Search results for: Cristina Wong

Authors: Cristina Opris, Bogdan Cojocaru, Madalina Tudorache, Simona M. Coman, Vasile I. Parvulescu, Camelia Bala, Bahir Duraki, Jeroen A. Van Bokhoven

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The tremendous achievements in the development of the society concretized by more sophisticated materials and systems are merely based on non-renewable resources. Consequently, after more than two centuries of intensive development, among others, we are faced with the decrease of the fossil fuel reserves, an increased impact of the greenhouse gases on the environment, and economic effects caused by the fluctuations in oil and mineral resource prices. The use of biomass may solve part of these problems, and recent analyses demonstrated that from the perspective of the reduction of the emissions of carbon dioxide, its valorization may bring important advantages conditioned by the usage of genetic modified fast growing trees or wastes, as primary sources. In this context, the abundance and complex structure of lignin may offer various possibilities of exploitation. However, its transformation in fuels or chemicals supposes a complex chemistry involving the cleavage of C-O and C-C bonds and altering of the functional groups. Chemistry offered various solutions in this sense. However, despite the intense work, there are still many drawbacks limiting the industrial application. Thus, the proposed technologies considered mainly homogeneous catalysts meaning expensive noble metals based systems that are hard to be recovered at the end of the reaction. Also, the reactions were carried out in organic solvents that are not acceptable today from the environmental point of view. To avoid these problems, the concept of this work was to investigate the synthesis of superparamagnetic core shell catalysts for the fragmentation of lignin directly in the aqueous phase. The magnetic nanoparticles were covered with a nanoshell of an oxide (niobia) with a double role: to protect the magnetic nanoparticles and to generate a proper (acidic) catalytic function and, on this composite, cobalt nanoparticles were deposed in order to catalyze the C-C bond splitting. With this purpose, we developed a protocol to prepare multifunctional and magnetic separable nano-composite Co@Nb2O5@Fe3O4 catalysts. We have also established an analytic protocol for the identification and quantification of the fragments resulted from lignin depolymerization in both liquid and solid phase. The fragmentation of various lignins occurred on the prepared materials in high yields and with very good selectivity in the desired fragments. The optimization of the catalyst composition indicated a cobalt loading of 4wt% as optimal. Working at 180 oC and 10 atm H2 this catalyst allowed a conversion of lignin up to 60% leading to a mixture containing over 96% in C20-C28 and C29-C37 fragments that were then completely fragmented to C12-C16 in a second stage. The investigated catalysts were completely recyclable, and no leaching of the elements included in the composition was determined by inductively coupled plasma optical emission spectrometry (ICP-OES).

Keywords: superparamagnetic core-shell catalysts, environmental production of fuels, renewable lignin, recyclable catalysts

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Authors: Cristina Diaz, Lucia Perez, Simone Visigalli, Giuseppe Di Florio, Gonzalo Fuentes, Roberto Canziani, Paolo Gronchi

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During the last 50 years, the selection of materials is one of the main decisions in machine design for different industrial applications. It is due to numerous physical, chemical, mechanical and technological factors to consider in it. Corrosion effects are related with all of these factors and impact in the life cycle, machine incidences and the costs for the life of the machine. Corrosion affects the deterioration or destruction of metals due to the reaction with the environment, generally wet. In food industry, dewatering industry, concrete industry, paper industry, etc. corrosion is an unsolved problem and it might introduce some alterations of some characteristics in the final product. Nowadays, depending on the selected metal, its surface and its environment of work, corrosion prevention might be a change of metal, use a coating, cathodic protection, use of corrosion inhibitors, etc. In the vast majority of the situations, use of a corrosion resistant material or in its defect, a corrosion protection coating is the solution. Stainless steels are widely used in machine design, because of their strength, easily cleaned capacity, corrosion resistance and appearance. Typical used are AISI 304 and AISI 316. However, their benefits don’t fit every application, and some coatings are required against corrosion such as some paintings, galvanizing, chrome plating, SiO₂, TiO₂ or ZrO₂ coatings, etc. In this work, some coatings based in a bilayer made of Titanium-Tantalum, Titanium-Niobium, Titanium-Hafnium or Titanium-Zirconium, have been developed used magnetron sputtering configuration by PVD (Physical Vapor Deposition) technology, for trying to reduce corrosion effects on AISI 304, AISI 316 and comparing it with Titanium alloy substrates. Ti alloy display exceptional corrosion resistance to chlorides, sour and oxidising acidic media and seawater. In this study, Ti alloy (99%) has been included for comparison with coated AISI 304 and AISI 316 stainless steel. Corrosion tests were conducted by a Gamry Instrument under ASTM G5-94 standard, using different electrolytes such as tomato salsa, wine, olive oil, wet compost, a mix of sand and concrete with water and NaCl for testing corrosion in different industrial environments. In general, in all tested environments, the results showed an improvement of corrosion resistance of all coated AISI 304 and AISI 316 stainless steel substrates when they were compared to uncoated stainless steel substrates. After that, comparing these results with corrosion studies on uncoated Ti alloy substrate, it was observed that in some cases, coated stainless steel substrates, reached similar current density that uncoated Ti alloy. Moreover, Titanium-Zirconium and Titanium-Tantalum coatings showed for all substrates in study including coated Ti alloy substrates, a reduction in current density more than two order in magnitude. As conclusion, Ti-Ta, Ti-Zr, Ti-Nb and Ti-Hf coatings have been developed for improving corrosion resistance of AISI 304 and AISI 316 materials. After corrosion tests in several industry environments, substrates have shown improvements on corrosion resistance. Similar processes have been carried out in Ti alloy (99%) substrates. Coated AISI 304 and AISI 316 stainless steel, might reach similar corrosion protection on the surface than uncoated Ti alloy (99%). Moreover, coated Ti Alloy (99%) might increase its corrosion resistance using these coatings.

Keywords: coatings, corrosion, PVD, stainless steel

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Authors: Cristina Chaminade, Mandar Kulkarni, Balaji Parthasarathy, Monica Plechero

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The characteristics of a regional innovation system (RIS) and the specificity of the knowledge base of an industry may contribute to create peculiar paths for innovation and development of firms’ geographic extended innovation networks. However, the relative empirical evidence in emerging economies remains underexplored. The paper aims to fill the research gap by means of some recent qualitative research conducted in 2016 in Beijing (China) and Bangalore (India). It analyzes cases studies of firms in the new media industry, a sector that merges different IT competences with competences from other knowledge domains and that is emerging in those RIS. The results show that while in Beijing the new media sector results to be more in line with the existing institutional setting and governmental goals aimed at targeting specific social aspects and social problems of the population, in Bangalore it remains a more spontaneous firms-led process. In Beijing what matters for the development of innovation networks is the governmental setting and the national and regional strategies to promote science and technology in this sector, internet and mass innovation. The peculiarities of recent governmental policies aligned to the domestic goals may provide good possibilities for start-ups to develop innovation networks. However, due to the specificities of those policies targeting the Chinese market, networking outside the domestic market are not so promoted. Moreover, while some institutional peculiarities, such as a culture of collaboration in the region, may be favorable for local networking, regulations related to Internet censorship may limit the use of global networks particularly when based on virtual spaces. Mainly firms with already some foreign experiences and contact take advantage of global networks. In Bangalore, the role of government in pushing networking for the new media industry at the present stage is quite absent at all geographical levels. Indeed there is no particular strategic planning or prioritizing in the region toward the new media industry, albeit one industrial organization has emerged to represent the animation industry interests. This results in a lack of initiatives for sustaining the integration of complementary knowledge into the local portfolio of IT specialization. Firms actually involved in the new media industry face institutional constrains related to a poor level of local trust and cooperation, something that does not allow for full exploitation of local linkages. Moreover, knowledge-provider organizations in Bangalore remain still a solid base for the IT domain, but not for other domains. Initiatives to link to international networks seem therefore more the result of individual entrepreneurial actions aimed at acquiring complementary knowledge and competencies from different domains and exploiting potentiality in different markets. From those cases, it emerges that role of government, soft institutions and organizations in the two RIS differ substantially in the creation of barriers and opportunities for the development of innovation networks and their specific aim.

