A fast, easy, cost-free method to remove excess dye or drug from small extracellular vesicle solution.

Tracking small extracellular vesicles (sEVs), such as exosomes, requires staining them with dyes that penetrate their lipid bilayer, a process that leaves excess dye that needs to be mopped up to achieve high specificity. Current methods to remove superfluous dye have limitations, among them that they are time-intensive, carry the risk of losing sample and can require specialized equipment and materials. Here we present a fast, easy-to-use, and cost-free protocol for cleaning excess dye from stained sEV samples by adding their parental cells to the mixture to absorb the extra dye much like sponges do. Since sEVs are considered a next-generation drug delivery system, we further show the success of our approach at removing excess chemotherapeutic drug, daunorubicin, from the sEV solution.

APRF1 Interactome Reveals HSP90 as a New Player in the Complex That Epigenetically Regulates Flowering Time in Arabidopsis thaliana.

WD40 repeat proteins (WDRs) are present in all eukaryotes and include members that are implicated in numerous cellular activities. They act as scaffold proteins and thus as molecular "hubs" for protein-protein interactions, which mediate the assembly of multifunctional complexes that regulate key developmental processes in Arabidopsis thaliana, such as flowering time, hormonal signaling, and stress responses. Despite their importance, many aspects of their putative functions have not been elucidated yet. Here, we show that the late-flowering phenotype of the anthesis promoting factor 1 (aprf1) mutants is temperature-dependent and can be suppressed when plants are grown under mild heat stress conditions. To gain further insight into the mechanism of APRF1 function, we employed a co-immunoprecipitation (Co-IP) approach to identify its interaction partners. We provide the first interactome of APRF1, which includes proteins that are localized in several subcellular compartments and are implicated in diverse cellular functions. The dual nucleocytoplasmic localization of ARRF1, which was validated through the interaction of APRF1 with HEAT SHOCK PROTEIN 1 (HSP90.1) in the nucleus and with HSP90.2 in the cytoplasm, indicates a dynamic and versatile involvement of APRF1 in multiple biological processes. The specific interaction of APRF1 with the chaperon HSP90.1 in the nucleus expands our knowledge regarding the epigenetic regulation of flowering time in A. thaliana and further suggests the existence of a delicate thermoregulated mechanism during anthesis.

Fabrication of a three-dimensional bone marrow niche-like acute myeloid Leukemia disease model by an automated and controlled process using a robotic multicellular bioprinting system.

BACKGROUND: Acute myeloid leukemia (AML) is a hematological malignancy that remains a therapeutic challenge due to the high incidence of disease relapse. To better understand resistance mechanisms and identify novel therapies, robust preclinical models mimicking the bone marrow (BM) microenvironment are needed. This study aimed to achieve an automated fabrication process of a three-dimensional (3D) AML disease model that recapitulates the 3D spatial structure of the BM microenvironment and applies to drug screening and investigational studies. METHODS: To build this model, we investigated a unique class of tetramer peptides with an innate ability to self-assemble into stable hydrogel. An automated robotic bioprinting process was established to fabricate a 3D BM (niche-like) multicellular AML disease model comprised of leukemia cells and the BM's stromal and endothelial cellular fractions. In addition, monoculture and dual-culture models were also fabricated. Leukemia cell compatibility, functionalities (in vitro and in vivo), and drug assessment studies using our model were performed. In addition, RNAseq and gene expression analysis using TaqMan arrays were also performed on 3D cultured stromal cells and primary leukemia cells. RESULTS: The selected peptide hydrogel formed a highly porous network of nanofibers with mechanical properties similar to the BM extracellular matrix. The robotic bioprinter and the novel quadruple coaxial nozzle enabled the automated fabrication of a 3D BM niche-like AML disease model with controlled deposition of multiple cell types into the model. This model supported the viability and growth of primary leukemic, endothelial, and stromal cells and recapitulated cell-cell and cell-ECM interactions. In addition, AML cells in our model possessed quiescent characteristics with improved chemoresistance attributes, resembling more the native conditions as indicated by our in vivo results. Moreover, the whole transcriptome data demonstrated the effect of 3D culture on enhancing BM niche cell characteristics. We identified molecular pathways upregulated in AML cells in our 3D model that might contribute to AML drug resistance and disease relapse. CONCLUSIONS: Our results demonstrate the importance of developing 3D biomimicry models that closely recapitulate the in vivo conditions to gain deeper insights into drug resistance mechanisms and novel therapy development. These models can also improve personalized medicine by testing patient-specific treatments.

DNA-binding protein PfAP2-P regulates parasite pathogenesis during malaria parasite blood stages.

