Brassica oleracea var. sabellica, plants were grown either at 400 μmol m−2 s−1 or 100 μmol m−2 s−1 at 10°C, or at 400 μmol m−2 s−1 with 5 or 15°C. The higher light intensity overall increased flavonol content of leaves, favoring the main quercetin glycosides, a caffeic acid monoacylated kaempferol triglycoside, and disinapoyl-gentiobiose. The higher temperature mainly increased the hydroxycinnamic acid derivative disinapoyl-gentiobiose, while at lower temperature synthesis is in favor of very complex sinapic acid acylated flavonol tetraglycosides such as kaempferol-3-O-sinapoyl-sophoroside-7-O-diglucoside. A global analysis of light and temperature dependent alterations of gene expression in B. oleracea var. sabellica leaves was performed with the most comprehensive Brassica microarray. When compared to the light experiment much less genes were differentially expressed in kale leaves grown at 5 or 15°C. A structured evaluation of differentially expressed genes revealed the expected enrichment in the functional categories of e.g. protein degradation at different light intensities or phytohormone metabolism at different temperature. Genes of the secondary metabolism namely phenylpropanoids are significantly enriched with both treatments. Thus, the genome of B. oleracea was screened for predicted genes putatively involved in the biosynthesis of flavonoids and hydroxycinnamic acid derivatives. All identified B. oleracea genes were analyzed for their most specific 60-mer oligonucleotides present on the 2 × 105 K format Brassica microarray. Expression differences were correlated to the structure-dependent response of flavonoid glycosides and hydroxycinnamic acid derivatives to alterations in either light or temperature. The altered metabolite accumulation was mainly reflected on gene expression level of core biosynthetic pathway genes and gave further hints to an isoform specific functional specialization.Light intensity and temperature are very important signals for the regulation of plant growth and development. Plants subjected to less favorable light or temperature conditions often respond with accumulation of secondary metabolites. Some of these metabolites have been identified as bioactive compounds, considered to exert positive effects on human health when consumed regularly. In order to test a typical range of growth parameters for the winter crop  Brassica oleracea var. sabellica) is an increasingly popular leafy vegetable of the Brassicaceae, the plant family with many important food crops. Eating these vegetables is supposed to be health-supporting since a number of epidemiological studies have identified a positive association between its consumption and human health   Edreva, . The redn. So far, 27 non-acylated, 30 monoacylated and 14 diacylated glycosides have been described based on the flavonol aglycones quercetin, kaempferol and isorhamnetin  in growth chambers the specific effect of low temperature and moderate PAR levels on structurally different flavonoid glycosides was established further  and temperature levels (5 or 15°C at 400 μmol m−2 s−1) on structurally different flavonoid glycosides and hydroxycinnamic acid derivatives in adult kale plants under defined conditions in climate chambers. These conditions are comparable to conditions in the field when kale is cultivated during central European winter thus resembling typical light and temperature conditions in a growing season. We want to (II) further differentiate the plant's response to PAR and temperature on expression level using the most comprehensive 2 × 104 k format Brassica Array. Consequently, the focus of the transcriptome analysis will be directed toward genes putatively involved in the phenylpropanoid pathway.The aim of this study was to (I) distinguish the effect of ecologically relevant PAR (400 μmol mB. oleracea var. sabellica) plants of the cultivar Winterbor  were grown in pots under greenhouse conditions for 12 weeks. For both experiments, the plants were cultivated in potting soil . Water supply was given as needed by the plants and fertilization was given as nutrient solution  every 4 weeks. The adult kale plants were then transferred to climate chambers. The plants were treated either with (I) different PAR, with 100 μmol m−2 s−1 and 400 μmol m−2 s−1, measured with light meter Li-250 , at 10°C or with (II) different temperatures, at 5 and 15°C with 400 μmol m−2 s−1. The PAR radiation was applied from 8 am to 6 pm each day. These conditions are comparable to conditions in the field during cultivation in central European winter season. The temperature was measured with a Pt1000 sensor integrated in the climate chamber with a fluctuation range of ±0.3°C independent from the PAR level. The relative humidity in the climate chambers was 70%. The duration of each experiment was 4 weeks. Leaves of six fully developed plants were harvested using three repetitions per treatment.Kale (A mixed sample of leaves (without petiole and midrib) taken from six fully developed plants per replication was frozen (−40°C), lyophilized for 72 h and subsequently ground to a fine powder. The samples were either stored in darkness at room temperature less than 6 months until required for analysis or they were immediately used for total RNA extraction. To analyze flavonol glycosides and hydroxycinnamic acid derivatives samples were extracted with 60% aqueous methanol as described  and nebulizer gas (40 psi). Helium was the inert, collision gas in the ion trap. The flavonol glycosides and hydroxycinnamic acid derivatives were separated on a Phenomenex Prodigy column  with a C18 security guard column  at a temperature of 30°C using a water/acetonitrile gradient  were used to obtain an external calibration curve ranging from 0.1 to 10 mg 100 ml−1. Mass optimization for the ion optics of the mass spectrometer was performed for quercetin m/z 301. In addition, arbitrary m/z 1000 was used as the target mass in the auto mode to include higher mass fragments. The MSn experiments were performed in auto or manual mode up to MS4 in a scan from m/z 200 to 2000.An HPLC series 1100 by Agilent , consisting of a degaser, binary pump, autosampler, column oven and photodiode array detector, was used to quantify the flavonol glycosides and hydroxycinnamic acid derivatives. An ion trap (Agilent series 1100 MSD) with an electrospray ionization (ESI) ion source in negative ionization mode was used as the mass spectrometer. Nitrogen was used as the dry gas  or at 10°C and 400 μmol m−2 s−1 , and plants grown at 400 μmol m−2 s−1 and 5°C  or at 400 μmol m−2 s−1 and 15°C . Agilent One-Color Gene Expression Microarray analysis following the recommendation of MIAME (www.mged.org) was performed at Beckman Coulter Genomics  using their 2 × 105 K format Brassica Array . The Open Source Microarray Processing Software Robin was used  including quality control tools using the default settings. Here the data were shifted to be zero centered and scaled to have unit variance. The PCA was performed by singular value decomposition. The analysis settings were as follows: Normalization with background correction “subtraction” and between array normalization method “scale” with the analysis strategy of linear models (package limma). Results were given as log fold change of expression in relation to different light or temperature treatment. The assignment of the different genes was done by comparison of the translated protein sequences of the 95 K Brassica unigene set with the Arabidopsis TAIR9 database using the newly developed Mercator pipeline for automated sequence annotation . For each identifier the gene with the highest homology was provided with identifier and description. The respective bitscores were classified as follows: very weakly similar ; weakly similar (bitscore 101–200); moderately similar (bitscore 201–500); highly similar (bitscore greater than 500). Assignment of the different genes represented by identifiers to respective bins and visualization of data sets was realized using MapMan 3.5.1 . Complete lists of significant alterations of mRNA abundance induced by either LL, or HL, and either LT, or HT are given as Supplementary Tables The microarray analysis was performed as described . Differences due to temperature or radiation were statistically analyzed by a one-factorial ANOVA by a comparison of the factor levels using Tukey's HSD test. The individual values of flavonol gylcosides and hydroxycinnamic acid derivative represent the mean of 3 biological replicates . All tests were conducted at a significance level of 5%.−2 s−1 and 400 μmol m−2 s−1 at 10°C) and temperatures , the plant's response was evaluated on expression level using microarrays. In addition to the global analysis of transcriptomic alterations, a specific focus of the expression analysis was directed toward genes putatively involved in the phenylpropanoid pathway of B. oleracea.As a distinctive winter crop, kale has to handle low levels of PAR and also low temperature. However, under field conditions there was a certain interaction of PAR and temperature as previously shown for the formation of flavonol glycosides  using default settings for quality assessment and normalization of data. The principal component analysis of all expression data demonstrates that 54% of expression variation can be explained by the two components PC1 and PC2  and HL data  indicated that light is the factor that mainly determines PC1. On the other hand the rather good separation of the LT data  and the HT data  indicated that temperature is the factor that mainly determines PC2. This was further supported by the fact that the HL data of the other experiments at 10°C and 400 μmol m−2 s−1 positioned between LT and HT data. This data reduction shows that in the two experimental setups with different PAR or different temperature typical expression patterns can be received for the respective situation.The comprehensive gene expression analysis was performed using the 2 × 105 K format Brassica chip were significantly altered in kale leaves when plants were grown either at 400 μmol m−2 s−1 or 100 μmol m−2 s−1. Gene expression was reliably detectable for 89,621 identifier represented on the 105 K format Brassica Array. Among these, 16,000 reliably detectable elements showed significant differences in expression between growth at 400 μmol m−2 s−1 and 100 μmol m−2 s−1, with 12,666 upregulated at 400 PAR and 3334 upregulated at 100 PAR. The pure numbers of differentially expressed genes in this experiment showed that higher light intensities require almost 4 times more genes to be induced than at low light intensity.Since light is a very important environmental factor it does not astonish that the expression of almost 18% of the genes represented on the −2 s−1 and 100 μmol m−2 s−1 were in the functional categories of protein degradation, transport processes, amino acid metabolism, and in secondary metabolism  and hydroxycinnamic acid derivatives (3 compounds)  were decreased or not affected. The concentrations of other investigated minor flavonol glycosides were also decreased or unchanged at higher PAR levels. It is known that quercetin glycosides have a higher antioxidant activity than their corresponding kaempferol glycosides , increased at 400 μmol m−2 s−1 while sinapoyl-feruloyl-gentiobiose remained unaffected. In summary, the higher light intensity resulted in structure-dependent responses favoring the main quercetin glycoside quercetin-3-O-sinapoyl-sophoroside-7-O-D-glucoside/quercetin-3-O-sophoroside-7-O-sinapoyl-diglucoside, the caffeic acid monoacylated kaempferol triglycoside kaempferol-3-O-caffeoyl-sophoroside-7-O-D-glucoside and the disinapoyl-gentiobiose.Higher sunlight due to an 8-fold higher reflection caused by a reflective mulch resulted in higher concentrations of flavonoids and anthocyanins in apple fruit   independent of the acylation pattern. In general, glycosylation causes better water solubility and higher concentrations of sugars act as cryo-protectans in plants at lower temperatures  whereas disinapoyl-gentiobiose was decreased at the same time assuming them to be better cryo-protectants.Lower temperatures resulted in higher concentrations of quercetin glycosides in the investigated kale. Especially the minor compounds quercetin-3-B. oleracea genome could be screened for respective homologs. All relevant genes  of Glycine max and flavonoid 3′5′-hydroxylase (F3′5′H) of Vitis vinifera were also included, however, putative homologs to FNSII and F3′5′H were not found in the B. oleracea genome. Predicted B. oleracea genes putatively involved in the biosynthesis of flavonol aglycons , cinnamate 4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL)  (Table HCT)  Table .PAL1, Bo4g03091, Bo4g186590) are strongly induced with higher light and unaffected by temperature changes, one or both of the PAL2 genes  were unaffected by light and stronger expressed at low temperature. A functional specialization has been shown previously for PAL1 and PAL2 in Arabidopsis for an abiotic environmental-triggered flavonoid biosynthesis  for funding (Projects KR-2066/3-2). The Leibniz Association is gratefully acknowledged for supporting this work by an additional grant.