Search results for: isolation of agent

Authors: Sahar Rakhshan, Simonetta Geninatti Crich, Diego Alberti, Rachele Stefania

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The combination of imaging and therapeutic agents in the same smart nanoparticle is a promising option to perform a minimally invasive imaging guided therapy. In this study, Low density lipoproteins (LDL), one of the most attractive biodegradable and biocompatible nanoparticles, were used for the simultaneous delivery of Paclitaxel (PTX), a hydrophobic antitumour drug and an amphiphilic contrast agent, Gd-AAZTA-C17, in B16-F10 melanoma cell line. These cells overexpress LDL receptors, as assessed by Flow cytometry analysis. PTX and Gd-AAZTA-C17 loaded LDLs (LDL-PTX-Gd) have been prepared, characterized and their stability was assessed under 72 h incubation at 37 ◦C and compared to LDL loaded with Gd-AAZTA-C17 (LDL-Gd) and LDL-PTX. The cytotoxic effect of LDL-PTX-Gd was evaluated by MTT assay. The anti-tumour drug loaded into LDLs showed a significantly higher toxicity on B16-F10 cells with respect to the commercially available formulation Paclitaxel Kabi (PTX Kabi) used in clinical applications. It was possible to demonstrate a high uptake of LDL-Gd in B16-F10 cells. As a consequence of the high cell uptake, melanoma cells showed significantly high cytotoxic effect when incubated in the presence of PTX (LDL-PTX-Gd). Furthermore, B16-F10 have been used to perform Magnetic Resonance Imaging. By the analysis of the image signal intensity, it was possible to extrapolate the amount of internalized PTX indirectly by the decrease of relaxation times caused by Gd, proportional to its concentration. Finally, the treatment with PTX loaded LDL on B16-F10 tumour bearing mice resulted in a marked reduction of tumour growth compared to the administration of PTX Kabi alone. In conclusion, LDLs are selectively taken-up by tumour cells and can be successfully exploited for the selective delivery of Paclitaxel and imaging agents.

Keywords: low density lipoprotein, melanoma cell lines, MRI, paclitaxel, personalized medicine application, theragnostic System

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Authors: Shahida Mohd Sharif, Norsidah Ujang

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Recent research has shown that many of the urban population in Kuala Lumpur, especially from the lower-income group, suffer from socio-psychological problems. They are reported as experiencing anxiety, depression, and stress, which is made worst by the recent COVID-19 pandemic. Much of the population was forced to observe the Movement Control Order (MCO), which is part of pandemic mitigation measures, pushing them to live in isolation as the new normal. The study finds the need to strategize for a better approach to help these people coping with the socio-psychological condition, especially the population from the lower-income group. In Kuala Lumpur, as part of the Local Agenda 21 programme, the Kuala Lumpur City Hall has introduced Green Initiative: Urban Farming, which among the approaches is the community garden. The local authority promotes the engagement to be capable of improving the social environment of the participants. Research has demonstrated that social interaction within community gardens can help the members improve their socio-psychological conditions. Therefore, the study explores the residents’ experience from low-cost flats participating in the community gardening initiative from a social attachment perspective. The study will utilise semi-structured interviews to collect the participants’ experience with community gardening and how the social interaction exchange between the members' forms and develop their ties and trust. For a context, the low-cost flats are part of the government social housing program (Program Perumahan Rakyat dan Perumahan Awam). Meanwhile, the community gardening initiative (Projek Kebun Kejiranan Bandar LA21 KL) is part of the local authority initiative to address the participants’ social, environmental, and economic issues. The study will conduct thematic analysis on the collected data and use the ATLAS.ti software for data organization and management purposes. The findings could help other researchers and stakeholders understand the social interaction experience within community gardens and its relation to ties and trusts. The findings could shed some light on how the participants could improve their social environment, and its report could provide the local authority with evidence-based documentation.

Keywords: community gardening participation, lower-income population, social attachment, social interaction

Procedia PDF Downloads 134

Authors: Thanusha D. Abeywickrama, Inoka C. Perera, Genji Kurisu

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Tuberculosis, considered being as the ninth leading cause of death worldwide, cause from a single infectious agent M. tuberculosis and the drug resistance nature of this bacterium is a continuing threat to the world. Therefore TB preventing treatment is expanding, where this study designed to analyze the regulatory mechanism of GntR transcriptional regulator gene Rv0792c, which lie between several genes codes for some hypothetical proteins, a monooxygenase and an oxidoreductase. The gene encoding Rv0792c was cloned into pET28a and expressed protein was purified to near homogeneity by Nickel affinity chromatography. It was previously reported that the protein binds within the intergenic region (BS region) between Rv0792c gene and monooxygenase (Rv0793). This resulted in binding of three protein molecules with the BS region suggesting tight control of monooxygenase as well as its own gene. Since monooxygenase plays a key role in metabolism, this gene may have a global regulatory role. The natural ligand for this regulator is still under investigation. In relation to the Rv0792 protein structure, a Circular Dichroism (CD) spectrum was carried out to determine its secondary structure elements. Percentage-wise, 17.4% Helix, 21.8% Antiparallel, 5.1% Parallel, 12.3% turn and 43.5% other were revealed from CD spectrum data under room temperature. Differential Scanning Calorimetry (DSC) was conducted to assess the thermal stability of Rv0792, which the melting temperature of protein is 57.2 ± 0.6 °C. The graph of heat capacity (Cp) versus temperature for the best fit was obtained for non-two-state model, which concludes the folding of Rv0792 protein occurs through stable intermediates. Peak area (∆HCal ) and Peak shape (∆HVant ) was calculated from the graph and ∆HCal / ∆HVant was close to 0.5, suggesting dimeric nature of the protein.

Keywords: CD spectrum, DSC analysis, GntR transcriptional regulator, protein structure

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Authors: Hafiz Husnain Nawaz

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Anthracnose disease in olive, caused by the fungal pathogen Colletotrichum acutatum, is considered one of the most critical issues in olive orchards in Pakistan. This disease poses a significant threat as it results in infections that can lead to the complete damage of olive plants, affecting leaves, stems, and fruits in the field. Controlling this disease is particularly challenging due to the absence of an effective fungicide that does not pose risks to farmer health and the environment. To address this challenge, our study aimed to evaluate the antagonistic activity of a biosurfactant produced by the Bacillus subtilis PE-07 strain against the anthracnose-causing agent in olive plants. This strain was selected after screening sixty rhizobacteria strains. Additionally, we assessed the heat stability, pH range, and toxicity of the biosurfactant produced by strain PE-07. Our results revealed that the biosurfactant exhibited maximum antifungal activity against C. acutatum. In vitro studies indicated that the biosurfactant could reduce fungal activity by inhibiting the spore germination of C. acutatum. Furthermore, the biosurfactant demonstrated a wide pH and temperature range, displaying antifungal activity at pH levels ranging from 5 to 10 and a temperature range from room temperature to 110°C. To evaluate the biosurfactant's safety, we conducted toxicity tests on zebra fish (Danio rerio). The results showed that the biosurfactant had minimal harmful effects, even at maximum concentrations. In conclusion, our study confirmed that the biosurfactant produced by B. subtilis exhibited high pH and heat stability with minimal harmful effects. Therefore, it presents a promising alternative to chemical pesticides for effectively controlling olive anthracnose in Pakistan.

