Search results for: buffer box

Authors: S. Junaid S. Qazi, Denice C. Bay, Raymond Chew, Raymond J. Turner

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EmrE, a member of the small multidrug resistance protein family in bacteria is considered to be the archetypical member of its family. It confers host resistance to a wide variety of quaternary cation compounds (QCCs) driven by proton motive force. Generally, purification yield is a challenge in all membrane proteins because of the difficulties in their expression, isolation and solubilization. EmrE is extremely hydrophobic which make the purification yield challenging. We have purified EmrE protein using two different approaches: organic solvent membrane extraction and hexahistidine (his6) tagged Ni-affinity chromatographic methods. We have characterized changes present between ligand affinity of untagged and his6-tagged EmrE proteins in similar membrane mimetic environments using biophysical experimental techniques. Purified proteins were solubilized in a buffer containing n-dodecyl-β-D-maltopyranoside (DDM) and the conformations in the proteins were explored in the presence of four QCCs, methyl viologen (MV), ethidium bromide (EB), cetylpyridinium chloride (CTP) and tetraphenyl phosphonium (TPP). SDS-Tricine PAGE and dynamic light scattering (DLS) analysis revealed that the addition of QCCs did not induce higher multimeric forms of either proteins at all QCC:EmrE molar ratios examined under the solubilization conditions applied. QCC binding curves obtained from the Trp fluorescence quenching spectra, gave the values of dissociation constant (Kd) and maximum specific one-site binding (Bmax). Lower Bmax values to QCCs for his6-tagged EmrE shows that the binding sites remained unoccupied. This lower saturation suggests that the his6-tagged versions provide a conformation that prevents saturated binding. Our data demonstrate that tagging an integral membrane protein can significantly influence the protein.

Keywords: small multidrug resistance (SMR) protein, EmrE, integral membrane protein folding, quaternary ammonium compounds (QAC), quaternary cation compounds (QCC), nickel affinity chromatography, hexahistidine (His6) tag

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Authors: Michelle R. Sullivan

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Positive romantic relationships and marriages offer a buffer against a host of physical and emotional difficulties. Conversely, poor relationship quality and marital discord can have deleterious consequences for individuals and families. Research has described non-monogamy, infidelity, and consensual non-monogamy, as both consequential and causal of relationship difficulty, or as a unique way a couple strives to make a relationship work. Much research on consensual non-monogamy has built on feminist theory and critique. To the author’s best knowledge, to date, no studies have examined the predictive relationship between individual and relationship characteristics and expectations of non-monogamy. The current longitudinal study: 1) estimated the prevalence of expectations of partner non-monogamy and 2) evaluated whether gender, sexual identity, age, education, how a couple met, and relationship quality were predictive expectations of partner non-monogamy. This study utilized the publically available longitudinal dataset, How Couples Meet and Stay Together. Adults aged 18- to 98-years old (n=4002) were surveyed by phone over 5 waves from 2009-2014. Demographics and how a couple met were gathered through self-report in Wave 1, and relationship quality and expectations of partner non-monogamy were gathered through self-report in Waves 4 and 5 (n=1047). The prevalence of expectations of partner non-monogamy (encompassing both infidelity and consensual non-monogamy) was 4.8%. Logistic regression models indicated that sexual identity, gender, education, and relationship quality were significantly predictive of expectations of partner non-monogamy. Specifically, male gender, lower education, identifying as lesbian, gay, or bisexual, and a lower relationship quality scores were predictive of expectations of partner non-monogamy. Male gender was not predictive of expectations of partner non-monogamy in the follow up logistic regression model. Age and whether a couple met online were not associated with expectations of partner non-monogamy. Clinical implications include awareness of the increased likelihood of lesbian, gay, and bisexual individuals to have an expectation of non-monogamy and the sequelae of relationship dissatisfaction that may be related. Future research directions could differentiate between non-monogamy subtypes and the person and relationship variables that lead to the likelihood of consensual non-monogamy and infidelity as separate constructs, as well as explore the relationship between predicting partner behavior and actual partner behavioral outcomes.

Keywords: open relationship, polyamory, infidelity, relationship satisfaction

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Authors: Bruna Lima Gomes, Vanessa Caroline De Barros Bonato, Luciano Freschi, Eduardo Purgatto

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Plant hormones are long known to be tightly associated with fruit development and are involved in controlling various aspects of fruit ripening. For fleshy fruits, ripening is characterized for changes in texture, color, aroma and other parameters that markedly contribute to its quality. Ethylene is one of the major players regulating the ripening-related processes, but emerging evidences suggest that auxin is also part of this dynamic control. Thus, the aim of this study was providing new insights into the auxin role during ripening and the hormonal interplay between auxin and ethylene. For that, tomato fruits (Micro-Tom) were collected at mature green stage and separated in four groups: one for indole-3-acetic acid (IAA) treatment, one for ethylene, one for a combination of IAA and ethylene, and one for control. Hormone solution was injected through the stylar apex, while mock samples were injected with buffer only. For ethylene treatments, fruits were exposed to gaseous hormone. Then, fruits were left to ripen under standard conditions and to assess ripening development, hue angle was reported as color indicator and ethylene production was measured by gas chromatography. The transcript levels of three ripening-related ethylene receptors (LeETR3, LeETR4 and LeETR6) were evaluated by RT-qPCR. Results showed that ethylene treatment induced ripening, stimulated ethylene production, accelerated color changes and induced receptor expression, as expected. Nonetheless, auxin treatment showed the opposite effect once fruits remained green for longer time than control group and ethylene perception has changed, taking account the reduced levels of receptor transcripts. Further, treatment with both hormones revealed that auxin effect in delaying ripening was predominant, even with higher levels of ethylene. Altogether, the data suggest that auxin modulates several aspects of the tomato fruit ripening modifying the ethylene perception. The knowledge about hormonal control of fruit development will help design new strategies for effective manipulation of ripening regarding fruit quality and brings a new level of complexity on fruit ripening regulation.

Keywords: ethylene, auxin, fruit ripening, hormonal crosstalk

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Authors: Constanza Monterrubio-Solis, Jose Tomas Ibarra

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The fertile interdependence of social-ecological systems reveals itself in the interactions between native forests and seeds, home gardens, kitchens, foraging activities, local knowledge, and food practices, creating particular flavors and food meanings as part of cultural identities within territories. Resilience in local-food systems, from a relational perspective, can be understood as the balance between persistence and adaptability to change. Food growing, preparation, and consumption are constantly changing and adapting as expressions of agency of female and male indigenous peoples and peasants. This paper explores local food systems’ expressions of resilience in the la Araucanía region of Chile, namely: diversity, redundancy, buffer capacity, modularity, self-organization, governance, learning, equity, and decision-making. Applying ethnographic research methods (participant observation, focus groups, and semi-structured interviews), this work reflects on the experience developed through work with Mapuche women cultivating home gardens in the region since 2012; it looks to material and symbolic elements of resilience in the local indigenous food systems. Local food systems show indeed indicators of social-ecological resilience. The biocultural memory is expressed in affection to particular flavors and recipes, the cultural importance of seeds and reciprocity networks, as well as an accurate knowledge about the indicators of the seasons and weather, which have allowed local food systems to thrive with a strong cultural foundation. Furthermore, these elements turn into biocultural resistance in the face of the current institutional pressures for rural specialization, processes of cultural assimilation such as agroecosystems and diet homogenization, as well as structural threats towards the diversity and freedom of native seeds. Thus, the resilience-resistance dynamic shown by the social-ecological systems of the southern Andes is daily expressed in the local food systems and flavors and is key for diverse and culturally sound social-ecological health.

