N = 18) or reproduction (N = 30) over time in late life. We found no evidence that developmental environment affected rates of survival senescence (βmean = –1.2 × 10−4 ± 0.022SE). However, a better developmental environment was associated with slower rates of reproductive senescence in late life (βmean = 0.062 ± 0.023SE), indicating a small, but significant, “silver‐spoon” effect of early‐life conditions that persisted through to late life. Our results illustrate how the effects of environmental conditions during development can persist throughout life, and indicate one possible cause of phenotypic plasticity in senescence.What determines variation between individuals in how they senesce, and are environmental conditions experienced during development relevant to late‐life performance? We report a meta‐analysis of studies of wild populations to determine how the quality of the environment experienced during development affects rates of survival and reproductive senescence. From studies of 14 bird or mammal species, we calculated effect sizes for the interaction between the effects of environmental quality during development and age in predicting survival ( Can the quality of the environment experienced during the first stage of life, the developmental period, have persistent effects that last into the final stage of life, affecting rates of senescence? Senescence, or the decline in reproduction or survival in old age, is common in both wild and domestic populations, but may also vary substantially between individuals in a population for different environmental and genetic reasons. Several recent analyses of data from detailed long‐term studies of wild animal populations have now tested the effect of developmental environment on senescence, across a range of species. Here, we used a meta‐analysis of these studies to summarize the effect of developmental environmental quality on senescence rates in the wild. Across 14 species of birds and mammals, there was no evidence that developmental environment influenced survival senescence; however, there was a significant influence on reproductive senescence. Being exposed to a higher quality environment during the developmental period provided a “silver‐spoon,” whereby individuals experienced slower declines in reproductive ability at the very last stage of their lives. Our results illustrate how the very first phase of life can shape the very last phase of life.Long‐standing evolutionary questions surround the ultimate causes and consequences of senescence, defined as the decline in fitness‐related traits with age Medawar . One aspThe extrinsic environment may play a key role in shaping rates of senescence in both reproduction and survival  survival senescence, and (ii) reproductive senescence. We then investigated the influence of study characteristics and biological factors in moderating the observed effects.Web of Science to identify relevant articles by conducting a key word search with the terms: (senescence OR ageing OR aging) AND . We then conducted forward and backward searches on six classic senescence articles as well as five recently relevant and influential review articles (see Appendix S1 for details). This resulted in a pool of 6766 unique articles. From this pool, we extracted all articles that met the criteria that the study should: (i) use individual‐level data from a population of wild animals to investigate the effect of the environment experienced during development on senescence rates in either survival probability and/or reproductive output (ii) include one or more linear model(s) testing the effect of an interaction between age  and a measure of developmental environmental quality, on senescence rates (measured in the same units of time as “age”); (iii) use a metric of environmental quality that was extrinsic to the individual, but that could theoretically have a causal influence on the fitness of the individual. Examples of suitable metrics of environmental quality included weather variables, conspecific population density, food availability, or predation risk. As we were interested in the influence of environmental conditions experienced during development, we only included articles that quantified the metric of environmental conditions during the period of gestation or the period of juvenility .We performed a broad search of the scientific literature in order to identify published tests of the effect of developmental environment on rates of senescence. For a detailed account of the parameters and methods involved in our literature search, see Appendix S1. In brief, we first used From the published material, we identified 10 articles that measured the effect of developmental environment on senescence rates as per our inclusion criteria. Several additional articles from our initial search pool indicated the availability of the data necessary to complete the required analysis, but did not present results in such a way that they met our criteria. In these cases, we contacted the authors to enquire about the relevant analyses for their study system. From this, we obtained additional results for four species that were previously unpublished.In total, we extracted 48 effect sizes from 14 independent studies of six bird and eight mammal species , whereas across effect sizes, weather conditions were the most frequently used . Direct measures of food availability accounted for the third most common metric (six effect sizes from four studies). All the other metrics used to measure environmental quality, including acute pollution exposure, birth date, cohort birth‐rate, social group size, anthropogenic habitat exposure, maternal age, sibling number, and predation risk, were each used in only one or two studies, generating one or two effect sizes each.We followed the predictions of the authors in assigning the directionality of each measure of environment to represent either “better” or “worse” environmental quality. Increasing numerical values of some environmental measures were inferred to represent increasing environmental quality , whereas others implied decreasing environmental quality . To standardize effect sizes, we corrected the direction of each effect size so that the increasing numerical value of the metric of environmental quality reflected increasing environmental quality.type of environmental measure, class, time of environmental measure, and sex. Due to the large variety of measures of environmental quality, we did not have appropriate statistical power to determine the independent influence of all 11 measures. Instead, we categorized type of environmental measure into three groups: measures of population density (density), measures related to weather variables (weather), and all other measures . Class was categorized as either mammal (32 effect sizes from eight species), or bird (16 effect sizes from six species). We categorized time of environmental measure, as gestation if the measure of the environment was taken before birth (eight effect sizes from three species), or juvenility if the measure of the environment was taken within or at the first year of life (40 effect sizes from 12 species). Sex had three categories . We later reduced analysis of sex to only two categories .We further categorized effect sizes by several study‐design and biological factors: r) using the relevant inference (test) statistic . Then, to avoid a skewed distribution near r = ±1, we transformed correlation coefficients with Fisher's z‐transformation  as standardized effect sizes in all meta‐analyses (see below). We calculated the variance for each effect as 1/(N−3), where N is the number of individuals in the analysis  of developmental environment on both reproductive senescence rates and survival senescence rates. We used a multilevel structure to control for potential nonindependence of effect sizes. Studies contributed multiple effect sizes to either models of reproductive or survival senescence if they contained analyses of both males and females, modeled more than one measure of environmental quality, or modeled environmental quality over each of the time periods (gestation and juvenility). Within studies, a single model also contributed multiple effect sizes if different measures of environmental quality were analyzed within the same model. To control for potential nonindependence within studies and within models, we ran three‐level meta‐analyses with a random effect of model, nested within a random effect of study  was investigated independently as the single categorical moderator term in a meta‐regression. For the meta‐regression with moderator sex, we removed effect sizes that considered both sexes due to small sample size . For each meta‐regression, we determined if the mean effects of categories were different from each other using a Wald‐type chi‐square test (mQ). We also determined the mean effect for each category (β) and tested if the effect within each category was different than 0 using a z‐test.After determining the global mean effect on rates of survival senescence and reproductive senescence, we used meta‐regressions to investigate if the mean effect sizes varied dependent on each biological or study‐design factor (see “Data synthesis”). Due to the relatively small sample of effect sizes, each categorizing factor  among effect sizes , and the third attributable to residual variation. The sum of the percentages of total variation due to these three sources equals the traditional 2I of Higgins et al. . Since our dataset included both published and unpublished effect size estimates, we also conducted bias detection tests on the subset of effect sizes from published materials to differentiate apparent publication bias from other causes of bias.Biases within a dataset used for meta‐analyses can result from a multitude of factors, including unequal research attention across taxonomy, publication biases toward significant results or predicted outcomes, or limitations in the comprehensiveness of literature searches methods , time , and social structure  were also sometimes, but not always, controlled for in the primary source models as random effects (Appendix S2). Since the importance of these various factors as sources of nonindependence will depend on the specifics of each individual study system, we did not make any assumptions about whether or not the inclusion of these random effects was necessary to each individual study. It is important to note however, that if sources of nonindependence were not adequately accounted for in the models from which effect sizes were extracted, then this could influence our results by falsely inflating N and reducing the variance as it was calculated.As an additional potential shortcoming of using partial effect sizes to derive estimates of metafor package  = –0.043 to 0.043, Table mean = 0.062, 95% CI = 0.016 to 0.107, Table We found no effect of developmental environment on survival senescence [β 2I, was low for reproductive senescence  and negligible for survival senescence .Heterogeneity between effect sizes not accounted for by sampling error, t21 = 2.19, P = 0.04, Table S1, Fig. S2), but not in models of the complete dataset, in which there was no significant asymmetry in funnel plots for survival senescence , or reproductive senescence . There was no effect of adding the year of publication of the study (or time of acquirement for unpublished data) as a moderator variable in meta‐regressions in either the model of survival  or reproductive senescence , indicating no discernable shift in results with time.The results of the two meta‐analyses were robust to Egger's