Search results for: polylactic acid (PLA).

Authors: Ankita Singh, Chandan Gorain, Amirul I. Mallick

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Successful adherence of the mucosal epithelial cells is the key early step for Campylobacter jejuni pathogenesis (C. jejuni). A set of Surface Exposed Colonization Proteins (SECPs) are among the major factors involved in host cell adherence and invasion of C. jejuni. Among them, constitutively expressed surface-exposed lipoprotein adhesin of C. jejuni, JlpA, interacts with intestinal heat shock protein 90 (hsp90α) and contributes in disease progression by triggering pro-inflammatory response via activation of NF-κB and p38 MAP kinase pathway. Together with its ability to express in the bacterial surface, higher sequence conservation and predicted predominance of several B cells epitopes, JlpA protein reserves its potential to become an effective vaccine candidate against wide range of Campylobacter sps including C. jejuni. Given that chickens are the primary sources for C. jejuni and persistent gut colonization remain as major cause for foodborne pathogenesis to humans, present study explicitly used chickens as model to test the immune-protective efficacy of JlpA protein. Taking into account that gastrointestinal tract is the focal site for C. jejuni colonization, to extrapolate the benefit of mucosal (intragastric) delivery of JlpA protein, a food grade Nisin inducible Lactic acid producing bacteria, Lactococcus lactis (L. lactis) was engineered to express recombinant JlpA protein (rJlpA) in the surface of the bacteria. Following evaluation of optimal surface expression and functionality of recombinant JlpA protein expressed by recombinant L. lactis (rL. lactis), the immune-protective role of intragastric administration of live rL. lactis was assessed in commercial broiler chickens. In addition to the significant elevation of antigen specific mucosal immune responses in the intestine of chickens that received three doses of rL. lactis, marked upregulation of Toll-like receptor 2 (TLR2) gene expression in association with mixed pro-inflammatory responses (both Th1 and Th17 type) was observed. Furthermore, intragastric delivery of rJlpA expressed by rL. lactis, but not the injectable form, resulted in a significant reduction in C. jejuni colonization in chickens suggesting that mucosal delivery of live rL. lactis expressing JlpA serves as a promising vaccine platform to induce strong immune-protective responses against C. jejuni in chickens.

Keywords: chickens, lipoprotein adhesion of Campylobacter jejuni, immuno-protection, Lactococcus lactis, mucosal delivery

Procedia PDF Downloads 139

Authors: Beate Englich, Linda Schlittenbauer, Christiane Pfeifer, Isabel Kratochvil, Michael Borte, Gabriele I. Stangl, Martin von Bergen, Thorsten Reemtsma, Irina Lehmann, Kristin M. Junge

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The worldwide production of endocrine disrupting compounds (EDC) has risen dramatically over the last decades, as so has the prevalence for obesity. Many EDCs are believed to contribute to this obesity epidemic, by enhancing adipogenesis or disrupting relevant metabolism. This effect is most tremendous in the early prenatal period when priming effects find a highly vulnerable time window. Therefore, we investigate the impact of parabens on childhood overweight development and adipogenesis in general. Parabens are ester of 4-hydroxy-benzoic acid and part of many cosmetic products or food packing. Therefore, ubiquitous exposure can be found in the westernized world, with exposure already starting during the sensitive prenatal period. We assessed maternal cosmetic product consumption, prenatal paraben exposure and infant BMI z-scores in the prospective German LINA cohort. In detail, maternal urinary concentrations (34 weeks of gestation) of methyl paraben (MeP), ethyl paraben (EtP), n-propyl paraben (PrP) and n-butyl paraben (BuP) were quantified using UPLC-MS/MS. Body weight and height of their children was assessed during annual clinical visits. Further, we investigated the direct influence of those parabens on adipogenesis in-vitro using a human mesenchymal stem cell (MSC) differentiation assay to mimic a prenatal exposure scenario. MSC were exposed to 0.1 – 50 µM paraben during the entire differentiation period. Differentiation outcome was monitored by impedance spectrometry, real-time PCR and triglyceride staining. We found that maternal cosmetic product consumption was highly correlated with urinary paraben concentrations at pregnancy. Further, prenatal paraben exposure was linked to higher BMI Z-scores in children. Our in-vitro analysis revealed that especially the long chained paraben BuP stimulates adipogenesis by increasing the expression of adipocyte specific genes (PPARγ, ADIPOQ, LPL, etc.) and triglyceride storage. Moreover, we found that adiponectin secretion is increased whereas leptin secretion is reduced under BuP exposure in-vitro. Further mechanistic analysis for receptor binding and activation of PPARγ and other key players in adipogenesis are currently in process. We conclude that maternal cosmetic product consumption is linked to prenatal paraben exposure of children and contributes to the development of infant overweight development by triggering key pathways of adipogenesis.

Keywords: adipogenesis, endocrine disruptors, paraben, prenatal exposure

Procedia PDF Downloads 274

Authors: Farah Diab, Mohamad Khalil, Francesca Storace, Francesca Baldini, Piero Portincasaa, Giulio Lupidi, Laura Vergani

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Thymbra spicata is a thyme-like plant belonging to the Lamiaceae family that shows a global distribution, especially in the eastern Mediterranean region. Leaves of T. spicata contain large amounts of phenols such as phenolic acids (rosmarinic acid), phenolic monoterpenes (carvacrol), and flavonoids. In Lebanon, T. spicata is currently used as a culinary herb in salad and infusion, as well as for traditional medicinal purposes. Carvacrol (5-isopropyl-2-methyl phenol), the most abundant polyphenol in the organic extract and essential oils, has a great array of pharmacological properties. In fact, carvacrol is largely employed as a food additive and neutraceutical agent. Our aim is to investigate the beneficial effects of T. spicata ethanolic extract (TE) and its main component, carvacrol, using in vitro models of hepatic steatosis and endothelial dysfunction. As a further point, we focused on investigating if and how the binding of carvacrol to albumin, the physiological transporter for drugs in the blood, might be altered by the presence of high levels of fatty acids (FAs), thus impairing the carvacrol bio-distribution in vivo. For that reason, hepatic FaO cells treated with exogenous FAs such as oleate and palmitate mimic hepatosteatosis; endothelial HECV cells exposed to hydrogen peroxide are a model of endothelial dysfunction. In these models, we measured lipid accumulation, free radical production, lipoperoxidation, and nitric oxide release before and after treatment with carvacrol. The carvacrol binding to albumin with/without high levels of long-chain FAs was assessed by absorption and emission spectroscopies. Our findings show that both TE and carvacrol (i) counteracted lipid accumulation in hepatocytes by decreasing the intracellular and extracellular lipid contents in steatotic FaO cells; (ii) decreased oxidative stress in endothelial cells by significantly reducing lipoperoxidation and free radical production, as well as, attenuating the nitric oxide release; (ii) high levels of circulating FAs reduced the binding of carvacrol to albumin. The beneficial effects of TE and carvacrol on both hepatic and endothelial cells point to a nutraceutical potential. However, high levels of circulating FAs, such as those occurring in metabolic disorders, might hinder the carvacrol transport, bio-distribution, and pharmacodynamics.

Keywords: carvacrol, endothelial dysfunction, fatty acids, non-alcoholic fatty liver diseases, serum albumin

Procedia PDF Downloads 192

Authors: E. Salem

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Combustion has been used for a long time as a means of energy extraction. However, in recent years, there has been a further increase in air pollution, through pollutants such as nitrogen oxides, acid etc. In order to solve this problem, there is a need to reduce carbon and nitrogen oxides through learn burning modifying combustors and fuel dilution. A numerical investigation has been done to investigate the effectiveness of several reduced mechanisms in terms of computational time and accuracy, for the combustion of the hydrocarbons/air or diluted with hydrogen in a micro combustor. The simulations were carried out using the ANSYS Fluent 19.1. To validate the results “PREMIX and CHEMKIN” codes were used to calculate 1D premixed flame based on the temperature, composition of burned and unburned gas mixtures. Numerical calculations were carried for several hydrocarbons by changing the equivalence ratios and adding small amounts of hydrogen into the fuel blends then analyzing the flammable limit, the reduction in NOx and CO emissions, then comparing it to experimental data. By solving the conservations equations, several global reduced mechanisms (2-9-12) were obtained. These reduced mechanisms were simulated on a 2D cylindrical tube with dimensions of 40 cm in length and 2.5 cm diameter. The mesh of the model included a proper fine quad mesh, within the first 7 cm of the tube and around the walls. By developing a proper boundary layer, several simulations were performed on hydrocarbon/air blends to visualize the flame characteristics than were compared with experimental data. Once the results were within acceptable range, the geometry of the combustor was modified through changing the length, diameter, adding hydrogen by volume, and changing the equivalence ratios from lean to rich in the fuel blends, the results on flame temperature, shape, velocity and concentrations of radicals and emissions were observed. It was determined that the reduced mechanisms provided results within an acceptable range. The variation of the inlet velocity and geometry of the tube lead to an increase of the temperature and CO2 emissions, highest temperatures were obtained in lean conditions (0.5-0.9) equivalence ratio. Addition of hydrogen blends into combustor fuel blends resulted in; reduction in CO and NOx emissions, expansion of the flammable limit, under the condition of having same laminar flow, and varying equivalence ratio with hydrogen additions. The production of NO is reduced because the combustion happens in a leaner state and helps in solving environmental problems.

