Search results for: in vitro experiments

Authors: A. Kampker, K. Kreisköther, C. Reinders

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Since additive manufacturing technologies constantly advance, the use of this technology in mold making seems reasonable. Many manufacturers of additive manufacturing machines, however, do not offer any suggestions on how to parameterize the machine to achieve optimal results for mold making. The purpose of this research is to determine the interdependencies of different materials and parameters within the PolyJet process by using design of experiments (DoE), to additively manufacture molds, e.g. for thermoforming and injection molding applications. Therefore, the general requirements of thermoforming molds, such as heat resistance, surface quality and hardness, have been identified. Then, different materials and parameters of the PolyJet process, such as the orientation of the printed part, the layer thickness, the printing mode (matte or glossy), the distance between printed parts and the scaling of parts, have been examined. The multifactorial analysis covers the following properties of the printed samples: Tensile strength, tensile modulus, bending strength, elongation at break, surface quality, heat deflection temperature and surface hardness. The key objective of this research is that by joining the results from the DoE with the requirements of the mold making, optimal and tailored molds can be additively manufactured with the PolyJet process. These additively manufactured molds can then be used in prototyping processes, in process testing and in small to medium batch production.

Keywords: additive manufacturing, design of experiments, mold making, PolyJet, 3D-Printing

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Authors: Dan Yan, Yunuo Zhang, Yuhan Huang, Weijie Ouyang

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The cornea serves as a vital protective barrier for the eye; however, it is prone to injury and damage that can disrupt corneal epithelium and nerves, triggering inflammation. Therefore, understanding the biological effects and molecular mechanisms involved in corneal wound healing and identifying drugs targeting these pathways is crucial for researchers in this field. This study aimed to investigate the therapeutic potential of progranulin (PGRN) in treating corneal injuries. Our findings demonstrated that PGRN significantly enhanced corneal wound repair by accelerating corneal re-epithelialization and re-innervation. In vitro experiments with cultured epithelial cells and trigeminal ganglion cells further revealed that PGRN stimulated corneal epithelial cell proliferation and promoted axon growth in trigeminal ganglion cells. Through RNA-sequencing (RNA-seq) analysis and other experimental techniques, we discovered that PGRN exerted its healing effects by modulating the Wnt signaling pathway, which played a critical role in repairing epithelial cells and promoting axon regeneration in trigeminal neurons. Importantly, our study highlighted the anti-inflammatory properties of PGRN by inhibiting the NF-κB signaling pathway, leading to decreased infiltration of macrophages. In conclusion, our findings underscored the potential of PGRN in facilitating corneal wound healing by promoting corneal epithelial cell proliferation, trigeminal ganglion cell axon regeneration, and suppressing ocular inflammation. These results suggest that PGRN could potentially expedite the healing process and improve visual outcomes in patients with corneal injuries.

Keywords: cornea, wound healing, progranulin, corneal epithelial cells, trigeminal ganglion cells

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Authors: Brajesh Tiwari, Yuvraj Dangi, Abhishek Jain, Ashok Jain

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The purpose of the investigation was to evaluate the potential of sphingosomes as nanoscale drug delivery units for site-specific delivery of anti-cancer agents. Doxorubicin Hydrochloride (DOX) was selected as a model anti-cancer agent. Sphingosomes were prepared and loaded with DOX and optimized for size and drug loading. The formulations were characterized by Malvern zeta-seizer and Transmission Electron Microscopy (TEM) studies. Sphingosomal formulations were further evaluated for in-vitro drug release study under various pH profiles. The in-vitro drug release study showed an initial rapid release of the drug followed by a slow controlled release. In vivo studies of optimized formulations and free drug were performed on albino rats for comparison of drug plasma concentration. The in- vivo study revealed that the prepared system enabled DOX to have had enhanced circulation time, longer half-life and lower elimination rate kinetics as compared to free drug. Further, it can be interpreted that the formulation would selectively enter highly porous mass of tumor cells and at the same time spare normal tissues. To summarize, the use of sphingosomes as carriers of anti-cancer drugs may prove to be a fascinating approach that would selectively localize in the tumor mass, increasing the therapeutic margin of safety while reducing the side effects associated with anti-cancer agents.

Keywords: sphingosomes, anti-cancer, doxorubicin, formulation

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Authors: Khaled M. Khleifat

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Pseudomonas aeruginosa was isolated from infected burn patients and characterized by standard biochemical tests. The in vitro copper uptake was compared between this isolated pathogenic strain and two non-pathogenic control strains of Gram-positive bacteria Bacillusthuringiensis strain Israelisas well as Gram-negative bacteria Enterobacter aerogenes. Maximum copper uptake of 470 ppm/g biomass was obtained by P. aeruginosa strain, while the control strains B. thuringiensis and Enterobacter aerogenes had copper uptake of 350 and 383 ppm/g biomass, respectively. However, the lowest copper uptake (60 ppm/g biomass) was observed with another control the saprophytic strain Pseudomonas (Shewanella) putrefaciens. A further investigation regarding the effect of copper toxicity on bacterial growth, gave an MIC score of 600 ppm for P. aeruginosa strain compared to 460 and 300 ppm for the two Gram positive and Gram negative control strains, respectively. In tandem with these in vitro findings, blood analysis on burn patients infected with P. aeruginosa has indicated a selective decrease of copper (hypocupremia) and ceruloplasmin plasma levels. The iron metabolism was also affected by this copper deprivation leading to a similar decrease in plasma levels of PCV, iron, total iron-binding capacity, and transferrin. All these hematological changes were significantly different (P < 0.05) from the matched group of non-infected burn patients. The observed hypocupremia in infected burn patients was attributed to demanding scavenger ability by P. aeruginosa strain for the copper of plasma.

