Search results for: oral production

Authors: Michael Tupý, Jaroslav Císař, Pavel Mokrejš, Dagmar Měřínská, Alice Tesaříková-Svobodová

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The worldwide problem is that the recycled PVB is wildly stored in landfills. However, PVB have very similar chemical properties such as PVC. Moreover, both of them are used in plasticized form. Thus, the thermal properties of plasticized PVC obtained from primary production and the PVB was obtained by recycling of windshields are compared. It is carried out in order to find degradable conditions and decide if blend of PVB/PVC can be processable together. Tested PVC contained 38 % of plasticizer diisononyl phthalate (DINP) and PVB was plasticized with 28 % of triethylene glycol, bis(2-ethylhexanoate) (3GO). Thermal and thermo-oxidative decomposition of both vinyl polymers are compared such as DSC and OOT analysis. The tensile strength analysis is added.

Keywords: polyvinyl chloride, polyvinyl butyral, recycling, reprocessing, thermal analysis, decomposition

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Authors: Jyoti Manandhar Shrestha, Saurab Raj Joshi, Maya Shrestha, Prashanna Shrestha, Kshitij Chaulagain

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Background: The widespread use of non-steroidal anti-inflammatory drugs has increased the incidence of ulcer and serious complications, such as perforation and bleeding. Although, the H2 receptor blockers and proton pump inhibitors decrease the acid secretion and promote healing of ulcer, their value in preventing relapse, recurrence, “acid rebound” after cessation of therapy and associated long term adverse effects limit their utility. So to minimize this, the herbal plant Aloe vera having anti-oxidant, anti-inflammatory, mucus secreting, cyto-protective and healing property is believed to cure the peptic ulcer. Objectives: To observe whether oral treatment with Aloe vera gel can prevent peptic ulcer. Indomethacin and ethanol were used to induce gastric ulcers. Thirty six albino rats of either sex were randomly allotted to six groups of six animals each. The negative control was pretreated with normal saline, the positive controls received ranitidine (20 mg/kg) and the test group received Aloe vera gel (300 mg/kg) orally for eight days. Then, after a 24 hour fast Indomethacin (20 mg/kg) or 80% ethanol (2ml) was administered orally to induce ulceration. At the end of the study, the rats were sacrificed, their stomachs opened, the ulcer index studied and tissues sent for histopathological examination. Results: It was observed that, in indomethacin treated group, the ulcer index in control group was 8.167 ± 1.72.In the Aloe vera pretreated animals, the ulcer index was 2.83 ± 1.72 and the standard ranitidine pretreated group ulcer index was 1.67 ± 1.36. In ethanol treated group, the ulcer index in control group was 7.5 ± 2.73. In the Aloe vera pretreated animals, the ulcer index was 2.67 ± 1.75 and the standard ranitidine pretreated group ulcer index was 1.33±1.21. Both ranitidine and Aloe vera gel significantly prevented stomach from gastric ulceration induced by indomethacin and ethanol. Conclusion: The results indicated that Aloe vera gel is effective against indomethacin and ethanol mediated gastric ulcer.

Keywords: Aloe vera gel, ethanol, indomethacin, peptic ulcer, ranitidine

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Authors: V. Ziegler, F. Schneider, M. Pesch

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With the EDM264, GRIMM offers a solution for mobile short- and long-term measurements in outdoor areas and at production sites. For research as well as permanent areal observations on a near reference quality base. The model EDM264 features a powerful and robust measuring cell based on optical particle counting (OPC) principle with all the advantages that users of GRIMM's portable aerosol spectrometers are used to. The system is embedded in a compact weather-protection housing with all-weather sampling, heated inlet system, data logger, and meteorological sensor. With TSP, PM10, PM4, PM2.5, PM1, and PMcoarse, the EDM264 provides all fine dust fractions real-time, valid for outdoor applications and calculated with the proven GRIMM enviro-algorithm, as well as six additional dust mass fractions pm10, pm2.5, pm1, inhalable, thoracic and respirable for IAQ and workplace measurements. This highly versatile instrument performs real-time monitoring of particle number, particle size and provides information on particle surface distribution as well as dust mass distribution. GRIMM's EDM264 has 31 equidistant size channels, which are PSL traceable. A high-end data logger enables data acquisition and wireless communication via LTE, WLAN, or wired via Ethernet. Backup copies of the measurement data are stored in the device directly. The rinsing air function, which protects the laser and detector in the optical cell, further increases the reliability and long term stability of the EDM264 under different environmental and climatic conditions. The entire sample volume flow of 1.2 L/min is analyzed by 100% in the optical cell, which assures excellent counting efficiency at low and high concentrations and complies with the ISO 21501-1standard for OPCs. With all these features, the EDM264 is a world-leading dust monitor for precise monitoring of particulate matter and particle number concentration. This highly reliable instrument is an indispensable tool for many users who need to measure aerosol levels and air quality outdoors, on construction sites, or at production facilities.

Keywords: aerosol research, aerial observation, fence line monitoring, wild fire detection

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Authors: Sam Yurdakul, Nick Baverstock, Jim Mills

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TDT 064 (FLEXISEQ®) is a drug-free gel used to treat osteoarthritis (OA)-associated pain and joint stiffness. It contains ultra-deformable phospholipid Sequessome™ vesicles, which can pass through the skin barrier intact. In six randomized OA studies, topical TDT 064 was well tolerated and improved joint pain, physical function and stiffness. In the largest study, these TDT 064-mediated effects were statistically significantly greater than oral placebo and equivalent to celecoxib. To understand the therapeutic effects of TDT 064, we investigated the localisation of the drug-free vesicles within rat synovial joints. TDT 064 containing DiO-labelled Sequessome™ vesicles was applied to the knees of four 6-week-old CD® hairless rats (10 mg/kg/ joint), 2–3 times/day, for 3 days (representing the recommended clinical dose). Eighteen hours later, the animals and one untreated control were sacrificed, and the knee joints isolated, flash frozen and embedded in Acrytol Mounting Media™. Approximately 15 sections (10 µm) from each joint were analysed by fluorescence microscopy. To investigate whether the localisation of DiO fluorescence was associated with intact vesicles, an anti-PEG monoclonal antibody (mAb) was used to detect Tween, a constituent of Sequessome™ vesicles. Sections were visualized at 484 nm (DiO) and 647 nm (anti-PEG mAb) and analysed using inForm 1.4 (Perkin Elmer, Inc.). Significant fluorescence was observed at 484 nm in sections from TDT 064-treated animals. No non-specific fluorescence was observed in control sections. Fluorescence was detected as discrete vesicles on the cartilage surfaces, inside the cartilaginous matrix and within the synovial space. The number of DiO-labelled vesicles in multiple fields of view was consistent and >100 in sections from four different treated knees. DiO and anti-PEG mAb co-localised within the collagenous tissues in four different joint sections. Under higher magnification (40x), vesicles were seen in the intercellular spaces of the synovial joint tissue, but no fluorescence was seen inside cells. These data suggest that the phospholipid vesicles in TDT 064 localize at the surface of the joint cartilage; these vesicles may therefore be supplementing the phospholipid deficiency reported in OA and acting as a biolubricant within the synovial joint.

