Search results for: calcium salt of palm oil fatty acid

Authors: Niharika Kaushal, Minni Singh

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Citrus fruits fall into the category of those commercially grown fruits that constitute an excellent repository of phytochemicals with health-promoting properties. Fruits belonging to the citrus family, when processed by industries, produce tons of agriculture by-products in the form of peels, pulp, and seeds, which normally have no further usage and are commonly discarded. In spite of this, such residues are of paramount importance due to their richness in valuable compounds; therefore, agro-waste is considered a valuable bioresource for various purposes in the food sector. A range of biological properties, including anti-oxidative, anti-cancerous, anti-inflammatory, anti-allergenicity, and anti-aging activity, have been reported for these bioactive compounds. Taking advantage of these inexpensive residual sources requires special attention to extract bioactive compounds. Mandarin (Citrus nobilis X Citrus deliciosa) is a potential source of bioflavonoids with antioxidant properties, and it is increasingly regarded as a functional food. Despite these benefits, flavonoids suffer from a barrier of pre-systemic metabolism in gastric fluid, which impedes their effectiveness. Therefore, colloidal delivery systems can completely overcome the barrier in question. This study involved the extraction and identification of key flavonoids from mandarin biomass. Using a green chemistry approach, supercritical fluid extraction at 330 bar, temperature 40C, and co-solvent 10% ethanol was employed for extraction, and the identification of flavonoids was made by mass spectrometry. As flavonoids are concerned with a limitation, the obtained extract was encapsulated in polylactic-co-glycolic acid (PLGA) matrix using a solvent evaporation method. Additionally, the antioxidant potential was evaluated by the 2,2-diphenylpicrylhydrazyl (DPPH) assay. A release pattern of flavonoids was observed over time using simulated gastrointestinal fluids. From the results, it was observed that the total flavonoids extracted from the mandarin biomass were estimated to be 47.3 ±1.06 mg/ml rutin equivalents as total flavonoids. In the extract, significantly, polymethoxyflavones (PMFs), tangeretin and nobiletin were identified, followed by hesperetin and naringin. The designed flavonoid-PLGA nanoparticles exhibited a particle size between 200-250nm. In addition, the bioengineered nanoparticles had a high entrapment efficiency of nearly 80.0% and maintained stability for more than a year. Flavonoid nanoparticles showed excellent antioxidant activity with an IC50 of 0.55μg/ml. Morphological studies revealed the smooth and spherical shape of nanoparticles as visualized by Field emission scanning electron microscopy (FE-SEM). Simulated gastrointestinal studies of free extract and nanoencapsulation revealed the degradation of nearly half of the flavonoids under harsh acidic conditions in the case of free extract. After encapsulation, flavonoids exhibited sustained release properties, suggesting that polymeric encapsulates are efficient carriers of flavonoids. Thus, such technology-driven and biomass-derived products form the basis for their use in the development of functional foods with improved therapeutic potential and antioxidant properties. As a result, citrus processing waste can be considered a new resource that has high value and can be used for promoting its utilization.

Keywords: citrus, agrowaste, flavonoids, nanoparticles

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Authors: R. Di Lorenzo, S. Laneri, A. Sacchi

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Hair surface properties affect hair texture and shine, whereas the healthy state of the hair cortex sways hair ends. Even if cosmetic treatments are intrinsically safe, there is potentially damaging action on the hair fibers. Loss of luster, frizz, split ends, and other hair problems are particularly prevalent among people who repeatedly alter the natural style of their hair or among people with intrinsically weak hair. Technological and scientific innovations in hair care thus become invaluable allies to preserve their natural well-being and shine. The study evaluated restoring keratin-like ingredients that improve hair fibers' structural integrity, increase tensile strength, improve hair manageability and moisturizing. The hair shaft is composed of 65 - 95% of keratin. It gives the hair resistance, elasticity, and plastic properties and also contributes to their waterproofing. Providing exogenous keratin is, therefore, a practical approach to protect and nourish the hair. By analyzing the amino acid composition of keratin, we find a high frequency of hydrophobic amino acids. It confirms the critical role interactions, mainly hydrophobic, between cosmetic products and hair. The active ingredient analyzed comes from vegetable proteins through an enzymatic cut process that selected only oligo- and polypeptides (> 3500 KDa) rich in amino acids with hydrocarbon side chains apolar or sulfur. These chemical components are the most expressed amino acids at the level of the capillary keratin structure, and it determines the most significant possible compatibility with the target substrate. Given the biological variability of the sources, it isn't easy to define a constant and reproducible molecular formula of the product. Still, it consists of hydroxypropiltrimonium vegetable peptides with keratin-like performances. 20 natural hair tresses (30 cm in length and 0.50 g weight) were treated with the investigated products (5 % v/v aqueous solution) following a specific protocol and compared with non-treated (Control) and benchmark-keratin-treated strands (Benchmark). Their brightness, moisture content, cortical and surface integrity, and tensile strength were evaluated and statistically compared. Keratin-like treated hair tresses showed better results than the other two groups (Control and Benchmark). The product improves the surface with significant regularization of the cuticle closure, improves the cortex and the peri-medullar area filling, gives a highly organized and tidy structure, delivers a significant amount of sulfur on the hair, and is more efficient moisturization and imbibition power, increases hair brightness. The hydroxypropyltrimonium quaternized group added to the C-terminal end interacts with the negative charges that form on the hair after washing when disheveled and tangled. The interactions anchor the product to the hair surface, keeping the cuticles adhered to the shaft. The small size allows the peptides to penetrate and give body to the hair, together with a conditioning effect that gives an image of healthy hair. Results suggest that the product is a valid ally in numerous restructuring/conditioning, shaft protection, straightener/dryer-damage prevention hair care product.

Keywords: conditioning, hair damage, hair, keratin, polarized light microscopy, scanning electron microscope, thermogravimetric analysis

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Authors: E. Locatelli, I. Monaco, R. C. Martin, Y. Li, R. Pini, M. Chiariello, M. Comes Franchini

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Despite the many advantages of application of nanomaterials in the field of nanomedicine, increasing concerns have been expressed on their potential adverse effects on human health. There is urgency for novel green strategies toward novel materials with enhanced biocompatibility using safe reagents. Photothermal ablation therapy, which exploits localized heat increase of a few degrees to kill cancer cells, has appeared recently as a non-invasive and highly efficient therapy against various cancer types; anyway new agents able to generate hyperthermia when irradiated are needed and must have precise biocompatibility in order to avoid damage to healthy tissues and prevent toxicity. Recently, there has been increasing interest in magnesium as a biomaterial: it is the fourth most abundant cation in the human body, and it is essential for human metabolism. However magnesium nanoparticles (Mg NPs) have had limited diffusion due to the high reduction potential of magnesium cations, which makes NPs synthesis challenging. Herein, we report the synthesis of Mg NPs and their surface functionalization for the obtainment of a stable and biocompatible nanomaterial suitable for photothermal ablation therapy against cancer. We synthesized the Mg crystals by reducing MgCl2 with metallic lithium and exploiting naphthalene as an electron carrier: the lithium–naphthalene complex acts as the real reducing agent. Firstly, the nanocrystal particles were coated with the ligand 12-ethoxy ester dodecanehydroxamic acid, and then entrapped into water-dispersible polymeric micelles (PMs) made of the FDA-approved PLGA-b-PEG-COOH copolymer using the oil-in-water emulsion technique. Lately, we developed a more straightforward methodology by introducing chitosan, a highly biocompatible natural product, at the beginning of the process, simultaneously using lithium–naphthalene complex, thus having a one-pot procedure for the formation and surface modification of MgNPs. The obtained MgNPs were purified and fully characterized, showing diameters in the range of 50-300 nm. Notably, when coated with chitosan the particles remained stable as dry powder for more than 10 months. We proved the possibility of generating a temperature rise of a few to several degrees once MgNPs were illuminated using a 810 nm diode laser operating in continuous wave mode: the temperature rise resulted significant (0-15 °C) and concentration dependent. We then investigated potential cytotoxicity of the MgNPs: we used HN13 epithelial cells, derived from a head and neck squamous cell carcinoma and the hepa1-6 cell line, derived from hepatocellular carcinoma and very low toxicity was observed for both nanosystems. Finally, in vivo photothermal therapy was performed on xenograft hepa1-6 tumor bearing mice: the animals were treated with MgNPs coated with chitosan and showed no sign of suffering after the injection. After 12 hours the tumor was exposed to near-infrared laser light. The results clearly showed an extensive damage to tumor tissue after only 2 minutes of laser irradiation at 3Wcm-1, while no damage was reported when the tumor was treated with the laser and saline alone in control group. Despite the lower photothermal efficiency of Mg with respect to Au NPs, we consider MgNPs a promising, safe and green candidate for future clinical translations.

Keywords: chitosan, magnesium nanoparticles, nanomedicine, photothermal therapy

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Authors: Roshni Raha, Kavya P., Gayathri M.

