Search results for: molecular recognition

Authors: Jin Young Kim, Kwon Kyoo Kang

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The SGR1 (STAYGREEN1) protein is a critical regulator of plant leaves in chlorophyll degradation and senescence. The functions and mechanisms of tomato SGR1 action are poorly understood and worthy of further investigation. To investigate the function of the SGR1 gene, we generated a SGR1-knockout (KO) null line via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene editing and conducted RNA sequencing and gas chromatography tandem mass spectrometry (GC-MS/MS) analysis to identify the differentially expressed genes. The SlSGR1 (Solanum lycopersicum SGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid content compared to wild-type (WT) fruit. Differential gene expression analysis revealed 728 differentially expressed genes (DEGs) between WT and sgr1 #1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, for which fold change was >2, and the adjusted p-value was <0.05. Most of the DEGs were related to photosynthesis and chloroplast function. In addition, the pigment, carotenoid changes in sgr1 #1-6 line was accumulated of key primary metabolites such as sucrose and its derivatives (fructose, galactinol, raffinose), glycolytic intermediates (glucose, G6P, Fru6P) and tricarboxylic acid cycle (TCA) intermediates (malate and fumarate). Taken together, the transcriptome and metabolite profiles of SGR1-KO lines presented here provide evidence for the mechanisms underlying the effects of SGR1 and molecular pathways involved in chlorophyll degradation and carotenoid biosynthesis.

Keywords: tomato, CRISPR/Cas9, null line, RNA-sequencing, metabolite profiling

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Authors: Chi-Chang Lin, Jian-Rong Wu, Jiun-Yan Chiu

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Myopia, ubiquitous symptom that is necessary to correct the eyesight by optical lens struggles many people for their daily life. Recent years, younger people raise interesting on using contact lens because of its convenience and aesthetics. In clinical, the risk of eye infections increases owing to the behavior of incorrectly using contact lens unsupervised cleaning which raising the infection risk of cornea, named ocular keratitis. In order to overcome the identification needs, new detection or analysis method with rapid and more accurate identification for clinical microorganism is importantly needed. In our study, we take advantage of Raman spectroscopy having unique fingerprint for different functional groups as the distinct and fast examination tool on microorganism. As we know, Raman scatting signals are normally too weak for the detection, especially in biological field. Here, we applied special SERS enhancement substrates to generate higher Raman signals. SERS filter we designed in this article that prepared by deposition of silver nanoparticles directly onto cellulose filter surface and suspension nanoparticles - gold nanostars (AuNSs) also be introduced together to achieve better enhancement for lower concentration analyte (i.e., various bacteria). Research targets also focusing on studying the shape effect of synthetic AuNSs, needle-like surface morphology may possible creates more hot-spot for getting higher SERS enhance ability. We utilized new designed SERS technology to distinguish the bacteria from ocular keratitis under strain level, and specific Raman and SERS fingerprint were grouped under pattern recognition process. We reported a new method combined different SERS substrates can be applied for clinical microorganism detection under strain level with simple, rapid preparation and low cost. Our presenting SERS technology not only shows the great potential for clinical bacteria detection but also can be used for environmental pollution and food safety analysis.

Keywords: bacteria, gold nanostars, Raman spectroscopy surface-enhanced Raman scattering filter

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Authors: Pavel Damborsky, Martina Zamorova, Jaroslav Katrlik

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Prostate cancer is annually the most common newly diagnosed cancer among men. An extensive number of evidence suggests that traditional serum Prostate-specific antigen (PSA) assay still suffers from a lack of sufficient specificity and sensitivity resulting in vast over-diagnosis and overtreatment. Thus, the early-stage detection of prostate cancer (PCa) plays undisputedly a critical role for successful treatment and improved quality of life. Over the last decade, particular altered glycans have been described that are associated with a range of chronic diseases, including cancer and inflammation. These glycans differences enable a distinction to be made between physiological and pathological state and suggest a valuable biosensing tool for diagnosis and follow-up purposes. Aberrant glycosylation is one of the major characteristics of disease progression. Consequently, the aim of this study was to develop a more reliable tool for early-stage PCa diagnosis employing lectins as glyco-recognition elements. Biosensor and biochip technology putting to use lectin-based glyco-profiling is one of the most promising strategies aimed at providing fast and efficient analysis of glycoproteins. The proof-of-concept experiments based on sandwich assay employing anti-PSA antibody and an aptamer as a capture molecules followed by lectin glycoprofiling were performed. We present a lectin-based biosensing assay for glycoprofiling of serum biomarker PSA using different biosensor and biochip platforms such as label-free surface plasmon resonance (SPR) and microarray with fluorescent label. The results suggest significant differences in interaction of particular lectins with PSA. The antibody-based assay is frequently associated with the sensitivity, reproducibility, and cross-reactivity issues. Aptamers provide remarkable advantages over antibodies due to the nucleic acid origin, stability and no glycosylation. All these data are further step for construction of highly selective, sensitive and reliable sensors for early-stage diagnosis. The experimental set-up also holds promise for the development of comparable assays with other glycosylated disease biomarkers.

Keywords: biomarker, glycosylation, lectin, prostate cancer

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Authors: Sarah Gaßmeyer, Nadine Hülsemann, Raphael Stoll, Kenji Miyamoto, Robert Kourist

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Enzymatic racemization allows the smooth interconversion of stereocenters under very mild reaction conditions. Racemases find frequent applications in deracemization and dynamic kinetic resolutions. Arylmalonate decarboxylase (AMDase) from Bordetella Bronchiseptica has high structural similarity to amino acid racemases. These cofactor-free racemases are able to break chemically strong CH-bonds under mild conditions. The racemase-like catalytic machinery of mutant G74C conveys it a unique activity in the racemisation of pharmacologically relevant derivates of 2-phenylpropionic acid (profenes), which makes AMDase G74C an interesting object for the mechanistic investigation of cofactor-independent racemases. Structure-guided protein engineering achieved a variant of this unique racemase with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. By saturation–transfer–difference NMR spectroscopy (STD-NMR), substrate binding during catalysis was investigated. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose variation increased the activity of G74C. While single-amino acid exchanges increased the activity moderately, structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering.

