Search results for: time-lapse incubator

Authors: Aydan Atalar, Nurcan Cetinkaya

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Corn stover is leftover of the leaves, stalk, husks and tassels in the field after harvesting the grain combined. Corn stover is a low-quality roughage but has mostly been used as roughage source for feeding ruminant animals in developing countries including Turkey; however, it can also be used to make biofuels as in developed countries. The objectives of the present study were to improve the digestibility of corn stover by the treatment of white rod fungus mainly Pleurotus osteritus (PO), Pleurotus eryingii (PE) and Lantinula edudes (LE) at different incubation times and also to determine the most effective fungus and incubation time to prepare fermeted corn stover for ruminant nutrition. The choped corn stover was treated with PO, PE and LE and incubated for 10, 20, 30 and 40 days in incubator at 26 0C. After each incubation time dry matter(DM), organic matter(OM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), neutral detergent lignin (ADL), in-vitro true dry matter digestibility (IVTDMD) and organic matter digestibility (IVTOMD) were determined. The mean IVTDMD and IVTOMD levels were increased by PO, PE and LE treatments in increasing order of incubation times. The obtained IVTDM values were 59.45, 60.51, 60.82 and 60.18 %; 59.45, 70.55, 67.18 and 66.96 %; 59.45, 70.55, 67.18 and 66,96 %; 59.45, 74.90, 69.18 % ; 59.45, 76.50, 71.24 and 73.04 for control, PO, PE and LE treatments at 0, 10, 20, 30 and 40 days incubation times respectively. The obtained IVTOMD values were 56.45,60.26,60.82and 60.18 %; 56.45, 68.70, 67.18 and 66.96 %; 56.45, 71.26, 69.18 and 69.28 %; 56.45, 73.23, 71.24 and 73.04 % for control, PO, PE and LE treatments at 0, 10, 20, 30 and 40 days incubation times respectively. The most effective fungus was PO and the incubation time was 30 days. In conclusion, PO treatment of corn stover with 30 days incubation may be used to prepare fermented corn stover for ruminant nutrition.

Keywords: biological treatment, corn stover, digestibility, Lantinula edudes, Pleurotus eryingii, Pleurotus osteritus

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Authors: Behnam Mahdiyan Nasl

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In a lab-scale research, the effects of feeding whey into the biogas system and how to solve the probable problems arising were analysed. In the study a semi-continuous glass reactor, having a total capacity of 13 liters and having a working capacity of 10 liters, was placed in an incubator, and the temperature was tried to be held at 38 °C. At first, the reactor was operated by adding 5 liters of animal manure and water with a ratio of 1/1. By passing time, the production rate of the gas reduced intensively that on the fourth day there was no production of gas and the system stopped working. In this condition, the pH was adjusted and by adding NaOH, it was increased from 5.4 to 7. On 48th day, the first gas measurement was done and an amount of 12.07 % of CH₄ was detected. After making buffer in the ambient, the number of bacteria existing in the cattle’s manure and contributing to the gas production was thought to be not adequate, and up to 20 % of its volume 2 liters of mud was added to the reactor. 7 days after adding the anaerobic mud, second gas measurement was carried out, and biogas including 43 % CH₄ was obtained. From the 61st day of the study, the cheese whey with the animal manure was started to be added with an amount of 40 mL per day. However, by passing time, the raising of the microorganisms existed in the whey (especially Ni and Co), the percent of methane in the biogas decreased. In fact, 2 weeks after adding PAS, the gas measurement was done and 36,97 % CH₄ was detected. 0,06 mL Ni-Co (to gain a concentration of 0.05 mg/L in the reactor’s mixture) solution was added to the system for 15 days. To find out the effect of the solution on archaea, 7 days after stopping addition of the solution, methane gas was found to have a 9,03 % increase and reach 46 %. Lastly, the effects of adding molasses to the reactor were investigated. The effects of its activity on the bacteria was analysed by adding 4 grams of it to the system. After adding molasses in 10 days, according to the last measurement, the amount of methane gas reached up to 49%.

Keywords: biogas, cheese whey, cattle manure, energy

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Authors: Lisete Barlach, Guilherme Ary Plonski

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Innovative ideas are not always synonymous with business opportunities. Any idea can be creative and not recognized as a potential project in which money and time will be invested, among other resources. Even in firms that promote and enhance innovation, there are two 'check-points', the first corresponding to the acknowledgment of the idea as creative and the second, its consideration as a business opportunity. Both the recognition of new business opportunities or new ideas involve cognitive and psychological frameworks which provide individuals with a basis for noticing connections between seemingly independent events or trends as if they were 'connecting the dots'. It also involves prototypes-representing the most typical member of a certain category–functioning as 'templates' for this recognition. There is a general assumption that these kinds of evaluation processes develop through experience, explaining why expertise plays a central role in this process: the more experienced a professional, the easier for him (her) to identify new opportunities in business. But, paradoxically, an increase in expertise can lead to the inflexibility of thought due to automation of procedures. And, besides this, other cognitive biases can also be present, because new ideas or business opportunities generally depend on heuristics, rather than on established algorithms. The paper presents a literature review about the Einstellung effect by tracking famous cases of rejected ideas, extracted from historical records. It also presents the results of empirical research, with data upon rejected ideas gathered from two different environments: projects rejected during first semester of 2017 at a large incubator center in Sao Paulo and ideas proposed by employees that were rejected by a well-known business company, at its Brazilian headquarter. There is an implicit assumption that Einstellung effect tends to be more and more present in contemporaneity, due to time pressure upon decision-making and idea generation process. The analysis discusses desirability, viability, and feasibility as elements that affect decision-making.

Keywords: cognitive biases, Einstellung effect, recognition of business opportunities, rejected ideas

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Authors: Sauradipta Ganguly, Akhato Sumi, Sanjeet Kumar Hom, Ajan T. Lotha

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Populus deltoides is a hardwood used predominantly for the manufacturing of plywood, matchsticks, and paper in India and hence has a higher economical significance. Wood-decaying fungi cause serious damage to Populus deltoides products, as the wood itself is perishable and vulnerable to decaying agents, decreasing their aesthetical value which in return results in significant monetary loss for the wood industries concerned. The aim of the study was to determine the antifungal activity of Cedrus deodara oil against three primary wood-decaying fungi namely white-rot fungi (Trametes versicolor), brown-rot fungi (Oligoporus placentus) and soft-rot fungi (Xylaria acuminata) on Populus deltoides samples under optimum laboratory conditions. The susceptibility of Populus deltoides samples on the fungal attack and the ability of deodar oil to control colonization of the wood rotting fungi on the samples were assessed. Three concentrations of deodar oil were considered for the study as treating solutions, i.e., 4%, 5%, and 6%. The Populus deltoides samples were treated with treating solutions, and the ability of the same to prevent a fungal attack on the samples were assessed using accelerated test in the laboratory at Biochemical Oxygen Demand incubator at temperature (25 ± 2°C) and relative humidity 70 ± 4%. Efficacy test and statistical analysis of deodar oil against Trametes versicolor, Oligoporus placentus, and Xylariaacuminataon P. deltoides samples exhibited light, minor and negligible mycelia growth at 4 %, 5% and 6% concentrations of deodar oil, respectively. Whereas, moderate to heavy attack was observed on the surface of the control samples. Statistical analysis further established that the treatments were statistically significant and had significantly inhibited fungal growth of all the three fungus spp by almost 3 to 5 times.

