Search results for: spent Pleurotus ostreatus and Pleurotus sajor-caju mushroom substrate

Authors: A. Moghadasein, M. Ghasemi, S. Fazelifar

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Aim: Chemerin is a novel adipokine that play an important role in regulating lipid metabolism and abiogenesis. Chemerin is dependent on autocrine and paracrine signals for the differentiation and maturation of fat cells; it also regulates glucose uptake in fat cells and stimulates lipolysis. It has been reported that in adipocytes, chemerin enhances the insulin-stimulated glucose and causes the phosphorylation of tyrosine in Insulin receptor substrate. According to the studies, Chemerin may increase insulin sensitivity in adipose tissue and is largely associated with Body mass index, triglycerides, and blood pressure in those with normal glucose tolerance. There is limited information available regarding the effect of exercise training on serum chemerin concentrations. The purpose of this study was to investigate the effect of endurance training on serum chemerin levels and lipids of plasma in overweight women. Methodology: This study was a quasi-experimental research with a pre-post test design. After required examination and verification of high pressure by the physician, 22 obese subjects (age: 35.64±5.55 yr, weight: 75.62±9.30 kg, body mass index: 32.4±1.6 kg/m2) were randomly assigned to aerobic training (n= 12) and control (n= 12) groups. Participants completed a questionnaire indicating the lack of sports history during the past six months, the lack of anti-hypertension drugs use, hormone therapy, cardiovascular problems, and complete stoppage of menstrual cycle. Aerobic training was performed 3 times weekly for 8 weeks. Resting levels of chemerin plasma, metabolic parameters were measured prior to and after the intervention. The control group did not participate in any training program. In this study, ethical considerations included the complete description of the objectives to the study participants, ensuring the confidentiality of their information. Kolmogorov-Smirnov and Levin test were used for determining the normal distribution of data and homogeneity of variances, respectively. Analyze of variance with repeated measure were used to investigate the changes in the intra-group and the differences in inter-group of variables. Statistical operations were performed using SPSS 16 and the significance level of the tests was considered at P < 0.05. Results: After an 8 week aerobic training, levels of chemerin plasma were significantly decreased in aerobic trained group when compared with their control groups (p < 0.05).Concurrently, levels of HDL-c were significantly decreased (p < 0.05) whereas, levels of cholesterol, TG and LDL-c, showed no significant changes (p > 0.05). No significant correlations between chemerin levels and weight loss were observed in subjects with overweight women. Conclusion: The present study demonstrated, 8 weeks aerobic training, reduced serum chemerin concentrations in overweight women. Whereas, aerobic training exercise programmers affected the lipid profile response of obese subjects differently. However further research is warranted in order to unravel the molecular mechanism for the range of responses and the role of serum chemerin.

Keywords: chemerin, aerobic training, lipid profile, obese women

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

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

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

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Authors: E Tolufase

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The demand, high costs and health implications of using energy derived from hydrocarbon compound have necessitated the continuous search for alternative source of energy. The World energy market is facing some challenges viz: depletion of fossil fuel reserves, population explosion, lack of energy security, economic and urbanization growth and also, in Nigeria some rural areas still depend largely on wood, charcoal, kerosene, petrol among others, as the sources of their energy. To overcome these short falls in energy supply and demand, as well as taking into consideration the risks from global climate change due to effect of greenhouse gas emissions and other pollutants from fossil fuels’ combustion, brought a lot of attention on efficiently harnessing the renewable energy sources. A very promising among the renewable energy resources for a clean energy technology for power production, vehicle and domestic usage is biogas. Therefore, optimization of biogas yield and quality is imperative. Hence, this study investigated yield and quality of biogas using low cost bio-digester and combination of various feed stocks referred to as co-digestion. Batch/Discontinuous Bio-digester type was used because it was cheap, easy, plausible and appropriate for different substrates used to get the desired results. Three substrates were used; cow dung, chicken droppings and lemon grass digested in five separate 21 litre digesters, A, B, C, D, and E and the gas collection system was designed using locally available materials. For single digestion we had; cow dung, chicken droppings, lemon grass, in Bio-digesters A, B, and C respectively, the co-digested three substrates in different mixed ratio 7:1:2 in digester D and E in ratio 5:3:2. The respective feed-stocks materials were collected locally, digested and analyzed in accordance with standard procedures. They were pre-fermented for a period of 10 days before being introduced into the digesters. They were digested for a retention period of 28 days, the physiochemical parameters namely; pressure, temperature, pH, volume of the gas collector system and volume of biogas produced were all closely monitored and recorded daily. The values of pH and temperature ranged 6.0 - 8.0, and 220C- 350C respectively. For the single substrate, bio-digester A(Cow dung only) produced biogas of total volume 0.1607m3(average volume of 0.0054m3 daily),while B (Chicken droppings ) produced 0.1722m3 (average of 0.0057m3 daily) and C (lemon grass) produced 0.1035m3 (average of 0.0035m3 daily). For the co-digested substrates in bio-digester D the total biogas produced was 0.2007m³ (average volume of 0.0067m³ daily) and bio-digester E produced 0.1991m³ (average volume of 0.0066m³ daily) It’s obvious from the results, that combining different substrates gave higher yields than when a singular feed stock was used and also mixing ratio played some roles in the yield improvement. Bio-digesters D and E contained the same substrates but mixed with different ratios, but higher yield was noticed in D with mixing ratio of 7:1:2 than in E with ratio 5:3:2.Therefore, co-digestion of substrates and mixing proportions are important factors for biogas production optimization.

Keywords: anaerobic, batch, biogas, biodigester, digestion, fermentation, optimization

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Authors: Chetna Dhand, Venkatesh Mayandi, Silvia Marrero Diaz, Roger W. Beuerman, Seeram Ramakrishna, Rajamani Lakshminarayanan

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Sturdy Insect Cuticle Sclerotization, Incredible Substrate independent Mussel’s bioadhesion, Tanning of Leather are some of catechol(amine)s mediated natural processes. Chemical contemplation spots toward a mechanism instigated with the formation of the quinone moieties from the respective catechol(amine)s, via oxidation, followed by the nucleophilic addition of the amino acids/proteins/peptides to this quinone leads to the development of highly strong, cross-linked and water-resistant proteinacious structures. Inspired with this remarkable catechol(amine)s chemistry towards amino acids/proteins/peptides, we attempted to design highly stable and water-resistant antifungal wound dressing mats with exceptional durability using collagen (protein), dopamine (catecholamine) and antifungal drugs (Amphotericin B and Caspofungin) as the key materials. Electrospinning technique has been used to fabricate desired nanofibrous mat including Collagen (COLL), COLL/Dopamine (COLL/DP) and calcium incorporated COLL/DP (COLL-DP-Ca2+). The prepared protein-based scaffolds have been studied for their microscopic investigations (SEM, TEM, and AFM), structural analysis (FT-IR), mechanical properties, water wettability characteristics and aqueous stability. Biocompatibility of these scaffolds has been analyzed for dermal fibroblast cells using MTS assay, Cell TrackerTM Green CMFDA and confocal imaging. Being the winner sample, COLL-DP-Ca2+ scaffold has been selected for incorporating two antifungal drugs namely Caspofungin (Peptide based) and Amphotericin B (Non-Peptide based). Antifungal efficiency of the designed mats has been evaluated for eight diverse fungal strains employing different microbial assays including disc diffusion, cell-viability assay, time kill kinetics etc. To confirm the durability of these mats, in term of their antifungal activity, drug leaching studies has been performed and monitored using disc diffusion assay each day. Ex-vivo fungal infection model has also been developed and utilized to validate the antifungal efficacy of the designed wound dressings. Results clearly reveal dopamine mediated crosslinking within COLL-antifungal scaffolds that leads to the generation of highly stable, mechanical tough, biocompatible wound dressings having the zone of inhabitation of ≥ 2 cm for almost all the investigated fungal strains. Leaching studies and Ex-vivo model has confirmed the durability of these wound dressing for more than 3 weeks and certified their suitability for commercialization. A model has also been proposed to enlighten the chemical mechanism involved for the development of these antifungal wound dressings with exceptional robustness.

Keywords: catecholamine chemistry, electrospinning technique, antifungals, wound dressings, collagen

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Authors: Deb Kumar Shah, M. Shaheer Akhtar, Ha Ryeon Lee, O-Bong Yang, Chong Yeal Kim

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The crystalline silicon (Si) solar cells are highly renowned photovoltaic technology and well-established as the commercial solar technology. Most of the solar panels are globally installed with the crystalline Si solar modules. At the present scenario, the major photovoltaic (PV) market is shared by c-Si solar cells, but the cost of c-Si panels are still very high as compared with the other PV technology. In order to reduce the cost of Si solar panels, few necessary steps such as low-cost Si manufacturing, cheap antireflection coating materials, inexpensive solar panel manufacturing are to be considered. It is known that the antireflection (AR) layer in c-Si solar cell is an important component to reduce Fresnel reflection for improving the overall conversion efficiency. Generally, Si wafer exhibits the 30% reflection because it normally poses the two major intrinsic drawbacks such as; the spectral mismatch loss and the high Fresnel reflection loss due to the high contrast of refractive indices between air and silicon wafer. In recent years, researchers and scientists are highly devoted to a lot of researches in the field of searching effective and low-cost AR materials. Silicon nitride (SiNx) is well-known AR materials in commercial c-Si solar cells due to its good deposition and interaction with passivated Si surfaces. However, the deposition of SiNx AR is usually performed by expensive plasma enhanced chemical vapor deposition (PECVD) process which could have several demerits like difficult handling and damaging the Si substrate by plasma when secondary electrons collide with the wafer surface for AR coating. It is very important to explore new, low cost and effective AR deposition process to cut the manufacturing cost of c-Si solar cells. One can also be realized that a nano-texturing process like the growth of nanowires, nanorods, nanopyramids, nanopillars, etc. on Si wafer can provide a low reflection on the surface of Si wafer based solar cells. The above nanostructures might be enhanced the antireflection property which provides the larger surface area and effective light trapping. In this work, we report on the development of crystalline Si solar cells without using the AR layer. The Silicon wafer was modified by growing nanowires like Si nanostructures using the wet controlled etching method and directly used for the fabrication of Si solar cell without AR. The nanostructures over Si wafer were optimized in terms of sizes, lengths, and densities by changing the etching conditions. Well-defined and aligned wires like structures were achieved when the etching time is 20 to 30 min. The prepared Si nanostructured displayed the minimum reflectance ~1.64% at 850 nm with the average reflectance of ~2.25% in the wavelength range from 400-1000 nm. The nanostructured Si wafer based solar cells achieved the comparable power conversion efficiency in comparison with c-Si solar cells with SiNx AR layer. From this study, it is confirmed that the reported method (controlled wet etching) is an easy, facile method for preparation of nanostructured like wires on Si wafer with low reflectance in the whole visible region, which has greater prospects in developing c-Si solar cells without AR layer at low cost.