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human immunodeficiency virus (HIV) type 1 dysregulates γδ T cells as part of an immune evasion mechanism. Nearly three decades of research defined the effects of HIV on γδ T cells and how this impacts disease. With highly effective antiretroviral therapy providing virus suppression and longer survival, we expected a return to normal for γδ T cells. This is not the case. Even in patients with CD4 T cell reconstitution, normal γδ T cell levels and function are not recovered. The durable damage to Vδ2 T cells is paralleled by defects in NK, CD8 T cells, and dendritic cells. Whether these consequences of HIV stem from similar or distinct mechanisms are not known and effective means for recovering the full range of cellular immunity have not been discovered. These unanswered questions receive too little attention in the overall program of efforts to cure HIV this disease. Approved drugs capable of increasing Vδ2 T cell function are being tested in clinical trials for cancer and hold promise for restoring normal function in patients with HIV disease. The impetus for conducting clinical trials will come from understanding the significance of γδ T cells in HIV disease and what might be gained from targeted immunotherapy. This review traces the history and current progress of AIDS-related research on γδ T cells. We emphasize the damage to γδ T cells that persists despite effective virus suppression. These chronic immune deficits may be linked to the comorbidities of AIDS  and will hinder efforts to eradicate HIV by cytotoxic T or NK cell killing. Here, we focus on one subset of T cells that may be critical in the pathogenesis of HIV and an attractive target for new immune-based therapies. Human immunodeficiency virus (HIV) is an aggressive, lymphotropic virus known for CD4 depletion and immune suppression. In addition to killing CD4 T cells, HIV affects several other lymphocyte subsets  and impa3 of blood  chain usage showed that HIV-driven expansion of Vδ1 cells did not select for specific Vδ chain rearrangements or individual Vγ chains . SequencMajor activities of Vγ9Vδ2 T cells include a strong response to Mycobacterium-infected human PBMC, even though these cells do not react to purified mycobacterial 65-kd heat shock protein . The Vγ9in vitro responses to phosphoantigen are also similar  and African-origin (low) peoples but repertoire complexity is similar among these groups and  similar . Positiv similar –34.in vitro phosphoantigen stimulation. A second group led by Malkovsky confirmed the functional anergy in Vδ2 T cells from HIV patients by documenting decreased responses to phosphoantigen or to the prototypical cell target Daudi B cell  Inversion of the Vδ2:Vδ1 cell ratio was an early event, occurring prior to inversion of the CD4:CD8 T cell ratio. (2) Vδ1 cells are increased in patients with HIV. (3) The Vδ2 cell depletion was accompanied by decreased responsiveness to phosphoantigens or tumor cells. (4) Loss of Vδ2 cells was greatest in patients with low CD4+ T cells, high viremia, opportunistic infections and late stage disease (AIDS). Consequently, HIV-mediated changes in γδ T cells appear to be part of the mechanism for evading antiviral immunity and establishing persistent infection with chronic disease. Persistent infection is essential for viruses like HIV that are transmitted with relatively low efficiency and require direct person-to-person contact. These studies highlighted the need to understand mechanisms for γδ T cell dysregulation, define impacts of these changes on immunity to HIV and look more broadly at “unintended consequences” of the viral immune evasion strategy.Mycobacterium leprae, but we can expect humans and non-human primate models to dominate this field for the foreseeable future.Model studies in non-human primates have helped to explain some of the γδ T cell changes during disease. Because rodents lack the TCR sequences needed for phosphoantigen recognition, studies on Vγ9Vδ2 T cells have been restricted to human beings and non-human primates. A recent genome mining study revealed that functional genes for Vγ9Vδ2 and butyrophilin 3A1 were actually present in a few other placental mammals including alpaca, sloth, bottlenose dolphin, killer whale, horse, and armadillo . Most ofin vitro  they were directly cytotoxic for SIV-infected target cells . There win vitro . The Vγ9in vitro . It was in vitro  that werin vitro . The relus entry , 48. Theus entry  meaning us entry . Circulaus entry .Candida albicans  but not with CD4 T cell count . This ad3 had no detectable Vγ9JP cells in PBMC  and effector function in antibody-dependent cellular cytotoxicity  or nucleotypes (nucleotide sequences for these peptides) within a sample of the total population of TCR sequences in a blood or tissue specimen. These sequences are classified as public  or private . For healthy, HIV-negative adults most of the clonotypes are public and many of the nucleotypes are also public indicating a high degree of similarity among unrelated individuals. This pattern is consistent with the use of monomorphic antigen-presenting molecules and is probably biased by nucleotide sequences in the germline regions of Vγ9 and JP that provide alternate routes to expressing the “germline” Vγ9JP rearrangement  using the marker  that is e marker . Vδ2 celtoxicity . DespiteA very small fraction of HIV patients actually have normal Vδ2 cell levels and function; these are elite controllers or natural virus suppressors defined as HIV patients with undetectable viremia except for occasional blips and no history of antiretroviral therapy . Among nNearly three decades of research produced many insights into HIV and γδ T cells. Studying HIV disease proved that Vγ9JPVδ2 cell depletion explained the defective response to phosphoantigen. Mechanisms were described for specific loss of Vγ9JPVδ2 cells that account for the relationship between depletion and phosphoantigen responsiveness. The findings showing that therapy reconstituted the Vγ9 repertoire but did not restore normal cell counts or function, were both encouraging and cautionary. Changes in γδ T cells are clearly related to HIV immune evasion but the long-term defects pose substantial challenges to the clinical management of HIV and efforts to reduce comorbid diseases.Natural history studies of HIV infection documented a brief, violent interval of acute infection characterized by extraordinarily high viremia and dramatic changes in immune cell populations, which continued until a stable viral set-point  was estaBoth viral and immunological set-points designate the phase of disease when acute HIV infection gives way to a stable, persistent infection with slower progression and more time for virus to spread by sexual contact. In some virus examples, the acute to persistent transition reflects changes in viral gene regulation but in HIV disease the most important mechanisms are viral evasion of host immunity. Once persistence is established, chronic disease progression causes death around 11 years later in untreated HIV disease, as declining CD4 T cell counts and collapsing innate immunity increase the incidence and severity of lethal opportunistic infections, cancer, cardiovascular disease, and other terminal conditions. Evolutionary selection of HIV created a pathogen capable of spreading in the human population by relatively inefficient sexual transmission even when the median life expectancy after infection was only 11 years for adults and shorter for infected infants or elderly persons. Now, in the era of effective ART and much longer survival times, we are seeing a new scenario that was not part of the selection pressures driving natural evolution of HIV. Therapy with effective virus suppression repairs some of the earlier immune damage and likely reduces transmission rates  but key 5 vRNA copies per ml of plasma lower that what is found during acute viremia. Accordingly, when the γ chain repertoire is reconstituted by new cell synthesis  involving γδ T cells were implicated in the activation of dendritic cells , enhanceA second example of Vδ2 T cell costimulation of NK is enhancement of effector activity capable of eliminating immature or mature, antigen-presenting dendritic cells Figure . Again, When Vδ2 T cell costimulation of NK cells is lost we might expect increased cancer risk among HIV patients, despite effective viral suppression by therapy. In HIV clinics of the University of Maryland, Baltimore, more than 15% of all HIV patients will have a cancer diagnosis and in 85% of those cases, cancer will be the cause of death . The greVδ2 T cell depletion, loss of costimulation, and the consequent reduction in dendritic cell killing may have important effects on several HIV-associated comorbidities. Chronic immune activation with inflammation is a hallmark of HIV disease and a danger to patients even after virus replication is suppressed. The lingering defect in Vδ2 T cells and the linked failure to co-stimulate NK for dendritic cell killing, likely increases the risk for chronic activation/inflammation as potent antigen-presenting cells accumulate in the absence of normal control mechanisms. Indeed, Fauci’s group noted the lack of potent dendritic cell editing by NK in HIV-infected and treated patients  and defeThe examples for defective Vδ2 T cells in HIV-infected and treated patients have important parallels in the NK literature. There are many reports about the phenotype of circulating NK cells in patients where HIV is controlled by antiretroviral therapy. In these patients, NK have lower density of NKp46, NKp30, and NKp44 receptors  and ofteWith highly effective antiretroviral therapy and increasing interest in eradicating HIV, it is important to look at the future of research on γδ T cells. Surely, Vδ2 T cells provide an attractive target for immunotherapy, probably using aminobisphosphonate compounds plus IL-2 or IL-15 to increase cell levels and functions. Patterned on a number of ongoing clinical studies in cancer –105, amiClinical research on therapeutic restoration of γδ T cells in HIV disease will require innovative clinical trial strategies. It may be difficult to launch therapeutic interventions such as aminobisphosphonate plus IL-2 for 2 or 3 months, then wait 20 years or more to accumulate significant data on changing rates of pulmonary arterial hypertension (found in <5% of HIV patients) or other comorbidities. While each of the comorbid conditions affects only a portion of the