Titer Plate Research Articles

Screening of Drug-Transporter Interactions in a 3D Microfluidic Renal Proximal Tubule on a Chip

Drug-transporter interactions could impact renal drug clearance and should ideally be detected in early stages of drug development to avoid toxicity-related withdrawals in later stages. This requires reliable and robust assays for which current high-throughput screenings have, however, poor predictability. Kidney-on-a-chip platforms have the potential to improve predictability, but often lack compatibility with high-content detection platforms. Here, we combined conditionally immortalized proximal tubule epithelial cells overexpressing organic anion transporter 1 (ciPTEC-OAT1) with the microfluidic titer plate OrganoPlate to develop a screenings assay for renal drug-transporter interactions. In this platform, apical localization of F-actin and intracellular tight-junction protein zonula occludens-1 (ZO-1) indicated appropriate cell polarization. Gene expression levels of the drug transporters organic anion transporter 1 (OAT1; SLC22A6), organic cation transporter 2 (OCT2; SLC22A2), P-glycoprotein (P-gp; ABCB1), and multidrug resistance-associated protein 2 and 4 (MRP2/4; ABCC2/4) were similar levels to 2D static cultures. Functionality of the efflux transporters P-gp and MRP2/4 was studied as proof-of-concept for 3D assays using calcein-AM and 5-chloromethylfluorescein-diacetate (CMFDA), respectively. Confocal imaging demonstrated a 4.4 ± 0.2-fold increase in calcein accumulation upon P-gp inhibition using PSC833. For MRP2/4, a 3.0 ± 0.2-fold increased accumulation of glutathione-methylfluorescein (GS-MF) was observed upon inhibition with a combination of PSC833, MK571, and KO143. Semi-quantitative image processing methods for P-gp and MRP2/4 was demonstrated with corresponding Z′-factors of 0.1 ± 0.3 and 0.4 ± 0.1, respectively. In conclusion, we demonstrate a 3D microfluidic PTEC model valuable for screening of drug-transporter interactions that further allows multiplexing of endpoint read-outs for drug-transporter interactions and toxicity.

Antibacterial, antibiofilm, hemolytic activity and phytochemical study of various aerial parts of Moringa oleifera in Pakistan

Theamethanolicoextractsaofffiveeaerial parts ofaMoringa oleifera Lamwwereascreened for their antibacterial, antibiofilm and hemolytic activity.”Theaphytochemical screening of the methanolic extracts of aerial parts was also evaluated. The antibacterial, antibiofilmiactivityiof the extractssof various aerial parts of M. oleifera was evaluated by disc diffusion and micro titer plate assay method against two selected bacterial species. Hemolyticaactivity of the methanoliciextracts was screened against normal human erythrocytes. Comparatively the methanolic extracts of all aerial parts were more effective against Gram positive bacteria Bacillus subtilis and Gram negative bacteria Escherichia coli (E. coli) .The present study suggests that all aerial parts of this plant can be used as an antibacterial, anti-biofilm agent in treating ailments caused by the tested organisms (Bacillus subtilis and Escherichia coli). Methanolic extracts of aerial parts possessed minimum hemolytic activity. Significant antimicrobial, antibiofilm activity of the extract was found in this study.Asian J. Med. Biol. Res. March 2018, 4(1): 77-85

Evaluation of Acute Toxicity and In vitro Antitrypanosomal Activity of the Methanolic Leaves Extract of Ficus exasperata