Keywords: populus deltoides, Trametes versicolor, Oligoporus placentus, Xylaria acuminata, Deodar oil, treatment

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Authors: Lorenzo Cocola, Massimo Fedel, Dragiša Savić, Bojana Danilović, Luca Poletto

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An instrument for the detection and evaluation of gaseous carbon dioxide in the headspace of closed containers has been developed in the context of Packsensor Italian-Serbian joint project. The device is based on Tunable Diode Laser Absorption Spectroscopy (TDLAS) with a Wavelength Modulation Spectroscopy (WMS) technique in order to accomplish a non-invasive measurement inside closed containers of fermented dairy products (yogurts and fermented cheese in cups and bottles). The purpose of this instrument is the continuous monitoring of carbon dioxide concentration during incubation and storage of products over a time span of the whole shelf life of the product, in the presence of different microorganisms. The instrument’s optical front end has been designed to be integrated in a thermally stabilized incubator. An embedded computer provides processing of spectral artifacts and storage of an arbitrary set of calibration data allowing a properly calibrated measurement on many samples (cups and bottles) of different shapes and sizes commonly found in the retail distribution. A calibration protocol has been developed in order to be able to calibrate the instrument on the field also on containers which are notoriously difficult to seal properly. This calibration protocol is described and evaluated against reference measurements obtained through an industry standard (sampling) carbon dioxide metering technique. Some sets of validation test measurements on different containers are reported. Two test recordings of carbon dioxide concentration evolution are shown as an example of instrument operation. The first demonstrates the ability to monitor a rapid yeast growth in a contaminated sample through the increase of headspace carbon dioxide. Another experiment shows the dissolution transient with a non-saturated liquid medium in presence of a carbon dioxide rich headspace atmosphere.

Keywords: TDLAS, carbon dioxide, cups, headspace, measurement

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Authors: Aurora Gitto, Philipp Proksch

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The presence of various microorganisms (bacteria, fungi) in surface water and groundwater represents an important issue for human health worldwide. The matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) has emerged as a promising and reliable tool for bacteria identification in clinical diagnostic microbiology and environmental strains thanks to an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation. The study aims first to conceptualise and set up library information and create a comprehensive database of MALDI-TOF-MS spectra from environmental water samples. The samples were analysed over a year (2021-2022) using membrane filtration methodology (0.45 μm and 0.22 μm) and then isolated on R2A agar for a period of 5 days and Yeast extract agar growing at 22 °C up to 4 days and 37 °C for 48 hours. The undetected organisms by MALDI-TOF-MS were analysed by PCR and then sequenced. The information obtained by the sequencing was further implemented in the MALDI-TOF-MS library. Among the culturable bacteria, the results show how the incubator temperature affects the growth of some genera instead of others, as demonstrated by Pseudomonas sp., which grows at 22 °C, compared to Bacillus sp., which is abundant at 37 °C. The bacteria community shows a variation in composition also between the media used, as demonstrated with R2A agar which has been defined by a higher presence of organisms not detected compared to YEA. Interesting is the variability of the Genus over one year of sampling and how the seasonality impacts the bacteria community; in fact, in some sampling locations, we observed how the composition changed, moving from winter to spring and summer. In conclusion, the bacteria community in groundwater and river bank filtration represents important information that needs to be added to the library to simplify future water quality analysis but mainly to prevent potential risks to human health.

Keywords: water quality, MALDI-TOF-MS, sequencing, library

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Authors: Cristobal Miralles Insa

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The network of chairs of the "Valencian Public System of Social Services" (VPSSS) is sponsored by the Valencian Institute of Training, Research, and Quality in Social Services and aims to promote research, dissemination, and evaluation of the needs that arise in the field of the public system of social services. It also seeks to transfer knowledge to foster the development of public policies in this field. Given that it is an Interuniversity Chair among the five public universities in Valencia, there is coordination of complementary themes and roles for this objective, with Universitat Politènica de València focusing primarily on promoting innovation and social entrepreneurship to address multiple social challenges through its platform INSSPIRA. This approach is aimed at the entire university community and its various interest groups, carrying out research, teaching, and dissemination activities that promote social inclusion, personal development, and autonomy for groups in situations of vulnerability, lack of protection, dependence, or social urgency. Although it focuses on the Valencian context, both the issues in this context and the tools in process to address them, often have a universal and scalable character and has been inspiring for the innovation system of UPV. This entrepreneurial incubator goes along from early stages of students on the campus until the so-called “StartUPV” system, where students are challenged with social problems that require creative solutions. Therefore, the Chair is conceived with a holistic spirit and an inspiring vocation that engages the whole university community. In this communication, it is described the entities and individuals participating in this UPV Chair of VPSSS, followed by the presentation of different work lines and objectives for the chair. Subsequently, a description of various activities undertaken to promote innovation in social services are described, where support to teaching and extracurricular activities in this field are exposed. It must be noted that some awareness and dissemination of activities are carried out in a transversal mode as they contribute to different objectives simultaneously; with special focus on Learning-Service approaches that achieved very good results which are also summarized.

