Search results for: assay parameters

Authors: Taru Singh, Shukla Das, V. G. Ramachandran

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Objectives: To develop SYBR green based real-time PCR assay to detect carbapenemases (NDM, IMP) genes in E. coli. Methods: A total of 40 E. coli from stool samples were tested. Six were previously characterized as resistant to carbapenems and documented by PCR. The remaining 34 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial RNA was extracted using manual method. The real-time PCR was performed using the Light Cycler III 480 instrument (Roche) and specific primers for each carbapenemase target were used. Results: Each one of the two carbapenemase gene tested presented a different melting curve after PCR amplification. The melting temperature (Tm) analysis of the amplicons identified was as follows: blaIMP type (Tm 82.18°C), blaNDM-1 (Tm 78.8°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. Conclusions: The new assay was able to detect the presence of two different carbapenemase gene type by real-time PCR.

Keywords: resistance, b-lactamases, E. coli, real-time PCR

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Authors: Suphaket Saenthaweesuk, Nutiya Somparn, Atcharaporn Thewmore

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The present study aims to investigate the effects of Cymbopogon citratus, Stapf (CS) or lemon grass ethanol extract on antioxidant and vascular disorders parameters in rat. The CS ethanol extract was screened for its phytochemical contents and antioxidant activity in vitro. Moreover, the extract was studied in rats to evaluate its effects in vivo. Rats were orally administered with CS at 1,000 mg/kg/day for 30 days. Phytochemical screening of CS extract indicated the presence of tannins, flavonoids and phenolic compounds. The extract contained phenolic compounds 1,400.10 ± 0.47 mg of gallic acid equivalents per gram CS extract. The free radical scavenging activity assessed by DPPH assay gave IC50 of 168.77 ± 3.32µg/mL, which is relatively lower than that of BHT with IC50 of 12.34 ± 1.14 µg/mL. In the animals, the protein expression of antioxidant enzymes, γ-glutamylcysteine ligase (γ-GCL) in liver was significantly increased. This was consistent with elevation of serum catalase (CAT) and superoxide dismutase (SOD) activities. However, Protein expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM-1) and endothelial nitric oxide synthase (eNOS) in heart and aorta were not differenced from normal control. Taken together, the present study provides evidence that CCS water extract exhibits direct antioxidant properties and can induce cytoprotective enzymes in vivo.

Keywords: antioxidant, Cymbopogon citratus Stapf, VCAM-1, γ-glutamylcysteine ligase

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Authors: Jumazulhisham Abdul Shukor, Mohd. Sazali Said, Roshanizah Harun, Shuib Husin, Ahmad Razlee Ab Kadir

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This paper applies Taguchi Optimization Method in determining the best machining parameters for pocket milling process on Polypropylene (PP) using CNC milling machine where the surface roughness is considered and the Carbide inserts cutting tool are used. Three machining parameters; speed, feed rate and depth of cut are investigated along three levels; low, medium and high of each parameter (Taguchi Orthogonal Arrays). The setting of machining parameters were determined by using Taguchi Method and the Signal-to-Noise (S/N) ratio are assessed to define the optimal levels and to predict the effect of surface roughness with assigned parameters based on L9. The final experimental outcomes are presented to prove the optimization parameters recommended by manufacturer are accurate.

Keywords: inserts, milling process, signal-to-noise (S/N) ratio, surface roughness, Taguchi Optimization Method

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Authors: Mei Han, Jinkou Zhao, Yuan Yao, Liangui Feng, Xianbin Ding, Guohui Wu, Chao Zhou, Lin Ouyang, Rongrong Lu, Bo Zhang

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To compare the HIV incidence estimated using BED capture enzyme linked immunosorbent assay (BED-CEIA) and observed in a cohort against the HIV incidence among men who have sex with men (MSM) measured by pooling polymerase chain reaction (pooling-PCR). A total of 617 MSM subjects were included in a respondent driven sampling survey in Chongqing in 2008. Among the 129 that were tested HIV antibody positive, 102 were defined with long-term infection, 27 were assessed for recent HIV infection (RHI) using BED-CEIA. The remaining 488 HIV negative subjects were enrolled to the prospective cohort and followed-up every 6 months to monitor HIV seroconversion. All of the 488 HIV negative specimens were assessed for acute HIV infection (AHI) using pooling-PCR. Among the 488 negative subjects in the open cohort, 214 (43.9%) were followed-up for six months, with 107 person-years of observation and 14 subjects seroconverted. The observed HIV incidence was 12.5 per 100 person-years (95% CI=9.1-15.7). Among the 488 HIV negative specimens, 5 were identified with acute HIV infection using pooling-PCR at an annual rate of 14.02% (95% CI=1.73-26.30). The estimated HIV-1 incidence was 12.02% (95% CI=7.49-16.56) based on BED-CEIA. The HIV incidence estimated with three different approaches was different among subgroups. In the highly HIV prevalent MSM, it costs US$ 1724 to detect one AHI case, while detection of one case of RHI with BED assay costs only US$ 42. Three approaches generated comparable and high HIV incidences, pooling PCR and prospective cohort are more close to the true level of incidence, while BED-CEIA seemed to be the most convenient and economical approach for at-risk population’s HIV incidence evaluation at the beginning of HIV pandemic. HIV-1 incidences were alarmingly high among MSM population in Chongqing, particularly within the subgroup under 25 years of age and those migrants aged between 25 to 34 years.

