Altered synaptic connectivity in an in vitro human model of STXBP1 encephalopathy

Early infantile epileptic encephalopathies are devastating conditions, generally of genetic origin, but the pathological mechanisms often remain obscure. A major obstacle in this field of research is the difficulty of studying cortical brain development in humans, in utero. To address this, we established an in vitro assay to study the impact of gene variants on the developing human brain, using living organotypic cultures of the human subplate and neighbouring cortical regions, prepared from ethically sourced, 14-17 post conception week brain tissue (www.hdbr.org). We were able to maintain cultures for several months, during which time, the gross anatomical structures of the cortical plate, subplate and marginal zone persisted, while neurons continued to develop morphologically, and form new synaptic networks. This preparation thus permits the study of genetic manipulations, and their downstream effects, upon an intact developing human cortical network. We focused upon STXBP1 haploinsufficiency, which is among the most common genetic causes of infantile epileptic encephalopathy. This was induced using shRNA interference, leading to impaired synaptic function and a drop in the number of glutamatergic synapses. We thereby provide a critical proof-of-principle for how to study the impact of any gene of interest on the development of the human cortex.

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Development and Application of a Transcriptomic Signature of Bioactivation in an Advanced In Vitro Liver Model to Reduce Drug-induced Liver Injury Risk Early in the Pharmaceutical Pipeline

Toxicological Sciences ◽

10.1093/toxsci/kfaa094 ◽

2020 ◽

Vol 177 (1) ◽

pp. 121-139 ◽

Cited By ~ 4

Author(s):

Wen Kang ◽

Alexei A Podtelezhnikov ◽

Keith Q Tanis ◽

Stephen Pacchione ◽

Ming Su ◽

...

Keyword(s):

Liver Injury ◽

Human Model ◽

Reactive Metabolites ◽

In Vitro Test ◽

Drug Induced ◽

Vitro Assay ◽

Drug Induced Liver Injury ◽

Drug Candidates ◽

Transcriptomic Signature

Abstract Early risk assessment of drug-induced liver injury (DILI) potential for drug candidates remains a major challenge for pharmaceutical development. We have previously developed a set of rat liver transcriptional biomarkers in short-term toxicity studies to inform the potential of drug candidates to generate a high burden of chemically reactive metabolites that presents higher risk for human DILI. Here, we describe translation of those NRF1-/NRF2-mediated liver tissue biomarkers to an in vitro assay using an advanced micropatterned coculture system (HEPATOPAC) with primary hepatocytes from male Wistar Han rats. A 9-day, resource-sparing and higher throughput approach designed to identify new chemical entities with lower reactive metabolite-forming potential was qualified for internal decision making using 93 DILI-positive and -negative drugs. This assay provides 81% sensitivity and 90% specificity in detecting hepatotoxicants when a positive test outcome is defined as the bioactivation signature score of a test drug exceeding the threshold value at an in vitro test concentration that falls within 3-fold of the estimated maximum drug concentration at the human liver inlet following highest recommended clinical dose administrations. Using paired examples of compounds from distinct chemical series and close structural analogs, we demonstrate that this assay can differentiate drugs with lower DILI risk. The utility of this in vitro transcriptomic approach was also examined using human HEPATOPAC from a single donor, yielding 68% sensitivity and 86% specificity when the aforementioned criteria are applied to the same 93-drug test set. Routine use of the rat model has been adopted with deployment of the human model as warranted on a case-by-case basis. This in vitro transcriptomic signature-based strategy can be used early in drug discovery to derisk DILI potential from chemically reactive metabolites by guiding structure-activity relationship hypotheses and candidate selection.