Keywords: combustor, equivalence-ratio, hydrogenation, premixed flames

Procedia PDF Downloads 114

Authors: Fatemeh Mohsennezhad

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Air pollution is one of major problems for environment. Providing healthy food and protecting water sources from pollution has been one of the concerns of human societies and decision-making centers so that protecting food from pollution, detecting sources of pollution and measuring them become important. Nutritive and political significance of grape in this area, extensive use of leaf and fruit of this plant and development of urban areas around grape gardens and construction of Tabriz – Miandoab road, which is the most important link between East and West Azarbaijan, led us to examine the impact of this road construction and urban environment pollutants such as lead chromium and cadmium on the quality of this valuable crop. First, the samples were taken from different adjacent places and medium distances from the road, each place being located exactly by Google earth and GPS. Digestion was done through burning dry material and hydrochloric acid and their ashes were analyzed by atomic absorption to determine (Pb, Cr, Cd) accumulations. In this experiments effects of 2 following factors were examined as a variable: Garden distance from the main road with levels 1: For 50 meters, 2: For 120-200 meters, 3: For above 800 meters, and plant organ with levels 1: For fruit, 2: For leaves. At the end, the results were processed by SPSS software. 3.54 ppm, the most lead quantity, was at sample No. 54 in fruits with 800 meters distance from the road and 1.00 ppm was the least lead quantity at sample No. 50 in fruits with 1000 meters from the road. In leaves, the most lead quantity was 19.16 ppm at sample No. 15 with 50 meters distance from the road and the least quantity was 1.41 ppm at sample No. 31 with 50 meters from the road. Pb uptake is significantly different at 50 meters and 200 meters distance. It means that Pb uptake near the main road is the highest. But this result is not true for others elements. Distance has not a meaningful effect on Cr uptake. The result of analysis of variation in distance and plant organ for Cd showed that between fruit and leaf, Cd uptake is significantly different. But distance and interaction between distance and plant organ is not meaningful. There is neither meaningful interaction between these elements uptakes in fruits nor in leaves. If leaves and fruits, assumed all together, showed a very meaningful integration between heavy metal accumulations. It means that each of these elements causes uptake others without considering special organs. In the tested area, it became clear that, from the accumulation of heavy metals perspective, there is no meaningful difference in existing distance between road and garden. There is a meaningful difference among heavy metals accumulation. In other words, increase ratio of one metal to another was different from the resulted differences shown in corresponding graphs. Interaction among elements and distance between garden and road was not meaningful.

Keywords: Vitis vinifera L., phytoremediation, heavy metals accumulation, lead, chromium, cadmium

Procedia PDF Downloads 354

Authors: Stephen Osakue Amiandamhen, Schwaller Andreas, Martina Meincken, Luvuyo Tyhoda

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Invasive alien tree species are currently being cleared in South Africa as a result of the forest and water imbalances. These species grow wildly constituting about 40% of total forest area. They compete with the ecosystem for natural resources and are considered as ecosystem engineers by rapidly changing disturbance regimes. As such, they are harvested for commercial uses but much of it is wasted because of their form and structure. The waste is being sold to local communities as fuel wood. These species can be considered as potential feedstock for the production of phosphate bonded composites. The presence of bark in wood-based composites leads to undesirable properties, and debarking as an option can be cost implicative. This study investigates the potentials of these invasive species processed without debarking on some fundamental properties of wood-based panels. Some invasive alien tree species were collected from EC Biomass, Port Elizabeth, South Africa. They include Acacia mearnsii (Black wattle), A. longifolia (Long-leaved wattle), A. cyclops (Red-eyed wattle), A. saligna (Golden-wreath wattle) and Eucalyptus globulus (Blue gum). The logs were chipped as received. The chips were hammer-milled and screened through a 1 mm sieve. The wood particles were conditioned and the quantity of bark in the wood was determined. The binding matrix was prepared using a reactive magnesia, phosphoric acid and class S fly ash. The materials were mixed and poured into a metallic mould. The composite within the mould was compressed at room temperature at a pressure of 200 KPa. After initial setting which took about 5 minutes, the composite board was demoulded and air-cured for 72 h. The cured product was thereafter conditioned at 20°C and 70% relative humidity for 48 h. Test of physical and strength properties were conducted on the composite boards. The effect of binder formulation and fly ash content on the properties of the boards was studied using fitted response surface technology, according to a central composite experimental design (CCD) at a fixed wood loading of 75% (w/w) of total inorganic contents. The results showed that phosphate/magnesia ratio of 3:1 and fly ash content of 10% was required to obtain a product of good properties and sufficient strength for intended applications. The proposed products can be used for ceilings, partitioning and insulating wall panels.

Keywords: invasive alien tree species, phosphate bonded composites, physical properties, strength

Procedia PDF Downloads 295

Authors: Razvan Mercut, Mihaela Ionescu, Vlad Parvanescu, Razvan Ghita, Tudor-Gabriel Caragea, Cristina Simionescu, Marius-Eugen Ciurea

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Introduction: Over the past years, the incidence of non-melanoma skin cancer (NMSC) has continuously increased, being one of the most commonly diagnosed carcinomasofthe cephalic extremity. NMSC regroups basal cell carcinoma (BCC), squamous cell carcinoma (SCC), Merkel cell carcinoma, cutaneous lymphoma, and sarcoma. The most common forms are BCC and SCC, both still implying a significant level of morbidity due to local invasion (especially BCC), even if the overall death rates are declining. The objective of our study was the evaluation of clinical and histological aspects of NMSC for a group of patients with BCC and SCC, from Craiova, a south-western major city in Romania. Materialand method: Our study lot comprised 65 patients, with an almost equal distribution of sexes, and ages between 23-91 years old (mean value±standard deviation62.61±16.67), all treated within the Clinic of Plastic Surgery and Reconstructive Microsurgery, Clinical Emergency County Hospital Craiova, Romania, between 2019-2020. In order to determine the main morphological characteristics of both studied cancers, we used paraffin embedding techniques, with various staining methods:hematoxylin-eosin, Masson's trichrome stain with aniline blue, and Periodic acid-schiffAlcian Blue. The statistical study was completed using Microsoft Excel (Microsoft Corp., Redmond, WA, USA), with XLSTAT (Addinsoft SARL, Paris, France). Results: The overall results of our study indicate that BCC accounts for 67.69% of all NMSC forms; SCC covers 27.69%, while 4.62% are representedby other forms. The most frequent site is the nose for BCC (27.69%, 18 patients), being followed by preauricular regions, forehead, and periorbital areas. For patients with SCC, tumors were mainly located at lips level (66.67%, 12 patients). The analysis of NMSC histological forms indicated that nodular BCC is predominant (45.45%, 20 patients), as well as ulcero-vegetant SCC (38.89%, 7 patients). We have not identified any topographic characteristics or NMSC forms significantly related to age or sex. Conclusions: The most frequent NMSC form identified for our study lot was BCC. The preferred location was the nose for BCC. For SCC, the oral cavity is the most frequent anatomical site, especially the lips level. Nodular BCC and ulcero-vegetant SCC were the most commonly identified histological types. Our findings emphasize the need for periodic screening, in order to improve prevention and early treatment for these malignancies.

Keywords: non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, histological

Procedia PDF Downloads 189

Authors: Nadhiya N. Subramony, Jenifer Vaughan, Lesley E. Scott

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In South Africa, the World Health Organisation estimated 454000 new cases of Mycobacterium tuberculosis (M.tb) infection (MTB) in 2015. Disseminated tuberculosis arises from the haematogenous spread and seeding of the bacilli in extrapulmonary sites. The gold standard for the detection of MTB in bone marrow is TB culture which has an average turnaround time of 6 weeks. Histological examinations of trephine biopsies to diagnose MTB also have a time delay owing mainly to the 5-7 day processing period prior to microscopic examination. Adding to the diagnostic delay is the non-specific nature of granulomatous inflammation which is the hallmark of MTB involvement of the bone marrow. A Ziehl-Neelson stain (which highlights acid-fast bacilli) is therefore mandatory to confirm the diagnosis but can take up to 3 days for processing and evaluation. Owing to this delay in diagnosis, many patients are lost to follow up or remain untreated whilst results are awaited, thus encouraging the spread of undiagnosed TB. The Xpert® MTB/RIF (Cepheid, Sunnyvale, CA) is the molecular test used in the South African national TB program as the initial diagnostic test for pulmonary TB. This study investigates the optimisation and performance of the Xpert® MTB/RIF on bone marrow aspirate specimens (BMA), a first since the introduction of the assay in the diagnosis of extrapulmonary TB. BMA received for immunophenotypic analysis as part of the investigation into disseminated MTB or in the evaluation of cytopenias in immunocompromised patients were used. Processing BMA on the Xpert® MTB/RIF was optimised to ensure bone marrow in EDTA and heparin did not inhibit the PCR reaction. Inactivated M.tb was spiked into the clinical bone marrow specimen and distilled water (as a control). A volume of 500mcl and an incubation time of 15 minutes with sample reagent were investigated as the processing protocol. A total of 135 BMA specimens had sufficient residual volume for Xpert® MTB/RIF testing however 22 specimens (16.3%) were not included in the final statistical analysis as an adequate trephine biopsy and/or TB culture was not available. Xpert® MTB/RIF testing was not affected by BMA material in the presence of heparin or EDTA, but the overall detection of MTB in BMA was low compared to histology and culture. Sensitivity of the Xpert® MTB/RIF compared to both histology and culture was 8.7% (95% confidence interval (CI): 1.07-28.04%) and sensitivity compared to histology only was 11.1% (95% CI: 1.38-34.7%). Specificity of the Xpert® MTB/RIF was 98.9% (95% CI: 93.9-99.7%). Although the Xpert® MTB/RIF generates a faster result than histology and TB culture and is less expensive than culture and drug susceptibility testing, the low sensitivity of the Xpert® MTB/RIF precludes its use for the diagnosis of MTB in bone marrow aspirate specimens and warrants alternative/additional testing to optimise the assay.