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India, with a humongous population, remains the world's poorest developing nation in terms of nutritional status, with iron deficiency anaemia (IDA) affecting the population. Despite efforts over the past decades, India's anaemia prevalence has not been reduced. Researchers are interested in developing therapies that will minimize the typical side effects of oral iron and optimize iron salts-based treatment through delivery methods based on the physiology of hepcidin regulation. However, they need to come up with iron therapies that will prevent making the infection worse. This article explores using bio-nanotechnology as the alternative, promising substitution of providing iron supplements for the treatment of diarrhoea and gut inflammation in kids and pregnant women. This article is an exploratory study using a literature survey and secondary research from review papers. In the realm of biotechnology, nanoparticles have become extremely famous due to unexpected variations in surface characteristics caused by particle size. Particle size distribution and shape exhibit unusual, enhanced characteristics when reduced to nanoscale. The article attempts to develop a model for a nanotechnology based solution in iron fortification to combat the problems of diarrhoea and gut inflammation. Certain dimensions that have been considered in the model include the size, shape, source, and biosynthesis of the iron nanoparticles. Another area of investigation addressed in the article is the cost-effective biocompatible production of these iron nanoparticles. Studies have demonstrated that a substantial reduction of metal ions to form nanoparticles from the bulk metal occurs in plants because of the presence of a wide diversity of biomolecules. Using this concept, the paper investigates the effectiveness and impact of how similar sources can be used for the biological synthesis of iron nanoparticles. Results showed that iron particles, when prepared in nano-metre size, offer potential advantages. When the particle size of the iron compound decreases and attains nano configuration, its surface area increases, which further improves its solubility in the gastric acid, leading to higher absorption, higher bioavailability, and producing the least organoleptic changes in food. It has no negative effects and possesses a safe, effective profile to reduce IDA. Considering all the parameters, it has been concluded that iron particles in nano configuration serve as alternative iron supplements for the complete treatment of IDA. Nanoparticles of ferric phosphate, ferric pyrophosphate, and iron oxide are the choices of iron supplements. From a sourcing perspective, the paper concludes green sources are the primary sources for the biological synthesis of iron nanoparticles. It will also be a cost-effective strategy since our goal is to treat the target population in rural India. Bio-nanotechnology serves as an alternative and promising substitution for iron supplements due to its low cost, excellent bioavailability, and strong organoleptic properties. One area of future research can be to explore the type of size and shape of iron nanoparticles that would be suitable for the different age groups of pregnant women and children and whether it would be influenced based on the topography in certain areas.

Keywords: anemia, bio-nanotechnology, iron-fortification, nanoparticle

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Authors: Arifur Rahman, Ardeshir Amirkhani, Honghua Hu, Mark Molloy, Karen Vickery

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Introduction and Objectives: Staphylococcus aureus and coagulase-negative staphylococci comprises approximately 65% of infections associated with medical devices and are well known for their biofilm formatting ability. Biofilm-related infections are extremely difficult to eradicate owing to their high tolerance to antibiotics and host immune defences. Currently, there is no efficient method for early biofilm detection. A better understanding to enable detection of biofilm specific proteins in vitro and in vivo can be achieved by studying planktonic and different growth phases of biofilms using a proteome analysis approach. Our goal was to construct a reference map of planktonic and biofilm associated proteins of S. aureus. Methods: S. aureus reference strain (ATCC 25923) was used to grow 24 hours planktonic, 3-day wet biofilm (3DWB), and 12-day wet biofilm (12DWB). Bacteria were grown in tryptic soy broth (TSB) liquid medium. Planktonic growth was used late logarithmic bacteria, and the Centres for Disease Control (CDC) biofilm reactor was used to grow 3 days, and 12-day hydrated biofilms, respectively. Samples were subjected to reduction, alkylation and digestion steps prior to Multiplex labelling using Tandem Mass Tag (TMT) 10-plex reagent (Thermo Fisher Scientific). The labelled samples were pooled and fractionated by high pH RP-HPLC which followed by loading of the fractions on a nanoflow UPLC system (Eksigent UPLC system, AB SCIEX). Mass spectrometry (MS) data were collected on an Orbitrap Elite (Thermo Fisher Scientific) Mass Spectrometer. Protein identification and relative quantitation of protein levels were performed using Proteome Discoverer (version 1.3, Thermo Fisher Scientific). After the extraction of protein ratios with Proteome Discoverer, additional processing, and statistical analysis was done using the TMTPrePro R package. Results and Discussion: The present study showed that a considerable proteomic difference exists among planktonic and biofilms from S. aureus. We identified 1636 total extracellular secreted proteins, of which 350 and 137 proteins of 3DWB and 12DWB showed significant abundance variation from planktonic preparation, respectively. Of these, simultaneous up-regulation in between 3DWB and 12DWB proteins such as extracellular matrix-binding protein ebh, enolase, transketolase, triosephosphate isomerase, chaperonin, peptidase, pyruvate kinase, hydrolase, aminotransferase, ribosomal protein, acetyl-CoA acetyltransferase, DNA gyrase subunit A, glycine glycyltransferase and others we found in this biofilm producer. On the contrary, simultaneous down-regulation in between 3DWB and 12DWB proteins such as alpha and delta-hemolysin, lipoteichoic acid synthase, enterotoxin I, serine protease, lipase, clumping factor B, regulatory protein Spx, phosphoglucomutase, and others also we found in this biofilm producer. In addition, we also identified a big percentage of hypothetical proteins including unique proteins. Therefore, a comprehensive knowledge of planktonic and biofilm associated proteins identified by S. aureus will provide a basis for future studies on the development of vaccines and diagnostic biomarkers. Conclusions: In this study, we constructed an initial reference map of planktonic and various growth phase of biofilm associated proteins which might be helpful to diagnose biofilm associated infections.

Keywords: bacterial biofilms, CDC bioreactor, S. aureus, mass spectrometry, TMT

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Authors: Marian Mihalik, Kornel Csach, Martin Kovalik, Matúš Mihalik, Martina Kubovčíková, Maria Zentková, Martin Vavra, Vladimír Girman, Jaroslav Briančin, Marija Perovic, Marija Boškovic, Magdalena Fitta, Robert Pelka

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Current nanomaterials for use in biomedicine are based mainly on iron oxides and on present knowledge on magnetic nanostructures. Manganites can represent another material which can be used optionally. Manganites and their unique electronic properties have been extensively studied in the last decades not only due to fundamental interest but to possible applications of colossal magnetoresistance, magnetocaloric effect, and ferroelectric properties. It was found that the oxygen-reduction reaction on perovskite oxide is intimately connected with metal ion e.g., orbital occupation. The effect of oxygen deviation from the stoichiometric composition on crystal structure was studied very carefully by many authors on LaMnO₃. Depending on oxygen content, the crystal structure changes from orthorhombic one to rhombohedric for oxygen content 3.1. In the case of hole-doped manganites, the change from the orthorhombic crystal structure, which is typical for La1-xCaxMnO3 based manganites, to the rhombohedric crystal structure (La1-xMxMnO₃ where M = K, Ag, and Sr based materials) results in an enormous increase of the Curie temperature. In our paper, we study the effect of oxygen content on crystal structure, thermal, and magnetic properties (including magnetocaloric effect) of La1-xAgxMnO₃nano particle system. The content of oxygen in samples was tuned by heat treatment in different thermal regimes and in various environment (air, oxygen, argon). Water nanosuspensions based on La0.80Ag0.15MnO₃ magnetic particles with the Curie temperature of about 43oC were prepared by two different approaches. First, by using a laboratory circulation mill for milling of powder in the presence of sodium dodecyl sulphate (SDS) and subsequent centrifugation. Second nanosuspension was prepared using an agate bowl, etching in citric acid and HNO3, ultrasound homogeniser, centrifugation, and dextran 40 kDA or 15 kDA as surfactant. Electrostatic stabilisation obtained by the first approach did not offer long term kinetic and aggregation colloidal stability and was unable to compensate for attractive forces between particles under a magnetic field. By the second approach, we prepared suspension oversaturated by dextran 40 kDA for steric stabilisation, with evidence of the presence of superparamagnetic behaviour. Low concentration of nanoparticles and not ideal coverage of nanoparticles impacting the stability of ferrofluids was the disadvantage of this approach. Strong steric stabilisation was observable at alcaic conditions under pH = ~10. Application of dextran 15 kDA leads to relatively stable ferrofluid with pH around physiological conditions, but desegregation of powder by HNO₃ was not effective enough, and the average size of fragments was to large of about 150 nm, and we did not see any signature of superparamagnetic behaviour. The prepared ferrofluids were characterised by scanning and transition microscope method, thermogravimetry, magnetization, and AC susceptibility measurements. Specific Absorption Rate measurements were undertaken on powder as well on ferrofluids in order to estimate the potential application of La₀.₈₀Ag₀.₁₅MnO₃ magnetic particles based ferrofluid for hyperthermia. Our complex study contains an investigation of biocompatibility and potential biohazard of this material.

Keywords: manganites, magnetic nanoparticles, oxygen content, magnetic phase transition, magnetocaloric effect, ferrofluid, hyperthermia

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Authors: Patricia Shirley Almeida Prado, Liz Brito, Selma Paixão Argollo, Gracie Moreira, Leticia Matos Sobrinho

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Diffuse idiopathic skeletal hyperostosis (DISH) is a degenerative bone disease also known as Forestier´s disease and ankylosing hyperostosis of the spine is characterized by a tendency toward ossification of half the anterior longitudinal spinal ligament without intervertebral disc disease. DISH is not considered to be osteoarthritis, although the two conditions commonly occur together. Diagnostic criteria include fusion of at least four vertebrae by bony bridges arising from the anterolateral aspect of the vertebral bodies. These vertebral bodies have a 'dripping candle wax' appearance, also can be seen periosteal new bone formation on the anterior surface of the vertebral bodies and there is no ankylosis at zygoapophyseal facet joint. Clinically, patients with DISH tend to be asymptomatic some patients mention moderate pain and stiffness in upper back. This disease is more common in man, uncommon in patients younger than 50 years and rare in patients under 40 years old. In modern populations, DISH is found in association with obesity, (type II) diabetes; abnormal vitamin A metabolism and also associated with higher levels of serum uric acid. There is also some association between the increase of risk of stroke or other cerebrovascular disease. The DISH condition can be confused with Heterotopic Ossification, what is the bone formation in the soft tissues as the result of trauma, wounding, surgery, burnings, prolonged immobility and some central nervous system disorder. All these conditions have been described extensively as myositis ossificans which can be confused with the fibrodysplasia (myositis) ossificans progressive. As in the DISH symptomatology it can be asymptomatic or extensive enough to impair joint function. A third confusion osteoarthritis disease that can bring confusion are the enthesopathies that occur in the entire skeleton being common on the ischial tuberosities, iliac crests, patellae, and calcaneus. Ankylosis of the sacroiliac joint by bony bridges may also be found. CASE 1: this case is skeletal remains presenting skull, some vertebrae and scapulae. This case remains unidentified and due to lack of bone remains. Sex, age and ancestry profile was compromised, however the DISH pathognomonic findings and diagnostic helps to estimate sex and age characteristics. Moreover to presenting DISH these skeletal remains also showed some bone alterations and non-metrics as fusion of the first vertebrae with occipital bone, maxillae and palatine torus and scapular foramen on the right scapulae. CASE 2: this skeleton remains shows an extensive bone heterotopic ossification on the great trochanter area of left femur, right fibula showed a healed fracture in its body however in its inteosseous crest there is an extensive bone growth, also in the Ilium at the region of inferior gluteal line can be observed some pronounced bone growth and the skull presented a pronounced mandibular, maxillary and palatine torus. Despite all these pronounced heterotopic ossification the whole skeleton presents moderate bone overgrowth that is not linked with aging, since the skeleton belongs to a young unidentified individual. The appropriate osteopathological diagnosis support the human identification process through medical reports and also assist with epidemiological data that can strengthen vulnerable anthropological estimates.

