Search results for: Plaque assay

Authors: L. Fonseca-Géigel, M. Alvarez, G. García, R. Cox, L. Morier, L. Fonte, M. G. Guzmán

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Toxoplasma gondii is an intracellular parasite capable of infecting all nucleated cells in a diverse array of species. Toxoplasma plaque assay have been described using Bacto Agar. Because of its experimental advantages carboxymethyl cellulose overlay, medium viscosity was choosing and the aim of this work was to develop alternative method for formation of T. gondii plaques. Tachyzoites were inoculated onto monolayers of Vero cells and cultured at 37° C under 5 % CO2. The cultures were followed up by microscopy inspection. Small plaques were visible by naphtol blue stain 4 days after infection. Larger plaques could be observed by day 10 of culture. The carboxymethyl cellulose is a cheap reagent and the methodology is easier, faster than assays under agar overlay. This is the first description of the carboxymethyl cellulose overlay use for obtaining the formation of T. gondii plaques and may be useful in consequent obtaining tachyzoites for detailed studies.

Keywords: Carboxymethyl cellulose, Cell culture, Plaque assay, Toxoplasma gondii.

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Authors: B. Prashantha, S. Anish

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The aim of the present study is to computationally evaluate the hemodynamic factors which affect the formation of atherosclerosis and plaque rupture in the human artery. An increase of atherosclerosis disease in the artery causes geometry changes, which results in hemodynamic changes such as flow separation, reattachment, and adhesion of new cells (chemotactic) in the artery. Hence, geometry plays an important role in the determining the nature of hemodynamic patterns. Influence of stenosis in the non-bifurcating artery, under pulsatile flow condition, has been studied on an idealized geometry. Analysis of flow through symmetric and asymmetric stenosis in the artery revealed the significance of oscillating shear index (OSI), flow separation, low WSS zones and secondary flow patterns on plaque formation. The observed characteristic of flow in the post-stenotic region highlight the importance of plaque eccentricity on the formation of secondary stenosis on the arterial wall.

Keywords: Atherosclerotic plaque, Oscillatory Shear Index, Stenosis nature, Wall Shear Stress.

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Authors: Siobhan O’Shea, Sangeetha Vijaysri Nair, Hee Cheol Kim, Charles Thomas Nugent, Cheuk Yan William Tong, Sam Douthwaite, Andrew Worlock

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The Aptima® HIV-1 Quant Dx Assay is a fully automated assay on the Panther system. It is based on Transcription- Mediated Amplification and real time detection technologies. This assay is intended for monitoring HIV-1 viral load in plasma specimens and for the detection of HIV-1 in plasma and serum specimens. Nine-hundred and seventy nine specimens selected at random from routine testing at St Thomas’ Hospital, London were anonymised and used to compare the performance of the Aptima HIV-1 Quant Dx assay and Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0. Two-hundred and thirty four specimens gave quantitative HIV-1 viral load results in both assays. The quantitative results reported by the Aptima Assay were comparable to those reported by the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 with a linear regression slope of 1.04 and an intercept on -0.097. The Aptima assay detected HIV-1 in more samples than the COBAS assay. This was not due to lack of specificity of the Aptima assay because this assay gave 99.83% specificity on testing plasma specimens from 600 HIV-1 negative individuals. To understand the reason for this higher detection rate a side-by-side comparison of low level panels made from the HIV-1 3rd international standard (NIBSC10/152) and clinical samples of various subtypes were tested in both assays. The Aptima assay was more sensitive than the COBAS assay. The good sensitivity, specificity and agreement with other commercial assays make the HIV-1 Quant Dx Assay appropriate for both viral load monitoring and detection of HIV-1 infections.

Keywords: HIV viral load, Aptima, Roche, Panther system.

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Authors: Anis S. Shuib, Peter R. Hoskins, William J. Easson

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Cardiovascular diseases, principally atherosclerosis, are responsible for 30% of world deaths. Atherosclerosis is due to the formation of plaque. The fatty plaque may be at risk of rupture, leading typically to stroke and heart attack. The plaque is usually associated with a high degree of lumen reduction, called a stenosis.It is increasingly recognized that the initiation and progression of disease and the occurrence of clinical events is a complex interplay between the local biomechanical environment and the local vascular biology. The aim of this study is to investigate the flow behavior through a stenosed artery. A physical experiment was performed using an artery model and blood analogue fluid. An axisymmetric model constructed consists of contraction and expansion region that follow a mathematical form of cosine function. A 30% diameter reduction was used in this study. The flow field was measured using particle image velocimetry (PIV). Spherical particles with 20μm diameter were seeded in a water-glycerol-NaCl mixture. Steady flow Reynolds numbers are 250. The area of interest is the region after the stenosis where the flow separation occurs. The velocity field was measured and the velocity gradient was investigated. There was high particle concentration in the recirculation zone. High velocity gradient formed immediately after the stenosis throat created a lift force that enhanced particle migration to the flow separation area.

