All organisms are exposed constantly to a variety of infectious and injurious stimuli. These induce inflammatory responses tailored to the threat posed. While the innate immune system is the front line of response to each stimulant, it has been considered traditionally to lack memory, acting in a generic fashion until the adaptive immune arm can take over. This outmoded simplification of the roles of innate and acquired arms of the immune system has been challenged by evidence of myeloid cells altering their response to subsequent encounters based on earlier exposure. This concept of ‘innate immune memory’ has been known for nearly a century, and is accepted among myeloid biologists. In recent years other innate immune cells, such as natural killer cells, have been shown to display memory, suggesting that innate immune memory is a trait common to several cell types. During the last 30 years, evidence has slowly accumulated in favour of not only haematopoietic cells, but also stromal cells, being imbued with memory following inflammatory episodes. A recent publication showing this also to be true in epithelial cells suggests innate immune memory to be widespread, if under‐appreciated, in non‐haematopoietic cells. In this review, we will examine the evidence supporting the existence of innate immune memory in stromal cells. We will also discuss the ramifications of memory in long‐lived tissue‐resident cells. Finally, we will pose questions we feel to be important in the understanding of these forgotten cells in the field of innate memory. In an immune setting, the term ‘memory’ evokes vaccines, memory T cells and the antibody response. Memory in mammals is not, however, solely the remit of lymphocytes. The field of immunology is changing, and evidence in both arms of the immune system is breaking down old concepts of innate and adaptive roles.Drosophila and lower metazoans all points towards inflammatory responses altered by prior exposure to infectious agents  vaccine was found to save more children than died of tuberculosis, indicating an off‐target effect Nature news and views article, which stated: ‘This is the first identification of inflammatory memory in a non‐immune cell’ Despite the abundance of research on innate memory, stroma is still largely overlooked when considering repeat challenges in immune or inflammatory episodes. Indeed, the recent discovery of epithelial innate memory Given the recent resurgence of interest in stromal memory responses, and the latest observations showing that haematopoietic, epithelial and stromal cell types all alter their responses based on previous insults, we believe it is time to discuss the field of stromal innate memory. In this review, we will collate three decades of stromal memory research, discuss its strengths and unanswered questions and suggest key avenues of enquiry. Innate memory is proving ever more important to our understanding of inflammation biology, and it is time that stroma, the forgotten cells of the memory field, were remembered.Because some of the important terms in this review may be subject to different interpretations, we will begin with some definitions. The ‘stroma’ comprises ubiquitous structural cells, providing extracellular matrix (ECM) and support functions in every organ. In this review we take ‘stromal cells’ to exclude cells of haematopoietic origin; for example, tissue‐resident macrophages. Macrophages and other myeloid cells are discussed below merely by way of introduction to the concept of memory. To our knowledge, the vast majority of the research relevant to stromal memory has been performed on fibroblasts and endothelial cells. We therefore focus on these cells, but we consider it very likely that memory also exists in other stromal cell types. We define ‘memory’ as a state of altered responsiveness that does not depend upon the continued presence of the stimulus by which it is evoked. A key characteristic of this form of memory is that it outlasts the primary signalling response of the cell. In the experiments discussed below memory lasts from days to weeks, or even longer. There will clearly be some overlap between memory and differentiation, for example in a few experimental designs discussed below, where stromal cells are exposed to persistent inflammatory stimulation rather than being stimulated, rested, then stimulated again. As far as this review is concerned, we define ‘inflammatory memory’ as a change in the capacity of stromal cells to respond to inflammatory stimuli, as opposed to an inflammation‐induced change in steady‐state gene expression.During the last three decades, our understanding and appreciation of the roles played by stromal cells in healthy and pathological inflammation has increased exponentially. Two well‐studied stromal cells [endothelial cells (EC) and fibroblasts] are described below.EC line the blood and lymph vessels, and thus have an important role in inflammation. Both host and foreign cells must interact with EC in order to extravasate into tissue. Their release of chemokines recruits leucocytes to a site of infection or injury, and their expression of adhesion molecules facilitates adhesion, rolling and diapedesis of circulating cells  as providing wide‐scale protection against a range of secondary agents. This protection takes the form of lower mortality rates and better pathogen clearance As an intermediary between the two arms of our immune system it is perhaps not surprising that, of all innate cells, macrophages should have memory. This may take either positive or negative forms, in that the response to a second challenge may be either enhanced or diminished by memory of a prior challenge. Both short‐ and long‐term forms of myeloid cell memory have been described. Long‐lived myeloid cell memory that underpins the beneficial off‐target effects of vaccines such as BCG has been coined ‘trained immunity’ by Netea and colleagues The other form of memory is a transient reduction of the inflammatory response. In 1947 bacterial substances were found to have diminishing pyrogenic effects in rabbits receiving successive doses The study of fibroblast memory goes back to 1992 The earlier years of the new millennium saw no new publications on the subject, and it appeared forgotten. In the interim, appreciation of fibroblast involvement in chronic inflammatory diseases was continuing to grow. With the greater understanding of stromal epigenetic alterations resulting in long‐term increases or decreases of chromatin accessibility, authors began to suggest that a ‘fibroblast inflammatory memory’ may be involved in the perpetuation et al. displayed inflammatory memory of T cell chemoattractants in fibroblast‐like synoviocytes (FLS) derived from patients with rheumatoid arthritis (RA) At the same time, the primary publications on fibroblast memory were re‐emerging. In 2009 gingival fibroblasts were shown to maintain their inflammatory response to re‐challenge with LPS, whereas macrophages became refractory et al. also demonstrated the tropism of fibroblast memory and its gene‐specificity et al. showed that tendon fibroblasts from chronic tendinopathy patients mounted stronger inflammatory responses in vitro than those of healthy tendons, thus demonstrating the longevity of innate immune memory in fibroblasts Recently, Klein Research into the concept of endothelial cell memory is newer than that of fibroblast memory, but a range of challenges have been examined. This field dates at least to 2002 The anatomical site of origin and inflammatory stimulus have been shown to dictate EC memory responses, and the vessel architecture has also been demonstrated as important. Shear stress  is higher in straight vessel sections than arches or junctions. Work from the Evans laboratory has shown that EC under low shear stress have constitutively active c‐Jun N‐terminal kinase (JNK) Schistosoma mansoni increases leucocyte adherence through the endothelial layer Studies have confirmed the relevance of EC memory in both infectious and non‐infectious inflammatory contexts. Prior infection by et al. found that EC progenitors performed better as therapeutic cells in models of ischaemic injury if they were primed ex vivo with C‐X‐C motif chemokine ligand 12 (CXCL12) before donation et al. found the endotoxin tolerance seen in EC could be manipulated. An initial challenge with LPS induced an inflammatory response, which was followed by non‐responsiveness to subsequent LPS challenges. If, however, monophosphoryl lipid A (MPLA) was used as the initial stimulus, the non‐responsiveness to LPS was achieved without an initial inflammatory event In 2008, Zemani The mechanisms behind innate memory have been investigated intensively in the myeloid lineage. The effects of Toll‐like receptor (TLR)‐4 signalling in particular have received most attention, and have been reviewed comprehensively The proinflammatory TLR‐4 response is, as with all healthy inflammatory episodes, curtailed by in built feedback loops et al. et al. Escherichia coli LPS nor Porphyromonas gingivalis LPS  could induce tolerance in gingival fibroblasts. They also showed that human gingival fibroblasts lack expression of classic negative regulators. During myeloid endotoxin tolerance, negative regulators are up‐regulated, leading to disrupted signalling through both pathways. While the above fibroblast studies showed a deficiency in negative regulators, neither showed explicitly that this was the reason for propagation of the inflammatory response. Future studies are necessary to link these observations with alterations in memory responses, as has been confirmed in myeloid cells Two