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Soaps and Detergents: Alternatives to Animal Eye Irritation Tests

Journal of the American College of Toxicology ◽

10.3109/10915819609008705 ◽

1996 ◽

Vol 15 (1) ◽

pp. 1-44 ◽

Cited By ~ 17

Author(s):

Mildred S. Christian ◽

Robert M. Diener

Keyword(s):

False Negative ◽

Screening Tools ◽

Current Status ◽

In Vitro Assays ◽

Computer Search ◽

Eye Irritation ◽

Vitro Assay ◽

Predictive Values ◽

The Impact

An extensive computer search was conducted, and a comprehensive overview of the current status of alternatives to animal eye irritation tests was obtained. A search of Medline and Toxline databases (1988 to present) was supplemented with references from sources regarding in vitro eye irritation. Particular attention was paid to soap and detergent products and related ingredients. Eighty-five references are included in the review; the in vitro assays are categorized, and their predictive values for assessing acute ocular irritation are evaluated and compared with the Draize rabbit eye irritation assay and with each other. The present review shows that the increased activity of scientists from academia, industry, and regulatory agencies has resulted in substantial progress in developing alternative in vitro procedures and that a number of large, interlaboratory evaluations and international workshops have assisted in the selection process. However, none of these methodologies has obtained acceptance for regulatory classification purposes. Conclusions drawn from this review include that (a) no single in vitro assay is considered capable of replacing the Draize eye irritation test; (b) the chorioallantoic membrane vascular assay (CAMVA) or the hen egg test-chorio-allantoic membrane test (HET-CAM), the chicken or bovine enucleated eye test, the neutral red and plasminogen activation assays for cytotoxicity, and the silicon microphysiometer appear to have the greatest potential as screening tools for eye irritation; and (c) choosing a specific assay or series of assays will depend on the type of agent tested and the impact of false-negative or false-positive results. New assays will continue to be developed and should be included in future evaluations, when sufficient data are available.

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Impact of Glucosinolate Content in Broccoli (Brassica oleracea (Italica Group)) on Growth of Pseudomonas marginalis, a Causal Agent of Bacterial Soft Rot

Plant Disease ◽

10.1094/pdis.2002.86.6.629 ◽

2002 ◽

Vol 86 (6) ◽

pp. 629-632 ◽

Cited By ~ 8

Author(s):

Craig S. Charron ◽

Carl E. Sams ◽

Craig H. Canaday

Keyword(s):

Brassica Oleracea ◽

Degradation Products ◽

Linear Regression Analysis ◽

Soft Rot ◽

Causal Agent ◽

Glucosinolate Content ◽

Bacterial Soft Rot ◽

Vitro Assay ◽

The Impact

Glucosinolate degradation products are known to suppress microbes. Brassica species produce glucosinolates. Previous investigations determined that susceptibility to bacterial soft rot of broccoli (Brassica oleracea (Italica group)) varied significantly by cultivar. To evaluate the impact of glucosinolates on Pseudomonas marginalis, a causal agent of bacterial soft rot, glucosinolates were measured in lyophilized florets from broccoli ‘Arcadia’, ‘Emperor’, ‘Green Comet’, ‘Green Valiant’, ‘Marathon’, ‘Packman’, ‘Premium Crop’, and ‘Shogun’. Total glucosinolate content was highest in ‘Shogun’ (29.8 μmol/g) and lowest in ‘Emperor’ (0.5 μmol/g). In an in vitro assay, simple linear regression analysis showed that 48% of differences in suppression of P. marginalis growth could be explained by differences in total glucosinolate content (P ≤ 0.01). Plant breeding efforts should include glucosinolate levels as a factor in selecting for disease resistance.

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A COMPARATIVE ANALYSIS OF PLANT GROWTH-PROMOTING TRAITS OF Pseudomonas AND Bacillus STRAINS ISOLATED FROM Lolium perenne RHIZOSPHERIC SOIL IN VOJVODINA (SERBIA) AND THEIR EFFECT ON THE PLANT YIELD

Acta Scientiarum Polonorum Hortorum Cultus ◽

10.24326/asphc.2020.3.4 ◽

2020 ◽

Vol 19 (3) ◽

pp. 37-45

Author(s):

Dragana Stamenov ◽

Timea I. Hajnal-Jafari ◽

Biljana Najvirt ◽

Snežana Anđelković ◽

Jelena Tomić ◽

...