Keywords: biocultural heritage, indigenous food systems, social-ecological resilience, southern Andes

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Authors: Grzegorz Swiderski, Agata Jablonska-Trypuc, Natalia Popow, Renata Swislocka, Wlodzimierz Lewandowski

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Doxorubicin belongs to the group of anthracycline antitumor antibiotics. Because of the wide spectrum of actions, it is one of the most widely used anthracycline antibiotics, including the treatment of breast, ovary, bladder, lung cancers as well as neuroblastoma, lymphoma, leukemia and myeloid leukemia. Antitumor activity of doxorubicin is based on the same mechanisms as for most anthracyclines. Like the metal ions affect the nucleic acids on many biological processes, so the environment of the metal chelates of antibiotics can have a significant effect on the pharmacological properties of drugs. Complexation of anthracyclines with metal ions may contribute to the production of less toxic compounds. In the framework of this study, the composition of complexes obtained in aqueous solutions of doxorubicin with metal ions (Cu2+ and Zn2+). Complexation was analyzed by spectrophotometric titration in aqueous solution at pH 7.0. The pH was adjusted with 0.02M Tris-HCl buffer. The composition of the complexes found was Cu: doxorubicin (1: 2) and a Zn: doxorubicin (1: 1). The effect of Dox, Dox-Cu and Dox-Zn was examined in MCF-7 breast cancer cell line, which were obtained from American Type Culture Collection (ATCC). The compounds were added to the cultured cells for a final concentration in the range of 0,01µM to 0,5µM. The number of MCF-7 cells with division into living and dead, was determined by direct counts of cells with the use of trypan blue dye using LUNA Logos Biosystems cell counter. ApoTox-Glo Triplex Assay (Promega, Madison, Wisconsin, USA) was used according to the manufacturer’s instructions to measure the MCF-7 cells’ viability, cytotoxicity and apoptosis. We observed a decrease in cells proliferation in a dose-dependent manner. An increase in cytotoxicity and decrease in viability in the ApoTox Triplex assay was also showed for all tested compounds. Apoptosis, showed as caspase 3/7 activation, was observed only in Dox treatment. In Dox-Zn and Dox-Cu caspase 3/7 activation was not observed. This work was financially supported by National Science Centre, Poland, under the research project number 2014/13/B/NZ7/02 352.

Keywords: anticancer properties, anthracycline antibiotic, doxorubicine, metal complexes

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Authors: Mark White

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The best practices for owners and urban planners to manage tsunami risk have evolved during the last fifty years, and related technical advances have created opportunities for them to obtain better performance than in earlier cataclysmic tsunami inundations. This basic pattern is illustrated at Hilo Bay, the waterfront area of Hilo, Hawaii, an urban seaport which faces the most severe tsunami hazard of the Hawaiian archipelago. Since April 1, 1946, Hilo Bay has endured tsunami waves with a maximum water height exceeding 2.5 meters following four severe earthquakes: Unimak Island (Mw 8.6, 6.1 m) in 1946; Valdiva (Mw 9.5, the largest earthquake of the 20th century, 10.6 m) in 1960; William Prince Sound (Mw 9.2, 3.8 m) in 1964; and Kalapana (Mw 7.7, the largest earthquake in Hawaii since 1868, 2.6 m) in 1975. Ignoring numerous smaller tsunamis during the same time frame, these four cataclysmic tsunamis have caused property losses in Hilo to exceed $1.25 billion and more than 150 deaths. It is reasonable to foresee another cataclysmic tsunami inundating the urban core of Hilo in the next 50 years, which, if unchecked, could cause additional deaths and losses in the hundreds of millions of dollars. Urban planners and individual owners are now in a position to reduce these losses in the next foreseeable tsunami that generates maximum water heights between 2.5 and 10 meters in Hilo Bay. Since 1946, Hilo planners and individual owners have already created buffer zones between the shoreline and its historic downtown area. As these stakeholders make inevitable improvements to the built environment along and adjacent to the shoreline, they should incorporate new methods for better managing the obvious tsunami risk at Hilo. At the planning level, new manmade land forms, such as tsunami parks and inundation reservoirs, should be developed. Individual owners should require their design professionals to include sacrificial seismic and tsunami fuses that will perform well in foreseeable severe events and that can be easily repaired in the immediate aftermath. These investments before the next cataclysmic tsunami at Hilo will yield substantial reductions in property losses and fatalities.

Keywords: hilo, tsunami parks, reservoirs, fuse systems, risk managment

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Authors: Ariel Vilchez, Francisca Acevedo, Rodrigo Navia

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The wound healing process can be accelerated and improved by the action of antioxidants such as curcumin (Cur) over the tissues; however, the efficacy of curcumin used through the digestive system is not enough to exploit its benefits. Electrospinning presents an alternative to carry curcumin directly to the wounds, and polyhydroxybutyrate (PHB) is proposed as the matrix to load curcumin owing to its biodegradable and biocompatible properties. PHB is among 150 types of Polyhydroxyalkanoates (PHAs) identified, it is a natural thermoplastic polyester produced by microbial fermentation obtained from microorganisms. The proposed objective is to develop electrospun bacterial PHB-based microfibers containing curcumin for possible biomedical applications. Commercial PHB was solved in Chloroform: Dimethylformamide (4:1) to a final concentration of 7% m/V. Curcumin was added to the polymeric solution at 1%, and 7% m/m regarding PHB. The electrospinning equipment (NEU-BM, China) with a rotary collector was used to obtain Cur-PHB fibers at different voltages and flow rate of the polymeric solution considering a distance of 20 cm from the needle to the collector. Scanning electron microscopy (SEM) was used to determine the diameter and morphology of the obtained fibers. Thermal stability was obtained from Thermogravimetric (TGA) analysis, and Fourier Transform Infrared Spectroscopy (FT-IR) was carried out in order to study the chemical bonds and interactions. A preliminary curcumin release to Phosphate Buffer Saline (PBS) pH = 7.4 was obtained in vitro and measured by spectrophotometry. PHB fibers presented an intact chemical composition regarding the original condition (dust) according to FTIR spectra, the diameter fluctuates between 0.761 ± 0.123 and 2.157 ± 0.882 μm, with different qualities according to their morphology. The best fibers in terms of quality and diameter resulted in sample 2 and sample 6, obtained at 0-10kV and 0.5 mL/hr, and 0-10kV and 1.5 mL/hr, respectively. The melting temperature resulted near 178 °C, according to the bibliography. The crystallinity of fibers decreases while curcumin concentration increases for the studied interval. The curcumin release reaches near 14% at 37 °C at 54h in PBS adjusted to a quasi-Fickian Diffusion. We conclude that it is possible to load curcumin in PHB to obtain continuous, homogeneous, and solvent-free microfibers by electrospinning. Between 0% and 7% of curcumin, the crystallinity of fibers decreases as the concentration of curcumin increases. Thus, curcumin enhances the flexibility of the obtained material. HPLC should be used in further analysis of curcumin release.

Keywords: antioxidant, curcumin, polyhydroxybutyrate, wound healing

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Authors: A. Dashti, M. Eskandari, L. Farahmand, P. Parvin, A. Jafargholi

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Breast Cancer is one of the most considerable diseases in the United States and other countries and is the second leading cause of death in women. Common breast cancer treatments would lead to adverse side effects such as loss of hair, nausea, and weakness. These complications arise because these cancer treatments damage some healthy cells while eliminating the cancer cells. In an effort to address these complications, laser radiation was utilized and tested as a targeted cancer treatment for breast cancer. In this regard, tissue engineering approaches are being employed by using an electrospun scaffold in order to facilitate the growth of breast cancer cells. Polycaprolacton (PCL) was used as a material for scaffold fabricating because of its biocompatibility, biodegradability, and supporting cell growth. The specific breast cancer cells have the ability to create a three-dimensional cell cluster due to the spontaneous accumulation of cells in the porosity of the scaffold under some specific conditions. Therefore, we are looking for a higher density of porosity and larger pore size. Fibers showed uniform diameter distribution and final scaffold had optimum characteristics with approximately 40% porosity. The images were taken by SEM and the density and the size of the porosity were determined with the Image. After scaffold preparation, it has cross-linked by glutaraldehyde. Then, it has been washed with glycine and phosphate buffer saline (PBS), in order to neutralize the residual glutaraldehyde. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor (MTT) results have represented approximately 91.13% viability of the scaffolds for cancer cells. In order to create a cluster, Michigan Cancer Foundation-7 (MCF-7, breast cancer cell line) and Michigan Cancer Foundation-10A (MCF-10A, human mammary epithelial cell line) cells were cultured on the scaffold in 24 well plate for five days. Then, we have exposed the cluster to the laser diode 808 nm radiation to investigate the effect of laser on the tumor with different power and time. Under the same conditions, cancer cells lost their viability more than the healthy ones. In conclusion, laser therapy is a viable method to destroy the target cells and has a minimum effect on the healthy tissues and cells and it can improve the other method of cancer treatments limitations.

