scholarly journals A High-Throughput Mechanical Activator for Cartilage Engineering Enables Rapid Screening of in vitro Response of Tissue Models to Physiological and Supra-Physiological Loads

2021 ◽  
pp. 1-19
Author(s):  
Elisa Capuana ◽  
Davide Marino ◽  
Roberto Di Gesù ◽  
Vincenzo La Carrubba ◽  
Valerio Brucato ◽  
...  
Keyword(s):  
High Throughput ◽  
Compressive Loading ◽  
Cartilage Degeneration ◽  
Early Response ◽  
Rapid Screening ◽  
New Device ◽  
Cartilage Engineering ◽  
Physiological Loading ◽  
Tgf Β1

Articular cartilage is crucially influenced by loading during development, health, and disease. However, our knowledge of the mechanical conditions that promote engineered cartilage maturation or tissue repair is still incomplete. Current in vitro models that allow precise control of the local mechanical environment have been dramatically limited by very low throughput, usually just a few specimens per experiment. To overcome this constraint, we have developed a new device for the high throughput compressive loading of tissue constructs: the High Throughput Mechanical Activator for Cartilage Engineering (HiT-MACE), which allows the mechanoactivation of 6 times more samples than current technologies. With HiT-MACE we were able to apply cyclic loads in the physiological (e.g., equivalent to walking and normal daily activity) and supra-physiological range (e.g., injurious impacts or extensive overloading) to up to 24 samples in one single run. In this report, we compared the early response of cartilage to physiological and supra-physiological mechanical loading to the response to IL-1β exposure, a common but rudimentary in vitro model of cartilage osteoarthritis. Physiological loading rapidly upregulated gene expression of anabolic markers along the TGF-β1 pathway. Notably, TGF-β1 or serum was not included in the medium. Supra-physiological loading caused a mild catabolic response while IL-1β exposure drove a rapid anabolic shift. This aligns well with recent findings suggesting that overloading is a more realistic and biomimetic model of cartilage degeneration. Taken together, these findings showed that the application of HiT-MACE allowed the use of larger number of samples to generate higher volume of data to effectively explore cartilage mechanobiology, which will enable the design of more effective repair and rehabilitation strategies for degenerative cartilage pathologies.

scholarly journals Recovery of NanoLuc Luciferase-Tagged Canine Distemper Virus for Facilitating Rapid Screening of Antivirals in vitro

2020 ◽  
Vol 7 ◽  
Author(s):  
Fuxiao Liu ◽  
Qianqian Wang ◽  
Yilan Huang ◽  
Ning Wang ◽  
Youming Zhang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Canine Distemper Virus ◽  
Reverse Genetics ◽  
Rapid Screening ◽  
Canine Distemper ◽  
Virus Propagation ◽  
The Family ◽  
Conventional Methods

Canine distemper virus (CDV), belonging to the genus Morbillivirus in the family Paramyxoviridae, is a highly contagious pathogen, affecting various domestic, and wild carnivores. Conventional methods are too cumbersome to be used for high-throughput screening of anti-CDV drugs. In this study, a recombinant CDV was rescued using reverse genetics for facilitating screening of anti-CDV drug in vitro. The recombinant CDV could stably express the NanoLuc® luciferase (NLuc), a novel enzyme that was smaller and “brighter” than others. The intensity of NLuc-catalyzed luminescence reaction indirectly reflected the anti-CDV effect of a certain drug, due to a positive correlation between NLuc expression and virus propagation in vitro. Based on such a characteristic feature, the recombinant CDV was used for anti-CDV assays on four drugs (ribavirin, moroxydine hydrochloride, 1-adamantylamine hydrochloride, and tea polyphenol) via analysis of luciferase activity, instead of via conventional methods. The result showed that out of these four drugs, only the ribavirin exhibited a detectable anti-CDV effect. The NLuc-tagged CDV would be a rapid tool for high-throughput screening of anti-CDV drugs.

scholarly journals Hematin Polymerization Assay as a High-Throughput Screen for Identification of New Antimalarial Pharmacophores

2000 ◽  
Vol 44 (10) ◽  
pp. 2638-2644 ◽  
Author(s):  
Yae Kurosawa ◽  
Arnulf Dorn ◽  
Michiko Kitsuji-Shirane ◽  
Hisao Shimada ◽  
Tomoko Satoh ◽  
...  
Keyword(s):  
High Throughput ◽  
Murine Model ◽  
Antimalarial Activity ◽  
Scintillation Counter ◽  
Rapid Screening ◽  
Malaria Pigment ◽  
Pharmacophore Hypothesis ◽  
Oral Activity ◽  
Hemoglobin Degradation

ABSTRACT Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of 14C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100,000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the “Catalyst” program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified “hit” compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in thePlasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program.