studies of fibroblasts are useful to note here. The first is that of Lee IFNs are induced in response to infectious agents, and also in response to tumour cells and proinflammatory cytokines. Their role in innate memory appears multi‐faceted. IFN‐γ, for example, can rescue endotoxin tolerant macrophages from their refractory state by opening condensed chromatin et al. developed a theory based on cross‐priming EC with TLR agonists Koch The NF‐κB pathway is well known for its involvement in the expression of proinflammatory genes. In macrophage endotoxin tolerance, the canonical NF‐κB pathway is refractory. Its inhibition is induced by the increase in inhibitory p50 homodimers at the loci of proinflammatory genes et al. found that the second challenge with TLR‐4 agonist increased EC cytokine production, and this correlated with increased NF‐κB pathway activity E. coli LPS for their experiments, so the explanation for the disparity is unclear. What is clear, however, is that the action of the NF‐κB pathway during the memory response correlated with the increase or decrease of inflammatory mediators elicited by the second challenge. There is also evidence for NF‐κB‐dependent altered responses in fibroblast memory. In fibroblasts stimulated with TNF‐α for 24 h then rested for 24 h, both NF‐κB activity and IL‐6 expression subsided to basal levels during the rest period. Restimulation of the rested cells caused more prolonged NF‐κB activation, and increased expression of IL‐6 that was dependent upon NF‐κB. At least in this case, the inflammatory memory of fibroblasts is not simply persistence of cell activation, but reflects priming of the NF‐κB signalling pathway for an augmented response Studies have shown that repeated TLR‐4 engagement leads to repression of the NF‐κB pathway in endothelial cells Epigenetic modification is generally regarded as key to the innate memory seen in macrophages. Trained immunity induces an augmented response to second challenge weeks or even months after the initial infection. Changes on such a time‐scale are likely to be epigenetic and, indeed, evidence abounds for such a theory. Permissive modifications have been described by several groups Endotoxin tolerance is a memory response of a much more short‐term nature. While previous examples show evidence for upstream mediators, the study of epigenetics has transferred well to the tolerance field. Examples of this include the length of endotoxin tolerance in macrophages, which has been shown to be from 24–48 to 5 days in duration The chromatin landscape in stromal memory is receiving increasing attention. When EC were primed with TLR‐3 agonist, the CXCL10 induction by subsequent TLR‐4 agonist was augmented and this was reduced by inhibiting histone deacetylases et al., who showed that chronic exposure of RA FLS to TNF‐α led to decreased histone H4 levels, increased H4 acetylation and increased NF‐κB and PolII occupancy at the promoter of genes which could be augmented subsequently upon rechallenge. Blocking TNF‐α with infliximab for 24 h before the rechallenge did not reduce the augmented response, suggesting that a permissive state had already been established. This may correlate well with the latent enhancers found to have prolonged transcription factor occupancy in trained macrophages et al. also showed the chromatin landscape to determine which genes became tolerant in macrophages and synovial fibroblasts derived from different anatomical sites More explicit evidence came from Sohn A generic example of the signalling events known to occur in stromal cells undergoing tolerance or augmented responses is illustrated in Fig. We are well aware of the deleterious nature of flawed adaptive immunology. The number and variety of autoantibodies contributing to a large range of autoimmune diseases displays the danger of what is ostensibly beneficial in infection.in vivo and in macrophages Endotoxin tolerance reduces the inflammatory response of myeloid cells to infectious triggers and thus preserves the host's life. Very low doses of LPS, however, do not induce tolerance, but rather a chronically inflamed state The concept of endogenous factors priming the response to damage‐associated molecular patterns (DAMPs) is established in the stromal memory field. Homocysteine (HCy) is a circulating amino acid used as a clinical marker for atherosclerosis risk. It has been shown to synergize with TNF‐α to induce stronger activation of EC The role of EC as inflammatory contributors to atherosclerosis is well established Research into fibroblast memory also suggests a pathological role. Our own research into non‐inflamed and RA synovial fibroblasts suggests that the augmented proinflammatory response is inherent to the joint Two publications have shown healthy skin fibroblasts to be incapable of the augmented response The concept of changes in fibroblasts contributing to chronic joint inflammation is well established et al. hypothesized A host of publications have recognized the modulating influence of prior inflammatory events Vaccines have proved beyond doubt the clinical benefit of harnessing adaptive immunity. In the same sense, macrophage innate memory is host‐protective in various infectious scenarios. Trained immunity protects the host by putting macrophages (and NK cells) on high alert for subsequent infections. Conversely, endotoxin tolerance reduces inflammatory secretions but maintains phagocytic function to defend the host from cytokine storm.These two sides of innate memory present an attractive therapeutic opportunity. Mihai and van der Meer have expounded the theory of an ‘innate vaccine’ in order to harness trained immunity ex‐vivo challenging of EC progenitors with CXCL12 induced greater homing to activated EC and greater tube formation et al. showed that in‐vitro endotoxin tolerance could be achieved in EC by stimulating with MPLA rather than LPS et al. showed that induction of tolerance could be achieved in gingival fibroblasts if IFN‐β was provided Although the field of stromal memory is still relatively understudied, clinical relevance has already been evidenced. As stated earlier, The increased interest in this field during the last few years has shed light upon several facets of stromal memory. It is, however, still a field in its in infancy. The aspects we feel to be most pressing are as follows.in‐vitro challenge and rechallenge experiments can shed light upon mechanisms of stromal cell memory, they model the situation poorly in vivo. Ground‐breaking experiments by Müller‐Ladner and colleagues demonstrated that RA synovial fibroblasts could be maintained in tissue culture without losing their erosive properties in an in‐vivo model of cartilage degradation in‐vitro approaches and elegant in‐vivo experiments of this kind. This may require the use of irradiated chimeras, or even models deficient in immune cells.Although in vitro and in vivoThe role played by metabolism is currently not known. The field of immunometabolism has expanded rapidly in recent years, and inflammatory triggers are known to alter metabolic processes in both haematopoietic and stromal cells in‐vitro repeat stimulation and real‐world inflammatory pressures. Therefore, although some features of inflammatory memory of stromal cells have been described, the adaptive value of the phenomenon remains unclear. Its gene‐specific nature suggests that it may be a mechanism to fine‐tune the recruitment of leucocytes during the evolution of an inflammatory episode, or to modify inflammation‐induced recruitment to sites that have previously experienced inflammation.As described earlier, there is disparity between The contribution to chronic inflammatory disease is also yet to be defined clearly. Why is it that stromal memory differs according to site of origin Finally, if cells from chronically inflamed tissue have memory, is it part of the cause for inflammation or is it acquired in the course of inflammation? If the former, we have a new susceptibility marker which may be more tissue and disease‐specific than most of the genome‐wide association study (GWAS) hits (which are often shared among many diseases). If the latter, this provides a new therapeutic target which could be applied to any chronic inflammatory disease where memory is acquired. Such a therapeutic would have the dual benefit of targeting more than one disease, and of leaving the patient immunocompetent.Innate memory in humans was recognized nearly a century ago, and for years was seen as the prerogative of myeloid cells. Innate memory, whether in the guise of endotoxin tolerance or trained immunity, is host protective. The former protects from tissue damage and cytokine storm, while the latter provides a generic augmented response to future infection. Recently non‐myeloid lineages were also recognized to exhibit memory, and stromal memory is starting to receive the attention it deserves.The roles of stromal cells in inflammation are manifold, and crucial in inflammatory responses to both infection and injury. The field of stromal memory is still in its infancy, and yet already promises to reshape our understanding of stromal cells’ roles in inflammatory episodes. Of the evidence presented herein, some suggest that stromal memory may be adaptive, and others deleterious. Some suggest a double‐edged sword, protecting against infection at the risk of autoinflammatory conditions. The data are varied, but the consensus in the field is that this under‐investigated mechanism will undoubtedly influence the nature, magnitude and duration of inflammatory episodes. Stromal memory, therefore, is well worth remembering.None declared.