Keywords: bone disease, DISH, human identification, human remains

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Authors: Rajeswari Raguraman, Sowmya Parameswaran, Krishnakumar Subramanian, Jagat Kanwar, Rupinder Kanwar

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Background: Tumor microenvironment has been implicated in several cancers to regulate cell growth, invasion and metastasis culminating in outcome of therapy. Tumor stroma consists of multiple cell types that are in constant cross-talk with the tumor cells to favour a pro-tumorigenic environment. Not much is known about the existence of tumor microenvironment in the pediatric intraocular malignancy, Retinoblastoma (RB). In the present study, we aim to understand the multiple stromal cellular subtypes and tumor stromal interactions expressed in RB tumors. Materials and Methods: Immunohistochemistry for stromal cell markers CD31, CD68, alpha-smooth muscle (α-SMA), vimentin and glial fibrillary acidic protein (GFAP) was performed on formalin fixed paraffin embedded tissues sections of RB (n=12). The differential expression of stromal target molecules; fibroblast activation protein (FAP), tenascin-C (TNC), osteopontin (SPP1), bone marrow stromal antigen 2 (BST2), stromal derived factor 2 and 4 (SDF2 and SDF4) in primary RB tumors (n=20) and normal retina (n=5) was studied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. The differential expression was correlated with the histopathological features of RB. The interaction between RB cell lines (Weri-Rb-1, NCC-RbC-51) and Bone marrow stromal cells (BMSC) was also studied using direct co-culture and indirect co-culture methods. The functional effect of the co-culture methods on the RB cells was evaluated by invasion and proliferation assays. Global gene expression was studied by using Affymetrix 3’ IVT microarray. Pathway prediction was performed using KEGG and the key molecules were validated using qRT-PCR. Results: The immunohistochemistry revealed the presence of several stromal cell types such as endothelial cells (CD31+;Vim+/-); macrophages (CD68+;Vim+/-); Fibroblasts (Vim+; CD31-;CD68- );myofibroblasts (α-SMA+/ Vim+) and invading retinal astrocytes/ differentiated retinal glia (GFAP+; Vim+). A characteristic distribution of these stromal cell types was observed in the tumor microenvironment, with endothelial cells predominantly seen in blood vessels and macrophages near actively proliferating tumor or necrotic areas. Retinal astrocytes and glia were predominant near the optic nerve regions in invasive tumors with sparse distribution in tumor foci. Fibroblasts were widely distributed with rare evidence of myofibroblasts in the tumor. Both gene and protein expression revealed statistically significant (P<0.05) up-regulation of FAP, TNC and BST2 in primary RB tumors compared to the normal retina. Co-culture of BMSC with RB cells promoted invasion and proliferation of RB cells in direct and indirect contact methods respectively. Direct co-culture of RB cell lines with BMSC resulted in gene expression changes in ECM-receptor interaction, focal adhesion, IL-8 and TGF-β signaling pathways associated with cancer. In contrast, various metabolic pathways such a glucose, fructose and amino acid metabolism were significantly altered under the indirect co-culture condition. Conclusion: The study suggests that the close interaction between RB cells and the stroma might be involved in RB tumor invasion and progression which is likely to be mediated by ECM-receptor interactions and secretory factors. Targeting the tumor stroma would be an attractive option for redesigning treatment strategies for RB.

Keywords: gene expression profiles, retinoblastoma, stromal cells, tumor microenvironment

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Authors: Piacentini Diego, Della Rovere Federica, Fattorini Laura, Lanni Francesca, Cittadini Martina, Altamura Maria Maddalena, Falasca Giuseppina

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Plants have evolved sophisticated mechanisms to cope with environmental cues. Changes in intracellular content and distribution of phytohormones, such as the auxin indole-3-acetic acid (IAA), have been involved in morphogenic adaptation to environmental stresses. In addition to phytohormones, plants can rely on a plethora of small signal molecules able to promptly sense and transduce the stress signals, resulting in morpho/physiological responses thanks also to their capacity to modulate the levels/distribution/reception of most hormones. Among these signaling molecules, nitrogen monoxide (nitric oxide – NO) is a critical component in several plant acclimation strategies to both biotic and abiotic stresses. Depending on its levels, NO increases plant adaptation by enhancing the enzymatic or non-enzymatic antioxidant systems or by acting as a direct scavenger of reactive oxygen/nitrogen (ROS/RNS) species produced during the stress. In addition, exogenous applications of NO-specific donor compounds showed the involvement of the signal molecule in auxin metabolism, transport, and signaling, under both physiological and stress conditions. However, the complex mechanisms underlying NO action in interacting with phytohormones, such as auxins, during metal stress responses are still poorly understood and need to be better investigated. Emphasis must be placed on the response of the root system since it is the first plant organ system to be exposed to metal soil pollution. The monocot Oryza sativa L. (rice) has been chosen given its importance as a stable food for some 4 billion people worldwide. In addition, increasing evidence has shown that rice is often grown in contaminated paddy soils with high levels of heavy metal cadmium (Cd) and metalloid arsenic (As). The facility through which these metals are taken up by rice roots and transported to the aerial organs up to the edible caryopses makes rice one of the most relevant sources of these pollutants for humans. This study aimed to evaluate if NO has a mitigatory activity in the roots of rice seedlings against Cd or As toxicity and to understand if this activity requires interactions with auxin. Our results show that exogenous treatments with the NO-donor SNP alleviate the stress induced by Cd, but not by As, in in-vitro-grown rice seedlings through increased intracellular root NO levels. The damages induced by the pollutants include root growth inhibition, root histological alterations and ROS (H2O2, O2●ˉ), and RNS (ONOOˉ) production. Also, SNP treatments mitigate both the root increase in root IAA levels and the IAA alteration in distribution monitored by the OsDR5::GUS system due to the toxic metal exposure. Notably, the SNP-induced mitigation of the IAA homeostasis altered by the pollutants does not involve changes in the expression of OsYUCCA1 and ASA2 IAA-biosynthetic genes. Taken together, the results highlight a mitigating role of NO in the rice root system, which is pollutant-specific, and involves the interaction of the signal molecule with both IAA and brassinosteroids at different (i.e., transport, levels, distribution) and multiple levels (i.e., transcriptional/post-translational levels). The research is supported by Progetti Ateneo Sapienza University of Rome, grant number: RG120172B773D1FF

Keywords: arsenic, auxin, cadmium, nitric oxide, rice, root system

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

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

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

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Authors: Martin Cvek, Miroslav Mrlik, Michal Sedlacik, Tomas Plachy

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Magnetorheological elastomers (MREs) are multi-phase composite materials containing micron-sized ferromagnetic particles dispersed in an elastomeric matrix. Their properties such as modulus, damping, magneto-striction, and electrical conductivity can be controlled by an external magnetic field and/or pressure. These features of the MREs are used in the development of damping devices, shock attenuators, artificial muscles, sensors or active elements of electric circuits. However, imperfections on the particle/matrix interfaces result in the lower performance of the MREs when compared with theoretical values. Moreover, magnetic particles are susceptible to corrosion agents such as acid rains or sea humidity. Therefore, the modification of particles is an effective tool for the improvement of MRE performance due to enhanced compatibility between particles and matrix as well as improvements of their thermo-oxidation and chemical stability. In this study, the carbonyl iron (CI) particles were controllably modified with poly(trimethylsilyloxyethyl methacrylate) (PHEMATMS) nano-grafts to develop magnetic core–shell structures exhibiting proper wetting with various elastomeric matrices resulting in improved performance within a frame of rheological, magneto-piezoresistance, pressure-piezoresistance, or radio-absorbing properties. The desired molecular weight of PHEMATMS nano-grafts was precisely tailored using surface-initiated atom transfer radical polymerization (ATRP). The CI particles were firstly functionalized using a 3-aminopropyltriethoxysilane agent, followed by esterification reaction with α-bromoisobutyryl bromide. The ATRP was performed in the anisole medium using ethyl α-bromoisobutyrate as a macroinitiator, N, N´, N´´, N´´-pentamethyldiethylenetriamine as a ligand, and copper bromide as an initiator. To explore the effect PHEMATMS molecular weights on final properties, two variants of core-shell structures with different nano-graft lengths were synthesized, while the reaction kinetics were designed through proper reactant feed ratios and polymerization times. The PHEMATMS nano-grafts were characterized by nuclear magnetic resonance and gel permeation chromatography proving information to their monomer conversions, molecular chain lengths, and low polydispersity indexes (1.28 and 1.35) as the results of the executed ATRP. The successful modifications were confirmed via Fourier transform infrared- and energy-dispersive spectroscopies while expected wavenumber outputs and element presences, respectively, of constituted PHEMATMS nano-grafts, were occurring in the spectra. The surface morphology of bare CI and their PHEMATMS-grafted analogues was further studied by scanning electron microscopy, and the thicknesses of grafted polymeric layers were directly observed by transmission electron microscopy. The contact angles as a measure of particle/matrix compatibility were investigated employing the static sessile drop method. The PHEMATMS nano-grafts enhanced compatibility of hydrophilic CI with low-surface-energy hydrophobic polymer matrix in terms of their wettability and dispersibility in an elastomeric matrix. Thus, the presence of possible defects at the particle/matrix interface is reduced, and higher performance of modified MREs is expected.