Keywords: Pseudomonas aeruginosa, hypocupremia, correlation, PCV

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Authors: Friday G. Okibe, Edit B. Agbaji, Victor O. Ajibola, Christain C. Onoyima

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Controlled drug delivery systems reduce dose-dependent toxicity associated with potent drugs, including anticancer drugs. In this research, hydroxyapatite (HA) and hydroxyapatite-sodium alginate nanocomposites (HASA) were successfully prepared and characterized using Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The FTIR result showed absorption peaks characteristics of pure hydroxyapatite (HA), and also confirmed the chemical interaction between hydroxyapatite and sodium alginate in the formation of the composite. Image analysis from SEM revealed nano-sized hydroxyapatite and hydroxyapatite-sodium alginate nanocomposites with irregular morphologies. Particle size increased with the formation of the nanocomposites relative to pure hydroxyapatite, with no significant change in particles morphologies. Drug loading and in-vitro drug release study were carried out using synthetic body fluid as the release medium, at pH 7.4 and 37 °C and under perfect sink conditions. The result shows that drug loading is highest for pure hydroxyapatite and decreased with increasing quantity of sodium alginate. However, the release study revealed that HASA-5%wt and HASA-20%wt presented better release profile than pure hydroxyapatite, while HASA-33%wt and HASA-50%wt have poor release profiles. This shows that Methotrexate-loaded hydroxyapatite-sodium alginate if prepared under optimal conditions is a potential carrier for effective delivery of Methotrexate.

Keywords: drug-delivery, hydroxyapatite, methotrexate, nanocomposites, sodium alginate

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Authors: Adria Rae Abigail R. Eda, Arvin C. Diesmos, Vance T. Vredenburg, Merab A. Chan

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Mitigating strategies using symbiotic cutaneous bacteria is one of the major concerns in the conservation of amphibian population. Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis associated with mass mortality and amphibian extinctions worldwide. In the Philippines, there is a lack of study on the cutaneous bacteria of Philippine amphibians that may have beneficial effects to ward off the deadly fungal infection. In this study, cutaneous bacteria from frogs were isolated and examined for anti-B. dendrobatidis activity. Eight species of frogs were collected at Mt. Palay-palay Mataas na Gulod National Park in Cavite, a site positive for the presence of B. dendrobatidis. Bacteria were isolated from the skin of frogs by swabbing the surfaces of the body and inoculated in Reasoner´s 2A (R2A) agar. Isolated bacteria were tested for potential inhibitory properties against B. dendrobatidis through zoospore inhibition assay. Results showed that frog cutaneous bacteria significantly inhibited the growth of B. dendrobatidis in vitro. By means of 16S rRNA gene primers, the anti-B. dendrobatidis bacteria were identified to be Enterobacter sp., Alcaligenes faecalis and Pseudomonas sp. Cutaneous bacteria namely Enterobacter sp. (isolates PLd33 and PCv4) and Pseudomonas (isolate PLd31) remarkably cleared the growth of B. dendrobatidis zoospore in 1% tryptone agar. Therefore, frog cutaneous bacteria inhibited B. dendrobatidis in vitro and could possibly contribute to the immunity and defense of frogs against the lethal chytridiomycosis.

Keywords: Batrachochytrium dendrobatidis, cutaneous bacteria, frogs, zoospore inhibition assay

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Authors: C. Müller, T. Ortmann, S. Scholl, H. J. Jördening

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Multienzymatic catalysis can be used as an alternative to chemical synthesis or hydrolysis of polysaccharides for the production of high value oligosaccharides from cheap resources such as sucrose. However, development of multienzymatic processes is complex, especially with respect to suitable conditions for enzymes originating from different organisms. Furthermore, an optimal configuration of the catalysts in a reaction cascade has to be found. These challenges can be approached by design of experiments. The system investigated in this study is a trienzymatic catalyzed reaction which results in laminaribiose production from sucrose and comprises covalently immobilized sucrose phosphorylase (SP), glucose isomerase (GI) and laminaribiose phosphorylase (LP). Operational windows determined with design of experiments and kinetic data of the enzymes were used to optimize the enzyme ratio for maximum product formation and minimal production of byproducts. After adjustment of the enzyme activity ratio to 1: 1.74: 2.23 (SP: LP: GI), different process options were investigated in silico. The considered options included substrate dependency, the use of glucose as co-substrate and substitution of glucose isomerase by glucose addition. Modeling of batch operation in a stirred tank reactor led to yields of 44.4% whereas operation in a continuous stirred tank reactor resulted in product yields of 22.5%. The maximum yield in a bienzymatic system comprised of sucrose phosphorylase and laminaribiose phosphorylase was 67.7% with sucrose and different amounts of glucose as substrate. The experimental data was in good compliance with the process model for batch operation. The continuous operation will be investigated in further studies. Simulation of operational process possibilities enabled us to compare various operational modes regarding different aspects such as cost efficiency, with the minimum amount of expensive and time-consuming practical experiments. This gives us more flexibility in process implementation and allows us, for example, to change the production goal from laminaribiose to higher oligosaccharides.

Keywords: design of experiments, enzyme kinetics, multi-enzymatic system, in silico process development

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Authors: Z. H. Shi, T. J. Dou, H. Zhang, H. X. Huang, N. Zeng

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The contamination of significant quantities of soils and sediments with uranium and other actinide elements as a result of nuclear activity poses many environmental risks. The electrokinetic process is one of the most promising remediation techniques for sludge, sediment, and saturated or unsaturated soils contaminated with heavy metals and radionuclides. However, secondary waste is a major concern for soil contaminated with nuclides. To minimize the generation of secondary wastes, this study used the anion and cation exchange membranes to improve the performance of the experimental apparatus. Remediation experiments of uranium-contaminated soil were performed with different agents. The results show that using acetic acid and EDTA as chelating agents clearly enhances the migration ability of the uranium. The ion exchange membranes (IEMs) used in the experiments not only reduce secondary wastes, but also, keep the soil pH stable.

Keywords: electrokinetic remediation, ion exchange membranes, soil, uranium

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Authors: Parnaz Boodagh, Danila Vella, Antonio Damore, Laura Modica De Mohac, Sang-Ho Ye, Garret Coyan, Gaetano Burriesci, William Wagner, Federica Cosentino

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The use of cardiac patches is among the main therapeutic solution for cardiovascular diseases, a leading mortality cause in the world with an increasing trend, responsible of 19 millions deaths in 2020. Several classes of biomaterials serve that purpose, both of synthetic origin and biological derivation, and many bioengineered treatment alternatives were proposed to satisfy two main requirements, providing structural support and promoting tissue remodeling. The objective of this paper is to compare the mechanical properties and the characterization of four cardiac patches: the Adeka, PhotoFix, CorPatch, and CardioCel patches. In vitro and in vivo tests included: biaxial, uniaxial, ball burst, suture retention for mechanical characterization; 2D surface topography, 3D volume and microstructure, and histology assessments for structure characterization; in vitro test to evaluate platelet deposition, calcium deposition, and macrophage polarization; rat right ventricular outflow tract (RVOT) models at 8- and 16-week time points to characterize the patch-host interaction. Lastly, the four patches were used to produce four stented aortic valve prosthesis, subjected to hydrodynamic assessment as well as durability testing to verify compliance with the standard ISO.

