scholarly journals In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1422
Author(s):  
Ousama Al Shanaa ◽  
Andrey Rumyantsev ◽  
Elena Sambuk ◽  
Marina Padkina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Potential Model ◽  
Model Organism ◽  
Rna Aptamers ◽  
Early Stages ◽  
Near Future

RNA aptamers are becoming increasingly attractive due to their superior properties. This review discusses the early stages of aptamer research, the main developments in this area, and the latest technologies being developed. The review also highlights the advantages of RNA aptamers in comparison to antibodies, considering the great potential of RNA aptamers and their applications in the near future. In addition, it is shown how RNA aptamers can form endless 3-D structures, giving rise to various structural and functional possibilities. Special attention is paid to the Mango, Spinach and Broccoli fluorescent RNA aptamers, and the advantages of split RNA aptamers are discussed. The review focuses on the importance of creating a platform for the synthesis of RNA nanoparticles in vivo and examines yeast, namely Saccharomyces cerevisiae, as a potential model organism for the production of RNA nanoparticles on a large scale.

scholarly journals A Combined Proteomics, Metabolomics and In Vivo Analysis Approach for the Characterization of Probiotics in Large-Scale Production

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 157 ◽  
Author(s):  
Laura Bianchi ◽  
Luca Laghi ◽  
Virginia Correani ◽  
Emily Schifano ◽  
Claudia Landi ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Profile ◽  
Model Organism ◽  
Streptococcus Thermophilus ◽  
Scale Production ◽  
High Concentration ◽  
Protein Patterns ◽  
Nuclear Magnetic Spectroscopy ◽  
In Vivo Analysis

The manufacturing processes of commercial probiotic strains may be affected in different ways in the attempt to optimize yield, costs, functionality, or stability, influencing gene expression, protein patterns, or metabolic output. Aim of this work is to compare different samples of a high concentration (450 billion bacteria) multispecies (8 strains) formulation produced at two different manufacturing sites, United States of America (US) and Italy (IT), by applying a combination of functional proteomics, metabolomics, and in vivo analyses. Several protein-profile differences were detected between IT- and US-made products, with Lactobacillus paracasei, Streptococcus thermophilus, and Bifidobacteria being the main affected probiotics/microorganisms. Performing proton nuclear magnetic spectroscopy (1H-NMR), some discrepancies in amino acid, lactate, betaine and sucrose concentrations were also reported between the two products. Finally, we investigated the health-promoting and antiaging effects of both products in the model organism Caenorhabditis elegans. The integration of omics platforms with in vivo analysis has emerged as a powerful tool to assess manufacturing procedures.

scholarly journals In vivo characterisation of fluorescent proteins in budding yeast

2018 ◽  
Author(s):  
Dennis Botman ◽  
Daan Hugo de Groot ◽  
Phillipp Schmidt ◽  
Joachim Goedhart ◽  
Bas Teusink
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Proteins ◽  
Codon Optimization ◽  
Budding Yeast ◽  
Model Organism ◽  
Ph Sensitivity ◽  
Future Studies ◽  
Signal Readout

AbstractFluorescent proteins (FPs) are widely used in many organisms, but are commonly characterised in vitro. However, the in vitro properties may poorly reflect in vivo performance. Therefore, we characterised 27 FPs in vivo using Saccharomyces cerevisiae as model organism. We linked the FPs via a T2A peptide to a control FP, producing equimolar expression of the 2 FPs from 1 plasmid. Using this strategy, we characterised the FPs for brightness, photostability, photochromicity and pH-sensitivity, achieving a comprehensive in vivo characterisation. Many FPs showed different in vivo properties compared to existing in vitro data. Additionally, various FPs were photochromic, which affects readouts due to complex bleaching kinetics. Finally, we codon optimized the best performing FPs for optimal expression in yeast, and found that codon-optimization alters FP characteristics. These FPs improve experimental signal readout, opening new experimental possibilities. Our results may guide future studies in yeast that employ fluorescent proteins.

scholarly journals Evaluating potential drug targets through human loss-of-function genetic variation

2019 ◽  
Author(s):  
Eric Vallabh Minikel ◽  
Konrad J Karczewski ◽  
Hilary C Martin ◽  
Beryl B Cummings ◽  
Nicola Whiffin ◽  
...  
Keyword(s):  
Drug Targets ◽  
Large Scale ◽  
Human Genetics ◽  
Positional Distribution ◽  
Model Organism ◽  
New Drugs ◽  
Potential Drug ◽  
Loss Of Function ◽  
Potential Drug Targets