Keywords: chemical etching, conversion efficiency, silicon nanostructures, silicon solar cells, surface modification

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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: Iryna Atamaniuk, Hannah Boysen, Nils Wieczorek, Natalia Politaeva, Iuliia Bazarnova, Kerstin Kuchta

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Over the last decade due to climate change and a strategy of natural resources preservation, the interest for the aquatic biomass has dramatically increased. Along with mitigation of the environmental pressure and connection of waste streams (including CO₂ and heat emissions), microalgae bioeconomy can supply food, feed, as well as the pharmaceutical and power industry with number of value-added products. Furthermore, in comparison to conventional biomass, microalgae can be cultivated in wide range of conditions without compromising food and feed production, thus addressing issues associated with negative social and the environmental impacts. This paper presents the state-of-the art technology for microalgae bioeconomy from cultivation process to production of valuable components and by-streams. Microalgae Chlorella sorokiniana were cultivated in the pilot-scale innovation concept in Hamburg (Germany) using different systems such as race way pond (5000 L) and flat panel reactors (8 x 180 L). In order to achieve the optimum growth conditions along with suitable cellular composition for the further extraction of the value-added components, process parameters such as light intensity, temperature and pH are continuously being monitored. On the other hand, metabolic needs in nutrients were provided by addition of micro- and macro-nutrients into a medium to ensure autotrophic growth conditions of microalgae. The cultivation was further followed by downstream process and extraction of lipids, proteins and saccharides. Lipids extraction is conducted in repeated-batch semi-automatic mode using hot extraction method according to Randall. As solvents hexane and ethanol are used at different ratio of 9:1 and 1:9, respectively. Depending on cell disruption method along with solvents ratio, the total lipids content showed significant variations between 8.1% and 13.9 %. The highest percentage of extracted biomass was reached with a sample pretreated with microwave digestion using 90% of hexane and 10% of ethanol as solvents. Proteins content in microalgae was determined by two different methods, namely: Total Kejadahl Nitrogen (TKN), which further was converted to protein content, as well as Bradford method using Brilliant Blue G-250 dye. Obtained results, showed a good correlation between both methods with protein content being in the range of 39.8–47.1%. Characterization of neutral and acid saccharides from microalgae was conducted by phenol-sulfuric acid method at two wavelengths of 480 nm and 490 nm. The average concentration of neutral and acid saccharides under the optimal cultivation conditions was 19.5% and 26.1%, respectively. Subsequently, biomass residues are used as substrate for anaerobic digestion on the laboratory-scale. The methane concentration, which was measured on the daily bases, showed some variations for different samples after extraction steps but was in the range between 48% and 55%. CO₂ which is formed during the fermentation process and after the combustion in the Combined Heat and Power unit can potentially be used within the cultivation process as a carbon source for the photoautotrophic synthesis of biomass.

Keywords: bioeconomy, lipids, microalgae, proteins, saccharides

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Authors: A. Chávez, A. Rodríguez, F. Pinzón

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Society is concerned about measures of environmental, economic and social impacts generated in the solid waste disposal. These places of confinement, also known as landfills, are locations where problems of pollution and damage to human health are reduced. They are technically designed and operated, using engineering principles, storing the residue in a small area, compact it to reduce volume and covering them with soil layers. Problems preventing liquid (leachate) and gases produced by the decomposition of organic matter. Despite planning and site selection for disposal, monitoring and control of selected processes, remains the dilemma of the leachate as extreme concentration of pollutants, devastating soil, flora and fauna; aggressive processes requiring priority attention. A biological technology is the activated sludge system, used for tributaries with high pollutant loads. Since transforms biodegradable dissolved and particulate matter into CO2, H2O and sludge; transform suspended and no Settleable solids; change nutrients as nitrogen and phosphorous; and degrades heavy metals. The microorganisms that remove organic matter in the processes are in generally facultative heterotrophic bacteria, forming heterogeneous populations. Is possible to find unicellular fungi, algae, protozoa and rotifers, that process the organic carbon source and oxygen, as well as the nitrogen and phosphorus because are vital for cell synthesis. The mixture of the substrate, in this case sludge leachate, molasses and wastewater is maintained ventilated by mechanical aeration diffusers. Considering as the biological processes work to remove dissolved material (< 45 microns), generating biomass, easily obtained by decantation processes. The design consists of an artificial support and aeration pumps, favoring develop microorganisms (denitrifying) using oxygen (O) with nitrate, resulting in nitrogen (N) in the gas phase. Thus, avoiding negative effects of the presence of ammonia or phosphorus. Overall the activated sludge system includes about 8 hours of hydraulic retention time, which does not prevent the demand for nitrification, which occurs on average in a value of MLSS 3,000 mg/L. The extended aeration works with times greater than 24 hours detention; with ratio of organic load/biomass inventory under 0.1; and average stay time (sludge age) more than 8 days. This project developed a pilot system with sludge leachate from Doña Juana landfill - RSDJ –, located in Bogota, Colombia, where they will be subjected to a process of activated sludge and extended aeration through a sequential Bach reactor - SBR, to be dump in hydric sources, avoiding ecological collapse. The system worked with a dwell time of 8 days, 30 L capacity, mainly by removing values of BOD and COD above 90%, with initial data of 1720 mg/L and 6500 mg/L respectively. Motivating the deliberate nitrification is expected to be possible commercial use diffused aeration systems for sludge leachate from landfills.

Keywords: sludge, landfill, leachate, SBR

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Authors: M. Shiva Prasad, S. R. Atchuta, T. Vijayaraghavan, S. Sakthivel

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The world is in need of efficient energy conversion technologies which are affordable, accessible, and sustainable with eco-friendly nature. Solar energy is one of the cornerstones for the world’s economic growth because of its abundancy with zero carbon pollution. Among the various solar energy conversion technologies, solar thermal technology has attracted a substantial renewed interest due to its diversity and compatibility in various applications. Solar thermal systems employ concentrators, tracking systems and heat engines for electricity generation which lead to high cost and complexity in comparison with photovoltaics; however, it is compatible with distinct thermal energy storage capability and dispatchable electricity which creates a tremendous attraction. Apart from that, employing cost-effective solar selective receiver tube in a concentrating solar thermal (CST) system improves the energy conversion efficiency and directly reduces the cost of technology. In addition, the development of solar receiver tubes by low cost methods which can offer high optical properties and corrosion resistance in an open-air atmosphere would be beneficial for low and medium temperature applications. In this regard, our work opens up an approach which has the potential to achieve cost-effective energy conversion. We have developed a highly selective tandem absorber coating through a facile wet chemical route by a combination of chemical oxidation, sol-gel, and nanoparticle coating methods. The developed tandem absorber coating has gradient refractive index nature on stainless steel (SS 304) and exhibited high optical properties (α ≤ 0.95 & ε ≤ 0.14). The first absorber layer (Cr-Mn-Fe oxides) developed by controlled oxidation of SS 304 in a chemical bath reactor. A second composite layer of ZrO₂-SiO₂ has been applied on the chemically oxidized substrate by So-gel dip coating method to serve as optical enhancing and corrosion resistant layer. Finally, an antireflective layer (MgF₂) has been deposited on the second layer, to achieve > 95% of absorption. The developed tandem layer exhibited good thermal stability up to 250 °C in open air atmospheric condition and superior corrosion resistance (withstands for > 200h in salt spray test (ASTM B117)). After the successful development of a coating with targeted properties at a laboratory scale, a prototype of the 1 m tube has been demonstrated with excellent uniformity and reproducibility. Moreover, it has been validated under standard laboratory test condition as well as in field condition with a comparison of the commercial receiver tube. The presented strategy can be widely adapted to develop highly selective coatings for a variety of CST applications ranging from hot water, solar desalination, and industrial process heat and power generation. The high-performance, cost-effective medium temperature receiver tube technology has attracted many industries, and recently the technology has been transferred to Indian industry.