HIV population, their combined impact is substantial but clinical trials rarely lump disparate conditions into single endpoints. We might overcome this problem by using immunological endpoints that are related to chronic disease mechanisms. For example, direct immunotherapy targeting γδ T cells might be tested as a way to activate Vδ2 T cells that would normalize NK cell phenotype and function. Recovering of normal properties in two distinct lymphocyte subsets may be sufficient to document a clinical benefit, even though neither change is likely to result in short term changes to the incidence, prevalence, or severity of comorbid diseases.in vivo with high specificity, and a rich pipeline of compounds under development that may do even better. The missing link is a commitment to small interventional clinical trials testing the capacity for γδ T cell-targeted immunotherapies to alter the profile of immunity and recover normal control in diseases like HIV/AIDS.The Vγ9Vδ2 T cells in HIV-infected individuals contain the historical record of disease, documenting the intensity of initial depletion and reconstitution during therapy by their abundance, activity, and sequences of their TCR. They are barometers for current disease status, reflecting the capacity for resisting opportunistic infections, natural tumor surveillance, the control of immune activation/inflammation, and likely several other conditions critical to the clinical management of HIV disease. In addition, reconstitution of the TCR repertoire may be a measure for treatment success and help to identify patients with the highest potential response to immunotherapies targeted at Vδ2 T cell activation. Their compact TCR repertoire and extraordinarily high proportion of public Vγ9 chains facilitates comparisons among individuals or groups of patients. When combined with the sophisticated knowledge about cell surface marker expression including costimulatory receptors/ligands, and their patterns of cytokine/chemokine expression, these cells become critical tools for the immunology of infectious diseases. Adding to the interest is the availability of approved drugs that activate Vγ9Vδ2 T cells The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CCL2, SEPT4, LAMP3, RTP4, MT1G and OAS3) that can be used to define these subjects. We have used this gene set to predict symptomatic infection from an independent study. This analysis gives further insight into host-pathogen interactions during influenza infection. However, the major potential value is in the clinical trial setting by providing a more quantitative method to better classify symptomatic individuals post influenza challenge.Despite increases in vaccination coverage, reductions in influenza-related mortality have not been observed. Better vaccines are therefore required and influenza challenge studies can be used to test the efficacy of new vaccines. However, this requires the accurate post-challenge classification of subjects by outcome, which is limited in current methods that use artificial thresholds to assign ‘symptomatic’ and ‘asymptomatic’ phenotypes. We present data from an influenza challenge study in which 22 healthy adults (11 vaccinated) were inoculated with H3N2 influenza (A/Wisconsin/67/2005). We generated genome-wide gene expression data from peripheral blood taken immediately before the challenge and at 12, 24 and 48 h post-challenge. Variation in symptomatic scoring was found amongst those with laboratory confirmed influenza. By combining the dynamic transcriptomic data with the clinical parameters this variability can be reduced. We identified four subjects with severe laboratory confirmed influenza that show differential gene expression in 1103 probes 48 h post-challenge compared to the remaining subjects. We have further reduced this profile to six genes  contains supplementary material, which is available to authorized users. Seasonal influenza is common, affecting 5–10 % of the 900 million people in the European Region . In ordeWe sought to investigate the utility of genome-wide gene expression profiling in classification of volunteers following challenge. Application of a transcriptomic approach in the context of a challenge study is also a powerful approach to gain new insights into the biology of disease, as concordance in the exposure history and detailed clinical phenotyping before and after infection reduce the heterogeneity inherent to study of naturally acquired infection. Gene expression microarrays have been used to characterise the aberrant nature of the immune response to the 1918 pandemic influenza virus in macaques . A numbeHere, we describe application of gene expression profiling to an influenza challenge study involving a vaccine called MVA-NP+M1. This viral-vectored vaccine has the potential to induce heterosubtypic immunity through the induction of cytotoxic T cells with specificity for epitopes within conserved influenza proteins. We have found MVA-NP+M1 to be safe and immunogenic , 13 with8 plaque forming units (pfu) of MVA-NP+M1. Thirty days after vaccination, all 22 volunteers underwent intranasal challenge with H3N2 influenza (A/Wisconsin/67/2005) at a dose of 1 ml of 105.25 TCID50/ml in a quarantine facility. Volunteers were all challenged within a 2-h period. All subjects had haemagglutination inhibition (HI) titres of <1:10 to the challenge virus on admission to the quarantine unit.Detailed description of the subjects and methods used in the clinical trial are provided in Lillie et al. . The triAfter virus challenge, volunteers had a physical examination by a trial physician daily and self-reported their symptoms using a modified Jackson scoring system , 17 twicWhole blood was collected in PAXgene tubes prior to influenza challenge (day 30) and then at three further time points . All nasal washing and blood collections for viral shedding and gene expression analysis were taken in the same order that the volunteers were challenged in.TM-Human kit (Ambion) following the manufacturer’s instructions. Briefly, total RNA from whole blood is mixed with a biotinylated capture oligo mix specific for human globin mRNA. Streptavidin magnetic beads capture the globin mRNA. Placing the tube against a magnet then allows the globin mRNA depleted RNA to be transferred to a fresh tube. Globin-depleted RNA is further purified using a rapid, magnetic bead-based clean up procedure. Globin mRNA depleted samples were quantified using a NanoDrop spectrophotometer, and the quality for a subset was verified using a Bioanalyzer following the manufacturer’s instructions .Total RNA was extracted from whole blood using the PAXgene Blood RNA Kit (Qiagen) according to the manufacturer’s instructions. The abundance of globin mRNA present in whole blood reduces the sensitivity of expression array data, and therefore, globin mRNA depletion was carried out using the GLOBINclearGenome-wide gene expression analysis was carried out on 500-ng globin depleted RNA using the Illumina HumanHT-12 v4 Expression BeadChip gene expression platform comprising 47,231 probes . The report for analysis was generated by Illumina’s GenomeStudio version 1.6.0. Background signals were subtracted and 11,158 low expression probes  were removed. The raw data were transformed and normalised using the Variance Stabilization and Normalization method . Qualitywww.ingenuity.com) analysing probes differentially expressed between the four moderate/severe LCI and the remaining samples, 48 h post-challenge. The pamr R package [Differential gene expression analysis was carried out on 36,073 probes using three different R packages: limma , maSigPr package  was used package  was norm package , accessiFollowing influenza challenge, two individuals in the vaccinated group and five individuals in the unvaccinated group developed LCI as defined in the clinical trial protocol  Fig. 1)1). WithiPrincipal component analysis (PCA) and hierarchical clustering was performed to visualise variation in the dataset Fig. . This deq < 0.05 controlling for multiple testing using Benjamini and Hochberg’s method, Online Resource, Table We proceeded to analyse differential gene expression using a linear regression model. The likelihood of observed differences in gene expression was defined using an empirical Bayes approach in which prior distribution was estimated from the overall variance in the dataset, allowing a more stable inference given small numbers of arrays . ImplemeIn further analyses, the most significant 100 of these differentially expressed probes (9 %) were selected and almost all were found to be upregulated at 48 h post-challenge in the moderate/severe LCI individuals Fig. . In contp value = 6.4 × 10−10), role of pattern recognition receptors (PRR) in recognition of bacteria and viruses (p = 4.0 × 10−6), activation of IRF by cytosolic PRR (p = 7.7 × 10−4), eIF2 signalling (p = 8.1 × 10−3) and death receptor signalling (p = 8.1 × 10−3) . Network analysis further highlighted the significant upregulation of genes involved in the IFN response seen in these patients . IFNs play a critical role in host defence to viral infection with the significance of IFNλ recently recognised [P = 9.3 × 10−17).To assess the functional significance of the differentially expressed genes in subjects with moderate/severe LCI at 48 h post-challenge, we performed pathway and network analysis  which alone classified our samples into two groups with 100 % accuracy. These two groups comprised those from subjects with moderate/severe LCI taken at 48 h post-challenge in one group and the remaining 18 samples (with either mild LCI or without LCI) in the second group. The six selected genes are strongly upregulated in individuals with moderate/severe LCI with network analysis showing relationships involving interferon activation .Although the analysis of over 1000 differentially expressed probes can provide insight into host-pathogen interactions, this magnitude of variables does not lend itself well to use in diagnostics. We therefore sought to find the minimum number of genes that could be used to successfully classify our data. Using the tool ‘prediction analysis for microarrays’ (pamr) , we idenIn order to assess the validity of this six gene signal, we identified a data set from an independent but similarly conducted influenza challenge study, published by Huang et al. . This stFor each of the subjects in the Huang study, we included six samples taken at different time points: two samples prior to challenge were included, and the remaining four samples were from consecutive post-challenge time points taken at 45, 53, 60 and 69 h. For all the post-challenge samples in the eight asymptomatic volunteers, the six gene set classified the samples with 100 % accuracy. For 89 % (8/9) of the symptomatic volunteers, pamr correctly classified at least one of these six post-challenge timepoints . This enabled us to classify our samples into two distinct groups (symptomatic and asymptomatic) with 100 % accuracy providing a potentially convenient, cost effective and quantitative method for determining post-challenge outcome during vaccine and challenge trials. The reproducibility of this gene set has been demonstrated using samples from an independent vaccine study [CCL2, LAMP3, RTP4 and OAS3) are represented in the list of the top 50 genes from this H1N1 study. Two studies in 2013 examined genome-wide gene expression in patients with influenza acquired in the community. While none of these six genes were amongst the best discriminators of respiratory syncytial virus (RSV) from influenza, four of the six genes  were represented in a gene list of 161 interferon-related differentially expressed genes [OAS3, LAMP3, SEPT4 and MT1G).We were able to reduce the 48 h time point gene expression signature to just six genes Below is the link to the electronic supplementary material.