Keywords: breast cancer, electrospun scaffold, polycaprolacton, laser diode, cancer treatment

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Authors: S. Fröhlich, M. Herold, M. Allmer

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Early stage quantitative analysis of host cell protein (HCP) variations is challenging yet necessary for comprehensive bioprocess development. High resolution mass spectrometry (HRMS) provides a high-end technology for accurate identification alongside with quantitative information. Hereby we describe a flexible HRMS assay platform to quantify HCPs relevant in microbial expression systems such as E. Coli in both up and downstream development by means of MVDA tools. Cell pellets were lysed and proteins extracted, purified samples not further treated before applying the SMART tryptic digest kit. Peptides separation was optimized using an RP-UHPLC separation platform. HRMS-MSMS analysis was conducted on an Orbitrap Velos Elite applying CID. Quantification was performed label-free taking into account ionization properties and physicochemical peptide similarities. Results were analyzed using SIEVE 2.0 (Thermo Fisher Scientific) and SIMCA (Umetrics AG). The developed HRMS platform was applied to an E. Coli expression set with varying productivity and the corresponding downstream process. Selected HCPs were successfully quantified within the fmol range. Analysing HCP networks based on pattern analysis facilitated low level quantification and enhanced validity. This approach is of high relevance for high-throughput screening experiments during upstream development, e.g. for titer determination, dynamic HCP network analysis or product characterization. Considering the downstream purification process, physicochemical clustering of identified HCPs is of relevance to adjust buffer conditions accordingly. However, the technology provides an innovative approach for label-free MS based quantification relying on statistical pattern analysis and comparison. Absolute quantification based on physicochemical properties and peptide similarity score provides a technological approach without the need of sophisticated sample preparation strategies and is therefore proven to be straightforward, sensitive and highly reproducible in terms of product characterization.

Keywords: process analytical technology, mass spectrometry, process characterization, MVDA, pattern recognition

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Authors: Mark Wenyi Lai

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The very lethal weapon of nuclear warhead had threatened the survival of the world for half of the 20th century. When most of the countries have already denounced and stopped the development, one country is eager to produce and use them. Since 2006, Pyongyang has launched six times of nuclear tests. The most recent one in September 2017 signaled North Korea’s military capability to project the mass destruction through ICBM (Intercontinental Ballistic Missile) over Seoul, Tokyo, Guam, Hawaii, Alaska or probably the West Coast of the United States with the explosive energy ten times of the atom bombing of Hiroshima in 1945. This research paper adopted time-series content analysis focusing on the related countries responses to North Korea’s tests in 2006, 2009, 2013, and 2016. The preliminary hypotheses are first, North Korea determined to protect the regime by having triad nuclear capability. Negotiations are mere means to this end. Second, South Korea is paralyzed by its ineffective domestic politics and unable to develop its independent strategy toward the North. Third, Japan was using the external threat to campaign for its rearmament plan and brought instability in foreign relations. Fourth, China found herself in the strange position of defending the loyal buffer state meanwhile witnessing the fourth and dangerous neighboring country gaining the card into nuclear club. Fifth, the United States had admitted that North Korea’s going nuclear is unstoppable. Therefore, to keep the regional stability in the East Asia, the US relied on the new balance of power formed by everyone versus Pyongyang. But, countries in East Asia actually have problems getting along with each other. Sixth, Russia distanced herself from the North Kore row but benefitted by advancing its strategic importance in the Far East. Tracing back the history of nuclear states, this research paper concluded that North Korea will head on becoming a more confident country. The regional stability will restore once related countries deal with the new fact and treat Pyongyang regime with a new strategy. The gradual opening and economic reform are on the way for the North Korea in the near future.

Keywords: nuclear test, North Korea, six party talk, US foreign policy

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Authors: Marie-Claude Roy, David Locky, Ermias Azeria, Jim Schieck

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It is estimated that up to 70% of the wetlands in the Parkland and Grassland natural regions of Alberta have been lost due to various land-use activities. These losses include ecosystem function and services they once provided. Those wetlands remaining are often embedded in a matrix of human-modified habitats and despite efforts taken to protect them the effects of land-uses on wetland condition and function remain largely unknown. We used biophysical field data and remotely-sensed human footprint data collected at 322 open-water wetlands by the Alberta Biodiversity Monitoring Institute (ABMI) to evaluate the impact of surrounding land use on the physico-chemistry characteristics and plant functional traits of wetlands. Eight physio-chemistry parameters were assessed: wetland water depth, water temperature, pH, salinity, dissolved oxygen, total phosphorus, total nitrogen, and dissolved organic carbon. Three plant functional traits were evaluated: 1) origin (native and non-native), 2) life history (annual, biennial, and perennial), and 3) habitat requirements (obligate-wetland and obligate-upland). Intensity land-use was quantified within a 250-meter buffer around each wetland. Ninety-nine percent of wetlands in the Grassland and Parkland regions of Alberta have land-use activities in their surroundings, with most being agriculture-related. Total phosphorus in wetlands increased with the cover of surrounding agriculture, while salinity, total nitrogen, and dissolved organic carbon were positively associated with the degree of soft-linear (e.g. pipelines, trails) land-uses. The abundance of non-native and annual/biennial plants increased with the amount of agriculture, while urban-industrial land-use lowered abundance of natives, perennials, and obligate wetland plants. Our study suggests that land-use types surrounding wetlands affect the physicochemical and biological conditions of wetlands. This research suggests that reducing human disturbances through reclamation of wetland buffers may enhance the condition and function of wetlands in agricultural landscapes.

Keywords: wetlands, biophysical assessment, land use, grassland and parkland natural regions

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Authors: I. V. Palamarchuk, N. V. Zaichko

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Background and Aims: Galectin-3 is a marker of subclinical cardiac injury and is elevated in individuals with type 2 diabetes mellitus; while hydrogen sulfide (H₂S), metabolite of sulfur-containing amino acids, is considered having antifibrogenic effects. This study was designed to investigate whether metformin and its combination with NaHS can influence plasma galectin-3 and cystathionine-γ-lyase/hydrogen sulfide (CSE/H₂S) system in diabetic rat’s heart. Methods: 32 healthy male rats (180-250 g) were divided into 4 groups. To induct diabetes, rats (group 2-4) were injected with streptozotocin (STZ, 40 mg/kg/i.p., 0.1 M citrate buffer (pH 4.5). Rats from 3d (STZ+Metf) and 4th (STZ+Metf+NaHS) groups were given metformin (500 mg/kg/day) orally, and rats from 4th (STZ+Metf+NaHS) group were injected sodium hydrosulfide (NaHS, 3 mg/kg/i.p.) once per day starting from 3 to 28 day after streptozotocin injection. Rats of first group (control) were administered the equivalent volumes of 0.9% NaCl. Plasma galectin-3 was measured by ELISA. Rats’ hearts were sampled for determination of H2S by reaction with N,N-Dimethyl-p-phenylenediamine. Determination of CSE gene expression was performed in real time using PCR in the presence of SYBR Green I, using DT-Light detecting amplifier ('DNA-technology', Russia). Results: Induction of streptozotocin diabetes (STZ-diabetes, group 2) was followed by low myocardial H2S concentration and CSE expression (by 35%, p < 0.05 and 60.5%, p < 0.001 respectively, than that in controls), while plasma galectin-3 in this group was significantly higher than in controls (by 3.8 times, p < 0.05). Administration of metformin (group 3) resulted in significantly higher H₂S concentration (by 28.5%, p < 0.05), whereas CSE expression was only by 6% more than that in STZ-diabetes, as well as plasma galectin-3 was only by 14.8% lower in comparison with untreated diabetic rats. The inhibition of H₂S generation and CSE activity by diabetes was greatly attenuated in STZ+Metf+NaHS group. The combination of metformin with NaHS significantly stimulated H₂S production (by 48%, p < 0.05 and 15%, p < 0.05 more than STZ-diabetes and STZ+Metf respectively) and CSE gene expression (by 64.8%, p < 0.05 compared to STZ-diabetes and by 55.4%,p < 0.05 compared to STZ+Metf). Besides, plasma galectin-3 in rats receiving metformin and NaHS was significantly lower by 42%, p < 0.05 and 32.5%, p < 0.05 compared to STZ-diabetes and STZ+Metf groups respectively. Conclusions: To summarize, dysfunction of CSE/H2S system and galectin-3 stimulation was found in streptozotocin-induced diabetic rats. Metformin and its combination with exogenous H2S effectively prevented the development of metabolic changes induced by diabetes. These findings suggest that CSE/H₂S system can be integrated into pathogenesis of diabetic complications through modulation of pro-inflammatory and pro-fibrogenic mediator galectin-3.