Keywords: joint pain, osteoarthritis, phospholipid vesicles, TDT 064

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Authors: Fiorenzo Lenzi, Doris Abt, Besnik Bytyqi

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Recent developments in composite materials for the interior cabin market provide more sustainable solutions for industrial applications. One contribution comes from epoxy-based prepregs recently developed to substitute phenolic prepregs in order to reduce the environmental impact of their production process and to eliminate health and safety issues related to their handling. Another example is the use of Mica-based products for improving the fire protection of interior cabin parts. Minerals, such as Mica, can be used as reinforcement in composites to reduce the heat release rate or, more traditionally, to improve the burn-through performance of fuselage and cargo lining components.

Keywords: prepreg, epoxy, Mica, battery protection

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Authors: Kim Kennedy, Daren Gibson, Stephanie Flukes, Chandra Diwakarla, Lisa Spalding, Leanne Pilkington, Andrew Redfern

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Introduction: It is well known that patients with Head and Neck Cancer (HNC) are at increased risk of subsequent head and neck cancers due to various aetiologies. Aim: We sought to determine the factors contributing to an increased risk of subsequent HNC primaries, and also to evaluate whether Aboriginal patients are at increased risk. Methods: We performed a retrospective cohort analysis of 320 HNC patients from a single centre in Western Australia, identifying 80 Aboriginal patients and 240 non-Aboriginal patients matched on a 1:3 ratio by site, histology, rurality, and age. We collected patient data including smoking and alcohol consumption, tumour and treatment data, and data on subsequent HNC primaries. Results: A subsequent HNC primary was seen in 37 patients (11.6%) overall. There was no significant difference in the rate of second primary HNCs between Aboriginal patients (12.5%) and nonAboriginal patients (11.2%) (p=0.408). Subsequent HNCs, were strongly associated with smoking and alcohol consumption however, with 95% of patients with a second primary being ever-smokers, and 54% of patients with a second primary having a history of excessive alcohol consumption. In the 37 patients with multiple HNC primaries, there were a total of 57 HNCs, with 29 patients having two primaries, six patients having 3 HNC primaries, one patient with four, and one with six. 54 out of the 57 cancers were in ever smokers (94.7%). There were only two multiple HNC primaries in a never smoker, non-drinker, and these cases were of unknown etiology with HPV/p16 status unknown in both cases. In the whole study population, there were 32 HPV-positive HNCs, and 67 p16-positive HNCs, with only two 2 nd HNCs in a p16-positive case, giving a rate of 3% in the p16+ population, which is actually much lower than the rate of second primaries seen in the overall population (11.6%), and was highest in the p16-negative population (15.7%). This suggests that p16-positivity is not a strong risk factor for subsequent primaries, and in fact p16-negativity appeared to be associated with increased risk, however this data is limited by the large number of patients without documented p16 status (45.3% overall, 12% for oropharyngeal, and 59.6% for oral cavity primaries had unknown p16 status). Summary: Subsequent HNC primaries were strongly associated with smoking and alcohol excess. Second and later HNC primaries did not appear to occur at increased rates in Aboriginal patients compared with non-Aboriginal patients, and p16-positivity did not predict increased risk, however p16-negativity was associated with an increased risk of subsequent HNCs.

Keywords: head and neck cancer, multiple primaries, aboriginal, p16 status, smoking, alcohol

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Authors: Zarine Avetisyan

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The process of speech production and transmission naturally implies the occurrence of certain defective assumptions and erroneous formulations which may be both spontaneous, caused by haste, carelessness, etc., or deliberate. Whether deliberate or not, fallacies always act by way of “faux pas”. In the latter case, we deal with fake or deceptive arguments which are the focus of the given paper. The paper departs from the assumption that fallacies are arguments that prove nothing. Additionally and more importantly, political discourse becomes the main domain for scholarly “cultivation” while pinning down fallacies. The fallacy of telling the truth but deliberately omitting important key details in order to falsify the larger picture called “the half truth” captures special attention in the given paper.

Keywords: break in the information chain, fallacy, half truth, political discourse

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Authors: Ajit Rai, Hamza Deroui, Blandine Vacher, Khwansiri Ninpan, Arthur Aumont, Francesco Vitillo, Robert Plana

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Large-scale critical industrial scheduling problems are based on Resource-Constrained Project Scheduling Problems (RCPSP), that necessitate integration with other optimization problems (e.g., vehicle routing, supply chain, or unique industrial ones), thus requiring practical solutions (i.e., modular, computationally efficient with feasible solutions). To the best of our knowledge, the current industrial state of the art is not addressing this holistic problem. We propose an original modular solution that answers the issues exhibited by the delivery of complex projects. With three interlinked entities (project, task, resources) having their constraints, it uses a greedy heuristic with a dynamic cost function for each task with a situational assessment at each time step. It handles large-scale data and can be easily integrated with other optimization problems, already existing industrial tools and unique constraints as required by the use case. The solution has been tested and validated by domain experts on three use cases: outage management in Nuclear Power Plants (NPPs), planning of future NPP maintenance operation, and application in the defense industry on supply chain and factory relocation. In the first use case, the solution, in addition to the resources’ availability and tasks’ logical relationships, also integrates several project-specific constraints for outage management, like, handling of resource incompatibility, updating of tasks priorities, pausing tasks in a specific circumstance, and adjusting dynamic unit of resources. With more than 20,000 tasks and multiple constraints, the solution provides a feasible schedule within 10-15 minutes on a standard computer device. This time-effective simulation corresponds with the nature of the problem and requirements of several scenarios (30-40 simulations) before finalizing the schedules. The second use case is a factory relocation project where production lines must be moved to a new site while ensuring the continuity of their production. This generates the challenge of merging job shop scheduling and the RCPSP with location constraints. Our solution allows the automation of the production tasks while considering the rate expectation. The simulation algorithm manages the use and movement of resources and products to respect a given relocation scenario. The last use case establishes a future maintenance operation in an NPP. The project contains complex and hard constraints, like on Finish-Start precedence relationship (i.e., successor tasks have to start immediately after predecessors while respecting all constraints), shareable coactivity for managing workspaces, and requirements of a specific state of "cyclic" resources (they can have multiple states possible with only one at a time) to perform tasks (can require unique combinations of several cyclic resources). Our solution satisfies the requirement of minimization of the state changes of cyclic resources coupled with the makespan minimization. It offers a solution of 80 cyclic resources with 50 incompatibilities between levels in less than a minute. Conclusively, we propose a fast and feasible modular approach to various industrial scheduling problems that were validated by domain experts and compatible with existing industrial tools. This approach can be further enhanced by the use of machine learning techniques on historically repeated tasks to gain further insights for delay risk mitigation measures.

