Search results for: protein synthesis

Authors: Qi LI, Xu Lin, Nengming Lin

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Objective: The aim of this study was to investigate the role of desmocollin 2 (DSC2) protein in the proliferation, migration and drug resistance of lung cancer cells. Method: CCK-8 assays and colony formation assays were used to evaluate the effect of dsc2 regulation on cancer cell viability and colony formation. Transwell assays and wound healing assays were also performed. Cell flow double staining was used to detect the apoptosis rate of cells with DSC2, which was added cisplatin. Western blot assay was used to detect cell cycle, PI3k/Akt and apoptosis-related proteins. Results: Our data showed that dsc2 is upregulated in clinical lung cancer tissues compared with pericarcinomatous tissues, and it is differentially expressed in lung cancer cell lines. The down-regulation of dsc2 in A549 and H358 lung cancer cells significantly suppressed the cell proliferation, metastasis, and motility. In contrast, the opposite effects were observed in overexpression of dsc2 both in H23 and PC9 cell lines. In addition to lung adenocarcinoma cell lines, we also examined its expression in lung squamous cell lines, such as H226. Western blotting showed that dsc2 could reduce the level of phosphorylated Akt (Ser 473) and p-mTOR. Thus, it is speculated that dsc2 up-regulation promotes proliferation and invasiveness through activation of the PI3K/AKT pathway. Also, knockdown of dsc2 in A549 and H226 could significantly decreased in the levels of cyclinB and wee1 protein. Additionally, flow cytometry showed that dsc2 knockdown combined with cisplatin could significantly enhance cell apoptosis rate. Conclusion: These data suggest that dsc2 promotes the proliferation and migration of lung cancer cells in vitro. Also, the results suggested that dsc2 could affect the cell cycle and apoptosis of lung cells. Furthermore, knockdown of dsc2 could sensitize cisplatin in both lung adenocarcinoma and lung squamous cell lines. Thus we suggested that dsc2 can be used as a therapeutic target for lung cancer.

Keywords: desmocollin 2, cisplatin, lung cancer, PI3K/AKT, lung squamous cell

Procedia PDF Downloads 76

Authors: Andrés Quiroz, Emilio Hormazabal, Ana Mutis, Fernando Ortega, Manuel Chacón-Fuentes, Leonardo Parra

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Red clover (Trifolium pratense L.) is an important forage species in temperate regions of the world. The main limitation of this species worldwide is a lack of persistence related to the high mortality of plants due to a complex of biotic and abiotic factors, determining a life span of two or three seasons. Because of the importance of red clover in Chile, a red clover breeding program was started at INIA Carillanca Research Center in 1989, with the main objective of improving the survival of plants, forage yield, and persistence. The main selection criteria for selecting new varieties have been based on agronomical parameters and biotic factors. The main biotic factor associated with red clover mortality in Chile is Hylastinus obscurus (Coleoptera: Curculionidae). Both larval and adults feed on the roots, causing weakening and subsequent death of clover plants. Pesticides have not been successful for controlling infestations of this root borer. Therefore, alternative strategies for controlling this pest are a high priority for red clover producers. Currently, the role of semiochemical in the interaction between H. obscurus and red clover plants has been widely studied for our group. Specifically, from the red clover foliage has been identified limonene is eliciting repellency from the root borer. Limonene is generated in the plant from two independent biosynthetic pathways, the mevalonic acid, and deoxyxylulose pathway. Mevalonate pathway enzymes are localized in the cytosol, whereas the deoxyxylulose phosphate pathway enzymes are found in plastids. In summary, limonene can be determinated by enzymatic bioassay using GPP as substrate and by limonene synthase expression. Therefore, the main objective of this work was to study genetic variation of limonene in material provided by INIA´s Red Clover breeding program. Protein extraction was carried out homogenizing 250 mg of leave tissue and suspended in 6 mL of extraction buffer (PEG 1500, PVP-30, 20 mM MgCl2 and antioxidants) and stirred on ice for 20 min. After centrifugation, aliquots of 2.5 mL were desalted on PD-10 columns, resulting in a final volume of 3.5 mL. Protein determination was performed according to Bradford with BSA as a standard. Monoterpene synthase assays were performed with 50 µL of protein extracts transferred into gas-tight 2 mL crimp seal vials after addition of 4 µL MgCl₂ and 41 µL assay buffer. The assay was started by adding 5 µL of a GPP solution. The mixture was incubated for 30 min at 40 °C. Biosynthesized limonene was quantified in a GC equipped with a chiral column and using synthetic R and S-limonene standards. The enzymatic the production of R and S-limonene from different Superqueli-Carillanca genotypes is shown in this work. Preliminary results showed significant differences in limonene content among the genotypes analyzed. These results constitute an important base for selecting genotypes with a high content of this repellent monoterpene towards H. obscurus.

Keywords: head space, limonene enzymatic determination, red clover, Hylastinus obscurus

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Authors: Minhal Abidi, Moinuddin

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Diabetes mellitus (DM) induced metabolic abnormalities causes oxidative stress which leads to the pathogenesis of complications associated with diabetes like retinopathy, nephropathy periodontitis etc. Combination of glycation and oxidation 'glycoxidation' occurs when oxidative reactions affect the early state of glycation products. Low density lipoprotein (LDL) is prone to glycoxidative attack by sugars and methylglyoxal (MGO) being a strong glycating agent may have severe impact on its structure and consequent role in diabetes. Pro-inflammatory cytokines like IL1β and TNFα produced by the action of gram negative bacteria in periodontits (PD) can in turn lead to insulin resistance. This work discusses modifications to LDL as a result of glycoxidation. The changes in the protein molecule have been characterized by various physicochemical techniques and the immunogenicity of the modified molecules was also evaluated as they presented neo-epitopes. Binding of antibodies present in diabetes patients to the native and glycated LDL has been evaluated. Role of modified epitopes in the generation of antibodies in diabetes and periodontitis has been discussed. The structural perturbations induced in LDL were analyzed by UV–Vis, fluorescence, circular dichroism and FTIR spectroscopy, molecular docking studies, thermal denaturation studies, Thioflavin T assay, isothermal titration calorimetry, comet assay. MALDI-TOF, ketoamine moieties, carbonyl content and HMF content were also quantitated in native and glycated LDL. IL1β and TNFα levels were also measured in the type 2 DM and PD patients. We report increased carbonyl content, ketoamine moieties and HMF content in glycated LDL as compared to native analogue. The results substantiate that in hyperglycemic state MGO modification of LDL causes structural perturbations making the protein antigenic which could obstruct normal physiological functions and might contribute in the development of secondary complications in diabetic patients like periodontitis.

