Search results for: molecular differentiation

Authors: Masauso Moses Phiri

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Frequent glucose monitoring is essential to the management of diabetes. Plasmonic enzyme-based glucose biosensors have the advantages of greater specificity, simplicity and rapidity. The aim of this study was to develop a rapid plasmonic colorimetric glucose biosensor based on biocatalytic enlargement of AuNS guided by GOx. Gold nanoparticles of 18 nm in diameter were synthesized using the citrate method. Using these as seeds, a modified seeded method for the synthesis of monodispersed gold nanostars was followed. Both the spherical and star-shaped nanoparticles were characterized using ultra-violet visible spectroscopy, agarose gel electrophoresis, dynamic light scattering, high-resolution transmission electron microscopy and energy-dispersive X-ray spectroscopy. The feasibility of a plasmonic colorimetric assay through growth of AuNS by silver coating in the presence of hydrogen peroxide was investigated by several control and optimization experiments. Conditions for excellent sensing such as the concentration of the detection solution in the presence of 20 µL AuNS, 10 mM of 2-(N-morpholino) ethanesulfonic acid (MES), ammonia and hydrogen peroxide were optimized. Using the optimized conditions, the glucose assay was developed by adding 5mM of GOx to the solution and varying concentrations of glucose to it. Kinetic readings, as well as color changes, were observed. The results showed that the absorbance values of the AuNS were blue shifting and increasing as the concentration of glucose was elevated. Control experiments indicated no growth of AuNS in the absence of GOx, glucose or molecular O₂. Increased glucose concentration led to an enhanced growth of AuNS. The detection of glucose was also done by naked-eye. The color development was near complete in ± 10 minutes. The kinetic readings which were monitored at 450 and 560 nm showed that the assay could discriminate between different concentrations of glucose by ± 50 seconds and near complete at ± 120 seconds. A calibration curve for the qualitative measurement of glucose was derived. The magnitude of wavelength shifts and absorbance values increased concomitantly with glucose concentrations until 90 µg/mL. Beyond that, it leveled off. The lowest amount of glucose that could produce a blue shift in the localized surface plasmon resonance (LSPR) absorption maxima was found to be 10 – 90 µg/mL. The limit of detection was 0.12 µg/mL. This enabled the construction of a direct sensitivity plasmonic colorimetric detection of glucose using AuNS that was rapid, sensitive and cost-effective with naked-eye detection. It has great potential for transfer of technology for point-of-care devices.

Keywords: colorimetric, gold nanostars, glucose, glucose oxidase, plasmonic

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Authors: Andrei Bejan, Luminita Marin, Dalila Belei

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Phenothiazine with electron-rich nitrogen and sulfur heteroatoms has a high electron-donating ability which promotes a good conjugation and therefore low band-gap with consequences upon charge carrier mobility improving and shifting of light emission in visible domain. Moreover, its non-planar butterfly conformation inhibits molecular aggregation and thus preserves quite well the fluorescence quantum yield in solid state compared to solution. Therefore phenothiazine and its derivatives are promising hole transport materials for use in organic electronic and optoelectronic devices as light emitting diodes, photovoltaic cells, integrated circuit sensors or driving circuits for large area display devices. The objective of this paper was to obtain a series of new phenothiazine derivatives by introduction of different electron withdrawing substituents as formyl, carboxyl and cyanoacryl units in order to create a push pull system which has potential to improve the electronic and optical properties. Bromine atom was used as electrono-donor moiety to extend furthermore the existing conjugation. The understudy compounds were structural characterized by FTIR and 1H-NMR spectroscopy and single crystal X-ray diffraction. Besides, the single crystal X-ray diffraction brought information regarding the supramolecular architecture of the compounds. Photophysical properties were monitored by UV-vis and photoluminescence spectroscopy, while the electrochemical behavior was established by cyclic voltammetry. The absorption maxima of the studied compounds vary in a large range (322-455 nm), reflecting the different electronic delocalization degree, depending by the substituent nature. In a similar manner, the emission spectra reveal different color of emitted light, a red shift being evident for the groups with higher electron withdrawing ability. The emitted light is pure and saturated for the compounds containing strong withdrawing formyl or cyanoacryl units and reach the highest quantum yield of 71% for the compound containing bromine and cyanoacrilic units. Electrochemical study show reversible oxidative and reduction processes for all the compounds and a close correlation of the HOMO-LUMO band gap with substituent nature. All these findings suggest the obtained compounds as promising materials for optoelectronic devices.

Keywords: electrochemical properties, phenothiazine derivatives, photoluminescence, quantum yield

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Authors: Sima Shakoorjavan, Mahdieh Eskafi, Dawid Stawski, Somaye Akbari

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In this study, poly (amidoamine) dendritic material (PAMAM) and poly (acrylic acid) (PAA) as polycation and polyanion were deposited on surface charged polylactide (PLA) nonwoven to study the relationship of dye absorption capacity of layered-PLA with the number of deposited layers. To produce negatively charged-PLA, acrylic acid (AA) was grafted on the PLA surface (PLA-g-AA) through a chemical redox reaction with the strong oxidizing agent. Spectroscopy analysis, water contact measurement, and FTIR-ATR analysis confirm the successful grafting of AA on the PLA surface through the chemical redox reaction method. In detail, an increase in dye absorption percentage by 19% and immediate absorption of water droplets ensured hydrophilicity of PLA-g-AA surface; and the presence of new carbonyl bond at 1530 cm-¹ and a wide peak of hydroxyl between 3680-3130 cm-¹ confirm AA grafting. In addition, PLA as linear polyester can undergo aminolysis, which is the cleavage of ester bonds and replacement with amid bonds when exposed to an aminolysis agent. Therefore, to produce positively charged PLA, PAMAM as amine-terminated dendritic material was introduced to PLA molecular chains at different conditions; (1) at 60 C for 0.5, 1, 1.5, 2 hours of aminolysis and (2) at room temperature (RT) for 1, 2, 3, and 4 hours of aminolysis. Weight changes and spectrophotometer measurements showed a maximum in weight gain graph and K/S value curve indicating the highest PAMAM attachment at 60 C for 1 hour and RT for 2 hours which is considered as an optimum condition. Also, the emerging new peak around 1650 cm-1 corresponding to N-H bending vibration and double wide peak at around 3670-3170 cm-1 corresponding to N-H stretching vibration confirm PAMAM attachment in selected optimum condition. In the following, regarding the initial surface charge of grafted-PLA, lbl deposition was performed and started with PAA or PAMAM. FTIR-ATR results confirm chemical changes in samples due to deposition of the first layer (PAA or PAMAM). Generally, spectroscopy analysis indicated that an increase in layer number costed dye absorption capacity. It can be due to the partial deposition of a new layer on the previously deposited layer; therefore, the available PAMAM at the first layer is more than the third layer. In detail, in the case of layer-PLA starting lbl with negatively charged, having PAMAM as the first top layer (PLA-g-AA/PAMAM) showed the highest dye absorption of both cationic and anionic model dye.

Keywords: surface modification, layer-by-layer technique, dendritic materials, PAMAM, dye absorption capacity, PLA nonwoven

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Authors: Ghirmay Teklemicheal, Samsom Mehari, Sara Tesfay

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Objectives: A total of 1074 suspected chikungunya cases were reported in Tesseney Province, Gash Barka region, Eritrea, during an outbreak. This study was aimed to assess the possible association of chikungunya severity among ABO blood groups and other potential determinants. Methods: A sex-matched and age-matched case-control study was conducted during the outbreak. For each case, one control subject had been selected from the mild Chikungunya cases. Along the same line of argument, a second control subject had also been designated through which neighborhood of cases were analyzed, scrutinized, and appeared to the scheme of comparison. Time is always the most sacrosanct element in pursuance of any study. According to the temporal calculation, this study was pursued from October 15, 2018, to November 15, 2018. Coming to the methodological dependability, calculating odds ratios (ORs) and conditional (fixed-effect) logistic regression methods were being applied. As a consequence of this, the data was analyzed and construed on the basis of the aforementioned methodological systems. Results: In this outbreak, 137 severe suspected chikungunya cases and 137 mild chikungunya suspected patients, and 137 controls free of chikungunya from the neighborhood of cases were analyzed. Non-O individuals compared to those with O blood group indicated as significant with a p-value of 0.002. Separate blood group comparison among A and O blood groups reflected as significant with a p-value of 0.002. However, there was no significant difference in the severity of chikungunya among B, AB, and O blood groups with a p-value of 0.113 and 0.708, respectively, and a strong association of chikungunya severity was found with hypertension and diabetes (p-value of < 0.0001); whereas, there was no association between chikungunya severity and asthma with a p-value of 0.695 and also no association with pregnancy (p-value =0.881), ventilator (p-value =0.181), air conditioner (p-value = 0.247), and didn’t use latrine and pit latrine (p-value = 0.318), among individuals using septic and pit latrine (p-value = 0.567) and also among individuals using flush and pit latrine (p-value = 0.194). Conclusions: Non- O blood groups were found to be at risk more than their counterpart O blood group individuals with severe form of chikungunya disease. By the same token, individuals with chronic disease were more prone to severe forms of the disease in comparison with individuals without chronic disease. Prioritization is recommended for patients with chronic diseases and non-O blood group since they are found to be susceptible to severe chikungunya disease. Identification of human cell surface receptor(s) for CHIKV is quite necessary for further understanding of its pathophysiology in humans. Therefore, molecular and functional studies will necessarily be helpful in disclosing the association of blood group antigens and CHIKV infections.

