Search results for: methyl acetate

Authors: Siddhant Sethi, Yasuharu Takashima, Shigetaka Nakamura, Kenzo Fujimoto

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Site-directed mutagenesis is a renowned technique to introduce specific mutations in the genome. To achieve site-directed mutagenesis, many chemical and enzymatic approaches have been reported in the past like disulphite induced genome editing, CRISPR-Cas9, TALEN etc. The chemical methods are invasive whereas the enzymatic approaches are time-consuming and expensive. Most of these techniques are unusable in the cellular application due to their toxicity and other limitations. Photo-chemical cytosine deamination, introduced in 2010, is one of the major technique for enzyme-free single-point mutation of cytosine to uracil in DNA and RNA, wherein, 3-cyanovinylcarbazole nucleoside (CNVK) containing oligodeoxyribonucleotide (ODN) having CNVK at -1 position to that of target cytosine is reversibly crosslinked to target DNA strand using 366 nm and then incubated at 90ºC to accommodate deamination. This technique is superior to enzymatic methods of site-directed mutagenesis but has a disadvantage that it requires the use of high temperature for the deamination step which restricts its applicability in the in vivo applications. This study has been focused on improving the technique by reducing the temperature required for deamination. Firstly, the photo-cross-linker, CNVK has been modified by replacing cyano group attached to vinyl group with methyl ester (OMeVK), amide (NH2VK), and carboxylic acid (OHVK) to observe the acceleration in the deamination of target cytosine cross-linked to vinylcarbazole derivative. Among the derivatives, OHVK has shown 2 times acceleration in deamination reaction as compared to CNVK, while the other two derivatives have shown deceleration towards deamination reaction. The trend of rate of deamination reaction follows the same order as that of hydrophilicity of the vinylcarbazole derivatives. OHVK being most hydrophilic has shown highest acceleration while OMeVK is least hydrophilic has proven to be least active for deamination. Secondly, in the related study, the counter-base of the target cytosine, guanine has been replaced by inosine, 2-aminopurine, nebularine, and 5-nitroindole having distinct hydrogen bonding patterns with target cytosine. Among the ODNs with these counter bases, ODN with inosine has shown 12 fold acceleration towards deamination of cytosine cross-linked to CNVK at physiological conditions as compared to guanosine. Whereas, when 2-aminopurine, nebularine, and 5-nitroindole were used, no deamination reaction took place. It can be concluded that inosine has potential to be used as the counter base of target cytosine for the CNVK mediated photo-cross-linking induced deamination of cytosine. The increase in rate of deamination reaction has been attributed to pattern and number of hydrogen bonding between the cytosine and counter base. One of the important factor is presence of hydrogen bond between exo-cyclic amino group of cytosine and the counter base. These results will be useful for development of more efficient technique for site-directed mutagenesis for C → U transformations in the DNA/RNA which might be used in the living system for treatment of various genetic disorders and genome engineering for making designer and non-native proteins.

Keywords: C to U transformation, DNA editing, genome engineering, ultra-fast photo-cross-linking

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Authors: Srijata Mitra, Mobina Parveen, Pranab Roy, Narayan Chandra Chattopadhyay

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Chlorinated hydrocarbon can be a major pollution problem in groundwater as well as soil. Many people interact with these chemicals on daily accidentally or by professionally in the laboratory. One of the most common sources for Chlorinated hydrocarbon contamination of soil and groundwater are industrial effluents. The wide use and discharge of Trichloroethylene (TCE), a volatile chlorohydrocarbon from chemical industry, led to major water pollution in rural areas. TCE is an mainly used as an industrial metal degreaser in industries. Biotransformation of TCE to the potent carcinogen vinyl chloride (VC) by consortia of anaerobic bacteria might have role for the above purpose. For these reasons, the aim of current study was to isolate and characterized the genes involved in TCE metabolism and also to investigate the in silico study of those genes. To our knowledge, only one aromatic dioxygenase system, the toluene dioxygenase in Pseudomonas putida F1 has been shown to be involved in TCE degradation. This is first instance where Bacillus cereus group being used in biodegradation of trichloroethylene. A novel bacterial strain 2479 was isolated from oil depot site at Rajbandh, Durgapur (West Bengal, India) by enrichment culture technique. It was identified based on polyphasic approach and ribotyping. The bacterium was gram positive, rod shaped, endospore forming and capable of degrading trichloroethylene as the sole carbon source. On the basis of phylogenetic data and Fatty Acid Methyl Ester Analysis, strain 2479 should be placed within the genus Bacillus and species cereus. However, the present isolate (strain 2479) is unique and sharply different from the usual Bacillus strains in its biodegrading nature. Fujiwara test was done to estimate that the strain 2479 could degrade TCE efficiently. The gene for TCE biodegradation was PCR amplified from genomic DNA of Bacillus cereus 2479 by using todC1 gene specific primers. The 600bp amplicon was cloned into expression vector pUC I8 in the E. coli host XL1-Blue and expressed under the control of lac promoter and nucleotide sequence was determined. The gene sequence was deposited at NCBI under the Accession no. GU183105. In Silico approach involved predicting the physico-chemical properties of deduced Tce1 protein by using ProtParam tool. The tce1 gene contained 342 bp long ORF encoding 114 amino acids with a predicted molecular weight 12.6 kDa and the theoretical pI value of the polypeptide was 5.17, molecular formula: C559H886N152O165S8, total number of atoms: 1770, aliphatic index: 101.93, instability index: 28.60, Grand Average of Hydropathicity (GRAVY): 0.152. Three differentially expressed proteins (97.1, 40 and 30 kDa) were directly involved in TCE biodegradation, found to react immunologically to the antibodies raised against TCE inducible proteins in Western blot analysis. The present study suggested that cloned gene product (TCE1) was capable of degrading TCE as verified chemically.

Keywords: cloning, Bacillus cereus, in silico analysis, TCE

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Authors: G. Magalhães-Mota, C. Magro, S. Sério, E. Mateus, P. A. Ribeiro, A. B. Ribeiro, M. Raposo

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Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol], belonging to the class of Pharmaceuticals and Personal Care Products (PPCPs), is a broad-spectrum antimicrobial agent and bactericide. Because of its antimicrobial efficacy, it is widely used in personal health and skin care products, such as soaps, detergents, hand cleansers, cosmetics, toothpastes, etc. However, it has been considered to disrupt the endocrine system, for instance, thyroid hormone homeostasis and possibly the reproductive system. Considering the widespread use of triclosan, it is expected that environmental and food safety problems regarding triclosan will increase dramatically. Triclosan has been found in river water samples in both North America and Europe and is likely widely distributed wherever triclosan-containing products are used. Although significant amounts are removed in sewage plants, considerable quantities remain in the sewage effluent, initiating widespread environmental contamination. Triclosan undergoes bioconversion to methyl-triclosan, which has been demonstrated to bio accumulate in fish. In addition, triclosan has been found in human urine samples from persons with no known industrial exposure and in significant amounts in samples of mother's milk, demonstrating its presence in humans. The action of sunlight in river water is known to turn triclosan into dioxin derivatives and raises the possibility of pharmacological dangers not envisioned when the compound was originally utilized. The aim of this work is to detect low concentrations of triclosan in an aqueous complex matrix through the use of a sensor array system, following the electronic tongue concept based on impedance spectroscopy. To achieve this goal, we selected the appropriate molecules to the sensor so that there is a high affinity for triclosan and whose sensitivity ensures the detection of concentrations of at least nano-molar. Thin films of organic molecules and oxides have been produced by the layer-by-layer (LbL) technique and sputtered onto glass solid supports already covered by gold interdigitated electrodes. By submerging the films in complex aqueous solutions with different concentrations of triclosan, resistance and capacitance values were obtained at different frequencies. The preliminary results showed that an array of interdigitated electrodes sensor coated or uncoated with different LbL and films, can be used to detect TCS traces in aqueous solutions in a wide range concentration, from 10⁻¹² to 10⁻⁶ M. The PCA method was applied to the measured data, in order to differentiate the solutions with different concentrations of TCS. Moreover, was also possible to trace a curve, the plot of the logarithm of resistance versus the logarithm of concentration, which allowed us to fit the plotted data points with a decreasing straight line with a slope of 0.022 ± 0.006 which corresponds to the best sensitivity of our sensor. To find the sensor resolution near of the smallest concentration (Cs) used, 1pM, the minimum measured value which can be measured with resolution is 0.006, so the ∆logC =0.006/0.022=0.273, and, therefore, C-Cs~0.9 pM. This leads to a sensor resolution of 0.9 pM for the smallest concentration used, 1pM. This attained detection limit is lower than the values obtained in the literature.