Keywords: concentrated solar thermal system, solar selective coating, tandem absorber, ultralow refractive index

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Authors: Lesly Y Carmona-Sarabia, Luisa Barraza-Vergara, Vilmalí López-Mejías, Wandaliz Torres-García, Maribella Domenech-Garcia, Madeline Torres-Lugo

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Biosensors are used in many applications providing real-time monitoring to treat long-term conditions. Thus, understanding the physicochemical properties and biological side effects on the skin of polymers (e. g., poly(methyl methacrylate), PMMA) employed in the fabrication of wearable biosensors is crucial for the selection of manufacturing materials within this field. The PMMA (hydrophobic and thermoplastic polymer) is commonly employed as a coating material or substrate in the fabrication of wearable devices. The cytotoxicityof PMMA (including residual monomers or degradation products) on the skin, in terms of cells and tissue, is required to prevent possible adverse effects (cell death, skin reactions, sensitization) on human health. Within this work, accelerated aging of PMMA (Mw ~ 15000) through thermal and photochemical degradation was under-taken. The accelerated aging of PMMA was carried out by thermal (200°C, 1h) and photochemical degradation (UV-Vis, 8-15d) adapted employing ISO protocols (ISO-10993-12, ISO-4892-1:2016, ISO-877-1:2009, ISO-188: 2011). In addition, in vitro cytotoxicity evaluation of PMMA degradation products was performed using NIH3T3 fibroblast cells to assess the response of skin tissues (in terms of cell viability) exposed with polymers utilized to manufacture wearable biosensors, such as PMMA. The PMMA (Mw ~ 15000) before and after accelerated aging experiments was characterized by thermal gravimetric analysis (TGA), differential scanning calorimetric (DSC), powder X-ray diffractogram (PXRD), and scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) to determine and verify the successful degradation of this polymer under the specific conditions previously mention. The degradation products were characterized through nuclear magnetic resonance (NMR) to identify possible byproducts generated after the accelerated aging. Results demonstrated a percentage (%) weight loss between 1.5-2.2% (TGA thermographs) for PMMA after accelerated aging. The EDS elemental analysis reveals a 1.32 wt.% loss of carbon for PMMA after thermal degradation. These results might be associated with the amount (%) of PMMA degrade after the accelerated aging experiments. Furthermore, from the thermal degradation products was detected the presence of the monomer and methyl formate (low concentrations) and a low molecular weight radical (·COOCH3) in higher concentrations by NMR. In the photodegradation products, methyl formate was detected in higher concentrations. These results agree with the proposed thermal or photochemical degradation mechanisms found in the literature.1,2 Finally, significant cytotoxicity on the NIH3T3 cells was obtained for the thermal and photochemical degradation products. A decrease in cell viability by > 90% (stock solutions) was observed. It is proposed that the presence of byproducts (e.g. methyl formate or radicals such as ·COOCH₃) from the PMMA degradation might be responsible for the cytotoxicity observed in the NIH3T3 fibroblast cells. Additionally, experiments using skin models will be employed to compare with the NIH3T3 fibroblast cells model.

Keywords: biosensors, polymer, skin irritation, degradation products, cell viability

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Authors: Sivananth Murugesan, I. Regupathi, B. Vishwas Prabhu, Ankit Devatwal, Vishnu Sivan Pillai

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Pectinase is an important enzyme which has a wide range of applications including textile processing and bioscouring of cotton fibers, coffee and tea fermentation, purification of plant viruses, oil extraction etc. Selective separation and purification of pectinase from fermentation broth and recover the enzyme form process stream for reuse are cost consuming process in most of the enzyme based industries. It is difficult to identify a suitable medium to enhance enzyme activity and retain its enzyme characteristics during such processes. The cost effective, selective separation of enzymes through the modified Liquid-liquid extraction is of current research interest worldwide. Reverse micellar extraction, globally acclaimed Liquid-liquid extraction technique is well known for its separation and purification of solutes from the feed which offers higher solute specificity and partitioning, ease of operation and recycling of extractants used. Surfactant concentrations above critical micelle concentration to an apolar solvent form micelles and addition of micellar phase to water in turn forms reverse micelles or water-in-oil emulsions. Since, electrostatic interaction plays a major role in the separation/purification of solutes using reverse micelles. These interaction parameters can be altered with the change in pH, addition of cosolvent, surfactant and electrolyte and non-electrolyte. Even though many chemical based commercial surfactant had been utilized for this purpose, the biosurfactants are more suitable for the purification of enzymes which are used in food application. The present work focused on the partitioning of pectinase from the synthetic aqueous solution within the reverse micelle phase formed by a biosurfactant, Soybean Lecithin dissolved in chloroform. The critical micelle concentration of soybean lecithin/chloroform solution was identified through refractive index and density measurements. Effect of surfactant concentrations above and below the critical micelle concentration was considered to study its effect on enzyme activity, enzyme partitioning within the reverse micelle phase. The effect of pH and electrolyte salts on the partitioning behavior was studied by varying the system pH and concentration of different salts during forward and back extraction steps. It was observed that lower concentrations of soybean lecithin enhanced the enzyme activity within the water core of the reverse micelle with maximizing extraction efficiency. The maximum yield of pectinase of 85% with a partitioning coefficient of 5.7 was achieved at 4.8 pH during forward extraction and 88% yield with a partitioning coefficient of 7.1 was observed during backward extraction at a pH value of 5.0. However, addition of salt decreased the enzyme activity and especially at higher salt concentrations enzyme activity declined drastically during both forward and back extraction steps. The results proved that reverse micelles formed by Soybean Lecithin and chloroform may be used for the extraction of pectinase from aqueous solution. Further, the reverse micelles can be considered as nanoreactors to enhance enzyme activity and maximum utilization of substrate at optimized conditions, which are paving a way to process intensification and scale-down.

Keywords: pectinase, reverse micelles, soybean lecithin, selective partitioning

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Authors: Yusuf Hesham Mohamed

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Introduction: Three-dimensional printing technology includes multiple procedures that fabricate three-dimensional objects through consecutively layering two-dimensional cross-sections on top of each other. 3D bioprinting is a promising field of 3D printing, which fabricates tissues and organs by accurately controlling the proper arrangement of diverse biological components. 3D bioprinting uses software and prints biological materials and their supporting components layer-by-layer on a substrate or in a tissue culture plate to produce complex live tissues and organs. 3D food printing is an emerging field of 3D bioprinting in which the 3D printed products are food products that are cheap, require less effort to produce, and have more desirable traits. The Aim of the Study is the development of an affordable 3D bioprinter by altering a locally made CNC instrument with an open-source platform to suit the 3D bio-printer purposes. Later, we went through applying the prototype in several applications regarding food technology and drug testing, including the organ-On-Chip. Materials and Methods: An off-the-shelf 3D printer was modified by designing and fabricating the syringe unit, which was designed on the basis of the Milli-fluidics system. Sodium alginate and gelatin hydrogels were prepared, followed by leaf cell suspension preparation from narrow sections of Fragaria’s viable leaves. The desired 3D structure was modeled, and 3D printing preparations took place. Cell-free and cell-laden hydrogels were printed at room temperature under sterile conditions. Post printing curing process was performed. The printed structure was further studied. Results: Positive results have been achieved using the altered 3D bioprinter where a 3D hydrogel construct of two layers made of the combination of sodium alginate to gelatin (15%: 0.5%) has been printed. DLP 3D printer was used to design the syringe component with a transparent PLA-Pro resin for the creation of a microfluidics system having two channels altered to the double extruder. The hydrogel extruder’s design was based on peristaltic pumps, which utilized a stepper motor. The design and fabrication were made using DIY-3D printed parts. Hard plastic PLA was the material utilized for printing. SEM was used to carry out the porous 3D construct imaging. Multiple physical and chemical tests were performed in order to ensure that the cell line was suitable for hosting. Fragaria plant was developed by suspending Fragaria’s cells from its leaves using the 3D bioprinter. Conclusion: 3D bioprinting is considered to be an emerging scientific field that can facilitate and improve many scientific tests and studies. Thus, having a 3D bioprinter in labs is considered to be an essential requirement. 3D bioprinters are very expensive; however, the fabrication of a 3D printer into a 3D bioprinter can lower the cost of the bioprinter. The 3D bioprinter implemented made use of peristaltic pumps instead of syringe-based pumps in order to extend the ability to print multiple types of materials and cells.

Keywords: scaffold, eco on chip, 3D bioprinter, DLP printer

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Authors: Sudip Kumar Sinha, Saptarshi Ghosh

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While there’s a perpetual buzz around zinc oxide (ZnO) superstructures for their unique optical features, the versatile material has been constantly utilized to manifest tailored electronic properties through rendition of distinct morphologies. And yet, the unorthodox approach of implementing the novel 1D nanostructures of ZnO (pristine or doped) for volatile sensing applications has ample scope to accommodate new unconventional morphologies. In the last two decades, solid-state sensors have attracted much curiosity for their relevance in identifying pollutant, toxic and other industrial gases. In particular gas sensors based on metal oxide semiconducting (wide Eg) nanomaterials have recently attracted intensive attention owing to their high sensitivity and fast response and recovery time. These materials when exposed to air, the atmospheric O2 dissociates and get absorb on the surface of the sensors by trapping the outermost shell electrons. Finally a depleted zone on the surface of the sensors is formed, that enhances the potential barrier height at grain boundary . Once a target gas is exposed to the sensor, the chemical interaction between the chemisorbed oxygen and the specific gas liberates the trapped electrons. Therefore altering the amount of adsorbate is a considerable approach to improve the sensitivity of any target gas/vapour molecule. Likewise, this study presents a spontaneous but self catalytic creation of Sn-doped ZnO hexagonal nanoprisms on Si (100) substrates through thermal evaporation-condensation method, and their subsequent deployment for volatile sensing. In particular, the sensors were utilized to detect molecules of ethanol, acetone and ammonia below their permissible exposure limits which returned sensitivities of around 85%, 80% and 50% respectively. The influence of Sn concentration on the growth, microstructural and optical properties of the nanoprisms along with its role in augmenting the sensing parameters has been detailed. The single-crystalline nanostructures have a typical diameter ranging from 300 to 500 nm and a length that extends up to few micrometers. HRTEM images confirmed the hexagonal crystallography for the nanoprisms, while SAED pattern asserted the single crystalline nature. The growth habit is along the low index <0001>directions. It has been seen that the growth mechanism of the as-deposited nanostructures are directly influenced by varying supersaturation ratio, fairly high substrate temperatures, and specified surface defects in certain crystallographic planes, all acting cooperatively decide the final product morphology. Room temperature photoluminescence (PL) spectra of this rod like structures exhibits a weak ultraviolet (UV) emission peak at around 380 nm and a broad green emission peak in the 505 nm regime. An estimate of the sensing parameters against dispensed target molecules highlighted the potential for the nanoprisms as an effective volatile sensing material. The Sn-doped ZnO nanostructures with unique prismatic morphology may find important applications in various chemical sensors as well as other potential nanodevices.