Keywords: regional innovation system, emerging economies, innovation network, institutions, organizations, Bangalore, Beijing

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Authors: Blaga Alexandra Cristina, Kloetzer Lenuta, Bompa Amalia Stela, Galaction Anca Irina, Cascaval Dan

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Vitamin B9 is an important member of vitamins B group, being a growth factor, important for making genetic material as DNA and RNA, red blood cells, for building muscle tissues, especially during periods of infancy, adolescence and pregnancy. Its production by biosynthesis is based on the high metabolic potential of mutant Bacillus subtilis, due to a superior biodisponibility compared to that obtained by chemical pathways. Pertraction, defined as the extraction and transport through liquid membranes consists in the transfer of a solute between two aqueous phases of different pH-values, phases that are separated by a solvent layer of various sizes. The pertraction efficiency and selectivity could be significantly enhanced by adding a carrier in the liquid membrane, such as organophosphoric compounds, long chain amines or crown-ethers etc., the separation process being called facilitated pertraction. The aim of the work is to determine the impact of the presence of two extractants/carriers in the bulk liquid membrane, i.e. di(2-ethylhexyl) phosphoric acid (D2EHPA) and lauryltrialkylmetilamine (Amberlite LA2) on the transport kinetics of vitamin B9. The experiments have been carried out using two pertraction equipments for a free liquid membrane or bulk liquid membrane. One pertraction cell consists on a U-shaped glass pipe (used for the dichloromethane membrane) and the second one is an H-shaped glass pipe (used for h-heptane), having 45 mm inner diameter of the total volume of 450 mL, the volume of each compartment being of 150 mL. The aqueous solutions are independently mixed by means of double blade stirrers with 6 mm diameter and 3 mm height, having the rotation speed of 500 rpm. In order to reach high diffusional rates through the solvent layer, the organic phase has been mixed with a similar stirrer, at a similar rotation speed (500 rpm). The area of mass transfer surface, both for extraction and for reextraction, was of 1.59x10-³ m2. The study on facilitated pertraction with the mixture of two carriers, namely D2EHPA and Amberlite LA-2, dissolved in two solvents with different polarities: n-heptane and dichloromethane, indicated the possibility to obtain the synergic effect. The synergism has been analyzed by considering the vitamin initial and final mass flows, as well as the permeability factors through liquid membrane. The synergic effect has been observed at low D2EHPA concentrations and high Amberlite LA-2 concentrations, being more important for the low-polar solvent (n-heptane). The results suggest that the mechanism of synergic pertraction consists on the reaction between the organophosphoric carrier and vitamin B9 at the interface between the feed and membrane phases, while the aminic carrier enhances the hydrophobicity of this compound by solvation. However, the formation of this complex reduced the reextraction rate and, consequently, affects the synergism related to the final mass flows and permeability factor. For describing the influences of carriers concentrations on the synergistic coefficients, some equations have been proposed by taking into account the vitamin mass flows or permeability factors, with an average deviations between 4.85% and 10.73%.

Keywords: pertraction, synergism, vitamin B9, Amberlite LA-2, di(2-ethylhexyl) phosphoric acid

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Authors: Márcia Afonso, Cristina Fael, Marisa Dinis-Almeida

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Population growth in cities has led to an increase in the infrastructures construction, including buildings and roadways. This aspect leads directly to the soils waterproofing. In turn, changes in precipitation patterns are developing into higher and more frequent intensities. Thus, these two conjugated aspects decrease the rainwater infiltration into soils and increase the volume of surface runoff. The practice of green and sustainable urban solutions has encouraged research in these areas. The porous asphalt pavement, as a green infrastructure, is part of practical solutions set to address urban challenges related to land use and adaptation to climate change. In this field, permeable pavements with porous asphalt mixtures (PA) have several advantages in terms of reducing the runoff generated by the floods. The porous structure of these pavements, compared to a conventional asphalt pavement, allows the rainwater infiltration in the subsoil, and consequently, the water quality improvement. This green infrastructure solution can be applied in cities, particularly in streets or parking lots to mitigate the floods effects. Over the years, the pores of these pavements can be filled by sediment, reducing their function in the rainwater infiltration. Thus, double layer porous asphalt (DLPA) was developed to mitigate the clogging effect and facilitate the water infiltration into the lower layers. This study intends to deepen the knowledge of the performance of DLPA when subjected to clogging. The experimental methodology consisted on four evaluation phases of the DLPA infiltration capacity submitted to three precipitation events (100, 200 and 300 mm/h) in each phase. The evaluation first phase determined the behavior after DLPA construction. In phases two and three, two 500 g/m² clogging cycles were performed, totaling a 1000 g/m² final simulation. Sand with gradation accented in fine particles was used as clogging material. In the last phase, the DLPA was subjected to simple sweeping and vacuuming maintenance. A precipitation simulator, type sprinkler, capable of simulating the real precipitation was developed for this purpose. The main conclusions show that the DLPA has the capacity to drain the water, even after two clogging cycles. The infiltration results of flows lead to an efficient performance of the DPLA in the surface runoff attenuation, since this was not observed in any of the evaluation phases, even at intensities of 200 and 300 mm/h, simulating intense precipitation events. The infiltration capacity under clogging conditions decreased about 7% on average in the three intensities relative to the initial performance that is after construction. However, this was restored when subjected to simple maintenance, recovering the DLPA hydraulic functionality. In summary, the study proved the efficacy of using a DLPA when it retains thicker surface sediments and limits the fine sediments entry to the remaining layers. At the same time, it is guaranteed the rainwater infiltration and the surface runoff reduction and is therefore a viable solution to put into practice in permeable pavements.

Keywords: clogging, double layer porous asphalt, infiltration capacity, rainfall intensity

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Authors: Teresa Naves, Katy Pallas, Carme Florit, Cristina Anton, Joan Collado, Diana Millan

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Although gender-fair language is relatively novel in Spain, in Catalonia, the Department of Education, as well as LGBT Associations, have been promoting several innovative programmes aimed at implementing gender-inclusive schools. These Rainbow School communities are ideal for looking at how these programmes affect the use of gender-fair language and the balanced representation of gender. The students' and teachers' perceptions and attitudes have been compared to those analysed in schools that have never implemented such programmes in primary or secondary education. Spanish and Catalan, unlike English, are gendered languages in which masculine forms have traditionally been used as the unmarked gender and have been claimed to be inclusive of all genders. While the Royal Spanish Academy (RAE) rejects the use of inclusive language and thus deems all variables of inclusion of double gender as unnecessary, the vast majority of universities are promoting not only inclusive language but also gender-inclusive curricula. Adopting gender-fair language policies and including gender perspective in the curricula is an innovative trend at university level and in primary and secondary school education. Inclusion in education is a basic human right and the foundation for a more just and equal society. Educators can facilitate the process of welcoming by ensuring handbooks, forms, and other communications are inclusive of all family structures and gender identities. Using gendered language such as 'girls and boys' can be alienating for gender non-conforming and gender diverse students; on the other hand, non-gendered words like 'students' are regarded as inclusive of all identities. The paper discusses the results of mixed method research (survey, interviews, and experiment) conducted in Rainbow and non-Rainbow schools in Alacant and Barcelona (Spain). The experiment aimed at checking the role of gender-fair language in learners' perception of gender balance. It was conducted in Spanish, Catalan, and English. Students aged 10 to 16 (N > 600) were asked to draw pictures of people using specific prompts. The prompts in Spanish and Catalan were written using the generic masculine, 'los presidentes' 'els presidents' (presidents); using double gendered language such as 'ninos y ninas', 'nens i nenes' (boys and girls); and using non-gendered words like 'alumnado' 'alumnat' (students). The prompts were subdivided into people in school contexts participants could identify with, such as students and teachers; occupations mostly associated with men, such as pilots and firefighters; and occupations associated with women, such as ballet dancers and nurses. As could be expected, the participants only drew approximately the same percentage of female and male characters when double-gendered language or non-gendered words such as 'students' or 'teachers' were used, regardless of the language used in the experiment. When they were asked to draw people using the so-called generic masculine in Spanish or Catalan, 'los estudiantes' 'els estudiants' (students), less than 35% of the drawings contained female characters. The differences between the results for Rainbow and Non-Rainbow schools will be discussed in the light of the innovative coeducation programmes and learners' perceptions on gender-fair language gathered in the surveys and interviews.