Keywords: social innovation, entrepeneurship, university, vulnerable sectors

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Authors: Dong-An Wang

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All kinds of microspheres have been extensively employed as carriers for drug, gene and therapeutic cell delivery. Most therapeutic cell delivery microspheres rely on a two-step methodology: fabrication of microspheres and subsequent seeding of cells onto them. In this study, we have developed a novel one-step cell encapsulation technique using a convenient and instant water-in-oil single emulsion approach to form cell-encapsulated gelatin microspheres. This technology is adopted for hyaline cartilage tissue engineering, in which autologous chondrocytes are used as therapeutic cells. Cell viability was maintained throughout and after the microsphere formation (75-100 µm diameters) process that avoids involvement of any covalent bonding reactions or exposure to any further chemicals. Further encapsulation of cell-laden microspheres in alginate gels were performed under 4°C via a prompt process. Upon the formation of alginate constructs, they were immediately relocated into CO2 incubator where the temperature was maintained at 37°C; under this temperature, the cell-laden gelatin microspheres dissolved within hours to yield similarly sized cavities and the chondrocytes were therefore suspended within the cavities inside the alginate gel bulk. Hence, the gelatin cell-laden microspheres served two roles: as cell delivery vehicles which can be removable through temperature curing, and as porogens within an alginate hydrogel construct to provide living space for cell growth and tissue development as well as better permeability for mutual diffusions. These cell-laden microspheres, namely “temperature-cured dissolvable gelatin microsphere based cell carriers” (tDGMCs), were further encapsulated in a chondrocyte-laden alginate scaffold system and analyzed by WST-1, gene expression analyses, biochemical assays, histology and immunochemistry stains. The positive results consistently demonstrated the promise of tDGMC technology in delivering these non-anchorage dependent cells (chondrocytes). It can be further conveniently translated into delivery of other non-anchorage dependent cell species, including stem cells, progenitors or iPS cells, for regeneration of tissues in internal organs, such as engineered hepatogenesis or pancreatic regeneration.

Keywords: biomaterials, tissue engineering, microsphere, hydrogel, porogen, anchorage dependence

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Authors: Boukrouh Soumaya, Cabaraux Jean-François, Avril Claire, Noutfia Ali, Chentouf Mouad

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The introduction of marginal leguminous forage species in the diet of ruminants are of great importance. Bitter vetch is a good source of proteins, highly resistant against drought and poor soil conditions. Accordingly; two years field trials (2018/2019 and 2019-2020) were conducted to determine the digestibility of straw and grains of 17 promising bitter vetch ecotypes(Vicia ervilia) in the north of Morocco. In vitro dry and organic matter digestibility, gas production, and kinetics of fermentation of grains and straw were evaluated using gas production technique, pepsin-cellulase enzymatic digestibility of DM (CDDM)and OM (CDOM), as well as protease enzymatic CP degradation (CPD) and in vitro true digestibility, were performed using DAISYII Incubator. In vitro digestibility was performed using gas production method of (Menke et al., 1979) improved by Menke and Steingass (1988). Samples were incubated in glass syringes that contained rumen fluid and incubation solution that conserved in water bath in 39°C during 72 hours. Gas production was recorded after 2, 4, 8, 12, 24, 48, and 72 hours. Studied digestibility parameters were dry and organic matter digestibility, microbial biomass production, partitioning factor, and volatile fatty acids. Enzymatic dry matter digestibility was different (p < 0.05) among grains and straw for all ecotypes. It varied from 804.1 to 957.7 g/kg DM and 270.4 to 412.3 g/kg DM for grains and straw, respectively. Metabolizable energy varied between 11.7 to 14.3 MJ/kg DM and 2.6 to 5.0 MJ/kg DM for grains and straw, respectively. Potential gas production (A), the rate constants (c and d), and lag times of grains and straws from different bitter vetch ecotypes were different (p > 0.05). The results emphasized that in any evaluation of bitter vetch ecotypes, where straw of this legume seed is used as an animal feed, not only seed yield but also yield and quality of straw should be taken into consideration, particularly in areas where straw from this legume is considered as an important feedstuff for ruminants. Enzymatic digestibility was lower than in vitro digestibility by gaz production and by the DAISYII method because rumen fluid contains bacteria than increase digestibility. There was no difference between in vitro digestibility by gaz production and the DAISY II method. The DAISY II method can be used to increase labor efficiency in the in vitro DM digestibility analysis if gaz production is not necessary for analysis.

Keywords: bitter vetch, grains, straw, ecotype, in vitro digestibility, gaz production, enzymatic digestibility

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Authors: Ning Zhang, Kateřina Grabicová, Pavel Horký, Zuzana Linhartová, Jan Turek, Martin Pšenička, Linan Gao, Roman Grabic, Tomáš Randák

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Sewage treatment plant (STP) effluent is a significant source of pharmaceuticals and personal care products (PPCPs) in the aquatic environment. Exposure laboratory studies have a limited reporting capacity to capture the complex effect of actual pollution on exposed organisms in the aquatic environment. This study aimed to study the impact of contamination originating from the STP's effluent on early life stages development and sex determination of brown trout (Salmo trutta m. fario) – a native coldwater species with a long period of embryo-larval development – by using special floating egg incubators usually used to support native fish species within fisheries management. Brown trout eggs were placed upstream (the control group) and downstream (the exposure group) of the STP Prachatice situated on the Zivny stream. Up to 72 PPCPs were detected in at least one water sample with higher concentration downstream than upstream. Compared with the control group, the mortality of brown trout in the exposure group was significantly higher; after statistical correction of temperature, the size, growth rate, and metabolic rate of brown trout in the exposure group were significantly lower than those in the control group. After several months of natural growth in the stream, the male-to-female ratio of randomly caught brown trout in the exposure group was 1: 1.9, showing the imbalance of sex ratio. These results suggest that STP effluents can negatively affect the early growth and development of fish in watersheds, and these negative effects may further affect the population density of aquatic organisms and the balance of the whole aquatic ecosystem. The use of floating egg incubators proved to be a promising approach for studying the effect of pollution on the early developmental stages of fish in natural conditions.

Keywords: fish egg incubator, PPCPs, real exposure scenario, STP

Procedia PDF Downloads 33

Authors: Yumeho Obata, Mariko Era, Takayoshi Kawahara, Takahide Kanyama, Hiroshi Morita