Keywords: gas sensor, HRTEM, photoluminescence, ultraviolet, zinc oxide

Procedia PDF Downloads 240

Authors: Carlos E. Rodríguez-Rodríguez, Karla Ruiz-Hidalgo, Kattia Madrigal-Zúñiga, Juan Salvador Chin-Pampillo, Mario Masís-Mora, Elizabeth Carazo-Rojas

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One of the main causes of contamination linked to agricultural activities is the spillage and disposal of pesticides, especially during the loading, mixing or cleaning of agricultural spraying equipment. One improvement in the handling of pesticides is the use of biopurification systems (BPS), simple and cheap degradation devices where the pesticides are biologically degraded at accelerated rates. The biologically active core of BPS is the biomixture, which is constituted by soil pre-exposed to the target pesticide, a lignocellulosic substrate to promote the activity of ligninolitic fungi and a humic component (peat or compost), mixed at a volumetric proportion of 50:25:25. Considering the known ability of lignocellulosic fungi to degrade a wide range of organic pollutants, and the high amount of lignocellulosic waste used in biomixture preparation, the bioaugmentation of biomixtures with these fungi represents an interesting approach for improving biomixtures. The present work aimed at evaluating the effect of the bioaugmentation of rice husk based biomixtures with the fungus Trametes versicolor in the removal of the insectice/nematicide carbofuran (CFN) and to optimize the composition of the biomixture to obtain the best performance in terms of CFN removal and mineralization, reduction in formation of transformation products and decrease in residual toxicity of the matrix. The evaluation of several lignocellulosic residues (rice husk, wood chips, coconut fiber, sugarcane bagasse or newspaper print) revealed the best colonization by T. versicolor in rice husk. Pre-colonized rice husk was then used in the bioaugmentation of biomixtures also containing soil pre-exposed to CFN and either peat (GTS biomixture) or compost (GCS biomixture). After spiking with 10 mg/kg CBF, the efficiency of the biomixture was evaluated through a multi-component approach that included: monitoring of CBF removal and production of CBF transformation products, mineralization of radioisotopically labeled carbofuran (14C-CBF) and changes in the toxicity of the matrix after the treatment (Daphnia magna acute immobilization test). Estimated half-lives of CBF in the biomixtures were 3.4 d and 8.1 d in GTS and GCS, respectively. The transformation products 3-hydroxycarbofuran and 3-ketocarbofuran were detected at the moment of CFN application, however their concentration continuously disappeared. Mineralization of 14C-CFN was also faster in GTS than GCS. The toxicological evaluation showed a complete toxicity removal in the biomixtures after 48 d of treatment. The composition of the GCS biomixture was optimized using a central composite design and response surface methodology. The design variables were the volumetric content of fungally pre-colonized rice husk and the volumetric ratio compost/soil. According to the response models, maximization of CFN removal and mineralization rate, and minimization in the accumulation of transformation products were obtained with an optimized biomixture of composition 30:43:27 (pre-colonized rice husk:compost:soil), which differs from the 50:25:25 composition commonly employed in BPS. Results suggest that fungal bioaugmentation may enhance the performance of biomixtures in CFN removal. Optimization reveals the importance of assessing new biomixture formulations in order to maximize their performance.

Keywords: bioaugmentation, biopurification systems, degradation, fungi, pesticides, toxicity

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Authors: Priyanka Jain, Ashish K. Sharma, Esha Shukla, K. J. Mukherjee

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We propose a paradigm shift in our approach to design improved platforms for recombinant protein production, by addressing system level issues rather than the individual steps associated with recombinant protein synthesis like transcription, translation, etc. We demonstrate that by controlling and modulating the cellular stress response (CSR), which is responsible for feedback control of protein synthesis, we can generate hyper-producing strains. We did transcriptomic profiling of post-induction cultures, expressing different types of protein, to analyze the nature of this cellular stress response. We found significant down-regulation of substrate utilization, translation, and energy metabolism genes due to generation CSR inside the host cell. However, transcription profiling has also shown that many genes are up-regulated post induction and their role in modulating the CSR is unclear. We hypothesized that these up-regulated genes trigger signaling pathways, generating the CSR and concomitantly reduce the recombinant protein yield. To test this hypothesis, we knocked out the up-regulated genes, which did not have any downstream regulatees, and analyzed their impact on cellular health and recombinant protein expression. Two model proteins i.e., GFP and L-Asparaginase were chosen for this analysis. We observed a significant improvement in expression levels, with some knock-outs showing more than 7-fold higher expression compared to control. The 10 best single knock-outs were chosen to make 45 combinations of all possible double knock-outs. A further increase in expression was observed in some of these double knock- outs with GFP levels being highest in a double knock-out ΔyhbC + ΔelaA. However, for L-Asparaginase which is a secretory protein, the best results were obtained using a combination of ΔelaA+ΔcysW knock-outs. We then tested all the knock outs for their ability to enhance the expression of a 'difficult-to-express' protein. The Rubella virus E1 protein was chosen and tagged with sfGFP at the C-terminal using a linker peptide for easy online monitoring of expression of this fusion protein. Interestingly, the highest increase in Rubella-sGFP levels was obtained in the same double knock-out ΔelaA + ΔcysW (5.6 fold increase in expression yield compared to the control) which gave the highest expression for L-Asparaginase. However, for sfGFP alone, the ΔyhbC+ΔmarR knock-out gave the highest level of expression. These results indicate that there is a fair degree of commonality in the nature of the CSR generated by the induction of different proteins. Transcriptomic profiling of the double knock out showed that many genes associated with the translational machinery and energy biosynthesis did not get down-regulated post induction, unlike the control where these genes were significantly down-regulated. This confirmed our hypothesis of these genes playing an important role in the generation of the CSR and allowed us to design a strategy for making better expression hosts by simply knocking out key genes. This strategy is radically superior to the previous approach of individually up-regulating critical genes since it blocks the mounting of the CSR thus preventing the down-regulation of a very large number of genes responsible for sustaining the flux through the recombinant protein production pathway.

Keywords: cellular stress response, GFP, knock-outs, up-regulated genes

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Authors: Chunyang Miao, Bingxin Li, Shanglong Ning, Christopher J. B. Ford

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While it is challenging to fabricate electronic devices close to atomic dimensions in conventional top-down lithography, molecular electronics is promising to help maintain the exponential increase in component densities via using molecular building blocks to fabricate electronic components from the bottom up. It offers smaller, faster, and more energy-efficient electronic and photonic systems. A self-assembled monolayer (SAM) of molecules is a layer of molecules that self-assembles on a substrate. They are mechanically flexible, optically transparent, low-cost, and easy to fabricate. A large-area multi-layer structure has been designed and investigated by the team, where a SAM of designed molecules is sandwiched between graphene and gold electrodes. Each molecule can act as a quantum dot, with all molecules conducting in parallel. When a source-drain bias is applied, significant current flows only if a molecular orbital (HOMO or LUMO) lies within the source-drain energy window. If electrons tunnel sequentially on and off the molecule, the charge on the molecule is well-defined and the finite charging energy causes Coulomb blockade of transport until the molecular orbital comes within the energy window. This produces ‘Coulomb diamonds’ in the conductance vs source-drain and gate voltages. For different tunnel barriers at either end of the molecule, it is harder for electrons to tunnel out of the dot than in (or vice versa), resulting in the accumulation of two or more charges and a ‘Coulomb staircase’ in the current vs voltage. This nanostructure exhibits highly reproducible Coulomb-staircase patterns, together with additional oscillations, which are believed to be attributed to molecular vibrations. Molecules are more isolated than semiconductor dots, and so have a discrete phonon spectrum. When tunnelling into or out of a molecule, one or more vibronic states can be excited in the molecule, providing additional transport channels and resulting in additional peaks in the conductance. For useful molecular electronic devices, achieving the optimum orbital alignment of molecules to the Fermi energy in the leads is essential. To explore it, a drop of ionic liquid is employed on top of the graphene to establish an electric field at the graphene, which screens poorly, gating the molecules underneath. Results for various molecules with different alignments of Fermi energy to HOMO have shown highly reproducible Coulomb-diamond patterns, which agree reasonably with DFT calculations. In summary, this large-area SAM molecular junction is a promising candidate for future electronic circuits. (1) The small size (1-10nm) of the molecules and good flexibility of the SAM lead to the scalable assembly of ultra-high densities of functional molecules, with advantages in cost, efficiency, and power dissipation. (2) The contacting technique using graphene enables mass fabrication. (3) Its well-observed Coulomb blockade behaviour, narrow molecular resonances, and well-resolved vibronic states offer good tuneability for various functionalities, such as switches, thermoelectric generators, and memristors, etc.

Keywords: molecular electronics, Coulomb blokade, electron-phonon coupling, self-assembled monolayer

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Authors: Saeid H. Mobini, Monika Lulsdorf, Thomas D. Warkentin

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The length of the breeding cycle from seed to seed is a limiting factor in the development of improved homozygous lines for breeding or recombinant inbred lines (RILs) for genetic analysis. The objective of this research was to accelerate the production of field pea RILs through application of rapid generation technology (RGT). RGT is based on the principle of growing miniature plants in an artificial medium under controlled conditions, and allowing them to produce a few flowers which develop seeds that are harvested prior to normal seed maturity. We aimed to maintain population size and genetic diversity in regeneration cycles. The effects of flurprimidol (a gibberellin synthesis inhibitor), plant density, hydroponic system, scheduled fertilizer applications, artificial light spectrum, photoperiod, and light/dark temperature were evaluated in the development of RILs from a cross between cultivars CDC Dakota and CDC Amarillo. The main goal was to accelerate flowering while reducing maintenance and space costs. In addition, embryo rescue of immature seeds was tested for shortening the seed fill period. Data collected over seven generations included plant height, the percentage of plant survival, flowering rate, seed setting rate, the number of seeds per plant, and time from seed to seed. Applying 0.6 µM flurprimidol reduced the internode length. Plant height was decreased to approximately 32 cm allowing for higher plant density without a delay in flowering and seed setting rate. The three light systems (T5 fluorescent bulbs, LEDs, and High Pressure Sodium +Metal-halide lamp) evaluated did not differ significantly in terms of flowering time in field pea. Collectively, the combination of 0.6 µM flurprimidol, 217 plant. m-2, 20 h photoperiod, 21/16 oC light/dark temperature in a hydroponic system with vermiculite substrate, applying scheduled fertilizer application based on growth stage, and 500 µmole.m-2.s-1 light intensity using T5 bulbs resulted in 100% of plants flowering within 34 ± 3 days and 96.5% of plants completed seed setting in 68.2 ± 3.6 days, i.e., 30-45 days/generation faster than conventional single seed descent (SSD) methods. These regeneration cycles were reproducible consistently. Hence, RGT could double (5.3) generations per year, using 3% occupying space, compared to SSD (2-3 generation/year). Embryo rescue of immature seeds at 7-8 mm stage, using commercial fertilizer solutions (Holland’s Secret™) showed seed setting rate of 95%, while younger embryos had lower germination rate. Mature embryos had a seed setting rate of 96.5% without either hormones or sugar added. So, considering the higher cost of embryo rescue using a procedure which requires skill, additional materials, and expenses, it could be removed from RGT with a further cost saving, and the process could be stopped between generations if required.