Keywords: Stenosis artery, Biofluid mechanics, PIV

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Authors: Anis S. Shuib, Peter R. Hoskins, William J. Easson

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Cardiovascular disease mostly in the form of atherosclerosis is responsible for 30% of all world deaths amounting to 17 million people per year. Atherosclerosis is due to the formation of plaque. The fatty plaque may be at risk of rupture, leading typically to stroke and heart attack. The plaque is usually associated with a high degree of lumen reduction, called a stenosis. The initiation and progression of the disease is strongly linked to the hemodynamic environment near the vessel wall. The aim of this study is to validate the flow of blood mimic through an arterial stenosis model with computational fluid dynamics (CFD) package. In experiment, an axisymmetric model constructed consists of contraction and expansion region that follow a mathematical form of cosine function. A 30% diameter reduction was used in this study. Particle image velocimetry (PIV) was used to characterize the flow. The fluid consists of rigid spherical particles suspended in waterglycerol- NaCl mixture. The particles with 20 μm diameter were selected to follow the flow of fluid. The flow at Re=155, 270 and 390 were investigated. The experimental result is compared with FLUENT simulated flow that account for viscous laminar flow model. The results suggest that laminar flow model was sufficient to predict flow velocity at the inlet but the velocity at stenosis throat at Re =390 was overestimated. Hence, a transition to turbulent regime might have been developed at throat region as the flow rate increases.

Keywords: Atherosclerosis, Particle-laden flow, Particle imagevelocimetry, Stenosis artery

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Authors: Ilma Robo, Saimir Heta, Nedja Hysi, Vera Ostreni

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Introduction: Marginal gingivitis is a disease with considerable frequency among patients who present routinely for periodontal control and treatment. In fact, this disease may not have alarming symptoms in patients and may go unnoticed by themselves when personal hygiene conditions are optimal. The aim of this study was to collect retrograde data on the prevalence of marginal gingiva in the respective group of patients, evaluated according to specific periodontal diagnostic tools. Materials and methods: The study was conducted in two patient groups. The first group was with 34 patients, during December 2019-January 2020, and the second group was with 64 patients during 2010-2018 (each year in the mentioned monthly period). Bacterial plaque index, hemorrhage index, amount of gingival fluid, presence of xerostomia and candidiasis were recorded in patients. Results: Analysis of the collected data showed that susceptibility to marginal gingivitis shows higher values according to retrograde data, compared to cross-sectional ones. Susceptibility to candidiasis and the occurrence of xerostomia, even in the combination of both pathologies, as risk factors for the occurrence of marginal gingivitis, show higher values ​​according to retrograde data. The female are presented with a reduced bacterial plaque index than the males, but more importantly, this index in the females is also associated with a reduced index of gingival hemorrhage, in contrast to the males. Conclusions: Cross-sectional data show that the prevalence of marginal gingivitis is more reduced, compared to retrograde data, based on the hemorrhage index and the bacterial plaque index together. Changes in production in the amount of gingival fluid show a higher prevalence of marginal gingivitis in cross-sectional data than in retrograde data; this is based on the sophistication of the way data are recorded, which evolves over time and also based on professional sensitivity to this phenomenon.

Keywords: Marginal gingivitis, cross-sectional, retrograde, prevalence.

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Authors: Ibrahim Aly , Rabab Zalat, Bahaa EL Deen W. El Aswad, Ismail M. Moharm , Basam M. Masoud, Tarek Diab

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The objective of the present study was to evaluate the novel nanomagnetic beads based–latex agglutination assay (NMB-LAT) as a simple test for diagnosis of S. haematobium as well as standardize the novel nanomagnetic beads based –ELISA (NMB-ELISA). According to urine examination this study included 85 S. haematobium infected patients, 30 other parasites infected patients and 25 negative control samples. The sensitivity of novel NMB-LAT was 82.4% versus 96.5% and 88.2% for NMB-ELISA and currently used sandwich ELISA respectively. The specificity of NMB-LAT was 83.6% versus 96.3% and 87.3% for NMB-ELISA and currently used sandwich ELISA respectively. In conclusion, the novel NMB-ELISA is a valuable applicable diagnostic technique for diagnosis of human schistosomiasis haematobium. The novel NMB-ELISA assay is a suitable applicable diagnostic method in field survey especially when followed by ELISA as a confirmatory test in query false negative results. Trials are required to increase the sensitivity and specificity of NMB-ELISA assay.

Keywords: Diagnosis, Latex agglutination, Nanomagnetic beads, Sandwich ELISA.

