A liquid-culture-based screening approach to study compounds affecting inflammatory processes in Caenorhabditis elegans

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Vu Thu Thuy Nguyen ◽  
Sarah Christina Meyer ◽  
Hanna Rieger ◽  
Kristina Endres
Keyword(s):  
Caenorhabditis Elegans ◽  
Liquid Culture ◽  
Human Cell Line ◽  
Small Scale ◽  
Initial Screening ◽  
Inflammatory Processes ◽  
Biological Sources ◽  
Selection Of ◽  
Screening Approaches

Abstract Discovery of biomedical drugs makes use of novel biological sources of limited availability and is often in need of fast, small-scale initial screening approaches. Here, we present a screening, based on the reporter Caenorhabditis elegans strain IG692, for identification of anti- and pro-inflammatory properties. The elaborated workflow is based on cultivation in fluid and by this, allows fast and reproducible seeding in 96 well plates. LPS and dexamethasone served as reliable controls, comparable to application in the human cell line THP-1. This in vivo approach offers a first step for selection of e.g. natural products or for repurposing of compounds from drug libraries and by this can serve as a tool in drug discovery for inflammatory human diseases.

scholarly journals Neurotoxicity evaluation of meloxicam in the alternative in vivo model, Caenorhabditis elegans

2020 ◽  
Vol 8 (8) ◽  
pp. 319-325
Author(s):  
Juliana Cyrillo Guimarães da Silva ◽  
Cassiana Bigolin ◽  
Laura Cé da Silva ◽  
Thalia Emmanoella Sebulsqui Saraiva ◽  
Julia Machado Menezes ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
In Vivo Model ◽  
High Dose ◽  
Neurological Effect ◽  
Inflammatory Processes ◽  
The Central Nervous System ◽  
Acute Neurotoxicity ◽  
Low Toxicity ◽  
Inflammatory Drugs

Inflammatory processes cause changes in the permeability of the blood brain barrier. Non-steroidal anti-inflammatory drugs (NSAID) are most commonly used to treat these inflammatory processes, including meloxicam, and they can reach the central nervous system (CNS) and cause neurotoxicity. Since there are no studies evaluating the neurotoxicity of NSAID in alternative models of toxicity, the aim of this study was to evaluate the acute neurotoxicity (through nematodes changes in behavior) of meloxicam in an alternative in vivo model, Caenorhabditis elegans, as well as, to determine meloxicam toxicity through LD50 and development assessments. Meloxicam LD50 was high (50.03 mg/mL) and only the highest dose (100 mg/mL) caused a decrease in the nematode body size, indicating low toxicity in this alternative model. Besides, a neurological effect was observed only in the highest dose. Meloxicam showed neurotoxicity only at a very high dose, suggesting low potential to cause toxicity in the CNS. However, further studies are necessary to evaluate meloxicam neurotoxicity.

scholarly journals Biosynthesis and genetic selection of oligopeptides with potentially therapeutic effects against Alzheimer' s disease

2018 ◽  
Author(s):  
Ηλίας Μάτης
Keyword(s):  
Caenorhabditis Elegans ◽  
Genetic Selection ◽  
Therapeutic Effects ◽  
E Coli ◽  
Selection Of

