Search results for: transfection reagents

Authors: Biswabandhu Bankura, Srikanta Guria, Madhusudan Das

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Purpose: Thyroid peroxidase (TPO) is the key enzyme in the biosynthesis of thyroid hormones. We aimed to identify the spectrum of mutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of the disease. Methods: 200 hypothyroid patients (case) and their corresponding sex and age matched 200 normal individuals (control) were screened depending on their clinical manifestations. Genomic DNA was isolated from peripheral blood samples and TPO gene (Exon 7 to Exon 14) was amplified by PCR. The PCR products were subjected to sequencing to identify mutations. Results: Single nucleotide changes such as Glu 641 Lys, Asp 668 Asn, Thr 725 Pro, Asp 620 Asn, Ser 398 Thr, and Ala 373 Ser were found. Changes in the TPO were assayed in vitro to compare mutant and wild-type activities. Five mutants were enzymatically inactive in the guaiacol and iodide assays. This is a strong indication that the mutations are present at crucial positions of the TPO gene, resulting in inactivated TPO. Key Findings: The results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population of West Bengal.

Keywords: thyroid peroxidase, hypothyroidism, mutation, in vitro assay, transfection

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Authors: Biswabandhu Bankura, Srikanta Guria, Madhusudan Das

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Purpose: Thyroid peroxidase (TPO) is the key enzyme in the biosynthesis of thyroid hormones. We aimed to identify the spectrum of mutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of the disease. Methods: 200 hypothyroid patients (case) and their corresponding sex and age matched 200 normal individuals (control) were screened depending on their clinical manifestations. Genomic DNA was isolated from peripheral blood samples and TPO gene (Exon 7 to Exon 14) was amplified by PCR. The PCR products were subjected to sequencing to identify mutations. Results: Single nucleotide changes such as Glu 641 Lys, Asp 668 Asn, Thr 725 Pro, Asp 620 Asn, Ser 398 Thr, and Ala 373 Ser were found. Changes in the TPO were assayed in vitro to compare mutant and wild-type activities. Five mutants were enzymatically inactive in the guaiacol and iodide assays. This is a strong indication that the mutations are present at crucial positions of the TPO gene, resulting in inactivated TPO. Key Findings: The results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population of West Bengal.

Keywords: thyroid peroxidase, hypothyroidism, mutation, in vitro assay, transfection

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Authors: Jer-Yuh Liu, Je-Chiuan Ye, Jin-Ming Hwang

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Protein kinase C alpha (PKCα) is a key signaling molecule in human cancer development. As a therapeutic strategy, targeting PKCα is difficult because the molecule is ubiquitously expressed in non-malignant cells. PKCα is regulated by the cooperative interaction of the transcription factors myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) in human cancer cells. By conducting tissue array analysis, herein, we determined the protein expression of MZF-1/Elk-1/PKCα in various cancers. The data show that the expression of MZF-1/Elk-1 is correlated with that of PKCα in hepatocellular carcinoma (HCC), but not in bladder and lung cancers. In addition, the PKCα down-regulation by shRNA Elk-1 was only observed in the HCC SK-Hep-1 cells. Blocking the interaction between MZF-1 and Elk-1 through the transfection of their binding domain MZF-160–72 decreased PKCα expression. This step ultimately depressed the epithelial-mesenchymal transition potential of the HCC cells. These findings could be used to develop an alternative therapeutic strategy for patients with the PKCα-derived HCC.

Keywords: protein kinase C alpha, myeloid zinc finger 1, ets-like protein-1, hepatocellular carcinoma

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Authors: Dipti Raj, Himanshu Mishra

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Wetting of a solid surface by a liquid is an extremely common yet subtle phenomenon in natural and applied sciences. A clear understanding of both short and long-term wetting behaviors of surfaces is essential for creating robust anti-biofouling coatings, non-wetting textiles, non-fogging mirrors, and preventive linings against dirt and icing. In this study, silica beads (diameter, D ≈ 100 μm) functionalized using different silane reagents were employed to modify the wetting characteristics of smooth polydimethylsiloxane (PDMS) surfaces. Resulting composite surfaces were found to be super-hydrophobic, i.e. contact angle of water,

Keywords: contact angle, Cassie-Baxter, PDMS, silica, texture, wetting

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Authors: Kumaran Narayanan, Andrew N. Osahor

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E. coli has been engineered by our group and by others as a vector to deliver DNA into cultured human and animal cells. However, so far conditions to improve gene delivery using this vector have not been investigated, resulting in a major gap in our understanding of the requirements for this vector to function optimally. Our group recently published novel data showing that simple addition of the DNA transfection reagent Lipofectamine increased the efficiency of the E. coli vector by almost 3-fold, providing the first strong evidence that further optimization of bactofection is possible. This presentation will discuss advances that demonstrate the effects of several intracellular strategies that improve the efficiency of this vector. Conditions that promote endosomal escape of internalized bacteria to evade lysosomal destruction after entry in the cell, a known obstacle limiting this vector, are elucidated. Further, treatments that increase bacterial lysis so that the vector can release its transgene into the mammalian environment for expression will be discussed. These experiments will provide valuable new insight to advance this E. coli system as an important class of vector technology for genetic correction of human disease models in cells and whole animals.