Keywords: Chikungunya, Chikungunya virus, disease outbreaks, case-control studies, Eritrea

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Authors: Z. Terzopoulou, I. Koliakou, D. Bikiaris

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Tissue engineering offers a new approach to regenerate diseased or damaged tissues such as bone. Great effort is devoted to eliminating the need of removing non-degradable implants at the end of their life span, with biodegradable polymers playing a major part. Poly(ε-caprolactone) (PCL) is one of the best candidates for this purpose due to its high permeability, good biodegradability and exceptional biocompatibility, which has stimulated extensive research into its potential application in the biomedical fields. However, PCL degrades much slower than other known biodegradable polymers and has a total degradation of 2-4 years depending on the initial molecular weight of the device. This is due to its relatively hydrophobic character and high crystallinity. Consequently, much attention has been given to the tunable degradation of PCL to meet the diverse requirements of biomedicine. Poly(ε-caprolactone) (PCL) is a biodegradable polyester that lacks bioactivity, so when used in bone tissue engineering, new bone tissue cannot bond tightly on the polymeric surface. Therefore, it is important to incorporate reinforcing fillers into PCL matrix in order to result in a promising combination of bioactivity, biodegradability, and strength. Natural clay halloysite nanotubes (HNTs) were incorporated into PCL polymeric matrix, via in situ ring-opening polymerization of caprolactone, in concentrations 0.5, 1 and 2.5 wt%. Both unmodified and modified with aminopropyltrimethoxysilane (APTES) HNTs were used in this study. The effect of nanofiller concentration and functionalization with end-amino groups on the physicochemical properties of the prepared nanocomposites was studied. Mechanical properties were found enhanced after the incorporation of nanofillers, while the modification increased further the values of tensile and impact strength. Thermal stability of PCL was not affected by the presence of nanofillers, while the crystallization rate that was studied by Differential Scanning Calorimetry (DSC) and Polarized Light Optical Microscopy (POM) increased. All materials were subjected to enzymatic hydrolysis in phosphate buffer in the presence of lipases. Due to the hydrophilic nature of HNTs, the biodegradation rate of nanocomposites was higher compared to neat PCL. In order to confirm the effect of hydrophilicity, contact angle measurements were also performed. In vitro biomineralization test confirmed that all samples were bioactive as mineral deposits were detected by X-ray diffractometry after incubation in SBF. All scaffolds were tested in relevant cell culture using osteoblast-like cells (MG-63) to demonstrate their biocompatibility

Keywords: biomaterials, nanocomposites, scaffolds, tissue engineering

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Authors: L. Sánchez-Abella, I. Loinaz, H-J. Grande, D. Dupin

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Aseptic loosening remains as the principal cause of revision in total hip arthroplasty (THA). For long-term implantations, submicron particles are generated in vivo due to the inherent wear of the prosthesis. When this occurs, macrophages undergo phagocytosis and secretion of bone resorptive cytokines inducing osteolysis, hence loosening of the implanted prosthesis. Therefore, new technologies are required to reduce the wear of the bearing materials and hence increase the life-span of the prosthesis. Our strategy focuses on surface modification of the bearing materials with a hydrophilic coating based on cross-linked water-soluble (meth)acrylic monomers to improve their tribological behavior. These coatings are biocompatible, with high swelling capacity and antifouling properties, mimicking the properties of natural cartilage, i.e. wear resistance with a permanent hydrated layer that prevents prosthesis damage. Cartilage mimicking based coatings may be also used to protect medical device surfaces from damage and scratches that will compromise their integrity and hence their safety. However, there are only a few reports on the mechanical and tribological characteristics of this type of coatings. Clear beneficial advantages of this coating have been demonstrated in different conditions and different materials, such as Ultra-high molecular weight polyethylene (UHMWPE), Polyethylene (XLPE), Carbon-fiber-reinforced polyetheretherketone (CFR-PEEK), cobalt-chromium (CoCr), Stainless steel, Zirconia Toughened Alumina (ZTA) and Alumina. Using routine tribological experiments, the wear for UHMWPE substrate was decreased by 75% against alumina, ZTA and stainless steel. For PEEK-CFR substrate coated, the amount of material lost against ZTA and CrCo was at least 40% lower. Experiments on hip simulator allowed coated ZTA femoral heads and coated UHMWPE cups to be validated with a decrease of 80% of loss material. Further experiments on hip simulator adding abrasive particles (1 micron sized alumina particles) during 3 million cycles, on a total of 6 million, demonstrated a decreased of around 55% of wear compared to uncoated UHMWPE and uncoated XLPE. In conclusion, CIDETEC‘s hydrogel coating technology is versatile and can be adapted to protect a large range of surfaces, even in abrasive conditions.

Keywords: cartilage, hydrogel, hydrophilic coating, joint

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Authors: Carlos Teodoro, Oscar Bautista

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In this work, we analyze theoretically the mass transport in a time-periodic electroosmotic flow through a parallel flat plate microchannel under different periodic functions of the applied external electric field. The microchannel connects two reservoirs having different constant concentrations of an electro-neutral solute, and the zeta potential of the microchannel walls are assumed to be uniform. The governing equations that allow determining the mass transport in the microchannel are given by the Poisson-Boltzmann equation, the modified Navier-Stokes equations, where the Debye-Hückel approximation is considered (the zeta potential is less than 25 mV), and the species conservation. These equations are nondimensionalized and four dimensionless parameters appear which control the mass transport phenomenon. In this sense, these parameters are an angular Reynolds, the Schmidt and the Péclet numbers, and an electrokinetic parameter representing the ratio of the half-height of the microchannel to the Debye length. To solve the mathematical model, first, the electric potential is determined from the Poisson-Boltzmann equation, which allows determining the electric force for various periodic functions of the external electric field expressed as Fourier series. In particular, three different excitation wave forms of the external electric field are assumed, a) sawteeth, b) step, and c) a periodic irregular functions. The periodic electric forces are substituted in the modified Navier-Stokes equations, and the hydrodynamic field is derived for each case of the electric force. From the obtained velocity fields, the species conservation equation is solved and the concentration fields are found. Numerical calculations were done by considering several binary systems where two dilute species are transported in the presence of a carrier. It is observed that there are different angular frequencies of the imposed external electric signal where the total mass transport of each species is the same, independently of the molecular diffusion coefficient. These frequencies are called crossover frequencies and are obtained graphically at the intersection when the total mass transport is plotted against the imposed frequency. The crossover frequencies are different depending on the Schmidt number, the electrokinetic parameter, the angular Reynolds number, and on the type of signal of the external electric field. It is demonstrated that the mass transport through the microchannel is strongly dependent on the modulation frequency of the applied particular alternating electric field. Possible extensions of the analysis to more complicated pulsation profiles are also outlined.

Keywords: electroosmotic flow, mass transport, oscillatory flow, species separation

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Authors: Huixia Shi, Can Hu, Jun Zhu, Hongling Guo, Haiyan Li, Hongyan Du

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The identification of human body fluids during forensic investigations is a critical step to determine key details, and present strong evidence to testify criminal in a case. With the popularity of DNA and improved detection technology, the potential question must be revolved that whether the suspect’s DNA derived from saliva or semen, menstrual or peripheral blood, how to identify the red substance or aged blood traces on the spot is blood; How to determine who contribute the right one in mixed stains. In recent years, molecular approaches have been developing increasingly on mRNA, miRNA, DNA methylation and microbial markers, but appear expensive, time-consuming, and destructive disadvantages. Physicochemical methods are utilized frequently such us scanning electron microscopy/energy spectroscopy and X-ray fluorescence and so on, but results only showing one or two characteristics of body fluid itself and that out of working in unknown or mixed body fluid stains. This paper focuses on using chemistry methods Raman spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to discriminate species of peripheral blood, menstrual blood, semen, saliva, vaginal secretions, urine or sweat. Firstly, non-destructive, confirmatory, convenient and fast Raman spectroscopy method combined with more accurate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method can totally distinguish one from other body fluids. Secondly, 11 spectral signatures and specific metabolic molecules have been obtained by analysis results after 70 samples detected. Thirdly, Raman results showed peripheral and menstrual blood, saliva and vaginal have highly similar spectroscopic features. Advanced statistical analysis of the multiple Raman spectra must be requested to classify one to another. On the other hand, it seems that the lactic acid can differentiate peripheral and menstrual blood detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, but that is not a specific metabolic molecule, more sensitivity ones will be analyzed in a forward study. These results demonstrate the great potential of the developed chemistry methods for forensic applications, although more work is needed for method validation.