Approximately 3%–5% of lung adenocarcinoma is driven by anaplastic lymphoma kinase (ALK) fusion oncogene, whose activity can be suppressed by multiple ALK inhibitors. Crizotinib and ceritinib have demonstrated superior efficacy to platinum‐based chemotherapy as front‐line treatment for patients with ALK‐positive advanced non‐small cell lung cancer (NSCLC). However, the direct comparison between them in the front‐line setting remains lacking.A total of 48 patients with ALK‐positive, previously untreated advanced NSCLC, who received crizotinib and ceritinib as front‐line treatment were retrospectively investigated. The efficacy and pattern of disease progression were analyzed.P = 0.016). The median progression‐free survival (PFS) was significantly longer with ceritinib than with crizotinib treatment ; the hazard ratio for disease progression or death, 0.27 . An objective response was noted in all patients in the ceritinib group and in 23 patients in the crizotinib group . The rate of systemic progression was significantly lower over time with ceritinib treatment compared to crizotinib treatment . Serious adverse events were noted in one (2.9%) patient showing elevated liver function in the crizotinib group and three (23.1%) patients showing diarrhea in the ceritinib group. Dose reduction was needed in five out of 13 (38.5%) patients receiving ceritinib treatment.Patients receiving ceritinib treatment were significantly younger than those receiving crizotinib treatment (52.0 vs. 63.0, Ceritinib showed higher efficacy associated with a better control of systemic progression compared to crizotinib for the front‐line treatment of ALK‐positive advanced NSCLCs. Advanced non‐small cell lung cancer (NSCLC) is a deadly disease with a dismal prognosis.Crizotinib, the first FDA‐approved ALKi for the treatment of advanced ALK‐positive NSCLC, has shown superior efficacy compared to the standard of care chemotherapy docetaxel and pemetrexed‐cisplatin doublet in the second‐ and first‐line setting, respectively.Apart from alectinib which features a promising brain tissue penetration, another second generation ALKi ceritinib is characterized by a relatively high potency against ALK tyrosine kinase compared to crizotinib and alectinib.On the other hand, the adequate penetration of ceritinib to brain tissues for the control and prevention of brain metastasis remains a controversial issue. A previous study of a mouse model had shown that the penetration of ceritinib, as measured by the ratio of plasma to cerebral spinal fluid concentration, was only around 15% in which the active efflux of ceritinib by the drug transporters ABCB1 and ABCG2 still accounted for its reduced penetration to brain tissues.In the meantime, no dedicated clinical trial which directly compares the therapeutic outcome between ceritinib and crizotinib is available, except a few reports which indirectly compare the two drugs using the patients that propensity score‐matched from individual clinical trials originally conducted for the study of crizotinib and ceritinib, respectively.Between January 2015 and August 2018, a total of 110 patients with advanced or metastatic ALK fusion NSCLCs diagnosed by Ventana ALK (D5F3) CDx immunohistochemistry assay  at Chang Gung Memorial Hospital were retrospectively reviewed using Chang Gung Research Database. A total of 48 patients had ALK inhibitors as the front‐line treatment, in which 35 patients received crizotinib 250 mg twice daily and 13 patients received ceritinib 750 mg or 450 mg once daily. Progression‐free survival (PFS) was defined as the interval between the date of the start of ALK inhibitors and the date of either radiologically‐documented progression or death. The treatment response, defined as complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD), was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. The pattern of post‐ALK inhibitor disease progression was also reviewed and defined as either systemic progression without prior CNS progression/death or CNS progression without prior systemic progression/death. The toxicities noted during ALK inhibitor treatment were systemically reviewed and the toxicity was graded according to the National Cancer Institute Common Toxicity Criteria, version 5.0. The study was approved by the Ethics Committee of Chang Gung Memorial Hospital.P‐values were two‐sided, and a P‐value less than 0.05 was considered to be statistically significant. All the data were analyzed using SPSS 10.1 .The Mann‐Whitney test was used to determine the statistical significance between two groups of continuous variables; Fisher's exact tests were used for categorical variables. The Kaplan‐Meier survival curve was analyzed using the R package survival, and the hazard ratio was analyzed using the Cox regression model. For the propensity score matching analysis, the ceritinib versus the crizotinib group served as the dependent variable; the covariates used included age, smoking history, Eastern Cooperative Oncology Group performance status (ECOG PS) and brain metastasis. The coefficient for each covariate was determined by logistic regression analysis, and the propensity score of each individual was calculated as the sum of the product of each coefficient and the value of each covariate. The pairs of ceritinib and crizotinib individuals with equivalent propensity scores were selected in a 1:2 manner using the R package MatchIt. The patterns of post‐ALK inhibitor disease progression were treated as competing risk events of which the cumulative incidence functions were calculated.P = 0.016) and the frequency of smoking history in crizotinib group was lower than the ceritinib group  were extracted from the Chang Gung Research Database. Most of the baseline clinical characteristics, including sex, ECOG PS, brain metastasis and treatment for brain metastasis, were well balanced between the crizotinib and ceritinib groups (Table P = 0.033); and the median PFS with ceritinib treatment was 32.3 months , as compared with 12.9 months . The 18‐month PFS rate was significantly higher in the ceritinib group than in the crizotinib group , we performed propensity score matching analysis. After 1:2 matching according to the individual's propensity score—one who belonged to the ceritinib group and two who belonged to the crizotinib group—patients with balanced clinical profiles were selected Table . Among tP = 0.014, Fig P = 0.730, Fig The pattern of post‐treatment disease progression, either systemic or CNS progression, was analyzed in a fashion of competing risk based on a cumulative incidence rate. The rate of systemic progression was significantly lower over time with ceritinib treatment as compared to crizotinib treatment , diarrhea  and elevated AST/ALT . In the ceritinib group, the most commonly noted all grade adverse events included diarrhea , nausea/poor appetite  and vomiting  mutated NSCLC treated by osimertinib relative to gefitinib/erlotinib in the front‐line setting.EGFR‐mutated tumor and thereby significantly improves tumor control relative to the front‐line use of gefitinib/erlotinib. The successful suppression of the T790M tumor clone by the front‐line use of osimertinib can also be suggested by the subsequent mechanisms of acquired resistance in which the T790M‐mediated resistance can no longer be identified.The better therapeutic efficacy of ceritinib relative to crizotinib can be partly associated with a broader coverage of ceritinib treatment for multiple tumor clones, particularly for those with crizotinib‐resistant ALK mutations. This can be understood by considering a similar scenario of the epidermal growth factor receptor gene (In line with the above mentioned context, an in vitro study has shown that ceritinib was highly active against tumor clones L1196M, G1269A, C1156Y and I1171T/N/S, which were the top four crizotinib‐resistant mechanisms associated with the acquired ALK mutations.Although the present study had its inherent limitations of being of a retrospective nature with a small sample size, it is of value given the lack of a direct head to head comparison between ceritinib and crizotinib. In addition, this analysis also suggested that the differential therapeutic efficacy between the two drugs when they were administered in the front‐line to Asian patients should actually be massive, and therefore the difference could be easily discerned, even if the analysis only involved a small group of patients.In conclusion, this study demonstrated that the administration of ceritinib in the front‐line for the treatment of ALK‐positive NSCLC had superior efficacy than crizotinib, in which a better control of systemic progression could be attributed to this finding.No authors report any conflict of interest.