Platelet-rich gel-incorporated silk scaffold promotes meniscus regeneration in a rabbit total meniscectomy model

2019 ◽  
Vol 14 (8) ◽  
pp. 753-768 ◽  
Author(s):  
Yifan Wu ◽  
Jianqiao Hong ◽  
Guangyao Jiang ◽  
Sihao Li ◽  
Shiming Chen ◽  
...  
Keyword(s):  
Cartilage Degeneration ◽  
Functional Regeneration ◽  
Scaffold Materials ◽  
Silk Scaffold ◽  
Meniscus Regeneration ◽  
Tgf Β1

Aim: To investigate whether platelet-rich gel (PRG) incorporation could promote meniscal regeneration of the silk scaffold. Materials & methods: A PRG-incorporated silk sponge was fabricated for reconstruction of the meniscus in a rabbit meniscectomy model. Subsequently, characterization of the scaffold, as well as the in vitro cytocompatibility and in vivo function was evaluated. Results: Our results showed that the PRG-incorporated silk scaffold provided a sustained release of TGF-β1 over 1 week. The PRG enhanced the cytocompatibility in vitro and cell infiltration in vivo of the silk sponge. Meanwhile, the implantation of the composite in situ ameliorated the cartilage degeneration in knee at 3 months. Conclusion: These findings indicated that PRG-incorporated silk scaffold could promote functional regeneration of the meniscus and effectively prevented subsequent osteoarthritis after meniscectomy.

In vitro models of TGF-β-induced fibrosis suitable for high-throughput screening of antifibrotic agents

2007 ◽  
Vol 293 (2) ◽  
pp. F631-F640 ◽  
Author(s):  
Qihe Xu ◽  
Jill T. Norman ◽  
Shashi Shrivastav ◽  
Javier Lucio-Cazana ◽  
Jeffrey B. Kopp
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Matrix Protein ◽  
Mesangial Cells ◽  
In Vitro Models ◽  
Nodule Formation ◽  
Dependent Manner ◽  
Gas Treatment ◽  
Tgf Β1

Progressive fibrosis is a cause of progressive organ dysfunction. Lack of quantitative in vitro models of fibrosis accounts, at least partially, for the slow progress in developing effective antifibrotic drugs. Here, we report two complementary in vitro models of fibrosis suitable for high-throughput screening. We found that, in mesangial cells and renal fibroblasts grown in eight-well chamber slides, transforming growth factor-β1 (TGF-β1) disrupted the cell monolayer and induced cell migration into nodules in a dose-, time- and Smad3-dependent manner. The nodules contained increased interstitial collagens and showed an increased collagen I:IV ratio. Nodules are likely a biological consequence of TGF-β1-induced matrix overexpression since they were mimicked by addition of collagen I to the cell culture medium. TGF-β1-induced nodule formation was inhibited by vacuum ionized gas treatment of the plate surface. This blockage was further enhanced by precoating plates with matrix proteins but was prevented, at least in part, by poly-l-lysine (PLL). We have established two cell-based models of TGF-β-induced fibrogenesis, using mesangial cells or fibroblasts cultured in matrix protein or PLL-coated 96-well plates, on which TGF-β1-induced two-dimensional matrix accumulation, three-dimensional nodule formation, and monolayer disruption can be quantitated either spectrophotometrically or by using a colony counter, respectively. As a proof of principle, chemical inhibitors of Alk5 and the antifibrotic compound tranilast were shown to have inhibitory activities in both assays.

scholarly journals New Device for High-Throughput Viability Screening of Flow Biofilms

2010 ◽  
Vol 76 (13) ◽  
pp. 4136-4142 ◽  
Author(s):  
Michael R. Benoit ◽  
Carolyn G. Conant ◽  
Cristian Ionescu-Zanetti ◽  
Michael Schwartz ◽  
A. Matin
Keyword(s):  
Fluid Flow ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Accurate Determination ◽  
Microtiter Plate ◽  
Rapid Screening ◽  
Microfluidic Channels ◽  
Screening Methods ◽  
New Device ◽  
Plate Reader