Keywords: cystathionine-γ-lyase, diabetic heart, galectin-3, hydrogen sulfide, metformin, sodium hydrosulfide

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Authors: Stefanie Behncke

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This paper analyses the effects of different macroprudential policy measures that have recently been implemented in Switzerland. Among them is the activation and the increase of the countercyclical capital buffer (CCB) and a tightening of loan-to-value (LTV) requirements. These measures were introduced to limit systemic risks in the Swiss mortgage and real estate markets. They were meant to affect mortgage growth, mortgage risks, and banks’ capital buffers. Evaluation of their quantitative effects provides insights for Swiss policymakers when reassessing their policy. It is also informative for policymakers in other countries who plan to introduce macroprudential instruments. We estimate the effects of the different macroprudential measures with a Differences-in-Differences estimator. Banks differ with respect to the relative importance of mortgages in their portfolio, their riskiness, and their capital buffers. Thus, some of the banks were more affected than others by the CCB, while others were more affected by the LTV requirements. Our analysis is made possible by an unusually informative bank panel data set. It combines data on newly issued mortgage loans and quantitative risk indicators such as LTV and loan-to-income (LTI) ratios with supervisory information on banks’ capital and liquidity situation and balance sheets. Our results suggest that the LTV cap of 90% was most effective. The proportion of new mortgages with a high LTV ratio was significantly reduced. This result does not only apply to the 90% LTV, but also to other threshold values (e.g. 80%, 75%) suggesting that the entire upper part of the LTV distribution was affected. Other outcomes such as the LTI distribution, the growth rates of mortgages and other credits, however, were not significantly affected. Regarding the activation and the increase of the CCB, we do not find any significant effects: neither LTV/LTI risk parameters nor mortgage and other credit growth rates were significantly reduced. This result may reflect that the size of the CCB (1% of relevant residential real estate risk-weighted assets at activation, respectively 2% at the increase) was not sufficiently high enough to trigger a distinct reaction between the banks most likely to be affected by the CCB and those serving as controls. Still, it might be have been effective in increasing the resilience in the overall banking system. From a policy perspective, these results suggest that targeted macroprudential policy measures can contribute to financial stability. In line with findings by others, caps on LTV reduced risk taking in Switzerland. To fully assess the effectiveness of the CCB, further experience is needed.

Keywords: banks, financial stability, macroprudential policy, mortgages

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Authors: Ann Rousseau, Steven Eggermont

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The current study sought to explain pre-adolescents’ differential susceptibility to the internalization of mediated appearance ideals, using a three-wave panel survey of preadolescent girls and boys (N = 973, Mage = 11.14). Based on the premises of objectification theory and sexual script theory, we proposed a double role for pubertal timing and cross-sex interactions in preadolescents’ media internalization. More specifically, we expected pubertal timing and cross-sex interactions to (a) trigger higher levels of media internalization, directly and indirectly via body surveillance, and (b) positively moderate the relationship between objectifying media exposure and girls’ and boys’ media internalization. A first cross-lagged model tested whether the pubertal timing and cross-sex interactions could trigger preadolescents media internalization and body surveillance. Structural equation analysis indicated that pubertal timing (Wave1) positively predicted body surveillance and media internalization (both Wave3). Cross-sex involvement (Wave1) was positively linked to media internalization (Wave2), but body surveillance (Wave2) was not associated with cross-sex interactions. Results also showed a reciprocal interaction between media internalization (Wave 2 and 3) and body surveillance (Wave2 and 3). Multiple group analysis showed that the observed relationships did not vary by gender. A second moderated moderation model examined whether (a) the relationship between objectifying media exposure (television and magazines, both Wave1) and media internalization (Wave3) depended on pubertal timing (Wave1), and (b) the two-way interaction between objectifying media exposure (Wave1) and pubertal timing (Wave1) varied depending on cross-sex interactions (Wave1). Results revealed that cross-sex interactions functioned as a buffer against media internalization. For preadolescents who had fewer cross-sex interactions, early puberty (relative to peers) positively moderated the relationship between magazine exposure and the internalization of mediated appearance ideals. No significant relationships were found for television. Again, no gender difference could be observed. The present study suggests a double role for pubertal timing and cross-sex interactions in preadolescents media internalization, and indicate that early developers with few cross-sex experiences are particularly vulnerable for media internalization. Additionally, the current findings suggest that there is relative gender equity in magazines’ ability to cultivate media internalization among preadolescents.

Keywords: cross-sex interactions, media effects, objectification theory, pubertal timing

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Authors: Olubukayo-Opeyemi Oyetayo, Oscar Mendez-Lucio, Andreas Bender, Hans Kiefer

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The thermal melting curve of a protein provides information on its conformational stability and could provide cues on its aggregation behavior. Naturally-occurring osmolytes have been shown to improve the thermal stability of most proteins in a concentration-dependent manner. They are therefore commonly employed as additives in therapeutic protein purification and formulation. A number of intertwined and seemingly conflicting mechanisms have been put forward to explain the observed stabilizing effects, the most prominent being the preferential exclusion mechanism. We attempted to probe and summarize molecular mechanisms for thermal stabilization of a monoclonal antibody (mAb) by developing quantitative structure-activity relationships using a rationally-selected library of 120 osmolyte-like compounds in the polyhydric alcohols, amino acids and methylamines classes. Thermal stabilization potencies were experimentally determined by thermal shift assays based on differential scanning fluorimetry. The cross-validated QSAR model was developed by partial least squares regression using descriptors generated from Molecular Operating Environment software. Careful evaluation of the results with the use of variable importance in projection parameter (VIP) and regression coefficients guided the selection of the most relevant descriptors influencing mAb thermal stability. For the mAb studied and at pH 7, the thermal stabilization effects of tested compounds correlated positively with their fractional polar surface area and inversely with their fractional hydrophobic surface area. We cannot claim that the observed trends are universal for osmolyte-protein interactions because of protein-specific effects, however this approach should guide the quick selection of (de)stabilizing compounds for a protein from a chemical library. Further work with a large variety of proteins and at different pH values would help the derivation of a solid explanation as to the nature of favorable osmolyte-protein interactions for improved thermal stability. This approach may be beneficial in the design of novel protein stabilizers with optimal property values, especially when the influence of solution conditions like the pH and buffer species and the protein properties are factored in.