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Dermatophagoides farinae is major mite allergens in indoors. D. farinae is often swarm over powder products (e.g. wheat flour), because it feeds on starch or protein that are included in them. Eating powder products which are mixed D.farinae causes various allergic symptoms. Therefore, the creation of food additive agents with high safety and control of mite effect is required. Fatty acid salts and spices are known that have pesticidal activities. This study describes the effects of fatty acid salts and spices against Dermatophagoides farinae. Materials and Methods: Potassium salts of 9 fatty acids (C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C18:1, C18:2, C18:3) were prepared by mixing the fatty acid with the appropriate amount of KOH solution to a concentration of 175 mM and pH 10.5. C12Cu and C12Zn were selected as other fatty acid salts. Cayenne pepper, habanero, Japanese pepper, mustard, jalapeno pepper, curry aroma and cinnamon were selected as spices. D. farina, have been cultured in laboratory. To rear the mites, double-soled dishes containing of sterilized food were put on the big plastic container (30.0 × 20.0 × 20.0cm) which had 100% ammonium nitrate solution in the bottom. Plastic container was placed on incubator at 25 °C and 64 % relative humidity (RH) under dark condition. Sterilized food composed of dried bonito flakes and dried yeast (Ebios), 1:1 by weight. The antiproliferative method, sample and medium culture were mixed in double-soled dish and kept at 25 °C and 64 % RH. Decrease rates were determined 1 week and 4 week after treatment under microscope. D. farina was considered to be dead if appendages did not move when prodded with a pin. Results and Conclusions: The results show that the fatty acids potassium showed no antiproliferative effects against D. farinae. On the other hand, Japanese pepper, mustard, curry aroma and cinnamon were effective to decrease propagative rate (over 80 %) after treatment for 1 week against D. farina. Japanese pepper, curry aroma and cinnamon were effective to decrease propagative rate (approximately 100 %) after treatment for 4 weeks against D. farina. Especially, Japanese pepper and cinnamon showed the fasted and the most consecutive antiproliferative effects. These results indicate that Japanese pepper and cinnamon have high antiproliferative effects against D. farina and suggest spices will be used as a food additive agent.

Keywords: fatty acid salts, spices, antiproliferative effects, dermatophagoides farinae

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Authors: T. Hamaishi, Y. Morinaga, H. Morita

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Introduction: Rice consumption in Japan has decreased, and Japanese government has recommended use of rice flour in order to expand the consumption of rice. There are two major protein components present in flour, called gliadin and glutenin. Gluten forms when water is added to flour and is mixed. As mixing continues, glutenin interacts with gliadin to form viscoelastic matrix of gluten. Rice flour bread does not expand as much as wheat flour bread. Because rice flour is not included gluten, it cannot construct gluten network in the dough. In recent years, some food additives have been used for dough-improving agent in bread making, especially surfactants has effect in order to improve dough extensibility. Therefore, we focused to fatty acid salt which is one of anionic surfactants. Fatty acid salt is a salt consist of fatty acid and alkali, it is main components of soap. According to JECFA(FAO/WHO Joint Expert Committee on Food Additives), salts of Myristic(C14), Palmitic(C16) and Stearic(C18) could be used as food additive. They have been evaluated ADI was not specified. In this study, we investigated to improving bread-making properties of rice flour dough adding fatty acid salt. Materials and methods: The sample of fatty acid salt is myristic (C14) dissolved in KOH solution to a concentration of 350 mM and pH 10.5. Rice dough was consisted of 100 g of flour using rice flour and wheat gluten, 5 g of sugar, 1.7 g of salt, 1.7g of dry yeast, 80 mL of water and fatty acid salt. Mixing was performed for 500 times by using hand. The concentration of C14K in the dough was 10 % relative to flour weight. Amount of gluten in the dough was 20 %, 30 % relative to flour weight. Dough expansion ability test was performed to measure physical property of bread dough according to the methods of Baker’s Yeast by Japan Yeast Industry Association. In this test, 150 g of dough was filled from bottom of the cylinder and fermented at 30 °C，85 % humidity for 120 min on an incubator. The height of the expansion in the dough was measured and determined its expansion ability. Results and Conclusion: Expansion ability of rice dough with gluten content of 20 %, 30% showed 316 mL, 341 mL for 120 min. When C14K adding to the rice dough, dough expansion abilities were 314 mL, 368 mL for 120 min, there was no significant difference. Conventionally it has been known that the rice flour dough contain gluten of 20 %. The considerable improvement of dough expansion ability was achieved when added C14K to wheat flour. The experimental result shows that c14k adding to the rice dough with gluten content more than 20 % was not improving bread-making properties. In conclusion, rice bread made with gluten content more than 20 % without C14K has been suggested to contribute to the formation of the sufficient gluten network.

Keywords: expansion ability, fatty acid salt, gluten, rice flour dough

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Authors: Ogemdi Chinwendu Anika, Anna Strzelecka, Yadira Bajón-Fernández, Raffaella Villa

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Anaerobic digestion (AD) has been used since time in memorial to take care of organic wastes in the environment, especially for sewage and wastewater treatments. Recently, the rising demand/need to increase renewable energy from organic matter has caused the AD substrates spectrum to expand and include a wider variety of organic materials such as agricultural residues and farm manure which is annually generated at around 140 billion metric tons globally. The problem, however, is that agricultural wastes are composed of materials that are heterogeneous and too difficult to degrade -particularly lignin, that make up about 0–40% of the total lignocellulose content. This study aimed to evaluate the impact of varying concentrations of lignin on biogas yields and their subsequent response to a commercial yeast-based bioadditive in batch anaerobic digesters. The experiments were carried out in batches for a retention time of 56 days with different lignin concentrations (200 mg, 300 mg, 400 mg, 500 mg, and 600 mg) treated to different conditions to first determine the concentration of the bioadditive that was most optimal for overall process improvement and yields increase. The batch experiments were set up using 130 mL bottles with a working volume of 60mL, maintained at 38°C in an incubator shaker (150rpm). Digestate obtained from a local plant operating at mesophilic conditions was used as the starting inoculum, and commercial kraft lignin was used as feedstock. Biogas measurements were carried out using the displacement method and were corrected to standard temperature and pressure using standard gas equations. Furthermore, the modified Gompertz equation model was used to non-linearly regress the resulting data to estimate gas production potential, production rates, and the duration of lag phases as indicatives of degrees of lignin inhibition. The results showed that lignin had a strong inhibitory effect on the AD process, and the higher the lignin concentration, the more the inhibition. Also, the modelling showed that the rates of gas production were influenced by the concentrations of the lignin substrate added to the system – the higher the lignin concentrations in mg (0, 200, 300, 400, 500, and 600) the lower the respective rate of gas production in ml/gVS.day (3.3, 2.2, 2.3, 1.6, 1.3, and 1.1), although the 300 mg increased by 0.1 ml/gVS.day over that of the 200 mg. The impact of the yeast-based bioaddition on the rate of production was most significant in the 400 mg and 500 mg as the rate was improved by 0.1 ml/gVS.day and 0.2 ml/gVS.day respectively. This indicates that agricultural residues with higher lignin content may be more responsive to inhibition alleviation by yeast-based bioadditive; therefore, further study on its application to the AD of agricultural residues of high lignin content will be the next step in this research.

