Chemistry of floral rewards: intra- and interspecific variability of nectar and pollen secondary metabolites across taxa

Floral chemistry mediates plant interactions with pollinators, pathogens, and herbivores, with major consequences for fitness of both plants and flower visitors. The outcome of such interactions often depends on compound dose and chemical context. However, chemical diversity and intraspecific variation of nectar and pollen secondary chemistry are known for very few species, precluding general statements about their composition. We analyzed methanol extracts of flowers, nectar, and pollen from 31 cultivated and wild plant species, including multiple sites and cultivars, by liquid chromatography-mass spectrometry. To depict the 29 chemical niche of each tissue type, we analyzed differences in nectar and pollen chemical richness, absolute and proportional concentrations, and intraspecific variability. We hypothesized that pollen would have higher concentrations and more compounds than nectar, consistent with Optimal Defense Theory and pollen’s importance as a male gamete. To investigate chemical correlations across and within tissues, which could reflect physiological constraints, we quantified chemical overlap between conspecific nectar and pollen, and phenotypic integration of individual compounds within tissue types Nectar and pollen were chemically differentiated both across and within species. Of 102 compounds identified, most occurred in only one species. Machine-learning algorithms assigned samples to the correct species and tissue type with 98.6% accuracy. Consistent with our hypothesis, pollen had 23.8- to 235-fold higher secondary chemical concentrations and 63% higher chemical richness than nectar. The most common secondary compound classes were flavonoids, alkaloids, terpenoids, and phenolics (primarily phenylpropanoids including chlorogenic acid). The most common specific compound types were quercetin and kaempferol glycosides, known to mediate biotic and abiotic effects. Pollens were distinguished from nectar by high concentrations of hydroxycinnamoyl-spermidine conjugates, which affect plant development, abiotic stress tolerance, and herbivore resistance. Although chemistry was qualitatively consistent within species and tissue types, concentrations varied across cultivars and sites, which could influence pollination, herbivory, and disease in wild and agricultural plants. Analyses of multivariate trait space showed greater overlap across sites and cultivars in nectar than pollen chemistry; this overlap reflected greater within-site and within-cultivar variability of nectar. Our analyses suggest different ecological roles of nectar and pollen mediated by chemical concentration, composition, and variability

Substrate Micropatterning as a New in Vitro Cell Culture System to Study Myelination

Artículo de publicación ISIMyelination is a highly regulated developmental process whereby oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system ensheathe axons with a multilayered concentric membrane. Axonal myelination increases the velocity of nerve impulse propagation. In this work, we present a novel in vitro system for coculturing primary dorsal root ganglia neurons along with myelinating cells on a highly restrictive and micropatterned substrate. In this new coculture system, neurons survive for several weeks, extending long axons on defined Matrigel tracks. On these axons, myelinating cells can achieve robust myelination, as demonstrated by the distribution of compact myelin and nodal markers. Under these conditions, neurites and associated myelinating cells are easily accessible for studies on the mechanisms of myelin formation and on the effects of axonal damage on the myelin sheath.Regenerative Medicine and Nanomedicine Initiative of the Canadian Institutes of Health Research (CIHR) RMF-7028 FONDECYT 1080252 CIHR Ministry of Industry of Canada Rio Tinto Alcan Molson Foundatio

In vitro analysis of the effects on wound healing of high- and low-molecular weight chains of hyaluronan and their hybrid H-HA/L-HA complexes

Abstract Background: Recent studies have reported the roles of Hyaluronic acid (HA) chains of diverse length in wound repair, especially considering the simultaneous occurrence in vivo of both high- (H-HA) and low-molecular weight (L-HA) hyaluronan at an injury site. It has been shown that HA fragments (5 ≤ MW ≤ 20 kDa) usually trigger an inflammatory response that, on one hand, is the first signal in the activation of a repair mechanism but on the other, when it’s overexpressed, it may promote unwanted side effects. The present experimental research has aimed to investigate H-HA, L-HA and of a newly developed complex of the two (H-HA/L-HA) for stability (e.g. hyaluronidases digestion), for their ability to promote wound healing of human keratinocytes in vitro and for their effect on cellular biomarker expression trends. Results: Time-lapse video microscopy studies proved that the diverse HA was capable of restoring the monolayer integrity of HaCat. The H-HA/L-HA complex (0.1 and 1%w/v) proved faster in regeneration also in co-culture scratch test where wound closure was achieved in half the time of H-HA stimulated cells and 2.5-fold faster than the control. Gene expression was evaluated for transformation growth factor beta 1 (TGF-β1) proving that L-HA alone increased its expression at 4 h followed by restoration of similar trends for all the stimuli. Depending on the diverse stimulation (H-HA, L-HA or the complex), metalloproteinases (MMP-2, -9, -13) were also modulated differently. Furthermore, type I collagen expression and production were evaluated. Compared to the others, persistence of a significant higher expression level at 24 h for the H-HA/L-HA complex was found. Conclusions: The outcomes of this research showed that, both at high and low concentrations, hybrid complexes proved to perform better than HA alone thus suggesting their potential as medical devices in aesthetic and regenerative medicine. Keywords: Wound healing, Hyaluronan, MMPs, Hybrid complexe