Keywords: biological control, heat stability and PH range, toxicity, Danio rerio

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Authors: Khadijat O. Abdulwahab, Mohammad A. Malik, Paul O’Brien, Grigore A. Timco, Floriana Tuna

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The synthesis of spinel ferrite nanoparticles with a narrow size distribution is very crucial in their numerous applications including information storage, hyperthermia treatment, drug delivery, contrast agent in magnetic resonance imaging, catalysis, sensors, and environmental remediation. Ferrites have the general formula MFe2O4 (M = Fe, Co, Mn, Ni, Zn etc.) and possess remarkable electrical and magnetic properties which depend on the cations, method of preparation, size and their site occupancies. To the best of our knowledge, there are no reports on the use of a single source precursor to synthesise quaternary ferrite nanoparticles. Herein, we demonstrated the use of trimetallic iron pivalate cluster [CrCoFeO(O2CtBu)6(HO2CtBu)3] as a single source precursor to synthesise monodisperse cobalt chromium ferrite (FeCoCrO4) nanoparticles by the hot injection thermolysis method. The precursor was thermolysed in oleylamine, oleic acid, with diphenyl ether as solvent at its boiling point (260°C). The effect of concentration on the stoichiometry, phases or morphology of the nanoparticles was studied. The p-XRD patterns of the nanoparticles obtained at both concentrations were matched with cubic iron cobalt chromium ferrite (FeCoCrO4). TEM showed that a more monodispersed spherical ferrite nanoparticles of average diameter 4.0 ± 0.4 nm were obtained at higher precursor concentration. Magnetic measurements revealed that all the ferrite particles are superparamagnetic at room temperature. The nanoparticles were characterised by Powder X-ray Diffraction (p-XRD), Transmission Electron Microscopy (TEM), Inductively Coupled Plasma (ICP), Electron Probe Microanalysis (EPMA), Energy Dispersive Spectroscopy (EDS) and Super Conducting Quantum Interference Device (SQUID).

Keywords: quaternary ferrite nanoparticles, single source precursor, monodisperse, cobalt chromium ferrite, colloidal, hot injection thermolysis

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Authors: Eduardo Kanagushiku Pereira, Frank Gregory Cavalcante da Silva, Barbara Soares Gonçalves, Ana Lúcia Bergamasco Galastri, Ronei Luciano Mamoni

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The inflammatory response initiates after the recognition of pathogens by receptors expressed by innate immune cells. Among these receptors, the NLRP3 was associated with the recognition of pathogenic fungi in experimental models. NLRP3 operates forming a multiproteic complex called inflammasome, which actives caspase-1, responsible for the production of the inflammatory cytokines IL-1beta and IL-18. In this study, we aimed to investigate the involvement of NLRP3 in the inflammatory response elicited in macrophages against Paracoccidioides brasiliensis (Pb), the etiologic agent of PCM. Macrophages were differentiated from THP-1 cells by treatment with phorbol-myristate-acetate. Following differentiation, macrophages were stimulated by Pb yeast cells for 24 hours, after previous treatment with specific NLRP3 (3,4-methylenedioxy-beta-nitrostyrene) and/or caspase-1 (VX-765) inhibitors, or specific inhibitors of pathways involved in NLRP3 activation such as: Reactive Oxigen Species (ROS) production (N-Acetyl-L-cysteine), K+ efflux (Glibenclamide) or phagossome acidification (Bafilomycin). Quantification of IL-1beta and IL-18 in supernatants was performed by ELISA. Our results showed that the production of IL-1beta and IL-18 by THP-1-derived-macrophages stimulated with Pb yeast cells was dependent on NLRP3 and caspase-1 activation, once the presence of their specific inhibitors diminished the production of these cytokines. Furthermore, we found that the major pathways involved in NLRP3 activation, after Pb recognition, were dependent on ROS production and K+ efflux. In conclusion, our results showed that NLRP3 participates in the recognition of Pb yeast cells by macrophages, leading to the activation of the NLRP3-inflammasome and production of IL-1beta and IL-18. Together, these cytokines can induce an inflammatory response against P. brasiliensis, essential for the establishment of the initial inflammatory response and for the development of the subsequent acquired immune response.

Keywords: inflammation, IL-1beta, IL-18, NLRP3, Paracoccidioidomycosis

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Authors: Ubaid Ur Rahman, Waqar Younas, Sooraj Kumar Chhabira

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Environmentally sustainable building construction has experienced significant growth during the past 10 years at international level. This paper shows that the conceptual framework adopts sustainability techniques in construction to develop environment friendly building called green building. Waste occurs during the different construction phases which causes the environmental problems like, deposition of waste on ground surface creates major problems such as bad smell. It also gives birth to different health diseases and produces toxic waste agent which is specifically responsible for making soil infertile. Old recycled building material is used in the construction of new building. Sustainable construction is economical and saves energy sources. Sustainable construction is the major responsibility of designer and project manager. The designer has to fulfil the client demands while keeping the design environment friendly. Project manager has to deliver and execute sustainable construction according to sustainable design. Steel is the most appropriate sustainable construction material. It is more durable and easily recyclable. Steel occupies less area and has more tensile and compressive strength than concrete, making it a better option for sustainable construction as compared to other building materials. New technology like green roof has made the environment pleasant, and has reduced the construction cost. It minimizes economic, social and environmental issues. This paper presents an overview of research related to the material use of green building and by using this research recommendation are made which can be followed in the construction industry. In this paper, we go through detailed analysis on construction material. By making suitable adjustments to project management practices it is shown that a green building improves the cost efficiency of the project, makes it environmental friendly and also meets future generation demands.

Keywords: sustainable construction, green building, recycled waste material, environment

Procedia PDF Downloads 240

Authors: Hind Bouami

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Handling life-critical systems complexity requires to be equipped with appropriate technology and the right human agents’ functions such as knowledge, experience, and competence in problem’s prevention and solving. Human agents are involved in the management and control of human-machine system’s performance. Documenting human agent’s situation awareness is crucial to support human-machine designers’ decision-making. Knowledge about risks, critical parameters and factors that can impact and threaten automation system’s performance should be collected using preventive and retrospective approaches. This paper aims to document operators’ situation awareness through the analysis of automated organizations’ feedback. The analysis of automated hospital pharmacies feedbacks helps to identify and control critical parameters influencing human machine interaction in order to enhance system’s performance and security. Our human machine system evaluation approach has been deployed in Macon hospital center’s pharmacy which is equipped with automated drug dispensing systems since 2015. Automation’s specifications are related to technical aspects, human-machine interaction, and human aspects. The evaluation of drug delivery automation performance in Macon hospital center has shown that the performance of the automated activity depends on the performance of the automated solution chosen, and also on the control of systemic factors. In fact, 80.95% of automation specification related to the chosen Sinteco’s automated solution is met. The performance of the chosen automated solution is involved in 28.38% of automation specifications performance in Macon hospital center. The remaining systemic parameters involved in automation specifications performance need to be controlled.

Keywords: life-critical systems, situation awareness, human-machine interaction, decision-making

Procedia PDF Downloads 179

Authors: Mailin Misson, Bo Jin, Sheng Dai, Hu Zhang

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Advances in biotechnology have witnessed a growing interest in enzyme applications for the development of green and sustainable bio processes. While known as powerful bio catalysts, enzymes are no longer of economic value when extended to large commercialization. Alternatively, immobilization technology allows enzyme recovery and continuous reuse which subsequently compensates high operating costs. Employment of enzymes on nano structured materials has been recognized as a promising approach to enhance enzyme catalytic performances. High porosity, inter connectivity and self-assembling behaviors endow nano fibers as exciting candidate for enzyme carrier in bio reactor systems. In this study, nano fibers were successfully fabricated via electro spinning system by optimizing the polymer concentration (10-30 %, w/v), applied voltage (10-30 kV) and discharge distance (11-26 cm). Microscopic images have confirmed the quality as homogeneous and good fiber alignment. The nano fibers surface was modified using strong oxidizing agent to facilitate bio molecule binding. Bovine serum albumin and β-galactosidase enzyme were employed as model bio catalysts and immobilized onto the oxidized surfaces through covalent binding. Maximum enzyme adsorption capacity of the modified nano fibers was 3000 mg/g, 3-fold higher than the unmodified counterpart (1000 mg/g). The highest immobilization yield was 80% and reached the saturation point at 2 mg/ml of enzyme concentration. The results indicate a significant increase of activity retention by the enzyme-bound modified nano fibers (80%) as compared to the nascent one (60%), signifying excellent enzyme-nano carrier bio compatibility. The immobilized enzyme was further used for the bio conversion of dairy wastes into value-added products. This study demonstrates great potential of acid-modified electrospun polystyrene nano fibers as enzyme carriers.