Keywords: triclosan, layer-by-layer, impedance spectroscopy, electronic tongue

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Authors: Mihaela Beregoi, Horia Iovu, Cristina Busuioc, Alexandru Evanghelidis, Elena Matei, Monica Enculescu, Ionut Enculescu

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Nanomaterials field is very attractive for all researchers who are attempting to develop new devices with the same or improved properties than the micro-sized ones, while reducing the reagents and power consumptions. In this way, a wide range of nanomaterials were fabricated and integrated in applications for electronics, optoelectronics, solar cells, tissue reconstruction and drug delivery. Obviously, the most appealing ones are those dedicated to the medical domain. Different types of nano-sized materials, such as particles, fibers, films etc., can be synthesized by using physical, chemical or electrochemical methods. One of these techniques is electrospinning, which enable the production of fibers with nanometric dimensions by pumping a polymeric solution in a high electric field; due to the electrostatic charging and solvent evaporation, the precursor mixture is converted into nonwoven meshes with different fiber densities and mechanical properties. Moreover, polyaniline is a conducting polymer with interesting optical properties, suitable for displays and electrochromic windows. Otherwise, polyaniline is an electroactive polymer that can contract/expand by applying electric stimuli, due to the oxidation/reduction reactions which take place in the polymer chains. These two main properties can be exploited in order to synthesize smart materials that change their dimensions, exhibiting in the same time good electrochromic properties. In the context aforesaid, a poly(methyl metacrylate) solution was spun to get webs composed of fibers with diameter values between 500 nm and 1 µm. Further, the polymer meshes were covered with a gold layer in order to make them conductive and also appropriate as working electrode in an electrochemical cell. The gold shell was deposited by DC sputtering. Such metalized fibers can be transformed into smart materials by covering them with a thin layer of conductive polymer. Thus, the webs were coated with a polyaniline film by the electrochemical route, starting from and aqueous solution of aniline and sulfuric acid, where sulfuric acid acts as oxidant agent. For the polymerization of aniline, a saturated calomel electrode was employed as reference, a platinum plate as counter electrode and the gold covered webs as working electrode. Chronoamperometry was selected as deposition method for polyaniline, by modifying the deposition time. Metalized meshes with different fiber densities were used, the transmission ranging between 70 and 80 %. The morphological investigation showed that polyaniline layer has a granular structure for all deposition experiments. As well, some preliminary optical tests were done by using sulfuric acid as electrolyte, which revealed the modification of polyaniline colour from green to dark blue when applying a voltage. In conclusion, new multilayered materials were obtained by a simple approach: the merge of the electrospinning method benefits with polyaniline chemistry. This synthesis method allows the fabrication of structures with reproducible characteristics, suitable for display or tissue substituents.

Keywords: electrospinning, fibers, smart materials, polyaniline

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Authors: Aurica P. Chiriac, Vera Balan, Mihai Asandulesa, Elena Butnaru, Nita Tudorachi, Elena Stoleru, Loredana E. Nita, Iordana Neamtu, Alina Diaconu, Liliana Mititelu-Tartau

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The interest for studying ‘smart’ materials is entirely justified and in this context were realized investigations on poly(2-hydroxyethylmethacrylate-co-3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane), which is a macromolecular compound with sensibility at pH and temperature, gel formation capacity, binding properties, amphilicity, good oxidative and thermal stability. Physico-chemical characteristics in terms of the molecular weight, temperature-sensitive abilities and thermal stability, as well rheological, dielectric and spectroscopic properties were evaluated in correlation with further coupling capabilities. Differential scanning calorimetry investigation indicated Tg at 36.6 °C and a melting point at Tm=72.8°C, for the studied copolymer, and up to 200oC two exothermic processes (at 99.7°C and 148.8°C) were registered with losing weight of about 4 %, respective 19.27%, which indicate just processes of thermal decomposition (and not phenomena of thermal transition) owing to scission of the functional groups and breakage of the macromolecular chains. At the same time, the rheological studies (rotational tests) confirmed the non-Newtonian shear-thinning fluid behavior of the copolymer solution. The dielectric properties of the copolymer have been evaluated in order to investigate the relaxation processes and two relaxation processes under Tg value were registered and attributed to localized motions of polar groups from side chain macromolecules, or parts of them, without disturbing the main chains. According to literature and confirmed as well by our investigations, β-relaxation is assigned with the rotation of the ester side group and the γ-relaxation corresponds to the rotation of hydroxy- methyl side groups. The fluorescence spectroscopy confirmed the copolymer structure, the spiroacetal moiety getting an axial conformation, more stable, with lower energy, able for specific interactions with molecules from environment, phenomena underlined by different shapes of the emission spectra of the copolymer. Also, the copolymer was used as template for indomethacin incorporation as model drug, and the biocompatible character of the complex was confirmed. The release behavior of the bioactive compound was dependent by the copolymer matrix composition, the increasing of 3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane comonomer amount attenuating the drug release. At the same time, the in vivo studies did not show significant differences of leucocyte formula elements, GOT, GPT and LDH levels, nor immune parameters (OC, PC, and BC) between control mice group and groups treated just with copolymer samples, with or without drug, data attesting the biocompatibility of the polymer samples. The investigation of the physico-chemical characteristics of poly(2-hydrxyethyl methacrylate-co-3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro (5.5) undecane) in terms of temperature-sensitive abilities, rheological and dielectrical properties, are bringing useful information for further specific use of this polymeric compound.

Keywords: bioapplications, dielectric and spectroscopic properties, dual sensitivity at pH and temperature, smart materials

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Authors: Aurora Sheehy, Caitlin Prince

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Within current orthodox medical protocols, trauma and mental health issues are deemed to reside within the realm of cognitive or psychological therapists and are marginalised in these areas, in part due to limited drugs option available, mostly manipulating neurotransmitters or sedating patients to reduce symptoms. By contrast, this research presents examples from the clinical practice of how trauma can be assessed and treated physiologically. Adverse Childhood Experiences (ACEs) are a tally of different types of abuse and neglect. It has been used as a measurable and reliable predictor of the likelihood of the development of autoimmune disease. It is a direct way to demonstrate reliably the health impact of traumatic life experiences. A second assessment tool is Allostatic Load, which refers to the cumulative effects that chronic stress has on mental and physical health. It records the decline of an individual’s physiological capacity to cope with their experience. It uses a specific grouping of serum testing and physical measures. It includes an assessment of neuroendocrine, cardiovascular, immune and metabolic systems. Allostatic load demonstrates the health impact that trauma has throughout the body. It forms part of an initial intake assessment in clinical practice and could also be used in research to evaluate treatment. Examining medicinal plants for their physiological, neurological and somatic effects through the lens of Polyvagal theory offers new opportunities for trauma treatments. In situations where Polyvagal theory recommends activities and exercises to enable parasympathetic activation, many herbs that affect Effector Memory T (TEM) cells also enact these responses. Traditional or Indigenous European herbs show the potential to support the polyvagal tone, through multiple mechanisms. As the ventral vagal nerve reaches almost every major organ, plants that have actions on these tissues can be understood via their polyvagal actions, such as monoterpenes as agents to improve respiratory vagal tone, cyanogenic glycosides to reset polyvagal tone, volatile oils rich in phenyl methyl esters improve both sympathetic and parasympathetic tone, bitters activate gut function and can strongly promote parasympathetic regulation. Auricular Acupuncture uses a system of somatotopic mapping of the auricular surface overlaid with an image of an inverted foetus with each body organ and system featured. Given that the concha of the auricle is the only place on the body where the Vagus Nerve neurons reach the surface of the skin, several investigators have evaluated non-invasive, transcutaneous electrical nerve stimulation (TENS) at auricular points. Drawn from an interdisciplinary evidence base and developed through clinical practice, these assessment and treatment tools are examples of practitioners in the field innovating out of necessity for the best outcomes for patients. This paper draws on case studies to direct future research.