Keywords: thermal stability, monoclonal antibodies, quantitative structure-activity relationships, osmolytes

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Authors: William E. Bauta, Jennifer Rebeles, Reggie Jacob

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Porphyrins are noteworthy in biomedical science for their cancer tissue accumulation and photophysical properties. The preferential accumulation of some porphyrins in cancerous tissue has been known for many years. This, combined with their characteristic photophysical and photochemical properties, including their strong fluorescence and their ability to generate reactive oxygen species in vivo upon laser irradiation, has led to much research into the application of porphyrins as cancer diagnostic and therapeutic agents. Porphyrins have been used as dyes to detect cancer cells both in vivo and, less commonly, in vitro. In one example, human sputum samples from lung cancer patients and patients without the disease were dissociated and stained with the porphyrin TCPP (5,10,15,20-tetrakis-(4-carboxyphenyl)-porphine). Cells were analyzed by flow cytometry. Cancer samples were identified by their higher TCPP fluorescence intensity relative to the no-cancer controls. However, quantitative analysis of fluorescence in cell suspensions stained with multiple fluorophores requires particles stained with each of the individual fluorophores as controls. Fluorescent control particles must be compatible in size with flow cytometer fluidics and have favorable hydrodynamic properties in suspension. They must also display fluorescence comparable to the cells of interest and be stable upon storage amine-functionalized spherical polystyrene beads in the 5 to 20-micron diameter range that was reacted with TCPP and EDC in aqueous pH six buffer overnight to form amide bonds. Beads were isolated by centrifugation and tested by flow cytometry. The 10-micron amine-functionalized beads displayed the best combination of fluorescence intensity and hydrodynamic properties, such as lack of clumping and remaining in suspension during the experiment. These beads were further optimized by varying the stoichiometry of EDC and TCPP relative to the amine. The reaction was accompanied by the formation of a TCPP-related particulate, which was removed, after bead centrifugation, using a microfiltration process. The resultant TCPP-functionalized beads were compatible with flow cytometry conditions and displayed a fluorescence comparable to that of stained cells, which allowed their use as fluorescence standards. The beads were stable in refrigerated storage in the dark for more than eight months. This work demonstrates the first preparation of porphyrin-functionalized flow cytometry control beads.

Keywords: tetraaryl porphyrin, polystyrene beads, flow cytometry, peptide coupling

Procedia PDF Downloads 63

Authors: Alija Uzunovic, Sasa Pilipovic, Aida Sapcanin, Zahida Ademovic, Berina Pilipović

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Capsaicin is a naturally occurring alkaloid extracted from capsicum fruit extracts of different of Capsicum species. It has been employed topically to treat many diseases such as rheumatoid arthritis, osteoarthritis, cancer pain and nerve pain in diabetes. The high degree of pre-systemic metabolism of intragastrical capsaicin and the short half-life of capsaicin by intravenous administration made topical application of capsaicin advantageous. In this study, we have evaluated differences in the dissolution characteristics of capsaicin patch 11 mg (purchased from market) at different dissolution rotation speed. The proposed patch area is 308 cm2 (22 cm x 14 cm; it contains 36 µg of capsaicin per square centimeter of adhesive). USP Apparatus 5 (Paddle Over Disc) is used for transdermal patch testing. The dissolution study was conducted using USP apparatus 5 (n=6), ERWEKA DT800 dissolution tester (paddle-type) with addition of a disc. The fabricated patch of 308 cm2 is to be cut into 9 cm2 was placed against a disc (delivery side up) retained with the stainless-steel screen and exposed to 500 mL of phosphate buffer solution pH 7.4. All dissolution studies were carried out at 32 ± 0.5 °C and different rotation speed (50± 5; 100± 5 and 150± 5 rpm). 5 ml aliquots of samples were withdrawn at various time intervals (1, 4, 8 and 12 hours) and replaced with 5 ml of dissolution medium. Withdrawn were appropriately diluted and analyzed by reversed-phase liquid chromatography (RP-LC). A Reversed Phase Liquid Chromatography (RP-LC) method has been developed, optimized and validated for the separation and quantitation of capsaicin in a transdermal patch. The method uses a ProntoSIL 120-3-C18 AQ 125 x 4,0 mm (3 μm) column maintained at 600C. The mobile phase consisted of acetonitrile: water (50:50 v/v), the flow rate of 0.9 mL/min, the injection volume 10 μL and the detection wavelength 222 nm. The used RP-LC method is simple, sensitive and accurate and can be applied for fast (total chromatographic run time was 4.0 minutes) and simultaneous analysis of capsaicin and dihydrocapsaicin in a transdermal patch. According to the results obtained in this study, we can conclude that the relative difference of dissolution rate of capsaicin after 12 hours was elevated by increase of dissolution rotation speed (100 rpm vs 50 rpm: 84.9± 11.3% and 150 rpm vs 100 rpm: 39.8± 8.3%). Although several apparatus and procedures (USP apparatus 5, 6, 7 and a paddle over extraction cell method) have been used to study in vitro release characteristics of transdermal patches, USP Apparatus 5 (Paddle Over Disc) could be considered as a discriminatory test. would be able to point out the differences in the dissolution rate of capsaicin at different rotation speed.

Keywords: capsaicin, in vitro, patch, RP-LC, transdermal

Procedia PDF Downloads 198

Authors: Hamza Javar Magnier, Robin Curtis

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There have been rapid advances in the upstream processing of protein therapeutics, which has shifted the bottleneck to downstream purification and formulation. Finding liquid formulations with shelf lives of up to two years is increasingly difficult for some of the newer therapeutics, which have been engineered for activity, but their formulations are often viscous, can phase separate, and have a high propensity for irreversible aggregation1. We explore means to develop improved predictive ability from a better understanding of how protein-protein interactions on formulation conditions (pH, ionic strength, buffer type, presence of excipients) and how these impact upon the initial steps in protein self-association and aggregation. In this work, we study the initial steps in the aggregation pathways using a minimal protein model based on square-well potentials and discontinuous molecular dynamics. The effect of model parameters, including range of interaction, stiffness, chain length, and chain sequence, implies that protein models fold according to various pathways. By reducing the range of interactions, the folding- and collapse- transition come together, and follow a single-step folding pathway from the denatured to the native state2. After parameterizing the model interaction-parameters, we developed an understanding of low-temperature conformational properties and fluctuations, and the correlation to the folding transition of proteins in isolation. The model fluctuations increase with temperature. We observe a low-temperature point, below which large fluctuations are frozen out. This implies that fluctuations at low-temperature can be correlated to the folding transition at the melting temperature. Because proteins “breath” at low temperatures, defining a native-state as a single structure with conserved contacts and a fixed three-dimensional structure is misleading. Rather, we introduce a new definition of a native-state ensemble based on our understanding of the core conservation, which takes into account the native fluctuations at low temperatures. This approach permits the study of a large range of length and time scales needed to link the molecular interactions to the macroscopically observed behaviour. In addition, these models studied are parameterized by fitting to experimentally observed protein-protein interactions characterized in terms of osmotic second virial coefficients.

Keywords: protein folding, native-ensemble, conformational fluctuation, aggregation

Procedia PDF Downloads 332

Authors: Aishwarya K. V., Shreya Sudesh

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Built along the edges of a 11th century CE man-made lake, the city of Bhopal has stood witness to historic layers dating back to Palaeolithic times; early and medieval kingdoms ranging from the Parmaras, Pratiharas to tribal Gonds; the Begum-Nawabs and finally became the Capital of Madhya Pradesh, post-Independence. The lake more popularly called the Upper Lake was created by the King Raja Bhoj from the Parmara dynasty in 1010 AD when he constructed a bund wall across the Kolans river. Atop this bund wall lies the Kamlapati Mahal - which was part of the royal enclosure built in 1702 belonging to the Gond Kingdom. The Mahal is the epicentre of development in the city because it lies in the centre of the axis joining the Old core and New City. Rapid urbanisation descended upon the city once it became the administrative capital of Madhya Pradesh, a newly-formed state of an Independent India. Industrial pockets began being set up and refugees from the Indo-Pakistan separation settled in various regions of the city. To cater to these sudden growth, there was a boom in infrastructure development in the late twentieth century which included precarious decisions made in terms of handling heritage sites causing the destruction of significant parts of the historic fabric. And this practice continues to this day as buffer/ protected zones are breached through exemptions and the absence of robust regulations allow further deterioration of urban heritage. The aim of the research is to systematically study in detail the effect of the urban infrastructure development of the city and its adverse effect on the existing heritage fabric. Through the paper, an attempt to study the parameters involved in preparing the Masterplan of the city and other development projects is done. The research would follow a values-led approach to study the heritage fabric where the significance of the place is assessed based on the values attributed by stakeholders. This approach will involve collection and analysis of site data, assessment of the significance of the site and listing of potential. The study would also attempt to arrive at a solution to deal with urban development along with the protection of the heritage fabric.