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Authors: Anita, Sari Septiani Tangke, Rusdina Bte Ladju, Nasrum Massi

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New diagnostic tools are urgently needed to interrupt the transmission of tuberculosis and multidrug-resistant tuberculosis. The microscopic-observation drug-susceptibility (MODS) assay is a rapid, accurate and simple liquid culture method to detect multidrug-resistant tuberculosis (MDR-TB). MODS were evaluated to determine a lower and same concentration of isoniazid and rifampin for detection of MDR-TB. Direct drug-susceptibility testing was performed with the use of the MODS assay. Drug-sensitive control strains were tested daily. The drug concentrations that used for both isoniazid and rifampin were at the same concentration: 0.16, 0.08 and 0.04μg per milliliter. We tested 56 M. tuberculosis clinical isolates and the control strains M. tuberculosis H37RV. All concentration showed same result. Of 53 M. tuberculosis clinical isolates, 14 were MDR-TB, 38 were susceptible with isoniazid and rifampin, 1 was resistant with isoniazid only. Drug-susceptibility testing was performed with the use of the proportion method using Mycobacteria Growth Indicator Tube (MGIT) system as reference. The result of MODS assay using lower concentration was significance (P<0.001) compare with the reference methods.

A lower and same concentration of isoniazid and rifampin can be used to detect MDR-TB. Operational cost and application can be more efficient and easier in resource-limited environments. However, additional studies evaluating the MODS using lower and same concentration of isoniazid and rifampin must be conducted with a larger number of clinical isolates.

Keywords: Isoniazid, MODS assay, MDR-TB, Rifampin.

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Authors: Darren C-W. Tan, Partha Roy

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To determine if the murine insulinoma, β-TC-6, is a suitable substitute for primary pancreatic β-cells in the study of β- cell functional heterogeneity, we used three distinct functional assays to ascertain the cell line-s response to glucose or a glucose analog. These assays include: (i) a 2-NBDG uptake assay; (ii) a calcium influx assay, and; (iii) a quinacrine secretion assay. We show that a population of β-TC-6 cells endocytoses the glucose analog, 2- NBDG, at different rates, has non-uniform intracellular calcium ion concentrations and releases quinacrine at different rates when challenged with glucose. We also measured the Km for β-TC-6 glucose uptake to be 46.9 mM and the Vm to be 8.36 x 10-5 mmole/million cells/min. These data suggest that β-TC-6 might be used as an alternative to primary pancreatic β-cells for the study of glucose-dependent β-cell functional heterogeneity.

Keywords: 2-NBDG, Fura-2/AM, functional heterogeneity, quinacrine.

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Authors: W. Samruan, A. Oonsivilai, R. Oonsivilai

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Today, people are more interested in the foods beneficial on their health. However, there are still lacks of accurate knowledge in the field of biological properties, functional properties, including the application of legume in foods. This study focused on antioxidant activity of soybean (SB) and fermented soybean (FSB) crude extracts evaluating to have more information in fortification SB and FSB crude extracts in food products and/or dietary supplement. SB and FSB crude extracts were prepared by infusion with water and ethanol. The antioxidant activity of crude extracts was studied with DPPH and ABTS assay including commercial standard. From both DPPH and ABTS assay, the antioxidant activity of SB and FSB water crude extract showed higher antioxidant activity than ethanol crude extract, and FSB crude extract showed higher antioxidant activity than SB crude extract. In DPPH assay, BHT and vitamin C showed IC50 values at 0.241, 0.039 mg/ml, in ABTS assay. In addition, Trolox showed IC50 at 0.058 mg/ml respectively. FSB water crude extract showed high antioxidant activity. Finally, the functional properties study of both water and ethanol crude extracts should be done for beneficial in application of these extracts in food products and dietary supplement in the near future.

Keywords: Antioxidant activity, Fermented soybean (FSB) crude extracts, soybean (SB) crude extracts.

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Authors: Mehrdad Hashemi, Mitra Behrooz Aghdam, Reza Mahdian, Ahmad Reza Kamyab

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Cytogenetic analysis still remains the gold standard method for prenatal diagnosis of trisomy 21 (Down syndrome, DS). Nevertheless, the conventional cytogenetic analysis needs live cultured cells and is too time-consuming for clinical application. In contrast, molecular methods such as FISH, QF-PCR, MLPA and quantitative Real-time PCR are rapid assays with results available in 24h. In the present study, we have successfully used a novel MGB TaqMan probe-based real time PCR assay for rapid diagnosis of trisomy 21 status in Down syndrome samples. We have also compared the results of this molecular method with corresponding results obtained by the cytogenetic analysis. Blood samples obtained from DS patients (n=25) and normal controls (n=20) were tested by quantitative Real-time PCR in parallel to standard G-banding analysis. Genomic DNA was extracted from peripheral blood lymphocytes. A high precision TaqMan probe quantitative Real-time PCR assay was developed to determine the gene dosage of DSCAM (target gene on 21q22.2) relative to PMP22 (reference gene on 17p11.2). The DSCAM/PMP22 ratio was calculated according to the formula; ratio=2 -ΔΔCT. The quantitative Real-time PCR was able to distinguish between trisomy 21 samples and normal controls with the gene ratios of 1.49±0.13 and 1.03±0.04 respectively (p value <0.001). These results represent the presence of 3 copies of target gene in DS samples Vs 2 copies in normal controls. The results of quantitative Real-time PCR were in complete agreement with results of cytogenetic analysis. This study confirms previous reports regarding successful implementation of quantitative Real-time PCR for detection of trisomy 21. However, the assay has been improved by using MGB probes and more accurate data analysis. This assay, in particular, when performed in combination with another molecular assay such as QF-PCR or MLPA, can be used as a reliable technique for rapid prenatal diagnosis of trisomy 21.