Keywords: BED-CEIA, HIV, incidence, pooled PCR, prospective cohort

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Authors: Soledad Carinelli, Iñigo Fernández, José Luis González-Mora, Pedro A. Salazar-Carballo

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Mastitis is the most relevant inflammatory disease in cattle, affecting the animal health and causing important economic losses on dairy farms. This disease takes place in the mammary gland or udder when some opportunistic microorganisms, such as Staphylococcus aureus, Streptococcus agalactiae, Corynebacterium bovis, etc., invade the teat canal. According to the severity of the inflammation, mastitis can be classified as sub-clinical, clinical and chronic. Standard methods for mastitis detection include counts of somatic cells, cell culture, electrical conductivity of the milk, and California test (evaluation of “gel-like” matrix consistency after cell lysed with detergents). However, these assays present some limitations for accurate detection of subclinical mastitis. Currently, haptoglobin, an acute phase protein, has been proposed as novel and effective biomarker for mastitis detection. In this work, an electrochemical biosensor based on polydopamine-modified magnetic nanoparticles (MNPs@pDA) for haptoglobin detection is reported. Thus, MNPs@pDA has been synthesized by our group and functionalized with hemoglobin due to its high affinity to haptoglobin protein. The protein was labeled with specific antibodies modified with alkaline phosphatase enzyme for its electrochemical detection using an electroactive substrate (1-naphthyl phosphate) by differential pulse voltammetry. After the optimization of assay parameters, the haptoglobin determination was evaluated in milk. The strategy presented in this work shows a wide range of detection, achieving a limit of detection of 43 ng/mL. The accuracy of the strategy was determined by recovery assays, being of 84 and 94.5% for two Hp levels around the cut off value. Milk real samples were tested and the prediction capacity of the electrochemical biosensor was compared with a Haptoglobin commercial ELISA kit. The performance of the assay has demonstrated this strategy is an excellent and real alternative as screen method for sub-clinical bovine mastitis detection.

Keywords: bovine mastitis, haptoglobin, electrochemistry, magnetic nanoparticles, polydopamine

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Authors: N. Hoosain, S. Nene, B. Pearce, C. Jacobs, M. Du Plessis, M. Benjeddou

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Organic Cation Transporters play a vital role in the absorption, tissue distribution and elimination of various substrates. Numerous studies have suggested that variations in non-synonymous single nucleotide polymorphisms (SNPs) of SLC22A2 could influence an individual’s response to various treatments, including clinically important drugs. This study is the first to determine the baseline frequency distribution for twenty SNPs of SLC22A2in the Zulu population. DNA was collected from 101 unrelated “healthy” Zulu participants. Genotypes of all samples were determined using a multiplex PCR and SNaPshot assay followed by the generation of the haplotype structure. This is the first time that the baseline frequency distribution of SNPs is reported for the Zulu population. Data from this study could be used in in vitro and in vivo pharmacogenetic and pharmacokinetic studies to evaluate the potential role the studied SNPs play in the therapeutic efficacy of clinically important drugs.

Keywords: SLC22A2 gene, SNaPshot assay, PCR, Zulu population

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Authors: Huseyin Apdik, Aysegul Dogan, Selami Demirci, Ezgi Avsar Apdik, Fikrettin Sahin

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Mesenchymal stem cells (MSCs) are crucial cell types for bone maintenance and growth along with resident bone progenitor cells providing bone tissue integrity during osteogenesis and skeletal growth. Any deficiency in this regulation would result in vital bone diseases. Of those, osteoporosis, characterized by a reduction in bone mass and mineral density, is a critical skeletal disease for especially elderly people. The commonly used drugs for the osteoporosis treatment are bisphosphonates (BPs). The most prominent role of BPs is to prevent bone resorption arisen from high osteoclast activity. However, administrations of bisphosphonates may also cause bisphosphonate-induced osteonecrosis of the jaw (BIONJ). Up to the present, the researchers have proposed several circumstances for BIONJ. However, effects of long-term and/or high dose usage of BPs on stem cell’s proliferation, survival, differentiation or maintenance capacity have not been evaluated yet. The present study will be held to; figure out BPs’ effects on MSCs in vitro in the aspect of cell proliferation and toxicity, migration, angiogenic activity, lineage specific gene and protein expression levels, mesenchymal stem cell properties and potential signaling pathways affected by BP treatment. Firstly, mesenchymal stem cell characteristics of Dental Pulp Stem Cells (DPSCs) and Periodontal Ligament Stem Cells (PDLSCs) were proved using flow cytometry analysis. Cell viability analysis was completed to determine the cytotoxic effects of BPs (Zoledronate (Zol), Alendronate (Ale) and Risedronate (Ris)) on DPSCs and PDLSCs by the 3-(4,5-di-methyl-thiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) assay. Non-toxic concentrations of BPs were determined at 24 h under growth condition, and at 21 days under osteogenic differentiation condition for both cells. The scratch assay was performed to evaluate their migration capacity under the usage of determined of BPs concentrations at 24 h. The results revealed that while the scratch closure is 70% in the control group for DPSCs, it was 57%, 66% and 66% in Zol, Ale and Ris groups, respectively. For PDLSs, while wound closure is 71% in control group, it was 65%, 66% and 66% in Zol, Ale and Ris groups, respectively. As future experiments, tube formation assay and aortic ring assay will be done to determinate angiogenesis abilities of DPSCs and PDLSCs treated with BPs. Expression levels of osteogenic differentiation marker genes involved in bone development will be determined using real time-polymerase change reaction (RT-PCR) assay and expression profiles of important proteins involved in osteogenesis will be evaluated using western blotting assay for osteogenically differentiated MSCs treated with or without BPs. In addition to these, von Kossa staining will be performed to measure calcium mineralization status of MSCs.

