Cell-based high-throughput screening of polysaccharide biosynthesis hosts

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.

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

Nature Communications ◽

10.1038/s41467-021-23954-8 ◽

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.

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High Throughput Phenotypic Screening of The Human Spermatozoon

Reproduction ◽

10.1530/rep-21-0372 ◽

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.

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Resorbable Beads Provide Extended Release of Antifungal Medication: In Vitro and In Vivo Analyses

Pharmaceutics ◽

10.3390/pharmaceutics11110550 ◽

2019 ◽

Vol 11 (11) ◽

pp. 550 ◽

Cited By ~ 1

Author(s):

Yung-Heng Hsu ◽

Huang-Yu Chen ◽

Jin-Chung Chen ◽

Yi-Hsun Yu ◽

Ying-Chao Chou ◽

...

Keyword(s):

High Performance ◽

Antifungal Agents ◽

High Dose ◽

Molding Method ◽

Potential Applications ◽

Radical Debridement ◽

Fungal Osteomyelitis ◽

High Level

Fungal osteomyelitis has been difficult to treat, with first-line treatments consisting of implant excision, radical debridement, and local release of high-dose antifungal agents. Locally impregnated antifungal beads are another popular treatment option. This study aimed to develop biodegradable antifungal-agent-loaded Poly(d,l-lactide-co-glycolide) (PLGA) beads and evaluate the in vitro/in vivo release patterns of amphotericin B and fluconazole from the beads. Beads of different sizes were formed using a compression-molding method, and their morphology was evaluated via scanning electron microscopy. Intrabead incorporation of antifungal agents was evaluated via Fourier-transform infrared spectroscopy, and in vitro fluconazole liberation curves of PLGA beads were inspected via high-performance liquid chromatography. When we implanted the drug-incorporated beads into the bone cavity of rabbits, we found that a high level of fluconazole (beyond the minimum therapeutic concentration [MTC]) was released for more than 49 d in vivo. Our results indicate that compression-molded PLGA/fluconazole beads have potential applications in treating bone infections.

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A Novel High-Throughput Technique for Identifying Monoclonal Antibodies capable of Death Receptor Induced Apoptosis

Journal of Cell Death ◽

10.4137/jcd.s3660 ◽

2009 ◽

Vol 2 ◽

pp. JCD.S3660

Author(s):

Hang Fai Kwok ◽

Julie A. Gormley ◽

Christopher J. Scott ◽

James A. Johnston ◽

Shane A. Olwill

Keyword(s):

Cell Death ◽

High Throughput ◽

High Throughput Screening ◽

False Negative ◽

Death Receptor ◽

Annexin V ◽

Fas Receptor ◽

Induced Apoptosis

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

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High-Throughput Screening of Novel Peptide Inhibitors of an Integrin Receptor from the Hexapeptide Library by Using a Protein Microarray Chip

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104268125 ◽

2004 ◽

Vol 9 (8) ◽

pp. 687-694 ◽

Cited By ~ 31

Author(s):

Yoonsuk Lee ◽

Dong-Ku Kang ◽

Soo-Ik Chang ◽

Moon Hi Han ◽

In-Cheol Kang

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Competitive Inhibition ◽

Protein Microarray ◽

Dependent Manner ◽

High Throughput Analysis ◽

Microarray Chip ◽

New Drug

Protein microarray is an emerging technology that makes high-throughput analysis possible for protein-protein interactions and analysis of proteome and biomarkers in parallel. The authors investigated the application of a novel protein microarray chip, Proteo Chip, in new drug discovery. Integrin αvβ3 microarray immobilized on the Proteo Chip was employed to screen new active peptides against the integrin from multiple hexapeptide sublibraries of a positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin αvβ3-vitronectin interaction was successfully demonstrated on the integrin microarray in a dose-dependent manner andwas inhibited not only by the syntheticRGDpeptide but also by various integrin antagonists on the integrin microarray chip. Novel peptide ligands with high affinity to the integrin were also identified from the peptide libraries with this chip-based screening system by a competitive inhibition assay in a simultaneous and highthroughput fashion. The authors have confirmed antiangiogenic functions of the novel peptides thus screened through an in vitro and in vivo angiogenesis assay. These results provide evidence that the Proteo Chip is a promising tool for highthroughput screening of lead molecules in new drug development.