Keywords: body fluids, identification, Raman spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

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Authors: Lillian Musila, Valerie Oundo, Daniel Erwin, Willie Sang

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Staphylococcus aureus infections are a major cause of nosocomial infections in Kenya. Methicillin resistant S. aureus (MRSA) infections are a significant burden to public health and are associated with considerable morbidity and mortality. At a hospital in Western Kenya two clusters of MRSA cases emerged within short periods of time. In this study we explored whether these clusters represented a nosocomial outbreak by characterizing the isolates using phenotypic and molecular assays and examining epidemiological data to identify possible transmission patterns. Specimens from the site of infection of the subjects were collected, cultured and S. aureus isolates identified phenotypically and confirmed by APIStaph™. MRSA were identified by cefoxitin disk screening per CLSI guidelines. MRSA were further characterized based on their antibiotic susceptibility patterns and spa gene typing. Characteristics of cases with MRSA isolates were compared with those with MSSA isolated around the same time period. Two cases of MRSA infection were identified in the two week period between 21 April and 4 May 2015. A further 2 MRSA isolates were identified on the same day on 7 September 2015. The antibiotic resistance patterns of the two MRSA isolates in the 1st cluster of cases were different suggesting that these were distinct isolates. One isolate had spa type t2029 and the other had a novel spa type. The 2 isolates were obtained from urine and an open skin wound. In the 2nd cluster of MRSA isolates, the antibiotic susceptibility patterns were similar but isolates had different spa types: one was t037 and the other a novel spa type different from the novel MRSA spa type in the first cluster. Both cases in the second cluster were admitted into the hospital but one infection was community- and the other hospital-acquired. Only one of the four MRSA cases was classified as an HAI from an infection acquired post-operatively. When compared to other S. aureus strains isolated within the same time period from the same hospital only one spa type t2029 was found in both MRSA and non-MRSA strains. None of the cases infected with MRSA in the two clusters shared any common epidemiological characteristic such as age, sex or known risk factors for MRSA such as prolonged hospitalization or institutionalization. These data suggest that the observed MRSA clusters were multi strain clusters and not an outbreak of a single strain. There was no clear relationship between the isolates by spa type suggesting that no transmission was occurring within the hospital between these cluster cases but rather that the majority of the MRSA strains were circulating in the community. There was high diversity of spa types among the MRSA strains with none of the isolates sharing spa types. Identification of disease clusters in space and time is critical for immediate infection control action and patient management. Spa gene typing is a rapid way of confirming or ruling out MRSA outbreaks so that costly interventions are applied only when necessary.

Keywords: cluster, Kenya, MRSA, spa typing

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Authors: Manisha, Manisha Mathur, Jay K. Desai, Shesh Asopa, Manisha Mehra

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The present study was carried out for the comprehensive analysis of lumpy skin disease (LSD) in cattle and to elucidate the histopathology of vital organs in natural outbreaks. Lumpy skin disease (LSD) is a viral infection that primarily affects cattle. It is caused by a Capri pox virus and is characterized by the formation of skin nodules or lesions. For this study, a postmortem of 20 cows who died of Lumpy skin disease in different regions of Rajasthan was conducted. This study aimed to examine a cow's external and internal organs to confirm if lumpy skin disease was the cause of death. Accurate diagnosis is essential for improving disease surveillance, understanding the disease's progression, and informing control measures. Pathological examinations reveal virus-induced changes across organs, while histopathological analyses provide crucial insights into the disease's pathogenesis, aiding in the development of advanced diagnostics and effective prevention strategies. Histopathological examination of nodular skin lesions revealed edema, hyperemia, acanthosis, severe hydropic degeneration/ballooning degeneration, and hyperkeratosis in the epidermis. In the lungs, congestion, oedema, emphysema, and atelectasis were observed grossly. Microscopically changes were suggestive of interstitial pneumonia, suppurative pneumonia, bronchopneumonia post pneumonic fibrosis, and stage of resolution. Grossely liver showed congestion and necrotic foci microscopically in most of the cases, and the liver showed acute viral hepatitis. Microscopically in kidneys, multifocal interstitial nephritis was observed. There was marked interstitial inflammation and zonal fibrosis with cystically dilated tubules and bowman's capsules. Microscopically, most of the heart tissue section showed normal histology with few sarcocysts in between cardiac muscles. In some cases, loss of cross striation, sarcoplasmic vacuolation, fregmentation, and disintegration of cardiac fibres were observed. The present study revealed the characteristic gross and histopathological changes in different organs in natural cases of lumpy skin disease. Further, the disease was confirmed based on the molecular diagnosis and transmission electron microscopy of capripox infection in the affected cattle in the study area.

Keywords: Capripoxvirus, lumpy skin disease, polymerage chain reaction, transmission electron microscopy

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Authors: Olumuyiwa Ayoola Kokapi, Olugbenga Solomon Bello

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Dyes are organic compounds with complex aromatic molecular structure that resulted in fast colour on a substance. Dye effluent found in wastewater generated from the dyeing industries is one of the greatest contributors to water pollution. Groundnut hull (GH) is an agricultural material that constitutes waste in the environment. Environmental contamination by hazardous organic chemicals is an urgent problem, which is partially solved through adsorption technologies. The choice of groundnut hull was promised on the understanding that some materials of agricultural origin have shown potentials to act as Adsorbate for hazardous organic chemicals. The aim of this research is to evaluate the potential of groundnut hull to adsorb Crystal violet dye through kinetic, isotherm and thermodynamic studies. The prepared groundnut hulls was characterized using Brunauer, Emmett and Teller (BET), Fourier transform infrared (FTIR) and scanning electron microscopy (SEM). Operational parameters such as contact time, initial dye concentration, pH, and effect of temperature were studied. Equilibrium time for the adsorption process was attained in 80 minutes. Adsorption isotherms used to test the adsorption data were Langmuir and Freundlich isotherms model. Thermodynamic parameters such as ∆G°, ∆H°, and ∆S° of the adsorption processes were determined. The results showed that the uptake of dye by groundnut hulls occurred at a faster rate, corresponding to an increase in adsorption capacity at equilibrium time of 80 min from 0.78 to 4.45 mg/g and 0.77 to 4.45mg/g with an increase in the initial dye concentration from 10 to 50 mg/L for pH 3.0 and 8.0 respectively. High regression values obtained for pseudo-second-order kinetic model, sum of square error (SSE%) values along with strong agreement between experimental and calculated values of qe proved that pseudo second-order kinetic model fitted more than pseudo first-order kinetic model. The result of Langmuir and Freundlich model showed that the adsorption data fit the Langmuir model more than the Freundlich model. Thermodynamic study demonstrated the feasibility, spontaneous and endothermic nature of the adsorption process due to negative values of free energy change (∆G) at all temperatures and positive value of enthalpy change (∆H) respectively. The positive values of ∆S showed that there was increased disorderliness and randomness at the solid/solution interface of crystal violet dye and groundnut hulls. The present investigation showed that, groundnut hulls (GH) is a good low-cost alternative adsorbent for the removal of Crystal Violet (CV) dye from aqueous solution.

Keywords: adsorption, crystal violet dye, groundnut halls, kinetics

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Authors: Alaaeddeen M. Seufi

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Many have been published on therapeutic derivatives from living organisms including insects. In addition to traditional maggot therapy, more than 900 therapeutic products were isolated from insects. Most people look at insects as enemies and others believe that insects are friends. Many beneficial insects rather than Honey Bees, Silk Worms and Shellac insect could insure human-insect friendship. In addition, insects could be MicroFactories, Biosensors or Bioreactors. InsectFarm is an amazing example of the applied research that transfers insects from laboratory to market by Prof Mircea Ciuhrii and co-workers. They worked for 18 years to derive therapeutics from insects. Their research resulted in production of more than 30 commercial medications derived from insects (e.g. Imunomax, Noblesse, etc.). Two general approaches were followed to discover drugs from living organisms. Some laboratories preferred biochemical approach to purify components of the innate immune system of insects and insect metabolites as well. Then the purified components could be tested for many therapeutic trials. Other researchers preferred molecular approach based on proteomic studies. Components of the innate immune system of insects were then tested for their medical activities. Our Laboratory team preferred to induce insect immune system (using oral, topical and injection routes of administration), then a transcriptomic study was done to discover the induced genes and to identify specific biomarkers that can help in drug discovery. Biomarkers play an important role in medicine and in drug discovery and development as well. Optimum biomarker development and application will require a team approach because of the multifaceted nature of biomarker selection, validation, and application. This team uses several techniques such as pharmacoepidemiology, pharmacogenomics, and functional proteomics; bioanalytical development and validation; modeling and simulation to improve and refine drug development. Our Achievements included the discovery of four components of the innate immune system of Spodoptera littoralis and Musca domestica. These components were designated as SpliDef (defesin), SpliLec (lectin), SpliCec (cecropin) and MdAtt (attacin). SpliDef, SpliLec and MdAtt were confirmed as antimicrobial peptides, while SpliCec was additionally confirmed as anticancer peptide. Our current research is going on to achieve something in antioxidants and anticoagulants from insects. Our perspective is to achieve something in the mass production of prototypes of our products and to reach it to the commercial level. These achievements are the integrated contributions of everybody in our team staff.