Cyclic adenosine 3′,5′-monophosphate (cAMP) modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for β-adrenergic receptors' signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefly discuss the complexity of cAMP synthesis and degradation in the cardiac context, describe the way to detect it and review the main pharmacological arsenal to modulate its availability. The depolarizing current through LTCC (ICa) contributes to the plateau phase of the cardiac action potential as well as to pacemaker activity in nodal cells , which results in activation of myofilaments contraction. Alterations in density or function of LTCC have been implicated in a variety of cardiovascular diseases, including atrial fibrillation    Bers, . Howevers) Bers, . Thus, tgy Table . Once cAgy Table . From thgy Table , a varieBaillie, . RecentlBaillie, . In this2+ or Mg2+  throught its binding to a putative helical structure on the C1b region  in the submicromolar range  are ubiquitous enzymes that catalyze the conversion of Adenosine triphosphate (ATP) into cAMP and pyrophosphate. ACs structure consists in 12 transmembrane domains divided into 2 hydrophobics domains (6 transmembrane domains each) and 2 main intracellular loops called C1 and C2 that naturally dimerize to form the catalytic domain Figure . In mamm2+ currents in frog ventricular myocytes   occurs when the heart is unable to pump sufficiently to maintain blood flow to meet the body's needs. Around 2% of adults have HF and this percentage increases to 6–10% for people over the age of 65 . FSK, a diterpene extracted from the plant As schemed in Figure βγ protein associates with the receptor thus allowing GDP/GTP exchange on the Gα GTPase domain, leading to subsequent Gα-GTP and Gβγ dissociation both regulating downstream specific signaling targets  according to the secondary effector of the Gα subunit  family. According to conventional knowledge, 7 transmembrane GPCRs at the plasma membrane convert extracellular signals into intracellular ones through canonical heterotrimeric G proteins which transduce signals from GPCRs to secondary effectors thus leading to the second messengers production and the propagation of the signal through ensuing regulation of numerous downstream intracellular signaling targets Gilman, . G protein vivo has been largely hampered by lack of specific antibodies against this class of receptors. Thus, over the years, microarray technology allowed researchers to monitor the mRNA expression levels of thousands of GPCRs encoding genes. Based on the available genomic data , produced by toxigenic strains of the Gram-negative Pasteurella multocida bacteria, was identified as a potent and selective activator of Gαq, Gαi, and Gα13 by deamidating a glutamine residue in the switch II region of the Gα-GTPase domain  in cardiac fibroblasts to aggravate cardiac hypertrophy and fibrosis  is a specific Gαs potent activator secreted by the bacteria Bordetella pertussis in an inactive form. PTX catalyzes the ADP-ribosylation of the Gαi subunit of the heterotrimeric G protein. The Gαi subunit remains locked in its GDP-bound inactive state, thus unable to interact with the receptor and to inhibit adenylyl cyclase activity  than for Gαo (IC50 ≈ 2 μM) or Gαi (IC50 ≈ 5 μM). Suramin exerts its effects by binding the effectors binding site on the Gα proteins  that suppress the Gαs activation coupled to β-adrenergic receptors, whereas they affect the Gαi/Gαo- and Gαq-coupled receptors  to a much lesser extent . PTX is a protein complex released by the bacterium in vitro and reduced β-AR–mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes  , are long known to act as competitive nonselective PDE inhibitors  transporter superfamily (subfamily C)] regulates cAMP efflux into the extracellular space to decrease cAMP availability. Three of them  are expressed in cardiac tissue but ABCC4 is the most studied and has been shown to enhance cAMP formation, contractility, and cardiac hypertrophy  type light sensor domain. This photoactivatatable adenylyl cyclase (bPAC) shows a low cyclase activity in the dark but that increases about 300-fold upon light activation , the exchange protein activated by cAMP (EPAC), the cyclic nucleotide gated ion channels (CNGC) and the popeye domain containing protein (POPDC). cAMP output signaling can be modulated by targeting its effectors.Inactive PKA relies on an heterotretamer consisting of two regulatory (R) and two catalytic (C) subunits Figure . Two priNocardiopsis sp.) act as competitive antagonists of the cAMP nucleotide for the binding site on the PKA regulatory subunit  and KT5720  constitutes with PKA the main direct cAMP effector and has been identified by two independent group in 1998  comprises two groups: cyclic nucleotide gated (CNG) channels and the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Both types are members of the six transmembrane channel superfamily and contain a cyclic nucleotide binding domain in their cytosolic C-Terminus that serves as an activation domain. Upon cyclic nucleotide binding, CNG channels gates the flow of monovalent cations such as Na50) of 120 nM, which is comparable to the affinities reported for PKA (100 nM)  encoding a 3 transmembrane proteins that bind cAMP through their conserved cytoplasmic Popeye domain with an affinity  upon cAMP production which induces a reporter gene under the control of a CRE element (cAMP Response Element) promoter. Various reporter genes have been used over the years: fluorescent proteins, luciferase, β-galactosidase or β-lactamase. Far downstream of the cAMP activation cascade, this method is sensitive but unable to give kinetics or localization information.The methods described above are unable to define the cellular localizations of cAMP production. Thus, visualization was achieved using resonance energy transfer (RET) techniques described by the Theodor Förster in 1940's Forster, . RET is et al developed a Cu-free click reaction with comparable kinetics to Cu dependent cycloaddition, but adapted for dynamic in vivo imaging  as a reagent  catalyst is one of the most popular reactions within the Click chemistry concept between an azide and a terminal or internal alkyne to give a 1,2,3-triazole (Rostovtsev et al., The development of optical methods that allow monitoring of cAMP dependent signaling in living cells and the growing list of molecules (summarized in Figure This work was supported and funded by “Fondation Bettencourt Schueller and Institut National de la santé et de la Recherche Médicale.”The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CRTC1, FOXP1, and BARX1. Furthermore, they replicated an association at the FOXF1 gene that has been previously found in a GWAS on Barrett’s esophagus. We aimed at further replicating the association at these and other loci that showed suggestive association with P <  10−4 in the BEACON sample. In total, we tested 88 SNPs in an independent sample consisting of 1065 EAC cases and 1019 controls of German descent. We could replicate the association at FOXP1, BARX1, and FOXF1 with nominal significance and thereby confirm that genetic variants at these genes confer EAC risk. In addition, we found association of variants near the genes XRCC2 and GATA6 that were strongly (P < 10−5) although not genome-wide significantly associated with the BEACON GWAS. Therefore, both variants and corresponding genes represent promising candidates for future EAC association studies on independent samples.The Barrett’s and Esophageal Adenocarcinoma Consortium (BEACON) recently performed a genome-wide association study (GWAS) on esophageal adenocarcinoma (EAC) and Barrett’s esophagus. They identified genome-wide significant association for variants at three genes, namely  P < 10−4 and were followed up in 874 EAC and 759 Barrett’s esophagus cases  as well as 6911 controls from the United Kingdom (replication sample). In total, SNPs at three different loci showed genome-wide significant disease association in the combined sample. The first is on chromosome 19p13 (top SNP marker rs10419226) containing the gene CREB-regulated transcription coactivator (CRTC1), whose aberrant activation has been associated with oncogenic activity homeobox protein BarH-like 1b (BARX1), which encodes a transcription factor important in esophageal differentiation forkhead box protein P1 (FOXP1), which encodes a transcription factor regulating esophagus development and acts as therapeutic target in cancer Recently, the Barrett’s and Esophageal Adenocarcinoma Consortium (BEACON) presented their genome-wide association study (GWAS) on 1516 cases with esophageal adenocarcinoma (EAC), 2416 cases with nonneoplastic Barrett’s esophagus, and 3209 healthy individuals (discovery sample), all of European ancestry forkhead box F1 (FOXF1) gene on chromosome 16q24. They used the most significant associated SNP marker in their discovery sample, rs3950627, and performed a stepwise conditional analysis to test for independent SNP associations within this region. In total, they identified three additional association signals  pointing to a complex genetic risk architecture at 16q24. The association of the originally reported risk conferring SNP, rs9936833, was thereby explained by all four SNP markers that showed association in the stepwise conditional analysis.Furthermore, the BEACON study refined a previously reported association with Barrett’s esophagus P < 10−4 to EAC/Barrett’s esophagus in the BEACON GWAS sample FOXF1 SNPs that have been previously implicated in the GWAS on Barrett’s esophagus In the present study, we aimed at further replicating the associations obtained by BEACON. We genotyped all 87 SNP markers in an independent German sample of 1065 EAC cases and 1019 controls that showed association with 2O) on each 384-well plate to exclude contamination. Clusterplot of each SNP was visually checked and manually corrected if necessary. Genotyping data underwent different quality control steps . After applying these criteria, 88 SNPs remained for association testing . Although none of the controls had been diagnosed with EAC, they have not been screened regarding Barrett’s esophagus status. In all, 127 cases were females and 938 were males. In controls, 521 were females and 498 were males. Of note, due to the small size of the female sample our statistical power was only moderate to detect sex-specific risk variants. Genotyping of all 90 markers was done using the Sequenom MassARRAY iPlex Gold® system . For quality control we genotyped intra- and interplate duplicates. In addition, we added negative controls  and 11 SNPs were EAC associated when one-sided tested , which was more pronounced in female compared to male cases  was EAC associated with our sample  between our and the BEACON study and observed that they are all in the same range  that have been highlighted in the BEACON study and in another GWAS on Barrett’s esophagus FOXP1, which were also genome-wide significantly associated with EAC/Barrett’s esophagus in the BEACON study. Although our findings do not withstand a Bonferroni correction, they confirm that genetic variability at FOXP1 confers risk to EAC. Furthermore, our study supports the involvement of two previously reported risk loci in the EAC pathology. When one-sided tested, variants at BARX1 and FOXF1 showed disease association. However, given that rs3950627, which was the strongest associated SNP at FOXF1 in the BEACON study P = 0.121, CRTC1  in the combined GWAS sample (discovery and replication cohort) showed EAC association within this study. SNP rs11771429 is located nearby XRCC2 and showed disease association in the BEACON study with P = 8.40 × 10−6GATA6 showed disease association in the BEACON study with P = 2.69 × 10−7GATA6 represents a plausible EAC candidate gene as it encodes a transcription factor with an important role in the regulation of cellular differentiation. Of note, GATA6 has already been implicated in the development of EAC CRCC2 and GATA6 represent interesting candidate genes, independent replications at these loci are needed before adding the respective SNPs to the list of confirmed EAC risk variants.In the present study, we aimed at replicating associations to EAC/Barrett’s esophagus which have been previously reported in the BEACON GWAS FOXP1, BARX1, and FOXF1 confer risk for the development of EAC. In addition, we found association with variants near XRCC2 and GATA6 that were strongly disease associated with the BEACON GWAS, although this was not genome-wide significant. Thus, both genes represent promising candidates for future EAC association studies on independent samples.In conclusion, we provide supportive evidence that genetic variants at