Keywords: bisphosphonates, bisphosphonate-induced osteonecrosis of the jaw, mesenchymal stem cells, osteogenesis

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Authors: Nassim Belkacem, Amina Azzam, Dalila Haouchine, Kahina Bennacer, Samira Soufit

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Objective: To evaluate the antioxidant activity of the methanolic extract along with the antimicrobial activity of the essential oil of the aerial parts of Rosmarinus officinalis L. collected in the region of Bejaia (northern center of Algeria). Materials and methods: The polyphenols and flavonoids contents of the methanolic extract were measured. The antioxidant activity was evaluated using two methods: the ABTS method and DPPH assay. The antimicrobial activity was studied by the agar diffusion method against five bacterial strains (Three Gram positive strains and two Gram negative strains) and one fungus. Results: The total polyphenol and flavonoid content was about 43.8 mg gallic acid equivalent per gram (GA Eq/g) and 7.04 mg quercetin equivalent per gram (Q Eq/g), respectively. In the ABTS assay, the rosemary extract has shown an inhibition of 98.02% at the concentration of 500ug/ml with a half maximal inhibitory concentration value (IC50) of 194.92ug/ml. The results of DPPH assay have shown that the rosemary extract has an inhibition of 94.67 % with an IC50 value of 17.87ug/ml, which is lower than that of Butylhydroxyanisol (BHA) about 6.03ug/ml and ascorbic acid about 1.24μg/ml. The yield in essential oil of rosemary obtained by hydrodistillation was 1.42%. Based on the determination of the diameter of inhibition, different antimicrobial activity of the essential oil was revealed against the six tested microbes. Escherichia coli from the University Hospital (UH), Streptococcus aureus (UH) and Pseudomonas aeruginosa ATCC have a minimum inhibitory concentration value (MIC) of 62.5µl/ml. However, Bacillus sp (UH) and Staphylococcus aureus ATCC have an MIC value of 125μl/ml. The inhibition zone against Candida sp was about 24 mm. The aromatograms showed that the essential oil of rosemary exercises an antifungal activity more important than the antibacterial one.

Keywords: Rosmarinus officinalis L., maceration, essential oil, antioxidant, antimicrobial activity

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Authors: Mayuva Youngsabanant, Suphaphorn Rabiab, Hatairuk Tungkasen, Nongnuch Gumlungpat, Mayuree Pumipaiboon

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The porcine follicular fluid proteins (pFF) of healthy small size ovarian follicles (1-3 mm in diameters) of Large White pig ovaries were collected by sterile technique. They were used for testing the effect on cell viability and characterization of Vero cell lines using MTT assay. Two hundred microliter of round shape Vero cell lines were culture in 96 well plates with DMEM for 24 h. After that, they were attachment to substrate and some changed into fibroblast shape and spread over the surface after culture for 48 h. Then, Vero cell lines were treated with pFF at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins/mL for 24 h. Yields of the best results were analyzed by using one-way ANOVA. MTT assay reviewed an increasing in percentage of viability of Vero cell lines indicated that at concentration of 400-600 µg proteins/mL showed higher percentage of viability (115.64 ± 6.95, 106.91 ± 5.27 and 116.73 ± 20.15) than control group. They were significantly different from the control group (p < 0.05) but lower than the positive control group (DMEM with 10% heat treated fetal bovine serum). Cell lines showed normal character in fibroblast elongate shape after treated with pFF except in high concentration of pFF. This result implies that pFF of small size ovarian follicle at concentration of 400-600 µg proteins/mL could be optimized concentration for using as a supplement in Vero cell line culture medium to promote cell viability instead of growth hormone from fetal bovine serum. This merit could be applied in other cell biotechnology researches. Acknowledgements: This work was funded by a grant from Silpakorn University and Faculty of Science, Silpakorn University, Thailand.

Keywords: cell viability, porcine follicular fluid, MTT assay, Vero cell line

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Authors: Assem Sallam, Aly Elzahaby, Ahmed Makled, Mohamed Khalil

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Change in design parameters of launch vehicle affects its overall flight path trajectory. In this paper, several design parameters are introduced to study their effect. Selected parameters are the launch vehicle mass, which is presented in the form of payload mass, the maximum allowable angle of attack the launch vehicle can withstand, the flight path angle that is predefined for the launch vehicle second stage, the required inclination and its effect on the launch azimuth and finally by changing the launch pad coordinate. Selected design parameters are studied for their effect on the variation of altitude, ground range, absolute velocity and the flight path angle. The study gives a general mean of adjusting the design parameters to reach the required launch vehicle performance.

