Search results for: equivalent mutant

Authors: Ozlem Oral, Emre Taskin, Aysel Yuce, Serap Dokmeci, Devrim Gozuacik

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Autophagy is an evolutionary conserved lysosome-dependent catabolic pathway, responsible for the degradation of long-lived proteins, abnormal aggregates and damaged organelles which cannot be degraded by the ubiquitin-proteasome system. Lysosomes degrade the substrates through the activity of lysosomal hydrolases and lysosomal membrane-bound proteins. Mutations in the coding region of these proteins cause malfunctional lysosomes, which contributes to the pathogenesis of lysosomal storage diseases. Gaucher disease is a lysosomal storage disease resulting from the mutation of a lysosomal membrane-associated glycoprotein called glucocerebrosidase and its cofactor saposin C. The disease leads to intracellular accumulation of glucosylceramide and other glycolipids. Because of the essential role of lysosomes in autophagic degradation, Gaucher disease may directly be linked to this pathway. In this study, we investigated the expression of autophagy and/or lysosome-related genes and proteins in fibroblast cells isolated from patients with different mutations. We carried out confocal microscopy analysis and examined autophagic flux by utilizing the differential pH sensitivities of RFP and GFP in mRFP-GFP-LC3 probe. We also evaluated lysosomal pH by active lysosome staining and lysosomal enzyme activity. Beside lysosomes, we also performed proteasomal activity and cell death analysis in patient samples. Our data showed significant attenuation in the expression of key autophagy-related genes and accumulation of their proteins in mutant cells. We found decreased the ability of autophagosomes to fuse with lysosomes, associated with elevated lysosomal pH and reduced lysosomal enzyme activity. Proteasomal degradation and cell death analysis showed reduced proteolytic activity of the proteasome, which consequently leads to increased susceptibility to cell death. Our data indicate that the major degradation pathways are affected by multifunctional lysosomes in mutant patient cells and may underlie in the mechanism of clinical severity of Gaucher patients. (This project is supported by TUBITAK-3501-National Young Researchers Career Development Program, Project No: 112T130).

Keywords: autophagy, Gaucher's disease, glucocerebrosidase, mutant fibroblasts

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Authors: Swati Gautam, Salman Akhtar, S. P. Jaiswar, Amita Jain

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Background: FGTB is now a major global health problem mostly in developing countries including India. In humans, Mycobacterium Tuberculosis (M.tb) is a causating agent of infection. High index of suspicion is required for early diagnosis due to asymptomatic presentation of FGTB disease. In macrophages Toll Like Receptor-2 (TLR-2) is one which mediated host’s immune response to M.tb. The expression of TLR-2 on macrophages is important to determine the fate of innate immune responses to M.tb. TLR-2 have two work. First its high expression on macrophages worsen the outer of infection and another side, it maintains M.tb to its dormant stage avoids activation of M.tb from latent phase. Single Nucleotide Polymorphism (SNP) of TLR-2 gene plays an important role in susceptibility to TB among different populations and subsequently, in the development of infertility. Methodology: This Case-Control study was done in the Department of Obs and Gynae and Department of Microbiology at King George’s Medical University, U.P, Lucknow, India. Total 300 subjects (150 Cases and 150 Controls) were enrolled in the study. All subjects were enrolled only after fulfilling the given inclusion and exclusion criteria. Inclusion criteria: Age 20-35 years, menstrual-irregularities, positive on Acid-Fast Bacilli (AFB), TB-PCR, (LJ/MGIT) culture in Endometrial Aspiration (EA). Exclusion criteria: Koch’s active, on ATT, PCOS, and Endometriosis fibroid women, positive on Gonococal and Chlamydia. Blood samples were collected in EDTA tubes from cases and healthy control women (HCW) and genomic DNA extraction was carried out by salting-out method. Genotyping of TLR2 genetic variants (Arg753Gln and Arg677Trp) were performed by using single amplification refractory mutation system (ARMS) PCR technique. PCR products were analyzed by electrophoresis on 1.2% agarose gel and visualized by gel-doc. Statistical analysis of the data was performed using the SPSS 16.3 software and computing odds ratio (OR) with 95% CI. Linkage Disequiliribium (LD) analysis was done by SNP stats online software. Results: In TLR-2 (Arg753Gln) polymorphism significant risk of FGTB observed with GG homozygous mutant genotype (OR=13, CI=0.71-237.7, p=0.05), AG heterozygous mutant genotype (OR=13.7, CI=0.76-248.06, p=0.03) however, G allele (OR=1.09, CI=0.78-1.52, p=0.67) individually was not associated with FGTB. In TLR-2 (Arg677Trp) polymorphism a significant risk of FGTB observed with TT homozygous mutant genotype (OR= 0.020, CI=0.001-0.341, p < 0.001), CT heterozygous mutant genotype (OR=0.53, CI=0.33-0.86, p=0.014) and T allele (OR=0.463, CI=0.32-0.66, p < 0.001). TT mutant genotype was only found in FGTB cases and frequency of CT heterozygous more in control group as compared to FGTB group. So, CT genotype worked as protective mutation for FGTB susceptibility group. In haplotype analysis of TLR-2 genetic variants, four possible combinations, i.e. (G-T, A-C, G-C, and A-T) were obtained. The frequency of haplotype A-C was significantly higher in FGTB cases (0.32). Control group did not show A-C haplotype and only found in FGTB cases. Conclusion: In conclusion, study showed a significant association with both genetic variants of TLR-2 of FGTB disease. Moreover, the presence of specific associated genotype/alleles suggest the possibility of disease severity and clinical approach aimed to prevent extensive damage by disease and also helpful for early detection of disease.

