A substantial body of credible evidence has accumulated that suggest that cannabis use is an important potentially preventable risk factor for the development of psychotic illness and its worse prognosis following the onset of psychosis. Here we summarize the relevant evidence to argue that the time has come to investigate the neurobiological effects of cannabis in patients with psychotic disorders. In the first section we summarize evidence from longitudinal studies that controlled for a range of potential confounders of the association of cannabis use with increased risk of developing psychotic disorders, increased risk of hospitalization, frequent and longer hospital stays, and failure of treatment with medications for psychosis in those with established illness. Although some evidence has emerged that cannabis-using and non-using patients with psychotic disorders may have distinct patterns of neurocognitive and neurodevelopmental impairments, the biological underpinnings of the effects of cannabis remain to be fully elucidated. In the second and third sections we undertake a systematic review of 70 studies, including over 3000 patients with psychotic disorders or at increased risk of psychotic disorder, in order to delineate potential neurobiological and neurochemical mechanisms that may underlie the effects of cannabis in psychotic disorders and suggest avenues for future research. Psychotic illnesses have an annual prevalence of around 0.4% of the population and a cost to the economy of £13.5 billion a year in England alone . Early-oThis article will restrict itself to the association between cannabis and psychotic disorders. When discussing the association between cannabis and psychosis a clear distinction must be made between several psychosis outcomes: (a) risk of developing enduring psychotic disorder, (b) risk of relapse once a psychotic disorder has developed and (c) the transient psychotomimetic states that cannabis induces . In partIt is worth bearing in mind, in the discussion that follows, that cannabis is not a homogenous entity and is constituted of a variety of chemical constituents which may have varying and, at times, opposing effects . ParticuMuch of the focus to date has been towards examining whether cannabis use is a risk factor for psychotic illness . This haLongitudinal studies have also demonstrated a dose-response relationship between cannabis use and psychosis risk: Moore and colleagues undertook a major meta-analysis which demonstrated an increased Odds Ratio (OR) of 1.41 for cannabis users (OR 2.09 in more frequent users) of developing a psychotic outcome . It has A further important consideration is a shared vulnerability for psychotic disorders and use of cannabis through genetic vulnerability or stress . An impop=0.064) after adjusting for alcohol use.It has been therefore argued that cannabis represents a component cause for enduring psychotic disorder, that is, by itself neither necessary nor sufficient to cause disorder but implicated in the patho-aetiology of illness alongside other factors which increase psychosis risk . One appFinally, a further argument against a causal association between cannabis and psychosis relates to the incidence of psychosis . It has In addition to increasing the risk of onset of psychosis, cannabis use also has an adverse effect on outcome in those who develop psychosis. Of the 50089 participants of the Swedish conscript study, 401 (0.8%) had schizophrenia-related inpatient admissions in the subsequent 34 years. Of those in whom baseline cannabis data were available, 78/357 (20%) had a history of cannabis use. This group had had more hospital admissions compared with never users over the 34-year period (10 vs. four) and had spent a year longer in hospital compared with the never users (547 days vs. 184) .Other studies confirm adverse prognostic features of cannabis use. In patients with an established psychotic illness, continued cannabis use has been shown to be associated with adverse outcomes. A recent meta-analysis using pooled data from over 16500 patients with psychosis, who were at various stages of psychotic illness, demonstrated that continued cannabis use is associated with greater risk of relapse, hospitalization and longer inpatient admissions . Discontn=1027) following FEP patients for a year after referral to Early Intervention Services also demonstrated poorer outcome in cannabis-using patients which remained after adjusting for other substance misuse  followed up to 5 years after presentation, cannabis use has been shown to be associated with an increased number of unique medications for psychosis prescribed, a proxy measure for decreased tolerability and poor treatment response in a naturalistic setting  than psychosis risk, for which debates about the extent of a causal link remain. Hence, for patients with established psychotic disorders, it seems that the time has come to move beyond debates about associations of cannabis and psychosis to developing an understanding of the underlying neurobiology in order to develop interventions that work.Why cannabis-using psychosis patients have a worse prognosis is as yet unclear. Emerging evidence suggests that cannabis-using psychosis patients may have distinct clinical, prognostic and neurocognitive features compared with non-users . A largeInterestingly, poorer prognosis and younger age of onset in cannabis-using patients contrasts with the suggestion of their having a less impaired neurodevelopmental profile. Three lines of evidence converge to show this: (a) fewer neurological soft signs (NSS) in cannabis-using patients versus non-using patients ; (b) impn=92) demonstrated this in a group of daily cannabis users versus infrequent or non-existent use (n=61) demonstrated similar findings in daily or dependent cannabis users as opposed to less frequent users when tobacco, alcohol but not other substance misuse had been adjusted for (n=61) found findings in the same direction after adjusting for tobacco and alcohol  found worse outcomes in continued cannabis users, while lifetime users were neurocognitively spared  due to the effect of cannabis itself; (ii) due to the effect of addiction or substance misuse on reward pathways; or (iii) this may represent cannabis-using patients having fewer pre-existing impairments and better cognitive abilities to start with. If cannabis users and non-users with psychosis are indeed distinct groups they may represent separate pathways to psychosis. Determining the biological underpinnings of such differences, particularly the neurochemical and neurophysiological abnormalities that may distinguish cannabis-using from non-using psychosis patients, may have implications in terms of unravelling potential alternative therapeutic targets in the cannabis-using group, a group with particularly worse outcomes.In this and the following section we undertake a systematic review of neurobiological and neurochemical differences specifically between cannabis and non-using patients with psychosis as well as those at increased risk of developing psychosis. Previous reviews have extrapolated findings from preclinical studies and/or reported on findings from non-psychotic cannabis users , or haveA systematic search strategy was undertaken with PubMed between November and December 2017 (last full search 28 December 2017), with results restricted to human studies. Search terms were: (cannabis OR marijuana) AND (psychosis OR schizo*) AND . Articles were selected by the following criteria: (a) human studies; (b) relevant to neurobiological measures of psychosis; (c) including a cannabis-using group of patients with psychotic disorder (P-C); (d) including a non-cannabis-using group of patients with psychotic disorder (P-NC). Some 325 articles were identified on initial search and abstracts reviewed. Identified articles were hand-searched for further articles and relevant references for reviews in the area . In totaor (b) as demonstrated in the data provided there were no statistically significant baseline differences between P-C (group with psychosis or high risk of psychosis who use cannabis) and P-NC (group with psychosis or high risk of psychosis who do not use cannabis) for the given confounder; or (c) the confounder was adjusted for statistically (for example using regression methods); or (d) the confounder was demonstrated not to have an effect on the measure of interest; or (e) a sensitivity analysis was undertaken to consider the effect of the results when the confounder of interest was removed. Because of the heterogeneity of outcome measures, this systematic review represents a qualitative synthesis of the data and meta-analysis was not undertaken.Data extracted from all selected articles included: (a) measure of interest; (b) numbers in psychosis cannabis-using and non-using groups; (c) numbers in control cannabis-using and non-using groups; (d) whether longitudinal or cross-sectional study; (e) psychosis population of interest ; (f) relevant findings. To assess risk of bias the following were extracted: (a) definition of cannabis use; (b) limitations of article; and (c) confounders considered in the study design – namely alcohol, tobacco, other recreational substance misuse and exposure to medications for psychosis. If data were not available from the main manuscript, supplementary data were searched. A confounder was noted as having been considered in any of the following instances: (a) the said confounder had been excluded at the inclusion/exclusion stage; n=261; P-NC n=539); 16 considered structural MRI studies in the first 5 years of psychosis ; eight considered structural findings after the first 5 years ; six considered functional MRI findings ; 14 considered other neurobiological measures including peripheral findings and electrophysiology ; 12 considered the endocannabinoid system ; five considered the dopamine system ; three studies to date have investigated the glutamate system ; and two studies have investigated the GABAergic system . Collectively, therefore, this systematic review collates data for over 1253 participants for P-C and 1843 participants for P-NC (including both patients and those at high risk). The results of the complete systematic review can be seen in Of the 70 articles extracted (numbers of patients given are approximate as we have tried to account for overlapping cohorts), eight studies considered structural magnetic resonance imaging (MRI) studies in high-risk states for psychosis  and brain derived neurotrophic factor on initial admission , 2004. BA number of distinct electrophysiological alterations have also been noted in P-C versus P-NC indicating pre-attentional, attentional and reward cue deficits. Although one study demonstrated no differences between cannabis-using and non-using patients, in P50 sensory gating , differeOne further body of work has found PPI deficits in cannabis-induced psychotic disorder intermediate between healthy controls and those of schizophrenia with cannabis use. These studies were not included because of the lack of a P-NC group; however, they have been used to argue a neurobiological basis to a differentiation between cannabis-induced psychotic disorder (CIPD) and schizophrenia with cannabis use , 2017. F1. MRI in high-risk statesTwo main cohorts have reported on structural MRI measures in those at high risk who use cannabis and who do not. The Edinburgh High Risk Study recruited first-degree family members of patients. Cross-sectional and longitudinal analysis demonstrated cannabis to be associated with loss of brain volume and subcortical structures including thalamic, mediotemporal and frontal areas , b, 