Trypanosomosis is one of the tropical diseases in which new and better drugs are needed. Effective treatment for African trypanosomosis is beset with problems of drug resistance and toxicity. In this study acute toxicity and in vitro antitrypanosomal activity of the methanolic leaves extract of Ficus exasperata were investigated. The acute toxicity study was conducted in two phases. Doses of 10, 100 and 1000 mg/kg were administered to 3 groups of 3 mice each at the initial phase, while doses of 600, 1,000, 1,600 and 2,900 were administered to 4 groups of 1 mouse each at the final phase. The median lethal dose (LD ) was determined to be 775 mg/kg intraperitoneally in mice. The 50 antitrypanosomal activity of the extract was evaluated in vitro using a micro titre plate. Blood obtained from infected rat with T. brucei brucei (8.4 x 106) was incubated at room temperature (27 0C) for 4 hours with methanolic leaves extract at 40 and 80 mg/ml and 3.5 mg/ml of diminazene aceturate concentrations respectively. The parasites were inactivated and became uninfective to mice at both the extract and the standard drug concentrations used, while the parasites remained active and infective to mice in the negative control. The current study revealed that the methanolic leaves extract of of Ficus exasperata could possess trypanocidal principle that may require further scientific ellucidations.

Endophytic fungi of Tinospora cordifolia with anti-gout properties.

Targeting xanthine oxidase inhibition has been a prime strategy for exploration and development of non-purine based drugs for anti-hyperuricemic therapy. Recent studies from our laboratory have suggested that fungal endophytes from medicinal plants could play a pivotal role in exploration of novel therapeutic moieties. In the present study, 19 fungal endophytes were isolated from the medicinal plant Tinospora cordifolia and evaluated for their XO inhibitory activity. In the qualitative assay, Out of 19 fungal endophytes, 7 endophytes exhibited > 30% XO inhibition, of which isolates #1 TCSTITPLM, #53 TCSTITPLM, #105 TCSTITPLM and #83 TCSTITPLM were found to exhibit XO inhibition in the range of 38-45%. Further, ethyl acetate extract of #1TCSTITPLM exhibited 69% XO inhibitory activity followed by chloroform extract of #53 TCSTITPLM with 63% XOI activity in the in vitro uric acid estimation assay. In the quantitative NBT titer plate assay, both extracts exhibited 85% of XO inhibition. The present study is the first report of fungal endophytes isolated from T. cordifolia exhibiting XO inhibitory activity. Ethyl acetate and chloroform extract of #1 TCSTITPLM and #53 TCSTITPLM demonstrated potent XO inhibitory action. Further studies are warranted for purification and characterization of the XO inhibiting moieties.

Evaluation of Anti biofilm and Antibiotic Potentiation Activities of Silver Nanoparticles Against some Nosocomial Pathogens

Nowadays silver nanoparticles (AgNPs) are used as antimicrobial due to its well known physical, chemical, and biological properties. A large collection of bacterial cells adhering to a surface is called bacterial biofilm. Exposure to silver nano particles (AgNPs) may prevent colonization of new bacteria onto the biofilm. In the present work, we have investigated whether the biofilm formation of some hospital isolates of pathogenic bacteria can be influenced by AgNPs. Also we examined the susceptibility of the isolates to some antibiotics in combination with AgNPs. Standard strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecalis and Staphylococcus aureus, in addition to nine hospital isolates for each species were used. Tube double serial dilution method for determination of MIC of AgNPs against isolates was performed. Biofilm formation was evaluated by end smooth 96 cells micro titer plates. A disc diffusion method was used to assay the various antibiotics and combinations for bactericidal activity against the isolates. Mean MICs of AgNPs for Ps. aeruginosa, A. baumannii, E. faecalis and S. aureus were 3.125, 6.25, 6.25 and 12.5 ug/mL. respectively. AgNPs exhibited more than % 90 inhibitory effect on biofilm formation of the examined species. The results showed that AgNPs have strong anti bacterial and anti biofilm activity against the examined pathogens. Synergistic effects of AgNPs in combination with gentamicin, kanamycine, cephalosporin and penicillin were observed in different cases. Ps. aeruginosa and S. aureus showed more sensitivity (increase in fold) to examined antibiotics plus AgNPs compared to A. baumannii and E. faecalis. Our results demonstrated that AgNPs showing promising anti biofilm activity on hospital isolates of Ps. aeruginosa, A. baumannii, E. faecalis and S. aureus. The study also showed a possible combination of examined antibiotics with AgNPs which concluded as synergism.