Keywords: heritage management, infrastructure development, urban conservation, urban heritage

Procedia PDF Downloads 205

Authors: Katherine Karayianis

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Children with Attention Deficit Hyperactivity Disorder (ADHD) have trouble learning in traditional classrooms. These children miss out on important developmental opportunities in school, which leads to challenges starting in early childhood, and these problems persist throughout their adult lives. Despite receiving supplemental support in school, children with ADHD still perform below their non-ADHD peers. Thus, there is a great need to find better ways of facilitating learning in children with ADHD. Evidence has shown that children with ADHD learn best through interactive engagement, but this is not always possible in schools, given classroom restraints and the large student-to-teacher ratio. Redesigning classrooms may not be feasible, so informal learning opportunities provide a possible alternative. One popular informal learning opportunity is educational TV shows like Sesame Street. These types of educational shows can teach children foundational skills taught in pre-K and early elementary school. One downside to these shows is the lack of interactive dialogue between the TV characters and the child viewers. Pseudo-interaction is often deployed, but the benefits are limited if the characters can neither understand nor contingently respond to the child. AI technology has become extremely advanced and is now popular in many electronic devices that both children and adults have access to. AI has been successfully used to create interactive dialogue in children’s educational TV shows, and results show that this enhances children’s learning and engagement, especially when children perceive the character as a reliable teacher. It is likely that children with ADHD, whose minds may otherwise wander, may especially benefit from this type of interactive technology, possibly to a greater extent depending on their perception of the animated dialogic agent. To investigate this issue, I have begun examining the moderating role of inattention among children’s learning from an educational TV show with different types of dialogic interactions. Preliminary results have shown that when character interactions are neither immediate nor accurate, children who are more easily distracted will have greater difficulty learning from the show, but contingent interactions with a TV character seem to buffer these negative effects of distractibility by keeping the child engaged. To extend this line of work, the moderating role of the child’s perception of the dialogic agent as a reliable teacher will be examined in the association between children’s attention and the type of dialogic interaction in the TV show. As such, the current study will investigate this moderated moderation.

Keywords: attention, dialogic TV, informal learning, educational TV, perception of teacher

Procedia PDF Downloads 40

Authors: Virag Vass, Ilona Bereczki, Erzsebet Szabo, Nora Debreczeni, Aniko Borbas, Pal Herczegh, Arpad Tosaki

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Hydrogen sulfide (H₂S) is a toxic gas, but it is produced by certain tissues in a small quantity. According to earlier studies, ibuprofen and H₂S has a protective effect against damaging heart tissue caused by ischemia-reperfusion. Recently, we have been investigating the effect of a new water-soluble H₂S releasing ibuprofen molecule administered after artificially generated ischemia-reperfusion on isolated rat hearts. The H₂S releasing property of the new ibuprofen derivative was investigated in vitro in medium derived from heart endothelial cell isolation at two concentrations. The ex vivo examinations were carried out on rat hearts. Rats were anesthetized with an intraperitoneal injection of ketamine, xylazine, and heparin. After thoracotomy, hearts were excised and placed into ice-cold perfusion buffer. Perfusion of hearts was conducted in Langendorff mode via the cannulated aorta. In our experiments, we studied the dose-effect of the H₂S releasing molecule in Langendorff-perfused hearts with the application of gradually increasing concentration of the compound (0- 20 µM). The H₂S releasing ibuprofen derivative was applied before the ischemia for 10 minutes. H₂S concentration was measured with an H₂S detecting electrochemical sensor from the coronary effluent solution. The 10 µM concentration was chosen for further experiments when the treatment with this solution was occurred after the ischemia. The release of H₂S is occurred by the hydrolyzing enzymes that are present in the heart endothelial cells. The protective effect of the new H₂S releasing ibuprofen molecule can be confirmed by the infarct sizes of hearts using the Triphenyl-tetrazolium chloride (TTC) staining method. Furthermore, we aimed to define the effect of the H₂S releasing ibuprofen derivative on autophagic and apoptotic processes in damaged hearts after investigating the molecular markers of these events by western blotting and immunohistochemistry techniques. Our further studies will include the examination of LC3I/II, p62, Beclin1, caspase-3, and other apoptotic molecules. We hope that confirming the protective effect of new H₂S releasing ibuprofen molecule will open a new possibility for the development of more effective cardioprotective agents with exerting fewer side effects. Acknowledgment: This study was supported by the grants of NKFIH- K-124719 and the European Union and the State of Hungary co- financed by the European Social Fund in the framework of GINOP- 2.3.2-15-2016-00043.

Keywords: autophagy, hydrogen sulfide, ibuprofen, ischemia, reperfusion

Procedia PDF Downloads 118

Authors: H. Minor-Pérez, A. M. Mota-Silva, S. Ortiz-Barrios

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Substances of vegetable origin with antioxidant capacity have a high potential for application on the conservation of some foods, can prevent or reduce for example oxidation of lipids. However a food is a complex system whose wide variety of components wich can reduce or eliminate this antioxidant capacity. The antioxidant activity can be determined with the ABTS technique. The radical ABTS+ is generated from the acid 2, 2´ - Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). This radical is a composite color bluish-green, stable and with a spectrum of absorption into the UV-visible. The addition of antioxidants causes discoloration, value that can be reported as a percentage of inhibition of the cation radical ABTS+. The objective of this study was evaluated the effect of the combination of the pH and the essential oil of cinnamon (EOC) on inhibition of the radical ABTS+, using statistical design of central point (Design Expert) to obtain mathematical models that describe this phenomenon. Were evaluated 17 treatments with combinations of pH 5, 6 and 7 (citrate-phosphate buffer) and the concentration of essential oil of cinnamon (C): 0 µg/mL, 100 µg/mL and 200 µg/mL. The samples were analyzed using the ABTS technique. The reagent was dissolved in methanol 80% to standardized the absorbance to 0.7 +/- 0.1 at 754 nm. Then samples were mixed with reagent standardized ABTS and after 1 min and 7 min absorbance was read for each treatment at 754 nm. Was used a curve pattern with vitamin C and reported the values as inhibition (%) of radical ABTS+. The statistical analysis shows the experimental results were adjusted to a quadratic model, to the times of 1 min and 7 min. This model describes the influence of the factors investigated independently: pH and cinnamon essential oil (µg/mL) and the effect of the interaction between pH*C, as well as the square of the pH2 and C2. The model obtained was Y = 10.33684 - 3.98118*pH + 1.17031*C + 0.62745*pH2 - 3.26675*10-3*C2 - 0.013112*pH*C, where Y is the response variable. The coefficient of determination was 0.9949 for 1 min. The equation was obtained at 7 min and = - 10.89710 + 1.52341*pH + 1.32892*C + 0.47953*pH2 - 3.56605*10- *C2 - 0.034687*pH*C. The coefficient of determination was 0.9970. This means that only 1% of the total variation is not explained by the developed models. At 100 µg/mL of EOC was obtained an inhibition percentage of 80%, 84% and 97% for the pH values of 5,6 and 7 respectively, while a value of 200 µg/mL the inhibition (%) was very similar for the treatments. In these values of pH was obtained an inhibition close 97%. In conclusion the pH does not have a significant effect on the antioxidant capacity, while the concentration of EOC was decisive for the antioxidant capacity. The authors acknowledge the funding provided by the CONACYT for the project 131998.