Keywords: polyvagal, auricular acupuncture, trauma, herbs

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Authors: Alireza Shams, Ali Zamanian, Atefehe Shamosi, Farnaz Ghorbani

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Introduction: Although the application of a new strategy remains a remarkable challenge for treatment of disabilities due to neuronal defects, progress in Nanomedicine and tissue engineering, suggesting the new medical methods. One of the promising strategies for reconstruction and regeneration of nervous tissue is replacing of lost or damaged cells by specific scaffolds after Compressive, ischemic and traumatic injuries of central nervous system. Furthermore, ultrastructure, composition, and arrangement of tissue scaffolds are effective on cell grafts. We followed implantation and differentiation of mesenchyme stem cells to neural cells on Gelatin Polylactic-co-glycolic acid (PLGA) scaffolds coated with iron nanoparticles. The aim of this study was to evaluate the capability of stem cells to differentiate into motor neuron-like cells under topographical cues and morphogenic factors. Methods and Materials: Bone marrow mesenchymal stem cells (BMMSCs) was obtained by primary cell culturing of adult rat bone marrow got from femur bone by flushing method. BMMSCs were incubated with DMEM/F12 (Gibco), 15% FBS and 100 U/ml pen/strep as media. Then, BMMSCs seeded on Gel/PLGA scaffolds and tissue culture (TCP) polystyrene embedded and incorporated by Fe Nano particles (FeNPs) (Fe3o4 oxide (M w= 270.30 gr/mol.). For neuronal differentiation, 2×10 5 BMMSCs were seeded on Gel/PLGA/FeNPs scaffolds was cultured for 7 days and 0.5 µ mol. Retinoic acid, 100 µ mol. Ascorbic acid,10 ng/ml. Basic fibroblast growth factor (Sigma, USA), 250 μM Iso butyl methyl xanthine, 100 μM 2-mercaptoethanol, and 0.2 % B27 (Invitrogen, USA) added to media. Proliferation of BMMSCs was assessed by using MTT assay for cell survival. The morphology of BMMSCs and scaffolds was investigated by scanning electron microscopy analysis. Expression of neuron-specific markers was studied by immunohistochemistry method. Data were analyzed by analysis of variance, and statistical significance was determined by Turkey’s test. Results: Our results revealed that differentiation and survival of BMMSCs into motor neuron-like cells on Gel/PLGA/FeNPs as a biocompatible and biodegradable scaffolds were better than those cultured in Gel/PLGA in absence of FeNPs and TCP scaffolds. FeNPs had raised physical power but decreased capacity absorption of scaffolds. Well defined oriented pores in scaffolds due to FeNPs may activate differentiation and synchronized cells as a mechanoreceptor. Induction effects of magnetic FeNPs by One way flow of channels in scaffolds help to lead the cells and can facilitate direction of their growth processes. Discussion: Progression of biological properties of BMMSCs and the effects of FeNPs spreading under magnetic field was evaluated in this investigation. In vitro study showed that the Gel/PLGA/FeNPs scaffold provided a suitable structure for motor neuron-like cells differentiation. This could be a promising candidate for enhancing repair and regeneration in neural defects. Dynamic and static magnetic field for inducing and construction of cells can provide better results for further experimental studies.

Keywords: differentiation, mesenchymal stem cells, nano particles, neuronal defects, Scaffolds

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

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

Keywords: mealworms, chitosan, chitosan oligosaccharide, prebiotics

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Authors: Natia Jalagonia, Tinatin Kuchukhidze

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Polymer electrolytes (PE) play an important part in electrochemical devices such as batteries and fuel cells. To achieve optimal performance, the PE must maintain a high ionic conductivity and mechanical stability at both high and low relative humidity. The polymer electrolyte also needs to have excellent chemical stability for long and robustness. According to the prevailing theory, ionic conduction in polymer electrolytes is facilitated by the large-scale segmental motion of the polymer backbone, and primarily occurs in the amorphous regions of the polymer electrolyte. Crystallinity restricts polymer backbone segmental motion and significantly reduces conductivity. Consequently, polymer electrolytes with high conductivity at room temperature have been sought through polymers which have highly flexible backbones and have largely amorphous morphology. The interest in polymer electrolytes was increased also by potential applications of solid polymer electrolytes in high energy density solid state batteries, gas sensors and electrochromic windows. Conductivity of 10-3 S/cm is commonly regarded as a necessary minimum value for practical applications in batteries. At present, polyethylene oxide (PEO)-based systems are most thoroughly investigated, reaching room temperature conductivities of 10-7 S/cm in some cross-linked salt in polymer systems based on amorphous PEO-polypropylene oxide copolymers.. It is widely accepted that amorphous polymers with low glass transition temperatures Tg and a high segmental mobility are important prerequisites for high ionic conductivities. Another necessary condition for high ionic conductivity is a high salt solubility in the polymer, which is most often achieved by donors such as ether oxygen or imide groups on the main chain or on the side groups of the PE. It is well established also that lithium ion coordination takes place predominantly in the amorphous domain, and that the segmental mobility of the polymer is an important factor in determining the ionic mobility. Great attention was pointed to PEO-based amorphous electrolyte obtained by synthesis of comb-like polymers, by attaching short ethylene oxide unit sequences to an existing amorphous polymer backbone. The aim of presented work is to obtain of solid polymer electrolyte membranes using PMHS as a matrix. For this purpose the hydrosilylation reactions of α,ω-bis(trimethylsiloxy)methyl¬hydrosiloxane with allyl triethylene-glycol mo¬nomethyl ether and vinyltriethoxysilane at 1:28:7 ratio of initial com¬pounds in the presence of Karstedt’s catalyst, platinum hydrochloric acid (0.1 M solution in THF) and platinum on the carbon catalyst in 50% solution of anhydrous toluene have been studied. The synthesized olygomers are vitreous liquid products, which are well soluble in organic solvents with specific viscosity ηsp ≈ 0.05 - 0.06. The synthesized olygomers were analysed with FTIR, 1H, 13C, 29Si NMR spectroscopy. Synthesized polysiloxanes were investigated with wide-angle X-ray, gel-permeation chromatography, and DSC analyses. Via sol-gel processes of doped with lithium trifluoromethylsulfonate (triflate) or lithium bis¬(trifluoromethylsulfonyl)¬imide polymer systems solid polymer electrolyte membranes have been obtained. The dependence of ionic conductivity as a function of temperature and salt concentration was investigated and the activation energies of conductivity for all obtained compounds are calculated

Keywords: synthesis, PMHS, membrane, electrolyte

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Authors: E. Heintz, H. J. van Lent, K. Glass, J. Lim

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The trend for sodium reduction in food products is clear. Following the World Health Organization (WHO) publication on sodium usage and intake, several countries have introduced initiatives to reduce food-related sodium intake. As salt is a common food preservative, this trend motivates the formulation of a suitable additive with comparable benefits of shelf life extension and microbial safety. Organic acid derivatives like acetates are known as generic microbial growth inhibitors and are commonly applied as additives to meet food safety demands. However, modern consumers have negative perceptions towards -synthetic-derived additives and increasingly prefer natural alternatives. Vinegar, for example, is a well-known natural fermentation product used in food preservation. However, the high acidity of vinegar often makes it impractical for direct use in meat products and a neutralized form would be desirable. This research demonstrates the efficacy of powdered vinegar (Provian DV) in inhibiting Listeria and spoilage organisms (LAB) to increase safety and shelf life of meat products. For this, the efficacy of Provian DV was compared to the efficacy of Provian K, a commonly used sodium free acetate-based preservative, which is known for its inhibition against Listeria. Materials & methods— Cured pork hams: Ingredients: Pork ham muscle, water, salt, dextrose, sodium tripolyphosphate, carrageenan, sodium nitrite, sodium erythorbate, and starch. Targets: 73-74% moisture, 1.75+0.1% salt, and pH 6.4+0.1. Treatments: Control (no antimicrobials), Provian®K 0.5% and 0.75%, Provian®DV 0.5%, 0.65%, 0.8% and 1.0%. Meat formulations in casings were cooked reaching an internal temperature of 73.9oC, cooled overnight and stored for 4 days at 4oC until inoculation. Inoculation: Sliced products were inoculated with approximately 3-log per gram of a cocktail of L. monocytogenes (including serotypes 4b, 1/2a and 1/2b) or LAB-cocktail (C. divergens and L. mesenteroides). Inoculated slices were vacuum packaged and stored at 4oC and 7°C. Samples were incubated 28 days (LAB) or 12 weeks (L. monocytogenes) Microbial analysis: Microbial populations were enumerated in rinsate obtained after adding 100ml of sterile Butterfield’s phosphate buffer to each package and massaging the contents externally by hand. L. monocytogenes populations were determined on triplicate samples by surface plating on Modified Oxford agar whereas LAB plate counts were determined on triplicate samples by surface plating on All Purpose Tween agar with 0.4% bromocresol purple. Proximate analysis: Triplicate non-inoculated ground samples were analyzed for the moisture content, pH, aw, salt, and residual nitrite. Results—The results confirmed the no growth of Listeria on cured ham with 0.5% Provian K stored at 4°C and 7°C for 12 weeks, whereas the no-antimicrobial control showed a 1-log increase within two weeks. 0.5% Provian DV demonstrated similar efficacy towards Listeria inhibition at 4°C while 0.65% Provian DV was required to match the Listeria control at 7°C. 0.75% Provian K and 1% Provian DV were needed to show inhibition of the LAB for 4 weeks at both temperatures. Conclusions—This research demonstrated that it is possible to increase safety and shelf life of cured ready-to-eat ham using preservatives that meet current food trends, like sodium reduction and natural origin.