Keywords: DNA, E. coli, gene expression, vector

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Authors: H. A. Taner, V. Önen

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A suitable choice of flotation parameters and reagents have a strong effect on the effectiveness of flotation process. The objective of this paper is to give an overview of the flotation of chalcopyrite with the different conditions and dispersants. Flotation parameters such as grinding time, pH, type, and dosage of dispersant were investigated. In order to understand the interaction of some dispersants, sodium silicate, sodium hexametaphosphate and sodium polyphosphate were used. The optimum results were obtained at a pH of 11.5 and a grinding time of 10 minutes. A copper concentrate was produced assaying 29.85% CuFeS₂ and 65.97% flotation recovery under optimum rougher flotation conditions with sodium silicate.

Keywords: chalcopyrite, dispersant, flotation, reagent

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Authors: S. Mamoucha, J. Rahul, N. Christodoulakis

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Introduction: Nature has been the source of medicinal agents since the dawn of the human existence on Earth. Currently, millions of people, in the developing world, rely on medicinal plants for primary health care, income generation and lifespan improvement. In Greece, more than 5500 plant taxa are reported while about 250 of them are considered to be of great pharmaceutical importance. Among the plants used for medical purposes, Vitex agnus-castus L. (Verbenaceae) is known since ancient times. It is a small tree or shrub, widely distributed in the Mediterranean basin up to the Central Asia. It is also known as chaste tree or monks pepper. Theophrastus mentioned the shrub several times, as ‘agnos’ in his ‘Enquiry into Plants’. Dioscorides mentioned the use of V. agnus-castus for the stimulation of lactation in nursing mothers and the treatment of several female disorders. The plant has important medicinal properties and a long tradition in folk medicine as an antimicrobial, diuretic, digestive and insecticidal agent. Materials and methods: Leaves were cleaned, detached, fixed, sectioned and investigated with light and Scanning Electron Microscopy (SEM). Histochemical tests were executed as well. Specific histochemical reagents (osmium tetroxide, H2SO4, vanillin/HCl, antimony trichloride, Wagner’ s reagent, Dittmar’ s reagent, potassium bichromate, nitroso reaction, ferric chloride and di methoxy benzaldehyde) were used for the sub cellular localization of secondary metabolites. Results: Light microscopical investigations of the elongated leaves of V. agnus-castus revealed three layers of palisade parenchyma, just below the single layered adaxial epidermis. The spongy parenchyma is rather loose. Adaxial epidermal cells are larger in magnitude, compared to those of the abaxial epidermis. Four different types of capitate, secreting trichomes, were localized among the abaxial epidermal cells. Stomata were observed at the abaxial epidermis as well. SEM revealed the interesting arrangement of trichomes. Histochemical treatment on fresh and plastic embedded tissue sections revealed the nature and the sites of secondary metabolites accumulation (flavonoids, steroids, terpenes). Acknowledgment: This work was supported by IKY - State Scholarship Foundation, Athens, Greece.

Keywords: Vitex agnus-castus, leaf anatomy, histochemical reagents, secondary metabolites

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Authors: Ali Oztuna, Meral Sarper, Deniz Torun, Fatma Bayrakdar, Selcuk Kilic, Mehmet Baysallar

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Bacillus anthracis is one of the biological agents most likely to be used in case of bioterrorist attack as well as being the cause of anthrax. The bacterium's major virulence factors are the anthrax toxins and an antiphagocytic polyglutamic capsule. TEM8 (ANTXR1) and CMG2 (ANTXR2) are ubiquitously expressed type I transmembrane proteins, and ANTXR2 is the major receptor for anthrax toxins. MicroRNAs are 21-24 bp small noncoding RNAs that regulate gene expression by base pairing with the 3' UTR (untranslated regions) of their target mRNAs resulting in mRNA degradation and/or translational repression. MicroRNAs contribute to regulation of most biological processes and influence numerous pathological states like infectious disease. In this study, post-exposure (toxins) protective effect of the hsa-miR-124-3p against Bacillus anthracis was examined. In this context, i) THP-1 and U937 cells were differentiated to MΦ macrophage, ii) miRNA transfection efficiencies were evaluated by flow cytometry and qPCR, iii) protection against Bacillus anthracis toxins were investigated by XTT, cAMP ELISA and MEK2 cleavage assays. Acknowledgements: This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under Grant SBAG-218S467.

Keywords: ANTXR2, hsa-miR-124-3p, MΦ macrophage, THP-1, U937

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Authors: Pesaru Vigneshwar Reddy, Parvathaneni Pavan

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Green organic chemistry which is the latest and one of the most researched topics now-a- days has been in demand since 1990’s. Majority of the research in green organic chemistry chemicals are some of the important starting materials for greater number of major chemical industries. The production of organic chemicals has raw materials (or) reagents for other application is major sector of manufacturing polymers, pharmaceuticals, pesticides, paints, artificial fibers, food additives etc. organic synthesis on a large scale compound to the labratory scale, involves the use of energy, basic chemical ingredients from the petro chemical sectors, catalyst and after the end of the reaction, seperation, purification, storage, packing distribution etc. During these processes there are many problems of health and safety for workers in addition to the environmental problems caused there by use and deposition as waste. Green chemistry with its 12 principles would like to see changes in conventional way that were used for decades to make synthetic organic chemical and the use of less toxic starting materials. Green chemistry would like to increase the efficiency of synthetic methods, to use less toxic solvents, reduce the stage of synthetic routes and minimize waste as far as practically possible. In this way, organic synthesis will be part of the effort for sustainable development Green chemistry is also interested for research and alternatives innovations on many practical aspects of organic synthesis in the university and research labaratory of institutions. By changing the methodologies of organic synthesis, health and safety will be advanced in the small scale laboratory level but also will be extended to the industrial large scale production a process through new techniques. The three key developments in green chemistry include the use of super critical carbondioxide as green solvent, aqueous hydrogen peroxide as an oxidising agent and use of hydrogen in asymmetric synthesis. It also focuses on replacing traditional methods of heating with that of modern methods of heating like microwaves traditions, so that carbon foot print should reduces as far as possible. Another beneficiary of this green chemistry is that it will reduce environmental pollution through the use of less toxic reagents, minimizing of waste and more bio-degradable biproducts. In this present paper some of the basic principles, approaches, and early achievements of green chemistry has a branch of chemistry that studies the laws of passing of chemical reactions is also considered, with the summarization of green chemistry principles. A discussion about E-factor, old and new synthesis of ibuprofen, microwave techniques, and some of the recent advancements also considered.

