Automated Plate Reader Research Articles

CccDNA-Targeted Drug Screen Reveals a Class of Antihistamines as Suppressors of HBV Genome Levels.

Chronic infection with hepatitis B virus (HBV) is incurable, as the current therapeutics cannot eliminate its persistent genomic material, cccDNA. Screening systems for cccDNA-targeting therapeutics are unavailable, as low copies of cccDNA in vitro complicate detection. To address this, cccDNA copies were massively increased to levels detectable via automated plate readers. This was achieved via continuous infection in a contact-free co-culture of an HBV generator (clone F881), which stably produced clinically relevant amounts of HBV, and HBV acceptors selected to carry high cccDNA loads. cccDNA-targeted therapeutics were then identified via reduced cccDNA-specific fluorescence, taking differences in the cell numbers and viability into account. Amongst the drugs tested, the H1 antihistamine Bilastine, HBVCP inhibitors and, surprisingly, current HBV therapeutics downregulated the cccDNA significantly, reflecting the assay's accuracy and sensitivity in identifying drugs that induce subtle changes in cccDNA levels, which take years to manifest in vivo. Bilastine was the only therapeutic that did not reduce HBV production from F881, indicating it to be a novel direct suppressor of cccDNA levels. When further assessed, only the structurally similar antihistamines Pitolisant and Nizatidine suppressed cccDNA levels when other H1 antihistamines could not. Taken together, our rapid fluorescence cccDNA-targeted drug screen successfully identified a class of molecules with the potential to treat hepatitis B.

Comparison of the APAS Independence Automated Plate Reader System with the Manual Standard of Care for Processing Urine Culture Specimens.

ABSTRACTUrine cultures are among the highest-volume tests in clinical microbiology laboratories and usually require considerable manual labor to perform. We evaluated the APAS Independence automated plate reader system and compared it to our manual standard of care (SOC) for processing urine cultures. The APAS device provides automated image interpretation of urine culture plate growth and sorts those images that require further evaluation. We examined 1,519 specimens over a 4-month period and compared the APAS growth interpretations to our SOC. We found that 72 of the 1,519 total specimens (4.74%) had growth discrepancies, where these specimens were interpreted differently by the APAS and the technologist, which required additional evaluation of plate images on the APAS system. Overall, there were 56 discrepancies in pathogen identification, which were present in 3.69% of the cultures. An additional pathogen was uncovered in a majority of these discrepancies; 12 (21.4%) identified an additional pathogen for the SOC, and 40 (71.4%) identified an additional pathogen for the APAS workflow. We found 214 (2.69%) antimicrobial susceptibility test (AST) discrepancies; 136 (1.71%) minor errors (mEs), 41 (0.52%) major errors (MEs), and 36 (0.45%) very major errors (VMEs). Many of the MEs and VMEs occurred in only a small subset of 13 organisms, suggesting that the specimen may have had different strains of the same pathogens with differing AST results. Given the significant labor required to perform urine cultures, the APAS Independence system has the potential to reduce manual labor while maintaining the identity and AST results of urinary pathogens.IMPORTANCE Urine cultures are among the highest-volume tests performed in clinical microbiology facilities and require considerable manual labor to perform. We compared the results of our manual SOC workflow with that of the APAS Independence system, which provides automated image interpretation and sorting of urine culture plates based on growth. We examined 1,519 urine cultures processed using both workflows and found that only 4.74% had growth pattern discrepancies and 3.69% pathogen identification discrepancies. There was substantial agreement in AST results between workflows, with only 2.69% having discrepancies. Only 1.71% of the ASTs had mEs, 0.52% had MEs, and 0.45% had VMEs, with most of the MEs and VMEs belonging to a small subset of organisms. The APAS system significantly decreased manual urine culture processing, while providing similar results to the SOC. As such, incorporating such automation into laboratory workflows has the potential to significantly improve efficiency.

ELISpot Assay for the Detection of ASFV-Specific Interferon-Gamma (IFN-γ)-Producing Cells.

