Development of a high throughput method for screening readily biodegradable chemicals.

Automation Highlights from the Literature

The objective of the present study was to develop and evaluate a high-throughput (HT) precipitation inhibitor screening method that can be used for the identification of the excipient-mediated precipitation inhibition of poorly soluble drugs. The impact of incubation temperature, shaking intensity, phase separation, inter- and intraday variability, cosolvent, and plate selection on the HT screening method performance was investigated. Additionally, the pipetting quality of the automated workstation, the correlation with the classical laboratory approach, and the practical implementation of the developed HT screening method using two model compounds are disclosed. Investigation of the HT method resulted in optimized experimental conditions, which showed low inter- and intraday variability (relative standard deviation [RSD]<5.88%). Higher shaking intensity (7 Hz) and incubation temperature (37°C) resulted in a lower likelihood of obtaining false-negative results. The acceptable dimethyl sulfoxide concentration in the precipitation inhibitor screening assay was set to ≤1% (v/v). All liquid dispensing steps resulted in an RSD of <3.4%, and an excellent correlation (R(2)=0.96, P<0.01) with the classical laboratory method was obtained. The practical implementation of the developed HT method was demonstrated by investigating the impact of 23 diverse excipients on the precipitation inhibition of two poorly soluble drugs (fenofibrate and carbamazepine). The screen resulted in the identification of hit excipients, which were not identical for fenofibrate and carbamazepine. This outcome emphasized that the HT screening approach is a reasonable starting point for searching for effective precipitation inhibitors, especially because the excipient-mediated precipitation inhibition effect is case specific and cannot be predicted in a straightforward manner.

Two high throughput methods are described for the synthesis of graded composition electrode arrays of carbon-coated by anaerobic pyrolysis of solution precursors at in the presence of sucrose as a carbon-coating additive. In the first method, direct in situ calcination, the array was prepared directly on the current collectors by a one-step heat-treatment of the solutions. In the second method, post-synthesis array transfer (PoSAT), the samples were first pyrolyzed in an array of individual microtubes, before mixing with solvent, binder solution, and carbon black then depositing onto the current collector array. High throughput X-ray diffraction clearly showed impurity phases for samples containing sucrose per mol iron(III) nitrate or less, but mainly olivine phase for sucrose contents above . Superior results from the PoSAT method showed up to 80% theoretical capacity appearing sharply at the point where the sucrose produced a carbon coating and the characteristic decay in capacity with excess carbon. The PoSAT method is not restricted to and is recommended as a useful method for high throughput screening of positive electrode materials.

1. A high throughput screening (HTS) method for the evaluation of the seven major human hepatic CYP isoform activities was developed on a 96-well format, with automation. The method utilized pooled human liver microsomes and seven probe substrates, generic conditions for incubation, reaction termination and metabolite extraction with solid phase extraction (SPE) plates. Metabolites from the seven reactions were pooled and quantified using a generic liquid chromatography and tandem mass spectrometry (LCMS/MS) method. 2. The HTS method was validated based on Km values obtained, which were in agreement with literature data. 3. The isoform inhibition profiles of ketoconazole, quinidine, sulfaphenazole, tranylcypromine, alpha-naphthoflavone, and 4-methylpyrazole against CYPs 3A4, 2D6, 2C9, 2A6 (and 2C19), 1A2 and 2E1, respectively, were obtained by this HTS method. Graphically obtained IC50 values are in agreement with literature reported values. 4. The HTS method represents a significant efficiency and selectivity improvement over traditional methods, and can be used for CYP inhibition assay and can be extended for liver activity profiling.

A high-throughput microtiter plate-based screening method for the detection of full-length recombinant proteins

Mosquitoes that transmit human diseases are of major importance to the international public health community. Pesticides remain a major component of integrated programs to control these medically important species. However, very few types of pesticides are currently registered for mosquito control. A high-throughput screening method using first-instar larvae of Aedes aegypti was created and evaluated in our laboratory to quickly screen large numbers of chemicals for activity against mosquitoes. LC50 values of a representative group of compounds were determined using this high-throughput screening method and compared with LD50 values determined by topical application against female adults of Ae. aegypti. Our results show that this high-throughput screening method is suitable for screening large numbers of candidate chemicals quickly to identify effective compounds.