regression bias detection tests. We detected a significant bias toward positive results from the subset of effect sizes extracted from published material , or survival senescence rates .In the meta‐regressions of both the effect on survival and reproductive senescence rates, there were no significant differences in mean effect between categories (P(mQ) values, Tables The number of covariates in the model from which each effect size was derived had no effect on ZOur meta‐analysis of eight mammal and six bird species indicated that variation in early environmental conditions affected rates of reproductive senescence, but found no evidence of effects of early environment on survival senescence.The significant mean effect of developmental environment on reproductive senescence indicates that the environmental conditions during development have far‐reaching effects all the way into the last stage of life. The positive direction of this effect implies that good developmental conditions provide a silver‐spoon, whereby the rates of decline of reproductive output in late life are slower when the quality of the environment experienced during development is better.Although the effect of developmental environment on reproductive senescence was significant, the effect size was relatively small, as per the conventional benchmarks proposed by Cohen . This isIn an evolutionary context, we assume that senescence is partially genetically determined  current environmental conditions were controlled for in the regression models from which effect size was extracted. For Mauritius kestrels, the environmental measure used had no association with fitness when measured in adults I = 37%) indicates that there may be some differences in the true effect of developmental environment on reproductive senescence rates based on biological or study design factors. Although none of the meta‐regressions found significant differences between the categories within moderator terms , there were some suggestive trends. The reproductive senescence of birds, but not mammals, was significantly affected by developmental environment. Additionally, environmental measures of weather had roughly twice the magnitude of effect in predicting reproductive senescence rates as compared to environmental measures of population density. Although there were apparent differences between categories in sex  and time of environmental measure , in these two cases the differences were likely driven by the relatively small sample sizes available for the nonsignificant categories.The low level of heterogeneity amongst effect sizes I < 0.1%). However, the 2I statistic is known to have low power when sample sizes are small  on the magnitude of effect observed in reproductive senescence rates. A greater number of studies would allow for a more robust test of the biological significance of these trends.Several questions remain regarding the influence of developmental environment on senescence rates. Most notably, we found no relevant studies of amphibians, reptiles, fishes, or any invertebrates, which is presumably reflective of the relative lack of long‐term individual‐based studies of these species in the wild (Clutton‐Brock and Sheldon (i) To differentiate the influence of environmental quality during development and during other periods of life‐history, the measure of environmental quality should be contained to only some predefined period, ending before sexual maturity. (ii) The effect should be analyzed using a model predicting either survival probability or reproductive output within a discreet time sequence . As a predictor term, age should interact with the specific measure of developmental quality to specifically determine an effect on senescence rates. (iii) These models must only include individuals above the age of the onset of senescence , otherwise, any perceivable effect may be reliant on altering survival or reproductive rates in another period of life. (iv) Models with repeat measures of individuals should include individual ID as a random effect. An effort to control for other expected codependences through time and space should be made where ever possible . (v) Metrics of environmental quality should be chosen with care, and ideally, a metric should be demonstrated to scale linearly with fitness before being used.Through our literature search, we identified several studies with data that would have been adequate to investigate this question, but the specific analyses had not been done. In the hope of encouraging a more extensive synthesis in the future, we have formulated some suggestions for specific analytical methods to investigate the influence of developmental environment on senescence in wild populations. We recommend that: We hope that publication of additional studies, and in particular, across other taxa, will further clarify our understanding of the long‐term effects of developmental environment on late‐life performance.Associate Editor: A. CharmantierTable S1. Eggers regression models for the subset of effect estimates from the survival senescence model, the reproductive senescence model, and all published effect sizes  combined.Click here for additional data file.   Click here for additional data file.   Click here for additional data file.   Click here for additional data file.   Click here for additional data file.


Microglia are resident immune cells that play multiple roles in central nervous system (CNS) development and disease. Although the classical concept of microglia/macrophage activation is based on a biphasic beneficial‐versus‐deleterious polarization, growing evidence now suggests a much more heterogenous profile of microglial activation that underlie their complex roles in the CNS. To date, the majority of data are focused on microglia in gray matter. However, demyelination is a prominent pathologic finding in a wide range of diseases including multiple sclerosis, Alzheimer's disease, and vascular cognitive impairment and dementia. In this mini‐review, we discuss newly discovered functional subsets of microglia that contribute to white matter response in CNS disease onset and progression. Microglia show different molecular patterns and morphologies depending on disease type and brain region, especially in white matter. Moreover, in later stages of disease, microglia demonstrate unconventional immuno‐regulatory activities such as increased phagocytosis of myelin debris and secretion of trophic factors that stimulate oligodendrocyte lineage cells to facilitate remyelination and disease resolution. Further investigations of these multiple microglia subsets may lead to novel therapeutic approaches to treat white matter pathology in CNS injury and disease. Filling in nearly half of the human brain,Clinical observations of these white matter–related diseases have commonly reported activation of microglia. PET studies in MS and postmortem brain section studies from AD patients showed that the increased population of activated microglial cells correlates with their clinical disability.Microglia are resident immune cells that constitute up to 10 ~ 15% of the cells in CNS. These cells contribute to brain homeostasis by surveying their surrounding microenvironment for indicators of injury or infection known as damage‐associated molecular patterns (DAMPs) or pathogen‐associated molecular patterns (PAMPs).In this mini‐review, we survey the diverse microglial changes in white matter during CNS injury and disease, with an emphasis on understanding how these responses affect disease progression and resolution, and the development of potential therapeutic opportunities.2As previously mentioned, microglia are found to be “activated” in various white matter–related diseases in a classical way. As competent presenters of antigen, highly activated microglia express molecules for antigen presentation such as major histocompatibility complex II (MHC II) and its costimulatory factors CD40 and CD86 , classical markers of microglia activation, in MS and AD patients.Similar phenomena were observed in ischemic dementia models as well. Inducing white matter lesions in rats by clipping the bilateral carotid arteries to mimic ischemic dementia revealed that microglia and macrophages in white matter showed elevated expression of MHC‐I/II or matrix metalloprotease‐2 (MMP‐2) at 3 days hypoperfusion, indicating their early activation after the onset of cerebral hypoperfusion.apoE (apolipoprotein E) and yet showed unique expressions as well such as Ccl4 or Cxcl10 depending on the clusters, indicating that microglia can have multiple forms of activation where both generalized and selective transcriptional programs are delicately orchestrated. These findings were able to be translated to human disease as at least one of the subsets shared the similar gene expression profile to the previously discovered microglia group in human MS lesions,Interestingly, recent studies have revealed that microglia activation is not a singular phenotypical change but rather a dynamic response that involves spatially and transcriptionally distinct subpopulations. Mrdjen et al performed high‐dimensional single‐cell mapping in EAE mouse CNS and discovered a unique activation profile of microglia.Evidence of different activation status of microglia was also observed in ischemic dementia as well depending on the region within the white matter. Simpson et al investigated the microglia of white matter lesions of aged human brains and discovered that periventricular lesions contained significantly more activated microglia as they expressed MHC II, CD40, and B7‐2 than either control white matter or deep subcortical lesions.Moreover, recent studies have revealed that microglial subtype can be altered depending on the disease pathology. Locatelli et al utilized a real‐time in vivo imaging in EAE mice and observed the evolution of microglia and mononuclear macrophages in a spatiotemporal manner.Collectively, these findings suggest that multiple subpopulations of activated microglia exist depending on the disease type or spatiotemporal status of the disease and they contribute to progression and resolution of various white matter–related diseases.3After white matter damage, clearance of myelin debris from demyelination is critical for oligodendrocyte precursor cell (OPC) recruitment and their maturation into oligodendrocytes.Similar results were observed in animal models as well. In TgAPP21 transgenic rats, an AD model, microglial activation and accumulation was significantly stronger in white matter tract including supraventricular corpus callosum (SVCC) than gray matter.To investigate how white and gray matter may contribute to this difference, Poel et al performed a transcriptional profiling of microglia from white and gray matter using postmortem tissues of both control and MS donors.vivo by inducing conditional depletion of OPCs in adult mouse brain, suggesting that OPC has a crucial influence onto cellular states of microglia which are relevant to neurodegenerative diseases. Although a small population of OPCs exists in the gray matter as well,The differential profile of microglia may be due to its surrounding environment. Huizinga et al conditioned mouse macrophage cell line J774.2 by incubating with either gray or white matter from humans and measured their phagocytic activity and found that the number of cells containing myelin or neuronal antigens was significantly higher in cells incubated with white matter.Overall, the above findings demonstrate that activities of microglia in white matter could be differentially regulated from gray matter, hence, may