scholarly journals Monitoring Parallel Robotic Cultivations with Online Multivariate Analysis

Processes ◽  
2020 ◽  
Vol 8 (5) ◽  
pp. 582 ◽  
Author(s):  
Sebastian Hans ◽  
Christian Ulmer ◽  
Harini Narayanan ◽  
Trygve Brautaset ◽  
Niels Krausch ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Signal To Noise Ratio ◽  
Model Organism ◽  
Principal Component ◽  
Optimal Operation ◽  
Aeration System ◽  
Microbial Screening ◽  
Good Signal

In conditional microbial screening, a limited number of candidate strains are tested at different conditions searching for the optimal operation strategy in production (e.g., temperature and pH shifts, media composition as well as feeding and induction strategies). To achieve this, cultivation volumes of >10 mL and advanced control schemes are required to allow appropriate sampling and analyses. Operations become even more complex when the analytical methods are integrated into the robot facility. Among other multivariate data analysis methods, principal component analysis (PCA) techniques have especially gained popularity in high throughput screening. However, an important issue specific to high throughput bioprocess development is the lack of so-called golden batches that could be used as a basis for multivariate analysis. In this study, we establish and present a program to monitor dynamic parallel cultivations in a high throughput facility. PCA was used for process monitoring and automated fault detection of 24 parallel running experiments using recombinant E. coli cells expressing three different fluorescence proteins as the model organism. This approach allowed for capturing events like stirrer failures and blockage of the aeration system and provided a good signal to noise ratio. The developed application can be easily integrated in existing data- and device-infrastructures, allowing automated and remote monitoring of parallel bioreactor systems.

scholarly journals Paper-Based SERS Platform for One-Step Screening of Tetracycline in Milk

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ana Marques ◽  
Bruno Veigas ◽  
Andreia Araújo ◽  
Beatriz Pagará ◽  
Pedro Viana Baptista ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Point Of Care ◽  
Dairy Industry ◽  
Principal Component ◽  
Surface Enhanced Raman Spectroscopy ◽  
Economic Losses ◽  
Screening Methods ◽  
Sers Substrate ◽  
Molecular Signatures

AbstractThroughout the last decade, the expansion of food testing has been gradually moving towards ordinary high throughput screening methods performed on-site. The demand for point-of-care testing, able to distinguish molecular signatures with high accuracy, sensitivity and specificity has been significantly increasing. This new requirement relies on the on-site detection and monitorization of molecular signatures suitable for the surveillance of food production and processing. The widespread use of antibiotics has contributed to disease control of livestock but has also created problems for the dairy industry and consumers. Its therapeutic and subtherapeutic use has increased the risk of contamination in milk in enough concentrations to cause economic losses to the dairy industry and have a health impact in highly sensitive individuals. This study focuses on the development of a simple Surface-Enhanced Raman Spectroscopy (SERS) method for fast high throughput screening of tetracycline (TET) in milk. For this, we integrate a paper-based low-cost, fully recyclable and highly stable SERS platform, with a minimal sample preparation protocol. A two-microliter sample of milk solutions spiked with TET (from 0.01 to 1000 ppm) is dried on a silver nanoparticle coated cardboard substrate and measured via a Raman spectrophotometer. The SERS substrate showed to be extremely stable with a shelf life of several months. A global spectrum principal component analysis approach was used to test all the detected vibrational modes and their correlation with TET concentration. Peak intensity ratios (455 cm−1/1280 cm−1 and 874 cm−1/1397 cm−1) were found to be correlated with TET concentrations in milk, achieving a sensitivity as low as 0.1 ppm. Results indicate that this SERS method combined with portable Raman spectrometer is a potential tool that can be used on-site for the monitoring of TET residues and other antibiotics.

scholarly journals Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter

2003 ◽  
Vol 47 (12) ◽  
pp. 3682-3687 ◽  
Author(s):  
Chartchai Changsen ◽  
Scott G. Franzblau ◽  
Prasit Palittapongarnpim
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antimicrobial Agents ◽  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Enhanced Fluorescence ◽  
Signal To Noise ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

ABSTRACT The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc2155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.

scholarly journals Microbiological High Throughput Screening: An Opportunity for the Lead Discovery Process

2000 ◽  
Vol 5 (1) ◽  
pp. 13-21 ◽  
Author(s):  
Marie-Helene Beydon ◽  
Alain Fournier ◽  
Lionel Drugeault ◽  
Jerome Becquart
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Discovery Process ◽  
Robotic Platform ◽  
Lead Discovery ◽  
Innovative Solutions ◽  
Different Types ◽  
Microbial Screening ◽  
Low Costs