Keywords: advanced glycation end products, diabetes mellitus, glycation, glycoxidation, low density lipoprotein, periodontitis

Procedia PDF Downloads 191

Authors: Z. Kolacinski, L. Szymanski. G. Raniszewski, D. Koza, L. Pietrzak

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Magnetic Bio-Nano-Fluid (BNF) can be composed of a buffer fluid such as plasma and magnetic nanoparticles such as iron, nickel, cobalt and their oxides. However iron is one of the best elements for magnetization by electromagnetic radiation. It can be used as a tool for medical diagnosis and treatment. Radio frequency (RF) radiation is able to heat iron nanoparticles due to magnetic hysteresis. Electromagnetic heating of iron nanoparticles and ferro-fluids BNF can be successfully used for non-invasive thermal ablation of cancer cells. Moreover iron atoms can be carried by carbon nanotubes (CNTs) if iron is used as catalyst for CNTs synthesis. Then CNTs became the iron containers and they screen the iron content against oxidation. We will present a method of CNTs addressing to the required cells. For thermal ablation of cancer cells we use radio frequencies for which the interaction with human body should be limited to minimum. Generally, the application of RF energy fields for medical treatment is justified by deep tissue penetration. The highly iron doped CNTs as the carriers creating magnetic fluid will be presented. An excessive catalyst injection method using electrical furnace and microwave plasma reactor will be presented. This way it is possible to grow the Fe filled CNTs on a moving surface in continuous synthesis process. This also allows producing uniform carpet of the Fe filled CNTs carriers. For the experimental work targeted to cell ablation we used RF generator to measure the increase in temperature for some samples like: solution of Fe2O3 in BNF which can be plasma-like buffer, solutions of pure iron of different concentrations in plasma-like buffer and in buffer used for a cell culture, solutions of carbon nanotubes (MWCNTs) of different concentrations in plasma-like buffer and in buffer used for a cell culture. Then the targeted therapies which can be effective if the carriers are able to distinguish the difference between cancerous and healthy cell’s physiology are considered. We have developed an approach based on ligand-receptor or antibody-antigen interactions for the case of colon cancer.

Keywords: cancer treatment, carbon nano tubes, drag delivery, hyperthermia, iron

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Authors: Denis Mustafov, Emmanouil Karteris, Maria Braoudaki

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Glioblastoma multiform (GBM) is a heterogenous primary brain tumour that kills most affected patients. To the authors best knowledge, despite all research efforts there is no early diagnostic biomarker for GBM. MicroRNAs (miRNAs) are short non-coding RNA molecules which are deregulated in many cancers. The aim of this research was to determine miRNAs with a diagnostic impact and to potentially identify promising therapeutic targets for glioblastoma multiform. In silico analysis was performed to identify deregulated miRNAs with diagnostic relevance for glioblastoma. The expression profiles of the chosen miRNAs were then validated in vitro in the human glioblastoma cell lines A172 and U-87MG. Briefly, RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed using the mirVANA miRNA isolation kit. Quantitative Real-Time Polymerase Chain Reaction was performed to verify their expression. The presence of five target proteins within the A172 cell line was evaluated by Western blotting. The expression of the CORO1C protein within 32 GBM cases was examined via immunohistochemistry. The miRNAs identified in silico included miR-21-5p, miR-34a and miR-128a. These miRNAs were shown to target deregulated GBM genes, such as CDK6, E2F3, BMI1, JAG1, and CORO1C. miR-34a and miR-128a showed low expression profiles in comparison to a control miR-RNU-44 in both GBM cell lines suggesting tumour suppressor properties. Opposing, miR-21-5p demonstrated greater expression indicating that it could potentially function as an oncomiR. Western blotting revealed expression of all five proteins within the A172 cell line. In silico analysis also suggested that CORO1C is a target of miR-128a and miR-34a. Immunohistochemistry demonstrated that 75% of the GBM cases showed moderate to high expression of CORO1C protein. Greater understanding of the deregulated expression of miR-128a and the upregulation of CORO1C in GBM could potentially lead to the identification of a promising diagnostic biomarker signature for glioblastomas.

Keywords: non-coding RNAs, gene expression, brain tumours, immunohistochemistry

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Authors: P. Behera, K. K. Singh, D. K. Saini, M. De

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Implementation of 2D materials in the detection of bio analytes is highly advantageous in the field of sensing because of its high surface to volume ratio. We have designed our sensor array with different cationic two-dimensional MoS₂, where surface modification was achieved by cationic thiol ligands with different functionality. Green fluorescent protein (GFP) was chosen as signal transducers for its biocompatibility and anionic nature, which can bind to the cationic MoS₂ surface easily, followed by fluorescence quenching. The addition of bio-analyte to the sensor can decomplex the cationic MoS₂ and GFP conjugates, followed by the regeneration of GFP fluorescence. The fluorescence response pattern belongs to various analytes collected and transformed to linear discriminant analysis (LDA) for classification. At first, 15 different proteins having wide range of molecular weight and isoelectric points were successfully discriminated at 50 nM with detection limit of 1 nM. The sensor system was also executed in biofluids such as serum, where 10 different proteins at 2.5 μM were well separated. After successful discrimination of protein analytes, the sensor array was implemented for bacteria sensing. Six different bacteria were successfully classified at OD = 0.05 with a detection limit corresponding to OD = 0.005. The optimized sensor array was able to classify uropathogens from non-uropathogens in urine medium. Further, the technique was applied for discrimination of bacteria possessing resistance to different types and amounts of drugs. We found out the mechanism of sensing through optical and electrodynamic studies, which indicates the interaction between bacteria with the sensor system was mainly due to electrostatic force of interactions, but the separation of native bacteria from their drug resistant variant was due to Van der Waals forces. There are two ways bacteria can be detected, i.e., through bacterial cells and lysates. The bacterial lysates contain intracellular information and also safe to analysis as it does not contain live cells. Lysates of different drug resistant bacteria were patterned effectively from the native strain. From unknown sample analysis, we found that discrimination of bacterial cells is more sensitive than that of lysates. But the analyst can prefer bacterial lysates over live cells for safer analysis.