Keywords: racemase, rational protein design, STD-NMR, structure guided saturation mutagenesis

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Authors: Ivan Zupan, Tomislav Saric, Suzana Tkalcic

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IMUNO-2865® is a commercially available, β–glucan based, natural hemicellulose compound with proven immunostimulative properties in people, domestic and some aquatic animals. During the experimental feeding trial with IMUNO-2865® in juvenile wild-caught chub under laboratory conditions, supplementation resulted in overall higher growth performance for all experimental groups regardless of the concentration of the added compound. The maximum, 5% concentration of the supplement, resulted in highest weight gain and calculated specific growth rate. In sea bream, as economically most important species in the Mediterranean aquaculture, significant increases in numbers of monocytes and heterophils were observed in the group supplemented with 2.5 % of IMUNO-2865® in the feed. An overall increase of erythrocytes was noted by the end of the experiment, although with variable distribution among groups. Blood Ca++ levels, total proteins, and total NH₃ were significantly higher after 60 days of feeding in all treatment groups compared to the control and remained elevated in the treated group following the secession of supplementation. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and serum paraoxonase PON1 (U/L) showed similar trends. All these parameters are playing a significant role in either oxygen supplementation of tissues, or anabolic and catabolic processes that on molecular levels contribute to the overall health and immune-building capacity of cells and tissues. The complete lack of mortality in sea bream and presented increases in cellular, biochemical and oxidative stress parameters in the blood suggest that the IMUNO-2865® represents a safe dietary supplement for in aquaculture, with an overall positive and potentially immunostimulative effect on farmed fish.

Keywords: IMUNO-2865®, β–glucans, Mediterranean aquaculture, fish imunnostimulans

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Authors: Sruthi Ramagiri, Rajeev Taliyan

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Background and purpose: Recent investigations on ischemia and reperfusion injury postulate that transient ischemia of remote organs after a prolonged ischemic insult confers neuroprotection. However, the molecular mechanisms of the remote limb ischemic post-conditioning (RIPOC) are yet to be elucidated. The current study was designed to investigate the protective mechanism of RIPOC against cerebral ischemic injury using global model of stroke. Materials and methods: Global ischemic reperfusion injury (IR) was achieved by 30 minutes ischemia of cerebral artery, followed by reperfusion for 24 hours. Induction of global ischemia was followed by 4 brief episodes (30 seconds each) of ischemia and reperfusion of femoral artery to accomplish RIPOC. 5-Hydroxy Decanoic acid (5-HD), a KATP channel blocker (20 mg/kg) was administered after induction of global ischemia and RIPOC intervention. Results: IR injury ensue significant behavioural deficits as manifested by rotarod performance and spontaneous locomotor activity when compared to sham control. Furthermore, IR injury significantly increased oxidonitrative stress and infarct volume as evidenced by biochemical parameters (MDA, GSH, Nitrite, SOD) and 2,3,5-triphenyltetrazolium chloride (TTC) staining respectively. Moreover, RIPOC intervention ameliorated the behavioural performance, attenuated the oxidative stress and infarct volume when compared to IR injury group. However, administration of 5-HD increased the oxidative stress and infarct size while deteriorating the behavioural parameters when compared to RIPOC group. Conclusions: In a nutshell, cerebral IR injury has significantly induced the neuronal damage, whereas RIPOC intervention decreased the neuronal injury. Moreover, 5-HD abolished the neuroprotection offered by RIPOC indicating the putative role of KATP channel opening in RIPOC against cerebral ischemic injury.

Keywords: RIPOC, cerebral injury, KATP channel, neuroprotection

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Authors: Mohammad Aladwan, Adelia Skripova

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Bioremediation aspects of crude oil-polluted fields can be achieved by isolation and identification of bacterial species from oil-contaminated soil in order to choose the most active isolates and increase the strength of others. In this study, oil-degrading bacteria were isolated and identified from oil-contaminated soil samples in northeastern Jordan. The bacterial growth count (CFU/g) was between 1.06×10⁵ and 0.75×10⁹. Eighty-two bacterial isolates were characterized by their morphology and biochemical tests. The identified bacterial genera included: Klebsiella, Staphylococcus, Citrobacter, Lactobacillus, Alcaligenes, Pseudomonas, Hafnia, Micrococcus, Rhodococcus, Serratia, Enterobacter, Bacillus, Salmonella, Mycobacterium, Corynebacterium, and Acetobacter. Molecular identification of a universal primer 16S rDNA gene was used to identify four bacterial isolates: Microbacterium esteraromaticum strain L20, Pseudomonas stutzeri strain 13636M, Klebsilla pneumoniae, and uncultured Klebsilla sp., known as new strains. Our results indicate that their specific oil-degrading bacteria isolates might have a high strength of oil degradation from oil-contaminated sites. Staphylococcus intermedius (75%), Corynebacterium xerosis (75%), and Pseudomonas fluorescens (50%) showed a high growth rate on different types of hydrocarbons, such as crude oil, toluene, naphthalene, and hexane. In addition, monooxygenase and catechol 2,3-dioxygenase were detected in 17 bacterial isolates, indicating their superior hydrocarbon degradation potential. Total petroleum hydrocarbons were analyzed using gas chromatography for soil samples. Soil samples M5, M7, and M8 showed the highest levels (43,645, 47,805, and 45,991 ppm, respectively), and M4 had the lowest level (7,514 ppm). All soil samples were analyzed for heavy metal contamination (Cu, Cd, Mn, Zn, and Pb). Site M7 contains the highest levels of Cu, Mn, and Pb, while Site M8 contains the highest levels of Mn and Zn. In the future, these isolates of bacteria can be used for the cleanup of oil-contaminated soil.

Keywords: bioremediation, 16S rDNA gene, oil-degrading bacteria, hydrocarbons

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Authors: Yasir Arafat, Asma Shah, Hua Shao

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

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

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Authors: Amin Abbasi, Fariborz Shekari, Seyed Bahman Mousavi

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Seed aging during storage is a complex biochemical and physiological processes that can lead to reduce seed germination. This phenomenon associated with increasing of total antioxidant activity during aging. To study the effects of hormones on seed aging, aged wheat seeds (control, 90 and 80% viabilities) were treated with GA3, Salicylic Acid, and paclobutrazol and antioxidant system were investigated as molecular biomarkers for seed vigor. The results showed that, seed priming treatment significantly affected germination percentage, normality seedling percentage, H2O2, MDA, CAT, APX, and GPX activates. Maximum germination percentage achieve in GA3 priming in control treatment. Germination percentage and normal seedling percentage increased in other GA3 priming treatment compared with other hormones. Also aging increased MDA, H2O2 content. MDA is considered sensitive marker commonly used for assessing membrane lipid peroxidation and H2O2result in toxicity to cellular membrane system and damages to plant cells. Amount of H2O2 and MDA declined in GA3 treatment. CAT, GPX and APX activities were reduced by increasing the aging time and at different levels of priming. The highest APX activity was observed in Salicylic Acid control treatment and the highest GPX and CAT activity was obtained in GA3 control treatment. The lowest MDA and H2O2 showed in GA3 control treatment, too. Hormone priming increased Antioxidant enzyme activity and decreased amount of reactive oxygen space and malondialdehyde (MDA) under aging treatment. Also, GA3 priming treatments have a significant effect on germination percentage and number of normal seedling. Generally aging seed, increase ROS and lipid peroxidation. Antioxidant enzymes activity of aged seeds increased after hormone priming.