Microbial HTS has been implemented at Rhone-Poulenc Rorer through the development of a dedicated robotic platform. This robot (Turbo) has been designed with the aim of fully integrating microbial HTS into the lead discovery processes. Innovative solutions have been found to reach high throughput as well as flexibility. This opens up new prospects for solid-phase microbial screening, taking advantage of the easy implementation and the very low costs of such screens. The different types of microbial screens done in our laboratory, as well as the throughputs and outputs obtained, are described. Some of the specific aspects of microbial HTS, as compared to biochemical and cell-based assays, are also discussed.

scholarly journals A Reliable High-Throughput Screening Model for Antidepressant

2021 ◽  
Vol 22 (17) ◽  
pp. 9505
Author(s):  
Rui Zhang ◽  
Caili Qiao ◽  
Qiuyan Liu ◽  
Jingwen He ◽  
Yifan Lai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Screening Method ◽  
Model Organism ◽  
Mild Stress ◽  
Mice Model ◽  
Zebrafish Model ◽  
Screening Model ◽  
Tyrosine Metabolism

Depression is the most frequent affective disorder and is the leading cause of disability worldwide. In order to screen antidepressants and explore molecular mechanisms, a variety of animal models were used in experiments, but there is no reliable high-throughput screening method. Zebrafish is a common model organism for mental illness such as depression. In our research, we established chronic unpredictable mild stress (CUMS) models in C57BL/6 mice and zebrafish; the similarities in behavior and pathology suggest that zebrafish can replace rodents as high-throughput screening organisms. Stress mice (ip., 1 mg/kg/d, 3 days) and zebrafish (10 mg/L, 20 min) were treated with reserpine. As a result, reserpine caused depression-like behavior in mice, which was consistent with the results of the CUMS mice model. Additionally, reserpine reduced the locomotor ability and exploratory behavior of zebrafish, which was consistent with the results of the CUMS zebrafish model. Further analysis of the metabolic differences showed that the reserpine-induced zebrafish depression model was similar to the reserpine mice model and the CUMS mice model in the tyrosine metabolism pathway. The above results showed that the reserpine-induced depression zebrafish model was similar to the CUMS model from phenotype to internal metabolic changes and can replace the CUMS model for antidepressants screening. Moreover, the results from this model were obtained in a short time, which can shorten the cycle of drug screening and achieve high-throughput screening. Therefore, we believe it is a reliable high-throughput screening model.

scholarly journals Mapping Out Receptors: High Throughput Screening of Endothelial Monocyte Activating Polypeptide II (EMAPII) Using PRESTO-TANGO

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Nathan Lam ◽  
Bernhard Maier ◽  
Sarvesh Chelvanambi ◽  
Takashi Hato ◽  
Matthias Clauss
Keyword(s):  
Colorectal Cancer ◽  
Cardiovascular Disease ◽  
Receptor Binding ◽  
High Throughput ◽  
High Throughput Screening ◽  
Neutralizing Antibodies ◽  
Signal To Noise Ratio ◽  
Receptor System ◽  
Gpcr Activation ◽  
Cxcr3 Ligand

Background: Secreted endothelial monocyte activating polypeptide II (EMAPII/AIMP1) is a pro-apoptotic, pro-inflammatory ligand implicated in diseases such as colorectal cancer, cardiovascular disease, and emphysema. Thus, EMAPII has been shown to induce apoptosis through CXCR3 receptor binding. However, not all EMAPII functions have been attributed to CXCR3. Discovery of new receptors interacting with EMAPII could lead to development of new therapies blocking cognate ligand-receptor binding. We hypothesize the existence of at least one unknown secondary receptor for EMAPII.  Methods: The PRESTO-TANGO assay, a construct which converts G-protein coupled receptor (GPCR) ligand binding into luciferase activity measurable by luminometer, was validated using transfection with TANGO-modified CXCR3 and S1P1R as test receptors in HTLA cells. Protocols for cell transfection, adherence, and cultivation were optimized with IP10, EMAPII, and S1P as test ligands.  Results: The assay was successfully validated using several GPCR activation readouts. Binding curves were generated for S1P/S1P1 (EC50= 569nM), IP10/CXCR3 (EC50= 47.1 nM), and EMAPII/CXCR3 (EC50= 628 nM). Conditions for the PRESTO-TANGO assay were further refined for maximal signal-to-noise ratio and robust inter-assay reproducibility in preparation for high-throughput screening. We are currently testing 314 TANGO-modified GPCRs for EMAPII affinity.  Conclusion: We have validated the Tango assay for the known EMAPII-CXCR3 ligand-receptor system, a valuable tool for evaluation of anti-EMAPII therapeutics. Discovery of a novel EMAPII receptor would allow for the development of therapies including neutralizing antibodies (analogous to the PD1 receptor antibody Pembrolizumab for solid tumors) in diseases such as colorectal cancer, cardiovascular disease, and emphysema.

scholarly journals Microfluidic Technologies for High Throughput Screening Through Sorting and On-Chip Culture of C. elegans

