Search results for: anions of carboxylic acids

Authors: Muhammad Zahid, Ilker S. Bayer, Athanassia Athanassiou

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Fluorinated polymers having C8 chemistry (chemicals with 8 fluorinated carbon atoms) are well renowned for their excellent low surface tension and water repelling properties. However, these polymers degrade into highly toxic heavy perfluoro acids in the environment. When the C8 chemistry is reduced to C6 chemistry, this environmental concern is eliminated at the expense of reduced liquid repellent performance. In order to circumvent this, in this study, we demonstrate pre-treatment of woven cotton fabrics with a fluorinated acrylic copolymer with C6 chemistry and subsequently with a silicone polymer to render them hydrophobic. A commercial fluorinated acrylic copolymer was blended with silica nanoparticles to form hydrophobic nano-roughness on cotton fibers and a second coating layer of polydimethylsiloxane (PDMS) was applied on the fabric. A static water contact angle (for 5µl) and rolling angle (for 12.5µl) of 147°±2° and 31° were observed, respectively. Hydrostatic head measurements were also performed to better understand the performance with 26±1 cm and 2.56kPa column height and static pressure respectively. Fabrication methods (with rod coater etc.) were kept simple, reproducible, and scalable and cost efficient. Moreover, the robustness of applied coatings was also evaluated by sonication cleaning and abrasion methods. Water contact angle (WCA), water shedding angle (WSA), hydrostatic head, droplet bouncing-rolling off and prolonged staining tests were used to characterize hydrophobicity of materials. For chemical and morphological analysis, various characterization methods were used such as attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), atomic force microscopy (AFM) and scanning electron microscopy (SEM).

Keywords: fluorinated polymer, hydrophobic, polydimethylsiloxane, water contact angle

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Authors: Kyung Hwa Hong, Eunmi Koh

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Wool and cotton fabrics are pretreated by a tannic acid aqueous solution to increase their dyeability and then dyed by Purple-Fleshed Sweet Potato (PSP) extract. The dyed fabrics are then investigated by various analysis techniques. The results revealed that wool and cotton fabrics can be dyed bluish red through the pretreatment and dyeing process. Both wool and cotton fabrics only pretreated with tannic acid display decreased L* value but no significant changes in a* and b* values as the concentration of tannic acid increases. And, as expected, the pretreated fabrics are even darker and show a richer purple color after the dyeing process with the PSP extract. With regard to the colorfastness of wool and cotton fabrics dyed by PSP extract in cleaning circumstances, such as dry-cleaning (for wool) and washing (for cotton), the wool and cotton fabrics had a 4.0 and 4.0 grade of colorfastness to dry-cleaning and washing, respectively. However, they both exhibited significantly inferior colorfastness to light (grade of 1.5). Thus, it was found that there is still a need for improvement with regard to color fastness, particularly against light. On the other hand, the wool and cotton fabrics also showed antibacterial and antioxidant characteristics. In addition, both the wool and cotton fabrics showed potential antibacterial ability (>99%) against Staphylococcus aureus; however, they showed somewhat insufficient antibacterial ability (60.8% for wool and 94.8% for cotton) against Klebsiella pneumoniae. Also, their antioxidant abilities increased up to ca. 90% with an increase in the tannic acid concentration (up to 0.5%). However, after the dyeing process, the antibacterial and antioxidant ability tended to decrease. This is assumed to have occurred because functional moieties such as phenolic acids were detached from the pretreated fabrics into the hot water (the dyeing solution) during the dyeing process. Therefore, further study would be necessary to derive the optimum treatment and dyeing conditions so as to maximize the coloring effect and functionalities of the fabrics.

Keywords: antibacterial activity, antioxidant activity, purple-fleshed sweet potato, fabrics

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Authors: Sara Arafeh, Kovriguine Evguine

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Cytochromes P450 are enzymes that require a supply of electrons to catalyze the synthesis of steroid hormones, fatty acids, and prostaglandin hormone. Cytochrome P450 Oxidoreductase (CYPOR), a membrane bound enzyme, provides these electrons in its open conformation. CYPOR has two cytosolic domains (FAD domain and FMN domain) and an N-terminal in the membrane. In its open conformation, electrons flow from NADPH, FAD, and finally to FMN where cytochrome P450 picks up these electrons. In the closed conformation, cytochrome P450 does not bind to the FMN domain to take the electrons. It was found that when the cytosolic domains are isolated, CYPOR could not bind to cytochrome P450. This suggested that the membrane environment is important for CYPOR function. This project takes the initiative to better understand the dynamics of CYPOR in its full length. Here, we determine the distance between specific sites in the FAD and FMN binding domains in CYPOR by Forster Resonance Energy Transfer (FRET) and Ultrafast TA spectroscopy with and without NADPH. The approach to determine these distances will rely on labeling these sites with red and infrared fluorophores. Mimic membrane attachment is done by inserting CYPOR in lipid nanodiscs. By determining the distances between the donor-acceptor sites in these domains, we can observe the open/closed conformations upon reducing CYPOR in the presence and absence of cytochrome P450. Such study is important to better understand CYPOR mechanism of action in various endosomal membranes including hepatic CYPOR which is vital in plasma cholesterol homeostasis. By investigating the conformational cycles of CYPOR, we can synthesize drugs that would be more efficient in affecting the steroid hormonal levels and metabolism of toxins catalyzed by Cytochrome P450.

Keywords: conformational cycle of CYPOR, cytochrome P450, cytochrome P450 oxidoreductase, FAD domain, FMN domain, FRET, Ultrafast TA Spectroscopy

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Authors: Agatha Swasti Ayuning Tyas

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Hyperglycemia in Diabetes Mellitus (DM) is an important factor in cellular and vascular damage, which is caused by activation of C Protein Kinase, polyol and hexosamine track, and production of Advanced Glycation End-Products (AGE). Those mentioned before causes the accumulation of Reactive Oxygen Species (ROS). Oxidative stress increases the expression of proinflammatory factors IL-6 as one of many signs of endothelial disfunction. Genistein in soy milk has a high immunomodulator potential. Goat milk contains amino acids which have antioxidative potential. Fermented kefir has an anti-inflammatory activity which believed will also contribute in potentiating goat milk and soy milk. This study is a quasi-experimental posttest-only research to 30 Wistar mice. This study compared the levels of IL-6 between healthy Wistar mice group (G1) and 4 DM Wistar mice with intervention and grouped as follows: mice without treatment (G2), mice treated with 100% goat milk kefir (G3), mice treated with combination of 50% goat milk kefir and 50% soy milk kefir (G4), and mice treated with 100% soy milk kefir (G5). DM animal models were induced with Streptozotocin & Nicotinamide to achieve hyperglycemic condition. Goat milk kefir and soy milk kefir are given at a dose of 2 mL/kg body weight/day for four weeks to intervention groups. Blood glucose was analyzed by the GOD-POD principle. IL-6 was analyzed by enzyme-linked sandwich ELISA. The level of IL-6 in DM untreated control group (G2) showed a significant difference from the group treated with the combination of 50% goat milk kefir and 50% soy milk kefir (G3) (p=0,006) and the group treated with 100% soy milk kefir (G5) (p=0,009). Whereas the difference of IL-6 in group treated with 100% goat milk kefir (G3) was not significant (p=0,131). There is also synergism between glucose level and IL-6 in intervention groups treated with combination of 50% goat milk kefir and 50% soy milk kefir (G3) and the group treated with 100% soy milk kefir (G5). Combination of 50 % goat milk kefir and 50% soy milk kefir and administration of 100% soy milk kefir alone can control the level of IL-6 remained low in DM Wistar mice induced with streptozocin and nicotinamide.