Keywords: anaerobic digestion, renewable energy, lignin valorisation, biogas

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Authors: K. J. Keerthi, Vasundhara Kamineni, A. Ravi Shanker, T. Rammurthy, A. Vijaya Lakshmi, Q. Hasan

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Pancreatic β-cells are the predominant insulin-producing cell types within the Islets of Langerhans and insulin is the primary hormone which regulates carbohydrate and fat metabolism. Apoptosis of β-cells or insufficient insulin production leads to Diabetes Mellitus (DM). Current therapy for diabetes includes either medical management or insulin replacement and regular monitoring. Replacement of β- cells is an attractive treatment option for both Type-1 and Type-2 DM in view of the recent paper which indicates that β-cells apoptosis is the common underlying cause for both the Types of DM. With the development of Edmonton protocol, pancreatic β-cells allo-transplantation became possible, but this is still not considered as standard of care due to subsequent requirement of lifelong immunosuppression and the scarcity of suitable healthy organs to retrieve pancreatic β-cell. Fetal pancreatic cells from abortuses were developed as a possible therapeutic option for Diabetes, however, this posed several ethical issues. Hence, in the present study Mesenchymal stem cells (MSCs) were differentiated into insulin producing cells which were isolated from Human Umbilical cord (HUC) tissue. MSCs have already made their mark in the growing field of regenerative medicine, and their therapeutic worth has already been validated for a number of conditions. HUC samples were collected with prior informed consent as approved by the Institutional ethical committee. HUC (n=26) were processed using a combination of both mechanical and enzymatic (collagenase-II, 100 U/ml, Gibco ) methods to obtain MSCs which were cultured in-vitro in L-DMEM (Low glucose Dulbecco's Modified Eagle's Medium, Sigma, 4.5 mM glucose/L), 10% FBS in 5% CO2 incubator at 37°C. After reaching 80-90% confluency, MSCs were characterized with Flowcytometry and Immunocytochemistry for specific cell surface antigens. Cells expressed CD90+, CD73+, CD105+, CD34-, CD45-, HLA-DR-/Low and Vimentin+. These cells were differentiated to β-cells by using H-DMEM (High glucose Dulbecco's Modified Eagle's Medium,25 mM glucose/L, Gibco), β-Mercaptoethanol (0.1mM, Hi-Media), basic Fibroblast growth factor (10 µg /L,Gibco), and Nicotinamide (10 mmol/L, Hi-Media). Pancreatic β-cells were confirmed by positive Dithizone staining and were found to be functionally active as they released 8 IU/ml insulin on glucose stimulation. Isolating MSCs from usually discarded, abundantly available HUC tissue, expanding and differentiating to β-cells may be the most feasible cell therapy option for the millions of people suffering from DM globally.

Keywords: diabetes mellitus, human umbilical cord, mesenchymal stem cells, differentiation

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Authors: S. Agarwal, A. Rani

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Catechol is a natural polyphenolic compound that widely exists in higher plants such as teas, vegetables, fruits, tobaccos, and some traditional Chinese medicines. The fly ash-based zeolites are capable of absorbing a wide range of pollutants. But the process of zeolite synthesis is time-consuming and requires technical setups by the industries. The marketed costs of zeolites are quite high restricting its use by small-scale industries for the removal of phenolic compounds. The present research proposes a simple method of alkaline treatment of FA to produce an effective adsorbent for catechol removal from wastewater. The experimental parameter such as pH, temperature, initial concentration and adsorbent dose on the removal of catechol were studied in batch reactor. For this purpose the adsorbent materials were mixed with aqueous solutions containing catechol ranging in 50 – 200 mg/L initial concentrations and then shaken continuously in a thermostatic Orbital Incubator Shaker at 30 ± 0.1 °C for 24 h. The samples were withdrawn from the shaker at predetermined time interval and separated by centrifugation (Centrifuge machine MBL-20) at 2000 rpm for 4 min. to yield a clear supernatant for analysis of the equilibrium concentrations of the solutes. The concentrations were measured with Double Beam UV/Visible spectrophotometer (model Spectrscan UV 2600/02) at the wavelength of 275 nm for catechol. In the present study, the use of low-cost adsorbent (BTFA) derived from coal fly ash (FA), has been investigated as a substitute of expensive methods for the sequestration of catechol. The FA and BTFA adsorbents were well characterized by XRF, FE-SEM with EDX, FTIR, and surface area and porosity measurement which proves the chemical constituents, functional groups and morphology of the adsorbents. The catechol adsorption capacities of synthesized BTFA and native material were determined. The adsorption was slightly increased with an increase in pH value. The monolayer adsorption capacities of FA and BTFA for catechol were 100 mg g⁻¹ and 333.33 mg g⁻¹ respectively, and maximum adsorption occurs within 60 minutes for both adsorbents used in this test. The equilibrium data are fitted by Freundlich isotherm found on the basis of error analysis (RMSE, SSE, and χ²). Adsorption was found to be spontaneous and exothermic on the basis of thermodynamic parameters (ΔG°, ΔS°, and ΔH°). Pseudo-second-order kinetic model better fitted the data for both FA and BTFA. BTFA showed large adsorptive characteristics, high separation selectivity, and excellent recyclability than FA. These findings indicate that BTFA could be employed as an effective and inexpensive adsorbent for the removal of catechol from wastewater.

Keywords: catechol, fly ash, isotherms, kinetics, thermodynamic parameters

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Authors: Khrystyna Pavlyk

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In the OECD countries, percentage of adults with higher education degrees has increased by 10 % during 2000-2010. Continuously increasing demand for higher education gives universities a chance of becoming key players in socio-economic development of a territory (region or city) via knowledge creation, knowledge transfer, and knowledge spillovers. During previous decade, universities have tried to support spin-offs and start-ups, introduced courses on sustainability and corporate social responsibility. While much has been done, new trends are starting to emerge in search of better approaches. Recently a number of universities created centers that conduct research in a field social entrepreneurship, which in turn underpin educational programs run at these universities. The list includes but is not limited to the Centre for Social Economy at University of Liège, Institute for Social Innovation at ESADE, Skoll Centre for Social Entrepreneurship at Oxford, Centre for Social Entrepreneurship at Rosklide, Social Entrepreneurship Initiative at INSEAD. Existing literature already examined social entrepreneurship centers in terms of position in the institutional structure, initial and additional funding, teaching initiatives, research achievements, and outreach activities. At the same time, Universities can become social enterprises themselves. Previous research revealed that universities use both business and social entrepreneurship models. Universities which are mainly driven by a social mission are more likely to transform into social entrepreneurial institutions. At the same time, currently, there is no clear understanding of what social entrepreneurship in higher education is about and thus social entrepreneurship in higher education needs to be studied and promoted at the same time. Main roles which socially oriented university can play in city development include: buyer (implementation of socially focused local procurement programs creates partnerships focused on local sustainable growth.); seller (centers created by universities can sell socially oriented goods and services, e.g. in consultancy.); employer (Universities can employ socially vulnerable groups.); business incubator (which will help current student to start their social enterprises). In the paper, we will analyze these in more detail. We will also examine a number of indicators that can be used to assess the impact, both direct and indirect, that universities can have on city's economy. At the same time, originality of this paper mainly lies not in methodological approaches used, but in countries evaluated. Social entrepreneurship is still treated as a relatively new phenomenon in post-transitional countries where social services were provided only by the state for many decades. Paper will provide data and example’s both from developed countries (the US and EU), and those located in CIS and CEE region.