Malaria-associated pathogenesis such as parasite invasion, egress, host cell remodelling and antigenic variation requires concerted action by many proteins, but the molecular regulation is poorly understood. Here we have characterized an essential Plasmodium-specific Apicomplexan AP2 transcription factor in Plasmodium falciparum (PfAP2-P; pathogenesis) during the blood-stage development with two peaks of expression. An inducible knockout of gene function showed that PfAP2-P is essential for trophozoite development, and critical for var gene regulation, merozoite development and parasite egress. Chromatin immunoprecipitation sequencing data collected at timepoints matching the two peaks of pfap2-p expression demonstrate PfAP2-P binding to promoters of genes controlling trophozoite development, host cell remodelling, antigenic variation and pathogenicity. Single-cell RNA sequencing and fluorescence-activated cell sorting revealed de-repression of most var genes in Δpfap2-p parasites. Δpfap2-p parasites also overexpress early gametocyte marker genes, indicating a regulatory role in sexual stage conversion. We conclude that PfAP2-P is an essential upstream transcriptional regulator at two distinct stages of the intra-erythrocytic development cycle.

PfAP2-MRP DNA-binding protein is a master regulator of parasite pathogenesis during malaria parasite blood stages.

Malaria pathogenicity results from the parasite's ability to invade, multiply within and then egress from the host red blood cell (RBC). Infected RBCs are remodeled, expressing antigenic variant proteins (such as PfEMP1, coded by the var gene family) for immune evasion and survival. These processes require the concerted actions of many proteins, but the molecular regulation is poorly understood. We have characterized an essential Plasmodium specific Apicomplexan AP2 (ApiAP2) transcription factor in Plasmodium falciparum (PfAP2-MRP; Master Regulator of Pathogenesis) during the intraerythrocytic developmental cycle (IDC). An inducible gene knockout approach showed that PfAP2-MRP is essential for development during the trophozoite stage, and critical for var gene regulation, merozoite development and parasite egress. ChIP-seq experiments performed at 16 hour post invasion (h.p.i.) and 40 h.p.i. matching the two peaks of PfAP2-MRP expression, demonstrate binding of PfAP2-MRP to the promoters of genes controlling trophozoite development and host cell remodeling at 16 h.p.i. and antigenic variation and pathogenicity at 40 h.p.i. Using single-cell RNA-seq and fluorescence-activated cell sorting, we show de-repression of most var genes in Δpfap2-mrp parasites that express multiple PfEMP1 proteins on the surface of infected RBCs. In addition, the Δpfap2-mrp parasites overexpress several early gametocyte marker genes at both 16 and 40 h.p.i., indicating a regulatory role in the sexual stage conversion. Using the Chromosomes Conformation Capture experiment (Hi-C), we demonstrate that deletion of PfAP2-MRP results in significant reduction of both intra-chromosomal and inter-chromosomal interactions in heterochromatin clusters. We conclude that PfAP2-MRP is a vital upstream transcriptional regulator controlling essential processes in two distinct developmental stages during the IDC that include parasite growth, chromatin structure and var gene expression.

CD34+ HSPCs-derived exosomes contain dynamic cargo and promote their migration through functional binding with the homing receptor E-selectin.

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.

Nanoscopic Characterization of Cell Migration under Flow Using Optical and Electron Microscopy.

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.

Refining the migration and engraftment of short-term and long-term HSCs by enhancing homing-specific adhesion mechanisms.

In contrast to the short-term (ST) CD34+ stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSCs) found in the CD34- stem cell pool have trouble migrating and engrafting when introduced through IV. To understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by ST and LT-HSCs to migrate to the bone marrow(BM). Specifically focusing on murine ST-HSCs (Flk2-CD34+) and LT-HSCs (Flk2-CD34-), we observed a distinctive expression pattern of BM homing effectors necessary for the first step, namely sialyl Lewis-X (sLex) (ligand for E-selectin), and the second step, namely CXCR4 chemokine receptor (receptor for SDF-1). sLex expression was higher on Flk2-CD34+ ST-HSCs (>60%) compared with Flk2-CD34- LT-HSCs (<10%), which correlated to binding to E-selectin. Higher concentrations of CXCR4 were observed on Flk2-CD34+ ST-HSCs compared with Flk2-CD34- LT-HSCs. Interestingly, the expression of CD26, a peptidase known to deactivate chemokines (ie, SDF-1), was higher on Flk2-CD34- LT-HSCs. Given that both E-selectin-binding and CXCR4-mediated migration are compromised in Flk2-CD34- LT-HSCs, we aimed to enhance their ability to migrate using recombinant human fucosyltransferase 6 (rhFTVI) and the CD26 inhibitor, Dip A (diprotin A). To this end, we observed that although LT-HSCs expressed low concentrations of sLex, they were able to engraft when transplanted into recipient mice. Moreover, although both CD26 inhibition and fucosylation enhanced migration of both HSC populations in vitro, only pretreatment of LT-HSCs with Dip A enhanced engraftment in vivo after transplantation into recipient mice. Remarkably, fucosylation of Flk2-CD34+ ST-HSCs consistently led to their ability to transplant secondary recipients. These data suggest that using fucosylation and Dip A 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 promotes the ability of ST-HSCs to engraft secondary recipient mice, the gold standard for testing functionality of LT-HSCs.

