<title>Novel europium-tetracycline probe for phosphate determination in microtiter plate</title>

SummaryActivated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va. APC is inhibited by several members of the serpin family as well a by α2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 μl of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate.The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period. The coefficient of variation was 5.9% at 35 ng/ml and 8.8% at 350 ng/ml APC. The sensitivity of the assay could be increased by measuring the amount of color produced after longer incubation times in the endpoint mode. The measured APC activity levels were little affected by varying protein C or prothrombin over the extremes of 0 to 150% of normal plasma concentrations. By constructing the standard curve in protein C-deficient plasma, the concentration of APC activity in normal pooled plasma was determined to be 2.8 ng/ml (45 pM), which represents 0.08% of the protein C concentration. The assay was approximately 50-fold more sensitive than the identical assay, but using Mab-coated microtiter wells rather than immunosorbent beads as the capture step.

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Development of 96 Multiple Injection-GC-MS Technique and Its Application in Protein Engineering of Natural and Non-Natural Enzymatic Reactions

10.26434/chemrxiv.10314239 ◽

2019 ◽

Author(s):

Anja Knorrscheidt ◽

Pascal Püllmann ◽

Eugen Schell ◽

Dominik Homann ◽

Erik Freier ◽

...

Keyword(s):

Microtiter Plate ◽

Axial Ligand ◽

Cell System ◽

Enzymatic Reactions ◽

The Novel ◽

Internal Standards ◽

E Coli ◽

Enzyme Screening ◽

Plate Analysis ◽

Microtiter Plate Analysis

Directed evolution requires the screening of enzyme libraries in biological matrices. Available assays are mostly substrate or enzyme specific. Chromatographic techniques like LC and GC overcome this limitation, but require long analysis times. The herein developed multiple injections in a single experimental run (MISER) using GC coupled to MS allows the injection of samples every 33 s resulting in 96-well microtiter plate analysis within 50 min. This technique is implementable in any GC-MS system with autosampling. Since the GC-MS is far less prone to ion suppression than LCMS, no chromatographic separation is required. This allows the utilisation of an internal standards and the detection of main and side-product. To prove the feasibility of the system in enzyme screening, two libraries were assessed: i) YfeX library in an E. coli whole cell system for the carbene-transfer reaction on indole revealing the novel axial ligand tryptophan, ii) a library of 616 chimeras of fungal unspecific peroxygenase (UPO) in S. cerevisiae supernatant for hydroxylation of tetralin resulting in novel constructs. The data quality and representation are automatically assessed by a new R-script.

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Investigate of the Ability of Cronobacter sakazakii Isolated from Clinical Samples of Children Under Two Years to Induce Swimming, Swarming and Biofilm

Ibn AL- Haitham Journal For Pure and Applied Science ◽

10.30526/2017.ihsciconf.1780 ◽

2018 ◽

pp. 123

Author(s):

Luma Abdal Hady Zwein ◽

Tharieyt Abdulrahman Motlag ◽

Mohamed Mousa

Keyword(s):

Cerebrospinal Fluid ◽

Congo Red ◽

Biofilm Formation ◽

Microtiter Plate ◽

Clinical Samples ◽

Cronobacter Sakazakii ◽

Yellow Color ◽

Golden Yellow ◽

Vitek 2 ◽

Biochemical Examination

The study included 200 samples were collected from children under two years included (50 samples from each of Cerebrospinal fluid, Blood, Stool and Urine) from, Central Children Hospital and Children's Protections Educational Hospital. Isolates bacterial were obtained cultural, microscopic and biochemical examination and diagnosed to the species by using vitek2 system. The results showed there were contamination in 6.5% of clinical samples. The diagnosed colonies which gave pink color on the MacConkey agar , golden yellow color on the Trypton Soy agar and green color on the Birillent Enterobacter sakazakii agar and gave a probability of 99% in the vitek 2 and were identified as Cronobacter sakazakii. The identification revealed of thirteen isolates: 6(46.16%) isolated from Cerebrospinal fluid samples, 7(53.84%) isolated from blood samples and not isolated bacteria from stool and urine samples. The results of the investigation of some virulence factors showed that all bacteria isolates were able to swimming with a diameter ranging (1-9 mm) and swarming with a diameter ranging (1-40 mm) and their ability to biofilm formation by using three methods. The results show the ability of isolates to form biofilm by using Congo red media methods where it is 12 (92.30 %) out of 13 isolated bacteria belonging to C. sakazakii able to form biofilm on the Congo red media which is 3 (23.07%) were strong production biofilm , 8 (61.53%) were intermediate production biofilm and 1 (7.69% ) were weak biofilm formation , while the 1 (7.69%) unable to form biofilm. Tubes method were all isolates were able to form biofilm, it were found that 3 (23.07%) isolates strong, and 8 (61.53%) intermediate and 2( 15.38%) weak biofilm formation. Microtiter plate method gave 5 (38.46 %) isolates strong, 6 (46.15%) intermediate and 1 (7.69%) weak biofilm formation.