We use the optimal foraging theory to study coexistence between two plant species and a generalist pollinator. We compare conditions for plant coexistence for non-adaptive vs. adaptive pollinators that adjust their foraging strategy to maximize fitness. When pollinators have fixed preferences, we show that plant coexistence typically requires both weak competition between plants for resources  and pollinator preferences that are not too biased in favour of either plant. We also show how plant coexistence is promoted by indirect facilitation via the pollinator. When pollinators are adaptive foragers, pollinator’s diet maximizes pollinator’s fitness measured as the per capita population growth rate. Simulations show that this has two conflicting consequences for plant coexistence. On the one hand, when competition between pollinators is weak, adaptation favours pollinator specialization on the more profitable plant which increases asymmetries in plant competition and makes their coexistence less likely. On the other hand, when competition between pollinators is strong, adaptation promotes generalism, which facilitates plant coexistence. In addition, adaptive foraging allows pollinators to survive sudden loss of the preferred plant host, thus preventing further collapse of the entire community. Et il se sentit très malheureux. Sa fleur lui avait raconté qu’elle était seule de son espèce dans l’univers. Et voici qu’il en était cinq mille, toutes semblables, dans un seul jardin!Le Petit Prince, Chapitre XX – Antoine de Saint-ExupéryThe diversity and complexity of mutualistic networks motivate ecologists to investigate how they can remain stable and persistent over time. Mathematical models and simulations show that some properties of mutualistic networks  make them more resistant against cascading extinctions , more liIn this article we consider a mutualistic module with two plant species and one pollinator species . This moFirst, we will assume that pollinator preferences for plants are fixed. In this case, there is a negative effect of one plant on the other by direct competition and a positive indirect effect that is mediated by the shared pollinators, called facilitation –10. As oSecond, we will assume that pollinator preferences are adaptive. We will assume that pollinator fitness is defined as the per capita pollinator population growth rate that depends on plant (that produce resources for pollinators) as well as on pollinator densities. First, pollinators benefit from nectar quality and nectar abundance (which correlates with plant population density). Second, pollinators compete for resources. This competition will play an important effect when pollinator population densities are high. A game theoretical approach to determine the optimal pollinator strategy is the Ideal Free Distribution (IFD) , 12. ThiOur main goal is to study how pollinator preferences and plant competition affect plant coexistence. First, we study the dynamics of the plant–pollinator module when pollinator preferences are fixed. Second, we calculate the pollinator’s evolutionarily stable foraging strategy (ESS) at fixed plant and pollinator population densities, and we study plant coexistence assuming pollinators instantaneously track their ESS. This case corresponds to time scale separation where population dynamics operate on a slow time scale, while pollinator foraging preferences operate on a fast time scale. Finally, we consider the situation without time scale separation and we model preference dynamics with the replicator equation. Overall, we show that pollinator foraging adaptation has complex effects, sometimes equivocal, on plant coexistence. On the one hand pollinator adaptation increases competitive asymmetries among plants, promoting competitive exclusion. On the other hand competition for plant resources among pollinators promotes generalism over specialization, which can prevent the loss of pollination services for some plants and promote coexistence.Pi  and one pollinator species A  such as nectar at a rate ai per plant. Resources not consumed by the pollinator decrease with rate wi . Resources are consumed by pollinators at rate bi per resource per pollinator. Pollinator’s relative preferences for either plant are denoted by ui with u1 + u2 = 1. Plant birth rates are proportional to the rate of pollen transfer that is concomitant with resource consumption. Thus, we assume that plant birth rates are proportional to pollinator resource consumption rates (uibiFiA) multiplied by conversion efficiency ri. Pollinator birth rates are proportional to resource consumption with corresponding conversion efficiency ei. Plants and pollinators die with the per capita mortality rate mi  and d, respectively.Let us consider two plant species pecies A . Plants dFi/dt = 0, plants and pollinator population dynamics are described by the following model , and Ki stands for the habitat carrying capacity  and initial pollinator density is kept at A(0) = 2.Community dynamics and the dynamics of pollinator preferences can be sensitive to initial conditions. There are four degrees of freedom for the initial conditions  influences the outcome. For scenario II we also used A(0) = 50, but we did not find important qualitative differences. Thus, for both scenarios we simulate Models   I and II. Models  and (4) P1 = P2 = A = 0) is always locally asymptotically stable [y stable , 25, becdP1/dt = dA/dt = 0 with P1 > 0, P2 = 0, A > 0 in D1 = −4b1de1K1m1r1u1w1 + (b1e1K1(m1 − a1r1)u1 + dr1w1)2. These two equilibria are feasible (positive) if a1r1 > m1 and D1 > 0. The first is a growth requirement: if not met, even an infinite number of specialized pollinators (with u1 = 1) cannot prevent plant 1 extinction. The second condition is met when pollinator preference for plant 1 (u1) is above a critical valueBy setting P2±, A2±). They are feasible if a2r2 > m2 and D2 > 0 (D2 is like D1 with interchanged sub-indices). The second condition is met when pollinator preferences for plant 2 are strong enough  so that u1 is below a critical value ub1P1−, A1−) when plant 2 is missing and  when plant 1 is missing) is unstable. This instability indicates critical threshold densities. When plant i and pollinator densities are above these thresholds, coexistence is possible. Otherwise, the system converges on the extinction equilibrium mentioned before. This is a mutualistic Allee effect [By symmetry, there are two non-trivial plant 2–pollinator equilibria  when plant 2 is missing and  when plant 1 is missing) will be called the resident equilibrium. Resident equilibria are stable with respect to small changes in resident plant and pollinator densities, but may be unstable against invasion of small densities of the missing plant species. In the case of the plant 1–pollinator equilibrium , plant 2 invades  if the competitive effect of plant 1 on plant 2 (c1), is smaller thanP2+,A2+) if the competitive effect of plant 2 on plant 1 (c2), is smaller thanThe equilibrium that is farther from the origin ((α(u1) and β(u1) are real when D1 > 0 and D2 > 0, respectively. In other words, invasibility only makes sense when the plant 1–pollinator resident equilibrium exists (u1 > ua1) or, when the plant 2–pollinator resident equilibrium exists (u1 < ub1), respectively. The graphs of Eqs (c = c1 = c2). Notice that because α and β are only feasible to the right of ua1 and to the left of ub1, respectively, their graphs may or may not intersect depending on the position of ua1 and ub1 (cf. panel a vs. b). We show this by setting a common plant carrying capacity K = K1 = K2 and making it larger or smaller than a critical value , s of Eqs  divide tal value K*=(b1e1K > K*) support coexistence of both plant–pollinator resident equilibria for intermediate pollinator preferences. This is not so in unproductive environments (K < K*), where resident equilibria occur within separated ranges of pollinator preferences (see below).Productive environments  and β(u1) intersect like in ua1 < u1 < ub1. Such intermediate pollinator preferences allow each species to coexist with the pollinator at a stable equilibrium. If competition is weak enough  pollinator preference for plant 2 is so low that plant 2 alone cannot support pollinators at a positive density. It is only due to presence of plant 1 that allows plant 2 survival through facilitation (α and to the right of ub1) plant 2 cannot invade. Similarly, when pollinator preferences for plant 1 are too low , coexistence relies on facilitation provided by plant 2 (resident) to plant 1 (invader) and on plant competition being not too strong (below curve β); if competition is too strong (above β) plant 1 cannot invade.First, we assume a high plant carrying capacity satisfying litation . Indeed,K < K*. Then ua1 > ub1, and α(u1) and β(u1) never intersect  that further divides the parameter space (ub1 < u1 < ua1) where neither plant can be a resident, and below γ curve (“P1 + P2 or EXT”), coexistence is achievable if both plants and the pollinator are initially at high enough densities. This is an extreme example of plant facilitation. If combined plant abundances are not large enough, then both plants and the pollinator go extinct as already predicted by the invasion analysis. Also, if one plant species is suddenly removed, extinction of pollinator and the other plant follows. Above the γ curve, plant competition is too strong to allow any coexistence and the outcome is always global extinction (“EXT”). When preference for plant 1 is low (u1 < ub1), the γ curve is slightly above the β curve so that the possible coexistence region is slightly larger than the coexistence region obtained by invasion of the rare plant (“P2 or P1 + P2”). However, plant coexistence in the region between the two curves depends on the initial density of plant 1: if P1(0) is very low, plant 2 wins as predicted by the invasion analysis, but if P1(0) is large enough, plant 1 will invade and coexist at an interior equilibrium with plant 2. In the opposite situation, where preference for plant 1 is very high (u1 > ua1), γ divides the region where plant 2 can invade (assuming c < α) as follows. Below γ, competition is weak and plant 2 invasion is followed by stable coexistence thanks to resident facilitation. Above γ, competition is strong and plant 2 invasion causes plant 1 extinction followed by plant 2 extinction. This is because pollinator preference for plant 1 is too strong (u1 > ub1) which does not allow pollinators to survive on plant 2. Thus, invasion by plant 2 leads to global extinction (“EXT”). c < α and c < β predict the existence of a locally stable interior equilibrium at which both plants coexist with the pollinator.When er space . For theV1 = V2, which yieldsV1(u1)>V2(u1) for all u1, the ESS is V1(u1)<V2(u1) for all u1, the ESS is We calculate the evolutionarily stable strategy for fitness defined by nvasions , i.e., W the ESS . We remae2a2P2)A,provided the ESS .e1a1P1/(e1a1P1 + e2a2P2), i.e., pollinators tend towards generalism, with relative preferences reflecting differences in resource supply rates and quality. This is because at higher pollinator densities fitness decreases due to intra-specific competition among pollinators for plant resources, which is compensated for by interacting with the less profitable plant. In contrast, when pollinator densities become very low, It follows from function . In thisν with which pollinator preferences change in c1 = c2 = 0). Thus, a plant can cause the decrease of the other plant only by influencing pollinator preferences. Let us assume that at time t = 0 both plants have equal densities and pollinators are rare , as shown in u1 = 1) which is the most profitable (e1 > e2), causing plant 2 to decline and to go extinct, eventually. However, as pollinator densities start to increase relative to plant densities, pollinators can become generalists which favours plant coexistence. We start with the assumption that pollinator preferences track instantaneously population numbers (panel a: ν = ∞), i.e., t ≈ 3, which is fast enough to prevent plant 2 extinction, and population densities will tend to an interior equilibrium. When pollinators preference is described by the replicator equation (panel b: ν = 1), we observe that pollinators will become generalists at a latter time (t ≈ 11) due to the time lag with which pollinators preferences follow population abundances. Even with this delay, the decline of plant 2 stops and we obtain convergence