Exposure of Induced Pluripotent Stem Cell-Derived Vascular Endothelial and Smooth Muscle Cells in Coculture to Hemodynamics Induces Primary Vascular Cell-Like Phenotypes

Human induced pluripotent stem cells (iPSCs) can be differentiated into vascular endothelial (iEC) and smooth muscle (iSMC) cells. However, because iECs and iSMCs are not derived from an intact blood vessel, they represent an immature phenotype. Hemodynamics and heterotypic cell:cell communication play important roles in vascular cell phenotypic modulation. Here we tested the hypothesis that hemodynamic exposure of iECs in coculture with iSMCs induces an in vivo-like phenotype. iECs and iSMCs were cocultured under vascular region-specific blood flow hemodynamics, and compared to hemodynamic cocultures of blood vessel-derived endothelial (pEC) and smooth muscle (pSMC) cells. Hemodynamic flow-induced gene expression positively correlated between pECs and iECs as well as pSMCs and iSMCs. While endothelial nitric oxide synthase 3 protein was lower in iECs than pECs, iECs were functionally mature as seen by acetylated-low-density lipoprotein (LDL) uptake. SMC contractile protein markers were also positively correlated between pSMCs and iSMCs. Exposure of iECs and pECs to atheroprone hemodynamics with oxidized-LDL induced an inflammatory response in both. Dysfunction of the transforming growth factor β (TGFβ) pathway is seen in several vascular diseases, and iECs and iSMCs exhibited a transcriptomic prolife similar to pECs and pSMCs, respectively, in their responses to LY2109761-mediated transforming growth factor β receptor I/II (TGFβRI/II) inhibition. Although there are differences between ECs and SMCs derived from iPSCs versus blood vessels, hemodynamic coculture restores a high degree of similarity in their responses to pathological stimuli associated with vascular diseases. Thus, iPSC-derived vascular cells exposed to hemodynamics may provide a viable system for modeling rare vascular diseases and testing new therapeutic approaches.This work was supported by a grant from the British Heart Foun-dation (FS/13/29/30024) (to F.S. and S.S.). All other authors were supported by the SBIR Grant NCATS R43TR001206

Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites

The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth

Plasmodium falciparum Reticulocyte Binding-Like Homologue Protein 2 (PfRH2) Is a Key Adhesive Molecule Involved in Erythrocyte Invasion

Erythrocyte invasion by Plasmodium merozoites is a complex, multistep process that is mediated by a number of parasite ligand-erythrocyte receptor interactions. One such family of parasite ligands includes the P. falciparum reticulocyte binding homologue (PfRH) proteins that are homologous with the P. vivax reticulocyte binding proteins and have been shown to play a role in erythrocyte invasion. There are five functional PfRH proteins of which only PfRH2a/2b have not yet been demonstrated to bind erythrocytes. In this study, we demonstrated that native PfRH2a/2b is processed near the N-terminus yielding fragments of 220 kDa and 80 kDa that exhibit differential erythrocyte binding specificities. The erythrocyte binding specificity of the 220 kDa processed fragment of native PfRH2a/2b was sialic acid-independent, trypsin resistant and chymotrypsin sensitive. This specific binding phenotype is consistent with previous studies that disrupted the PfRH2a/2b genes and demonstrated that PfRH2b is involved in a sialic acid independent, trypsin resistant, chymotrypsin sensitive invasion pathway. Interestingly, we found that the smaller 80 kDa PfRH2a/2b fragment is processed from the larger 220 kDa fragment and binds erythrocytes in a sialic acid dependent, trypsin resistant and chymotrypsin sensitive manner. Thus, the two processed fragments of PfRH2a/2b differed with respect to their dependence on sialic acids for erythrocyte binding. Further, we mapped the erythrocyte binding domain of PfRH2a/2b to a conserved 40 kDa N-terminal region (rPfRH240) in the ectodomain that is common to both PfRH2a and PfRH2b. We demonstrated that recombinant rPfRH240 bound human erythrocytes with the same specificity as the native 220 kDa processed protein. Moreover, antibodies generated against rPfRH240 blocked erythrocyte invasion by P. falciparum through a sialic acid independent pathway. PfRH2a/2b thus plays a key role in erythrocyte invasion and its conserved receptor-binding domain deserves attention as a promising candidate for inclusion in a blood-stage malaria vaccine