Keywords: launch vehicle azimuth, launch vehicle trajectory, launch vehicle payload, launch pad location

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Authors: Kittipong Tripetch

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This paper proposes for the first time symbolic formula of the power spectrum of cross couple oscillator and its modified circuit. Many principle existed to derived power spectrum in microwave textbook such as impedance, admittance parameters, ABCD, H parameters, etc. It can be compared by graph of power spectrum which methodology is the best from the point of view of practical measurement setup such as condition of impedance parameter which used superposition of current to derived (its current injection of the other port of the circuit is zero, which is impossible in reality). Four Graphs of impedance parameters of cross couple oscillator is proposed. After that four graphs of Scattering parameters of cross couple oscillator will be shown.

Keywords: optimization, power spectrum, impedance parameters, scattering parameter

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Authors: Sung-Hee Hwang, Michael Lee

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Upregulated Src activity has been implicated in a variety of cancers. Thus, Src family tyrosine kinase (SFK) inhibitors are often effective cancer treatments. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas mediated genome editing. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence specific double-stranded DNA (dsDNA) cleavage. Interestingly, ATG5 KO cells clearly showed a greater proliferation rate than WT NIH 3T3 cells, implying that autophagy induction is cytotoxic. Also, the clonogenic survival of ATG5 KO cells was greater than WT cells. The MTT assay revealed that the cytotoxic effect of PP2 was weaker on ATG5 knockout cells than that WT cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. Furthermore, Cyto-ID autophagy assay also revealed that PP2 failed to induce autophagy in ATG5 knockout cells. Together, our findings suggest that the resistance to PP2 in ATG5 knockout cells is associated with defective autophagy.

Keywords: ATG5 knockout, Autophagy, CRISPR/Cas9, PP2

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Authors: Pankaj Chandna, Dinesh Kumar

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The present work analyses different parameters of end milling to minimize the surface roughness for AISI D2 steel. D2 Steel is generally used for stamping or forming dies, punches, forming rolls, knives, slitters, shear blades, tools, scrap choppers, tyre shredders etc. Surface roughness is one of the main indices that determines the quality of machined products and is influenced by various cutting parameters. In machining operations, achieving desired surface quality by optimization of machining parameters, is a challenging job. In case of mating components the surface roughness become more essential and is influenced by the cutting parameters, because, these quality structures are highly correlated and are expected to be influenced directly or indirectly by the direct effect of process parameters or their interactive effects (i.e. on process environment). In this work, the effects of selected process parameters on surface roughness and subsequent setting of parameters with the levels have been accomplished by Taguchi’s parameter design approach. The experiments have been performed as per the combination of levels of different process parameters suggested by L9 orthogonal array. Experimental investigation of the end milling of AISI D2 steel with carbide tool by varying feed, speed and depth of cut and the surface roughness has been measured using surface roughness tester. Analyses of variance have been performed for mean and signal-to-noise ratio to estimate the contribution of the different process parameters on the process.

Keywords: D2 steel, orthogonal array, optimization, surface roughness, Taguchi methodology

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Authors: Ratna Siti Khodijah, Mirzam Abdurrachman

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Gold is recovered from gold ores. Within the ores, there are not only gold but also several types of precious metals. Copper, silver, and platinum group elements (ruthenium, rhodium, palladium, rhenium, osmium, and iridium) are metals commonly found in the ores. These metals combine to form an ore because they have the same properties. It is due to their position in periodic-system-of-elements are near to gold. However, the presence of mercury in every gold ore has not been mentioned, even though it is located right next to gold in the periodic-system-of-elements and they are located in the same block, d-block. Thus, it is possible that mercury is contained in the ores. Moreover, the elements of the same group with mercury—zinc and cadmium—sometimes can be found in the ores. It is suspected that mercury can not be detected because the processing of gold ores usually using fire assay method. Before the ores melting, mercury would evaporate because it has the lowest boiling point of all precious metal in the ores. Therefore, it suggested doing research on the presence of mercury in gold ores by CVAAS method. The results of this study would obtain the amount of mercury in gold ores that should be purified. So it can be produced economically if possible.

Keywords: boiling point, d-block, fire assay, precious metal

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Authors: Lie-Sian Yap, Ming-Chien Yang

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This study demonstrated a novel injectable hydrogel scaffold composing of Pluronic F127, carboxymethyl hexanoyl chitosan (CA) and glutaraldehyde (GA) for encapsulating human fetal osteoblastic cells (hFOB) 1.19. The hydrogel was prepared by mixing F127 and GA in CA solution at 4°C. The mechanical properties and cytotoxicity of this hydrogel were determined through rheological measurements and MTT assay, respectively. After encapsulation process, the hFOB 1.19 cells morphology was examined using fluorescent and confocal imaging. The results indicated that the Tgel of this system was around 30°C, where sol-gel transformation occurred within 90s and F127/CA/GA gel was able to remain intact in the medium for more than 1 month. In vitro cell culture assay revealed that F127/CA/GA hydrogels were non-cytotoxic. Encapsulated hFOB 1.19 cells not only showed the spherical shape and formed colonies, but also reduced their size. Moreover, the hFOB 1.19 cells showed that cells remain alive after the encapsulation process. Based on these results, these F127/CA/GA hydrogels can be used to encapsulate cells for tissue engineering applications.