Keywords: immobilization, enzyme, nanocarrier, nanofibers

Procedia PDF Downloads 288

Authors: Mannar R. Maurya, Bhawna Uprety, Fernando Avecilla, Pedro Adão, J. Costa Pessoa

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The reaction of the tripodal tetradentate dibasic ligand 6,6'–(2–(pyridin–2–yl)ethylazanediyl)bis(methylene)bis(2,4–di–tert–butylphenol), H2L1 I, with [VIVO(acac)2] in CH3CN gives the VVO–complex, [VVO(acac)(L1)] 1. Crystallization of 1 in CH3CN at ~0 ºC, gives dark blue crystals of 1, while at room temperature it affords dark green crystals of [{VVO(L1)}2µ–O] 3. Upon prolonged treatment of 1 in MeOH [VVO(OMe)(MeOH)(L1)] 2 is obtained. All three complexes are analyzed by single–crystal X–ray diffraction, depicting distorted octahedral geometry around vanadium. In the reaction of H2L1 with VIVOSO4 partial hydrolysis of the tripodal ligand results in elimination of the pyridyl fragment of L1 and the formation of H[VVO2(L2)] 4, containing the ONO tridentate ligand 6,6'–azanediylbis(methylene)bis(2,4–di–tert–butylphenol), H2L2 II. Compound 4, which was not fully characterized, undergoes dimerization in acetone yielding the hydroxido–bridged [{VVO(L2)}2µ–(HO)2] 5, having distorted octahedral geometry around each vanadium. In contrast, from a solution of 4 in acetonitrile, the dinuclear compound [{VVO(L2)}2µ–O] 6 is obtained, with trigonal bipyramidal geometry around each vanadium. The methoxido complex 2 is successfully employed as a functional catechol–oxidase mimic in the oxidation of catechol to o–quinone under air. The process is confirmed to follow a Michaelis–Menten type kinetics with respect to catechol, the Vmax and KM values obtained being 7.66×10–6 M min -1 and 0.0557 M, respectively, and the turnover frequency is 0.0541 min–1. Complex 2 is also used as a catalyst precursor for the oxidative bromination of thymol in aqueous medium. The selectivity shows quite interesting trends, namely when not using excess of primary oxidizing agent, H2O2 the para mono–brominated product corresponds to ~93 % of the products and no dibromo derivative is formed.

Keywords: oxidovanadium (V) complexes, tripodal ligand, crystal structure, catechol oxidase mimetic activity

Procedia PDF Downloads 335

Authors: A. Díaz-Roa, P. I. Silva Junior, F. J. Bello

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Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Dipterous maggots release diverse proteins and peptides contained in larval excretion and secretion (ES) products playing a key role in digestion. The most important mechanism for combating infection using larval therapy depends on larval ES. These larvae are protected against infection by a diverse spectrum of antimicrobial peptides (AMPs), one already known like lucifensin. Special interest in these peptides has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue and kill different bacteria during larval therapy. The action of larvae on wounds occurs through 3 mechanisms of action: removal of necrotic tissue, stimulation of granulation tissue, and antibacterial action of larval ES. Some components of the ES include calcium, urea, allantoin ammonium bicarbonate and reducing the viability of Gram positive and Gram negative bacteria. The Lucilia sericata fly larvae have been the most used, however, we need to evaluate new species that could potentially be similar or more effective than fly above. This study was thus aimed at identifying and characterizing S. magellanica AMPs contained in ES products for the first time and compared them with the common fly used L. sericata. These products were obtained from third-instar larvae taken from a previously established colony. For the first analysis, ES fractions were separate by Sep-Pak C18 disposable columns (first step). The material obtained was fractionated by RP-HPLC by using Júpiter C18 semi-preparative column. The products were then lyophilized and their antimicrobial activity was characterized by incubation with different bacterial strains. The first chromatographic analysis of ES from L. sericata gives 6 fractions with antimicrobial activity against Gram-positive bacteria Micrococus luteus, and 3 fractions with activity against Gram-negative bacteria Pseudomonae aeruginosa while the one from S. magellanica gaves 1 fraction against M. luteus and 4 against P. aeruginosa. Maybe one of these fractions could correspond to the peptide already known from L. sericata. These results show the first work for supporting further experiments aimed at validating S. magellanica use in larval therapy. We still need to search if we find some new molecules, by making mass spectrometry and ‘de novo sequencing’. Further studies are necessary to identify and characterize them to better understand their functioning.

Keywords: antimicrobial peptides, larval therapy, Lucilia sericata, Sarconesiopsis magellanica

Procedia PDF Downloads 364

Authors: Jawaher F. Alsharef, Abdullah A. Ghaddaf, Mohammed S. Alomari, Abdullah A. Al Qurashi, Ahmed S. Abdulhamid, Mohammed S. Alshehri, Majed Alosaimi

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Botulinum toxin type A (BTX-A) is the most popular therapeutic agent for muscle relaxation and pain control. Lately, BTX-A injection received great interest as a part of multimodal pain management for lower limb lengthening and deformity correction. This systematic review aimed to determine the role of BTX-A injection in pain management for during lower limb lengthening and/or deformity correction. We searched Medline, Embase, and CENTRAL. We included randomized controlled trials (RCTs) that compared the BTX-A injection to placebo for individuals undergoing lower limb lengthening and/or deformity correction. We sought to evaluate the following outcomes: pain on visual analogue scale (VAS), range of motion parameters, average opioid consumption, and adverse events. The standardized mean difference (SMD) was used to represent continuous outcomes while risk ratio (RR) was used to represent dichotomous outcomes. A total of 4 RCTs that enrolled 257 participants (337 limbs) deemed eligible. Adjuvant BTX-A injection showed a significant reduction in post-operative pain compared to placebo (SMD=–0.28, 95% CI –0.53 to –0.04). No difference was found between BTX-A injection and placebo in terms of range of motion parameters, average opioid consumption, or adverse events after surgical limb lengthening and/or deformity correction (RR= 0.77, 95% CI –0.58 to 1.03). Conclusions: Adjuvant BTX-A injection conferred a discernible reduction in post-operative pain during surgical limb lengthening and/or deformity without increasing the risk of adverse events.

Keywords: botulinum toxin type A, limb lengthening, distraction osteogenesis, deformity correction, pain management

Procedia PDF Downloads 138

Authors: Omaima A. Sharaf, Tarek A. Moussa, Said M. Badr El-Din, H. Moawad

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Background: Polycyclic aromatic hydrocarbons (PAHs) pose great environmental and human health concerns for their widespread occurrence, persistence, and carcinogenic properties. PAHs releases due to anthropogenic activities to the wider environment have led to higher concentrations of these contaminants than would be expected from natural processes alone. This may result in a wide range of environmental problems that can accumulate in agricultural ecosystems, which threatened to become a negative impact on sustainable agricultural development. Thus, this study aimed to evaluate the physico-chemical, and microbial properties of the compost leachate (CL) to assess its role as nutrient and microbial source (biostimulation/bioaugmentation) for developing a cost-effective bioremediation technology for PAHs contaminated sites. Material and Methods: PAHs-degrading bacteria were isolated from CL that was collected from a composting site located in central Scotland, UK. Isolation was carried out by enrichment using phenanthrene (PHR), pyrene (PYR) and benzo(a)pyrene (BaP) as the sole source of carbon and energy. The isolates were characterized using a variety of phenotypic and molecular properties. Six different isolates were identified based on the difference in morphological and biochemical tests. The efficiency of these isolates in PAHs utilization was assessed. Further analysis was performed to define taxonomical status and phylogenic relation between the most potent PAHs-utilizing bacterial strains and other standard strains, using molecular approach by partial 16S rDNA gene sequence analysis. Results indicated that the 16S rDNA sequence analysis confirmed the results of biochemical identification, as both of biochemical and molecular identification of the isolates assigned them to Bacillus licheniformis, Pseudomonas aeruginosa, Alcaligenes faecalis, Serratia marcescens, Enterobacter cloacae and Providenicia which were identified as the prominent PAHs-utilizers isolated from CL. Conclusion: This study indicates that the CL samples contain a diverse population of PAHs-degrading bacteria and the use of CL may have a potential for bioremediation of PAHs contaminated sites.