Keywords: bone marrow aspirate , extrapulmonary TB, low sensitivity, Xpert® MTB/RIF

Procedia PDF Downloads 172

Authors: D. Lopes, I. I. R. Baptista, R. F. Vieira, J. Vaz, H. Varela, O. M. Freitas, V. F. Domingues, R. Jorge, C. Delerue-Matos, S. A. Figueiredo

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Deodorization is nowadays a need in wastewater treatment plants. Nitrogen and sulphur compounds, volatile fatty acids, aldehydes and ketones are responsible for the unpleasant odours, being ammonia, hydrogen sulphide and mercaptans the most common pollutants. Although chemical treatments of the air extracted are efficient, these are more expensive than biological treatments, namely due the use of chemical reagents (commonly sulphuric acid, sodium hypochlorite and sodium hydroxide). Biofiltration offers the advantage of avoiding the use of reagents (only in some cases, nutrients are added in order to increase the treatment efficiency) and can be considered a sustainable process when the packing medium used is of natural origin. In this work the application of some natural materials locally available was studied both at laboratory and pilot scale, in a real wastewater treatment plant. The materials selected for this study were indigenous Portuguese forest materials derived from eucalyptus and pinewood, such as woodchips and bark, and coconut fiber was also used for comparison purposes. Their physico-chemical characterization was performed: density, moisture, pH, buffer and water retention capacity. Laboratory studies involved batch adsorption studies for ammonia and hydrogen sulphide removal and evaluation of microbiological activity. Four pilot-scale biofilters (1 cubic meter volume) were installed at a local wastewater treatment plant treating odours from the effluent receiving chamber. Each biofilter contained a different packing material consisting of mixtures of eucalyptus bark, pine woodchips and coconut fiber, with added buffering agents and nutrients. The odour treatment efficiency was monitored over time, as well as other operating parameters. The operation at pilot scale suggested that between the processes involved in biofiltration - adsorption, absorption and biodegradation - the first dominates at the beginning, while the biofilm is developing. When the biofilm is completely established, and the adsorption capacity of the material is reached, biodegradation becomes the most relevant odour removal mechanism. High odour and hydrogen sulphide removal efficiencies were achieved throughout the testing period (over 6 months), confirming the suitability of the materials selected, and mixtures thereof prepared, for biofiltration applications.

Keywords: ammonia hydrogen sulphide and removal, biofiltration, natural materials, odour control in wastewater treatment plants

Procedia PDF Downloads 302

Authors: Yew Mun Yip, Dawei Zhang

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Proteins are the biological machinery that executes specific vital functions in every cell of the human body by folding into their 3D structures. When a protein misfolds from its native structure, the machinery will malfunction and lead to misfolding diseases. Although in vitro experiments are able to conclude that the mutations of the amino acid sequence lead to incorrectly folded protein structures, these experiments are unable to decipher the folding process. Therefore, molecular dynamic (MD) simulations are employed to simulate the folding process so that our improved understanding of the folding process will enable us to contemplate better treatments for misfolding diseases. MD simulations make use of force fields to simulate the folding process of peptides. Secondary structures are formed via the hydrogen bonds formed between the backbone atoms (C, O, N, H). It is important that the hydrogen bond energy computed during the MD simulation is accurate in order to direct the folding process to the native structure. Since the atoms involved in a hydrogen bond possess very dissimilar electronegativities, the more electronegative atom will attract greater electron density from the less electronegative atom towards itself. This is known as the polarization effect. Since the polarization effect changes the electron density of the two atoms in close proximity, the atomic charges of the two atoms should also vary based on the strength of the polarization effect. However, the fixed atomic charge scheme in force fields does not account for the polarization effect. In this study, we introduce the polarized structure-specific backbone charge (PSBC) model. The PSBC model accounts for the polarization effect in MD simulation by updating the atomic charges of the backbone hydrogen bond atoms according to equations derived between the amount of charge transferred to the atom and the length of the hydrogen bond, which are calculated from quantum-mechanical calculations. Compared to other polarizable models, the PSBC model does not require quantum-mechanical calculations of the peptide simulated at every time-step of the simulation and maintains the dynamic update of atomic charges, thereby reducing the computational cost and time while accounting for the polarization effect dynamically at the same time. The PSBC model is applied to two different β-peptides, namely the Beta3s/GS peptide, a de novo designed three-stranded β-sheet whose structure is folded in vitro and studied by NMR, and the trpzip peptides, a double-stranded β-sheet where a correlation is found between the type of amino acids that constitute the β-turn and the β-propensity.

Keywords: hydrogen bond, polarization effect, protein folding, PSBC

Procedia PDF Downloads 270

Authors: E. Nakamachi, K. Matsumoto, K. Yamamoto, Y. Morita, H. Sakamoto

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In recently, electromagnetic and mechanical stimulations have been recognized as the effective extracellular environment stimulation technique to enhance the defected peripheral nerve tissue regeneration. In this study, we developed a new hybrid bioreactor by adopting 50 Hz uniform alternative current (AC) magnetic stimulation and 4% strain mechanical stimulation. The guide tube for nerve regeneration is mesh structured tube made of biodegradable polymer, such as polylatic acid (PLA). However, when neural damage is large, there is a possibility that peripheral nerve undergoes necrosis. So it is quite important to accelerate the nerve tissue regeneration by achieving enhancement of nerve axonal extension rate. Therefore, we try to design and fabricate the system that can simultaneously load the uniform AC magnetic field stimulation and the stretch stimulation to cells for enhancement of nerve axonal extension. Next, we evaluated systems performance and the effectiveness of each stimulation for rat adrenal pheochromocytoma cells (PC12). First, we designed and fabricated the uniform AC magnetic field system and the stretch stimulation system. For the AC magnetic stimulation system, we focused on the use of pole piece structure to carry out in-situ microscopic observation. We designed an optimum pole piece structure using the magnetic field finite element analyses and the response surface methodology. We fabricated the uniform AC magnetic field stimulation system as a bio-reactor by adopting analytically determined design specifications. We measured magnetic flux density that is generated by the uniform AC magnetic field stimulation system. We confirmed that measurement values show good agreement with analytical results, where the uniform magnetic field was observed. Second, we fabricated the cyclic stretch stimulation device under the conditions of particular strains, where the chamber was made of polyoxymethylene (POM). We measured strains in the PC12 cell culture region to confirm the uniform strain. We found slightly different values from the target strain. Finally, we concluded that these differences were allowable in this mechanical stimulation system. We evaluated the effectiveness of each stimulation to enhance the nerve axonal extension using PC12. We confirmed that the average axonal extension length of PC12 under the uniform AC magnetic stimulation was increased by 16 % at 96 h in our bio-reactor. We could not confirm that the axonal extension enhancement under the stretch stimulation condition, where we found the exfoliating of cells. Further, the hybrid stimulation enhanced the axonal extension. Because the magnetic stimulation inhibits the exfoliating of cells. Finally, we concluded that the enhancement of PC12 axonal extension is due to the magnetic stimulation rather than the mechanical stimulation. Finally, we confirmed that the effectiveness of the uniform AC magnetic field stimulation for the nerve axonal extension using PC12 cells.

Keywords: nerve cell PC12, axonal extension, nerve regeneration, electromagnetic-mechanical stimulation, bioreactor

Procedia PDF Downloads 265

Authors: Amelia Frickey, Timothy (TJ) Sheridan, Angelica Rossi, Bahar Aliakbarian

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The growth of large food supply chains demanded improved end-to-end traceability of food products, which has led to companies being increasingly interested in using smart technologies such as Radio Frequency Identification (RFID)-enabled packaging to track items. As technology is being widely used, there are several technological or economic issues that should be overcome to facilitate the adoption of this track-and-trace technology. One of the technological challenges of RFID technology is its sensitivity to different environmental form factors, including packaging materials and the content of the packaging. Although researchers have assessed the performance loss due to the proximity of water and aqueous solutions, there is still the need to further investigate the impacts of food products on the reading range of RFID tags. However, to the best of our knowledge, there are not enough studies to determine the correlation between RFID tag performance and food beverages properties. The goal of this project was to investigate the effect of the solution properties (pH and conductivity) and different packaging materials filled with food-like water-based solutions on the performance of an RFID tag. Three commercially available ultra high-frequency RFID tags were placed on three different bottles and filled with different concentrations of water-based solutions, including sodium chloride, citric acid, sucrose, and ethanol. Transparent glass, Polyethylneterephtalate (PET), and Tetrapak® were used as the packaging materials commonly used in the beverage industries. Tag readability (Theoretical Read Range, TRR) and sensitivity (Power on Tag Forward, PoF) were determined using an anechoic chamber. First, the best place to attach the tag for each packaging material was investigated using empty and water-filled bottles. Then, the bottles were filled with the food-like solutions and tested with the three different tags and the PoF and TRR at the fixed frequency of 915MHz. In parallel, the pH and conductivity of solutions were measured. The best-performing tag was then selected to test the bottles filled with wine, orange, and apple juice. Despite various solutions altering the performance of each tag, the change in tag performance had no correlation with the pH or conductivity of the solution. Additionally, packaging material played a significant role in tag performance. Each tag tested performed optimally under different conditions. This study is the first part of comprehensive research to determine the regression model for the prediction of tag performance behavior based on the packaging material and the content. More investigations, including more tags and food products, are needed to be able to develop a robust regression model. The results of this study can be used by RFID tag manufacturers to design suitable tags for specific products with similar properties.