to the same population and evolution equilibrium. However, when adaptation is yet slower, the pollinator preferences will follow changing population densities with a longer time delay (panel c: ν = 0.25), and we get a qualitatively different outcome with plant 2 extinction. This is because when pollinators start to behave as generalists (t ≈ 100), plant 2 abundance is already so low that it is more profitable for pollinators to switch back to pollinate plant 1 only. We obtain similar results as in c1 = c2 = 0.4, but coexistence becomes impossible when direct competition becomes too strong.This is especially easy to observe when there is no direct competition between plants (ci > 0), and time scales on plant coexistence.In the next section we study combined effects of initial conditions, plant competition for resources (P1(0) + P2(0) = 50). We contrast these predictions with the situation where population densities are fixed, i.e., when population dynamics are not considered and pollinator preferences are at the ESS. In this latter case the necessary condition for both plants to survive is that pollinators behave as generalists which corresponds to the region between the two curves δ0 and δ1 in A, when Here we study the combined effects of population dynamics δ0 (δ1) lead to exclusion of plant 1 (plant 2) because pollinators specialize on plant 2 (plant 1). Population and pollinator preference dynamics do change these predictions. The main pattern observed in the simulations is that plant coexistence becomes less likely as the plant competition coefficient (c) increases. This is not surprising because a higher inter-specific competition between plants decreases plant population abundance which makes coexistence of both plants less likely or impossible . In fact, when inter-specific plant competition is too strong and the pollinators do not adapt, plant densities can become so low that the system collapses due to mutualistic Allee effects .If population densities do not change, initial conditions to the left (right) of ν) on coexistence is more complex, in particular when plant competition is moderate or weak . At low adaptation rates  increasing the adaptation rate makes the region of coexistence smaller. With faster adaptation rates , increasing the adaptation rate further narrows the region of coexistence for large initial pollinator densities, but widens this region for smaller initial pollinator densities (see the pink areas below the δ0 and δ1 curves). Although initially pollinators specialize on the more profitable plant, the inter-specific competition among pollinators leads to generalism, and provided the adaptation rate is fast enough, to plant coexistence. This is the same effect as in The effect of pollinator adaptation rate (c = 0.8), the main effect of pollinator adaptation is that coexistence entirely disappears . This is because plant densities are reduced and pollinator densities do not reach high enough densities that would lead to pollinator generalism. Finally, when competition is very strong (c = 1.2) global extinctions do not happen . This is because adaptation allows pollinators to switch fast enough towards the most profitable plant before competition drives total plant abundance below the Allee threshold that would lead to global extinction.When competition is strong . We examined the corresponding time series to confirm that they display damped oscillations or limit cycles like in For non-adaptive pollinators, community collapse is more widespread than in the scenario ν = 1), initial preferences have little influence on the outcome (unless grossly biased towards 0 or 1) and initial plant composition is more important. The effect is more sharp when adaptation rate is infinitely fast (ν = ∞), because plant coexistence is entirely independent of the initial pollinator preferences.Fast enough pollinator adaptation prevents community collapse, by enabling pollinators to abandon initially maladaptive diets before it is too late .Under fixed pollinator preferences  j can invade the plant i and the pollinator community, but plant j alone cannot coexist with the pollinator (left or right “P1 + P2” regions in i) facilitates the other plant (j) via pollinator sharing, by increasing the pollinator density , the invader can indirectly increase the resident’s plant density. This is another manifestation of pollinator mediated facilitation [ei) that cause the subsequent collapse of the whole community . In our simulations, we determined the region of initial population densities and pollinator preferences leading towards plant coexistence, as a function of these two factors. The larger this region, the more likely plant coexistence. As competition strength increases, coexistence becomes less likely as expected from competition theory [I, we see that the region of coexistence is generally wider when pollinator densities are initially large and more narrow when pollinators are initially rare (c ≤ 0.4). This agrees with the pattern outlined in ν, panel b) the extinction of the less preferred plant can be prevented. However, if the change is too slow  the less preferred plant declines too fast to be rescued from extinction. Thus, the results from scenario I tells us that time lags in pollinator adaptation with respect to population dynamics have important consequences for plant coexistence.Whether or not adaptive pollinator preferences promote plant coexistence depends critically on the strength of competition  and the rate of pollinator adaptation . This is likely to happen if external perturbations  makes the most preferred plant too rare and the less preferred too common, in a very short time. If pollinators cannot turn into generalists fast enough they will go extinct because of the mutualistic Allee effect. This leads to the collapse of the community (white regions in ν = 0). When pollinators are able to adapt, such global extinctions can be prevented, sometimes at the price of one plant going extinct. We also observe global extinction in scenario II when initial plant ratios and fixed pollinator preferences are both not too biased  as welllinators ), can ple1 > e2) while we keep all the other plant-specific parameters equal. We also ran simulations with plant 1 being better with respect to other plant-specific parameters  affect plants only indirectly, by influencing pollinator preferences. In contrast, other plant-specific parameters affect plant dynamics directly. Finally, the larger ci and Ki the more likely plant i always win in competition, but this is a natural result expected in models derived from the Lotka–Volterra competitive equations.Our simulations assume that plant 1 is richer in energy rewards when compared to plant 2 (Raphanus raphanistrum) is exposed to increasing numbers of an alternative plant (Cirsium arvense). In the same study, the relative visitation frequency of a plant (Raphanus) declines faster than predicted by the decline in the relative proportion of its flowers [Some predictions from our model are in qualitative agreement with experiments. For example  shows tr flowers . The ESS flowers ). The ef flowers . Other s flowers . Finally flowers .u1 strictly between 0 or 1) a requisite for coexistence. However, when pollinators are generalists, rare plants would experience decreasing pollination quality, due to the lack of constancy of individual pollinators delivering non-specific pollen or losing con-specific pollen [I and II again, but replacing our c pollen –39. We dc pollen , pollen c pollen  or pollic pollen , 40. NevThere is no question that IPT affects pollination efficiency. However, the relative importance of IPT may also depend on the structure of the environment where interactions occur. A survey of field and laboratory results  reports The scope of our work is limited to adaptation in a single pollinator species only. In real life settings adaptation can be affected by (i) competition among several pollinator species, and by (ii) plant–pollinator co-evolution.With respect to point (i), large community simulations  indicateei) indirectly affect plant dynamics ri, mi, ci). Plant adaptation likely happens over generations, so a replicator equation approach or adaptive dynamics [Addressing point (ii) will require trade-offs in plant traits. We showed how differences in pollinator efficiencies Click here for additional data file.S1 File(ZIP)Click here for additional data file.


A modified cyclopentadienyl ligand greatly improves regioselectivity in Rh(iii) catalyzed alkene insertion/C–H activation. O-pivaloyl benzhydroxamic acids mediated by a Rh(iii) precatalyst bearing sterically bulky substituents. While the prototypical Cp* ligand provides product with low selectivity, sterically bulky Cpt affords product with excellent regioselectivity for a range of benzhydroxamic acids and alkenes. Crystallographic evidence offers insight as to the source of the increased regioselectivity.We report the regioselective synthesis of dihydroisoquinolones from aliphatic alkenes and  When the substituents are electronically similar, the larger group resides β- to Rh in the metallacycle to avoid unfavorable steric interactions .4 When the substituents are electronically different, the more electron-donating group prefers being α- to rhodium in the metallacycle, presumably to stabilize the electron poor metal.6 The type of C–H bond being activated also plays an important role in the regioselectivity of migratory insertion; aromatic substrates typically provide synthetically useful regioselectivities when electronically different alkynes are used (>10 : 1) but alkenyl C–H activation leads to products with lower regioselectivities, presumably due to minimal steric interactions during migratory insertion.8 We found that sterically bulky di-tert-butylcyclopentadienyl ligand (Cpt) enhances the regioselectivity of the alkyne migratory insertion event in these cases, delivering regioselectivities (>10 : 1) modestly above those achievable by Cp* ligated Rh complexes (<6 : 1). However, when the alkyne migratory insertion was poorly selective with RhCp* (<3 : 1), RhCpt complex was ineffective at providing synthetically useful levels of selectivity. Furthermore, the Cpt ligand was only effective with aryl substituted alkynes, presumably because of strong steric interactions between the ligand and alkyne in the insertion event.Steric and electronic effects are understood to control migratory insertion of unsymmetrical alkynes in Rh (eqn 2).12 We hypothesized competing steric and electronic effects cause the low regioselectivity, with steric effects favoring the formation of a 4-substituted product and electronics favoring the formation of a 3-substituted product.13 As a temporary solution to this problem, our group and others have employed tethering strategies to increase the regioselectivity of the migratory insertion event (eqn 3).15 Of course, regioselectivity controlled by the ligand on Rh would be the optimal solution to the selectivity problem (eqn 4).16 Consequently, we focused our attention toward developing an intermolecular variant of this reaction that would provide product with improved regioselectivity.Migratory insertion of alkenes to access heterocycles using C–H activation chemistry is still relatively rare, with seminal studies by Glorius and Fagnou reporting the synthesis of dihydroisoquinolones.O-pivaloyl-benzhydroxamic acid 1a with 1-decene 2a to provide dihydroisoquinolones 3a and 3a′. When Cp* is used as a ligand, the desired products are isolated in excellent yield but poor selectivity (2.4 : 1 3a : 3a′)  , entry 1CF3)17 and found that this catalyst provides 3a and 3a′ products in good yield but without an increase in selectivity (2.4 : 1)  and were pleased to find a remarkable increase in selectivity from 2.4 : 1 to 12 : 1 (3a : 3a′). Pleased by this improvement, we tested the sterically bulky di-tert-butyl Cp ligand Cpt and were surprised to find that RhCpt provides the desired product in 91% yield with exquisite regioselectivity (15 : 1)  , entry 2(15 : 1) , entry 4t is able to substantially increase the regioselectivity of alkene migratory insertion, we explored the scope of O-pivaloyl benzyhydroxamic acids amenable to this reaction. We were pleased to find para-substituted benzhydroxamic acids are well tolerated (50–76% yields) with both Cp* and Cpt ligands. Notably, the low regioselectivity of <2.2 : 1 seen using [RhCp*Cl2]2 is dramatically improved with [RhCptCl2]2 giving excellent regioselectivities of >14 : 1   . Interes2b furnishes a 1.6 : 1 ratio of dihydroisoquinolone with