result in different outcome in terms of remyelination and disease resolution Table .42) production by microglia and death of oligodendrocytes was observed in rat cells, suggesting NO2 ‐induced damage as the cytotoxic mechanism for oligodendrocytes.Aside from its phagocytic activities, multiple studies have shown that activated microglia secrete various factors that directly affect the fate of OPCs and oligodendrocytes, key component cells of white matter homeostasis and repair. Traditionally, activated microglia were considered to secrete pro‐inflammatory factors involved in the initiation and propagation of inflammatory cascade thus promoting demyelination in neural disorders. LPS‐activated microglia, polarized to pro‐inflammatory status, secreted tumor necrosis factor alpha (TNFα) and interleukin‐1beta (IL‐1β), both known to be cytotoxic to oligodendrocytes.Cxcl10, Tgfb1, Pdgfa, and Pdgfb were found to be expressed which are known to recruit OPCs to the lesion site and promote their differentiation, thus possibly facilitating the remyelination process.On the other hand, recent studies have revealed protective roles of microglia during the remyelination phase. After myelin damage in MS model, a genome‐wide gene expression analysis was performed for the microglia in corpus callosum during remyelination. A repertoire of cytokines and chemokines such as Msx3), levels of activin A, and IGF1 mRNAs were increased and facilitated remyelination in mice MS models. Moreover, Wang et al discovered that treatment of histone deacetylase (HDAC) inhibitor scriptaid also triggered the beneficial switch as it resulted in less production of TNFα and NO by microglia and macrophages with increased preservation of co‐cultured oligodendrocytes in vitro and prevented white matter damage in vivo.This regulation of secretion factors could be influenced by different activation states of microglia. Miron et al discovered that the microglial and macrophage switch from pro‐inflammatory to immuno‐regulatory occurred over time after inducing focal demyelination by LPC in the mouse corpus callosum. These regulatory populations secreted higher levels of IGF1 and activin A than those of pro‐inflammatory phenotypes and promoted oligodendrocyte differentiation.These findings above demonstrate the untraditional subset of microglia that secretes trophic factors for OPC generation, migration and maturation, revealing the complex nature of microglia population. Altogether, depending on the pathological stage of white matter damage, different subsets of microglia could affect the fate of OPCs and oligodendrocytes by secreting factors of various effects Figure .5Currently, no therapies designed to improve remyelination are approved, especially none targeted directly to microglia.In mice induced with ischemic stroke model, hypothermia decreased the inflammatory phenotype and increased the immuno‐regulatory type of microglia along with decrease in their total number compared to the control group, which promoted the long‐term integrity of the white matter.Ccl4 or Cxcl10 in MS‐related microglia,For the microglial phenotype switching strategy to be developed successfully, fully understanding the difference between various microglial subtypes will be beneficial. For instance, if unique biomarkers of specific subset of microglia in certain disease could be validated, specifically ameliorating this subset and maintaining other beneficial population could facilitate the resolution of the disease. As recent single‐cell sequencing studies have already discovered genes that can potentially be used as biomarkers such as Another potential strategy is the targeted drug delivery to specific brain regions. As activated microglial subpopulation is also diverse depending on their location within the brain, bioengineering of lesion‐specific drug delivery platforms should be considered as well. As several studies have reported that white matter residing microglia tend to have different activation status compared to those of the gray matter, specific delivery of microglia‐targeted drug to white matter could prove to be beneficial to the disease resolution. Moreover, as studies have revealed not only inter‐regional but also intra‐regional differences exist between the microglial subsets, uncovering the region‐specific markers and mechanisms and region‐specific drug delivery could prove to be a novel and efficient strategy. Of course, there is always a risk that phenomena observed in experimental models can often be different from actual human disease pathology. Therefore, the thorough identification of microglia subtypes in cell or animal experimental models must be carefully investigated with clinical samples first to confirm whether they could actually be translated in human disease in order to develop these strategies.6Microglia were traditionally considered as deleterious cells in CNS disease. In white matter, excessive microglial activation damages neuronal axons and myelin sheaths. However, an emerging literature now suggests a more nuanced scenario with both damaging and beneficial microglia phenotypes. As discussed in this mini‐review, recent advances in molecular technologies now allow one to detect multiple subsets of microglia within the broadly defined “activated” states. After white matter damage, microglia are activated in multiple forms depending on various conditions such as their proximity to the lesion, location within the brain, and the type of disease. During the acute stage, deleterious microglia promote an inflammatory microenvironment involving antigen presentation and secretion of pro‐inflammatory molecules that trigger demyelination and neuronal damage. During the later recovery period, however, excessive microglial activation is toned down and their phagocytic activity contributes to clearance of myelin debris and dead cells. Immuno‐regulatory microglia also secrete factors that promote OPC migration and their maturation into oligodendrocytes, thus facilitating the remyelination process. Although accumulating evidence of these multiple damaging vs beneficial roles has been reported,The authors declare no conflict of interest.


MECP2.The clinical spectrum of Rett syndrome  in males is considered to be wider than previously expected. Therefore, the existence of RTT with a normal male karyotype is still controversial. Here, we report the first case of a male patient presenting with an early seizure type of Rett‐like phenotypes with a missense variant of An 8‐month‐old male was admitted to the pediatric department due to an initial seizure event following aspiration pneumonia and was referred to our clinic for the evaluation of unexplained neuroregression. Genomic DNA was prepared from venous blood by standard procedures and was processed at the Yale Center for Genome Analysis (YCGA) for whole exome sequencing (WES). Processing of sequence data, variant calling, and the identification of de novo mutations were then performed. Direct Sanger sequencing was performed following PCR amplification.In this patient with a normal karyotype, WES analysis led to the identification of a novel, de novo missense variant of MECP2 (p.Arg133His) that is not observed in the normal population.MECP2 missense mutation could guide future treatment and follow‐up plans for RETT‐like phenotypes.This rare case of an p.Arg133His hemizygous  He had one brother (12‐year‐old) and sister (8‐year‐old). None of the family members had any medical history during the growth period.He was admitted to the pediatric department due to an initial seizure event following aspiration pneumonia and was referred to our clinic for the evaluation of unexplained neuroregression. Although he was hypotonic from birth, he achieved a social smile at 3 months and started head control during the first 4 months. He rolled over, and nearly grasped his toys with prone position at 6 months. Generalized tonic–clonic type seizures at 6 months were his first clinical symptom, a detailed history revealed delays in developmental milestones after that. Electroencephalogram (EEG) findings showed abnormal awake and sleep recordings due to slow background activity, suggestive of diffuse cerebral dysfunction with symptomatic or cryptogenic seizures. Magnetic resonance imaging showed cerebral hypoplasia especially in the frontal and temporal lobes at approximately 4 years of age. He was observed at the outpatient clinic for developmental delays associated with encephalopathy and seizure events, which occurred hundreds of times for 2 years and were fairly well‐controlled with valproic acid, phenobarbital, and clonazepam.At 26 months after surgery for bilateral cryptorchidism, progressive respiratory difficulty persisted and weaning from the ventilator was not possible; repetitive aspiration pneumonia occurred as he was unable to proceed with sputum expectoration. Therefore, tracheostomy was performed and night‐time breathing using a ventilator was maintained subsequently. At the time of admission, repetitive hand flipping without purpose and lip smacking was observed during examination, although epileptiform discharges were not observed during EEG, we decided to proceed with additional evaluation other than that previously considered at this point. The various clinical features of the patient are described in Table There were no abnormal findings based on laboratory investigation, and genetic analysis of mutations including Prader‐Willi gene, spinal muscular atrophy gene, and other chromosomal aberrations. Chromosome analysis revealed a 46, XY karyotype. A muscle biopsy also demonstrated no abnormal findings.3This study was reviewed by the appropriate ethics committee. Blood samples from the patient and parents were obtained using standard protocols after receiving informed written consent. Genomic DNA was prepared from venous blood using standard procedures and was processed at Yale Center for Genome Analysis (YCGA) for whole exome sequencing (WES) as described previously . Abrogation of MECP2 might affect all cells of the male patient and it could lead to the presentation of RTT‐like phenotypes including early‐onset seizure and breathing problems. Unlike typical RTT with commonly known clinical symptoms in girls, young males with severe progressive developmental difficulty should be considered as a variant form of RTT as part of the differential diagnosis. In early‐onset seizure variant of RTT, distinct phenotypes including early seizure variant with early seizure onset prior to regression may present like our patient , which was not observed in the normal population. Interestingly, several studies have reported other amino acid changes in MECP2  at the same residue, but few male cases with same mutation  have been reported in RettBASS (http://mecp2.chw.edu.au). In our case, which was associated with an R133H variant, gene expression could be affected as the ring‐shaped histidine might interfere with the protein function, resulting in less affinity, as compared to that with the Y‐shaped arginine during the RNA transcription process  and transcriptional repression domains (TRDs), which deacetylase histone in DNA to inactivate the chromosome. Similar to that concluded previously by Hoffbuhr et al. , our pat6The existence of RTT‐like symptoms with a normal male karyotype is still controversial; thus, it is important to report information regarding the rare genotypes of male RTT patients in publications. This rare case of an R133H missense mutation in a patient who was hemizygous for MECP2 could help to guide future treatment and follow‐up plans for this condition.None.