Keywords: polycyclic aromatic hydrocarbons, physico-chemical analyses, compost leachate, microbial and biochemical analyses, phylogenic relations, 16S rDNA sequence analysis

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Authors: Miaomiao Zhang, Sabrina Beckmann, Haluk Ertan, Rocky Chau, Mike Manefield

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Phenazines are a large class of nitrogen-containing aromatic heterocyclic compounds, which are almost exclusively produced by bacteria from diverse genera including Pseudomonas and Streptomyces. Phenazine-1-carboxylic acid (PCA) as one of 'core' phenazines are converted from chorismic acid before modified to other phenazine derivatives in different cells. Phenazines have attracted enormous interests because of their multiple roles on biocontrol, bacterial interaction, biofilm formation and fitness of their producers. However, in spite of ecological importance, degradation as a part of phenazines’ fate only have extremely limited attention now. Here, to isolate PCA-degrading bacteria, 200 mg L-1 PCA was supplied as sole carbon, nitrogen and energy source in minimal mineral medium. Quantitative PCR and Reverse-transcript PCR were employed to study abundance and activity of functional gene MFORT 16269 in PCA degradation, respectively. Intermediates and products of PCA degradation were identified with LC-MS/MS. After enrichment and isolation, a PCA-degrading strain was selected from soil and was designated as Rhodanobacter sp. PCA2 based on full 16S rRNA sequencing. As determined by HPLC, strain PCA2 consumed 200 mg L-1 (836 µM) PCA at a rate of 17.4 µM h-1, accompanying with significant cells yield from 1.92 × 105 to 3.11 × 106 cells per mL. Strain PCA2 was capable of degrading other phenazines as well, including phenazine (4.27 µM h-1), pyocyanin (2.72 µM h-1), neutral red (1.30 µM h-1) and 1-hydroxyphenazine (0.55 µM h-1). Moreover, during the incubation, transcript copies of MFORT 16269 gene increased significantly from 2.13 × 106 to 8.82 × 107 copies mL-1, which was 2.77 times faster than that of the corresponding gene copy number (2.20 × 106 to 3.32 × 107 copies mL-1), indicating that MFORT 16269 gene was activated and played roles on PCA degradation. As analyzed by LC-MS/MS, decarboxylation from the ring structure was determined as the first step of PCA degradation, followed by cleavage of nitrogen-containing ring by dioxygenase which catalyzed phenazine to nitrosobenzene. Subsequently, phenylhydroxylamine was detected after incubation for two days and was then transferred to aniline and catechol. Additionally, genomic and proteomic analyses were also carried out for strain PCA2. Overall, the findings presented here showed that a newly isolated strain Rhodanobacter sp. PCA2 was capable of degrading phenazines through decarboxylation and cleavage of nitrogen-containing ring, during which MFORT 16269 gene was activated and played important roles.

Keywords: decarboxylation, MFORT16269 gene, phenazine-1-carboxylic acid degradation, Rhodanobacter sp. PCA2

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Authors: Marco Furinghetti, Alberto Pavese, Michele Rinaldi

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Concave Surface Slider devices have been more and more used in real applications for seismic protection of both bridge and building structures. Several research activities have been carried out, in order to investigate the lateral response of such a typology of devices, and a reasonably high level of knowledge has been reached. If radial analysis is performed, the frictional force is always aligned with respect to the restoring force, whereas under bidirectional seismic events, a bi-axial interaction of the directions of motion occurs, due to the step-wise projection of the main frictional force, which is assumed to be aligned to the trajectory of the isolator. Nonetheless, if non-linear time history analyses have to be performed, standard codes provide precise rules for the definition of an averagely spectrum-compatible set of accelerograms in radial conditions, whereas for bidirectional motions different combinations of the single components spectra can be found. Moreover, nowadays software for the adjustment of natural accelerograms are available, which lead to a higher quality of spectrum-compatibility and to a smaller dispersion of results for radial motions. In this endeavor a simplified design procedure is defined, for building structures, base-isolated by means of Concave Surface Slider devices. Different case study structures have been analyzed. In a first stage, the capacity curve has been computed, by means of non-linear static analyses on the fixed-base structures: inelastic fiber elements have been adopted and different direction angles of lateral forces have been studied. Thanks to these results, a linear elastic Finite Element Model has been defined, characterized by the same global stiffness of the linear elastic branch of the non-linear capacity curve. Then, non-linear time history analyses have been performed on the base-isolated structures, by applying seven bidirectional seismic events. The spectrum-compatibility of bidirectional earthquakes has been studied, by considering different combinations of single components and adjusting single records: thanks to the proposed procedure, results have shown a small dispersion and a good agreement in comparison to the assumed design values.

Keywords: concave surface slider, spectrum-compatibility, bidirectional earthquake, base isolation

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Authors: Samantha R. Dutra

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There are many issues that students face in college, such as academic-based struggles, financial issues, family responsibilities, and vocational problems. Students with dyslexia struggle even more with these problems compared to other students. This qualitative study examines faculty perceptions of instructing students with dyslexia. This study is important to the human services and post-secondary educational fields due to the increase in disabled students enrolled in college. This study is also substantial because of the reported bias faced by students with dyslexia and their academic failure. When students with LDs such as dyslexia experience bias, discrimination, and isolation, they are more apt to not seek accommodations, lack communication with faculty, and are more likely to drop out or fail. College students with dyslexia often take longer to complete their post-secondary education and are more likely to withdraw or drop out without earning a degree. Faculty attitudes and academic cultures are major barriers to the success and use of accommodations as well as modified instruction for students with disabilities, which leads to student success. Faculty members are often uneducated or misinformed regarding students with dyslexia. More importantly, many faculty members are unaware of the many ethical and legal implications that they face regarding accommodating students with dyslexia. Instructor expectations can generally be defined as the understanding and perceptions of students regarding their academic success. Skewed instructor expectations can affect how instructors interact with their students and can also affect student success. This is true for students with dyslexia in that instructors may have lower and biased expectations of these students and, therefore, directly impact students’ academic successes and failures. It is vital to understand how instructor attitudes affect the academic achievement of dyslexic students. This study will examine faculty perceptions of instructing students with dyslexia and faculty attitudes towards accommodations and institutional support. The literature concludes that students with dyslexia have many deficits and several learning needs. Furthermore, these are the students with the highest dropout and failure rates, as well as the lowest retention rates. Disabled students generally have many reasons why accommodations and supports just do not help. Some research suggests that accommodations do help students and show positive outcomes. Many improvements need to be made between student support service personnel, faculty, and administrators regarding providing access and adequate supports for students with dyslexia. As the research also suggests, providing more efficient and effective accommodations may increase positive student as well as faculty attitudes in college, and may improve student outcomes overall.