Keywords: food safety, natural preservation, listeria control, shelf life extension

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Authors: Anitha Kandasamy, Thirumurugan Ramaiah

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Interestingly, organic materials exhibit large optical nonlinearity with quick responses and having the flexibility of molecular tailoring using computational modelling and favourable synthetic methodologies. Pyridine based organic compounds and carboxylic acid contained aromatic compounds play a crucial role in crystal engineering of NCS complexes that displays admirable optical nonlinearity with fast response and favourable physicochemical properties such as low dielectric constant, wide optical transparency and large laser damage threshold value requires for optoelectronics device applications. Based on these facts, it was projected to form an acentric molecule of π-conjugated system interaction with appropriately replaced electron donor and acceptor groups for achieving higher SHG activity in which quinoline-2-carboyxlic acid is chosen as an electron acceptor and capable of acting as an acid as well as a base molecule, while 2-amino-4-methylpyridine is used as an electron donor and previously employed in numerous proton transfer complexes for synthesis of NLO materials for optoelectronic applications. 2-amino-4-mehtylpyridinium quinoline-2-carboxylate molecular complex (2AQ) is having π-donor-acceptor groups in which 2-amino-4-methylpyridine donates one of its electron to quinoline -2-carboxylic acid thereby forming a protonated 2-amino-4-methyl pyridinium moiety and mono ionized quinoline-2-carboxylate moiety which are connected via N-H…O intermolecular interactions with non-centrosymmetric crystal packing arrangement at microscopic scale is accountable to the enhancement of macroscopic second order NLO activity. The 2AQ crystal was successfully grown by a slow evaporation solution growth technique and its structure was determined in orthorhombic crystal system with acentric, P212121, space group. Hirshfeld surface analysis reveals that O…H intermolecular interactions primarily contributed with 31.0 % to the structural stabilization of 2AQ. The molecular structure of title compound has been confirmed by 1H and 13C NMR spectral studies. The vibrational modes of functional groups present in 2AQ have been assigned by using FTIR and FT-Raman spectroscopy. The grown 2AQ crystal exhibits high optical transparency with lower cut-off wavelength (275 nm) within the region of 275-1500 nm. The laser study confirmed that 2AQ exhibits high SHG efficiency of 12.6 times greater than that of KDP. TGA-DTA analysis revealed that 2AQ crystal had a thermal stability of 223 °C. The low dielectric constant and low dielectric loss at higher frequencies confirmed good crystalline nature with fewer defects of grown 2AQ crystal. The grown crystal exhibits soft material and positive photoconduction behaviour. Mulliken atomic distribution and FMOs analysis suggested that the strong intermolecular hydrogen bonding which lead to the enhancement of NLO activity. These properties suggest that 2AQ crystal is a suitable material for optoelectronic and laser frequency conversion applications.

Keywords: crystal growth, NLO activity, proton transfer complex, quantum chemical investigation

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Authors: Yi-Hao Wong, Li-Li Chan, Chee-Onn Leong, Stephen Ambu, Joon-Wah Mak, Priyasashi Sahu

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Background: Acanthamoeba is a genus of amoebae which lives as a free-living in nature or as a human pathogen that causes severe brain and eye infections. Virulence potential of Acanthamoeba is not constant and can change with growth conditions. DNA methylation, an epigenetic process which adds methyl groups to DNA, is used by eukaryotic cells, including several human parasites to control their gene expression. We used qPCR, siRNA gene silencing, and RNA sequencing (RNA-Seq) to study DNA-methyltransferase gene family (DNMT) in order to indicate the possibility of its involvement in programming Acanthamoeba virulence potential. Methods: A virulence-attenuated Acanthamoeba isolate (designation: ATCC; original isolate: ATCC 50492) was subjected to mouse passages to restore its pathogenicity; a virulence-reactivated isolate (designation: AC/5) was generated. Several established factors associated with Acanthamoeba virulence phenotype were examined to confirm the succession of reactivation process. Differential gene expression of DNMT between ATCC and AC/5 isolates was performed by qPCR. Silencing on DNMT gene expression in AC/5 isolate was achieved by siRNA duplex. Total RNAs extracted from ATCC, AC/5, and siRNA-treated (designation: si-146) were subjected to RNA-Seq for comparative transcriptomic analysis in order to identify the genome-wide effect of DNMT in regulating Acanthamoeba gene expression. qPCR was performed to validate the RNA-Seq results. Results: Physiological and cytophatic assays demonstrated an increased in virulence potential of AC/5 isolate after mouse passages. DNMT gene expression was significantly higher in AC/5 compared to ATCC isolate (p ≤ 0.01) by qPCR. si-146 duplex reduced DNMT gene expression in AC/5 isolate by 30%. Comparative transcriptome analysis identified the differentially expressed genes, with 3768 genes in AC/5 vs ATCC isolate; 2102 genes in si-146 vs AC/5 isolate and 3422 genes in si-146 vs ATCC isolate, respectively (fold-change of ≥ 2 or ≤ 0.5, p-value adjusted (padj) < 0.05). Of these, 840 and 1262 genes were upregulated and downregulated, respectively, in si-146 vs AC/5 isolate. Eukaryotic orthologous group (KOG) assignments revealed a higher percentage of downregulated gene expression in si-146 compared to AC/5 isolate, were related to posttranslational modification, signal transduction and energy production. Gene Ontology (GO) terms for those downregulated genes shown were associated with transport activity, oxidation-reduction process, and metabolic process. Among these downregulated genes were putative genes encoded for heat shock proteins, transporters, ubiquitin-related proteins, proteins for vesicular trafficking (small GTPases), and oxidoreductases. Functional analysis of similar predicted proteins had been described in other parasitic protozoa for their survival and pathogenicity. Decreased expression of these genes in si146-treated isolate may account in part for Acanthamoeba reduced pathogenicity. qPCR on 6 selected genes upregulated in AC/5 compared to ATCC isolate corroborated the RNA sequencing findings, indicating a good concordance between these two analyses. Conclusion: To the best of our knowledge, this study represents the first genome-wide analysis of DNA methylation and its effects on gene expression in Acanthamoeba spp. The present data indicate that DNA methylation has substantial effect on global gene expression, allowing further dissection of the genome-wide effects of DNA-methyltransferase gene in regulating Acanthamoeba pathogenicity.

Keywords: Acanthamoeba, DNA methylation, RNA sequencing, virulence

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Authors: A. Newman-Tancredi, R. Depoortère, P. Gruca, E. Litwa, M. Lason, M. Papp