Keywords: AMPs, insect, innate immunitty, therappeutics

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Authors: Tais Basaco, Stefanie Pektor, Josue A. Moreno, Matthias Miederer, Andreas Türler

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Radiopharmaceuticals based in monoclonal anti-body (mAb) via chemical linkers have become a potential tool in nuclear medicine because of their specificity and the large variability and availability of therapeutic radiometals. It is important to identify the conjugation sites and number of attached chelator to mAb to obtain radioimmunoconjugates with required immunoreactivity and radiostability. Girentuximab antibody (G250) is a potential candidate for radioimmunotherapy of clear cell carcinomas (RCCs) because it is reactive with CAIX antigen, a transmembrane glycoprotein overexpressed on the cell surface of most ( > 90%) (RCCs). G250 was conjugated with the bifunctional chelating agent DOTA (1,4,7,10-Tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid) via a benzyl-thiocyano group as a linker (p-SCN-Bn-DOTA). DOTA-G250 conjugates were analyzed by size exclusion chromatography (SE-HPLC) and by electrophoresis (SDS-PAGE). The potential site-specific conjugation was identified by liquid chromatography–mass spectrometry (LC/MS-MS) and the number of linkers per molecule of mAb was calculated using the molecular weight (MW) measured by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The average number obtained in the conjugates in non-reduced conditions was between 8-10 molecules of DOTA per molecule of mAb. The average number obtained in the conjugates in reduced conditions was between 1-2 and 3-4 molecules of DOTA per molecule of mAb in the light chain (LC) and heavy chain (HC) respectively. Potential DOTA modification sites of the chelator were identified in lysine residues. The biological activity of the conjugates was evaluated by flow cytometry (FACS) using CAIX negative (SKRC-18) and CAIX positive (SKRC-52). The DOTA-G250 conjugates were labelled with 177Lu with a radiochemical yield > 95% reaching specific activities of 12 MBq/µg. The stability in vitro of different types of radioconstructs was analyzed in human serum albumin (HSA). The radiostability of 177Lu-DOTA-G250 at high specific activity was increased by addition of sodium ascorbate after the labelling. The immunoreactivity was evaluated in vitro and in vivo. Binding to CAIX positive cells (SK-RC-52) at different specific activities was higher for conjugates with less DOTA content. Protein dose was optimized in mice with subcutaneously growing SK-RC-52 tumors using different amounts of 177Lu- DOTA-G250.

Keywords: mass spectrometry, monoclonal antibody, radiopharmaceuticals, radioimmunotheray, renal cancer

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Authors: Ratneev Kaur, Tajinder Kaur, Anupam Kaur

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Introduction: Polycystic Ovary Syndrome (PCOS) is a heterogenous disorder of endocrine system among women of reproductive age. PCOS is characterized by hyperandrogenism, anovulation, polycystic ovaries, hirsutism, obesity, and hyperinsulinemia. Several pathways are implicated in its etiology including the metabolic pathway of steroid hormone synthesis regulatory pathways. PCOS is an androgen excess disorder, genes operating in steroidogenesis may alter pathogenesis of PCOS. The cytochrome P450scc is a cholesterol side chain cleavage enzyme coded by CYP11A1 gene and catalyzes conversion of cholesterol to pregnenolone, the initial and rate-limiting step in steroid hormone synthesis. It is postulated that polymorphisms in this gene may play an important role in the regulation of CYP11A1 expression and leading to increased or decreased androgen production. The present study will be the first study from north India to best of our knowledge, to analyse the association of CYP11A1 (rs11632698) polymorphism in women suffering from PCOS. Methodology: The present study was approved by ethical committee of Guru Nanak Dev University in consistent with declaration of Helsinki. A total of 300 samples (150 PCOS cases and 150 controls) were recruited from Hartej hospital, for the present study. Venous blood sample (3ml) was withdrawn from women diagnosed with PCOS by doctor, according to Rotterdam 2003 criteria and from healthy age matched controls only after informed consent and detailed filled proforma. For molecular genetics analysis, blood was stored in EDTA vials. After DNA isolation by organic method, PCR-RFLP approach was used for genotyping and association analysis of rs11632698 polymorphism. Statistical analysis was done to check for significance of selected polymorphism with PCOS. Results: In 150 PCOS cases, the frequency of AA, AG and GG genotype was found to be 48%, 35%, and 13% compared to 62%, 27% and 8% in 150 controls. The major allele (A) and minor allele (G) frequency was 68% and 32% in cases and 78% and 22% in controls. Minor allele frequency was higher in cases as compared to controls, as well as the distribution of genotype was observed to be statistically significant (ᵡ²=6.525, p=0.038). Odds ratio in dominant, co-dominant and recessive models observed was 1.81 (p=0.013), 1.54 (p=0.012) and 1.77 (p=0.132) respectively. Conclusion: The present study showed statistically significant association of rs11632698 with PCOS (p=0.038) in North Indian women.

Keywords: polycystic ovary syndrome, CYP11A1, rs11632698, hyperandrogenism

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Authors: Shenghong Huang, Weirong Wang, Xisheng Luo, Xinzhu Li, Xinwen Zhao

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Understanding of material mixing induced by Richtmyer-Meshkov instability (RMI) at extreme shock compressing conditions (high energy density environment: P >> 100GPa, T >> 10000k) is of great significance in engineering and science, such as inertial confinement fusion(ICF), supersonic combustion, etc. Turbulent mixing induced by RMI is a kind of complex fluid dynamics, which is closely related with hydrodynamic conditions, thermodynamic states, material physical properties such as compressibility, strength, surface tension and viscosity, etc. as well as initial perturbation on interface. For phenomena in ordinary thermodynamic conditions (low energy density environment), many investigations have been conducted and many progresses have been reported, while for mixing in extreme thermodynamic conditions, the evolution may be very different due to ionization as well as large difference of material physical properties, which is full of scientific problems and academic interests. In this investigation, the first principle based molecular dynamic method is applied to study metal Lithium and gas Hydrogen (Li-H2) interface mixing in micro/meso scale regime at different shock compressing loading speed ranging from 3 km/s to 30 km/s. It's found that, 1) Different from low-speed shock compressing cases, in high-speed shock compresing (>9km/s) cases, a strong acceleration of metal/gas interface after strong shock compression is observed numerically, leading to a strong phase inverse and spike growing with a relative larger linear rate. And more specially, the spike growing rate is observed to be increased with shock loading speed, presenting large discrepancy with available empirical RMI models; 2) Ionization is happened in shock font zone at high-speed loading cases(>9km/s). An additional local electric field induced by the inhomogeneous diffusion of electrons and nuclei after shock font is observed to occur near the metal/gas interface, leading to a large acceleration of nuclei in this zone; 3) In conclusion, the work of additional electric field contributes to a mechanism of RMI in micro/meso scale regime at extreme shock compressing conditions, i.e., a Rayleigh-Taylor instability(RTI) is induced by additional electric field during RMI mixing process and thus a larger linear growing rate of interface spike.

Keywords: ionization, micro/meso scale, material mixing, shock

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Authors: Marie Davin, Marie-Laure Fauconnier, Gilles Colinet

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In Wallonia, there are 6,000 estimated brownfields (rising to over 3.5 million in Europe) that require remediation. Polycyclic Aromatic Hydrocarbons (PAHs) are a class of recalcitrant carcinogenic/mutagenic organic compounds of major concern as they accumulate in the environment and represent 17% of all encountered pollutants. As an alternative to environmentally aggressive, expensive and often disruptive soil remediation strategies, a lot of research has been directed to developing techniques targeting organic pollutants. The following experiment, based on the observation that PAHs soil content decreases in the presence of plants, aimed at improving our understanding of the underlying mechanisms involved in phytoremediation. It focusses on plant root exudates and whether they improve PAHs solubilization, which would make them more available for bioremediation by soil microorganisms. The effect of saponin, a natural surfactant found in some plant roots such as members of the Fabaceae family, on PAHs solubilization was also investigated as part of the implementation of the experimental protocol. The experiments were conducted on soil collected from a brownfield in Saint-Ghislain (Belgium) and presenting weathered PAHs contamination. Samples of soil were extracted with different solutions containing either plant root exudates or commercial saponin. Extracted PAHs were determined in the different aqueous solutions using High-Performance Liquid Chromatography and Fluorimetric Detection (HPLC-FLD). Both root exudates of alfalfa (Medicago sativa L.) or red clover (Trifolium pratense L.) and commercial saponin were tested in different concentrations. Distilled water was used as a control. First of all, results show that PAHs are more extracted using saponin solutions than distilled water and that the amounts generally rise with the saponin concentration. However, the amount of each extracted compound diminishes as its molecular weight rises. Also, it appears that passed a certain surfactant concentration, PAHs are less extracted. This suggests that saponin might be investigated as a washing agent in polluted soil remediation techniques, either for ex-situ or in-situ treatments, as an alternative to synthetic surfactants. On the other hand, preliminary results on experiments using plant root exudates also show differences in PAHs solubilization compared to the control solution. Further results will allow discussion as to whether or not there are differences according to the exudates provenance and concentrations.