Keywords: dyslexia, faculty perception, higher education, learning disability

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Authors: Susana P. Dias, Andrea Gomes, Fernanda M. Ferreira, Marta F. Henriques

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Thymus mastichina L. is an endogenous medicinal and aromatic plant of the Mediterranean flora. It has been used empirically over the years as a natural preservative in food. Nowadays, the antimicrobial activity of its bioactive compounds, such as essential oils and extracts, has been well recognized. The main purpose of this study was to evaluate the antimicrobial effect of Thymus mastichina ethanolic and aqueous extracts on pathogens and spoilage microorganisms present in cheese during ripening. The effect that the extract type and its concentration has on the development of Staphylococcus aureus, Escherichia coli, and Yarrowia lipolytica populations during 24 hours, was studied 'in vitro' using appropriate culture media. The results achieved evidenced the antimicrobial activity of T. mastichina extracts against the studied strains, and the concentration of 2 mg/mL (w/v) was selected and used directly on the cheese surface during ripening. In addition to the microbiological evaluation in terms of total aerobic bacteria, Enterobacteriaceae, yeasts (particularly Y. lipolytica) and molds, the treated cheeses physicochemical evaluation (humidity, aw, pH, colour, and texture) was also performed. The results were compared with cheeses with natamicyn (positive control) and without any treatment (negative control). The physicochemical evaluation showed that the cheeses treated with ethanolic extract of Thymus mastichina, except the fact that they lead to a faster water loss during ripening, did not present considerable differences when compared to controls. The study revealed an evident antimicrobial power of the extracts, although less effective than the one shown by the use of natamycin. For this reason, the improvement of the extraction methods and the adjustment of the extract concentrations will contribute to the use of T. mastichina as a healthier and eco-friendly alternative to natamycin, that is also more attractive from an economic point of view.

Keywords: antimicrobial activity, cheese, ethanolic extract, Thymus mastichina

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Authors: Byung Lee, Charles Perschau

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This study analyzes the development of a communication journal, the Journal of Advertising Education (JAE) over the past 20 years by examining citations of all research articles there. The purpose of a journal is to offer a stable and transparent forum for the presentation, scrutiny, and discussion of research in a targeted domain. This study asks whether JAE has fulfilled this purpose. The authors and readers who are involved in a journal need to have common research topics of their interest. In the case of the discipline of communication, scholars have a variety of backgrounds beyond communication itself since the social scientific study of communication is a relatively recent development, one that emerged after World War II, and the discipline has been heavily indebted to other social sciences, such as psychology, sociology, social psychology, and political science. When authors impart their findings and knowledge to others, their work is not done in isolation. They have to stand on previous studies, which are listed as sources in the bibliography. Since communication has heavily piggybacked on other disciplines, cited sources should be as diverse as the resources it taps into. This paper analyzes 4,244 articles that were cited by JAE articles in the past 36 issues. Since journal article authors reveal their intellectual linkage by using bibliographic citations, the analysis of citations in journal articles will reveal various networks of relationships among authors, journal types, and fields in an objective and quantitative manner. The study found that an easier access to information sources because of the development of electronic databases and the growing competition among scholars for publication seemed to influence authors to increase the number of articles cited even though some variations existed during the examined period. The types of articles cited have also changed. Authors have more often cited journal articles, periodicals (most of them available online), and web site sources, while decreased their dependence on books, conference papers, and reports. To provide a forum for discussion, a journal needs a common topic or theme. This can be realized when an author writes an article about a topic, and that article is cited and discussed in another article. Thus, the citation of articles in the same journal is vital for a journal to form a forum for discussion. JAE has gradually increased the citations of in-house articles with a few fluctuations over the years. The study also examines not only specific articles that are often cited, but also specific authors often cited. The analysis of citations in journal articles shows how JAE has developed into a full academic journal while offering a communal forum even though the speed of its formation is not as fast as desired probably because of its interdisciplinary nature.

Keywords: citation, co-citation, the Journal of Advertising Education, development of a journal

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Authors: Chad Van Schoelandt, Chara Kokkiou

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This paper discusses Socrates’ fundamental views of morality and freedom in Plato’s Phaedo through examining the fittingness of resentment and related emotional responses. In different parts of the dialogue there seems to be two kinds of emotional justification, which seem to explain different types of appeal that Socrates makes in order to defend his own emotional responses and make recommendations to others. The upshot of this paper is to bring out the connection between different emotional responses and beliefs. In particular, it focuses on the unfittingness of the Strawsonian resentment. If one, taking a rationalistic approach, agrees that some emotions, such as resentment, have a cognitive or belief-like component, then people reacting differently to the same situation suggests differences in their judgments and beliefs. However, at times, including in Socrates’s direction to his friends in the Phaedo, emotions are justified by pragmatic appeal, independent of the beliefs associated with the emotion. In any case, there are both fittingness-based and pragmatic factors that determine and condition the warrant of an emotional response. Overall, an emotion is fitting when the agent’s beliefs indicate that the conditions of appropriatedness are met. Socrates views resentment and sorrow as unfitting due to the mismatch with his own moral beliefs and his teaching to others. At the same time, Socrates argues that his friends’ expression of sorrow at his last moments is unseemly because it is not included in the widely accepted social practices, though the emotion itself is not necessarily unfitting. Socrates’s unexpected emotional response to his death, namely his lack of resentment and sorrow, implies a different belief system and indicates his students’ lack of understanding of the actual implications of his views. Thus, the paper will bring out how complicated Socrates’s ideas were even for people who had a sustained engagement with his ideas. Overall, the paper will illuminate how these two parties (Socrates – friends) view different moral duties, namely the individual duty to philosophy, which signifies a meaningful life, and the civic duty to obey the law, which signals Socrates’ death.

Keywords: Emotions, freedom, morality, Plato

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Authors: Leda Maria Costa Pereira, Maria Denise Lopes, Nucharin Songsasen

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Mammalian ovaries contain thousands of follicles that eventually degenerate or die after culture in vitro. Caspase-3 is a key enzyme that regulating cell death. Our objective was to examine the influence of anti-apoptotic drug Z-VAD-FMK (pan-caspase inhibitor) on in vitro viability of dog follicles within the ovarian cortex. Ovaries were obtained from prepubertal (age, 2.5–6 months) and adult (age, 8 months to 2 years) bitches and ovarian cortical fragments were recovered. The cortices were then incubated on 1.5% (w/v) agarose gel blocks within a 24-wells culture plate (three cortical pieces/well) containing Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM) supplemented with 4.2 µg/ml insulin, 3.8 µg/ml transferrin, 5 ng/ml selenium, 2 mM L-glutamine, 100 µg/mL of penicillin G sodium, 100 µg/mL of streptomycin sulfate, 0.05 mM ascorbic acid, 10 ng/mL of FSH and 0.1% (w/v) polyvinyl alcohol in humidified atmosphere of 5% CO2 and 5% O2. The cortices were divided in six treatment groups: 1) 10 ng/mL EGF (EGF V0); 2) 10 ng/mL of EGF plus 1 mM Z-VAD-FMK (EGF V1); 3) 10 ng/mL of EGF and 10 mM Z-VAD-FMK (EGF V10); 4) 1 mM Z-VAD-FMK; 5) 10 mM Z-VAD-FMK and (6) no EGF and Z-VAD-FMK supplementation. Ovarian follicles within the tissues were processed for histology and assessed for follicle density, viability (based on morphology) and diameter immediately after collection (Control) or after 3 or 7 days of in vitro incubation. Comparison among fresh and culture treatment group was performed using ANOVA test. There were no differences (P > 0.05) in follicle density and viability among different culture treatments. However, there were differences in this parameter between culture days. Specifically, culturing tissue for 7 days resulted in significant reduction in follicle viability and density, regardless of treatments. We found a difference in size between culture days when these follicles were cultured using 10 mM Z-VAD-FMK or 10 ng/mL EGF (EGF V0). In sum, the finding demonstrated that Z-VAD-FMK at the dosage used in the present study does not provide the protective effect to ovarian tissue during in vitro culture. Future studies should explore different Z-VAD-FMK dosages or other anti-apoptotic agent, such as surviving in protecting ovarian follicles against cell death.