Keywords: gender-fair language, gender-inclusive schools, learners’ and teachers’ perceptions and attitudes, rainbow coeducation programmes

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Authors: Mengjiao Fan, Thomson W. L. Wong

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It is unavoidable that older adults may have to deal with aging-related motor problems. Aging is highly likely to affect motor learning and control as well. For example, older adults may suffer from poor motor function and quality of life due to age-related eye changes. These adverse changes in vision results in impairment of movement automaticity. Reaching is a fundamental component of various complex movements, which is therefore beneficial to explore the changes and adaptation in visuomotor behaviors. The current study aims to explore how aging affects visuomotor behaviors by comparing motor performance and gaze behaviors between two age groups (i.e., young and older adults). Visuomotor behaviors in reaching under providing or blocking online visual feedback (simulated visual deficiency) conditions were investigated in 60 healthy young adults (Mean age=24.49 years, SD=2.12) and 37 older adults (Mean age=70.07 years, SD=2.37) with normal or corrected-to-normal vision. Participants in each group were randomly allocated into two subgroups. Subgroup 1 was provided with online visual feedback of the hand-controlled mouse cursor. However, in subgroup 2, visual feedback was blocked to simulate visual deficiency. The experimental task required participants to complete 20 times of reaching to a target by controlling the mouse cursor on the computer screen. Among all the 20 trials, start position was upright in the center of the screen and target appeared at a randomly selected position by the tailor-made computer program. Primary outcomes of motor performance and gaze behaviours data were recorded by the EyeLink II (SR Research, Canada). The results suggested that aging seems to affect the performance of reaching tasks significantly in both visual feedback conditions. In both age groups, blocking online visual feedback of the cursor in reaching resulted in longer hand movement time (p < .001), longer reaching distance away from the target center (p<.001) and poorer reaching motor accuracy (p < .001). Concerning gaze behaviors, blocking online visual feedback increased the first fixation duration time in young adults (p<.001) but decreased it in older adults (p < .001). Besides, under the condition of providing online visual feedback of the cursor, older adults conducted a longer fixation dwell time on target throughout reaching than the young adults (p < .001) although the effect was not significant under blocking online visual feedback condition (p=.215). Therefore, the results suggested that different levels of visual feedback during movement execution can affect gaze behaviors differently in older and young adults. Differential effects by aging on visuomotor behaviors appear on two visual feedback patterns (i.e., blocking or providing online visual feedback of hand-controlled cursor in reaching). Several specific gaze behaviors among the older adults were found, which imply that blocking of visual feedback may act as a stimulus to seduce extra perceptive load in movement execution and age-related visual degeneration might further deteriorate the situation. It indeed provides us with insight for the future development of potential rehabilitative training method (e.g., well-designed errorless training) in enhancing visuomotor adaptation for our aging population in the context of improving their movement automaticity by facilitating their compensation of visual degeneration.

Keywords: aging effect, movement automaticity, reaching, visuomotor behaviors, visual degeneration

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Authors: Mihaela Beregoi, Horia Iovu, Cristina Busuioc, Alexandru Evanghelidis, Elena Matei, Monica Enculescu, Ionut Enculescu

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Nanomaterials field is very attractive for all researchers who are attempting to develop new devices with the same or improved properties than the micro-sized ones, while reducing the reagents and power consumptions. In this way, a wide range of nanomaterials were fabricated and integrated in applications for electronics, optoelectronics, solar cells, tissue reconstruction and drug delivery. Obviously, the most appealing ones are those dedicated to the medical domain. Different types of nano-sized materials, such as particles, fibers, films etc., can be synthesized by using physical, chemical or electrochemical methods. One of these techniques is electrospinning, which enable the production of fibers with nanometric dimensions by pumping a polymeric solution in a high electric field; due to the electrostatic charging and solvent evaporation, the precursor mixture is converted into nonwoven meshes with different fiber densities and mechanical properties. Moreover, polyaniline is a conducting polymer with interesting optical properties, suitable for displays and electrochromic windows. Otherwise, polyaniline is an electroactive polymer that can contract/expand by applying electric stimuli, due to the oxidation/reduction reactions which take place in the polymer chains. These two main properties can be exploited in order to synthesize smart materials that change their dimensions, exhibiting in the same time good electrochromic properties. In the context aforesaid, a poly(methyl metacrylate) solution was spun to get webs composed of fibers with diameter values between 500 nm and 1 µm. Further, the polymer meshes were covered with a gold layer in order to make them conductive and also appropriate as working electrode in an electrochemical cell. The gold shell was deposited by DC sputtering. Such metalized fibers can be transformed into smart materials by covering them with a thin layer of conductive polymer. Thus, the webs were coated with a polyaniline film by the electrochemical route, starting from and aqueous solution of aniline and sulfuric acid, where sulfuric acid acts as oxidant agent. For the polymerization of aniline, a saturated calomel electrode was employed as reference, a platinum plate as counter electrode and the gold covered webs as working electrode. Chronoamperometry was selected as deposition method for polyaniline, by modifying the deposition time. Metalized meshes with different fiber densities were used, the transmission ranging between 70 and 80 %. The morphological investigation showed that polyaniline layer has a granular structure for all deposition experiments. As well, some preliminary optical tests were done by using sulfuric acid as electrolyte, which revealed the modification of polyaniline colour from green to dark blue when applying a voltage. In conclusion, new multilayered materials were obtained by a simple approach: the merge of the electrospinning method benefits with polyaniline chemistry. This synthesis method allows the fabrication of structures with reproducible characteristics, suitable for display or tissue substituents.

Keywords: electrospinning, fibers, smart materials, polyaniline

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Authors: S. F. Afzali, W. L. Wong

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The epizootic ulcerative syndrome (EUS) causes by the oomycete fungus, Aphanomyces invadans; known to be one of the infectious fish diseases for farmed and wild fishes in fresh and brackish-water from the Asia-pacific region, America and Africa. Although, EUS had been documented by the Office International des Epizooties (OIE) since 1995, hitherto, there is neither standard chemical agents that can be used for successful treatment of this destructive infection in the time of outbreak; nor available vaccine for prevention. Plant-based remedies in controlling fish diseases are gaining much attention recently as an alternative to chemical treatments, which possess negative effects to the environment and human. In present study, Sonneratia alba, a mangrove plant belongs to the Sonneratiaceae family, was screened in vitro and in vivo for its antifungal activity against A. invadans mycelium growth and its effects on fish innate immune system and disease resistant. The in vitro tests was performed using the disc diffusion methods with measurements of minimum inhibitory concentration (MIC) and inhibition zone. For in vivo study, the S. alba extract supplemented diets were administrated at 0.0, 1.0%, 3.0%, and 5.0% on healthy goldfish, Carassius auratus, which challenged with A. invadans zoospores (100 spores/ml). To compare the significant differences in the hematological and immunological parameters obtained from the experiments, the data were analysed using the SPSS. The methanol extract of S. alba effectively inhibited the mycelial growth of A. invadans at a minimum concentration of 1000 ppm for agar and filter paper diffusion experiments. In the agar diffusion test, 500 ppm of the extract inhibited the fungus mycelial growth up to 96 hours after exposure. The mycelial growth from the edge of the pre-inoculated A. invadans agar discs treated with S. alba extracts at concentrations of 100, 500 and 1000 ppm were 15, 8 and 0 mm respectively. The results of the filter paper disc test showed that the S. alba extract at its minimal inhibitory concentration (1000 ppm) has similar qualitative inhibitory effect as malachite green at 1 ppm and formalin at 250 ppm. According to the in vivo tests findings, in the infected fish fed with 3.0% and 5.0% supplementation diet, the numbers of white blood cell and myeloperoxidase activity significantly increased after the second week of treatment. Whilst the numbers of red blood cell significantly decreased in the infected fish fed with 0.0 and 1.0% supplementation diet. After the third week of feeding, significant increases in the total protein, albumin level, lysozyme activity were recorded in the infected fish fed with 3.0% and 5.0% supplementation diet. Also, the enriched diets increased the survival rate as compared to the untreated group that suffered from 90% mortality. The present study indicated that S. alba extract may inhibit the mycelial growth of A. invadans effectively, suggesting an alternative to other chemotherapeutic agents, which brought much environmental and health concerns to the public, for EUS treatment.