MUTAGENIC ACTIVITY OF TILORONE – ACTIVE PHARMACEUTICAL SUBSTANCE OF AMIXIN IN MICROTITER PLATE VARIANT OF AMES TEST

Aim – to discover potential mutagenic activity of tilorone – active pharmaceutical substance of amixin. Methods. Mutagenic activity of tilorone was studied in the Ames test on Salmonella typhimurium ТА 98 (frame-shift mutations) and ТА 100 (Dot mutations – base pars change). Tilorone was examined in concentrations 5, 10, 50, 100 and 250 µg/ml. As standard mutagens, 2-nitrofluorene was used for Salmonella typhimurium TA 98 and sodium azide for Salmonella typhimurium TA 100 in tests without metabolic activation. In tests with metabolic activation, mutagen for both strains served as 2-aminoanthracene. The experiment was carried out with and without metabolic activation. In this work Muta-ChromoPlate kit (Biotoxicity, Canada) was used. Tilorone toxicity against salmonella strains was calculated at the same concentrations. Results. The obtained data show that in presence of standard mutagenic compounds amount of wells with mutated cells were 82–91%. At the same time in negative control this amount was not higher than 4.2%. In presence of tilorone amount of wells where mutated cells were detected, it was 4–16% – 3.3–13.0 times lower then the minimum value – 52%, which, according to protocol of micro titer plate test, is the evidence of mutagenic activity. In presence of all tilorone concentrations there is not any inhibition of bath growth of used Salmonella typhimurium strains. Conclusions. Tilorone in the range of used concentration did not posses any mutagenic activity or toxicity on S. typhimurium ТА 98 та ТА 100.

Automatic microfluidic enzyme-linked immunosorbent assay based on CLOCK-controlled autonomous centrifugal microfluidics

This paper reports on the development of an autonomous centrifugal microfluidic device, which automatically executes an enzyme-linked immunosorbent assay (ELISA) on a steady rotational frequency. The device was designed on the principles of CLOCK (Control of Liquid Operation on Centrifugal hydroKinetics) to achieve autonomous centrifugal microfluidics. These demonstrations confirmed that the device achieves accurate flow control of an automated ELISA with fluctuations of the interval time for implementation of each unit (antigen-antibody reaction, wash and injection of detection substrate) being less than 5%. The most optimal method for the immobilization of primary antibodies was investigated for the developed platform. We compared three methods of antibody immobilization on the developed platform, by using polystyrene microbeads, a polyurethane foam structure, and direct immobilization to the reaction chamber. We chose the use of polyurethane foam as the best immobilization method based on reaction efficiency and the convenient handling of the immobilized antibody on the solid phase. Measurement of human albumin was demonstrated using the developed device, and a calibration curve of human albumin detection was successfully prepared with an overall assay operation time of 18 min. The limit of detection (LOD) was estimated to be 0.516 ng/ml, which is comparable with the LOD of 0.707 ng/ml obtained by the assay on a conventional titer plate assay platform.

Study of the Anti-Staphylococcal Potential of Honeys Produced in Northern Poland.

The antimicrobial activity of 144 samples of honeys including 95 products from apiaries located in Northern Poland was evaluated. The antibacterial activity of those natural products, their thermal stability, and activity in the presence of catalase was investigated by microdilution assays in titration plates. The MTT assay was performed for the determination of anti-biofilm activity. Spectrophotometric assays were used for the determination of antioxidant potential, total phenolic content, and ability to generate hydrogen peroxide. Some of the investigated honeys exhibited surprisingly high antimicrobial, especially anti-staphylococcal, potential, with Minimal Inhibitory Concentration (MIC) values of only 1.56% (v/v). Much higher resistance was observed in the case of staphylococci growing as biofilms. Lower concentrations of the product, up to 12.5% (v/v) stimulated its growth and effective eradication of biofilm required concentration of at least 25% (v/v). Hydrogen peroxide has been identified as a crucial contributor to the antimicrobial activity of honeys supplied by Polish beekeepers. However, some of the results suggest that phytochemicals, especially polyphenols, play an important role depending on botanical source (both positive, e.g., in the case of buckwheat honeys as well as negative, e.g., in the case of some rapeseed honeys) in their antimicrobial potential.