Keywords: ARMS, EDTA, FGTB, TLR

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Authors: Rania M. Abdallah, Ahmed A. S. Dessouki, Moustafa H. Aly

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This paper has presented a new simple small signal model for a resonant tunnelling diode device. The resonant tunnelling diode equivalent circuit elements were calculated and the results led to good agreement between the calculated equivalent circuit elements and the measurement results.

Keywords: resonant tunnelling diode, small signal model, negative differential conductance, electronic engineering

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Authors: H. Sakamoto, J. Tochimoto, S. Kurosawa, M. Suzuki, S. Oguri

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Salt stress adversely affects plant growth at various stages of development including seed germination, seedling establishment, vegetative growth and finally reproduction. Because of their immobile nature, plants have evolved mechanisms to sense and respond to salt stress. Seed dormancy is an adaptive trait that enables seed germination to coincide with favorable environmental conditions. We identified a novel locus of Arabidopsis, designated SHG1 (salt hypersensitive germination 1), whose disruption leads to reduced germination rate under moderate salt stress conditions. SHG1 encodes a transmembrane protein with an ankyrin repeat motif that has been implicated in diverse cellular processes such as signal transduction. The SGH1-disrupted Arabidopsis mutant died at the cotyledon stage when sown on salt-containing medium, although wild type plants could form true leaves under the same conditions. On the other hand, this mutant showed similar phenotypes to wild type plants when sown on medium without salt and transferred to salt-containing medium at the vegetative stage. These results suggested that SHG1 played indispensable role in the seed germination and seedling establishment under moderate salt stress conditions. SHG1 may be involved in the release of seed dormancy.

Keywords: germination, ankyrin repeat, arabidopsis, salt tolerance

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Authors: F. Ahwide, Y. Bouker, K. Hatem

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This paper presents long term wind data analysis in terms of annual and diurnal variations at different areas of Libya. The data of the wind speed and direction are taken each ten minutes for a period, at least two years, are used in the analysis. ‘WindPRO’ software and Excel workbook were used for the wind statistics and energy calculations. As for Derna, average speeds are 10 m, 20 m, and 40 m, and respectively 6.57 m/s, 7.18 m/s, and 8.09 m/s. Highest wind speeds are observed at SSW, followed by S, WNW and NW sectors. Lowest wind speeds are observed between N and E sectors. Most frequent wind directions are NW and NNW. Hence, wind turbines can be installed against these directions. The most powerful sector is NW (29.4 % of total expected wind energy), followed by 19.9 % SSW, 11.9% NNW, 8.6% WNW and 8.2% S. Furthermore in Al-Maqrun: the most powerful sector is W (26.8 % of total expected wind energy), followed by 12.3 % WSW and 9.5% WNW. While in Goterria: the most powerful sector is S (14.8 % of total expected wind energy), followed by SSE, SE, and WSW. And Misalatha: the most powerful sector is S, by far represents 28.5% of the expected power, followed by SSE and SE. As for Tarhuna, it is by far SSE and SE, representing each one two times the expected energy of the third powerful sector (NW). In Al-Asaaba: it is SSE by far represents 50% of the expected power, followed by S. It can to be noted that the high frequency of the south direction winds, that come from the desert could cause a high frequency of dust episodes. This fact then, should be taken into account in order to take appropriate measures to prevent wind turbine deterioration. In Excel workbook, an estimation of annual energy yield at position of Derna, Al-Maqrun, Tarhuna, and Al-Asaaba meteorological mast has been done, considering a generic wind turbine of 1.65 MW. (mtORRES, TWT 82-1.65MW) in position of meteorological mast. Three other turbines have been tested. At 80 m, the estimation of energy yield for Derna, Al-Maqrun, Tarhuna, and Asaaba is 6.78 GWh or 3390 equivalent hours, 5.80 GWh or 2900 equivalent hours, 4.91 GWh or 2454 equivalent hours and 5.08 GWh or 2541 equivalent hours respectively. It seems a fair value in the context of a possible development of a wind energy project in the areas, considering a value of 2400 equivalent hours as an approximate limit to consider a wind warm economically profitable. Furthermore, an estimation of annual energy yield at positions of Misalatha, Azizyah and Goterria meteorological mast has been done, considering a generic wind turbine of 2 MW. We found that, at 80 m, the estimation of energy yield is 3.12 GWh or 1557 equivalent hours, 4.47 GWh or 2235 equivalent hours and 4.07GWh or 2033 respectively . It seems a very poor value in the context of possible development of a wind energy project in the areas, considering a value of 2400 equivalent hours as an approximate limit to consider a wind warm economically profitable. Anyway, more data and a detailed wind farm study would be necessary to draw conclusions.

Keywords: wind turbines, wind data, energy yield, micrositting

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Authors: Reshu Saxena, R. K. Tripathi