Keywords: energy, e-factor, carbon foot print, micro-wave, sono-chemistry, advancement

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Authors: Arunas Stirke, Neringa Bakute, Gatis Mozolevskis

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A technology of microfluidics is an emerging tool in the field of biology, medicine and chemistry. Microfluidic device is also known as ‘lab-on-a-chip’ technology [1]. In moving from macro- to microscale, there is unprecedented control over spatial and temporal gradients and patterns that cannot be captured in conventional Petri dishes and well plates [2]. However, there is not a single standard microfluidic chip designated for all purposes – every different field of studies needs a specific microchip with certain geometries, inlet/outlet, channel depth and other parameters to precisely regulate the required function. Since our group is studying an effect of pulsed electric field (PEF) to the cells, we have manufactured a microfluidic chip designated for high-throughput electroporation of cells. In our microchip, a cell culture chamber is divided into two parallel channels by a membrane, meanwhile electrodes for electroporation are attached to the wall of the channels. Both microchannels have their own inlet and outlet, enabling injection of transfection material separately. Our perspective is to perform electroporation of mammalian cells in two different ways: (1) plasmid and cells are injected in the same microchannel and (2) injected into separate microchannels. Moreover, oxygen and pH sensors are integrated on order to analyse cell viability parameters after PEF treatment.

Keywords: microfluidics, chip, fabrication, electroporation

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Authors: D. Lopes, I. I. R. Baptista, R. F. Vieira, J. Vaz, H. Varela, O. M. Freitas, V. F. Domingues, R. Jorge, C. Delerue-Matos, S. A. Figueiredo

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Deodorization is nowadays a need in wastewater treatment plants. Nitrogen and sulphur compounds, volatile fatty acids, aldehydes and ketones are responsible for the unpleasant odours, being ammonia, hydrogen sulphide and mercaptans the most common pollutants. Although chemical treatments of the air extracted are efficient, these are more expensive than biological treatments, namely due the use of chemical reagents (commonly sulphuric acid, sodium hypochlorite and sodium hydroxide). Biofiltration offers the advantage of avoiding the use of reagents (only in some cases, nutrients are added in order to increase the treatment efficiency) and can be considered a sustainable process when the packing medium used is of natural origin. In this work the application of some natural materials locally available was studied both at laboratory and pilot scale, in a real wastewater treatment plant. The materials selected for this study were indigenous Portuguese forest materials derived from eucalyptus and pinewood, such as woodchips and bark, and coconut fiber was also used for comparison purposes. Their physico-chemical characterization was performed: density, moisture, pH, buffer and water retention capacity. Laboratory studies involved batch adsorption studies for ammonia and hydrogen sulphide removal and evaluation of microbiological activity. Four pilot-scale biofilters (1 cubic meter volume) were installed at a local wastewater treatment plant treating odours from the effluent receiving chamber. Each biofilter contained a different packing material consisting of mixtures of eucalyptus bark, pine woodchips and coconut fiber, with added buffering agents and nutrients. The odour treatment efficiency was monitored over time, as well as other operating parameters. The operation at pilot scale suggested that between the processes involved in biofiltration - adsorption, absorption and biodegradation - the first dominates at the beginning, while the biofilm is developing. When the biofilm is completely established, and the adsorption capacity of the material is reached, biodegradation becomes the most relevant odour removal mechanism. High odour and hydrogen sulphide removal efficiencies were achieved throughout the testing period (over 6 months), confirming the suitability of the materials selected, and mixtures thereof prepared, for biofiltration applications.

Keywords: ammonia hydrogen sulphide and removal, biofiltration, natural materials, odour control in wastewater treatment plants

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Authors: Vanja Misic, Mohamed El-Mogy, Yousef Haj-Ahmad

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Endonuclease G (EndoG) is a well conserved mitochondrio-nuclear nuclease with dual lethal and vital roles in the cell. The aim of our study was to examine whether EndoG exerts its nuclease activity on exogenous DNA substrates such as plasmid DNA (pDNA), considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus, targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. These findings make it unlikely that targeting of EndoG is a viable strategy for improving the duration and level of transgene expression from non-viral DNA vectors in gene therapy efforts.

Keywords: EndoG, silencing, exogenous DNA stability, HeLa cells

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Authors: Hanjun Zhao

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Limited efficacy of current antivirals and antiviral-resistant mutations impair anti-influenza treatment. Here, we evaluated the in vitro and in vivo antiviral effect of three defective interfering genes (DIG-3) of influenza virus. Virus replication was significantly reduced in 293T and A549 cells transfected with DIG-3. Mice transfected with DIG-3 encoded by jetPEI-vector, as prophylaxis and therapeutics against A(H7N7) virus respectively, had significantly better survivals (80% and 50%) than control mice (0%). We further developed a dual-functional peptide TAT-P1, which delivers DIG-3 with high transfection efficiency and concomitantly exerts antiviral activity by preventing endosomal acidification. TAT-P1/DIG-3 was more effective than jetPEI/DIG-3 in treating A(H7N7) or A(H1N1)pdm09-infected mice and showed potent prophylactic protection on A(H7N7) or A(H1N1)pdm09-infected mice. The addition of P1 peptide, preventing endosomal acidification, could enhance the protection of TAT-P1/DIG-3 on A(H1N1)pdm09-infected mice. Dual-functional TAT-P1 with DIG-3 can effectively protect or treat mice infected by avian and seasonal influenza virus infection.