Keywords: brownfield, Medicago sativa, phytoremediation, polycyclic aromatic hydrocarbons, root exudates, saponin, solubilization, Trifolium pratense

Procedia PDF Downloads 253

Authors: Ami Okabe, Daisuke Gondo, Akira Ogawa, Yasuhisa Hasegawa, Koichi Sato, Sadao Araki, Hideki Yamamoto

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Membrane separation draws attention as the energy-saving technology. Pervaporation (PV) uses hydrophobic ceramic membranes to separate organic compounds from industrial wastewaters. PV makes it possible to separate organic compounds from azeotropic mixtures and from aqueous solutions. For the PV separation of low concentrations of organics from aqueous solutions, hydrophobic ceramic membranes are expected to have high separation performance compared with that of conventional hydrophilic membranes. Membrane separation performance is evaluated based on the pervaporation separation index (PSI), which depends on both the separation factor and the permeate flux. Ingenuity is required to increase the PSI such that the permeate flux increases without reducing the separation factor or to increase the separation factor without reducing the flux. A thin separation layer without defects and pinholes is required. In addition, it is known that the flux can be increased without reducing the separation factor by reducing the diffusion resistance of the membrane support. In a previous study, we prepared hydrophobic silica membranes by a molecular templating sol−gel method using cetyltrimethylammonium bromide (CTAB) to form pores suitable for permitting the passage of organic compounds through the membrane. We separated low-concentration organics from aqueous solutions by PV using these membranes. In the present study, hydrophobic silica membranes were prepared on a porous alumina hollow fiber support that is thinner than the previously used alumina support. Ethyl acetate (EA) is used in large industrial quantities, so it was selected as the organic substance to be separated. Hydrophobic silica membranes were prepared by dip-coating porous alumina supports with a -alumina interlayer into a silica sol containing CTAB and vinyltrimethoxysilane (VTMS) as the silica precursor. Membrane thickness increases with the lifting speed of the sol in the dip-coating process. Different thicknesses of the γ-alumina layer were prepared by dip-coating the support into a boehmite sol at different lifting speeds (0.5, 1, 3, and 5 mm s-1). Silica layers were subsequently formed by dip-coating using an immersion time of 60 s and lifting speed of 1 mm s-1. PV measurements of the EA (5 wt.%)/water system were carried out using VTMS hydrophobic silica membranes prepared on -alumina layers of different thicknesses. Water and EA flux showed substantially constant value despite of the change of the lifting speed to form the γ-alumina interlayer. All prepared hydrophobic silica membranes showed the higher PSI compared with the hydrophobic membranes using the previous alumina support of hollow fiber.

Keywords: membrane separation, pervaporation, hydrophobic, silica

Procedia PDF Downloads 404

Authors: Ante Turudic, Filip Varga, Zlatko Liber, Jernej Jakse, Zlatko Satovic, Ivan Radosavljevic, Martina Grdisa

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Microsatellites (SSRs) are highly informative repetitive sequences of 2-6 base pairs, which are the most used molecular markers in assessing the genetic diversity of plant species. Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip) is an outcrossing diploid (2n = 18) endemic to the eastern Adriatic coast and source of the natural insecticide pyrethrin. Due to the high repetitiveness and large size of the genome (haploid genome size of 9,58 pg), previous attempts to develop microsatellite markers using the standard methods were unsuccessful. A next-generation sequencing (NGS) approach was applied on genomic DNA extracted from fresh leaves of Dalmatian pyrethrum. The sequencing was conducted using NovaSeq6000 Illumina sequencer, after which almost 400 million high-quality paired-end reads were obtained, with a read length of 150 base pairs. Short reads were assembled by combining two approaches; (1) de-novo assembly and (2) joining of overlapped pair-end reads. In total, 6.909.675 contigs were obtained, with the contig average length of 249 base pairs. Of the resulting contigs, 31.380 contained one or multiple microsatellite sequences, in total 35.556 microsatellite loci were identified. Out of detected microsatellites, dinucleotide repeats were the most frequent, accounting for more than half of all microsatellites identifies (21,212; 59.7%), followed by trinucleotide repeats (9,204; 25.9%). Tetra-, penta- and hexanucleotides had similar frequency of 1,822 (5.1%), 1,472 (4.1%), and 1,846 (5.2%), respectively. Contigs containing microsatellites were further filtered by SSR pattern type, transposon occurrences, assembly characteristics, GC content, and the number of occurrences against the draft genome of T. cinerariifolium published previously. After the selection process, 50 microsatellite loci were used for primer design. Designed primers were tested on samples from five distinct populations, and 25 of them showed a high degree of polymorphism. The selected loci were then genotyped on 20 samples belonging to one population resulting in 17 microsatellite markers. Availability of codominant SSR markers will significantly improve the knowledge on population genetic diversity and structure as well as complex genetics and biochemistry of this species. Acknowledgment: This work has been fully supported by the Croatian Science Foundation under the project ‘Genetic background of Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir/ Sch. Bip.) insecticidal potential’ - (PyrDiv) (IP-06-2016-9034).

Keywords: genome assembly, NGS, SSR, Tanacetum cinerariifolium

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Authors: K. V. Ramanaiah, R. Jagan, N. N. Murthy

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Trinuclear oxo-centered carboxylate-bridged iron complexes, [Fe3(µ3-O)(µ2-O2CR)L¬3]+/0 (where R = alkyl or aryl; L = H2O, ROH, Py, solvent) have attracted tremendous attention because of their interesting structural and magnetic properties, exhibit mixed-valent trapped and de-trapped states, and have bioinorganic relevance. The presence of a trinuclear iron binding center has been implicated in the formation of both bacterial and human iron storage protein, Ft. They are used as precursors for the synthesis of models for the active-site structures of non-heme proteins, hemerythrin (Hr), methane monooxygenase (MMO) and polyiron storage protein, ferritin (Ft). Used as important building blocks for the design and synthesis of supramolecules this can exhibit single molecular magnetism (SMM). Such studies have often employed simple and compact carboxylate ligands and the use of bulky carboxylates is scarce. In the present study, we employed two different type of sterically hindered carboxylates and synthesized a series of novel oxo-centered, carboxylate-bridged triiron complexes of general formula [Fe3(O)(O2CCPh3)6L3]X (L = H2O, 1; py, 2; 4-NMe2py, 3; X = ClO4; L = CH3CN, 4; X = FeCl4) and [Fe3(O)(O2C-anth)6L3]X (L = H2O, 5; X = ClO4; L = CH3OH, 6; X = Cl). Along with complex [Fe(OMe)2(O2CCPh3)]10, 7 was prepared by the self-assemble of anhydrous FeCl3, sodium triphenylacetate and sodium methoxide at ratio of 1:1:2 in CH3OH. The Electronic absorption spectra of these complexes 1-6, in CH2Cl2 display weak bands at near FTIR region (970-1135 nm, ε > 15M-1cm-1). For complex 7, one broad band centered at ~670nm and also an additional intense charge transfer (L→M or O→M) bands between 300 to 550nm observed for all the complexes. Paramagnetic 1H NMR is introduced as a good probe for the characterization of trinuclear oxo - cantered iron compounds in solution when the L ligand coordinated to iron varies as: H2O, py, 4-NMe2py, and CH3OH. The solution state magnetic moment values calculated by using Evans method for all the complexes and also solid state magnetic moment value of complex, 7 was calculated by VSM method, which is comparable with solution state value. These all magnetic moment values indicate there is a spin exchange process through oxo and carboxylate bridges in between two irons (d5). The ESI-mass data complement the data obtained from single crystal X-ray structure. Further purity of the compounds was confirmed by elemental analysis. Finally, structural determination of complexes 1, 3, 4, 5, 6 and 7 were unambiguously conformed by single crystal x-ray studies.