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HIV-1 transmission and spread involves significant host-virus interaction. Potential targets for prevention of HIV-1 lies at the site of mucosal barriers. Thus a better understanding of how HIV-1 infects target cells at such sites and lead their invasion is required, with prime focus on the host determinants regulating HIV-1 spread. HIV-1 Nef is important for viral infectivity and pathogenicity. It promotes HIV-1 replication, facilitating immune evasion by interacting with various host factors and altering cellular pathways via multiple protein-protein interactions. In this study nef was sequenced from HIV-1 patients, and showed specific mutations revealing sequence variability in nef. To explore the difference in Nef functionality based on sequence variability we have studied the effects of HIV-1 Nef in human SupT1 T cell line and (THP-1) monocyte-macrophage cell lines through proteomics approach. 2D-Gel Electrophoresis in control and Nef-transfected SupT1 cells demonstrated several differentially expressed proteins with significant modulation of alpha-enolase. Through further studies, effects of Nef on alpha-enolase regulation were found to be cell lineage-specific, being stimulatory in macrophages/monocytes, inhibitory in T cells and without effect in HEK-293 cells. Cell migration and invasion studies were employed to determine biological function affected by Nef mediated regulation of alpha-enolase. Cell invasion was enhanced in THP-1 cells but was inhibited in SupT1 cells by wildtype nef. In addition, the modulation of enolase and cell invasion remained unaffected by a unique nef variant. These results indicated that regulation of alpha-enolase expression and invasive property of host cells by Nef is sequence specific, suggesting involvement of a particular motif of Nef. To precisely determine this site, we designed a heptapeptide including the suggested alpha-enolase regulating sequence of nef and a nef mutant with deletion of this site. Macrophages/monocytes being the major cells affected by HIV-1 at mucosal barriers, were particularly investigated by the nef mutant and peptide. Both the nef mutant and heptapeptide led to inhibition of enhanced enolase expression and increased invasiveness in THP-1 cells. Together, these findings suggest a possible mechanism of host invasion by HIV-1 through Nef mediated regulation of alpha-enolase and identifies a potential therapeutic target for HIV-1 entry at mucosal barriers.

Keywords: HIV-1 Nef, nef variants, host-virus interaction, tissue invasion

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Authors: Omar Youssef, Aija Knuuttila, Paivi Piirilä, Virinder Sarhadi, Sakari Knuutila

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Exhaled breath condensate (EBC) is a unique sample that allows studying different genetic changes in lung carcinoma through a non-invasive way. With the aid of next generation sequencing (NGS) technology, analysis of genetic mutations has been more efficient with increased sensitivity for detection of genetic variants. In order to investigate the possibility of applying this method for cancer diagnostics, mutations in EBC DNA from lung cancer patients and healthy individuals were studied by using NGS. The key aim is to assess the feasibility of using this approach to detect clinically important mutations in EBC. EBC was collected from 20 healthy individuals and 9 lung cancer patients (four lung adenocarcinomas, four 8 squamous cell carcinoma, and one case of mesothelioma). Mutations in hotpot regions of 22 genes were studied by using Ampliseq Colon and Lung cancer panel and sequenced on Ion PGM. Results demonstrated that all nine patients showed a total of 19 cosmic mutations in APC, BRAF, EGFR, ERBB4, FBXW7, FGFR1, KRAS, MAP2K1, NRAS, PIK3CA, PTEN, RET, SMAD4, and TP53. In controls, 15 individuals showed 35 cosmic mutations in BRAF, CTNNB1, DDR2, EGFR, ERBB2, FBXW7, FGFR3, KRAS, MET, NOTCH1, NRAS, PIK3CA, PTEN, SMAD4, and TP53. Additionally, 45 novel mutations not reported previously were also seen in patients’ samples, and 106 novel mutations were seen in controls’ specimens. KRAS exon 2 mutations G12D was identified in one control specimen with mutant allele fraction of 6.8%, while KRAS G13D mutation seen in one patient sample showed mutant allele fraction of 17%. These findings illustrate that hotspot mutations are present in DNA from EBC of both cancer patients and healthy controls. As some of the cosmic mutations were seen in controls too, no firm conclusion can be drawn on the clinical importance of cosmic mutations in patients. Mutations reported in controls could represent early neoplastic changes or normal homeostatic process of apoptosis occurring in lung tissue to get rid of mutant cells. At the same time, mutations detected in patients might represent a non-invasive easily accessible way for early cancer detection. Follow up of individuals with important cancer mutations is necessary to clarify the significance of these mutations in both healthy individuals and cancer patients.

Keywords: exhaled breath condensate, lung cancer, mutations, next generation sequencing

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Authors: Ying Han, Weirong Chen, Qi Li

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An energy management method based on equivalent hydrogen consumption minimum strategy is proposed in this paper aiming at the direct-current (DC) microgrid consisting of photovoltaic cells, fuel cells, energy storage devices, converters and DC loads. The rational allocation of fuel cells and battery devices is achieved by adopting equivalent minimum hydrogen consumption strategy with the full use of power generated by photovoltaic cells. Considering the balance of the battery’s state of charge (SOC), the optimal power of the battery under different SOC conditions is obtained and the reference output power of the fuel cell is calculated. And then a droop control method based on time-varying droop coefficient is proposed to realize the automatic charge and discharge control of the battery, balance the system power and maintain the bus voltage. The proposed control strategy is verified by RT-LAB hardware-in-the-loop simulation platform. The simulation results show that the designed control algorithm can realize the rational allocation of DC micro-grid energy and improve the stability of system.

Keywords: DC microgrid, equivalent minimum hydrogen consumption strategy, energy management, time-varying droop coefficient, droop control

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Authors: Chao Yang, Rosalind Bueckert, Jeff Schoenau, Axel Diederichsen, Hossein Zakeri, Tom Warkentin

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Pea is a very popular pulse crop that widely grew in Western Canadian prairie. However, the N fixation capabilities of these pea lines were not well evaluated under local environmental conditions. In this study, 2 supernodulating mutants Frisson P64 Sym29, Frisson P88 Sym28 along with their wild parent Frisson, 1 hypernodulating mutant Rondo-nod3 (fix+) along with its wild parent Rondo, 1 non-nodulating mutant Frisson P56 (nod-) and 2 commercial pea cultivar CDC Meadow and CDC Dakota which are widely planted in Western Canada were selected in order to evaluate the capabilities of their BNF, biomass, and yield production in symbiosis with R. leguminosarumbv. viciae, Our results showed different environmental conditions and variation of pea lines could both significantly impact days to flowering (DTF), days to podding (DTP), biomass and yield of tested pea lines (P < 0.0001), suggesting consideration of environmental factors could be important when selecting pea cultivar for local farming under different soil zones in Western Canada. Significant interaction effects between environmental conditions and pea lines were found on pea N fixation as well (P = 0.001), suggesting changes in N fixation capability of the same pea cultivar when grown under different environmental conditions. Our results provide useful information for farming and better opportunity for selection of pea cultivars with higher N-fixing capacity during breeding programs in Western Canada.