Keywords: deterministic scheduling, optimization coupling, modular scheduling, RCPSP

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Authors: Akhigbe Mark, Abang Roger, Mwoltu Nanaribet, Edet Blessing

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Introduction.: The introduction of pre- exposure prophylaxis (PrEP) as a biomedical prevention method for HIV/AIDS has been around for more than a decade since the first confirmed evidence of its effectiveness when used daily as an oral pill. It is now a very valuable addition for people who are at higher risk of contracting HIV. Although globalacceptanceof PrEP hasincreased, PrEP is still highly concentrated in a small number of countries and within a small sub-population, with Kenya and South Africa accounting for only 19% of people who have received PrEP in Africa region, there is still a significant regionGap in PrEP availability and use, with only 28% of the target of 3 million in low-and middle countries currently using PrEP. Description: The purpose of this study is to find out if serum creatinine could be used as an incentive to improve PrEP uptake among Key population.Numerous approaches to increasing the uptake ofPrEP as a prevention mechanism for HIV in KPs has beenemployed, and one of them is serum creatinine. This approach is a biomarker of renal function, which was used in study as an incentive to increase PrEP uptake among key population groups (female sex workers, men who have sex with men, persons who inject drugs, transgender) in 3 states from South-South Nigeria. Whole blood samples are collected from clients, analysis of the samples is done using the clinical chemistry analyzer before they are initiated onto PrEP. Lessons learned and Recommendations: Secondary data was extracted from 3 states of HALG Implementing facilities in Southern part of Nigeria, PrEP uptake before and afterthe introduction of serum creatinine between March 2020 and August 2020 among key populationsin Nigeria. A total of 5664 patients were initiated on PrEP before, and after the introduction of serum creatinine, the PrEP uptake rate before (March 2020 to May 2020) introduction of serum creatinine accounted for only 5% of the total onset, and after (June 2020 to August 2020) introduction of serum creatinine, the uptake rate accounted for 95% of the total onset. These finding shows that increased uptake of PrEP before/after serum creatineindicates that serum creatine may be an effective stimulus for promoting PrEP in key populations.

Keywords: serum creatinine, incentives, PrEP, key populations, Nigeria

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Authors: Sontana Mimapan, Phattarawadee Wattanasuntorn, Phanom Saijit

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Aflatoxin contamination in maize, one of several agriculture crops grown for livestock feeding, is still a problem throughout the world mainly under hot and humid weather conditions like Thailand. In this study Aspergillus flavus (A. Flavus), the key fungus for aflatoxin production especially aflatoxin B1 (AFB1), isolated from naturally infected maize were identified and characterized according to colony morphology and PCR using ITS, Beta-tubulin and calmodulin genes. The strains were analysed for the presence of four aflatoxigenic biosynthesis genes in relation to their capability to produce AFB1, Ver1, Omt1, Nor1, and aflR. Aflatoxin production was then confirmed using immunoaffinity column technique. A loop-mediated isothermal amplification (LAMP) was applied as an innovative technique for rapid detection of target nucleic acid. The reaction condition was optimized at 65C for 60 min. and calcein flurescent reagent was added before amplification. The LAMP results showed clear differences between positive and negative reactions in end point analysis under daylight and UV light by the naked eye. In daylight, the samples with AFB1 producing A. Flavus genes developed a yellow to green color, but those without the genes retained the orange color. When excited with UV light, the positive samples become visible by bright green fluorescence. LAMP reactions were positive after addition of purified target DNA until dilutions of 10⁻⁶. The reaction products were then confirmed and visualized with 1% agarose gel electrophoresis. In this regards, 50 maize samples were collected from dairy farms and tested for the presence of four aflatoxigenic biosynthesis genes using LAMP technique. The results were positive in 18 samples (36%) but negative in 32 samples (64%). All of the samples were rechecked by PCR and the results were the same as LAMP, indicating 100% specificity. Additionally, when compared with the immunoaffinity column-based aflatoxin analysis, there was a significant correlation between LAMP results and aflatoxin analysis (r= 0.83, P < 0.05) which suggested that positive maize samples were likely to be a high- risk feed. In conclusion, the LAMP developed in this study can provide a simple and rapid approach for detecting AFB1 producing A. Flavus genes from maize and appeared to be a promising tool for the prediction of potential aflatoxigenic risk in livestock feedings.

Keywords: Aflatoxin B1, Aspergillus flavus genes, maize, loop-mediated isothermal amplification

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Authors: Mimoza Canga, Irene Malagnino, Giulia Malagnino, Alketa Qafmolla, Ruzhdie Qafmolla, Vito Antonio Malagnino