The enzyme-linked immunospot (ELISpot) assay is a technique of unparalleled sensitivity to determine the frequency of antigen-specific immune cells secreting an immunomodulatory mediator upon recall antigen stimulation, making it a valuable tool in vaccine research. Typically done in multi-well microplate format, it also allows a high-throughput analysis of numerous immune cell samples, e.g., from different donor subjects, especially with the help of automated plate readers and specialized software that currently exist in most laboratories. IFN-γ is a hallmark cytokine secreted especially by T-cell subsets in recall response to pathogens, and consequently the IFN-γ ELISpot assay is one of the most widely used. The cellular arm of the immune response is known to be fundamental in protection against virulent ASFV, and therefore this assay is frequently employed in ASFV vaccine research to evaluate the results from immunization experiments.The technique involves the use of plates with wells that have a membrane for base with a strong binding capacity for amino acids that thus can be densely coated with an antibody for IFN-γ. Upon adding cells and specific antigen or other control stimuli, responding cells will release IFN-γ that is captured by the antibody in close proximity and revealed using a second antibody (sandwich method) through either chromogenic or fluorescent methods, leading to the detection of a "spot" on the membrane for each positive cell. Here we detail our protocol to detect the frequency of ASFV antigen-specific IFN-γ-producing cells in immunized pig lymphocytes and give an example of a typical result using the technique.

High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening.

Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters. The method is based on dynamic self-assembly of commercially available chiral amines, 2-formylphenylboronic acid, and chiral diols in acetonitrile to form fluorescent diastereomeric complexes. Each analyte enantiomer engenders a diastereomer with distinct fluorescence wavelength/intensity originating from enantiopure fluorescent ligands. In this assay, enantiomers of amines and amine derivatives assemble with diol-type ligands containing a binaphthol moiety (BINOL and VANOL), whereas diol enantiomers form complexes with the enantiopure amine-type fluorescent ligand tryptophanol. The differential fluorescence is utilized to determine the amount of each enantiomer in the mixture with an error of <1% e.e. This method enables high-throughput real-time evaluation of enantiomeric/diastereomeric excess (e.e./d.e.) and product yield of crude asymmetric reaction products. The procedure comprises high-throughput liquid dispensing of three components into 384-well plates and recording of fluorescence using an automated plate reader. The approach enables scaling up the screening of combinatorial libraries and, together with parallel synthesis, creates a robust platform for discovering chiral catalysts or auxiliaries for asymmetric transformations and chiral drug development. The procedure takes ~4-6 h and requires 10-20 ng of substrate per well. Our fluorescence-based assay offers distinct advantages over existing methods because it is not sensitive to the presence of common additives/impurities or unreacted/incompletely utilized reagents or catalysts.

Enzyme-Linked Immunosorbent Assay for Detection of Serum or Mucosal Isotype-Specific IgG and IgA Whole-Virus Antibody to Influenza A Virus in Swine.

Enzyme-linked immunosorbent assays can be used to detect isotype-specific anti-influenza antibodies in biological samples to characterize the porcine immune response to influenza A virus (IAV). The isotype antibody assay is based on an indirect ELISA using whole influenza virus as antigen and commercial antibodies directed against porcine IgG and IgA. Samples such as serum, nasal wash, and bronchoalveolar lavage fluid allow for evaluation of systemic, upper, and lower respiratory tract mucosal antibody responses, respectively. The isotype ELISA assay is performed in a 96-well format using IAV test antigen and anti-swine IgG or IgA detection antibodies conjugated to an enzyme that catalyze a color change reaction. The optical density of the sample is measured using an automated plate reader. The assay is useful to characterize the IgG or IgA immune response to challenge or vaccination against specific IAV isolates in different compartments of the immune system.

Multi-well plate cell contraction assay detects negatively correlated cellular responses to pharmacological inhibitors in contractility and migration

To enable large-scale screening of signaling molecules and drugs that regulate cellular contractility-associated mechanotransduction, we previously modified, particularly in terms of the capability of efficiently collecting big data, conventional methodologies using wrinkled substrates to determine the cellular contractility. Here, we present a new system to perform the wrinkle-based cell force assay in a multi-well plate format conformed to standardized geometric configurations and compatible with available technologies such as automated plate readers. With this highly improved throughput in terms of hardware as well as software using a deep learning-based technology, we evaluated the effect of treating cells with various types of pharmacological inhibitors on the cellular contractility. We found opposite responses of cells to the inhibitors between the contractility and collective migration activities. While similar inverse relationships between the contractility and migration have been reported in separate studies, our results here with the high-throughput screening system more broadly generalized these observations.