Keywords: atom transfer radical polymerization, core-shell, particle modification, wettability

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Authors: Pauline Nyssen, Ange Mouithys-Mickalad, Maryse Hoebeke

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Inflammation is a complex physiological phenomenon involving chemical and enzymatic mechanisms. Polymorphonuclear neutrophil leukocytes (PMNs) play an important role by producing reactive oxygen species (ROS) and releasing myeloperoxidase (MPO), a pro-oxidant enzyme. Released both in the phagolysosome and the extracellular medium, MPO produces during its peroxidase and halogenation cycles oxidant species, including hypochlorous acid, involved in the destruction of pathogen agents, like bacteria or viruses. Inflammatory pathologies, like rheumatoid arthritis, atherosclerosis induce an excessive stimulation of the PMNs and, therefore, an uncontrolled release of ROS and MPO in the extracellular medium, causing severe damages to the surrounding tissues and biomolecules such as proteins, lipids, and DNA. The treatment of chronic inflammatory pathologies remains a challenge. For many years, MPO has been used as a target for the development of effective treatments. Numerous studies have been focused on the design of new drugs presenting more efficient MPO inhibitory properties. However, some designed inhibitors can be toxic. An alternative consists of assessing the potential inhibitory action of clinically-known molecules, having antioxidant activity. Propofol, 2,6-diisopropyl phenol, which is used as an intravenous anesthetic agent, meets these requirements. Besides its anesthetic action employed to induce a sedative state during surgery or in intensive care units, propofol and its injectable form Diprivan indeed present antioxidant properties and act as ROS and free radical scavengers. A study has also evidenced the ability of propofol to inhibit the formation of the neutrophil extracellular traps fibers, which are important to trap pathogen microorganisms during the inflammation process. The aim of this study was to investigate the potential inhibitory action mechanism of propofol and Diprivan on MPO activity. To go into the anti-inflammatory action of propofol in-depth, two of its oxidative derivatives, 2,6-diisopropyl-1,4-p-benzoquinone (PPFQ) and 3,5,3’,5’-tetra isopropyl-(4,4’)-diphenoquinone (PPFDQ), were studied regarding their inhibitory action. Specific immunological extraction followed by enzyme detection (SIEFED) and molecular modeling have evidenced the low anti-catalytic action of propofol. Stopped-flow absorption spectroscopy and direct MPO activity analysis have proved that propofol acts as a reversible MPO inhibitor by interacting as a reductive substrate in the peroxidase cycle and promoting the accumulation of redox compound II. Overall, Diprivan exhibited a weaker inhibitory action than the active molecule propofol. In contrast, PPFQ seemed to bind and obstruct the enzyme active site, preventing the trigger of the MPO oxidant cycles. PPFQ induced a better chlorination cycle inhibition at basic and neutral pH in comparison to propofol. PPFDQ did not show any MPO inhibition activity. The three interest molecules have also demonstrated their inhibition ability on an important step of the inflammation pathway, the PMNs superoxide anions production, thanks to EPR spectroscopy and chemiluminescence. In conclusion, propofol presents an interesting immunomodulatory activity by acting as a reductive substrate in the peroxidase cycle of MPO, slowing down its activity, whereas PPFQ acts more as an anti-catalytic substrate. Although PPFDQ has no impact on MPO, it can act on the inflammation process by inhibiting the superoxide anions production by PMNs.

Keywords: Diprivan, inhibitor, myeloperoxidase, propofol, spectroscopy

Procedia PDF Downloads 149

Authors: Danyi Wang, Andrew Schriefer, Dennis O'Rourke, Brajendra Kumar, Yang Liu, Fei Zhong, Juergen Scheuenpflug, Zheng Feng

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Purpose: It has been recognized that the microbiome plays critical roles in disease pathogenesis, including cancer, autoimmune disease, and multiple sclerosis. To develop a clinical-grade assay for exploring microbiome-derived clinical biomarkers across disease areas, a two-phase approach is implemented. 1) Identification of the optimal sample preparation reagents using pre-mixed bacteria and healthy donor stool samples coupled with proprietary Sigma-Aldrich® bioinformatics solution. 2) Exploratory analysis of patient samples for enabling precision medicine. Study Procedure: In phase 1 study, we first compared the 16S sequencing results of two ATCC® microbiome standards (MSA 2002 and MSA 2003) across five different extraction kits (Kit A, B, C, D & E). Both microbiome standards samples were extracted in triplicate across all extraction kits. Following isolation, DNA quantity was determined by Qubit assay. DNA quality was assessed to determine purity and to confirm extracted DNA is of high molecular weight. Bacterial 16S ribosomal ribonucleic acid (rRNA) amplicons were generated via amplification of the V3/V4 hypervariable region of the 16S rRNA. Sequencing was performed using a 2x300 bp paired-end configuration on the Illumina MiSeq. Fastq files were analyzed using the Sigma-Aldrich® Microbiome Platform. The Microbiome Platform is a cloud-based service that offers best-in-class 16S-seq and WGS analysis pipelines and databases. The Platform and its methods have been extensively benchmarked using microbiome standards generated internally by MilliporeSigma and other external providers. Data Summary: The DNA yield using the extraction kit D and E is below the limit of detection (100 pg/µl) of Qubit assay as both extraction kits are intended for samples with low bacterial counts. The pre-mixed bacterial pellets at high concentrations with an input of 2 x106 cells for MSA-2002 and 1 x106 cells from MSA-2003 were not compatible with the kits. Among the remaining 3 extraction kits, kit A produced the greatest yield whereas kit B provided the least yield (Kit-A/MSA-2002: 174.25 ± 34.98; Kit-A/MSA-2003: 179.89 ± 30.18; Kit-B/MSA-2002: 27.86 ± 9.35; Kit-B/MSA-2003: 23.14 ± 6.39; Kit-C/MSA-2002: 55.19 ± 10.18; Kit-C/MSA-2003: 35.80 ± 11.41 (Mean ± SD)). Also, kit A produced the greatest yield, whereas kit B provided the least yield. The PCoA 3D visualization of the Weighted Unifrac beta diversity shows that kits A and C cluster closely together while kit B appears as an outlier. The kit A sequencing samples cluster more closely together than both the other kits. The taxonomic profiles of kit B have lower recall when compared to the known mixture profiles indicating that kit B was inefficient at detecting some of the bacteria. Conclusion: Our data demonstrated that the DNA extraction method impacts DNA concentration, purity, and microbial communities detected by next-generation sequencing analysis. Further microbiome analysis performance comparison of using healthy stool samples is underway; also, colorectal cancer patients' samples will be acquired for further explore the clinical utilities. Collectively, our comprehensive qualification approach, including the evaluation of optimal DNA extraction conditions, the inclusion of positive controls, and the implementation of a robust qualified bioinformatics pipeline, assures accurate characterization of the microbiota in a complex matrix for deciphering the deep biology and enabling precision medicine.

Keywords: 16S rRNA sequencing, analytical validation, bioinformatics pipeline, metagenomics

Procedia PDF Downloads 170

Authors: Loreta Serniene, Dalia Sekmokiene, Justina Tomkeviciute, Lina Lauciene, Vaida Andruleviciute, Ingrida Sinkeviciene, Kristina Kondrotiene, Neringa Kasetiene, Mindaugas Malakauskas

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One of the most important areas of product development and research in the dairy industry is the product enrichment with active ingredients as well as leading to increased product safety and sustainability. The most expanding field of the active ingredients is the various plants' CO₂ extracts with aromatic, antioxidant and antimicrobial properties. In this study, 15 plant extracts were evaluated based on their antioxidant, antimicrobial properties as well as sensory acceptance indicators for the development of new dairy products. In order to increase the total antioxidant capacity of the milk products, it was important to determine the content of phenolic compounds and antioxidant activity of CO₂ extract. The total phenolic content of fifteen different commercial CO₂ extracts was determined by the Folin-Ciocalteu reagent and expressed as milligrams of the Gallic acid equivalents (GAE) in gram of extract. The antioxidant activities were determined by 2.2′-azinobis-(3-ethylbenzthiazoline)-6-sulfonate (ABTS) methods. The study revealed that the antioxidant activities of investigated CO₂ extract vary from 4.478-62.035 µmole Trolox/g, while the total phenolic content was in the range of 2.021-38.906 mg GAE/g of extract. For the example, the estimated antioxidant activity of Chinese cinnamon (Cinammonum aromaticum) CO₂ extract was 62.023 ± 0.15 µmole Trolox/g and the total flavonoid content reached 17.962 ± 0.35 mg GAE/g. These two parameters suggest that cinnamon could be a promising supplement for the development of new cheese. The inhibitory effects of these essential oils were tested by using agar disc diffusion method against pathogenic bacteria, most commonly found in dairy products. The obtained results showed that essential oil of lemon myrtle (Backhousia citriodora) and cinnamon (Cinnamomum cassia) has antimicrobial activity against E. coli, S. aureus, B. cereus, P. florescens, L. monocytogenes, Br. thermosphacta, P. aeruginosa and S. typhimurium with the diameter of inhibition zones variation from 10 to 52 mm. The sensory taste acceptability of plant extracts in combination with a dairy product was evaluated by a group of sensory evaluation experts (31 individuals) by the criteria of overall taste acceptability in the scale of 0 (not acceptable) to 10 (very acceptable). Each of the tested samples included 200g grams of natural unsweetened greek yogurt without additives and 1 drop of single plant extract (essential oil). The highest average of overall taste acceptability was defined for the samples with essential oils of orange (Citrus sinensis) - average score 6.67, lemon myrtle (Backhousia citriodora) – 6.62, elderberry flower (Sambucus nigra flos.) – 6.61, lemon (Citrus limon) – 5.75 and cinnamon (Cinnamomum cassia) – 5.41, respectively. The results of this study indicate plant extracts of Cinnamomum cassia and Backhousia citriodora as a promising additive not only to increase the total antioxidant capacity of the milk products and as alternative antibacterial agent to combat pathogenic bacteria commonly found in dairy products but also as a desirable flavour for the taste pallet of the consumers with expressed need for safe, sustainable and innovative dairy products. Acknowledgment: This research was funded by the European Regional Development Fund according to the supported activity 'Research Projects Implemented by World-class Researcher Groups' under Measure No. 01.2.2-LMT-K-718.