Keywords: carboxymethyl hexanoyl chitosan, cell encapsulation, hFOB 1.19, Pluronic F127

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Authors: R. Fegas, S. Zerkout, S. Taberkokt, M. Righezza

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The present work demonstrate the potential of Betacyclodextrine (BCD) for the chiral analysis of a drug .Various separation mechanisms were applied and several parameters affecting the separation were studied, including the type and concentration of chiral selector, and pH of buffer. A simple and sensitive high-performance liquid chromatography (HPLC) method was developed as an assay for fexofenadine enantiomers in pharmaceutical preparation. Fexofenadine enantiomers were separated using a mobile phase of 0.25mM NaH2PO4–acetonitrile (65:35, v/v) – Betacyclodextrine on achiral phenyl-urea column at a flow rate of 1ml/min and measurement at 220nm. The chiral mechanism of separation was mainly based on specific interaction between the solute and the stationary phase. The retention was directly controlled by mobile phase composition but not the selectivity which results of the two mechanisms, electrostatic interactions and partition mechanism.

Keywords: fexofenadine enantiomer, HPLC, achiral phenyl-urea column

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Authors: A. F. Agboola, B. R. O. Omidiwura, A. Oyeyemi, E. A. Iyayi, A. S. Adelani

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Dietary cholesterol has elicited the most public interest as it relates with coronary heart disease. Thus, humans have been paying more attention to health, thereby reducing consumption of cholesterol enriched food. Egg is considered as one of the major sources of human dietary cholesterol. However, an alternative way to reduce the potential cholesterolemic effect of eggs is to modify the fatty acid composition of the yolk. The effect of palm oil (PO), soybean oil (SO), sesame seed oil (SSO) and fish oil (FO) supplementation in the diets of layers on egg yolk fatty acid, cholesterol, egg production and egg quality parameters were evaluated in a 42-day feeding trial. One hundred and five Isa Brown laying hens of 34 weeks of age were randomly distributed into seven groups of five replicates and three birds per replicate in a completely randomized design. Seven corn-soybean basal diets (BD) were formulated: BD+No oil (T1), BD+1.5% PO (T2), BD+1.5% SO (T3), BD+1.5% SSO (T4), BD+1.5% FO (T5), BD+0.75% SO+0.75% FO (T6) and BD+0.75% SSO+0.75% FO (T7). Five eggs were randomly sampled at day 42 from each replicate to assay for the cholesterol, fatty acid profile of egg yolk and egg quality assessment. Results showed that there were no significant (P>0.05) differences observed in production performance, egg cholesterol and egg quality parameters except for yolk height, albumen height, yolk index, egg shape index, haugh unit, and yolk colour. There were no significant differences (P>0.05) observed in total cholesterol, high density lipoprotein and low density lipoprotein levels of egg yolk across the treatments. However, diets had effect (P<0.05) on TAG (triacylglycerol) and VLDL (very low density lipoprotein) of the egg yolk. The highest TAG (603.78 mg/dl) and VLDL values (120.76 mg/dl) were recorded in eggs of hens on T4 (1.5% sesame seed oil) and was similar to those on T3 (1.5% soybean oil), T5 (1.5% fish oil) and T6 (0.75% soybean oil + 0.75% fish oil). However, results revealed a significant (P<0.05) variations on eggs’ summation of polyunsaturated fatty acid (PUFA). In conclusion, it is suggested that dietary oils could be included in layers’ diets to produce designer eggs low in cholesterol and high in PUFA especially omega-3 fatty acids.

Keywords: dietary oils, egg cholesterol, egg fatty acid profile, egg quality parameters

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Authors: Awatif Boumaza, Abderraouf Hilali, Hayat Talbi, Houda Sbayou

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The methanolic extract of Zygophyllum cornutun coss, an Algerian medicinal plant, was screened to the presence of mutagenic activity and genotoxic effect using the Ames test (Salmonella/microsome) and the micronucleus assay respectively. Positive results were obtained with both tests. The Ames test showed mutagenic activity in the presence of microsomal activation, while negative result was observed without microsomal activation. In the micronucleus test, two parameters were evaluated: the frequency of the micronucleus that increased in a dose dependent way and the proliferation index that decreased according to the micronucleus frequency. Even that further studies must be carried out, the mutagenic activity and the genotoxic effect of Zygophyllum cornutum should be taken in consideration when used as therapeutic plant.

Keywords: ames test, micronucleus test, mutagenic activity, genotoxicity, Zygophyllum cornutum

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Authors: Assia Galleze, Abdurrahim Kocyigit, Nacira Cherif, Nidel Benhalilou, Nabila Attal, Chafia Touil Boukkoffa, Rachida Raache

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Introduction and aims: Chemotherapeutic agents used to inhibit cell division and reduce tumor growth, increase reactive oxygen species levels, which contributes to their genotoxicity [1]. The comet assay is an inexpensive and rapid method to detect the damage at cellular levels and has been used in various cancer populations undergoing chemotherapy [2,3]. The present study aim to assess the oxidative stress and the genotoxicity induced by chemotherapy by the determination of plasma malondialdehyde (MDA) level, protein carbonyl (PC) content, superoxide dismutase (SOD) activity and lymphocyte DNA damage in Algerian children with lymphoma. Materials and Methods: For our study, we selected thirty children with lymphoma treated in university hospital of Beni Messous, Algeria, and fifty unrelated subjects as controls, after obtaining the informed consent in accordance with the Declaration of Helsinki (1964). Plasma levels of MDA, PC and SOD activity were spectrophotometrically measured, while DNA damage was assessed by alkaline comet assay in peripheral blood leukocytes. Results and Discussion: Plasma MDA, PC levels and lymphocyte DNA damage, were found to be significantly higher in lymphoma patients than in controls (p < 0.001). Whereas, SOD activity in lymphoma patients was significantly lower than in healthy controls (p < 0.001). There were significant positive correlations between DNA damage, MDA and PC in patients (r = 0.96, p < 0.001, r = 0.97, p < 0.001, respectively), and negative correlation with SOD (r = 0.87, p < 0.01). Conclusion and Perspective: Our results indicated that, leukocytes DNA damage and oxidative stress were significantly higher in lymphoma patients, suggesting that the direct effect of chemotherapy and the alteration of the redox balance may influence oxidative/antioxidative status.