When listening to a narrative, the verbal expressions translate into meanings and flow of mental imagery. However, the same narrative can be heard quite differently based on differences in listeners' previous experiences and knowledge. We capitalized on such differences to disclose brain regions that support transformation of narrative into individualized propositional meanings and associated mental imagery by analyzing brain activity associated with behaviorally assessed individual meanings elicited by a narrative.Sixteen right‐handed female subjects were instructed to list words that best described what had come to their minds while listening to an eight‐minute narrative during functional magnetic resonance imaging (fMRI). The fMRI data were analyzed by calculating voxel‐wise intersubject correlation (ISC) values. We used latent semantic analysis (LSA) enhanced with Wordnet knowledge to measure semantic similarity of the produced words between subjects. Finally, we predicted the ISC with the semantic similarity using representational similarity analysis.We found that semantic similarity in these word listings between subjects, estimated using LSA combined with WordNet knowledge, predicting similarities in brain hemodynamic activity. Subject pairs whose individual semantics were similar also exhibited similar brain activity in the bilateral supramarginal and angular gyrus of the inferior parietal lobe, and in the occipital pole.Our results demonstrate, using a novel method to measure interindividual differences in semantics, brain mechanisms giving rise to semantics and associated imagery during narrative listening. During listening to a captivating narrative, the inferior parietal lobe and early visual cortical areas seem, thus, to support elicitation of individual meanings and flow of mental imagery. The intriguing question of how the human brain codes the semantics of language has been under investigation for decades  investigating brain mechanisms during listening (audio‐only), reading (time‐locked text‐only), and lipreading (silent video) a narrative  was used for presenting the stimuli. The audio stimuli were played with an MRI‐compatible in‐ear earbuds (Sensimetrics S14 insert earphones). In addition, MRI‐safe protecting earmuffs were placed over the earbuds for noise removal and safety. Sound intensity was adjusted for each subject during a dummy echo‐planar imaging (EPI) sequence before the actual experiment to be loud enough to be heard over the scanner noise by playing example stimuli that were normalized to the same level as the auditory stories. In the MRI scanner, the stimulus videos and texts were back‐projected on a semitransparent screen, using a Panasonic PT‐DZ110XEJ projector . The viewing distance was 35 cm.During narrative presentation, the subjects' brain hemodynamic activity was recorded with fMRI  at the Advanced Magnetic Imaging Centre of the Aalto University. Anatomical T1‐weighted structural images were acquired with 1 × 1 × 1 mm resolution .After the fMRI session, the subjects were presented the narrative again in writing, divided into 128 consecutive coherent phrases (3–5 s in duration), and were instructed to try to recall their interpretation (“what came to your mind”) during listening to the narrative when they first heard it in the scanner and to list, within 20–30 s, words best describing what had come to their minds. There were no limitations as to the type of words  or the amount of words, other than the time limit per segment.2.32.3.1The words were synonyms .One word was the direct hypernym of the other .One word was the two‐link indirect hypernym of the other .One adjective had a direct similar‐to relation with the other (handsome and beautiful).One adjective had a two‐link indirect similar‐to relation with the other .One word was a derivationally related form of the other .The words had the same stem but belonged to different parts of speech .First, the Finnish conjunctions were removed from the words and all words were translated into English. We then utilized LSA  is the path distance between x and y. The parameter α was set to 0.25 following previous recommendations  exceeded one, the excess was simply cut and the value set to one. The similarity measure between subjects was obtained by first calculating the similarity in each of the 128 segments by taking the average of the similarity values of all (3*3 = 9) word pairs, and then taking the average of these segment‐wise similarity values.where 2.42.4.1www.fmrib.ox.ac.uk/fsl) using the BRAMILA parallel preprocessing pipeline . First, after correcting for slice‐timing during acquisition, the EPI volumes were spatially realigned to the middle scan by rigid body transformations to correct for head movements using FSL MCFLIRT. EPI and structural images were coregistered and normalized to each individual's anatomical scan  followed by a linear transformation from anatomical to standard MNI template space . Finally, BOLD time series were detrended (linear detrend), motion parameters were regressed out .To reveal the brain areas related to semantic similarity, we predicted the ISC during listening against the semantic similarity (LSA combined with Wordnet). The significance was tested by conducting a representational similarity analysis using the Mantel test Mantel, . For eac3Behavioral responses of the subjects revealed that while some individuals perceived the story semantically similarly  and the second half to calculate ISC  as well as in cuneus in the visual cortex. The SMG and AG belong to the semantic network laid out in a previous meta‐analysis of the semantic system of human brain  after presenting a 7‐min narrative via two different modalities: an animated film without spoken dialogue and an audio description of the animation. By using LSA, they compared across subjects semantic similarity of free recalls of the animation and of the audio description, and observed that greater semantic similarity between subject pairs in their interpretations of the narrative, largely irrespective of modality, predicted ISC in the primary visual areas, premotor cortex, right AG, left SMG, and bilateral superior frontal gyrus. This approach, together with the present one, show that it is possible to quantify interindividual differences of semantic representations and mental imagery during narrative listening in the human brain. Our control analysis also suggests that a subject pair's tendency to elicit similar associations to segments during the first half of the narrative correlates with the pair's tendency to elicit associations to other segments of the story  that have been previously associated with semantic processing at the narrative level  measures, to estimate the “mental contents” of experimental subjects. This could include, for example, usability research where the recording of testing a human–machine interface is played back to the test subjects afterwards and they are asked to produce word lists describing what was on their minds during the testing. Clinical research might also benefit from the method, as it is possible to assess the thought patterns of patients compared to healthy volunteers while they, for example, watch a movie containing social interactions during neuroimaging, as significant differences in brain activity have been observed between, for example, high‐functioning autistic and neurotypical subjects, yet specific behavioural measures of differences in interpretation have been lacking . Click here for additional data file.


We report the case of an 85‐year‐old man who was surgically diagnosed with lung adenocarcinoma (pT2aN1M0 stage IIA). He was administered platinum combination chemotherapy as first‐line treatment for lung cancer recurrence. The patient's pleural fluid sample was obtained and analysed using a next‐generation sequencer, which demonstrated the presence of mesenchymal–epithelial transition gene (MET) exon 14 skipping mutations. As the patient developed progressive disease after receiving first‐line chemotherapy, crizotinib was administered as the second‐line treatment. The treatment was effective, and the patient had a stable disease for 7 months. This case suggests that crizotinib is effective against non‐small cell lung cancer with MET exon 14 alterations. Recently, various molecular targeted therapies have been developed and have shown positive effects against non‐small cell lung cancer (NSCLC) with a driver oncogene. Crizotinib, a multi‐targeted tyrosine kinase inhibitor, has been approved by regulatory agencies as a treatment for advanced NSCLC with anaplastic lymphoma kinase (ALK) or ROS1 rearrangements. Although crizotinib is not yet approved for use in NSCLC patients with mesenchymal–epithelial transition gene (MET) mutations, it was originally developed as a MET inhibitor. Hence, we report the case of a patient who had lung adenocarcinoma recurrence with a MET exon 14 (METex14) skipping mutation treated with crizotinib as a secondary chemotherapy drug. In this case, the administration of crizotinib was sufficient to control disease progression for 7 months.The patient was a non‐smoking 85‐year‐old Japanese man. He underwent lobectomy of the left upper lung and was diagnosed with lung adenocarcinoma (pT2N1M0 stage IIA) in October 2014. His lung cancer recurred in the left lower lobe, second lumbar bone, and left pleura in June 2016. A pleural fluid specimen was obtained and examined for the presence of genetic alterations using a next‐generation sequencer (NGS) and identified METex14 skipping mutations. Although the patient received four cycles of combination chemotherapy with carboplatin and pemetrexed (PEM), followed by one cycle of PEM as maintenance chemotherapy, in addition to pleurodesis with talc for malignant pleural effusion, computed tomography (CT) scan demonstrated progressive disease (PD) due to an increase in the number of multiple nodules in the lower left lobe and a left‐sided pleural effusion  was administered as secondary chemotherapy without health insurance coverage after receiving the patient's consent on 27 April 2017 and after obtaining approval from the ethics committee of our hospital. On 4 May, the patient experienced impairment in taste perception (grade 2), anorexia (grade 2), and photophobia (grade 1). However, these adverse events eventually resolved without any specific treatments. The dosage of crizotinib was reduced from 500 to 400 mg/day on 25 May due to the occurrence of renal dysfunction . On 31 July, the target lesion was slightly reduced as shown on the patient's CT scan, and the patient's disease remained stable. On 20 November, the target lesion was slightly increased in size as shown on the patient's CT scan, but there was no evidence of disease progression. Although his disease was controlled with crizotinib for 7 months, the treatment was discontinued as the patient developed anorexia and experienced financial difficulties  as a ligand. The activation of HGF/MET signalling pathway is involved in the survival, proliferation, migration, and invasion of cancer cells. METex14 alterations are very diverse. These alterations can be caused by loss, insertion, or replacement of the nucleotide sequence at the splicing site of MET exon 14 RNA and lead to increased MET protein stability and oncogenic potential Several studies reported the efficacy of crizotinib in NSCLC patients with METex14 alterations (Table Here, we report the case of a Japanese NSCLC patient with METex14 alterations who was successfully treated with crizotinib. This Asian elderly patient had stable disease for at least 7 months and developed minor adverse events after treatment with crizotinib alone. This case suggests that crizotinib is also effective in this patient population as reported in previous studies. After the ongoing phase II multicentre clinical trial in Japan, crizotinib might be approved for use in patients with advanced NSCLC with METex14 alterations.Appropriate written informed consent was obtained for publication of this case report and accompanying images.