Keywords: smart food packaging, supply chain management, food waste, radio frequency identification

Procedia PDF Downloads 114

Authors: Sulalit Bandyopadhyay, Muhammad Awais Ashfaq Alvi, Anuvansh Sharma, Wilhelm R. Glomm

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Release of drugs from nanogels and nanogel-based systems can occur under the influence of external stimuli like temperature, pH, magnetic fields and so on. pNIPAm-AAc nanogels respond to the combined action of both temperature and pH, the former being mostly determined by hydrophilic-to-hydrophobic transitions above the volume phase transition temperature (VPTT), while the latter is controlled by the degree of protonation of the carboxylic acid groups. These nanogels based systems are promising candidates in the field of drug delivery. Combining nanogels with magneto-plasmonic nanoparticles (NPs) introduce imaging and targeting modalities along with stimuli-response in one hybrid system, thereby incorporating multifunctionality. Fe@Au core-shell NPs possess optical signature in the visible spectrum owing to localized surface plasmon resonance (LSPR) of the Au shell, and superparamagnetic properties stemming from the Fe core. Although there exist several synthesis methods to control the size and physico-chemical properties of pNIPAm-AAc nanogels, yet, there is no comprehensive study that highlights the dependence of incorporation of one or more layers of NPs to these nanogels. In addition, effective determination of volume phase transition temperature (VPTT) of the nanogels is a challenge which complicates their uses in biological applications. Here, we have modified the swelling-collapse properties of pNIPAm-AAc nanogels, by combining with Fe@Au NPs using different solution based methods. The hydrophilic-hydrophobic transition of the nanogels above the VPTT has been confirmed to be reversible. Further, an analytical method has been developed to deduce the average VPTT which is found to be 37.3°C for the nanogels and 39.3°C for nanogel coated Fe@Au NPs. An opposite swelling –collapse behaviour is observed for the latter where the Fe@Au NPs act as bridge molecules pulling together the gelling units. Thereafter, Cyt C, a model protein drug and L-Dopa, a drug used in the clinical treatment of Parkinson’s disease were loaded separately into the nanogels and nanogel coated Fe@Au NPs, using a modified breathing-in mechanism. This gave high loading and encapsulation efficiencies (L Dopa: ~9% and 70µg/mg of nanogels, Cyt C: ~30% and 10µg/mg of nanogels respectively for both the drugs. The release kinetics of L-Dopa, monitored using UV-vis spectrophotometry was observed to be rather slow (over several hours) with highest release happening under a combination of high temperature (above VPTT) and acidic conditions. However, the release of L-Dopa from nanogel coated Fe@Au NPs was the fastest, accounting for release of almost 87% of the initially loaded drug in ~30 hours. The chemical structure of the drug, drug incorporation method, location of the drug and presence of Fe@Au NPs largely alter the drug release mechanism and the kinetics of these nanogels and Fe@Au NPs coated with nanogels.

Keywords: controlled release, nanogels, volume phase transition temperature, l-dopa

Procedia PDF Downloads 331

Authors: Evanka Madan, Madhu Puri, Dan Zilberstein, Rohini Muthuswami, Rentala Madhubala

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The extensive interaction between the host and pathogen metabolic networks decidedly shapes the outcome of infection. Utilization of arginine by the host and pathogen is critical for determining the outcome of pathogenic infection. Infections with L. donovani, an intracellular parasite, will lead to an extensive competition of arginine between the host and the parasite donovani infection. One of the major amino acid (AA) sensing signaling pathways in mammalian cells are the mammalian target of rapamycin complex I (mTORC1) pathway. mTORC1, as a sensor of nutrient, controls numerous metabolic pathways. Arginine is critical for mTORC1 activation. SLC38A9 is the arginine sensor for the mTORC1, being activated during arginine sufficiency. L. donovani transport arginine via a high-affinity transporter (LdAAP3) that is rapidly up-regulated by arginine deficiency response (ADR) in intracellular amastigotes. This study, to author’s best knowledge, investigates the interaction between two arginine sensing systems that act in the same compartment, the lysosome. One is important for macrophage defense, and the other is essential for pathogen virulence. We hypothesize that the latter modulates lysosome arginine to prevent host defense response. The work presented here identifies an upstream regulatory role of LdAAP3 in regulating the expression of SLC38A9-mTORC1 pathway, and consequently, their function in L. donovani infected THP-1 cells cultured in 0.1 mM and 1.5 mM arginine. It was found that in physiological levels of arginine (0.1 mM), infecting THP-1 with Leishmania leads to increased levels of SLC38A9 and mTORC1 via an increase in the expression of RagA. However, the reversal was observed with LdAAP3 mutants, reflecting the positive regulatory role of LdAAP3 on the host SLC38A9. At the molecular level, upon infection, mTORC1 and RagA were found to be activated at the surface of phagolysosomes which was found to form a complex with phagolysosomal localized SLC38A9. To reveal the relevance of SLC38A9 under physiological levels of arginine, endogenous SLC38A9 was depleted and a substantial reduction in the expression of host mTORC1, its downstream active substrate, p-P70S6K1 and parasite LdAAP3, was observed, thereby showing that silencing SLC38A9 suppresses ADR. In brief, to author’s best knowledge, these results reveal an upstream regulatory role of LdAAP3 in manipulating SLC38A9 arginine sensing in host macrophages. Our study indicates that intra-macrophage survival of L. donovani depends on the availability and transport of extracellular arginine. An understanding of the sensing pathway of both parasite and host will open a new perspective on the molecular mechanism of host-parasite interaction and consequently, as a treatment for Leishmaniasis.

Keywords: arginine sensing, LdAAP3, L. donovani, mTORC1, SLC38A9, THP-1

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Authors: M. S. Stepanova, V. V. Zakharov, P. D. Khavlyuk, I. D. Skurlov, A. Y. Dubovik, A. L. Rogach

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Carbon dots are small carbon-based spherical nanoparticles, which are typically less than 10 nm in size that can be modified with surface passivation and heteroatoms doping. The light-absorbing ability of carbon dots has attracted a significant amount of attention in photoluminescence for bioimaging and fluorescence sensing applications owing to their advantages, such as tunable fluorescence emission, photo- and thermostability and low toxicity. In this study, carbon dots were synthesized by the solvothermal method from citric acid and ethylenediamine dissolved in water. The solution was heated for 5 hours at 200°C and then cooled down to room temperature. The carbon dots films were obtained by evaporation from a high-concentration aqueous solution. The increase of both luminescence intensity and light transmission was obtained as a result of a 405 nm laser exposure to a part of the carbon dots film, which was detected using a confocal laser scanning microscope (LSM 710, Zeiss). Blueshift up to 35 nm of the luminescence spectrum is observed as luminescence intensity, which is increased more than twofold. The exact value of the shift depends on the time of the laser exposure. This shift can be caused by the modification of surface groups at the carbon dots, which are responsible for long-wavelength luminescence. In addition, a shift of the absorption peak by 10 nm and a decrease in the optical density at the wavelength of 350 nm is detected, which is responsible for the absorption of surface groups. The obtained sample was also studied with time-resolved confocal fluorescence microscope (MicroTime 100, PicoQuant), which made it possible to receive a time-resolved photoluminescence image and construct emission decays of the laser-exposed and non-exposed areas. 5 MHz pulse rate impulse laser has been used as a photoluminescence excitation source. Photoluminescence decay was approximated by two exhibitors. The laser-exposed area has the amplitude of the first-lifetime component (A1) twice as much as before, with increasing τ1. At the same time, the second-lifetime component (A2) decreases. These changes evidence a modification of the surface groups of carbon dots. The detected effect can be used to create thermostable fluorescent marks, the physical size of which is bounded by the diffraction limit of the optics (~ 200-300 nm) used for exposure and to improve the optical properties of carbon dots or in the field of optical encryption. Acknowledgements: This work was supported by the Ministry of Science and Higher Education of Russian Federation, goszadanie no. 2019-1080 and financially supported by Government of Russian Federation, Grant 08-08.