Keywords: hormones priming, wheat, aging seed, antioxidant, lipid peroxidation

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Authors: Mohammad K. Parvez, Mohammed S. Al-Dosari

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Hepatitis E virus (HEV) is an emerging RNA virus that causes acute and chronic liver disease with a global mortality rate of about 2%. Despite milestone developments in understanding of HEV biology, there is still lack of a robust culture system or animal model. Therefore, in a novel approach, two recombinant-baculoviruses (vBac-ORF2 and vBac-ORF3) that could overexpress HEV ORF2 (structural/capsid) and ORF3 (nonstructural/regulatory) proteins, respectively were constructed. The established HEV-SAR55 (genotype 1) replicon that contained GFP gene, in place of ORF2/ORF3 sequences was in vitro transcribed, and GFP production in RNA transfected S10-3 cells was scored by FACS. Enhanced infectivity, if any, of nascent virions produced by exogenously-supplied ORF2 and viral RNA by co-expression of ORF3 was tested on naïve HepG2 cells. Co-transduction with vBac-ORF2/vBac-ORF3 (108 pfu/microL) produced high amounts of native ORF2/ORF3 in approximately 60% of S10-3 cells, determined by immunofluorescence microscopy and Western analysis. FACS analysis showed about 9% GFP positivity of S10-3 cells on day6 post-transfection (i.e, day5 post-transduction). Further, FACS scoring indicated that lysates from S10-3 cultures receiving the RNA plus vBac-ORF2 were capable of producing HEV particles with about 4% infectivity in HepG2 cells. However, lysates of cultures co-transduced with vBac-ORF3, were found to further enhance virion infectivity by approximately 17%. This supported a previously proposed role of ORF3 as a minor-structural protein in HEV virion assembly and infectivity. In conclusion, the present model for efficient genomic RNA packaging and production of infectious virions could be a valuable tool to study various aspects of HEV molecular biology, in vitro.

Keywords: chronic liver disease, hepatitis E virus, ORF2, ORF3, replicon

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Authors: Thomas Bryan, Veton Kepuska, Ivica Kostanic

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Speaker recognition is performed in high Additive White Gaussian Noise (AWGN) environments using principals of Computational Auditory Scene Analysis (CASA). CASA methods often classify sounds from images in the time-frequency (T-F) plane using spectrograms or cochleargrams as the image. In this paper atomic decomposition implemented by matching pursuit performs a transform from time series speech signals to the T-F plane. The atomic decomposition creates a sparsely populated T-F vector in “weight space” where each populated T-F position contains an amplitude weight. The weight space vector along with the atomic dictionary represents a denoised, compressed version of the original signal. The arraignment or of the atomic indices in the T-F vector are used for classification. Unsupervised feature learning implemented by a sparse autoencoder learns a single dictionary of basis features from a collection of envelope samples from all speakers. The approach is demonstrated using pairs of speakers from the TIMIT data set. Pairs of speakers are selected randomly from a single district. Each speak has 10 sentences. Two are used for training and 8 for testing. Atomic index probabilities are created for each training sentence and also for each test sentence. Classification is performed by finding the lowest Euclidean distance between then probabilities from the training sentences and the test sentences. Training is done at a 30dB Signal-to-Noise Ratio (SNR). Testing is performed at SNR’s of 0 dB, 5 dB, 10 dB and 30dB. The algorithm has a baseline classification accuracy of ~93% averaged over 10 pairs of speakers from the TIMIT data set. The baseline accuracy is attributable to short sequences of training and test data as well as the overall simplicity of the classification algorithm. The accuracy is not affected by AWGN and produces ~93% accuracy at 0dB SNR.

Keywords: time-frequency plane, atomic decomposition, envelope sampling, Gabor atoms, matching pursuit, sparse dictionary learning, sparse autoencoder

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Authors: Tettey Afi Pamela, Sotaro Fujii, Hidetoshi Saito, Kawaii Koichiro

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Introduction: Organisms employ adaptive mechanisms when faced with any stressor or risk of being wiped out. This has made it possible for them to survive in harsh environmental conditions such as increasing temperature, low pH, and anoxia. Some of the mechanisms they utilize include the expression of heat shock proteins, synthesis of cryoprotectants, and anhydrobiosis. Heat shock proteins (HSPs) have been widely studied to determine their involvement in stress tolerance among various organism, of which chironomid species have been no exception. We examined the survival and expression of genes encoding five (5) heat shock proteins (HSP70, HSP67, HSP60, HSP27, and HSP23) from Chironomus sulfurosus larvae reared from 1st instar at 25°C, 30°C, 35°C, and 40°C. Results: The highest survival rate was recorded at 30°C, followed by 25°C, then 35°C. Only a small percentage of C. sulfurosus survived at 40°C (14.5%). With regards to HSPs expression, some HSPs responded to an increase in high temperature. The relative expression levels were lowest at 30°C for HSP70, HSP60, HSP27, and HSP23. At 25°C and 40°C, HSP70, HSP67, HSP60, HSP27, and HSP23 had the highest expression. At 35°C, all had the lowest expression. Discussion: The expression of heat shock proteins varies from one species to another. We designated the genes HSP 70, HSP 67, HSP 60, HSP 27, and HSP 23 genes based on transcriptome analysis of C. sulfurosus. Our study can be termed as a long-heat shock study as C. sulfurosus was reared from the first instar to the fourth instar, and this might have led to a continuous induction of HSPs at 25°C. 40°C had the lowest survival but highest HSPs expression as C. sulfurosus larvae had to utilize HSPs for sustenance. These results and future high-throughput studies at both the transcriptome and proteome level will improve the information needed to predict the future geographic distribution of these species within the context of global warming.