20132. Structural MRI in the first 5 years of psychotic disorderThere also appears to be differences detectable by neuroimaging approaches between cannabis-using and non-using patients in the first 5 years of psychotic disorder. Broadly speaking, given that both psychosis and cannabis use are known to be associated with atrophic changes and functional deficits compared with healthy non-using controls, there could alternative explanations to neurobiological differences between patients who use cannabis and those who do not: (a) there could be no difference between the groups (the null hypothesis); (b) there could be evidence of increased deficit or impairment in the P-C group compared with the P-NC group which may be accounted for by deleterious effects of cannabis use (i.e. an additive interaction of cannabis × psychosis); (c) there could be decreased deficit or impairment in the P-C group compared with the P-NC group (i.e. a sparing interaction of cannabis × psychosis).In line with the null hypothesis, some studies have failed to find structural differences between groups . AlthougAs against those, other studies have reported fewer grey matter  and whitCollectively, there are varying findings from neuroimaging studies in early psychosis. The majority of studies indicate there are structural brain differences between cannabis-using patients and non-using patients. There are likely to be altered effects of cannabis on patients, with CB1 receptor-rich areas such as the cingulate particularly prone to atrophic change. As against this there is some evidence of sparing of brain regions such as mediotemporal or striatal regions. Which regions are affected or spared and the extent of these are likely to be a function of propensity to develop psychosis, patterns of cannabis use and duration and age of first use. Currently, the functional correlates and clinical significant of these brain differences remain to be fully elucidated.3. Structural MRI after the first 5 years of psychotic disorderIn contrast to the conflicting evidence from studies in the early stages of psychosis, all studies after 5 years of psychotic disorder point towards degenerative changes in the cannabis-using patient group. These include two longitudinal studies of early psychosis followed up for 5 years , 2010. A4. Evidence from functional MRI studiesA few studies have examined neurophysiological differences between P-C and P-NC using functional MRI. In general, these studies have been modest in size, have studied varying paradigms and have been limited by a failure to include a cannabis-using control group and therefore require further replication. Nonetheless, they indicate differences between cannabis-using patients and those who do not use cannabis. These include differences in regional brain activation in emotional memory  ; functio1. Evidence from the endocannabinoid systemThe interaction of exogenous phytocannabinoids is through perturbation of the endocannabinoid system . The endocannabinoid receptor system comprises the CB1 and CB2 receptors, and G-protein coupled receptors with relative, but not complete segregation of CB1 receptors to the central nervous system, whereas CB2 receptor is expressed in the peripheral nervous system and believed to be involved in inflammatory processes . These an=39, cannabis users excluded), associated with concomitant increase in anandamide levels  showing improved response versus placebo after 6 weeks as adjunctive therapy to patients’ usual medication for psychosis  showed alterations in lipid precursors of endocannabinoids between users and non-users; however, given the small size this needs further replication  for rs12199654. Both genotypes were demonstrated to have independent and additive effects .2. Evidence from the dopamine systemOne would expect cannabis, a robustly implicated risk factor for psychosis, to affect dopamine signalling in the human brain. The dopamine hypothesis is the pre-eminent explanation for psychotic illness . Indeed,Yet in human studies the evidence is far less conclusive. Studies using functional MRI in healthy volunteers have demonstrated that transient psychotic symptoms induced by experimental administration of THC is associated with its effects on activation in key brain areas rich in dopaminergic innervation, such as the striatum and midbrain , b. Howen=5)  or tyrosine hydroxylase between P-C and P-NC; however, this was a small study and classified participants only on blood THC levels at time of death . Anothern=5) . Three sn=5) . Interesn=5) . One stun=5) . Kuepper3. Evidence from the glutamatergic and GABAergic systemsIf dopamine abnormalities do not fully explain psychosis linked to cannabis use, an alternate explanation must be sought. CB1 receptor activation, the mechanism through which THC induces psychotic symptoms under experimental conditions, mediates inhibition of glutamate and γ-aminobutyric acid (GABA) through retrograde transmission in cortical, hippocampal and midbrain regions . While tTo date there appears some suggestion that cannabis use, independent of psychosis, may be associated with perturbation of the glutamatergic system. Decreased glutamatergic indices in cortical and subcortical areas in cannabis users compared with non-users have been reported . FurtherEvidence of GABAergic deficits mimicking psychotic-like symptoms and deficits in GABAergic markers in patients on post-mortem studies  supportsCollectively, there is therefore a compelling case for investigating glutamatergic indices in psychosis patients with cannabis use. Interaction with the GABAergic system also requires further elaboration. Future work should therefore investigate whether cannabis use is associated with alterations in glutamatergic and/or GABAergic indices. These can be investigated through MRS and single photon emission tomography (SPET) or positron emission tomography (PET) approaches. MRS is widely used in investigating both glutamate and GABA levels in psychosis, but is limited by its inability to differentiate between metabolic and neurotransmitter pools of glutamatergic markers . PET/SPERegarding the endocannabinoid system, particular promise may be related to the effects of cannabidiol. As yet no randomized controlled trial has determined whether there is an increased efficacy and acceptability of CBD as a medication for psychosis in cannabis users and the neurobiological mechanisms, including perturbation of the endocannabinoid system which may underpin this.Imaging techniques may be applied to differentiate cannabis-using and non-using patients across the trajectory of illness, including clinical high-risk groups, unmedicated and medicated patients with early psychosis and patients with enduring illness. Longitudinal study design will likely help establish the precise relationship between cannabis use, alterations in the glutamate/GABA signal and outcome. Understanding the neurochemical mechanisms would enable alternative treatment strategies to be developed for this patient group, or may indeed point towards a ‘endocannabinoid dysfunction pathway’ to psychosis pathway, not fully explained by dopaminergic, glutamatergic or GABAergic changes.


Conformationally constrained peptidomimetics have been developed to mimic interfacial epitopes and target a wide selection of protein-protein interactions. ß-Hairpin mimetics based on constrained macrocyclic peptides have provided access to excellent structural mimics of ß-hairpin epitopes and found applications as interaction inhibitors in many areas of biology and medicinal chemistry. Recently, ß-hairpin peptidomimetics and naturally occurring ß-hairpin-shaped peptides have also been discovered with potent antimicrobial activity and novel mechanisms of action, targeting essential outer membrane protein (OMP) complexes in Gram-negative bacteria. This includes the Lpt complex, required for transporting LPS to the cell surface during OM biogenesis and the BAM complex that folds OMPs and inserts them into the OM bilayer. The Lpt complex is a macromolecular superstructure comprising seven different proteins (LptA-LptG) that spans the entire bacterial cell envelope, whereas the BAM complex is a folding machine comprising a ß-barrel OMP (BamA) and four different lipoproteins (BamB-BamE). Folded synthetic and natural ß-hairpin-shaped peptides appear well-suited for interacting with proteins within the Lpt and BAM complexes that are rich in ß-structure. Recent progress in identifying antibiotics targeting these complexes are reviewed here. Already a clinical candidate has been developed (murepavadin) that targets LptD, with potent antimicrobial activity specifically against pseudmonads. The ability of folded synthetic ß-hairpin epitope mimetics to interact with ß-barrel and ß-jellyroll domains in the Lpt and Bam complexes represent new avenues for antibiotic discovery, which may lead to the development of much needed new antimicrobials to combat the rise of drug-resistant pathogenic Gram-negative bacteria. Mimics of protein epitopes mediating macromolecular interactions have attracted great interest in chemical biology and drug design. One of the early drivers of protein epitope mimetic design was in targeting protein-protein interactions (PPIs)  biogenesis in Gram-negative bacteria. This research is set against the backdrop of the growing global health threat caused by the rapid evolution and spread of antibiotic resistance, particularly in Gram-negative bacteria such as p. Rice,  as well 2+ or Ca2+ ions, thereby strengthening the OM, which is important for OM function and stability. It was shown recently that not only the peptidoglycan layer but also the OM contributes as a load-bearing structure with mechanical strength to help resist osmotic forces occurring across the cell envelope  is a typical symmetric glycerophospholipid bilayer, whereas the OM is an asymmetric bilayer containing an inner leaflet of glycerophospholipid and an outer leaflet containing lipopolysaccharide (LPS) , interacts with LptC in the IM and with the LptD/E complex anchored in the OM  fold into transmembrane ß-barrel domains, with their N and C termini facing the periplasm. The C-terminal region of LptD contains one of the largest ß-barrels so far characterized, with 26 ß-strands integrated into the OM bilayer . ImportaGiven its essential role for OM biogenesis in Gram-negative bacteria and its in-part exposed position on the cell surface, it is perhaps remarkable that during the golden age of antibiotic discovery in the latter half of the twentieth century, no antibiotics were reported that target the Lpt complex.The ß-hairpin secondary structure is found in many naturally occurring cationic antimicrobial peptides (CAMPs) produced by the innate immune systems in a wide variety of organisms  and the other containing the LptD/E complex   loses much of its activity against all the Gram-negative strains tested, indicating a likely chiral target.The synthetic peptidomimetic antibiotics described earlier were until recently the only compounds known to target the Lpt complex and inhibit LPS transport in pseudomonads. Their discovery not only validated the Lpt complex as a valuable target in antibiotic discovery but raised the intriguing question whether antimicrobial natural products might exist with a similar mechanism of action. The first example of a natural product targeting the Lpt complex in thanatin . Thanatiin-vivo photoaffinity interaction mapping with E. coli. Potential interaction partners were identified following photolabeling, using a powerful mass spectrometry (MS)-based proteomic analysis. Three photolabeled OMPs revealed by this study were LptD, LptA, and