Keywords: chemotherapy, comet assay, DNA damage, lymphoma

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Authors: Hussain Mustatab Wahedi, Sun You Kim

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Skin acts as a barrier against the harmful environmental factors. Integrity and timely recovery of the skin from injuries and harmful effects of radiations is thus very important. This study aimed to investigate the importance of Sirt1 in the recovery of skin from UVB-induced damage and cutaneous wounds by using natural and synthetic novel Sirt1 activators. Juglone, known as a natural Pin1 inhibitor, and NED416 a novel synthetic Sirt1 activator were checked for their ability to regulate the expression and activity of Sirt1 and hence photo-damage and wound healing in cultured skin cells (NHDF and HaCaT cells) and mouse model by using Sirt1 siRNA knockdown, cell migration assay, GST-Pulldown assay, western blot analysis, tube formation assay, and immunohistochemistry. Interestingly, Sirt1 knockdown inhibited skin cell migration in vitro. Juglone up regulated the expression of Sirt1 in both the cell lines under normal and UVB irradiated conditions, enhanced Sirt1 activity and increased the cell viability by reducing reactive oxygen species synthesis and apoptosis. Juglone promoted wound healing by increasing cell migration and angiogenesis through Cdc42/Rac1/PAK, MAPKs and Smad pathways in skin cells. NED416 upregulated Sirt1 expression in HaCaT and NHDF cells as well as increased Sirt1 activity. NED416 promoted the process of wound healing in early as well as later stages by increasing macrophage recruitment, skin cell migration, and angiogenesis through Cdc42/Rac1 and MAPKs pathways. So, both these compounds activated Sirt1 and promoted the process of wound healing thus pointing towards the possible role of Sirt1 in skin regeneration and wound healing.

Keywords: skin regeneration, wound healing, Sirt1, UVB light

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Authors: Adebisi Adunola Demehin, Wanlaya Thamnarak, Jaruwan Chatwichien, Chatchakorn Eurtivong, Kiattawee Choowongkomon, Somsak Ruchirawat, Nopporn Thasana

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The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein involved in cellular signalling processes and, its aberrant activity is crucial in the development of many cancers such as lung cancer. Selaginellaceae are fern allies that have long been used in Chinese traditional medicine to treat various cancer types, especially lung cancer. Biflavonoids, the major secondary metabolites in Selaginellaceae, have numerous pharmacological activities, including anti-cancer and anti-inflammatory. For instance, amentoflavone induces a cytotoxic effect in the human NSCLC cell line via the inhibition of PARP-1. However, to the best of our knowledge, there are no studies on biflavonoids as EGFR inhibitors. Thus, this study aims to investigate the EGFR inhibitory activities of biflavonoids isolated from Selaginella siamensis and Selaginella bryopteris. Amentoflavone, tetrahydroamentoflavone, sciadopitysin, robustaflavone, robustaflavone-4-methylether, delicaflavone, and chrysocauloflavone were isolated from the ethyl-acetate extract of the whole plants. The structures were determined using NMR spectroscopy and mass spectrometry. In vitro study was conducted to evaluate their cytotoxicity against A549, HEPG2, and T47D human cancer cell lines using the MTT assay. In addition, a target-based assay was performed to investigate their EGFR inhibitory activity using the kinase inhibition assay. Finally, a molecular docking study was conducted to predict the binding modes of the compounds. Robustaflavone-4-methylether and delicaflavone showed the best cytotoxic activity on all the cell lines with IC50 (µM) values of 18.9 ± 2.1 and 22.7 ± 3.3 on A549, respectively. Of these biflavonoids, delicaflavone showed the most potent EGFR inhibitory activity with an 84% relative inhibition at 0.02 nM using erlotinib as a positive control. Robustaflavone-4-methylether showed a 78% inhibition at 0.15 nM. The docking scores obtained from the molecular docking study correlated with the kinase inhibition assay. Robustaflavone-4-methylether and delicaflavone had a docking score of 72.0 and 86.5, respectively. The inhibitory activity of delicaflavone seemed to be linked with the C2”=C3” and 3-O-4”’ linkage pattern. Thus, this study suggests that the structural features of these compounds could serve as a basis for developing new EGFR-TK inhibitors.

Keywords: anticancer, biflavonoids, EGFR, molecular docking, Selaginellaceae

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Authors: Sebaa H., Cherifi F., Djabeur Abderrezak M.