RhCp* -catalyzed synthesis of 4-substituted dihydroisoquinolones is with potassium vinyltrifluoroborates where electronics are believed to control regioselectivity.20 We found that when vinylcyclohexane was submitted to a reaction with [RhCp*Cl2]2 as the precatalyst, the 3-substituted dihydroisoquinolone 4a was isolated in 90% yield with 11 : 1 regioselectivity (tCl2]2 the opposite isomer 4b was isolated in 75% yield and 10 : 1 (4b : 4a) regioselectivity. Given this unexpected discovery, we were interested in gleaning insight into how Cpt influences regioselectivity of alkene migratory insertion. A competition experiment between vinyl cyclohexane 2m and 1-decene 2a run to 10% conversion favored the formation of dihydroisoquinolone 3a in >19 : 1 ratio as determined by 1H NMR. This experiment suggests that enhanced steric interactions between the substrate and ligand slow the rate of migratory insertion.While it is desirable to achieve high regioselectivity for a single regioisomer, it is even more attractive to use a ligand to access alternate regioisomers. Currently, the only example of Rh and subsequent insertion to give the β-substituted product 3a′ (t-butyl group finds minimized steric interactions during coordination (II). Subsequent migratory insertion from II places the alkyl substituent α to Rh in the transition state, which we propose is able to stabilize a buildup of partial positive charge, making the α-substituted product 3a both sterically and electronically favored with Cpt. In the case of the Cp* ligand with small alkyl alkenes, neither steric nor electronic interactions dominate so low selectivity is observed.Based on the X-ray crystal structure and regioselectivity data, we propose the following model for regioselectivity of the 1,2-migratory insertion of alkenes, where steric contributions from the duct 3a′ . Coordint favors the opposite regioisomer. While certainly a puzzling result, we propose that the selectivity can be explained by Cpt rotation such that the t-butyl groups both occupy the space above the metallacycle. Cpt rotation gears the O-piv toward the alkene coordination site disfavoring alkene coordination to this side (IV) favoring the α-substituted product 3a. At the same time, alkene coordination (III) with the cyclohexyl opposite the O-piv minimizes steric interactions enabling insertion of the large alkene and preferential formation of β-substituted product 3a′. While not conclusive, the observation that cyclohexyl alkene reacts significantly slower than n-octyl alkene suggests that migratory insertion of the cyclohexyl alkene proceeds through a higher energy and potentially highly ordered transition state, such as Cpt rotation.However if the size of the alkene substituent is significantly increased, as in the case of vinyl cyclohexane, then Cptert-butyl cyclopentadienyl ligand (Cpt) is effective at increasing regioselectivity of alkene migratory insertion in the synthesis of dihydroisoquinolones by Rh(iii) C–H activation catalysis. In contrast to previous cases where Cp* delivers modest levels of regioselectivity of migratory insertion , Cpt renders previously non-selective reactions  highly regioselective. Furthermore, ligand control (Cp* vs. Cpt) enables the highly selective synthesis of two regioisomeric products with vinylcyclohexanes. Finally, crystallographic evidence lends support for a possible explanation of the enhanced regioselectivity.In conclusion, we found that sterically bulky di-


Ensembles of fcc nickel nanowires have been synthesized with defined mean sizes in the interior of single-wall carbon nanotubes. The method allows the intrinsic nature of single-domain magnets to emerge with large coercivity as their size becomes as small as the exchange length of nickel. By means of X-ray magnetic circular dichroism we probe electronic interactions at nickel-carbon interfaces where nickel exhibit no hysteresis and size-dependent spin magnetic moment. A manifestation of the interacting two subsystems on a bulk scale is traced in the nanotube’s magnetoresistance as explained within the framework of weak localization. Magnetic metal clusters continue to attract focus in a broad range of research fields from low dimensional physics123459134in situ with dimensions smaller than the exchange length of nickelIn this contribution we report a neat method for magnetic nanostructuring within a single-wall carbon nanotube (SWCNT)12131415161719From transmission electron microscopy (TEM), X-ray diffraction and bulk magnetization measurements we first show that the nickel clusters are controlled in size and exhibit superparamagnetism that manifests itself as spin glass-like states at low temperatures whose net magnetization is reduced at most upon cooling without an external magnetic field (zero field cooling). The hysteretic behaviours, i.e. finite coercivity and superparamagnetic blocking temperature, are scaled by the cluster size. The coercivity become as large as ~42.5 mT for the smallest cluster, much larger than around 0.1 mT of bulk nickelX-ray magnetic circular dichroism (XMCD) is an exclusive method that allows orbital and spin magnetic moments of minority elements in compounds and alloys to be evaluated23TB at which the magnetization exhibits a maximum can be defined. We obtain TB = 18, 40 and 42 K for the 3, 7 and 10 nm clusters that can be inversely scaled by the cluster length as plotted in the 26282930The temperature dependence of the magnetization measured in 100 mT, Encapsulated in SWCNTs, the single domain nickel clusters aren’t affected by environmental factors, allowing these heterogeneous nanostructures to become stable hard magnets. Magnetic properties of each cluster are dominated by the size effect as well as SWCNT-cluster interactions at metal-carbon interfaces. Both together can be accessed exquisitely via XMCD that is intrinsically a surface sensitive method25μ+ and μ− of the 3 nm cluster collected at the Ni L3,2 edges with photon spins parallel (+) and anti-parallel (−) to the direction of the magnetic field applied to the sample, respectively. Both Ni L3,2 peaks are skewed right and the 6 eV satellite is observed at about 859 eV, characteristic of metallic nickel. The XMCD spectrum is the difference between them, Δμ = μ+ − μ−. The orbital d hole can be derived by using the sum rules3334andnh is the number of holes in the Ni 3d states, p and q are the area intensities of a μ+ − μ− spectrum over the Ni L3 and L2 edge, respectively, and r is the area intensity of a μ+ + μ− spectrum over the Ni L3,2 edges, see the where d magnetic moments derived from the XMCD data plotted against temperature. The two data sets can be compared since the spin polarization of the Ni 4s and 4p bands is very small35Superposed onto the SQUID data in H −1/ where n = 3 corresponds to the dimensionality, the The resistance has been measured as a function of temperature without and with magnetic fields up to ~1 T applied to a direction normal to the current direction. The SWCNT’s electronic conduction can be explained within the variable range hopping (VRH) model as the resistance vs temperature M = [R − R]/R, where R is the resistance at temperature T and magnetic field B, and hence R in zero field. In low fields B < 0.1 T where hysteretic spin glass states emerge at low temperatures, the SWCNT film containing nickel clusters shows only a faint magnetoresistance, which means that the spin disorder among magnetic clusters observed below the blocking temperature in ZFC magnetisation measurements hardly affect the magnetotransport, otherwise alternating direction of cluster spins should give rise to a pronounced negative magnetoresistance if conduction electrons get spin polarized within each magnetic domain. This supports our conclusion that cluster spins interact with conduction electrons only at the metal-carbon interfaces where there is no magnetic order.The magnetoresistance is defined by: M measured at 9.5 K. The magnetoresistance stays negative and increases as the field increases. No hysteresis observed in the magnetoresistance upon bipolar field scans within ±1 T support the above-mentioned conclusion that the negative magnetoresistance does not originate from the spin dependent electron scattering, nor the domain wall scatteringThe field dependence can be fitted well to the following function predicted for a weakly localized three dimensional systemD is diffusion coefficient and τε the energy relaxation time of an electron due to inelastic scattering.where The negative magnetoresistance observed for the SWCNT sheet at low temperatures can be understood as a result of weak localization associated with enhanced backscattering when the elastic scattering dominates the electron conduction, as expected and previously observed for carbon nanotube films in similar field ranges. In higher fields a positive magnetoresistance is known to dictate due to the shrinkage of wave functions within the variable range hopping regime46Upon the encapsulation of nickel clusters the negative magnetoresistance is reduced to 37% of the value for the empty SWCNT as the resistance is increased at low temperatures. The fact that more electron-doped SWCNTsTo conclude, we have demonstrated that the nickel nanomagnets created in SWCNTs outperform their bulky counterparts. With SWCNTs’ advanced properties  the method allows nanomagnets to be implemented in various materials and devices. A large change in diffusive conduction of SWCNT webs via the inclusion of nanomagnets indicates much greater impact on the ballistic transport of a single SWCNT that are yet to be studied. A deeper understanding of the magnetic interaction at the metal-carbon interface gained in the present study would help us overcome the superparamagnetic limit in magnetic recording10H14NiO4, followed by heating in vacuum: The filling process was commenced by heating the SWCNT film in air at 400 °C for 30 minutes and subsequently in vacuum for 2 hours. Then, the SWCNT film was sealed together with Ni(II) acetylacetonate powder (Sigma Aldrich) in an evacuated glass ampule. The sealed ampule was heated at 130 °C for 8 days12The SWCNT films were filtered out of an ethanol solution of an e-Dips SWCNT material with tube diameters of 1.7 ± 0.1 nmNickel-filled SWCNT samples were dispersed in n-hexane by sonication and dropped onto molybdenum microgrids covered with holey amorphous carbon films for STEM. ADF-STEM images were obtained by using a JEOL JEM-2100F microscope equipped with Delta spherical aberration correctors at an electron accelerating voltage of 60 kVπsin(θ)/λ, where 2θ is the scattering angle and λ = 0.1542 nm the X-ray wavelength.XRD measurements were carried out at a Bruker Nanostar equipped with a pinhole camera, a 2D gas detector (Vantec 2000) and an image plate (Fuji FLA 7000) for simultaneous measurement of small-angle and wide-angle X-ray scattering patterns. The X-ray patterns were radially averaged to obtain the scattering intensities in dependence on the scattering vector = 4p edge with the photon helicity parallel (μ+) or antiparallel (μ−) to the magnetic field applied to the sample. The experimental end-station of this beamline allowed cooling the sample down to 5 K in magnetic fields up to 6 T.The Raman data were recorded at room temperature using a Bruker FT Raman spectrometer with an excitation energy (wavelength) of 1.16 eV (1064 nm). Bulk magnetisation measurements were carried out at Atominstitut, Vienna University of Technology, using a SQUID magnetometer with magnetic fields up to 1 T and temperatures in a range of 5 to 300 K. XMCD measurements were carried out at UE46-PGM-1 variable polarization undulator beamline at BESSY II synchrotron facility, Helmholtz-Zentrum Berlin (HZB). XMCD spectra were recorded by measuring the sample drain current over the Ni 2The resistance was measured in four point geometry at temperatures ranging from 9 to 310 K and in external magnetic fields up to 1 T in a setup equipped with a closed-cycle helium cryocooler with the sample attached onto a temperature-controlled cold finger in vacuum. The data were collected with the magnetic field applied normal to the current direction. Switching the magnetic field orientation parallel to the current direction caused no change in the resistance and magnetoresistance. This confirms that the Lorentz force on the carrier doesn’t lead to a significant contribution to the magnetoresistance.How to cite this article: Shiozawa, H. et al. Nickel clusters embedded in carbon nanotubes as high performance magnets. Sci. Rep.5, 15033; doi: 10.1038/srep15033 (2015).