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The N-methyl-D-aspartic acid (NMDA) receptor antagonist, ketamine, can elicit rapid-acting antidepressant (RAAD) effects in treatment-resistant patients, but it requires parenteral co-administration with a classical antidepressant under medical supervision. In addition, ketamine can also produce serious side effects that limit its long-term use, and there is much interest in identifying RAADs based on ketamine’s mechanism of action but with safer profiles. Ketamine elicits GABAergic interneuron inhibition, glutamatergic neuron stimulation, and, notably, activation of serotonin 5-HT1A receptors in the prefrontal cortex (PFC). Direct activation of the latter receptor subpopulation with selective ‘biased agonists’ may therefore be a promising strategy to identify novel RAADs and, consistent with this hypothesis, the prototypical cortical biased agonist, NLX-101, exhibited robust RAAD-like activity in the chronic mild stress model of depression (CMS). The present study compared the effects of a novel, selective 5-HT1A receptor-biased agonist, NLX-204, with those of ketamine and NLX-101. Materials and methods: CMS procedure was conducted on Wistar rats; drugs were administered either intraperitoneally (i.p.) or by bilateral intracortical microinjection. Ketamine: 10 mg/kg i.p. or 10 µg/side in PFC; NLX-204 and NLX-101: 0.08 and 0.16 mg/kg i.p. or 16 µg/side in PFC. In addition, interaction studies were carried out with systemic NLX-204 or NLX-101 (each at 0.16 mg/kg i.p.) in combination with intracortical WAY-100635 (selective 5-HT1A receptor antagonist; 2 µg/side) or muscimol (GABA-A receptor agonist, 12.5 ng/side). Anhedonia was assessed by CMS-induced decrease in sucrose solution consumption; anxiety-like behavior was assessed using the Elevated Plus Maze (EPM), and cognitive impairment was assessed by the Novel Object Recognition (NOR) test. Results: A single administration of NLX-204 was sufficient to reverse the CMS-induced deficit in sucrose consumption, similarly to ketamine and NLX-101. NLX-204 also reduced CMS-induced anxiety in the EPM and abolished CMS-induced NOR deficits. These effects were maintained (EPM and NOR) or enhanced (sucrose consumption) over a subsequent 2-week period of treatment. The anti-anhedonic response of the drugs was also maintained for several weeks Following treatment discontinuation, suggesting that they had sustained effects on neuronal networks. A single PFC administration of NLX-204 reversed deficient sucrose consumption, similarly to ketamine and NLX-101. Moreover, the anti-anhedonic activities of systemic NLX-204 and NLX 101 were abolished by coadministration with intracortical WAY-100635 or muscimol. Conclusions: (i) The antidepressant-like activity of NLX-204 in the rat CMS model was as rapid as that of ketamine or NLX-101, supporting targeting cortical 5-HT1A receptors with selective, biased agonists to achieve RAAD effects. (ii)The anti-anhedonic activity of systemic NLX-204 was mimicked by local administration of the compound in the PFC, confirming the involvement of cortical circuits in its RAAD-like effects. (iii) Notably, the effects of systemic NLX-204 and NLX-101 were abolished by PFC administration of muscimol, indicating that they act by (indirectly) eliciting a reduction in cortical GABAergic neurotransmission. This is consistent with ketamine’s mechanism of action and suggests that there are converging NMDA and 5-HT1A receptor signaling cascades in PFC underlying the RAAD-like activities of ketamine and NLX-204. Acknowledgements: The study was financially supported by NCN grant no. 2019/35/B/NZ7/00787.

Keywords: depression, ketamine, serotonin, 5-HT1A receptor, chronic mild stress

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Authors: Oluwaseun Adeleke, Samuel O. Ikani, Fidelis Edet, Anthony Nwala, Mopelola Raji, Simeon Christian Chukwu

Abstract:

Background: The DISC project collaborated with partners to implement demand creation and service delivery interventions, including the MoT (Moment of Truth) innovation, in over 500 health facilities across 15 states. This has increased the voluntary conversion rate to self-injection among women who opt for injectable contraception. While some facilities recorded an increasing trend in key performance indicators, few others persistently performed sub-optimally due to provider and system-related barriers. Methodology: Twenty-two facilities performing sub-optimally were selected purposively from three Nigerian states. Low productivity was appraised using low reporting rates and poor SI conversion rates as indicators. Interviews were conducted with health providers across these health facilities using a rapid diagnosis tool. The project also conducted a data quality assessment that evaluated the veracity of data elements reported across the three major sources of family planning data in the facility. Findings: The inability and sometimes refusal of providers to support clients to self-inject effectively was associated with the misunderstanding of its value to their work experience. It was also observed that providers still held a strong influence over clients’ method choices. Furthermore, providers held biases and misconceptions about DMPA-SC that restricted the access of obese clients and new acceptors to services – a clear departure from the recommendations of the national guidelines. Additionally, quality of care standards was compromised because job aids were not used to inform service delivery. Facilities performing sub-optimally often under-reported DMPA-SC utilization data, and there were multiple uncoordinated responsibilities for recording and reporting. Additionally, data validation meetings were not regularly convened, and these meetings were ineffective in authenticating data received from health facilities. Other reasons for sub-optimal performance included poor documentation and tracking of stock inventory resulting in commodity stockouts, low client flow because of poor positioning of health facilities, and ineffective messaging. Some facilities lacked adequate human and material resources to provide services effectively and received very few supportive supervision visits. Supportive supervision visits and Data Quality Audits have been useful to address the aforementioned performance barriers. The project has deployed digital DMPA-SC self-injection checklists that have been aligned with nationally approved templates. During visits, each provider and community mobilizer is accorded special attention by the supervisor until he/she can perform procedures in line with best practice (protocol). Conclusion: This narrative provides a summary of a range of factors that identify health facilities performing sub-optimally in their provision of DMPA-SC services. Findings from this assessment will be useful during project design to inform effective strategies. As the project enters its final stages of implementation, it is transitioning high-impact activities to state institutions in the quest to sustain the quality of service beyond the tenure of the project. The project has flagged activities, as well as created protocols and tools aimed at placing state-level stakeholders at the forefront of improving productivity in health facilities.

Keywords: family planning, contraception, DMPA-SC, self-care, self-injection, barriers, opportunities, performance

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Authors: Sajeli A. Begum, Ameer Basha, Kirti Hira, Rukaiyya Khan

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Cyclic dipeptides are simple diketopiperazine derivatives being investigated by several scientists for their biological effects which include anticancer, antimicrobial, haematological, anticonvulsant, immunomodulatory effect, etc. They are potentially active microbial metabolites having been synthesized too, for developing into drug candidates. Cultures of Pseudomonas species have earlier been reported to produce cyclic dipeptides, helping in quorum sensing signals and bacterial–host colonization phenomena during infections, causing cell anti-proliferation and immunosuppression. Fluorescing Pseudomonas species have been identified to secrete lipid derivatives, peptides, pyrroles, phenazines, indoles, aminoacids, pterines, pseudomonic acids and some antibiotics. In the present work, results of investigation on the cyclic dipeptide metabolites secreted by the culture broth of Pseudomonas species as potent pro-inflammatory cytokine inhibitors are discussed. The bacterial strain was isolated from the rhizospheric soil of groundnut crop and identified as Pseudomonas aeruginosa by 16S rDNA sequence (GenBank Accession No. KT625586). Culture broth of this strain was prepared by inoculating into King’s B broth and incubating at 30 ºC for 7 days. The ethyl acetate extract of culture broth was prepared and lyophilized to get a dry residue (EEPA). Lipopolysaccharide (LPS)-induced ELISA assay proved the inhibition of tumor necrosis factor-alpha (TNF-α) secretion in culture supernatant of RAW 264.7 cells by EEPA (IC50 38.8 μg/mL). The effect of oral administration of EEPA on plasma TNF-α level in rats was tested by ELISA kit. The LPS mediated plasma TNF-α level was reduced to 45% with 125 mg/kg dose of EEPA. Isolation of the chemical constituents of EEPA through column chromatography yielded ten cyclic dipeptides, which were characterized using nuclear magnetic resonance and mass spectroscopic techniques. These cyclic dipeptides are biosynthesized in microorganisms by multifunctional assembly of non-ribosomal peptide synthases and cyclic dipeptide synthase. Cyclo (Gly-L-Pro) was found to be more potentially (IC50 value 4.5 μg/mL) inhibiting TNF-α production followed by cyclo (trans-4-hydroxy-L-Pro-L-Phe) (IC50 value 14.2 μg/mL) and the effect was equal to that of standard immunosuppressant drug, prednisolone. Further, the effect was analyzed by determining mRNA expression of TNF-α in LPS-stimulated RAW 264.7 macrophages using quantitative real-time reverse transcription polymerase chain reaction. EEPA and isolated cyclic dipeptides demonstrated diminution of TNF-α mRNA expression levels in a dose-dependent manner under the tested conditions. Also, they were found to control the expression of other pro-inflammatory cytokines like IL-1β and IL-6, when tested through their mRNA expression levels in LPS-stimulated RAW 264.7 macrophages under LPS-stimulated conditions. In addition, significant inhibition effect was found on Nitric oxide production. Further all the compounds exhibited weak toxicity to LPS-induced RAW 264.7 cells. Thus the outcome of the study disclosed the effectiveness of EEPA and the isolated cyclic dipeptides in down-regulating key cytokines involved in pathophysiology of autoimmune diseases.In another study led by the investigators, microbial cyclic dipeptides were found to exhibit excellent antimicrobial effect against Fusarium moniliforme which is an important causative agent of Sorghum grain mold disease. Thus, cyclic dipeptides are emerging small molecular drug candidates for various autoimmune diseases.