Keywords: cardiac patch, cardiovascular disease, cardiac repair, blood contact biomaterial

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Authors: Abdul Karim Zulkarnain, Marchaban, Subagus Wahyono, Ratna Asmah Susidarti

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Mahkota Dewa contains phalerin which has activity as sun screen. In this study, 13 formulations of cream oil in water (o/w) were prepared and tested for their physical characteristics. The physical characteristics were then used for determining the optimum formula. This study aimed to explore the physical stability of optimized formulation of cream, its sun protecting factor (SPF) values using in vitro and in vivo tests. The optimum formula of o/w cream were prepared based on Simplex Lattice Design (LSD) method using software Design Expert®. The formulation of o/w cream were varied based on the proportion of cetyl alcohol, mineral oil and tween 80. The difference of physical characteristic of optimum and predicted formula was tested using t-test with significant level of 95%. The optimum formula of o/w cream was the formula which consists of cetyl alcohol 9.71%, mineral oil, 29%, and tween 80 3.29. Based on t-test, there was no significant difference of physical characteristics of optimum and predicted formulation. Viscosity, spread power, adhesive power, and separation volume ratio of o/w at week 0-4 were relatively stable. The o/w creams were relatively stable at extreme temperature. The o/w creams from mahkota dewa, phalerin, and benzophenone have SPF values of 21.32, 33.12, and 42.49, respectively. The formulas did not irritate the skin based on in vivo test.

Keywords: cream, stability, In vitro, In vivo

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Authors: Katarzyna Drela, Miroslaw Wielgos, Mikolaj Wrobel, Barbara Lukomska

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Mesenchymal stem cells (MSCs) have a great potential in regenerative medicine. Since the initial number of isolated MSCs is limited, in vitro propagation is often required to reach sufficient numbers of cells for therapeutic applications. During long-term culture MSCs may undergo genetic or epigenetic alterations that subsequently increase the probability of spontaneous malignant transformation. Thus, factors that influence genomic stability of MSCs following long-term expansions need to be clarified before cultured MSCs are employed for clinical application. The aim of our study was to investigate the potential for spontaneous transformation of human neonatal cord blood (HUCB-MSCs) and adult bone marrow (BM-MSCs) derived MSCs. Materials and Methods: HUCB-MSCs and BM-MSCs were isolated by standard Ficoll gradient centrifugations method. Isolated cells were initially plated in high density 106 cells per cm2. After 48 h medium were changed and non-adherent cells were removed. The malignant transformation of MSCs in vitro was evaluated by morphological changes, proliferation rate, ability to enter cell senescence, the telomerase expression and chromosomal abnormality. Proliferation of MSCs was analyzed with WST-1 reduction method and population doubling time (PDT) was calculated at different culture stages. Then the expression pattern of genes characteristic for mesenchymal or epithelial cells, as well as transcriptions factors were examined by RT-PCR. Concomitantly, immunocytochemical analysis of gene-related proteins was employed. Results: Our studies showed that MSCs from all bone marrow isolations ultimately entered senescence and did not undergo spontaneous malignant transformation. However, HUCB-MSCs from one of the 15 donors displayed an increased proliferation rate, failed to enter senescence, and exhibited an altered cell morphology. In this sample we observed two different cell phenotypes: one mesenchymal-like exhibited spindle shaped morphology and express specific mesenchymal surface markers (CD73, CD90, CD105, CD166) with low proliferation rate, and the second one with round, densely package epithelial-like cells with significantly increased proliferation rate. The PDT of epithelial-like populations was around 1day and 100% of cells were positive for proliferation marker Ki-67. Moreover, HUCB-MSCs showed a positive expression of human telomerase reverse transcriptase (hTERT), cMYC and exhibit increased number of CFU during the long-term culture in vitro. Furthermore, karyotype analysis revealed chromosomal abnormalities including duplications. Conclusions: Our studies demonstrate that HUCB-MSCs are susceptible to spontaneous malignant transformation during long-term culture. Spontaneous malignant transformation process following in vitro culture has enormous effect on the biosafety issues of future cell-based therapies and regenerative medicine regimens.

Keywords: mesenchymal stem cells, spontaneous, transformation, long-term culture

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Authors: Hameed B. Mahood, Ali Sh. Baqir

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Vaporization of a single n-pentane drop in a direct contact with another flowing immiscible liquid (warm water) has been experimentally investigated. The experiments were carried out utilising a cylindrical Perspex tube of diameter 10 cm and height and 150 cm. Saturated liquid n-pentane and warm water at 45oC were used as the dispersed and continuous phases, respectively. Photron FASTCAM SA 1.1high speed camera (75,000f/s) with software V. 321 was implemented during the experiments. Five different continuous phase flow rates (warm water) (10, 20, 30, 40, and 46 L⁄h) were used in the study. The results indicated that the increase of the continuous phase (warm water) flow rate results in increasing of the drop/bubble diameter.

Keywords: drop evaporation, direct contact heat transfer, drop/bubble growth, experimental technique

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Authors: L. Mouzai, M. Bouhadef

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Rills begin to be generated once overland flow shear capacity overcomes the soil surface resistance. This resistance depends on soil texture, the arrangement of soil particles and on chemical and physical properties. The rill density could affect soil erosion, especially when the distance between the rills (interrill) contributes to the variation of the rill characteristics, and consequently on sediment concentration. To investigate this point, agricultural sandy soil, a soil tray of 0.2x1x3m³ and a piece of hardwood rectangular in shape to build up rills were the base of this work. The results have shown that small lines have been developed between the rills and the flow acceleration increased in comparison to the flow on the flat surface (interrill). Sediment concentration increased with increasing rill number (density).