Keywords: Trisomy 21, Real-time PCR, MGB-TaqMan Probes, Gene Dosage.

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Authors: S. M. Abdul Khader, Anurag Ayachit, Raghuvir Pai, K. A. Ahmed, V. R. K. Rao, S. Ganesh Kamath

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The numerical simulation has made tremendous advances in investigating the blood flow phenomenon through elastic arteries. Such study can be useful in demonstrating the disease progression and hemodynamics of cardiovascular diseases such as atherosclerosis. In the present study, patient specific case diagnosed with partially stenosed complete right ICA and normal left carotid bifurcation without any atherosclerotic plaque formation is considered. 3D patient specific carotid bifurcation model is generated based on CT scan data using MIMICS-4.0 and numerical analysis is performed using FSI solver in ANSYS-14.5. The blood flow is assumed to be incompressible, homogenous and Newtonian, while the artery wall is assumed to be linearly elastic. The two-way sequentially coupled transient FSI analysis is performed using FSI solver for three pulse cycles. The hemodynamic parameters such as flow pattern, Wall Shear Stress, pressure contours and arterial wall deformation are studied at the bifurcation and critical zones such as stenosis. The variation in flow behavior is studied throughout the pulse cycle. Also, the simulation results reveal that there is a considerable increase in the flow behavior in stenosed carotid in contrast to the normal carotid bifurcation system. The investigation also demonstrates the disturbed flow pattern especially at the bifurcation and stenosed zone elevating the hemodynamics, particularly during peak systole and later part of the pulse cycle. The results obtained agree well with the clinical observation and demonstrates the potential of patient specific numerical studies in prognosis of disease progression and plaque rupture.

Keywords: Fluid-Structure Interaction, arterial stenosis, Wall Shear Stress, Carotid Artery Bifurcation.

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Authors: R. Sullivan, T. Holden, G. Tremberger, Jr, E. Cheung, C. Branch, J. Burrero, G. Surpris, S. Quintana, A. Rameau, N. Gadura, H. Yao, R. Subramaniam, P. Schneider, S. A. Rotenberg, P. Marchese, A. Flamhlolz, D. Lieberman, T. Cheung

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Migration in breast cancer cell wound healing assay had been studied using image fractal dimension analysis. The migration of MDA-MB-231 cells (highly motile) in a wound healing assay was captured using time-lapse phase contrast video microscopy and compared to MDA-MB-468 cell migration (moderately motile). The Higuchi fractal method was used to compute the fractal dimension of the image intensity fluctuation along a single pixel width region parallel to the wound. The near-wound region fractal dimension was found to decrease three times faster in the MDA-MB- 231 cells initially as compared to the less cancerous MDA-MB-468 cells. The inner region fractal dimension was found to be fairly constant for both cell types in time and suggests a wound influence range of about 15 cell layer. The box-counting fractal dimension method was also used to study region of interest (ROI). The MDAMB- 468 ROI area fractal dimension was found to decrease continuously up to 7 hours. The MDA-MB-231 ROI area fractal dimension was found to increase and is consistent with the behavior of a HGF-treated MDA-MB-231 wound healing assay posted in the public domain. A fractal dimension based capacity index has been formulated to quantify the invasiveness of the MDA-MB-231 cells in the perpendicular-to-wound direction. Our results suggest that image intensity fluctuation fractal dimension analysis can be used as a tool to quantify cell migration in terms of cancer severity and treatment responses.

Keywords: Higuchi fractal dimension, box-counting fractal dimension, cancer cell migration, wound healing.

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Authors: H. Bouaziz, M. Sefi, J. de Lapuente, M. Borras, N. Zeghal

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Although, arsenic trioxide has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by arsenic trioxide in human keratinocytes (HaCaT) using the MTT [3-(4, 5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. Human keratinocytes were treated with different doses of arsenic trioxide for 4 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that arsenic trioxide significantly reduced the viability of HaCaT cells in a dose-dependent manner, showing an IC50 value of 34.18 ± 0.6 μM. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HaCaT cells associated with arsenic trioxide exposure. We observed a significant increase in comet tail length and tail moment, showing an evidence of arsenic trioxide -induced genotoxic damage in HaCaT cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by arsenic.

Keywords: Arsenic trioxide, cytotoxixity, genotoxicity, HaCaT.

