scholarly journals Bioprospecting the solar panel microbiome: high-throughput screening for antioxidant bacteria in aCaenorhabditis elegansmodel

2018 ◽  
Author(s):  
Kristie Tanner ◽  
Patricia Martorell ◽  
Salvador Genovés ◽  
Daniel Ramón ◽  
Lorenzo Zacarías ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
High Throughput ◽  
High Throughput Screening ◽  
Extreme Environment ◽  
Bacterial Isolates ◽  
Solar Panels ◽  
Potential Applications ◽  
Ultraviolet Protection ◽  
Pigmented Bacteria

AbstractMicrobial communities that are exposed to sunlight typically share a series of adaptations to deal with the radiation they are exposed to, including efficient DNA repair systems, pigment production and protection against oxidative stress, which makes these environments good candidates for the search of novel antioxidant microorganisms. In this research project, we isolated potential antioxidant pigmented bacteria from a dry and highly-irradiated extreme environment: solar panels. High-throughputin vivoassays usingCaenorhabiditis elegansas an experimental model demonstrated the high antioxidant and ultraviolet-protection properties of these bacterial isolates that proved to be rich in carotenoids. Our results suggest that solar panels harbor a microbial community that includes strains with potential applications as antioxidants.

scholarly journals Epileptic brain fluorescent imaging reveals apigenin can relieve the myeloperoxidase-mediated oxidative stress and inhibit ferroptosis

2020 ◽  
Vol 117 (19) ◽  
pp. 10155-10164 ◽  
Author(s):  
Chenwen Shao ◽  
Jiwen Yuan ◽  
Yani Liu ◽  
Yajuan Qin ◽  
Xueao Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Real Time ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescence Probe ◽  
Ex Vivo ◽  
Fluorescent Imaging ◽  
Flavonoid Compound ◽  
Antiepileptic Agents

Myeloperoxidase (MPO)-mediated oxidative stress has been suggested to play an important role in the pathological dysfunction of epileptic brains. However, there is currently no robust brain-imaging tool to detect real-time endogenous hypochlorite (HClO) generation by MPO or a fluorescent probe for rapid high-throughput screening of antiepileptic agents that control the MPO-mediated chlorination stress. Herein, we report an efficient two-photon fluorescence probe (named HCP) for the real-time detection of endogenous HClO signals generated by MPO in the brain of kainic acid (KA)-induced epileptic mice, where HClO-dependent chlorination of quinolone fluorophore gives the enhanced fluorescence response. With this probe, we visualized directly the endogenous HClO fluxes generated by the overexpression of MPO activity in vivo and ex vivo in mouse brains with epileptic behaviors. Notably, by using HCP, we have also constructed a high-throughput screening approach to rapidly screen the potential antiepileptic agents to control MPO-mediated oxidative stress. Moreover, from this screen, we identified that the flavonoid compound apigenin can relieve the MPO-mediated oxidative stress and inhibit the ferroptosis of neuronal cells. Overall, this work provides a versatile fluorescence tool for elucidating the role of HClO generation by MPO in the pathology of epileptic seizures and for rapidly discovering additional antiepileptic agents to prevent and treat epilepsy.

scholarly journals Cell-based high-throughput screening of polysaccharide biosynthesis hosts

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zi-Xu Liu ◽  
Si-Ling Huang ◽  
Jin Hou ◽  
Xue-Ping Guo ◽  
Feng-Shan Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Polysaccharide Biosynthesis ◽  
Effective Strategies ◽  
Cell Factories ◽  
Pathway Optimization ◽  
Potential Applications ◽  
High Level

AbstractValuable polysaccharides are usually produced using wild-type or metabolically-engineered host microbial strains through fermentation. These hosts act as cell factories that convert carbohydrates, such as monosaccharides or starch, into bioactive polysaccharides. It is desirable to develop effective in vivo high-throughput approaches to screen cells that display high-level synthesis of the desired polysaccharides. Uses of single or dual fluorophore labeling, fluorescence quenching, or biosensors are effective strategies for cell sorting of a library that can be applied during the domestication of industrial engineered strains and metabolic pathway optimization of polysaccharide synthesis in engineered cells. Meanwhile, high-throughput screening strategies using each individual whole cell as a sorting section are playing growing roles in the discovery and directed evolution of enzymes involved in polysaccharide biosynthesis, such as glycosyltransferases. These enzymes and their mutants are in high demand as tool catalysts for synthesis of saccharides in vitro and in vivo. This review provides an introduction to the methodologies of using cell-based high-throughput screening for desired polysaccharide-biosynthesizing cells, followed by a brief discussion of potential applications of these approaches in glycoengineering.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

scholarly journals A Microfluidic Spheroid Culture Device with a Concentration Gradient Generator for High-Throughput Screening of Drug Efficacy