Keyword(s):

Plant Growth ◽

Lolium Perenne ◽

Biochemical Characterization ◽

Total Yield ◽

Forage Crops ◽

Vitro Assay ◽

Plant Growth Promoting ◽

Growth Promoting ◽

The Impact

The objective of this work was to do a comparative study of Pseudomonas and Bacillus isolates for their plant growth-promoting (PGP) potential, monitoring the impact of selected isolates on the yield of English ryegrass (Lolium perenne). Isolation, physiological and biochemical characterization, in vitro assay of enzymatic and plant-growth promoting activities of isolates were done. Pseudomonas isolates have been shown to have the ability to use different sources of carbon, to live in the condition of low pH as well as temperature and to produce siderophore. On the other hand, Bacillus isolates have the ability to solubilize phosphate, to produce a greater amount of indol-3-acetic acid (IAA) than Pseudomonas isolates and have an inhibitory effect on the growth of phytopathogenic fungi. In other investigated traits, isolates were similar. The use of Pseudomonas sp. P12 and Bacillus sp. B1 isolates had a positive effect on the plant mass and total yield, which indicate that the use of these isolates can result in a better yield of forage crops.

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Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314013 ◽

2020 ◽

Author(s):

Patrick A. Murphy ◽

Noor Jailkhani ◽

Sarah-Anne Nicholas ◽

Amanda M. Del Rosario ◽

Jeremy L. Balsbaugh ◽

...

Keyword(s):

Extracellular Matrix ◽

Alternative Splicing ◽

Low Flow ◽

Matrix Composition ◽

Splice Isoforms ◽

Vitro Assay ◽

Disturbed Flow ◽

The Impact

Objective: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. Conclusions: Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.

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Mitotic arrest affects clustering of tumor cells

Cell Division ◽

10.1186/s13008-021-00070-z ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Julia Bonnet ◽

Lise Rigal ◽

Odile Mondesert ◽

Renaud Morin ◽

Gaëlle Corsaut ◽

...

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Cancer Cell ◽

Cell Aggregation ◽

Metastatic Potential ◽

Mitotic Arrest ◽

Vitro Assay ◽

The Impact ◽

Mcf 7

Abstract Background Cancer cell aggregation is a key process involved in the formation of tumor cell clusters. It has recently been shown that clusters of circulating tumor cells (CTCs) have an increased metastatic potential compared to isolated circulating tumor cells. Several widely used chemotherapeutic agents that target the cytoskeleton microtubules and cause cell cycle arrest at mitosis have been reported to modulate CTC number or the size of CTC clusters. Results In this study, we investigated in vitro the impact of mitotic arrest on the ability of breast tumor cells to form clusters. By using live imaging and quantitative image analysis, we found that MCF-7 cancer cell aggregation is compromised upon incubation with paclitaxel or vinorelbine, two chemotherapeutic drugs that target microtubules. In line with these results, we observed that MCF-7 breast cancer cells experimentally synchronized and blocked in metaphase aggregated poorly and formed loose clusters. To monitor clustering at the single-cell scale, we next developed and validated an in vitro assay based on live video-microscopy and custom-designed micro-devices. The study of cluster formation from MCF-7 cells that express the fluorescent marker LifeAct-mCherry using this new assay allowed showing that substrate anchorage-independent clustering of MCF-7 cells was associated with the formation of actin-dependent highly dynamic cell protrusions. Metaphase-synchronized and blocked cells did not display such protrusions, and formed very loose clusters that failed to compact. Conclusions Altogether, our results suggest that mitotic arrest induced by microtubule-targeting anticancer drugs prevents cancer cell clustering and therefore, could reduce the metastatic potential of circulating tumor cells.

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Mitotic arrest affects clustering of tumor cells

10.21203/rs.3.rs-47408/v1 ◽

2020 ◽

Author(s):

Julia Bonnet ◽

Lise Rigal ◽

Odile Mondesert ◽

Renaud Morin ◽

Gaelle Corsaut ◽

...