Keywords: chironomid, heat shock proteins, high temperature, heat shock protein expression

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Authors: K. Nikovics, D. Riccobono, M. Oger, H. Morin, L. Barbier, T. Poyot, X. Holy, A. Bendahmane, M. Drouet, A. L. Favier

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Muscle regeneration after injury (as irradiation) is of great importance. However, the molecular and cellular mechanisms are still unclear. Cytokines are believed to play fundamental role in the different stages of muscle regeneration. They are secreted by many cell populations, but the predominant producers are macrophages and helper T cells. On the other hand, it has been shown that adipose tissue derived stromal/stem cell (ASC) injection could improve muscle regeneration. Stem cells probably induce the coordinated modulations of gene expression in different macrophage cells. Therefore, we investigated the patterns and timing of changes in gene expression of different cytokines occurring upon stem cells loading. Muscle regeneration was studied in an irradiated muscle of minipig animal model in presence or absence of ASC treatment (irradiated and treated with ASCs, IRR+ASC; irradiated not-treated with ASCs, IRR; and non-irradiated no-IRR). We characterized macrophage populations by immunolabeling in the different conditions. In our study, we found mostly M2 and a few M1 macrophages in the IRR+ASC samples. However, only few M2b macrophages were noticed in the IRR muscles. In addition, we found intensive fibrosis in the IRR samples. With in situ hybridization and immunolabeling, we analyzed the cytokine expression of the different macrophages and we showed that M2d macrophage are the most abundant in the IRR+ASC samples. By in situ hybridization, strong expression of the transforming growth factor β (TGF-β) was observed in the IRR+ASC but very week in the IRR samples. But when we analyzed TGF-β level with immunolabeling the expression was very different: many M2 macrophages showed week expression in IRR+ASC and few cells expressing stronger level in IRR muscles. Therefore, we investigated the MMP expressions in the different muscles. Our data showed that the M2 macrophages of the IRR+ASC muscle expressed MMP2 proteins. Our working hypothesis is that MMP2 expression of the M2 macrophages can decrease fibrosis in the IRR+ASC muscle by capturing TGF-β.

Keywords: adipose tissue derived stromal/stem cell, cytokine, macrophage, muscle regeneration

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Authors: Suchart Tripopsakul

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This paper uses data from the Global Entrepreneurship Monitor (GEM) to investigate the difference of attitudes towards entrepreneurial intention (EI). EI is generally assumed to be the single most relevant predictor of entrepreneurial behavior. The aim of this paper is to examine a range of attitudes effect on individual’s intent to start a new venture. A cross-cultural comparison between Asia and Europe is used to further investigate the possible differences between potential entrepreneurs from these distinct national contexts. The empirical analysis includes a GEM data set of 10 countries (n = 10,306) which was collected in 2013. Logistic regression is used to investigate the effect of individual’s attitudes on EI. Independent variables include individual’s perceived capabilities, the ability to recognize business opportunities, entrepreneurial network, risk perceptions as well as a range of socio-cultural attitudes. Moreover, a cross-cultural comparison of the model is conducted including six ASEAN (Malaysia, Indonesia, Philippines, Singapore, Vietnam and Thailand) and four European nations (Spain, Sweden, Germany, and the United Kingdom). The findings support the relationship between individual’s attitudes and their entrepreneurial intention. Individual’s capability, opportunity recognition, networks and a range of socio-cultural perceptions all influence EI significantly. The impact of media attention on entrepreneurship and was found to influence EI in ASEAN, but not in Europe. On the one hand, Fear of failure was found to influence EI in Europe, but not in ASEAN. The paper develops and empirically tests attitudes toward Entrepreneurial Intention between ASEAN and Europe. Interestingly, fear of failure was found to have no significant effect in ASEAN, and the impact of media attention on entrepreneurship and was found to influence EI in ASEAN. Moreover, the resistance of ASEAN entrepreneurs to the otherwise high rates of fear of failure and high impact of media attention are proposed as independent variables to explain the relatively high rates of entrepreneurial activity in ASEAN as reported by GEM. The paper utilizes a representative sample of 10,306 individuals in 10 countries. A range of attitudes was found to significantly influence entrepreneurial intention. Many of these perceptions, such as the impact of media attention on entrepreneurship can be manipulated by government policy. The paper also suggests strategies by which Asian economy in particular can benefit from their apparent high impact of media attention on entrepreneurship.

Keywords: an entrepreneurial intention, attitude, GEM, ASEAN and Europe

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Authors: Mary Carmen Peloche Barrera

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Since its early years as an independent nation, the United States has been one of the main promoters regarding the recognition, legislation, and protection of human rights. In the matter of freedom, the founding father Thomas Jefferson envisioned the role of the U.S. as a defender of freedom and equality throughout the world. This founding ideal shaped America’s domestic and foreign policy in the 19th and the 20th century and became an aspiration of the ideals of the country to expand its values and institutions. The history of the emergence of human rights cannot be studied without making reference to leaders such as Woodrow Wilson, Franklin, and Eleanor Roosevelt, as well as Martin Luther King. Throughout its history, this country has proclaimed that the protection of the freedoms of men, both inside and outside its borders, is practically the reason for its existence. Although the United States was one of the first countries to recognize the existence of inalienable rights for individuals, as well as the main promoter of the Universal Declaration of Human Rights of 1948, the country has gone through critical moments that had led to questioning its commitment to the issue. Racial segregation, international military interventions, national security strategy, as well as national legislation on immigration, are some of the most controversial issues related to decisions and actions driven by the United States, which at the same time mismatched with its role as an advocate of human rights, both in the Americas and in the rest of the world. The aim of this paper is to study the swinging of the efforts and commitments of the United States towards human rights. The paper will analyze the history and evolution of human rights in the United States, to study the greatest challenges for the country in this matter. The paper will focus on both the domestic policy (related to demographic issues) and foreign policy (about its role in a post-war world). Currently, more countries are joining the multilateral efforts for the promotion and protection of human rights. At the same time, the United States is one of the least committed countries in this respect, having ratified only 5 of the 18 treaties emanating from the United Nations. The last ratification was carried out in 2002 and, since then, the country has been losing ground, in an increasingly vertiginous way, in its credibility and, even worse, in its role as leader of 'the free world'. With or without the United States, the protection of human rights should remain the main goal of the international community.

Keywords: United States, human rights, foreign policy, domestic policy

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Authors: J. DeBoard, R. Dietrich, J. Hughes, K. Yurko, G. Harms

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Alzheimer’s disease (AD) is a predominant type of dementia and is likely a major cause of neural network impairment. The pathogenesis of this neurodegenerative disorder has yet to be fully elucidated. There are currently no known cures for the disease, and the best hope is to be able to detect it early enough to impede its progress. Beyond age and genetics, another prevalent risk factor for AD might be traumatic brain injury (TBI), which has similar neurodegenerative hallmarks. Our research focuses on obtaining information and methods to be able to predict when neurodegenerative effects might occur at a clinical level by observation of events at a cellular and molecular level in model mice. First, we wish to introduce our evidence that brain damage can be observed via brain imaging prior to the noticeable loss of neuromuscular control in model mice of AD. We then show our evidence that some blood biomarkers might be able to be early predictors of AD in the same model mice. Thus, we were interested to see if we might be able to predict which mice might show long-term neurodegenerative effects due to differing degrees of TBI and what level of TBI causes further damage and earlier death to the AD model mice. Upon application of TBIs via an apparatus to effectively induce extremely mild to mild TBIs, wild-type (WT) mice and AD mouse models were tested for cognition, neuromuscular control, olfactory ability, blood biomarkers, and brain imaging. Experiments are currently still in process, and more results are therefore forthcoming. Preliminary data suggest that neuromotor control diminishes as well as olfactory function for both AD and WT mice after the administration of five consecutive mild TBIs. Also, seizure activity increases significantly for both AD and WT after the administration of the five TBI treatment. If future data supports these findings, important implications about the effect of TBI on those at risk for AD might be possible.