Η νόσος Alzheimer αποτελεί την πλέον ολέθρια νευροεκφυλιστική νόσο και την πιο διαδεδομένη μορφή άνοιας. Μέχρις στιγμής, υποψήφιες ενώσεις για την αντιμετώπιση της νόσου δεν έχουν επιδείξει ικανότητα αναχαίτησης της προόδου της νόσου, με αποτέλεσμα την αυξημένη αναγκαιότητα εύρεσης νέων θεραπευτικών ενώσεων. Η παθογένεια της νόσου Alzheimer έχει συσχετιστεί με το αμυλοειδές-β (Αβ), ένα πεπτίδιο 37-43 αμινοξέων το οποίο εμφανίζει εγγενή τάση σχηματισμού ολιγομερών και αδιάλυτων συσσωματωμάτων με νευροτοξικές ιδιότητες. Κατά συνέπεια, η ανακάλυψη μικρομοριακών βιολογικών αναστολέων της συσσωμάτωσης του Αβ αποτελεί μία ελκυστική θεραπευτική προσέγγιση για την αντιμετώπιση της νόσου, η οποία έχει αποτελέσει βασική επιδίωξη της φαρμακευτικής βιομηχανίας.Η συγκεκριμένη διδακτορική διατριβή περιγράφει το σχεδιασμό και ανάπτυξη ενός βακτηριακού συστήματος το οποίο δύναται να λειτουργήσει ως πλατφόρμα ανακάλυψης βιοδραστικών ενώσεων, με αποτρεπτική δράση ως προς τη συσσωμάτωση του Αβ, καθώς και τη συνεπαγόμενη νευροτοξικότητά του. Για αυτό το σκοπό, βιβλιοθήκες κυκλικών πεπτιδίων παράγονται βιοσυνθετικά σε κύτταρα E. coli ενώ παράλληλα υποβάλλονται σε διαλογή με κριτήριο την ικανότητά τους να παρεμποδίζουν τη συσσωμάτωση του Αβ, χρησιμοποιώντας μια γενετική δοκιμασία υψηλού ρυθμού απόδοσης. Η εν λόγω γενετική δοκιμασία παρέχει τη δυνατότητα εντοπισμού βιοδραστικών κυκλικών πεπτιδίων τα οποία αυξάνουν το επίπεδο φθορισμού της πρωτεϊνικής χίμαιρας του Αβ με την πράσινη φθορίζουσα πρωτεΐνη GFP. Η παραγωγή της χίμαιρας και των βιβλιοθηκών των κυκλικών πεπτιδίων πραγματοποιούνται παράλληλα σε τροποποιημένα βακτήρια, ενώ η διαλογή των μεμονωμένων φθοριζόντων κλώνων επιτελείται με τη χρήση κυτταρομετρίας ροής.Το τροποποιημένο βακτηριακό σύστημα που χρησιμοποιήθηκε, επέτρεψε τη βιοσύνθεση και τη γρήγορη διαλογή 10 εκατομμυρίων κυκλικών πεπτιδίων, καθώς και την επιλογή εκατοντάδων βιοδραστικών πεπτιδίων με εν δυνάμει ρυθμιστικό ρόλο έναντι της συσσωμάτωσης του Αβ. Από αυτά, δύο πεπτίδια που έφεραν τις αλληλουχίες cyclo-SASPT και cyclo-TAFDR και ονομάστηκαν AβC5-34 και AβC5-116 αντίστοιχα, παρήχθησαν μέσω σύνθεσης και χρησιμοποιήθηκαν για τη μελέτη της δράσης τους πάνω στη συσσωμάτωση του Αβ, μέσω βιοχημικών, βιοφυσικών και βιολογικών δοκιμασιών, in vitro. Οι δοκιμασίες αυτές φανέρωσαν ότι τα συγκεκριμένα κυκλικά πεπτίδια παρεμβάλλονται στη φυσιολογική συσσωμάτωση του Αβ, οδηγώντας στο σχηματισμό μη τυπικών συσσωματώσεων οι οποίες εμφανίζουν περιορισμένη νευροτοξικότητα. Επιπλέον, η προστατευτική δράση των ΑβC5-34 και ΑβC5-116 έναντι της συσσωμάτωσης και κυτταροτοξικότητας του Αβ αξιολογήθηκαν in vivo σε διαγονιδιακά στελέχη Caenorhabditis elegans που παράγουν Αβ. Οι δοκιμασίες αυτές έδειξαν πως τα πεπτίδια AβC5-34 και AβC5-116 παρουσιάζουν προστατευτικές ιδιότητες έναντι της συσσωμάτωσης και της κυτταροτοξικότητας του Αβ. Συνεπώς, τα πεπτίδια AβC5-34 και AβC5-116 παρουσιάζουν φαρμακολογικό ενδιαφέρον ενώ η περαιτέρω αξιολόγηση και βελτιστοποίηση τους θα μπορούσε να οδηγήσει σε ενώσεις με θεραπευτικές ιδιότητες έναντι της νόσου Alzheimer. Τέλος, ο συνδυασμός στοχευμένης μεταλλαξιγένεσης και ανάλυσης DNA νέας γενιάς, επέτρεψε την ανακάλυψη των σχέσεων δομής και δραστικότητας για τον πληθυσμό των επιλεγμένων κυκλικών πεπτιδίων, καθώς και την ταυτοποίηση διακριτών οικογενειών βιοδραστικών πεπτιδίων.

scholarly journals Caenorhabditis elegans as an indicator of toxicity of Bacillus thuringiensis strains to Meloidogyne incognita race 3

2018 ◽  
Vol 48 (7) ◽  
Author(s):  
Sandro Coelho Linhares Montalvão ◽  
Marcelo Tavares de Castro ◽  
Carlos Marcelo Silveira Soares ◽  
Luiz Eduardo Bassay Blum ◽  
Rose Gomes Monnerat
Keyword(s):  
Caenorhabditis Elegans ◽  
Meloidogyne Incognita ◽  
Root Knot Nematode ◽  
C Elegans ◽  
Antagonistic Microorganisms ◽  
Large Populations ◽  
Deep Plowing ◽  
Selection Of