Keywords: diabetes mellitus, goat milk kefir, soy milk kefir, interleukin 6

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Authors: Maria L. G. Lourenco, Viviane Y. Hibaru, Keylla H. N. P. Pereira, Fabiana F. Souza, Joao C. P. Ferreira, Simone B. Chiacchio, Luiz H. A. Machado

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Neonatal conjunctivitis, or ophthalmia, is an infection of the conjunctiva or cornea before opening the eyelids. It is believed that immunodeficiency contributes to the development of the condition. This study aims at reporting a case of ophthalmia neonatorum in a dog, in addition to its diagnosis and treatment. A litter of five pug neonates was admitted to the Sao Paulo State University (UNESP) Veterinary Hospital, Botucatu, Sao Paulo, Brazil, with complaints of ocular secretion. The neonates were five days old. The clinical examination revealed that three newborns presented swelling in the ocular region and a purulent secretion in the medial corner of the eye that was exerting pressure on the ocular globes, which are compatible with the description of this disease. The diagnosis was made based on the clinical signs and bacterial culture of the secretion, which revealed the presence of bacteria belonging to the genus Staphylococcus sp. The laboratory assays did not reveal any alterations. The treatment was instituted gently, opening the eyelids early and cleaning the purulent ocular secretion with saline solution. An ophthalmic ointment with retinol, amino acids, methionine, and chloramphenicol (Epitezan®) was prescribed four times a day for seven days. Blood plasma (2 mL/100 g) was administered subcutaneously because bacterial infections in neonates may represent a failure in the transference of passive immunity. A more thorough cleaning of the environment was also recommended. Neonatal conjunctivitis has a simple diagnosis and treatment. If not treated early, it can evolve to adherence of the eyelids to the cornea, ulceration, and perforation of the cornea. Therefore, the prognosis is favorable as long as the condition is diagnosed early, and the treatment is instituted quickly.

Keywords: ophthalmia neonatorum, neonatal infection, puppy, newborn

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Authors: Leila Karshenas, Hamidreza Khodaei, Behnaz Mahdavi

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Ovulation is a physiologic process with an inflammatory response that depends on a coordinated activity of gonadotropins and steroid hormones, as well as inflammatory mediators such as cytokines, prostaglandins, leptin, nitric oxide (NO), etc. Conjugated linoleic acid (CLA) is composed of polyunsaturated fatty acids (PUFA) found in dairy products, beef and lamb. There is strong evidence that dietary CLA affects mediators involved in ovulation. The aim of this study was to determine the effects of different doses of dietary CLA on systemic and local hormones and factors involved in ovulation. In this case-control study, 80 (50±2-day old) female mice were randomly divided into four groups (C as the controls and T1, T2 and T3 as the treatment groups). There were four replicates in each group and there were five mice in every replicate (20 mice, in total). The mice in the control group were fed with no CLA in their diet but the ones in the treatment group received 0.1, 0.3 and 0.5g/kg of CLA (replacing corn oil in the diet), respectively for 120 days. Later on, blood samples were obtained from the tails of animals that displayed estrus signs and estradiol (E2), progesterone (P4), LH, FSH, NO, leptin and TNFα were measured. Furthermore, the effects of CLA on the ovarian production of prostaglandins (PGs) and NO were investigated. The data were analyzed by SAS software.CLA significantly decreased serum levels of FSH (p<0.05), LH, estradiol, NO, leptin and TNFα (p<0.01). In addition, CLA decreased progesterone levels but this effect was statistically insignificant. The significantly negative effects of CLA were seen on the ovarian production of PGE2 and PGF2α (p<0.01).It seems that CLA may play an effective role in reducing the ovulation rate in mice as CLA adversely affected female reproduction and it had negative effects on systemic and local hormones involved in ovulation.

Keywords: conjugated linoleic acid, nitric oxide, ovary, ovulation, prostaglandin, gonadotropin

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Authors: Abu Salim Mustafa

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Rv3873 is a relatively large size protein (371 amino acids in length) and its gene is located in the immunodominant genomic region of difference (RD)1 that is present in the genome of Mycobacterium tuberculosis but deleted from the genomes of all the vaccine strains of Bacillus Calmette Guerin (BCG) and most other mycobacteria. However, when tested for cellular immune responses using peripheral blood mononuclear cells from tuberculosis patients and BCG-vaccinated healthy subjects, this protein was found to be a major stimulator of cell mediated immune responses in both groups of subjects. In order to further identify the sequence of immunodominant epitopes and explore their Human Leukocyte Antigen (HLA)-restriction for epitope recognition, 24 peptides (25-mers overlapping with the neighboring peptides by 10 residues) covering the sequence of Rv3873 were synthesized chemically using fluorenylmethyloxycarbonyl chemistry and tested in cell mediated immune responses. The results of these experiments helped in the identification of an immunodominant peptide P9 that was recognized by people expressing varying HLA-DR types. Furthermore, it was also predicted to be a promiscuous binder with multiple epitopes for binding to HLA-DR, HLA-DP and HLA-DQ alleles of HLA-class II molecules that present antigens to T helper cells, and to HLA-class I molecules that present antigens to T cytotoxic cells. In addition, the evaluation of peptide P9 using an immunogenicity predictor server yielded a high score (0.94), which indicated a greater probability of this peptide to elicit a protective cellular immune response. In conclusion, P9, a peptide with multiple epitopes and ability to bind several HLA class I and class II molecules for presentation to cells of the cellular immune response, may be useful as a peptide-based vaccine against tuberculosis.