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Argania spinosa, Sapotaceae sole representative in Algeria and Morocco; hence it is endemic in these regions. However, it is a recognised oil, forage, and timber tree highly adapted to aridity. The exploitation of the argan fruits produces considerable amounts of under or related products. These products, such as the endocarps of a fruit, recuperated after the use of kernels to extract oil. This research studies in detail the contents of total phenolic content was determined by Folin Ciocalteu reagent and Flavonoids by aluminum chloride colorimetric assay). Antioxidant activity of extracts was expressed as the percentage of DPPH radical inhibition and IC50 values (μg/mL). Antimicrobial activity evaluated using agar disk diffusion method against reference Pseudomonas aeruginosa ATTC 27453, Escherichia coli ATCC 23922. Immature endocarps showed a higher polyphenol content than mature endocarps. The total phenolic content in immature endocarps was found to vary from 983,75+ /- 0.45 to 980,1 +/- 0.43 mg gallic acid equivalents/g dry weight, whereas in mature endocarps, the polyphenol content ranged from 100,58 mg/g +/- 0.42 to 105 +/- 0.55% mg gallic acid equivalent / g dry weight. The flavonoid content was 16.5 mg equivalent catechin/g dry weight and 9.81mg equivalent catechin /g dry weight for immature and mature endocarp fruits, respectively. DPPH assay of the endocarps extract yielded a half-maximal effective concentration (IC50) value in the immature endocarps (549.33 μg/mL) than in mature endocarps (322 μg/mL). This result can be attributed to the higher phenolics and flavonoid compounds in the immature endocarps. Methanol extract of immature endocarps exhibited antibacterial activity against E.colie (inhibition zone, 11mm).

Keywords: antioxidant activity, antimicrobial activity, total phenolic content, DPPH assay

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Authors: Sorayya Malek, Mogeeb Mosleh, Sharifah M. Syed

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In this study, Support Vector Machine (SVM) technique was applied to predict the dichotomized value of Dissolved oxygen (DO) from two freshwater lakes namely Chini and Bera Lake (Malaysia). Data sample contained 11 parameters for water quality features from year 2005 until 2009. All data parameters were used to predicate the dissolved oxygen concentration which was dichotomized into 3 different levels (High, Medium, and Low). The input parameters were ranked, and forward selection method was applied to determine the optimum parameters that yield the lowest errors, and highest accuracy. Initial results showed that pH, water temperature, and conductivity are the most important parameters that significantly affect the predication of DO. Then, SVM model was applied using the Anova kernel with those parameters yielded 74% accuracy rate. We concluded that using SVM models to predicate the DO is feasible, and using dichotomized value of DO yields higher prediction accuracy than using precise DO value.

Keywords: dissolved oxygen, water quality, predication DO, support vector machine

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Authors: Saeeda Nadir Ali, Najma Sultana, Muhammad Saeed Arayne, Amtul Qayoom

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Present study describes a simple and a fast liquid chromatographic method using ultraviolet detector for simultaneous determination of anxiety relief medicine alprazolam with ACE inhibitors i.e; lisinopril, captopril and enalapril employing purospher star C18 (25 cm, 0.46 cm, 5 µm). Separation was achieved within 5 min at ambient temperature via methanol: water (8:2 v/v) with pH adjusted to 2.9, monitoring the detector response at 220 nm. Optimum parameters were set up as per ICH (2006) guidelines. Calibration range was found out to be 0.312-10 µg mL-1 for alprazolam and 0.625-20 µg mL-1 for all the ACE inhibitors with correlation coefficients > 0.998 and detection limits 85, 37, 68 and 32 ng mL-1 for lisinopril, captopril, enalapril and alprazolam respectively. Intra-day, inter-day precision and accuracy of the assay were in acceptable range of 0.05-1.62% RSD and 98.85-100.76% recovery. Method was determined to be robust and effectively useful for the estimation of studied drugs in dosage formulations and human serum without obstruction of excipients or serum components.

Keywords: alprazolam, ACE inhibitors, RP HPLC, serum

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Authors: Mohammad Zamzam, Wesam Bachir, Imad Asaad

Abstract:

Several studies have produced estimates of Er:YAG laser parameters and specifications but there is still insufficient data for reliable selection of laser parameters. As a consequence, there is a heightened need for ideal specifications of Er:YAG laser to reduce the amount of enamel ablation. The objective of this paper is to investigate the influence of Er:YAG laser parameters, energy level and pulse duration, on orthodontic composite removal after bracket debonding. The sample consisted of 45 cuboids of orthodontic composite made by plastic moulds. The samples were divided into three groups, each was irradiated with Er:YAG laser set at different energy levels and three values for pulse durations (50 µs, 100 µs, and 300 µs). Geometrical parameters (depth and area) of cavities formed by laser irradiation were determined. ANCOVA test showed statistically significant difference (p < 0.0.5) between the groups indicating a potential effect of laser pulse duration on the geometrical parameters after controlling laser energy level. A post-hoc Bonferroni test ranked the 50µ Er:YAG laser pulse as the most influential factor for all geometrical parameters in removing remnant composite from enamel surface. Also, 300 mJ laser pulses caused the largest removal of the composite. The results of the present study demonstrated the efficacy of 50 µs and 300 mJ Er:YAG laser pulse for removal of remnant orthodontic composite.