B-Cell Epitope Mapping Using a Library of Overlapping Synthetic Peptides in an Enzyme-Linked Immunosorbent Assay.

This chapter describes a strategy for mapping linear B-cell epitopes of proteins using synthetic biotinylated peptides in an ELISA.A set of overlapping peptides were designed based upon a known amino acid sequence of the target protein, VapA (Virulence-associated Protein A) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals. The peptides synthesized as biotinylated peptides were coated directly onto micro titer plates which had been pre-coated with NeutrAvidin™ and used to screen sera from foals confirmed to have R. equi disease. A linear B-cell epitope was identified which corresponded to a 20mer sequence of the VapA protein.

Phage therapy of corrosion-producing bacterium Stenotrophomonas maltophilia using isolated lytic bacteriophages

PurposeCorrosion-producing microorganisms have different physiology and include sulfate-reducing bacteria, iron oxidizers and magnesium oxidizers. Biocorrosion has been seen in various industries, especially the petrochemicals and oil industries. One proposal to solve this problem is the use of bacteriophages to treat the bacteria-caused corrosion. The aims of this study were isolation and identification of corrosion-producing bacteria from petroleum pipeline corrosion as well as finding their specific bacteriophages for phage therapy purposes.Design/methodology/approachThe sample pipes with the corrosion were obtained from the Gandomkar petroleum pipeline station, Chaharmahal and Bakhtiari, Iran. For screening the corrosion-producing bacteria, the rusted pipe samples were cultured in a selective culture medium, manganese agar. The purified individual colonies were subjected to molecular examinations. For isolating bacteriophages from silversmithing workshops wastewater in Isfahan, whole plate titration methods and transmission electron microscopy were used to isolate and detect phages.FindingsThe cultivation of corrosion-based material on manganese agar after 18 hours incubation at 30°C resulted in the isolation of cream-colored colonies. The microscopic examinations showed Gram-negative coccobacilli. Based on molecular examinations, the isolated bacteria were identified as Stenotrophomonas maltophilia strain PBM-IAUF-2 with Genebank accession number of KU145278.1. The found bacteriophage was related to the Siphoviridae family of phages.Originality/valueThis paper is the first report of isolation and identification of corrosion-producing bacteria and its specific lytic phages from Gandomkar petroleum pipeline station, Chaharmahal and Bakhtiari, Iran. The biological procedures for preventing the microbial corrosion could be an asset and considered as a potential in the petroleum and industrial microbiology. Phage therapy is considered as one of the economical methods for reducing the biocorrosion.

Influence of subinhibitory-concentration (sub-MIC) Cefetoxime on biofilm formation. SEM study of ESBL-producing Salmonella typhi

Objectives: In the present study, we have analyzed ESBL-producing S. typhi’s capability in forming a significant amount of biofilm on plastic and glass surface, and the influence of cefetoxime on biofilm development at subinhibitory (Sub-MIC) concentration. Methods: Nine strains of cefetoxime-mediated ESBL-producing S. typhi were used in the study. S. typhi formed biofilm on plastic and glass materials; it was demonstrated using micro titre plate (MTP) and standard test tube methods. Comparative study of the influence of cefetoxime on biofilm formation in its MIC (128 µg/ml) and at sub-MIC (64 µg/ml) was demonstrated by microtitre plate method. The biofilm production was observed in SEM images, statistical analysis (ANOVA) showed significant increase in cell surface and volume due to the influence of Cefetoxime. Results: Of the nine selected isolates, two S. typhi strains, namely BST 51 and BST 130, produced relatively strong biofilm in the presence of cefetoxime at sub-MIC level (64 µg/ml), comparatively weak biofilm formation at MIC level (128 µg/ml). Typical morphological changes were observed in cefetoxime-resistant strains, S. typhi BST 51 and BST 130, in comparison to cefetoxime-sensitive strain S. typhi BST 63 used as a control. We found an increase in surface and volume of a cell in response to cefetoxime and statistical data (ANOVA) proved that resistant strains were significantly different from control strains. Conclusion: The above study clearly shows that cefetoxime at sub-MIC level efficiently induces biofilm formation and promotes changes in morphology of the cell. J Microbiol Infect Dis 2017; 7(2): 67-75

One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm.