The prognosis of hepatocellular carcinoma (HCC) is poor because of high incidence of recurrence and metastasis. JAK/STAT signalling pathway regulates cell proliferation, apoptosis, differentiation and migration and epithelial‐mesenchymal transition (EMT) is also considered to contribute to invasion and metastasis of epithelial malignant tumours. Scutellarin is an active component found in many traditional Chinese herbs and has been regularly used in anti‐inflammatory and antitumour medicine. This study aimed to identify the effect of scutellarin and its possible mechanism of action in HCC cells. Proliferation, colony‐forming, apoptosis and cell migration assays were used to examine the effect of scutellarin on HCC cells. Quantitative real‐time PCR and Western blotting were performed to study the molecular mechanisms of action of scutellarin. Light and electron microscopy and immunofluorescence analysis were performed to study the effect of scutellarin on cellular mechanics. We show that scutellarin potentially suppresses invasiveness of HepG2 and MHCC97‐H cells in vitro by remodelling their cytoskeleton. The molecular mechanism behind it might be the inhibition of the EMT process, which could be attributed to the down‐regulation of the JAK2/STAT3 pathway. These findings may provide new clinical ideas for the treatment of liver cancer. In recent years, many studies have shown that scutellarin shows antitumour effects in colon, breast and tongue cancers and acts by different mechanisms including promoting apoptosis and inhibiting invasion.22 and 95% air at 37°C. Scutellarin was purchased from Melone Pharmaceutical Co., Ltd. Scutellarin was dissolved in DMSO for all experiments. All other reagents, if not specifically noted, were purchased from Sigma.Human HCC cell lines were purchased from Shanghai Cell Biological Institute of the Chinese Academy of Science and cultured in DMEM supplemented with 10% foetal calf serum and 1% penicillin–streptomycin in a humidified atmosphere of 5% COMTT assay was conducted to evaluate the effect of scutellarin on proliferation of HCC cells. Colony‐forming assay was conducted to detect the colony‐forming ability of HCC cells after treated with scutellarin. Apoptosis assay was conducted to evaluate the effect of scutellarin on apoptosis of HCC cells. Cell migration assay was performed to evaluate the effect of scutellarin on migration of HCC cells. Quantitative real‐time PCR and Western blot analysis were performed to detect mRNA expression and protein expression of JAK2, STAT3 and EMT‐related genes in HCC cells. Light and electron microscopy and immunofluorescence were applied to visualize the cytoskeleton in HCC cells.t test or an analysis of variance. All statistical analyses were performed with SPSS version 17.0 and all figures were generated using GraphPad Prism 5.01. The significance level was set at P < 0.05.The data are presented as mean ± SD. Intra‐group comparisons were made by employing Student's 3As shown in Figure JAK2 and STAT3 did not change significantly with increase in scutellarin concentration. However, we observed an increase in the mRNA expression of E‐cadherin, which is representative of epithelial cell lineage, while the transcript levels of Snail and Vimentin genes expressed in the mesenchymal cells decreased (Figure As shown in Figure Cytoskeletal elements play important roles in cell mobility and invasiveness.4JAK2 is a member of the JAK family of protein tyrosine kinases, which performs diverse functional roles in carcinogenesis.Cancer cells break down cell‐cell contacts and remodel cell‐matrix adhesion sites to detach from the primary site and to invade into the surrounding tissue. These processes are also observed in various non‐pathological conditions, such as EMT which is observed in developmental stages. Our results demonstrate that scutellarin can inhibit the invasive potential of HCC cells and the mechanism for the same involves the suppression of JAK2/STAT3 pathway signalling and also EMT in HCC cells.The cytoskeleton has been shown to modulate the movement of tumour cells. Yilmaz and Christofori showed that actin cytoskeleton remodelling corresponds to EMT and thereby regulates cell motility.Scutellaria barbata extracts before, however, the extract is a heterogeneous mixture, which makes its further application difficult. Our study has a unique advantage, since we studied the antitumour effect of scutellarin, the main ingredient of S barbata. Thus, our findings confer reliability to the existing research. Moreover, we explored the cellular mechanics by visualization of the cytoskeleton structure and F‐actin filaments in HepG2 and MHCC97‐H cells upon scutellarin treatment for the first time.Many studies have shown the antitumour effects of In conclusion, we observed that scutellarin can potentially inhibit the invasive potential of HepG2 and MHCC97‐H cells in vitro, by possibly remodelling the cytoskeleton. The molecular mechanism behind it might be an inhibition of EMT, which could be attributed to the down‐regulation of JAK2/STAT3 pathway signalling. These findings may provide new ideas for the treatment of liver cancer in clinical settings.The authors declare no conflict of interest.


Activation of antiviral innate immune responses depends on the recognition of viral components or viral effectors by host receptors. This virus recognition system can activate two layers of host defence, pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI). While ETI has long been recognized as an efficient plant defence against viruses, the concept of antiviral PTI has only recently been integrated into virus–host interaction models, such as the RNA silencing‐based defences that are triggered by viral dsRNA PAMPs produced during infection. Emerging evidence in the literature has included the classical PTI in the antiviral innate immune arsenal of plant cells. Therefore, our understanding of PAMPs has expanded to include not only classical PAMPS, such as bacterial flagellin or fungal chitin, but also virus‐derived nucleic acids that may also activate PAMP recognition receptors like the well‐documented phenomenon observed for mammalian viruses. In this review, we discuss the notion that plant viruses can activate classical PTI, leading to both unique antiviral responses and conserved antipathogen responses. We also present evidence that virus‐derived nucleic acid PAMPs may elicit the NUCLEAR SHUTTLE PROTEIN‐INTERACTING KINASE 1 (NIK1)‐mediated antiviral signalling pathway that transduces an antiviral signal to suppress global host translation. To cope with invading pathogens, plant cells evolved a sophisticated immune system under constant pressure for dominance over the pathogens’ virulence strategies, which in turn coevolved to escape the host recognition system  and Oilseed rape mosaic virus  and the potexvirus Potato virus X  depend on ligand (PAMP/DAMP)‐induced dimerization or oligomerization with coreceptors for activation  from the bipartite begomoviruses, et al., in vitro at conserved threonine residues, positions Thr‐468, Thr‐469 and Thr‐474 within the activation loop, and mediates substrate phosphorylation in vivo /Arabidopsis and Tomato yellow spot virus (ToYCV)/Tomato] indicates that virus infection both activates and suppresses NIK1‐mediated antiviral signalling  plants at the seven‐leaf stage were inoculated by biolistic delivery with infectious clones of CaLCuV DNA‐A, and DNA‐B and the accumulation of viral DNA was monitored by PCR  undergoes dsRNA ligand‐mediated dimerization and activation upon direct association of viral dsRNA produced during infection with its kinase domain  of plant viruses use the endocytic recycling pathway to move viral RNA– and viral DNA–protein complexes to the plasmodesmata for the MP‐assisted cell‐to‐cell spread of the virus (Lewis and Lazarowitz, Although this endocytic recycling pathway is an attractive hypothesis for virus perception by plant PRRs, similar to mammalian endosomal TLRs, it may not be possible to synchronize PTI activation as a primary, early response to pathogen attack, with the later event of virus movement to adjacent cells. Therefore, only with the identification of the origin and nature of virus‐derived PAMPs and the characterization of nucleic acid‐sensing PRRs will it be possible to address these possibilities for virus perception by plasma membrane‐anchored RLKs.Recent studies with SERK‐like coreceptors and known PRRs, which function in plant innate immunity, have demonstrated that the classical PTI is part of the antiviral defence arsenal of plant cells. Several components of classic PTI, including the co‐receptor SERKs, BAK1 and SERK1, and the MAPK4 negative regulator, in addition to common immune responses, co‐exist as part of antiviral PTI. As for viral PAMPs, dsRNAs isolated from virus‐infected plants have been demonstrated to induce typical PTI responses dependent on the coreceptor SERK1. Likewise, begomovirus‐derived nucleic acids were shown here to induce the NIK1‐mediated antiviral signalling in a NIK1‐ and NIK2‐dependent manner and were included as a piece of new complementary evidence for this review. However, the nature and origin of the nucleic acid PAMPs have not been deciphered, and the nucleic acid sensors have yet to be identified. Furthermore, as viruses are intracellular obligate parasites and may not have access to the apoplast, it remains to be determined how PRRs would sense viruses and viral PAMPS extracellularly. The identification of the origin and nature of virus‐derived nucleic acid PAMPs and the discovery of the cognate PRRs will shed light on the mechanism of antiviral PTI activation and its role in resistance against viruses.The authors declare no conflict of interest.