Keywords: Canadian prairie, environmental condition, N fixation, pea cultivar

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Authors: Sifi Ibrahim, Benaddou Fatima Zohra, Yousfi Mohamed

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The Marrubium deserti and Ammodaucus leucotrichus L. an Algerian endemic species, has several applications in traditional medicine for example as a remedy for asthma and diabetes, and was found to have antibacterial properties. In this work, an antioxidant and antimicrobial activities was performed on phenolic extracts of Marrubium deserti, Ammodaucus leucotrichus plants. The yield of methanol maceration of these plants is 12.4% and 20.4% respectively. The content of total polyphenols, flavonoids and anthocyanin in methanolic extracts, are varied between 19.52±1.88 and 59.24±3.45 mg/g gallic acid equivalent, and 2.08±0.29 to 1.46±0.39 mg/g quercetin equivalent, and 0.395 to 1.934µmol/g respectively. The total chlorophylls and carotenoids were be ranged from 0.149±0.20 to 1.537±0.20 g/ml and 1.537±0.20 to 0.149 ± 0.20 g/ml, respectively. According to DPPH and FRAP test, the values of EC50 was shows a higher activity of Marrubium deserti than Ammodaucus leucotrichus with EC50 values (DPPH) were 34.53±0.71 μg/mL and 258.60±15.67 mg/ml respectively. The TEAC values of FRAP test was a highly superior for Marrubium deserti 209.66±0.26 mg Equivalent Trolox/g dry residue than Ammodaucus leucotrichus 45.88±2.93 mg Trolox Equivalent/g dry residue. The antimicrobial activity against nine strains of bacteria (Staphylococcus aureus(+), Staphylococcus aureus (-), Bacillus cereus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi), was showed that the tested extracts are a significant antibacterial activity with inhibition zones ranging from 10 to 50 mm. the value of CMI were ranging from 0.89 to 14.29 mg/ml.

Keywords: phenolic extract, antioxidant activity, antimicrobial activity, Marrubium deserti, Ammodaucus leucotrichus

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Authors: W. Abu Rayyan, A. Singh, A. M. Al-Jaafreh, W. Abu Dayyih, M. Bustami, S. Salem, N. Seder, K. Schröppel

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Hyphal growth and the transcriptional regulation to the host environment are key issues during the pathogenesis of C. albicans. Tec1p is the C. albicans homolog of a TEA transcription factor family, which share a conserved DNA-binding TEA domain in their N-terminal. In order to define a structure-function relationship of the C. albicans Tec1p protein, we constructed several mutations on the N terminal, C terminal or in the TEA binding domain itself by homologous recombination technology. The modifications in the open reading frame of TEC1 were tested for reconstitution of the morphogenetic development of the tec1/tec1 mutant strain CaAS12. Mutation in the TEA consensus sequence did not confer transition to hyphae whereas the reconstitution of the full-length Tec1p has reconstituted hyphal development. A deletion in one of glutamine-rich regions either in the Tec1p N-terminal or the C-terminal in regions of 53-212 or 637–744 aa, respectively, did not restore morphological development in mutant CaAS12 strain. Whereas, the reconstitution with Tec1p mutants other than the glutamate-rich region has restored the morphogenetic switch. Additionally, the deletion of the glutamine-rich region has attenuated the invasive growth and the heat shock resistance of C. albicans. In conclusion, we show that a glutamine-rich region of Tec1p is essential for the hyphal development and mediating adaptation to the host environment of C. albicans.

Keywords: Candida albicans, morphogenetic development, TEA domain, hyphal formation, TEC1

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Authors: B. G. Muhammad, S. M. Jafar

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Neutron Activation Analysis (NAA) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) were employed to analyze the level of trace elements concentrations in sediment samples and their bioaccumulation in some aquatic species selected randomly from surface water resources in the Northern peninsula of Malaysia. The NAA results of the sediment samples indicated a wide range in concentration of different elements were observed. Fe, K, and Na were found to have major concentration values that ranges between 61,000 ± 1400 to 4,500 ± 100 ppm, 20100±1000 to 3100±600 and 3,100±600 and 200±10 ppm, respectively. Traces of heavy metals with much more contamination health concern, such as Cr and As, were also identified in many of the samples analyzed. The average specific activities of 40K, 232Th and 226Ra in soil and the corresponding radium equivalent activity and the external hazard index were all found to be lower than the maximum permissible limits (370 Bq kg-1 and 1).

Keywords: external hazard index, Neutron Activation Analysis, radium equivalent, trace elements concentrations

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Authors: Hamidreza Bayat, Arash Mirabdolah Lavasani, Meysam Bolhasani, Sajad Moosavi

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Flow around a flat tube is studied numerically. Reynolds number is defined base on equivalent circular tube and it is varied in range of 100 to 300. Equations are solved by using finite volume method and results are presented in form of drag and lift coefficient. Results show that drag coefficient of flat tube is up to 66% lower than circular tube with equivalent diameter. In addition, by increasing l/D from 1 to 2, the drag coefficient of flat tube is decreased about 14-27%.