ABSTRACT: The cotton plant (Gossypium hirsutum) is affected by several diseases of economic importance, among them root-knot nematode (Meloidogyne incognita races 3 and 4). Methods to control this disease include the application of nematicides, solarization, deep plowing, crop rotation and use of antagonistic microorganisms. Among species of Bacillus, there are strains that act as bioregulators and antagonists of several pathogens. Tests to identify these strains are hampered by the difficulty of obtaining large populations of the pathogen and by the time of execution of the in vivo tests that should be conducted for about 90 days. The objective of this research was to compare the toxicity of B. thuringiensis strains to two nematodes, M. incognita and Caenorhabditis elegans, evaluating the possibility of using C. elegans as an indicator for the selection of strains with biocontrol potential against M. incognita. Therefore, the toxicity of nine B. thuringiensis strains on C. elegans and M. incognita was evaluated under laboratory and greenhouse conditions. Most strains toxic to C. elegans in vitro were also toxic to M. incognita, and three of them (S906, S1192, S2036) significantly reduced the populations of the two nematodes. The toxic effect of B. thuringiensis strains on C. elegans was like that reported for the same bacterial isolates on M. incognita in vivo. These results suggested that it is plausible to use C. elegans as an indicator of toxicity for selection of B. thuringiensis strains toxic to M. incognita.

scholarly journals In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Merricka C. Livingstone ◽  
Alexis A. Bitzer ◽  
Alish Giri ◽  
Kun Luo ◽  
Rajeshwer S. Sankhala ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Monoclonal Antibodies ◽  
Disease Burden ◽  
Vaccine Candidate ◽  
Specific Binding ◽  
Circumsporozoite Protein ◽  
Target Epitope ◽  
Selection Of

AbstractPlasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

scholarly journals A Region of the Myosin Rod Important for Interaction With Paramyosin in Caenorhabditis elegans Striated Muscle

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 631-643
Author(s):  
Pamela E Hoppe ◽  
Robert H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Heavy Chain ◽  
Molecular Interaction ◽  
Striated Muscle ◽  
Genetic Interaction ◽  
Specific Interaction ◽  
Thick Filament ◽  
Parallel Arrangement ◽  
Myosin Rod

Abstract The precise arrangement of molecules within the thick filament, as well as the mechanisms by which this arrangement is specified, remains unclear. In this article, we have exploited a unique genetic interaction between one isoform of myosin heavy chain (MHC) and paramyosin in Caenorhabditis elegans to probe the molecular interaction between MHC and paramyosin in vivo. Using chimeric myosin constructs, we have defined a 322-residue region of the MHC A rod critical for suppression of the structural and motility defects associated with the unc-15(e73) allele. Chimeric constructs lacking this region of MHC A either fail to suppress, or act as dominant enhancers of, the e73 phenotype. Although the 322-residue region is required for suppression activity, our data suggest that sequences along the length of the rod also play a role in the isoform-specific interaction between MHC A and paramyosin. Our genetic and cell biological analyses of construct behavior suggest that the 322-residue region of MHC A is important for thick filament stability. We present a model in which this region mediates an avid interaction between MHC A and paramyosin in parallel arrangement in formation of the filament arms.

scholarly journals Adenosine A2A receptor signaling promotes FoxO associated autophagy in chondrocytes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin Friedman ◽  
Carmen Corciulo ◽  
Cristina M. Castro ◽  
Bruce N. Cronstein
Keyword(s):  
Cell Line ◽  
Metabolic Stress ◽  
Human Cell Line ◽  
Adenosine A2a Receptor ◽  
Autophagic Flux ◽  
A2a Receptor ◽  
Adenosine A2a ◽  
A2ar Agonist

AbstractAutophagy, a homeostatic pathway upregulated during cellular stress, is decreased in osteoarthritic chondrocytes and this reduction in autophagy is thought to contribute to the development and progression of osteoarthritis (OA). The adenosine A2A receptor (A2AR) is a potent anti-inflammatory receptor and deficiency of this receptor leads to the development of OA in mice. Moreover, treatment using liposomally conjugated adenosine or a specific A2AR agonist improved joint scores significantly in both rats with post-traumatic OA (PTOA) and mice subjected to a high fat diet obesity induced OA. Importantly, A2AR ligation is beneficial for mitochondrial health and metabolism in vitro in primary and the TC28a2 human cell line. An additional set of metabolic, stress-responsive, and homeostatic mediators include the Forkhead box O transcription factors (FoxOs). Data has shown that mouse FoxO knockouts develop early OA with reduced cartilage autophagy, indicating that FoxO-induced homeostasis is important for articular cartilage. Given the apparent similarities between A2AR and FoxO signaling, we tested the hypothesis that A2AR stimulation improves cartilage function through activation of the FoxO proteins leading to increased autophagy in chondrocytes. We analyzed the signaling pathway in the human TC28a2 cell line and corroborated these findings in vivo in a metabolically relevant obesity-induced OA mouse model. We found that A2AR stimulation increases activation and nuclear localization of FoxO1 and FoxO3, promotes an increase in autophagic flux, improves metabolic function in chondrocytes, and reduces markers of apoptosis in vitro and reduced apoptosis by TUNEL assay in vivo. A2AR ligation additionally enhances in vivo activation of FoxO1 and FoxO3 with evidence of enhanced autophagic flux upon injection of the liposome-associated A2AR agonist in a mouse obesity-induced OA model. These findings offer further evidence that A2AR may be an excellent target for promoting chondrocyte and cartilage homeostasis.

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