The precursor–ligand complexes are stable for months, and thus perovskite NCs can be readily prepared prior to use. Moreover, we show that this versatile synthetic process is scalable and generally applicable for perovskite NCs of different compositions.The growing demand for perovskite nanocrystals (NCs) for various applications has stimulated the development of facile synthetic methods. Perovskite NCs have often been synthesized by either ligand‐assisted reprecipitation (LARP) at room temperature or by hot‐injection at high temperatures and inert atmosphere. However, the use of polar solvents in LARP affects their stability. Herein, we report on the spontaneous crystallization of perovskite NCs in nonpolar organic media at ambient conditions by simple mixing of precursor–ligand complexes without application of any external stimuli. The shape of the NCs can be controlled from nanocubes to nanoplatelets by varying the ratio of monovalent (e.g. formamidinium Faster than fast: Shape‐controlled lead halide perovskite nanocrystals crystallize spontaneously at ambient conditions when precursor–ligand complexes are mixed directly in a nonpolar organic medium. Nanocubes and nanoplatelets have been obtained. This versatile synthetic process is generally applicable for a wide range of halide perovskite nanocrystals. Over the last few years, halide perovskite nanocrystals (NCs) have emerged as a new class of semiconductor materials and efficient color‐tunable light sources for a wide range of applications such as displays, light‐emitting devices, lasers, broadband photodetectors, phototransistors, and photovoltaics owing to their extraordinary optical and optoelectronic properties, which are easily tunable by their composition and morphology.N,N‐dimethylformide (DMF), greatly affects the stability of NCs.+) and Cs+) to divalent cation (Pb2+) precursors in the reaction medium. Mechanistic studies reveal that the NCs are formed through seed‐mediated growth. Furthermore, we show that this simple synthetic approach is versatile.Since the first colloidal synthesis of perovskite NCs was reported in 2014, great efforts have been devoted to the fabrication of high‐quality perovskite NCs  by various synthetic methods such as ligand‐assisted reprecipitation (LARP),+)–ligand and lead halide (PbBr2)–ligand) in a nonpolar solvent under ambient conditions, and at room temperature . This approach is different from the conventional LARP method. Here, the precursors are directly dissolved in nonpolar solvents through metal ion complexation of ligands, and the crystallization occurs spontaneously when the two precursors are mixed. Importantly, we found that the precursor solutions are extremely stable for months and the perovskite NCs can be readily prepared in large quantities a few minutes prior to use. In principle, this facile synthetic approach could be easily applied to a range of ABX3 perovskite NCs  with different types of A and X compositions as depicted in Figure 2–ligand complex under continuous stirring at room temperature (see the Supporting Information for details). The color of the reaction medium changes to orange and the reaction medium starts to fluoresce under UV light immediately after the two precursors are mixed without application of heat or use of polar solvent. This suggests that the precursor–ligand complexes break down and spontaneously crystallize to form FAPbI3 colloidal perovskite NCs in the reaction medium. In addition, the orange emission of FAPbI3 NCs indicates that charge carriers are quantum confined, since the bulk FAPbI3 exhibits near‐infrared emission (≈800 nm).−1) added to the PbI2–ligand solution yields a dark‐brown solution with red luminescence under UV light illumination, which indicates the formation of FAPbI3 colloidal NCs with weaker quantum confinement.As shown in Figure 3 colloidal solutions were characterized by UV/Vis absorption and PL spectroscopy . This is in a way similar to the synthesis of CsPbBr3 NCs which show tunable emission when the ratio of Cs to Pb precursor is varied.The optical properties of the obtained FAPbIy Figure  c. The c3 NCs has been characterized by transmission electron microscopy (TEM). The corresponding images of the NCs obtained with the addition of 50 μL and 200 μL FA–oleate show nearly monodisperse nanoplatelets and nanocubes  with a PL peak at 751 nm. The high‐resolution TEM image shows that the nanocubes are single‐crystalline . The nanoplatelets have broader XRD peaks compared to the nanocubes indicating a smaller grain size. The PLQYs of FAPbI3 nanoplatelets and nanocubes were found to be 30 % and 65 %, respectively. The lower PLQY of the nanoplatelets is because those are more susceptible to surface defects compared to nanocubes as they have larger surface‐to‐volume ratio. Furthermore, we demonstrate the scalability of this simple synthesis method by increasing each reaction component's volume by 50 times . Our results clearly suggest that simple mixing of two precursors in different ratios results in shape control with excellent monodispersity and scalability. Additionally, we found that the NCs prepared by this approach exhibit higher long‐term stability than the NCs prepared by conventional LARP synthesis under UV illumination  at ambient conditions .The morphology of the FAPbIs Figure  d,e, res3 perovskite NCs upon mixing the two precursors, we performed the reaction in a cuvette and monitored the PL evolution of nanocrystals formed in the reaction medium over a period of time. As depicted in Figure In general, the growth of most metal and semiconductor NCs is seed‐mediated or stimulated by a template, while the shape of the NCs can be controlled through precursor concentrations, ligands, and reaction temperature. In order to elucidate the growth of FAPbIAlthough the PL evolution of the product gives insight into the possible growth mechanism, it is unclear how the precursors crystallize into perovskite nuclei when they are mixed in a nonpolar organic solution. To gain a better understanding of this process, one has to consider the dissolution of precursor salts in organic media. Generally, precursor metal ions are not soluble in organic solvents. However they can be solubilized using coordinating ligands through metal–ligand complexation, meaning that the ligand solution acts like a good solvent for the precursors. Here, perovskite complexes are made by dissolving corresponding salts in a ligand solution, which is then injected into toluene, a bad solvent for metal ion precursors. Therefore, the precursors crystallize into perovskite nuclei immediately when they are mixed in an organic solvent, like in the conventional LARP method Figure  b. Furth3 and CsPbBr3. This has been achieved by selecting the respective monovalent cation–oleate  and PbX2–ligand complexes as reactants. As shown in Figure 3 and CsPbI3 nanoplatelets were successfully prepared by simply mixing Cs–oleate with a PbX2–ligand complex (X=Br or I) in toluene. The TEM images , in contrast to the room‐temperature formation of FAPbI3 NCs. This is likely due to a higher energy barrier between perovskite precursors and CsPbBr3 nanocubes. Nevertheless, this reaction temperature is still far lower than the temperature  of the hot‐injection synthesis used to produce CsPbBr3 nanocubes in organic solvents.3, FAPbBr3, and MAPbBr3 NCs .We further verified the versatility of this facile synthesis approach by applying it to different kinds of perovskite NCs, such as CsPbI2+ precursors. Importantly, we have demonstrated the versatility of this synthesis approach by applying it to both organic–inorganic hybrid and all‐inorganic perovskite NCs of different halide compositions. We foresee that this facile method could be easily extended to obtain other morphologies such as nanorods or nanowires by varying the concentration and temperature of the Pb2+ precursor. This versatile and facile synthesis method not only opens new avenues toward the shape‐controlled perovskite NCs with excellent scalability, but also expands our current understanding of the crystallization of perovskite NCs directly in nonpolar solvents.In conclusion, we have presented a facile scalable synthesis of perovskite NCs at ambient conditions by spontaneous crystallization in a nonpolar organic solvent. Compared to the classical LARP method, no polar solvent is needed to dissolve the precursors; instead, ligands act as coordinating solvents. The morphology of perovskite NCs is easily tunable from 3D nanocubes to 2D nanoplatelets by decreasing the ratio between the monovalent cation and PbThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