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Drug Design of Inhibitors of Alzheimer’s Disease (AD): POM and DFT Analyses of Cholinesterase Inhibitory Activity of β-amino di-Carbonyl Derivatives

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557518666181102102816 ◽

2019 ◽

Vol 19 (8) ◽

pp. 688-705

Author(s):

Taibi Ben Hadda ◽

Abdur Rauf ◽

Hsaine Zgou ◽

Fatma Sezer Senol ◽

Ilkay Erdogan Orhan ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cholinesterase Inhibitors ◽

Metal Accumulation ◽

Microtiter Plate ◽

Metal Chelation ◽

Carbonyl Derivatives ◽

Cholinesterase Inhibitory Activity ◽

Inhibitory Potential

Background:Since deficit of acetylcholine has been evidenced in the Alzheimer’s disease (AD) patients, cholinesterase inhibitors are currently the most specified drug category for the remediation of AD.Method:In the present study, 16 compounds (1-16) with dicarbonyl skeletons have been synthesized and tested for their inhibitory potential in vitro against AChE and BChE using ELISA microtiter plate assays at 100 μg/mL. Since metal accumulation is related to AD, the compounds were also tested for their metal-chelation capacity.Results and Conclusion:All the investigated dicarbonyl compounds exerted none or lower than 30% inhibition against both cholinesterases, whereas compounds 2, 8 and 11 showed 37, 42, 41% of inhibition towards BChE, being the most active. The highest metal-chelation capacity was observed with compound 8 (53.58 ± 2.06%). POM and DFT analyses are in good harmonization with experimental data.

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Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates

Microbial Cell Factories ◽

10.1186/s12934-021-01541-2 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Tobias Habicher ◽

Tobias Klein ◽

Jacqueline Becker ◽

Andreas Daub ◽

Jochen Büchs

Keyword(s):

Controlled Release ◽

Bacillus Licheniformis ◽

Protease Activity ◽

Microtiter Plate ◽

Fed Batch ◽

Batch Mode ◽

Screening Process ◽

Microtiter Plates ◽

Batch Fermenter ◽

Production Conditions

Abstract Background Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. Results To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. Conclusion The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

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waveRAPID—A Robust Assay for High-Throughput Kinetic Screens with the Creoptix WAVEsystem

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211013827 ◽

2021 ◽

pp. 247255522110138

Author(s):

Önder Kartal ◽

Fabio Andres ◽

May Poh Lai ◽

Rony Nehme ◽

Kaspar Cottier

Keyword(s):

Drug Discovery ◽

Fold Increase ◽

Microtiter Plate ◽

Experimental Conditions ◽

Short Pulses ◽

Cycle Times ◽

Fragment Screening ◽

New Instrument ◽

Single Well ◽

Compound Screening

Surface-based biophysical methods for measuring binding kinetics of molecular interactions, such as surface plasmon resonance (SPR) or grating-coupled interferometry (GCI), are now well established and widely used in drug discovery. Increasing throughput is an often-cited need in the drug discovery process and this has been achieved with new instrument generations where multiple interactions are measured in parallel, shortening the total measurement times and enabling new application areas within the field. Here, we present the development of a novel technology called waveRAPID for a further—up to 10-fold—increase in throughput, consisting of an injection method using a single sample. Instead of sequentially injecting increasing analyte concentrations for constant durations, the analyte is injected at a single concentration in short pulses of increasing durations. A major advantage of the new method is its ability to determine kinetics from a single well of a microtiter plate, making it uniquely suitable for kinetic screening. We present the fundamentals of this approach using a small-molecule model system for experimental validation and comparing kinetic parameters to traditional methods. By varying experimental conditions, we furthermore assess the robustness of this new technique. Finally, we discuss its potential for improving hit quality and shortening cycle times in the areas of fragment screening, low-molecular-weight compound screening, and hit-to-lead optimization.

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Microtiter plate assays to assess antibiofilm activity against bacteria

Nature Protocols ◽

10.1038/s41596-021-00515-3 ◽

2021 ◽

Author(s):

Evan F. Haney ◽

Michael J. Trimble ◽

Robert E. W. Hancock

Keyword(s):

Microtiter Plate ◽

Antibiofilm Activity

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Refactoring of a synthetic raspberry ketone pathway with EcoFlex

Microbial Cell Factories ◽

10.1186/s12934-021-01604-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Simon J. Moore ◽

Yonek B. Hleba ◽

Sarah Bischoff ◽

David Bell ◽

Karen M. Polizzi ◽

...

Keyword(s):

Gene Expression ◽

Synthetic Biology ◽

Combinatorial Libraries ◽

Value Added ◽

Microtiter Plate ◽

Fine Tuning ◽

Golden Gate ◽

E Coli ◽

Fine Chemical ◽

Raspberry Ketone

Abstract Background A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg−1) fine chemical farmed from raspberry (Rubeus rubrum) fruit. Results By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10β, as a routine cloning host. The use of E. coli DH10β facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture. Conclusions Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.

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Europium tetracycline biosensor for the determination of cholesterol

10.1117/12.697956 ◽

2007 ◽

Author(s):

Lilia Coronato Courrol ◽

Flávia Rodrigues de Oliveira Silva ◽

Ricardo Elgul Samad ◽

Ronaldo Domingues Mansano ◽

Nilson Dias Vieira, Jr.

Keyword(s):

Europium Tetracycline

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