Keywords: anti apoptotic drug, bitches, follicles, Z-VAD-FMK

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Authors: Rawan Al-Nemari, Abdulrahman Al-Senaidy, Abdelhabib Semlali

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Background and objectives: The most common cause of death in women worldwide is breast cancer. Regarding all chemotherapy disadvantages and side effects, it’s becoming necessary to identify natural products that target cancer cells with lesser harmful side effects on non-targeted cells and biological environment. Different parts of A. squamosa L., commonly known as custard apple, show varied therapeutic effects. The objective of this study is to investigate in vitro and in vivo, the anti-cancer activity of A. squamosa leaves extract. Methods: The physiological responses using MTT, nucleus staining, and LDH assays were used to evaluate cell survival and proliferation in both ER+ and ER- cells when they were exposed to extract. Monolayer wound repair assay was used to investigate the effect of extracts on cell migration. Apoptotic gene’s expression was investigated by real-time polymerase chain reaction. To study the effect of the extract on the size of tumor, breast cancer induced rats were used. Results: A. squamosa leaves extract showed high anti-proliferative and cytotoxicity effects against different breast cancer cell lines with high concentration, 100 ug/ml. The extracts have reduced the cells wound closure. Polymerase chain reaction revealed downregulation of Bcl-2 and upregulation of Bax. In breast cancer model animal developed in our laboratory, after 4 weeks treatment, treated groups have shown smaller tumor size in comparison with control group (n=4). Conclusion: These results suggest that A. squamosa leaves extract has anti-cancer activity against breast cancer in both in vitro and in vivo, and it may be developed as a potential novel agent to treat breast cancer.

Keywords: apoptosis, breast cancer, migration, proliferation

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Authors: Fazil Hasan, M. Shafiq Ansari, Mohammad Muslim

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Zygogramma bicolorata Pallister (Coleoptera: Chrysomelidae) is an exotic insect and effective biocontrol agent of Parthenium hysterophorus L. (Asteraceae). Our study aimed to determine the induction and termination of diapause, in response to abiotic (temperature and moisture) and biotic factors (age and reproductive status) and the effect of diapause on adult longevity and female fecundity. The adults burrowed into the soil about 1–6 cm below the surface for diapause at any time from July to December with a peak of 70% in the 2nd week of December at Aligarh region, India. The termination of diapause took place in May and June with the commencement of monsoon rains. Non-diapausing adults were also capable of breeding during winter under laboratory conditions. There was a significantly increased in the percentage of diapaused adults in subsequent generation i.e. 4% in F1 generation and 90% in F7 generation. The percentage of diapause was also significantly increased with age of adults. It has a positive effect on female fecundity as compared to the fecundity in pre-diapaused duration. Experiments proved that soil moisture played an important role in providing the conditions for initiation and termination of diapause. The adults which undergone diapause in January and February were continuously exposed to 35º, 40º and 45º C for one week and a daily dose of 10 and 8 hours for 6 and 5 days, respectively resulting in termination of diapause. This method may be used to initiate mass multiplication for carrying out releases early in the season. Exposure of adults to extremely low temperatures i.e. 5º and 10º C induced 94.3% and 92.5% diapause, respectively with no adult mortality. Therefore, low temperatures can also be used as a medium for the storage of mass reared beetles for a long time without having negative effect on their longevity and fecundity. Thus, our findings are of great utility in the biological suppression of P. hysterophorus as it will enhance the effectiveness of this beetle through manipulation of diapause.

Keywords: Zygogramma bicolorata, environmental factors, age, sex, diapause, Parthenium hysterophorus, biocontrol

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Authors: Masaaki Ii, Takashi Saito, Yasuhiko Tabata, Shintaro Nemoto

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Background: Clinical trials of autologous adipose-derived stem cell (AdSC) therapy for ischemic heart diseases (IHD) are now on-going. We have investigated the hypothesis that combination of AdSCs and statin, an agent with pleiotropic effects, could augment the therapeutic effect on myocardial infarction (MI). Methods and Results: Human AdSC functions with different doses of simvastatin-conjugated nanoparticle (STNP) uptake were evaluated by in vitro assays. STNP promoted the migration activity without changing the proliferation activity, and also up-regulated growth factors. Next, MI was induced by LAD ligation in nude mice, and the mice were assigned in the following groups 3 days after MI: 1) PBS (control), 2) NP-AdSCs (50000 cells), 3) STNP, and 4) STNP-AdSCs (50000 cells). Cardiac functional recovery assessed by echocardiography was improved at 4 weeks after surgery in STNP-AdSC group. Masson’s trichrome-stained sections revealed that LV fibrosis length was reduced, and the number of TUNEL-positive cardiomyocytes was less in STNP-AdSC group. Surprisingly, a number of de novo endogenous Nkx-2.5/GATA4 positive immature cardiomyocytes as well as massive vascular formation were observed in outer layer of infarcted myocardium despite of a few recruited/retained transfused STNP-AdSCs 4 weeks after MI in STNP-AdSC group. Finally, massive myocardial regeneration was observed 8 weeks after MI. Conclusions: Intravenously injected small number of statin nanoparticle-loaded hAdSCs exhibited a potent therapeutic effect inducing endogenous cardiac tissue regeneration.

Keywords: statin, drug delivery system, stem cells, cardiac regeneration

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Authors: R. M. S. Sachini Amararathne

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This research is striving to develop a thermo degradable starch plastic compound/ masterbatch for industrial packaging applications. A native corn starch-modified with an esterification reaction of lauric acid is melt blent with an unsaturated elastomer (styrene-butadiene-rubber/styrene-butadiene-styrene). A trace amount of metal salt is added into the internal mixer to study the effect of pro-oxidants in a thermo oxidative environment. Then the granulated polymer composite which is consisted with 80-86% of polyolefin (LLDP/LDPE/PP) as the pivotal agent; is extruded with processing aids, antioxidants and some other additives in a co-rotating twin-screw extruder. The pelletized composite is subjected to compression molding/ Injection molding or blown film extrusion processes to acquire the samples/specimen for tests. The degradation process is explicated by analyzing the results of fourier transform infrared spectroscopy (FTIR) measurements, thermo oxidative aging studies (placing the dumb-bell specimen in an air oven at 70 °C for four weeks of exposure.) governed by tensile and impact strength test reports. Furthermore, the samples were elicited into manifold outdoors to inspect the degradation process. This industrial process is implemented to reduce the volume of fossil-based garbage by achieving the biodegradability and compostability in the natural cycle. Hence the research leads to manufacturing a degradable plastic packaging compound which is now available in the Sri Lankan market.

Keywords: blown film extrusion, compression moulding, polyolefin, pro-oxidant, styrene-butadine-rubber, styrene-butadiene-styrene, thermo oxidative aging, unsaturated elastomer

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Authors: Hamza Javar Magnier, Robin Curtis

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There have been rapid advances in the upstream processing of protein therapeutics, which has shifted the bottleneck to downstream purification and formulation. Finding liquid formulations with shelf lives of up to two years is increasingly difficult for some of the newer therapeutics, which have been engineered for activity, but their formulations are often viscous, can phase separate, and have a high propensity for irreversible aggregation1. We explore means to develop improved predictive ability from a better understanding of how protein-protein interactions on formulation conditions (pH, ionic strength, buffer type, presence of excipients) and how these impact upon the initial steps in protein self-association and aggregation. In this work, we study the initial steps in the aggregation pathways using a minimal protein model based on square-well potentials and discontinuous molecular dynamics. The effect of model parameters, including range of interaction, stiffness, chain length, and chain sequence, implies that protein models fold according to various pathways. By reducing the range of interactions, the folding- and collapse- transition come together, and follow a single-step folding pathway from the denatured to the native state2. After parameterizing the model interaction-parameters, we developed an understanding of low-temperature conformational properties and fluctuations, and the correlation to the folding transition of proteins in isolation. The model fluctuations increase with temperature. We observe a low-temperature point, below which large fluctuations are frozen out. This implies that fluctuations at low-temperature can be correlated to the folding transition at the melting temperature. Because proteins “breath” at low temperatures, defining a native-state as a single structure with conserved contacts and a fixed three-dimensional structure is misleading. Rather, we introduce a new definition of a native-state ensemble based on our understanding of the core conservation, which takes into account the native fluctuations at low temperatures. This approach permits the study of a large range of length and time scales needed to link the molecular interactions to the macroscopically observed behaviour. In addition, these models studied are parameterized by fitting to experimentally observed protein-protein interactions characterized in terms of osmotic second virial coefficients.