Keywords: fungal pathogen, goldfish, organic extract, treatment

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Authors: Cristina Maria Mendoza Sanchez, Maria Angeles Navarro Perán

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Introduction: Nursing as a profession, from its initial formation and after its development in practice, has been built and identified mainly from its technical competence and professionalization within the positivist approach of the XIX century that provides a conception of the disease built on the basis of to the biomedical paradigm, where the care provided is more focused on the physiological processes and the disease than on the suffering person understood as a whole. The main issue that is in need of study here is a review of the nursing profession's history to get to know how the nursing profession was before the XIX century. It is unclear if there were organizations or people with knowledge about looking after others or if many people survived by chance. The holistic care, in which the appearance of the disease directly affects all its dimensions: physical, emotional, cognitive, social and spiritual. It is not a concept from the 21st century. It is common practice, most probably since established life in this world, with the final purpose of covering all these perspectives through quality care. Objective: In this paper, we describe and analyze the history of education in nursing learning in terms of reviewing and analysing theoretical foundations of clinical teaching and learning in nursing, with the final purpose of determining and describing the development of the nursing profession along the history. Method: We have done a descriptive systematic review study, doing a systematically searched of manuscripts and articles in the following health science databases: Pubmed, Scopus, Web of Science, Temperamentvm and CINAHL. The selection of articles has been made according to PRISMA criteria, doing a critical reading of the full text using the CASPe method. A compliment to this, we have read a range of historical and contemporary sources to support the review, such as manuals of Florence Nightingale and John of God as primary manuscripts to establish the origin of modern nursing and her professionalization. We have considered and applied ethical considerations of data processing. Results: After applying inclusion and exclusion criteria in our search, in Pubmed, Scopus, Web of Science, Temperamentvm and CINAHL, we have obtained 51 research articles. We have analyzed them in such a way that we have distinguished them by year of publication and the type of study. With the articles obtained, we can see the importance of our background as a profession before modern times in public health and as a review of our past to face challenges in the near future. Discussion: The important influence of key figures other than Nightingale has been overlooked and it emerges that nursing management and development of the professional body has a longer and more complex history than is generally accepted. Conclusions: There is a paucity of studies on the subject of the review to be able to extract very precise evidence and recommendations about nursing before modern times. But even so, as more representative data, an increase in research about nursing history has been observed. In light of the aspects analyzed, the need for new research in the history of nursing emerges from this perspective; in order to germinate studies of the historical construction of care before the XIX century and theories created then. We can assure that pieces of knowledge and ways of care were taught before the XIX century, but they were not called theories, as these concepts were created in modern times.

Keywords: nursing history, nursing theory, Saint John of God, Florence Nightingale, learning, nursing education

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Authors: Elaine Yee Sing Wong, Jonine Jancey, Andy H. Lee, Anthony P. James

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Singapore has a rapidly aging population, where the majority of older women aged 50 years and above, are physically inactive and have unhealthy dietary habits, placing them at ‘high risk’ of non-communicable diseases. Given the multiplicity of less than optimal dietary habits and high levels of physical inactivity among Singaporean women, it is imperative to develop appropriate lifestyle interventions at recreational centres to enhance both their physical and nutritional knowledge, as well as provide them with the opportunity to develop skills to support behaviour change. To the best of our knowledge, this proposed study is the first physical activity and nutrition cluster randomised controlled trial conducted in Singapore for older women. Findings from this study may provide insights and recommendations for policy makers and key stakeholders to create new healthy living, recreational centres with supportive environments. This 6-month community-based cluster randomised controlled trial will involve the implementation and evaluation of physical activity and nutrition program for community dwelling Singaporean women, who currently attend recreational centres to promote social leisure activities in their local neighbourhood. The intervention will include dietary education and counselling sessions, physical activity classes, and telephone contact by certified fitness instructors and qualified nutritionists. Social Cognitive Theory with Motivational Interviewing will inform the development of strategies to support health behaviour change. Sixty recreational centres located in Singapore will be randomly selected from five major geographical districts and randomly allocated to the intervention (n=30) or control (n=30) cluster. A sample of 600 (intervention n=300; control n=300) women aged 50 years and above will then be recruited from these recreational centres. The control clusters will only undergo pre and post data collection and will not receive the intervention. It is hypothesised that by the end of the intervention, the intervention group participants (n = 300) compared to the control group (n = 300), will show significant improvements in the following variables: lipid profile, body mass index, physical activity and dietary behaviour, anthropometry, mental and physical health. Data collection will be examined and compared via the Statistical Package for the Social Science version 23. Descriptive and summary statistics will be used to quantify participants’ characteristics and outcome variables. Multi-variable mixed regression analyses will be used to confirm the effects of the proposed health intervention, taking into account the repeated measures and the clustering of the observations. The research protocol was approved by the Curtin University Human Research Ethics Committee (approval number: HRE2016-0366). The study has been registered with the Australian and New Zealand Clinical Trial Registry (12617001022358).

Keywords: community based, healthy aging, intervention, nutrition, older women, physical activity

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Authors: Fernanda Piculo, Giovana Vesentini, Gabriela Marini, Debora Cristina Damasceno, Angelica Mercia Pascon Barbosa, Marilza Vieira Cunha Rudge

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Introduction: Women with previous gestational diabetes mellitus (GDM) were significantly more likely to have urinary incontinence (UI) and pelvic floor muscle dysfunction compared to non-diabetic women two years after a cesarean section. Additional results demonstrated that induced diabetes causes detrimental effects on pregnant rat urethral muscle. These results indicate the need for exploration of the mechanistic role of a recovery factor in female UI. Chemokine ligand 7 (CCL7) was significantly over expressed in rat serum, urethral and vaginal tissues immediately following induction of stress UI in a rat model simulating birth trauma. CCL7 over expression has shown potency for stimulating targeted stem cell migration and provide a translational link (clinical measurement) which further provide opportunities for treatment. The aim of this study was to investigate the CCL7 levels profile in diabetic pregnant women with urinary incontinence during pregnancy over the first year postpartum. Methods: This study was conducted in the Perinatal Diabetes Research Center of the Botucatu Medical School/UNESP, and was approved by the Research Ethics Committee of the Institution (CAAE: 20639813.0.0000.5411). The diagnosis of GDM was established between 24th and 28th gestational weeks, by the 75 g-OGTT test according to ADA’s criteria. Urinary incontinence was defined according to the International Continence Society and the CCL7 levels was measured by ELISA (R&D Systems, Catalog Number DCC700). Two hundred twelve women were classified into four study groups: normoglycemic continent (NC), normoglycemic incontinent (NI), diabetic continent (DC) and diabetic incontinent (DI). They were evaluated at six-time-points: 12-18, 24-28 and 34-38 gestational weeks, 24-48 hours, 6 weeks and 6-12 months postpartum. Results: At 12-18 weeks, it was possible to consider only two groups, continent and incontinent, because at this early gestational period has not yet been the diagnosis of GDM. The group with GDM and UI (DI group) showed lower levels of CCL7 in all time points during pregnancy and postpartum, compared to normoglycemic groups (NC and NI), indicating that these women have not recovered from child birth induced UI during the 6-12 months postpartum compared to their controls, and that the progression of UI and/or lack of recovery throughout the first postpartum year can be related with lower levels of CCL7. Instead, serum CCL7 was significantly increased in the NC group. Taken together, these findings of overexpression of CCL7 in the NC group and decreased levels in the DI group, could confirm that diabetes delays the recovery from child birth induced UI, and that CCL7 could potentially be used as a serum marker of injury. Conclusion: This study demonstrates lower levels of CCL7 in the DI group during pregnancy and postpartum and suggests that the progression of UI in diabetic women and/or lack of recovery throughout the first postpartum year can be related with low levels of CCL7. This provides a translational potential where CCL7 measurement could be used as a surrogate for injury after delivery. Successful controlled CCL7 mediated stem cell homing to the lower urinary tract could one day introduce the potential for non-operative treatment or prevention of stress urinary incontinence.