Keywords: laminar flow, flat-tube, drag coefficient, cross-flow, heat exchanger

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Authors: Soujanya Pasumarthi

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Inverse docking is a relatively new technique that has been used to identify potential receptor targets of small molecules. Our docking software package MDock is well suited for such an application as it is both computationally efficient, yet simultaneously shows adequate results in binding affinity predictions and enrichment tests. As a validation study, we present the first stage results of an inverse-docking study which seeks to identify potential direct targets of PRIMA-1. PRIMA-1 is well known for its ability to restore mutant p53's tumor suppressor function, leading to apoptosis in several types of cancer cells. For this reason, we believe that potential direct targets of PRIMA-1 identified in silico should be experimentally screened for their ability to inhibitcancer cell growth. The highest-ranked human protein of our PRIMA-1 docking results is oxidosqualene cyclase (OSC), which is part of the cholesterol synthetic pathway. The results of two followup experiments which treat OSC as a possible anti-cancer target are promising. We show that both PRIMA-1 and Ro 48-8071, a known potent OSC inhibitor, significantly reduce theviability of BT-474 breast cancer cells relative to normal mammary cells. In addition, like PRIMA-1, we find that Ro 48-8071 results in increased binding of mutant p53 to DNA in BT- 474cells (which highly express p53). For the first time, Ro 48-8071 is shown as a potent agent in killing human breast cancer cells. The potential of OSC as a new target for developing anticancer therapies is worth further investigation.

Keywords: inverse docking, in silico screening, protein-ligand interactions, molecular docking

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Authors: Thi Thao Ninh, Yuri Trusov, José Ramón Botella

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Heterotrimeric G proteins, comprised of three subunits, α, β and γ, are involved in signal transduction pathways that mediate a vast number of processes across the eukaryotic kingdom. 23 Gα subunits are present in humans whereas most plant genomes encode for only one canonical Gα. The disparity observed between Arabidopsis, rice, and maize Gα-deficient mutant phenotypes suggest that Gα functions have diversified between eudicots and monocots during evolution. Alternatively, since the only Gα mutations available in dicots have been produced in Arabidopsis, the possibility exists that this species might be an exception to the rule. In order to test this hypothesis, we studied the G protein α subunit (TGA1) in tomato. Four tga1 knockout lines were generated in tomato cultivar Moneymaker using CRISPR/Cas9. The tga1 mutants exhibit a number of auxin-related phenotypes including changes in leaf shape, reduced plant height, fruit size and number of seeds per fruit. In addition, tga1 mutants have increased sensitivity to abscisic acid during seed germination, reduced sensitivity to exogenous auxin during adventitious root formation from cotyledons and excised hypocotyl explants. Our results suggest that Gα mutant phenotypes in tomato are very similar to those observed in monocots, i.e. rice and maize, and cast doubts about the validity of using Arabidopsis as a model system for plant G protein studies.

Keywords: auxin-related phenotypes, CRISPR/Cas9, G protein α subunit, heterotrimeric G proteins, tomato

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Authors: Kachout Mnaouer, Bel Hadj Tahar Jamel, Choubani Fethi

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In this paper, three electric equivalent models to evaluate crosstalk between three-conductor transmission lines are proposed. First, electric equivalent models for three-conductor transmission lines are presented. Secondly, rigorous equations to calculate the per-unit length inductive and capacitive parameters are developed. These models allow us to calculate crosstalk between conductors. Finally, to validate the presented models, we compare the theoretical results with simulation data. Obtained results show that proposed models can be used to predict crosstalk performance.

Keywords: near-end crosstalk, inductive parameter, L, Π, T models

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Authors: Sarah Devi Silvian, Hanna Shobrina Iqomatul Haq, Fara Cholidatun Nabila, Agustin Krisna Wardani

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Bacteria ability to survive in antibiotic is controlled by the presence of gene that encodes the antibiotic resistance protein. The instability of the antibiotic resistance gene can be observed by exposing the bacteria under the lethal dose of antibiotic. Low concentration of antibiotic can induce mutation, which may take a role in bacterial adaptation through the antibiotic concentration. Lactobacillus casei FNCC 0090 is one of the probiotic bacteria that has an ability to survive in tetracycline by expressing the tetM gene. The aims of this study are to observe the possibilities of mutation happened in L.casei FNCC 0090 by exposing in sub-lethal dose of tetracycline and also observing the instability of the tetM gene by comparing the sequence between the wild type and mutant. L.casei FNCC 0090 has a lethal dose in 60 µg/ml, low concentration is applied to induce the mutation, the range from 10 µg/ml, 15 µg/ml, 30 µg/ml, 45 µg/ml, and 50 µg/ml. L.casei FNCC 0090 is exposed to the low concentration from lowest to the highest concentration to induce the adaptation. Plasmid is isolated from the highest concentration culture which is 50 µg/ml by using modified alkali lysis method with the addition of lysozyme. The tetM gene is isolated by using PCR (Polymerase Chain Reaction) method, then PCR amplicon is purified and sequenced. Sequencing is done on both samples, wild type and mutant. Both sequences are compared and the mutations can be traced in the presence of nucleotides changes. The changing of the nucleotides means that the tetM gene is instable.

Keywords: L. casei FNCC 0090, probiotic, tetM, tetracycline

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Authors: D. Shehu, Z. Alias

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Glutathione S-transferases (GSTs) are family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. The gene for KKSG9 was cloned, purified and biochemically characterized. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide (CuOOH). The enzyme also displayed dehalogenation function against dichloroacetate (a common substrate for zeta class GSTs) in addition to permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.