Keywords: cyclic dipeptides, cytokines, Fusarium moniliforme, Pseudomonas, TNF-alpha

Procedia PDF Downloads 183

Authors: Patrizia Firmani, Sara Perucchini, Irene Rapone, Raffella Borrelli, Stefano Chiaberge, Manuela Grande, Rosamaria Marrazzo, Alberto Savoini, Andrea Siviero, Silvia Spera, Fabio Vago, Davide Deriu, Sergio Fanutti, Alessandro Oldani

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According to the Renewable Energy Directive II, the use of palm oil in diesel will be gradually reduced from 2023 and should reach zero in 2030 due to the deforestation caused by its production. Eni aims at finding alternative feedstocks for its biorefineries to eliminate the use of palm oil by 2023. Therefore, the ideal vegetable oils to be used in bio-refineries are those obtainable from plants that grow in marginal lands and with low impact on food-and-feed chain; hence, Eni research is studying the possibility of using oleaginous seeds, such as castor, croton, and cotton, to extract the oils to be exploited as feedstock in bio-refineries. To verify their suitability for the upgrading processes, an analytical protocol for their characterization has been drawn up and applied. The analytical characterizations include a step of water and ashes content determination, elemental analysis (CHNS analysis, X-Ray Fluorescence, Inductively Coupled Plasma - Optical Emission Spectroscopy, ICP– Mass Spectrometry), and total acid number determination. Gas chromatography coupled to flame ionization detector (GC-FID) is used to quantify the lipid content in terms of free fatty acids, mono-, di- and triacylglycerols, and fatty acids composition. Eventually, Nuclear Magnetic Resonance and Fourier Transform-Infrared spectroscopies are exploited with GC-MS and Fourier Transform-Ion Cyclotron Resonance to study the composition of the oils. This work focuses on the GC-FID analysis of the lipid fraction of these oils, as the main constituent and of greatest interest for bio-refinery processes. Specifically, the lipid component of the extracted oil was quantified after sample silanization and transmethylation: silanization allows the elution of high-boiling compounds and is useful for determining the quantity of free acids and glycerides in oils, while transmethylation leads to a mixture of fatty acid esters and glycerol, thus allowing to evaluate the composition of glycerides in terms of Fatty Acids Methyl Esters (FAME). Cotton oil was extracted from cotton oilcake, croton oil was obtained by seeds pressing and seeds and oilcake ASE extraction, while castor oil comes from seed pressing (not performed in Eni laboratories). GC-FID analyses reported that the cotton oil is 90% constituted of triglycerides and about 6% diglycerides, while free fatty acids are about 2%. In terms of FAME, C18 acids make up 70% of the total and linoleic acid is the major constituent. Palmitic acid is present at 17.5%, while the other acids are in low concentration (<1%). Both analyzes show the presence of non-gas chromatographable compounds. Croton oils from seed pressing and extraction mainly contain triglycerides (98%). Concerning FAME, the main component is linoleic acid (approx. 80%). Oilcake croton oil shows higher abundance of diglycerides (6% vs ca 2%) and a lower content of triglycerides (38% vs 98%) compared to the previous oils. Eventually, castor oil is mostly constituted of triacylglycerols (about 69%), followed by diglycerides (about 10%). About 85.2% of total FAME is ricinoleic acid, as a constituent of triricinolein, the most abundant triglyceride of castor oil. Based on the analytical results, these oils represent feedstocks of interest for possible exploitation as advanced biofuels.

Keywords: analytical protocol, biofuels, biorefinery, gas chromatography, vegetable oil

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Authors: Mary-Ann N. Jallad, Dalal F. Jaber, Alexander M. Abdelnoor

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Introduction: Current evidence confirms that both innate and adaptive immune systems are capable of recognizing and abolishing malignant cells. The emergence of cancerous tumors in patients is, therefore, an indication that certain cancer cells can resist elimination by the immune system through a process known as “immune evasion”. In fact, cancer cells often exploit regulatory mechanisms to escape immunity. Such mechanisms normally exist to control the immune responses and prohibit exaggerated or autoimmune reactions. Recently, immunotherapies have shown promising yet limited results. Therefore this study investigates several immunotherapeutic combinations and devises a triple immunotherapy which harnesses the innate and acquired immune responses towards the annihilation of malignant cells through overcoming their ability of immune evasion, consequently hampering malignant progression and eliminating established tumors. The aims of the study are to rule out acute/chronic toxic effects of the proposed treatment combinations, to assess the effect of these combinations on tumor growth and survival rates, and to investigate potential mechanisms underlying the phenotypic results through analyzing serum levels of anti-tumor cytokines, angiogenic factors and tumor progression indicator, and the tumor-infiltrating immune-cells populations. Methodology: For toxicity analysis, cancer-free C57BL/6 mice are randomized into 9 groups: Group 1 untreated, group 2 treated with sterile saline (solvent of used treatments), group 3 treated with Monophosphoryl-lipid-A, group 4 with anti-CTLA4-antibodies, group 5 with 1-Methyl-Tryptophan (Indolamine-Dioxygenase-1 inhibitor), group 6 with both MPLA and anti-CTLA4-antibodies, group 7 with both MPLA and 1-MT, group 8 with both anti-CTLA4-antibodies and 1-MT, and group 9 with all three: MPLA, anti-CTLA4-antibodies and 1-MT. Mice are monitored throughout the treatment period and for three following months. At that point, histological sections from their main organs are assessed. For tumor progression and survival analysis, a murine melanoma model is generated by injecting analogous mice with B16F10 melanoma cells. These mice are segregated into the listed nine groups. Their tumor size and survival are monitored. For a depiction of underlying mechanisms, melanoma-bearing mice from each group are sacrificed at several time-points. Sera are tested to assess the levels of Interleukin-12 (IL-12), Vascular-Endothelial-Growth Factor (VEGF), and S100B. Furthermore, tumors are excised for analysis of infiltrated immune cell populations including T-cells, macrophages, natural killer cells and immune-regulatory cells. Results: Toxicity analysis shows that all treated groups present no signs of neither acute nor chronic toxicity. Their appearance and weights were comparable to those of control groups throughout the treatment period and for the following 3 months. Moreover, histological sections from their hearts, kidneys, lungs, and livers were normal. Work is ongoing for completion of the remaining study aims. Conclusion: Toxicity was the major concern for the success of the proposed comprehensive combinational therapy. Data generated so far ruled out any acute or chronic toxic effects. Consequently, ongoing work is quite promising and may significantly contribute to the development of more effective immunotherapeutic strategies for the treatment of cancer patients.

Keywords: cancer immunotherapy, check-point blockade, combination therapy, melanoma

Procedia PDF Downloads 91

Authors: Irina Alexandra Paun, Mona Mihailescu, Marian Zamfirescu, Catalin Romeo Luculescu, Adriana Maria Acasandrei, Cosmin Catalin Mustaciosu, Roxana Cristina Popescu, Maria Dinescu

Abstract:

A major concern in bone tissue engineering is to develop complex 3D architectures that mimic the natural cells environment, facilitate the cells growth in a defined manner and allow the flow transport of nutrients and metabolic waste. In particular, porous structures of controlled pore size and positioning are indispensable for growing human-like bone structures. Another concern is to monitor both the structures and the seeded cells with high spatial resolution and without interfering with the cells natural environment. The present approach relies on laser-based technologies employed for fabricating porous biomimetic structures that support the growth of osteoblast-like cells and for their non-invasive 3D imaging. Specifically, the porous structures were built by two photon polymerization –direct writing (2PP_DW) of the commercially available photoresists IL-L780, using the Photonic Professional 3D lithography system. The structures consist of vertical tubes with micrometer-sized heights and diameters, in a honeycomb-like spatial arrangement. These were fabricated by irradiating the IP-L780 photoresist with focused laser pulses with wavelength centered at 780 nm, 120 fs pulse duration and 80 MHz repetition rate. The samples were precisely scanned in 3D by piezo stages. The coarse positioning was done by XY motorized stages. The scanning path was programmed through a writing language (GWL) script developed by Nanoscribe. Following laser irradiation, the unexposed regions of the photoresist were washed out by immersing the samples in the Propylene Glycol Monomethyl Ether Acetate (PGMEA). The porous structures were seeded with osteoblast like MG-63 cells and their osteogenic potential was tested in vitro. The cell-seeded structures were analyzed in 3D using the digital holographic microscopy technique (DHM). DHM is a marker free and high spatial resolution imaging tool, where the hologram acquisition is performed non-invasively i.e. without interfering with the cells natural environment. Following hologram recording, a digital algorithm provided a 3D image of the sample, as well as information about its refractive index, which is correlated with the intracellular content. The axial resolution of the images went down to the nanoscale, while the temporal scales ranged from milliseconds up to hours. The hologram did not involve sample scanning and the whole image was available in one frame recorded going over 200μm field of view. The digital holograms processing provided 3D quantitative information on the porous structures and allowed a quantitative analysis of the cellular response in respect to the porous architectures. The cellular shape and dimensions were found to be influenced by the underlying micro relief. Furthermore, the intracellular content gave evidence on the beneficial role of the porous structures in promoting osteoblast differentiation. In all, the proposed laser-based protocol emerges as a promising tool for the fabrication and non-invasive imaging of porous constructs for bone tissue engineering. Acknowledgments: This work was supported by a grant of the Romanian Authority for Scientific Research and Innovation, CNCS-UEFISCDI, project PN-II-RU-TE-2014-4-2534 (contract 97 from 01/10/2015) and by UEFISCDI PN-II-PT-PCCA no. 6/2012. A part of this work was performed in the CETAL laser facility, supported by the National Program PN 16 47 - LAPLAS IV.