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The goal of the present study was to analyze the risk factors regarding dental fear. This observational study was conducted during the period of February 2020 - April 2022 in Albania. The sample was composed of 200 participants, of which 40% were males and 60% were females. The participants' age range varied from 35 to 75 years old. We divided them into four age groups: 35-45, 46-55, 56-65, and 66-75 years old. Statistical analysis was performed using IBM SPSS Statistics 23.0. Data were scrutinized by the Post Hoc LSD test in analysis of variance (ANOVA). The P ≤ 0.05 values were considered significant. Data analysis included Confidence Interval (95% CI). The prevailing age range in the sample was mostly from 55 to 65 years old, 35.6% of the patients. In all, 50% of the patients had extreme fear about the fact that the dentist may be infected with Covid-19, 12.2% of them had low dental fear, and 37.8% had extreme dental fear. However, data collected from the current study indicated that a large proportion of patients 49.5% of them had high dental fear regarding the dentist not respecting the quarantine due to COVID-19, in comparison with 37.2% of them who had low dental fear and 13.3% who had extreme dental fear. The present study confirmed that 22.2% of the participants had an extreme fear of poor hygiene practices of the dentist that have been associated with the transmission of COVID-19 infection, 57.8% had high dental fear, and 20% of them had low dental fear. The present study showed that 50% of the patients stated that another factor that causes extreme fear was that the patients feel pain after interventions in the oral cavity. Strong associations were observed between dental fear and pain 95% CI; 0.24-0.52, P-value ˂ .0001. The results of the present study confirmed strong associations between dental fear and the fact that the dentist may be infected with Covid-19 (95% CI; 0.46-0.70, P-value ˂ .0001). Similarly, the analysis of the present study demonstrated that there was a statistically significant correlation between dental fear and poor hygiene practices of the dentist with 95% CI; 0.82-1.02, P-value ˂ .0001. On the basis of our statistical data analysis, the dentist did not respect the quarantine due to COVID-19 having a significant impact on dental fear with a P-value of ˂ .0001. This study shows important risk factors that significantly increase dental fear.

Keywords: Covid-19, dental fear, pain, past dreadful experiences

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Authors: Malay K. Das, Bhupen Kalita

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The poor oral bioavailability of resveratrol (RSV) due to presystemic metabolism can be avoided via dermal route of administration. The hydrophilic-lipophilic nature of resveratrol-phospholipid complex (RSVPs) favors the delivery of resveratrol via the skin. The RSVPs embedded polymeric patch with moderate adhesiveness was developed for dermal application for sustained anti-inflammatory effect. The prepared patches were evaluated for various physicochemical properties, surface morphology by SEM, TEM, and compatibility of patch components by FT-IR and DSC studies. The dermal flux of the optimized patch formulation was found to be at 4.28 ± 0.48 mg/cm2/24 h. The analysis of skin extract after permeation study revealed the presence of resveratrol, which confirmed the localization of RSVPs in the skin. The stability of RSVPs in the polymeric patch and the physiologic environment was confirmed by FE-SEM studies on the patches after drug release and skin permeation studies. The RSVPs particles released from the polymer matrix maintaining the structural integrity and permeate the keratinized horney layer of skin. The optimized patch formulation showed sustained anti-inflammatory effect (84.10% inhibition of inflammation at 24 h) in carrageenan-induced rat paw edema model compared to marketed diclofenac sodium gel (39.58% inhibition of inflammation at 24 h). The CLSM study confirmed the localization of RSVPs for a longer period, thus enabling drug targeting to the dermis for sustained anti-inflammatory effect. Histological studies with phase contrast trinocular microscope suggested no alteration of skin integrity and no evidence of the presence of inflammatory cells after exposure to the permeants. The patch was found to be safe for skin application as evaluated by Draize method for skin irritation scoring in a rabbit model. These results suggest the therapeutic efficacy of the developed patch in both acute and chronic inflammatory diseases.

Keywords: resveratrol-phospholipid complex, skin delivery, sustained anti-inflammatory effect, inflammatory diseases, dermal patch

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Authors: Mehrnaz Mostafavi

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Perovskite nanocrystals and quantum dots have emerged as leaders in the field of photovoltaic technologies, demonstrating exceptional light-harvesting abilities and stability. This study investigates the substantial progress and potential of these nano-sized materials in transforming solar energy conversion. The research delves into the foundational characteristics and production methods of perovskite nanocrystals and quantum dots, elucidating their distinct optical and electronic properties that render them well-suited for photovoltaic applications. Specifically, it examines their outstanding light absorption capabilities, enabling more effective utilization of a wider solar spectrum compared to traditional silicon-based solar cells. Furthermore, this paper explores the improved durability achieved in perovskite nanocrystals and quantum dots, overcoming previous challenges related to degradation and inconsistent performance. Recent advancements in material engineering and techniques for surface passivation have significantly contributed to enhancing the long-term stability of these nanomaterials, making them more commercially feasible for solar cell usage. The study also delves into the advancements in device designs that incorporate perovskite nanocrystals and quantum dots. Innovative strategies, such as tandem solar cells and hybrid structures integrating these nanomaterials with conventional photovoltaic technologies, are discussed. These approaches highlight synergistic effects that boost efficiency and performance. Additionally, this paper addresses ongoing challenges and research endeavors aimed at further improving the efficiency, stability, and scalability of perovskite nanocrystals and quantum dots in photovoltaics. Efforts to mitigate concerns related to material degradation, toxicity, and large-scale production are actively pursued, paving the way for broader commercial application. In conclusion, this paper emphasizes the significant role played by perovskite nanocrystals and quantum dots in advancing photovoltaic technologies. Their exceptional light-harvesting capabilities, combined with increased stability, promise a bright future for next-generation solar cells, ushering in an era of highly efficient and cost-effective solar energy conversion systems.

Keywords: perovskite nanocrystals, quantum dots, photovoltaic technologies, light-harvesting, solar energy conversion, stability, device designs

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Authors: Shoji Katagiri, Hugang Han

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This paper clarifies the role of ICT capital in Economic Growth. Albeit ICT remarkably contributes to economic growth, there are few studies on ICT capital in ICT sector from theoretical point of view. In this paper, production function of ICT which is used as input of intermediate good in final good and ICT sectors is incorporated into our model. In this setting, we analyze the role of ICT on balance growth path and show the possibility of general equilibrium solutions for this model. Through the simulation of the equilibrium solutions, we find that when ICT impacts on economy and economic growth increases, it is necessary that increases of efficiency at ICT sector and of accumulation of non-ICT and ICT capitals occur simultaneously.