Keywords: carbon dots, photoactivation, optical properties, photoluminescence and absorption spectra

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Authors: Gyaltsen Dakpa, K. J. Senthil Kumar, Nai-Wen Tsao, Sheng-Yang Wang

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Context: Coronavirus disease 2019 (COVID-19) is a severe respiratory illness caused by the SARS-CoV-2 virus. One of the hallmarks of COVID-19 is a change in metabolism, which can lead to increased severity and mortality. The mechanism of SARS-CoV-2-mediated perturbations of metabolic pathways has yet to be fully understood. Research Aim: This study aimed to investigate the metabolic alteration caused by SARS-CoV-2 spike protein in Phorbol 12-myristate 13-acetate (PMA)-induced human monocytes (THP-1) and to examine the regulatory effect of natural compounds like Antcins A on SARS-CoV-2 spike protein-induced metabolic alteration. Methodology: The study used a combination of proton nuclear magnetic resonance (1H-NMR) and MetaboAnalyst 5.0 software. THP-1 cells were treated with SARS-CoV-2 spike protein or control, and the metabolomic profiles of the cells were compared. Antcin A was also added to the cells to assess its regulatory effect on SARS-CoV-2 spike protein-induced metabolic alteration. Findings: The study results showed that treatment with SARS-CoV-2 spike protein significantly altered the metabolomic profiles of THP-1 cells. Eight metabolites, including glycerol-phosphocholine, glycine, canadine, sarcosine, phosphoenolpyruvic acid, glutamine, glutamate, and N, N-dimethylglycine, were significantly different between control and spike-protein treatment groups. Antcin A significantly reversed the changes in these metabolites. In addition, treatment with antacid A significantly inhibited SARS-CoV-2 spike protein-mediated up-regulation of TLR-4 and ACE2 receptors. Theoretical Importance The findings of this study suggest that SARS-CoV-2 spike protein can cause significant metabolic alterations in THP-1 cells. Antcin A, a natural compound, has the potential to reverse these metabolic alterations and may be a potential candidate for developing preventive or therapeutic agents for COVID-19. Data Collection: The data for this study was collected from THP-1 cells that were treated with SARS-CoV-2 spike protein or a control. The metabolomic profiles of the cells were then compared using 1H-NMR and MetaboAnalyst 5.0 software. Analysis Procedures: The metabolomic profiles of the THP-1 cells were analyzed using 1H-NMR and MetaboAnalyst 5.0 software. The software was used to identify and quantify the cells' metabolites and compare the control and spike-protein treatment groups. Questions Addressed: The question addressed by this study was whether SARS-CoV-2 spike protein could cause metabolic alterations in THP-1 cells and whether Antcin A can reverse these alterations. Conclusion: The findings of this study suggest that SARS-CoV-2 spike protein can cause significant metabolic alterations in THP-1 cells. Antcin A, a natural compound, has the potential to reverse these metabolic alterations and may be a potential candidate for developing preventive or therapeutic agents for COVID-19.

Keywords: SARS-CoV-2-spike, ¹H-NMR, metabolomics, antcin-A, taiwanofungus camphoratus

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Authors: Igor Jerkovic

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Biodiversity of flora provides many different nectar sources for the bees. Unifloral honeys possess distinctive flavours, mainly derived from their nectar sources (characteristic volatile organic components (VOCs)). Specific or nonspecific VOCs (chemical markers) could be used for unifloral honey characterisation as addition to the melissopalynologycal analysis. The main honey volatiles belong, in general, to three principal categories: terpenes, norisoprenoids, and benzene derivatives. Some of these substances have been described as characteristics of the floral source, and other compounds, like several alcohols, branched aldehydes, and furan derivatives, may be related to the microbial purity of honey processing and storage conditions. Selection of the extraction method for the honey volatiles profiling should consider that heating of the honey produce different artefacts and therefore conventional methods of VOCs isolation (such as hydrodistillation) cannot be applied for the honey. Two-way approach for the isolation of the honey VOCs was applied using headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE). The extracts were analysed by gas chromatography and mass spectrometry (GC-MS). HS-SPME (with the fibers of different polarity such as polydimethylsiloxane/ divinylbenzene (PDMS/DVB) or divinylbenzene/carboxene/ polydimethylsiloxane (DVB/CAR/PDMS)) enabled isolation of high volatile headspace VOCs of the honey samples. Among them, some characteristic or specific compounds can be found such as 3,4-dihydro-3-oxoedulan (in Centaurea cyanus L. honey) or 1H-indole, methyl anthranilate, and cis-jasmone (in Citrus unshiu Marc. honey). USE with different solvents (mainly dichloromethane or the mixture pentane : diethyl ether 1 : 2 v/v) enabled isolation of less volatile and semi-volatile VOCs of the honey samples. Characteristic compounds from C. unshiu honey extracts were caffeine, 1H-indole, 1,3-dihydro-2H-indol-2-one, methyl anthranilate, and phenylacetonitrile. Sometimes, the selection of solvent sequence was useful for more complete profiling such as sequence I: pentane → diethyl ether or sequence II: pentane → pentane/diethyl ether (1:2, v/v) → dichloromethane). The extracts with diethyl ether contained hydroquinone and 4-hydroxybenzoic acid as the major compounds, while (E)-4-(r-1’,t-2’,c-4’-trihydroxy-2’,6’,6’-trimethylcyclo-hexyl)but-3-en-2-one predominated in dichloromethane extracts of Allium ursinum L. honey. With this two-way approach, it was possible to obtain a more detailed insight into the honey volatile and semi-volatile compounds and to minimize the risks of compound discrimination due to their partial extraction that is of significant importance for the complete honey profiling and identification of the chemical biomarkers that can complement the pollen analysis.

Keywords: honey chemical biomarkers, honey volatile compounds profiling, headspace solid-phase microextraction (HS-SPME), ultrasonic solvent extraction (USE)

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Authors: Mitakshara Sharma, S. K. Nema, Sanjeev Narang

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Diabetes mellitus (DM) is a group of metabolic disorders characterized by metabolic abnormalities, chronic hyperglycaemia and long term macrovascular & microvascular complications. Vascular complications are due to platelet hyperactivity and dysfunction, increased inflammation, altered coagulation and endothelial dysfunction. Large proportion of patients with Type II DM suffers from preventable vascular angiopathies, and there is need to develop risk factor modifications and interventions to reduce impact of complications. These complications are attributed to platelet activation, recognised by increase in Platelet Volume Indices (PVI) including Mean Platelet Volume (MPV) and Platelet Distribution Width (PDW). The current study is prospective analytical study conducted over 2 years. Out of 1100 individuals, 930 individuals fulfilled inclusion criteria and were segregated into three groups on basis of glycosylated haemoglobin (HbA1C): - (a) Diabetic, (b) Non-Diabetic and (c) Subjects with Impaired fasting glucose (IFG) with 300 individuals in IFG and non-diabetic groups & 330 individuals in diabetic group. Further, diabetic group was divided into two groups on the basis of presence or absence of known diabetes related vascular complications. Samples for HbA1c and PVI were collected using Ethylene diamine tetraacetic acid (EDTA) as anticoagulant and processed on SYSMEX-X-800i autoanalyser. The study revealed gradual increase in PVI from non-diabetics to IFG to diabetics. PVI were markedly increased in diabetic patients. MPV and PDW of diabetics, IFG and non diabetics were (17.60 ± 2.04)fl, (11.76 ± 0.73)fl, (9.93 ± 0.64)fl and (19.17 ± 1.48)fl, (15.49 ± 0.67)fl, (10.59 ± 0.67)fl respectively with a significant p value 0.00 and a significant positive correlation (MPV-HbA1c r = 0.951; PDW-HbA1c r = 0.875). MPV & PDW of subjects with diabetes related complications were higher as compared to those without them and were (17.51±0.39)fl & (15.14 ± 1.04)fl and (20.09 ± 0.98) fl & (18.96 ± 0.83)fl respectively with a significant p value 0.00. There was a significant positive correlation between PVI and duration of diabetes across the groups (MPV-HbA1c r = 0.951; PDW-HbA1c r = 0.875). However, a significant negative correlation was found between glycaemic levels and total platelet count (PC- HbA1c r =-0.164). This is multi-parameter and comprehensive study with an adequately powered study design. It can be concluded from our study that PVI are extremely useful and important indicators of impending vascular complications in all patients with deranged glycaemic control. Introduction of automated cell counters has facilitated the availability of PVI as routine parameters. PVI is a useful means for identifying larger & active platelets which play important role in development of micro and macro angiopathic complications of diabetes leading to mortality and morbidity. PVI can be used as cost effective markers to predict and prevent impending vascular events in patients with Diabetes mellitus especially in developing countries like India. PVI, if incorporated into protocols for management of diabetes, could revolutionize care and curtail the ever increasing cost of patient management.

Keywords: diabetes, IFG, HbA1C, MPV, PDW, PVI

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Authors: Sebastiano Candamano, Antonio Iorfida, Patrizia Frontera, Anastasia Macario, Fortunato Crea

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Alkali-activated materials are promising binders obtained by an alkaline attack on fly-ashes, metakaolin, blast slag among others. In order to guarantee the highest ecological and cost efficiency, a proper selection of precursors and alkaline activators has to be carried out. These choices deeply affect the microstructure, chemistry and performances of this class of materials. Even if, in the last years, several researches have been focused on mix designs and curing conditions, the lack of exhaustive activation models, standardized mix design and curing conditions and an insufficient investigation on shrinkage behavior, efflorescence, additives and durability prevent them from being perceived as an effective and reliable alternative to Portland. The aim of this study is to develop alkali-activated cements mortars containing high amounts of industrial by-products and waste, such as ground granulated blast furnace slag (GGBFS) and ashes obtained from the combustion process of forest biomass in thermal power plants. In particular, the experimental campaign was performed in two steps. In the first step, research was focused on elucidating how the workability, mechanical properties and shrinkage behavior of produced mortars are affected by the type and fraction of each precursor as well as by the composition of the activator solutions. In order to investigate the microstructures and reaction products, SEM and diffractometric analyses have been carried out. In the second step, their durability in harsh environments has been evaluated. Mortars obtained using only GGBFS as binder showed mechanical properties development and shrinkage behavior strictly dependent on SiO2/Na2O molar ratio of the activator solutions. Compressive strengths were in the range of 40-60 MPa after 28 days of curing at ambient temperature. Mortars obtained by partial replacement of GGBFS with metakaolin and forest biomass ash showed lower compressive strengths (≈35 MPa) and shrinkage values when higher amount of ashes were used. By varying the activator solutions and binder composition, compressive strength up to 70 MPa associated with shrinkage values of about 4200 microstrains were measured. Durability tests were conducted to assess the acid and thermal resistance of the different mortars. They all showed good resistance in a solution of 5%wt of H2SO4 also after 60 days of immersion, while they showed a decrease of mechanical properties in the range of 60-90% when exposed to thermal cycles up to 700°C.