Keywords: decanuclear, paramagnetic NMR, trinuclear, uv-visible

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Authors: Judit Perjessy, Zsolt Zalan, Ferenc Hegyi, Eniko Horvath-Szanics, Krisztina Takacs, Andras Nagy, Adel Klupacs, Erika Koppany-Szabo, Zhirong Wang, Kaituo Wang, Muying Du, Jianquan Kan

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The post-harvest diseases cause great economic losses in the fruit and vegetables; the prevention of these deterioration has great importance. Against the fungi, which cause most of the diseases, are extensively used the fungicides. However, there are increasing consumer concerns over the presence of pesticide residues in food. An alternative and in recent years, increasingly studied method for the prevention of the diseases is biocontrol, where antagonistic microorganisms are used for the control of fungi. The genera of Lactobacillus is well known and extensively studied, but its applicability as biocontrol agents in post-harvest preservation of fruit and vegetables is poorly investigated. However these bacteria can be found on the surface of the plants and have great antimicrobial activity. In our study we have investigated the chitinase activity, the antifungal effect and the applicability of several Lactobacillus strains to select potential biocontrol agents. We investigated the determination of the environmental parameters of a gene (encoding chitinase) expression and we also investigated the relationship between actual antifungal activity and potential chitinase activity. Mixed cultures were also developed to enhance the antifungal activity and determined the optimal mold spore and bacteria concentration ratio for the appropriate efficacy. Five Lactobacillus strains (L. acidophilus N2, L. delbrueckii subsp. bulgaricus B397, L. sp. 2231, L. sake subsp. sake 2471, L. buchneri 1145) possess chitinase-coding gene from the 43 investigated Lactobacillus strains. Proteins with similar molecular weight and separation properties like bacterial chitinases were detected from these strains, which also possess chitin-binding property. Nevertheless, they were inactive, lacks the chitinolytic activity. In point of the cumulative activity of inhibition, our results showed that certain strains were statistically significant in a positive direction compared to other strains, e.g., L. rhamnosus VT1 and L. Casey 154 have shown great general antifungal effect against 11 molds from the genera Penicillium and Botrytis and isolated from spoiled fruit and vegetables. Also, some mixed cultures (L. rhamnosus VT1 - L. Plantarum 299v) showed significant antifungal effects against the indigenous molds on the surface of apple fruit during the industrial storage experiment. Thus, they could be promising for post-harvest biopreservation.

Keywords: biocontrol, chitinase, Lactobacillus, post-harvest

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Authors: Emine Aydin, Duygu Gocmen

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Pumpkin flour (PF) has a long shelf life and can be used as a nutritive, functional (antioxidant properties, phenolic contents, etc.) and coloring agent in many food items, especially in bakery products, sausages, instant noodles, pasta and flour mixes. Pre-treatment before drying is one of the most important factors affecting the quality of a final powdered product. Pretreatment, such as soaking in a bisulfite solution, provides that total carotenoids in raw materials rich in carotenoids, especially pumpkins, are retained in the dried product. This is due to the beneficial effect of antioxidant additives in the protection of carotenoids in the dehydrated plant foods. The oxygen present in the medium is removed by the radical SO₂, and thus the carotene degradation caused by the molecular oxygen is inhibited by the presence of SO₂. In this study, pumpkin flours (PFs) produced by two different applications (with or without metabisulfite pre-treatment) and then dried in a freeze dryer. The phenolic contents and antioxidant activities of pumpkin flour were determined. In addition to this, the compound of bioavailable phenolic substances which is obtained by PF has also been investigated using in vitro methods. As a result of researches made in recent years, it has been determined that all nutrients taken with foodstuffs are not bioavailable. Bioavailability changes depending on physical properties, chemical compounds, and capacities of individual digestion of foods. Therefore in this study; bioaccessible phenolics and phenolic bioaccessibility were also determined. The phenolics of the samples with metabisulfite application were higher than those of the samples without metabisulfite pre-treatment. Soaking in metabisulfite solution might have a protective effect for phenolic compounds. Phenolics bioaccessibility of pumpkin flours was investigated in order to assess pumpkin flour as sources of accessible phenolics. The higher bioaccessible phenolics (384.19 mg of GAE 100g⁻¹ DW) and phenolic bioaccessibility values (33.65 mL 100 mL⁻¹) were observed in the pumpkin flour with metabisulfite pre-treatment. Metabisulfite application caused an increase in bioaccessible phenolics of pumpkin flour. According to all assay (ABTS, CUPRAC, DPPH, and FRAP) results, both free and bound phenolics of pumpkin flour with metabisulfite pre-treatment had higher antioxidant activity than those of the sample without metabisulfite pre-treatment. The samples subjected to MS pre-treatment exhibited higher antioxidant activities than those of the samples without MS pre-treatment, this possibly due to higher phenolic contents of the samples with metabisulfite applications. As a result, metabisulfite application caused an increase in phenolic contents, bioaccessible phenolics, phenolic bioaccessibility and antioxidant activities of pumpkin flour. It can be said that pumpkin flour can be used as an alternative functional and nutritional ingredient in bakery products, dairy products (yoghurt, ice-cream), soups, sauces, infant formulae, confectionery, etc.

Keywords: pumpkin flour, bioaccessible phenolics, phenolic bioaccessibility, antioxidant activity

Procedia PDF Downloads 325

Authors: Santi Maneewatchararangsri, Wanpen Chaicumpa, Patcharin Saengjaruk, Urai Chaisri

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Leptospirosis is a globally neglected disease that continues to be a significant public health and veterinary burden, with millions of cases reported each year. Early and accurate differential diagnosis of leptospirosis from other febrile illnesses and the development of a broad spectrum of leptospirosis vaccines are needed. The LipL32 outer membrane lipoprotein is a member of Leptospira adhesive matrices and has been found to exert hemolytic activity to erythrocytes in vitro. Therefore, LipL32 is regarded as a potential target for diagnosis, broad-spectrum leptospirosis vaccines, and for passive immunotherapy. In this study, we established LipL32-specific mouse monoclonal antibodies, mAbLPF1 and mAbLPF2, and their respective mouse- and humanized-engineered single chain variable fragment (ScFv). Their antibodies’ neutralizing activities against Leptospira-mediated hemolysis in vitro, and the therapeutic efficacy of mAbs against heterologous Leptospira infected hamsters were demonstrated. The epitope peptide of mAb LPF1 was mapped to a non-contiguous carboxy-terminal β-turn and amphipathic α-helix of LipL32 structure contributing to phospholipid/host cell adhesion and membrane insertion. We found that the mAbLPF2 epitope was located on the interacting loop of peptide binding groove of the LipL32 molecule responsible for interactions with host constituents. Epitope sequences are highly conserved among Leptospira spp. and are absent from the LipL32 superfamily of other microorganisms. Both epitopes are surface-exposed, readily accessible by mAbs, and immunogenic. However, they are less dominant when revealed by LipL32-specific immunoglobulins from leptospirosis-patient sera and rabbit hyperimmune serum raised by whole Leptospira. Our study also demonstrated an adhesion inhibitory activity of LipL32 protein to host membrane components and cells mediated by mAbs as well as an anti-hemolytic activity of the respective antibodies. The therapeutic antibodies, particularly the humanized-ScFv, have a potential for further development as non-drug therapeutic agent for human leptospirosis, especially in subjects allergic to antibiotics. The epitope peptides recognized by two therapeutic mAbs have potential use as tools for structure-function studies. Finally, protective peptides may be used as a target for epitope-based vaccines for control of leptospirosis.

Keywords: leptospira lipl32-specific antibodies, therapeutic epitopes, epitopes characterization, immunotherapy

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Authors: Maierhaba Mijiti

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Objective：The aim is to understand the relationship between the expression level of the long-non-coding RNA LINC AC008063 and the clinicopathological parameters of patients with head and neck squamous cell carcinoma (HNSCC), and to clarify the biological function of LINC AC008063 in HNSCC cells. Moreover, it provides a potential biomarker for the diagnosis, treatment, and prognosis evaluation of HNSCC. Methods: The expression level of LINC AC008063 in the HNSCC was analyzed using transcriptome sequencing data from the TCGA (The cancer genome atlas) database. The expression levels of LINC AC008063 in human embryonic lung diploid cells 2BS, human immortalized keratinocytes HACAT, HNSCC cell lines CAL-27, Detroit562, AMC-HN-8, FD-LSC-1, FaDu and WSU-HN30 were determined by real-time quantitative PCR (qPCR). RNAi (RNA interference) was introduced for LINC AC008063 knockdown in HNSCC cell lines, the localization and abundance analysis of LINC AC008063 was determined by RT-qPCR, and the biological functions were examined by CCK-8, clone formation, flow cytometry, transwell invasion and migration assays, Seahorse assay. Results: LINC AC008063 was upregulated in HNSCC tissue (P<0.001), and verified b CCK-8, clone formation, flow cytometry, transwell invasion and migration assays, Seahorse assayy qPCR in HNSCC cell lines. The survival analysis revealed that the overall survival rate (OS) of patients with high LINC AC008063 expression group was significantly lower than that in the LINC AC008063 expression group, the median survival times for the two groups were 33.10 months and 61.27 months, respectively (P=0.002). The clinical correlation analysis revealed that its expression was positively correlated with the age of patients with HNSCC (P<0.001) and positively correlated with pathological state (T3+T4＞T1+T2, P=0.03). The RT-qPCR results showed that LINC AC008063 was mainly enriched in cytoplasm (P=0.01). Knockdown of LINC AC008063 inhibited proliferation, colony formation, migration and invasion; the glycolytic capacity was significantly decreased in HNSCC cell lines (P＜0.05). Conclusion: High level of LINC AC008063 was associated with the malignant progression of HNSCC as well as promoting the important biological functions of proliferation, colony formation, migration and invasion; in particular, the glycolytic capacity was decreased in HNSCC cells. Therefore, LINC AC008063 may serve as a potential biomarker for HNSCC and a distinct molecular target to inhibit glycolysis.