Keywords: biomimetic, holography, laser, osteoblast, two photon polymerization

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Authors: Yogesh Dalvi, Ruby Varghese, Nibu Varghese, C. K. Krishnan Nair

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Introduction: The Genus Phellinus, locally known as Phansomba is a well-known traditional folk medicine. Especially, in Western Ghats of India, many tribes use several species of Phellinus for various ailments related to teeth, throat, tongue, stomach and even wound healing. It is one of the few mushrooms which play a pivotal role in Ayurvedic Dravyaguna. Aim: The present study focuses on to investigate phytochemical analysis, antioxidant, and antitumor (in vitro and in vivo) potential of Phellinus robinae from South India, Kerala Material and Methods: The present study explores the following: 1. Phellinus samples were collected from Ranni, Pathanamthitta district of Kerala state, India from Artocarpus heterophyllus Lam. and species were identified using rDNA region. 2. The fruiting body was shadow dried, powdered and extracted with 50% alcohol using water bath at 60°C which was further condensed by rotary evaporator and lyophilized at minus 40°C temperature. 3. Secondary metabolites were analyzed by using various phytochemical screening assay (Hager’s Test, Wagner’s Test, Sodium hydroxide Test, Lead acetate Test, Ferric chloride Test, Folin-ciocalteu Test, Foaming Test, Benedict’s test, Fehling’s Test and Lowry’s Test). 4. Antioxidant and free radical scavenging activity were analyzed by DPPH, FRAP and Iron chelating assay. 5. The antitumor potential of Water alcohol extract of Phellinus (PAWE) is evaluated through In vitro condition by Trypan blue dye exclusion method in DLA cell line and In vivo by murine model. Result and Discussion: Preliminary phytochemical screening by various biochemical tests revealed presence of a variety of active secondary molecules like alkaloids, flavanoids, saponins, carbohydrate, protein and phenol. In DPPH and FRAP assay PAWE showed significantly higher antioxidant activity as compared to standard Ascorbic acid. While, in Iron chelating assay, PAWE exhibits similar antioxidant activity that of Butylated Hydroxytoluene (BHT) as standard. Further, in the in vitro study, PAWE showed significant inhibition on DLA cell proliferation in dose dependent manner and showed no toxicity on mice splenocytes, when compared to standard chemotherapy drug doxorubicin. In vivo study, oral administration of PAWE showed dose dependent tumor regression in mice and also raised the immunogenicity by restoring levels of antioxidant enzymes in liver and kidney tissue. In both in vitro and in vivo gene expression studies PAWE up-regulates pro-apoptotic genes (Bax, Caspases 3, 8 and 9) and down- regulates anti-apoptotic genes (Bcl2). PAWE also down regulates inflammatory gene (Cox-2) and angiogenic gene (VEGF). Conclusion: Preliminary phytochemical screening revealed that PAWE contains various secondary metabolites which contribute to its antioxidant and free radical scavenging property as evaluated by DPPH, FRAP and Iron chelating assay. PAWE exhibits anti-proliferative activity by the induction of apoptosis through a signaling cascade of death receptor-mediated extrinsic (Caspase8 and Tnf-α), as well as mitochondria-mediated intrinsic (caspase9) and caspase pathways (Caspase3, 8 and 9) and also by regressing angiogenic factor (VEGF) without any inflammation or adverse side effects. Hence, PAWE serve as a potential antioxidant and antitumor agent.

Keywords: antioxidant, antitumor, Dalton lymphoma ascites (DLA), fungi, Phellinus robinae

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Authors: Ipsita Pujari, Abitha Thomas, Vidhu S. Babu, K. Satyamoorthy

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Orchids are esteemed as celebrities in cut flower industry globally, due to their long-lasting fragrance and freshness. Apart from splendor, the unique metabolites endowed with pharmaceutical potency have made them one of the most hunted in plant kingdom. This had led to their trafficking, resulting in habitat loss, subsequently making them occupiers of IUCN red list as RET species. Many of the orchids especially wild varieties still remain undiscovered. In view to protect and conserve the wild germplasm, researchers have been inventing novel micropropagation protocols; thereby conserving Orchids. India is overflowing with exclusive wild cultivars of Orchids, whose pharmaceutical properties remain untapped and are not marketed owing to relatively small flowers. However, their germplasm is quite pertinent to be preserved for making unusual hybrids. Dendrobium genus is the second largest among Orchids exists in India and has highest demand attributable to enduring cut flowers and significant therapeutic uses in traditional medicinal system. Though the genus is quite endemic in Western Ghat regions of the country, many species are still anonymous with their unknown curative properties. A standard breeding cycle in Orchids usually takes five to seven years (Dendrobium hybrids taking a long juvenile phase of two to five years reaching maturity and flowering stage) and this extensive life cycle has always hindered the development of Dendrobium breeding. Dendrobium is reported with essential therapeutic plant bio-chemicals and ‘Moscatilin’ is one, found exclusive to this famous Dendrobium genus. Moscatilin is reported to have anti-mutagenic and anti-cancer properties, whose positive action has very recently been demonstrated against a range of cancers. Our preliminary study here established a simple and economic small-scale propagation protocol of Dendrobium ovatum describing in vitro production of Moscatilin. Subsequently for enhancing the content of Moscatilin, an efficient experimental related to the organization of transgenic (hairy) D. ovatum root cultures through infection of Agrobacterium rhizogenes 2364 strain on MS basal medium is being reported in the present study. Hairy roots generated on almost half of the explants used (spherules, in vitro plantlets and calli) maintained through suspension cultures, after 8 weeks of co-cultivation with Agrobacterium rhizogenes. GFP assay performed with isolated hairy roots has confirmed the integrative transformation which was further positively confirmed by PCR using rolB gene specific primers. Reverse phase-high performance liquid chromatography and mass spectrometry techniques were used for quantification and accurate identification of Moscatilin respectively from transgenic systems. A noticeable ~3 fold increase in contents were observed in transformed D. ovatum root cultures as compared to the simple in vitro culture, callus culture and callus regeneration plantlets. Role of elicitors e.g., Methyl jasmonate, Salicylic acid, Yeast extract and Chitosan were tested for elevating the Moscatilin content to obtain a comprehensive optimized protocol facilitating the in vitro production of valuable Moscatilin with larger yield. This study would provide evidence towards the in vitro assembly of Moscatilin within a short time-period through not a so-expensive technology for the first time. It also serves as an appropriate basis for bioreactor scale-up resulting in commercial bioproduction of Moscatilin.

Keywords: bioproduction, Dendrobium ovatum, hairy root culture, moscatilin

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Authors: Agman Gupta, Rajashekar Badam, Noriyoshi Matsumi