Keywords: alkali activated cement, biomass ash, durability, shrinkage, slag

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Authors: Mehrnaz Mostafavi

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Recent advances in single-molecule protein identification (ID) and quantification techniques are poised to revolutionize proteomics, enabling researchers to delve into single-cell proteomics and identify low-abundance proteins crucial for biomedical and clinical research. This paper introduces a different approach to single-molecule protein ID and quantification using tri-color amino acid tags and a plasmonic nanopore device. A comprehensive simulator incorporating various physical phenomena was designed to predict and model the device's behavior under diverse experimental conditions, providing insights into its feasibility and limitations. The study employs a whole-proteome single-molecule identification algorithm based on convolutional neural networks, achieving high accuracies (>90%), particularly in challenging conditions (95–97%). To address potential challenges in clinical samples, where post-translational modifications affecting labeling efficiency, the paper evaluates protein identification accuracy under partial labeling conditions. Solid-state nanopores, capable of processing tens of individual proteins per second, are explored as a platform for this method. Unlike techniques relying solely on ion-current measurements, this approach enables parallel readout using high-density nanopore arrays and multi-pixel single-photon sensors. Convolutional neural networks contribute to the method's versatility and robustness, simplifying calibration procedures and potentially allowing protein ID based on partial reads. The study also discusses the efficacy of the approach in real experimental conditions, resolving functionally similar proteins. The theoretical analysis, protein labeler program, finite difference time domain calculation of plasmonic fields, and simulation of nanopore-based optical sensing are detailed in the methods section. The study anticipates further exploration of temporal distributions of protein translocation dwell-times and the impact on convolutional neural network identification accuracy. Overall, the research presents a promising avenue for advancing single-molecule protein identification and quantification with broad applications in proteomics research. The contributions made in methodology, accuracy, robustness, and technological exploration collectively position this work at the forefront of transformative developments in the field.

Keywords: nano proteomics, nanopore-based optical sensing, deep learning, artificial intelligence

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Authors: Paula K. Novelli, Margarida M. Barros, Luciana F. Flueri

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Utilization of agricultural by-products, wheat ban and soybean bran, as substrate for solid state fermentation (SSF) was studied, aiming the achievement of different enzymes from Aspergillus sp. with distinct biological characteristics and its application and improvement on animal nutrition. Aspergillus niger and Aspergillus oryzea were studied as they showed very high yield of phytase and protease production, respectively. Phytase activity was measure using p-nitrophenilphosphate as substrate and a standard curve of p-nitrophenol, as the enzymatic activity unit was the quantity of enzyme necessary to release one μmol of p-nitrophenol. Protease activity was measure using azocasein as substrate. Activity for phytase and protease substantially increased when the different biochemical characteristics were considered in the study. Optimum pH and stability of the phytase produced by A. niger with wheat bran as substrate was between 4.0 - 5.0 and optimum temperature of activity was 37oC. Phytase fermented in soybean bran showed constant values at all pHs studied, for optimal and stability, but low production. Phytase with both substrates showed stable activity for temperatures higher than 80oC. Protease from A. niger showed very distinct behavior of optimum pH, acid for wheat bran and basic for soybean bran, respectively and optimal values of temperature and stability at 50oC. Phytase produced by A. oryzae in wheat bran had optimum pH and temperature of 9 and 37oC, respectively, but it was very unstable. On the other hand, proteases were stable at high temperatures, all pH’s studied and showed very high yield when fermented in wheat bran, however when it was fermented in soybean bran the production was very low. Subsequently the upscale production of phytase from A. niger and proteases from A. oryzae were applied as an enzyme additive in fish fed for digestibility studies. Phytases and proteases were produced with stable enzyme activity of 7,000 U.g-1 and 2,500 U.g-1, respectively. When those enzymes were applied in a plant protein based fish diet for digestibility studies, they increased protein, mineral, energy and lipids availability, showing that these new enzymes can improve animal production and performance. In conclusion, the substrate, as well as, the microorganism species can affect the biochemical character of the enzyme produced. Moreover, the production of these enzymes by SSF can be up to 90% cheaper than commercial ones produced with the same fungi species but submerged fermentation. Add to that these cheap enzymes can be easily applied as animal diet additives to improve production and performance.

Keywords: agricultural by-products, animal nutrition, enzymes production, solid state fermentation

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Authors: Rajeev Ravindran, Amit K. Jaiswal

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Waste from the food-processing industry is produced in large amount and contains high levels of lignocellulose. Due to continuous accumulation throughout the year in large quantities, it creates a major environmental problem worldwide. The chemical composition of these wastes (up to 75% of its composition is contributed by polysaccharide) makes it inexpensive raw material for the production of value-added products such as biofuel, bio-solvents, nanocrystalline cellulose and enzymes. In order to use lignocellulose as the raw material for the microbial fermentation, the substrate is subjected to enzymatic treatment, which leads to the release of reducing sugars such as glucose and xylose. However, the inherent properties of lignocellulose such as presence of lignin, pectin, acetyl groups and the presence of crystalline cellulose contribute to recalcitrance. This leads to poor sugar yields upon enzymatic hydrolysis of lignocellulose. A pre-treatment method is generally applied before enzymatic treatment of lignocellulose that essentially removes recalcitrant components in biomass through structural breakdown. Present study is carried out to find out the best pre-treatment method for the maximum liberation of reducing sugars from spent coffee waste (SPW). SPW was subjected to a range of physical, chemical and physico-chemical pre-treatment followed by a sequential, combinatorial pre-treatment strategy is also applied on to attain maximum sugar yield by combining two or more pre-treatments. All the pre-treated samples were analysed for total reducing sugar followed by identification and quantification of individual sugar by HPLC coupled with RI detector. Besides, generation of any inhibitory compounds such furfural, hydroxymethyl furfural (HMF) which can hinder microbial growth and enzyme activity is also monitored. Results showed that ultrasound treatment (31.06 mg/L) proved to be the best pre-treatment method based on total reducing content followed by dilute acid hydrolysis (10.03 mg/L) while galactose was found to be the major monosaccharide present in the pre-treated SPW. Finally, the results obtained from the study were used to design a sequential lignocellulose pre-treatment protocol to decrease the formation of enzyme inhibitors and increase sugar yield on enzymatic hydrolysis by employing cellulase-hemicellulase consortium. Sequential, combinatorial treatment was found better in terms of total reducing yield and low content of the inhibitory compounds formation, which could be due to the fact that this mode of pre-treatment combines several mild treatment methods rather than formulating a single one. It eliminates the need for a detoxification step and potential application in the valorisation of lignocellulosic food waste.

Keywords: lignocellulose, enzymatic hydrolysis, pre-treatment, ultrasound

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Authors: Nazish Badar

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In early 2009, Pandemic type A (H1N1) Influenza virus emerged globally. Since then, it has continued circulation causing considerable morbidity and mortality. The purpose of this study was to evaluate the evolutionary changes in Influenza A (H1N1) pdm09 viruses from 2009-15 and their relevance with the current vaccine viruses. Methods: Respiratory specimens were collected with influenza-like illness and Severe Acute Respiratory Illness. Samples were processed according to CDC protocol. Sequencing and phylogenetic analysis of Haemagglutinin (HA) and neuraminidase (NA) genes was carried out comparing representative isolates from Pakistan viruses. Results: Between Jan2009 - Feb 2016, 1870 (13.2%) samples were positive for influenza A out of 14086. During the pandemic period (2009–10), Influenza A/ H1N1pdm 09 was the dominant strain with 366 (45%) of total influenza positives. In the post-pandemic period (2011–2016), a total of 1066 (59.6%) cases were positive Influenza A/ H1N1pdm 09 with co-circulation of different Influenza A subtypes. Overall, the Pakistan A(H1N1) pdm09 viruses grouped in two genetic clades. Influenza A(H1N1)pdm09 viruses only ascribed to Clade 7 during the pandemic period whereas viruses belong to clade 7 (2011) and clade 6B (2015) during the post-pandemic years. Amino acid analysis of the HA gene revealed mutations at positions S220T, I338V and P100S specially associated with outbreaks in all the analyzed strains. Sequence analyses of post-pandemic A(H1N1)pdm09 viruses showed additional substitutions at antigenic sites; S179N,K180Q (SA), D185N, D239G (CA), S202A (SB) and at receptor binding sites; A13T, S200P when compared with pandemic period. Substitution at Genetic markers; A273T (69%), S200P/T (15%) and D239G (7.6%) associated with severity and E391K (69%) associated with virulence was identified in viruses isolated during 2015. Analysis of NA gene revealed outbreak markers; V106I (23%) among pandemic and N248D (100%) during post-pandemic Pakistan viruses. Additional N-Glycosylation site; HA S179N (23%), NA I23T(7.6%) and N44S (77%) in place of N386K(77%) were only found in post-pandemic viruses. All isolates showed histidine (H) at position 275 in NA indicating sensitivity to neuraminidase inhibitors. Conclusion: This study shows that the Influenza A(H1N1)pdm09 viruses from Pakistan clustered into two genetic clades, with co-circulation of some variants. Certain key substitutions in the receptor binding site and few changes indicative of virulence were also detected in post-pandemic strains. Therefore, it is imperative to continue monitoring of the viruses for early identification of potential variants of high virulence or emergence of drug-resistant variants.