Keywords: antiviral peptide, dual-functional peptide, defective interfering genes, influenza virus

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Authors: António Fiúza, Aurora Futuro, Joana Monteiro, Joaquim Góis

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Lepidolite is an important lithium mineral that, to the author’s best knowledge, has not been used to produce lithium hydroxide, which is necessary for energy conversion to electric vehicles. Alkaline leaching of lithium concentrates allows the establishment of a production diagram avoiding most of the environmental drawbacks that are associated with the usage of acid reagents. The tested processes involve a pretreatment by digestion at high temperatures with additives, followed by leaching at hot atmospheric pressure. The solutions obtained must be compatible with solutions from the leaching of spodumene concentrates, allowing the development of a common treatment diagram, an important accomplishment for the feasible exploitation of Portuguese resources. Statistical programming and interpretation techniques minimize the laboratory effort required by conventional approaches and allow phenomenological comprehension.

Keywords: alkaline leaching, lithium, research design, statistical interpretation

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Authors: Fiúza A., Góis J. Leite M., Braga H., Lima A., Jorge P., Moutela P., Martins L., Futuro A.

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Lepidolite is an important lithium mineral that, to the author’s best knowledge, has not been used to produce lithium hydroxide, necessary for energy conversion to electric vehicles. Alkaline leaching of lithium concentrates allows the establishment of a production diagram avoiding most of the environmental drawbacks that are associated with the usage of acid reagents. The tested processes involve a pretreatment by digestion at high temperatures with additives, followed by leaching at hot atmospheric pressure. The solutions obtained must be compatible with solutions from the leaching of spodumene concentrates, allowing the development of a common treatment diagram, an important accomplishment for the feasible exploitation of Portuguese resources. Statistical programming and interpretation techniques are used to minimize the laboratory effort required by conventional approaches and also allow phenomenological comprehension.

Keywords: artificial intelligence, tailings free process, ferroelectric electrolyte battery, life cycle assessment

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Authors: Kartikaningsih Danis, Yao-Hui Huang

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Various techniques including conventional and advanced have been employed for the boron treatment from water and wastewater. The electrocoagulation involves an electrolytic reactor for coagulation/flotation with aluminium as anode and cathode. There is aluminium as coagulant to be used for removal which may induce secondary pollution in chemical coagulation. The purpose of this study is to investigate and compare the performance between electrocoagulation and chemical coagulation on boron removal from synthetic wastewater. The effect of different parameters, such as pH reaction, coagulant dosage, and initial boron concentration were examined. The results show that the boron removal using chemical coagulation was lower. At the optimum condition (e.g. pH 8 and 0.8 mol coagulant dosage), boron removal efficiencies for chemical coagulation and electrocoagulation were 61% and 91%, respectively. In addition, the electrocoagulation needs no chemical reagents and makes the boron treatment easy for application.

Keywords: boron removal, chemical coagulation, aluminum, electro-coagulation

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Authors: V. Ivanov, J. Chu, V. Stabnikov

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Different biotechnological methods for the production of construction materials and for the performance of construction processes in situ are developing within a new scientific discipline of Construction Biotechnology. The aim of this research was to develop and test new biotechnologies and biotechnological grouts for the minimization of the hydraulic conductivity of the fractured rocks and porous soil. This problem is essential to minimize flow rate of groundwater into the construction sites, the tunneling space before and after excavation, inside levies, as well as to stop water seepage from the aquaculture ponds, agricultural channels, radioactive waste or toxic chemicals storage sites, from the landfills or from the soil-polluted sites. The conventional fine or ultrafine cement grouts or chemical grouts have such restrictions as high cost, viscosity, sometime toxicity but the biogrouts, which are based on microbial or enzymatic activities and some not expensive inorganic reagents, could be more suitable in many cases because of lower cost and low or zero toxicity. Due to these advantages, development of biotechnologies for biogrouting is going exponentially. However, most popular at present biogrout, which is based on activity of urease- producing bacteria initiating crystallization of calcium carbonate from calcium salt has such disadvantages as production of toxic ammonium/ammonia and development of high pH. Therefore, the aim of our studies was development and testing of new biogrouts that are environmentally friendly and have low cost suitable for large scale geotechnical, construction, and environmental applications. New microbial biotechnologies have been studied and tested in the sand columns, fissured rock samples, in 1 m3 tank with sand, and in the pack of stone sheets that were the models of the porous soil and fractured rocks. Several biotechnological methods showed positive results: 1) biogrouting using sequential desaturation of sand by injection of denitrifying bacteria and medium following with biocementation using urease-producing bacteria, urea and calcium salt decreased hydraulic conductivity of sand to 2×10-7 ms-1 after 17 days of treatment and consumed almost three times less reagents than conventional calcium-and urea-based biogrouting; 2) biogrouting using slime-producing bacteria decreased hydraulic conductivity of sand to 1x10-6 ms-1 after 15 days of treatment; 3) biogrouting of the rocks with the width of the fissures 65×10-6 m using calcium bicarbonate solution, that was produced from CaCO3 and CO2 under 30 bars pressure, decreased hydraulic conductivity of the fissured rocks to 2×10-7 ms-1 after 5 days of treatment. These bioclogging technologies could have a lot of advantages over conventional construction materials and processes and can be used in geotechnical engineering, agriculture and aquaculture, and for the environmental protection.