Objective(s):Pseudomonas aeruginosa is a Gram-negative and aerobic rod bacterium that displays mucoid and non-mucoid phenotype. Mucoid strains secrete alginate, which is the main agent of biofilms in chronic P. aeruginosa infections, show high resistance to antibiotics; consequently, the biological disruption of mucoid P. aeruginosa biofilms is an attractive area of study for researchers. Alginate lyase gene (algl) is a member of alginate producing operon which by glycosidase activity produces primer for other enzymes in this cluster. Also this activity can destroy the extracellular alginate; therefore this enzyme participates in alginate production and destruction pathway. Alginate lyase causes detachment of a biofilm by reducing its adhesion to the surfaces, and increases phagocytosis and antibiotic susceptibility. In this study, alginate lyase was purified in just one step and its properties were investigated.Materials and Methods:The purification was done by affinity chromatography, analysed by SDS-PAGE, and its effect on P. aeruginosa biofilms was surveyed by micro titer plate assay and SEM. The substrate specificity of the enzyme was determined by PCR.Results:Alginate lyase from isolate 48 was purified in one step. It is more thermally resistant than alginate lyase from Pseudomonas aeruginosa PAO1 and poly M, poly G and poly MG alginate were the substrate of this enzyme. Moreover, it has an eradication effect on biofilms from P. aeruginosa 48 and PAO1.Conclusion:In this study an alginate lyase with many characteristics suitable in medicine such as thermal stability, effective on poly M alginate, and bacterial biofilm destructive was introduced and purified.

Potential application of synthesized ferrocenylimines compounds for the elimination of bacteria in water

This work reports a study towards a search for environmentally friendly water disinfectant. The most common method for water treatment is based on chlorine which had a wide application over the years as a water disinfectant, but suffer the disadvantage of reacting with natural organic matter to form disinfection by products. In this study, the potential application of novel ferrocenylimines compounds, namely 4-ferrocenylaniline (1), N-(3-bromo-2-hydroxylbenzylidene)-4-ferrocenylimine (2) and N-(3-bromo-5-chlorosalicyl)-4-ferrocenylimine (3) for the elimination of bacteria in water was investigated by evaluating their antibacterial properties against twelve different bacterial strains using microdilution method in sterile 96 well micro titer plates. The in vitro antibacterial activity revealed that the ferrocenylimines compound exhibit higher antibacterial activity than ferrocene, which is one of the starting materials towards the synthesis of this novel ferrocenylimines compounds. The most active ferrocenylimines compound was compound 3 with a minimal inhibitory concentration (MIC) value of 0.30 mg/ml against S. sonnei. In addition, all the ferrocenylimines compounds possessed excellent antibacterial activity against B. cereus with the same MIC value of 0.31 mg/ml. The results obtained so far show great potential in the three tested ferrocenylimines compounds for use in water treatment in killing bacteria in water.

Synthesis and Characterization of as-formed and Calcined MnFe2O4 nanoparticles: A comparative study of their Antibacterial Activities

In this study, MnFe2O4 NPs were synthesized through Solution combustion method at a temperature of about 450°C using Oxalyl Dihydrazide (ODH) as a chemical fuel. The synthesized MnFe2O4 nanomaterial was calcined at a temperature of 700°C. The structural properties and surface morphology of the synthesized NPs were characterized by Powder X-ray Diffraction technique (PXRD) and Scanning Electron Microscopy (SEM). Further these NPs were subjected to antibacterial studies with different strains of Gram-positive and Gram-negative bacteria by Agar well diffusion method and Microbroth dilution technique. The comparison between the bacterial inhibition potential of the synthesized NPs were investigated by the zone of inhibition obtained around the agar wells and the colour change obtained in the titre plate wells containing the bacterial suspension with respect to different concentrations of the as-formed and calcined MnFe2O4 NPs.