The impact of transient heat transfer on tissue culture cell distribution

It is common practice to subculture adherent eukaryotic cells in multi-well plates at room temperature before 37 °C incubation for growth. Under these conditions, cell distribution was non-uniform with a higher density of cells near the edges of the wells. Non-uniform cell density can alter cell behaviour in numerous biological assays and can result in variability when using automated plate readers on intact cells. This study investigated the possibility that the non-uniform cell distribution was caused by temperature gradients in the growth medium and well walls. Cell density analyses revealed significantly greater cell densities near well walls. Temperature distribution was documented using infrared imaging and temperature-sensitive LCD films, and a transient heat transfer mathematical model was developed to characterise the system and compared to the cell density results. The model predicts that an initial less-than-1 °C temperature gradient is present in the well shortly after initiating 37 °C incubation, leading to preferential cell adhesion to the warmer edges of wells in the first ∼30 min of incubation. Techniques to remediate non-uniform cell distribution were evaluated, and a simple method proved effective to promote uniform cell densities across wells of 24-well plates.

A Semi-automated High-Throughput Microtitration Assay for Filoviruses.

The 50% tissue culture infectious dose (TCID50) endpoint dilution assay is one of the gold standard methods for measuring filovirus infectivity. We have increased virology microtitration assay throughput at biosafety level (BSL)-4 by implementing automated liquid handling and semi-automated assay endpoint readout. Utilization of automated liquid handling for cell plating and virus dilution along with optimization of the assay endpoint readout, using a luminescent-based cell viability assay and an automated plate reader, has improved workflow efficiency, reduced operator burden and assay time, decreased assay variability, and increased data return.

Principal component analysis and kinetic model adjustment to describe the community-level physiological profiles (CLPP) in the sediments of the river Minho estuary (Northern Portugal)

Principal component analysis and kinetic model adjustment to describe the community-level physiological profiles (CLPP) in the sediments of the river Minho estuary (Northern Portugal)

PO271 THE PLASMA ADIPONECTIN LEVELS IN ELDERLY PATIENTS WITH PREDIABETES

Background: Ginseng have been proposed to mediate antidiabetic effect. The active components of ginseng are thought to be ginsenoside and distributed in many parts of the ginseng plant. Rg3, one of the active ingredients of ginseng saponins, has been known about the effects on molecularmechanism related to diabetes including inhibition the palmitate-induced beta-cell apoptosis and enhancement the glucose uptake. Glucose variability could induce a higher degree of apoptosis and decreased the insulin secretory capacity in beta-cells. In the present study, we investigated whether Rg3 suppresses intermittent high glucose(IHG)induced beta-cell apoptosis and improved insulin secretion in INS-1 pancreatic beta-cells. Method: To perform Glucose Fluctuation and protection effect by ginsenoside Rg3, mediums were aspirated and new medium containing Glucose Fluctuation or/and Rg3 concentrations was added and incubated for 2days. Annexin V staining for apoptosis detection analyzed by FACsort. Cell viability was determined by CCK-8 assay. The optical density was measured at 450 nm by automatic plate reader. Quantification of secreted insulin protein by ELISA was measured using Rat/mouse Insulin ELISA kit. BrdU staining was added in and quantification of secreted insulin proteins were analyzed by FACSort. Result: After 48hrs, INS-1 cell treated with alternative high glucose concentration(33mM glucose) and low glucose concentration(5mM) increased beta-cells apoptosis by 5.37 fold. But culturing with Rg3 1mM, 5mM,10mM, the percentages of apoptotic cells were reduced by dose dependently. Cell viability was reduced 63% by IHG. This toxic effect was also attenuated by culturing with Rg3. Additionally, when we added BrdU to culture medium and labeling of proliferating cells, cell proliferation was tended to be increased. And the ratio of quantification of secreted insulin protein/total protein was also tended to be increased with Rg3 dose dependently. Conclusion: Ginsenoside Rg3 suppresses intermittent high glucose-induced apoptosis and increases cell viability. Also it has tendancy to increase cell proliferation and the ratio of insulin secreting protein/total protein in INS-1 pancreatic beta-cells.