Keywords: endogenous economic growth, ICT, intensity, capital accumulation

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Authors: Hassan Nour, Nouh Mounadi, Oussama Abchir, Belaidi Salah, Samir Chtita

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Cholinesterase enzymes are biological catalysts essential for the transformation of acetylcholine, which is a neurotransmitter implicated in memory and learning, into acetic acid and choline, altering the neurotransmission process in Alzheimer’s disease patients. Therefore, inhibition of cholinesterase enzymes is a relevant strategy for the symptomatic treatment of Alzheimer’s disease. The current investigation aims to explore potential cholinesterase (ChE) inhibitors through a comprehensive computational approach. Forty-nine phytoconstituents extracted from Cannabis sativa L. were in-silico screened using molecular docking and pharmacokinetic and toxicological analysis to evaluate their possible inhibitory effect on the cholinesterase enzymes. Two phytoconstituents belonging to cannabinoid derivatives were revealed to be promising candidates for Alzheimer's therapy by acting as cholinesterase inhibitors. They have exhibited high binding affinities towards the cholinesterase enzymes and showed their ability to interact with key residues involved in cholinesterase enzymatic activity. In addition, they presented good ADMET profiles allowing them to be promising oral drug candidates. Furthermore, molecular dynamics (MD) simulations were executed to explore their interaction stability under mimetic biological conditions and thus support our findings. To corroborate the docking results, the binding free energy corresponding to the more stable ligand-ChE complexes was re-estimated by applying the MM-PBSA method. MD and MM-PBSA studies affirmed that the ligand-ChE recognition is a spontaneous reaction leading to stable complexes. The conducted investigations have led to great findings that would strongly guide the pharmaceutical industries toward the rational development of potent anti-Alzheimer agents.

Keywords: Alzheimer’s disease, molecular docking, Cannabis sativa L., cholinesterase inhibitors, molecular dynamics, ADMET, MM-PBSA

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Authors: Ranjot Kaur, Om P. Katare, Anupama Sharma, Sarah R. Dennison, Kamalinder K. Singh, Bhupinder Singh

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Respiratory microbial infections being among the top leading cause of death worldwide are difficult to treat as the microbes reside deep inside the airways, where only a small fraction of drug can access after traditional oral or parenteral routes. As a result, high doses of drugs are required to maintain drug levels above minimum inhibitory concentrations (MIC) at the infection site, unfortunately leading to severe systemic side-effects. Therefore, delivering antimicrobials directly to the respiratory tract provides an attractive way out in such situations. In this context, current study embarks on the systematic development of lung lia pid-modified chitosan nanoparticles for inhalation of voriconazole. Following the principles of quality by design, the chitosan nanoparticles were prepared by ionic gelation method and further coated with major lung lipid by precipitation method. The factor screening studies were performed by fractional factorial design, followed by optimization of the nanoparticles by Box-Behnken Design. The optimized formulation has a particle size range of 170-180nm, PDI 0.3-0.4, zeta potential 14-17, entrapment efficiency 45-50% and drug loading of 3-5%. The presence of a lipid coating was confirmed by FESEM, FTIR, and X-RD. Furthermore, the nanoparticles were found to be safe upto 40µg/ml on A549 and Calu-3 cell lines. The quantitative and qualitative uptake studies also revealed the uptake of nanoparticles in lung epithelial cells. Moreover, the data from Spraytec and next-generation impactor studies confirmed the deposition of nanoparticles in lower airways. Also, the interaction of nanoparticles with DPPC monolayers signifies its biocompatibility with lungs. Overall, the study describes the methodology and potential of lipid-coated chitosan nanoparticles in futuristic inhalation nanomedicine for the management of pulmonary aspergillosis.

Keywords: dipalmitoylphosphatidylcholine, nebulization, DPPC monolayers, quality-by-design

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Authors: Luz Estrada

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The currents of evolutionary materialistic thought have argued that knowledge about an object is not obtained through the abstractive method. That is, the object cannot come to be understood if founded upon itself, nor does it take place by the encounter between form and matter. According to this affirmation, the research presented here identified as a problematic situation the absence of comprehension of the formal creation as a generative operation. This has been referred to as a recurrent lack in the production of objects and corresponds to the need to conceive the configurative process from the reality of its genesis. In this case, it is of interest to explore ways of creation that consider the object as if it were a living organism, as well as responding to the object’s experience as embodied in the designer since it unfolds its genesis simultaneously to the ways of existence of those who are involved in the generative experience.

Keywords: architecture, theoretical graphics, evolutionary genetics, formal perception

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Authors: Chittapon Jantararussamee, Malai Taweechotipatr, Udomsri Showpittapornchai, Wisuit Pradidarcheep

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Hepatic fibrosis is characterized by collagen accumulation in hepatic lobules following wound healing process. If lefts untreated, it could progress into hepatic cirrhosis, portal hypertension, and liver failure. Probiotics comprise of lactic acid bacteria which are crucial components of the intestinal microflora and possess many beneficial properties. The objective of this study is to investigate the hepatoprotective effects of probiotic lactic acid bacteria (mixture of Lactobacillus paracasei, Lactobacillus casei, and Lactobacillus confusus at a ratio of 1: 1: 1) on thioacetamide-induced liver fibrotic rats in term of histomorphology study. Twenty-four male Wistar rats were randomly divided into four groups with 6 rats each: (A) control, (B) fibrotic, (C) fibrotic+probiotic, and (D) probiotic. Group (A) received daily oral administration of distilled water. Group (B and C) were induced by intraperitoneal injection of thioacetamide (TAA) (200 mg/kg BW) 3 times per week for consecutive 8 weeks. In probiotic-treated group (C and D), the number of a mixture of the viable microbial cells at 10⁹ CFU/ml was administered orally daily. After sacrifice, liver tissues were collected and processed for routine histological technique and stained with Sirius red. It was found that the fibrotic rats showed hepatic injury marked by area of inflammation, hydropic degeneration of hepatocytes, and accumulation of myofibroblast-like cells. The collagen fibers were substantially accumulated in the hepatic lobules. Moreover, probiotic-treated group significantly reduced the accumulation of collagen in rats treated by TAA. The liver damage was found to be lesser in the probiotic-treated group. It was noted that the liver tissues of control and probiotics groups were shown to be normal. Administration with probiotic lactic acid bacteria could improve the histomorphology in fibrotic liver and be useful for prevention of hepatic disorders.