Keywords: mycobacterium tuberculosis, PPE68, peptides, vaccine

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Authors: Yixin Yan, Miao Yan, Irini Angelidaki, Ioannis Fotidis

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Ammonia, which derives from the degradation of urea and protein-substrates, is the major toxicant of the commercial anaerobic digestion reactors causing loses of up to 1/3 of their practical biogas production, which reflects directly on the overall revenue of the plants. The current experimental work is aiming to alleviate the ammonia inhibition in anaerobic digestion (AD) process by developing an innovative bioaugmentation method of ammonia tolerant methanogenic consortia. The ammonia tolerant consortia were cultured in batch reactors and immobilized together with biochar in agar (customized inocula). Three continuous stirred-tank reactors (CSTR), fed with the organic fraction of municipal solid waste at a hydraulic retention time of 15 days and operated at thermophilic (55°C) conditions were assessed. After an ammonia shock of 4 g NH4+-N L-1, the customized inocula were bioaugmented into the CSTR reactors to alleviate ammonia toxicity effect on AD process. Recovery rate of methane production and methanogenic activity will be assessed to evaluate the bioaugmentation performance, while 16s rRNA gene sequence will be used to reveal the difference of microbial community changes through bioaugmentation. At the microbial level, the microbial community structures of the four reactors will be analysed to find the mechanism of bioaugmentation. Changes in hydrogen formation potential will be used to predict direct interspecies electron transfer (DIET) between ammonia tolerant methanogens and syntrophic bacteria. This experimental work is expected to create bioaugmentation inocula that will be easy to obtain, transport, handled and bioaugment in AD reactors to efficiently alleviate the ammonia toxicity, without alternating any of the other operational parameters including the ammonia-rich feedstocks.

Keywords: artisanal fishing waste, acidogenesis, volatile fatty acids, pH, inoculum/substrate ratio

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Authors: Hongquan Zhang

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Inspired by endogenous protein motors, researchers have constructed various synthetic DNA motors based on the specificity and predictability of Watson-Crick base pairing. However, the application of DNA motors to signal amplification and biosensing is limited because of low mobility and difficulty in real-time monitoring of the walking process. The objective of our work was to construct a new type of DNA motor termed target-triggered DNA motors that can walk for hundreds of steps in response to a single target binding event. To improve the mobility and processivity of DNA motors, we used gold nanoparticles (AuNPs) as scaffolds to build high-density, three-dimensional tracks. Hundreds of track strands are conjugated to a single AuNP. To enable DNA motors to respond to specific protein and nucleic acid targets, we adapted the binding-induced DNA assembly into the design of the target-triggered DNA motors. In response to the binding of specific target molecules, DNA motors are activated to autonomously walk along AuNP, which is powered by a nicking endonuclease or DNAzyme-catalyzed cleavage of track strands. Each moving step restores the fluorescence of a dye molecule, enabling monitoring of the operation of DNA motors in real time. The motors can translate a single binding event into the generation of hundreds of oligonucleotides from a single nanoparticle. The motors have been applied to amplify the detection of proteins and nucleic acids in test tubes and live cells. The motors were able to detect low pM concentrations of specific protein and nucleic acid targets in homogeneous solutions without the need for separation. Target-triggered DNA motors are significant for broadening applications of DNA motors to molecular sensing, cell imagining, molecular interaction monitoring, and controlled delivery and release of therapeutics.

Keywords: biosensing, DNA motors, gold nanoparticles, signal amplification

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Authors: Muhammad Iqbal, Hafiz Muhammad Tauqeer, Hamza Rafaqat, Muhammad Naveed, Muhammad Awais Irshad

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Soil pollution with chromium (Cr) has become one of the most important concerns due to its toxicity for humans. To date, various remediation approaches have been employed for the remediation and management of Cr contaminated soils. Phytoextraction is an eco-friendly and emerging remediation approach which has gained attention due to several advantages over conventional remediation approach. The use of energy crops for phytoremediation is an emerging trend worldwide. These energy crops have high tolerance against various environmental stresses, the potential to grow in diverse ecosystems and high biomass production make them a suitable candidate for phytoremediation of contaminated soils. The removal efficiency of plants in phytoextraction depends upon several soil and plant factors including solubility, bioavailability and metal speciation in soils. A pot scale experiment was conducted to evaluate the phytoextraction potential of Arundo donax L. with the application of acetic acid (A.A) in Cr contaminated soils. Plants were grown in pots filled with 5 kg soils for 90 days. After 30 days plants acclimatization in pot conditions, plants were treated with various levels of Cr (2.5 mM, 5 mM, 7.5 mM, 10 mM) and A.A (Cr 2.5 mM + A.A 2.5 mM, Cr 5 mM + A.A 2.5 mM, Cr 7.5 mM + A.A 2.5 mM, Cr 10 mM + A.A 2.5 mM). The application of A.A significantly increased metal uptake and in roots and shoots of A. donax. This increase was observed at Cr 7.5 mM + A.A 2.5 mM but at high concentrations, visual symptoms of Cr toxicity were observed on leaves. Similarly, A.A applications also affect the activities of key enzymes including catalase (CAT), superoxidase dismutase (SOD), and ascorbate peroxidase (APX) in leaves of A. donax. Based on results it is concluded that the applications of A.A acid for phytoextraction is an alternative approach for the management of Cr affected soils and synthetic chelators should be replaced with organic acids.

Keywords: acetic acid, A. donax, chromium, energy crop, phytoextraction

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Authors: Mariame Taibi, Marouane Aouiji, Rachid Bengueddour

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The Moroccan coastline is particularly rich in algae and constitutes a reserve of species with considerable economic, social and ecological potential. This work focuses on the research and characterization of algae bioactive compounds that can be used in pharmacology or phytopathology. The biochemical composition of the green alga Ulva lactuca (Ulvophyceae) was studied by determining the content of moisture, ash, phenols, flavonoids, total tannins, and chlorophyll. Seven solvents: distilled water, methanol, ethyl acetate, chloroform, benzene, petroleum ether, and hexane, were tested for their effectiveness in recovering chemical compounds. The identification of functional groupings, as well as the bioactive chemical compounds, was determined by FT-IR and GC-MS. The moisture content of the alga was 77%, while the ash content was 15%. Phenol content differed from one solvent studied to another, while chlorophyll a, b, and total chlorophyll were determined at 14%, 9.52%, and 25%, respectively. Carotenoid was present in a considerable amount (8.17%). The experimental results show that methanol is the most effective solvent for recovering bioactive compounds, followed by water. Moreover, the green alga Ulva lactuca is characterized by a high level of total polyphenols (45±3.24 mg GAE/gDM), average levels of total tannins and flavonoids (22.52±8.23 mg CE/gDM, 15.49±0.064 mg QE/gDM) respectively. The results of Fourier transform infrared spectroscopy (FT-IR) confirmed the presence of alcohol/phenol and amide functions in Ulva lactuca. The GC-MS analysis gave precisely the compounds contained in the various extracts, such as phenolic compounds, fatty acids, terpenoids, alcohols, alkanes, hydrocarbons, and steroids. All these results represent only a first step in the search for biologically active natural substances from seaweed. Additional tests are envisaged to confirm the bioactivity of seaweed.