Keywords: Acidovorax sp. KKS102, bioremediation, glutathione s-transferase, site-directed mutagenesis, zeta

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Authors: Gajanan M. Sonwane

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Nowadays, the entire pharmaceutical industry is facing the challenge of increasing efficiency and innovation. The major hurdles are the growing cost of research and development and a concurrent stagnating number of new chemical entities (NCEs). Hence, the challenge is to select the most druggable targets and to search the equivalent drug-like compounds, which also possess specific pharmacokinetic and toxicological properties that allow them to be developed as drugs. The present research work includes the studies of developing new anticancer heterocycles by using molecular modeling techniques. The heterocycles synthesized through such methodology are much effective as various physicochemical parameters have been already studied and the structure has been optimized for its best fit in the receptor. Hence, on the basis of the literature survey and considering the need to develop newer anticancer agents, new phenazinamine derivatives were designed by subjecting the nucleus to molecular modeling, viz., GQSAR analysis and docking studies. Simultaneously, these designed derivatives were subjected to in silico prediction of biological activity through PASS studies and then in silico toxicity risk assessment studies. In PASS studies, it was found that all the derivatives exhibited a good spectrum of biological activities confirming its anticancer potential. The toxicity risk assessment studies revealed that all the derivatives obey Lipinski’s rule. Amongst these series, compounds 4c, 5b and 6c were found to possess logP and drug-likeness values comparable with the standard Imatinib (used for anticancer activity studies) and also with the standard drug methotrexate (used for antimitotic activity studies). One of the most notable mutations is the threonine to isoleucine mutation at codon 315 (T315I), which is known to be resistant to all currently available TKI. Enzyme assay planned for confirmation of target selective activity.

Keywords: drug design, tyrosine kinases, anticancer, Phenazinamine

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Authors: Jae Bok Heo, Yun Mi Lee, Hee Rang Yun

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Several GTPases are required for ribosome biogenesis and assembly. We recently characterized rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), belonging to the YlqF/YawG family of GTPases, as playing a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating the function of OsNug2, yeast two-hybrid screens were carried out using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a potential interacting protein candidate. In vitro pull down and bimolecular fluorescence complementation assays confirmed the interaction between OsNug2 and OsSTK1, and like green fluorescent protein-tagged OsNug2, green fluorescent protein-tagged OsSTK1 was targeted to the nucleus of Arabidopsis protoplasts. OsSTK1 was not found to affect the GTP-binding activity of OsNug2; however, when recombinant OsSTK1 was included in OsNug2 assay reaction mixtures, OsSTK1 increased the GTPase activity of OsNug2. To test whether OsSTK1 phosphorylates OsNug2 in vitro, a kinase assay was performed. OsSTK1 was found to have weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Yeast complementation testing resulted in a GAL::OsNug2(S209N) mutant-harboring yeast strain exhibiting a growth-defective phenotype on galactose medium at 39°C, divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of mutant OsNug2(S209N) was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings reported here indicate that OsSTK1 functions as a positive regulator protein of OsNug2 by enhancing the GTPase activity of OsNug2, and that the phosphorylation of serine 209 of OsNug2 is essential for the complete function of OsNug2 in ribosome biogenesis.

Keywords: OsSTK1, OsNug2, GTPase activity, GTP binding activity, phosphorylation

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Authors: S. Kamil Akin, Turgut Acikara

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In recent years the world and our country has experienced several explosion events occurred due to terrorist attacks and accidents. In these explosion events many people have lost their lives and many buildings have been damaged. If structures were designed taking the blast loads into account, these results may not have happened or the casualties would have been less. In this thesis analysis of the protection walls have been conducted to prevent the building damage from blast loads. These analyzes was carried out for two different types of wall, concrete and reinforced concrete. Analyses were carried out on four different thicknesses of each wall element. In each wall element the stresses and displacements of the exposed surface due to the detonation charge has been calculated. The limit shear stress and displacement of the wall element according to their material properties has been taken into account. As the result of the analyses the standoff distances and TNT equivalent amount has been determined. According to equivalent TNT amounts and standoff distances the structural response of the protective wall elements has been observed. These structural responses have been observed by ABAQUS finite element package. Explosion loads were brought into effect to the protective wall element models by using the ABAQUS / CONWEP.

Keywords: blast loading, blast wave, TNT equivalent method, CONWEP, finite element analysis, detonation

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Authors: Lidiia Nazarenko, Henryk Stolarski, Holm Altenbach

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In the paper, a (hierarchical) approach to analysis of thermo-elastic properties of random composites with interphases is outlined and illustrated. It is based on the statistical homogenization method – the method of conditional moments – combined with recently introduced notion of the energy-equivalent inhomogeneity which, in this paper, is extended to include thermal effects. After exposition of the general principles, the approach is applied in the investigation of the effective thermo-elastic properties of a material with randomly distributed nanoparticles. The basic idea of equivalent inhomogeneity is to replace the inhomogeneity and the surrounding it interphase by a single equivalent inhomogeneity of constant stiffness tensor and coefficient of thermal expansion, combining thermal and elastic properties of both. The equivalent inhomogeneity is then perfectly bonded to the matrix which allows to analyze composites with interphases using techniques devised for problems without interphases. From the mechanical viewpoint, definition of the equivalent inhomogeneity is based on Hill’s energy equivalence principle, applied to the problem consisting only of the original inhomogeneity and its interphase. It is more general than the definitions proposed in the past in that, conceptually and practically, it allows to consider inhomogeneities of various shapes and various models of interphases. This is illustrated considering spherical particles with two models of interphases, Gurtin-Murdoch material surface model and spring layer model. The resulting equivalent inhomogeneities are subsequently used to determine effective thermo-elastic properties of randomly distributed particulate composites. The effective stiffness tensor and coefficient of thermal extension of the material with so defined equivalent inhomogeneities are determined by the method of conditional moments. Closed-form expressions for the effective thermo-elastic parameters of a composite consisting of a matrix and randomly distributed spherical inhomogeneities are derived for the bulk and the shear moduli as well as for the coefficient of thermal expansion. Dependence of the effective parameters on the interphase properties is included in the resulting expressions, exhibiting analytically the nature of the size-effects in nanomaterials. As a numerical example, the epoxy matrix with randomly distributed spherical glass particles is investigated. The dependence of the effective bulk and shear moduli, as well as of the effective thermal expansion coefficient on the particle volume fraction (for different radii of nanoparticles) and on the radius of nanoparticle (for fixed volume fraction of nanoparticles) for different interphase models are compared to and discussed in the context of other theoretical predictions. Possible applications of the proposed approach to short-fiber composites with various types of interphases are discussed.