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Authors: Wiriya Loongyai, Kiettisak Promphet, Nilubol Kangsukul, Ratchawat Noppha

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Polymerase chain reaction (PCR) assay and conventional microbiological methods were used to detect bacterial contamination of egg shells and egg content in different commercial housing systems, open house system and evaporative cooling system. A PCR assay was developed for direct detection using a set of primers specific for the invasion by A gene (invA) of Salmonella spp. PCR detected the presence of Salmonella in 2 samples of shell egg from the evaporative cooling system, while conventional cultural methods detected no Salmonella from the same samples.

Keywords: egg content, egg shell, invA gene, PCR, Salmonellaspp.

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Authors: Shishen Xie, Yingda L. Xie

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Tuberculosis (TB) is a potentially serious infectious disease that remains a health concern. The Interferon Gamma Release Assay (IGRA) is a blood test to find out if an individual is tuberculous positive or negative. This study applies statistical analysis to the clinical data of interferon-gamma levels of seventy-three subjects who diagnosed pulmonary TB in an anti-tuberculous treatment. Data analysis is performed to determine if there is a significant decline in interferon-gamma levels for the subjects during a period of six months, and to infer if the anti-tuberculous treatment is effective.

Keywords: Data analysis, interferon gamma release assay, statistical methods, tuberculosis infection.

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Authors: Asma Meziti, Hicham Meziti, Kaouthar Boudiaf, Benboubetra Mustapha, Hemama Bouriche.

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Nigella sativa L. is an aromatic plant belonging to the family Ranunculaceae. It has been used traditionally, especially in the middle East and India, for the treatment of asthma, cough, bronchitis, headache, rheumatism, fever, influenza and eczema. Several biological activities have been reported in Nigella sativa seeds, including antioxidant. In this context we tried to estimate the antioxidant activity of various extracts prepared from Nigella sativa seeds, methanolic extract (ME), chloroformic extract (CE), hexanic extract (HE : fixed oil), ethyl acetate extract (EAE) water extract (WE). The Folin-Ciocalteu assay showed that CE and EAE contained high level of phenolic compounds 81.31 and 72.43μg GAE/mg of extract respectively. Similarly, the CE and EAE exhibited the highest DPPH radical scavenging activity, with IC50 values of 106.56μg/ml and 121.62μg/ml respectively. In addition, CE and HE showed the most scavenging activity against superoxide radical generated in the PMS-NADH-NBT system with respective IC50 values of 361.86 μg/ml and 371.80 μg/ml, which is comparable to the activity of the standard antioxidant BHT (344.59 μg/ml). Ferrous ion chelating capacity assay showed that WE, EAE and ME are the most active with 40.57, 39.70 and 22.02 mg EDTA-E/g of extract. The inhibition of linoleic acid/ß-carotene coupled oxidation was estimated by ßcarotene bleaching assay, this showed a highest relative antioxidant activity with CE and EAE (69.82% of inhibition). The antioxidant activities of the methanolic extract and the fixed oil are confirmed by an in vivo assay in mice, the daily oral administration of methanolic extract (500 and 800 mg/kg/day) and fixed oil (2 and 4 ml/kg/day) during 21 days, resulted in a significant enhancement of the blood total antioxidant capacity (measured by KRL test) and the plasmatic antioxidant capacity towards DPPH radical.

Keywords: Antioxidant Capacity, Chelating, Phenolic Compounds, Nigella Sativa, Scavenger

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Authors: S. Techaoei, K. Jarmkom, P. Eakwaropas, W. Khobjai

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The objective of this research was focused on investigating in vitro antimicrobial activity of Phellinus linteus fruiting body extracts on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. Phellinus linteus fruiting body was extracted with ethanol and ethyl acetate and was vaporized. The disc diffusion assay was used to assess antimicrobial activity against tested bacterial strains. Primary screening of chemical profile of crude extract was determined by using thin layer chromatography. The positive control and the negative control were used as erythromycin and dimethyl sulfoxide, respectively. Initial screening of Phellinus linteus crude extract with the disc diffusion assay demonstrated that only ethanol had greater antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. The MIC assay showed that the lower MIC was observed with 0.5 mg/ml of Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus and 0.25 mg/ml. of Escherichia coli and Staphylococcus aureus, respectively. TLC chemical profile of extract was represented at R_f ≈ 0.71-0.76.

Keywords: Staphylococcus aureus, Phellinus linteus, methicillin-resistant Staphylococcus aureus, antimicrobial activity, Escherichia coli.

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Authors: Jung-hoon Ro, Soo-young Ye, Jae-hee Jung, Ah-young Jeon, Yun-jin KimIn-cheol Kim, Chul-han Kim, Gye-rok Jeon

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As the increase of intraoral acidity due to ingestion of sweet foods and acidic beverages usually bring forth a dental caries and a erosion, the measurement of intraoral pH is essential in the study of oral environment. The indwelling intraoral pH telemetry for lasting longer than 24 hours in the mouth was developed to overcome the limits of conventional wire electrode method previously used for salivary and plaque pH measurement, and to assess its effectiveness.