Keywords: social enterprise, university, regional economic development, comparative study

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Authors: Surekha Barkur, Aseefhali Bankapur, Santhosh Chidangil

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Raman Tweezers technique has become prevalent in single cell studies. This technique combines Raman spectroscopy which gives information about molecular vibrations, with optical tweezers which use a tightly focused laser beam for trapping the single cells. Thus Raman Tweezers enabled researchers analyze single cells and explore different applications. The applications of Raman Tweezers include studying blood cells, monitoring blood-related disorders, silver nanoparticle-induced stress, etc. There is increased interest in the toxic effect of nanoparticles with an increase in the various applications of nanoparticles. The interaction of these nanoparticles with the cells may vary with their size. We have studied the effect of silver nanoparticles of sizes 10nm, 40nm, and 100nm on erythrocytes using Raman Tweezers technique. Our aim was to investigate the size dependence of the nanoparticle effect on RBCs. We used 785nm laser (Starbright Diode Laser, Torsana Laser Tech, Denmark) for both trapping and Raman spectroscopic studies. 100 x oil immersion objectives with high numerical aperture (NA 1.3) is used to focus the laser beam into a sample cell. The back-scattered light is collected using the same microscope objective and focused into the spectrometer (Horiba Jobin Vyon iHR320 with 1200grooves/mm grating blazed at 750nm). Liquid nitrogen cooled CCD (Symphony CCD-1024x256-OPEN-1LS) was used for signal detection. Blood was drawn from healthy volunteers in vacutainer tubes and centrifuged to separate the blood components. 1.5 ml of silver nanoparticles was washed twice with distilled water leaving 0.1 ml silver nanoparticles in the bottom of the vial. The concentration of silver nanoparticles is 0.02mg/ml so the 0.03mg of nanoparticles will be present in the 0.1 ml nanoparticles obtained. The 25 ul of RBCs were diluted in 2 ml of PBS solution and then treated with 50 ul (0.015mg) of nanoparticles and incubated in CO2 incubator. Raman spectroscopic measurements were done after 24 hours and 48 hours of incubation. All the spectra were recorded with 10mW laser power (785nm diode laser), 60s of accumulation time and 2 accumulations. Major changes were observed in the peaks 565 cm-1, 1211 cm-1, 1224 cm-1, 1371 cm-1, 1638 cm-1. A decrease in intensity of 565 cm-1, increase in 1211 cm-1 with a reduction in 1224 cm-1, increase in intensity of 1371 cm-1 also peak disappearing at 1635 cm-1 indicates deoxygenation of hemoglobin. Nanoparticles with higher size were showing maximum spectral changes. Lesser changes observed in case of 10nm nanoparticle-treated erythrocyte spectra.

Keywords: erythrocytes, nanoparticle-induced toxicity, Raman tweezers, silver nanoparticles

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Authors: Krzysztof Buczak, Sonia Lachowska, Pawel Kucharski, Agnieszka Antonczyk

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Tiletamine - zolazepam combination is increasingly used in veterinary anaesthesiology in wild animals, including snakes. The available literature shows a lack of information about anesthesia in this mixture in ovoviviparous snakes. The studies show the possibility of using the combination at a dose of 20 mg/kg or more for snake immobilization. This paper presents an anesthetic protocol with the use of a combination of tiletamine - zolazepam at the dose of 10 mg/kg intramuscularly and maintenance with inhalant anesthesia with isoflurane in pure oxygen. The objective of this study was to evaluate the usefulness of the anesthetic protocol to proceed with coeliotomy in Amazon Tree Boa. The patient was a five years old bicolor female Amazon Tree Boa (Corallus hortulanus) with dystocia. The clinical examination reveals significant emaciation (bodyweight 520g), high degree of dehydration, heart rate (HR = 60 / min), pale mucous membranes and poor reactivity. Meloxicam (1 mg/kg) and tramadol (10 mg/kg) were administered subcutaneously and the patient was placed in an incubator with access to fresh oxygen. Four hours later, the combination of tiletamine - zolazepam (10 mg/kg) was administered intramuscularly for induction of anesthesia. The snake was intubated and connected to inhalant anesthesia equipment. For maintenance, the anesthesia isoflurane in pure oxygen was used due to apnea, which occurs 30 minutes after the induction semi-closed system was attached and the ventilator was turned on (PCV system, four breaths per minute, 8 cm of H2O). Cardiopulmonary parameters (HR, RR, SPO2, ETCO2, ETISO) were assessed throughout the procedure. During the entire procedure, the operating room was heated to a temperature of 26 degrees Celsius. Additionally, the hose was placed on a heating mat, which maintained a temperature of 30 degrees Celsius. For 15 minutes after induction, the loss of muscle tone was observed from the head to the tail. Induction of general anesthesia was scored as good because of the possibility of intubation. During the whole procedure, the heart rate was at the rate of 58 beats per minute (bpm). Ventilation parameters were stable throughout the procedure. The recovery period lasts for about 4 hours after the end of general anesthesia. The muscle tension returned from tail to head. The snake started to breathe spontaneously within 1,5 hours after the end of general anesthesia. The protocol of general anesthesia with the combination of tiletamine- zolazepam with a dose of 10 mg/kg is useful for proceeding with the emergency coeliotomy in maintenance with isoflurane in oxygen. Further study about the impact of the combination of tiletamine- zolazepam for the recovery period is needed.