Keywords: artificial rainfall, experiments, rills, soil erosion, transport capacity

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Authors: Liu Si, Wang Chunmei, Liu Huangmei

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A prominent component of the semantic versus pragmatic debate, scalar implicature (SI) has been gaining great attention ever since it was proposed by Horn. The constant debate is between the structural and pragmatic approach. The former claims that generation of SI is costless, automatic, and dependent mostly on the structural properties of sentences, whereas the latter advocates both that such generation is largely dependent upon context, and that the process is costly. Many experiments, among which Katsos’s text comprehension experiments are influential, have been designed and conducted in order to verify their views, but the results are not conclusive. Besides, most of the experiments were conducted in English language materials. Katsos conducted one off-line and three on-line text comprehension experiments, in which the previous shortcomings were addressed on a certain extent and the conclusion was in favor of the pragmatic approach. We intend to test the results of Katsos’s experiment in Chinese scalar implicature. Four experiments in both off-line and on-line conditions to examine the generation and response time of SI in Chinese "yixie" (some) and "quanbu (dou)" (all) will be conducted in order to find out whether the structural or the pragmatic approach could be sustained. The study mainly aims to answer the following questions: (1) Can SI be generated in the upper- and lower-bound contexts as Katsos confirmed when Chinese language materials are used in the experiment? (2) Can SI be first generated, then cancelled as default view claimed or can it not be generated in a neutral context when Chinese language materials are used in the experiment? (3) Is SI generation costless or costly in terms of processing resources? (4) In line with the SI generation process, what conclusion can be made about the cognitive processing model of language meaning? Is it a parallel model or a linear model? Or is it a dynamic and hierarchical model? According to previous theoretical debates and experimental conflicts, presumptions could be made that SI, in Chinese language, might be generated in the upper-bound contexts. Besides, the response time might be faster in upper-bound than that found in lower-bound context. SI generation in neutral context might be the slowest. At last, a conclusion would be made that the processing model of SI could not be verified by either absolute structural or pragmatic approaches. It is, rather, a dynamic and complex processing mechanism, in which the interaction of language forms, ad hoc context, mental context, background knowledge, speakers’ interaction, etc. are involved.

Keywords: cognitive linguistics, pragmatics, scalar implicture, experimental study, Chinese language

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Authors: Tamer El-Messery, Teresa Sanchez-Moya, Ruben Lopez-Nicolas, Gaspar Ros, Esmat Aly

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Olive leaves (OLs), the main by-product of the olive oil industry, have a considerable amount of phenolic compounds. The exploitation of these compounds represents the current trend in food processing. In this study, OLs polyphenols were microencapsulated with polycaprolactone (PCL) and utilized in formulating novel functional yoghurt. PCL-microcapsules were characterized by scanning electron microscopy, and Fourier transform infrared spectrometry analysis. Their total phenolic (TPC), total flavonoid (TFC) contents, and antioxidant activities (DPPH, FRAP, ABTS), and polyphenols bioaccessibility were measured after oral, gastric, and intestinal steps of in vitro digestion. The four yoghurt formulations (containing 0, 25, 50, and 75 mg of PCL-microsphere/100g yoghurt) were evaluated for their pH, acidity, syneresis viscosity, and color during storage. In vitro digestion significantly affected the phenolic composition in non-encapsulated extract while had a lower impact on encapsulated phenolics. Higher protection was provided for encapsulated OLs extract, and their higher release was observed at the intestinal phase. Yoghurt with PCL-microsphere had lower viscosity, syneresis, and color parameters, as compared to control yoghurt. Thus, OLs represent a valuable and cheap source of polyphenols which can be successfully applied, in microencapsulated form, to formulate functional yoghurt.

Keywords: yoghurt quality attributes, olive leaves, phenolic and flavonoids compounds, antioxidant activity, polycaprolactone as microencapsulant

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Authors: Vahid Changizi, Aram Rostami, Akbar Mosavi

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Purpose of study: Critical macromolecules of cells such as DNA are in exposure to damage of free radicals that induced from interaction of ionizing radiation with biological systems. Selenium and vitamin-E are natural compound that has been shown to be a direct free radical scavenger. The aim of this study was to investigate the in vivo/in vitro radioprotective effect of selenium and vitamin-E separately and synergistically against genotoxicity induced by 6MV x-rays irradiation in cultured blood lymphocytes from 15 human volunteers. Methods: Fifteen volunteers were divided in three groups include A, B and C. These groups were given slenium(800 IU), vitamin-E(100 mg) and selenium(400 IU) + vitamin-E(50 mg), respectively. Peripheral blood samples were collected from each group before(0 hr) and 1, 2 and 3 hr after selenium and vitamin-E administration (separately and synergistically). Then the blood samples were irradiated to 200 cGy of 6 Mv x-rays. After that, lymphocyte samples were cultured with mitogenic stimulation to determine the chromosomal aberrations wih micronucleus assay in cytokinesis-blocked binucleated cells. Results: The lymphocytes in the blood samples collected at 1 hr after ingestion selenium and vitamin-E, exposed in vitro to x-rays exhibited a significant decrease in the incidence of micronuclei, compared with control group at 0 hr. The maximum protection and decrease in frequency of micronuclei(50%) was observed at 1 hr after administration of selenium and vitamin-E synergistically. Conclusion: The data suggest that ingestion of selenium and vitamin-E as a radioprotector substances before exposures may reduce genetic damage caused by x-rays irradiation.

Keywords: x-rays, selenium, vitamin-e, lymphocyte, micronuclei

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Authors: Akbar Esmaeili, Zahra Salarieh

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Cerium oxide nanoparticles (nano-series) are used as catalysts in industrial applications due to their free radical scavenging properties. Given that free radicals play an essential role in the pathology of many neurological diseases, we investigated the use of nanocrystals as a potential therapeutic agent for oxidative damage. This project synthesized nano-series from a new and environmentally friendly bio-pathway. Investigation of cerium nitrate in culture medium containing inoculated Lactobacillus acidophilus strain before incubation produces nano-series. Loaded with glatiramer acetate (GA) was formed by coating carboxymethylcellulose (CMC) and CeO2. FE-SEM analysis showed nano-series in the 9-11 nm range, spherical shape, and uniform particle size distribution. Cubic nanoparticles containing anti-multiple sclerosis (anti-Ms) treatment called GA were used. Glycerol monostearate (GMS) was used as a fat base, and evening primrose extract was used as an anti-inflammatory in cubosomes. Design-Expert® software was used to study the effects of different formulation factors on the properties of GAloaded cubic dispersions. Thirty GA-labeled cubic dispersions were prepared with GA-labeled carboxymethylcellulose and evaluated in vitro. The results showed an average nano-series size of 89.02 and a zeta potential of -49.9. Cubosomes containing GA-CMC/CeO2 showed a stable release profile for 180 min. The results showed that cubosomes containing GA-CMC/CeO2 could be a promising drug carrier with normal release behavior.