Keywords: antioxidant activity, ABTS technique, essential oil of cinnamon, mathematical models

Procedia PDF Downloads 376

Authors: Wenqian Bai

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Background: In response to the post-pandemic directives of Xi Jinping concerning the new era of ecological civilization, China has embarked on ecological restoration projects across its territorial spaces. This initiative faces challenges such as complex evaluation metrics and subpar informatization standards. Methodology: This research focuses on Mianzhu City, Sichuan Province, to assess its resource and environmental carrying capacities and the appropriateness of land use for development from ecological, agricultural, and urban perspectives. The study incorporates a range of spatial data to evaluate factors like ecosystem services (including water conservation, soil retention, and biodiversity), ecological vulnerability (addressing issues like soil erosion and desertification), and resilience. Utilizing the Minimum Cumulative Resistance model along with the ‘Three Zones and Three Lines’ strategy, the research maps out ecological corridors and significant ecological networks. These frameworks support the ecological restoration and environmental enhancement of the area. Results: The study identifies critical ecological zones in Mianzhu City's northwestern region, highlighting areas essential for protection and particularly crucial for water conservation. The southeastern region is categorized as a generally protected ecological zone with respective ratings for water conservation functionality and ecosystem resilience. The research also explores the spatial challenges of three ecological functions and underscores the substantial impact of human activities, such as mining and agricultural expansion, on the ecological baseline. The proposed spatial arrangement for ecological restoration, termed ‘One Mountain, One Belt, Four Rivers, Five Zones, and Multiple Corridors’, strategically divides the city into eight major restoration zones, each with specific tasks and projects. Conclusion: With its significant ‘mountain-plain’ geography, Mianzhu City acts as a crucial ecological buffer for the Yangtze River's upper reaches. Future development should focus on enhancing ecological corridors in agriculture and urban areas, controlling soil erosion, and converting farmlands back to forests and grasslands to foster ecosystem rehabilitation.

Keywords: ecological restoration, resource and environmental carrying capacity, land development suitability, ecosystem services, ecological vulnerability, ecological networks

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Authors: Kun Hua O., Dong Woon Kim, Won Hyung Lee

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Introduction: Neuropathic pain induced by spinal or peripheral nerve injury is highly resistant to common painkillers, nerve blocks, and other pain management approaches. Recently, several new therapeutic drug candidates have been developed to control neuropathic pain. In this study, we used the spinal nerve L5 ligation (SNL) model to investigate the ability of intrathecal or intravenous Evans blue to decrease pain behavior and to study the relationship between Evans blue and the neural structure of pain transmission. Method: Neuropathic pain (allodynia) of the left hind paw was induced by unilateral SNL in Sprague-Dawley rats(n=10) in each group. Evans blue (5, 15, 50μg/10μl) or phosphate buffer saline(PBS,10μl) was injected intrathecally at 3days post-ligation or intravenously(1mg/200 μl) 3days and 5days post-ligation . Mechanical sensitivity was assessed using Von Frey filaments at 3 days post-ligation and at 2 hours, days 1, 2, 3, 5,7 after intrathecal Evans blue injection, and on days 2, 4, 7, and 11 at 14 days after intravenous injection. In the intrathecal group, microglia and glutaminergic neurons in the dorsal horn and VNUT(vesicular nucleotide transporter) in the dorsal root ganglia were tested to evaluate co-staining with Evans blue. The experimental procedures were performed in accordance with the animal care guideline of the Korean Academy of Medical Science(Animal ethic committee of Chungnam National University Hospital: CNUH-014-A0005-1). Results: Tight ligation of the L5 spinal nerve induced allodynia in the left hind paw 3 days post-ligation. Intrathecal Evans blue most significantly(P＜0.001) alleviated allodynia at 2 days after intrathecal, but not an intravenous injection. Glutaminergic neurons in the dorsal horn and VNUT in the dorsal root ganglia were co-stained with Evans blue. On the other hand, microglia in the dorsal horn were partially co-stained with Evans blue. Conclusion: We confirmed that Evans blue might have an analgesic effect through the central nervous system, not another system in neuropathic pain of the SNL animal model. These results suggest Evans blue may be a potential new drug for the treatment of chronic pain. This research was supported by the National Research Foundation of Korea (NRF-2020R1A2C100757512), funded by the Ministry of Education.

Keywords: neuropathic pain, Evas blue, intrathecal, intravenous

Procedia PDF Downloads 65

Authors: E. Didaskalou, C. Roussi-Vergou, E. Andreou, G. Skrzypiec, P. Slee

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There has been growing interest lately, in the study of victimization among adolescent students in Greece and elsewhere with a view to improve school policies concerning anti-bullying practices. Researchers have recently focused on investigating the relationships between the extent of students’ victimization and the distinct mechanisms that they employ for coping with this particular problem. In particular, the emphasis has been placed on exploring the relationship between the coping strategies students use to counteract bullying, their sense of belonging at school, and extent of their victimization. Methods: Within the research framework outlined above, we set out to: a) examine the frequency of self-reported victimization among secondary school students, b) investigate the coping strategies employed by students when confronted with school bullying and c) explore any differences between bullied and non-bullied students with regard to coping strategies and school belongingness. The sample consisted of 860 from fifteen secondary public schools in central Greece. The schools were typical Greek secondary schools and the principals volunteered to participate in this study. Participants’ age ranged from 12 to 16 years. Measures: a) Exposure to Victimization: The frequency of victimization was directly located by asking students the question: ‘Over the last term, how often have you been bullied or harassed by a student or students at this high school?’ b) Coping Strategies: The ‘Living and Learning at School: Bullying at School’ was administered to students, c) School belongingness was assessed by the Psychological Sense of School Membership Scale, that students completed. Results: Regarding the frequency of self-reported victimization, 1.5% of the students reported being victimized every day, 2.8% most days of the week, 2.1% one or more days a week, 2.9% about once a week, 22.6% less than once a week and 68.1% never. The coping strategies that the participants employed for terminating their victimization included: a) adult support seeking, b) emotional coping/keep away from school, c) keeping healthy and fit, d) demonstrating a positive attitude towards the bully, d) peer support seeking, e) emotional out bursting, f) wishful thinking and self-blaming, g) pretending as if it is not happening, h) displaying assertive behaviors and i) getting away from the bullies. Bullied from non-bullied children did not differ as much in coping, as in feelings of being rejected in school. Discussion: The findings are in accordance with accumulated research evidence which points to a strong relationship between student perceptions of school belongingness and their involvement in bullying behaviors. We agree with the view that a positive school climate is likely to serve as a buffer that mitigates wider adverse societal influences and institutional attitudes which favor violence and harassment among peers.

Keywords: school bullying, school belonging, student coping strategies, victimization

Procedia PDF Downloads 217

Authors: Vimal Kumar Dewangan, T. S. Sampath Kumar, Mukesh Doble, Viju Daniel Varghese

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The apatite-based, i.e., calcium-deficient hydroxyapatite (CDHAp) bone cement is well-known potential bone graft/substitute in orthopaedics due to its similar chemical composition with natural bone minerals. Therefore, an easy approach was attempted to fabricate the apatite-based (CDHAp) bone cement with improved injectability, bioresorbability, and macroporosity. In this study, the desired bone cement was developed by mixing the solid phase (consisting of wet chemically synthesized nanocrystalline hydroxyapatite and commercially available (synthetic) tricalcium phosphate) and the liquid phase (consisting of cement binding accelerator with few biopolymers in a dilute acidic solution) along with a liquid porogen as polysorbate or a solid porogen as mannitol (for comparison) in an optimized liquid-to-powder ratio. The fabricated cement sets within clinically preferred setting time (≤20 minutes) are better injectable (>70%) and also stable at ~7.3-7.4 (physiological pH). The CDHAp phased bone cement was resulted by immersing the fabricated after-set cement in phosphate buffer solution and other similar artificial body fluids and incubated at physiological conditions for seven days, confirmed through the X-ray diffraction and Fourier transform-infrared spectroscopy analyses. The so-formed synthetic apatite-based bone cement holds the acceptable compressive strength (within the range of trabecular bone) with average interconnected pores size falls in a macropores range (~50-200μm) inside the cement, verified by scanning electron microscopy (SEM), mercury intrusion porosimetry and micro-CT analysis techniques. Also, it is biodegradable (degrades ~19-22% within 10-12 weeks) when incubated in artificial body fluids under physiological conditions. The biocompatibility study of the bone cement, when incubated with MG63 cells, shows a significant increase in the cell viability after 3rd day of incubation compared with the control, and the cells were well-attached and spread completely on the surface of the bone cement, confirmed through SEM and fluorescence microscopy analyses. With this all, we can conclude that the developed synthetic macroporous apatite-based bone cement may have the potential to become promising material used as a trabecular bone substitute.