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Cancer Cell ◽

Cell Aggregation ◽

Metastatic Potential ◽

Mitotic Arrest ◽

Vitro Assay ◽

The Impact ◽

Mcf 7

Abstract BackgroundCancer cell aggregation is a key process involved in the formation of tumor cells clusters. It has recently been shown that clusters of circulating tumor cells (CTCs) have an increased metastatic potential compared to isolated circulating tumor cells. Several widely used chemotherapeutic agents that target the cytoskeleton microtubules and cause cell cycle arrest at mitosis have been reported to modulate the number of CTCs or the size of CTC clusters.ResultsIn this study, we investigated in vitro the impact of mitotic arrest on the ability of breast tumor cells to form clusters. By using live imaging and quantitative image analysis, we found that MCF-7 cancer cell aggregation is compromised upon incubation with paclitaxel or vinorelbine, two chemotherapeutic drugs that target microtubules. In line with these results, we found that MCF-7 breast cancer cells experimentally synchronized and blocked in metaphase aggregated poorly with strongly reduced cohesion. To monitor clustering at the single-cell scale, we next developed and validated an in vitro assay based on live video-microscopy and custom-designed micro-devices. The study of cluster formation from MCF-7 cells that express the fluorescent marker LifeAct-mCherry using this new assay allowed showing that substrate anchorage-independent clustering of MCF-7 cells was associated with the formation of actin-dependent highly dynamic cell protrusions. Metaphase-synchronized and blocked cells did not display such protrusions, and formed very loose clusters that failed to compact.ConclusionsAltogether, our results suggest that mitotic arrest induced by microtubule-targeting anticancer drugs prevents cancer cell clustering and therefore, could reduce the metastatic potential of circulating tumor cells.

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Biallelic variants in ADARB1, encoding a dsRNA-specific adenosine deaminase, cause a severe developmental and epileptic encephalopathy

Journal of Medical Genetics ◽

10.1136/jmedgenet-2020-107048 ◽

2020 ◽

pp. jmedgenet-2020-107048 ◽

Cited By ~ 1

Author(s):

Reza Maroofian ◽

Jiří Sedmík ◽

Neda Mazaheri ◽

Marcello Scala ◽

Maha S Zaki ◽

...

Keyword(s):

Enzymatic Activity ◽

Rna Editing ◽

Stop Codon ◽

Neurodevelopmental Disorder ◽

Premature Stop Codon ◽

Epileptic Encephalopathy ◽

In Vitro Assays ◽

Global Developmental Delay ◽

The Impact

BackgroundAdenosine-to-inosine RNA editing is a co-transcriptional/post-transcriptional modification of double-stranded RNA, catalysed by one of two active adenosine deaminases acting on RNA (ADARs), ADAR1 and ADAR2. ADARB1 encodes the enzyme ADAR2 that is highly expressed in the brain and essential to modulate the function of glutamate and serotonin receptors. Impaired ADAR2 editing causes early onset progressive epilepsy and premature death in mice. In humans, ADAR2 dysfunction has been very recently linked to a neurodevelopmental disorder with microcephaly and epilepsy in four unrelated subjects.MethodsWe studied three children from two consanguineous families with severe developmental and epileptic encephalopathy (DEE) through detailed physical and instrumental examinations. Exome sequencing (ES) was used to identify ADARB1 mutations as the underlying genetic cause and in vitro assays with transiently transfected cells were performed to ascertain the impact on ADAR2 enzymatic activity and splicing.ResultsAll patients showed global developmental delay, intractable early infantile-onset seizures, microcephaly, severe-to-profound intellectual disability, axial hypotonia and progressive appendicular spasticity. ES revealed the novel missense c.1889G>A, p.(Arg630Gln) and deletion c.1245_1247+1 del, p.(Leu415PhefsTer14) variants in ADARB1 (NM_015833.4). The p.(Leu415PhefsTer14) variant leads to incorrect splicing resulting in frameshift with a premature stop codon and loss of enzyme function. In vitro RNA editing assays showed that the p.(Arg630Gln) variant resulted in a severe impairment of ADAR2 enzymatic activity.ConclusionIn conclusion, these data support the pathogenic role of biallelic ADARB1 variants as the cause of a distinctive form of DEE, reinforcing the importance of RNA editing in brain function and development.