Keywords: ciochemistry, biotechnology, molecular, biology

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Authors: Carlos Amnael Orozco-Diaz, Robert David Moorehead, Gwendolen Reilly, Fiona Gilchrist, Cheryl Ann Miller

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The current gold-standard for maxillofacial reconstruction surgery (MRS) utilizes auto-grafted cancellous bone as a filler. This study was aimed towards developing a polylactic-acid/hydroxyapatite (PLA-HA) composite suitable for fused-deposition 3D printing. Functionalization of the polymer through the addition of HA was directed to promoting bone-regeneration properties so that the material can rival the performance of cancellous bone grafts in terms of bone-lesion repair. This kind of composite enables the production of MRS implants based off 3D-reconstructions from image studies – namely computed tomography – for anatomically-correct fitting. The present study encompassed in-vitro degradation and in-vitro biocompatibility profiling for 3D-printed PLA and PLA-HA composites. PLA filament (Verbatim Co.) and Captal S hydroxyapatite micro-scale HA powder (Plasma Biotal Ltd) were used to produce PLA-HA composites at 5, 10, and 20%-by-weight HA concentration. These were extruded into 3D-printing filament, and processed in a BFB-3000 3D-Printer (3D Systems Co.) into tensile specimens, and were mechanically challenged as per ASTM D638-03. Furthermore, tensile specimens were subjected to accelerated degradation in phosphate-buffered saline solution at 70°C for 23 days, as per ISO-10993-13-2010. This included monitoring of mass loss (through dry-weighing), crystallinity (through thermogravimetric analysis/differential thermal analysis), molecular weight (through gel-permeation chromatography), and tensile strength. In-vitro biocompatibility analysis included cell-viability and extracellular matrix deposition, which were performed both on flat surfaces and on 3D-constructs – both produced through 3D-printing. Discs of 1 cm in diameter and cubic 3D-meshes of 1 cm3 were 3D printed in PLA and PLA-HA composites (n = 6). The samples were seeded with 5000 MG-63 osteosarcoma-like cells, with cell viability extrapolated throughout 21 days via resazurin reduction assays. As evidence of osteogenicity, collagen and calcium deposition were indirectly estimated through Sirius Red staining and Alizarin Red staining respectively. Results have shown that 3D printed PLA loses structural integrity as early as the first day of accelerated degradation, which was significantly faster than the literature suggests. This was reflected in the loss of tensile strength down to untestable brittleness. During degradation, mass loss, molecular weight, and crystallinity behaved similarly to results found in similar studies for PLA. All composite versions and pure PLA were found to perform equivalent to tissue-culture plastic (TCP) in supporting the seeded-cell population. Significant differences (p = 0.05) were found on collagen deposition for higher HA concentrations, with composite samples performing better than pure PLA and TCP. Additionally, per-cell-calcium deposition on the 3D-meshes was significantly lower when comparing 3D-meshes to discs of the same material (p = 0.05). These results support the idea that 3D-printable PLA-HA composites are a viable resorbable material for artificial grafts for bone-regeneration. Degradation data suggests that 3D-printing of these materials – as opposed to other manufacturing methods – might result in faster resorption than currently-used PLA implants.

Keywords: bone regeneration implants, 3D-printing, in vitro testing, biocompatibility, polymer degradation, polymer-ceramic composites

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Authors: Frank Kuebler, Rolf Steinhilper

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Artificial neural networks (ANN) as well as Design of Experiments (DOE) based regression analysis (RA) are mainly used for modeling of complex systems. Both methodologies are commonly applied in process and quality control of manufacturing processes. Due to the fact that resource efficiency has become a critical concern for manufacturing companies, these models needs to be extended to predict resource-consumption of manufacturing processes. This paper describes an approach to use neural networks as well as DOE based regression analysis for predicting resource consumption of manufacturing processes and gives a comparison of the achievable results based on an industrial case study of a turning process.

Keywords: artificial neural network, design of experiments, regression analysis, resource efficiency, manufacturing process

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Authors: Mike Wei, Nebu Koshy, Koen van Besien, Giorgio Inghirami, Steven M. Horwitz

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T-cell prolymphocytic leukemia (T-PLL) is a rare hematologic disease characterized by a T-cell phenotype, rapid progression, and poor prognosis with median survival of less than a year. Alemtuzumab-based chemotherapy has increased the rate of complete remissions but these are often short-lived, and allogeneic transplant is considered the only curative therapy. In recent studies, JAK3 activating mutations have been identified in T-cell cancers, with T-PLL having the highest rate of JAK3 mutations (30 – 42%). As such, T-PLL is a model disease for evaluating the utility of JAK3 inhibitors. We present a case of a 64-year-old man with relapsed-refractory T-PLL. He was initially treated with alemtuzumab and obtained complete response and was consolidated with matched unrelated donor stem cell transplant. His disease stayed in remission for approximately 1.5 years before relapse, which was then treated with a clinical trial of romidepsin-lenalidomide (partial responses then progression at 6 months) and later alemtuzumab. Due to complications of myelosuppression and CMV reactivation, his treatment was interrupted leading to disease progression. The doubling time of lymphocyte count was approximately 20 days and over a span of 60 days the lymphocyte count rose from 8 x 109/L to 68 x 109/L. Exon sequencing showed a JAK3 mutation. The patient consented to and was treated with FDA-approved tofacitinib (initially 5 mg BID, increased to 10 mg BID after 15 days of treatment). An initial decrease in lymphocyte count was followed by progression. In vitro treatment of the patient’s cells showed modest effects of tofacitinib and ruxolitinib as single agents, in the range of doxorubicin, but synergy between the agents. After 40 days of treatment with tofacitinib and with a lymphocyte count of 150 x 109/L, ruxolitinib (5mg BID) was added. Over the 60 days since dual inhibition was started, the lymphocyte count has stabilized. The patient has remained completely asymptomatic during treatment with tofacitinib and ruxolitinib. Neutrophil count has remained normal. Platelet count and hemoglobin have however declined from ~50 x109/L to ~30 x109/L and from 11 g/dL to 8.1 g/dL respectively, since the introduction of ruxolitinib. The stabilization in lymphocyte count confirms the clinical activity of JAK inhibitors in T-PLL as suggested by the presence of JAK3 mutations and by in-vitro assays. It also suggests clinical synergy between ruxolitinib and tofacitinib in this setting. Prospective studies of JAK inhibitors in PLL patients with formal dose-finding studies are needed.