Keywords: biocementation, bioclogging, biogrouting, fractured rocks, porous soil, tunneling space

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Authors: Irina Veselova, Maria Makedonskaya, Olga Eremina, Alexandr Sidorov, Eugene Goodilin, Tatyana Shekhovtsova

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Such catecholamines as dopamine, norepinephrine, and epinephrine are the principal neurotransmitters in the sympathetic nervous system. Catecholamines and their metabolites are considered to be important markers of socially significant diseases such as atherosclerosis, diabetes, coronary heart disease, carcinogenesis, Alzheimer's and Parkinson's diseases. Currently, neurotransmitters can be studied via electrochemical and chromatographic techniques that allow their characterizing and quantification, although these techniques can only provide crude spatial information. Besides, the difficulty of catecholamine determination in biological materials is associated with their low normal concentrations (~ 1 nM) in biomaterials, which may become even one more order lower because of some disorders. In addition, in blood they are rapidly oxidized by monoaminooxidases from thrombocytes and, for this reason, the determination of neurotransmitter metabolism indicators in an organism should be very rapid (15—30 min), especially in critical states. Unfortunately, modern instrumental analysis does not offer a complex solution of this problem: despite its high sensitivity and selectivity, HPLC-MS cannot provide sufficiently rapid analysis, while enzymatic biosensors and immunoassays for the determination of the considered analytes lack sufficient sensitivity and reproducibility. Fluorescent and SERS-sensors remain a compelling technology for approaching the general problem of selective neurotransmitter detection. In recent years, a number of catecholamine sensors have been reported including RNA aptamers, fluorescent ribonucleopeptide (RNP) complexes, and boronic acid based synthetic receptors and the sensor operated in a turn-off mode. In this work we present the fluorescent and SERS turn-on sensor systems based on the bio- or chemorecognizing nanostructured films {chitosan/collagen-Tb/Eu/Cu-nanoparticles-indicator reagents} that provide the selective recognition, visualization, and sensing of the above mentioned catecholamines on the level of nanomolar concentrations in biomaterials (cell cultures, tissue etc.). We have (1) developed optically transparent porous films and gels of chitosan/collagen; (2) ensured functionalization of the surface by molecules-'recognizers' (by impregnation and immobilization of components of the indicator systems: biorecognizing and auxiliary reagents); (3) performed computer simulation for theoretical prediction and interpretation of some properties of the developed materials and obtained analytical signals in biomaterials. We are grateful for the financial support of this research from Russian Foundation for Basic Research (grants no. 15-03-05064 a, and 15-29-01330 ofi_m).

Keywords: biomaterials, fluorescent and SERS-recognition, neurotransmitters, solid-phase turn-on sensor system

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Authors: Rasha M. Hussein, Reem M. Hashem, Laila A. Rashed

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Alzheimer’s disease is the most common dementia disease in the elderly. It is characterized by the accumulation of extracellular amyloid β (Aβ) peptides and intracellular hyper-phosphorylated tau protein. In addition, recent evidence indicates that accumulation of intracellular amyloid β peptides may play a role in Alzheimer’s disease pathogenesis. This suggests that intracellular Heat Shock Proteins (HSP) that maintain the protein quality control in the cell might be potential candidates for disease amelioration. DNAJB6, a member of DNAJ family of HSP, effectively prevented the aggregation of poly glutamines stretches associated with Huntington’s disease both in vitro and in cells. In addition, DNAJB6 was found recently to delay the aggregation of Aβ42 peptides in vitro. In the present study, we investigated the ability of DNAJB6 to prevent the aggregation of both intracellular and extracellular Aβ peptides using transfection of HEK293 cells with Aβ-GFP and recombinant Aβ42 peptides respectively. We performed western blotting and immunofluorescence techniques. We found that DNAJB6 can prevent Aβ-GFP aggregation, but not the seeded aggregation initiated by extracellular Aβ peptides. Moreover, DNAJB6 required interaction with HSP70 to prevent the aggregation of Aβ-GFP protein and its J-domain was essential for this anti-aggregation activity. Interestingly, overexpression of other DNAJ proteins as well as HSPB1 suppressed Aβ-GFP aggregation efficiently. Our findings suggest that DNAJB6 is a promising candidate for the inhibition of Aβ-GFP mediated aggregation through a canonical HSP70 dependent mechanism.

Keywords: Aβ, Alzheimer’s disease, chaperone, DNAJB6, aggregation

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Authors: Mohit Batra, Noel Sarkar, Jayeeta Mitra

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In this study silica nanoparticles were synthesized using different methods and different silica sources namely Tetraethyl ortho silicate (TEOS), Sodium Silicate, Rice husk while chitosan nanoparticles were prepared with ionic gelation method using Sodium tripolyphosphate (TPP). Size and texture of silica nanoparticles were studied using field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) along with the effect of change in concentration of various reagents in different synthesis processes. Size and dispersion of Silica nanoparticles prepared from TEOS using stobber’s method were found better than other methods while nanoparticles prepared using rice husk were cheaper than other ones. Catalyst found to play a very significant role in controlling the size of nanoparticles in all methods.