Keywords: head and neck squamous cell carcinoma, oncogene, long non-coding RNA, LINC AC008063, invasion and metastasis

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Authors: Engy Shokry, Giancarlo La Marca, Maria Luisa Della Bona

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Newborn screening for Congenital Adrenal Hyperplasia (CAH) relies on quantification of 17α-hydroxyprogesterone using enzyme immunoassays. These assays, in spite of being rapid, readily available and easy to perform, its reliability was found questionable due to lack of selectivity and specificity resulting in large number of false-positives, consequently family anxiety and associated hospitalization costs. To improve specificity of conventional 17α-hydroxyprogesterone screening which may experience false transient elevation in preterm, low birth weight or acutely ill neonates, steroid profiling by LC-MS/MS as a second-tier test was implemented. Unlike the previously applied LC-MS/MS methods, with the disadvantage of requiring a relatively high number of blood drops. Since newborn screening tests are increasing, it is necessary to minimize the sample volume requirement to make the maximum use of blood samples collected on filter paper. The proposed new method requires just one 3.2 mm dried blood spot (DBS) punch. Extraction was done using methanol: water: formic acid (90:10:0.1, v/v/v) containing deuterium labelled internal standards. Extracts were evaporated and reconstituted in 10 % acetone in water. Column switching strategy for on-line sample clean-up was applied to improve the chromatographic run. The first separative step retained the investigated steroids and passed through the majority of high molecular weight impurities. After the valve switching, the investigated steroids are back flushed from the POROS® column onto the analytical column and separated using gradient elution. Found quantitation limits were 5, 10 and 50 nmol/L for 17α-hydroxyprogesterone, androstenedione and cortisol respectively with mean recoveries of between 98.31-103.24 % and intra-/ inter-assay CV% < 10 % except at LLOQ. The method was validated using standard addition calibration and isotope dilution strategies. Reference ranges were determined by analysing samples from 896 infants of various ages at the time of sample collection. The method was also applied on patients with confirmed CAH. Our method represents an attractive combination of low sample volume requirement, minimal sample preparation time without derivatization and quick chromatography (5 min). The three steroid profile and the concentration ratios (17OHP + androstenedione/cortisol) allowed better screening outcomes of CAH reducing false positives, associated costs and anxiety.

Keywords: congenital adrenal hyperplasia (CAH), 17α-hydroxyprogesterone, androstenedione, cortisol, LC-MS/MS

Procedia PDF Downloads 439

Authors: Claudio Lamilla, Andrés Santos, Vicente Llanquinao, Jocelyn Hermosilla, Leticia Barrientos

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The industry in general is very interested in improving and optimizing industrial processes in order to reduce the costs involved in obtaining raw materials and production. Thus, an interesting and cost-effective alternative is the incorporation of bioactive metabolites in such processes, being an example of this enzymes which catalyze efficiently a large number of enzymatic reactions of industrial and biotechnological interest. In the search for new sources of these active metabolites, Antarctica is one of the least explored places on our planet where the most drastic cold conditions, salinity, UVA-UVB and liquid water available are present, features that have shaped all life in this very harsh environment, especially bacteria that live in different Antarctic ecosystems, which have had to develop different strategies to adapt to these conditions, producing unique biochemical strategies. In this work the production of cellulolytic enzymes of seven bacterial strains isolated from marine sediments at different sites in the Antarctic was evaluated. Isolation of the strains was performed using serial dilutions in the culture medium at M115°C. The identification of the strains was performed using universal primers (27F and 1492R). The enzyme activity assays were performed on R2A medium, carboxy methyl cellulose (CMC)was added as substrate. Degradation of the substrate was revealed by adding Lugol. The results show that four of the tested strains produce enzymes which degrade CMC substrate. The molecular identifications, showed that these bacteria belong to the genus Streptomyces and Pseudoalteromonas, being Streptomyces strain who showed the highest activity. Only some bacteria in marine sediments have the ability to produce these enzymes, perhaps due to their greater adaptability to degrade at temperatures bordering zero degrees Celsius, some algae that are abundant in this environment and have cellulose as the main structure. The discovery of new enzymes adapted to cold is of great industrial interest, especially for paper, textiles, detergents, biofuels, food and agriculture. These enzymes represent 8% of industrial demand worldwide and is expected to increase their demand in the coming years. Mainly in the paper and food industry are required in extraction processes starch, protein and juices, as well as the animal feed industry where treating vegetables and grains helps improve the nutritional value of the food, all this clearly puts Antarctic microorganisms and their enzymes specifically as a potential contribution to industry and the novel biotechnological applications.

Keywords: antarctic, bacteria, biotechnological, cellulolytic enzymes

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Authors: Rasha Al-Saedi, Anthony Meijer

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The progress of new porous materials has increased rapidly over the past decade for use in applications such as catalysis, gas storage and removal of environmentally unfriendly species due to their high surface area and high thermal stability. In this work, a theoretical study of the zirconium-based metal organic framework (MOFs) were examined in order to determine their potential for gas adsorption of various guest molecules: CO2, N2, CH4 and H2. The zirconium cluster consists of an inner Zr6O4(OH)4 core in which the triangular faces of the Zr6- octahedron are alternatively capped by O and OH groups which bound to nine formate groups and three benzoate groups linkers. General formula is [Zr(μ-O)4(μ-OH)4(HCOO)9((phyO2C)3X))] where X= CH2OH, CH2NH2, CH2CONH2, n(NH2); (n = 1-3). Three types of adsorption sites on the Zr metal center have been studied, named according to capped chemical groups as the ‘−O site’; the H of (μ-OH) site removed and added to (μ-O) site, ‘–OH site’; (μ-OH) site removed, the ‘void site’ where H2O molecule removed; (μ-OH) from one site and H from other (μ-OH) site, in addition to no defect versions. A series of investigations have been performed aiming to address this important issue. First, density functional theory DFT-B3LYP method with 6-311G(d,p) basis set was employed using Gaussian 09 package in order to evaluate the gas adsorption performance of missing-linker defects in zirconium cluster. Next, study the gas adsorption behaviour on different functionalised zirconium clusters. Those functional groups as mentioned above include: amines, alcohol, amide, in comparison with non-substitution clusters. Then, dispersion-corrected density functional theory (DFT-D) calculations were performed to further understand the enhanced gas binding on zirconium clusters. Finally, study the water effect on CO2 and N2 adsorption. The small functionalized Zr clusters were found to result in good CO2 adsorption over N2, CH4, and H2 due to the quadrupole moment of CO2 while N2, CH4 and H2 weakly polar or non-polar. The adsorption efficiency was determined using the dispersion method where the adsorption binding improved as most of the interactions, for example, van der Waals interactions are missing with the conventional DFT method. The calculated gas binding strengths on the no defect site are higher than those on the −O site, −OH site and the void site, this difference is especially notable for CO2. It has been stated that the enhanced affinity of CO2 of no defect versions is most likely due to the electrostatic interactions between the negatively charged O of CO2 and the positively charged H of (μ-OH) metal site. The uptake of the gas molecule does not enhance in presence of water as the latter binds to Zr clusters more strongly than gas species which attributed to the competition on adsorption sites.