FSWs) have strong economic incentives for sexual risk‐taking behaviour. We test whether knowledge of HIV status affects such behaviours among FSWs.Female sex workers  and experienced a change in knowledge of HIV status (71%). Knowledge of HIV status did not affect participants’ number of clients but did affect their consistent condom use. After controlling for individual fixed effects, study round and calendar month, knowledge of HIV‐negative status was associated with a significant increase in consistent condom use by 9.5 percentage points , while knowledge of HIV‐positive status was not associated with a significant change in consistent condom use .Most of the 960 participants tested for FSWs engaged in safer sex with clients when they perceived that they themselves were not living with HIV. Even in communities with very high HIV prevalence, the majority of the population will test HIV‐negative. Our results thus imply that expansion of HIV testing programmes may serve as a behavioural HIV prevention measure among FSWs.In urban Uganda,  To begin with, all previous studies among members of the general population were conducted prior to the emergence of evidence on the efficacy of TasP With this study, we aim to substantially strengthen the evidence on the effect of knowledge of HIV status on sexual behaviour among FSWs in the current era. We do so using cohort data, which allows us to use a quasi‐experimental estimation approach: individual fixed effects estimation. The individual fixed effects remove potential bias due to all individual‐level confounding factors – whether observed or unobserved – that are stable over the study period of four months. In addition, we control for study round and calendar time, adding further causal strength by removing bias due to study round‐ and time‐varying confounding factors.Understanding the effect of knowledge of HIV status on sexual behaviours among FSWs is important for policy and practice. In general, it can support the design of interventions to ensure that HIV testing initiatives are best leveraged for sexual behaviour risk reduction. It can also specifically help ensure that HIV testing interventions targeted at FSWs decrease, rather than increase, the risk of HIV transmission between FSWs and their clients.22.1The cohort of FSWs we are using for the analyses in this paper was formed as part of a three‐arm cluster‐randomized controlled trial of HIV self‐testing delivery models in urban Uganda, which was carried out from 2016 to 2017 Over the duration of the trial, participants had ample opportunity for HIV testing. All participants were referred to free standard of care testing services by a FSW peer educator. Participants in the HIV self‐testing intervention arms received either an HIV self‐test kit or a coupon for an HIV self‐test kit from their peer educator shortly after enrolment and again three months later. We used the OraQuick Rapid HIV‐1/2 Antibody Tests  in this study, which have 93% sensitivity and 99% specificity compared to standard‐of‐care blood‐based HIV testing Ethical approval for the trial was granted by the institutional review board at the Harvard T.H. Chan School of Public Health and the Mildmay Uganda Research Ethics Committee. We obtained written informed consent from all participants.2.2Kampala, the capital city of Uganda, is a major centre of commerce in East Africa. Many migrants move to Kampala to pursue economic opportunities including sex work. There are approximately 13,000 FSWs in Kampala; one in three is estimated to be living with HIV 2.3Participants were recruited for the trial by FSW peer educators and assessed for eligibility by research assistants 2.4Participants completed three study rounds of data collection: at baseline, one month, and four months. The timing of these rounds was the same for all participants. All data were collected electronically  by research assistants in face‐to‐face interviews at private locations selected by participants .2.5We measured two sexual behaviour outcomes: (i) participants’ number of clients on an average working night and (ii) participants’ consistent condom use with clients. At all study rounds participants were asked to report the number of clients they have sex with on an average working night and the number of clients with whom they use a condom. If participants reported using condoms with all their clients, their condom use was categorized as consistent.2.6We categorized participants’ knowledge of HIV status into three states: (i) knowledge of HIV‐negative status, (ii) knowledge of unknown HIV status, and (iii) knowledge of HIV‐positive status. The knowledge we refer to is the perception of HIV status. We chose this measurement for knowledge of HIV status because it is the perception of HIV status that will primarily determine sexual behaviour. For instance, if a FSW is in fact HIV‐negative but believes that she is HIV‐positive, it is the belief in her HIV status that will guide her behavioural choices rather than the biological status. Pathways through which perceived knowledge of HIV status are likely to affect sexual behaviour include emotions and considerations such as “I am HIV‐positive and thus need to protect my HIV‐negative partners” or “I am already HIV‐positive and thus do not need to protect myself.”In this study, participants’ self‐perceived knowledge of HIV status may not match the results of their most recent HIV test for a number of reasons including, (i) sexual encounters after the most recent HIV test, (ii) mistrust of a new HIV testing technology (i.e. self‐testing), (iii) uncertainty surrounding their own ability to correctly interpret HIV self‐test results, and (iv) outside interventions intended to cure individuals of HIV  To capture participants’ knowledge of HIV status, at each study round we asked participants to estimate the likelihood that they were currently living with HIV using a 10‐rung ladder scale – an approach adapted from a previous study that measured participants’ perceived risk of HIV acquisition 2.7We measured sociodemographic characteristics at baseline. At each study round participants reported recent HIV testing  and the results of their most recent HIV test. Our electronic data collection platform automatically captured the study round  and calendar month of observation. Because of the spacing of the study rounds, there were some calendar months between the second and last study round when no data were collected. At four months, all participants who gave consent completed a blood‐based rapid HIV test.2.8We used individual fixed effects analysis of longitudinal data to estimate the association between participants' knowledge of HIV status and sexual behaviours. Individual fixed effects estimation controls for all observed and unobserved confounders that do not vary over time within an individual However, individual fixed effects do not control for confounders that vary over time within an individual. We thus added two time‐varying factors, study round and calendar month, as independent variables. The study round fixed effects control for confounders that may have changed with study round in similar ways across participants. Such confounders may include changes in social desirability bias due to participation in this study and associations between HIV status knowledge and sexual behaviour that arise because participants in the study are encouraged to both test for HIV and to reduce HIV risk‐related sexual behaviour. The calendar month fixed effects then control for time‐varying changes, such as social marketing campaigns, health system reforms or general underlying societal trends, which affect all participants. To maximize the statistical efficiency of our analyses, we selected the category with the greatest number of observations as the reference for each of these variables We used linear panel regression for estimation. Because peer educators were involved in the recruitment of participants and trained participants on how to interpret HIV self‐test results, we adjusted our standard errors for clustering at the level of the peer educator.To understand how the direction of change in HIV status knowledge is associated with changes in participants’ sexual behaviours, we conducted a number of sub‐group analyses. We divided participants into sub‐groups based on their knowledge of HIV status  and sexual behaviours (low risk and high risk) at baseline. By dividing participants by their knowledge of HIV status at baseline, we can better understand how specific changes in knowledge of HIV status from prior knowledge states may be differentially associated with changes in sexual behaviours. For example, a change in knowledge of status from HIV‐negative to HIV‐positive may lead to different sexual behaviours than a change in knowledge of HIV status from unknown to HIV‐positive. We then further divided participants by HIV risk‐related sexual behaviours at baseline , because we can only observe changes in sexual behaviours among participants that are able to change their sexual behaviours from baseline. Examples of directional changes in sexual behaviour following a particular directional change in HIV status knowledge include (i) FSWs who learn that they are HIV‐negative decreasing HIV risk‐related sexual behaviours and (ii) FSWs who learn that they are HIV‐positive increasing HIV risk‐related sexual behaviours. For all sub‐group analyses, we used linear panel regressions with individual fixed effects, controlling for study round and calendar month. We adjusted standard errors for clustering at the level of the peer educator.We used Stata 13.1  for all analyses in this study.33.1From October to November 2016, 1587 potential participants were phone‐screened for eligibility, of which 997 were invited for in‐person eligibility assessment, and 960 were enrolled. Some of the most common reasons for study exclusion were reported HIV testing within the past three months  and self‐reported HIV‐positive status .The descriptive characteristics and sexual behaviours of the 960 study participants at baseline are shown in Table Loss to follow‐up was low in this cohort 3.2Figure At four months, 92% (340/368) of participants who reported knowledge of HIV‐negative status tested HIV‐negative in a blood‐based rapid test, while 83% (104/126) of participants who reported knowledge of HIV‐positive status tested HIV‐positive in a blood‐based rapid test.3.3p < 0.001) higher among participants with knowledge of HIV‐negative status, and 2.5 percentage points  lower among participants with knowledge of HIV‐positive status. Knowledge of HIV‐positive status, however, was not significantly associated with participants’ condom use with clients.Figure We conducted four sensitivity analyses to confirm the association between participants’ knowledge of HIV status and sexual behaviours with clients. First, we tested the robustness of our linear regressions with Poisson regressions for number of clients per average working night and logistic regressions for consistent condom use with clients. Second, we tested the influence of adjusting for time‐varying confounders  in our linear regressions by removing these variables from our analyses and running unadjusted models. Third, we tested the robustness of our measure of knowledge of HIV status. Instead of three categories of status knowledge, we used the full 10‐point scale that we used to elicit this information. We used ladder rung 5 as a reference category for these analyses, because it contained the greatest number of observations and thus improved the efficiency of our estimations p < 0.001), (ii) when we used unadjusted linear regressions , (iii) when we used a 10‐point HIV knowledge scale , and (iv) when we limited our sample to participants who reported HIV testing since the start of the study . Additionally, knowledge of HIV‐negative status significantly decreased participants’ number of clients on an average working night in the sensitivity analysis with unadjusted linear regressions (ii), but none of the other sensitivity analyses: (i), (iii), (iv). Knowledge of HIV‐positive status was not significantly associated with participants’ condom use or number of clients on an average working night in sensitivity analyses (i) to (iv).The findings from our main analyses remained consistent in all four sensitivity analyses (results shown in the Table S1). Knowledge of HIV‐negative status was significantly associated with an increase in participants’ consistent condom use with clients (i) when we used logistic regressions  and consistent condom use with clients . It is also possible that the different peer educators differentially affected both uptake of HIV testing and social desirability in reporting sexual behaviours, leading to time‐varying confounding of the relationship between knowledge of HIV status and condom use Another limitation of our study is that there may have been measurement error in participants’ reported sexual behaviours. In this study, participants were asked to report the number of sexual clients they have on an average working night. Reporting averages may have been difficult for some participants, especially considering that the majority of participants had either no formal education or only some primary education. Since condom use was measured in relation to number of clients on an average working night, this measurement might reflect more of an attitude towards condom use than a precise estimate. There also may have been some ambiguity about the definition of a sexual client, and depending on the nature of the sex act (which we did not specify in this question) condom use may have been less or more frequent. Despite these limitations, we still saw a significant association between knowledge of HIV‐negative status and participants’ condom use with clients. Future analyses should consider using more precise estimates for number of clients and condom use with clients (e.g. number of clients and condom use within a defined time period).The participants in our study may also have been more likely to change their sexual behaviours over time compared to other FSWs because of their participation in study‐related activities Participants in our study were followed‐up for a duration of four months. This duration of follow‐up is adequate to determine the short‐term effects of knowledge of HIV status on sexual behaviours. The FSWs in our sample – unlike people in the general population – have many sexual encounters almost every day and changes in knowledge of HIV status can thus lead to observable changes in sexual behaviours nearly immediately. Hence, the four‐month follow‐up period is able to detect the associations of interest, and it is relatively long considering the potential for sexual behaviours to change nearly every day.However, it is possible that longer‐term effects – for instance, years after a change in knowledge of HIV status – differ substantially from those that evolve over a few months’ time. For example, a FSW who recently learned she was living with HIV might be in shock or feel angry, and thus may not be able or inclined to use condoms to protect her sexual partners from HIV infection. Over time, however, this FSW might come to terms with her HIV status and consequently increase condom use with clients and other sexual partners. Future studies with longer duration of follow‐up should be conducted to gain insights into the effects of HIV status knowledge over several years’ time.Finally, our results may have limited generalizability. The FSWs who participated in our cohort may have self‐selected on characteristics that affect the relationship between HIV status knowledge and sexual behaviours. Moreover, our associations are limited to participants who changed their knowledge of HIV status over the study period. The latter limitation is unlikely to be severe, however, because the majority of participants (>70%) did change their knowledge of HIV status over the study period. These common changes were likely caused by the increased encouragement and opportunities for HIV testing due to participation in the HIV self‐testing trial.More broadly, FSW populations across sub‐Saharan Africa are highly diverse 5Frequent HIV testing among FSWs is an HIV prevention strategy recommended by the World Health Organization The authors of this study declare no conflicts of interest.KFO, MAM, JEH, JAS, CEO and TB conceptualized the paper. KFO conducted the analysis and wrote the first draft. MAM, JEH, JAS, CEO and TB supervised the methodology. KFO, CEO, TN, AN and MC oversaw study administration and the collection of quality data. All authors edited the draft and provided insights into the manuscript.Table S1. Sensitivity analyses: the effect of knowledge of HIV status on HIV risk‐related sexual behavioursClick here for additional data file.


Picea sitchensis (Bong.) Carr.) by analyzing a total of 276 Gb of nuclear DNA from the tops and bottoms of 20 old‐growth trees averaging 76 m in height. We estimate a somatic base substitution rate of 2.7 × 10−8 per base pair within a generation. To date, this is one of the highest estimated per‐generation rates of mutation among eukaryotes, indicating that somatic mutations contribute substantially to the total per‐generation mutation rate in conifers. Nevertheless, as the sampled trees are centuries old, the per‐year rate is low in comparison with nontree taxa. We argue that although somatic mutations raise genetic load in conifers, they generate important genetic variation and enable selection both among cell lineages within individual trees and among offspring.The rates and biological significance of somatic mutations have long been a subject of debate. Somatic mutations in plants are expected to accumulate with vegetative growth and time, yet rates of somatic mutations are unknown for conifers, which can reach exceptional sizes and ages. We investigated somatic mutation rates in the conifer Sitka spruce ( The long lifespans of trees hamper their ability to adapt to a changing environment; and yet, paradoxically, tree populations are often well adapted to local climates and evolve effective responses to changing stresses such as herbivorous insects. A plausible explanation is that trees produce many more mutations in nonreproductive cells, called somatic mutations, that can be inherited because trees do not have a segregated germline . Somatic mutations could alter how trees evolve by generating more genetic variation upon which natural selection can act. We document a very high rate of somatic mutation between the base and tip of Sitka spruce trees that are centuries old.Long‐lived trees face an evolutionary challenge: they must adapt despite generation times that are decades or centuries long. While facing substantial environmental change per generation, conifers often display strong adaptation to their local environments  is roughly 25 times higher than in short‐lived barley (Hordeum vulgare) and buckwheat (Fagopyrum esculentum). However, indirect estimates of the total mutation rate, including meiosis and the mitoses required for gamete production, are generally low for trees when calculated per year , identifying ∼40 nucleotide differences , estimated to be 220 to 500 years old . We climbed each tree to obtain a sample of young foliage from a single third‐ or fourth‐order twig as high in the crown as possible, which we dried in silica beads and divided into two biological replicates . To infer a consensus genotype of the tree as a seedling, we took the bark samples from opposite sides of the trunk. Because the most recent common ancestor of the bark samples was a cell in the center of the tree, near its meristem as a young seedling, any genotype shared by both bark samples is likely to represent the genotype of the young seedling itself Fig. B. We reqGenetic differences between the bark (lower) and foliage (upper) samples thus represent somatic mutations that occurred in the apical meristem during height growth, suggesting that—in the absence of chimerism in the apical meristem—they are likely heritable. Although one might initially think that a somatic mutation rate could also be estimated by comparing the two bark samples from a tree, the radial growth pattern of trees implies that a bark sample will consist of cells that have descended according to an almost star‐like phylogeny, with a most recent common ancestor also near the center of the tree, reducing the genetic difference expected to accumulate between the two bark samples (Supporting Information Note 1).Picea glauca × engelmannii hybrids  sequenced was filtered in two steps, one filtering out sites with poor‐quality sequence data (“site‐level filters”) and the next filtering out weakly supported sample genotypes  across all trees were less reliable—presumably because they indicate alignment errors—we divided variable sites into two categories, rare (MAF ≤ 0.05) and frequent (MAF > 0.05), applying more stringent filters on sites where the minor allele was frequent. We first discarded indels and multiallelic sites and then applied a series of filters that do not alter the relative proportions of mutant and nonmutant sites: the site‐level filters labeled “all sites” in Table For the genotype‐level filters, we defined homozygous‐reference, heterozygous, and homozygous‐alternate genotypes (relative