Keywords: array-based sensing, drug resistant bacteria, linear discriminant analysis, two-dimensional MoS₂

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Authors: Mehmet Doğan, Mahir Alkan, Yasemin Turhan, Zürriye Gündüz, Pinar Beyli, Serap Doğan

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Advances and new discoveries in the field of the material science on the basis of technological developments have played an important role. Today, material science is branched the lower branches such as metals, nonmetals, chemicals, polymers. The polymeric nano composites have found a wide application field as one of the most important among these groups. Many polymers used in the different fields of the industry have been desired to improve the thermal stability. One of the ways to improve this property of the polymers is to form the nano composite products of them using different fillers. There are many using area of boron compounds and is increasing day by day. In order to the further increasing of the variety of using area of boron compounds and industrial importance, it is necessary to synthesis of nano-products and to find yourself new application areas of these products. In this study, PMMA/boronoxide nano composites were synthesized using solution intercalation, polymerization and melting methods; and PAA/boronoxide nano composites using solution intercalation method. Furthermore, rheological properties of nano composites synthesed according to melting method were also studied. Nano composites were characterized by XRD, FTIR-ATR, DTA/TG, BET, SEM, and TEM instruments. The effects of filler material amount, solvent types and mediating reagent on the thermal stability of polymers were investigated. In addition, the rheological properties of PMMA/boronoxide nano composites synthesized by melting method were investigated using High Pressure Capillary Rheometer. XRD analysis showed that boronoxide was dispersed in polymer matrix; FTIR-ATR that there were interactions with boronoxide between PAA and PMMA; and TEM that boronoxide particles had spherical structure, and dispersed in nano sized dimension in polymer matrix; the thermal stability of polymers was increased with the adding of boronoxide in polymer matrix; the decomposition mechanism of PAA was changed. From rheological measurements, it was found that PMMA and PMMA/boronoxide nano composites exhibited non-Newtonian, pseudo-plastic, shear thinning behavior under all experimental conditions.

Keywords: boronoxide, polymer, nanocomposite, rheology, characterization

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Authors: Héctor González Espinosa, Ricardo Ivan Cordova Chávez, Alejandra Contreras Ramos, Itzia Irene Padilla Martínez, José Guadalupe Trujillo Ferrara, Marvin Antonio Soriano Ursúa

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Boronic acids are able to create diester bonds with carbohydrates because of their hydroxyl groups; in nature, there are some organoborates with these characteristics, such as the calcium fructoborate, formed by the union of two fructose molecules and a boron atom, synthesized by plants. In addition, it has been observed that, in animal cells only the compounds with cis-diol functional groups are capable of linking to boric or boronic acids. The formation of these organoboron compounds could impair the physical and chemical properties of the precursors, even their acute toxicity. In this project, two carbohydrate-derived boron-containing compounds from D-fructose and D-arabinose and phenylboronic acid are analyzed by different spectroscopy techniques such as Raman, Infrared with Fourier Transform Infrared (FT-IR), Nuclear Magnetic Resonance (NMR) and X-ray diffraction crystallography to describe their chemical characteristics. Also, an acute toxicity test was performed to determine their LD50 using the Lorke’s method. It was confirmed by multiple spectra the formation of the adducts by the generation of the diester bonds with a β-D-pyranose of fructose and arabinose. The most prominent findings were the presence of signals corresponding to the formation of new bonds, like the stretching of B-O bonds, or the absence of signals of functional groups like the hydroxyls presented in the reagents used for the synthesis of the adducts. The NMR spectra yielded information about the stereoselectivity in the synthesis reaction, observed by the interaction of the protons and their vicinal atoms in the anomeric and second position carbons; but also, the absence of a racemic mix by the finding of just one signal in the range for the anomeric carbon in the 13C NMR spectra of both adducts. The acute toxicity tests by the Lorke’s method showed that the LD50 value for both compounds is 1265 mg/kg. Those results let us to propose these adducts as highly safe agents for further biological evaluation with medical purposes.

Keywords: acute toxicity, adduct, boron, carbohydrate, diester bond

Procedia PDF Downloads 65

Authors: Natesan Balasubramanian, Palpandi Pounpandi, Venkatraman Thamil Priya, Vellasamy Shanmugaiah, Karubbiah Balakrishnan, Mandayam Anandam Thirunarayan

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Streptococcus agalactiae is commonly known as Group B Streptococcus (GBS) and it is the most common cause of life-threatening bacterial infection. GBS first considered as a veterinary pathogen causing mastitis in cattle later becomes a human pathogen for severe neonatal infections. In this present study, a total of 20 new clinical isolates of S. agalactiae were collected from male (6) and female patient (14) with different age group. The isolates were from Urinary tract infection (UTI), blood, pus and eye ulcer. All the 20 S. agalactiae isolates has clear hemolysis properties on blood agar medium and were identified by serogrouping and MALTI-TOF-MS analysis. Antibiotic susceptibility/resistance test was performed for 20 S. agalactiae isolates, further phenotypic resistance pattern was observed for tetracycline, vancomycin, ampicillin and penicillin. Genotypically we found two antibiotic resistance genes such as Betalactem antibiotic resistance gene (Tem) (70%) and tetracycline resistance gene Tet(O) 15% in our isolates. Six virulence factors encoding genes were performed by PCR in twenty GBS isolates, cfb gene (100%), followed by, cylE(90.47%), lmp(85.7%), bca(71.42%), rib (38%) and low frequency in bac gene (4.76%) were determined. Most of the S. agalactiae isolates produced strong biofilm in the polystyrene surface (hydrophobic), and low-level biofilm formation was found in glass tube (hydrophilic) surface. lytR is secreted protein and localized in bacterial cell wall, extra cellular membrane, and cytoplasm. In silico docking studies were performed for lytR protein with four antibiofilm compounds, including a peptide (PR39) with the docking study showed peptide has strong interaction followed by ellagic acid and interaction length is 2.95, 2.97 and 2.95 A°. In ligand EGCGO10 and O11 two atoms intract with lytR (Leu271), with binding bond affinity length is 3.24 and 3.14. The aminoacid Leu 271 is act as an impartant aminoacid, since ellagic acid and EGCG interact with same aminoacid.