Search filter development for adverse effects has tended to focus on retrieving studies of drug interventions. However, a different approach is required for surgical interventions.medline and Embase for the adverse effects of surgical interventions.To develop and validate search filters for Systematic reviews of surgical interventions where the primary focus was to evaluate adverse effect(s) were sought. The included studies within these reviews were divided randomly into a development set, evaluation set and validation set. Using word frequency analysis we constructed a sensitivity maximising search strategy and this was tested in the evaluation and validation set.medline and 348 were available on Embase. Generic adverse effects search strategies in medline and Embase could achieve approximately 90% relative recall. Recall could be further improved with the addition of specific adverse effects terms to the search strategies.Three hundred and fifty eight papers were included from 19 surgical intervention reviews. Three hundred and fifty two papers were available on medline and Embase. However, we appreciate the limitations of our methods, and recommend further research on larger sample sizes and prospective systematic reviews.We have derived and validated a novel search filter that has reasonable performance for identifying adverse effects of surgical interventions in  Searching for information on adverse effects can be problematic. This is because there are a number of specific challenges which need to be overcome. The first problem is that adverse effects are generally considered to be either a secondary or even tertiary outcome in health research  or study design (such as RCTs). Search filter development in the arena of adverse effects has tended to focus on identifying studies that report on adverse drug effects , the adverse effects of surgical interventions are the next most popular type of intervention evaluated   . No automated search strategy was implemented, as previous research has indicated that even very broad search strings would miss relevant records  for a surgical intervention were the primary outcome.No adverse effects search terms  had been used by the review authors. Typically, such reviews rely on search terms for the population or condition and intervention only. This enabled an unselected cohort to be built, where relevant articles are not chosen because of the presence of adverse effects terms.The search included either handsearching or reference checking in addition to database searches.The author and another researcher independently screened titles and abstracts in DARE and selected systematic reviews for potential inclusion. Any discrepancies between the researchers were resolved by discussion and consensus. The full articles of potentially relevant systematic reviews were also independently screened, with again discrepancies resolved by discussion and consensus.The full text of the included articles within these systematic reviews was checked to confirm the presence of adverse effects data that had been used in the systematic review. The use of included papers from systematic reviews has been shown to be effective in identifying a reference standard set of records for the use in studies evaluating search strategies . We calculated relative recall as a measure of the percentage of known records retrieved using the filter because it provides an estimate of sensitivity. The relative recall of the relevant search terms identified from frequency analysis was calculated using the following formula;Individual word and multiple‐word frequency analysis on the development set was undertaken using WriteWords to identify commonly occurring terms related to adverse effects. WriteWords is freely available on the Internet and allows you to count the frequency of usage of words or phrases in text  would have been successful in retrieval. We also noted any database records with no indication that the full text contained information on adverse effects.This process was first undertaken in medline and 348 were available from Embase.From 9129 DARE records screened, 451 full reports were retrieved and of these 348 reviews were about adverse effects with 79 evaluating surgical interventions. Of these 79 reviews, only 19 did not include any adverse effects terms in their search strategies and had carried out reference checking and/or hand searching. These 19 reviews included 358 unique studies. Of these 358 studies, 352 were available on medline were randomly allocated into a development set with 118 records and an evaluation and validation set of records containing 117 records each.The 352 records comprising the gold standard set on surgical adverse effects available in The development set was used to identify the search terms or phrases for the filter. Word and phrase frequency analysis on the development set was undertaken using WriteWords. We started by searching the search term with the highest recall, ‘complication*’ in the title and abstract Table . We thenA search strategy which retrieved 101/118 records (86%) was achieved using the combinations of terms presented in Box Box 11 complication*.ti,ab. (75)2 ae.fs. (89) [adverse effects]3 safe*.ti,ab. (97)4 co.fs. (100) [complications]5 post‐operative complications/(101)6 or/1–5This search strategy Box  was thenThe search strategy Box  performeWe conducted post‐hoc analysis to identify factors that may have affected the recall. When we explored the records that had not been retrieved from the validation set, there was one additional record that could have been retrieved if the phrase ‘procedure related’ was added to the search strategy in the title and abstract field. After adding the phrase ‘procedure related’ to the search strategy we then retrieved 103/117 records (88%) of the records in the validation set with the revised search strategy. Of the 14 records not retrieved six contained terms related to specific adverse effects such as ‘postoperative pain’, ‘adverse oncological consequences’, ‘postoperative death’, ‘postoperative mortality’, ‘wound infections’, ‘surgical wound infection/’, and ‘sensory or motor nerve loss’. A search strategy which incorporates both generic and specific adverse effects terms could therefore potentially achieve 93% (109/117) recall in the validation set of records.medline were estimated to be ‘procedure related’, ‘complication’ and ‘safe’ in the title and abstract.The terms which gave the highest precision in The 348 records comprising the gold standard set on surgical adverse effects available in Embase were randomly divided into a development set of 116 records and an evaluation and validation set with 116 records in each set.The development set was used to identify the search terms or phrases for the filter. Word and phrase frequency analysis on the development set was undertaken using WriteWords. We started by searching the search term with the highest recall ‘complication*’ in the title and abstract Table . We thenA search strategy which retrieved 102/116 records (88%) was achieved using the combination of search terms presented in Box Box 21 complication*.ti,ab. (73)2 co.fs. (91) [Complication]3 safe*.ti,ab. (96)4 ae.fs. (99) [adverse drug reaction]5 post‐operative morbidity.ti,ab. (100)6 surgical risk/(101)7 complication/(102)8 or/1–7This search strategy Box  was thenThis revised search strategy Box  was thenBox 31 complication*.ti,ab.2 co.fs. [Complication]3 safe*.ti,ab.4 ae.fs. [adverse drug reaction]5 post‐operative morbidity.ti,ab.6 surgical risk/7 complication/8 post‐operative complication/9 procedure related.ti,ab.10 or/1–9The terms which gave the highest precision in Embase were estimated to be the Emtree term ‘complication/’, followed by ‘postoperative morbidity’ or ‘procedure related’ in the title and abstract, then Emtree terms ‘surgical risk/’ and ‘postoperative complication/’.medline search filter in Box In summary therefore, the proposed medline with the addition of specific adverse effects terms the recall would have been 94%, 96% and 93% in the developmental, evaluation and validation sets of records, respectively. In Embase with the addition of specific adverse effects terms the recall would have been 91%, 95% and 95% in the developmental, evaluation and validation sets of records, respectively  the recall could be raised to 93% in medline and 95% in Embase.We have used a cohort of published surgical studies to derive and validate a novel search filter for the adverse effects of surgical interventions. The results here give a clear indication of the relevant search terms that work in terms of relative recall performance. Use of the filter will also increase the precision of searches for adverse effects. The recall of searches using solely generic adverse effects terms was 87% in medline and Embase was achieved without specific named adverse effects and 93% in medline and 96% in Embase when specific adverse effects terms were added  and further research with larger sample sizes of relevant papers.medline and 92% in Embase and 93% in medline and 95% in Embase when specific adverse effects search terms are added.This is the first proposed search filter for surgical adverse effects. The use of search filters for the adverse effects of surgical interventions is both feasible and advisable in instances where unmanageable numbers of records would otherwise be retrieved. Sensitivity with the proposed generic adverse effects search filters achieved 87% in Further research on larger datasets is required in order to measure the precision of searching for adverse effects of surgical interventions and to test the suggested search filters with more rigour.We declare that we have no conflicts of interest.This report is independent research arising from a Postdoctoral Research Fellowship, Su Golder PDF‐2014‐07‐041 supported by the National Institute for Health Research. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.