Keywords: silica nanoparticles, gelatin, bio-nanocomposites, SEM, TEM, chitosan

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Authors: A. Boveiri Monji, H. Yousefnia, M. Haji Hosseini, S. Zolghadri

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A clean spectrophotometric method for the determination of hafnium by using a green reagent, acidic extract of Ficus carica tree leaves is developed. In 6-M hydrochloric acid, hafnium reacts with this reagent to form a yellow product. The formed product shows maximum absorbance at 421 nm with a molar absorptivity value of 0.28 × 104 l mol⁻¹ cm⁻¹, and the method was linear in the 2-11 µg ml⁻¹ concentration range. The detection limit value was found to be 0.312 µg ml⁻¹. Except zirconium and iron, the selectivity was good, and most of the ions did not show any significant spectral interference at concentrations up to several hundred times. The proposed method was green, simple, low cost, and selective.

Keywords: spectrophotometric determination, Ficus caricatree leaves, synthetic reagents, hafnium

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Authors: Hilary Rutto

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Leaching with acids are the most commonly reagents used to remove copper ions from its copper ores. It is important that the process conditions are optimized to improve the leaching efficiency. In the present study the effects of pH, oxidizing agent (hydrogen peroxide), stirring speed, solid to liquid ratio and acid concentration on the leaching of copper ions from it ore were investigated using a pH Stat apparatus. Copper ions were analyzed at the end of each experiment using Atomic Absorption (AAS) machine. Results showed that leaching efficiency improved with an increase in acid concentration, stirring speed, oxidizing agent, pH and decreased with an increase in the solid to liquid ratio.

Keywords: leaching, copper, oxidizing agent, pH stat apparatus

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Authors: Fawzyah Albaldi

Abstract:

Urinary tract infections (UTIs) are the most prevalent of infectious diseases and > 80% are caused by uropathogenic E. coli (UPEC). Infection occurs following adhesion to urothelial plaques on bladder epithelial cells, whose major protein constituent are the uroplakins (UPs). Two of the four uroplakins (UPIa and UPIb) are members of the tetraspanin superfamily. The UPEC adhesin FimH is known to interact directly with UPIa. Tetraspanins are a diverse family of transmembrane proteins that generally act as “molecular organizers” by binding different proteins and lipids to form tetraspanin enriched microdomains (TEMs). Previous work by our group has shown that TEMs are involved in the adhesion of many pathogenic bacteria to human cells. Adhesion can be blocked by tetraspanin-derived synthetic peptides, suggesting that tetraspanins may be valuable drug targets. In this study, we investigate the role of tetraspanins in UPEC adherence to bladder epithelial cells. Human bladder cancer cell lines (T24, 5637, RT4), commonly used as in-vitro models to investigate UPEC infection, along with primary human bladder cells, were used in this project. The aim was to establish a model for UPEC adhesion/infection with the objective of evaluating the impact of tetraspanin-derived reagents on this process. Such reagents could reduce the progression of UTI, particularly in patients with indwelling catheters. Tetraspanin expression on the bladder cells was investigated by q-PCR and flow cytometry, with CD9 and CD81 generally highly expressed. Interestingly, despite these cell lines being used by other groups to investigate FimH antagonists, uroplakin proteins (UPIa, UPIb and UPIII) were poorly expressed at the cell surface, although some were present intracellularly. Attempts were made to differentiate the cell lines, to induce cell surface expression of these UPs, but these were largely unsuccessful. Pre-treatment of bladder epithelial cells with anti-CD9 monoclonal antibody significantly decreased UPEC infection, whilst anti-CD81 had no effects. A short (15aa) synthetic peptide corresponding to the large extracellular region (EC2) of CD9 also significantly reduced UPEC adherence. Furthermore, we demonstrated specific binding of that fluorescently tagged peptide to the cells. CD9 is known to associate with a number of heparan sulphate proteoglycans (HSPGs) that have also been implicated in bacterial adhesion. Here, we demonstrated that unfractionated heparin (UFH)and heparin analogs significantly inhibited UPEC adhesion to RT4 cells, as did pre-treatment of the cells with heparinases. Pre-treatment with chondroitin sulphate (CS) and chondroitinase also significantly decreased UPEC adherence to RT4 cells. This study may shed light on a common pathogenicity mechanism involving the organisation of HSPGs by tetraspanins. In summary, although we determined that the bladder cell lines were not suitable to investigate the role of uroplakins in UPEC adhesion, we demonstrated roles for CD9 and cell surface proteoglycans in this interaction. Agents that target these may be useful in treating/preventing UTIs.

Keywords: UTIs, tspan, uroplakins, CD9

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Authors: Sonia Kudlacik-Kramarczyk, Anna Drabczyk, Dagmara Malina, Bozena Tyliszczak, Agnieszka Sobczak-Kupiec

Abstract:

Introduction: In the preparation of polymer materials, compounds of natural origin are currently gaining more and more interest. This is particularly noticeable in the case of synthesis of materials considered for biomedical use. Then, selected material has to meet many requirements. It should be characterized by non-toxicity, biodegradability and biocompatibility. Therefore special attention is directed to substances such as polysaccharides, proteins or substances that are the basic building components of proteins, i.e. amino acids. These compounds may be crosslinked with other reagents that leads to the preparation of polymer matrices. Such amino acids as e.g. cysteine or histidine. On the other hand, previously mentioned requirements may be met by polymers obtained as a result of biosynthesis, e.g. polyhydroxybutyrate. This polymer belongs to the group of aliphatic polyesters that is synthesized by microorganisms (selected strain of bacteria) under specific conditions. It is possible to modify matrices based on given polymer with substances of various origin. Such a modification may result in the change of their properties or/and in providing the material with new features desirable in viewpoint of specific application. Described materials are synthesized using UV radiation. Process of photopolymerization is fast, waste-free and enables to obtain final products with favorable properties. Methodology: Polymer matrices have been prepared by means of photopolymerization. First step involved the preparation of solutions of particular reagents and mixing them in the appropriate ratio. Next, crosslinking agent and photoinitiator have been added to the reaction mixture and the whole was poured into the Petri dish and treated with UV radiation. After the synthesis, polymer samples were dried at room temperature and subjected to the numerous analyses aimed at the determining their physicochemical properties. Firstly, sorption properties of obtained polymer matrices have been determined. Next, mechanical properties have been characterized, i.e. tensile strength. The ability to deformation under applied stress of all prepared polymer matrices has been checked. Such a property is important in viewpoint of the application of analyzed materials e.g. as wound dressings. Wound dressings have to be elastic because depending on the location of the wound and its mobility, such a dressing has to adhere properly to the wound. Furthermore, considering the use of the materials for biomedical purposes it is essential to determine its behavior in environments simulating these ones occurring in human body. Therefore incubation studies using selected liquids have also been conducted. Conclusions: As a result of photopolymerization process, polymer matrices based on natural compounds have been prepared. These exhibited favorable mechanical properties and swelling ability. Moreover, biocompatibility in relation to simulated body fluids has been stated. Therefore it can be concluded that analyzed polymer matrices constitute an interesting materials that may be considered for biomedical use and may be subjected to the further more advanced analyses using specific cell lines.

Keywords: photopolymerization, polymer matrices, simulated body fluids, swelling properties

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Authors: Joanna Drzeżdżon

Abstract:

Polyvinyl alcohol and its derivatives such as 2-chloro-2-propen-1-ol have found application in many industries. They are mainly used for the production of adhesives, thickeners and stabilizers of emulsion paints, and surgical threads. Moreover, polyvinyl alcohol derivatives are indispensable reagents in the synthesis of hemiacetals. Polyolefins derived from polyvinyl alcohol are obtained by using catalysts belonging to complex compounds of transition metal ions. The aim of the studies is to the synthesis of a new complex, i.e. dipicolinate oxovanadium(IV) complex with 4,4’-dimethoxy-2,2’-bipyridyl, and to determine its catalytic activities. Moreover, the another aim of the studies was to set conditions for 2-chloro-2-propen-1-ol oligomerization. The catalytic system has been based on the dipicolinate complex of oxovanadium(IV) with 4,4’-dimethoxy-2,2’-bipyridyl and MMAO-12. The results of the studies showed that how a new oxovanadium(IV) complex compound effects on the 2-chloro-2-propen-1-ol oligomerization. Moreover, the results revealed that new catalytic material is a highly active catalyst for the investigated oligomerization.

Keywords: 2-chloro-2-propen-1-ol, oligomerization, dipicolinate, vanadium, methylaluminoxane

Procedia PDF Downloads 121

Authors: Yazan S. Khaled, Shazana Shamsuddin, Jim Tiernan, Mike McPherson, Thomas Hughes, Paul Millner, David G. Jayne

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Introduction: There is a need for real-time imaging of colorectal cancer (CRC) to allow tailored surgery to the disease stage. Fluorescence guided laparoscopic imaging of primary colorectal cancer and the draining lymphatics would potentially bring stratified surgery into clinical practice and realign future CRC management to the needs of patients. Fluorescent nanoparticles can offer many advantages in terms of intra-operative imaging and therapy (theranostic) in comparison with traditional soluble reagents. Nanoparticles can be functionalised with diverse reagents and then targeted to the correct tissue using an antibody or Affimer (artificial binding protein). We aimed to develop and test fluorescent silica nanoparticles and targeted against CRC using an anti-carcinoembryonic antigen (CEA) Affimer (Aff). Methods: Anti-CEA and control Myoglobin Affimer binders were subcloned into the expressing vector pET11 followed by transformation into BL21 Star™ (DE3) E.coli. The expression of Affimer binders was induced using 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells were harvested, lysed and purified using nickle chelating affinity chromatography. The photosensitiser Foslip (soluble analogue of 5,10,15,20-Tetra(m-hydroxyphenyl) chlorin) was incorporated into the core of silica nanoparticles using water-in-oil microemulsion technique. Anti-CEA or control Affs were conjugated to silica nanoparticles surface using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo SMCC) chemical linker. Binding of CEA-Aff or control nanoparticles to colorectal cancer cells (LoVo, LS174T and HC116) was quantified in vitro using confocal microscopy. Results: The molecular weights of the obtained band of Affimers were ~12.5KDa while the diameter of functionalised silica nanoparticles was ~80nm. CEA-Affimer targeted nanoparticles demonstrated 9.4, 5.8 and 2.5 fold greater fluorescence than control in, LoVo, LS174T and HCT116 cells respectively (p < 0.002) for the single slice analysis. A similar pattern of successful CEA-targeted fluorescence was observed in the maximum image projection analysis, with CEA-targeted nanoparticles demonstrating 4.1, 2.9 and 2.4 fold greater fluorescence than control particles in LoVo, LS174T, and HCT116 cells respectively (p < 0.0002). There was no significant difference in fluorescence for CEA-Affimer vs. CEA-Antibody targeted nanoparticles. Conclusion: We are the first to demonstrate that Foslip-doped silica nanoparticles conjugated to anti-CEA Affimers via SMCC allowed tumour cell-specific fluorescent targeting in vitro, and had shown sufficient promise to justify testing in an animal model of colorectal cancer. CEA-Affimer appears to be a suitable targeting molecule to replace CEA-Antibody. Targeted silica nanoparticles loaded with Foslip photosensitiser is now being optimised to drive photodynamic killing, via reactive oxygen generation.