Keywords: antioxidant properties, antimicrobial properties, cinnamon, CO₂ plant extracts, dairy products, essential oils, lemon myrtle

Procedia PDF Downloads 206

Authors: Glauco M. M. M. Lustosa, João Paulo C. Costa, Sonia M. Zanetti, Mario Cilense, Leinig Antônio Perazolli, Maria Aparecida Zaghete

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The contamination of the environment has been one of the biggest problems of our time, mostly due to developments of many industries. SnO2 is an n-type semiconductor with band gap about 3.5 eV and has its electrical conductivity dependent of type and amount of modifiers agents added into matrix ceramic during synthesis process, allowing applications as sensing of gaseous pollutants on ambient. The chemical synthesis by polymeric precursor method consists in a complexation reaction between tin ion and citric acid at 90 °C/2 hours and subsequently addition of ethyleneglycol for polymerization at 130 °C/2 hours. It also prepared polymeric resin of zinc, cobalt and niobium ions. Stoichiometric amounts of the solutions were mixed to obtain the systems (Zn, Nb)-SnO2 and (Co, Nb) SnO2 . The metal immobilization reduces its segregation during the calcination resulting in a crystalline oxide with high chemical homogeneity. The resin was pre-calcined at 300 °C/1 hour, milled in Atritor Mill at 500 rpm/1 hour, and then calcined at 600 °C/2 hours. X-Ray Diffraction (XDR) indicated formation of SnO2 -rutile phase (JCPDS card nº 41-1445). The characterization by Scanning Electron Microscope of High Resolution showed spherical ceramic powder nanostructured with 10-20 nm of diameter. 20 mg of SnO2 -based powder was kept in 20 ml of isopropyl alcohol and then taken to an electrophoretic deposition (EPD) system. The EPD method allows control the thickness films through the voltage or current applied in the electrophoretic cell and by the time used for deposition of ceramics particles. This procedure obtains films in a short time with low costs, bringing prospects for a new generation of smaller size devices with easy integration technology. In this research, films were obtained in an alumina substrate with interdigital electrodes after applying 2 kV during 5 and 10 minutes in cells containing alcoholic suspension of (Zn, Nb)-SnO2 and (Co, Nb) SnO2 of powders, forming a sensing layer. The substrate has designed integrated micro hotplates that provide an instantaneous and precise temperature control capability when a voltage is applied. The films were sintered at 900 and 1000 °C in a microwave oven of 770 W, adapted by the research group itself with a temperature controller. This sintering is a fast process with homogeneous heating rate which promotes controlled growth of grain size and also the diffusion of modifiers agents, inducing the creation of intrinsic defects which will change the electrical characteristics of SnO2 -based powders. This study has successfully demonstrated a microfabricated system with an integrated micro-hotplate for detection of CO and NO2 gas at different concentrations and temperature, with self-heating SnO2 - based nanoparticles films, being suitable for both industrial process monitoring and detection of low concentrations in buildings/residences in order to safeguard human health. The results indicate the possibility for development of gas sensors devices with low power consumption for integration in portable electronic equipment with fast analysis. Acknowledgments The authors thanks to the LMA-IQ for providing the FEG-SEM images, and the financial support of this project by the Brazilian research funding agencies CNPq, FAPESP 2014/11314-9 and CEPID/CDMF- FAPESP 2013/07296-2.

Keywords: chemical synthesis, electrophoretic deposition, self-heating, gas sensor

Procedia PDF Downloads 276

Authors: Elsie M. Nolte, Annie M. Joubert, Roy Lakier, Maryke Etsebeth, Jolene M. Helena, Marcel Verwey, Laurence Lafanechere, Anne E. Theron

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Treatment regimens for breast- and lung cancers may include both radiation- and chemotherapy. Ideally, a pharmaceutical agent which selectively sensitizes cancer cells to gamma (γ)-radiation would allow administration of lower doses of each modality, yielding synergistic anti-cancer benefits and lower metastasis occurrence, in addition to decreasing the side-effect profiles. A range of 2-methoxyestradiol (2-ME) analogues, namely 2-ethyl-3-O-sulphamoyl-estra-1,3,5 (10) 15-tetraene-3-ol-17one (ESE-15-one), 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) were in silico-designed by our laboratory, with the aim of improving the parent compound’s bioavailability in vivo. The main effect of these compounds is the disruption of microtubule dynamics with a resultant mitotic accumulation and induction of programmed cell death in various cancer cell lines. This in vitro study aimed to determine the cellular responses involved in the radiation sensitization effects of these analogues at low doses in breast- and lung cancer cell lines. The oestrogen receptor positive MCF-7-, oestrogen receptor negative MDA-MB-231- and triple negative BT-20 breast cancer cell lines as well as the A549 lung cancer cell line were used. The minimal compound- and radiation doses able to induce apoptosis were determined using annexin-V and cell cycle progression markers. These doses (cell line dependent) were used to pre-sensitize the cancer cells 24 hours prior to 6 gray (Gy) radiation. Experiments were conducted on samples exposed to the individual- as well as the combination treatment conditions in order to determine whether the combination treatment yielded an additive cell death response. Morphological studies included light-, fluorescence- and transmission electron microscopy. Apoptosis induction was determined by flow cytometry employing annexin V, cell cycle analysis, B-cell lymphoma 2 (Bcl-2) signalling, as well as reactive oxygen species (ROS) production. Clonogenic studies were performed by allowing colony formation for 10 days post radiation. Deoxyribonucleic acid (DNA) damage was quantified via γ-H2AX foci and micronuclei quantification. Amplification of the p53 signalling pathway was determined by western blot. Results indicated that exposing breast- and lung cancer cells to nanomolar concentrations of these analogues 24 hours prior to γ-radiation induced more cell death than the compound- and radiation treatments alone. Hypercondensed chromatin, decreased cell density, a damaged cytoskeleton and an increase in apoptotic body formation were observed in cells exposed to the combination treatment condition. An increased number of cells present in the sub-G1 phase as well as increased annexin-V staining, elevation of ROS formation and decreased Bcl-2 signalling confirmed the additive effect of the combination treatment. In addition, colony formation decreased significantly. p53 signalling pathways were significantly amplified in cells exposed to the analogues 24 hours prior to radiation, as was the amount of DNA damage. In conclusion, our results indicated that pre-treatment of breast- and lung cancer cells with low doses of 2-ME analogues sensitized breast- and lung cancer cells to γ-radiation and induced apoptosis more so than the individual treatments alone. Future studies will focus on the effect of the combination treatment on non-malignant cellular counterparts.

Keywords: cancer, microtubule dynamics, radiation therapy, radiosensitization

Procedia PDF Downloads 209

Authors: Gousilin Leandra Rocha Da Silva, Stéfani T. A. Dantas, Bruna F. Rossi, Erika R. Bonsaglia, Ivana G. Castilho, Terue Sadatsune, Ary Fernandes Júnior, Vera l. M. Rall

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Kidney failure causes decreased diuresis and accumulation of nitrogenous substances in the body. To increase patient survival, hemodialysis is used as a partial substitute for renal function. However, contamination of the water used in this treatment, causing bacteremia in patients, is a worldwide concern. The Burkholderia cepacia complex (Bcc), a group of bacteria with more than 20 species, is frequently isolated from hemodialysis water samples and comprises opportunistic bacteria, affecting immunosuppressed patients, due to its wide variety of virulence factors, in addition to innate resistance to several antimicrobial agents, contributing to the permanence in the hospital environment and to the pathogenesis in the host. The objective of the present work was to characterize molecularly and phenotypically Bcc isolates collected from the water and dialysate of the Hemodialysis Unit and from the blood of patients at a Public Hospital in Botucatu, São Paulo, Brazil, between 2019 and 2021. We used 33 Bcc isolates, previously obtained from blood cultures from patients with bacteremia undergoing hemodialysis treatment (2019-2021) and 24 isolates obtained from water and dialysate samples in a Hemodialysis Unit (same period). The recA gene was sequenced to identify the specific species among the Bcc group. All isolates were tested for the presence of some genes that encode virulence factors such as cblA, esmR, zmpA and zmpB. Considering the epidemiology of the outbreak, the Bcc isolates were molecularly characterized by Multi Locus Sequence Type (MLST) and by pulsed-field gel electrophoresis (PFGE). The verification and quantification of biofilm in a polystyrene microplate were performed by submitting the isolates to different incubation temperatures (20°C, average water temperature and 35°C, optimal temperature for group growth). The antibiogram was performed with disc diffusion tests on agar, using discs impregnated with cefepime (30µg), ceftazidime (30µg), ciprofloxacin (5µg), gentamicin (10µg), imipenem (10µg), amikacin 30µg), sulfametazol/trimethoprim (23.75/1.25µg) and ampicillin/sulbactam (10/10µg). The presence of ZmpB was identified in all isolates, while ZmpA was observed in 96.5% of the isolates, while none of them presented the cblA and esmR genes. The antibiogram of the 33 human isolates indicated that all were resistant to gentamicin, colistin, ampicillin/sulbactam and imipenem. 16 (48.5%) isolates were resistant to amikacin and lower rates of resistance were observed for meropenem, ceftazidime, cefepime, ciprofloxacin and piperacycline/tazobactam (6.1%). All isolates were sensitive to sulfametazol/trimethoprim, levofloxacin and tigecycline. As for the water isolates, resistance was observed only to gentamicin (34.8%) and imipenem (17.4%). According to PFGE results, all isolates obtained from humans and water belonged to the same pulsotype (1), which was identified by recA sequencing as B. cepacia¸, belonging to sequence type ST-767. By observing a single pulse type over three years, one can observe the persistence of this isolate in the pipeline, contaminating patients undergoing hemodialysis, despite the routine disinfection of water with peracetic acid. This persistence is probably due to the production of biofilm, which protects bacteria from disinfectants and, making this scenario more critical, several isolates proved to be multidrug-resistant (resistance to at least three groups of antimicrobials), turning the patient care even more difficult.