Keywords: tofacitinib, ruxolitinib, T-cell prolymphocytic leukemia, JAK3

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Authors: Masoud Sakhinia, Sajjad Ahmad

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Introduction: Though knowledge of the compositional makeup and structure of the limbal niche has progressed exponentially during the past decade, much is yet to be understood. Identifying the precise profile and role of the stromal makeup which spans the ocular surface may inform researchers of the most optimum conditions needed to effectively expand LESCs in vitro, whilst preserving their differentiation status and phenotype. Limbal fibroblasts, as opposed to corneal fibroblasts are thought to form an important component of the microenvironment where LESCs reside. Methods: The corneal stroma was tissue engineered in vitro using both limbal and corneal fibroblasts embedded within a tissue engineered 3D collagen matrix. The effect of these two different fibroblasts on LESCs and hTCEpi corneal epithelial cell line were then subsequently determined using phase contrast microscopy, histolological analysis and PCR for specific stem cell markers. The study aimed to develop an in vitro model which could be used to determine whether limbal, as opposed to corneal fibroblasts, maintained the stem cell phenotype of LESCs and hTCEpi cell line. Results: Tissue culture analysis was inconclusive and required further quantitative analysis for remarks on cell proliferation within the varying stroma. Histological analysis of the tissue-engineered cornea showed a comparable structure to that of the human cornea, though with limited epithelial stratification. PCR results for epithelial cell markers of cells cultured on limbal fibroblasts showed reduced expression of CK3, a negative marker for LESC’s, whilst also exhibiting a relatively low expression level of P63, a marker for undifferentiated LESCs. Conclusion: We have shown the potential for the construction of a tissue engineered human cornea using a 3D collagen matrix and described some preliminary results in the analysis of the effects of varying stroma consisting of limbal and corneal fibroblasts, respectively, on the proliferation of stem cell phenotype of primary LESCs and hTCEpi corneal epithelial cells. Although no definitive marker exists to conclusively illustrate the presence of LESCs, the combination of positive and negative stem cell markers in our study were inconclusive. Though it is less traslational to the human corneal model, the use of conditioned medium from that of limbal and corneal fibroblasts may provide a more simple avenue. Moreover, combinations of extracellular matrices could be used as a surrogate in these culture models.

Keywords: cornea, Limbal Stem Cells, tissue engineering, PCR

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Authors: Priyanka Sharma, Niti Puri

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Anemia develops when blood lacks enough healthy erythrocytes. Past studies indicated that anemia, inflammatory process, and oxidative stress are interconnected. Erythrocytes are continuously exposed to reactive oxygen species (ROS) during circulation, due to normal aerobic cellular metabolism and also pathology of inflammatory diseases. Systemic mastocytosis and genetic depletion of mast cells have been shown to affect anaemia. In the present study, we attempted to reveal whether mast cells have a direct role in clearance or erythrophagocytosis of normal or oxidatively damaged erythrocytes. Murine erythrocytes were treated with tert-butyl hydroperoxidase (t-BHP), an agent that induces oxidative damage and mimics in vivo oxidative stress. Normal and oxidatively damaged erythrocytes were labeled with carboxyfluorescein succinimidyl ester (CFSE) to track erythrophagocytosis. We show, for the first time, direct erythrophagocytosis of oxidatively damaged erythrocytes in vitro by RBL-2H3 mast cells as well as in vivo by murine peritoneal mast cells. Also, activated mast cells, as may be present in inflammatory conditions, showed a significant increase in the uptake of oxidatively damaged erythrocytes than resting mast cells. This suggests the involvement of mast cells in erythrocyte clearance during oxidative stress or inflammatory disorders. Partial inhibition of phagocytosis by various inhibitors indicated that this process may be controlled by several pathways. Hence, our study provides important evidence for involvement of mast cells in severe anemia due to inflammation and oxidative stress and might be helpful to circumvent the adverse anemic disorders.

Keywords: mast cells, anemia, erythrophagocytosis, oxidatively damaged erythrocytes

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Authors: Jamila Fliou, Federica Spinola, Ouassima Riffi, Asmaa Zriouel, Ali Amechrouq, Luca Nalbone, Alessandro Giuffrida, Filippo Giarratana

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This study performed a preliminary evaluation of the phytochemical composition and in vitro antibacterial activity of ethanolic extracts of Lavandula x intermedia leaves and flowers collected in the Fez-Meknes region of Morocco. Phytochemical analyses comprised qualitative colourimetric determinations of alkaloids, anthraquinones, and terpenes and quantitative analysis of total polyphenols, flavonoids, and condensed tannins by UV spectrophotometer. Antibacterial activity was evaluated by determining minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against different ATCC bacterial strains. The phytochemical analysis showed a high amount of total polyphenols, flavonoids, and tannins in the leaf extract and a higher amount of terpenes based on colourimetric reaction than the flower extract. A positive colourimetric reaction for alkaloids and anthraquinones was detected for both extracts. The antibacterial activity of leaves and flower extract was not different against Gram-positive and Gram-negative strains (p<0.05). The results of the present study suggest the possible use of ethanolic extracts of L. x intermedia collected in the Fez-Meknes region of Morocco as a natural agent against bacterial pathogens.