Whether this relationship is preserved in response to running training (changes in running economy and V̇O2max) has yet to be explored. This study examined the relationships of (i) running economy and V̇O2max between runners, and (ii) the changes in running economy and V̇O2max that occur within runners in response to habitual training. 168 trained distance runners  performed a discontinuous submaximal running test to determine running economy . A continuous incremental treadmill running test to volitional exhaustion was used to determine V̇O2max 54 participants  also completed at least one follow up assessment. Partial correlation analysis revealed small positive relationships between running economy and V̇O2max , in addition to moderate positive relationships between the changes in running economy and V̇O2max in response to habitual training . In conclusion, the current investigation demonstrates that only a small to moderate relationship exists between running economy and V̇O2max in highly trained distance runners. With >85% of the variance in these parameters unexplained by this relationship, these findings reaffirm that running economy and V̇O2max are primarily determined independently.A positive relationship between running economy and maximal oxygen uptake (V̇O As the margin of success is extremely small in elite distance running, subtle enhancements in either parameter could result in substantial performance gains. Therefore, understanding the relationship of RE and V̇O2max both between and within individuals is necessary to understand and optimise performance.Running economy (RE) and maximal oxygen uptake ; creating a common divisor that is known to produce spurious correlations [2max. Furthermore, studies have solely employed oxygen cost (OC) as a measure of RE, rather than the more valid and comprehensive measurement of energy cost , anelations . Partialost . Thus, 2max that occur within athletes over time with training might further reveal if there is an inherent association between these variables, whilst also informing the optimisation of both variables and thus performance. Previous investigations in well trained athletes have noted enhancements in cycling efficiency following short-term, intensive endurance training, but with no change in V̇O2max evident [2max in response to endurance training and across a competitive season; despite no change in mean group V̇O2max [2max might only be achievable at the expense of the other variable. However, this previous investigation was limited to measurements of gross efficiency, with no data presented on movement economy. Moreover, analysis of this longitudinal relationship was restricted to observations within small cohorts of athletes, and with responses to run training yet to be explored.Moreover, the concurrent alterations in RE and V̇O evident ,12,13. I V̇O2max . These p2max and RE, quantified as EC , within a large cohort of highly trained distance runners. The secondary aim was to examine the longitudinal relationship between the changes in V̇O2max and RE occurring within athletes in response to endurance training.The primary aim of the current investigation was to explore the cross-sectional relationship between V̇O2max have been noted between sexes [The cross-sectional investigation involved retrospective analysis of data from 168 healthy endurance trained athletes with competitive distances ranging from 800m to the marathon , summating to 182 assessments in total. No evidence is currently available regarding sex differences in the concurrent alterations in RE and V̇O2max in response to habitual endurance training, thus data for males and females were combined for longitudinal analysis.The longitudinal aspect of the study was based on 54 participants  from amongst the larger cohort of 168 runners, that had completed at least one follow up assessment, with a median trial separation of 203 days (range: 37–2567 days) in order to assess within-athlete changes in both RE and V̇O-1), participants completed a discontinuous submaximal incremental test consisting of six to nine stages of 3 minutes continuous running, with increments of 1 km∙h-1 on a motorised treadmill of known belt speeds  interspersed by 30 s rest periods for blood sampling. As the speeds assessed were typically between 10.5 km∙h-1 and 18 km∙h-1, treadmill gradient was maintained at 1% throughout submaximal assessments in order to reflect the energetic cost of outdoor running [C and OC  and pulmonary gas exchange (detailed below) were monitored throughout the test.Following a warm-up . Moreovncreases . Recent 2max was determined by a continuous incremental treadmill running ramp test to volitional exhaustion. After a warm-up, participants initially ran at a speed 2 km∙h-1 below the final speed of the submaximal test and at a 1% gradient. Each minute, the incline was increased by 1% until volitional exhaustion. The test duration was typically 6–8 minutes.V̇OPrior to exercise on laboratory visits, body mass was measured using digital scales  to the nearest 0.1 kg. Stature was recorded to the nearest 1 cm using a stadiometer .-1. Prior to each test, two point calibrations of both gas sensors were completed, using a known gas mixture  and ambient air. Ventilatory volume was calibrated using a 3 L (±0.4%) syringe. This system has previously been shown to be a valid apparatus for the determination of oxygen consumption (V̇O2) and carbon dioxide production (V̇CO2) at both low and maximal exercise intensities [2, and V̇CO2 is achieved within the first 2 minutes of each stage for highly trained endurance runners [2, carbon dioxide production V̇CO2, and RER.Breath-by-breath gas exchange data was quantified via an automated open circuit metabolic cart . Participants breathed through a low dead-space mask, with air sampled at 60 mL∙minensities . As prev runners , mean vab) . The LTP was identified via the modified Dmax method [b greater than 0.4 mmol.L-1 (lactate threshold) and the final stage. The four stages prior to LTP were identified for each participant, with an average of these four stages used to quantify OC and EC.A 20μL capillary blood sample was taken from the earlobe for analysis of blood lactate  during the monitored period. The energy derived from each substrate was then calculated by multiplying fat and carbohydrate usage by 9.75 kcal and 4.07 kcal, respectively, reflecting the mean energy content of the metabolised substrates during moderate to high intensity exercise [C was quantified as the sum of these values, expressed in kcal∙km-1. V̇O2 during the final minute of each submaximal stage was used to determine oxygen cost (OC) in mL∙km-1 to enable comparisons to previous investigations.V̇Oquations  were useexercise . EC was C, OC and V̇O2max. As body mass was strongly related to both RE measures  and V̇O2max, partial correlations controlling for body mass, and associated 95% confidence intervals (CI), were used to assess the relationship between absolute V̇O2max and both EC and OC. This method removes the influence of body mass on both RE and V̇O2max whilst avoiding spurious correlations created by correlating two variables with a common divisor [C and V̇O2max adjusted for body mass for each individual were calculated based on individual residuals. This involved summating the individual’s residual, in comparison to the cohort relationship with body mass (e.g. EC vs body mass), with the group mean for that variable [2max and the changes in both EC and OC over repeat visits, partial correlation coefficients were calculated using ANCOVA [Data are presented as mean±SD for all dependant variables. Data analysis was conducted using SPSS for windows . When an individual visited the laboratory for repeated assessments, an average of the assessments was calculated and used for the cross sectional analysis. Pearsons product-moment coefficients were calculated to assess the relationship between body mass and E divisor . For gravariable . For theg ANCOVA ; providig ANCOVA . Signifi2max and vLTP values for both males and females.Participant characteristics are shown in C and V̇O2max .Partial correlation analysis controlling for body mass, revealed small positive relationships between EC and V̇O2max over time .Partial correlation analysis from ANCOVA revealed moderate positive relationships between the changes in E2max in a large cohort of highly trained distance runners. The major contribution of this study to the field is that only a small to moderate association exists between RE and V̇O2max (R2 ~ 12%) when body mass is appropriately accounted for. With >85% of the variance in these parameters unexplained by this relationship, these findings reaffirm that RE and V̇O2max are primarily determined independently.The present investigation explored the cross-sectional and longitudinal relationships between RE and V̇O2max and the metabolic cost of running, when quantified as both EC (r ~ 0.25) and OC (r ~ 0.33). These results support the findings of Pate et al. [2 and V̇O2max in a similarly large cohort of habitual distance runners. Conversely, a stronger, moderate positive relationship has been reported between submaximal V̇O2 and V̇O2max in smaller cohorts of elite distance runners  and phy = 0.59; ). Howeve = 0.59; ,26, and 2max within participants in response to training has not previously been documented. Supporting the findings from our cross sectional analysis, a moderate positive relationship (r = 0.35) was observed between the changes in EC and V̇O2max over repeated assessments. Moreover, these findings support recent observations from competitive road cyclists that highlighted a similar moderate relationship (r = 0.44) between changes in gross efficiency and V̇O2max across a training season [The longitudinal analysis of the relationship between the changes in RE and the changes in V̇Og season .C and V̇O2max that some previous studies have documented; with a higher V̇O2max facilitating greater lipid oxidation and consequently a greater OC during sub-maximal exercise [C may be sensitive to lipid oxidation, the calculation of EC includes the RER and thus is insensitive to differences in substrate metabolism. The influence of substrate metabolism could conceivably explain the marginally stronger relationship observed between OC and V̇O2max, than EC and V̇O2max, in both the cross sectional (r ~ 0.33 vs r ~ 0.25) and longitudinal observations (r = 0.44 vs r = 0.35). More importantly, a positive relationship was documented between EC and V̇O2max that is clearly independent of variations in lipid metabolism.It has been postulated that variations in lipid oxidation rates between individuals might, in part, explain the relationship between Oexercise . Whilst C and V̇O2max remain unclear. It has been argued that for athletes of a similar, high performance level, there would be an inevitable relationship between EC and V̇O2max in order to produce a similar velocity at V̇O2max [2max. However there is considerable evidence that V̇O2max during whole body exercise such as running is largely dependent on oxygen delivery rather than utilisation [C and V̇O2max.The mechanisms that underpin the small relationship between E V̇O2max . Howeverlisation , which m2max was small. The current study found only ~ 7%  and 12%  of the variance in RE was explained by V̇O2max. This small association likely reflects the distinct nature of these variables and their physiological determinants. V̇O2max is known to be determined by factors such as cardiac output [2max, adaptations that lead to enhancements in one of these variables are unlikely to directly influence in the opposing variable.Though reaching statistical significance, the association between RE and V̇Oc output , total hc output , and mitc output ,36, vertc output  and grouc output . As ther2max in highly trained distance runners. With >85% of the variance in these parameters unexplained by this relationship, these findings reaffirm that running economy and V̇O2max are primarily determined independently.In conclusion, the current investigation demonstrates that only a small to moderate relationship exists between running economy and V̇O