Keywords: antibiotics, biofilms, clinical isolates, S. agalactiae, virulence

Procedia PDF Downloads 108

Authors: S. El Ballal, H. El Sabbagh, M. Abd El Gaber, A. Eisa, A. Al Gamal

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Cadmium is one of the most harmful heavy metals able to induce severe injury. In this study, sixty four male Sprague Dawley rats weighing (70-80 gm) were used. Rats were divided into 4 groups each group of 16 rats. Group A: served as control and received commercial ration and distilled water Group B: cadmium chloride was administered orally in water at dose of 300 ppm cadmium (560 mg/L as CdCl2). Group C: Animals received cadmium in drinking water in addition to administration of N-acetylcysteine (NAC) orally at a dose of 150 mg/kg body weight, equivalent to 1500 ppm in food. Group D: Animals received cadmium in drinking water in addition to administration of alpha lipoic acid (ALA) orally at a dose of 150 mg/kg body weight, equivalent to 1500 ppm in food. The experiment was continued for 2 months. Collection of blood and tissue samples was performed at 2, 4, 6, 8 weeks. Blood sample were collected for serum biochemical analysis including malondialdehyde (MDA), total antioxidants, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, albumin, urea and uric acid. Tissue specimens were collected for histopathological examination including liver, kidney, brain and testis. Histopathological examination revealed that cadmium choloride induces pathological alterations which increased in severity with time. The use of NAC and ALA can ameliorate toxic effect of CdCl2. The results showed significant decrease MDA and significant increase total antioxidants in group C and D compared to group B, Liver enzymes include AST and ALT showed significant decrease. Regarding to results of total protein and albumin, they revealed significant increase. Urea and uric acid showed significant decrease. From our study we conclude that NAC and ALA have protective effect against cadmium toxicity.

Keywords: ALA, cadmium, histopathology, NAC

Procedia PDF Downloads 338

Authors: S. Kudlacik-Kramarczyk, M. Kedzierska, B. Tyliszczak

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Recently, the continuous development of medicine and related sciences has been observed. Particular emphasis is directed on the development of biomaterials, i.e., non-toxic, biocompatible and biodegradable materials that may improve the effectiveness of treatment as well as the comfort of patients. This is particularly important in the case of cancer treatment. Currently, there are many methods of cancer treatment based primarily on chemotherapy and the surgical removal of the tumor, but it is worth noting that these therapies also cause many side effects. Among women, the most common cancer is breast cancer. It may be completely cured, but the consequence of treatment is partial or complete breast mastectomy and radiation therapy, which results in severe skin burns. The skin of the patient after radiation therapy is very burned, and therefore requires intensive care and high frequency of dressing changes. The traditional dressing adheres to the burn wounds and does not absorb adequate amount of exudate from injuries and the patient is forced to change the dressing every 2 hours. Therefore, the main purpose was to develop an innovative combination of dressing material with drug carriers that may be used in anti-cancer therapy. The innovation of this solution is the combination of these two products into one system, i.e., a transdermal system with the possibility of a controlled release of the drug- cytostatic. Besides, the possibility of modifying the hydrogel matrix with aloe vera juice provides this material with new features favorable from the point of view of healing processes of burn wounds resulting from the radiation therapy. In this study, hydrogel materials containing protein spheres with the active substance have been obtained as a result of photopolymerization process. The reaction mixture consisting of the protein (albumin) spheres incorporated with cytostatic, chitosan, adequate crosslinking agent and photoinitiator has been subjected to the UV radiation for 2 minutes. Prepared materials have been subjected to the numerous studies including the analysis of cytotoxicity using murine fibroblasts L929. Analysis was conducted based on the mitochondrial activity test (MTT reduction assay) which involves the determining the number of cells characterized by proper metabolism. Hydrogel materials obtained using different amount of crosslinking agents have been subjected to the cytotoxicity analysis. According to the standards, tested material is defined as cytotoxic when the viability of cells after 24 h incubation with this material is lower than 70%. In the research, hydrogel polymer materials containing protein spheres incorporated with the active substance, i.e. a cytostatic, have been developed. Such a dressing may support the treatment of cancer due to the content of the anti-cancer drug - cytostatic, and may also provide a soothing effect on the healing of the burn wounds resulted from the radiation therapy due to the content of aloe vera juice in the hydrogel matrix. Based on the conducted cytotoxicity studies, it may be concluded that the obtained materials do not adversely affect the tested cell lines, therefore they can be subjected to more advanced analyzes.

Keywords: hydrogel polymers, cytostatics, drug carriers, cytotoxicity

Procedia PDF Downloads 133

Authors: Vadim V. Zefirov, Victor E. Sizov, Marina A. Pigaleva, Igor V. Elmanovich, Mikhail S. Kondratenko, Marat O. Gallyamov

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This work presents a novel method for treating porous hydrophobic polyolefin membranes using supercritical carbon dioxide that allows usage of the modified membrane in redox flow batteries with an aqueous electrolyte. Polyolefin membranes are well known and widely used, however, they cannot be used as separators in redox flow batteries with an aqueous electrolyte since they have insufficient wettability, and therefore do not provide sufficient proton conductivity. The main aim of the presented work was the development of hydrophilic composites based on cheap membranes and precursors. Supercritical fluid was used as a medium for the deposition of the hydrophilic phase on the hydrophobic surface of the membrane. Due to the absence of negative capillary effects in a supercritical medium, a homogeneous composite is obtained as a result of synthesis. The in-situ synthesized silicon oxide nanoparticles and the chitosan polymer layer act as the hydrophilic phase and not only increase the affinity of the membrane towards the electrolyte, but also reduce the pore size of the polymer matrix, which positively affects the ion selectivity of the membrane. The composite material obtained as a result of synthesis has enhanced hydrophilic properties and is capable of providing proton conductivity in redox flow batteries. The morphology of the obtained composites was characterized by electron microscopy. To analyze the phase composition, infrared spectroscopy was used. The hydrophilic properties were studied by water contact angle measurements. In addition, the proton conductivity and ion selectivity of the obtained samples were studied, and tests in real redox flow batteries were performed. As a result, modified membrane was characterised in detail and moreover it was shown that modified cheap polyolefin membranes have pronounced proton conductivity and high ion selectivity, so their performance in a real redox flow battery approaches expensive commercial analogues, reaching 70% of energy efficiency.

Keywords: carbon dioxide, chitosan, polymer membrane, redox flow batteries, silica nanoparticles, supercritical fluid

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Authors: Snehal D. Ganjave, Hardik Dodia, Avinash V. Sunder, Swati Madhu, Pramod P. Wangikar

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Batch cultivation of recombinant bacteria in shake flasks results in low cell density due to nutrient depletion. Previous protocols on high cell density cultivation in shake flasks have relied mainly on controlled release mechanisms and extended cultivation protocols. In the present work, we report an optimized fed-batch process for high cell density cultivation of recombinant E. coli BL21(DE3) for protein production. A cybernetic model-based, multi-objective optimization strategy was implemented to obtain the optimum operating variables to achieve maximum biomass and minimized substrate feed rate. A syringe pump was used to feed a mixture of glycerol and yeast extract into the shake flask. Preliminary experiments were conducted with online monitoring of dissolved oxygen (DO) and offline measurements of biomass and glycerol to estimate the model parameters. Multi-objective optimization was performed to obtain the pareto front surface. The selected optimized recipe was tested for a range of proteins that show different extent soluble expression in E. coli. These included eYFP and LkADH, which are largely expressed in soluble fractions, CbFDH and GcanADH , which are partially soluble, and human PDGF, which forms inclusion bodies. The biomass concentrations achieved in 24 h were in the range 19.9-21.5 g/L, while the model predicted value was 19.44 g/L. The process was successfully reproduced in a standard laboratory shake flask without online monitoring of DO and pH. The optimized fed-batch process showed significant improvement in both the biomass and protein production of the tested recombinant proteins compared to batch cultivation. The proposed process will have significant implications in the routine cultivation of E. coli for various applications.