Keywords: hemodialysis, burkholderia cepacia, PFGE, MLST, multi drug resistance

Procedia PDF Downloads 101

Authors: Abbas Ahmadi, Nasser Aliasgharzad, Ali Asghar Jafarzadeh

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Nowadays, drying of the Urmia Lake as a largest saline lake in the world and emerging its saline bed from water has caused the risk of salty dune storms, which threats the health of human society and also plants and animal communities living in the region. Biological soil crusts (BSCs) as a dust stabilizer attracted the attention of Soil conservation experts in recent years. Although the presence of water by the impenetrable lake bed and endorheic basin can be an advantage to create BSCs, but the extraordinary of the lake bed salinity is a factor for prevention of its establishment in the region. Therefore, the present research work has been carried out to investigate the effects of inoculating the Cyanobacteria, algae and their combination to create BSCs for dust control. In this study, an algae attributed to Chlamydomonas sp and a cyanobacteria attributed to Anabaena sp isolated from the soils of Urmia Lake margin were used to create BSC in four soil samples which collected from 0-10 cm of the current margin (A), the previous bed (B), affected lands by lake (C) and Quomtappe sand dune (D). The main characteristics of the A, B and C soil samples are their highly salinity (their ECe are 108, 140 and 118 dS/m, respectively) and sodicity. Also, texture class of the soil A was loamy sand, and other two soils had clay textures. Soil D was Non-saline, but it was sodic with a sandy texture class. This study was conducted separately in each soil in a completely randomized design under four inoculation treatments of non-inoculated (T0), Algae (T1), cyanobacteria (T2) and equal mixture of algae and cyanobacteria (T3) with three replications. In the experiment, the soil was placed into wind tunnel trays, and a suspension containing microorganisms mixed with the trays surface soil. During the experiment, water was sprayed to the trays at the morning and evening of every day. After passing the incubation period (30 days), some characteristics of samples such as pH, EC, cold water extractable carbohydrate (CWEC), hot water extractable carbohydrate (HWEC), sulfuric acid extractable carbohydrate (SAEC), organic matter, crust thickness, penetration resistance, wind erosion threshold velocity and soil loss in the wind tunnel were measured, and Correlation between the measured characteristics was obtained through the SPSS software. Analysis of variance and so comparison between the means of treatments were analyzed with MSTATC software. In this research, Chlorophyll, an amount, was used as an indicator of the microorganism's population in the samples. Based on obtained results, the amount of Chlorophyll a in the T2 treatment of soil A and all treatments of soil D was significantly increased in comparison to the control and crust thickness showed increase in all treatments by microorganism’s inoculation. But effect of the treatments was significant in soils A and D. At all treatment’s inoculation of microorganisms in soil A caused to increase %46, %34 and %55 of the wind erosion threshold velocity in T1, T2 and T3 treatments in comparison to the control, respectively, and in soil D all treatments caused wind erosion threshold velocity became two times more than control. However, soil loss in the wind tunnel experiments was significant in T2 and T3 treatments of these soils and T1 treatment had no effect in reducing soil loss. Correlation between Chlorophyll a and salinity shows the important role of salinity in microbial growth prevention and formation of BSCs in the studied samples. In general, according to the obtained results, it can be concluded that salinity reduces the growth of microorganisms in saline soils of the region, and in soils with fine textures, salinity role in prevention of the microbial growth is clear. Also, using the mix of algae and cyanobacteria together caused the synergistic growth of them and consequently, better protection of the soil against wind erosion was provided.

Keywords: wind erosion, algae, cyanobacteria, carbohydrate

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Authors: Agnieszka E. Laudy, Karolina Stępien, Sergiusz Lulinski, Krzysztof Durka, Stefan Tyski

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1,3-dihydro-1-hydroxy-2,1-benzoxaborole and its derivatives are a particularly interesting group of synthetic agents and were successfully employed in supramolecular chemistry medicine. The first important compounds, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole and 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole were identified as potent antifungal agents. In contrast, (S)-3-(aminomethyl)-7-(3-hydroxypropoxy)-1-hydroxy-1,3-dihydro-2,1-benzoxaborole hydrochloride is in the second phase of clinical trials as a drug for the treatment of Gram-negative bacterial infections of the Enterobacteriaceae family and Pseudomonas aeruginosa. Equally important and difficult task is to search for compounds active against Gram-negative bacilli, which have multi-drug-resistance efflux pumps actively removing many of the antibiotics from bacterial cells. We have examined whether halogen-substituted benzoxaborole-based derivatives and their analogues possess antibacterial activity and are substrates for multi-drug-resistance efflux pumps. The antibacterial activity of 1,3-dihydro-3-hydroxy-1,1-dimethyl-1,2,3-benzosiloxaborole and 10 halogen-substituted its derivatives, as well as 1,2-phenylenediboronic acid and 3 synthesised fluoro-substituted its analogs, were evaluated. The activity against the reference strains of Gram-positive (n=5) and Gram-negative bacteria (n=10) was screened by the disc-diffusion test (0.4 mg of tested compounds was applied onto paper disc). The minimal inhibitory concentration values and the minimal bactericidal concentration values were estimated according to The Clinical and Laboratory Standards Institute and The European Committee on Antimicrobial Susceptibility Testing recommendations. During the minimal inhibitory concentration values determination with or without phenylalanine-arginine beta-naphthylamide (50 mg/L) efflux pump inhibitor, the concentrations of tested compounds ranged 0.39-400 mg/L in the broth medium supplemented with 1 mM magnesium sulfate. Generally, the studied benzosiloxaboroles and benzoxadiboroles showed a higher activity against Gram-positive cocci than against Gram-negative rods. Moreover, benzosiloxaboroles have the higher activity than benzoxadiboroles compounds. In this study, we demonstrated that substitution (mono-, di- or tetra-) of 1,3-dihydro-3-hydroxy-1,1-dimethyl-1,2,3-benzosiloxaborole with halogen groups resulted in an increase in antimicrobial activity as compared to the parent substance. Interestingly, the 6,7-dichloro-substituted parent substance was found to be the most potent against Gram-positive cocci: Staphylococcus sp. (minimal inhibitory concentration 6.25 mg/L) and Enterococcus sp. (minimal inhibitory concentration 25 mg/L). On the other hand, mono- and dichloro-substituted compounds were the most actively removed by efflux pumps present in Gram-negative bacteria mainly from Enterobacteriaceae family. In the presence of efflux pump inhibitor the minimal inhibitory concentration values of chloro-substituted benzosiloxaboroles decreased from 400 mg/L to 3.12 mg/L. Of note, the highest increase in bacterial susceptibility to tested compounds in the presence of phenylalanine-arginine beta-naphthylamide was observed for 6-chloro-, 6,7-dichloro- and 6,7-difluoro-substituted benzosiloxaboroles. In the case of Escherichia coli, Enterobacter cloacae and P. aeruginosa strains at least a 32-fold decrease in the minimal inhibitory concentration values of these agents were observed. These data demonstrate structure-activity relationships of the tested derivatives and highlight the need for further search for benzoxaboroles and related compounds with significant antimicrobial properties. Moreover, the influence of phenylalanine-arginine beta-naphthylamide on the susceptibility of Gram-negative rods to studied benzosiloxaboroles indicate that some tested agents are substrates for efflux pumps in Gram-negative rods.

Keywords: antibacterial activity, benzosiloxaboroles, efflux pumps, phenylalanine-arginine beta-naphthylamide

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Authors: Farideh Namvar, Rosfarizan Mohamed

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In the field of nanotechnology, the use of various biological units instead of toxic chemicals for the reduction and stabilization of nanoparticles, has received extensive attention. This use of biological entities to create nanoparticles has designated as “Green” synthesis and it is considered to be far more beneficial due to being economical, eco-friendly and applicable for large-scale synthesis as it operates on low pressure, less input of energy and low temperatures. The lack of toxic byproducts and consequent decrease in degradation of the product renders this technique more preferable over physical and classical chemical methods. The variety of biomass having reduction properties to produce nanoparticles makes them an ideal candidate for fabrication. Metal oxide nanoparticles have been said to represent a "fundamental cornerstone of nanoscience and nanotechnology" due to their variety of properties and potential applications. However, this also provides evidence of the fact that metal oxides include many diverse types of nanoparticles with large differences in chemical composition and behaviour. In this study, iron oxide nanoparticles (Fe3O4-NPs) were synthesized using a rapid, single step and completely green biosynthetic method by reduction of ferric chloride solution with brown seaweed (Sargassum muticum) water extract containing polysaccharides as a main factor which acts as reducing agent and efficient stabilizer. Antimicrobial activity against six microorganisms was tested using well diffusion method. The resulting S-IONPs are crystalline in nature, with a cubic shape. The average particle diameter, as determined by TEM, was found to be 18.01 nm. The S-IONPs were efficiently inhibited the growth of Listeria monocytogenes, Escherichia coli and Candida species. Our favorable results suggest that S-IONPs could be a promising candidate for development of future antimicrobial therapies. The nature of biosynthesis and the therapeutic potential by S-IONPs could pave the way for further research on design of green synthesis therapeutic agents, particularly nanomedicine, to deal with treatment of infections. Further studies are needed to fully characterize the toxicity and the mechanisms involved with the antimicrobial activity of these particles. Antioxidant activity of S-IONPs synthesized by green method was measured by ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (IC50= 1000µg) radical scavenging activity. Also, with the increasing concentration of S-IONPs, catalase gene expression compared to control gene GAPDH increased. For anti-angiogenesis study the Ross fertilized eggs were divided into four groups; the control and three experimental groups. The gelatin sponges containing albumin were placed on the chorioalantoic membrane and soaked with different concentrations of S-IONPs. All the cases were photographed using a photo stereomicroscope. The number and the lengths of the vessels were measured using Image J software. The crown rump (CR) and weight of the embryo were also recorded. According to the data analysis, the number and length of the blood vessels, as well as the CR and weight of the embryos reduced significantly compared to the control (p < 0.05), dose dependently. The total hemoglobin was quantified as an indicator of the blood vessel formation, and in the treated samples decreased, which showed its inhibitory effect on angiogenesis.