Keywords: liver fibrosis, probiotics, lactic acid bacteria, thioacetamide

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Authors: Sadeq Al Yaari, Muhammad Alkhunayn, Aayah Al Yaari, Ayman Al Yaari, Montaha Al Yaari, Sajedah Al Yaari, Fatehi Eissa

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The recent rise of smart educational aids and the move away from traditional listening aids are leading to a fundamental shift in the way in which individuals with attention and listening problems (ALP) manipulate listening inputs and/or act appropriately to the spoken information presented to them. A total sample of twenty-six ALP pupils (m=20 and f=6) between 7-12 years old was selected from different strata based on gender, region and school. In the sample size, thirteen (10 males and 3 females) received the treatment in terms of smart classes provided with smart educational aids in a listening course that lasted for four month-semester while others did not (they studied the same course by the same instructor but in ordinary class). A pretest was administered to assess participants’ levels, and a posttest was given to evaluate their attention and listening comprehension performance, namely in phonetic and phonological tests with sociolinguistic themes that have been designed for this purpose. Test results were analyzed both psychoneurolinguistically and statistically. Results reveal a remarkable change in pupils’ behavioral listening where scores witnessed a significant difference in the performance of the experimental ALP group in the pretest compared to the posttest (Pupils performed better at the pretest-posttest on phonetics than at the two tests on phonology). It is concluded that smart educational aids designed for listening skills help not only increase the listening command of pupils with ALP to understand what they listen to but also develop their interactive listening capability and, at the same rate, are responsible for increasing concentrated and in-depth listening capacity. Plus, ALP pupils become able to grasp the audio content of text recordings, including educational audio recordings, news, oral stories and tales, views, spiritual/religious text and general knowledge. However, the pupils have not experienced individual smart audio-visual aids that connect listening to other language receptive and productive skills, which could be the future area of research.

Keywords: language skills, implementing, listening skill, attention, smart aids

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Authors: Baishali Deb, Hari Om Pandey, Shrilla Elangbam, Mukesh Singh, Ayon Tarafdar, A. K. S. Tomar, A. K. Pandey, Triveni Dutt

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The goal of the present investigation was to determine the impact of social stress on behavioural characteristics, physiological responses, and haemato-biochemical indicators under various social environments in Tharparkar cattle. Serum cortisol and faecal cortisol metabolites analysis were used to determine the stress level of Tharparkar cattle. Social isolation and social mixing were the two different social circumstances used to evaluate the animals. In both the experiments i.e., social isolation and social mixing, the lying period of animals decreased significantly (p<0.05) while standing period significantly (p<0.05) increased. Frequency and duration of activities like idling, walking, exploration, oral manipulation, and elimination increased significantly (p<0.05) in Tharparkar cattle after being subjected to social isolation and social mixing. Time spent in grooming (self-grooming and allo-grooming) in respect to social isolation significantly increased during isolation and post-reunion, whereas there was a significant (p<0.05) decline in the grooming behaviour especially allo-grooming during mixing of the animals. Feeding and rumination time also decreased significantly (p<0.05) in animals during both the experiments. Physiological parameters such as respiration rate, heart rate and pulse rate increased during the treatment periods. There was no significant difference in the haematological parameters for both the experiments. There was significant (p<0.05) increase in serum cortisol and faecal cortisol metabolites (FCM) concentration in animals subjected to social stress. Therefore, it can be concluded that social stress strongly impacts the behaviour and physiological parameters of the animals, causing stress and nervousness, proving that social stress is a valid psychological stress in animals. The higher concentration of FCM in Tharparkar cattle subjected to social stress, further supported by higher serum cortisol and behaviour manifestations, suggest that FCM could be used to assess stress response as a non-invasive method.

Keywords: social stress, fecal cortisol metabolites, non-invasive, animal welfare, behaviour

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Authors: Shashank Gupta, Shiva Garg

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The purpose of the present paper is to study the changes in the strength characteristics of autoclaved aerated concrete (AAC) and also the density when different expansion agents are used. The expansion agent so used releases air in the concrete thereby making it lighter by reducing its density. It also increases the workability of the concrete. The various air entraining agents used for this study are hydrogen peroxide, oleic acid, and olive oil. The addition of these agents causes the concrete to rise like cake but it reduces the strength of concrete due to the formation of air voids. The amount of agents chosen for concrete production are 0.5%, 1%, 1.5% by weight of cement.

Keywords: AAC, olive oil, hydrogen peroxide, oleic acid, steam curing

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Authors: Mohammadjavad Sotoudeheian, Navid Farahmandian

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Kawasaki disease (KD) is the primary cause of acquired pediatric heart disease as an acute vasculitis. In children with prolonged fever, rash, and inflammation of the mucosa KD must be considered as a clinical diagnosis. There is a persuasive suggestion of immune-mediated damage as the pathophysiologic cascade of KD. For example, the invasion of cytotoxic T-cells supports a viral etiology and the inflammasome of the innate immune system is a critical component in the vasculitis formation in KD. Animal models of KD propose the cytokine profiles, such as increased IL-1 and GM-CSF, which cause vascular damage. CRP and IFN-γ elevated expression and the upregulation of IL-6, and IL-10 production are also described in previous studies. Untreated KD is a critical risk factor for coronary artery diseases and myocardial infarction. Vascular damage may encompass amplified T-cell activity. SMAD3 is an essential molecule in down-regulating T-cells and increasing expression of FoxP3. It has a critical effect in the differentiation of regulatory T-cells. The discrepancy of regulatory T-cells and pro-inflammatory Th17 has been studied in acute coronary syndrome during KD. However in the coronary artery damaged lymphocytes and IgA plasma cells are seen at the lesion locations, the major immune cells in the coronary lesions are monocytes/macrophages and neutrophils. These cells secrete TNF-α, and activates matrix metalloprotease (MMP)-9, reducing the integrity of vessels and prompting patients to arise aneurysm. MMPs can break down the components of the extracellular matrix and assist immune cell movement. IVIG as an effective form of treatment clarified the role of the immune system, which may target pathogenic antigens and regulate cytokine production. Several reports have revealed that in the coronary arteries, high expression of MMP-9 in monocyte/macrophage results in pathologic cascades. Curcumin is a potent antioxidant and anti-inflammatory molecule. Curcumin decreases the production of reactive oxygen and nitrogen species and inhibits transcription factors like AP-1 and NF-κB. Curcumin also contains the characteristics of inhibitory effects on MMPs, especially MMP-9. The upregulation of MMP-9 is an important cellular response. Curcumin treatment caused a reverse effect and down-regulates MMP-9 gene expression which may fund the anti-inflammatory effect. Curcumin inhibits MMP-9 expression via PKC and AMPK-dependent pathways in Human monocytes cells. Elevated expression and activity of MMP-9 are correlated with advanced vascular lesions. AMPK controls lipid metabolism and oxidation, and protein synthesis. AMPK is also necessary for the MMP-9 activity and THP-1 cell adhesion to endothelial cells. Curcumin was shown to inhibit the activation of AMPKα. Compound C (AMPK inhibitor) inhibits MMP-9 expression level. Therefore, through inactivating AMPKs and PKC, curcumin decreases the MMP-9 level, which results in inhibiting monocyte/macrophage differentiation. Compound C also suppress the phosphorylation of three major classes of MAP kinase signaling, suggesting that curcumin may suppress MMP-9 level by inactivation of MAPK pathways. MAPK cascades are activated to induce the expression of MMP-9. Curcumin inhibits MAPKs phosphorylation, which contributes to the down-regulation of MMP-9. This study demonstrated that the potential inhibitory properties of curcumin over MMP-9 lead to a therapeutic strategy to reduce the risk of coronary artery involvement during KD.