Keywords: Influenza A (H1N1) pdm09, evolutionary analysis, post pandemic period, Pakistan

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Authors: Xi Chen, King-Yip Cheng

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Hypercholesterolemia, characterized by high low-density lipoprotein cholesterol (LDL-C), raises cardiovascular events in patients with type 2 diabetes (T2D). Although several drugs, such as statin and PCSK9 inhibitors, are available for the treatment of hypercholesterolemia, they exert detrimental effects on glucose metabolism and hence increase the risk of T2D. On the other hand, the drugs used to treat T2D have minimal effect on improving the lipid profile. Therefore, there is an urgent need to develop treatments that can simultaneously improve glucose and lipid homeostasis. Adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 2 (APPL2) causes insulin resistance in the liver and skeletal muscle via inhibiting insulin and adiponectin actions in animal models. Single-nucleotide polymorphisms in the APPL2 gene were associated with LDL-C, non-alcoholic fatty liver disease, and coronary artery disease in humans. The aim of this project is to investigate whether APPL2 antisense oligonucleotide (ASO) can alleviate dietary-induced T2D and hypercholesterolemia. High-fat diet (HFD) was used to induce obesity and insulin resistance in mice. GalNAc-conjugated APPL2 ASO (GalNAc-APPL2-ASO) was used to silence hepatic APPL2 expression in C57/BL6J mice selectively. Glucose, lipid, and energy metabolism were monitored. Immunoblotting and quantitative PCR analysis showed that GalNAc-APPL2-ASO treatment selectively reduced APPL2 expression in the liver instead of other tissues, like adipose tissues, kidneys, muscle, and heart. The glucose tolerance test and insulin sensitivity test revealed that GalNAc-APPL2-ASO improved glucose tolerance and insulin sensitivity progressively. Blood chemistry analysis revealed that the mice treated with GalNAc-APPL2-ASO had significantly lower circulating levels of total cholesterol and LDL cholesterol. However, there was no difference in circulating levels of high-density lipoprotein (HDL) cholesterol, triglyceride, and free fatty acid between the mice treated with GalNac-APPL2-ASO and GalNAc-Control-ASO. No obvious effect on food intake, body weight, and liver injury markers after GalNAc-APPL2-ASO treatment was found, supporting its tolerability and safety. We showed that selectively silencing hepatic APPL2 alleviated insulin resistance and hypercholesterolemia and improved energy metabolism in the dietary-induced obese mouse model, indicating APPL2 as a therapeutic target for metabolic diseases.

Keywords: APPL2, antisense oligonucleotide, hypercholesterolemia, type 2 diabetes

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Authors: A. Hamza, H. Kathyayini, N. Nagaraju

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Aluminophosphates, both amorphous and crystalline materials find applications as adsorbents, ceramics, and pigments and as catalysts/catalyst supports in organic fine chemical synthesis. Most of the applications are varied depending on the type of metal incorporated, particle size, surface area, porosity and morphology of aluminophosphate. The porous and surface properties of these materials are normally fine-tuned by adopting various preparation methodologies. Numerous crystalline microporous and mesoporous aluminophosphates and metal-aluminophosphates have been reported in literature, in which the synthesis has been carried out by using structure directing organic molecules/surfactants. In present work, amorphous aluminophosphate (AlP) and metal-aluminophosphates MAlP (M = Cu, Zn, Cr, Fe, Ce and Zr) and their mixed forms M-1M2AlP are prepared under a typical precipitation condition, i.e. at low temperature in order to keep the Von-Weirmann relative super saturation of the precipitating medium and obtain small size precipitate particles. These materials are prepared without using any surfactants. All materials are thoroughly characterised for surface and bulk properties by N2 adsorption-desorption technique, XRD, FT-IR, TG and SEM. The materials are also analysed for the amount and the strength of their surface acid sites, by NH3-TPD and CO2-TPD techniques respectively. All the materials prepared in the work are investigated for their catalytic activity in following applications in the synthesis of industrially important Jasminaldehyde via, aldol condensation of n-heptanal and benzaldehyde, in the synthesis of biologically important chalcones by Claisen-shmidth condensation of benzaldehyde and substituted chalcones. The effect of the amount of the catalysts, duration of the reaction, temperature of the reaction, molar ratio of the reactants has been studied. The porosity of pure aluminophosphate is found to be changed significantly by the incorporation of transition metals during preparation of aluminophosphate. The pore size increased from microporous to mesoporous and finally to macroporous by following order of metals Cu = Zn < Cr < Ce < Fe = Zr. The change in surface area and porosity of double metal-aluminophosphates depended on the concentration of both the metals. The acidity of aluminophosphate is either increased or decreased which depended on the type and valence of metals loaded. A good number of basic sites are created in metal-aluminophosphates irrespective of the metals used. A maximum catalytic activity for synthesis of both jasminaldehyde and chalcone is obtained by FeAlP as catalysts; these materials are characterized by decreased strength and concentration of acidic sites with optimum level basic sites.

Keywords: amorphous metal-aluminophosphates, surface properties, acidic-basic properties, Aldol, Claisen-Shmidth condensation, jasminaldehyde, chalcone

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Authors: Buddha Ratna Shrestha, Jimmy Faivre, Xavier Banquy

Abstract:

By precisely controlling the molecular interactions between anti-wear macromolecules and bottle-brush lubricating molecules in the solution state, we obtained a fluid with excellent lubricating and wear protection capabilities. The reason for this synergistic behavior relies on the subtle interaction forces between the fluid components which allow the confined macromolecules to sustain high loads under shear without rupture. Our results provide rational guides to design such fluids for virtually any type of surfaces. The lowest friction coefficient and the maximum pressure that it can sustain is 5*10-3 and 2.5 MPa which is close to the physiological pressure. Lubricating and protecting surfaces against wear using liquid lubricants is a great technological challenge. Until now, wear protection was usually imparted by surface coatings involving complex chemical modifications of the surface while lubrication was provided by a lubricating fluid. Hence, we here research for a simple, effective and applicable solution to the above problem using surface force apparatus (SFA). SFA is a powerful technique with sub-angstrom resolution in distance and 10 nN/m resolution in interaction force while performing friction experiment. Thus, SFA is used to have the direct insight into interaction force, material and friction at interface. Also, we always know the exact contact area. From our experiments, we found that by precisely controlling the molecular interactions between anti-wear macromolecules and lubricating molecules, we obtained a fluid with excellent lubricating and wear protection capabilities. The reason for this synergistic behavior relies on the subtle interaction forces between the fluid components which allow the confined macromolecules to sustain high loads under shear without rupture. The lowest friction coefficient and the maximum pressure that it can sustain in our system is 5*10-3 and 2.5 GPA which is well above the physiological pressure. Our results provide rational guides to design such fluids for virtually any type of surfaces. Most importantly this process is simple, effective and applicable method of lubrication and protection as until now wear protection was usually imparted by surface coatings involving complex chemical modifications of the surface. Currently, the frictional data that are obtained while sliding the flat mica surfaces are compared and confirmed that a particular mixture of solution was found to surpass all other combination. So, further we would like to confirm that the lubricating and antiwear protection remains the same by performing the friction experiments in synthetic cartilages.

Keywords: bottle brush polymer, hyaluronic acid, lubrication, tribology

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Authors: I. Berger, N. Machour, F. Portet-Koltalo

Abstract:

Polycyclic aromatic hydrocarbons (PAHs) are toxic and carcinogenic pollutants produced in majority by incomplete combustion processes in industrialized and urbanized areas. After being emitted in atmosphere, these persistent contaminants are deposited to soils or sediments. Even if persistent, some can be partially degraded (photodegradation, biodegradation, chemical oxidation) and they lead to oxygenated metabolites (oxy-PAHs) which can be more toxic than their parent PAH. Oxy-PAHs are less measured than PAHs in sediments and this study aims to compare different analytical tools in order to extract and quantify a mixture of four hydroxylated PAHs (OH-PAHs) and four carbonyl PAHs (quinones) in sediments. Methodologies: Two analytical systems – HPLC with on-line UV and fluorescence detectors (HPLC-UV-FLD) and GC coupled to a mass spectrometer (GC-MS) – were compared to separate and quantify oxy-PAHs. Microwave assisted extraction (MAE) was optimized to extract oxy-PAHs from sediments. Results: First OH-PAHs and quinones were analyzed in HPLC with on-line UV and fluorimetric detectors. OH-PAHs were detected with the sensitive FLD, but the non-fluorescent quinones were detected with UV. The limits of detection (LOD)s obtained were in the range (2-3)×10-4 mg/L for OH-PAHs and (2-3)×10-3 mg/L for quinones. Second, even if GC-MS is not well adapted to the analysis of the thermodegradable OH-PAHs and quinones without any derivatization step, it was used because of the advantages of the detector in terms of identification and of GC in terms of efficiency. Without derivatization, only two of the four quinones were detected in the range 1-10 mg/L (LODs=0.3-1.2 mg/L) and LODs were neither very satisfying for the four OH-PAHs (0.18-0.6 mg/L). So two derivatization processes were optimized, comparing to literature: one for silylation of OH-PAHs, one for acetylation of quinones. Silylation using BSTFA/TCMS 99/1 was enhanced using a mixture of catalyst solvents (pyridine/ethyle acetate) and finding the appropriate reaction duration (5-60 minutes). Acetylation was optimized at different steps of the process, including the initial volume of compounds to derivatize, the added amounts of Zn (0.1-0.25 g), the nature of the derivatization product (acetic anhydride, heptafluorobutyric acid…) and the liquid/liquid extraction at the end of the process. After derivatization, LODs were decreased by a factor 3 for OH-PAHs and by a factor 4 for quinones, all the quinones being now detected. Thereafter, quinones and OH-PAHs were extracted from spiked sediments using microwave assisted extraction (MAE) followed by GC-MS analysis. Several mixtures of solvents of different volumes (10-25 mL) and using different extraction temperatures (80-120°C) were tested to obtain the best recovery yields. Satisfactory recoveries could be obtained for quinones (70-96%) and for OH-PAHs (70-104%). Temperature was a critical factor which had to be controlled to avoid oxy-PAHs degradation during the MAE extraction process. Conclusion: Even if MAE-GC-MS was satisfactory to analyze these oxy-PAHs, MAE optimization has to be carried on to obtain a most appropriate extraction solvent mixture, allowing a direct injection in the HPLC-UV-FLD system, which is more sensitive than GC-MS and does not necessitate a previous long derivatization step.

Keywords: derivatizations for GC-MS, microwave assisted extraction, on-line HPLC-UV-FLD, oxygenated PAHs, polluted sediments

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Authors: Soma Banerjee, Aamen Talukdar, Argha Mandal, Dipankar Chaudhuri

Abstract:

Japanese Encephalitis is a major infectious disease with nearly half the world’s population living in areas where it is prevalent. Currently, treatment for it involves only supportive care and symptom management through vaccination. Due to the lack of antiviral drugs against Japanese Encephalitis Virus (JEV), the quest for such agents remains a priority. For these reasons, simulation studies of drug targets against JEV are important. Towards this purpose, docking experiments of the kinase inhibitors were done against the chosen target NS3 helicase as it is a nucleoside binding protein. Previous efforts regarding computational drug design against JEV revealed some lead molecules by virtual screening using public domain software. To be more specific and accurate regarding finding leads, in this study a proprietary software Schrödinger-GLIDE has been used. Druggability of the pockets in the NS3 helicase crystal structure was first calculated by SITEMAP. Then the sites were screened according to compatibility with ATP. The site which is most compatible with ATP was selected as target. Virtual screening was performed by acquiring ligands from databases: KinaseSARfari, KinaseKnowledgebase and Published inhibitor Set using GLIDE. The 25 ligands with best docking scores from each database were re-docked in XP mode. Protein structure alignment of NS3 was performed using VAST against MMDB, and similar human proteins were docked to all the best scoring ligands. The low scoring ligands were chosen for further studies and the high scoring ligands were screened. Seventy-three ligands were listed as the best scoring ones after performing HTVS. Protein structure alignment of NS3 revealed 3 human proteins with RMSD values lesser than 2Å. Docking results with these three proteins revealed the inhibitors that can interfere and inhibit human proteins. Those inhibitors were screened. Among the ones left, those with docking scores worse than a threshold value were also removed to get the final hits. Analysis of the docked complexes through 2D interaction diagrams revealed the amino acid residues that are essential for ligand binding within the active site. Interaction analysis will help to find a strongly interacting scaffold among the hits. This experiment yielded 21 hits with the best docking scores which could be investigated further for their drug like properties. Aside from getting suitable leads, specific NS3 helicase-inhibitor interactions were identified. Selection of Target modification strategies complementing docking methodologies which can result in choosing better lead compounds are in progress. Those enhanced leads can lead to better in vitro testing.

Keywords: antivirals, docking, glide, high-throughput virtual screening, Japanese encephalitis, ns3 helicase

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Authors: Valeria Dilcheva, Svetlozara Petkova, Ivelin Vladov

Abstract:

Nemathodes of the genus Trichinella are zoonotic parasites with a cosmopolitan distribution. More than 100 species of mammals, birds and reptiles are involved in the natural cycle of this nematode. At present, T. spiralis, T. pseudospiralis, and T. britovi have been found in Bulgaria. The existence of natural wildlife and domestic reservoirs of Trichinella spp. can be a serious threat to human health. Three trichinella isolates caused human trichinella infection outbreaks from three regions of Sofia City Province were used for the research: sample No. 1 - Ratus norvegicus, sample No. 2 – domestic pig (Sus scrofa domestica), sample No. 3 - domestic pig (Sus scrofa domestica). Trichinella larvae of the studied species were isolated via digestive method (pepsin, hydrochloric acid, water) at 37ºC by standard procedure and were determined by gender (male and female) based on their morphological characteristics. As a reference trichinella species were used: T. spiralis, T. pseudospiralis, T. nativa and T. britovi. Single male and female larvae of the three isolates were crossed with single male and female larvae of the reference trichinella species as well as reciprocally. As a result of cross-breeding, offspring of muscular larvae with T. spiralis and T. britovi were obtained, while in experiments with T. pseudospiralis and T. nativa, trichinella larvae were not found in the laboratory mice. The results obtained in the control groups indicate that the trichinella larvae used from the isolates and the four trichinella species are infective. Also, the infective ability of the F1 offspring from the successful cross-breeding between isolates and reference species was investigated. Through the data obtained in the experiment was found that isolates No. 1 and No. 2 belong to the species T. spiralis, and isolate No. 3 belongs to the species T. britovi. The results were confirmed by PCR and real-time PCR analysis. Thus the presence and circulation of the species T. spiralis and T. britovi in Bulgaria was confirmed. Probably the rodents (rats) are involved in the distribution of T. spiralis in urban environment. The species T. britovi found in a domestic pig speaks of some contact with wild animals for which T. britovi is characteristic. The probable reason is that a large number of farmers in Bulgaria practice the free-range breeding of domestic pigs. Part of the farmers also used as food for domestic pigs waste products from the game (foxes, jackals, bears, wolves) and probably thus the infection was obtained. The distribution range of trichinella species in Bulgaria is not strictly outlined. It is believed that T. spiralis is most common in domestic animals and T. britovi and T. pseudospiralis are characteristic of wildlife. To answer the question whether wild and synanthropic animals are infected with the same or different trichinella species, which species predominate in nature and what their distribution among different hosts is, further research is required.

Keywords: cross-breeding, Sofia, trichinellosis, Trichinella britovi, Trichinella spiralis

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Authors: Agnieszka Sobczak-Kupiec, Dagmara Malina, Klaudia Pluta, Wioletta Florkiewicz, Bozena Tyliszczak

Abstract:

Introduction: Request for compound of phosphorus grows continuously, thus, it is searched for alternative sources of this element. One of these sources could be by-products from meat industry which contain prominent quantity of phosphorus compounds. Hydroxyapatite, which is natural component of animal and human bones, is leading material applied in bone surgery and also in stomatology. This is material, which is biocompatible, bioactive and osteoinductive. Methodology: Hydroxyapatite preparation: As a raw material was applied deproteinized and defatted bone pulp called bone sludge, which was formed as waste in deproteinization process of bones, in which a protein hydrolysate was the main product. Hydroxyapatite was received in calcining process in chamber kiln with electric heating in air atmosphere in two stages. In the first stage, material was calcining in temperature 600°C within 3 hours. In the next stage unified material was calcining in three different temperatures (750°C, 850°C and 950°C) keeping material in maximum temperature within 3.0 hours. Bone sludge: Bone sludge was formed as waste in deproteinization process of bones, in which a protein hydrolysate was the main product. Pork bones coming from the partition of meat were used as a raw material for the production of the protein hydrolysate. After disintegration, a mixture of bone pulp and water with a small amount of lactic acid was boiled at temperature 130-135°C and under pressure4 bar. After 3-3.5 hours boiled-out bones were separated on a sieve, and the solution of protein-fat hydrolysate got into a decanter, where bone sludge was separated from it. Results of the study: The phase composition was analyzed by roentgenographic method. Hydroxyapatite was the only crystalline phase observed in all the calcining products. XRD investigation was shown that crystallization degree of hydroxyapatite was increased with calcining temperature. Conclusion: The researches were shown that phosphorus content is around 12%, whereas, calcium content amounts to 28% on average. The conducted researches on bone-waste calcining at the temperatures of 750-950°C confirmed that thermal utilization of deproteinized bone-waste was possible. X-ray investigations were confirmed that hydroxyapatite is the main component of calcining products, and also XRD investigation was shown that crystallization degree of hydroxyapatite was increased with calcining temperature. Contents of calcium and phosphorus were distinctly increased with calcining temperature, whereas contents of phosphorus soluble in acids were decreased. It could be connected with higher crystallization degree of material received in higher temperatures and its stable structure. Acknowledgements: “The authors would like to thank the The National Centre for Research and Development (Grant no: LIDER//037/481/L-5/13/NCBR/2014) for providing financial support to this project”.

Keywords: bone by-products, bone sludge, calcination, hydroxyapatite

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