Keywords: CCL7, gestational diabetes, pregnancy, urinary incontinence

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Authors: Jason Ceavers, Travis Thompson, Juan Torres, Ramanakumar Anam, Alan Wong, Andrei Carvalho, Shane Quo, Shawn Alonso, Moises Cintron, Ricardo C. Carrero, German Lopez, Vamsi Garimella, German Giese

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Introduction: Advance directives (AD) are essential for patients to communicate their wishes when they are not able to. Ideally, these discussions should not occur for the first time when a patient is hospitalized with an acute life-threatening illness. There is a large number of patients who do not have clearly documented ADs, resulting in the misutilization of resources and additional patient harm. This is a nationwide issue, and the Joint Commission has it as one of its national quality metrics. Presented here is a proposed protocol to increase the number of documented AD discussions in a community-based, internal medicine residency primary care clinic in South Florida. Methods: The SMART Aim for this quality improvement project is to increase documentation of AD discussions in the outpatient setting by 25% within three months in medicare patients. A survey was sent to stakeholders (clinic attendings, residents, medical assistants, front desk staff, and clinic managers), asking them for three factors they believed contributed most to the low documentation rate of AD discussions. The two most important factors were time constraints and systems issues (such as lack of a standard method to document ADs and ADs not being uploaded to the chart) which were brought up by 25% and 21.2% of the 32 survey responders, respectively. Pre-intervention data from clinic patients in 2020-2021 revealed 17.05% of patients had clear, actionable ADs documented. To address these issues, an AD pocket card was created to give to patients. One side of the card has a brief explanation of what ADs are. The other side has a column of interventions (cardiopulmonary resuscitation, mechanical ventilation, dialysis, tracheostomy, feeding tube) with boxes patients check off if they want the intervention done, do not want the intervention, do not want to discuss the topic, or need more information. These cards are to be filled out and scanned into their electronic chart to be reviewed by the resident before their appointment. The interventions that patients want more information on will be discussed by the provider. If any changes are made, the card will be re-scanned into their chart. After three months, we will chart review the patients seen in the clinic to determine how many medicare patients have a pocket card uploaded and how many have advance directives discussions documented in a progress note or annual wellness note. If there is not enough time for an AD discussion, a follow-up appointment can be scheduled for that discussion. Discussion: ADs are a crucial part of patient care, and failure to understand a patient’s wishes leads to improper utilization of resources, avoidable litigation, and patient harm. Time constraints and systems issues were identified as two major factors contributing to the lack of advance directive discussion in our community-based resident primary care clinic. Our project aims at increasing the documentation rate for ADs through a simple pocket card intervention. These are self-explanatory, easy to read and allow the patients to clearly express what interventions they desire or what they want to discuss further with their physician.

Keywords: advance directives, community-based, pocket card, primary care clinic

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Authors: Yi H. Wong, Li L. Chan, Chee O. Leong, Stephen Ambu, Joon W. Mak, Priyadashi S. Sahu

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Background: Acanthamoeba is a free-living opportunistic protist which is ubiquitously distributed in the environment. Virulent Acanthamoeba can cause fatal encephalitis in immunocompromised patients and potential blinding keratitis in immunocompetent contact lens wearers. Approximately 24 species have been identified but only the A. castellanii, A. polyphaga and A. culbertsoni are commonly associated with human infections. Until to date, the precise molecular basis for Acanthamoeba pathogenesis remains unclear. Previous studies reported that Acanthamoeba virulence can be diminished through prolonged axenic culture but revived through serial mouse passages. As no clear explanation on this reversible pathogenesis is established, hereby, we postulate that the epigenetic regulators, DNA-methyltransferases (DNMT) and histone-deacetylases (HDAC), could possibly be involved in granting the virulence plasticity of Acanthamoeba spp. Methods: Four rounds of mouse passages were conducted to revive the virulence potential of the virulence-attenuated Acanthamoeba castellanii strain (ATCC 50492). Briefly, each mouse (n=6/group) was inoculated intraperitoneally with Acanthamoebae cells (2x 105 trophozoites/mouse) and incubated for 2 months. Acanthamoebae cells were isolated from infected mouse organs by culture method and subjected to subsequent mouse passage. In vitro cytopathic, encystment and gelatinolytic assays were conducted to evaluate the virulence characteristics of Acanthamoebae isolates for each passage. PCR primers which targeted on the 2 members (DNMT1 and DNMT2) and 5 members (HDAC1 to 5) of the DNMT and HDAC gene families respectively were custom designed. Quantitative real-time PCR (qPCR) was performed to detect and quantify the relative expression of the two gene families in each Acanthamoeba isolates. Beta-tubulin of A. castellanii (Genbank accession no: XP_004353728) was included as housekeeping gene for data normalisation. PCR mixtures were also analyzed by electrophoresis for amplicons detection. All statistical analyses were performed using the paired one-tailed Student’s t test. Results: Our pathogenicity tests showed that the virulence-reactivated Acanthamoeba had a higher degree of cytopathic effect on vero cells, a better resistance to encystment challenge and a higher gelatinolytic activity which was catalysed by serine protease. qPCR assay showed that DNMT1 expression was significantly higher in the virulence-reactivated compared to the virulence-attenuated Acanthamoeba strain (p ≤ 0.01). The specificity of primers which targeted on DNMT1 was confirmed by sequence analysis of PCR amplicons, which showed a 97% similarity to the published DNA-methyltransferase gene of A. castellanii (GenBank accession no: XM_004332804.1). Out of the five primer pairs which targeted on the HDAC family genes, only HDAC4 expression was significantly difference between the two variant strains. In contrast to DNMT1, HDAC4 expression was much higher in the virulence-attenuated Acanthamoeba strain. Conclusion: Our mouse passages had successfully restored the virulence of the attenuated strain. Our findings suggested that DNA-methyltransferase (DNMT1) and histone deacetylase (HDAC4) expressions are associated with virulence potential of Acanthamoeba spp.

Keywords: acanthamoeba, DNA-methyltransferase, histone deacetylase, virulence-associated proteins

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Authors: Yi-Hao Wong, Li-Li Chan, Chee-Onn Leong, Stephen Ambu, Joon-Wah Mak, Priyasashi Sahu