NetCTLpan: pan-specific MHC class I pathway epitope predictions

Reliable predictions of immunogenic peptides are essential in rational vaccine design and can minimize the experimental effort needed to identify epitopes. In this work, we describe a pan-specific major histocompatibility complex (MHC) class I epitope predictor, NetCTLpan. The method integrates predictions of proteasomal cleavage, transporter associated with antigen processing (TAP) transport efficiency, and MHC class I binding affinity into a MHC class I pathway likelihood score and is an improved and extended version of NetCTL. The NetCTLpan method performs predictions for all MHC class I molecules with known protein sequence and allows predictions for 8-, 9-, 10-, and 11-mer peptides. In order to meet the need for a low false positive rate, the method is optimized to achieve high specificity. The method was trained and validated on large datasets of experimentally identified MHC class I ligands and cytotoxic T lymphocyte (CTL) epitopes. It has been reported that MHC molecules are differentially dependent on TAP transport and proteasomal cleavage. Here, we did not find any consistent signs of such MHC dependencies, and the NetCTLpan method is implemented with fixed weights for proteasomal cleavage and TAP transport for all MHC molecules. The predictive performance of the NetCTLpan method was shown to outperform other state-of-the-art CTL epitope prediction methods. Our results further confirm the importance of using full-type human leukocyte antigen restriction information when identifying MHC class I epitopes. Using the NetCTLpan method, the experimental effort to identify 90% of new epitopes can be reduced by 15% and 40%, respectively, when compared to the NetMHCpan and NetCTL methods. The method and benchmark datasets are available at http://www.cbs.dtu.dk/services/NetCTLpan/

Evidence for the Role of Horizontal Transfer in Generating pVT1, a Large Mosaic Conjugative Plasmid from the Clam Pathogen, Vibrio tapetis

The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600T reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae

Whole genome sequencing to investigate the emergence of clonal complex 23 Neisseria meningitidis serogroup Y disease in the United States

In the United States, serogroup Y, ST-23 clonal complex Neisseria meningitidis was responsible for an increase in meningococcal disease incidence during the 1990s. This increase was accompanied by antigenic shift of three outer membrane proteins, with a decrease in the population that predominated in the early 1990s as a different population emerged later in that decade. To understand factors that may have been responsible for the emergence of serogroup Y disease, we used whole genome pyrosequencing to investigate genetic differences between isolates from early and late N. meningitidis populations, obtained from meningococcal disease cases in Maryland in the 1990s. The genomes of isolates from the early and late populations were highly similar, with 1231 of 1776 shared genes exhibiting 100% amino acid identity and an average πN = 0.0033 and average πS = 0.0216. However, differences were found in predicted proteins that affect pilin structure and antigen profile and in predicted proteins involved in iron acquisition and uptake. The observed changes are consistent with acquisition of new alleles through horizontal gene transfer. Changes in antigen profile due to the genetic differences found in this study likely allowed the late population to emerge due to escape from population immunity. These findings may predict which antigenic factors are important in the cyclic epidemiology of meningococcal disease

Examining agency governance in the European Union financial sector – a case-study of the European Securities and Markets Authority

Ever since the outset of the financial crisis of 2009, agencies have emerged as key actors of European Union (EU) financial sector governance. As an organisational form that can be insulated from national political pressures, and committed to the Union interest, agencies proliferated in the financial sector ushering the agencification trend in finance. In this sense, the European Securities and Markets Authority (ESMA) – as part of the European Supervisory Authorities – practically embodies this trend. ESMA presents a radical shift in financial markets’ governance due to the nature of its soft law regulations and the direct impact it exerts on addressees’ behaviour in emergency circumstances. But ESMA’s success in optimising financial sector governance largely depends on its legitimacy, which is centred on independence. At the same time independence demands wider participation and inclusiveness of the decision-making process. This is not easy to achieve in a complex system with multiple stakeholders as is the governance of the EU financial sector (e.g., EU institutions, national actors, private sector). This paper examines ESMA’s interinstitutional relations and independence in light of publicly voiced criticism. We find that ESMA’s main executive bodies are still susceptible to influences by Member States as well as EU institutions (i.e., Commission), which undermines its operational independence

Phenotypic Overlap between MMP-13 and the Plasminogen Activation System during Wound Healing in Mice

BACKGROUND: Proteolytic degradation of extracellular matrix is a crucial step in the healing of incisional skin wounds. Thus, healing of skin wounds is delayed by either plasminogen-deficiency or by treatment with the broad-spectrum metalloproteinase (MP) inhibitor Galardin alone, while the two perturbations combined completely prevent wound healing. Both urokinase-type plasminogen activator and several matrix metallo proteinases (MMPs), such as MMP-3, -9 and -13, are expressed in the leading-edge keratinocytes of skin wounds, which may account for this phenotypic overlap between these classes of proteases. METHODOLOGY: To further test that hypothesis we generated Mmp13;Plau and Mmp13;Plg double-deficient mice in a cross between Mmp13- and Plau-deficient mice as well as Mmp13- and Plg-deficient mice. These mice were examined for normal physiology in a large cohort study and in a well-characterized skin wound healing model, in which we made incisional 20 mm-long full-thickness skin wounds. PRINCIPAL FINDINGS: While mice that are deficient in Mmp13 have a mean healing time indistinguishable to wild-type mice, wound healing in both Plau- and Plg-deficient mice is significantly delayed. Histological analysis of healed wounds revealed a significant increase in keratin 10/14 immunoreactive layers of kerationcytes in the skin surface in Mmp13;Plau double-deficient mice. Furthermore, we observe, by immunohistological analysis, an aberrant angiogenic pattern during wound healing induced by Plau-deficiency, which has not previously been described. CONCLUSIONS: We demonstrate a phenotypic overlap, defined as an additional delay in wound healing in the double-deficient mice compared to the individual single-deficient mice, between MMP-13 and the plasminogen activation system in the process of wound healing, but not during gestation and in postnatal development. Thus, a dual targeting of uPA and MMP-13 might be a possible future strategy in designing therapies aimed at tissue repair or other pathological processes, such as cancer invasion, where proteolytic degradation is a hallmark