Keywords: anesthesia, corallus hortulanus, ovoviparous, snake, tiletamine, zolazepam

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Authors: Abdolamir Allameh, Shahnaz Esmaeili, Mina Allameh, Safoura Khajeniazi

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Stem cells with therapeutic applications can be isolated from human placenta/umblical cord blood (UCB) as well as the cord tissue (UC). Stem cells in culture are vulnerable to oxidative stress, particularly when subjected to differentiation process. The aim of this study was to examine the chnages in the rate of oxidation that occurs to cellular macromolecules during hepatic differentiation of mononuclear cells (MSCs). In addition, the impact of the hepatic differentiation process of MSC on cellular and biological activity of the cells will be undertaken. For this purpose, first mononuclear cells (MNCs) were isolated from human UCB which was obtained from a healthy full-term infant. The cells were cultured at a density of 3×10⁵ cells/cm² in DMEM- low-glucose culture media supplemented with 20% FBS, 2 mM L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin. Cell cultures were then incubated at 37°C in a humidified 5% CO₂ incubator. After removing non-adherent cells by replacing culture medium, fibroblast-like adherent cells were resuspended in 0.25% trypsin-EDTA and plated in 25 cm² flasks (1×10⁴/ml). Characterization of the MSCs was routinely done by observing their morphology and growth curve. MSCs were subjected to a 2-step hepatocyte differentiation protocol in presence of hepatocyte growth factor (HGF), dexamethazone (DEX) and oncostatin M (OSM). The hepatocyte-like cells derived from MSCs were checked every week for 3 weeks for changes in lipid peroxidation, protein carbonyl formation and DNA oxidation i.e., 8-hydroxy-2'-deoxyguanosine (8-OH-dG) assay. During the 3-week differentiation process of MSCs to hepatocyte-like cells we found that expression liver-specific markers such as albumin, was associated with increased levels of lipid peroxidation and protein carbonyl formation. Whereas, undifferentiated MSCs has relatively low levels of lipid peroxidation products. There was a significant increase ( p < 0.05) in lipid peroxidation products in hepatocytes on days 7, 14, and 21 of differentiation. Likewise, the level of protein carbonyls in the cells was elevated during the differentiation. The level of protein carbonyls measured in hepatocyte-like cells obtained 3 weeks after differentiation induction was estimated to be ~6 fold higher compared to cells recovered on day 7 of differentiation. On the contrary, there was a small but significant decrease in DNA damage marker (8-OH-dG) in hepatocytes recovered 3 weeks after differentiation onset. The level of 8-OHdG which was in consistent with formation of reactive oxygen species (ROS). In conclusion, this data suggest that despite the elevation in oxidation of lipid and protein molecules during hepatocyte development, the cells were normal in terms of DNA integrity, morphology, and biologically activity.

Keywords: adult stem cells, DNA integrity, free radicals, hepatic differentiation

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Authors: Norhalim Yunus, Mohamad Husaini Dahalan, Nor Halina Ghazali

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Building technology start-ups from ground zero into world-class companies in form and substance present a rare opportunity for government-affiliated institutions in Malaysia. The challenge of building such start-ups becomes tougher when their core businesses involve commercialization of unproven technologies for the mass market. These simple truths, while difficult to execute, will go a long way in getting a business off the ground and flying high. Malaysian Technology Development Corporation (MTDC), a company founded to facilitate the commercial exploitation of R&D findings from research institutions and universities, and eventually help translate these findings of applications in the marketplace, is an excellent case in point. The purpose of this paper is to examine MTDC as an institution as it explores the concept of ‘it takes a village to raise a child’ in an effort to create and nurture start-ups into established world class Malaysian technology companies. With MTDC at the centre of Malaysia's innovative start-ups, the analysis seeks to specifically answer two questions: How has the concept been applied in MTDC? and what can we learn from this successful case? A key aim is to elucidate how MTDC's journey as a private limited company can help leverage reforms and achieve transformation, a process that might be suitable for other small, open, third world and developing countries. This paper employs a single case study, designed to acquire an in-depth understanding of how MTDC has developed and grown technology start-ups to world-class technology companies. The case study methodology is employed as the focus is on a contemporary phenomenon within a real business context. It also explains the causal links in real-life situations where a single survey or experiment is unable to unearth. The findings show that MTDC maximises the concept of it needs a village to raise a child in totality, as MTDC itself assumes the role of the innovator to 'raise' start-up companies into world-class stature. As the innovator, MTDC creates shared value and leadership, introduces innovative programmes ahead of the curve, mobilises talents for optimum results and aggregates knowledge for personnel advancement. The success of the company's effort is attributed largely to leadership, visionary, adaptability, commitment to innovate, partnership and networking, and entrepreneurial drive. The findings of this paper are however limited by the single case study of MTDC. Future research is required to study more cases of success or/and failure where the concept of it takes a village to raise a child have been explored and applied.

Keywords: start-ups, technology transfer, commercialization, technology incubator

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Authors: Mairaj Salim, Asma Zaheer

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Oman has accomplished as much or more than its fellow Gulf monarchies, despite starting from scratch considerably later, having less oil income to utilize, dealing with a larger and more rugged geography, and resolving a bitter civil war along the way. Of course, Oman's progress in the past 30-plus years has not been without problems and missteps, but the balance is squarely on the positive side of the ledger. Oil has been the driving force of the Omani economy since Oman began commercial production in 1967. The oil industry supports the country’s high standard of living and is primarily responsible for its modern and expansive infrastructure, including electrical utilities, telephone services, roads, public education and medical services. In addition to extensive oil reserves, Oman also has substantial natural gas reserves, which are expected to play a leading role in the Omani economy in the Twenty-first Century. To reduce the country’s dependence on oil revenues, the government is restructuring the economy by directing investment to non-oil activities. Since the 21st century IT has changed the performing tasks. To manage the affairs for the benefits of organizations and economy, the Omani government has adopted E-Business technologies for the development. E-Business is important because it allows • Transformation of old economy relationships (vertical/linear relationships) to new economy relationships characterized by end-to-end relationship management solutions (integrated or extended relationships) • Facilitation and organization of networks, small firms depend on ‘partner’ firms for supplies and product distribution to meet customer demands • SMEs to outsource back-end process or cost centers enabling the SME to focus on their core competence • ICT to connect, manage and integrate processes internally and externally • SMEs to join networks and enter new markets, through shortened supply chains to increase market share, customers and suppliers • SMEs to take up the benefits of e-business to reduce costs, increase customer satisfaction, improve client referral and attract quality partners • New business models of collaboration for SMEs to increase their skill base • SMEs to enter virtual trading arena and increase their market reach A national strategy for the advancement of information and communication technology (ICT) has been worked out, mainly to introduce e-government, e-commerce, and a digital society. An information technology complex KOM (Knowledge Oasis Muscat) had been established, consisting of section for information technology, incubator services, a shopping center of technology software and hardware, ICT colleges, E-Government services and other relevant services. So, all these efforts play a vital role in the development of Oman economy.