Spatial repellents are an essential tool for personal protection against mosquitoes that bite and transmit disease pathogens to humans. Current repellent screening methods, such as olfactometers and alternative choice tests, are complex systems that require a relatively large quantity of compound (mg). The present study validates a high-throughput spatial repellent screening method using a glass tube that has the ends covered with netting, in addition to treated filters and plastic end caps. The apparatus occupies relatively little space, is easy to decontaminate, and requires small amounts of compound (μg). In a horizontal tube orientation, DEET (N,N-diethyl-meta-toluamide), citronella oil and IR3535 had 1 h half repellent concentration (EC50 ) values of 32, 32 and 298 μg/cm2 , respectively, against the Orlando strain of Aedes aegypti (L.) (Diptera: Culicidae). Vertical tube orientation increased EC50 values by approximately two-fold, except IR3535, which remained essentially unchanged. Transfluthrin showed concentration-dependent spatial repellency (1 h EC50 = 0.5 μg/cm2 ) without any knockdown, although only in vertical tubes. Transfluthrin showed 50% knockdown in 1 h at 0.5 μg/cm2 and 50% mortality at 0.15 μg/cm2 in horizontal tubes. In conclusion, this high-throughput screening method is useful for assessing vapour toxicity and the spatial repellency of candidate molecules prior to semi-field and field studies.

The objective of this study was to screen glucoside 3-dehydrogenase (G3DH)-producing strain based on a high-throughput G3DH screening method. Optimization of culture conditions of the isolated strain was also applied in this study. This screening method employed electron transfer reaction in 96-well microtiter plates, α-methyl-D-glucoside, galactose, 2-deoxy-D-glucose, and 3-O-methyl-D-glucose were used as substrates. Using this screening method, one out of 78 strains isolated from different soil samples was obtained with high G3DH activity. The accuracy of the screening method was proved by alkaline treatment analysis of 3-keto sugars. The isolated strain was identified as Sphingobacterium faecium ZJF-D6 by phenotypic characterization and 16S rDNA sequence analysis. The culture conditions of S. faecium for G3DH production were optimized. Sucrose was found as the most suitable carbon source for the G3DH production. The highest G3DH production and cell growth were achieved using the medium at the initial pH of 7.0 at 25°C for 36h with activity of 8.03 × 10(-2)U/mL culture. This strain appears promising for potential application in the industry to produce 3-keto sugars. To our knowledge, this is the first report on S. faecium for G3DH production. The method described herein represents a useful tool for the high-throughput isolation of G3DH.

Automation Highlights from the Literature

A high-efficiency drug screening method is urgently needed due to the expanding number of potential targets and the extremely long time required to assess them. To date, high throughput and high content have not been successfully combined in image-based drug screening, which is the main obstacle to improve the efficiency. Here, we establish a high-throughput and high-content drug screening method by preparing a superhydrophobic microwell array plate (SMAP) and combining it with protein-retention expansion microscopy (proExM). Primarily, we described a flexible method to prepare the SMAP based on photolithography. Cells were cultured in the SMAP and treated with different drugs using a microcolumn-microwell sandwiching technology. After drug treatment, proExM was applied to realize super-resolution imaging. As a demonstration, a 7 × 7 image array of microtubules was successfully collected within 3 h with 68 nm resolution using this method. Qualitative and quantitative analyses of microtubule and mitochondria morphological changes after drug treatment suggested that more details were revealed after applying proExM, demonstrating the successful combination of high throughput and high content.

Chemical biology provides an alternative way to identify genes involved in a particular biological process. It has the potential to overcome issues such as redundancy or lethality often found in genetic approaches, since the chemical compounds can simultaneously target all homologous proteins that function at the same step, and chemicals can be applied conditionally. Even with a variety of genetic approaches, the molecular mechanisms of plant hypersensitive cell death that occurs during disease resistance responses remain unclear. Therefore, application of chemical biology should provide new insights into this phenomenon. Here we describe a high-throughput chemical screening procedure to detect hypersensitive cell death quantitatively, using a suspension cell culture of Arabidopsis thaliana and a well-studied avirulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 avrRpm1.

Protein engineering of aldolase LbDERA for enhanced activity toward real substrates with a high-throughput screening method coupled with an aldehyde dehydrogenase