Keywords: effective properties, energy equivalence, Gurtin-Murdoch surface model, interphase, random composites, spherical equivalent inhomogeneity, spring layer model

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Authors: Mehdi Ghatei, Masoomeh Lorestani

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Upstream Tailings Storage Facilities (TSFs) may experience slope instabilities due to soil liquefaction, especially in regions known to be seismically active. In this study, liquefaction susceptibility of an upstream-raised TSF in Western Australia was assessed using two different approaches. The first approach assessed liquefaction susceptibility using Cone Penetration Tests with pore pressure measurement (CPTu) as described by the National Centre for Earthquake Engineering Research (NCEER). This assessment was based on the four CPTu tests that were conducted on the perimeter embankment of the TSF. The second approach used the Finite Element (FE) method with application of an equivalent linear model to predict the undrained cyclic behavior, the pore water pressure and the liquefaction of the materials. The tailings parameters were estimated from the CPTu profiles and from the laboratory tests. The cyclic parameters were estimated from the literature where test results of similar material were available. The results showed that there was a good agreement, in the liquefaction susceptibility of the tailings material, between the NCEER and FE methods with equivalent linear model.

Keywords: liquefaction , CPTU, NCEER, finite element method, equivalent linear model

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Authors: Hyun-Woo Jun, Tae-Chul Jeong, Huai-Cong Liu, Ju Lee

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The paper presents the electromagnetic-thermal flow coupled analysis of permanent magnet synchronous motor (PMSM) which has cooling channel in stator core for forced air cooling. Unlike the general PMSM design, to achieve ohmic loss reduction for high efficiency, cooling channel actively used in the stator core. Equivalent thermal network model was made to analyze the effect of the formation of the additional flow path in the core. According to the shape and position changing of the channel design, electromagnetic-thermal coupled analysis results were reviewed.

Keywords: coupled problems, electric motors, equivalent circuits, fluid flow, thermal analysis

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Authors: Michal Kielbik, Izabela Szulc-Kielbik, Anna Brzostek, Jaroslaw Dziadek, Magdalena Klink

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The goal of many research groups in the world is to find new components that are important for survival of mycobacteria in the host cells. Mycobacterium tuberculosis (Mtb) possesses a number of enzymes degrading cholesterol that are considered to be an important factor for its survival and persistence in host macrophages. One of them - cholesterol oxidase (ChoD), although not being essential for cholesterol degradation, is discussed as a virulence compound, however its involvement in macrophages’ response to Mtb is still not sufficiently determined. The recognition of tubercle bacilli antigens by pathogen recognition receptors is crucial for the initiation of the host innate immune response. An important receptor that has been implicated in the recognition and/or uptake of Mtb is Toll-like receptor type 2 (TLR2). Engagement of TLR2 results in the activation and phosphorylation of intracellular signaling proteins including IRAK-1 and -4, TRAF-6, which in turn leads to the activation of target kinases and transcription factors responsible for bactericidal and pro-inflammatory response of macrophages. The aim of these studies was a detailed clarification of the role of Mtb cholesterol oxidase as a virulence factor affecting the TLR2 signaling pathway in human macrophages. As human macrophages the THP-1 differentiated cells were applied. The virulent wild-type Mtb strain (H37Rv), its mutant lacking a functional copy of gene encoding cholesterol oxidase (∆choD), as well as complimented strain (∆choD–choD) were used. We tested the impact of Mtb strains on the expression of TLR2-depended signaling proteins (mRNA level, cytosolic level and phosphorylation status). The cytokine and bactericidal response of THP-1 derived macrophages infected with Mtb strains in relation to TLR2 signaling pathway dependence was also determined. We found that during the 24-hours of infection process the wild-type and complemented Mtb significantly reduced the cytosolic level and phosphorylation status of IRAK-4 and TRAF-6 proteins in macrophages, that was not observed in the case of ΔchoD mutant. Decreasement of TLR2-dependent signaling proteins, induced by wild-type Mtb, was not dependent on the activity of proteasome. Blocking of TLR2 expression, before infection, effectively prevented the induced by wild-type strain reduction of cytosolic level and phosphorylation of IRAK-4. None of the strains affected the surface expression of TLR2. The mRNA level of IRAK-4 and TRAF-6 genes were significantly increased in macrophages 24 hours post-infection with either of tested strains. However, the impact of wild-type Mtb strain on both examined genes was significantly stronger than its ΔchoD mutant. We also found that wild-type strain stimulated macrophages to release high amount of immunosuppressive IL-10, accompanied by low amount of pro-inflammatory IL-8 and bactericidal nitric oxide in comparison to mutant lacking cholesterol oxidase. The influence of wild-type Mtb on this type of macrophages' response strongly dependent on fully active IRAK-1 and IRAK-4 signaling proteins. In conclusion, Mtb using cholesterol oxidase causes the over-activation of TLR2 signaling proteins leading to the reduction of their cytosolic level and activity resulting in the modulation of macrophages response to allow its intracellular survival. Supported by grant: 2014/15/B/NZ6/01565, National Science Center, Poland

Keywords: Mycobacterium tuberculosis, cholesterol oxidase, macrophages, TLR2-dependent signaling pathway

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Authors: Priya M. V., Thakar Singh

