater activated sludge treatment plant) was tested with a Microtox standardized toxicity test and respirometry. Results showed that Sb (III) is more toxic than Sb (V) for methanogenic microorganisms. Sb (V) caused a 50% decrease in methanogenic activity at 250 mg/L. In contrast, exposure to Sb (III) resulted in a 50% inhibition at a concentration of only 11 mg/L, and an almost complete inhibition (95%) at 25 mg/L. For hydrogen-producing microorganisms, Sb (III) and Sb (V) inhibited 50% of this production with 12.6 mg/L and 87.7 mg/L, respectively. The results for aerobic environments showed that 500 mg/L of Sb (V) do not inhibit the Allivibrio fischeri (Microtox) activity or specific oxygen uptake rate of activated sludge. In the case of Sb (III), this caused a loss of 50% of the respiration of the microorganisms at concentrations below 40 mg/L. The results obtained indicate that the toxicity of the antimony will depend on the speciation of this metalloid and that Sb (III) has a significantly higher inhibitory potential compared to Sb (V). It was shown that anaerobic microorganisms can reduce Sb (V) to Sb (III). Acknowledgments: This work was funded in part by grants from the UA-CONACYT Binational Consortium for the Regional Scientific Development and Innovation (CAZMEX), the National Institute of Health (NIH ES- 04940), and PAPIIT-DGAPA-UNAM (IN105220).

Keywords: aerobic inhibition, antimony reduction, hydrogen inhibition, methanogenic toxicity

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Authors: Nikolai A. Khlebnikov, Vladimir N. Krasilnikov, Evgenii V. Polyakov, Anastasia A. Maltceva

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Carbon materials with advanced surfaces are widely used both in modern industry and in environmental protection. The physical-chemical nature of these materials is determined by the morphology of primary atomic and molecular carbon structures, which are the basis for synthesizing the following materials: zero-dimensional (fullerenes), one-dimensional (fiber, tubes), two-dimensional (graphene) carbon nanostructures, three-dimensional (multi-layer graphene, graphite, foams) with unique physical-chemical and functional properties. Experience shows that the microscopic morphological level is the basis for the creation of the next mesoscopic morphological level. The dependence of the morphology on the chemical way and process prehistory (crystallization, colloids formation, liquid crystal state and other) is the peculiarity of the last called level. These factors determine the consumer properties of carbon materials, such as specific surface area, porosity, chemical resistance in corrosive environments, catalytic and adsorption activities. Based on the developed ideology of thin precursor synthesis, the authors discuss one of the approaches of the porosity control of carbon-containing materials with a given aggregates morphology. The low-temperature thermolysis of precursors in a gas environment of a given composition is the basis of the above-mentioned idea. The processes of carbothermic precursor synthesis of two different compounds: tungsten carbide WC:nC and zinc oxide ZnO:nC containing an impurity phase in the form of free carbon were selected as subjects of the research. In the first case, the transition metal (tungsten) forming carbides was the object of the synthesis. In the second case, there was selected zinc that does not form carbides. The synthesis of both kinds of transition metals compounds was conducted by the method of precursor carbothermic synthesis from the organic solution. ZnO:nC composites were obtained by thermolysis of succinate Zn(OO(CH2)2OO), formate glycolate Zn(HCOO)(OCH2CH2O)1/2, glycerolate Zn(OCH2CHOCH2OH), and tartrate Zn(OOCCH(OH)CH(OH)COO). WC:nC composite was synthesized from ammonium paratungstate and glycerol. In all cases, carbon structures that are specific for diamond- like carbon forms appeared on the surface of WC and ZnO particles after the heat treatment. Tungsten carbide and zinc oxide were removed from the composites by selective chemical dissolution preserving the amorphous carbon phase. This work presents the results of investigating WC:nC and ZnO:nC composites and carbon nanopowders with tubular, tape, plate and onion morphologies of aggregates that are separated by chemical dissolution of WC and ZnO from the composites by the following methods: SEM, TEM, XPA, Raman spectroscopy, and BET. The connection between the carbon morphology under the conditions of synthesis and chemical nature of the precursor and the possibility of regulation of the morphology with the specific surface area up to 1700-2000 m2/g of carbon-structured materials are discussed.