Keywords: pH telemetry, intraoral acidity, wireless.

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Authors: R. Kaur, Y. B. Pakade, J. K. Katnoria

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Considering the serious health hazards of air pollutants from automobiles, the present study was aimed to estimate the genotoxic/tumor inducing potential of three soil samples collected from junctions of Bus stand (BS), Crystal (CT) and Railway station (RS) of Amritsar, Punjab (India) using Allium cepa root chromosomal aberration assay (AlRCAA) and potato disc tumor assay (PDTA). The genotoxic potential in AlRCAA was 41.27% and 41.26% for BS; 37.89% and 43.38% for RS and 33.76% and 37.83% for CT during in situ and root dip treatments, respectively. The maximum number of tumors were induced in RS sample (64) followed by BS (21) and CT (9) during PDTA. The physicochemical parameters of soil sample were also studied and the concentration of lead was found to be 95.21 mg/Kg in RS, 35.30 mg/Kg in BS and 24.59 mg/Kg in CT samples.

Keywords: Automobiles, genotoxicity, Physicochemical parameters, pollutants.

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Authors: Harshani Uggallage, Kapila D. Dissanayaka

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A Sulforhodamine-B assay-guided fractionation on Nigella sativa seeds was conducted to determine the presence of cytotoxic compounds against human hepatoma (HepG2) cells. Initially, a freeze-dried sample of Nigella sativa seeds was sequentially extracted into solvents of increasing polarities. Crude extracts from the sequential extraction of Nigella sativa seeds in chloroform and ethyl acetate showed the highest cytotoxicity. The combined mixture of these two extracts was subjected to bioassay guided fractionation using a modified Kupchan method of partitioning, followed by Sephadex® LH-20 chromatography. This chromatographic separation process resulted in a column fraction with a convincing IC50 (half-maximal inhibitory concentration) value of 13.07 µg/ml, which is considerable for developing therapeutic drug leads against human hepatoma. Reversed phase High-Performance Liquid Chromatography (HPLC) was finally conducted for the same column fraction and the result indicates the presence of one or several main cytotoxic compounds against human HepG2 cells.

Keywords: Cytotoxic compounds, half-maximal inhibitory concentration, high-performance liquid chromatography, human HepG2 cells, Nigella sativa seeds, Sulforhodamine-B assay-guided fractionation.

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Authors: M. A. Okezue, K. L. Clase, S. R. Byrn

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The requirement for maintaining data integrity in laboratory operations is critical for regulatory compliance. Automation of procedures reduces incidence of human errors. Quality control laboratories located in low-income economies may face some barriers in attempts to automate their processes. Since data from quality control tests on pharmaceutical products are used in making regulatory decisions, it is important that laboratory reports are accurate and reliable. Zinc Sulphate (ZnSO₄) tablets is used in treatment of diarrhea in pediatric population, and as an adjunct therapy for COVID-19 regimen. Unfortunately, zinc content in these formulations is determined titrimetrically; a manual analytical procedure. The assay for ZnSO4 tablets involves time-consuming steps that contain mathematical formulae prone to calculation errors. To achieve consistency, save costs, and improve data integrity, validated spreadsheets were developed to simplify the two critical steps in the analysis of ZnSO4 tablets: standardization of 0.1M Sodium Edetate (EDTA) solution, and the complexometric titration assay procedure. The assay method in the United States Pharmacopoeia was used to create a process flow for ZnSO₄ tablets. For each step in the process, different formulae were input into two spreadsheets to automate calculations. Further checks were created within the automated system to ensure validity of replicate analysis in titrimetric procedures. Validations were conducted using five data sets of manually computed assay results. The acceptance criteria set for the protocol were met. Significant p-values (p < 0.05, α = 0.05, at 95% Confidence Interval) were obtained from students’ t-test evaluation of the mean values for manual-calculated and spreadsheet results at all levels of the analysis flow. Right-first-time analysis and principles of data integrity were enhanced by use of the validated spreadsheet calculators in titrimetric evaluations of ZnSO₄ tablets. Human errors were minimized in calculations when procedures were automated in quality control laboratories. The assay procedure for the formulation was achieved in a time-efficient manner with greater level of accuracy. This project is expected to promote cost savings for laboratory business models.

Keywords: Data integrity, spreadsheets, titrimetry, validation, zinc sulphate tablets.