Keywords: enamel, Er:YAG, geometrical parameters, orthodontic composite, remnant composite

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Authors: Shayli Varasteh Moradi, Wayne A. Johnston, Dejan Gagoski, Kirill Alexandrov

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The demand for a rapid and more efficient technique to identify protein-protein interaction particularly in the areas of therapeutics and diagnostics development is growing. The method described here is a rapid in vitro protein-protein interaction analysis approach based on AlphaLISA technology combined with Leishmania tarentolae cell-free protein production (LTE) system. Cell-free protein synthesis allows the rapid production of recombinant proteins in a multiplexed format. Among available in vitro expression systems, LTE offers several advantages over other eukaryotic cell-free systems. It is based on a fast growing fermentable organism that is inexpensive in cultivation and lysate production. High integrity of proteins produced in this system and the ability to co-express multiple proteins makes it a desirable method for screening protein interactions. Following the translation of protein pairs in LTE system, the physical interaction between proteins of interests is analysed by AlphaLISA assay. The assay is performed using unpurified in vitro translation reaction and therefore can be readily multiplexed. This approach can be used in various research applications such as epitope mapping, antigen-antibody analysis and protein interaction network mapping. The intra-viral protein interaction network of Zika virus was studied using the developed technique. The viral proteins were co-expressed pair-wise in LTE and all possible interactions among viral proteins were tested using AlphaLISA. The assay resulted to the identification of 54 intra-viral protein-protein interactions from which 19 binary interactions were found to be novel. The presented technique provides a powerful tool for rapid analysis of protein-protein interaction with high sensitivity and throughput.

Keywords: AlphaLISA technology, cell-free protein expression, epitope mapping, Leishmania tarentolae, protein-protein interaction

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Authors: Matheus Fernando Pereira, Varese Salvador Timoteo

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In this work, we have solved the two-dimensional advection-diffusion equation numerically for a spatially dependent solute dispersion along non-uniform flow with a pulse type source in order to make a systematic study on the influence of medium heterogeneity, initial flow velocity, and initial dispersion coefficient parameters on the solutions of the equation. The behavior of the solutions is then investigated as we change the three parameters independently. Our results show that even though the parameters represent different physical features of the system, the effect on their variation is very similar. We also observe that the effects caused by the parameters on the concentration depend on the distance from the source. Finally, our numerical results are in good agreement with the exact solutions for all values of the parameters we used in our analysis.

Keywords: advection-diffusion equation, dispersion, numerical methods, pulse-type source

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Authors: A. E. Kurtoglu, A. Cevik, M. Bilgehan

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In this parametric study, effect of soil and material characteristics on safety of structures is investigated. The soil parameters such as shear strength, unit weight; geometrical parameters of the structure such as foundation depth and height of building; and material properties such as weight of concrete were selected as input parameters. A real accelerogram of 1989 El-Centro earthquake recorded by the USGS in Imperial Valley is used for this study. It is contained in the standard Strong Motion CD-ROM (SMC) format, which can be recognized and interpreted by FEM software used. The soil-structure interaction model subjected to above-mentioned earthquake was analyzed for 729 cases. Effect of input parameters on safety factor of the soil-structure system was then investigated and the interaction between the input and output parameters is presented in graphical form. Findings showed that all input parameters have significant effects on factor of safety results.

Keywords: factor of safety, finite element method, safety of structures, soil structure interaction

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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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Authors: Shalu Jain, Anju Bansal, Anup Kumar, Sunita Saxena

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Objectives: The novel TMPRSS2:ERG gene fusion is a common somatic event in prostate cancer that in some studies is linked with a more aggressive disease phenotype. Thus, this study aims to determine whether clinical variables are associated with the presence of TMPRSS2:ERG-fusion gene transcript in Indian patients of prostate cancer. Methods: We evaluated the clinical variables with presence and absence of TMPRSS2:ERG gene fusion in prostate cancer and BPH association of clinical patients. Patients referred for prostate biopsy because of abnormal DRE or/and elevated sPSA were enrolled for this prospective clinical study. TMPRSS2:ERG mRNA copies in samples were quantified using a Taqman chemistry by real time PCR assay in prostate biopsy samples (N=42). The T2:ERG assay detects the gene fusion mRNA isoform TMPRSS2 exon1 to ERG exon4. Results: Histopathology report has confirmed 25 cases as prostate cancer adenocarcinoma (PCa) and 17 patients as benign prostate hyperplasia (BPH). Out of 25 PCa cases, 16 (64%) were T2: ERG fusion positive. All 17 BPH controls were fusion negative. The T2:ERG fusion transcript was exclusively specific for prostate cancer as no case of BPH was detected having T2:ERG fusion, showing 100% specificity. The positive predictive value of fusion marker for prostate cancer is thus 100% and the negative predictive value is 65.3%. The T2:ERG fusion marker is significantly associated with clinical variables like no. of positive cores in prostate biopsy, Gleason score, serum PSA, perineural invasion, perivascular invasion and periprostatic fat involvement. Conclusions: Prostate cancer is a heterogeneous disease that may be defined by molecular subtypes such as the TMPRSS2:ERG fusion. In the present prospective study, the T2:ERG quantitative assay demonstrated high specificity for predicting biopsy outcome; sensitivity was similar to the prevalence of T2:ERG gene fusions in prostate tumors. These data suggest that further improvement in diagnostic accuracy could be achieved using a nomogram that combines T2:ERG with other markers and risk factors for prostate cancer.

Keywords: prostate cancer, genetic rearrangement, TMPRSS2:ERG fusion, clinical variables

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