Keywords: Alzheimer's disease, blood biomarker, neurodegeneration, neuromuscular control, olfaction, traumatic brain injury

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Authors: Airy Huang, Fuji Foo, Aries Prasetya Wibowo

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In recent years, the focus of Natural Language Processing (NLP) development has been gradually shifting from the semantics-based approach to deep learning one, which performs faster with fewer resources. Although it performs well in many applications, the deep learning approach, due to the lack of semantics understanding, has difficulties in noticing and expressing a novel business case with a pre-defined scope. In order to meet the requirements of specific robotic services, deep learning approach is very labor-intensive and time consuming. It is very difficult to improve the capabilities of conversational AI in a short time, and it is even more difficult to self-learn from experiences to deliver the same service in a better way. In this paper, we present an adaptive conversational AI algorithm that combines both semantic knowledge and deep learning to address this issue by learning new business cases through conversations. After self-learning from experience, the robot adapts to the business cases originally out of scope. The idea is to build new or extended robotic services in a systematic and fast-training manner with self-configured programs and constructed dialog flows. For every cycle in which a chat bot (conversational AI) delivers a given set of business cases, it is trapped to self-measure its performance and rethink every unknown dialog flows to improve the service by retraining with those new business cases. If the training process reaches a bottleneck and incurs some difficulties, human personnel will be informed of further instructions. He or she may retrain the chat bot with newly configured programs, or new dialog flows for new services. One approach employs semantics analysis to learn the dialogues for new business cases and then establish the necessary ontology for the new service. With the newly learned programs, it completes the understanding of the reaction behavior and finally uses dialog flows to connect all the understanding results and programs, achieving the goal of self-learning process. We have developed a chat bot service mounted on a kiosk, with a camera for facial recognition and a directional microphone array for voice capture. The chat bot serves as a concierge with polite conversation for visitors. As a proof of concept. We have demonstrated to complete 90% of reception services with limited self-learning capability.

Keywords: conversational AI, chatbot, dialog management, semantic analysis

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Authors: Hao Yang, Lei Chen, Ting Mei, Jianbang Zheng

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Currently, the uncooled PbSe photodetectors in the mid-infrared range (2-5μm) with sensitization technology extract more photoelectric response than traditional ones, and enable the room temperature (300K) photo-detection with high detectivity, which have attracted wide attentions in many fields. This technology generally contains the film fabrication with vapor phase deposition (VPD) and a sensitizing process with doping of oxygen and iodine. Many works presented in the recent years almost provide and high temperature activation method with oxygen/iodine vapor diffusion, which reveals that oxygen or iodine plays an important role in the sensitization of PbSe material. In this paper, we provide our latest experimental results and discussions in the stoichiometry of oxygen and iodine and its influence on the polycrystalline structure and photo-response. The experimental results revealed that crystal orientation was transformed from (200) to (420) by sensitization, and the responsivity of 5.42 A/W was gained by the optimal stoichiometry of oxygen and iodine with molecular density of I2 of ~1.51×1012 mm-3 and oxygen pressure of ~1Mpa. We verified that I2 plays a role in transporting oxygen into the lattice of crystal, which is actually not its major role. It is revealed that samples sensitized with iodine transform atomic proportion of Pb from 34.5% to 25.0% compared with samples without iodine from XPS data, which result in the proportion of about 1:1 between Pb and Se atoms by sublimation of PbI2 during sensitization process, and Pb/Se atomic proportion is controlled by I/O atomic proportion in the polycrystalline grains, which is very an important factor for improving responsivity of uncooled PbSe photodetector. Moreover, a novel sensitization and dopant activation method is proposed using oxygen ion implantation with low ion energy of < 500eV and beam current of ~120μA/cm2. These results may be helpful to understanding the sensitization mechanism of polycrystalline lead salt materials.

Keywords: polycrystalline PbSe, sensitization, transport, stoichiometry

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Authors: Neslihan Ozman Say, Jim Gilmour, Pratik Desai, William Zimmerman

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Landfill leachate treatment has been attracting researchers recently for various environmental and economical reasons. Leachate discharge to receiving waterbodies without treatment causes serious detrimental effects including partial oxygen depletion due to high biological oxygen demand (BOD) and chemical oxygen demand (COD) concentrations besides toxicity of heavy metals it contains and high ammonia concentrations. In this study, it is aimed to show microalgal ammonia removal performances of a wild microalgae consortia as an alternative treatment method and determine the dominant leachate tolerant species for this consortia. For the microalgae species identification experiments a microalgal consortium which has been isolated from a local pond in Sheffield inoculated in %5 diluted raw landfill leachate and acclimated to the leachate by batch feeding for a month. In order to determine the most tolerant microalgal consortium, four different untreated landfill leachate samples have been used as diluted in four different ratios as 5%, 10%, 20%, and 40%. Microalgae cell samples have been collected from all experiment sets and have been examined by using 18S rDNA sequencing and specialised gel electrophoresis which are adapted molecular biodiversity methods. The best leachate tolerant algal consortium is being used in order to determine ammonia removal performances of the culture in a microbubble assisted photobioreactor (PBR). A porous microbubble diffuser which is supported by a fluidic oscillator is being used for dosing CO₂ and air mixture in the PBR. It is known that high mass transfer performance of microbubble technology provides a better removal efficiency and a better mixing in the photobioreactor. Ammonia concentrations and microalgal growth are being monitored for PBR currently. It is aimed to present all the results of the study in final paper submission.

Keywords: ammonia removal from leachate, landfill leachate treatment, microalgae species identification, microbubble assisted photobioreactors

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Authors: Moses Ikechukwu Benjamin

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The continued increase in methicillin-resistant Staphylococcuspseudintermedius (MRSP) among dogs and the zoonotic transmission event of MRSP from dogs to humans threaten veterinary medicine and public health. The cardinal objective of this study was to determine the antibiogram and frequency of toxingenes in MRSP obtained from shelter dogs with skin infections and dog owners in Abakaliki, Eastern Nigeria. Skinswabs from 61 shelter dogs with skin infections and 33 nasal swabs from dog owners were processed and analyzed using standard microbiological techniques. Susceptibility to antibiotics was determined by Kirby Bauer disc diffusion technique. The screening for Seccanine, lukD, siet, and exitoxin genes was carried out by PCR. A total of 23 (37.7 %) and 1 (3 %) MRSP strains were obtained from shelter dogs and dog owners, respectively. Generally, isolates exhibited high resistance to amoxicillin-clavulanic acid, ceftazidime, and cefepime (100 % - 66.7 %) but were very susceptible (100 % - 70.7 %) to chloramphenicol and doripenem. The only isolate from dog owners harbouredseccanine, lukD, and siet toxin genes while solatesfrom shelter dogs harbouredseccanine16 (69.6 %), lukD 17 (73.9 %), siet 20 (87 %), and exi1 (4.4 %) toxin genes. Isolates were generally observed to be more resistant than other reports from the literature. Interesting, there was a similarity in the resistance antibiotypes and frequency of toxin genes harboured by MRSP isolates between shelter dogs with skin infections and their owner in a sampled household, thus suggesting a likely zoonotic transmission event. This report of the occurrence of MRSP and high frequency of toxin genes (Seccanine,lukD, and siet) in shelter dogs and dog owners represent a major challenge, especially in terms of antibiotic therapy, and is a serious concern for both animal and public health.