Keywords: anti-angiogenesis, antimicrobial, antioxidant, biosynthesis, iron oxide (fe3o4) nanoparticles, sargassum muticum, seaweed

Procedia PDF Downloads 316

Authors: Tahani Mashikhi

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Countries worldwide rely on electric power as a critical economic growth and progress factor. Renewable energy sources, often referred to as alternative energy sources, such as wind, solar energy, geothermal energy, biomass, and hydropower, have garnered significant interest in response to the rising consumption of fossil fuels. Dye-sensitized solar cells (DSSCs) are a highly promising alternative for energy production as they possess numerous advantages compared to traditional silicon solar cells and thin-film solar cells. These include their low cost, high flexibility, straightforward preparation methodology, ease of production, low toxicity, different colors, semi-transparent quality, and high power conversion efficiency. A solar cell, also known as a photovoltaic cell, is a device that converts the energy of light from the sun into electrical energy through the photovoltaic effect. The Gratzel cell is the initial dye-sensitized solar cell made from colloidal titanium dioxide. The operational mechanism of DSSCs relies on various key elements, such as a layer composed of wide band gap semiconducting oxide materials (e.g. titanium dioxide [TiO₂]), as well as a photosensitizer or dye that absorbs sunlight to inject electrons into the conduction band, the electrolyte utilizes the triiodide/iodide redox pair (I− /I₃−) to regenerate dye molecules and a counter electrode made of carbon or platinum facilitates the movement of electrons across the circuit. Electrospray deposition permits the deposition of fragile, non-volatile molecules in a vacuum environment, including dye sensitizers, complex molecules, nanoparticles, and biomolecules. Surface science techniques, particularly X-ray photoelectron spectroscopy, are employed to examine dye-sensitized solar cells. This study investigates the possible application of electrospray deposition to build high-quality layers in situ in a vacuum. Two distinct categories of dyes can be employed as sensitizers in DSSCs: organometallic semiconductor sensitizers and purely organic dyes. Most organometallic dyes, including Ru533, RuC, and RuP, contain a ruthenium atom, which is a rare element. This ruthenium atom enhances the efficiency of dye-sensitized solar cells (DSSCs). These dyes are characterized by their high cost and typically appear as dark purple powders. On the other hand, organic dyes, such as SQ2, RK1, D5, SC4, and R6, exhibit reduced efficacy due to the lack of a ruthenium atom. These dyes appear in green, red, orange, and blue powder-colored. This study will specifically concentrate on metal-organic dyes. The adsorption of dye molecules onto the rutile TiO₂ (110) surface has been deposited in situ under ultra-high vacuum conditions by combining an electrospray deposition method with X-ray photoelectron spectroscopy. The X-ray photoelectron spectroscopy (XPS) technique examines chemical bonds and interactions between molecules and TiO₂ surfaces. The dyes were deposited at varying times, from 5 minutes to 40 minutes, to achieve distinct layers of coverage categorized as sub-monolayer, monolayer, few layers, or multilayer. Based on the O 1s photoelectron spectra data, it can be observed that the monolayer establishes a strong chemical bond with the Ti atoms of the oxide substrate by deprotonating the carboxylic acid groups through 2M-bidentate bridging anchors. The C 1s and N 1s photoelectron spectra indicate that the molecule remains intact at the surface. This can be due to the existence of all functional groups and a ruthenium atom, where the binding energy of Ru 3d is consistent with Ru2+.

Keywords: deposit, dye, electrospray, TiO₂, XPS

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Authors: Ling Ai, Ziyu Zhao, Zeyu Zhou, Xiaochen Yang, Heng Zhai, Stuart Holmes

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Optimizing the catalyst layer structure is crucial for enhancing the performance of proton exchange membrane fuel cells (PEMFCs) with low Platinum (Pt) loading. Current works focused on the utilization, durability, and site activity of Pt particles on support, and performance enhancement has been achieved by loading Pt onto porous support with different morphology, such as graphene, carbon fiber, and carbon black. Some schemes have also incorporated cost considerations to achieve lower Pt loading. However, the design of the catalyst layer (CL) structure in the membrane electrode assembly (MEA) must consider the interactions between the layers. Addressing the crucial aspects of water management, low contact resistance, and the establishment of effective three-phase boundary for MEA, multi-walled carbon nanotubes (MWCNTs) are promising CL support due to their intrinsically high hydrophobicity, high axial electrical conductivity, and potential for ordered alignment. However, the drawbacks of MWCNTs, such as strong agglomeration, wall surface chemical inertness, and unopened ends, are unfavorable for Pt nanoparticle loading, which is detrimental to MEA processing and leads to inhomogeneous CL surfaces. This further deteriorates the utilization of Pt and increases the contact resistance. Robust chemical oxidation or nitrogen doping can introduce polar functional groups onto the surface of MWCNTs, facilitating the creation of open tube ends and inducing defects in tube walls. This improves dispersibility and load capacity but reduces length and conductivity. Consequently, a trade-off exists between maintaining the intrinsic properties and the degree of functionalization of MWCNTs. In this work, MWCNTs were modified based on the operational requirements of the MEA from the viewpoint of interlayer interactions, including the search for the optimal degree of oxidation, N-doping, and micro-arrangement. MWCNT were functionalized by oxidizing, N-doping, as well as micro-alignment to achieve lower contact resistance between CL and proton exchange membrane (PEM), better hydrophobicity, and enhanced performance. Furthermore, this work expects to construct a more continuously distributed three-phase boundary by aligning MWCNT to form a locally ordered structure, which is essential for the efficient utilization of Pt active sites. Different from other chemical oxidation schemes that used HNO3:H2SO4 (1:3) mixed acid to strongly oxidize MWCNT, this scheme adopted pure HNO3 to partially oxidize MWCNT at a lower reflux temperature (80 ℃) and a shorter treatment time (0 to 10 h) to preserve the morphology and intrinsic conductivity of MWCNT. The maximum power density of 979.81 mw cm-2 was achieved by Pt loading on 6h MWCNT oxidation time (Pt-MWCNT6h). This represented a 59.53% improvement over the commercial Pt/C catalyst of 614.17 (mw cm-2). In addition, due to the stronger electrical conductivity, the charge transfer resistance of Pt-MWCNT6h in the electrochemical impedance spectroscopy (EIS) test was 0.09 Ohm cm-2, which was 48.86% lower than that of Pt/C. This study will discuss the developed catalysts and their efficacy in a working fuel cell system. This research will validate the impact of low-functionalization modification of MWCNTs on the performance of PEMFC, which simplifies the preparation challenges of CL and contributing for the widespread commercial application of PEMFCs on a larger scale.

Keywords: carbon nanotubes, electrocatalyst, membrane electrode assembly, proton exchange membrane fuel cell

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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: Eduardo Lanzagorta Garcia, Chaitra Venkatesh, Romina Pezzoli, Laura Gabriela Rodriguez Barroso, Declan Devine, Margaret E. Brennan Fournet

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New antimicrobial interventions are urgently required to combat rising global health and medical infection challenges. Here, an innovative antimicrobial technology, providing price competitive alternatives to antibiotics and readily integratable with currently technological systems is presented. Two cutting edge antimicrobial materials, antimicrobial peptides (AMPs) and uncompromised sustained Ag+ action from triangular silver nanoplates (TSNPs) reservoirs, are merged for versatile effective antimicrobial action where current approaches fail. Antimicrobial peptides (AMPs) exist widely in nature and have recently been demonstrated for broad spectrum of activity against bacteria, viruses, and fungi. TSNP’s are highly discrete, homogenous and readily functionisable Ag+ nanoreseviors that have a proven amenability for operation within in a wide range of bio-based settings. In a design for advanced antimicrobial sustainable plastics, antimicrobial TSNPs are formulated for processing within biodegradable biopolymers. Histone H5 AMP was selected for its reported strong antimicrobial action and functionalized with the TSNP (AMP-TSNP) in a similar fashion to previously reported TSNP biofunctionalisation methods. A synergy between the propensity of biopolymers for degradation and Ag+ release combined with AMP activity provides a novel mechanism for the sustained antimicrobial action of biopolymeric thin films. Nanoplates are transferred from aqueous phase to an organic solvent in order to facilitate integration within hydrophobic polymers. Extrusion is used in combination with calendering rolls to create thin polymerc film where the nanoplates are embedded onto the surface. The resultant antibacterial functional films are suitable to be adapted for food packing and biomedical applications. TSNP synthesis were synthesized by adapting a previously reported seed mediated approach. TSNP synthesis was scaled up for litre scale batch production and subsequently concentrated to 43 ppm using thermally controlled H2O removal. Nanoplates were transferred from aqueous phase to an organic solvent in order to facilitate integration within hydrophobic polymers. This was acomplised by functionalizing the TSNP with thiol terminated polyethylene glycol and using centrifugal force to transfer them to chloroform. Polycaprolactone (PCL) and Polylactic acid (PLA) were individually processed through extrusion, TSNP and AMP-TSNP solutions were sprayed onto the polymer immediately after exiting the dye. Calendering rolls were used to disperse and incorporate TSNP and TSNP-AMP onto the surface of the extruded films. Observation of the characteristic blue colour confirms the integrity of the TSNP within the films. Antimicrobial tests were performed by incubating Gram + and Gram – strains with treated and non-treated films, to evaluate if bacterial growth was reduced due to the presence of the TSNP. The resulting films successfully incorporated TSNP and AMP-TSNP. Reduced bacterial growth was observed for both Gram + and Gram – strains for both TSNP and AMP-TSNP compared with untreated films indicating antimicrobial action. The largest growth reduction was observed for AMP-TSNP treated films demonstrating the additional antimicrobial activity due to the presence of the AMPs. The potential of this technology to impede bacterial activity in food industry and medical surfaces will forge new confidence in the battle against antibiotic resistant bacteria, serving to greatly inhibit infections and facilitate patient recovery.