Keywords: MMP-9, coronary artery aneurysm, Kawasaki’s disease, curcumin, AMPK, immune system, NF-κB, MAPK

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Authors: Azeem Tariq, Stephane De Tourdonnet, Lars Stoumann Jensen, Reiner Wassmann, Bjoern Ole Sander, Quynh Duong Vu, Trinh Van Mai, Andreas De Neergaard

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Rice growing area is increasing to meet the food demand of increasing population. Mostly, rice is growing on lowland, small landholder fields in most part of the world, which is one of the major sources of greenhouse gases (GHG) emissions from agriculture fields. The strategies such as, altering water and residues (carbon) management practices are assumed to be essential to mitigate the GHG emissions from flooded rice system. The actual implementation and potential of these measures on small farmer fields is still challenging. A field study was conducted on red river delta in Northern Vietnam to identify the potential challenges and barriers to the small rice farmers for implementation of climate smart rice practices. The objective of this study was to develop and access the feasibility of climate smart rice prototypes under actual farmer conditions. Field and scientific oriented framework was used to meet our objective. The methodological framework composed of six steps: i) identification of stakeholders and possible options, ii) assessment of barrios, drawbacks/advantages of new technologies, iii) prototype design, iv) assessment of mitigation potential of each prototype, v) scenario building and vi) scenario assessment. A farm survey was conducted to identify the existing farm practices and major constraints of small rice farmers. We proposed the two water (pre transplant+midseason drainage and early+midseason drainage) and one straw (full residue incorporation) management option keeping in views the farmers constraints and barriers for implementation. To test new typologies with existing prototypes (midseason drainage, partial residue incorporation) at farmer local conditions, a participatory field experiment was conducted for two consecutive rice seasons at farmer fields. Following the results of each season a workshop was conducted with stakeholders (farmers, village leaders, cooperatives, irrigation staff, extensionists, agricultural officers) at local and district level to get feedbacks on new tested prototypes and to develop possible scenarios for climate smart rice production practices. The farm analysis survey showed that non-availability of cheap labor and lacks of alternatives for straw management influence the small farmers to burn the residues in the fields except to use for composting or other purposes. Our field results revealed that application of early season drainage significantly mitigates (40-60%) the methane emissions from residue incorporation. Early season drainage was more efficient and easy to control under cooperate manage system than individually managed water system, and it leads to both economic (9-11% high rice yield, low cost of production, reduced nutrient loses) and environmental (mitigate methane emissions) benefits. The participatory field study allows the assessment of adaptation potential and possible benefits of climate smart practices on small farmer fields. If farmers have no other residue management option, full residue incorporation with early plus midseason drainage is adaptable and beneficial (both environmentally and economically) management option for small rice farmers.

Keywords: adaptation, climate smart agriculture, constrainsts, smallholders

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Authors: Nenita Pambid Domingo

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The komedya otherwise known as moro-moro or pretending to be Moors, is a traditional Filipino play in the vernacular adapted from the Spanish comedia de capa y espada. It was used by Spanish colonizers in the Philippines, circa 1766 to evangelize and strengthen the faith of Indios or Filipino natives to Christianity. Unlike the Moros y Cristianos festival held all over Spain celebrating the Reconquista from the 8th to the 15th century, the Philippine Moro-Moro or Komedya is a romance between a Muslim and a Christian and the battles between Christians and Moros, where the Moros are always defeated and the Muslim prince is converted to the Christian faith and marries the Christian princess at the end of the play. For over 200 years, the komedya has been part of the Filipinos’ life and has been dubbed by some Philippine scholars as the Philippine’s national theater. Until now postings of performances in different parts of the Philippines in different Philippine languages are uploaded at youtube. In the US, “San Dionisio sa America (SDA),” an organization of natives from Barrio San Dionisio, Parañaque, Philippines has been performing the komedya for the past 16 years during their town’s fiesta, in honor of the barrio's patron saints St Denis of Paris, France and Saint Joseph whom the devotees fondly call "Tata Dune" and "Tata Hosep". The komedya performed in the US is infused with modern elements in the production and content, but retain the basic form in verse and the stylized war dance, marches, and singsong delivery of lines. Most of the Celebras or town fiestas and komedya performances are held at The Barnsdall Art Park and Gallery Theatre in Hollywood, Los Angeles. The presentation will focus on the linguistic and content analysis of the Tagalog verses in the 2010 komedya entitled Mga Prinsesa ng Cordova (The Princesses of Cordova) publicized as a modern komedya. The presentation will also touch on the healing function of the language and performance that is part of the town’s religious festivities. It will also look into the aesthetics of the production, audience reception, participation of the sponsors, producers called Hermana/Hermano Mayor, the performers who are a mix of Filipinos from the Philippines and Filipino-Americans who are starting to lose the Tagalog language and the non-Filipino participants, as well as the general audience who are from Parañaque and those not from Parañaque, who come to witness the event and enjoy the festivities.

Keywords: devotion, diaspora nationalism, komedya, st. denis of Paris, France, traditional Philippine theater

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Authors: Kira Rysakova, Vitaly Novikov

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An improved method of obtaining enzymatic collagen hydrolysate from the tissues of marine hydrobionts is proposed, which allows to obtain hydrolysate without pre-isolation of pure collagen. The method can be used to isolate enzymatic collagen hydrolysate from the waste of industrial processing of Red King crab and non-traditional objects - marine holothurias. Comparative analysis of collagen hydrolysates has shown the possibility of their use in a number of nutrient media, but this requires additional optimization of their composition and biological tests on wide sets of test strains of microorganisms.