Geminivirus-Mediated Delivery of Florigen Promotes Determinate Growth in Aerial Organs and Uncouples Flowering from Photoperiod in Cotton

This article discusses geminivirus-mediated delivery of florigen. Florigen acts as a general growth hormone, advancing determinate growth. The findings extend our understanding of florigen as a general growth hormone and could benefit crop management techniques

Response of Sunflower (Helianthus annuus L.) Leaf Surface Defenses to Exogenous Methyl Jasmonate

Helianthus annuus, the common sunflower, produces a complex array of secondary compounds that are secreted into glandular trichomes, specialized structures found on leaf surfaces and anther appendages of flowers. The primary components of these trichome secretions are sesquiterpene lactones (STL), a diverse class of compounds produced abundantly by the plant family Compositae and believed to contribute to plant defense against herbivory. We treated wild and cultivated H. annuus accessions with exogenous methyl jasmonate, a plant hormone that mediates plant defense against insect herbivores and certain classes of fungal pathogens. The wild sunflower produced a higher density of glandular trichomes on its leaves than the cultivar. Comparison of the profiles of glandular trichome extracts obtained by liquid chromatography–mass spectroscopy (LC-MS) showed that wild and cultivated H. annuus were qualitatively similar in surface chemistry, although differing in the relative size and proportion of various compounds detected. Despite observing consistent transcriptional responses to methyl jasmonate treatment, we detected no significant effect on glandular trichome density or LC-MS profile in cultivated or wild sunflower, with wild sunflower exhibiting a declining trend in overall STL production and foliar glandular trichome density of jasmonate-treated plants. These results suggest that glandular trichomes and associated compounds may act as constitutive defenses or require greater levels of stimulus for induction than the observed transcriptional responses to exogenous jasmonate. Reduced defense investment in domesticated lines is consistent with predicted tradeoffs caused by selection for increased yield; future research will focus on the development of genetic resources to explicitly test the ecological roles of glandular trichomes and associated effects on plant growth and fitness

Plasmodesmal receptor-like kinases identified through analysis of rice cell wall extracted proteins

In plants, plasmodesmata (PD) are intercellular channels that function in both metabolite exchange and the transport of proteins and RNAs. Currently, many of the PD structural and regulatory components remain to be elucidated. Receptor-like kinases (RLKs) belonging to a notably expanded protein family in plants compared to the animal kingdom have been shown to play important roles in plant growth, development, pathogen resistance, and cell death. In this study, cell biological approaches were used to identify potential PD-associated RLK proteins among proteins contained within cell walls isolated from rice callus cultured cells. A total of 15 rice RLKs were investigated to determine their subcellular localization, using an Agrobacterium-mediated transient expression system. Of these six PD-associated RLKs were identified based on their co-localization with a viral movement protein that served as a PD marker, plasmolysis experiments, and subcellular localization at points of wall contact between spongy mesophyll cells. These findings suggest potential PD functions in apoplasmic signaling in response to environmental stimuli and developmental inputs

New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties

The apple is the most common and culturally important fruit crop of temperate areas. The elucidation of its origin and domestication history is therefore of great interest. The wild Central Asian species Malus sieversii has previously been identified as the main contributor to the genome of the cultivated apple (Malus domestica), on the basis of morphological, molecular, and historical evidence. The possible contribution of other wild species present along the Silk Route running from Asia to Western Europe remains a matter of debate, particularly with respect to the contribution of the European wild apple. We used microsatellite markers and an unprecedented large sampling of five Malus species throughout Eurasia (839 accessions from China to Spain) to show that multiple species have contributed to the genetic makeup of domesticated apples. The wild European crabapple M. sylvestris, in particular, was a major secondary contributor. Bidirectional gene flow between the domesticated apple and the European crabapple resulted in the current M. domestica being genetically more closely related to this species than to its Central Asian progenitor, M. sieversii. We found no evidence of a domestication bottleneck or clonal population structure in apples, despite the use of vegetative propagation by grafting. We show that the evolution of domesticated apples occurred over a long time period and involved more than one wild species. Our results support the view that self-incompatibility, a long lifespan, and cultural practices such as selection from open-pollinated seeds have facilitated introgression from wild relatives and the maintenance of genetic variation during domestication. This combination of processes may account for the diversification of several long-lived perennial crops, yielding domestication patterns different from those observed for annual species