BamB, of which LptD and LptA were the most significantly labeled  mutants. Of several ThanR mutants isolated, all contained mutations in LptA, and the mutated lptA gene when introduced into wild-type E. coli could confer resistance to the antibiotic. Indirect evidence that thanatin inhibits LPS transport was provided by transmission electron microscopy (EM) of thanatin-treated E. coli cells, which revealed a major perturbation to the membrane architecture, and the accumulation of membrane-like material inside the cell, which is typical of effects seen when individual components of the Lpt complex are down-regulated. However, direct evidence for an effect on LPS transport might be forthcoming with the emergence of the powerful in-vitro assays discussed above.Evidence that thanatin interacts with the Lpt complex came again from E. coli LptA oligomer revealed how LptA subunits interact in a head-to-tail fashion through their N-and C-terminal ß-strands, by ß-strand complementation  of mitochondria is required for folding and insertion of ß-barrel precursors into the mitochondrial OM. The mitochondrial homolog of BamA is called Sam50, which appears to function with a similar folding mechanism. Recent investigations of ß-barrel folding by the mitochondrial SAM complex suggest that folding precursors adopt transient ß-hairpin-like elements , typically in a region corresponding to the C-terminal ß-strand, which targets them for initial interaction with the BAM complex  from treated cells. The lack of an effect on the levels of BamA in treated cells might be explained by up-regulation of bamA expression by an envelope stress response (σE). While the results reported are consistent with inhibition of the BAM complex by JB-95, direct proof for this is still outstanding.Another cyclic ß-hairpin-shaped peptide called JB-95  was repoE. coli  displaying a truncated LPS. MAB1 showed a bactericidal activity against this strain, in the low nanomolar range over the course of several hours. Indirect evidence that MAB1 inhibits BamA folding activity was indicated by the significantly lowered levels of several OMPs in E. coli in the presence of MAB1. The mAb also caused activation of the σE stress response. Evidence for a permeabilizing effect of MAB1 on the cell envelope was shown by enhanced uptake of ethidium bromide, a rapid loss of periplasmic mCherry and a slower release of cytoplasmic Green Fluorescent Protein (GFP) from cells exposed to MAB1. Epitope mapping studies revealed that MAB1 interacts with residues in BamA extracellular loop 4  called MAB1 was described recently, directed against BamA in Pseudomonas spp. (Ghequire et al., bamA mediates the killing effects of LlpA on sensitive strains. The results suggest that LlpA interacts with BamA loop 6, and this is facilitated and/or stabilized by interaction of the lectin-like domains of LlpA with specific carbohydrate moieties in LPS. The crystal structure of LlpA from Pseudomonas sp. shows the protein to be rich in ß-structure, with two mannose-binding lectin domains and a C-terminal ß-hairpin extension (Ghequire et al., E. coli (Aoki et al., A natural antimicrobial peptide has also been discovered that targets BamA. The lectin-like bacteriocin called LlpA, a protein of ca. 28 kDa, mediates killing of selective Gram-negative bacteria, by a mechanism that is not yet fully defined. However, BamA was recently identified as a receptor for LlpA-mediated killing in These examples demonstrate that molecules interacting with the exposed external loops of BamA may have antimicrobial activity. Conceivably, molecules smaller than mAbs might be developed with similar mechanisms of action, including not only engineered antibody fragments but also smaller folded peptidomimetics better suited to circumventing the bacterial carbohydrate shield around the OM. Although one swallow does not make a summer, the intriguing discoveries highlighted in this review provide encouragement and starting points for the development of a new generation of antimicrobial agents specifically targeting Gram-negative pathogens. The need and scope for innovation in this area certainly seems to be large.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although the effects of individual-level factors on wellbeing change following work exit have been identified, the role of welfare-state variables at the country level has yet to be investigated.Data on 8037 respondents aged 50 years and over in 16 European countries were drawn from the Survey of Health, Ageing and Retirement in Europe (SHARE) and the English Longitudinal Study of Ageing (ELSA). We employed multilevel models to assess determinants of change in wellbeing following work exit, using CASP-12 change scores. After adjusting for institutionally defined route and timing of work exit, in addition to other individual-level variables, we tested country-level variables including welfare-state regime and measures of disaggregated welfare spending to determine their associations with wellbeing change and the proportion of between-country variance explained.Individuals whose exit from paid work was involuntary or diverged from the typical retirement age experienced declines in wellbeing. Country effects accounted for 7% of overall variance in wellbeing change. Individuals residing in countries with a Mediterranean welfare regime experienced more negative changes in wellbeing, with a difference of –2.15  CASP-12 points compared with those in Bismarckian welfare states. Welfare regime explained 62% of between-country variance. National per-capita expenditure on non-healthcare in-kind benefits (services) was associated with more positive wellbeing outcomes.National expenditure on in-kind benefits, particularly non-healthcare services, is associated with more favourable wellbeing change outcomes following work exit in early old age. Welfare-state effects explain the majority of between-country differences in change in wellbeing. Compared with retirees, individuals who exit work in early old age via involuntary routes such as unemployment or disability experience declines in wellbeing.Welfare regime explained 62% of between-country differences in wellbeing change following work exit in this analysis of 16 European countries, although country effects only contributed to 7% of overall variation in wellbeing change.Individuals residing in countries with a Mediterranean welfare regime experience the most negative change in wellbeing.Total per-capita social-protection expenditure, and particularly expenditure on non-healthcare services, was associated with more favourable changes in wellbeing after exit from paid work.These results have important implications for welfare policy and underscore the importance of provision of welfare services as greater numbers of workers approach retirement age and exit the labour market. ,Work exit or retirement in early old age is an important socially constructed, age-graded transition with significant implications for health and wellbeing.,,Wellbeing change following work exit can be positive or negative.,The association between work exit and individual-level wellbeing is influenced by route of exit, age at exit and other factors present at the time of exit. Exit from work via dismissal, permanent sickness or unemployment results in reduced subjective wellbeing and increased psychological distress.,Attempts have been made to define typologies for grouping countries into welfare ‘regimes’ according to how social-protection benefits are granted, their generosity and duration,,IJE online, for definitions)22 and then further disaggregated into four primary components: old-age cash transfers (comprising pensions and survivors’ pensions), working-age cash transfers, health benefits in-kind and other service  expenditure. These components have been shown to be uncorrelated and can be considered distinct dimensions of welfare policy.Earlier welfare typologies, particularly those that employed overall welfare spending measures, manifested a one-sided focus on provision of social insurancePrevious work has quantified the degree to which welfare-state factors explain country-level differences in development indicators by partitioning of variance within a multilevel framework.15 and in countries where spending on social protection is higher experience a more favourable change in wellbeing after exit from paid work. We also aimed to determine whether cash transfers or in-kind benefits had a stronger association with positive wellbeing change, and whether these associations differ when welfare spending is operationalized using measures other than welfare effort.To our knowledge, no previous study has attempted to quantify country-level influences, in particular welfare-state measures, on wellbeing change in response to work exit in early old age. The present study sought to investigate associations between welfare regime and disaggregated measures of welfare spending with change in wellbeing following work exit, after adjustment for individual-level characteristics. It was hypothesized that respondents in countries such as Scandinavian Social Democratic welfare states with more generous criteria for receipt of benefits,t0) to any other state in the following wave (t1). Where individuals experienced multiple exit events, data on the last event were used. This yielded a total sample of 8548 respondents who had exited from work in the period 2002–13 with wellbeing measures (see IJE online). Of these, 511 (5.9%) had one or more missing observations for covariates and this yielded a complete sample of 8037 respondents.The analytic sample was drawn from respondents across 16 countries from Waves 1–5 (2004–13) of the Study of Health, Ageing and Retirement in Europe (SHARE) and Waves 1–6 (2002–13) of the English Longitudinal Study of Ageing (ELSA).IJE online), previously employed in studies of wellbeing across welfare states.,,,t0 to t1.Wellbeing was measured using CASP-12 —a shortened version of the validated CASP-19 wellbeing scale (t1. This was specified using the benefit categories in SHARE. ELSA responses were mapped onto these (see IJE online). The categories were: (i) disability insurance benefits, (ii) unemployment benefits, (iii) sickness benefits, (iv) social assistance benefits, (v) public early-retirement pension, (vi) public old-age pension and (vii) none of these. When an individual received multiple benefit types, they were assigned to the lowest-numbered category.Route of exit was defined based on institutional definitions and determined according to type of public benefit received at t0 and t1. Timing of exit was represented using a nominal variable with three categories: (i) work exit >12 months before pensionable age, (ii) work exit within 12 months of pensionable age and (iii) work exit >12 months after pensionable age.We obtained OECD data for ‘typical’ pensionable ages in each country, defined as the earliest point at which an individual can draw full pension entitlements based on a career starting at age 20 with contributions in each year until retirement.IJE online). This scale included 36 items and was calibrated to a range of 0 to 1. Models also adjusted for year of work exit, participation in social activities in the previous month (yes/no), birth outside country of residence (yes/no), partnership status (partnered/non-partnered), country-specific quartile of equivalized non-pension household net wealth and natural logarithm of equivalized gross household income. These variables were selected for inclusion in the fully adjusted model using backward stepwise selection (see IJE online). Individual-level financial variables were