Keywords: collagen hydrolysate, marine hydrobionts, red king crab, marine holothurias, enzymes, exclusive HPLC

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Authors: Ruina Xu, Peixue Jiang, Feng Luo

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The novel concept of enhanced geothermal system with CO2 instead of water as working fluid (CO2-EGS) has attracted wide attention due to additional benefit of CO2 geological storage during the power generation process. In this research, numerical investigation on a doublet CO2-EGS system is performed, focusing on the influence of the injection/production well perforation location in the targeted geothermal reservoir. Three different reservoir inlet and outlet boundary conditions are used in simulations since the well constrains are different in reality. The results show that CO2-EGS system performance of power generation and power cost vary greatly among cases of different wells perforation locations, and the optimum options under different boundary conditions are also different.

Keywords: Enhanced Geothermal System, supercritical CO2, heat transfer, CO2-EGS

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Authors: Shizrah Jamal, Robia Ghafoor, Farhan Raza

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Porcelain fused to metal crown is the most versatile variety of crown that is commonly placed worldwide. Various complications have been reported in the PFM crowns with use over the period of time. These include chipping of the porcelain, recurrent caries, loss of retention, open contacts, and tooth fracture. The objective of the present study was to determine the frequency of these complications in crowns cemented over a period of five years in a tertiary care hospital and also to report the survival of these crowns. A retrospective study was conducted in Dental clinics, Aga Khan University Hospital in which 150 PFM crowns cemented over a period of five years were evaluated. Patient demographics, oral hygiene habits, para-functional habits, crown insertion and follow-up dates were recorded in a specially designed proforma. All PFM crowns fulfilling the inclusion criteria were assessed both clinically and radiographically for the presence of any complication. SPSS version 22.0 was used for statistical analysis. Frequency distribution and proportion of complications were determined. Chi-square test was used to determine the association of complications of PFM crowns with multiple variables including tooth wear, opposing dentition and betel nut chewing. Kaplan- meier survival analysis was used to determine the survival of PFM crowns over the period of five years. Level of significance was kept at 0.05. A total of 107 patients, with a mean age of 43.51 + 12.4 years, having 150 PFM crowns were evaluated. The most common complication observed was open proximal contacts (8.7%) followed by porcelain chipping (6%), decementation (5.3%), and abutment fracture (1.3%). Chi square test showed that there was no statistically significant association of PFM crown complication with tooth wear, betel nut and opposing dentition (p-value <0.05). The overall success and survival rates of PFM crowns turned out to be 78.7 and 84.7% respectively. Within the limitations of the study, it can be concluded that PFM crowns are an effective treatment modality with high success and survival rates. Since it was a single centered study; the results should be generalized with caution.

Keywords: chipping, complication, crown, survival rate

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Authors: Sathvika Reddy, Devi Revathi

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Background: In India, deliberate self-harm (DSH) with poisoning agents carries a significant impact on morbidity and mortality. Changes in the patterns of poisoning vary across various geographical locations. It is important to know the patterns in a given region in order to facilitate rapid clinical diagnosis, appropriate treatment to reduce associated morbidity and mortality. Aim and Objective: To study the patterns, treatment outcomes of acute poisoning in patients admitted to emergency wards in a tertiary care hospital and to provide poison information services. Materials and Methods: This study was conducted at M.S Ramaiah Memorial and Teaching Hospital from November 2016 to March 2017. The patient’s data was obtained from patient case sheet, interaction with health care professionals, interviewing patients and their caretakers (if possible), and were documented in a suitably designed form. Results: The study involved 131 patients with a mean age of 27.76 ± 15.5 years. Majority of the patients were in the age group 21-30 years, literates (n=53) dwelling in urban (n=113) areas belonging to upper middle class (n=50). Analgesics and antipyretics were commonly utilized in intentional drug overdosage (n=49). Envenomation constituted n=21(16.03%). Furthermore, a significant relationship was observed between marital status and self-poisoning (n=64) (P < 0.001) which commonly occurred through oral ingestion. The outcomes were correlated with the GCS and PSS system and n=85 recovered, n=17 were discharged against medical advice, and n=4 died, and n=4 were lost to follow up respectively. The poison information queries include drug overdose (n=29) and management related queries (n=22) provided majorly by residents (n=45) to update knowledge (n=11) and for better patient care (n=40). Conclusion: The trend in poisoning is dynamic. Medications were identified as the main cause of poisoning in urban areas of India. Educational programs with more emphasis on preventive measures are necessary to create awareness among the general public.

Keywords: poisoning, suicides, clinical pharmacist, envenomation, poison information services

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Authors: Elena Listo Ubeda, Miguel Marchamalo Sacristan

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The demand for water doubles every twenty years, at a rate which is twice as fast as the world´s population growth. Despite it´s great importance, water is one of the most degraded natural resources in the world, mainly because of the reduction of natural vegetation coverage, population growth, contamination and changes in the soil use which reduces its capacity to collect water. This situation is especially serious in Central America, as reflected in the Human Development reports. The objective of this project is to assist in the improvement of water production and quality in Central America. In order to do these two watersheds in Costa Rica were selected as experiments: that of the Virilla-Durazno River, located in the extreme north east of the central valley which has an Atlantic influence; and that of the Jabillo River, which flows directly into the Pacific. The Virilla river watershed is located over andisols, and that of the Jabillo River is over alfisols, and both are of great importance for water supply to the Greater Metropolitan Area and the future tourist resorts respectively, as well as for the production of agriculture, livestock and hydroelectricity. The hydrological reaction in different soil-cover complexes, varying from the secondary forest to natural vegetation and degraded pasture, was analyzed according to the evaluation of the properties of the soil, infiltration, soil compaction, as well as the effects of the soil cover complex on erosion, calculated by the C factor of the Revised Universal Soil Loss Equation (RUSLE). A water balance was defined for each watershed, in which the volume of water that enters and leaves were estimated, as well as the evapotranspiration, runoff, and infiltration. Two future scenarios, representing the implementation of reforestation and deforestation plans, were proposed, and were analyzed for the effects of the soil cover complex on the water balance in each case. The results obtained show an increase of the ground water recharge in the humid forest areas, and an extension of the study of the dry areas is proposed since the ground water recharge here is diminishing. These results are of great significance for the planning, design of Payment Schemes for Environmental Services and the improvement of the existing water supply systems. In Central America spatial planning is a priority, as are the watersheds, in order to assess the water resource socially and economically, and securing its availability for the future.

Keywords: Costa Rica, infiltration, soil, water

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

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

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

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