Mimulus guttatus sensu lato) into a high‐elevation common garden during an extreme drought period in the Sierra Nevada mountains, California, USA. We use a demographic approach to quantify fitness and test for selection on life history traits in local versus lower‐elevation populations and in normal versus drought years to test the potential for up‐slope migration and phenotypic selection to alter the distribution of key life history traits in montane environments. We find that lower‐elevation populations tend to outperform local populations, confirming the potential for up‐slope migration. Although selection generally favored some local montane traits, including larger flowers and larger stem size at flowering, drought conditions tended to select for earlier flowering typical of lower‐elevation genotypes. Taken together, this suggests that monkeyflower lineages moving upward in elevation could experience selection for novel trait combinations, particularly under warmer and drier conditions that are predicted to occur with continued climate change.One of the strongest biological impacts of climate change has been the movement of species poleward and upward in elevation. Yet, what is not clear is the extent to which the spatial distribution of locally adapted lineages and ecologically important traits may also shift with continued climate change. Here, we take advantage of a transplant experiment mimicking up‐slope seed dispersal for a suite of ecologically diverse populations of yellow monkeyflower ( We use a high‐elevation common garden experiment to test the potential for up‐slope migration and corresponding patterns of phenotypic selection in yellow monkeyflowers. We find that lower‐elevation populations have higher fitness than local populations, but experience strong selection on life history traits in a high‐elevation environment. Lineages moving upward in elevation with continued climate change could experience selection for novel trait combinations. Shifts in distribution poleward or upward in elevation have already been observed in many species  during an extreme drought period in the Sierra Nevada mountains, USA, to test for selection on life history traits in a high‐elevation environment. In this study, we use trait and fitness data from a common garden field experiment in which annual and perennial populations across an elevation transect were transplanted into a high‐elevation montane meadow  species complex comprises extensive variation in morphological, floral, and life history traits . However, genomic studies suggest that genetic variation in this complex is partitioned geographically rather than according to life history divisions . Experimental seedlings were derived from field‐collected seeds  germinated in the UC Santa Cruz greenhouse in 2012 and randomly sampled from among the germinants  in a separate seed germination field experiment at the common garden site in 2013. To prevent genetic contamination of the native population through rhizomes, pollen, or seeds, we transplanted seedlings into 4‐inch round pots  buried within the local substrate, emasculated flowers of any experimental plants reaching the reproductive stage, and collected any maturing fruits prior to dehiscence. Emasculation is a common technique to prevent pollen contamination in transplant experiments and has been shown to not alter flower number or seed set in several plant species . Additional details on the common garden experiment are given in Peterson et al. .2.2.1We tracked the survival, phenology, and fecundity of all experimental individuals every 3–10 days from the time of transplant through the end of the growing season . We measured seedling size at transplant as a covariate and three life history traits relevant to the timing of and allocation to sexual reproduction: time to first flower, plant size at first flower, and flower size. We measured seedling size on the transplant date as the product of seedling diameter measured at the widest point and at a 90° angle to that point. We measured time to first flower as the number of days from transplant to the first census with at least one open flower. We measured plant size as the diameter of the primary flowering stem at the basal node and flower size as the width of the corolla at the widest point on the first census with an open flower.2.3λ) as our fitness measure as well as the elasticities, or the proportional contributions to λ, of underlying vital rates. These models are described in detail in Peterson et al.  = MN(t), where N(t) is a vector of stage‐classified individuals at time t and M is a 3 × 3 matrix of transition rates parameterized according to Figure S, defined as at least one vegetative rosette following snowmelt), clonal rosette production , and flower number . We recorded adult survival and counted the number of vegetative rosettes following snowmelt each year on 11 May 2013 and 1 May 2014. We also estimated ovule number per flower  by collecting second flowers from a subset of individuals per population . We used a separate seed germination experiment to estimate a mean germination rate (G) for each population based on 96 field‐collected seeds left to overwinter in plug trays  at the common garden site, to account for seed overwinter survival and early viability selection ,A = 6.7 × 10−4) across all populations and years  from a separate seed germination experiment in our population models  to produce population‐specific estimates of A. This alternative approach did not alter the elasticities of vital rates or the rank order of mean fitness (λ), so we chose the initial approach for consistency with Peterson et al. (D) based on a seed viability study using pooled M. guttatus seeds from multiple Sierra Nevada populations  with population means for ovule number (O) and germination rate (G) and a constant ovule‐to‐seedling rate (A) to estimate individual seed and seedling production  from seedlings in each year and used these values for both seedling and rosette transition rates  around each estimate as an indication of statistical significance.We constructed 95% bias‐corrected confidence intervals around all 2.4λ), of each life history trait  using three individual vital rates—flower number (F), ovule number per flower (O), and the number of rosettes following snowmelt (SR)—and integrating their contributions to population growth (see details below).We estimated the elasticity, or the proportional effect on population growth rate (F) and ovule number per flower (O) as separate vital rates because we only collected ovule data on a subset of flowering individuals. Mimulus guttatus has a mixed mating system with delayed self‐fertilization in the absence of outcross pollination, and ovule number per flower is proportional to potential seed set per flower   Figure  (Van Tie) Figure . This ism Figure , where am Figure . We comp2.4.1μβ) by dividing each vital rate and trait by the mean value for that life history group and year  and ovule number per flower (O). In the montane perennial group, too few individuals flowered to fit multiple regressions with all traits. Instead, we only tested for selection gradients for flowering time and size at flowering since these were the only traits with significant or marginally significant relationships in univariate regressions . Flower number (F) and rosette production (SR) are overdispersed counts  incorporating paths through correlated traits in addition to direct paths  based only on direct paths from a trait to s Figure . For eacs Figure , we wills Figure . One asss Figure . Thus, as Figure . Howevers Figure .33.1λ) in a mesic montane environment, followed by robust annuals and montane perennials; fast‐cycling annuals had the lowest fitness  and dormancy (1 − G) had greater effects on population growth due to poor seedling performance. Further, transitions to rosettes had a higher proportional effect on λ in montane perennials relative to low‐elevation perennials, reflecting differences in the relative importance of sexual versus vegetative reproduction among perennials. In general, low survival and fecundity in 2013 resulted in higher elasticities for transitions to more protected life stages, including seeds and belowground rhizomes in montane perennials.Environmental differences between years and life history differences among populations were reflected in the relative importance of different vital rates for population growth Table . In annu3.3Patterns of trait expression in the high‐elevation common garden were broadly consistent with genetically based life history divergence among populations Table . Fast‐cy3.43.4.1Selection gradients in this high‐elevation common garden varied among vital rates and years, but tended to favor larger flowers and larger stem size at flowering in both annual and perennial life history groups Figure . In geneSelection gradients for flowering time differed strongly among vital rates. Later flowering tended to decrease flower number but increase ovule number per flower Figure . In pere3.4.2By integrating selection gradients across multiple vital rates, trait elasticities represent the net pattern of selection for each trait. We found that larger flowers were consistently favored across all populations and years Figure a, and laIndirect selection through correlated traits tended to either have small effects on trait elasticities or magnify their effects by acting in the same direction Figure . Trait c4We used a demographic approach to explore how selection acts on life history traits through specific vital rates and also net population growth in annual and perennial monkeyflowers when experimentally transplanted upward in elevation into a montane site. We found that selection generally favored some perennial traits, including larger flowers and larger stem size at flowering, but that drought conditions selected for earlier flowering in all but the earliest‐flowering fast‐cycling annuals. Together, this suggests that upwardly migrating lineages in this system could experience selection for novel trait combinations in the warmer and drier conditions forecasted to occur with continued climate change , particularly for perennials. Hereford et al.  noted thAlthough many species have undergone poleward or upward range shifts in response to climate change, we have little understanding of how these species‐level patterns reflect the redistribution of locally adapted lineages and ecologically important traits. In particular, climate change may alter the distribution of ecologically important traits through multiple mechanisms, including phenotypic plasticity, evolutionary responses to shifting selection pressures, and migration of locally adapted lineages. By experimentally mimicking upward seed dispersal across a range of populations, we find evidence for adaptational lag coupled with strong selection on life history traits mediated by variation in local soil moisture regimes. Taken together, this suggests that low‐elevation populations in this system may be able to migrate upward in elevation as long as the local soil moisture regimes are similar, but will experience selection for more high‐elevation trait values, potentially resulting in novel trait combinations. Whether montane lineages in this system will be able to migrate beyond the current upper elevation limit—and the extent to which selection there may favor unique trait values—is as yet unknown, although the greater allocation to vegetative over sexual reproduction could limit both the dispersal capacity and evolutionary potential of montane perennials. Given the prevalence of local adaptation (Bocedi et al., The authors have no competing interests to declare.MLP, KMK, and ALA designed the study. MLP collected and analyzed the data. MLP wrote the first draft of the manuscript, and all authors contributed to revisions. Click here for additional data file. Click here for additional data file.