purchasing power parity (PPP)-adjusted (see IJE online) and equivalization was performed by dividing by the square root of the household size as per standard OECD methods.A physical frailty index based on the accumulation of deficits was operationalized using all survey items relating to medically diagnosed conditions, medical symptoms, functional activities and activities of daily living available in both datasetset al.47  and further disaggregated into old-age cash benefits, non-old-age cash benefits, in-kind health benefits and non-health in-kind benefits.Countries were classified into welfare regimes using the scheme used by Bambra et al.47 . This wa,t0 to correct for regression towards the mean.,,Random intercept multilevel models, with individuals nested within countries, were used to account for dependence of observations at the country level. The assumptions of multilevel models include normality of variances, homogeneity of variance and independence of observations at all levels. The small sample of countries available is problematic, however, as standard random intercepts models including fewer than 20–30 level-2 units are likely to yield biased estimates of random-effects parameters.t0 only). This provided an estimate of the percentage variance explained by country differences. The variance components obtained from this model were used as a baseline for comparison with subsequent models to estimate the percentage of country-level variance explained by groups of country-level variables. The percentage of variation attributable to each level  is estimated using the intraclass-correlation coefficient (ICC), defined as ‘the proportion of the variance explained by the grouping structure in the population’.To estimate the extent to which differences in wellbeing change between countries was explained by country-level variables, we fitted a minimally adjusted model for change in CASP-12  added groups of variables representing social-protection effort, emphasis and expenditure. Models 2 and 3 fitted total welfare effort, and welfare effort devoted to in-kind and cash benefits, respectively. Model 4 fitted emphasis on in-kind benefits as a percentage of total public social-protection spending. Model 5 fitted total per-capita public expenditure on social-protection benefits, whereas Model 6 disaggregated expenditures into in-kind and cash benefits. In Model 7, expenditures on cash and in-kind benefits were further classified according to whether these were age-related or health benefits. The percentage of variance due to country effects explained by the addition of each set of country-level variables was calculated. Residual plots for level-2 units were generated to show country deviations from the overall mean based on the minimally adjusted model and then the conditional model after adjustment for welfare typology.IJE online, summarizes country-level welfare measures of effort, emphasis and expenditure in 2011 and gives mean values by welfare regime.The individual-level characteristics of the analytic sample are shown in Exits from paid work for reasons related to unemployment or disability, and outside typical age windows for retirement, were associated with negative changes in wellbeing. The results of the minimally adjusted model (not shown) found that country of residence accounted for 7% of variance in change in CASP-12 scores following work exit. As shown in Welfare regime explained 62% of country-level variance in wellbeing change following work exit. Models 2–7  show the,Work exits occurring over 1 year before or after the typical pensionable age and via unemployment, disability or sickness were independently associated with declines in wellbeing. These adjusted changes in CASP-12 can be greater than or comparable in magnitude to other adverse events such as divorce or separation or diagnosis of a serious physical illness.Welfare regime explained a higher proportion of between-country differences than any measure of social-protection effort, emphasis or expenditure. Consequently, individuals in Scandinavian Social Democratic welfare states may experience more positive change in wellbeing due to not only higher expenditure on in-kind benefits, but also other institutional factors. These include rules that guide institutional patterns of work exit and individuals’ behaviour and differences in financing mechanisms, extent of benefit coverage and eligibility.Whereas earlier welfare state typologies focused on the cash-transfer component of welfare spending as the primary differentiator of welfare regimes, welfare services delivered via in-kind benefits have recently received greater attention.3 . The results imply that policymakers should prioritize universal provision of non-health services over cash transfers as a more cost-effective means of mitigating potential negative wellbeing consequences of exit. Adverse changes in wellbeing have the potential to negatively impact physiological health and mortality risk.,Expenditure devoted to non-healthcare services had the greatest effect on wellbeing following work exit, and this type of expenditure varies substantially between countries. By contrast, welfare effort devoted to in-kind health-related benefits is relatively similar across developed countries and is unlikely to represent a differentiating feature of welfare-state regimes. Rather, mechanisms of financing and delivery of health services are likely to constitute the primary drivers of national differences in health indicators.24 which indicate that welfare effort devoted to cash unemployment benefits is not associated with higher life satisfaction among unemployed individuals. Other studies used aggregated country-level wellbeing measures as their outcome and only considered welfare effort. Okulicz-Kozaryn et al.25 found that overall welfare effort had a positive effect on subjective life satisfaction in cross-section whereas Veenhoven26 found no effect.This is the first study to address country-level determinants of wellbeing change following work exit and to use a disaggregated spending approach. To date, few studies have considered the associations between welfare spending and wellbeing. Our results agree with those of Eichorn,The study’s strengths include its large sample size and adjustment for important individual-level determinants of wellbeing change following work exit. Another is its disaggregated spending approach and partitioning of variance components within a multilevel MCMC framework using comparable country-level indicators. This approach presents new avenues for investigating the influence of welfare-state policies across a range of outcome measures.One assumption of multilevel models is that level-2 units are randomly drawn from a representative sample.Our findings show that country-level welfare policies explain a large proportion of the variance in wellbeing change between countries and show associations with individual-level wellbeing change following work exit. Expenditure on non-healthcare services had the strongest positive association with wellbeing change.This work was supported by the Economic and Social Research Council . The Survey of Health, Ageing and Retirement in Europe data collection has been primarily funded by the European Commission through its fifth and sixth framework programmes . Additional funding was provided by the US National Institute on Aging  as well as by various national sources. Funding for the English Longitudinal Study of Ageing is provided by the National Institute of Aging  and a consortium of UK government departments coordinated by the Office for National Statistics.Supplementary DataClick here for additional data file.


Here we report the development of a unique spatial light-modulated stimulated Raman scattering (SLM-SRS) microscopy that tailors the broadband excitation beam with sparse-sampling masks designed for rapid multiplexed vibrational imaging to monitor real-time cancer treatment effects and Methods: We design an optimal mask pattern that enables selection of predominant windows in SRS spectrum for collective excitation at the highest possible peak power, thus providing an improved signal-to-noise ratio (SNR) without compromise of chemical specificity. The mask pattern generated is applied to the broad excitation beam using a flexible spatial light modulator. The SLM module further offers complementary function whereby rapid scanning of SRS spectrum can be facilitated prior to the mask generation, thereby making the SLM-SRS system a stand-alone imaging platform.Results: We demonstrate that SLM-SRS microscopy permits rapid multiplexed SRS imaging of polystyrene and polymethyl methacrylate beads in Brownian motion in dimethyl sulfoxide (DMSO) at 70 ms intervals without motion artiacts. We further apply SLM-SRS to monitor the therapeautic effect of mild alkaline solution on cancer cells, which shows immediate apoptotic response. Finally, we visualize in vivo penetration of DMSO into the plant tissue and evaluate acute toxicity of DMSO on cellulose and proteins within the tissue.Conclusion: We develop novel SLM-SRS microscopy and affirm its broad applicability for rapid monitoring of dynamic biological processes at the subcellular and molecular level. Conversely, a higher signal level can be enjoyed with the use of femtosecond (<100 fs) laser beam, but the broad range of the resonance frequencies (>200 cm-1) would indiscriminately excite the multiple components in the sample, resulting in the deterioration of chemical selectivity. On the other hand, a spectroscopic detection method may rely on a programmable optical mask that tailors the broadband emission of Raman scattering For observation of dynamic processes in living systems, high speed imaging is crucial to catering to the temporal resolution of the dynamic events. A simple strategy is to excite a single vibrational band in the Raman spectrum of each target component that offers the highest contrast against the interfering counterparts. The minimum spectral scanning steps used greatly shorten the overall data acquisition time, allowing rapid imaging of the dynamic processes such as protein metabolism in the mouse brain tissue and rapid uptake of antibiotics into biofilms osecond <0 fs lasein vivo penetration of dimethyl sulfoxide (DMSO) into a plant root for cytotoxicity assessment.Here we report the development of a unique spatial light-modulated stimulated Raman scattering (SLM-SRS) microscopy which tailors the broadband excitation beam for multiplexed vibrational imaging with higher sensitivity. The pulse-shaping is achieved by using a sparse-sampling mask designed that allows collective excitation of the predominant spectral windows against the overlapping spectra. Due to the inverse time-frequency relationship between the time and spectral domains, the modulated beam with selective frequency components can attain higher peak power to improve signal-to-noise ratio without compromise of chemical specificity. For implementation, we incorporate a pulse-shaping module comprising a spatial light modulator (SLM) with flexible array design which offers dual functions: i) spectral patterning based on the sparse-sampling mask, and ii) rapid acquisition of SRS spectrum as prior knowledge of the sample needed for pattern generation. Such versatility makes our SLM-SRS imaging system stand-alone to target any unknown biological samples. With the rapid refresh rate offered by the SLM module, we successfully acquire multiplexed 4D SRS images of the functional responses of live cells to the alkaline environment as well as monitor -1  microscopy system developed is depicted in Figure 1 Figure