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Authors: I. Savitskaya, A. Kistaubayeva, A. Zhubanova, I. Blavachinskaiya, D. Ibrayeva, M. Abdulzhanova, A. Otarbay, A.Isabekova

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This study was conducted for the investigation of number of cellulolytic bacteria and their ability in decomposition. Seven samples surface soil were collected on cellulose Zailiskii Alatau slopes. Cellulolitic activity of new strains of Bacillus, isolated from soil is determined. Isolated cellulose degrading bacteria were screened for determination of the highest cellulose activity by quantitative assay using Congo red, gravimetric assay and colorimetric DNS method trough of the determination of the parameters of sugar reduction. Strains are assigned to: B.subtilis, B.licheniformis, B. cereus and, В. megaterium. Bacillus strains consisting of several different types of cellulases have broad substrate specificity of cellulase complexes formed by them. Cellulolitic bacteria were recorded to have highest cellulase activity and selected for optimization of cellulase enzyme production.

Keywords: Cellulose-degrading bacteria, cellulase complex, foothills soil, screening.

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Authors: Rezvan Najafi, Korosh Heydari, Massoud Saidijam

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5-FU is a chemotherapeutic agent that has been used in colorectal cancer (CRC) treatment. However, it is usually associated with the acquired resistance, which decreases the therapeutic effects of 5-FU. miR-200c is involved in chemotherapeutic drug resistance, but its mechanism is not fully understood. In this study, the effect of inhibition of miR-200c in sensitivity of HCT-116 CRC cells to 5-FU was evaluated. HCT-116 cells were transfected with LNA-anti- miR-200c for 48 h. mRNA expression of miR-200c was evaluated using quantitative real- time PCR. The protein expression of phosphatase and tensin homolog (PTEN) and E-cadherin were analyzed by western blotting. Annexin V and propidium iodide staining assay were applied for apoptosis detection. The caspase-3 activation was evaluated by an enzymatic assay. The results showed LNA-anti-miR-200c inhibited the expression of PTEN and E-cadherin protein, apoptosis and activation of caspase 3 compared with control cells. In conclusion, these results suggest that miR-200c as a prognostic marker can overcome to 5-FU chemoresistance in CRC.

Keywords: Colorectal cancer, miR-200c, 5-FU resistance, E-cadherin, PTEN.

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Authors: M. Shahar Yar, M. Mustaqeem Abdullah, Jaseela Majeed

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A series of 1-(1H-benzimidazol-2-yl)-3-(substituted phenyl)-2-propen-1-one were allowed to react with hydrazine hydrate and phenyl hydrazine in submitted reactions to get pyrazoline and phenyl pyrazoline derivatives. All the compounds entered for screening at the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) for their in vitro antibacterial activity against Mycobacterium tuberculosis H37Rv strain (ATCC 27294) using Microplate Alamar Blue Assay (MABA) susceptibility test. The results expressed as MIC (minimum inhibitory concentration) in μg/mL. Among the fifteen compounds, eight compounds were found to have MIC values less than 10 μg/mL. These were subjected for cytotoxicity assay in VERO cells to determine CC50 (cytotoxic concentration 50%) values and finally SI (Selectivity Index) were calculated. Compound (XV) 2-[5-(4- fluorophenyl)-1-phenyl-4,5-dihydro-1H-3-pyrazolyl]-1Hbenzimidazole was considered the best candidate of the series that could be a good starting point to develop new lead compounds in the fight against tuberculosis.

Keywords: anti-tubercular activity, benzimidazole, pyrazoline.

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Authors: J. Carlos Kuri, Varun Vij, Raymond J. Turner, Orly Yadid-Pecht

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Celiac disease (CD) is an immune system disorder that is related to eating gluten. As gluten-free (GF) diet has become a concern of many people for health reasons, a gold standard had to be nominated. Enzyme-linked immunosorbent assay (ELISA) has taken the seat of this role. However, multiple limitations were discovered, and with that, the desire for an alternative method now exists. Nucleic acid based aptamers have become of great interest due to their selectivity, specificity, simplicity, and rapid-testing advantages. However, fluorescence-based aptasensors have been tagged as unstable, but lifespan details are rarely stated. In this work, the lifespan stability of a fluorescence-based aptasensor is shown over a 8-week long study displaying the accuracy of the sensor and false negatives. This study follows 22 different samples, including GF and gluten-rich (GR) and soy sauce products, off-the-shelf products, and reference material from laboratories; giving a total of 836 tests. The analysis shows an accuracy of correctly classifying GF and GR products of 96.30% and 100%, respectively, when the protocol is augmented with molecular sieves. The overall accuracy remains around 94% within the first 4 weeks and then decays to 63%.

Keywords: Aptasensor, PEG, rGO, FAM, RM, ELISA.