Keywords: cybernetic model, E. coli, high cell density cultivation, multi-objective optimization

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Authors: Nastaran Hazrati, Ali Akbar Miran Beigi, Majid Abdouss, Amir Vahid

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Hydrogen sulfide is the most toxic gas of crude oil. Adsorption is an energy-efficient process used to remove undesirable compounds such as H2S in gas or liquid streams by passing the stream through a media bed composed of an adsorbent. In this study, H2S of Iran crude oil was separated via cold stripping then zinc incorporated MCM-41 was synthesized via an in-situ method. ZnO functionalized mesoporous silica samples were characterized by XRD, N2 adsorption and TEM. The obtained results of adsorption of H2S showed superior ability of all the materials and with an increase in ZnO amount adsorption was increased.

Keywords: MCM-41, ZnO, H2S removal, adsorption

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Authors: Seungmin Shin, Jin-Baek Kim

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Hematite (α-Fe2O3) has very good semiconducting properties with a band gap of 2.1 eV and is antiferromagnetic. Due to its electrochemical stability, low toxicity, wide abundance, and low-cost, hematite, it is a particularly attractive material for photoelectrochemical cells. Additionally, hematite has also found applications in gas sensing, field emission, heterogeneous catalysis, and lithium-ion battery electrodes. Here, we discovered a new universal top-down method for the synthesis of one-dimensional hematite nanowire arrays. Various shapes and lengths of hematite nanowire have been easily fabricated over large areas by sequential processes. The obtained hematite nanowire arrays are promising candidates as photoanodes in photoelectrochemical solar cells.

Keywords: hematite, lithography, nanowire, top-down process

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Authors: Hugo Espinosa-Andrews

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Introduction: Yogurt is widely accepted worldwide due to its high nutritional value, consistency, and texture. Their rheological properties play a significant role in consumer acceptance and are related to the manufacturing process and formulation. Typically, the viscoelastic characteristics of yogurts are studied using the small amplitude oscillatory shear test; however, the initial stages of flow and oral processing are described in the nonlinear zone, in which a large amplitude oscillatory stress test is applied. The objective of this work was to analyze the nonlinear viscoelastic behavior of commercial yogurts using Lissajous curves. Methods: Two commercial yogurts were purchased in a local store in Guadalajara Jalisco Mexico: a natural Greek-style yogurt and a low-fat traditional yogurt. Viscoelastic properties were evaluated using a large amplitude oscillatory stress procedure (LAOS). A crosshatch geometry of 40 mm and a truncation of 1000 µm were used. Stress sweeps were performed at 6.28 rad/s from 1 to 250 Pa at 5°C. The nonlinear viscoelastic properties were analyzed using the Lissajous curves. Results: The yogurts showed strain-viscoelastic behavior related to deformation-dependent materials. In the low-strain region, the elastic modulus predominated over the viscous modulus, showing gel-elastic properties. The sol-gel transitions were observed at approximately 66.5 Pa for the Greek yogurt, double that detected for traditional yogurt. The viscoelastic behavior of the yogurts was characteristic of weak excess deformation: behavior indicating a stable molecular structure at rest, and moderate structure at medium shear-forces. The normalized Lissajous curves characterized viscoelastic transitions of the yogurt as the stress increased. Greater viscoelasticity deformation was observed in Greek yogurt than in traditional yogurt, which is related to the presence of a protein network with a greater degree of crosslinking. Conclusions: The yogurt composition influences the viscoelastic properties of the material. Yogurt with the higher percentage of protein has greater viscoelastic and viscous properties, which describe a product of greater consistency and creaminess.

Keywords: yogurt, viscoelastic properties, LAOS, elastic modulus

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Authors: S. Varnagiris, M. Urbonavicius, S. Tuckute, M. Lelis, K. Bockute

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Amongst many water purification techniques, TiO₂ photocatalysis is recognized as one of the most promising sustainable methods. It is known that for photocatalytical applications anatase is the most suitable TiO₂ phase, however heterojunction of anatase/rutile phases could improve the photocatalytical activity of TiO₂ even further. Despite the relative simplicity of TiO₂ different synthesis methods lead to the highly dispersed crystal phases and photocatalytic activity of the corresponding samples. Accordingly, suggestions and investigations of various innovative methods of TiO₂ synthesis are still needed. In this work structural and photocatalytical properties of TiO₂ films deposited by the unconventional method of simultaneous co-sputtering from two magnetrons powered by pulsed-Direct Current (pDC) and Radio Frequency (RF) power sources with negative bias voltage have been studied. More specifically, TiO₂ film thickness, microstructure, surface roughness, crystal structure, optical transmittance and photocatalytical properties were investigated by profilometer, scanning electron microscope, atomic force microscope, X-ray diffractometer and UV-Vis spectrophotometer respectively. The proposed unconventional two magnetron co-sputtering based TiO₂ film formation method showed very promising results for crystalline TiO₂ film formation while keeping process temperatures below 100 °C. XRD analysis revealed that by using proper combination of power source type and bias voltage various TiO₂ phases (amorphous, anatase, rutile or their mixture) can be synthesized selectively. Moreover, strong dependency between power source type and surface roughness, as well as between the bias voltage and band gap value of TiO₂ films was observed. Interestingly, TiO₂ films deposited by two magnetron co-sputtering without bias voltage had one of the highest band gap values between the investigated films but its photocatalytic activity was superior compared to all other samples. It is suggested that this is due to the dominating nanocrystalline anatase phase with various exposed surfaces including photocatalytically the most active {001}.

Keywords: films, magnetron co-sputtering, photocatalysis, TiO₂

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Authors: M. Obaid, Nasser A. M. Barakat, Fadali O.A

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Low stability in oil media and the hydrophobicity problems of the ploysulfone electrospun membranes could be overcome in the present study. Synthesis of super-hydrophilic and highly stable in oil polysulfone electrospun nanofiber membrane was achieved by electrospinning of polysulfone solution containing NaOH salt followed by activation of the dried electrospun membrane by deposition of polyamide layer on the surface using m-phenylenediamine and 1,3,5-benzenetricarbonyl chloride. The introduced membrane has super-hydrophilicity characteristic (contact angle=3o), excellent stability in oil media and distinct performance in oil-water separation process.