to the hybrid spruce reference) by setting criteria for the proportion of reads supporting the reference and alternate alleles. We also applied the genotype‐level filters listed in Table I and V, below).Only the filters labeled “all sites” in Table For sites that passed all the filters, tree genotypes were retained and checked for mutations only if all four sample genotypes passed the filters. We considered any tree genotype for which both bark (lower) samples have the same genotype, both foliage (upper) samples have the same genotype, and the bark and foliage genotypes differ to be a candidate somatic mutation. We thus obtained two sets of candidate mutations, one each from the higher‐ and lower‐confidence pools.All reads that showed the candidate mutant allele also showed a unique allele at a nearby site in the heterozygous samples, and at least one of the homozygous samples contained reads supporting the two alleles.At the candidate site and at a nearby polymorphic site, there were three haplotypes in the heterozygous samples, each supported by multiple reads.All four samples contained at least one read supporting the candidate mutant allele.For candidate mutations in the lower‐confidence pool, we first used the Integrative Genomics Viewer  for sequencing. If good‐quality Sanger sequence showed no mutant allele in a putatively heterozygous sample, we considered the candidate mutation to be a false positive. If the mutant allele was both confirmed in a putative heterozygous sample and absent in a putative homozygous sample from a tree, we considered the candidate mutation to be a true positive.Picea glauca × engelmannii transcriptome, from which the impact on the predicted protein was inferred  that were checked for mutations, and we then used the fraction of tree genotypes at variable sites that passed filters to determine how many tree genotypes from invariant sites to include as well. This procedure was necessary because some of the filters applied only to variable sites, including mutant sites .I is the number of tree genotypes at invariant sites that passed the all‐sites filters, V is the number of tree genotypes at variable sites that passed the all‐sites filters, and σ rare  and σ freq represent the number of tree genotypes at variable sites with MAF ≤ 0.05 and MAF > 0.05, respectively, that also passed the SNP‐only filters and were subsequently checked for mutations . The fraction in I and V depend on the choice of filters σ raTo calculate the mutation rates for the higher‐ and lower‐confidence pools, the number of verified mutations from a pool was divided by the size of the corresponding search space, and then divided by two to translate the mutation rate at diploid sites to a haploid rate. This mutation rate is given within a generation, rather than per generation, because the age of the trees is unlikely to be the average generation time for the species and because our estimate excludes mutations that arise during seed production or early development. We divided the mutation rate within a generation by 360 years, the midpoint of the age range for similar old growth trees in the same stand  with a depth of at least five in all four samples was 12,289,511. After applying all filters, there remained a total of 2,291,498 tree genotypes at variable sites in the higher‐confidence pool (18.6%) and 3,762,052 tree genotypes at variable sites in the lower‐confidence pool (30.6%). Many of these sites were in exons  was 124,959,176, and the number of tree genotypes at variable sites that passed the all sites filters (V) was 4,903,088 . After the heterozygosity correction . The cumulative search space across all trees was therefore estimated to be 56.5 Mb for the higher‐confidence pool and 84.1 Mb for the lower‐confidence pool, comprising both a subset of the target regions for each tree and also nontarget sites covered by off‐target reads.We then calculated the size of the search space using Comparing the consensus genotypes from the bark samples to the foliage samples using the conservative filters yielded 24 candidate base substitutions in the higher‐confidence pool. Of these, Sanger sequencing verified three, identified 16 as false positives, and was inconclusive in five cases. When we applied the IGV criteria (see Methods) to the 24 candidate mutations, only the three mutations later verified by Sanger sequencing were predicted to be true positives. Of the five candidate mutations that we could not Sanger sequence, two appeared to be paralogs with three haplotypes, and three had roughly equal numbers of reads supporting each allele in all four samples from the tree, suggesting that all five were false positives.Using more relaxed filters, we obtained 141 candidate somatic mutations in the lower‐confidence pool. For those which were not included in the higher‐confidence pool, we first used the IGV method to eliminate false positives and rejected all but three. For these three, Sanger sequencing verified two as somatic mutations but identified the third as a false positive. In total, across both the higher‐ and lower‐confidence pools, we verified five somatic base substitutions within the genomic regions scanned.Picea transcriptome assembly  per base pair within a generation for the high‐confidence pool, or 7.4 × 10−11  base substitutions per base pair per year  or 5.3 × 10−11 (500 years).We estimate a somatic base substitution rate of 2.7 × 10ear Fig. . If we uear Fig. , we obta−8  per base pair within a generation using the full set of five verified mutations.Extrapolating our estimated mutation rate from the number of postfiltered sites to the entire diploid genome of Sitka spruce  suggests that mutant genotypes are unlikely to have been miscalled as wild‐type during the SNP calling process (see Methods).P = 0.94; low confidence‐pool: P = 0.93), although this is admittedly based on very few mutations.The somatic base substitution rate that we report for old‐growth Sitka spruce is among the highest per‐generation mutation rates for any eukaryote Fig. A, althouDifferences in age and height among trees within a species  being 40% shorter than the average. The remaining discrepancy may be explained by episodes of faster mutation in the history of the genus , by the advanced age of the trees in this study, by purifying selection within meristems, or by our exclusion of the mutations that arise each generation during meiosis, as well as the mitoses involved in seed production and seedling growth.Our estimated mutation rate per year is, however, an order of magnitude lower than the neutral substitution rate inferred for spruce from molecular phylogenetics , we roughly estimated the total amount of genetic variation that somatic mutations might generate across all fertile branches of a tree within a generation. Approximating the number and length of first‐, second‐, and third‐order branches leading to seed or pollen cones over the life of an old‐growth Sitka spruce, we predict that on the order of 100,000 base substitutions could occur somewhere during development in total across all fertile branch lineages in these large trees. Indels, inversions, and structural changes would generate additional genetic variation. The vast majority of these mutations would occur late in development, however, affecting only a few cones, as the majority of the total branch length is amassed at the tips of branches.Selection within or among meristems could act on such variation within an individual tree. Such selection would skew the distribution of fitness effects for heritable mutations by removing variants that are deleterious at the cell or branch level, and by improving the probability that beneficial mutations fix in meristematic cell lineages Gaul . In partPissodes strobi; Mitchell et al. The architecture and development of conifers must affect the efficacy of selection within the individual. The relatively large number of apical initials in conifers . Strong selection among seedlings  would increase the probability that survivors bear very rare beneficial mutations. As a result of accessing a large pool of somatic mutations among seeds, conifers might display faster adaptive evolution than their long generation times would suggest.The high somatic mutation rate of Sitka spruce within a generation must also generate much of the genetic variation that creates and maintains local adaptation. Because conifers often have large effective population sizes , we would predict 135 mitoses within a generation for our samples from Sitka spruce. Consequently, cell divisions in a lineage of apical initials may happen years apart for ∼360‐year‐old trees. Our results are thus broadly consistent with the view that the apical meristems of tall conifers undergo fewer mitotic cell divisions than expected based on their height and, in so doing, bear fewer mutations per year.These comparisons suggest that the number of mitotic divisions in spruce is an order of magnitude smaller than 2000. Indeed, if we were to assume that the mutation rate in plants is constant at 2 × 10We offer a direct genomic estimate of a somatic mutation rate in a conifer. The somatic base substitution rate is very high in old‐growth Sitka spruce within a generation but very low per year. Somatic mutations will considerably increase genetic load, which depends on the per‐generation deleterious mutation rate. The relevance of somatic mutations for adaptation to fast‐changing conditions is, however, limited by the long generation times and strong apical dominance of conifers, unless beneficial variants increase the proliferation of cells or branches within a tree and thereby their odds of persistence. Additionally, somatic mutations may facilitate adaptation when the few seeds that bear locally adapted mutations are substantially more likely to survive.The authors declare no conflict of interest.Associate Editor: L. BromhamSupporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Table S1. Heights and branch lengths of the trees in this study.Figure S1. The fixation probability of a mutation beneficial at the cell level divided by the fixation probability of a neutral mutation Psel/Pneutral versus the mutation's selective advantage at the cell level (s).η is the effective number of replicating apical initial cells (see Supporting Information Note 4).Table S2. Data and references for Figure 2.Table S3. Values for the calculation of the heterozygosity correction used for the calculation of the reported mutation rates (see Supporting Information Note 2).Table S4. Alternative values for the calculation of the heterozygosity correction (see Supporting Information Note 2).Click here for additional data file.