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3355 ◽  
Author(s):  
Wanyoung Lim ◽  
Sungsu Park
Keyword(s):  
High Throughput ◽  
Concentration Gradient ◽  
High Throughput Screening ◽  
3D Cell Culture ◽  
Drug Efficacy ◽  
Cancer Drug ◽  
Spheroid Culture ◽  
Gradient Generator ◽  
Micro Channels

Three-dimensional (3D) cell culture is considered more clinically relevant in mimicking the structural and physiological conditions of tumors in vivo compared to two-dimensional cell cultures. In recent years, high-throughput screening (HTS) in 3D cell arrays has been extensively used for drug discovery because of its usability and applicability. Herein, we developed a microfluidic spheroid culture device (μFSCD) with a concentration gradient generator (CGG) that enabled cells to form spheroids and grow in the presence of cancer drug gradients. The device is composed of concave microwells with several serpentine micro-channels which generate a concentration gradient. Once the colon cancer cells (HCT116) formed a single spheroid (approximately 120 μm in diameter) in each microwell, spheroids were perfused in the presence of the cancer drug gradient irinotecan for three days. The number of spheroids, roundness, and cell viability, were inversely proportional to the drug concentration. These results suggest that the μFSCD with a CGG has the potential to become an HTS platform for screening the efficacy of cancer drugs.

scholarly journals High Throughput Phenotypic Screening of The Human Spermatozoon

Reproduction ◽  
2021 ◽  
Author(s):  
Zoe Claire Johnston ◽  
Franz S Gruber ◽  
Sean Brown ◽  
Neil R Norcross ◽  
Jason R Swedlow ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Hormonal Contraceptives ◽  
Diagnostic Tools ◽  
Sperm Function ◽  
Phenotypic Screening ◽  
Environmental Chemical ◽  
Potential Applications

Despite recent advances in male reproductive health research, there remain many elements of male (in)fertility where our understanding is incomplete. Consequently, diagnostic tools and treatments for men with sperm dysfunction, other than medically assisted reproduction, are limited. On the other hand, the gaps in our knowledge of the mechanisms which underpin sperm function have hampered the development of male non-hormonal contraceptives. The study of mature spermatozoa is inherently difficult. They are a unique and highly specialised cell type which does not actively transcribe or translate proteins and cannot be cultured for long periods of time or matured in vitro. One, large scale, approach to both increasing understanding of sperm function, and the discovery and development of compounds that can modulate sperm function, is to directly observe responses to compounds with phenotypic screening techniques. These target agnostic approaches can be developed into high-throughput screening platforms with the potential to drastically increase advances in the field. Here we discuss the rationale and development of high-throughput phenotypic screening platforms for mature human spermatozoa, and the multiple potential applications these present, as well as the current limitations and leaps in our understanding and capabilities needed to overcome them. Further development and use of these technologies could lead to the identification of compounds which positively or negatively affect sperm cell motility or function, or novel platforms for toxicology or environmental chemical testing among other applications. Ultimately, each of these potential applications is also likely to increase understanding within the field of sperm biology.

scholarly journals A High-Throughput Cellular Screening Assay for Small-Molecule Inhibitors and Activators of Cytoplasmic Dynein-1-Based Cargo Transport

2020 ◽  
Vol 25 (9) ◽  
pp. 985-999
Author(s):  
John Vincent ◽  
Marian Preston ◽  
Elizabeth Mouchet ◽  
Nicolas Laugier ◽  
Adam Corrigan ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Cytoplasmic Dynein ◽  
Lead Discovery ◽  
Screening Assay ◽  
Age Related ◽  
The Uk

Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein’s cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.

scholarly journals In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO)

Catalysts ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 935
Author(s):  
Sarah Maxel ◽  
Linyue Zhang ◽  
Edward King ◽  
Ana Paula Acosta ◽  
Ray Luo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Random Mutagenesis ◽  
Residual Activity ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Coli Strain ◽  
Screening Tools ◽  
Cyclohexanone Monooxygenase

Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is characterized as having wide substrate versatility for the biooxidation of (cyclic) ketones into esters and lactones with high stereospecificity. Despite industrial potential, CHMO usage is restricted by poor thermostability. Limited high-throughput screening tools and challenges in rationally engineering thermostability have impeded CHMO engineering efforts. We demonstrate the application of an aerobic, high-throughput growth selection platform in Escherichia coli (strain MX203) for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance. In the presence of the native substrate cyclohexanone, variant CHMO GV (A245G-A288V) was discovered from a random mutagenesis library screened at 42 °C. This variant retained native activity, exhibited ~4.4-fold improvement in residual activity after 30 °C incubation, and demonstrated ~5-fold higher cyclohexanone conversion at 37 °C compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and supports additional backbone hydrogen bonding. Further rational design resulted in CHMO A245G-A288V-T415C with improved thermostability at 45 °C. Our platform for oxygenase evolution enabled the rapid engineering of protein stability critical for industrial scalability.

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