Simultaneous titration and phenotypic antiviral drug susceptibility testing for herpes simplex virus 1 and 2

BackgroundMost herpes simplex virus (HSV) isolates from treatment-naïve patients are susceptible to antivirals. However, prolonged antiviral therapy can select for drug-resistant strains, especially in immunocompromised patients. Standard phenotypic methods for antiviral resistance testing are labor and time-intense and molecular resistance determinants are insufficiently understood for routine diagnostic use of genotypic resistance testing. ObjectiveTo enable rapid, scalable antiviral susceptibility testing and minimize viral passage, we developed a 7-day, 96-well assay for simultaneous HSV 1/2 titration and phenotypic resistance testing for acyclovir and foscarnet. Study designThe assay was optimized and validated by testing clinical isolates and laboratory strains (n=39) with known IC50 for acyclovir (23 resistant) and foscarnet (1 resistant) based on plaque reduction or dye-uptake assays. A chemiluminescent detection reagent is used for quantification of cytopathic effect instead of plaque counting or measuring dye-uptake. Drug concentrations inhibiting 50% of chemiluminescent signal reduction (IC50) were determined concurrently at each of three virus dilutions. ResultsResults agree for 92.3% (acyclovir) and 100% (foscarnet) of isolates. For all three discordant samples, results of reference testing by plaque reduction agreed with the chemiluminescent assay. Reproducibility studies showed 100% qualitative agreement and 3–37% coefficient of variation based on IC50. ConclusionsChemiluminescence detection as a surrogate for cellular viability with an automated plate reader provides improved throughput and workflow, as well as high accuracy and reproducibility for antiviral drug susceptibility testing.

Hierarchical Functional Specificity of Cytosolic Heat Shock Protein 70 (Hsp70) Nucleotide Exchange Factors in Yeast

Heat shock protein 70 (Hsp70) molecular chaperones play critical roles in protein homeostasis. In the budding yeast Saccharomyces cerevisiae, cytosolic Hsp70 interacts with up to three types of nucleotide exchange factors (NEFs) homologous to human counterparts: Sse1/Sse2 (Heat shock protein 110 (Hsp110)), Fes1 (HspBP1), and Snl1 (Bag-1). All three NEFs stimulate ADP release; however, it is unclear why multiple distinct families have been maintained throughout eukaryotic evolution. In this study we investigate NEF roles in Hsp70 cell biology using an isogenic combinatorial collection of NEF deletion mutants. Utilizing well characterized model substrates, we find that Sse1 participates in most Hsp70-mediated processes and is of particular importance in protein biogenesis and degradation, whereas Fes1 contributes to a minimal extent. Surprisingly, disaggregation and resolubilization of thermally denatured firefly luciferase occurred independently of NEF activity. Simultaneous deletion of SSE1 and FES1 resulted in constitutive activation of heat shock protein expression mediated by the transcription factor Hsf1, suggesting that these two factors are important for modulating stress response. Fes1 was found to interact in vivo preferentially with the Ssa family of cytosolic Hsp70 and not the co-translational Ssb homolog, consistent with the lack of cold sensitivity and protein biogenesis phenotypes for fes1Δ cells. No significant consequence could be attributed to deletion of the minor Hsp110 SSE2 or the Bag homolog SNL1. Together, these lines of investigation provide a comparative analysis of NEF function in yeast that implies Hsp110 is the principal NEF for cytosolic Hsp70, making it an ideal candidate for therapeutic intervention in human protein folding disorders.

Enzyme-linked immunosorbent assay for detection of serum or mucosal isotype-specific IgG and IgA whole-virus antibody to influenza A virus in swine.

Enzyme-linked immunosorbent assays can be used to detect isotype-specific anti-influenza antibodies in biological samples to characterize the porcine immune response to influenza A virus. The isotype antibody assay is based on an indirect ELISA using whole influenza virus as antigen and detection antibodies directed against porcine IgG and IgA. Samples such as serum, nasal wash, and bronchoalveolar lavage fluid allow for evaluation of systemic, upper, and lower respiratory tract mucosal antibody responses, respectively. The isotype ELISA assay is performed in a 96-well format using anti-swine detection antibodies conjugated to an enzyme that catalyze a color change reaction. The optical density of the sample is measured using an automated plate reader. The assay is useful to characterize the IgG or the IgA response to challenge or vaccination against specific influenza virus isolates in different compartments of the immune system.