Keywords: calcium deficient hydroxyapatite, synthetic apatite-based bone cement, injectability, macroporosity, trabecular bone substitute

Procedia PDF Downloads 61

Authors: Angelica Corcoran, Fredrik Lind, Pavleta Knutsson, Henrik Thunman

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Solid waste volumes are at current predominately deposited on landfill. Furthermore, the impending climate change requires new solutions for a sustainable future energy mix. Currently, solid waste is globally utilized to small extent as fuel during combustion for heat and power production. Due to its variable composition and size, solid waste is considered difficult to combust and requires a technology with high fuel flexibility. One of the commercial technologies used for combustion of such difficult fuels is circulating fluidized beds (CFB). In a CFB boiler, fine particles of a solid material are used as 'bed material', which is accelerated by the incoming combustion air that causes the bed material to fluidize. The chosen bed material has conventionally been silica sand with the main purpose of being a heat carrier, as it transfers heat released by the combustion to the heat-transfer surfaces. However, the release of volatile compounds occurs rapidly in comparison with the lateral mixing in the combustion chamber. To ensure complete combustion a surplus of air is introduced, which decreases the total efficiency of the boiler. In recent years, the concept of partly or entirely replacing the silica sand with an oxygen carrier as bed material has been developed. By introducing an oxygen carrier to the combustion chamber, combustion can be spread out both temporally and spatially in the boiler. Specifically, the oxygen carrier can take up oxygen from the combustion air where it is in abundance and release it to combustible gases where oxygen is in deficit. The concept is referred to as oxygen carrier aided combustion (OCAC) where the natural ore ilmenite (FeTiO3) has been the oxygen carrier used. The authors have validated the oxygen buffering ability of ilmenite during combustion of biomass in Chalmers 12-MWth CFB boiler in previous publications. Furthermore, the concept has been demonstrated on full industrial scale during combustion of municipal solid waste (MSW) in E.ON’s 75 MWth CFB boiler. The experimental campaigns have showed increased mass transfer of oxygen inside the boiler when combustion both biomass and MSW. As a result, a higher degree of burnout is achieved inside the combustion chamber and the plant can be operated at a lower surplus of air. Moreover, the buffer of oxygen provided by the oxygen carrier makes the system less sensitive to disruptions in operation. In conclusion, combusting difficult fuels with OCAC results in higher operation stability and an increase in boiler efficiency.

Keywords: OCAC, ilmenite, combustion, CFB

Procedia PDF Downloads 213

Authors: Branislav Kovačević, Andrej Pilipović, Zoran Novčić, Marina Milović, Lazar Kesić, Milan Drekić, Saša Pekeč, Leopold Poljaković Pajnik, Saša Orlović

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Poplars present one of the most important tree species used for phytoremediation in the northern hemisphere. They can be used either as direct “cleaners” of the contaminated soils or as buffer zones preventing the contaminant plume to the surrounding environment. In order to produce appropriate planting material for this purpose, there is a long process of the breeding of the most favorable candidates. Although the development of the poplar propagation technology has been evolving for decades, white poplar nursery production, as well as the establishment of short-rotation coppice plantations, still considerably depends on the success of hardwood cuttings’ survival. This is why easy rooting is among the most desirable properties in white poplar breeding. On the other hand, there are many opportunities for the optimization of the technological procedures in order to meet the demands of particular genotype (clonal technology). In this study the effect of the term of hardwood cuttings’ preparation of four white poplar clones on their survival and further growth of rooted cuttings in nursery conditions were tested. There were three terms of cuttings’ preparation: the beginning of February (2nd Feb 2023), the beginning of March (3rd Mar 2023) and the end of March (21nd Mar 2023), which is regarded as the standard term. The cuttings were stored in cool chamber at 2±2°C. All cuttings were planted on the same date (11th Apr 2023), in soil prepared with rotary tillage, and then cultivated by usual nursey procedures. According to the results obtained after the bud set (29th Sept 2023) there were significant differences in the survival and growth of rooted cuttings between examined terms of cutting preparation. Also, there were significant differences in the reaction of examined clones on terms of cutting preparation. In total, the best results provided cuttings prepared at the first term (2nd Feb 2023) (survival rate of 39.4%), while performance after two later preparation terms was significantly poorer (20.5% after second and 16.5% after third term). These results stress the significance of dormancy preservation in cuttings of examined white poplar clones for their survival, which could be especially important in context of climate change. Differences in clones’ reaction to term of cutting preparation suggest necessity of adjustment of the technology to the needs of particular clone i.e. design of clone specific technology.

Keywords: rooting, Populus alba, nursery, clonal technology

Procedia PDF Downloads 29

Authors: Jung-En Kuan, Whei-Fen Wu

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In this study, a symbiotic bacteria from yellow mealworm's (Tenebrio molitor) mid-gut was isolated with characteristics of growth on minimal-tributyrin medium. After a PCR-amplification of its 16s rDNA, the resultant nucleotide sequences were then analyzed by schemes of the phylogeny trees. Accordingly, it was designated as Pseudomonas nitroreducens D-01. Next, by searching the lipolytic enzymes in its protein data bank, one of those potential lipolytic α/β hydrolases was identified, again using PCR-amplification and nucleotide-sequencing methods. To construct an expression of this lipolytic gene in plasmids, the target-gene primers were then designed, carrying the C-terminal his-tag sequences. Using the vector pET21a, a recombinant lipolytic hydrolase D gene with his-tag nucleotides was successfully cloned into it, of which the lipolytic D gene is under a control of the T7 promoter. After transformation of the resultant plasmids into Eescherichia coli BL21 (DE3), an IPTG inducer was used for the induction of the recombinant proteins. The protein products were then purified by metal-ion affinity column, and the purified proteins were found capable of forming a clear zone on tributyrin agar plate. Shortly, its enzyme activities were determined by degradation of p-nitrophenyl ester(s), and the substantial yellow end-product, p-nitrophenol, was measured at O.D.405 nm. Specifically, this lipolytic enzyme efficiently targets p-nitrophenyl butyrate. As well, it shows the most reactive activities at 40°C, pH 8 in potassium phosphate buffer. In thermal stability assays, the activities of this enzyme dramatically drop when the temperature is above 50°C. In metal ion assays, MgCl₂ and NH₄Cl induce the enzyme activities while MnSO₄, NiSO₄, CaCl₂, ZnSO₄, CoCl₂, CuSO₄, FeSO₄, and FeCl₃ reduce its activities. Besides, NaCl has no effects on its enzyme activities. Most organic solvents decrease the activities of this enzyme, such as hexane, methanol, ethanol, acetone, isopropanol, chloroform, and ethyl acetate. However, its enzyme activities increase when DMSO exists. All the surfactants like Triton X-100, Tween 80, Tween 20, and Brij35 decrease its lipolytic activities. Using Lineweaver-Burk double reciprocal methods, the function of the enzyme kinetics were determined such as Km = 0.488 (mM), Vmax = 0.0644 (mM/min), and kcat = 3.01x10³ (s⁻¹), as well the total efficiency of kcat/Km is 6.17 x10³ (mM⁻¹/s⁻¹). Afterwards, based on the phylogenetic analyses, this lipolytic protein is classified to type IV lipase by its homologous conserved region in this lipase family.

Keywords: enzyme, esterase, lipotic hydrolase, type IV

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

Abstract:

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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