Keywords: colorectal cancer, silica nanoparticles, Affimers, antibodies, imaging

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Authors: Mohamed E. Khalifa, Adil A. Gobouri

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Potential synthesis of a series of 3-amino-4-arylazothiophene derivatives from reaction of 2-cyano-2-phenylthiocarbamoyl acetamide and the appropriate α-halogenated reagents, followed by coupling with different aryl diazonium salts (Japp-Klingemann reaction), and another series of 5-arylazo-thiazol-2-ylcarbamoyl-thiophene derivatives from base-catalyzed intramolecular condensation of 5-arylazo-2-(N-chloroacetyl)amino-thiazole with selected B-keto compounds (Thorpe-Ziegler reaction) was performed. The biological activity of the two series was studied in vitro. Their versatility for pharmaceutical purposes was reported, where they displayed remarkable activities against selected pathogenic microorganisms; Bacillus subtilize, Staphylococcus aureus (Gram positive bacteria), Escherichia coli, Pseudomonas aeruginosa (Gram negative bacteria) and Aspergillus flavus, Candida albicans (fungi) with various degrees related to their chemical structures.

Keywords: thiophene, 2-aminothiazole, compounds, antioxidant, antitumor, antimicrobial

Procedia PDF Downloads 336

Authors: Rathnayaka D. D. T. , Vidanage P. W. , Wasalathilake K. C. , Wickramasingha H. W. , Wijayarathne U. P. L. , Perera S. A. S.

Abstract:

This paper describes the research carried out to develop a process to increase the NaCl percentage of crude salt which is obtained from the conventional solar evaporation process. In this study refined salt was produced from crude solar salt by a chemico-physical method which consists of coagulation, precipitation and filtration. Initially crude salt crystals were crushed and dissolved in water. Optimum amounts of calcium hydroxide, sodium carbonate and Poly Aluminium Chloride (PAC) were added to the solution respectively. Refined NaCl solution was separated out by a filtration process. The solution was tested for Total Suspended Solids, SO42-, Mg2+, Ca2+. With optimum dosage of reagents, the results showed that a level of 99.60% NaCl could be achieved. Further this paper discusses the economic viability of the proposed process. A 83% profit margin can be achieved by this process and it is an increase of 112.3% compared to the traditional process.

Keywords: chemico-physical, economic, optimum, refined, solar salt

Procedia PDF Downloads 241

Authors: Boukhiar Aissa, Iguergaziz Nadia, Halladj Fatima, Lamrani Yasmina, Benamara Salem

Abstract:

Determining the alcohol content in alcoholic fermentation bioprocess has a great importance. In fact, it is a key indicator for monitoring this fermentation bioprocess. Several methodologies (chemical, spectrophotometric, chromatographic...) are used to the determination of this parameter. However, these techniques are very long and require: rigorous preparations, sometimes dangerous chemical reagents, and/or expensive equipment. In the present study, the date juice is used as a substrate of alcoholic fermentation. The extracted juice undergoes an alcoholic fermentation by Saccharomyces cerevisiae. The study of the possible use of refractometry as a sole means for the in situ control of this process revealed a good correlation (R2 = 0.98) between initial and final ° Brix: ° Brix f = 0.377× ° Brixi. In addition, we verified the relationship between the variation in final and initial ° Brix (Δ ° Brix) and alcoholic rate produced (A exp): CΔ° Brix / A exp = 1.1. This allows the tracing of abacus isoresponses that permit to determine the alcoholic and residual sugar rates with a mean relative error (MRE) of 5.35%.

Keywords: refractometry, alcohol, residual sugar, fermentation, brix, date, juice

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Authors: Dipranjan Laha, Parimal Karmakar

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Metal oxide nanoparticles are well known to generate oxidative stress and deregulate normal cellular activities. Among these, transition metals copper oxide nanoparticles (CuO NPs) are more compelling than others and able to modulate different cellular responses. In this work, we have synthesized and characterized CuO NPs by various biophysical methods. These CuO NPs (~30 nm) induce autophagy in human breast cancer cell line, MCF7 in a time and dose-dependent manner. Cellular autophagy was tested by MDC staining, induction of green fluorescent protein light chain 3 (GFP-LC3B) foci by confocal microscopy, transfection of pBABE-puro mCherry-EGFP-LC3B plasmid and western blotting of autophagy marker proteins LC3B, beclin1, and ATG5. Further, inhibition of autophagy by 3-Methyladenine (3-MA) decreased LD50 doses of CuO NPs. Such cell death was associated with the induction of apoptosis as revealed by FACS analysis, cleavage of PARP, dephosphorylation of Bad and increased cleavage product of caspase3. siRNA-mediated inhibition of autophagy-related gene beclin1 also demonstrated similar results. Finally, induction of apoptosis by 3-MA in CuO NPs treated cells were observed by TEM. This study indicates that CuO NPs are a potent inducer of autophagy which may be a cellular defense against the CuO NPs mediated toxicity and inhibition of autophagy switches the cellular response into apoptosis. A combination of CuO NPs with the autophagy inhibitor is essential to induce apoptosis in breast cancer cells. Acknowledgments: The authors would like to acknowledge for financial support for this research work to the Department of Biotechnology (No. BT/PR14661/NNT/28/494/2010), Government of India.

Keywords: nanoparticle, autophagy, apoptosis, siRNA-mediated inhibition

Procedia PDF Downloads 431