Keywords: electrospinning, oil-degradability, membrane, nanofibers

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Authors: Gwo-Hwa Wan, Tai-Ho Hung, Fen-Fang Chung, Wan-Ying Lee, Hui-Ching Yang

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Air pollutant exposure results in elevated concentrations of oxidative stress and inflammatory biomarkers in general populations. Increased concentrations of inflammatory biomarkers in pregnant women would be associated with preterm labor and low birth weight. To our best knowledge, the associations between air pollutants exposure and inflammation in pregnant women and fetuses are unknown, as well as their effects on fetal growth. This study aimed to evaluate the influences of outdoor air pollutants in northern Taiwan areas on the inflammatory biomarker (high sensitivity C-reactive protein, hs-CRP) concentration in the blood of healthy pregnant women and how the biomarker impacts fetal growth. In this study, 38 healthy pregnant women who are in their first trimester and live in northern Taiwan area were recruited from the Taipei Chang Gung Memorial Hospital. Personal characteristics and prenatal examination data (e.g., blood pressure) were obtained from recruited subjects. The concentrations of inflammatory mediators, hs-CRP, in the blood of healthy pregnant women were analyzed. Additionally, hourly data of air pollutants (PM10, SO2, NO2, O3, CO) concentrations were obtained from air quality monitoring stations in Taipei area, established by the Taiwan Environmental Protection Administration. The definition of lag 0 and lag 01 are the exposure to air pollutants on the day of blood withdrawal, and the average exposure to air pollutants one day before and on the day of blood withdrawal, respectively. The statistical analyses were conducted using SPSS software version 22.0 (SPSS, Inc., Chicago, IL, USA). This analytical result indicates that the healthy pregnant women aged between 28 and 42 years old. The body mass index before pregnancy averaged 21.51 (sd = 2.51) kg/m2. Around 90% of the pregnant women had never smoking habit, and 28.95% of them had allergic diseases. Approximately around 84% and 5.26% of the pregnant women worked at indoor and outdoor environments, respectively. The mean hematocrit level of the pregnant women was 37.10%, and the hemoglobin levels were ranged between 10.1 and 14.7 g/dL with 12.47 g/dL of mean value. The blood hs-CRP concentrations of healthy pregnant women in the first trimester ranged between 0.32 and 32.5 mg/L with 2.83 (sd = 5.69) mg/L of mean value. The blood hs-CRP concentrations were positively associated with ozone concentrations at lag 0-14 (r = 0.481, p = 0.017) in healthy pregnant women. Significant lag effects were identified in ozone at lag 0-14 with a positive excess concentration of blood hs-CRP.

Keywords: air pollutant, hs-CRP, pregnant woman, ozone, first trimester

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Authors: Hanan. Al Chaghouri, Mohammad Azad Malik, P. John Thomas, Paul O’Brien

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The process of assembling metal nanoparticles at the interface of two liquids has received a great interest over the past few years due to a wide range of important applications and their unusual properties compared to bulk materials. We present a low cost, simple and cheap synthesis of metal nanoparticles, core/shell structures and semiconductors followed by assembly of these particles between immiscible liquids. The aim of this talk is divided to three parts: firstly, to describe the achievement of a closed loop recycling for producing cadmium sulphide as powders and/or nanostructured thin films for solar cells or other optoelectronic devices applications by using a different chain length of commercially available secondary amines of dithiocarbamato complexes. The approach can be extended to other metal sulphides such as those of Zn, Pb, Cu, or Fe and many transition metals and oxides. Secondly, to synthesis significantly cheaper magnetic particles suited for the mass market. Ni/NiO nanoparticles with ferromagnetic properties at room temperature were among the smallest and strongest magnets (5 nm) were made in solution. The applications of this work can be applied to produce viable storage devices and the other possibility is to disperse these nanocrystals in solution and use it to make ferro-fluids which have a number of mature applications. The third part is about preparing and assembling of submicron silver, cobalt and nickel particles by using polyol methods and liquid/liquid interface, respectively. Noble metal like gold, copper and silver are suitable for plasmonic thin film solar cells because of their low resistivity and strong interactions with visible light waves. Silver is the best choice for solar cell application since it has low absorption losses and high radiative efficiency compared to gold and copper. Assembled cobalt and nickel as films are promising for spintronic, magnetic and magneto-electronic and biomedics.

Keywords: assembling nanoparticles, liquid/liquid interface, thin film, core/shell, solar cells, recording media

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Authors: A. Guemache, M. Omari

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Oxides with formula Ca1-xSrx MnO3(0≤x≤0.2) were synthesized using co precipitation method. The identification of the obtained phase was carried out using infrared spectroscopy and x-ray diffraction. Thermogravimetric and differential analysis was permitted to characterize different transformations of precursors which take place during one heating cycle. The study of electrochemical behavior was carried out by cyclic voltammetry and impedance spectroscopy. The obtained results show that apparent catalytic activity improved when increasing the concentration of strontium. Anodic current densities varies from 1.3 to 5.9 mA/cm2 at the rate scan of 20 mV.s-1 and a potential 0.8 V for oxides with composition x=0 to 0.2.

Keywords: oxide, co-precipitation, thermal analysis, electrochemical properties

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Authors: Talita Kato, Moises Grespan, Elza I. Ida, Massami Shimokomaki, Adriana L. Soares

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White Striping (WS) is becoming a concern for the poultry industry, as it affects the look of breast broiler chicken meat leading it to rejection by the consumers. It is characterized by the appearance of varying degrees of white striations on the Pectoralis major muscle surface following the direction of the muscle fiber. The etiology of this myopathy is still unknown, however it is suggested to be associated with increased weight gain rate and age of the bird, attributing the phenomenon to the genetically bird’s selection for efficiently higher meat production. The aim of this study was to evaluate the occurrence of Pectoralis major WS in a commercial plant in southern Brazil and its chemical characterization. The breast meat samples (n=660) from birds of 47 days of age, were classified as: Normal NG (no apparent white striations), Moderate MG (when the fillets present thin lines <1 mm) and Severe SG (white striations present ˃1 mm thick covering a large part of the fillet surface). Thirty samples (n = 10 for each level of severity) were analyzed for pH, color (L*, a*, b*), proximate chemical composition (moisture, protein, ash and lipids contents) and hydroxyproline in order to determine the collagen content. The results revealed the occurrence for NG group was 16.97%, 51.67% for MG group and 31.36% for SG group. Although the total protein content did not differ significantly, the collagen index was 42% higher in favor to SG in relation to NG. Also the lipid fraction was 27% higher for SG group. The NG presented the lowest values of the parameters L* and a* (P ≤ 0.05), as there was no white striations on its surface and highest b* value in SG, because of the maximum lipid contents. These results indicate there was a contribution of the SG muscle cells to oversynthesize connective tissue components on the muscle fascia. In conclusion, this study revealed a high incidence of White Striping on broiler commercial line in Brazil thus, there is a need to identify the causes of this abnormality in order to diminish or to eliminate it.

