Mutations in Transmembrane Domains 1, 4 and 9 of the Plasmodium falciparum Chloroquine Resistance Transporter Alter Susceptibility to Chloroquine, Quinine and Quinidine

Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) can result in verapamil-reversible CQ resistance and altered susceptibility to other antimalarials. PfCRT contains 10 membrane-spanning domains and is found in the digestive vacuole (DV) membrane of intraerythrocytic parasites. The mechanism by which PfCRT mediates CQ resistance is unclear although it is associated with decreased accumulation of drug within the DV. On the permissive background of the P. falciparum 106/1(K76) parasite line, we used single-step drug selection to generate isogenic clones containing unique pfcrt point mutations that resulted in amino acid changes in PfCRT transmembrane domains 1 (C72R, K76N, K76I and K76T) and 9 (Q352K, Q352R). The resulting changes of charge and hydropathy affected quantitative CQ susceptibility and accumulation as well as the stereospecific responses to quinine and quinidine. These results, together with a previously described S163R mutation in transmembrane domain 4, indicate that transmembrane segments 1, 4 and 9 of PfCRT provide important structural components of a substrate recognition and translocation domain. Charge-affecting mutations within these segments may affect the ability of PfCRT to bind different quinoline drugs and determine their net accumulation in the DV. © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Lt

Single-cell transcriptional diversity is a hallmark of developmental potential

Single-cell RNA sequencing (scRNA-seq) is a powerful approach for reconstructing cellular differentiation trajectories. However, inferring both the state and direction of differentiation is challenging. Here, we demonstrate a simple, yet robust, determinant of developmental potential-the number of expressed genes per cell-and leverage this measure of transcriptional diversity to develop a computational framework (CytoTRACE) for predicting differentiation states from scRNA-seq data. When applied to diverse tissue types and organisms, CytoTRACE outperformed previous methods and nearly 19,000 annotated gene sets for resolving 52 experimentally determined developmental trajectories. Additionally, it facilitated the identification of quiescent stem cells and revealed genes that contribute to breast tumorigenesis. This study thus establishes a key RNA-based feature of developmental potential and a platform for delineation of cellular hierarchies.Experimental cancer immunology and therap