Keywords: carbon morphology, composite materials, precursor synthesis, tungsten carbide, zinc oxide

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Authors: Jolene M. Helena, Annie M. Joubert, Peace Mabeta, Magdalena Coetzee, Roy Lakier, Anne E. Mercier

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Targeting the distant tumour and its microenvironment whilst preserving bone density is important in improving the outcomes of patients with bone metastases. 2-Ethyl-3-O-sulphamoyl-estra1,3,5(10)16-tetraene (ESE-16) is an in-silico-designed 2- methoxyestradiol analogue which aimed at enhancing the parent compound’s cytotoxicity and providing a more favourable pharmacokinetic profile. In this study, the potential radiosensitization effects of ESE-16 were investigated in an in vitro bone metastasis model consisting of murine pre-osteoblastic (MC3T3-E1) and pre-osteoclastic (RAW 264.7) bone cells, metastatic prostate (DU 145) and breast (MDA-MB-231) cancer cells, as well as human umbilical vein endothelial cells (HUVECs). Cytotoxicity studies were conducted on all cell lines via spectrophotometric quantification of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide. The experimental set-up consisted of flow cytometric analysis of cell cycle progression and apoptosis detection (Annexin V-fluorescein isothiocyanate) to determine the lowest ESE-16 and radiation doses to induce apoptosis and significantly reduce cell viability. Subsequent experiments entailed a 24-hour low-dose ESE-16-exposure followed by a single dose of radiation. Termination proceeded 2, 24 or 48 hours thereafter. The effect of the combination treatment was investigated on osteoclasts via tartrate-resistant acid phosphatase (TRAP) activity- and actin ring formation assays. Tumour cell experiments included investigation of mitotic indices via haematoxylin and eosin staining; pro-apoptotic signalling via spectrophotometric quantification of caspase 3; deoxyribonucleic acid (DNA) damage via micronuclei analysis and histone H2A.X phosphorylation (γ-H2A.X); and Western blot analyses of bone morphogenetic protein-7 and matrix metalloproteinase-9. HUVEC experiments included flow cytometric quantification of cell cycle progression and free radical production; fluorescent examination of cytoskeletal morphology; invasion and migration studies on an xCELLigence platform; and Western blot analyses of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor receptor 1 and 2. Tumour cells yielded half-maximal growth inhibitory concentration (GI50) values in the nanomolar range. ESE-16 concentrations of 235 nM (DU 145) and 176 nM (MDA-MB-231) and a radiation dose of 4 Gy were found to be significant in cell cycle and apoptosis experiments. Bone and endothelial cells were exposed to the same doses as DU 145 cells. Cytotoxicity studies on bone cells reported that RAW 264.7 cells were more sensitive to the combination treatment than MC3T3-E1 cells. Mature osteoclasts were more sensitive than pre-osteoclasts with respect to TRAP activity. However, actin ring morphology was retained. The mitotic arrest was evident in tumour and endothelial cells in the mitotic index and cell cycle experiments. Increased caspase 3 activity and superoxide production indicated pro-apoptotic signalling in tumour and endothelial cells. Increased micronuclei numbers and γ-H2A.X foci indicated increased DNA damage in tumour cells. Compromised actin and tubulin morphologies and decreased invasion and migration were observed in endothelial cells. Western blot analyses revealed reduced metastatic and angiogenic signalling. ESE-16-induced radiosensitization inhibits metastatic signalling and tumour cell survival whilst preferentially preserving bone cells. This low-dose combination treatment strategy may promote the quality of life of patients with metastatic bone disease. Future studies will include 3-dimensional in-vitro and murine in-vivo models.

Keywords: angiogenesis, apoptosis, bone metastasis, cancer, cell migration, cytoskeleton, DNA damage, ESE-16, radiosensitization.

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