Keywords: collagen content, commercial line, pectoralis major muscle, proximate composition

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Authors: Magdy G. Asaad

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Solitons are among the most beneficial solutions for science and technology for their applicability in physical applications including plasma, energy transport along protein molecules, wave transport along poly-acetylene molecules, ocean waves, constructing optical communication systems, transmission of information through optical fibers and Josephson junctions. In this talk, we will apply the bilinear technique to generate a class of soliton solutions to the (3+1)-dimensional nonlinear soliton equation of Jimbo-Miwa type. Examples of the resulting soliton solutions are computed and a few solutions are plotted.

Keywords: Pfaffian solutions, N-soliton solutions, soliton equations, Jimbo-Miwa

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Authors: Dong-Gi Lee, Suyeon Cha, Yong-Sun Bahn

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Sterol lipid is essential for cell membrane structure in eukaryotic cells. In mammalian cells, sterol regulatory element binding proteins (SREBPs) act as principal regulators of cellular cholesterol which is essential for proper cell membrane fluidity and structure. SREBP and sterol regulation are related to levels of cellular oxygen because it is a major substrate for sterol synthesis. Upon cellular sterol and oxygen levels are depleted, SREBP is translocated to the Golgi where it undergoes proteolytic cleavage of N terminus, then it travels to the nucleus to play a role as transcription factor. In yeast cells, synthesis of ergosterol is also highly oxygen consumptive, and Sre1 is a transcription factor known to play a central role in adaptation to growth under low oxygen condition and sterol homeostasis in Cryptococcus neoformans. In this study, we observed phenotypes in other strains of Cryptococcus species by constructing hob1Δ and sre1Δ mutants to confirm whether the functions of both genes are conserved in most serotypes. As a result, hob1Δ showed no noticeable phenotype under treatment of antifungal drugs and most environmental stresses in R265 (C. gattii) and XL280 (C. neoformans), suggesting that Hob1 is related to sterol regulation only in H99 (serotype A). On the other hand, the function of Sre1 was found to be conserved in most serotypes. Furthermore, mating experiment of hob1Δ or sre1Δ showed dramatic defects in serotype A (H99) and D (XL280). It revealed that Hob1 and Sre1 related to mating ability in Cryptococcus species, especially cell fusion efficiency. In conclusion, HOB1 and SRE1 play crucial role in regulating sterol-homeostasis and differentiation in C. neoformans, moreover, Hob1 is specific gene in Cryptococcus neoformans. It suggests that Hob1 is considered as potent factor-targeted new safety antifungal drug.

Keywords: cryptococcus neoformans, Hob1, Sre1, sterol regulatory element binding proteins

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Authors: Hamsasew Hankebo Lemago, Dóra Hessz, Tamás Igricz, Zoltán Erdélyi, , Imre Miklós Szilágyi

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Metal oxide inverse opals are a promising class of photocatalysts with a unique hierarchical structure. Atomic layer deposition (ALD) is a versatile technique for the synthesis of high-precision metal oxide thin films, including inverse opals. In this study, we report the synthesis of TiO₂, ZnO, and Al₂O₃ inverse opal and their composites photocatalysts using thermal or plasma-enhanced ALD. The synthesized photocatalysts were characterized using a variety of techniques, including scanning electron microscopy (SEM)-energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), Raman spectroscopy, photoluminescence (PL), ellipsometry, and UV-visible spectroscopy. The results showed that the ALD-synthesized metal oxide inverse opals had a highly ordered structure and a tunable pore size. The PL spectroscopy results showed low recombination rates of photogenerated electron-hole pairs, while the ellipsometry and UV-visible spectroscopy results showed tunable optical properties and band gap energies. The photocatalytic activity of the samples was evaluated by the degradation of methylene blue under visible light irradiation. The results showed that the ALD-synthesized metal oxide inverse opals exhibited high photocatalytic activity, even under visible light irradiation. The composites photocatalysts showed even higher activity than the individual metal oxide inverse opals. The enhanced photocatalytic activity of the composites can be attributed to the synergistic effect between the different metal oxides. For example, Al₂O₃ can act as a charge carrier scavenger, which can reduce the recombination of photogenerated electron-hole pairs. The ALD-synthesized metal oxide inverse opals and their composites are promising photocatalysts for a variety of applications, such as wastewater treatment, air purification, and energy production. The ALD-synthesized metal oxide inverse opals and their composites are promising photocatalysts for a variety of applications, such as wastewater treatment, air purification, and energy production.

Keywords: ALD, metal oxide inverse opals, photocatalysis, composites

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Authors: J. E. Mendes, T. T. de Barros, O. B. G. de Assis, J. D. C. Pessoa

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Chitosan, a natural polysaccharide of β-1,4-linked glucosamine residues, is a biopolymer obtained primarily from the exoskeletons of crustaceans. Interest in polymeric materials increases year by year. Chitosan is one of the most plentiful biomaterials, with a wide range of pharmaceutical, biomedical, industrial and agricultural applications. Chitosan nanoparticles were synthesized via the ionotropic gelation of chitosan with sodium tripolyphosphate (TPP). Two concentrations of chitosan microparticles (0.1 and 0.2%) were synthesized. In this study, it was possible to synthesize and characterize microparticles of chitosan biomaterial and this will be used for future applications in cell anchorage for 3D bioprinting.

