A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10−4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.
In contrast to the short-term(ST)-CD34pos stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSC) found in the CD34neg stem cell pool have trouble migrating and engrafting when introduced intravenously. We set out to fully elucidate the adhesion mechanisms used by ST/LT-HSCs to migrate to the bone marrow in order to understand these deficiencies. Focusing on murine ST-HSCs(Flk2negCD34pos) and LT-HSCs(Flk2negCD34neg), we observed a distinctive expression pattern of bone marrow homing effectors necessary for the first step, namely sialyl Lewis-X(sLex;ligand for E-selectin), and the second step, namely CXCR4 (receptor for SDF-1). sLex expression was higher on Flk2negCD34pos ST-HSCs(>60%) compared to Flk2negCD34neg LT-HSCs(<10%), which correlated to binding to E-selectin. Higher levels of CXCR4 were observed on Flk2negCD34pos ST-HSCs compared to Flk2negCD34neg LT-HSCs. Interestingly, expression of CD26, a peptidase known to deactivate chemokines (i.e.SDF-1), was higher on Flk2negCD34neg LT-HSCs. Given that migration is compromised in Flk2negCD34neg LT-HSCs, we aimed to enhance their ability to migrate using recombinant fucosyltransferase 6 (rhFTVI) and DiprotinA (CD26-inhibitor). We observed that although LT-HSCs expressed low levels of sLex, in vivo engraftment was not compromised. Moreover, although both treaments enhanced migration in vitro, only pre-treatment of LT-HSCs with DiprotinA enhanced engraftment in vivo. Remarkably, fucosylation of Flk2negCD34pos ST-HSCs consistently led to their ability to transplant secondary recipients, the gold standard for testing functionality of LT-HSCs. These data suggest that treatments to overcome the molecular disparity in adhesion mechanisms among ST-HSCs and LT-HSCs, differentially influences their abilities to migrate and engraft in vivo and boosts ST-HSCs engraftment in vivo.
Exosomes are tiny vesicles released by cells that carry communications to local and distant locations. Emerging research has revealed the role played by integrins found on the surface of exosomes in delivering information once they reach their destination. But until now, little has been known on the initial upstream steps of the migration process. Using biochemical and imaging approaches, we show here that exosomes isolated from both leukemic and healthy hematopoietic stem/progenitor cells can navigate their way from the cell of origin due to the presence of sialyl Lewis X modifications surface glycoproteins. This, in turn, allows binding to E-selectin at distant sites so the exosomes can deliver their messages. We show that when leukemic exosomes were injected into NSG mice, they traveled to the spleen and spine, sites typical of leukemic cell engraftment. This process, however, was inhibited in mice pre-treated with blocking E-selectin antibodies. Significantly, our proteomic analysis found that among the proteins contained within exosomes are signaling proteins, suggesting that exosomes are trying to deliver active cues to recipient cells that potentially alter their physiology. Intriguingly, the work outlined here also suggests that protein cargo can dynamically change upon exosome binding to receptors such as E-selectin, which thereby could alter the impact it has to regulate the physiology of the recipient cells. Furthermore, as an example of how miRNAs contained in exosomes can influence RNA expression in recipient cells, our analysis showed that miRNAs found in KG1a-derived exosomes target tumor suppressing proteins such as PTEN.
Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that takes place through essential interactions with adhesion molecules on an endothelial cell layer. The homing process of HSPCs begins with the tethering and rolling of the cells on the endothelial layer, which is achieved by the interaction between selectins on the endothelium to the ligands on HSPC/leukemic cells under shear stress of the blood flow. Although many studies have been based on in vitro conditions of the cells rolling over recombinant proteins, significant challenges remain when imaging HSPC/ leukemic cells on the endothelium, a necessity when considering characterizing cell-to-cell interaction and rolling dynamics during cell migration. Here, we report a new methodology that enables imaging of stem-cell-intrinsic spatiotemporal details during its migration on an endothelium-like cell monolayer. We developed optimized protocols that preserve transiently appearing structures on HSPCs/leukemic cells during its rolling under shear stress for fluorescence and scanning electron microscopy characterization. Our new experimental platform is closer to in vivo conditions and will contribute to indepth understanding of stem-cell behavior during its migration and cell-to-cell interaction during the process of homing.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.