092 Biophysical parameters of adherent human induced pluripotent stem sells for the rational design of cryoprotectant addition and removal procedures

Applications for induced pluripotent stem cells are extensive and include basic biology, drug discovery, toxicological assessment, tissue regeneration, and cancer therapy, among many others. However, reprogramming of somatic cells is an intensive and time consuming process and karyotypic abnormalities are known to occur after extended periods in culture. Therefore, the ability to cryopreserve iPSCs with a high survival rate would be advantageous. Although stem cells are most frequently cryopreserved as small cell clumps in suspension, colony dissociation is known to cause cell loss and increased differentiation. Thus cryopreservation in the adherent state would alleviate any need to dissociate stem cells from the culture surface and prevent any damage that is a result of dissociation. Vitrification methods are particularly promising for cryopreservation of adherent cells because damage due to extracellular ice is prevented. However, vitrification methods require the use of high CPA concentrations which increase the risk of osmotic and toxic damage. Recently, we developed a rational design algorithm for designing toxicity-minimized CPA addition and removal procedures. (J.D. Benson, A.J. Kearsley, A.Z. Higgins, Cryobiology 64 (2012) 144–151) To successfully apply these predicted procedures, accurate knowledge of cell biophysical parameters is required. The purpose of this study was to determine the necessary biophysical parameters for the rational design of CPA addition and removal procedures for vitrification of iPSCs. To determine membrane permeability parameters for human iPSCs, we adapted our calcein fluorescence quenching method (A.K. Fry, A.Z. Higgins, Cellular and Molecular Bioengineering 5 (2012) 287–298) for use with an automated plate reader. Permeability parameters were determined for dimethyl sulphoxide, ethylene glycol, glycerol, and propylene glycol at 4 °C, 21 °C, and 37 °C. Most notably, glycerol permeation was significantly slower than the other CPA types. To determine the osmotic tolerance limits of iPSCs, cells were exposed to test solutions, which included varying concentrations of hypotonic buffer and hypertonic sucrose, for 15 mins. Cell yield was assessed 24 h after cells were returned to culture using PrestoBlue®. Osmotic tolerance limits were determined for single cell suspensions and adherent iPSCs at 4 °C, 21 °C, and 37 °C. Using ANOVA analysis, the effect of the osmolality and temperature as well as the cross interaction between osmolality and temperature were significant for both hypotonic and hypertonic exposures. Also, the tolerable limits varied greatly depending on the test temperature. In particular, excessive cell volume changes were more damaging at 37 °C than 4 °C or 21 °C. The permeability parameters and osmotic tolerance limits presented in this study enable rational design of CPA addition and removal procedures. The information in this study is an important step toward development of successful vitrification strategies for adherent human iPSCs. Source of funding: This project was funded by a Collaborative Research Contract from Life Technologies with Oregon State University. Conflict of interest: None declared. adam.higgins@oregonstate.edu

Increased Activity of Prothrombinase Fgl-2 in Peripheral Blood Mononuclear Cells of Patients with B-Cell Lymphoma.