Molecules ◽  
2019 ◽  
Vol 24 (23) ◽  
pp. 4292 ◽  
Author(s):  
Daniel Midkiff ◽  
Adriana San-Miguel
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Model Organism ◽  
Large Data ◽  
Large Data Sets ◽  
Data Sets ◽  
C Elegans ◽  
Drug Candidates ◽  
On Chip ◽  
And Behavior

The nematode Caenorhabditis elegans is a powerful model organism that has been widely used to study molecular biology, cell development, neurobiology, and aging. Despite their use for the past several decades, the conventional techniques for growth, imaging, and behavioral analysis of C. elegans can be cumbersome, and acquiring large data sets in a high-throughput manner can be challenging. Developments in microfluidic “lab-on-a-chip” technologies have improved studies of C. elegans by increasing experimental control and throughput. Microfluidic features such as on-chip control layers, immobilization channels, and chamber arrays have been incorporated to develop increasingly complex platforms that make experimental techniques more powerful. Genetic and chemical screens are performed on C. elegans to determine gene function and phenotypic outcomes of perturbations, to test the effect that chemicals have on health and behavior, and to find drug candidates. In this review, we will discuss microfluidic technologies that have been used to increase the throughput of genetic and chemical screens in C. elegans. We will discuss screens for neurobiology, aging, development, behavior, and many other biological processes. We will also discuss robotic technologies that assist in microfluidic screens, as well as alternate platforms that perform functions similar to microfluidics.

A Generic High-Throughput Screening Assay for Kinases: Protein Kinase A as an Example

2003 ◽  
Vol 8 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Rommel Mallari ◽  
Elissa Swearingen ◽  
Wei Liu ◽  
Arnold Ow ◽  
Stephen W. Young ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Signal To Noise Ratio ◽  
Product Formation ◽  
Screening Assay ◽  
Chemical Libraries ◽  
Coulombic Interactions ◽  
High Throughput Screening Assay ◽  
Peptide Product

A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated 33P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5-to 9-fold and a Z′ factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors. ( Journal of Biomolecular Screening 2003:198-204)

scholarly journals Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish

2021 ◽  
Vol 22 (16) ◽  
pp. 8401
Author(s):  
Valentina Naef ◽  
Maria Marchese ◽  
Asahi Ogi ◽  
Gianluca Fichi ◽  
Daniele Galatolo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Motor Impairment ◽  
Model Organism ◽  
Cell Degeneration ◽  
Vertebrate Model ◽  
Therapeutic Compounds

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs−/− larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

2020 ◽  
Vol 25 (9) ◽  
pp. 1064-1071
Author(s):  
Maikel Izquierdo ◽  
De Lin ◽  
Sandra O’Neill ◽  
Martin Zoltner ◽  
Lauren Webster ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Trypanosoma Cruzi ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Signal To Noise Ratio ◽  
Screening Method ◽  
Cellular Functions ◽  
Screening Assay ◽  
High Throughput Screening Assay

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin–based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)–based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

scholarly journals Development of a Ca2+-Activated Photoprotein, Photina®, and Its Application to High-Throughput Screening

2007 ◽  
Vol 12 (5) ◽  
pp. 694-704 ◽  
Author(s):  
Silvia Bovolenta ◽  
Maria Foti ◽  
Stefan Lohmer ◽  
Sabrina Corazza
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Signal To Noise Ratio ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Number ◽  
Ovary Cells ◽  
Mammalian Cell Lines ◽  
Calcium Dyes ◽  
G Protein Coupled

The present work describes the engineering and characterization of a new Ca2+-activated photoprotein (Photina®) and its use in mammalian cell lines for implementation of flash luminescence cell-based assays for high-throughput screening (HTS). When used to measure the activation of 2 G protein—coupled receptors (GPCRs), targeting Photina® to the mitochondria increased the signal strength as compared to the normal cytoplasmic expression of Photina.® The mitochondrial-targeted Photina® also produced a higher signal-to-noise ratio than conventional calcium dyes and a consistently stronger signal than aequorin when tested under equivalent conditions. MitoPhotina® provided strong and reliable results when used to measure the activity of purinergic receptors endogenously expressed in the Chinese Hamster Ovary cells and heterologously expressed GPCRs in response to their cognate ligands. Several different types of flash luminescence plate readers (FLIPR3, FLIPRTETRA, CyBi®-®Lumax flash HT, Lumilux®, Lumibox) in different plate formats (96, 384, 1536 wells) were used to validate the use of Photina in HTS. The cell number had to be adjusted to correspond to the qualities of the different readers, but once so adjusted, it provided equivalent results on each device. The results obtained show robust and reproducible light signals that offer new possibilities for application of photoproteins to the generation of cell-based assays for HTS. ( Journal of Biomolecular Screening 2007:694-704)

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