BMI) (or waist circumference or percentage body fat). The aim of the Medical Research Council New Investigator Research Grant (MR/P023347/1) ‘Body size trajectories and cardio‐metabolic resilience to obesity in three United Kingdom birth cohorts’ is to reveal the body size trajectories, pubertal development patterns and other factors (e.g. early‐life adversity) that might attenuate the positive associations of adulthood obesity makers (e.g. BMI) with cardio‐metabolic disease risk factors and other outcomes, thereby providing some degree of protection against the adverse effects of obesity. This work builds on the Principle Investigator's previous research as part of the Cohort and Longitudinal Studies Enhancement Resources initiative and focuses on secondary data analysis in the nationally representative UK birth cohort studies , which have life course body size and exposure data and a biomedical sweep in adulthood. The grant will provide novel evidence on the life course processes and exposures that lead to some people developing a cardio‐metabolic complication or disease or dying while other people with the same BMI do not. This paper details the grant's scientific rationale, research objectives and potential impact.Individuals with obesity do not represent a single homogenous group in terms of cardio‐metabolic health prospects. The concept of metabolically healthy obesity is a crude way of capturing this heterogeneity and has resulted in a plethora of research linking to future outcomes to show that it is not a benign condition. By contrast, very few studies have looked back in time and modelled the life course processes and exposures that explain the heterogeneity in cardio‐metabolic health and morbidity and mortality risk among people with the same body mass index ( However, this group with so‐called ‘healthy obesity’ is always going to have higher average blood pressure than the much larger group who are not obese (BMI <30 kg/m2) and do not have hypertension (blood pressure <140/90 mmHg). Such inherent baseline differences in risk factors between the healthy obese and healthy non‐obese groups are reported in the descriptive statistics table of most papers on this topic and are largely responsible for known differences in mortality risk between these two groups  has been used to describe the sub‐group of individuals who are obese  but do not have any, or only minimal, cardio‐metabolic complications . The idea that someone can be obese yet metabolically healthy has been highly controversial and widely debated (Piepoli etc.) is undeniable, but the concept of MHO is a crude way of capturing this heterogeneity. A lot of work over the decades has looked forward in time, finding and using somewhat obvious results  to argue that healthy obesity doesn't really exist. By contrast, very few studies have looked back in time and modelled the life course processes and exposures that explain why some people develop a disease or die while other people with the same BMI do not.As surmised in a recent commentary Johnson , variatiet al.  to weight and health status at 31 years of age, but likely found spurious results because of the very low number of MHO individuals in their sample   from truncal adiposity and insulin resistance, which is intriguing given the evidence linking rapid infant weight gain to increased obesity risk  from the metabolic syndrome, whereas Howe et al. (n = 284). The two studies that have investigated early‐onset obesity also reported equivocal results  initiative, the Principle Investigator (WJ) has already harmonised the body size data across these (and other UK) birth cohorts studies to investigate secular trends in obesity development  New Investigator Research Grant (MR/P023347/1) ‘Body size trajectories and cardio‐metabolic resilience to obesity in three United Kingdom birth cohorts’ is to reveal the body size trajectories and pubertal development patterns that might attenuate the positive associations of adulthood obesity with cardio‐metabolic disease risk factors, thereby providing cardio‐metabolic resilience to obesity. This work will also quantify the trajectories and patterns that might result in some normal weight adults having poor health prospects. The focus will be on secondary data analysis in nationally representative UK birth cohort studies which have life course body size data and a biomedical sweep in adulthood, namely the MRC e.g. HbA1c) is modified by body size trajectories of pubertal development patterns. Using this type of interaction nods back to the Abraham et al. study (1971). Briefly, cardio‐metabolic risk factors will be considered separately and in composite outcome variables. Body size trajectories will be captured by (1) conditional change measures (that represent starting size and rate of change during different age periods), (2) traits from multilevel growth curve models  and (3) latent trajectory classes from mixture models  will quantify, and describe the contribution of between‐cohort differences in body size trajectories and pubertal development patterns to, any secular trend in cardio‐metabolic resilience to obesity.Rather than having to define MHO, which is an inherently limited construct, the analysis plan focuses on developing models that treat continuous variables as continuous and estimate how the relationship of adulthood BMI with some risk factors , there are plans to link these data to the CLOSER harmonised body size data set for epidemiological investigation. The NSHD arguably has the richest life course and biomedical data, and a PhD student is working with WJ at Loughborough University to investigate how early‐life adversity and lifestyle behaviours might provide cardio‐metabolic resilience to obesity in this cohort. Other regional birth cohort studies in the UK have more frequent body size data in childhood . Finally, while this evolving research programme will naturally lend itself to the use of longitudinal birth cohort data, large‐scale survey data could be used to describe worldwide and temporal variation in BMI‐risk factor associations.The grant will also be used as a springboard to develop a much larger and comprehensive programme of research in this field. Work using the Body size trajectories have played a central part in the evolution of life course epidemiology, and this grant is the next step in refining our understanding of their role in disease development. The results will constitute a significant advancement in our understanding of how, when and in whom some degree of cardio‐metabolic resilience to obesity develops. We envisage that the findings will inform on‐going research, including trials that aim to alter body size trajectories and stimulate new lines of investigation. This grant is also potentially relevant to a wide range of clinicians. Child growth assessment is a fundamental part of routine care in the UK and, by providing a better understanding of the role of growth and development in cardio‐metabolic disease aetiology, our findings have the potential to impact on this practice and the guidelines on what constitutes healthy growth and development. Obesity clinics and cardiologists alike may also benefit from an improved understanding of which patients are likely to suffer most from the cardio‐metabolic complications of obesity, based on their body size trajectory and other life course exposures. Findings could also potentially be used to augment recommendations to parents and their children on how to maximise long‐term cardio‐metabolic health. Of course, the end goal of this grant is to provide a stronger literature base to inform policy and practice, as a means to impact on public health and wellbeing. In particular, the results may be informative for characterising which individuals should be targeted for healthcare provision or therapeutic intervention and may be exploited for new evidence‐based interventions that improve disease processes.e.g. to UK PhD students and postdocs on ‘cross cohort analysis of secular trends in longitudinal processes and relationships’) and increase dissemination and discussion of results. The grant is fully integrated into CLOSER and the institutions who house the studies, which will help (1) WJ strengthen his link with the UK birth cohorts, (2) facilitate communication with the wider academic and non‐academic community and (3) hopefully benefit the end‐user of birth cohort study data (e.g. by feeding back harmonised data sets and guides to key stakeholders). Finally, the grant incorporates numerous training and learning opportunities for the Principal Investigator and employed Research Associate and, as such, will have significant impacts on their career trajectories.To ensure maximum coverage and impact on the scientific and medical community, the results will be disseminated in publications and at international conferences. A collaborative group has been formed, and their steering and mentorship will naturally increase the impact of these outputs. Further, planned communication with organisations such as Public Health England will help ensure that the results are translational. A well‐thought‐out series of three workshops has been designed to provide training (e.g. early‐life adversity) that lead to some people developing a cardio‐metabolic complication or disease or dying while other people with the same BMI do not.Individuals who are obese have different cardio‐metabolic health and disease risks, as do individuals who are normal weight, but very little is known about the life course exposures and processes that explain this heterogeneity. Primarily using data from the UK birth cohort studies, this grant will provide novel evidence on the body size trajectories, patterns of pubertal development, and other factors (The author reports no conflict of interest.