AbstractAbstract 2665 Background:Hematologic and solid tumors are associated with hypercoagulability the reason for which has not been delineated. Prothrombinase named fibrinogen-like protein 2 (FGL-2) is a 70 kD transmembrane protein that was found to have a quality of a serine protease capable of directly cleaving prothrombin to thrombin. FGL-2 is synthesized by monocytes, T-lymphocytes and endothelial cells. FGL-2 protein and its mRNA have been previously found within different tumor cells. Aim:To study the role of FGL-2 in patients with lymphoproliferative disorders. Our hypothesis is that upregulation of FGL-2 activity in patients with B-cell malignancies may contribute to tumorigenesis via generation of thrombin leading to increased angiogenesis and spread/metastasis of malignant cells. Methods:Thrombin generation reflecting FGL-2 activity was measured in homogenized peripheral blood mononuclear cells (PBMC) from 29 patients with active lymphoproliferative disorders and 107 normal controls. Informed consent was obtained from every participant. PBMC extracts were incubated with an equal volume of human prothrombin (final concentration10 μM) for 30 min at 37 °C. Thrombin generation was measured at 405 nm using an automated plate reader after addition of chromogen S-2238. The thrombin activity of each sample was calculated by comparison with absorbance curve generated by known concentrations of human thrombin. FGL-2 was immunoprecipitated (IP) from PBMC with an anti-FGL-2 antibody and EZview Red Protein A Affinity Gel (sigma # P6486). The activity of IP FGL-2 was measured by thrombin generation assay. The expression of FGL-2 was analyzed in HUVEC and PBMC in the presence or absence of IF-γ at 20 ng/ml. Total RNA was isolated using RNAqueous™ (Ambion #AM1912) and RT-PCR, analysis was performed using Rotor-gene RG-3000 (Corbett). The difference in cycle time (ΔCT) was measured by comparing FGL-2 gene with ABL-1 gene (house keeping gene). The relative quantification was calculated by the formula RQ= 2−ΔCT. HUVEC were transfected with 0.5 μM SiRNA synthesized complementary to FGL-2 (Target SiRNA) using Dharmacon ON-TARGET plus SMART pool reagent (Thermo Fisher Scientific) according to manufacturer instructions. FGL-2 expression following addition of target SiRNA was compared to that obtained following addition of non-specific (non-target) SiRNA. Student’s t-test was used for all comparisons. Results:As shown in the table, almost 3-fold increase in FGL-2 activity in PBMC was observed among patients with active B-cell lymphoma, either aggressive or indolent, as compared to that of healthy controls (p<0.0001 for all comparisons). No difference in FGL-2 activity in PBMC between patients with aggressive and indolent lymphoma was observed (p=0.68).Three-fold increase in thrombin generation was obtained in PBMC from the patients following IP, similarly to that observed in non-IP PBMC. A significant increase in mRNA of FGL-2 was found in either PBMC of lymphoma patient or HUVEC treated with IF-γ. In HUVEC the increase in FGL-2 mRNA was inhibited by 80% following treatment with specific SiRNA. Conclusions:1.There is increased activity of prothrombinase FGL-2 in PBMC of patients with B-cell lymphoma. Possible correlation between increased mRNA and protein activity of FGL-2 and B-cell lymphoma burden will be evaluated.2.The mechanistic effect of FGL-2 on tumorigenesisangiogenesis will be studied.Control subjects (N=107)Indolent B-cell lymphoma: FL, MCL, MZL, SLL (n=16)Aggressive B-cell lymphoma: DLBC, BL (n=13)All B-cell lymphoma (n=29)Mean ± SD (ng/ml)142.2 ± 118.4427.1 ± 219.2389.3 ± 267.5410.2 ± 238.3Range (ng/ml)0–439180–948153–1011153–1011P valueP<0.0001*P<0.0001*P<0.0001*FL – follicular lymphoma; MCL – mantle cell lymphoma; MZL – marginal cell lymphoma; SLL – small lymphocytic lymphoma; DLBCL – diffuse large B-cell lymphoma; BL – Burkitt’s lymphoma*compared to the control group. Disclosures:No relevant conflicts of interest to declare.

Effect of Application Times on Candida albicans for Denture Disinfection

Study background: This in vitro study aimed to evaluate the minimum application times of three denture disinfection procedures to significantly reduce initial Candida albicans colonization. Methods: Specimens of a soft denture relining material were incubated with Candida albicans for 2.5 h and allotted to three different disinfection regimes (sodium hypochlorite 1%, effervescent cleansing tabs, and microwaving immersed in water) with application times ranging from 30 to 600 s. Adhering fungi were quantified using a bioluminescence assay in combination with an automated plate reader for cell quantification. Results: Luminescence at baseline varied between 409 and 472 relative luminescence units (rlu). A significant reduction of luminescence was found after 120 s of soaking in sodium hypochlorite (31 rlu) and effervescent cleansing tabs (35 rlu), and after 60 s of microwave irradiation (26 rlu). Conclusions: Threshold minimum application times of 120 s for sodium hypochlorite and cleansing tabs, and 60 s for microwaving were determined. A further extension of application times did not have additional antifungal effects.