RNAi silencing of the Arabidopsis thaliana ULCS1 gene results in pleiotropic phenotypes during plant growth and development.

WD40-repeat-containing proteins (WDRs) are highly abundant in all eukaryotes. Several have been implicated as subunits of multi-protein CRL E3 ligase complexes that regulate ubiquitination mediated protein degradation and thus various cellular and developmental processes. Impairment of the WDR protein ULCS1 from Arabidopsis causes pleiotropic phenotypes during plant development, including reduced lignification, anther indehiscence, and sterility. Here we show that RNAi-mediated downregulation of ULCS1 results in a fast-growing phenotype during vegetative development. Due to accelerated growth, ulcs1i mutants reach their vegetative to reproductive transition point earlier than WT plants. However, their comparable germination rate and their similar number of secondary branches and rosette leaves at bolting indicate that ulcs1i is not an early flowering time mutant. GUS staining of progeny, obtained from crosses between ulcs1i and CYCB1::GUS plants, revealed an increased number of mitotic cell divisions in the root meristems of ulcs1i compared to WT. Immunolabeling of homogalacturonans (HGAs) epitopes showed significant fluorescent signal differences at the cell walls and the mucilage of the seeds between ulcs1i and WT. Furthermore, we demonstrate that ULCS1 interacts with the UBA-like protein in a yeast two-hybrid assay, suggesting a direct or indirect physical coupling of these proteins in Arabidopsis.

GA-Mediated Disruption of RGA/BZR1 Complex Requires HSP90 to Promote Hypocotyl Elongation.

Circuitries of signaling pathways integrate distinct hormonal and environmental signals, and influence development in plants. While a crosstalk between brassinosteroid (BR) and gibberellin (GA) signaling pathways has recently been established, little is known about other components engaged in the integration of the two pathways. Here, we provide supporting evidence for the role of HSP90 (HEAT SHOCK PROTEIN 90) in regulating the interplay of the GA and BR signaling pathways to control hypocotyl elongation of etiolated seedlings in Arabidopsis. Both pharmacological and genetic depletion of HSP90 alter the expression of GA biosynthesis and catabolism genes. Major components of the GA pathway, like RGA (REPRESSOR of ga1-3) and GAI (GA-INSENSITIVE) DELLA proteins, have been identified as physically interacting with HSP90. Interestingly, GA-promoted DELLA degradation depends on the ATPase activity of HSP90, and inhibition of HSP90 function stabilizes the DELLA/BZR1 (BRASSINAZOLE-RESISTANT 1) complex, modifying the expression of downstream transcriptional targets. Our results collectively reveal that HSP90, through physical interactions with DELLA proteins and BZR1, modulates DELLA abundance and regulates the expression of BZR1-dependent transcriptional targets to promote plant growth.

Simplified detection of polyhistidine-tagged proteins in gels and membranes using a UV-excitable dye and a multiple chelator head pair.

The polyhistidine tag (His-tag) is one of the most popular protein tags used in the life sciences. Traditionally, the detection of His-tagged proteins relies on immunoblotting with anti-His antibodies. This approach is laborious for certain applications, such as protein purification, where time and simplicity are critical. The His-tag can also be directly detected by metal ion-loaded nickel-nitrilotriacetic acid-based chelator heads conjugated to fluorophores, which is a convenient alternative method to immunoblotting. Typically, such chelator heads are conjugated to either green or red fluorophores, the detection of which requires specialized excitation sources and detection systems. Here, we demonstrate that post-run staining is ideal for His-tag detection by metal ion-loaded and fluorescently labeled chelator heads in PAGE and blot membranes. Additionally, by comparing the performances of different chelator heads, we show how differences in microscopic affinity constants translate to macroscopic differences in the detection limits in environments with limited diffusion, such as PAGE. On the basis of these results, we devise a simple approach, called UVHis-PAGE, that uses metal ion-loaded and fluorescently labeled chelator heads to detect His-tagged proteins in PAGE and blot membranes. Our method uses a UV transilluminator as an excitation source, and the results can be visually inspected by the naked eye.

Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR.

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.