Therapeutic Effect of Dendrobium candidum on Lupus Nephritis in Mice.

Context:Dendrobium candidum (D. candimum) widely is a functional drug. The curative effect of D. candidum on lupus nephritis has been studied in vivo.Materials and Method:The DBA/2 and B6D2F1 mice were used for this in vivo experiment. The 50% effective dose (ED50) was used to check the effective concentration for this study. Then the SCr, BUN, TC, TG, IL-6, IL-12, TNF-α, and IFN-γ levels were determined by kits. The output of urine protein was determined by means of Coomassie Brilliant Blue, and the auto-antibody dsDNA was determined with titer plate technology and indirect immunofluorescence. The NF-κB, IκB-α, TGF ‘β1, Fas, and FasL expressions were measured by RT-PCR and western blot assay. The component analysis of D. candidum was determined by nuclear magnetic resonance.Results:Based on the ED50 result at 329 mg/kg, 200 and 400 mg/kg doses were chosen for this study. SCr, BUN, TC and TG levels of 400 mg/kg D. candidum mice were lower than control mice, TP and ALB levels were higher than control mice. The control and 400 mg/kg treated mice tested positive for dsDNA at the end of sixth and tenth week after the experiment began. The glomerular number of 400 mg/kg treated mice was more than control group. Treatment with 400 mg/kg D. candidum reduced IL-6, IL-12, TNF-α and IFN-γcytokine levels as compared to control mice. D. candidum decreased NF-κb, TGF ‘β1, Fas, FasL and increased IκB-α expressions in kidney tissue. There were 11 compounds in dry D. candidum, these compounds might make the curative effects of lupus nephritis.Conclusion:D. candidum showed a potential curative effect on lupus nephritis. It could be used as a health medicine on lupus nephritis.SUMMARY D. candidum reduced the SCr, BUN, TC, TG serum levels and raised the TP, ALB levels compared to control group.The glomerular number of D. candidum treated mice was more than control group.D. candidum treated mice showed lower IL-6, IL-12, TNF-α and IFN-γ cytokine levels than control mice.D. candidum decreased NF-κb, TGF-β1, Fas, FasL and increased IκB-α expressions in kidney tissue. Abbreviations used: LN: Lupus nephritis, SLE: systemic lupus erythematosus, D. candidum: Dendrobium candidum; IL-6: interleukin-6, IL-12: interleukin-12, TNF-α: tumor necrosis factor alpha, IFN-γ: Interferon-gamma, SCr: serum creatinine, BUN: blood urea nitrogen, TC: total cholesterol, TG: triglyceride, TP: total protein, ALB: albumin.

Confocal and SEM imaging to demonstrate food pathogen- biofilm biocontrol by pyocyanin from Pseudomonas aeruginosa BTRY1

An assortment of redox-active phenazine compounds like pyocyanin with their characteristic blue-green colour are synthesized by Pseudomonas aeruginosa, Gram-negative opportunistic pathogens, which are also considered one of the most commercially valuable microorganisms. In this study, pyocyanin from Pseudomonas aeruginosa BTRY1 from food sample was assessed for its antibiofilm activity by micro titer plate assay against strong biofilm producers belonging to the genera Bacillus, Staphylococcus, Brevibacterium and Micrococcus. Pyocyanin inhibited biofilm activity in very minute concentrations. This was also confirmed by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM). Both SEM and CLSM helped to visualize the biocontrol of biofilm formation by eight pathogens. The imaging and quantification by CLSM also established the impact of pyocyanin on biofilm-biocontrol mainly in the food industry.

Study Gene Expression for phzA Gene for PHzA Gene Responsible of Pyocyanin Production of P. aeruginosaby qPCR.