Fluorescence‐PCR assays and isolation of luminescent bacterial clones using an automated plate reader

The genes responsible for luminescence in various species of the marine microorganism Photobacterium, have been used for many years as a tool by researchers and instructors. In particular, the lux operon of Photobacterium fischeri has been used by many instructors to teach recombinant DNA techniques. Two methods using an automated plate reader and multiwell plates were applied to a set of previously-published exercises. In these exercises that involve transfer of lux genes to Escherichia coli to create a luminescent phenotype, this technology was used to screen for Lux(+) colonies. It was found to be more convenient and more sensitive than the previously used method; that is, assaying bacterial plates by direct observation. Eight students synthesized four genomic libraries and isolated six Lux(+) clones. The fluorescent-detection feature of the plate reader was used to verify amplification of target sequence in polymerase chain reaction (PCR) reactions. Lux(+) E. coli colony lysates were examined. An exonuclease-activated, fluorescent DNA probe generated a signal on hybridization to an amplified portion of the luxA gene in each lysate tested. This method is suggested as a means of demonstrating the concept of real-time PCR without the expense of the specialized device typically used for this technique.

Expression, purification, and characterization of a recombinant flavin reductase from the luminescent marine bacterium Photobacterium leiognathi

In Photobacterium, the flavin reductase encoded by luxG regenerates the reduced form of flavin mononucleotide (FMN). Reduced FMN is one of the substrates of the luciferase enzyme that catalyzes a light-emitting reaction. A set of experiments, that employs a luxG-expression plasmid construct (pGhis) and is suitable for an undergraduate laboratory course, is presented. Hexahistidine-tagged protein is expressed in E. coli from pGhis, with the T7 RNA polymerase/lac repressor induction system. Bacteria are lysed by sonication and the tag allows for purification by immobilized metal ion affinity chromatography. A gel filtration column is used to remove ions and the other small molecules. The Bradford assay, with multiwell plates and an automated plate reader, is used to identify protein concentration peaks from both columns. The concentration of purified enzyme is then calculated from its A(280) using the predicted extinction coefficient. Yield and purity are further assayed with SDS-PAGE. Activity of purified enzyme is measured with riboflavin or FMN as substrate. Reaction rate is quantified by monitoring decrease in A(340) as the redox partner, NADH, is oxidized.

Strain array for cell culture: cyclic strain affects epithelial cell spreading

We have designed, fabricated and characterized a strain array (6 mm well plate format) for cell culture that applies different strain to cells plated in each row of wells on the device. This device is capable of applying 0%, 2%, 4%, 7% or 17% strain to distinct samples with separate perfusion paths using a pressure difference of 10kPa. Our device has 25 wells but mimics the geometry of a standard 96‐well plate (for compatibility with automated plate readers and other technologies) and leaves samples easily accessible to assay. Further, samples experiencing different strain fields have separate perfusion pathways. These capabilities are useful in experiments to examine mechanotransduction mechanisms, specifically sensitivity to applied strain. Madin‐Darby Canine Kidney epithelial cells were attached on the device for 3 hrs then cyclically strained at 1Hz. Cells exhibited different patterns of spreading and localization of GFP‐tagged vinculin in response to strain levels over time.This work is funded by NSF EFRI CBE0735551, NIH R21 HL089027, and CIRM RC1‐00151‐1. CS is funded by the Smittcamp Graduate Fellowship of the Stanford Cardiovascular Institute and an NSF Graduate Research Fellowship.

Effects of aging on surface properties and adhesion of Streptococcus mutans on various fissure sealants

The aim of the present study was the quantification of Streptococcus mutans adhesion on ten widely used pit and fissure sealant materials and the correlation of these findings to surface roughness (R(a)) and surface free energy (SFE). Additionally, changes in streptococcal adhesion and surface parameters after water immersion and artificial aging have been investigated. Circular specimens of ten fissure sealants (seven resin-based composites, two glass ionomers, and one compomer) were made and polished. Surface roughness was determined by perthometer and SFE by goniometer measurements. Sealant materials were incubated with S. mutans suspension (2.5 h, 37 degrees C), and adhering bacteria were quantified by using a biofluorescence assay in combination with an automated plate reader. Surface properties and S. mutans adhesion were measured prior to and after water immersion after 1 and 6 months and after additional thermocycling (5,000 cycles; 5 degrees C/55 degrees C). The tested sealants showed significant differences in S. mutans adhesion prior to and after the applied aging procedures. Aging resulted in slight increases (mostly <0.2 microm) in surface roughness, as well as in significant decreases in SFE and in significantly lower quantities of adhering bacteria. Ketac Bond and UltraSeal XT plus revealed the lowest adhesion potential after artificial aging. In general, the amount of adhering S. mutans was reduced after aging, which may be related to the decline in SFEs.