Keywords: algae, Ulva lactuca, phenolic compounds, FTIR, GC-MS

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Authors: Yu. Mateyshina, A. Ulihin, N. Uvarov

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Supercapacitors are one of the most promising devices for energy storage applications as they can provide higher power density than batteries and higher energy density than conventional dielectric capacitors. Carbon materials with various microtextures are considered as main candidates for supercapacitors in terms of high surface area, interconnected pore structure, controlled pore size, high electrical conductivity and environmental friendliness. The specific capacitance (C) of the electrode material of the Electrochemical Double Layer Capacitors (EDLC) is known to depend on the specific surface area (Ss) and the pore structure. Activated carbons are most commonly used in supercapacitors because of their high surface area (Ss ≥ 1000 m2/g), good adhesion to electrolytes and low cost. In this work, electrochemical properties of new microporous and mesoporous carbon electrode materials were studied. The aim of the work was to investigate the relationship between the specific capacitance and specific surface area in a series of materials prepared from different organic precursors.. As supporting matrixes different carbon samples with Ss = 100-2000 m2/g were used. The materials were modified by treatment in acids (H2SO4, HNO3, acetic acid) in order to enable surface hydrophilicity. Then nanoparticles of transition metal oxides (for example NiO) were deposited on the carbon surfaces using methods of salts impregnation, mechanical treatment in ball mills and the precursors decomposition. The electrochemical characteristics of electrode hybrid materials were investigated in a symmetrical two-electrode cell using an impedance spectroscopy, voltammetry in both potentiodynamic and galvanostatic modes. It was shown that the value of C for the materials under study strongly depended on the preparation method of the electrode and the type of electrolyte (1 M H2SO4, 6 M KOH, 1 M LiClO4 in acetonitryl). Specific capacity may be increased by the introduction of nanoparticles from 50-100 F/g for initial carbon materials to 150-300 F/g for nanocomposites which may be used in supercapacitors. The work is supported by the по SC-14.604.21.0013.

Keywords: supercapacitors, carbon electrode, mesoporous carbon, electrochemistry

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Authors: Tabussam Tufail

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Purpose: The core purpose of the current review article is to elaborate the phytochemicals present in turnip (brassica rapus l.) and also allied health claims. Plant-based foods contain a significant amount of bioactive compounds which provide desirable health benefits beyond the basic nutrition. Epidemiological evidence suggests that consumption of a diet rich in vegetables and fruits has positive implications for human health. Design: Potential of turnip peroxidase (TP) for the treatment of phenolic-contaminated solutions has been reviewed. However, issues of taste along with behavioral nutrition ought to be considered. So in the last decades, special attention has been paid towards edible plants, especially those that are rich in secondary metabolites (frequently called phytochemicals) and nowadays, there is an increasing interest in the antioxidant activity of such phytochemicals present in the diet. These chemicals favor nutritional and phytotherapy that is emerging as new concepts of health aid in recent years. Turnip is rich in these valuable ingredients though it can be employed as having health promoting and healing properties. Findings: Numerous bioactive components i.e. organic acids, phenolic compounds, turnip peroxidase, kaempeferol, vitamin-K, etc. are present in turnip. The review focused on the significance of plant derived (especially turnip) phenolic compounds as a source of certain beneficial compounds for human health. Owing to the presence of bioactive moieties, the turnip has high antioxidant activity, positive role in blood clotting, effectual in phenobarbital-induced sleeping time, effective against hepatic injury in diabetics and also have a good hepatoprotective role. Strong recommendations for consumption of nutraceuticals from turnip have become progressively popular to improve health, and to prevent from diseases.

Keywords: phytochemicals, turnip, antioxidants, health benefits

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Authors: G. Wojcik, J. Gindlhuber, A. Syarif, K. Hoetzenecker, P. Bohm, P. Vesely, V. Biasin, G. Kwapiszewska

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Pulmonary fibrosis is a rare disease where uncontrolled wound healing processes damage the lung structure. Intensive changes within the extracellular matrix (ECM) and its interaction with fibroblasts have a major role in pulmonary fibrosis development. Among others, collagen is one of the main components of the ECM, and it is important for lung structure. In IPF, constant production of collagen by fibroblast, through TGFβ1-SMAD2/3 pathways, leads to an uncontrolled deposition of matrix and hence lung remodeling. Abnormal changes in lipid production, alterations in fatty acids (FAs) metabolism, enhanced oxidative stress, and lipid peroxidation in fibrotic lung and fibrotic fibroblasts have been reported; however, the interplay between the collagen and lipids is not yet established. One of the FAs influx regulators is Angiopoietin-like 4 (ANGPTL4), which inhibits lipoprotein lipase work, decreasing the availability of FAs. We hypothesized that altered lipid composition or availability could have the capability to influence the phenotype of different fibroblast populations in the lung and hence influence lung fibrosis. To prove our hypothesis, we aim to investigate lipids and their influence on human, animal, and in vitro levels. In the bleomycin model, treatment with the well-known metabolic drugs Rosiglitazone or Metformin significantly lower collagen production. Similar results were noticed in ANGPTL4 KO animals, where the KO of ANGPTL4 leads to an increase of FAs availability and lower collagen deposition after the bleomycin challenge. Currently, we study the treatment of different FAs on human lung para fibroblasts (hPF) isolated from donors. To understand the lipid composition, we are collecting human lung tissue from donors and pulmonary fibrosis patients for Liquid chromatography-mass spectrometry. In conclusion, our results suggest the lipid influence on collagen deposition during lung fibrosis, but further research needs to be conducted to understand the matter of this relationship.