Keywords: field pea, flowering, rapid regeneration, recombinant inbred lines, single seed descent

Procedia PDF Downloads 365

Authors: L. Kutateldze, T. Urushadze, R. Khvedelidze, N. Zakariashvili, I. Khokhashvili, T. Sadunishvili

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Microbial cellulase/xylanase have shown their potential application in various industries including pulp and paper, textile, laundry, biofuel production, food and feed industry, brewing, and agriculture. Extremophilic micromycetes and their enzymes that are resistant to critical values of temperature and pH, and retaining enzyme activity for a long time are of great industrial interest. Among strains of microscopic fungi from the collection of S. Durmishidze Institute of Biochemistry and Biotechnology, strains isolated from different ecological niches of Southern Caucasus-active producers of cellulase/xylanase have been selected by means of screening under deep cultivation conditions. Extremophilic micromycetes and their enzymes that are resistant to critical values of temperature and pH, and retaining enzyme activity for a long time are of great industrial interest. Among strains of microscopic fungi from the collection of S. Durmishidze Institute of Biochemistry and Biotechnology, strains isolated from different ecological niches of Southern Caucasus-active producers of cellulase/xylanase have been selected by means of screening under deep cultivation conditions. Representatives of the genera Aspergillus, Penicillium and Trichoderma are outstanding by relatively high activities of these enzymes. Among the producers were revealed thermophilic strains, representatives of the genus Aspergillus-Aspergillus terreus, Aspergillus versicolor, Aspergillus wentii, also strains of Sporotrichum pulverulentum and Chaetomium thermophile. As a result of optimization of cultivation media and conditions, activities of enzymes produced by the strains have been increased by 4 -189 %. Two strains, active producers of cellulase/xylanase – Penicillium canescence E2 (mesophile) and Aspergillus versicolor Z17 (thermophile) were chosen for further studies. Cellulase/xylanase enzyme preparations from two different genera of microscopic fungi Penicillium canescence E2 and Aspergillus versicolor Z 17 were obtained with activities 220 U/g /1200 U/g and 125 U/g /940 U/g, correspondingly. Main technical characteristics were as follows: the highest enzyme activities were obtained for mesophilic strain Penicillium canescence E2 at 45-500C, while almost the same enzyme activities were fixed for the thermophilic strain Aspergillus versicolor Z 17 at temperature 60-65°C, exceeding the temperature optimum of the mesophile by 150C. Optimum pH of action of the studied cellulase/xylanases from mesophileic and thermophilic strains were similar and equaled to 4.5-5.0 It has been shown that cellulase/xylanase technical preparations from selected strains of Penicillium canescence E2 and Aspergillus versicolor Z17 hydrolyzed cellulose of untreated wheat straw to reducible sugars by 46-52%, and to glucose by 22-27%. However the thermophilic enzyme preparations from the thermophilic A.versicolor strains conducted the process at 600C higher by 100C as compared to mesophlic analogue. Rate of hydrolyses of the pretreated substrate by the same enzyme preparations to reducible sugars and glucose conducted at optimum for their action 60 and 500C was 52-61% and 29-33%, correspondingly. Thus, maximum yield of glucose and reducible sugars form untreated and pretreated wheat straw was achieved at higher temperature (600C) by enzyme preparations from thermophilic strain, which gives advantage for their industrial application.

Keywords: cellulase/xylanase, cellulose hydrolysis, microscopic fungi, thermophilic strain

Procedia PDF Downloads 288

Authors: Hyemi Kim, Jay Kim, Kyunghoon Han, Ra-Yeong Choi, In-Woo Kim, Hyung Joo Suh, Ki-Bae Hong, Sung Hee Han

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Chitosan is used in various industries such as food and medical care because it is known to have various functions such as anti-obesity, anti-inflammatory and anti-cancer benefits. Most of the commercial chitosan is extracted from crustaceans. As the harvest rate of snow crabs and red snow crabs decreases and safety issues arise due to environmental pollution, research is underway to extract chitosan from insects. In this study, we used Response Surface Methodology (RSM) to predict the optimal conditions to produce chitosan oligosaccharides from mealworms (MCOS), which can be absorbed through the intestine as low-molecular-weight chitosan. The experimentally confirmed optimal conditions for MCOS production using chitosanase were found to be a substrate concentration of 2.5%, enzyme addition of 30 mg/g and a reaction time of 6 hours. The chemical structure and physicochemical properties of the produced MCOS were measured using MALDI-TOF mass spectra and FTIR spectra. The MALDI-TOF mass spectra revealed peaks corresponding to the dimer (375.045), trimer (525.214), tetramer (693.243), pentamer (826.296), and hexamer (987.360). In the FTIR spectra, commercial chitosan oligosaccharides exhibited a weak peak pattern at 3500-2500 cm-1, unlike chitosan or chitosan oligosaccharides. There was a difference in the peak at 3200~3500 cm-1, where different vibrations corresponding to OH and amine groups overlapped. Chitosan, chitosan oligosaccharide, and commercial chitosan oligosaccharide showed peaks at 2849, 2884, and 2885 cm-1, respectively, attributed to the absorption of the C-H stretching vibration of methyl or methine. The amide I, amide II, and amide III bands of chitosan, chitosan oligosaccharide, and commercial chitosan oligosaccharide exhibited peaks at 1620/1620/1602, 1553/1555/1505, and 1310/1309/1317 cm-1, respectively. Furthermore, the solubility of MCOS was 45.15±3.43, water binding capacity (WBC) was 299.25±4.57, and fat binding capacity (FBC) was 325.61±2.28 and the solubility of commercial chitosan oligosaccharides was 49.04±9.52, WBC was 280.55±0.50, and FBC was 157.22±18.15. Thus, the characteristics of MCOS and commercial chitosan oligosaccharides are similar. The results of investigating the impact of chitosan oligosaccharide on the proliferation of probiotics revealed increased growth in L. casei, L. acidophilus, and Bif. Bifidum. Therefore, the major short-chain fatty acids produced by gut microorganisms, such as acetic acid, propionic acid, and butyric acid, increased within 24 hours of adding 1% (p<0.01) and 2% (p<0.001) MCOS. The impact of MCOS on the overall gut microbiota was assessed, revealing that the Chao1 index did not show significant differences, but the Simpson index decreased in a concentration-dependent manner, indicating a higher species diversity. The addition of MCOS resulted in changes in the overall microbial composition, with an increase in Firmicutes and Verrucomicrobia (p<0.05) compared to the control group, while Proteobacteria and Actinobacteria (p<0.05) decreased. At the genus level, changes in microbiota due to MCOS supplementation showed an increase in beneficial bacteria like lactobacillus, Romboutsia, Turicibacter, and Akkermansia (p<0.0001) while harmful bacteria like Enterococcus, Morganella, Proterus, and Bacteroides (p<0.0001) decreased. In this study, chitosan oligosaccharides were successfully produced under established conditions from mealworms, and these chitosan oligosaccharides are expected to have prebiotic effects, similar to those obtained from crabs.

Keywords: mealworms, chitosan, chitosan oligosaccharide, prebiotics

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Authors: Vladimir Smirnov, Roman Tominov, Vadim Avilov, Oleg Ageev

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The creation of a new generation micro- and nanoelectronics elements opens up unlimited possibilities for electronic devices parameters improving, as well as developing neuromorphic computing systems. Interest in the latter is growing up every year, which is explained by the need to solve problems related to the unstructured classification of data, the construction of self-adaptive systems, and pattern recognition. However, for its technical implementation, it is necessary to fulfill a number of conditions for the basic parameters of electronic memory, such as the presence of non-volatility, the presence of multi-bitness, high integration density, and low power consumption. Several types of memory are presented in the electronics industry (MRAM, FeRAM, PRAM, ReRAM), among which non-volatile resistive memory (ReRAM) is especially distinguished due to the presence of multi-bit property, which is necessary for neuromorphic systems manufacturing. ReRAM is based on the effect of resistive switching – a change in the resistance of the oxide film between low-resistance state (LRS) and high-resistance state (HRS) under an applied electric field. One of the methods for the technical implementation of neuromorphic systems is cross-bar structures, which are ReRAM cells, interconnected by cross data buses. Such a structure imitates the architecture of the biological brain, which contains a low power computing elements - neurons, connected by special channels - synapses. The choice of the ReRAM oxide film material is an important task that determines the characteristics of the future neuromorphic system. An analysis of literature showed that many metal oxides (TiO2, ZnO, NiO, ZrO2, HfO2) have a resistive switching effect. It is worth noting that the manufacture of nanocomposites based on these materials allows highlighting the advantages and hiding the disadvantages of each material. Therefore, as a basis for the neuromorphic structures manufacturing, it was decided to use ZnₓTiᵧHfzOᵢ nanocomposite. It is also worth noting that the ZnₓTiᵧHfzOᵢ nanocomposite does not need an electroforming, which degrades the parameters of the formed ReRAM elements. Currently, this material is not well studied, therefore, the study of the effect of resistive switching in forming-free ZnₓTiᵧHfzOᵢ nanocomposite is an important task and the goal of this work. Forming-free nanocomposite ZnₓTiᵧHfzOᵢ thin film was grown by pulsed laser deposition (Pioneer 180, Neocera Co., USA) on the SiO2/TiN (40 nm) substrate. Electrical measurements were carried out using a semiconductor characterization system (Keithley 4200-SCS, USA) with W probes. During measurements, TiN film was grounded. The analysis of the obtained current-voltage characteristics showed a resistive switching from HRS to LRS resistance states at +1.87±0.12 V, and from LRS to HRS at -2.71±0.28 V. Endurance test shown that HRS was 283.21±32.12 kΩ, LRS was 1.32±0.21 kΩ during 100 measurements. It was shown that HRS/LRS ratio was about 214.55 at reading voltage of 0.6 V. The results can be useful for forming-free nanocomposite ZnₓTiᵧHfzOᵢ films in neuromorphic systems manufacturing. This work was supported by RFBR, according to the research project № 19-29-03041 mk. The results were obtained using the equipment of the Research and Education Center «Nanotechnologies» of Southern Federal University.