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

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Organic cation transporter (OCT) 1could influence an individual’s response to various treatments and increase their susceptibility to diseases.Genotypic and allelic frequencies of nineteen non-synonymous and one intronic Single Nucleotide Polymorphism (SNP) from the OCT1 gene were determined in 101 unrelated healthy Zulu participants, using a SNaPshot^® multiplex assay. Minor allele frequencies (MAF)were compared to representative populations of Africa, Asia and Europe, from Ensembl. MAFs for S14F, V519F, rs622342 and P341L were 2.0%, 6.0%, 6.0% and 1.0%, respectively. Sixteen of nineteen investigated non-synonymous SNPs were monomorphic. No study participant harbored variant alleles for S189L, G220V, P283L, G401S, M420V, M440I, G465R, I542V, R61C, R287G, C88S, A306T, A413V, I421F, C436F and V501E. Haplotype, CGTCGCCGCGCAAGAGGTGA, was most frequently observed (81.23%).Further investigations are encouraged to evaluate potential roles these SNPs could play in the therapeutic efficacy of clinically important drugs and in the development of various diseases in the Zulu population.

Keywords: OCT1, PCR, SNaPshot assay, Zulu population.

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Authors: S. Ibrahim, U. B. Abubakar, S. Danbirni, A. Usman, F. M. Ballah, C. A. Kudi, L. Lawson, G. H. Abdulrazak, I. A. Abdulkadir

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Performing culture and characterization of mycobacteria in low resource settings like Nigeria is a very difficult task to undertake because of the very few and limited laboratories carrying out such an experiment; this is a largely due to stringent and laborious nature of the tests. Hence, a rapid, simple and accurate test for characterization is needed. The “SD BIOLINE TB Ag MPT 64 Rapid ®” is a simple and rapid immunochromatographic test used in differentiating Mycobacteria into Mycobacterium tuberculosis (NTM). The 100 sputa were obtained from patients suspected to be infected with tuberculosis and presented themselves to hospitals for check-up and treatment were involved in the study. The samples were cultured in a class III Biosafety cabinet and level III biosafety practices were followed. Forty isolates were obtained from the cultured sputa, and there were identified as Acid-fast bacilli (AFB) using Zeihl-Neelsen acid-fast stain. All the isolates (AFB positive) were then subjected to the SD BIOLINE Analyses. A total of 31 (77.5%) were characterized as MTBC, while nine (22.5%) were NTM. The total turnaround time for the rapid assay was just 30 minutes as compared to a few days of phenotypic and genotypic method. It was simple, rapid and reliable test to differentiate MTBC from NTM.

Keywords: Culture, mycobacteria, non-tuberculous mycobacteria, SD bioline.

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Authors: Eszter Tóth, Ervin Welker

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Shadoo protein (Sho) was described in 2003 as the newest member of Prion protein superfamily [1]. Sho has similar structural motifs like prion protein (PrP) that is known for its central role in transmissible spongiform enchephalopathies. Although a great number of functions have been proposed, the exact physiological function of PrP is not known yet. Investigation of the function and localization of Sho may help us to understand the function of the Prion protein superfamily. Analyzing the subcellular localization of YFP-tagged forms of Sho, we detected the protein in the plasma membrane and in the nucleus of various cell lines. To reveal the localization of the endogenous protein we generated antibodies against Shadoo as well as employed commercially available anti-Shadoo antibodies: i) EG62 anti-mouse Shadoo antibody generated by Eurogentec Ltd.; ii) S-12 anti-human Shadoo antibody by Santa Cruz Biotechnology Inc.; iii) R-12 anti-mouse Shadoo antibody by Santa Cruz Biotechnology Inc.; iv) SPRN antibody against human Shadoo by Abgent Inc. We carried out immunocytochemistry on non-transfected HeLa, Zpl 2-1, Zw 3-5, GT1-1, GT1-7 and SHSY5Y cells as well as on YFP-Sho, Sho-YFP, and YFP-GPI transfected HeLa cells. Their specificity (in antibody-peptide competition assay) and co-localization (with the YFP signal) were assessed.

Keywords: Shadoo, prion protein, immunocytochemistry, antibody-peptide competition assay, antibody.

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Authors: Donna M. Morrison, Lambert Chester, Coretta A. N. Samuels, David R. Ledoux

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A survey was conducted in the five rice-growing regions in Guyana to determine the presence of aflatoxins in multiple fractions of rice in June/October 2015 growing season. The fractions were paddy, steamed paddy, cargo rice, white rice and parboiled rice. Samples were analyzed by High Performance Liquid Chromatography. A subset of the samples was further analyzed by enzyme-linked immunosorbent assay (ELISA) for concurrence. All analyses were conducted at the University of Missouri, USA. Of the 186 samples tested, 16 had aflatoxin concentrations greater than 20 ppb the recommended limit for aflatoxins in food according to the United States Food and Drug Administration. An additional three samples had aflatoxin B₁ concentrations greater than the European Union Commission maximum levels for aflatoxin B₁ in rice at 5 µg/kg and total aflatoxins (B₁, B₂, G₁ and G₂) at 10 µg/kg. The survey indicates that there is no widespread aflatoxin problem in rice in Guyana. The incidence of aflatoxins appears to be localized.

Keywords: Aflatoxins, enzyme-linked immunosorbent assay, high-performance liquid chromatography, rice fractions.

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