Keywords: collagen, fibroblasts, lipidomics, lung, pulmonary fibrosis

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Authors: Ruphi Naz, Altaf Ahmad, Mohammad Anis

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Sialic acid (5-Acetylneuraminic acid, Neu5Ac) is a common sugar found as a terminal residue on glycoconjugates in many animals. Humans brain and the central nervous system contain the highest concentration of sialic acid (as N-acetylneuraminic acid) where these acids play an important role in neural transmission and ganglioside structure in synaptogenesis. Due to its important biological function, sialic acid is attracting increasing attention. To understand metabolic networks, fluxes and regulation, it is essential to be able to determine the cellular and subcellular levels of metabolites. Genetically-encoded fluorescence resonance energy transfer (FRET) sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. Taking this, we developed a genetically encoded FRET (fluorescence resonance energy transfer) based nanosensor to analyse the sialic acid level in living cells. Sialic acid periplasmic binding protein (sia P) from Haemophilus influenzae was taken and ligated between the FRET pair, the cyan fluorescent protein (eCFP) and Venus. The chimeric sensor protein was expressed in E. coli BL21 (DE3) and purified by affinity chromatography. Conformational changes in the binding protein clearly confirmed the changes in FRET efficiency. So any change in the concentration of sialic acid is associated with the change in FRET ratio. This sensor is very specific to sialic acid and found stable with the different range of pH. This nanosensor successfully reported the intracellular level of sialic acid in bacterial cell. The data suggest that the nanosensors may be a versatile tool for studying the in vivo dynamics of sialic acid level non-invasively in living cells

Keywords: nanosensor, FRET, Haemophilus influenzae, metabolic networks

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Authors: Ouchemoukh Salim, Amessis-Ouchemoukh Nadia, Guenaoui Nawel, Moumeni Lynda, Zaidi Hicham, Otmani Amar, Sadou Dyhia

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Honey is a hive food rich in carbohydrates and water and it also has a lot of nutrients (enzymes, minerals, organic acids, phytochemicals...). It is used in different nutritional and therapeutic fields. Algerian honey was studied for its physicochemical parameters, nutritional values (moisture, brix, pH, electrical conductivity, and amounts of HMF, proteins, proline, total phenolic compounds and flavonoids) and some biological activities (antioxidant, anti-inflammatory and enzymatic anti-browning). The antioxidant activities of the samples were estimated using different methods (ABTS, DPPH free radicals scavenging, reducing power, and chelating ferrous activity). All honeys were acidic (3.45≤pH≤4.65). The color varied from mimosa yellow to dark brown. The specific rotation was levorotatory in most honey samples, and the electrical conductivity, hydroxymethylfurfural, and proline values agreed with the international honey requirements. For anti-inflammatory activity, the results showed that the inhibiting capacity of the denaturation of the BSA of the honey analyzed varied from 15 to 75 % with a maximum of activity at the concentration of 0,5 mg/ml. All honey exhibited enzymatic anti-browning on different slices of fruits. In fact, the results showed that the controls have the greatest browning unit compared to the honeys studied and PPO and POD enzymes had the lowest enzyme activity. High significant correlations were found between the color of honey, its antioxidant content and its biological activities (antioxidant, anti-inflammatory and enzymatic anti-browning). The dark color of honey is a good indicator of the best biological properties, therefore, the best nutritional and therapeutic values.

Keywords: honey, physico-chemical parameters, bioactive compounds, biological properties

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Authors: Malihe Moeinin, Robert Hill, Ferranti Wong

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Background: A white spot lesion (WSL) is defined as subsurface enamel porosity from carious demineralisation on the smooth surfaces of the tooth. It appears as a milky white opacity. Lesions shown an apparently intact surface layer, followed underneath by the more porous lesion body. The small pores within the body of the lesion act as diffusion pathway for both acids and minerals, so allowing the demineralisation of enamel to occur at the advancing front of the lesion. Objectives: The objective is to mapthe porosity and its size on WSL with Focused Ion Bean- Scanning Electron Microscopy (FIB-SEM) Method: The basic method used for FIB-SEM consisted of depositing a one micron thick layer of platinum over 25μmx 25μm of the interest region of enamel. Then, making a rough cut (25μmx 5μmx 20μm) with 3nA current and 30Kv was applied with the help of drift suppression (DS), using a standard “cross-sectional” cutting pattern, which ended at the front of the deposited platinum layer. Two adjacent areas (25μmx 5μmx 20μm) on the both sides of the platinum layer were milled under the same conditions. Subsequent, cleaning cross-sections were applied to polish the sub-surface edge of interest running perpendicular to the surface. The "slice and view" was carried out overnight for milling almost 700 slices with 2Kv and 4nA and taking backscattered (BS) images. Then, images were imported into imageJ and analysed. Results: The prism structure is clearly apparent on FIB-SEM slices of WSL with the dissolution of prism boundaries as well as internal porosity within the prism itself. Porosity scales roughly 100-400nm, which is comparable to the light wavelength (500nm). Conclusion: FIB-SEM is useful to characterize the porosity of WSL and it clearly shows the difference between WSL and normal enamel.

Keywords: white spot lesion, FIB-SEM, enamel porosity, porosity

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Authors: Fatima Majeed Almonajim

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Panucum miliaceum L is a plant from the genus Gramineae, In the world, millets are regarded as a significant grain, however, they are very little exploited. Millet grain is abundant in nutrients and health-beneficial phenolic compounds, making it suitable as food and feed. The plant has received considerable attention for its high content of phenolic compounds, low glycemic index, the presence of unsaturated fats and lack of gluten which are beneficial to human health, and thus, have made the plant being effective in treating celiac disease, diabetes, lowering blood lipids (cholesterol) and preventing tumors. Moreover, the plant requires little water to grow, a property that is worth considering. This study provides an overview of the nutritional and health benefits provided by millet types grown in 2 areas Iraq and Iran, aiming to compare the effect of climate on the components of millet. In this research, millet samples collected from the both Babylon (Iraqi) and Isfahan (Iranian) types were extracted and after HPTLC, the resulted pattern of the two samples were compared. As a result, the Iranian millet showed more terpenoid compounds than Iraqi millet, and therefore, Iranian millet has a higher priority than Iraqi millet in increasing the human body's immunity. On the other hand, in view of the number of essential amino acids, the Iraqi millet contains more nutritional value compared to the Iranian millet. Also, due to the higher amount of histidine in the Iranian millet, compiled to the lack of gluten found from previous studies, we came to the conclusion that the addition of millet in the diet of children, more specifically those children with irritable bowel syndrome, can be considered beneficial. Therefore, as a component of dairy products, millet can be used in preparing food for children such as dry milk.