Keywords: nanotechnology, nanocomposites, neuromorphic systems, RRAM, pulsed laser deposition, resistive switching effect

Procedia PDF Downloads 132

Authors: Milica Carević, Katarina Banjanac, Marija ĆOrović, Ana Milivojević, Nevena Prlainović, Aleksandar Marinković, Dejan Bezbradica

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Nowadays, considering the growing awareness of functional food beneficial effects on human health, due attention is dedicated to the research in the field of obtaining new prominent products exhibiting improved physiological and physicochemical characteristics. Therefore, different approaches to valuable bioactive compounds synthesis have been proposed. β-Galactosidase, for example, although mainly utilized as hydrolytic enzyme, proved to be a promising tool for these purposes. Namely, under the particular conditions, such as high lactose concentration, elevated temperatures and low water activities, reaction of galactose moiety transfer to free hydroxyl group of the alternative acceptor (e.g. different sugars, alcohols or aromatic compounds) can generate a wide range of potentially interesting products. Up to now, galacto-oligosaccharides and lactulose have attracted the most attention due to their inherent prebiotic properties. The goal of this study was to obtain a novel product sorbitol galactoside, using the similar reaction mechanism, namely transgalactosylation reaction catalyzed by β-galactosidase from Aspergillus oryzae. By using sugar alcohol (sorbitol) as alternative acceptor, a diverse mixture of potential prebiotics is produced, enabling its more favorable functional features. Nevertheless, an introduction of alternative acceptor into the reaction mixture contributed to the complexity of reaction scheme, since several potential reaction pathways were introduced. Therefore, the thorough optimization using response surface method (RSM), in order to get an insight into different parameter (lactose concentration, sorbitol to lactose molar ratio, enzyme concentration, NaCl concentration and reaction time) influences, as well as their mutual interactions on product yield and productivity, was performed. In view of product yield maximization, the obtained model predicted optimal lactose concentration 500 mM, the molar ratio of sobitol to lactose 9, enzyme concentration 0.76 mg/ml, concentration of NaCl 0.8M, and the reaction time 7h. From the aspect of productivity, the optimum substrate molar ratio was found to be 1, while the values for other factors coincide. In order to additionally, improve enzyme efficiency and enable its reuse and potential continual application, immobilization of β-galactosidase onto tailored silica nanoparticles was performed. These non-porous fumed silica nanoparticles (FNS)were chosen on the basis of their biocompatibility and non-toxicity, as well as their advantageous mechanical and hydrodinamical properties. However, in order to achieve better compatibility between enzymes and the carrier, modifications of the silica surface using amino functional organosilane (3-aminopropyltrimethoxysilane, APTMS) were made. Obtained support with amino functional groups (AFNS) enabled high enzyme loadings and, more importantly, extremely high expressed activities, approximately 230 mg proteins/g and 2100 IU/g, respectively. Moreover, this immobilized preparation showed high affinity towards sorbitol galactoside synthesis. Therefore, the findings of this study could provided a valuable contribution to the efficient production of physiologically active galactosides in immobilized enzyme reactors.

Keywords: β-galactosidase, immobilization, silica nanoparticles, transgalactosylation

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Authors: Utpal Das, Sona Das

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InAs/GaSb type-II superlattice (T2SL) Mid-wave infrared (MWIR) focal plane arrays (FPAs) have recently seen rapid development. However, in small pixel size large format FPAs, the occurrence of high mesa sidewall surface leakage current is a major constraint necessitating proper surface passivation. A simple pixel isolation technique in InAs/GaSb T2SL detector arrays without the conventional mesa etching has been proposed to isolate the pixels by forming a more resistive higher band gap material from the SL, in the inter-pixel region. Here, a single step femtosecond (fs) laser anneal of the T2SL structure of the inter-pixel T2SL regions, have been used to increase the band gap between the pixels by QW-intermixing and hence increase isolation between the pixels. The p-i-n photodiode structure used here consists of a 506nm, (10 monolayer {ML}) InAs:Si (1x10¹⁸cm⁻³)/(10ML) GaSb SL as the bottom n-contact layer grown on an n-type GaSb substrate. The undoped absorber layer consists of 1.3µm, (10ML)InAs/(10ML)GaSb SL. The top p-contact layer is a 63nm, (10ML)InAs:Be(1x10¹⁸cm⁻³)/(10ML)GaSb T2SL. In order to improve the carrier transport, a 126nm of graded doped (10ML)InAs/(10ML)GaSb SL layer was added between the absorber and each contact layers. A 775nm 150fs-laser at a fluence of ~6mJ/cm² is used to expose the array where the pixel regions are masked by a Ti(200nm)-Au(300nm) cap. Here, in the inter-pixel regions, the p+ layer have been reactive ion etched (RIE) using CH₄+H₂ chemistry and removed before fs-laser exposure. The fs-laser anneal isolation improvement in 200-400μm pixels due to spatially selective quantum well intermixing for a blue shift of ~70meV in the inter-pixel regions is confirmed by FTIR measurements. Dark currents are measured between two adjacent pixels with the Ti(200nm)-Au(300nm) caps used as contacts. The T2SL quality in the active photodiode regions masked by the Ti-Au cap is hardly affected and retains the original quality of the detector. Although, fs-laser anneal of p+ only etched p-i-n T2SL diodes show a reduction in the reverse dark current, no significant improvement in the full RIE-etched mesa structures is noticeable. Hence for a 128x128 array fabrication of 8μm square pixels and 10µm pitch, SU8 polymer isolation after RIE pixel delineation has been used. X-n+ row contacts and Y-p+ column contacts have been used to measure the optical response of the individual pixels. The photo-response of these 8μm and other 200μm pixels under a 2ns optical pulse excitation from an Optical-Parametric-Oscillator (OPO), shows a peak responsivity of ~0.03A/W and 0.2mA/W, respectively, at λ~3.7μm. Temporal response of this detector array is seen to have a fast response ~10ns followed typical slow decay with ringing, attributed to impedance mismatch of the connecting co-axial cables. In conclusion, response times of a few ns have been measured in 8µm pixels of a 128x128 array. Although fs-laser anneal has been found to be useful in increasing the inter-pixel isolation in InAs/GaSb T2SL arrays by QW inter-mixing, it has not been found to be suitable for passivation of full RIE etched mesa structures with vertical walls on InAs/GaSb T2SL.

Keywords: band-gap blue-shift, fs-laser-anneal, InAs/GaSb T2SL, Inter-pixel isolation, ns-Response, photodiode array

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Authors: Chen Jiang, Eric Jordan, Maurice Gell, Balakrishnan Nair

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Nuclear fusion, one of the most promising options for reliably generating large amounts of carbon-free energy in the future, has seen a plethora of ground-breaking technological advances in recent years. An efficient and durable “breeding blanket”, needed to ensure a reactor’s self-sufficiency by maintaining the optimal coolant temperature as well as by minimizing radiation dosage behind the blanket, still remains a technological challenge for the various reactor designs for commercial fusion power plants. A relatively new dual-coolant lead-lithium (DCLL) breeder design has exhibited great potential for high-temperature (>700oC), high-thermal-efficiency (>40%) fusion reactor operation. However, the structural material, namely reduced activation ferritic-martensitic (RAFM) steel, is not chemically stable in contact with molten Pb-17%Li coolant. Thus, to utilize this new promising reactor design, the demand for effective corrosion-resistant coatings on RAFM steels represents a pressing need. Solution Spray Technologies LLC (SST) is developing a double-layer ceramic coating design to address the corrosion protection of RAFM steels, using a novel solution and solution/suspension plasma spray technology through a US Department of Energy-funded project. Plasma spray is a coating deposition method widely used in many energy applications. Novel derivatives of the conventional powder plasma spray process, known as the solution-precursor and solution/suspension-hybrid plasma spray process, are powerful methods to fabricate thin, dense ceramic coatings with complex compositions necessary for the corrosion protection in DCLL breeders. These processes can be used to produce ultra-fine molten splats and to allow fine adjustment of coating chemistry. Thin, dense ceramic coatings with chosen chemistry for superior chemical stability in molten Pb-Li, low activation properties, and good radiation tolerance, is ideal for corrosion-protection of RAFM steels. A key challenge is to accommodate its CTE mismatch with the RAFM substrate through the selection and incorporation of appropriate bond layers, thus allowing for enhanced coating durability and robustness. Systematic process optimization is being used to define the optimal plasma spray conditions for both the topcoat and bond-layer, and X-ray diffraction and SEM-EDS are applied to successfully validate the chemistry and phase composition of the coatings. The plasma-sprayed double-layer corrosion resistant coatings were also deposited onto simulated RAFM steel substrates, which are being tested separately under thermal cycling, high-temperature moist air oxidation as well as molten Pb-Li capsule corrosion conditions. Results from this testing on coated samples, and comparisons with bare RAFM reference samples will be presented and conclusions will be presented assessing the viability of the new ceramic coatings to be viable corrosion prevention systems for DCLL breeders in commercial nuclear fusion reactors.