Keywords: methicillin-resistant S. pseudintermedius, zoonotic transmission, antibiotic resistance, companion dogs, toxin genes

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Authors: Yanjun Chen, Chuanping Hu, Jie Shao, Lin Mei, Chongyang Zhang

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Traditional image classification is based on encoding scheme (e.g. Fisher Vector, Vector of Locally Aggregated Descriptor) with low-level image features (e.g. SIFT, HoG). Compared to these low-level local features, deep convolutional features obtained at the mid-level layer of convolutional neural networks (CNN) have richer information but lack of geometric invariance. For scene classification, there are scattered objects with different size, category, layout, number and so on. It is crucial to find the distinctive objects in scene as well as their co-occurrence relationship. In this paper, we propose a method to take advantage of both deep convolutional features and the traditional encoding scheme while taking object-centric and scene-centric information into consideration. First, to exploit the object-centric and scene-centric information, two CNNs that trained on ImageNet and Places dataset separately are used as the pre-trained models to extract deep convolutional features at multiple scales. This produces dense local activations. By analyzing the performance of different CNNs at multiple scales, it is found that each CNN works better in different scale ranges. A scale-wise CNN adaption is reasonable since objects in scene are at its own specific scale. Second, a fisher kernel is applied to aggregate a global representation at each scale and then to merge into a single vector by using a post-processing method called scale-wise normalization. The essence of Fisher Vector lies on the accumulation of the first and second order differences. Hence, the scale-wise normalization followed by average pooling would balance the influence of each scale since different amount of features are extracted. Third, the Fisher vector representation based on the deep convolutional features is followed by a linear Supported Vector Machine, which is a simple yet efficient way to classify the scene categories. Experimental results show that the scale-specific feature extraction and normalization with CNNs trained on object-centric and scene-centric datasets can boost the results from 74.03% up to 79.43% on MIT Indoor67 when only two scales are used (compared to results at single scale). The result is comparable to state-of-art performance which proves that the representation can be applied to other visual recognition tasks.

Keywords: deep convolutional features, Fisher Vector, multiple scales, scale-specific normalization

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Authors: Edna Gnomblerou

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Given the current scale of corruption epidemic in West African economies, governments are seeking for immediate and effective measures to reduce the likelihood of the plague within the region. Generally, accountants and auditors are expected to help organizations in detecting illegal practices. However, their role in the fight against corruption is sometimes limited due to the collusive nature of corruption. The Denmark anti-corruption model shows that the implementation of additional controls over public accounts and independent efficient audits improve transparency and increase the probability of detection. This study is aimed at reviewing the existing anti-corruption policies of the Economic Commission of West African States (ECOWAS) as to observe the role attributed to accounting, auditing and other managerial practices in their anti-corruption drive. It further discusses the usefulness of accounting and auditing in helping anti-corruption commissions in controlling misconduct and increasing the perception to detect irregularities within public administration. The purpose of this initiative is to identify and assess the relevance of accounting and auditing in curbing corruption. To meet this purpose, the study was designed to answer the questions of whether accounting and auditing processes were included in the reviewed anti-corruption strategies, and if yes, whether they were effective in the detection process. A descriptive research method was adopted in examining the role of accounting and auditing in West African anti-corruption strategies. The analysis reveals that proper recognition of accounting standards and implementation of financial audits are viewed as strategic mechanisms in tackling corruption. Additionally, codes of conduct, whistle-blowing and information disclosure to the public are among the most common managerial practices used throughout anti-corruption policies to effectively and efficiently address the problem. These observations imply that sound anti-corruption strategies cannot ignore the values of including accounting and auditing processes. On one hand, this suggests that governments should employ all resources possible to improve accounting and auditing practices in the management of public sector organizations. On the other hand, governments must ensure that accounting and auditing practices are not limited to the private sector, but when properly implemented constitute crucial mechanisms to control and reduce corrupt incentives in public sector.

Keywords: accounting, anti-corruption strategy, auditing, ECOWAS

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Authors: Marzia Bilkiss, Muhammad J. A. Shiddiky, Mostafa K. Masud, Prabhakaran Sambasivam, Ido Bar, Jeremy Brownlie, Rebecca Ford

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A greater achievement in the Integrated Disease Management (IDM) to prevent the loss would result from early diagnosis and quantitation of the causal pathogen species for accurate and timely disease control. This could significantly reduce costs to the growers and reduce any flow on impacts to the environment from excessive chemical spraying. Necrotrophic fungal disease botrytis grey mould, caused by Botrytis cinerea and Botrytis fabae, significantly reduce temperate legume yield and grain quality during favourable environmental condition in Australia and worldwide. Several immunogenic and molecular probe-type protocols have been developed for their diagnosis, but these have varying levels of species-specificity, sensitivity, and consequent usefulness within the paddock. To substantially improve speed, accuracy, and sensitivity, advanced nanoparticle-based biosensor approaches have been developed. For this, two sets of primers were designed for both Botrytis cinerea and Botrytis fabae which have shown the species specificity with initial sensitivity of two genomic copies/µl in pure fungal backgrounds using multiplexed quantitative PCR. During further validation, quantitative PCR detected 100 spores on artificially infected legume leaves. Simultaneously an electro-catalytic assay was developed for both target fungal DNA using functionalised magnetic nanoparticles. This was extremely sensitive, able to detect a single spore within a raw total plant nucleic acid extract background. We believe that the translation of this technology to the field will enable quantitative assessment of pathogen load for future accurate decision support of informed botrytis grey mould management.