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Background: Acanthamoeba is a genus of amoebae which lives as a free-living in nature or as a human pathogen that causes severe brain and eye infections. Virulence potential of Acanthamoeba is not constant and can change with growth conditions. DNA methylation, an epigenetic process which adds methyl groups to DNA, is used by eukaryotic cells, including several human parasites to control their gene expression. We used qPCR, siRNA gene silencing, and RNA sequencing (RNA-Seq) to study DNA-methyltransferase gene family (DNMT) in order to indicate the possibility of its involvement in programming Acanthamoeba virulence potential. Methods: A virulence-attenuated Acanthamoeba isolate (designation: ATCC; original isolate: ATCC 50492) was subjected to mouse passages to restore its pathogenicity; a virulence-reactivated isolate (designation: AC/5) was generated. Several established factors associated with Acanthamoeba virulence phenotype were examined to confirm the succession of reactivation process. Differential gene expression of DNMT between ATCC and AC/5 isolates was performed by qPCR. Silencing on DNMT gene expression in AC/5 isolate was achieved by siRNA duplex. Total RNAs extracted from ATCC, AC/5, and siRNA-treated (designation: si-146) were subjected to RNA-Seq for comparative transcriptomic analysis in order to identify the genome-wide effect of DNMT in regulating Acanthamoeba gene expression. qPCR was performed to validate the RNA-Seq results. Results: Physiological and cytophatic assays demonstrated an increased in virulence potential of AC/5 isolate after mouse passages. DNMT gene expression was significantly higher in AC/5 compared to ATCC isolate (p ≤ 0.01) by qPCR. si-146 duplex reduced DNMT gene expression in AC/5 isolate by 30%. Comparative transcriptome analysis identified the differentially expressed genes, with 3768 genes in AC/5 vs ATCC isolate; 2102 genes in si-146 vs AC/5 isolate and 3422 genes in si-146 vs ATCC isolate, respectively (fold-change of ≥ 2 or ≤ 0.5, p-value adjusted (padj) < 0.05). Of these, 840 and 1262 genes were upregulated and downregulated, respectively, in si-146 vs AC/5 isolate. Eukaryotic orthologous group (KOG) assignments revealed a higher percentage of downregulated gene expression in si-146 compared to AC/5 isolate, were related to posttranslational modification, signal transduction and energy production. Gene Ontology (GO) terms for those downregulated genes shown were associated with transport activity, oxidation-reduction process, and metabolic process. Among these downregulated genes were putative genes encoded for heat shock proteins, transporters, ubiquitin-related proteins, proteins for vesicular trafficking (small GTPases), and oxidoreductases. Functional analysis of similar predicted proteins had been described in other parasitic protozoa for their survival and pathogenicity. Decreased expression of these genes in si146-treated isolate may account in part for Acanthamoeba reduced pathogenicity. qPCR on 6 selected genes upregulated in AC/5 compared to ATCC isolate corroborated the RNA sequencing findings, indicating a good concordance between these two analyses. Conclusion: To the best of our knowledge, this study represents the first genome-wide analysis of DNA methylation and its effects on gene expression in Acanthamoeba spp. The present data indicate that DNA methylation has substantial effect on global gene expression, allowing further dissection of the genome-wide effects of DNA-methyltransferase gene in regulating Acanthamoeba pathogenicity.

Keywords: Acanthamoeba, DNA methylation, RNA sequencing, virulence

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Authors: Adenike Adesanya, Nonthaphat Wong, Xiang-Yun Lan, Shea Ping Yip, Chien-Ling Huang

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Background: The most challenging mutation of the oncokinase BCR-ABL protein T315I, which is commonly known as the “gatekeeper” mutation and is notorious for its strong resistance to almost all tyrosine kinase inhibitors (TKIs), especially imatinib. Therefore, this study aims to identify T315I-dependent downstream microRNA (miRNA) pathways associated with drug resistance in chronic myeloid leukemia (CML) for prognostic and therapeutic purposes. Methods: T315I-carrying K562 cell clones (K562-T315I) were generated by the CRISPR-Cas9 system. Imatinib-treated K562-T315I cells were subjected to small RNA library preparation and next-generation sequencing. Putative lncRNA-miRNA-mRNA networks were analyzed with (i) DESeq2 to extract differentially expressed miRNAs, using Padj value of 0.05 as cut-off, (ii) STarMir to obtain potential miRNA response element (MRE) binding sites of selected miRNAs on lncRNA H19, (iii) miRDB, miRTarbase, and TargetScan to predict mRNA targets of selected miRNAs, (iv) IntaRNA to obtain putative interactions between H19 and the predicted mRNAs, (v) Cytoscape to visualize putative networks, and (vi) several pathway analysis platforms – Enrichr, PANTHER and ShinyGO for pathway enrichment analysis. Moreover, mitochondria isolation and transcript quantification were adopted to determine the new mechanism involved in T315I-mediated resistance of CML treatment. Results: Verification of the CRISPR-mediated mutagenesis with digital droplet PCR detected the mutation abundance of ≥80%. Further validation showed the viability of ≥90% by cell viability assay, and intense phosphorylated CRKL protein band being detected with no observable change for BCR-ABL and c-ABL protein expressions by Western blot. As reported by several investigations into hematological malignancies, we determined a 7-fold increase of H19 expression in K562-T315I cells. After imatinib treatment, a 9-fold increment was observed. DESeq2 revealed 171 miRNAs were differentially expressed K562-T315I, 112 out of these miRNAs were identified to have MRE binding regions on H19, and 26 out of the 112 miRNAs were significantly downregulated. Adopting the seed-sequence analysis of these identified miRNAs, we obtained 167 mRNAs. 6 hub miRNAs (hsa-let-7b-5p, hsa-let-7e-5p, hsa-miR-125a-5p, hsa-miR-129-5p, and hsa-miR-372-3p) and 25 predicted genes were identified after constructing hub miRNA-target gene network. These targets demonstrated putative interactions with H19 lncRNA and were mostly enriched in pathways related to cell proliferation, senescence, gene silencing, and pluripotency of stem cells. Further experimental findings have also shown the up-regulation of mitochondrial transcript and lncRNA MALAT1 contributing to the lncRNA-miRNA-mRNA networks induced by BCR-ABL T315I mutation. Conclusions: Our results have indicated that lncRNA-miRNA regulators play a crucial role not only in leukemogenesis but also in drug resistance, considering the significant dysregulation and interactions in the K562-T315I cell model generated by CRISPR-Cas9. In silico analysis has further shown that lncRNAs H19 and MALAT1 bear several complementary miRNA sites. This implies that they could serve as a sponge, hence sequestering the activity of the target miRNAs.

Keywords: chronic myeloid leukemia, imatinib resistance, lncRNA-miRNA-mRNA, T315I mutation

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Authors: Lacramioara Ochiuz, Aurelia Vasile, Iulian Stoleriu, Cristina Ghiciuc, Maria Ignat

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Topical administration of chemotherapeutic agents (eg. carmustine, bexarotene, mechlorethamine etc.) in local treatment of cutaneous T-cell lymphoma (CTCL) is accompanied by multiple side effects, such as contact hypersensitivity, pruritus, skin atrophy or even secondary malignancies. A known method of reducing the side effects of anticancer agent is the development of modified drug release systems using drug incapsulation in biocompatible nanoporous inorganic matrices, such as mesoporous MCM-41 silica. Mesoporous MCM-41 silica is characterized by large specific surface, high pore volume, uniform porosity, and stable dispersion in aqueous medium, excellent biocompatibility, in vivo biodegradability and capacity to be functionalized with different organic groups. Therefore, MCM-41 is an attractive candidate for a wide range of biomedical applications, such as controlled drug release, bone regeneration, protein immobilization, enzymes, etc. The main advantage of this material lies in its ability to host a large amount of the active substance in uniform pore system with adjustable size in a mesoscopic range. Silanol groups allow surface controlled functionalization leading to control of drug loading and release. This study shows (I) the amino-grafting optimization of mesoporous MCM-41 silica matrix by means of co-condensation during synthesis and post-synthesis using APTES (3-aminopropyltriethoxysilane); (ii) loading the therapeutic agent (carmustine) obtaining a modified drug release systems; (iii) determining the profile of in vitro carmustine release from these systems; (iv) assessment of carmustine release kinetics by fitting on four mathematical models. Obtained powders have been described in terms of structure, texture, morphology thermogravimetric analysis. The concentration of the therapeutic agent in the dissolution medium has been determined by HPLC method. In vitro dissolution tests have been done using cell Enhancer in a 12 hours interval. Analysis of carmustine release kinetics from mesoporous systems was made by fitting to zero-order model, first-order model Higuchi model and Korsmeyer-Peppas model, respectively. Results showed that both types of highly ordered mesoporous silica (amino grafted by co-condensation process or post-synthesis) are thermally stable in aqueous medium. In what regards the degree of loading and efficiency of loading with the therapeutic agent, there has been noticed an increase of around 10% in case of co-condensation method application. This result shows that direct co-condensation leads to even distribution of amino groups on the pore walls while in case of post-synthesis grafting many amino groups are concentrated near the pore opening and/or on external surface. In vitro dissolution tests showed an extended carmustine release (more than 86% m/m) both from systems based on silica functionalized directly by co-condensation and after synthesis. Assessment of carmustine release kinetics revealed a release through diffusion from all studied systems as a result of fitting to Higuchi model. The results of this study proved that amino-functionalized mesoporous silica may be used as a matrix for optimizing the anti-cancer topical therapy by loading carmustine and developing prolonged-release systems.