Keywords: protein folding, native-ensemble, conformational fluctuation, aggregation

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Authors: Hosein Maghsoudi

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Rheumatois arthritis (RA) is progressive inflammatory autoimmune diseases that primarily affect the joints, characterized by synovial hyperplasia and inflammatory cell infiltration, deformed and painful joints, which can lead tissue destruction, functional disability systemic complications, and early dead and socioeconomic costs. The cause of rheumatoid arthritis is unknown, but genetic and environmental factors are contributory and the prognosis is guarded. However, advances in understanding the pathogenesis of the disease have fostered the development of new therapeutics, with improved outcomes. The current treatment strategy, which reflects this progress, is to initiate aggressive therapy soon after diagnosis and to escalate the therapy, guided by an assessment of disease activity, in pursuit of clinical remission. The pathobiology of RA is multifaceted and involves T cells, B cells, fibroblast-like synoviocyte (FLSc) and the complex interaction of many pro-inflammatory cytokine. Novel biologic agents that target tumor necrosis or interlukin (IL)-1 and Il-6, in addition T- and B-cells inhibitors, have resulted in favorable clinical outcomes in patients with RA. Despite this, at least 30% of RA patients are résistance to available therapies, suggesting novel mediators should be identified that can target other disease-specific pathway or cell lineage. Among the inflammatory cell population that might participated in RA pathogenesis, FLSc are crucial in initiaing and driving RA in concert of cartilage and bone by secreting metalloproteinase (MMPs) into the synovial fluid and by direct invasion into extracellular matrix (ECM), further exacerbating joint damage. Invasion of fibroblast-like synoviocytes (FLSc) is critical in the pathogenesis of rheumatoid-arthritis. The metalloproteinase (MMPs) and activator of Toll-like receptor 4 (TLR4)/nuclear factor- κB pthway play a critical role in RA-FLS invasion induced by lipopolysaccharide (LPS). The present study aimed to explore the anti-invasion activity of Glycyrrhizic Acid as a pharmacologically safe phytochemical agent with potent anti-inflammatory properties on IL-1beta and TNF-alpha signalling pathways in Bovine fibroblast-like synoviocyte ex- vitro, on LPS-stimulated bovine FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Results showed that Glycyrrhizic Acid suppressed LPS-stimulated bovine FLS migration and invasion by inhibition MMP-9 expression and activity. In addition our results revealed that Glycyrrhizic Acid inhibited the transcriptional activity of MMP-9 by suppression the nbinding activity of NF- κB in the MMP-9 promoter pathway. The extract of licorice (Glycyrrhiza glabra L.) has been widely used for many centuries in the traditional Chinese medicine as native anti-allergic agent. Glycyrrhizin (GL), a triterpenoidsaponin, extracted from the roots of licorice is the most effective compound for inflammation and allergic diseases in human body. The biological and pharmacological studies revealed that GL possesses many pharmacological effects, such as anti-inflammatory, anti-viral and liver protective effects, and the biological effects, such as induction of cytokines (interferon-γ and IL-12), chemokines as well as extrathymic T and anti-type 2 T cells. GL is known in the traditional Chinese medicine for its anti-inflammatory effect, which is originally described by Finney in 1959. The mechanism of the GL-induced anti-inflammatory effect is based on different pathways of the GL-induced selective inhibition of the prostaglandin E2 production, the CK-II- mediated activation of both GL-binding lipoxygenas (gbLOX; 17) and PLA2, an anti-thrombin action of GL and production of the reactive oxygen species (ROS; GL exerts liver protection properties by inhibiting PLA2 or by the hydroxyl radical trapping action, leading to the lowering of serum alanine and aspartate transaminase levels. The present study was undertaken to examine the possible mechanism of anti-inflammatory properties GL on IL-1beta and TNF-alpha signalling pathways in bovine fibroblast-like synoviocyte ex-vivo, on LPS-stimulated bovine FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Our results clearly showed that treatment of bovine fibroblast-like synoviocyte with GL suppressed LPS-induced cell migration and invasion. Furthermore, it revealed that GL inhibited the transcription activity of MMP-9 by suppressing the binding activity of NF-κB in the MM-9 promoter. MMP-9 is an important ECM-degrading enzyme and overexpression of MMPs in important of RA-FLSs. LPS can stimulate bovine FLS to secret MMPs, and this induction is regulated at the transcription and translational levels. In this study, LPS treatment of bovine FLS caused an increase in MMP-2 and MMP-9 levels. The increase in MMP-9 expression and secretion was inhibited by ex- vitro. Furthermore, these effects were mimicked by MMP-9 siRNA. These result therefore indicate the the inhibition of LPS-induced bovine FLS invasion by GL occurs primarily by inhibiting MMP-9 expression and activity. Next we analyzed the functional significance of NF-κB transcription of MMP-9 activation in Bovine FLSs. Results from EMSA showed that GL suppressed LPS-induced NF-κB binding to the MMP-9 promotor, as NF-κB regulates transcriptional activation of multiple inflammatory cytokines, we predicted that GL might target NF-κB to suppress MMP-9 transcription by LPS. Myeloid differentiation-factor 88 (MyD88) and TIR-domain containing adaptor protein (TIRAP) are critical proteins in the LPS-induced NF-κB and apoptotic signaling pathways, GL inhibited the expression of TLR4 and MYD88. These results demonstrated that GL suppress LPS-induced MMP-9 expression through the inhibition of the induced TLR4/NFκB signaling pathway. Taken together, our results provide evidence that GL exerts anti-inflammatory effects by inhibition LPS-induced bovine FLSs migration and invasion, and the mechanisms may involve the suppression of TLR4/NFκB –mediated MMP-9 expression. Although further work is needed to clarify the complicated mechanism of GL-induced anti-invasion of bovine FLSs, GL might be used as a further anti-invasion drug with therapeutic efficacy in the treatment of immune-mediated inflammatory disease such as RA.

Keywords: glycyrrhizic acid, bovine fibroblast-like synoviocyte, tlr4/nf-κb, metalloproteinase-9

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Authors: Mahnaz Zarein, Hamed Shoorei

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Background: Malathion is one of the most toxic agents widely used in agriculture throughout the world. This agent has adverse effects on the functions of multiple organs such as the reproductive system in both male and female genders. On the one hand, daily use of antioxidant supplementations such as hesperidin is capable to neutralize the deleterious impacts of malathion. Therefore, in this experimental study, the protective effects of hesperidin against ovarian toxicity induced by malathion were investigated. Material & Methods: Balb/c adult mice (n=32) were randomly divided into 4 groups including 1) the control group, treated with normal saline, 2) the Mal group, treated with 30mg/kg malathion, daily for 1 month, 3) Mal + Hes group, treated with 20 mg/kg malathion and 20 mg/kg hesperidin, daily for 1 month, and 5) Hes group, treated with 20 mg/kg hesperidin. At the end of the experimental period, mice were anesthetized and their drops of blood were collected to the assessment of some hormones such as LH, FSH, E2, and P4. Also, the right ovaries were used to H&E staining, and the left ovaries were used for IHC staining (PCNA and FSHR). Results: Histopathological assessments showed that the number of follicles, i.e. primordial, primary, and secondary, significantly decreased, while the atretic follicle counts remarkably increased compared to the control group (p<0.05). Hormonal levels revealed that the production of all mentioned hormones decreased in the Mal group in comparison with the control group (p<0.05). The expression of PCNA, as a proliferative marker, and FHSR, as a marker showing maturation, decreased when mice received malathion compared to the control group (p<0.05). Interestingly, treatment with hesperidin significantly neutralized the adverse effects of malathion on all mentioned parameters. Conclusion: Daily use of antioxidant supplementations such as hesperidin could alleviate the ovarian toxicity induced by malathion.