Keywords: breeding blanket, corrosion protection, coating, plasma spray

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Authors: Ved Vrat Verma, Rani Gupta, Manisha Goel

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γ-glutamyltranspeptidase (GGT: EC 2.3.2.2) is an N-terminal nucleophile hydrolase conserved in all three domains of life. GGT plays a key role in glutathione metabolism where it catalyzes the breakage of the γ-glutamyl bonds and transfer of γ-glutamyl group to water (hydrolytic activity) or amino acids or short peptides (transpeptidase activity). GGTs from bacteria, archaea, and eukaryotes (human, rat and mouse) are homologous proteins sharing >50% sequence similarity and conserved four layered αββα sandwich like three dimensional structural fold. These proteins though similar in their structure to each other, are quite diverse in their enzyme activity: some GGTs are better at hydrolysis reactions but poor in transpeptidase activity, whereas many others may show opposite behaviour. GGT is known to be involved in various diseases like asthma, parkinson, arthritis, and gastric cancer. Its inhibition prior to chemotherapy treatments has been shown to sensitize tumours to the treatment. Microbial GGT is known to be a virulence factor too, important for the colonization of bacteria in host. However, all known inhibitors (mimics of its native substrate, glutamate) are highly toxic because they interfere with other enzyme pathways. However, a few successful efforts have been reported previously in designing species specific inhibitors. We aim to leverage the diversity seen in GGT family (pathogen vs. eukaryotes) for designing specific inhibitors. Thus, in the present study, we have used DIVERGE software to identify sites in GGT proteins, which are crucial for the functional and structural divergence of these proteins. Since, type II divergence sites vary in clade specific manner, so type II divergent sites were our focus of interest throughout the study. Type II divergent sites were identified for pathogen vs. eukaryotes clusters and sites were marked on clade specific representative structures HpGGT (2QM6) and HmGGT (4ZCG) of pathogen and eukaryotes clade respectively. The crucial divergent sites within 15 A radii of the binding cavity were highlighted, and in-silico mutations were performed on these sites to delineate the role of these sites on the mechanism of catalysis and protein folding. Further, the amino acid network (AAN) analysis was also performed by Cytoscape to delineate assortative mixing for cavity divergent sites which could strengthen our hypothesis. Additionally, molecular dynamics simulations were performed for wild complexes and mutant complexes close to physiological conditions (pH 7.0, 0.1 M ionic strength and 1 atm pressure) and the role of putative divergence sites and structural integrities of the homologous proteins have been analysed. The dynamics data were scrutinized in terms of RMSD, RMSF, non-native H-bonds and salt bridges. The RMSD, RMSF fluctuations of proteins complexes are compared, and the changes at protein ligand binding sites were highlighted. The outcomes of our study highlighted some crucial divergent sites which could be used for novel inhibitors designing in a species-specific manner. Since, for drug development, it is challenging to design novel drug by targeting similar protein which exists in eukaryotes, so this study could set up an initial platform to overcome this challenge and help to deduce the more effective targets for novel drug discovery.

Keywords: γ-glutamyltranspeptidase, divergence, species-specific, drug design

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Authors: Anas Hallak, Latifa Seblini, Juergen Wilde

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In mechatronics or sensor technology, plastic housings are used to protect sensitive components from harmful environmental influences, such as moisture, media, or reactive substances. Connections, preferably in the form of metallic lead-frame structures, through the housing wall are required for their electrical supply or control. In this system, an insufficient connection between the plastic component, e.g., Polyamide66, and the metal surface, e.g., copper, due to the incompatibility is dominating. As a result, leakage paths can occur along with the plastic-metal interface. Since adhesive bonding has been established as one of the most important joining processes and its use has expanded significantly, driven by the development of improved high-performance adhesives and bonding techniques, this technology has been involved in metal-plastic hybrid structures. In this study, an epoxy bonding agent from DELO (DUALBOND LT2266) has been used to improve the mechanical and chemical binding between the metal and the polymer. It is an adhesion promoter with two reaction stages. In these, the first stage provides fixation to the lead frame directly after the coating step, which can be done by UV-Exposure for a few seconds. In the second stage, the material will be thermally hardened during injection molding. To analyze the two reaction stages of the primer, dynamic DSC experiments were carried out and correlated with Fourier-transform infrared spectroscopy measurements. Furthermore, the number of crosslinking bonds formed in the system in each reaction stage has also been estimated by a rheological characterization. Those investigations have been performed with different times of UV exposure: 12, 96 s and in an industrial preferred temperature range from -20 to 175°C. The shear viscosity values of primer have been measured as a function of temperature and exposure times. For further interpretation, the storage modulus values have been calculated, and the so-called Booij–Palmen plot has been sketched. The next approach in this study is the self-healing mechanisms in the hydride system in which the primer should flow into micro-damage such as interface, cracks, inhibit them from growing, and close them. The ability of the primer to flow in and penetrate defined capillaries made in Ultramid was investigated. Holes with a diameter of 0.3 mm were produced in injection-molded A3EG7 plates with 4 mm thickness. A copper substrate coated with the DUALBOND was placed on the A3EG7 plate and pressed with a certain force. Metallographic analyses were carried out to verify the filling grade, which showed an almost 95% filling ratio of the capillaries. Finally, to estimate the self-healing mechanism in metal-plastic hybrid systems, characterizations have been done on a simple geometry with a metal inlay developed by the Institute of Polymer Technology in Friedrich-Alexander-University. The specimens have been modified with tungsten wire which was to be pulled out after the injection molding to create a micro-hole in the specimen at the interface between the primer and the polymer. The capability of the primer to heal those micro-cracks upon heating, pressing, and thermal aging has been characterized through metallographic analyses.

Keywords: hybrid structures, self-healing, thermoplastic housing, adhesive

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Authors: Natalie Riley, Anita Quigley, Robert M. I. Kapsa, George W. Greene

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As the fields of medicine and bionics grow rapidly in technological advancement, the future and success of it depends on the ability to effectively interface between the artificial and the biological worlds. The biggest obstacle when it comes to implantable, electronic medical devices, is maintaining a ‘clean’, low noise electrical connection that allows for efficient sharing of electrical information between the artificial and biological systems. Implant fouling occurs with the adhesion and accumulation of proteins and various cell types as a result of the immune response to protect itself from the foreign object, essentially forming an electrical insulation barrier that often leads to implant failure over time. Lubricin (LUB) functions as a major boundary lubricant in articular joints, a unique glycoprotein with impressive anti-adhesive properties that self-assembles to virtually any substrate to form a highly ordered, ‘telechelic’ polymer brush. LUB does not passivate electroactive surfaces which makes it ideal, along with its innate biocompatibility, as a coating for implantable bionic electrodes. It is the aim of the study to investigate LUB’s anti-fouling properties and its potential as a safe, bioinspired material for coating applications to enhance the performance and longevity of implantable medical devices as well as reducing the frequency of implant replacement surgeries. Native, bovine-derived LUB (N-LUB) and recombinant LUB (R-LUB) were applied to gold-coated mylar surfaces. Fibroblast, chondrocyte and neural cell types were cultured and grown on the coatings under both passive and electrically stimulated conditions to test the stability and anti-adhesive property of the LUB coating in the presence of an electric field. Lactate dehydrogenase (LDH) assays were conducted as a directly proportional cell population count on each surface along with immunofluorescent microscopy to visualize cells. One-way analysis of variance (ANOVA) with post-hoc Tukey’s test was used to test for statistical significance. Under both passive and electrically stimulated conditions, LUB significantly reduced cell attachment compared to bare gold. Comparing the two coating types, R-LUB reduced cell attachment significantly compared to its native counterpart. Immunofluorescent micrographs visually confirmed LUB’s antiadhesive property, R-LUB consistently demonstrating significantly less attached cells for both fibroblasts and chondrocytes. Preliminary results investigating neural cells have so far demonstrated that R-LUB has little effect on reducing neural cell attachment; the study is ongoing. Recombinant LUB coatings demonstrated impressive anti-adhesive properties, reducing cell attachment in fibroblasts and chondrocytes. These findings and the availability of recombinant LUB brings into question the results of previous experiments conducted using native-derived LUB, its potential not adequately represented nor realized due to unknown factors and impurities that warrant further study. R-LUB is stable and maintains its anti-fouling property under electrical stimulation, making it suitable for electroactive surfaces.

Keywords: anti-fouling, bioinspired, cell attachment, lubricin

Procedia PDF Downloads 124

Authors: Majed Al Otaibi, Melissa Lessard-Beaudoin, Amel Loudghi, Raphael Chouinard-Watkins, Melanie Plourde, Frederic Calon, C. Alexandre Castellano, Stephen Cunnane, Helene Payette, Pierrette Gaudreau, Denis Gris, Rona K. Graham

Abstract:

Introduction: Alzheimer disease (AD) is a chronic disorder that affects millions of individuals worldwide. Symptoms include memory dysfunction, and also alterations in attention, planning, language and overall cognitive function. Olfactory dysfunction is a common symptom of several neurological disorders including AD. Studying the mechanisms underlying the olfactory dysfunction may therefore lead to the discovery of potential biomarkers and/or treatments for neurodegenerative diseases. Objectives: To determine if olfactory dysfunction predicts future cognitive impairment in the aging population and to characterize the olfactory system in a murine model expressing a genetic factor of AD. Method: For the human study, quantitative olfactory tests (UPSIT and OMT) have been done on 93 subjects (aged 80 to 94 years) from the Quebec Longitudinal Study on Nutrition and Successful Aging (NuAge) cohort accepting to participate in the ORCA secondary study. The telephone Modified Mini Mental State examination (t-MMSE) was used to assess cognition levels, and an olfactory self-report was also collected. In a separate cohort, olfactory cortical volume was calculated using MRI results from healthy old adults (n=25) and patients with AD (n=18) using the AAL single-subject atlas and performed with the PNEURO tool (PMOD 3.7). For the murine study, we are using Western blotting, RT-PCR and immunohistochemistry. Result: Human Study: Based on the self-report, 81% of the participants claimed to not suffer from any problem with olfaction. However, based on the UPSIT, 94% of those subjects showed a poor olfactory performance and different forms of microsmia. Moreover, the results confirm that olfactory function declines with age. We also detected a significant decrease in olfactory cortical volume in AD individuals compared to controls. Murine study: Preliminary data demonstrate there is a significant decrease in expression levels of the proform of caspase-3 and the caspase substrate STK3, in the olfactory bulb of mice expressing human APOE4 compared with controls. In addition, there is a significant decrease in the expression level of the caspase-9 proform and caspase-8 active fragment. Analysis of the mature neuron marker, NeuN, shows decreased expression levels of both isoforms. The data also suggest that Iba-1 immunostaining is increased in the olfactory bulb of APOE4 mice compared to wild type mice. Conclusions: The activation of caspase-3 may be the cause of the decreased levels of STK3 through caspase cleavage and may play role in the inflammation observed. In the clinical study, our results suggest that seniors are unaware of their olfactory function status and therefore it is not sufficient to measure olfaction using the self-report in the elderly. Studying olfactory function and cognitive performance in the aging population will help to discover biomarkers in the early stage of the AD.