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Recent Advances in In-Vitro Assays for Type 2 Diabetes Mellitus: An Overview

The Review of Diabetic Studies ◽

10.1900/rds.2020.16.13 ◽

2020 ◽

Vol 16 (1) ◽

pp. 13-23

Author(s):

Nazmina Vhora ◽

Ujjal Naskar ◽

Aishwarya Hiray ◽

Abhijeet S. Kate ◽

Alok Jain

Keyword(s):

Type 2 Diabetes ◽

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Antidiabetic Activity ◽

Screening Methods ◽

Selection Of

BACKGROUND: A higher rate of attenuation of molecules in drug discovery has enabled pharmaceutical companies to enhance the efficiency of their hit identification and lead optimization. Selection and development of appropriate in-vitro and in-vivo strategies may improve this process as primary and secondary screening utilize both strategies. In-vivo approaches are too relentless and expensive for assessing hits. Therefore, it has become indispensable to develop and implement suitable in-vitro screening methods to execute the required activities and meet the respective targets. However, the selection of an appropriate in-vitro assay for specific evaluation of cellular activity is no trivial task. It requires thorough investigation of the various parameters involved. AIM: In this review, we aim to discuss in-vitro assays for type 2 diabetes (T2D), which have been utilized extensively by researchers over the last five years, including target-based, non-target based, low-throughput, and high-throughput screening assays. METHODS: The literature search was conducted using databases including Scifinder, PubMed, ScienceDirect, and Google Scholar to find the significant published articles. DISCUSSION and CONCLUSION: The accuracy and relevance of in-vitro assays have a significant impact on the drug discovery process for T2D, especially in assessing the antidiabetic activity of compounds and identifying the site of effect in high-throughput screening. The report reviews the advantages, limitations, quality parameters, and applications of the probed invitro assays, and compares them with one another to enable the selection of the optimal method for any purpose. The information on these assays will accelerate numerous procedures in the drug development process with consistent quality and accuracy.

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

10.1101/423731 ◽

2018 ◽

Cited By ~ 1

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.

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High Throughput Screening of Pharmacokinetics and Metabolism in Drug Discovery (II) —Investigation on In vitro and In vivo Correlation in Drug Metabolism Screening—

YAKUGAKU ZASSHI ◽

10.1248/yakushi.125.131 ◽

2005 ◽

Vol 125 (1) ◽

pp. 131-139 ◽

Cited By ~ 3

Author(s):

Hiroshi KOMURA ◽

Kenichi MATSUDA ◽

Yukie SHIGEMOTO ◽

Iichiro KAWAHARA ◽

Rieko ANO ◽

...

Keyword(s):

Drug Discovery ◽

Drug Metabolism ◽

High Throughput ◽

High Throughput Screening

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High-throughput screening identifies microRNAs that target Nox2 and improve function after acute myocardial infarction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00685.2016 ◽

2017 ◽

Vol 312 (5) ◽

pp. H1002-H1012 ◽

Cited By ~ 23

Author(s):

Junyu Yang ◽

Milton E. Brown ◽

Hanshuo Zhang ◽

Mario Martinez ◽

Zhihua Zhao ◽

...

Keyword(s):

Myocardial Infarction ◽

Nadph Oxidase ◽

Cardiac Function ◽

High Throughput ◽

High Throughput Screening ◽

Heart Function ◽

In Vivo Studies ◽

Screening System

Myocardial infarction (MI) is the most common cause of heart failure. Excessive production of ROS plays a key role in the pathogenesis of cardiac remodeling after MI. NADPH with NADPH oxidase (Nox)2 as the catalytic subunit is a major source of superoxide production, and expression is significantly increased in the infarcted myocardium, especially by infiltrating macrophages. While microRNAs (miRNAs) are potent regulators of gene expression and play an important role in heart disease, there still lacks efficient ways to identify miRNAs that target important pathological genes for treating MI. Thus, the overall objective was to establish a miRNA screening and delivery system for improving heart function after MI using Nox2 as a critical target. With the use of the miRNA-target screening system composed of a self-assembled cell microarray (SAMcell), three miRNAs, miR-106b, miR-148b, and miR-204, were identified that could regulate Nox2 expression and its downstream products in both human and mouse macrophages. Each of these miRNAs were encapsulated into polyketal (PK3) nanoparticles that could effectively deliver miRNAs into macrophages. Both in vitro and in vivo studies in mice confirmed that PK3-miRNAs particles could inhibit Nox2 expression and activity and significantly improve infarct size and acute cardiac function after MI. In conclusion, our results show that miR-106b, miR-148b, and miR-204 were able to improve heart function after myocardial infarction in mice by targeting Nox2 and possibly altering inflammatory cytokine production. This screening system and delivery method could have broader implications for miRNA-mediated therapeutics for cardiovascular and other diseases. NEW & NOTEWORTHY NADPH oxidase (Nox)2 is a promising target for treating cardiovascular disease, but there are no specific inhibitors. Finding endogenous signals that can target Nox2 and other inflammatory molecules is of great interest. In this study, we used high-throughput screening to identify microRNAs that target Nox2 and improve cardiac function after infarction.

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