ABSTRACT Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.

scholarly journals Pterostilbene Alleviates Pulmonary Fibrosis by Regulating ASIC2

2020 ◽  
Author(s):  
Yanfang Peng ◽  
Yingwen Zhang ◽  
Yabing Zhang ◽  
Xiuping Wang ◽  
Yukun Xia ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Western Blot ◽  
High Throughput ◽  
Protein Level ◽  
High Throughput Sequencing ◽  
Epithelial Mesenchymal Transition ◽  
A549 Cells ◽  
Mesenchymal Transition ◽  
Tgf Β1

Abstract Background: Idiopathic pulmonary fibrosis (IPF) is a serious chronic disease of the respiratory system, and its current treatment have certain shortcomings and adverse effects. In this study, we evaluate the anti-fibrotic activity of pterostilbene (PTE) using an IPF model induced by TGF-β1 in vitro.Methods: A549 and AEC cells were incubated with 10 ng/ml TGF-β1 to induce lung fibroblast activation. 30 μmol/L PTE was used to treat the cells. The epithelial-mesenchymal transition (EMT), accumulation of extracellular matrix (ECM) and autophagy of cells were suggested by western blot. The apoptosis was proved by flow cytometry analysis and western blot. Transcriptome high-throughput sequencing on A549 cells incubated with TGF-β1 alone or TGF-β1 and PTE (TGF-β1+PTE) was performed, and differentially expressed genes caused by PTE were identified. The ASIC2 overexpression plasmid was used to rescue the protein level of ASIC2 in A549 and AEC cells.Results: TGF-β1 caused the EMT and accumulation of ECM, and blocked the autophagy and apoptosis of A549 and AEC cells. Most importantly, 30 μmol/L PTE inhibited the pulmonary fibrosis induced by TGF-β1. Compared with cells treated with TGF-β1, PTE treatment inhibited the EMT and accumulation of ECM, and rescued cell apoptosis and autophagy. The results of transcriptome high-throughput sequencing performed that PTE greatly reduced the protein level of ASIC2. In addition, compared with the TGF-β1+PTE group, the transfection of ASIC2 overexpression plasmid stimulated the EMT and accumulation of ECM, and inhibited apoptosis and autophagy, suggesting that PTE inhibited pulmonary fibrosis by down-regulating ASIC2. Conclusions: In conclusion, our study suggests that PTE and ASIC2 inhibitors may benefit future IPF treatments.

scholarly journals Detection of Banana Mild Mosaic Virus in Musa In Vitro Plants: High-Throughput Sequencing Presents Higher Diagnostic Sensitivity Than (IC)-RT-PCR and Identifies a New Betaflexiviridae Species

Plants ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 226
Author(s):  
Marwa Hanafi ◽  
Wei Rong ◽  
Lucie Tamisier ◽  
Chadi Berhal ◽  
Nicolas Roux ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Diagnostic Sensitivity ◽  
Rapid Screening ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
In Vitro Plants ◽  
Mild Mosaic Virus

: The banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus that infects Musa spp. and has a very wide geographical distribution. The current BanMMV indexing process for an accession requires the testing of no less than four plants cultivated in a greenhouse for at least 6 months and causes a significant delay for the distribution of the germplasm. We evaluated the sensitivity of different protocols for BanMMV detection from in vitro plants to accelerate the testing process. We first used corm tissues from 137 in vitro plants and obtained a diagnostic sensitivity (DSE) of only 61% when testing four plants per accession. After thermotherapy was carried out to eliminate BanMMV infection, the meristem was recovered and further grown in vitro. The same protocol was evaluated in parallel on the corm tissue surrounding the meristem, as a rapid screening to evaluate virus therapy success, and was compared to the results obtained following the standard protocol. The obtained results showed 28% false negatives when conducting testing from corm tissues, making this protocol unsuitable in routine processes. Furthermore, RT-PCR and high-throughput sequencing (HTS) tests were applied on tissues from the base (n = 39) and the leaves (n = 36). For RT-PCR, the average DSE per sample reached 65% from either the base or leaves. HTS was applied on 36 samples and yielded 100% diagnostic specificity (DSP) and 100% DSE, whatever the sampled tissue, allowing the identification of a new Betaflexiviridae species infecting Musa. These results suggest that a reliable diagnostic of BanMMV from in vitro plants using RT-PCR or HTS technologies might represent an efficient alternative for testing after greenhouse cultivation.

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