Cancer is a disease caused by uncontrolled proliferation or division of abnormal cells in an organism. Traditionally, cancer cell proliferation is measured using a two-dimensional (2D) in vitro model by culturing and treating the cells in standard microplates to study inhibition and cytotoxicity effect of the drug compounds. After further testing the potential cancer drug candidates in the 2D environment, the investigation is often transferred to an in vivo model by measuring the change in three-dimensional (3D) tumor sizes in animals. Although the drug candidates can demonstrate promising inhibitory or cytotoxicity results in a 2D environment, similar effects may not be observed in a 3D environment, which physiologically has a closer resemblance to a human body. Therefore, cancer drug discovery research has demonstrated poor translation from in vitro to in vivo. Previous publications have demonstrated that growing cancer cells in the form of 3D tumor spheroids can be more predictive of the in vivo study outcomes comparing to the 2D cell culture method. Therefore, by investigating the effect of cancer drugs on 3D tumor spheroids, may improve the successful translation rate to in vivo animal study. In this work, we developed an image-based high-throughput screening method for 3D tumor spheroids on 384-well ultra-low attachment (ULA) round bottom microplates using the Celigo image cytometer. In order to validate this screening method, five experiments were conducted demonstrating comparable results in both 3D and 2D models. First, optimal cell seeding density for tumor spheroid formation is determined by investigating U87MG (glioblastoma) cell seeding number in respect to the desired spheroid size. Second, the dose response effect of 17-AAG in respect to spheroid size is measured to determine the IC50 value. Third, the effect of 17-AAG on the viability of tumor spheroid is investigated in a time-course study. Next, the developed high-throughput method is used to perform dose response measurement of 4 drugs (17-AAG, Paclitaxel, TMZ, and Doxorubicin) in respect to the spheroid size. Finally, the effect of the 4 drugs on tumor spheroid viability is measured. Each experiment is performed simultaneously in the 2D model, where 384-well flat bottom microplates are used. In the 2D adherent cell model, confluence percentage and direct cell count-based viability results are generated to compare to the 3D model. This proposed method allows an efficient process for more qualified drug candidates, which can reduce time and the financial burden of animal testing, as well as the overall time of the cancer drug discovery project. Citation Format: Leo Li-Ying Chan, Olivier Déry, Scott Cribbes, Dmitry Kuksin, Sarah Kessel. A high-throughput image cytometry-based screening method for the cytotoxic effect of drug compounds on 3D tumor spheroid. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 603.

BackgroundSurvival time for lung cancer is poor with over 90% of patients dying within five years of diagnosis primarily due to detection at late stage. The main objective of this study was to evaluate Fourier transform infrared spectroscopy (FTIR) as a high throughput and cost effective method for identifying biochemical changes in sputum as biomarkers for detection of lung cancer.MethodsSputum was collected from 25 lung cancer patients in the Medlung observational study and 25 healthy controls. FTIR spectra were generated from sputum cell pellets using infrared wavenumbers within the 1800 to 950 cm-1 "fingerprint" region.ResultsA panel of 92 infrared wavenumbers had absorbances significantly different between cancer and normal sputum spectra and were associated with putative changes in protein, nucleic acid and glycogen levels in tumours. Five prominent significant wavenumbers at 964 cm-1, 1024 cm-1, 1411 cm-1, 1577 cm-1 and 1656 cm-1 separated cancer spectra from normal spectra into two distinct groups using multivariate analysis (group 1: 100% cancer cases; group 2: 92% normal cases). Principal components analysis revealed that these wavenumbers were also able to distinguish lung cancer patients who had previously been diagnosed with breast cancer. No patterns of spectra groupings were associated with inflammation or other diseases of the airways.ConclusionsOur results suggest that FTIR applied to sputum might have high sensitivity and specificity in diagnosing lung cancer with potential as a non-invasive, cost-effective and high-throughput method for screening.Trial RegistrationClinicalTrials.gov: NCT00899262

The present study was to isolate, cultivate, and screen plant growth-promoting bacteria (PGPB) from mangrove sediments at high throughput, and compare the differences in microbial diversity and PGPB screening efficiency between the high-throughput and traditional agar plate methods. A high-throughput method combining limiting dilution and two-sided barcode PCR was developed to effectively isolate and identify bacteria from mangrove sediments. Moreover, the metagenome and 16S rRNA amplicon sequencing were used for identification of potential PGPB genera and investigation of microbial diversity in different pooled cultures. The results showed that the microbial communities of the bacteria cultured by the high-throughput method had a significantly lower Simpson index (p<0.05) and a higher proportion of rare species than that by the agar plate method. A diverse bacterial consortium encompassing 79 species of 39 genera and 6 phyla were successfully isolated by the high-throughput method. Furthermore, three species of Bacillus and four species of Pseudomonas exhibiting plant growth-promoting properties were isolated and purified from a series of Bacillus and Pseudomonas genera, including a previously uncultured Pseudomonas strain. In contrast, all the PGPB screened by the agar plate method belonged to the Vibrio genus, which has been reported to be pathogenic to humans. The study demonstrated that the high-throughput method was superior to traditional plate methods for isolating a broader spectrum of microbial diversity from mangrove sediments, particularly rare species. When combined with metagenome sequencing, this approach enables a more efficient screening for PGPB potentially applicable for agriculture or environmental protection.