A. Subseq1, a2, . . ., ak, and j stands for the stream of photons from specific chemical component, aj (where ). In reality, the detector measures the mixed photons from all aj components in the sample. Thus, our goal is to unmix the detected photons and estimate all the individual jGiven that each aj has its own spectra, Sij (where n = number of wavenumber channels in the spectroscopic system), the stream of mixed photons,, detected over time t is:In this work, we aim to design a unique binary mask pattern so that the intensity transmitted through the mask can represent a direct measurement of the dominant signals from a specific component against the other components. The working principle is as follows: Assuming that the sample consists of k number of chemical components, a represents the rate of mixed photons experimentally detected by a detector.where ij, that allows dominant photon generation from aj over the other interfering components. Then, the measured signal  of the mixed photons from all components with the optical mask applied over T seconds can be modeled as:To retrieve the unmixed photons, we could design a binary mask, M and the measured mean signal of specific photons from aj as follows:The mask can be optimized by minimizing the variance between ) that makes close to  The details of the solution to the optimization problem are articulated in We can find an optimal mask  then can be taken in various forms depending on the peak position (peak wavelength) of the Gaussian excitation spectrum. We generate a set of sparse-sampling masks for each effective spectrum by scanning the peak wavelength (λ) of the excitation beam to find the optimal mask that gives the highest specificity for each component. At given effective spectra, we estimate the ratio (jλϕ) of spectral energy density between the target component and the sum of all other interfering components:In consideration of the sparse-sampling mask design for multiplexed SRS imaging, the resonant frequencies are excited with a pulse laser whose energy density follows Gaussian distribution. Thus, the input spectra used for the mask design should reflect the intensity modulation caused by the excitation beam. The effective spectra  while the 'block' region 255 (white). The array values are then directly transferred to the pixel array on the SLM, each pixel of which rotates the polarization state of the incident beam from 0 to π/2 according to the pixel value (0 to 255). As a result, the light landing on the 'pass' region is assigned as vertical polarization so that it can be reflected by the subsequent polarization beamsplitter. The rest remains horizontally polarized for separation at the polarization beamsplitter, thereby achieving intensity modulation of the beam into desired pattern. The mask transfer is facilitated by the PCIe board, which enables rapid communication within 5 ms, allowing rapid multiplexed SRS imaging.A mixture of 10 μm polystyrene (PS) and polymethyl methacrylate (PMMA) beads (Phosphorex) is prepared by the mixing microsphere solutions in water. 5 µl of solution is dropped on a coverslip and sealed for imaging.3 (0.5 M) is added to 3 mL of PBS media. Immediately, the 3D multiplexed SRS imaging at two channels (lipid and protein) is performed.HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing high glucose, glutamax, and 10% FBS. Cells were subcultured and plated on a bottom glass  for one day in the incubator. Before imaging, the cells are washed with PBS twice to remove excess media. To create the alkaline environment at pH 8.5, 1 mL of NaHCOin vivo plant imaging, Arabidopsis thaliana seedings were grown on petri dishes containing Murashige and Skoog medium  under conditions (16 hr light/8 hr dark) in a growth cabinet. The temperature of the cabinet is set at 23 degrees in Celsius. The SRS experiments are carried out with 3-day-old seedlings.For We firstly assess the performance of SLM-SRS microscopy with a mixture of 10 μm PS and PMMA beads prepared. Figure We compare the SLM-SRS images with the counterparts obtained by raster-scanning using the SLM. Figure -1) and broadband (~ 200 cm-1) excitation beams.Apart from the PS and PMMA beads mixture, we also evaluate the SNR improvement for other chemical mixtures with larger spectral overlap Figure . In geneTo validate the rapid multiplexed imaging capability of SLM-SRS microscopy, we simultaneously visualize 4.5 μm PS and 3.0 μm PMMA beads with Brownian motions in DMSO. Prior to SLM-SRS imaging, we generate the sparse-sampling masks for the three chemicals and confirm their chemical specificity in the SRS image  microscopy that spectrally shapes the broadband excitation beam to enhance the SNR without compromise of chemical selectivity. Our results show that SLM-SRS technique improves the SNR of PMMA and PS SRS images by 3- and 5-fold, respectively, compared to the narrowband excitation while retaining high chemical specificity. We also demonstrate that the pulse-shaping module based on the flexible SLM in the system can provide dual functions for spectral patterning and rapid acquisition of SRS spectra together, thus enabling our SLM-SRS technique to be independent of external instruments to gain prior knowledge of the unknown samples. We prove the capability of SLM-SRS microscopy for rapid 3-color vibrational imaging by visualizing the Brownian motion of the PS and PMMA beads in DMSO solutions at 70 ms intervals without any artifacts. Finally, we demonstrate the biological applicability of SLM-SRS microscopy by uncovering new biological insights associated with cell apoptosis and transport of toxic solute into the plant root Supplementary figures.Click here for additional data file.Movie S1.Click here for additional data file.Movie S2.Click here for additional data file.Movie S3.Click here for additional data file.Movie S4.Click here for additional data file.


This Research Topic aimed to provide a useful resource on our current understanding of the mechanisms that mediate antigen sampling at mucosal surfaces, and the influence they have on mucosal immunity. The body's mucosal surfaces such as those lining the gastrointestinal and respiratory tracts are continuously exposed to large burdens of commensal bacteria, as wells as pathogenic microorganisms and their toxins . As a coHara et al. describe the influence that dietary antigens may also have. They show that mice fed antigen free diets had reduced intestinal IgA production, and reduced numbers of germinal center B cells and T follicular helper (Tfh) cells in their Peyer's patches. Importantly, dietary antigens also appear to play a role in host protection against intestinal pathogens, affecting for example, immunity to Salmonella Typhimurium infection.Much is known of how the commensal microbiota can affect IgA production in the intestine , 5, but Zhao et al. define the mechanism by which intranasal chitosan-formulated DNA vaccination induces IgA secretion in the intestine. They show that chitosan-DNA is specifically targeted to CD103+ dendritic cells. These cells accumulate in the mesenteric lymph nodes and promote IgA-class switching in antigen-specific B cells via IL-6-IL-6R and BAFF-TACI signaling, as well as Th17/Tfh cell differentiation. These data suggest that intranasal chitosan delivery may have useful adjuvant application for the induction of intestinal IgA responses.In their study, in vitro systems. Wosen et al. show how IFNγ stimulation can induce MHC-II expression in human small intestinal enteroids (“mini guts”). Since enteroids could be derived from individual patients with distinct MHC-II genotypes this may represent a novel tractable in vitro system to interrogate the role of IEC-expressed MHC-II in celiac disease and other intestinal MHC-II-associated diseases.The expression of major histocompatibility complex II (MHC-II) by intestinal epithelial cells (IEC) raises the suggestion that IEC contribute to immunity or tolerance through MHC-II-mediated antigen presentation to T cells . HoweverDillon and Lo provide a detailed review of the cellular and molecular factors that regulate M-cell differentiation in the mucosal-associated lymphoid tissues. M cells constitutively sample the lumenal contents and rapidly transcytose particulate antigens across their short cytoplasm into the underlying basolateral pocket where they are sampled by leukocytes and lymphocytes. This review also contains an in-depth description of the unique molecular and morphological features that enable M cells achieve their important immune surveillance functions.In the intestine specialized antigen-sampling IEC known as M cells (or microfold cells) reside within the follicle-associated epithelia (FAE) covering the GALT. Due to many important recent advances it is perhaps not too surprising that articles covering M-cell immunobiology feature strongly in this Research Topic. Dillon and Lo also provide a comparative account of how M cells can be induced under certain circumstances in the chronically inflamed intestine. Complex interactions between host genetic and environmental factors can profoundly affect the pathogenesis of some autoimmune diseases. Since dysbiosis of the intestinal microbiota is increasingly identified as one of these factors, Kobayashi et al. discuss the novel hypothesis that dysbiosis and M-cell mediated bacterial transport into Peyer's patches may also affect susceptibility to, or the pathology of, some autoimmune diseases. For example, they propose how the transcytosis of segmented filamentous bacteria by M cells might induce Tfh cells differentiation in Peyer's patches, and how this might stimulate autoantibody production, and by doing so exacerbate the pathogenesis of arthritis. The exciting array of M cell-specific tools that are now available provide timely opportunities to test the many interesting hypotheses raised in this review.Much of our knowledge of M cells was derived from the analysis of cells and tissues during the steady-state. Kimura et al. describe the identification and features of M cells associated with the tracheal and bronchial epithelia of the lower respiratory tract. Interestingly, M cells were also observed in the epithelia associated with ectopically-induced lymphoid follicles in distinct mouse models of respiratory disease. Further studies are now required to determine the role these cells play in the induction of immunity in the respiratory tract and their influence in respiratory disease pathogenesis.Cells with M cell-like characteristics have also been described in the FAE lining the nasopharynx-associated lymphoid tissues of the upper respiratory tract . In theiBalic et al. report the surprising finding that M cell-like cells in the FAE covering the bursa of Fabricius of avian species such as the chicken express CSF1R highly. This raises the hypothesis that in contrast to mammals the differentiation avian M cell-like cells may directly depend on CSF1-CSF1R stimulation. The demonstration that avian M cell-like cells express CSF1R highly also provides a useful novel marker to specifically study their role in mucosal immunity and disease.Colony-stimulating factor 1 (CSF1) controls the growth and differentiation of macrophages . FurtherIn conclusion, we consider the novel advances and stimulating hypotheses presented within these articles will provide useful foundations for future studies on the factors that mediate antigen sampling at mucosal surfaces to improve tolerance, prevent disease and enhance mucosal immunity to pathogen infections.NM and KH wrote the manuscript, edited the manuscript, and approved the final submitted version of the text.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.