Keywords: malathion, ovary, antioxidant hesperidin, FSHR PCNA, ovary

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Authors: Naseem Fatima, A. N. Srivastava, Tasleem Raza, Vijay Kumar

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Introduction: Carcinoma gallbladder is third most common gastrointestinal lethal disease with the highest incidence and mortality rate among women in Northern India. Scientists have found several risk factors that make a person more likely to develop gallbladder cancer; among these risk factors, deregulation of miRNAs has been demonstrated to be one of the most crucial factors. The changes in the expression of specific miRNA genes result in the control of inflammation, cell cycle regulation, stress response, proliferation, differentiation, apoptosis and invasion thus mediate the process in tumorgenesis. The aim of this study was to investigate the role of MiRNA-335 and may as a molecular marker in early detection of gallbladder cancer in suspected cases. Material and Methods: A total of 20 consecutive patients with gallbladder cancer aged between 30-75 years were registered for the study. Total RNA was extracted from tissue by using the mirVANA MiRNA isolation Kit according to the manufacturer’s protocol. The MiRNA- 335 and U6 snRNA-specific cDNA were reverse-transcribed from total RNA using Taqman microRNA reverse-transcription kit according to the manufacturer’s protocol. TaqMan MiRNA probes hsa-miR-335 and Taqman Master Mix without AmpEase UNG, Individual real-time PCR assays were performed in a 20 μL reaction volume on a Real-Time PCR system (Applied Biosystems StepOnePlus™) to detect MiRNA-335 expression in tissue. Relative quantification of target MiRNA expression was evaluated using the comparative cycle threshold (CT) method. The correlation was done in between cycle threshold (CT Value) of target MiRNA in gallbladder cancer with respect to non-cancerous Cholelithiasis gallbladder. Each sample was examined in triplicate. The Newman-Keuls Multiple Comparison Test was used to determine the expression of miR-335. Results: MiRNA335 was found to be significantly downregulated in the gallbladder cancer tissue (P<0.001), when compared with non-cancerous Cholelithiasis gallbladder cases. Out of 20 cases, 75% showed reduced expression of MiRNA335, were at last stage of disease with low overall survival rate and remaining 25% were showed up-regulated expression of MiRNA335 with high survival rate. Conclusion: The present study showed that reduced expression of MiRNA335 is associated with the advancement of the disease, and its deregulation may provide important clues to understanding it as a prognostic marker and opportunities for future research.

Keywords: carcinoma gallbladder, downregulation, MiRNA-335, RT-PCR assay

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Authors: Virag Vass, Ilona Bereczki, Erzsebet Szabo, Nora Debreczeni, Aniko Borbas, Pal Herczegh, Arpad Tosaki

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Hydrogen sulfide (H₂S) is a toxic gas, but it is produced by certain tissues in a small quantity. According to earlier studies, ibuprofen and H₂S has a protective effect against damaging heart tissue caused by ischemia-reperfusion. Recently, we have been investigating the effect of a new water-soluble H₂S releasing ibuprofen molecule administered after artificially generated ischemia-reperfusion on isolated rat hearts. The H₂S releasing property of the new ibuprofen derivative was investigated in vitro in medium derived from heart endothelial cell isolation at two concentrations. The ex vivo examinations were carried out on rat hearts. Rats were anesthetized with an intraperitoneal injection of ketamine, xylazine, and heparin. After thoracotomy, hearts were excised and placed into ice-cold perfusion buffer. Perfusion of hearts was conducted in Langendorff mode via the cannulated aorta. In our experiments, we studied the dose-effect of the H₂S releasing molecule in Langendorff-perfused hearts with the application of gradually increasing concentration of the compound (0- 20 µM). The H₂S releasing ibuprofen derivative was applied before the ischemia for 10 minutes. H₂S concentration was measured with an H₂S detecting electrochemical sensor from the coronary effluent solution. The 10 µM concentration was chosen for further experiments when the treatment with this solution was occurred after the ischemia. The release of H₂S is occurred by the hydrolyzing enzymes that are present in the heart endothelial cells. The protective effect of the new H₂S releasing ibuprofen molecule can be confirmed by the infarct sizes of hearts using the Triphenyl-tetrazolium chloride (TTC) staining method. Furthermore, we aimed to define the effect of the H₂S releasing ibuprofen derivative on autophagic and apoptotic processes in damaged hearts after investigating the molecular markers of these events by western blotting and immunohistochemistry techniques. Our further studies will include the examination of LC3I/II, p62, Beclin1, caspase-3, and other apoptotic molecules. We hope that confirming the protective effect of new H₂S releasing ibuprofen molecule will open a new possibility for the development of more effective cardioprotective agents with exerting fewer side effects. Acknowledgment: This study was supported by the grants of NKFIH- K-124719 and the European Union and the State of Hungary co- financed by the European Social Fund in the framework of GINOP- 2.3.2-15-2016-00043.

Keywords: autophagy, hydrogen sulfide, ibuprofen, ischemia, reperfusion

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Authors: Prince Vivek, Vijay K. Bharti, Manishi Mukesh, Ankita Sharma, Om Prakash Chaurasia, Bhuvnesh Kumar

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High performance of endurance in equid requires adaptive changes involving physio-biochemical, and molecular responses in an attempt to regain homeostasis. We hypothesized that the identification of the suitable reference genes might be considered for assessing of endurance related traits in pony at high altitude and may ensure for individuals struggling to potent endurance trait in ponies at high altitude. A total of 12 mares of ponies, Zanskar breed, were divided into three groups, group-A (without load), group-B, (60 Kg) and group-C (80 Kg) on backpack loads were subjected to a load carry protocol, on a steep climb of 4 km uphill, and of gravel, uneven rocky surface track at an altitude of 3292 m to 3500 m (endpoint). Blood was collected before and immediately after the load carry on sodium heparin anticoagulant, and the peripheral blood mononuclear cell was separated for total RNA isolation and thereafter cDNA synthesis. Real time-PCR reactions were carried out to evaluate the mRNAs expression profile of a panel of putative internal control genes (ICGs), related to different functional classes, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β₂ microglobulin (β₂M), β-actin (ACTB), ribosomal protein 18 (RS18), hypoxanthine-guanine phosophoribosyltransferase (HPRT), ubiquitin B (UBB), ribosomal protein L32 (RPL32), transferrin receptor protein (TFRC), succinate dehydrogenase complex subunit A (SDHA) for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of native pony’s. Three different algorithms, geNorm, NormFinder, and BestKeeper software, were used to evaluate the stability of reference genes. The result showed that GAPDH was best stable gene and stability value for the best combination of two genes was observed TFRC and β₂M. In conclusion, the geometric mean of GAPDH, TFRC and β₂M might be used for accurate normalization of transcriptional data for assessing endurance related traits in Zanskar ponies during load carrying.

Keywords: endurance exercise, ubiquitin B (UBB), β₂ microglobulin (β₂M), high altitude, Zanskar ponies, reference gene

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