Development of Needed Tools for Babesia duncani, a Rising Cause of Human Babesiosis
Notice bibliographique
Résumé
Human babesiosis is a bloodborne, tick-transmitted zoonotic disease that has seen an increase in cases across the United States and Canada. Babesia duncani is the second most prevalent and most virulent cause of human babesiosis but little is known of its cellular biology, molecular pathogenesis, and molecular genetics. To remedy this and help accelerate research on Babesia duncani, we worked towards developing a functional Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system for Babesia duncani while identifying strong promoters to drive expression of genes, developing an optimized transfection protocol, and identifying suitable selectable markers. To identify strong promoters to drive expression of genes in transfections, reverse transcriptase (RT)-qPCR was performed on RNA extracted and synthesized into cDNA. To find a workable transfection protocol that could be optimized, established Babesia and Plasmodium transfection protocols from the literature were screened for their transfection efficacy (amount of nanoluciferase transcript produced) and impact on parasite viability following transfection by RT-qPCR readout. Once a workable transfection protocol had been identified, the protocol was optimized by varying the number of parasites and amount of DNA used per transfection. A CRISPR/Cas9 transfection was performed using the identified optimized transfection protocol. Lastly, to identify suitable selectable markers a novel 384-well high throughput compound screening format was developed. Babesia duncani were incubated for 48-hours with the compound of interest and lysed in the presence of propidium iodide before fluorescence was read 24-hours later. Histone 2B (H2B) promoter was identified as the strongest promoter of those tested. Enolase and elongation factor-1α (eF-1α) were the next strongest promoters but had roughly 3- and 5-fold less expression than H2B, respectively. Govindarajalu et al, 2019 Lyse-Reseal protocol gave the third highest amount of nanoluciferase transcript without negatively impacting parasite growth following transfection. It was selected as the protocol to be optimized, with results indicating increased number of parasites and increased amount of DNA used per transfection led to greater transfection efficiency. However the results also indicated there may also be an inherent limitation to the maximum efficiency obtainable with the protocol seen in the diminishing return when increasing the number of parasites or amount of DNA used. The results of CRISPR/Cas9 transfection were promising as a mCherry expressing Babesia duncani, the expected outcome, was observed 24-hours post transfection however no other ones were observed following that. Using the high throughput compound screening format a handful of compounds were tested, identifying potential novel selectable markers for Babesia duncani as well as avenues for drug development.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,002 | 0,002 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,001 |
| Méta-épidémiologie (sens large) | 0,001 | 0,001 |
| Bibliométrie | 0,001 | 0,001 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,001 | 0,001 |
| Science ouverte | 0,001 | 0,001 |
| Intégrité de la recherche | 0,001 | 0,002 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,005 | 0,005 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».