Emicizumab is a humanized bispecific antibody that binds simultaneously to factor (F) IXa and FX replacing the cofactor function of FVIIIa. Because emicizumab recognizes FIX/FIXa and FX/FXa, a question may arise whether emicizumab competes with antithrombin (AT) and/or tissue factor pathway inhibitor (TFPI), thereby enhancing overall hemostatic potential by blocking their antihemostatic effects. To address this question, we performed enzymatic assays using purified coagulation factors to confirm whether emicizumab interferes with the action of AT on FIXa or FXa, or with the action of TFPI on FXa. In those assays, we found no interference of emicizumab on the actions of AT and TFPI. We next assessed emicizumab's influences on the anticoagulation actions of AT or TFPI in thrombin generation assays triggered with FXIa or tissue factor (TF) in AT-depleted or TFPI-depleted plasma supplemented with AT or TFPI in vitro. In those assays, we employed anti-FIXa and anti-FX monospecific one-armed antibodies derived from emicizumab instead of emicizumab itself so as to prevent emicizumab's FVIIIa cofactor activity from boosting thrombin generation. Consequently, we found that neither anti-FIXa, anti-FX monospecific antibody, nor the mixture of the two interfered with the anticoagulation actions of AT or TFPI in plasma. Although emicizumab can bind to FIXa and FXa, our results showed no interference of emicizumab with the action of AT or TFPI on FIXa or FXa. This indicates that the presence of emicizumab is irrelevant to the action of AT and TFPI, and thus should not alter the coagulant/anticoagulant balance related to AT and TFPI. Emicizumab  is an asymmetric humanized bispecific antibody that bridges factors (F) IXa and FX, accelerating FIXa-catalyzed FX activation and facilitating thrombin burst in FVIII-deficient plasma.Regulating negative control of the coagulation process is a new approach to the treatment of hemophilia A. Approaches recently being tried in clinical studies include therapeutic RNA interference (RNAi) targeting antithrombin (AT)Thus, emicizumab and AT can bind to FIXa, and emicizumab, AT, and TFPI can bind to FXa. This raises the question of whether emicizumab may interfere with the actions of AT or TFPI; in other words, whether emicizumab's mechanism of action would include the lowering of AT or TFPI activities. We anticipated that emicizumab would be unlikely to interfere with the actions of AT or TFPI, because emicizumab binds the epidermal growth factor (EGF)-like domains of FIXa and FXa, while AT and TFPI bind the protease domains, and because its binding affinities are on the order of 1 micromolar.4) was produced from a Chinese hamster ovary cell line using recombinant DNA technology as previously reported.4antibodies having either of the Fabs of emicizumab were transiently expressed in HEK293 cells and purified.Emicizumab (recombinant humanized IgG2and 0.1% (w/v) bovine serum albumin (BSA), and incubated for 30 minutes at room temperature. After adding a chromogenic substrate specific to FIXa , we measured absorbance at 405 nm to determine FIXa activity. We performed the experiment in the presence of 30 IU/mL heparin under the same conditions.To evaluate FIXa inhibition, we conducted an enzymatic assay consisting of 89 nM human FIXa, 4 μM phospholipids, various concentrations of AT, and emicizumab in Tris-buffered saline  containing 1 mM CaCl2and 0.1% (w/v) BSA, and incubated for 30 minutes at room temperature. After adding a chromogenic substrate specific to FXa , we measured absorbance at 405 nm to determine FXa activity. In the enzymatic assay containing AT, we performed the experiment in the presence of 0.005 IU/mL heparin under the same conditions.To evaluate FXa inhibition, we conducted an enzymatic assay consisting of 1.25 nM human FXa, 4 μM phospholipids, various concentrations of either AT or TFPI, and emicizumab in Tris-buffered saline containing 1 mM CaClThrombin generation assays were performed with standard equipment and calibrated automated thrombography (CT) using a 96-well plate fluorometer  equipped with analyzing software . Thrombin generation was initiated with an intrinsic pathway triggering solution containing 0.04 nM human FXIa and 20 μM phospholipids in Tris-buffered saline solution containing 0.1% (w/v) BSA, or extrinsic pathway triggering solutions  in AT- or TFPI-depleted plasma reconstituted with AT or TFPI. In the intrinsic pathway triggering solution, we adopted the FXIa concentration which can detect the changes of thrombin generation parameters sensitively around the plasma concentration of emicizumab (around 50 μg/mL = 343 nM) in the HAVEN1 Phase III study see also. To iniWe first determined the appropriate conditions for the chromogenic assays for FIXa and FXa activities with which to assess AT or TFPI inhibitory action on FIXa or FXa. In the assays, the measured absorbance increased in a FIXa- or FXa-concentration–dependent manner . On theTo assess the inhibitory action of AT on FIXa and FXa, and any potential modulation of this action by emicizumab, we performed the chromogenic assays for FIXa and FXa activities under the conditions mentioned earlier. Because the inhibitory effect of AT on FIXa and FXa is strongly enhanced by heparin, we evaluated the inhibitory effect of AT on FIXa and FXa with or without heparin. We confirmed that AT decreased FIXa activity dose dependently, and that its inhibitory effect was enhanced in the presence of heparin ,B. VaWe next evaluated the effect of emicizumab on the inhibitory action of TFPI on FXa by using the chromogenic assay for FXa activity. We confirmed that C-terminus truncated TFPI decreased FXa activity dose dependently . VariouWe demonstrated in the enzymatic assays that emicizumab did not interfere with the action of AT or TFPI on FIXa or FXa. To evaluate these effects in the presence of other coagulation-related factors, we next evaluated the effects of emicizumab on thrombin generation in AT- and TFPI-depleted plasma. As emicizumab does not need to be activated to exert FVIIIa cofactor function on coagulation, it has potential to affect parameters in thrombin generation. Because thrombin generation assays cannot distinguish between the potential blocking effects of emicizumab on AT or TFPI's action versus the effect of emicizumab's FVIIIa cofactor function, both of which would increase thrombin generation, we first had to prevent the effect of emicizumab's FVIIIa cofactor function on coagulation. To accomplish this, we generated anti-FIXa and anti-FX monospecific one-armed antibodies derived from emicizumab for use in the thrombin generation assays instead of emicizumab.To assess the effects of anti-FIXa and anti-FX monospecific one-armed antibodies on thrombin generation in AT-depleted plasma, we first confirmed in AT-depleted plasma that AT dose dependently decreased thrombin peak height under three triggering conditions  to evaluate both intrinsic and extrinsic pathways  administered intravenously or subcutaneously to patients with hemophilia A and B showed a dose-dependent procoagulant effect.FIXa and FXa are inhibited by AT which binds to the active sites located on the heavy chain of these trypsin-like serine proteases, and FXa is inhibited by the Kunitz domain 2 of TFPI interacting with the serine protease domain located on the heavy chain of FXa.In this study, we demonstrated that activity of emicizumab does not include AT or TFPI inhibition, indicating that emicizumab does not interfere with coagulation feedback by AT or TFPI.Emicizumab had little influence on thrombin peak height under three triggering conditions  in normal plasma . We thiThese properties of emicizumab would eventually enhance the parameters in these thrombin generation assay conditions, and therefore would decrease the robustness to assess the effects of emicizumab on the action of AT and TFPI. Anti-FIXa and anti-FX monospecific one-armed antibodies derived from emicizumab do not possess FVIIIa cofactor function on coagulation; therefore, we consider that these one-armed antibodies are more preferable to be used in this study.Although the add-on effects of emicizumab on thrombin peak height were not observed in normal plasma, as it would reach a saturation state , in theKD-based simulation predicted that 32% of FX would form complex with emicizumab.We cannot exclude all of the effects of anti-FIXa and anti-FX monospecific one-armed antibodies on thrombin generation parameters; however, we think that we could evaluate the effect of one-armed antibodies that they do not inhibit the action of AT or TFPI because these one-armed antibodies showed almost same thrombin generation parameters compared with the antibody-absent control group and4. Heparin-like molecules, heparin sulfate proteoglycans, are closely associated with endothelial cells and enhance the action of circulating antithrombin. In chromogenic assays for FIXa and FXa activities, we examined the influence of heparin in this assay, and found that emicizumab did not interfere with the inhibitory effect of AT on FIXa or FXa in the presence of heparin ,D. ThThe inhibition of FXa by TFPI is a biphasic, slow tight-binding mechanism.In coagulation feedback system, activated protein C (APC) is also known as an important factor in addition to AT and TFPI. Unlike FVIIIa, emicizumab is not inactivated by APC. However, coagulation reaction enhanced by emicizumab could be downregulated through inactivation of FVa by APC.In conclusion, we demonstrated that emicizumab exerts its action without perturbing AT or TFPI, and thus should not alter the coagulant/anticoagulant balance related to AT and TFPI. These basic findings are important, for example, when considering the meanings of coagulation-related biomarkers in the presence of emicizumab.