AbstractHuman genetics has informed the clinical development of new drugs, and is beginning to influence the selection of new drug targets. Large-scale DNA sequencing studies have created a catalogue of naturally occurring genetic variants predicted to cause loss of function in human genes, which in principle should provide powerfulin vivomodels of human genetic “knockouts” to complement model organism knockout studies and inform drug development. Here, we consider the use of predicted loss-of-function (pLoF) variation catalogued in the Genome Aggregation Database (gnomAD) for the evaluation of genes as potential drug targets. Many drug targets, including the targets of highly successful inhibitors such as aspirin and statins, are under natural selection at least as extreme as known haploinsufficient genes, with pLoF variants almost completely depleted from the population. Thus, metrics of gene essentiality should not be used to eliminate genes from consideration as potential targets. The identification of individual humans harboring “knockouts” (biallelic gene inactivation), followed by individual recall and deep phenotyping, is highly valuable to study gene function. In most genes, pLoF alleles are sufficiently rare that ascertainment will be largely limited to heterozygous individuals in outbred populations. Sampling of diverse bottlenecked populations and consanguineous individuals will aid in identification of total “knockouts”. Careful filtering and curation of pLoF variants in a gene of interest is necessary in order to identify true LoF individuals for follow-up, and the positional distribution or frequency of true LoF variants may reveal important disease biology. Our analysis suggests that the value of pLoF variant data for drug discovery lies in deep curation informed by the nature of the drug and its indication, as well as the biology of the gene, followed by recall-by-genotype studies in targeted populations.

scholarly journals Tracking Diacylglycerol and Phosphatidic Acid Pools in Budding Yeast

2015 ◽  
Vol 8s1 ◽  
pp. LPI.S31781 ◽  
Author(s):  
Suriakarthiga Ganesan ◽  
Brittney N. Shabits ◽  
Vanina Zaremberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phosphatidic Acid ◽  
Physical Properties ◽  
Negative Curvature ◽  
Fluorescent Proteins ◽  
Budding Yeast ◽  
Model Organism ◽  
Current Evidence ◽  
C1 Domain

Phosphatidic acid (PA) and diacylglycerol (DAG) are key signaling molecules and important precursors for the biosynthesis of all glycerolipids found in eukaryotes. Research conducted in the model organism Saccharomyces cerevisiae has been at the forefront of the identification of the enzymes involved in the metabolism and transport of PA and DAG. Both these lipids can alter the local physical properties of membranes by introducing negative curvature, but the anionic nature of the phosphomonoester headgroup in PA sets it apart from DAG. As a result, the mechanisms underlying PA and DAG interaction with other lipids and proteins are notoriously different. This is apparent from the analysis of the protein domains responsible for recognition and binding to each of these lipids. We review the current evidence obtained using the PA-binding proteins and domains fused to fluorescent proteins for in vivo tracking of PA pools in yeast. In addition, we present original results for visualization of DAG pools in yeast using the C1 domain from mammalian PKCδ. An emerging first cellular map of the distribution of PA and DAG pools in actively growing yeast is discussed.

scholarly journals A Yeast-Based In Vivo Bioassay to Screen for Class I Phosphatidylinositol 3-Kinase Specific Inhibitors

2012 ◽  
Vol 17 (8) ◽  
pp. 1018-1029 ◽  
Author(s):  
Teresa Fernández-Acero ◽  
Isabel Rodríguez-Escudero ◽  
Francisca Vicente ◽  
Maria Cândida Monteiro ◽  
José R. Tormo ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Large Scale ◽  
Sodium Dodecyl ◽  
Model Organism ◽  
Pi3k Pathway ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Pi3k Inhibition ◽  
Phosphatidylinositol 3

The phosphatidylinositol 3-kinase (PI3K) pathway couples receptor-mediated signaling to essential cellular functions by generating the lipid second messenger phosphatidylinositol-3,4,5- trisphosphate. This pathway is implicated in multiple aspects of oncogenesis. A low-cost bioassay that readily measures PI3K inhibition in vivo would serve as a valuable tool for research in this field. Using heterologous expression, we have previously reconstituted the PI3K pathway in the model organism Saccharomyces cerevisiae. On the basis of the fact that the overproduction of PI3K is toxic in yeast, we tested the ability of commercial PI3K inhibitors to rescue cell growth. All compounds tested counteracted the PI3K-induced toxicity. Among them, 15e and PI-103 were the most active. Strategies to raise the intracellular drug concentration, specifically the use of 0.003% sodium dodecyl sulfate and the elimination of the Snq2 detoxification pump, optimized the bioassay by enhancing its sensitivity. The humanized yeast-based assay was then tested on a pilot scale for high-throughput screening (HTS) purposes using a collection of natural products of microbial origin. From 9600 extracts tested, 0.6% led to a recovery of yeast growth reproducibly, selectively, and in a dose-dependent manner. Cumulatively, we show that the developed PI3K inhibition bioassay is robust and applicable to large-scale HTS.