Identification of 66 bacterial isolates obtained from different sources has been shoud belonging to the bacterium P. aeruginosa in arate of 46 infection isolation and 20 environmental isolates which confirmed by Microscopy diagnosis and Biochemical tests.Micro titration plates method was (MTP) used for detection of isolates abilites to produce Pyocyanin dye. The isolates were differed in their production of Pyocyanin dye between productive with high efficiency, medium efficiency and low efficiency.For the study of gene expression of these isolates, extracted RNA was obtaind from of all isolates, then measure the amount of gene expression by qRT- PCR technology for gene PhzAresponsible for production of Pyocyanin dye, was studied the isolates incuded were 56 as treatment isolates and 10 as control isolates of each gene using the One Step Real time–PCR. It was found that the phzA gene expression by relative Quantification method ,within the range (0.93-2.12) ,with highly significant differences and probability (P

Micro system comprising 96 micro valves on a titer plate

A system of 96 micro valves has been developed and mounted on top of a 48-well micro titer plate providing two valves for each well controlling its air inlet and outlet. Testing of the valve system showed that all valves are working and are opened and closed reliably. A pneumatic system is switching inlet and outlet valves independently of each other. The geometry of the feed channels ensures an equal air flow through all wells, when the valves are open. Between the micro valves, one optical fibre was inserted through the lid of each well allowing measuring the oxygen partial pressure in the enclosed air volume by fluorescence sensor spots. Escherichia coli bacteria were grown inside the wells and their metabolism was observed by the oxygen partial pressure change due to respiration. In all 48 wells, the same oxygen transfer rate was observed within an averaged standard deviation of 1 mmol/L/h. The oxygen transfer rate differences compared to a macroscopic standard shake flask system were overall compatible within their uncertainties.

Calculation of the ELISA's cut-off based on the change-point analysis method for detection of Trypanosoma cruzi infection in Bolivian dogs in the absence of controls.

In ELISAs, sera of individuals infected by Trypanosoma cruzi show absorbance values above a cut-off value. The cut-off is generally computed by means of formulas that need absorbance readings of negative (and sometimes positive) controls, which are included in the titer plates amongst the unknown samples. When no controls are available, other techniques should be employed such as change-point analysis. The method was applied to Bolivian dog sera processed by ELISA to diagnose T. cruzi infection. In each titer plate, the change-point analysis estimated a step point which correctly discriminated among known positive and known negative sera, unlike some of the six usual cut-off formulas tested. To analyse the ELISAs results, the change-point method was as good as the usual cut-off formula of the form “mean + 3 standard deviation of negative controls”. Change-point analysis is therefore an efficient alternative method to analyse ELISA absorbance values when no controls are available.

Antibacterial activity of Tribulus terrestris methanol extract against clinical isolates of Escherichia coli

Summary Introduction: Tribulus terrestris L. is traditionally used for treatment of urinary tract infections. Escherichia coli, as the most prominent agent of urinary tract infections, can be sensitive to T. terrestris extract. Objectives: The aim of this study was to evaluate the antibacterial activity of T. terrestris methanol extract against clinical isolates of E. coli from urinary tract infections. Saponins were determined as main constituents of T. terrestris methanol extract. Methods: The antibacterial activities of T. terrestris methanol extract were evaluated by micro-broth dilution assay. The synergistic effects of T. terrestris methanol extract were screened with gentamicin by micro titer plate and disc diffusion assays. The isobologram curve was figured and the Fractional Inhibitory Concentration Index (FICI) was determined. Results: The saponin content of T. terrestris methanol extract was 54% (w/w). The means of MIC and MBC values for E. coli clinical isolates (n=51) were 3.5±0.27 and 7.4±0.5 mg/ml while these amounts were 3.9±1.3 and 6.4±1.8 μg/ml for gentamicin. T. terrestris methanol extract and gentamicin had synergistic effect with FICI equal to 0.1375. Conclusion: Therefore, T. terrestris can be applicable as alternative treatment in management of urinary tract infections.