Keywords: HPTLC, phytochemicals, specialty foods, Panucum miliaceum L, nutrition

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Authors: Valeriy M. Kondratev, Ekaterina A. Vyacheslavova, Talgat Shugabaev, Alexander S. Gudovskikh, Alexey D. Bolshakov

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Modern sensorics imposes strict requirements on the biosensors characteristics, especially technological feasibility, and selectivity. There is a growing interest in the analysis of human health biological markers, which indirectly testifying the pathological processes in the body. Such markers are acids and alkalis produced by the human, in particular - ammonia and hydrochloric acid, which are found in human sweat, blood, and urine, as well as in gastric juice. Biosensors based on modern nanomaterials, especially low dimensional, can be used for this markers detection. Most classical adsorption sensors based on metal and silicon oxides are considered non-selective, because they identically change their electrical resistance (or impedance) under the action of adsorption of different target analytes. This work demonstrates a feasible frequency-resistive method of electrical impedance spectroscopy data analysis. The approach allows to obtain of selectivity in adsorption sensors of a resistive type. The method potential is demonstrated with analyzis of impedance spectra of silicon nanowires in the presence of NH3 and HCl vapors with concentrations of about 125 mmol/L (2 ppm) and water vapor. We demonstrate the possibility of unambiguous distinction of the sensory signal from NH3 and HCl adsorption. Moreover, the method is found applicable for analysis of the composition of ammonia and hydrochloric acid vapors mixture without water cross-sensitivity. Presented silicon sensor can be used to find diseases of the gastrointestinal tract by the qualitative and quantitative detection of ammonia and hydrochloric acid content in biological samples. The method of data analysis can be directly translated to other nanomaterials to analyze their applicability in the field of biosensory.

Keywords: electrical impedance spectroscopy, spectroscopy data analysis, selective adsorption sensor, nanotechnology

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Authors: Yasir Arafat, Asma Shah, Hua Shao

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Background and Aims: Legume/cereal intercropping is used worldwide for enhancement in biomass and yield of cereal crops. However, because of intercropping, the belowground biological and chemical interactions and their effect on physiological parameters and yield of crops are limited. Methods: Wheat faba bean (WF) intercropping was designed to understand the underlying changes in the soil's chemical environment, soil microbial communities, and effect on growth and yield parameters. Experimental plots were established as having no root partition (NRP), semi-root partition (SRP), complete root partition (CRP), and their sole cropping (CK). Low molecular weight organic acids (LMWOAs) were determined by GC-MS, and high throughput sequencing of the 16S rRNA gene was carried out to screen microbial structure and composition in different root partitions of the WF intercropping system. Results: We show that intercropping induced a shift in the relative abundance of some genera of plant growth promoting rhizobacteria (PGPR) such as Allorhizobium, Neorhizobium, Pararhizobium, and Rhizobium species and resulted in better growth and yield performance of wheat. Moreover, as the plant's distance of wheat from faba beans decreased, the diversity of microbes increased, and a positive effect was observed on physiological traits and crop yield. Furthermore, an abundance and positive correlations of palmitic acid, arachidic acid, stearic acid, and 9-Octadecenoic with PGPR were recorded in the root zone of WF intercropping, which can play an important role in this facilitative mechanism of enhancing growth and yield of cereals. Conclusion: The two treatments clearly affected soil microbial and chemical composition, which can be reflected in growth and yield enhancement.

Keywords: intercropping, microbial community, LMWOAs, PGPR, soil chemical environment

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Authors: Szu-Chieh Yu, Pei-Chin Chiand, Chih-Yi Lin, Yi-Wen Chien

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UV radiation from sunlight cause numbers of acute and chronic skin damage which can result in inflammation, immune changes, physical changes and DNA damage that facilitates skin aging and the development of skin carcinogenesis. Reactive oxygen species (ROS) are generated by excessive solar UV radiation, resulting in oxidative damage to cellar components, proteins, lipids, and nucleic acids. Thus, antioxidation plays an important role that protects skin against ROS-induced injury. Safflower (Carthamus tinctorius L.) is an important Chinese medicine contained abundance flavones and hydroxysafflor yellow A (HSYA) which is main active ingredient. HSYA is part of quinochalcone and has unique structures of hydroxy groups that provided the antioxidant effect. In this study, the aim was to investigate the protective role of HYSA in human keratinocytes (HaCaT) against UVA-induced oxidative damage and the possible mechanism. The HaCaT cells were UVA-irradiated and the effects of HYSA on cell viability, reactive oxygen species generation, DNA fragmentation and lipid peroxidation were measured. The mRNA expression of matrix metalloproteinase Ι (MMP Ι), cyclooxygenase-2 (COX-2) were determined by RT-PCR. In this study, UVA exposure lead to decrease in cell viability and increase in reactive oxygen species generation in HaCaT cells. HYSA could effectively increase the viability of HaCaT cells after UVA exposure and protect them from UVA-induced oxidative stress. Moreover, HYSA can reduce inflammation through inhibition the mRNA expression of MMP Ι and COX-2. Our results suggest that HSYA can act as a free radical scavenger while keratinocytes were photodamaged. HYSA could be a useful natural medicine for the protection of epidermal cells from UVA-induced damage and will be developed into products for skin care.

Keywords: HaCaT keratinocytes, hydroxysafflor yellow A (HSYA), MMP Ι, oxidative stress

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Authors: Maira C. M. Fonseca, Maria Aparecida N. Sediyama, Rosana Goncalves R. das Dores, Sanzio Mollica Vidigal, Alberto C. P. Dias

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Among Brazilian plant diversity, many species are used as food and considered minor crops (non-conventional plant foods) (NCPF). Arrowroot (Maranta arundinacea) is a NCPF from which starch is extracted from rhizome do not have gluten. Thus, arrowroot flower starch can be consumed by celiac people. Additional, some medicinal and functional proprieties are assigned to arrowroot leaves which currently are underutilized. In Brazil, it’s cultivated mainly by small scale farmers and there is no specific recommendation for fertilization. This work aimed to determinate the best fertilization for rhizome production and to verify its influence in phenolic composition and antioxidant activity of leaf extracts. Two arrowroot varieties, “Common” and “Seta”, were cultivated in organic system at state of Minas Gerais, Brazil, using cattle manure with three levels of nitrogen (N) (0, 300 and 900 kg N ha-1). The experiment design was in randomized block with four replicates. The highest production of rhizomes in both varieties, “Common” (38198.24 kg ha-1) and “Seta” (43567.71 kg ha-1), were obtained with the use of 300 kg N ha-1. With this fertilization, the total aerial part, petiole and leaf production in the varieties were respectively: “Common” (190.312 kg ha-1; 159.312 kg ha-1; 31.100 kg ha-1) and “Seta” (207.656 kg ha-1; 180.539 kg ha-1; 27.062 kg ha-1). Methanolic leaf extracts were analysed by HPLC-DAD. The major phenolic compounds found were caffeioylquinic acids, p-coumaric derivatives and flavonoids. In general, the production of these compounds significantly decreases with the increase levels of nitrogen (900 kg N ha-1). With 300 kg N ha-1 the phenolic production was similar to control. The antioxidant activity was evaluated using DPPH method and was detected around 60% of radical scavenging when 0.1 mg/mL of plant extracts were used. We concluded that fertilization with 300 kg N ha-1 increased arrowroot rhizome production, maintaining phenolic compounds yield at leaves.