Keywords: ICT, ITA, CRM, SCM, ERP, KOM, SMEs, e-commerce and e-business

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Authors: Banafsheh Nikmehr, Mohsen Bahrami, Yueqiang Song, Anuradha Koduru, Ayse K. Vuruskan, Hongkun Lu, Mallory Pitts, Tolga B. Mesen, Tamer M. Yalcinkaya

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The number of people who receive in vitro fertilization (IVF) treatment has increased on a startling trajectory over the past two decades. Despite advances in this field, particularly the introduction of intracytoplasmic sperm injection (ICSI) and the preimplantation genetic screening (PGS), the IVF success remains low. A major factor contributing to IVF failure is embryonic aneuploidy (abnormal chromosome content), which often results in miscarriage and birth defects. Although PGS is often used as the standard diagnostic tool to identify aneuploid embryos, it is an invasive approach that could affect the embryo development, and yet inaccessible to many patients due its high costs. As such, there is a clear need for a non-invasive cost-effective approach to identify euploid embryos for single embryo transfer (SET). The reported differences between morphokinetic behaviors of aneuploid and euploid embryos has shown promise to address this need. However, current literature is inconclusive and further research is urgently needed to translate current findings into clinical diagnostics. In this ongoing study, we found significant differences between morphokinetic behaviors of euploid and aneuploid embryos that provides important insights and reaffirms the promise of such behaviors for developing non-invasive methodologies. Methodology—A total of 242 embryos (euploid: 149, aneuploid: 93) from 74 patients who underwent IVF treatment in Carolinas Fertility Clinics in Winston-Salem, NC, were analyzed. All embryos were incubated in an EmbryoScope incubator. The patients were randomly selected from January 2019 to June 2021 with most patients having both euploid and aneuploid embryos. All embryos reached the blastocyst stage and had known PGS outcomes. The ploidy assessment was done by a third-party testing laboratory on day 5-7 embryo biopsies. The morphokinetic variables of each embryo were measured by the EmbryoViewer software (Uniesense FertiliTech) on time-lapse images using 7 focal depths. We compared the time to: pronuclei fading (tPNf), division to 2,3,…,9 cells (t2, t3,…,t9), start of embryo compaction (tSC), Morula formation (tM), start of blastocyst formation (tSC), blastocyst formation (tB), and blastocyst expansion (tEB), as well as intervals between them (e.g., c23 = t3 – t2). We used a mixed regression method for our statistical analyses to account for the correlation between multiple embryos per patient. Major Findings— The average age of the patients was 35.04 yrs. The average patient age associated with euploid and aneuploid embryos was not different (P = 0.6454). We found a significant difference in c45 = t5-t4 (P = 0.0298). Our results indicated this interval on average lasts significantly longer for aneuploid embryos - c45(aneuploid) = 11.93hr vs c45(euploid) = 7.97hr. In a separate analysis limited to embryos from the same patients (patients = 47, total embryos=200, euploid=112, aneuploid=88), we obtained the same results (P = 0.0316). The statistical power for this analysis exceeded 87%. No other variable was different between the two groups. Conclusion— Our results demonstrate the importance of morphokinetic variables as potential biomarkers that could aid in non-invasively characterizing euploid and aneuploid embryos. We seek to study a larger population of embryos and incorporate the embryo quality in future studies.

Keywords: IVF, embryo, euploidy, aneuploidy, morphokinteic

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Authors: Seyedeh Nasim Mirbahari, Taha Azad, Mehdi Totonchi

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Oncolytic viruses, which only replicate in tumor cells, are being extensively studied for their use in cancer therapy. A particular virus known as the vaccinia virus, a member of the poxvirus family, has demonstrated oncolytic abilities glioma. Treating Glioma with traditional methods such as chemotherapy and radiotherapy is quite challenging. Even though oncolytic viruses have shown immense potential in cancer treatment, their effectiveness in glioblastoma treatment is still low. Therefore, there is a need to improve and optimize immunotherapies for better results. In this study, we have designed oncoVV-TT, which can more effectively target tumor cells while minimizing replication in normal cells by replacing the thymidine kinase gene with a luc-p2a-GFP gene expression cassette. Human glioblastoma cell line U251 MG, rat glioblastoma cell line C6, and non-tumor cell line HFF were plated at 105 cells in a 12-well plates in 2 mL of DMEM-F2 medium with 10% FBS added to each well. Then incubated at 37°C. After 16 hours, the cells were treated with oncoVV-TT at an MOI of 0.01, 0.1 and left in the incubator for a further 24, 48, 72 and 96 hours. Viral replication assay, fluorescence imaging and viability tests, including trypan blue and crystal violet, were conducted to evaluate the cytotoxic effect of oncoVV-TT. The finding shows that oncoVV-TT had significantly higher cytotoxic activity and proliferation rates in tumor cells in a dose and time-dependent manner, with the strongest effect observed in U251 MG. To conclude, oncoVV-TT has the potential to be a promising oncolytic virus for cancer treatment, with a more cytotoxic effect in human glioblastoma cells versus rat glioma cells. To assess the effectiveness of vaccinia virus-mediated viral therapy, we have tested U251mg and C6 tumor cell lines taken from human and rat gliomas, respectively. The study evaluated oncoVV-TT's ability to replicate and lyse cells and analyzed the survival rates of the tested cell lines when treated with different doses of oncoVV-TT. Additionally, we compared the sensitivity of human and mouse glioma cell lines to the oncolytic vaccinia virus. All experiments regarding viruses were conducted under biosafety level 2. We engineered a Vaccinia-based oncolytic virus called oncoVV-TT to replicate specifically in tumor cells. To propagate the oncoVV-TT virus, HeLa cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 10 MOI virus was added. After 48 h, cells were harvested by scraping, and viruses were collected by 3 sequential freezing and thawing cycles followed by removal of cell debris by centrifugation (1500 rpm, 5 min). The supernatant was stored at −80 ◦C for the following experiments. To measure the replication of the virus in Hela, cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 5 MOI virus or equal dilution of PBS was added. At the treatment time of 0 h, 24 h, 48 h, 72 h and 96 h, the viral titers were determined under the fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). Fluorescence intensity was quantified using the imagej software according to the manufacturer’s protocol. For the isolation of single-virus clones, HeLa cells seeded in six-well plates (5×105 cells/well). After 24 h (100% confluent), the cells were infected with a 10-fold dilution series of TianTan green fluorescent protein (GFP)virus and incubated for 4 h. To examine the cytotoxic effect of oncoVV-TT virus ofn U251mg and C6 cell, trypan blue and crystal violet assay was used.

Keywords: oncolytic virus, immune therapy, glioma, vaccinia virus

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