Keywords: antimicrobial activity, Lavandula spp., lavender, lavandin, UV spectrophotometric analysis

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Authors: Michael Dare Asemoloye, Segun Gbolagade Jonathan, Rafiq Ahmad, Odunayo Joseph Olawuyi, D. O. Adejoye

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Fungi have potentials for degrading hydrocarbons through the secretion of different enzymes. Crude oil tolerance and degradation by Trichoderma harzianum was investigated in this study with its ability to produce peroxidase enzymes (LiP and MnP). Many fungal strains were isolated from rhizosphere of grasses growing on a crude oil spilled site, and the most frequent strain based on percentage incidence was further characterized using morphological and molecular characteristics. Molecular characterization was done through the amplification of Ribosomal-RNA regions of 18s (1609-1627) and 28s (287-266) using ITS1 and ITS4 combinations and it was identified using NCBI BLAST tool. The selected fungus was also subjected to an in-vitro tolerance test at crude oil concentrations of 5, 10, 15, 20 and 25% while 0% served as control. In addition, lignin peroxidase genes (lig1-6) and manganese peroxidase gene (mnp) were detected and expressed in this strain using RT-PCR technique, its peroxidase producing activities was also studied in aliquots (U/ml). This strain had highest incidence of 80%, it was registered in NCBI as Trichoderma harzianum asemoJ KY488466. The strain KY488466 responded to crude oil concentrations as it increase, the dose inhibition response percentage (DIRP) increased from 41.67 to 95.41 at 5 to 25 % crude oil concentrations. All the peroxidase genes are present in KY488466, and expressed with amplified 900-1000 bp through RT-PCR technique. In this strain, lig2, lig4 and mnp genes were over-expressed, lig 6 was moderately expressed, while none of the genes was under-expressed. The strain also produced 90±0.87 U/ml lignin peroxidase and 120±1.23 U/mil manganese peroxidase enzymes in aliquots. These results imply that KY488466 can tolerate and survive high crude oil concentration and could be exploited for bioremediation of oil-spilled soils, the produced peroxidase enzymes could also be exploited for other biotechnological experiments.

Keywords: crude oil, enzymes, expression, peroxidase genes, tolerance, Trichoderma harzianum

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Authors: E. Duffin, L. Brownlow

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Arson is a crime which can provide exceptional problems to forensic specialists. Its destructive nature makes evidence much harder to find, especially when used to cover up another crime. There is a consistent potential threat of arsonists seeking new and easier ways to set fires. Existing research in this field primarily focuses on the use of accelerants such as petrol, with less attention to other more accessible and harder to detect materials. This includes the growing speculation of potato and maize-based snacks being used as fire accelerants. It was hypothesized that all ‘crisp-type’ snacks in foil packaging had the potential to act as accelerants and would burn readily in the various experiments. To test this hypothesis, a series of small lab-based experiments were undertaken, igniting samples of the snacks. Factors such as ingredients, shape, packaging and calorific value were all taken into consideration. The time (in seconds) spent on fire by the individual snacks was recorded. It was found that all of the snacks tested burnt for statistically similar amounts of time with a p-value of 0.0157. This was followed with a large mock real-life scenario using packets of crisps on fire and car seats to investigate as to the possibility of these snacks being verifiable tools to the arsonist. Here, three full packets of crisps were selected based on variations in burning during the lab experiments. They were each lit with a lighter to initiate burning, then placed onto a car seat to be timed and observed with video cameras. In all three cases, the fire was significant and sustained by the 200-second mark. On the basis of this data, it was concluded that potato and maize-based snacks were viable accelerants of fire. They remain an effective method of starting fires whilst being cheap, accessible, non-suspicious and non-detectable. The results produced supported the hypothesis that all ‘crisp-type’ snacks in foil packaging (that had been tested) had the potential to act as accelerants and would burn readily in the various experiments. This study serves to raise awareness and provide a basis for research and prevention of arson regarding maize and potato-based snacks as fire accelerants.

Keywords: arson, crisps, fires, food

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Authors: Lela Pintarić, Iva Rezić, Ana Vrsalović Presečki

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Considering an enormous increase of the use of metal nanoparticles with the exactly defined characteristics, the main goal of this research was to found the optimal and environmental friendly method of their synthesis. The synthesis of the inorganic core-shell nanoparticles was optimized as a model. The core-shell nanoparticles are composed of the enzyme core belted with the metal ions, oxides or salts as a shell. In this research, enzyme urease was the core catalyst and the shell nanoparticle was made of silver. Silver nanoparticles are widespread utilized and some of their common uses are: as an addition to disinfectants to ensure an aseptic environment for the patients, as a surface coating for neurosurgical shunts and venous catheters, as an addition to implants, in production of socks for diabetics and athletic clothing where they improve antibacterial characteristics, etc. Characteristics of synthesized nanoparticles directly depend on of their size, so the special care during this optimization was given to the determination of the size of the synthesized nanoparticles. For the purpose of the above mentioned optimization, sixteen experiments were generated by the Design of Experiments (DoE) method and conducted under various temperatures, with different initial concentration of the silver nitrate and constant concentration of the urease of two separate manufacturers. Synthesized nanoparticles were analyzed by the Nanoparticle Tracking Analysis (NTA) method on Malvern NanoSight NS300. Results showed that the initial concentration of the silver ions does not affect the concentration of the synthesized silver nanoparticles neither their size distribution. On the other hand, temperature of the experiments has affected both of the mentioned values.

Keywords: core-shell nanoparticles, optimization, silver, urease

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Authors: Nélio Veiga, Paula Ferreira, Tiago Correia, Maria J. Correia, Carlos Pereira, Odete Amaral, Ilídio J. Correia

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Introduction: The application of fissure sealants is considered to be an important primary prevention method used in dental medicine. However, the formation of microleakage gaps between tooth enamel and the fissure sealant applied is one of the most common reasons of dental caries development in teeth with fissure sealants. The association between various dental biomaterials may limit the major disadvantages and limitations of biomaterials functioning in a complementary manner. The present study consists in the incorporation of a cariostatic agent – silver diamine fluoride (SDF) – in a resin-based fissure sealant followed by the study of release kinetics by spectrophotometry analysis of the association between both biomaterials and assessment of the inhibitory effect on the growth of the reference bacterial strain Streptococcus mutans (S. mutans) in an in vitro study. Materials and Methods: An experimental in vitro study was designed consisting in the entrapment of SDF (Cariestop^® 12% and 30%) into a commercially available fissure sealant (Fissurit^®), by photopolymerization and photocrosslinking. The same sealant, without SDF was used as a negative control. The effect of the sealants on the growth of S. mutans was determined by the presence of bacterial inhibitory halos in the cultures at the end of the incubation period. In order to confirm the absence of bacteria in the surface of the materials, Scanning Electron Microscopy (SEM) characterization was performed. Also, to analyze the release profile of SDF along time, spectrophotometry technique was applied. Results: The obtained results indicate that the association of SDF to a resin-based fissure sealant may be able to increase the inhibition of S. mutans growth. However, no SDF release was noticed during the in vitro release studies and no statistical significant difference was verified when comparing the inhibitory halo sizes obtained for test and control group.  Conclusions: In this study, the entrapment of SDF in the resin-based fissure sealant did not potentiate the antibacterial effect of the fissure sealant or avoid the immediate development of dental caries. The development of more laboratorial research and, afterwards, long-term clinical data are necessary in order to verify if this association between these biomaterials is effective and can be considered for being used in oral health management. Also, other methodologies for associating cariostatic agents and sealant should be addressed.