Keywords: antioxidant activity, non-conventional plants, organic fertilization, phenolic compounds

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Authors: Narayana Murthy Akurathi, Vijaya Lakshmi Marella

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The literature is reviewed with regard to online Super critical fluid extraction (SFE) coupled directly with supercritical fluid chromatography (SFC) -mass spectrometry that have typically more sensitive than conventional LC-MS/MS and GC-MS/MS. It is becoming increasingly interesting to use on-line techniques that combine sample preparation, separation and detection in one analytical set up. This provides less human intervention, uses small amount of sample and organic solvent and yields enhanced analyte enrichment in a shorter time. The sample extraction is performed under light shielding and anaerobic conditions, preventing the degradation of thermo labile analytes. It may be able to analyze compounds over a wide polarity range as SFC generally uses carbon dioxide which was collected as a by-product of other chemical reactions or is collected from the atmosphere as it contributes no new chemicals to the environment. The diffusion of solutes in supercritical fluids is about ten times greater than that in liquids and about three times less than in gases which results in a decrease in resistance to mass transfer in the column and allows for fast high resolution separations. The drawback of SFC when using carbon dioxide as mobile phase is that the direct introduction of water samples poses a series of problems, water must therefore be eliminated before it reaches the analytical column. Hundreds of compounds analysed simultaneously by simple enclosing in an extraction vessel. This is mainly applicable for pharmaceutical industry where it can analyse fatty acids and phospholipids that have many analogues as their UV spectrum is very similar, trace additives in polymers, cleaning validation can be conducted by putting swab sample in an extraction vessel, analysing hundreds of pesticides with good resolution.

Keywords: super critical fluid extraction (SFE), super critical fluid chromatography (SFC), LCMS/MS, GCMS/MS

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Authors: Mustafa Durmuş, Yesim Ozogul, Esra Balıkcı, Saadet Gokdoğan, Fatih Ozogul, Ali Rıza Köşker, İlknur Yuvka

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Food scientists and researchers have paid attention to develop new ways for improving the nutritional value of foods. The application of nanotechnology techniques to the food industry may allow the modification of food texture, taste, sensory attributes, coloring strength, processability, and stability during shelf life of products. In this research, the effects of nanoemulsions based on commercial oils for vacuum-packed sea bass fillets stored at 2±2°C were investigated in terms of the sensory, chemical (total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA), peroxide value (PV) and free fatty acids (FFA), pH, water holding capacity (WHC)) and microbiological qualities (total anaerobic bacteria and total lactic acid bacteria). Physical properties of emulsions (viscosity, the particle size of droplet, thermodynamic stability, refractive index, and surface tension) were determined. Nanoemulsion preparation method was based on high energy principle, with ultrasonic homojenizator. Sensory analyses of raw fish showed that the demerit points of the control group were found higher than those of treated groups. The sensory score (odour, taste and texture) of the cooked fillets decreased with storage time, especially in the control. Results obtained from chemical and microbiological analyses also showed that nanoemulsions significantly (p<0.05) decreased the values of biochemical parameters and growth of bacteria during storage period, thus improving quality of vacuum-packed sea bass.

Keywords: quality parameters, nanoemulsion, sea bass, shelf life, vacuum packing

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Authors: L. Piñeiro, N. Cobas, L. Gómez-Limia, S. Martínez, I. Franco

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The swordfish (Xiphias gladius) is a large oceanic fish of high commercial value, which is widely distributed in waters of the world’s oceans. They are considered to be an important source of high quality proteins, vitamins and essential fatty acids, although only half of the population follows the recommendation of nutritionists to consume fish at least twice a week. Swordfish is consumed worldwide because of its low fat content and high protein content. It is generally sold as fresh, frozen, and as pieces or slices. The aim of this study was to evaluate the effect of salting and frying on the composition of the water-soluble vitamins (B₂, B₃, B₉ and B₁₂) and fat-soluble vitamins (A, D, and E) of swordfish. Three loins of swordfish from Pacific Ocean were analyzed. All the fishes had a weight between 50 and 70 kg and were transported to the laboratory frozen (-18 ºC). Before the processing, they were defrosted at 4 ºC. Each loin was sliced and salted in brine. After cleaning the slices, they were divided into portions (10×2 cm) and fried in olive oil. The identification and quantification of vitamins were carried out by high-performance liquid chromatography (HPLC), using methanol and 0.010% trifluoroacetic acid as mobile phases at a flow-rate of 0.7 mL min^-1. The UV-Vis detector was used for the detection of the water- and fat-soluble vitamins (A and D), as well as the fluorescence detector for the detection of the vitamin E. During salting, water and fat-soluble vitamin contents remained constant, observing an evident decrease in the values of vitamin B₂. The diffusion of salt into the interior of the pieces and the loss of constitution water that occur during this stage would be related to this significant decrease. In general, after frying water-soluble and fat-soluble vitamins showed a great thermolability with high percentages of retention with values among 50–100%. Vitamin B₃ is the one that exhibited higher percentages of retention with values close to 100%. However, vitamin B₉ presented the highest losses with a percentage of retention of less than 20%.

Keywords: frying, HPLC, salting, swordfish, vitamins

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Authors: Paula Buldres, Jorge Toledo

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Objectives: This study aimed to characterize potential probiotic strains isolated from canine breast milk for use in dogs with enteropathies. Methodology: Six canine breast milk strains, one canine colostrum strain, and one control porcine breast milk strain were characterized. According to its functional properties of resistance to acids, different concentrations of bile salts, and pancreatin, its presumptive properties of safety and inhibitory effect on pathogens, non-cytotoxic characteristics, and adhesion to the intestine. The immunomodulatory effect of formulations with better probiotic characterization in vitro and in vivo was also analyzed. Results: Two strains characterized as potential probiotics were obtained, which corresponded to the canine strains (TUCO-16 and TUCO-17), presenting resistance to acidic pH, bile salts, and pancreatin, as well as an inhibitory effect on pathogenic Escherichia coli, Salmonella sp., and Clostridium perfringens. Strains TUCO-16 and TUCO-17 induced a significant increase in the expression of TNF-α and IL-8 in canine macrophages, respectively. Expression analyses of pattern recognition receptors in DH82 cells suggest that TUCO-16 and TUCO-17 might increase the TLR2 expression marker, and porcine strain (TUCO-4) increases the NOD2 expression marker. Based on the count obtained and the encapsulation yield, the best formulations correspond to FOS-Inulin for the TUCO-17 and TUCO-4 strains; Maltodextrin-Inulin for TUCO-16. All the strains are non-cytotoxic. The strain that showed the highest adhesion to intestinal epithelial cells was TUCO-17 with the FOS-Inulin formulation. On the other hand, the probiotics decreased the expression of pro-inflammatory markers in vivo, both in the intestine and in the spleen of mice. Conclusion: The combination of these three strains under study (TUCO-16, TUCO-17, and TUCO-4) would cover the probiotic properties in formulation and immunomodulation of all the markers under study.