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8 How to drive efficiencies in pork production: What are the gaps in knowledge and obstacles?

Journal of Animal Science ◽

10.1093/jas/skaa054.043 ◽

2020 ◽

Vol 98 (Supplement_3) ◽

pp. 24-25

Author(s):

Steve J Kitt

Keyword(s):

Production Efficiency ◽

Scientific Discipline ◽

Growing Pigs ◽

Scientific Application ◽

Vitro Assay ◽

Pork Production ◽

The Impact ◽

Swine Nutrition ◽

Cross Fostering

Abstract Swine nutrition and production has been the cornerstone scientific discipline of the Midwest Section ASAS meeting for decades. During this time, the impact of scientists’ work and the scientific application by swine professionals has been profound. However, if we are to double our food supply by 2050, we have much to do. This presentation will attempt to illustrate contrasting examples of production efficiencies associated with differing production goals, and highlight examples of processes that lead to improved pork production efficiency within the discipline of swine nutrition. This presentation will also cover obstacles and gaps in knowledge that practicing nutritionists, pork producers, and other professionals regularly encounter. Some knowledge gaps are within a single scientific discipline and if resources were put towards the problem would be relatively easily solved. Examples include: development of an inexpensive in vitro assay to assess DDGS amino acid digestibility, re-defining the upper and lower critical effective temperature for modern pigs, defining the minimum and best equipment for growing pigs, identifying additivity effects of various dietary compounds, scientifically sound method(s) to understand and resolve mycotoxin contamination of grains. Other gaps in knowledge are more complex and require multi-disciplinary scientific approach. Examples include: defining root causes of sow mortality, defining factors influencing pig and piglet mortality especially during health challenges, developing models to assist optimization of litter size and post-wean performance, determining best practices of cross-fostering. Pork production will continue to be a viable yet challenging endeavor. It will require persistence, collaboration, and ingenuity from our scientific and pork production community in order to adequately feed the growing population’s demand for protein.

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Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro

10.1101/2021.05.31.446494 ◽

2021 ◽

Author(s):

Brandon M Trainor ◽

Dimitri G Pestov ◽

Natalia Shcherbik

Keyword(s):

Gene Expression ◽

Regulation Of Gene Expression ◽

Mrna Translation ◽

Protein Translation ◽

Transcription Control ◽

Vitro Assay ◽

Control Of Gene Expression ◽

Translational Machinery ◽

The Impact

The conventional view regarding regulation of gene expression is based on transcription control. However, a growing number of recent studies has revealed the important additional impact of translational regulation. Eukaryotic translational machinery appears to be capable of reprogramming mRNA translation to generate proteins required to maintain a healthy cellular proteostasis under particular physiological conditions or to adapt to stress. Although the mechanisms of such remarkable regulation are beginning to emerge, recent studies have identified the ribosome as one of the major constituents of translation-dependent control of gene expression that is especially important during stress. Built of RNA and proteins, ribosomes are susceptible to environmental and intracellular stresses. How stress-modified ribosomes regulate translation and whether they play a role in stress-induced gene expression remain largely elusive. This knowledge gap is likely due to the lack of an appropriate experimental system. Canonical approaches based on exposing cells or cell-free extracts to stressors provide inconclusive results due to off-target effects of modifying agents. Here we describe a robust and simple in vitro assay that allows separation of yeast ribosomes from other translational machinery constituents, followed by reconstitution of the translation reaction. This ribosome separation and reconstitution assay (RSR) is highly advantageous, as it allows modification of ribosomes without compromising other key translational components, followed by supplementing the ribosomes back into translation reactions containing undamaged, translationally-competent yeast lysate. Besides addressing the impact of ribosome-derived stress on translation, RSR can also be used to characterize mutated ribosomes and ribosomes devoid of associated factors.

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