Ferulic acid is a simple phenolic acid found mainly in cereals and grains, used as an antioxidant and food preservative. Recent evidence suggests that ferulic acid possess anti-inflammatory, anti-diabetic, anticancer, and cardioprotective properties. Several investigations also have shown that ferulic acid rich food might prevent hypertension. As a potent scavenger of free radicals , ferulic acid attenuates oxidative stress, which is responsible for lowering elevated blood-pressure through improved endothelial function and increased bioavailability of the nitric oxide in the arterial vasculature. This review article describes the role of ferulic acid in the pathophysiology of vascular dysfunction and hypertension along with highlighted the merit of further scientific and clinical exploration. Ferula foetida is the source of 3-methoxy-4-hydroxycinnamic acid which was isolated first by Austrian chemist Hlasiwetz Barth  and catalase (CAT) activities and mRNA expression were increased by ferulic acid and ric acid . Antioxiric acid  and highric acid . Anotherric acid –23.in vitro and in vivo models. The LPS-treated Raw 264.7 murine macrophage is widely used to study inflammation in vitro. Macrophages play a central role in inflammation and are responsible for the production of inflammatory mediators such as pro-inflammatory and inflammatory cytokines  production was decreased by ferulic acid and related ester derivatives  -releasing derivative of ferulic acid, inhibited iNOS mRNA and protein expression . The in-0 mg/kg) .In vitro intestinal alpha-glucosidase and porcine pancreatic alpha-amylase inhibitory activity have been reported recently by ferulic acid (in vivo (Ferulic acid showed useful anti-diabetic activities . Blood glic acid . In diablic acid . A recenlic acid  and adiplic acid . Improvelic acid  possiblylic acid . Previou(in vivo .in vitro (−/−) comparison with clofibrate in mice fed with a western diet , 4443, 4in vitro and in vivo studies showed that ferulic acid inhibited angiotensin-converting enzyme activity (−1) in SHRsp rats showed that systolic blood pressure (measured by tail-cuff method) was lowered below the baseline, which was correlated to the simultaneous reduction of plasma angiotensin-converting enzyme activity from 20.6 to 16.9 mU ml−1 at 2 h  at a dose of 0.1 μM were studied in cultured human umbilical vein endothelial cells (HUVEC), followed by measurement of gene expression by microarray and quantitative RT–PCR. This study suggested that these selected polyphenols up regulated 233 genes and down regulated 363 genes .Transcriptional regulator of the vascular cell was also modulated by ferulic acid supplementation. Nrf-2 is a key transcriptional factor that activates the antioxidant-reactive element (ARE). Nrf-2 and ARE also regulates the expression of antioxidant Phase II detoxifying enzymes. Ferulic acid supplementation may increase cardiac Nrf2 protein expression in rats  which maA recent investigation also reported that polyphenolics extract of whole wheat grains and ferulic acid protected doxorubicin-induced cardio-toxicity in rat cardiomyocytes . This st4 mediated inflammatory pathway (Cerebral ischemia is one of the deadly pathological conditions in which abrupt deprivation of oxygen and glucose is experienced by the brain tissues. As a result excessive production of free radicals, excess recruitment of inflammatory cells, and accumulation of intracellular calcium and initiation of apoptotic signals triggers ischemic damage to neuronal cells . Thus, a pathway . Protect pathway . Nitric  pathway . Nitric  pathway .Ferulic acid showed very promising activities on blood pressure regulation and modulated many of the known physiological system and molecular mechanisms. It is still not clear, how ferulic acid increased the bioavailability of nitric oxide in the vasculature. No information is available to get direct evidence of increasing nitric oxide from eNOS system or increasing the eNOS both in protein and mRNA level. Another noticeable research area would be the cardiac structure and function. Few experimental results were found whether ferulic acid modulates left ventricular dysfunction and hypertrophy of heart both in human and animal study. Metabolic syndrome and vascular dysfunction are also observed in many of the obese and diabetes patients. Ferulic acid has the potential to improve glucose disposal and metabolic syndrome . MoreoveAs discussed in this review, most of the beneficial effect of ferulic acid is due to its antioxidant and anti-inflammatory activities. However, the potential use of supplemental ferulic acid in the therapy of age-related human pathologies is still to be confirmed.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rituximab (RTX) is a chimeric B-cell-depleting monoclonal antibody against CD-20 positive cells that has been approved for the induction and maintenance of granulomatosis with polyangiitis (GPA). Reports have identified RTX to cause drug-related psoriasis. Many theories of underlying pathways have been proposed. However, further workup around the mechanism and treatment is required. Here, we present a 38-year-old woman known to have GPA that developed drug-related psoriasis that was successfully treated with treatment discontinuation and starting adalimumab along with a literature review. Drug-induced psoriasis is an increasingly recognized phenomenon, and different classes of therapies have been linked to drug-induced psoriasis such as antimalaria and beta-blockers . RecentlA 38-year-old female was diagnosed to have GPA manifested by recurrent epistaxis and one episode of pulmonary hemorrhage. Biopsy proved diffuse alveolar hemorrhage and capillaritis. She was treated with 1 gram of methylprednisolone for 3 days followed by oral prednisolone 60 mg along with 1 gram RTX infusion. No plasmapheresis was offered. She was doing well and maintaining remission on 10 mg of prednisolone and RTX courses. Three months after the third course of RTX (18 months from the first course), a scaly itchy rash erupted over the upper and lower extremities along with the abdomen. There was no joint pain or swelling. She denied the previous history of psoriatic rash, arthritis, uveitis, or chronic diarrhea. No family history of spondyloarthropathy or psoriasis was found. Examination revealed erythematous salivary scaly plaques over the abdomen  and exteA total of 13 reported cases in the literature described RTX-related new onset psoriasis or psoriatic arthritis in adults. The majority had underlying rheumatoid arthritis (RA) 8 patients) –10, two  patientsThe mechanism by which RTX can worsen or induce psoriasis is unclear, but it may be due to several reasons: It is postulated that B-cell depletion by RTX may have removed an unknown regulatory effect of B-cells on T-cells leading to T-cell activation . RTX mayIn conclusion, new onset of psoriasis related to certain drug exposure is not uncommon. However, the incidence after the exposure to RTX is considered a rare side effect. A hypothesis explaining these interactions is still obscure, and a subject of debate is whether withdrawal of the offending drug alone is sufficient in terms of the recurrence and outcome.


Nursing homes (NH) must implement best practices to improve care. The purpose of this study is to understand NH characteristics that help or hinder implementation of the Goals of Care (GOC) intervention, an evidence-based decision aid to guide decision-making in advanced dementia. Study design was a cross-sectional staff survey at 11 NHs in North Carolina that participated in the GOC trial. Questions measured the dependent variable of implementation effectiveness (IE) . NH organizational characteristics were measured using publicly available data and administrator surveys. Averages were obtained, IE and NH factors above the average were ranked as high and vice versa. Analysis consisted of pattern matching logic, predicted results are compared to actual results, using within-case and cross-case analyses. NHs with high IE were expected to have the majority (five or more) of NH characteristics to be high to confirm the within  and between  relationships. Within-case analysis expected results were met in 5 NHs, with 3 highs (IE high and five or more characteristics were high) and 2 lows (IE low and five or more characteristics were low). Among three NHs with high IE, the following characteristics were high: Medicare and SNF/ICF beds. Across 6 of the 11 NHs, high total beds and percent of White residents were related to high IE. NHs with enhanced resources may have fewer challenges implementing innovations compared to resource constrained NHs. Implementation strategies should account for resource limitations to promote successful implementation.


In recent years, mesenchymal stem cells have provoked much attentiveness in the field of regenerative medicine because of their differentiation potential and the capability to facilitate tissue repair via the emancipation of biologically active molecules. They have gained interest because of their distinctive curative properties. Mesenchymal stem cells are isolated from the Wharton’s jelly part of umbilical cord possessing higher proliferation capacity, immunomodulatory activity, plasticity, as well as self-renewal capacity than the mesenchymal stem cells from various origins, and it is considered to be the best resource for allogeneic transplantation. The isolated umbilical cord-derived mesenchymal stem cells are cultured in the Dulbecco’s Modified Eagle’s Medium, and thereby it begins to release soluble factors into the medium during the period of culture which is termed as conditioned medium. This conditioned media has both differentiation capacity and therapeutic functions. Thus, it can be able to differentiate the cells into different lineages and the paracrine effect of these cells helps in replacement of the damaged cells. This medium may accord to optimization of diagnostic and prognostic systems as well as the generation of novel and targeted therapeutic perspectives. Friedenstein et al. first published his report on explaining the expansion of adherent and spindle shaped cells derived from human bone marrow 2. These MSCs have the ability to differentiate into various lineages which include osteocytes (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes (fat cells) 3. The usual functioning role of mesenchymal stromal cells is maintaining the tissue homeostasis by differentiating towards the tissue specific cell types, proliferation and also by emancipating the growth factors and immunoregulatory agents 4. Some recent evidence stipulates that the MSCs assist in tissue regeneration through hindrance or inhibition of unwanted immune reactions and impart growth factors rather than directly restoring the damaged cells 5. Mesenchymal stem cells (MSCs) are considered to be multipotent stromal cells that are derived from the mesoderm with self-renewal capacity 6. These MSCs have immunosuppressive and anti-inflammatory effects, which might constitute an interesting source for curative applications. They also inhibit the maturation of dendritic cells, enhancement of anti-inflammatory functions and declination of the production of cytokines 2. Bone marrow is previously considered as the chief source of MSCs, but their number begins to decrease significantly with age and this has led to the discovery of numerous alternative sources. When compared to bone marrow-derived stem cells, the umbilical cord-mesenchymal stem cells (UC-MSCs) have less human leukocyte antigen-II (HLA-II) and major histocompatibility complex (MHC) class I molecules. These UC-MSCs have been proposed as a candidate for numerous clinical applications. The conditioned medium (CM) derived from MSCs was also found to have similar clinical applications as that of MSCs from the previous studies. So, it is imperative to understand the application and therapeutic properties of the CM.These multipotent adult stem cells have been isolated from different tissues, including bone marrow, adipose tissue, umbilical cord, cord blood, amniotic fluid, peripheral blood, placenta, fetal lung, etc Human umbilical cord derived-mesenchymal stem cells (UC-MSCs)The MSCs are obtained from various umbilical cord (UC) compartments or from the complete UC. This UC generally possesses about four compartments as a source of MSCs: 1. The whole UC is cut into minute pieces or fragments and thereby following the enzymatic digestion process or explant techniques. 