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A field experiment was carried out during the rabi season of 2017 and 2018 to evaluate the productivity and economic viability of bed-planted chickpea-based intercropping systems. The experiment was laid out in a randomized block design consisting of four replications with thirteen treatments. Results showed that sole chickpea recorded the highest seed yield, and it was statistically at par with seed yield obtained under chickpea + oats fodder (2:1), chickpea + oats fodder (4:1), and chickpea + linseed (4:1) intercropping systems. However, oilseed rape and barley as intercrops showed an adverse effect on yield and yield attributes of chickpea. Chickpea + oats fodder in 2:1 row ratio recorded the highest chickpea equivalent yield of 24.07 and 24.77 q/ha during 2017 and 2018, respectively. Higher net returns (Rs. 63098 and 70924/ha) and benefit-cost ratio (1.47 and 1.63) were also recorded in chickpea + oats fodder (2:1) intercropping system over sole chickpea (Rs. 44862 and 53769/ha and 1.21 and 1.41) during both the years. Chickpea + oats fodder (4:1), chickpea + linseed (2:1), and chickpea + linseed (4:1) also recorded significantly higher chickpea equivalent yield, net returns, and benefit-cost ratio as compared to sole chickpea.

Keywords: bed planted chickpea, chickpea equivalent yield, economic returns, intercropping system, productivity

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Authors: G. Ultanbekova, Zh. Suleimenova, Zh. Rakhmetova, G. Mombekova, S. Mantieva

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Plant Growth Promoting Rhizobacteria (PGPR) are a group of free-living bacteria that colonize the rhizosphere, enhance plant growth of many cereals and other important agricultural crops and protect plants from disease and abiotic stresses through a wide variety of mechanisms. The use of PGPR has been proven to be an environmentally sound way of increasing crop yields by facilitating plant growth. In the present study, strain improvement of PGPR isolates were carried out by chemical mutagenesis for the improvement of growth and yield of lima bean. Induced mutagenesis is widely used for the selection of microorganisms producing biologically active substances and further improving their activities. Strain improvement is usually done by classical mutagenesis which involves exposing the microbes to chemical or physical mutagens. The strains of Pseudomonas putida 4/1, Azotobacter chroococcum Р-29 and Bacillus subtilis were subjected to mutation process for strain improvement by treatment with a chemical agent (sodium nitrite) to cause mutation and were observed for its consequent action on the seeds germination and plant growth of lima bean (Phaseolus lunatus). Bacterial mutant strains of Pseudomonas putida M-1, Azotobacter chroococcum M-1 and Bacillus subtilis M-1, treated with sodium nitrite in the concentration of 5 mg/ml for 120 min, were found effective to enhance the germination of lima bean seeds compared to parent strains. Moreover, treatment of the lima bean seeds with a mutant strain of Bacillus subtilis M-1 had a significant stimulation effect on plant growth. The length of the stems and roots of lima bean treated with Bacillus subtilis M-1 increased significantly in comparison with parent strain in 1.6 and 1.3 times, respectively.

Keywords: chemical mutagenesis, germination, kidney bean, plant growth promoting rhizobacteria (PGPR)

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Authors: Masoud Rafiee

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Study of the effect of vermin compost on yield, and Land equivalent ration (LER) of chickpea-barley mixed cropping under normal dry land condition can be useful in order to increase qualitative and quantitative performance. In this case, two factors include fertilizer (vermicompost biological fertilizer, ammonium phosphate chemical fertilizer, vermicompost + %75 chemical fertilizer) and chickpea + barley mixed cropping (sole chickpea, %75 chickpea: %25 barley, %50 chickpea: %50 barley, %25 chickpea: %75 barley, and sole barley) in RCBD in three replications in two experiments include normal and dry land conditions were studied. Result showed that total LER base on dry matter was affected by environment and mixed cropping interaction and was more than 1 in all mixed cropping treatments. In different mixed cropping rates, wet forage yield decreased by decreasing chickpea ratio as well as increasing barley ratio. Total LER mean in base on forage dry matter in mixed-, chemical-, and vermicompost fertilizer treatments were 1.12, 1.05 and 1.10 in normal condition and 1.15, 1.08 and 1.14 in dry land condition, respectively, represented the important of biological fertilizer in mixed cropping systems.

Keywords: land equivalent ration, biological fertilizer, mixed cropping systems, water stress

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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: S. A. Anuar, N. H. Hamid, M. H. Hashim, S. M. D. Salleh

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This paper present the experimental work on the seismic performance of double unit tunnel form building (TFB) subjected to in-plane lateral cyclic loading. A one third scale of 3-storey double unit of TFB is tested at ±0.01%, ±0.1%, ±0.25%, ±0.5%, ±0.75% and ±1.0% drifts until the structure achieves its strength degradation. After that, the TFB is repaired and retrofitted using additional shear wall, steel angle and CFRP sheet. A similar testing approach is applied to the specimen after repair and retrofit. The crack patterns, lateral strength, stiffness, ductility and equivalent viscous damping (EVD) were analyzed and compared before and after repair and retrofit. The result indicates that the lateral strength increases by 22 in pushing direction and 27% in pulling direction. Moreover, the stiffness and ductility obtained before and after retrofit increase tremendously by 87.87% and 39.66%, respectively. Meanwhile, the energy absorption measured by equivalent viscous damping obtained after retrofit increase by 12.34% in pulling direction. It can be concluded that the proposed retrofit method is capable to increase the lateral strength capacity, stiffness and energy absorption of double unit TFB.

Keywords: tunnel form building, in-plane lateral cyclic loading, crack pattern, lateral strength, stiffness, ductility, equivalent viscous damping, repair and retrofit

Procedia PDF Downloads 331