Keywords: chitosan microparticles, biomaterial, scaffold, bioprinting

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Authors: Elodie Niemiec, Philippe Champagne, Jean-Francois Blach, Philippe Moreau, Anthony Thuault, Arnaud Tricoteaux

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Hygiene of equipment in contact with users is an important issue in the railroad industry. The numerous cleanings to eliminate bacteria and dirt cost a lot. Besides, mechanical solicitations on contact parts are observed daily. It should be interesting to elaborate on a self-cleaning and antibacterial coating with sufficient adhesion and good resistance against mechanical and chemical solicitations. Thus, a Hauts-de-France and Maubeuge Val-de-Sambre conurbation authority co-financed Ph.D. thesis has been set up since October 2017 based on anterior studies carried by the Laboratory of Ceramic Materials and Processing. To accomplish this task, a soft chemical route has been implemented to bring a lotus effect on metallic substrates. It involves nanometric liquid zinc oxide synthesis under 100°C. The originality here consists in a variation of surface texturing by modification of the synthesis time of the species in solution. This helps to adjust wettability. Nanostructured zinc oxide has been chosen because of the inherent photocatalytic effect, which can activate organic substance degradation. Two methods of heating have been compared: conventional and microwave assistance. Tested subtracts are made of stainless steel to conform to transport uses. Substrate preparation was the first step of this protocol: a meticulous cleaning of the samples is applied. The main goal of the elaboration protocol is to fix enough zinc-based seeds to make them grow during the next step as desired (nanorod shaped). To improve this adhesion, a silica gel has been formulated and optimized to ensure chemical bonding between substrate and zinc seeds. The last step consists of deposing a wide carbonated organosilane to improve the superhydrophobic property of the coating. The quasi-proportionality between the reaction time and the nanorod length will be demonstrated. Water Contact (superior to 150°) and Roll-off Angle at different steps of the process will be presented. The antibacterial effect has been proved with Escherichia Coli, Staphylococcus Aureus, and Bacillus Subtilis. The mortality rate is found to be four times superior to a non-treated substrate. Photocatalytic experiences were carried out from different dyed solutions in contact with treated samples under UV irradiation. Spectroscopic measurements allow to determinate times of degradation according to the zinc quantity available on the surface. The final coating obtained is, therefore, not a monolayer but rather a set of amorphous/crystalline/amorphous layers that have been characterized by spectroscopic ellipsometry. We will show that the thickness of the nanostructured oxide layer depends essentially on the synthesis time set in the hydrothermal growth step. A green, easy-to-process and control coating with self-cleaning and antibacterial properties has been synthesized with a satisfying surface structuration.

Keywords: antibacterial, biomimetism, soft-chemistry, zinc oxide

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Authors: Parya Nasehi, Mohammad Kazem Mohammadi

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3,4-dihydropyrimidin-2(1H)-thiones were synthesized in the presence of Ag nanoparticle/melamine sulfonic acid (MSA) supported on alumina. The reaction was carried out at 110 oC for 20 min under solvent free conditions. This method have some advantages such as good yield, mild reaction conditions, ease of operation and work up, short reaction time and high product purity.

Keywords: nanoparticle melamine sulfonic acid, Al2O3, Biginelli reaction, 3, 4-dihydropyrimidin-2(1H, solvent free

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Authors: Ji Hoi Heo, Jun Seong Park, Hyo Kim

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Geopolymer is an inorganic material synthesized by alkali activation of source materials rich in soluble SiO₂ and Al₂O₃. Many researches have studied the effect of aluminum species on the synthesis of geopolymer. However, it is still unclear about the influence of Al additives on the properties of geopolymer. The current study identified the role of the Al additive on the thermal performance of fly ash based geopolymer and observing the microstructure development of the composite. NaOH pellets were dissolved in water for 14 M (14 moles/L) sodium hydroxide solution which was used as an alkali activator. The weight ratio of alkali activator to fly ash was 0.40. Sodium aluminate powder was employed as an Al additive and added in amounts of 0.5 wt.% to 2 wt.% by the weight of fly ash. The mixture of alkali activator and fly ash was cured in a 75°C dry oven for 24 hours. Then, the hardened geopolymer samples were exposed to 300°C, 600°C and 900°C for 2 hours, respectively. The initial compressive strength after oven curing increased with increasing sodium aluminate content. It was also observed in SEM results that more amounts of geopolymer composite were synthesized as sodium aluminate was added. The compressive strength increased with increasing heating temperature from 300°C to 600°C regardless of sodium aluminate addition. It was consistent with the ATR-FTIR results that the peak position related to asymmetric stretching vibrations of Si-O-T (T: Si or Al) shifted to higher wavenumber as the heating temperature increased, indicating the further geopolymer reaction. In addition, geopolymer sample with higher content of sodium aluminate showed better compressive strength. It was also reflected on the IR results by more shift of the peak position assigned to Si-O-T toward the higher wavenumber. However, the compressive strength decreased after being exposed to 900°C in all samples. The degree of reduction in compressive strength was decreased with increasing sodium aluminate content. The deterioration in compressive strength was most severe in the geopolymer sample without sodium aluminate additive, while the samples with sodium aluminate addition showed better thermal durability at 900°C. This is related to the phase transformation with the occurrence of nepheline phase at 900°C, which was most predominant in the sample without sodium aluminate. In this work, it was concluded that sodium aluminate could be a good additive in the geopolymer synthesis by showing the improved compressive strength at elevated temperatures.

Keywords: compressive strength, fly ash based geopolymer, microstructure development, Na-aluminate

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Authors: Shabnam Abdi, Behzad Toosi

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Background: Melanoma is the most lethal type of malignant skin cancer in humans and dogs since it spreads rapidly throughout the body. Despite significant advances in treatment, cancer at an advanced stage has a poor prognosis. Hence, more effective treatments are needed to enhance outcomes with fewer side effects. Erythropoietin-producing hepatocellular receptors are the largest family of receptor tyrosine kinases and are divided into two subfamilies, EphA and EphB, both of which play a significant role in disease, especially cancer. Due to their association with proliferation and invasion in many aggressive types of cancer, Eph receptor tyrosine kinases (Eph RTKs) are promising cancer therapy molecules. Because these receptors have not been studied in canine melanoma, we investigated how EphA2 influences survival and tumorigenicity of melanoma cells. Methods: Expression of EphA2 protein in canine melanoma cell lines and human melanoma cell line was evaluated by Western blot. Melanoma cells were transduced with lentiviral particles encoding Eph-targeting shRNAs or non-silencing shRNAs (control) for silencing the expression of EphA2 receptor, and silencing was confirmed by Western blotting and immunofluorescence. The effect of siRNA treatment on cellular proliferation, colony formation, tumorsphere assay, invasion was analyzed by Resazurin assay Matrigel invasion assay, respectively. Results: Expression of EphA2 was detected in canine and human melanoma cell lines. Moreover, stably silencing EphA2 by specific shRNAs significantly and consistently decreased the expression of EphA2 protein in both human and canine melanoma cells. Proliferation, colony formation, tumorsphere and invasion of melanoma cells were significantly decreased in EphA2 siRNA-treated cells compared to control. Conclusion: Our data provide the first functional evidence that the EphA2 receptor plays a critical role in the malignant cellular behavior of melanoma in both human and dogs.

Keywords: ephA2, targeting, melanoma, human, canine

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