Keywords: probiotics, gastrointestinal infec, dog, probiotic formulation, immunomodulatory probiotics

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Authors: Michael Eickermann, Juergen Junk

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The Western honey (Apis mellifera) is threatened by a number of parasites, especially the devastating Varroa mite (Varroa destructor) is responsible for a high level of mortality over winter, e.g., in Europe and USA. While the use of synthetic pesticides or organic acids has been preferred so far to control this parasite, breeding strategies for less susceptible honey bees are in early stages. Hygienic behavior can be an important tool for controlling Varroa destructor. Worker bees with a high level of this behavior are able to detect infested brood in the cells under the wax lid during pupation and remove them out of the hive. The underlying processes of this behavior are only partly investigated, but it is for sure that hygienic behavior is heritable and therefore, can be integrated into commercial breeding lines. In a first step, breeding lines with a high level of phenotypic hygienic behavior have been identified by using a bioassay for accurate assessment of this trait in a long-term national breeding program in Luxembourg since 2015. Based on the artificial infestation of nucleus colonies with 150 phoretic Varroa destructor mites, the level of phenotypic hygienic behavior was detected by counting the number of mites in all stages, twelve days after infestation. A nucleus with a high level of hygienic behavior was overwintered and used for breeding activities in the following years. Artificial insemination was used to combine different breeding lines. Buckfast lines, as well as Carnica lines, were used. While Carnica lines offered only a low increase of hygienic behavior up to maximum 62.5%, Buckfast lines performed much better with mean levels of more than 87.5%. Some mating ends up with a level of 100%. But even with a level of 82.5% Varroa mites are not able to reproduce in the colony anymore. In a final step, a nucleus with a high level of hygienic behavior were build up to full colonies and located at two places in Luxembourg to build up a drone congregation area. Local beekeepers can bring their nucleus to this location for mating the queens with drones offering a high level of hygienic behavior.

Keywords: agiculture, artificial insemination, honey bee, varroa destructor

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Authors: Anusha Atmakuri, R. D. Tyagi, Patrick Drogui

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Landfilling is the most familiar and easy way to dispose solid waste. Landfill is generally received via wastes from municipal near to a landfill. The waste collected is from commercial, industrial, and residential areas and many more. Landfill leachate (LFL) is formed when rainwater passes through the waste placed in landfills and consists of several dissolved organic materials, for instance, aquatic humic substances (AHS), volatile fatty acids (VFAs), heavy metals, inorganic macro components, and xenobiotic organic matters, highly toxic to the environment. These components of LFL put a load on it, hence it necessitates the treatment of LFL prior to its discharge into the environment. Various methods have been used to treat LFL over the years, such as physical, chemical, biological, physicochemical, electrical, and advanced oxidation methods. This study focuses on the combination of biological and electrochemical methods- extracellular polymeric substances and electrocoagulation(EC). The coupling of electro-coagulation process with extracellular polymeric substances (EPS) (as flocculant) as pre and\or post treatment strategy provides efficient and economical process for the decontamination of landfill leachate contaminated with suspended matter, metals (e.g., Fe, Mn) and ammonical nitrogen. Electro-coagulation and EPS mediated coagulation approach could be an economically viable for the treatment of landfill leachate, along with possessing several other advantages over several other methods. This study utilised waste substrates such as activated sludge, crude glycerol and waste cooking oil for the production of EPS using fermentation technology. A comparison of different scenarios for the treatment of landfill leachate is presented- such as using EPS alone as bioflocculant, EPS and EC with EPS being the 1st stage, and EPS and EC with EC being the 1st stage. The work establishes the use of crude EPS as a bioflocculant for the treatment of landfill leachate and wastewater from a site near a landfill, along with EC being successful in removal of some major pollutants such as COD, turbidity, total suspended solids. A combination of these two methods is to be explored more for the complete removal of all pollutants from landfill leachate.

Keywords: landfill leachate, extracellular polymeric substances, electrocoagulation, bioflocculant.

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Authors: N. Umasuthan, S. D. N. K. Bathige, W. S. Thulasitha, I. Whang, J. Lee

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The MyD88 is an evolutionarily conserved host-expressed adaptor protein that is essential for proper TLR/ IL1R immune-response signaling. A previously identified complete cDNA (1626 bp) of OfMyD88 comprised an ORF of 867 bp encoding a protein of 288 amino acids (32.9 kDa). The gDNA (3761 bp) of OfMyD88 revealed a quinquepartite genome organization composed of 5 exons (with the sizes of 310, 132, 178, 92 and 155 bp) separated by 4 introns. All the introns displayed splice signals consistent with the consensus GT/AG rule. A bipartite domain structure with two domains namely death domain (24-103) coded by 1st exon, and TIR domain (151-288) coded by last 3 exons were identified through in silico analysis. Moreover, homology modeling of these two domains revealed a similar quaternary folding nature between human and rock bream homologs. A comprehensive comparison of vertebrate MyD88 genes showed that they possess a 5-exonic structure. In this structure, the last three exons were strongly conserved, and this suggests that a rigid structure has been maintained during vertebrate evolution. A cluster of TATA box-like sequences were found 0.25 kb upstream of cDNA starting position. In addition, putative 5'-flanking region of OfMyD88 was predicted to have TFBS implicated with TLR signaling, including copies of NFB1, APRF/ STAT3, Sp1, IRF1 and 2 and Stat1/2. Using qPCR technique, a ubiquitous mRNA expression was detected in liver and blood. Furthermore, a significantly up-regulated transcriptional expression of OfMyD88 was detected in head kidney (12-24 h; >2-fold), spleen (6 h; 1.5-fold), liver (3 h; 1.9-fold) and intestine (24 h; ~2-fold) post-Fla challenge. These data suggest a crucial role for MyD88 in antibacterial immunity of teleosts.

Keywords: MyD88, innate immunity, flagellin, genomic analysis

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Authors: Abanoub Mikhael, Darryl Hardie, Derek Smith, Helena Petrosova, Robert Ernst, David Goodlett

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Lipopolysaccharide (LPS) is a hallmark virulence factor of Gram-negative bacteria. It is a complex, structurally het- erogeneous mixture due to variations in number, type, and position of its simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of intact R-type lipopolysaccharide complex mixture (lipooligo- saccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and un- equivocal structural assignments. In addition to FAIMS gas phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na-H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families, i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 181 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.

Keywords: lipopolysaccharide, ion mobility MS, Kendrick mass defect, Tandem mass spectrometry

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