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Introduction: Recently, silicon has been recognized as one of the potential alternatives as anode active material in Li-ion batteries (LIBs) to replace the conventionally used graphite anodes. Silicon is abundantly present in the nature, it can alloy with lithium metal, and has a higher theoretical capacity (~4200 mAhg-1) that is approximately 10 times higher than graphite. However, because of a large volume expansion (~400%) upon repeated de-/alloying, the pulverization of Si particles causes the exfoliation of electrode laminate leading to the loss of electrical contact and adversely affecting the formation of solid-electrolyte interface (SEI).1 Functional polymers as binders have emerged as a competitive strategy to mitigate these drawbacks and failure mechanism of silicon anodes.1 A variety of aqueous/non-aqueous polymer binders like sodium carboxy-methyl cellulose (CMC-Na), styrene butadiene rubber (SBR), poly(acrylic acid), and other variants like mussel inspired binders have been investigated to overcome these drawbacks.1 However, there are only a few reports that mention the attempt of addressing all the drawbacks associated with silicon anodes effectively using a single novel functional polymer system as a binder. In this regard, here, we report a novel highly robust n-type bisiminoacenaphthenequinone (BIAN)-paraphenylene-based crosslinked polymer as a binder for Si anodes in lithium-ion batteries (Fig. 1). On its application, crosslinked-BIAN binder was evaluated to provide mechanical robustness to the large volume expansion of Si particles, maintain electrical conductivity within the electrode laminate, and facilitate in the formation of a thin SEI by restricting the extent of electrolyte decomposition on the surface of anode. The fabricated anodic half-cells were evaluated electrochemically for their rate capability, cyclability, and discharge capacity. Experimental: The polymerized BIAN (P-BIAN) copolymer was synthesized as per the procedure reported by our group.2 The synthesis of crosslinked P-BIAN: a solution of P-BIAN copolymer (1.497 g, 10 mmol) in N-methylpyrrolidone (NMP) (150 ml) was set-up to stir under reflux in nitrogen atmosphere. To this, 1,6-dibromohexane (5 mmol, 0.77 ml) was added dropwise. The resultant reaction mixture was stirred and refluxed at 150 °C for 24 hours followed by refrigeration for 3 hours at 5 °C. The product was obtained by evaporating the NMP solvent under reduced pressure and drying under vacuum at 120 °C for 12 hours. The obtained product was a black colored sticky compound. It was characterized by 1H-NMR, XPS, and FT-IR techniques. Results and Discussion: The N 1s XPS spectrum of the crosslinked BIAN polymer showed two characteristic peaks corresponding to the sp2 hybridized nitrogen (-C=N-) at 399.6 eV of the diimine backbone in the BP and quaternary nitrogen at 400.7 eV corresponding to the crosslinking of BP via dibromohexane. The DFT evaluation of the crosslinked BIAN binder showed that it has a low lying lowest unoccupied molecular orbital (LUMO) that enables it to get doped in the reducing environment and influence the formation of a thin (SEI). Therefore, due to the mechanically robust crosslinked matrices as well as its influence on the formation of a thin SEI, the crosslinked BIAN binder stabilized the Si anode-based half-cell for over 1000 cycles with a reversible capacity of ~2500 mAhg-1 and ~99% capacity retention as shown in Fig. 2. The dynamic electrochemical impedance spectroscopy (DEIS) characterization of crosslinked BIAN-based anodic half-cell confirmed that the SEI formed was thin in comparison with the conventional binder-based anodes. Acknowledgement: We are thankful to the financial support provided by JST-Mirai Program, Grant Number: JP18077239

Keywords: self-healing binder, n-type binder, thin solid-electrolyte interphase (SEI), high-capacity silicon anodes, low-LUMO

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Authors: Pejman Salimi, Sebastiano Tieuli, Somayeh Taghavi, Michela Signoretto, Remo Proietti Zaccaria

Abstract:

Increasing the growth of industrial waste due to the large quantities of production leads to numerous environmental and economic challenges, such as climate change, soil and water contamination, human disease, etc. Energy recovery of waste can be applied to produce heat or electricity. This strategy allows for the reduction of energy produced using coal or other fuels and directly reduces greenhouse gas emissions. Among different factories, leather manufacturing plays a very important role in the whole world from the socio-economic point of view. The leather industry plays a very important role in our society from a socio-economic point of view. Even though the leather industry uses a by-product from the meat industry as raw material, it is considered as an activity demanding integrated prevention and control of pollution. Along the entire process from raw skins/hides to finished leather, a huge amount of solid and water waste is generated. Solid wastes include fleshings, raw trimmings, shavings, buffing dust, etc. One of the most abundant solid wastes generated throughout leather tanning is shaving waste. Leather shaving is a mechanical process that aims at reducing the tanned skin to a specific thickness before tanning and finishing. This product consists mainly of collagen and tanning agent. At present, most of the world's leather processing is chrome-tanned based. Consequently, large amounts of chromium-containing shaving wastes need to be treated. The major concern about the management of this kind of solid waste is ascribed to chrome content, which makes the conventional disposal methods, such as landfilling and incineration, not practicable. Therefore, many efforts have been developed in recent decades to promote eco-friendly/alternative leather production and more effective waste management. Herein, shaving waste resulting from metal-free tanning technology is proposed as low-cost precursors for the preparation of carbon material as anodes for lithium-ion batteries (LIBs). In line with the philosophy of a reduced environmental impact, for preparing fully sustainable and environmentally friendly LIBs anodes, deionized water and carboxymethyl cellulose (CMC) have been used as alternatives to toxic/teratogen N-methyl-2- pyrrolidone (NMP) and to biologically hazardous Polyvinylidene fluoride (PVdF), respectively. Furthermore, going towards the reduced cost, we employed water solvent and fluoride-free bio-derived CMC binder (as an alternative to NMP and PVdF, respectively) together with LiFePO₄ (LFP) when a full cell was considered. These actions make closer to the 2030 goal of having green LIBs at 100 $ kW h⁻¹. Besides, the preparation of the water-based electrodes does not need a controlled environment and due to the higher vapour pressure of water in comparison with NMP, the water-based electrode drying is much faster. This aspect determines an important consequence, namely a reduced energy consumption for the electrode preparation. The electrode derived from leather waste demonstrated a discharge capacity of 735 mAh g⁻¹ after 1000 charge and discharge cycles at 0.5 A g⁻¹. This promising performance is ascribed to the synergistic effect of defects, interlayer spacing, heteroatoms-doped (N, O, and S), high specific surface area, and hierarchical micro/mesopore structure of the biochar. Interestingly, these features of activated biochars derived from the leather industry open the way for possible applications in other EESDs as well.

Keywords: biowaste, lithium-ion batteries, physical activation, waste management, leather industry

Procedia PDF Downloads 139

Authors: Oluwaseun Adeleke, Samuel O. Ikani, Simeon Christian Chukwu, Fidelis Edet, Anthony Nwala, Mopelola Raji, Simeon Christian Chukwu

Abstract:

Background: Health service providers are offered in-service training periodically to strengthen their ability to deliver services that are ethical, quality, timely and safe. Not all capacity-building courses have successfully resulted in intended service delivery outcomes because of poor training content, design, approach, and ambiance. The Delivering Innovations in Selfcare (DISC) project developed a Moment of Truth innovation, which is a proven training model focused on improving consumer/provider interaction that leads to an increase in the voluntary uptake of subcutaneous depot medroxyprogesterone acetate (DMPA-SC) self-injection among women who opt for injectable contraception. Methodology: Six months after training on a moment of truth (MoT) training manual, the project conducted two intensive rounds of qualitative data collection and triangulation that included provider, client, and community mobilizer interviews, facility observations, and routine program data collection. Respondents were sampled according to a convenience sampling approach, and data collected was analyzed using a codebook and Atlas-TI. Providers and clients were interviewed to understand their experience, perspective, attitude, and awareness about the DMPA-SC self-inject. Data were collected from 12 health facilities in three states – eight directly trained and four cascades trained. The research team members came together for a participatory analysis workshop to explore and interpret emergent themes. Findings: Quality-of-service delivery and performance outcomes were observed to be significantly better in facilities whose providers were trained directly trained by the DISC project than in sites that received indirect training through master trainers. Facilities that were directly trained recorded SI proportions that were twice more than in cascade-trained sites. Direct training comprised of full-day and standalone didactic and interactive sessions constructed to evoke commitment, passion and conviction as well as eliminate provider bias and misconceptions in providers by utilizing human interest stories and values clarification exercises. Sessions also created compelling arguments using evidence and national guidelines. The training also prioritized demonstration sessions, utilized job aids, particularly videos, strengthened empathetic counseling – allaying client fears and concerns about SI, trained on positioning self-inject first and side effects management. Role plays and practicum was particularly useful to enable providers to retain and internalize new knowledge. These sessions provided experiential learning and the opportunity to apply one's expertise in a supervised environment where supportive feedback is provided in real-time. Cascade Training was often a shorter and abridged form of MoT training that leveraged existing training already planned by master trainers. This training was held over a four-hour period and was less emotive, focusing more on foundational DMPA-SC knowledge such as a reorientation to DMPA-SC, comparison of DMPA-SC variants, counseling framework and skills, data reporting and commodity tracking/requisition – no facility practicums. Training on self-injection was not as robust, presumably because they were not directed at methods in the contraceptive mix that align with state/organizational sponsored objectives – in this instance, fostering LARC services. Conclusion: To achieve better performance outcomes, consideration should be given to providing training that prioritizes practice-based and emotive content. Furthermore, a firm understanding and conviction about the value training offers improve motivation and commitment to accomplish and surpass service-related performance outcomes.

Keywords: training, performance outcomes, innovation, family planning, contraception, DMPA-SC, self-care, self-injection.

Procedia PDF Downloads 49