Keywords: antimicrobial, biodegradable, peptide, polymer, nanoparticle

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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: B.L. España-Sánchez, E. Luna-Hernández, R.A. Mauricio-Sánchez, M.E. Cruz-Soto, F. Padilla-Vaca, R. Muñoz, L. Granados-López, L.R. Ovalle-Flores, J.L. Menchaca-Arredondo, G. Luna-Bárcenas

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Treatment of burn injured has been considered an important clinical problem due to the fluid control and the presence of microorganisms during the healing process. Conventional treatment includes antiseptic techniques, topical medication and surgical removal of damaged skin, to avoid bacterial growth. In order to accelerate this process, different alternatives for tissue regeneration have been explored, including artificial skin, polymers, hydrogels and hybrid materials. Some requirements consider a nonreactive organic polymer with high biocompatibility and skin adherence, avoiding bacterial infections. Chitin-derivative biopolymer such as chitosan (CS) has been used in skin regeneration following third-degree burns. The biological interest of CS is associated with the improvement of tissue cell stimulation, biocompatibility and antibacterial properties. In particular, antimicrobial properties of CS can be significantly increased when is blended with nanostructured materials. Silver-based nanocomposites have gained attention in medicine due to their high antibacterial properties against pathogens, related to their high surface area/volume ratio at nanomolar concentrations. Silver nanocomposites can be blended or synthesized with chitin-derivative biopolymers in order to obtain a biodegradable/antimicrobial hybrid with improved physic-mechanical properties. In this study, nanocomposites based on chitosan/silver nanoparticles (CS/nAg) were synthesized by the in situ chemical reduction method, improving their antibacterial properties against pathogenic bacteria and enhancing the healing process in thermal burn injuries produced in an animal model. CS/nAg was prepared in solution by the chemical reduction method, using AgNO₃ as precursor. CS was dissolved in acetic acid and mixed with different molar concentrations of AgNO₃: 0.01, 0.025, 0.05 and 0.1 M. Solutions were stirred at 95°C during 20 hours, in order to promote the nAg formation. CS/nAg solutions were placed in Petri dishes and dried, to obtain films. Structural analyses confirm the synthesis of silver nanoparticles (nAg) by means of UV-Vis and TEM, with an average size of 7.5 nm and spherical morphology. FTIR analyses showed the complex formation by the interaction of hydroxyl and amine groups with metallic nanoparticles, and surface chemical analysis (XPS) shows low concentration of Ag⁰/Ag⁺ species. Topography surface analyses by means of AFM shown that hydrated CS form a mesh with an average diameter of 10 µm. Antibacterial activity against S. aureus and P. aeruginosa was improved in all evaluated conditions, such as nAg loading and interaction time. CS/nAg nanocomposites films did not show Ag⁰/Ag⁺ release in saline buffer and rat serum after exposition during 7 days. Healing process was significantly enhanced by the presence of CS/nAg nanocomposites, inducing the production of myofibloblasts, collagen remodelation, blood vessels neoformation and epidermis regeneration after 7 days of injury treatment, by means of histological and immunohistochemistry assays. The present work suggests that hydrated CS/nAg nanocomposites can be formed a mesh, improving the bacterial penetration and the contact with embedded nAg, producing complete growth inhibition after 1.5 hours. Furthermore, CS/nAg nanocomposites improve the cell tissue regeneration in thermal burn injuries induced in rats. Synthesis of antibacterial, non-toxic, and biocompatible nanocomposites can be an important issue in tissue engineering and health care applications.

Keywords: antibacterial, chitosan, healing process, nanocomposites, silver

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Authors: Doriane Delafosse, Dominique Fontvieille

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Background: Wastewater treatment plants (WWTPs) generally display significant efficacy in virus retention yet, are sometimes highly variable, partly in relation to large fluctuating loads at the head of the plant and partly because of episodic dysfunctions in some treatment processes. The problem is especially sensitive when human enteric viruses, such as human Noroviruses Genogroup I or Adenoviruses, are in concern: their release downstream WWTP, in environments often interconnected to recreational areas, may be very harmful to human communities even at low concentrations. It points out the importance of WWTP permanent monitoring from which their internal treatment processes could be adjusted. One way to adjust primary treatments is to add coagulants and flocculants to sewage ahead settling tanks to improve decantation. In this work, sludge produced by three coagulants (two organics, one mineral), four flocculants (three cationic, one anionic), and their combinations were studied for their efficacy in human enteric virus retention. Sewage samples were coming from a WWTP in the vicinity of the laboratory. All experiments were performed three times and in triplicates in laboratory pilots, using Murine Norovirus (MNV-1), a surrogate of human Norovirus, as an internal control (spiking). Viruses were quantified by (RT-)qPCR after nucleic acid extraction from both treated water and sediment. Results: Low values of sludge virus retention (from 4 to 8% of the initial sewage concentration) were observed with each cationic organic flocculant added to wastewater and no coagulant. The largest part of the virus load was detected in the treated water (48 to 90%). However, it was not counterbalancing the amount of the introduced virus (MNV-1). The results pertained to two types of cationic flocculants, branched and linear, and in the last case, to two percentages of cations. Results were quite similar to the association of a linear cationic organic coagulant and an anionic flocculant, though suggesting that differences between water and sludges would sometimes be related to virus size or virus origins (autochthonous/allochthonous). FeCl₃, as a mineral coagulant associated with an anionic flocculant, significantly increased both auto- and allochthonous virus retention in the sediments (15 to 34%). Accordingly, virus load in treated water was lower (14 to 48%) but with a total that still does not reach the amount of the introduced virus (MNV-1). It also appeared that the virus retrieval in a bare 0.1M NaCl suspension varied rather strongly according to the FeCl₃ concentration, suggesting an inhibiting effect on the molecular analysis used to detect the virus. Finally, no viruses were detected in both phases (sediment and water) with the combination branched cationic coagulant-linear anionic flocculant, which was later demonstrated as an effect, here also, of polymers on the virus detection-molecular analysis. Conclusions: The combination of FeCl₃-anionic flocculant gave its highest performance to the decantation-based virus removal process. However, large unbalanced values in spiking experiments were observed, suggesting that polymers cast additional obstacles to both elution buffer and lysis buffer on their way to reach the virus. The situation was probably even worse with autochthonous viruses already embedded into sewage's particulate matter. Polymers and FeCl₃ also appeared to interfere in some steps of molecular analyses. More attention should be paid to such impediments wherever chemical additives are considered to be used to enhance WWTP processes. Acknowledgments: This research was supported by the ABIOLAB laboratory (Montbonnot Saint-Martin, France) and by the ASPOSAN association. Field experiments were possible thanks to the Grand Chambéry WWTP authorities (Chambéry, France).

Keywords: flocculants-coagulants, polymers, enteric viruses, wastewater sedimentation treatment plant

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Authors: Joanna Szymanska, Paulina Wawulska-Marek, Jaroslaw Mizera

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Nowadays, the rapid growth of global energy consumption and uncontrolled depletion of natural resources become a serious problem. Shale rocks are the largest and potential global basins containing hydrocarbons, trapped in closed pores of the shale matrix. Regardless of the shales origin, mining conditions are extremely unfavourable due to high reservoir pressure, great depths, increased clay minerals content and limited permeability (nanoDarcy) of the rocks. Taking into consideration such geomechanical barriers, effective extraction of natural gas from shales with plastic zones demands effective operations. Actually, hydraulic fracturing is the most developed technique based on the injection of pressurized fluid into a wellbore, to initiate fractures propagation. However, a rapid drop of pressure after fluid suction to the ground induces a fracture closure and conductivity reduction. In order to minimize this risk, proppants should be applied. They are solid granules transported with hydraulic fluids to locate inside the rock. Proppants act as a prop for the closing fracture, thus gas migration to a borehole is effective. Quartz sands are commonly applied proppants only at shallow deposits (USA). Whereas, ceramic proppants are designed to meet rigorous downhole conditions to intensify output. Ceramic granules predominate with higher mechanical strength, stability in strong acidic environment, spherical shape and homogeneity as well. Quality of ceramic proppants is conditioned by raw materials selection. Aim of this study was to obtain the proppants from aluminosilicates (the kaolinite subgroup) and mix of minerals with a high alumina content. These loamy minerals contain a tubular and platy morphology that improves mechanical properties and reduces their specific weight. Moreover, they are distinguished by well-developed surface area, high porosity, fine particle size, superb dispersion and nontoxic properties - very crucial for particles consolidation into spherical and crush-resistant granules in mechanical granulation process. The aluminosilicates were mixed with water and natural organic binder to improve liquid-bridges and pores formation between particles. Afterward, the green proppants were subjected to sintering at high temperatures. Evaluation of the minerals utility was based on their particle size distribution (laser diffraction study) and thermal stability (thermogravimetry). Scanning Electron Microscopy was useful for morphology and shape identification combined with specific surface area measurement (BET). Chemical composition was verified by Energy Dispersive Spectroscopy and X-ray Fluorescence. Moreover, bulk density and specific weight were measured. Such comprehensive characterization of loamy materials confirmed their favourable impact on the proppants granulation. The sintered granules were analyzed by SEM to verify the surface topography and phase transitions after sintering. Pores distribution was identified by X-Ray Tomography. This method enabled also the simulation of proppants settlement in a fracture, while measurement of bulk density was essential to predict their amount to fill a well. Roundness coefficient was also evaluated, whereas impact on mining environment was identified by turbidity and solubility in acid - to indicate risk of the material decay in a well. The obtained outcomes confirmed a positive influence of the loamy minerals on ceramic proppants properties with respect to the strict norms. This research is perspective for higher quality proppants production with costs reduction.

Keywords: aluminosilicates, ceramic proppants, mechanical granulation, shale gas

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