Keywords: density functional theory, gas adsorption, metal- organic frameworks, molecular simulation, porous materials, theoretical chemistry

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Authors: Luminita Marin, Dalila Belei, Carmen Dumea

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Aggregation induced emission (AIE) is an intriguing optical phenomenon recently evidenced by Tang and his co-workers, for which aggregation works constructively in the improving of light emission. The AIE challenging phenomenon is quite opposite to the notorious aggregation caused quenching (ACQ) of light emission in the condensed phase, and comes in line with requirements of photonic and optoelectronic devices which need solid state emissive substrates. This paper reports a series of ten new aggregation induced emission (AIE) low molecular weight compounds based on triazole and pyridine-N-oxide heterocyclic units bonded by short flexible chains, obtained by a „click” chemistry reaction. The compounds present extremely weak luminescence in solution but strong light emission in solid state. To distinguish the influence of the crystallinity degree on the emission efficiency, the photophysical properties were explored by UV-vis and photoluminescence spectroscopy in solution, water suspension, amorphous and crystalline films. On the other hand, the compound morphology of the up mentioned states was monitored by dynamic light scattering, scanning electron microscopy, atomic force microscopy and polarized light microscopy methods. To further understand the structural design – photophysical properties relationship, single crystal X-ray diffraction on some understudy compounds was performed too. The UV-vis absorption spectra of the triazole water suspensions indicated a typical behaviour for nanoparticle formation, while the photoluminescence spectra revealed an emission intensity enhancement up to 921-fold higher of the crystalline films compared to solutions, clearly indicating an AIE behaviour. The compounds have the tendency to aggregate forming nano- and micro- crystals in shape of rose-like and fibres. The crystals integrity is kept due to the strong lateral intermolecular forces, while the absence of face-to-face forces explains the enhanced luminescence in crystalline state, in which the intramolecular rotations are restricted. The studied flexible triazoles draw attention to a new structural design in which small biologically friendly luminophore units are linked together by small flexible chains. This design enlarges the variety of the AIE luminogens to the flexible molecules, guiding further efforts in development of new AIE structures for appropriate applications, the biological ones being especially envisaged.

Keywords: aggregation induced emission, pyridine-N-oxide, triazole

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Authors: Pukhrambam Helena Chanu, Janardan Yadav

Abstract:

This study investigates the potential of various fungal isolates to solubilize zinc and counteract rice pathogens, with the aim of mitigating zinc deficiency and disease prevalence in rice farming. Soil samples from the rhizosphere were collected, and zinc-solubilizing fungi were isolated and purified. Molecular analysis identified Talaromyces sp, Talaromyces versatilis, Talaromyces pinophilus, and Aspergillus terreus as effective zinc solubilizers. Through qualitative and quantitative assessments, it was observed that solubilization efficiencies varied among the isolates over time, with Talaromyces versatilis displaying the highest capacity for solubilization. This variability in solubilization rates may be attributed to differences in fungal metabolic activity and their ability to produce organic acids that facilitate zinc release from insoluble sources in the soil. In inhibition assays against rice pathogens, the fungal isolates exhibited antagonistic properties, with Talaromyces versatilis demonstrating the most significant inhibition rates. This antagonistic activity may be linked to the production of secondary metabolites, such as antibiotics or lytic enzymes by fungi, which inhibit the growth of rice pathogens. The ability of Talaromyces versatilis to outperform other isolates in both zinc solubilization and pathogen inhibition highlights its potential as a multifunctional biocontrol agent in rice cultivation systems. These findings emphasize the potential of fungi as natural solutions for enhancing zinc uptake and managing diseases in rice cultivation. Utilizing indigenous zinc-solubilizing fungi offers a sustainable and environmentally friendly approach to addressing zinc deficiency in soils, reducing the need for chemical fertilizers. Moreover, harnessing the antagonistic activity of these fungi can contribute to integrated disease management strategies, minimizing reliance on synthetic pesticides and promoting ecological balance in agroecosystems. Additionally, the study included the evaluation of dipping time under different concentrations, viz.,10 ppm, 20 ppm, and 30 ppm of biosynthesized nano ZnO on rice seedlings. This investigation aimed to optimize the application of nano ZnO for efficient zinc uptake by rice plants while minimizing potential risks associated with excessive nanoparticle exposure. Evaluating the effects of varying concentrations and dipping durations provides valuable insights into the safe and effective utilization of nano ZnO as a micronutrient supplement in rice farming practices.

Keywords: biosynthesized nano ZnO, rice, root dipping, zinc solubilizing fungi.

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Authors: Diana M. Torres, Anngie K. Hernandez, Andrea Villareal, Magda R. Gomez, Sadao Kobayashi

Abstract:

Colombian Garlic crops exhibited mild mosaic, yellow stripes, and deformation. This group of symptoms suggested a viral infection. Several viruses belonging to the genera Potyvirus, Carlavirus and Allexivirus are known to infect garlic and lower their yield worldwide, but in Colombia, there are no studies of viral infections in this crop, only leek yellow stripe virus (LYSV) has been reported to our best knowledge. In Colombia, there are no management strategies for viral diseases in garlic because of the lack of information about viral infections on this crop, which is reflected in (i) high prevalence of viral related symptoms in garlic fields and (ii) high dispersal rate. For these reasons, the purpose of the present study was to evaluate the viral status of garlic in Colombia, which can represent a major threat on garlic yield and quality for this country 55 symptomatic leaf samples were collected for virus detection by RT-PCR and mechanical inoculation. Total RNA isolated from infected samples were subjected to RT-PCR with primers 1-OYDV-G/2-OYDV-G for Onion yellow dwarf virus (OYDV) (expected size 774pb), 1LYSV/2LYSV for LYSV (expected size 1000pb), SLV 7044/SLV 8004 for Shallot latent virus (SLV) (expected size 960pb), GCL-N30/GCL-C40 for Garlic common latent virus (GCLV) (expected size 481pb) and EF1F/EF1R for internal control (expected size 358pb). GCLV, SLV, and LYSV were detected in infected samples; in 95.6% of the analyzed samples was detected at least one of the viruses. GCLV and SLV were detected in single infection with low prevalence (9.3% and 7.4%, respectively). Garlic generally becomes coinfected with several types of viruses. Four viral complexes were identified: three double infection (64% of analyzed samples) and one triple infection (15%). The most frequent viral complex was SLV + GCLV infecting 48.1% of the samples. The other double complexes identified had a prevalence of 7% (GCLV + LYSV and SLV + LYSV) and 5.6% of the samples were free from these viruses. Mechanical transmission experiments were set up using leaf tissues of collected samples from infected fields, different test plants were assessed to know the host range, but it was restricted to C. quinoa, confirming the presence of detected viruses which have limited host range and were detected in C. quinoa by RT-PCR. The results of molecular and biological tests confirm the presence of SLV, LYSV, and GCLV; this is the first report of SLV and LYSV in garlic plants in Colombia, which can represent a serious threat for this crop in this country.

Keywords: SLV, GCLV, LYSV, leek yellow stripe virus, Allium sativum

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Authors: Cedillo Ortiz Cesar Isaac, Marañón-Ruiz Virginia-Francisca, Lozano-Alvarez Juan Antonio, Jáuregui-Rincón Juan, Roger Chiu Zarate

Abstract:

Introduction: The contamination of water with the azo-dye is a problem worldwide as although wastewater contaminate is treated in a municipal sewage system, still contain a considerable amount of dyes. In the present, there are different processes denominated tertiary method in which it is possible to lower the concentration of the dye. One of these methods is by adsorption onto various materials which can be organic or inorganic materials. The xanthan is a biomaterial as removal agents to decrease the dye content in aqueous solution. The Zimm-Bragg model described the experimental isotherms obtained when this biopolymer was used in the removal of textile dyes. Nevertheless, it was not established if a possible correlation between dye structure and removal efficiency exists. In this sense, the principal objective of this report is to propose a qualitative relationship between the structure of three azo-dyes (Congo Red (CR), Methyl Red (MR) and Methyl Orange (MO)) and their removal efficiency from aqueous environment when xanthan are used as dye sequestering agents. Methods: The dyes were subjected to different pH and ionic strength values to obtain the conditions of maximum dye removal. Afterward, these conditions were used to perform the adsorption isotherm as was reported in the previous study in our group. The Zimm-Bragg model was used to describe the experimental data and the parameters of nucleation (Ku) and cooperativity (U) were obtained by optimization using the R statistical software. The spectra from UV-Visible (aqueous solution), Infrared absorption and Raman spectroscopies (dry samples) were obtained from the biopolymer-dye complex. Results: The removal percent with xanthan in each dye are as follows: with CR had 99.98 % when the pH is 12 and ionic strength is 10.12, with MR had 84.79 % when the pH is 9.5 and ionic strength is 43 and finally the MO had 30 % in pH 4 and 72. It can be seen that when xanthan is used to remove the dyes, exists a lower dependence between structure and removal efficiency. This may be due to the different tendency to form aggregates of each dye. This aggregation capacity and the charge of each dye resulting from the pH and ionic strength values of aqueous solutions are key factors in the dye removal. The experimental isotherm of MR was only that adequately described by Zimm-Bragg model. Because with the CR had the 100 % of remove thus is very difficult obtain de experimental isotherm and finally MO had results fluctuating and therefore was impossible get the accurate data. Conclusions: The study of the removal of three dyes with xanthan as dye sequestering agents suggests that aggregation capacity of dyes and the charge resulting from structural characteristics such as molecular weight and functional groups have a relationship with the removal efficiency. Acknowledgements: We are gratefully acknowledged support for this project by Consejo Nacional de Ciencia y Tecnología, México (CONACyT, Grant No. 632694.)

Keywords: adsorption, azo dyes, xanthan gum, Zimm Bragg theory

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