Keywords: Alzheimer's disease, APOE4, cognition, caspase, brain atrophy, neurodegenerative, olfactory dysfunction

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Authors: Shlomit Paz, Meir Broza

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Cholera is an acute intestinal infection caused by ingestion of food or water contaminated with the bacterium Vibrio cholerae. It has a short incubation period and produces an enterotoxin that causes copious, painless, watery diarrhoea that can quickly lead to severe dehydration and death if treatment is not promptly given. In an epidemic, the source of the contamination is usually the feces of an infected person. The disease can spread rapidly in areas with poor treatment of sewage and drinking water. Cholera remains a global threat and is one of the key indicators of social development. An estimated 3-5 million cases and over 100,000 deaths occur each year around the world. The relevance of climatic events as causative factors for cholera epidemics is well known. However, the examination of the involvement of winds in intra-continental disease distribution is new. The study explore the hypothesis that the spreading of cholera epidemics may be related to the dominant wind direction over land by presenting the influence of the wind direction on windborn dissemination by flying insects, which may serve as vectors. Chironomids ("non-biting midges“) exist in the majority of freshwater aquatic habitats, especially in estuarine and organic-rich water bodies typical to Vibrio cholerae. Chironomid adults emerge into the air for mating and dispersion. They are highly mobile, huge in number and found frequently in the air at various elevations. The huge number of chironomid egg masses attached to hard substrate on the water surface, serve as a reservoir for the free-living Vibrio bacteria. Both male and female, while emerging from the water, may carry the cholera bacteria. In experimental simulation, it was demonstrated that the cholera-bearing adult midges are carried by the wind, and transmit the bacteria from one body of water to another. In our previous study, the geographic diffusions of three cholera outbreaks were examined through their linkage with the wind direction: a) the progress of Vibrio cholerae O1 biotype El Tor in Africa during 1970–1971 and b) again in 2005–2006; and c) the rapid spread of Vibrio cholerae O139 over India during 1992–1993. Using data and map of cholera dissemination (WHO database) and mean monthly SLP and geopotential data (NOAA NCEP-NCAR database), analysis of air pressure data at sea level and at several altitudes over Africa, India and Bangladesh show a correspondence between the dominant wind direction and the intra-continental spread of cholera. The results support the hypothesis that aeroplankton (the tiny life forms that float in the air and that may be caught and carried upward by the wind, landing far from their origin) carry the cholera bacteria from one body of water to an adjacent one. In addition to these findings, the current follow-up study will present new results regarding the possible involvement of winds in the spreading of cholera in recent outbreaks (2010-2013). The findings may improve the understanding of how climatic factors are involved in the rapid distribution of new strains throughout a vast continental area. Awareness of the aerial transfer of Vibrio cholerae may assist health authorities by improving the prediction of the disease’s geographic dissemination.

Keywords: cholera, Vibrio cholerae, wind direction, Vibrio cholerae dissemination

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Authors: Shanna Swart, Caryn Fenner, Athanasios Kotsiopoulos, Susan Harrison

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Linear alkanes are produced as by-products from the increasing use of gas-to-liquid fuel technologies for synthetic fuel production and offer great potential for value addition. Their current use as low-value fuels and solvents do not maximize this potential. Therefore, attention has been drawn towards direct activation of these aliphatic alkanes to more useful products such as alcohols, aldehydes, carboxylic acids and derivatives. Cytochrome P450 monooxygenases (P450s) can be used for activation of these aliphatic alkanes using whole-cells or cell-free systems. Some limitations of whole-cell systems include reduced mass transfer, stability and possible side reactions. Since the P450 systems are little studied as cell-free systems, they form the focus of this study. Challenges of a cell-free system include co-factor regeneration, substrate availability and enzyme stability. Enzyme immobilization offers a positive outlook on this dilemma, as it may enhance stability of the enzyme. In the present study, 2 different P450s (CYP153A6 and CYP102A1) as well as the relevant accessory enzymes required for electron transfer (ferredoxin and ferredoxin reductase) and co-factor regeneration (glucose dehydrogenase) have been expressed in E. coli and purified by metal affinity chromatography. Glucose dehydrogenase (GDH), was used as a model enzyme to assess the potential of various enzyme immobilization strategies including; surface attachment on MagReSyn® microspheres with various functionalities and on electrospun nanofibers, using self-assembly based methods forming Cross Linked Enzymes (CLE), Cross Linked Enzyme Aggregates (CLEAs) and spherezymes as well as in a sol gel. The nanofibers were synthesized by electrospinning, which required the building of an electrospinning machine. The nanofiber morphology has been analyzed by SEM and binding will be further verified by FT-IR. Covalent attachment based methods showed limitations where only ferredoxin reductase and GDH retained activity after immobilization which were largely attributed to insufficient electron transfer and inactivation caused by the crosslinkers (60% and 90% relative activity loss for the free enzyme when using 0.5% glutaraldehyde and glutaraldehyde/ethylenediamine (1:1 v/v), respectively). So far, initial experiments with GDH have shown the most potential when immobilized via their His-tag onto the surface of MagReSyn® microspheres functionalized with Ni-NTA. It was found that Crude GDH could be simultaneously purified and immobilized with sufficient activity retention. Immobilized pure and crude GDH could be recycled 9 and 10 times, respectively, with approximately 10% activity remaining. The immobilized GDH was also more stable than the free enzyme after storage for 14 days at 4˚C. This immobilization strategy will also be applied to the P450s and optimized with regards to enzyme loading and immobilization time, as well as characterized and compared with the free enzymes. It is anticipated that the proposed immobilization set-up will offer enhanced enzyme stability (as well as reusability and easy recovery), minimal mass transfer limitation, with continuous co-factor regeneration and minimal enzyme leaching. All of which provide a positive outlook on this robust multi-enzyme system for efficient activation of linear alkanes as well as the potential for immobilization of various multiple enzymes, including multimeric enzymes for different bio-catalytic applications beyond alkane activation.

Keywords: alkane activation, cytochrome P450 monooxygenase, enzyme catalysis, enzyme immobilization

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Authors: Alex J. Summers, Jasmine P. Devadhasan, Douglas Montgomery, Brittany Fischer, Jian Gu, Frederic Zenhausern

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Immunoassays have been utilized for several applications, including the detection of pathogens. Our laboratory is in the development of a tier 1 biothreat panel utilizing Vertical Flow Assay (VFA) technology for simultaneous detection of pathogens and toxins. One method of manufacturing VFA membranes is with non-contact piezoelectric dispensing, which provides advantages, such as low-volume and rapid dispensing without compromising the structural integrity of antibody or substrate. Challenges of this processinclude premature discontinuation of dispensing and misaligned spotting. Preliminary data revealed the Yp 11C7 mAb (11C7)reagent to exhibit a large angle of failure during printing which may have contributed to variable printing outputs. A Design of Experiment (DOE) was executed using this reagent to investigate the effects of hydrostatic pressure and reagent concentration on microarray printing outputs. A Nano-plotter 2.1 (GeSIM, Germany) was used for printing antibody reagents ontonitrocellulose membrane sheets in a clean room environment. A spotting plan was executed using Spot-Front-End software to dispense volumes of 11C7 reagent (20-50 droplets; 1.5-5 mg/mL) in a 6-test spot array at 50 target membrane locations. Hydrostatic pressure was controlled by raising the Pressure Compensation Vessel (PCV) above or lowering it below our current working level. It was hypothesized that raising or lowering the PCV 6 inches would be sufficient to cause either liquid accumulation at the tip or discontinue droplet formation. After aspirating 11C7 reagent, we tested this hypothesis under stroboscope.75% of the effective raised PCV height and of our hypothesized lowered PCV height were used. Humidity (55%) was maintained using an Airwin BO-CT1 humidifier. The number and quality of membranes was assessed after staining printed membranes with dye. The droplet angle of failure was recorded before and after printing to determine a “stroboscope score” for each run. The DOE set was analyzed using JMP software. Hydrostatic pressure and reagent concentration had a significant effect on the number of membranes output. As hydrostatic pressure was increased by raising the PCV 3.75 inches or decreased by lowering the PCV -4.5 inches, membrane output decreased. However, with the hydrostatic pressure closest to equilibrium, our current working level, membrane output, reached the 50-membrane target. As the reagent concentration increased from 1.5 to 5 mg/mL, the membrane output also increased. Reagent concentration likely effected the number of membrane output due to the associated dispensing volume needed to saturate the membranes. However, only hydrostatic pressure had a significant effect on stroboscope score, which could be due to discontinuation of dispensing, and thus the stroboscope check could not find a droplet to record. Our JMP predictive model had a high degree of agreement with our observed results. The JMP model predicted that dispensing the highest concentration of 11C7 at our current PCV working level would yield the highest number of quality membranes, which correlated with our results. Acknowledgements: This work was supported by the Chemical Biological Technologies Directorate (Contract # HDTRA1-16-C-0026) and the Advanced Technology International (Contract # MCDC-18-04-09-002) from the Department of Defense Chemical and Biological Defense program through the Defense Threat Reduction Agency (DTRA).

Keywords: immunoassay, microarray, design of experiment, piezoelectric dispensing

Procedia PDF Downloads 183