Keywords: biomaterial, fissure sealant, primary prevention, silver diamine fluoride

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Authors: Khaled Khleifat, Muayyad Abboud, Amjad Khleifat, Humodi Saeed

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In this study, Pseudomonas aeruginosa was isolated from infected burn patients and characterized by standard biochemical tests. The in vitro copper uptake was compared between this isolated pathogenic strain and two non-pathogenic control strains of Gram positive bacteria Bacillusthuringiensis strain Israelisas well as Gram negative bacteria Enterobacter aerogenes. Maximum copper uptake of 470 ppm/g biomass was obtained by P. aeruginosa strain, while the control strains B. thuringiensis andEnterobacter aerogenes had copper uptake of 350 and 383 ppm/g biomass, respectively. However, the lowest copper uptake (60 ppm/g biomass) was observed with another control the saprophytic strain Pseudomonas (Shewanella) putrefaciens. A further investigation regarding the effect of copper toxicity on bacterial growth, gave an MIC score of 600 ppm for P. aeruginosa strain compared to 460 and 300 ppm for the two Gram positive and Gram negative control strains, respectively. In tandem with these in vitro findings, blood analysis on burn patients infected with P. aeruginosa has indicated a selective decrease of copper (hypocupremia) and ceruloplasmin plasma levels. The iron metabolism was also affected by this copper deprivation leading to a similar decrease in plasma levels of PCV, iron, total iron binding capacity, and transferrin. All these hematological changes were significantly different (P < 0.05) from the matched group of non-infected burn patients. The observed hypocupremia in infected burn patients was attributed to demanding scavenger ability by P. aeruginosa strain for the copper of plasma.

Keywords: pseudomonas, Cu uptake, burn patients, biosorption

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Authors: Payam Rasoulnia, Seyyed Mohammad Mousavi

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Heavy fuel oil firing power plants produce huge amounts of ashes as solid wastes, which seriously need to be managed and processed. Recycling precious metals of V and Ni from these oil-fired ashes which are considered as secondary sources of metals recovery, not only has a great economic importance for use in industry, but also it is noteworthy from the environmental point of view. Vanadium is an important metal that is mainly used in the steel industry because of its physical properties of hardness, tensile strength, and fatigue resistance. It is also utilized in oxidation catalysts, titanium–aluminum alloys and vanadium redox batteries. In the present study bioleaching of vanadium and nickel from an oil-fired ash sample was conducted using Aspergillus niger fungus. The experiments were carried out using spent-medium bioleaching method in both Erlenmeyer flasks and also bubble column bioreactor, in order to compare them together. In spent-medium bioleaching the solid waste is not in direct contact with the fungus and consequently the fungal growth is not retarded and maximum organic acids are produced. In this method the metals are leached through biogenic produced organic acids present in the medium. In shake flask experiments the fungus was cultured for 15 days, where the maximum production of organic acids was observed, while in bubble column bioreactor experiments a 7 days fermentation period was applied. The amount of produced organic acids were measured using high performance liquid chromatography (HPLC) and the results showed that depending on the fermentation period and the scale of experiments, the fungus has different major lixiviants. In flask tests, citric acid was the main produced organic acid by the fungus and the other organic acids including gluconic, oxalic, and malic were excreted in much lower concentrations, while in the bioreactor oxalic acid was the main lixiviant and it was produced considerably. In Erlenmeyer flasks during 15 days fermentation of Aspergillus niger, 8080 ppm citric acid and 1170 ppm oxalic acid was produced, while in bubble column bioreactor over 7 days of fungal growth, 17185 ppm oxalic acid and 1040 ppm citric acid was secreted. The leaching tests using the spent-media obtained from both of fermentation experiments, were performed at the same conditions of leaching duration of 7 days, leaching temperature of 60 °C and pulp density up to 3% (w/v). The results revealed that in Erlenmeyer flask experiments 97% of V and 50% of Ni were extracted while using spent medium produced in bubble column bioreactor, V and Ni recoveries were achieved to 100% and 33%, respectively. These recovery yields indicate that in both scales almost total vanadium can be recovered, while nickel recovery was lower. With help of the bioreactor spent-medium nickel recovery yield was lower than that of obtained from the flask experiments, which it could be due to precipitation of some values of Ni in presence of high levels of oxalic acid existing in its spent medium.

Keywords: Aspergillus niger, bubble column bioreactor, oil-fired ash, spent-medium bioleaching

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Authors: Atinderpal Kaur, Shweta Dang

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Our aim is to develop a nanoemulsion-based delivery system containing polyphenon 60 (P60) and ciprofloxacin (Cipro) for intravaginal delivery to treat urinary tract infection. In the present study Polyphenon 60 (P60) and ciprofloxacin (Cipro) were loaded in a single nano emulsion (NE) system via ultra-sonication technique and characterized for particle size, in vitro release and antibacterial efficacy against Bcl-2 level Escherichia coli bacteria. To determine in vivo pharmacokinetic parameters and intravaginal transportation of NE, gamma scintigraphy and biodistribution study was conducted by radiolabelling NE with technetium pertechnetate (99mTc). The preliminary antibacterial investigation showed synergy between these compounds with FICindex of 0.42. The developed formulation showed zeta potential +55.3 and particle size of 151.7 nm, with PDI of 0.196. The in vitro release percentage of P60 at the end of 7th hours was 94.8 ± 0.9 % whereas the release for Cipro was 75.1± 0.15 % in simulated vaginal media. MBC was identified and the findings demonstrated that in both ESBL (Extended Spectrum β- lactamase) and MBL (Metallo β- lactamase) cultures the P60+Cipro NE showed inhibition of growth of all the isolates at 2 mg/ml dilutions. The percentage per gram of radiolabelled drug was found (3.50±0.26) and (3.81±0.30) in kidney and urinary bladder, respectively at 3 h. From the findings, it was concluded that the developed P60+Cipro NE was transported efficiently throughout the target organs, had long duration of action and high biocompatibility via intravaginal administration as compared to oral administration.

Keywords: ciprofloxacin, gamma scintigraphy, intravaginal drug delivery, Polyphenon 60

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