scholarly journals Analysing the Yeast Complexome - The Complex Portal rising to the challenge

2020 ◽  
Author(s):  
Birgit H M Meldal ◽  
Carles Pons ◽  
Livia Perfetto ◽  
Noemi Del-Toro ◽  
Edith Wong ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Ontology ◽  
Nucleic Acid ◽  
Large Scale ◽  
Model Organism ◽  
Genetic Interactions ◽  
Specific Gene ◽  
Genome Database ◽  
Macromolecular Complexes ◽  
Literature Evidence

AbstractThe EMBL-EBI Complex Portal is a knowledgebase of macromolecular complexes providing persistent stable identifiers. Entries are linked to literature evidence and provide details of complex membership, function, structure and complex-specific Gene Ontology annotations. Data is freely available and downloadable in HUPO-PSI community standards and missing entries can be requested for curation. In collaboration with Saccharomyces Genome Database and UniProt, the yeast complexome, a compendium of all known heteromeric assemblies from the model organism Saccharomyces cerevisiae, was curated. This expansion of knowledge and scope has led to a 50% increase in curated complexes compared to the previously published dataset, CYC2008. The yeast complexome is used as a reference resource for the analysis of complexes from large-scale experiments. Our analysis showed that genes coding for proteins in complexes tend to have more genetic interactions, are co-expressed with more genes, are multifunctional, localize more often in the nucleus, and are more often involved in nucleic acid-related metabolic processes and processes where large machineries are the predominant functional drivers. A comparison to genetic interactions showed that about 40% of expanded co-complex pairs also have genetic interactions, suggesting strong functional links between complex members.

C. elegans models of neuromuscular diseases expedite translational research

2010 ◽  
Vol 1 (3) ◽  
Author(s):  
James Sleigh ◽  
David Sattelle
Keyword(s):  
Large Scale ◽  
Genetic Model ◽  
Muscular Atrophy ◽  
Neuromuscular Disorders ◽  
Neuromuscular Diseases ◽  
Model Organism ◽  
Disease Genes ◽  
Chemical Library ◽  
C Elegans

AbstractThe nematode Caenorhabditis elegans is a genetic model organism and the only animal with a complete nervous system wiring diagram. With only 302 neurons and 95 striated muscle cells, a rich array of mutants with defective locomotion and the facility for individual targeted gene knockdown by RNA interference, it lends itself to the exploration of gene function at nerve muscle junctions. With approximately 60% of human disease genes having a C. elegans homologue, there is growing interest in the deployment of lowcost, high-throughput, drug screens of nematode transgenic and mutant strains mimicking aspects of the pathology of devastating human neuromuscular disorders. Here we explore the contributions already made by C. elegans to our understanding of muscular dystrophies (Duchenne and Becker), spinal muscular atrophy, amyotrophic lateral sclerosis, Friedreich’s ataxia, inclusion body myositis and the prospects for contributions to other neuromuscular disorders. A bottleneck to low-cost, in vivo, large-scale chemical library screening for new candidate therapies has been rapid, automated, behavioural phenotyping. Recent progress in quantifying simple swimming (thrashing) movements is making such screening possible and is expediting the translation of drug candidates towards the clinic.

scholarly journals In vitro turnover numbers do not reflect in vivo activities of yeast enzymes

2021 ◽  
Vol 118 (32) ◽  
pp. e2108391118
Author(s):  
Yu Chen ◽  
Jens Nielsen
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Metabolic Model ◽  
Proteomics Data ◽  
Weak Correlation ◽  
E Coli ◽  
Activity Of Enzymes

Turnover numbers (kcat values) quantitatively represent the activity of enzymes, which are mostly measured in vitro. While a few studies have reported in vivo catalytic rates (kapp values) in bacteria, a large-scale estimation of kapp in eukaryotes is lacking. Here, we estimated kapp of the yeast Saccharomyces cerevisiae under diverse conditions. By comparing the maximum kapp across conditions with in vitro kcat we found a weak correlation in log scale of R2 = 0.28, which is lower than for Escherichia coli (R2 = 0.62). The weak correlation is caused by the fact that many in vitro kcat values were measured for enzymes obtained through heterologous expression. Removal of these enzymes improved the correlation to R2 = 0.41 but still not as good as for E. coli, suggesting considerable deviations between in vitro and in vivo enzyme activities in yeast. By parameterizing an enzyme-constrained metabolic model with our kapp dataset we observed better performance than the default model with in vitro kcat in predicting proteomics data, demonstrating the strength of using the dataset generated here.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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