Keywords: carmustine, silica, controlled, release

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Authors: Adriana Muñoz-Talavera, Ana Rosa Rincón-Sánchez, Abraham Escobedo-Moratilla, María Cristina Islas-Carbajal, Miguel Ángel Gómez-Lim

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The highest rates of diabetes incidence and prevalence worldwide will increase the number of diabetic patients requiring insulin or insulin analogues. Then, current production systems would not be sufficient to meet the future market demands. Therefore, developing efficient expression systems for insulin and insulin analogues are needed. In addition, insulin analogues with better pharmacokinetics and pharmacodynamics properties and without mitogenic potential will be required. SCI-57 (single chain insulin-57) is an insulin analogue having 10 times greater affinity to the insulin receptor, higher resistance to thermal degradation than insulin, native mitogenicity and biological effect. Plants as expression platforms have been used to produce recombinant proteins because of their advantages such as cost-effectiveness, posttranslational modifications, absence of human pathogens and high quality. Immunoglobulin production with a yield of 50% has been achieved by transient expression in Nicotiana benthamiana (Nb). The aim of this study is to produce SCI-57 by transient expression in Nb. Methodology: DNA sequence encoding SCI-57 was cloned in pICH31070. This construction was introduced into Agrobacterium tumefaciens by electroporation. The resulting strain was used to infiltrate leaves of Nb. In order to isolate SCI-57, leaves from transformed plants were incubated 3 hours with the extraction buffer therefore filtrated to remove solid material. The resultant protein solution was subjected to anion exchange chromatography on an FPLC system and ultrafiltration to purify SCI-57. Detection of SCI-57 was made by electrophoresis pattern (SDS-PAGE). Protein band was digested with trypsin and the peptides were analyzed by Liquid chromatography tandem-mass spectrometry (LC-MS/MS). A purified protein sample (20µM) was analyzed by ESI-Q-TOF-MS to obtain the ionization pattern and the exact molecular weight determination. Chromatography pattern and impurities detection were performed using RP-HPLC using recombinant insulin as standard. The identity of the SCI-57 was confirmed by anti-insulin ELISA. The total soluble protein concentration was quantified by Bradford assay. Results: The expression cassette was verified by restriction mapping (5393 bp fragment). The SDS-PAGE of crude leaf extract (CLE) of transformed plants, revealed a protein of about 6.4 kDa, non-present in CLE of untransformed plants. The LC-MS/MS results displayed one peptide with a high score that matches SCI-57 amino acid sequence in the sample, confirming the identity of SCI-57. From the purified SCI-57 sample (PSCI-57) the most intense charge state was 1069 m/z (+6) on the displayed ionization pattern corresponding to the molecular weight of SCI-57 (6412.6554 Da). The RP-HPLC of the PSCI-57 shows the presence of a peak with similar retention time (rt) and UV spectroscopic profile to the insulin standard (SCI-57 rt=12.96 and insulin rt=12.70 min). The collected SCI-57 peak had ELISA signal. The total protein amount in CLE from transformed plants was higher compared to untransformed plants. Conclusions: Our results suggest the feasibility to produce insulin analogue SCI-57 by transient expression in Nicotiana benthamiana. Further work is being undertaken to evaluate the biological activity by glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes.

Keywords: insulin analogue, mass spectrometry, Nicotiana benthamiana, transient expression

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Authors: Jia H. Wong, Jingli Zhang, Habsah Mohamad, Iswatun H. Abdullah Ripain, Muhammad Bilal, Amelia J. Lloyd, Abdul A. Mohamed Yusoff, Jafri M. Abdullah

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Epilepsy is one of the most common neurological diseases affecting more than 50 million of people worldwide. Epilepsy is a state of recurrent, spontaneous seizures with multiple syndromes and symptoms of different causes of brain dysfunction, prognosis, and treatments; characterized by transient, occasional and stereotyped interruptions of behavior whereby the excitatory-inhibitory activities within the central nervous system (CNS) are thrown out of balance due to various kinds of interferences. The goal of antiepileptic treatment is to enable patients to be free from seizures or to achieve control of seizures through surgical treatment and/or pharmacotherapy. Pharmacotherapy through AED plays an important role especially in countries with epilepsy treatment gap due to costs and availability of health facilities, skills and resources, yet there are about one-third of the people with epilepsy have drug-resistant seizures. Hence, this poses considerable challenges to the healthcare system and the effort in providing cost-effective treatment as well as the search for alternatives to treatment and management of epilepsy. Enhancement of γ-aminobutyric acid (GABA)-mediated inhibitory neurotransmission is one of the key mechanisms of actions of antiepileptic drugs. GABA type > a receptors (GABAAR) are ligand-gated ion channels that mediate rapid inhibitory neurotransmission upon the binding of GABA with a heteropentameric structure forming a central pore that is permeable to the influx of chloride ions in its activated state. The major isoform of GABAA receptors consists of two α1, two β2, and one γ2 subunit. It is the most abundantly expressed combinations in the brain and the most commonly researched through Xenopus laevis oocytes. With the advancing studies on ethnomedicine and traditional treatments using medicinal plants, increasing evidence reveal that spice and herb plants with medicinal properties play an important role in the treatment of ailments within communities across different cultures. Capsaicin is the primary natural capsaicinoid in hot peppers of plant genus Capsicum, consist of an aromatic ring, an amide linkage and a hydrophobic side chain. The study showed that capsaicins conferred neuroprotection in status epilepticus mouse models through anti-ictogenic, hypothermic, antioxidative, anti-inflammatory, and anti-apoptotic actions in a dose-dependent manner. In this study, five capsaicin derivatives were tested for their ability to increase the GABA-induced chloride current on α1β2γ2S of GABAAR expressed on Xenopus laevis oocytes using the method of two-microelectrode voltage clamp. Two of the capsaicin derivatives, IS5 (N-(4-hydroxy-3-methoxybenzyl)-3-methylbutyramide) and IS10 (N-(4-hydroxy-3-methoxybenzyl)-decanamide) at a concentration of 30µM were able to significantly increase the GABA-induced chloride current with p=0.002 and p=0.026 respectively. This study were able to show the enhancement effect of two capsaicin derivatives with moderate length of hydrocarbon chain on this receptor subtype, revealing the promising inhibitory activity of capsaicin derivatives through enhancement of GABA-induced chloride current and further investigations should be carried out to verify its antiepileptic effects in animal models.