2. The Wharton’s jelly (WJ) MSCs is acquired by removal of the UC vessels (arteries and vein). The umbilical cord arteries (UCA) and veins (UCV)  can also be minced into minute fragments and then plated for the growth of MSCs. 3. The sub-amnion part of the lining membrane is unfastened with a blade, and it is cut into minute pieces. 7. 4. The extracted vessels from the cord have to be tied at the two ends in a kind of loop. The loops will be presented in an enzymatic solution for a definite period of time to make the cells to dissociate from the perivascular part 2. Cell growth are analyzed by direct cell counts 2. Thus, these MSCs are generally isolated by enzyme digestion process as well as density gradient centrifugation. The isolated cells were plated in a complete culture medium with the supplements and maintained at 37°C under 5% COOther than the general advantages such as painless collection approach and rapid self-renewal ability, UC-MSCs has displayed the capacity to differentiate into three different germ layers to accumulate in the damaged tissue or inflamed regions and to provoke tissue repair. These MSCs which are isolated from Wharton’s jelly of UC have higher proliferation capacity, immunomodulatory activity, plasticity as well as self-renewal ability than MSCs derived from various origins and are considered to be the best resource for allogeneic transplantation. These UC-MSCs are characterized by high safety, abundance, less immunogenicity and shorter amplification times. Most of the research studies show that these circulating MSCs are attracted to the sites of damage or inflamed regions where it undergoes tissue-specific differentiation .Conditioned medium from UC-MSCsThe isolated UC-derived MSCs are cultured in the Dulbecco’s Modified Eagle’s Medium (DMEM) medium, and thereby it begins to emancipate certain soluble factors into the medium during the period of culture which is termed as CM .8. The growth factors and cytokines secreted from adipose tissue-derived MSCs have been elucidated in previous studies (Table 1) 9.These factors that are secreted thereby promote anti-oxidant, immunosuppressive and cell proliferative effects at the transplant regions. The CM has both differentiation capacity and therapeutic function. Thus, it can be able to differentiate the cells into different lineages and the paracrine effect of these cells helps in replacement of the damaged cells. This derived CM might intensify the positive effects of cell-based therapies. Since UC-CM contains increased levels of growth factors and chemokines, they normally accord to a chemoattractive environment of the adjacent tissues Clinical applications of conditioned medium10-12 -induced muscle atrophy in vitro and in vivo of conditioned serum-free medium (SFM) derived from UC-MSCs were assessed. This UC-SFM had a restorative impact on the human dermal fibroblast proliferation and decreased UV-A induced cell death. Furthermore, UCMSC-SFM chunked UV-A obstruction of superoxide dismutase activity. The topical application of UCMSC-SFM to the mouse skin prior to UV irradiation chunked the hampering of superoxide dismutase and glutathione peroxidase activities and lessened the up-regulation of malonaldehyde. Thus, UCMSC-SFM protects against photoaging instigated by UV-A and UV-B radiation and is found to be a much assuring candidate for the skin anti-photoaging treatments15. Taken together, all the previous existing studies reveal that the use of UC-CM might be suitable for regenerative medicine.Previous studies have also demonstrated the anti-photoaging effects following chronic ultraviolet (UV) irradiation in both  MSCs are population of stem cells with greater self-renewal and multipotentiality. MSCs are potential seed cells in regenerative medicine and have been used for the treatment of various diseases. Stem-cell therapy, especially UC-MSCs, is a promising alternative to treat on-going tissue damage by resetting the underlying disease process through alteration of the mucosal immune response. Moreover, they are proven to settle in the inflamed sites to repair injured tissues. Recent findings suggest that MSC-CM have similar properties like MSCs and favourable antitumor characteristics as well. The understanding of these mechanisms and CM may contribute to the optimization of diagnostic and prognostic systems as well as the generation of novel and targeted therapeutic perspectives.


Pinus pinaster and assessed whether such variation was associated with climate gradients. To that end, we characterized the resin duct system of 2-year-old saplings from 10 populations across the species’ distribution range. We measured axial resin duct features  and their inducibility in response to methyl jasmonate. Genotyping of single nucleotide polymorphisms allowed to account for the population genetic structure in our models in order to avoid spurious correlations between resin duct characteristics and climate. We found large inter-population variation in resin duct density and conductive area, but not in their inducibility. Our results suggest that population variation in the percentage conductive area of resin ducts likely arise from adaptation to local climate conditions. This study highlights the adaptive relevance of resin ducts and helps to shed light on the micro-evolutionary patterns of resin-based defenses in conifers.Resin ducts are important anatomical defensive traits related to biotic resistance in conifers. Previous studies have reported intraspecific genetic variation in resin duct characteristics. However, little is currently known about the micro-evolutionary patterns and adaptive value of these defensive structures. Here, we quantified inter-population genetic variation in resin duct features and their inducibility in  Several external stimuli have been reported to induce the formation of "traumatic" resin ducts, including insect attacks, pathogen infestation, or wounding alone (Endoconidiophora polonica (previously Ceratocystis polonica (Siemaszko) C. Moreau) or MJ application in Picea abies (L.) Karst trees enhanced traumatic resin duct production and biotic resistance to bark beetles and their associated pathogens in later infections  Carr. populations shaped genetic variation in resin duct characteristics. However, genetic variation includes not only adaptive variation resulting from diversifying selection in fitness-related traits, but also neutral variation due to processes such as genetic drift or gene flow  and their inducibility in Pinus pinaster Ait.) is a Mediterranean conifer species of high economic and ecological value in Southwest Europe and North Africa, inhabiting a wide range of heterogeneous environments, from wet-coastal to regions with prolonged summer drought  of the main stem at mid height was sampled and stored in FAA  and then transferred to 75% ethanol (v/v) as previously reported in ImageJ 1.51k image analysis software (2), the mean area of resin ducts (hereafter RD mean size – mm2), and the percentage of the total tissue area occupied by the lumen of resin ducts (hereafter RD conductive area – %)  coupled to a binocular stereoscope and analyzed using software . After csoftware Figure 2lmer function from lme4 package in R  and their response to MJ treatment (inducibility), we applied linear mixed models using the age in R . Populat2. The model of We obtained average climate data for the period 1950–2000 at the origin of populations from regional  and globLSmeans function from the homonym package in R . To that end, population least square means for each resin duct trait were obtained from the afore-mentioned model using the age in R .Q matrix) based on the genotyping of 126 SNPs from an independent set of clonal replicates (see more details in http://doi.org/10.5281/zenodo.1445313). The Q matrix assigns to each genotype a genetic membership coefficient for each of the five genetic clusters . However, RD mean size was a 10-fold higher in the phloem than in the xylem . Consequently, RD conductive area was higher in the phloem than in the xylem   and in the phloem . Moreover, RD density in the phloem was negatively correlated with RD mean size in the xylem  and vice versa, RD density in the xylem was negatively correlated with RD mean size in the phloem  (see RD density was negatively correlated to RD mean size both in the xylem (130) see Table S32 between populations. TAMR was the population with the highest RD density in the xylem whereas PLEU showed the lowest value for this trait . These results also agree with previous findings. In particular, Picea sp. Variation among Pinus sylvestris L. and P. nigra J.F. Arnold populations in resin duct characteristics has also been reported  . Abioticreported , althougP. pinaster populations suggests important implications in potential biotic resistance in this species. It has been suggested that resource allocation to resin ducts provides enhanced resistance to different antagonists. For instance, in North American pine species, trees that survived or avoided bark beetle attacks had more or larger resin ducts than trees that died (Picea sitchensis provenances previously characterized as resistant to the white pine weevil (Pissodes strobi) exhibited a denser network of resin ducts , which were analyzed in previous work  Voss introgression zone were found to be positively correlated with aridity and continentality at the origin of the populations . Phenotypic differences in resin duct characteristics among in ducts . Likewisulations .P. pinaster, like other European tree species, experienced severe bottlenecks during the ice ages . That seems to be the case for most plant secondary metabolites in P. pinaster, whose genetic differentiation among populations (STQ) was not different from that expected by genetic drift alone (STF)  .When making inferences in comparative ecology, not accounting for the genetic relatedness can lead to the misinterpretation of potential clinal patterns due to the non-independence of the populations . After aThe opposite situation would be those resin duct characteristics that were significantly correlated with climate even after accounting for the demographic history of the species. Such is the case of RD conductive area. In the xylem, this trait remained significantly and negatively correlated with the PC1 of Atlanticity after accounting for the neutral variation, suggesting that climate heterogeneity is contributing to inter-population differentiation of resin ducts in this tissue. In addition, a positive significant correlation between RD conductive area in the phloem and the PC2 emerged after accounting for the neutral variation. In both cases, adaptation to local climate conditions, alongside the demographic history of the species, seems to help explain the differentiation of populations in these traits. Current hypothesis on growth-defense trade-offs state that resource allocation to defenses is promoted in limiting environments to the detriment of growth capacity . Our resversus quantitative variation  in resin duct characteristics to broadly understand the evolutionary origin of this anatomical trait. Moreover, assessment of adaptive genetic variation in resin ducts should also be tested in mature trees, as previous studies showed that ontogeny plays an important role modulating resin-based defenses in conifers  which is likely associated to climate gradients .The SNP dataset analyzed in this study is publicly available in the Zenodo repository  grants FUTURPIN (AGL2015-68274-C03-02-R), AdapCon (CGL2011-30182-C02-01/02) and RESILPINE (RTI2018-094691-B-C33), and by the Xunta de Galicia-GAIN grant (IN607A2016/013). CVG received financial support from the MINECO FPI Grant program (MINECO-Spain BES-2016-076624). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).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.