Keywords: biosensor, botrytis grey mould, sensitive, species specific

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Authors: Yu-Chieh Chen, Li-Yun Wang, Chi-Chih Chen, Huy Hùng Đào, Ya-Mei Chen, Ming-Chu Cheng, Wen-Bin Chung, Hso-Chi Chaung, Guan-Ming Ke

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Many recent researches have demonstrated that CD154, a protein primarily expressed on activated T cell molecules, has potentially acted as a molecular adjuvant to improve the immunogenicity of subunit vaccines against viral infections. Classical swine fever (CSF) affects the swine industry worldwide that is one of the most devastating and highly contagious pig diseases. It is listed by the World Organization for Animal Health (OIE) as an infectious animal disease that must be reported. Although pigs vaccinated with subunit vaccines can be differentially diagnosed from those infected animals, subunit vaccines usually need adjuvants to enhance and elicit immune responses. In this study, CD154 was linked with CSFV E2 sequences and then expressed in CHO cells to produce the fusion protein as E2-CD154. The porcine specific CpG adjuvant was also used in one of the formulations. The specific pathogen-free pigs (SPF) at the age of 4-week-old were randomly separated into four groups, vaccinated with E2-CpG, E2-CD154, E2-CD154-CpG or the commercial Bayovac® CSF-E2 vaccine and boosted two weeks after primary vaccination. The results showed that the percentages of CD4+ and CD4+IL2+ in peripheral blood mononuclear cells (PBMC) in E2-CD154 vaccinated piglets seven days after primary vaccination were gained by 1-5% relative to the control group. In addition, the percentages of CD4+IFNγ+ T cells had slightly edged up 0.1-0.3% compared with the control group. Also, increased E2-specific IFNγ levels had edged up CD4+CD8+ T cells found in E2-CD154 and E2-CD154-CpG groups, particularly in the E2-CD154-CpG group. These results implicate that CD154 may enhance cellular immunity and synergistically act with species-specific CpG adjuvant as a dual-phase adjuvant. Therefore, the CD154 may be beneficial as a promising adjuvant in subunit vaccines.

Keywords: CD154, CpG adjuvant, cellular immunity, subunit vaccine, pig

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Authors: Jarmila Bernasovska, Pavel Ružbarský, Ivan Bernasovsky, Regina Lohajová Behulová

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The paper presents the results of the application of molecular genetics methods in sport research, with special emphasis on the most advanced methods and trends in diagnosing of motoric predispositions for the sake of identifying talented children. Genetic tests differ in principle from the traditional motoric tests, because the DNA of an individual does not change during life. Genetics is important in determining the capacity of an individual and for professional sport level. Genetic information can be used for individual genetic predispositions in early childhood. The phenotypes are influenced by a combination of genetic and environmental factors. The aim of the presented study was to examine physical condition, coordination skills, motoric docility and to determine the frequency of ACTN3 (R577X) gene in Romany children from Eastern Slovakia and compared their motoric performance with non-Romany children. This paper is not looking just for a performance, but also its association to genetic predispositions in relation to ACTN3 gene and its R577X polymorphism. Genotype data were obtained from 175 Romany children from 6 to 15 years old and 218 non-Romany children at the same age from Eastern Slovakia. Biological material for genetic analyses comprised samples of buccal swabs. Genotypes were determined using Real Time High resolution melting PCR method (Rotor Gene 6000 Corbett and LightCycler 480 Roche). Romany children of analyzed group legged to non-Romany children at the same age in all the compared tests. The % distribution of R and X alleles in children was different from controls. The frequency of XX genotype was 11,45% which is comparable to a frequency of an Indian population. Data were analysed with the ANOVA statistical programme and parametric and nonparametric tests. This work was supported by grants APVV-0716-10, ITMS 26220120023 and ITMS 26220120041.

Keywords: ACTN3 gene, R577X polymorphism, Romany children, sport performance, Slovakia

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Authors: Barbara Zapala, Marek Dziechciowski, Olaf Chmura, Monika Piwowar, Katarzyna Gawlik, Dorota Pawlicka-Gosiewska, Krzysztof Skotniczny, Bogdan Solnica, Kazimierz Pitynski

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BACKGROUND: Endometrial cancer is one of the most common gynecologic malignancy in developed countries.We hypothesized that amount of circulating micro-particles in blood may be connected with the development of endometrial hyperplasia and endometrial cancer. The aim of this study was to measure the micro-particles amount in uterine venous blood and in peripheral venous blood in women with atypical endometrial hyperplasia and endometrioid endometrial cancer. MATERIALS AND METHODS: By using flow cytometry (BD Canto II cytometer) we measured micro-particles amount in citrate plasma samples from peripheral and uterine venous blood of women with atypical hyperplasia of endometrium or endometrial cancer. We determined the amount of total (TF+), endothelial (CD144+) and monocytic (CD14+) micro- particles. RESULTS: Here we show statistically significant higher micro-particle levels in women with atypical hyperplasia of endometrium or endometrial cancer in comparison to healthy women. Performing measurements of the amounts of total, endothelial and monocytic microparticles allow for reliable differentiation between healthy, atypical hyperplasia and endometrial cancer groups. In blood samples from uterine veins the circulating micro-particle levels were significantly different from peripheral blood samples. The micro-particle levels in uterine blood samples were 7-fold higher than in those from peripheral blood of women with both atypical hyperplasia of endometrium and endometrial cancer when compared to the control group of healthy women. CONCLUSION: These results strongly suggested that the level of circulating micro-particles may be a sign of endometrial cancer development, however the detailed study is needed focusing on molecular processes passed through this small circulating molecules.

Keywords: endometrial cancer, endometrial hyperplasia, microvesicles, uterine blood

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Authors: S. Redjala, N. Ait Hocine, M. Gratton, N. Poirot, R. Ferhoum, S. Azem

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Polycarbonate (PC) is a promising polymer with high transparency in the range of the visible spectrum and is used in various fields, for example medical, electronic, automotive. Its low weight, chemical inertia, high impact resistance and relatively low cost are of major importance. In recent decades, some materials such as metals and ceramics have been replaced by polymers because of their superior advantages. However, some characteristics of the polymers are highly modified under the effect of ultraviolet (UV) radiation and temperature. The changes induced in the material by such aging depend on the exposure time, the wavelength of the UV radiation and the temperature level. The UV energy is sufficient to break the chemical bonds leading to a cleavage of the molecular chains. This causes changes in the mechanical, thermal, optical and morphological properties of the material. The present work is focused on the study of the effects of aging under ultraviolet (UV) radiation and under different temperature values on the physical-chemical and mechanical properties of a PC. Thus, various investigations, such as FTIR and XRD analyses, SEM and optical microscopy observations, micro-hardness measurements and monotonic and cyclic tensile tests, were carried out on the PC in the initial state and after aging. Results have shown the impact of aging on the properties of the PC studied. In fact, the MEB highlighted changes in the superficial morphology of the material by the presence of cracks and material de-bonding in the form of debris. The FTIR spectra reveal an attenuation of the peaks like the hydroxyl (OH) groups located at 3520 cm-1. The XRD lines shift towards a larger angle, reaching a maximum of 3°. In addition, Vickers micro-hardness measurements show that aging affects the surface and the core of the material, which results in different mechanical behaviours under monotonic and cyclic tensile tests. This study pointed out effects of aging on the macroscopic properties of the PC studied, in relationship with its microstructural changes.