Keywords: α1β2γ2 GABAA receptors, α1β2γ2S, antiepileptic, capsaicin derivatives, two-microelectrode voltage clamp, Xenopus laevis oocytes

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Authors: Federica Verna, Alessandra Pautasso, Maria Caramelli, Cristiana Maurella, Walter Mignone, Cristina Casalone

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Mosquito-Borne Diseases (MBDs) are dangerously increasing in prevalence, geographical distribution and severity, representing an emerging threat for both humans and animals. Interaction between multiple disciplines is needed for an effective early warning, surveillance and control of MBDs, according to the One Health concept. This work reports the integrated surveillance system enforced by IZSPLV in Piedmont, Liguria and Valle d’Aosta regions (Northwestern Italy) in order to control MDBs spread. Veterinary services and local human health authority are involved in an information network, to connect the surveillance of human clinical cases with entomological surveillance and veterinary monitoring in order to implement control measures in case of outbreak. A systematic entomological surveillance is carried out during the vector season using mosquitoes traps located in sites selected according to risk factors. Collected mosquitoes are counted, identified to species level by morphological standard classification keys and pooled by collection site, date and species with a maximum of 100 individuals. Pools are analyzed, after RNA extraction, by Real Time RT-PCR distinctive for West Nile Virus (WNV) Lineage 1 and Lineage 2, Real Time RT-PCR USUTU virus (USUV) and a traditional flavivirus End-point RT-PCR. Positive pools are sequenced and the related sequences employed to perform a basic local alignment search tool (BLAST) in the GenBank library. Positive samples are sent to the National Reference Centre for Animal Exotic Diseases (CESME, Teramo) for confirmation. With particular reference to WNV, after the confirmation, as provided by national legislation, control measures involving both local veterinary and human health services are activated: equine sera are randomly sampled within a 4 km radius from the positive collection sites and tested with ELISA kit and WNV NAT screening of blood donors is introduced. This surveillance network allowed to detect since 2011 USUV circulation in this area of Italy. WNV was detected in Piedmont and Liguria for the first time in 2014 in mosquitoes. During the 2015 vector season, we observed the expansion of its activity in Piedmont. The virus was detected in almost all Provinces both in mosquitoes (6 pools) and animals (19 equine sera, 4 birds). No blood bag tested resulted infected. The first neuroinvasive human case occurred too. Competent authorities should be aware of a potentially increased risk of MBDs activity during the 2016 vector season. This work shows that this surveillance network allowed to early detect the presence of MBDs in humans and animals, and provided useful information to public authorities, in order to apply control measures. Finally, an additional value of our diagnostic protocol is the ability to detect all viruses belonging to the Flaviviridae family, considering the emergence caused by other Flaviviruses in humans such as the recent Zika virus infection in South America. Italy has climatic and environmental features conducive to Zika virus transmission, the competent vector and many travellers from Brazil reported every year.

Keywords: integrated surveillance, mosquito borne disease, West Nile virus, Zika virus

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Authors: Irina Alexandra Paun, Mona Mihailescu, Marian Zamfirescu, Catalin Romeo Luculescu, Adriana Maria Acasandrei, Cosmin Catalin Mustaciosu, Roxana Cristina Popescu, Maria Dinescu

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A major concern in bone tissue engineering is to develop complex 3D architectures that mimic the natural cells environment, facilitate the cells growth in a defined manner and allow the flow transport of nutrients and metabolic waste. In particular, porous structures of controlled pore size and positioning are indispensable for growing human-like bone structures. Another concern is to monitor both the structures and the seeded cells with high spatial resolution and without interfering with the cells natural environment. The present approach relies on laser-based technologies employed for fabricating porous biomimetic structures that support the growth of osteoblast-like cells and for their non-invasive 3D imaging. Specifically, the porous structures were built by two photon polymerization –direct writing (2PP_DW) of the commercially available photoresists IL-L780, using the Photonic Professional 3D lithography system. The structures consist of vertical tubes with micrometer-sized heights and diameters, in a honeycomb-like spatial arrangement. These were fabricated by irradiating the IP-L780 photoresist with focused laser pulses with wavelength centered at 780 nm, 120 fs pulse duration and 80 MHz repetition rate. The samples were precisely scanned in 3D by piezo stages. The coarse positioning was done by XY motorized stages. The scanning path was programmed through a writing language (GWL) script developed by Nanoscribe. Following laser irradiation, the unexposed regions of the photoresist were washed out by immersing the samples in the Propylene Glycol Monomethyl Ether Acetate (PGMEA). The porous structures were seeded with osteoblast like MG-63 cells and their osteogenic potential was tested in vitro. The cell-seeded structures were analyzed in 3D using the digital holographic microscopy technique (DHM). DHM is a marker free and high spatial resolution imaging tool, where the hologram acquisition is performed non-invasively i.e. without interfering with the cells natural environment. Following hologram recording, a digital algorithm provided a 3D image of the sample, as well as information about its refractive index, which is correlated with the intracellular content. The axial resolution of the images went down to the nanoscale, while the temporal scales ranged from milliseconds up to hours. The hologram did not involve sample scanning and the whole image was available in one frame recorded going over 200μm field of view. The digital holograms processing provided 3D quantitative information on the porous structures and allowed a quantitative analysis of the cellular response in respect to the porous architectures. The cellular shape and dimensions were found to be influenced by the underlying micro relief. Furthermore, the intracellular content gave evidence on the beneficial role of the porous structures in promoting osteoblast differentiation. In all, the proposed laser-based protocol emerges as a promising tool for the fabrication and non-invasive imaging of porous constructs for bone tissue engineering. Acknowledgments: This work was supported by a grant of the Romanian Authority for Scientific Research and Innovation, CNCS-UEFISCDI, project PN-II-RU-TE-2014-4-2534 (contract 97 from 01/10/2015) and by UEFISCDI PN-II-PT-PCCA no. 6/2012. A part of this work was performed in the CETAL laser facility, supported by the National Program PN 16 47 - LAPLAS IV.

Keywords: biomimetic, holography, laser, osteoblast, two photon polymerization

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Authors: Qiaosi Li, Frankie Kwan Kit Wong, Tung Fung

Abstract:

Lightweight unmanned aerial vehicle (UAV) loading with novel sensors offers a low cost approach for data acquisition in complex environment. This study established a framework for applying UAV system in complex environment quick mapping and assessed the performance of UAV-based hyperspectral image and digital surface model (DSM) derived from photogrammetric point clouds for 13 species classification in wetland area Mai Po Inner Deep Bay Ramsar Site, Hong Kong. The study area was part of shallow bay with flat terrain and the major species including reedbed and four mangroves: Kandelia obovata, Aegiceras corniculatum, Acrostichum auerum and Acanthus ilicifolius. Other species involved in various graminaceous plants, tarbor, shrub and invasive species Mikania micrantha. In particular, invasive species climbed up to the mangrove canopy caused damage and morphology change which might increase species distinguishing difficulty. Hyperspectral images were acquired by Headwall Nano sensor with spectral range from 400nm to 1000nm and 0.06m spatial resolution image. A sequence of multi-view RGB images was captured with 0.02m spatial resolution and 75% overlap. Hyperspectral image was corrected for radiative and geometric distortion while high resolution RGB images were matched to generate maximum dense point clouds. Furtherly, a 5 cm grid digital surface model (DSM) was derived from dense point clouds. Multiple feature reduction methods were compared to identify the efficient method and to explore the significant spectral bands in distinguishing different species. Examined methods including stepwise discriminant analysis (DA), support vector machine (SVM) and minimum noise fraction (MNF) transformation. Subsequently, spectral subsets composed of the first 20 most importance bands extracted by SVM, DA and MNF, and multi-source subsets adding extra DSM to 20 spectrum bands were served as input in maximum likelihood classifier (MLC) and SVM classifier to compare the classification result. Classification results showed that feature reduction methods from best to worst are MNF transformation, DA and SVM. MNF transformation accuracy was even higher than all bands input result. Selected bands frequently laid along the green peak, red edge and near infrared. Additionally, DA found that chlorophyll absorption red band and yellow band were also important for species classification. In terms of 3D data, DSM enhanced the discriminant capacity among low plants, arbor and mangrove. Meanwhile, DSM largely reduced misclassification due to the shadow effect and morphological variation of inter-species. In respect to classifier, nonparametric SVM outperformed than MLC for high dimension and multi-source data in this study. SVM classifier tended to produce higher overall accuracy and reduce scattered patches although it costs more time than MLC. The best result was obtained by combining MNF components and DSM in SVM classifier. This study offered a precision species distribution survey solution for inaccessible wetland area with low cost of time and labour. In addition, findings relevant to the positive effect of DSM as well as spectral feature identification indicated that the utility of UAV-borne hyperspectral and photogrammetry deriving 3D data is promising in further research on wetland species such as bio-parameters modelling and biological invasion monitoring.

Keywords: digital surface model (DSM), feature reduction, hyperspectral, photogrammetric point cloud, species mapping, unmanned aerial vehicle (UAV)

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