Living systems are characterised by an ability to sustain chemical reaction networks far‐from‐equilibrium. It is likely that life first arose through a process of continual disruption of equilibrium states in recursive reaction networks, driven by periodic environmental changes. Herein, we report the emergence of proto‐enzymatic function from recursive polymerisation reactions using amino acids and glycolic acid. Reactions were kept out of equilibrium by diluting products 9:1 in fresh starting solution at the end of each recursive cycle, and the development of complex high molecular weight species is explored using a new metric, the Mass Index, which allows the complexity of the system to be explored as a function of cycle. This process was carried out on a range of different mineral environments. We explored the hypothesis that disrupting equilibrium via recursive cycling imposes a selection pressure and subsequent boundary conditions on products. After just four reaction cycles, product mixtures from recursive reactions exhibit greater catalytic activity and truncation of product space towards higher‐molecular‐weight species compared to non‐recursive controls. Biological entities can be viewed as self‐propagating networks of autocatalytic sets in a sustained far‐from‐equilibrium state, in which the stoichiometry of all functionally active components is maintained between generations.Chemical recursion is key not only for maintaining the dynamic, far‐from‐equilibrium states that characterise biology, but also for providing a means of imparting a “chemical history” on generations of cyclical chemical reactions, in which the outcome of one reaction cycle is partly influenced by those that have preceded it. Without such programmed history, there is no possibility for the emergence of evolving chemical systems and therefore life.Experimental frameworks for generating artificial life through recursive selection have been proposed;Confirming whether this has been achieved may be an equally, if not more, daunting task.l‐leucine, l‐glutamic acid, and l‐lysine) and one α‐hydroxy acid (glycolic acid) were chosen as a model reaction system. A fixed ratio of monomers was reacted on various mineral surfaces in a recursive manner, with approximately 10 % of the product mixture seeded on to fresh mineral and monomer feedstock solutions at the end of each reaction cycle. Nine environments were chosen to steer reaction outcomes: crushed glass, montmorillonite, gypsum, quartz, calcite, chalcopyrite, opal, and kernite, along with a mineral‐free environment. The selected minerals were chosen for their chemical and morphological diversity . In previous work, we had examined the ability of minerals and salts to influence the product distribution of similar uncontrolled condensation reactions.We carried out multiple recursive cycles of depsipeptide co‐polymerisation on various mineral substrates in order to 1) assess the robustness of any effects of recursive cycling and 2) provide a means of steering the reaction down different trajectories or “chemical histories”. Three amino acids , which breaks down into yellow p‐nitrophenol (pNP) and acetate, was used as an esterase substrate for the hydrolytic assay.Having observed the effect of recursion on the composition of depsipeptide products, the next step was to test for the emergence of function. A hydrolytic assay was used to determine the esterase activity of products. Ester bond hydrolysis is an important step in the ester–amide exchange reaction that enables depsipeptide elongation,The increase in esterase activity with chemical recursion suggests some form of selective advantage of functionally active products in a recursive system. Interestingly, this also correlates with the increase in lysine and depletion of leucine content of products .Structural characterisation in solution was carried out to assess any impact of this change in composition on secondary structure formation in products. Circular dichroism profiles of mixtures differed markedly from starting material controls, with a characteristic β‐sheet signal at 210 nm, indicating potential secondary structure formation consistent with lysine‐rich sequences.In this work, the emergence of selection and functionality from unconstrained, recursive depsipeptide polymerisation has been demonstrated for the first time. Further work is ongoing to determine the precise mechanism of this phenomenon. Recursive cycling was used to induce a selection pressure on the system. The ability of mineral surfaces to selectively stabilise small molecules is well documented, as is the heat lability of many depsipeptides.The 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.


This microreview details recent developments in stimuli‐responsive polymers with phosphorus in the main‐chain, in particular polyphosphazenes and polyphosphoesters. The presence of phosphorus in the polymers endows unique properties onto the macromolecules, which can be utilized for the preparation of materials capable of physically responding to specific stimuli. Achieving the desired responsiveness has been much facilitated by recent developments in synthetic polymer chemistry, in particular controlled synthesis and backbone functionalization phosphorus‐based polymers, in order to achieve the required properties and hence responsiveness of the materials. The development of phosphorus‐based polymers which respond to the most important stimuli are discussed, namely, pH, oxidation, reduction, temperature and biological triggers. The polymers are placed in the context not just of each other but also with reference to state‐of‐the‐art organic polymers. While the importance of polymers both as commodity and as high‐performance materials is long established, in recent decades a better fundamental understanding of structure property relationships has been combined with vast improvements in controlled polymerization methods. This has allowed for the preparation of materials with evermore finely tuned macromolecular structures and with it, more advanced and precise tailor‐made properties. Among these developments, stimuli‐responsive polymers,Phosphorus‐based polymers, while much less prevalent than their carbon‐based analogues, are of widespread interest in a number of areas in polymer science. Indeed since the biopolymers DNA and RNA are macromolecules with phosphorus in the main‐chain and poly(phosphate) and its cleavage products are responsible for a variety of essential pathways,Phosphorus‐containing moieties can be readily incorporated onto organic polymers, for example via vinyl monomers with phosphonic acid or acidic phosphates, or alternatively phosphorus can be incorporated as branching units in well‐defined dendrimers, with both routes leading to rich families of functional polymers. The reader's attention is directed elsewhere for a review of these substances  are of particular significance, for example as polymer therapeutics or for the encapsulation of active pharmaceutical ingredients. There are numerous examples of cleavable organic polymers incorporating acid‐labile moieties, for example acid‐sensitive acetal moietiesN‐ethylpyrrolidone substituted polyphosphazenes undergo accelerated degradation at pH 3 compared to pH 7.4.The polyphosphazene backbone shows an inherent tendency to hydrolyze, a process which has been confirmed in numerous studies to be significantly accelerated under acidic conditions.The reason for the selective backbone cleavage of polyphosphazenes at acidic pH values is due to protonation of the nucleophilic nitrogen in the backbone, which facilitates nucleophilic attack of water and hence hydrolysis of the P–N bonds. The proposed degradation mechanism is shown in Figure Polyphosphoesters are also well known to undergo hydrolytic main‐chain cleavage. In contrast to polyphosphazenes, a significantly faster rate of cleavage is usually observed under basic conditions,p‐methylphenoxy substituents which show a pH dependent release profile.Acid‐cleavable polyphosphoramidates have also been investigated in nanoformulations, for example with the anticancer drug campthothecin.For the design of triggered‐release polymeric systems, many drug linker strategies can be adapted from well‐established organic or indeed organometallic prodrug systems.Alternatively, hyperbranched polyphosphoesters, with their excellent aqueous solubility and ample end‐groups for functionalization have also been highly successfully applied with a wide range of release systems in this field.In a similar approach, our group, in collaboration with others recently investigated the covalent linkage of ruthenium half‐sandwich compounds with branched polyphosphazenes to prepare macromolecular prodrugsFigureà.35 The rMacromolecules are known to undergo cell uptake via endocytosis pathwaysIV prodrugs designed to undergo intracellular reduction.IV loaded macromolecular carriers. Attachment via axial ligands meant the Pt complexes underwent simultaneous release from the macromolecule upon reduction. Due to the endocytic pathway, not accesible to the small‐molecule Pt species, a considerable 30‐fold increase in the uptake was observed for the polymer‐bound drugs. While the PtIV drug showed minimal cytotoxic effects due to its low uptake, the loaded macromolecular prodrug was highly active against a number of cancer cell lines  moieties in a polymer backbone with hydrogen peroxide. D. P. Gates and co‐workers have shown, for example, that when The use of light as a stimulus to evoke a response in polymer materials is particularly attractive, enabling spatiotemporal control over the desired response. Such materials are highly sought after in fields such as photolithography, controlled drug release and for the preparation of advanced sensory materials.Polymers which can be cleaved or undergo backbone disassembly upon response to light in a spatiotemporal manner are of interest as positive photoresists or in controlled release applicationsThe optical transparency of the polyphosphazene main‐chain lies between the near‐infrared to below 220 nmIn an example from our laboratory, hypericin, a clinical photosensitizer could be conjugated to water‐soluble polyphosphazenes.The degradability and ease of loading means polyphosphoesters also lend themselves as macromolecular carriers for photosensitizers. Recently Yang and co‐workers have shown that the photosensitizer chlorin e6 could be loaded onto hyperbranched polyphosphoesters.It may be anticipated that polymers containing phosphate in the main‐chain would be susceptible to enzymatic cleavage, and it is often cited as a characteristic of polyphosphoesters. However,While there are no reports of polyphosphazene main‐chain itself undergoing enzymatic cleavage, polyphosphazenes with degradation rates accelerated by enzymes have been investigated by our groupPolymers that undergo sudden conformational transitions in response to small changes in temperature can be termed as thermosensitive or thermoresponsive. One of the most studied such materials are polymers which possess a lower critical solution temperature (LCST) transition in aqueous environments, that is the polymer chains collapse at the LCST leading to precipitation and/or gelation.A number of thermoresponsive polyphosphoesters have been prepared, the phosphorus functionalization allowing tuning of the LCST values.Polyphosphazenes demonstrating thermoresponsive behavior due to an LCST have also been widely reported, the main route being via backbone substitution with amphiphilic oligomers. For example our group showed a simple route to prepare polymers with LCST values in the range of 18 °C to 90 °CA broad family of polymers are described, which can respond to several stimuli, either as a direct consequence of phosphorus in the main‐chain or through its use as an easily functionalized connection point for responsive moieties. Although not the only field of use, a major development has been in the biomedical direction due to properties of biodegradability and biocompatibility, a desirable basis for materials intended for this field. In particular, the ability to combine controlled molecular weights with multivalency and post‐polymerization functionalization is important for the stimuli‐responsive materials discussed herein, since it is the chemical functionality and the molecular weight of the polymers that determine the responsive behavior of polymers. The stimuli‐responsive materials detailed herein demonstrate a number of possible gains for phosphorus‐based polymers over classical organic polymers and represent a burgeoning field with still much untapped potential.