Keywords: mechanical properties, physical-chemical properties, polycarbonate, UV aging, temperature aging

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Authors: Daina Jonkus, Solvita Petrovska, Dace Smiltina, Lasma Cielava

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The beta-lactoglobulin and kappa-casein are milk proteins which are important for milk composition. Cows with beta-lactoglobulin and kappa-casein gene BB genotypes have highest milk crude protein and fat content. The aim of the study was to determinate the frequencies of milk protein gene polymorphisms in local Latvian Brown (LB) cows breed and analyze the influence of beta-lactoglobulin and kappa-casein genotypes to milk productivity traits. 102 cows’ genotypes of milk protein genes were detected using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) and electrophoresis on 3% agarose gel. For beta-lactoglobulin were observed 2 types of alleles A and B and for kappa-casein 3 types: A, B and E. Highest frequency in beta-lactoglobulin gene was observed for B allele – 0.926. Molecular analysis of beta-lactoglobulin gene shows 86.3% of individuals are homozygous by B allele and animals are with genotypes BB and 12.7% of individuals are heterozygous with genotypes AB. The highest milk yield 4711.7 kg was for 1st lactation cows with AB genotypes, whereas the highest milk protein content (3.35%) and fat content (4.46 %) was for BB genotypes. Analysis of the kappa-casein locus showed a prevalence of the A allele – 0.750. The genetic variant of B was characterized by a low frequency – 0.240. Moreover, the frequency of E occurred in the LB cows’ population with very low frequency – 0.010. 54.9 % of cows are homozygous with genotypes AA, and only 4.9 % are homozygous with genotypes BB. 32.8 % of individuals are heterozygous with genotypes AB, and 2.0 % are with AE. The highest milk productivity was for 1st lactation cows with AB genotypes: milk yield 4620.3 kg, milk protein content 3.39% and fat content 4.53 %. According to the results, in local Latvian brown there are only 2.9% of cows are with BB-BB genotypes, which is related to milk coagulation ability and affected cheese production yield. Acknowledgment: the investigation is supported by VPP 2014-2017 AgroBioRes Project No. 3 LIVESTOCK.

Keywords: beta-lactoglobulin, cows, genotype frequencies, kappa-casein

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Authors: Md. Z. Alam, Amal A. Elgharbawy, Muhammad Moniruzzaman, Nassereldeen A. Kabbashi, Parveen Jamal

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The enzymatic hydrolysis of lignocellulosic biomass is one of the obstacles in the process of sugar production, due to the presence of lignin that protects the cellulose molecules against cellulases. Although the pretreatment of lignocellulose in ionic liquid (IL) system has been receiving a lot of interest; however, it requires IL removal with an anti-solvent in order to proceed with the enzymatic hydrolysis. At this point, introducing a compatible cellulase enzyme seems more efficient in this process. A cellulase enzyme that was produced by Trichoderma reesei on palm kernel cake (PKC) exhibited a promising stability in several ILs. The enzyme called PKC-Cel was tested for its optimum pH and temperature as well as its molecular weight. One among evaluated ILs, 1,3-diethylimidazolium dimethyl phosphate [DEMIM] DMP was applied in this study. Evaluation of six factors was executed in Stat-Ease Design Expert V.9, definitive screening design, which are IL/ buffer ratio, temperature, hydrolysis retention time, biomass loading, cellulase loading and empty fruit bunches (EFB) particle size. According to the obtained data, IL-enzyme system shows the highest sugar concentration at 70 °C, 27 hours, 10% IL-buffer, 35% biomass loading, 60 Units/g cellulase and 200 μm particle size. As concluded from the obtained data, not only the PKC-Cel was stable in the presence of the IL, also it was actually stable at a higher temperature than its optimum one. The reducing sugar obtained was 53.468±4.58 g/L which was equivalent to 0.3055 g reducing sugar/g EFB. This approach opens an insight for more studies in order to understand the actual effect of ILs on cellulases and their interactions in the aqueous system. It could also benefit in an efficient production of bioethanol from lignocellulosic biomass.

Keywords: cellulase, hydrolysis, lignocellulose, pretreatment

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Authors: Fadime Eroglu

Abstract:

Acanthamoeba genus belongs to kingdom protozoa, and it is known as free-living amoebae. Acanthamoeba genus has been isolated from human bodies, swimming pools, bottled mineral water, contact lens solutions, dust, and soil. The members of the genus Acanthamoeba causes Acanthamoeba Keratitis which is a painful sight-threatening disease of the eyes. In recent years, the prevalence of Acanthamoeba keratitis has been high rate reported. The eight different Acanthamoeba species are known to be effective in Acanthamoeba keratitis. These species are Acanthamoeba castellanii, Acanthamoeba polyphaga, Acanthamoeba griffini, Acanthamoeba hatchetti, Acanthamoeba culbertsoni and Acanhtamoeba rhysodes. The conventional diagnosis of Acanthamoeba Keratitis has relied on cytological preparations and growth of Acanthamoeba in culture. However molecular methods such as real-time PCR has been found to be more sensitive. The real-time PCR has now emerged as an effective method for more rapid testing for the diagnosis of infectious disease in decade. Therefore, a real-time PCR assay for the detection of Acanthamoeba keratitis and Acanthamoeba species have been developed in this study. The 18S rRNA sequences from Acanthamoeba species were obtained from National Center for Biotechnology Information and sequences were aligned with MEGA 6 programme. Primers and probe were designed using Custom Primers-OligoPerfectTMDesigner (ThermoFisherScientific, Waltham, MA, USA). They were also assayed for hairpin formation and degree of primer-dimer formation with Multiple Primer Analyzer ( ThermoFisherScientific, Watham, MA, USA). The eight different ATCC Acanthamoeba species were obtained, and DNA was extracted using the Qiagen Mini DNA extraction kit (Qiagen, Hilden, Germany). The DNA of Acanthamoeba species were analyzed using newly designed primer and probe set in real-time PCR assay. The early definitive laboratory diagnosis of Acanthamoeba Keratitis and the rapid initiation of suitable therapy is necessary for clinical prognosis. The results of the study have been showed that new primer and probes could be used for detection and distinguish for Acanthamoeba species. These new developing methods are helpful for diagnosis of Acanthamoeba Keratitis.

Keywords: Acathamoeba Keratitis, Acanthamoeba species, fast diagnosis, Real-Time PCR

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