Theobroma cacao leaves with Colletotrichum tropicale (E+), the dominant FEF species in healthy T. cacao, induces consistent changes in the expression of hundreds of host genes, including many with known defensive functions. Further, E+ plants exhibited increased lignin and cellulose content, reduced maximum rates of photosynthesis (Amax), and enrichment of nitrogen-15 and carbon-13 isotopes. These phenotypic changes observed in E+ plants correspond to changes in expression of specific functional genes in related pathways. Moreover, a cacao gene (Tc00g04254) highly up-regulated by C. tropicale also confers resistance to pathogen damage in the absence of endophytes or their products in host tissues. Thus, the benefits of increased pathogen resistance in E+ plants are derived in part from up-regulation of intrinsic host defense responses, and appear to be offset by potential costs including reduced photosynthesis, altered host nitrogen metabolism, and endophyte heterotrophy of host tissues. Similar effects are likely in most plant-endophyte interactions, and should be recognized in the design and interpretation of genetic and phenotypic studies of plants.It is increasingly recognized that macro-organisms  consist of both host tissues and multiple microbial symbionts that play essential roles in their host's ecological and evolutionary success. Consequently, identifying benefits and costs of symbioses, as well as mechanisms underlying them are research priorities. All plants surveyed under natural conditions harbor foliar endophytic fungi (FEF) in their leaf tissues, often at high densities. Despite producing no visible effects on their hosts, experiments have nonetheless shown that FEF reduce pathogen and herbivore damage. Here, combining results from three genomic, and two physiological experiments, we demonstrate pervasive genetic and phenotypic effects of the apparently asymptomatic endophytes on their hosts. Specifically, inoculation of endophyte-free (E−)  Although non-pathogenic, microbial symbionts are increasingly recognized for their often profoundly beneficial influences on hosts  offer a tractable experimental system in which the effects of endophytes on their host genetic and phenotypic expression can readily be assessed. All plants surveyed under natural conditions harbor FEF. Despite the fact that they are living in high density or abundance within plant tissues, they produce few, if any, visible effects on their hosts  consistently dominating the fungal assemblage, in any host plant that also includes many relatively rare species  and the completed sequences of the T. cacao and Arabidopsis thaliana genomes, we assessed changes in host gene expression in response to endophyte inoculation by comparing leaves inoculated with the foliar endophyte C. tropicale (E+) to endophyte-free, un-inoculated leaves (E−), in three experiments in which seedlings were grown under growth chamber conditions. In the leaf tissue from the first gene expression experiment we also determined changes in lignin and cellulose content. In addition, we used the functional annotation of significantly up- and down-regulated genes in the first two experiments as guides for expected changes in host phenotypic and physiological expression. Specifically, in two subsequent experiments examining phenotypic responses to endophyte inoculation, we measured photosynthetic responses and stable isotope ratios for nitrogen and carbon in E+ and E− leaves in plants grown under greenhouse conditions. In all inoculation experiments, E+ leaves showed significantly more endophyte colonization than E− leaves  that was among the most highly up-regulated genes in E+ treatments, we conducted transient gene expression experiments to assess the effect of increased expression of this gene on host pathogen resistance in the absence of endophyte treatment. Finally, we conducted a third (time course) microarray experiment to document the dynamics of host genetic expression in response to endophyte inoculation through time.Using 3347 and 17,247 unigenes C. tropicale, routinely the dominant species found in foliar endophytic fungal communities that establish in healthy T. cacao leaves  FEF are ubiquitous associates of plants worldwide, (2) that C. tropicale in particular is a dominant member of the endophyte community in many tropical plants  with those of control un-inoculated T. cacao leaves (E−). Tissue used for the first microarray experiment was also evaluated for lignin and cellulose content in the leaf epidermal cells of E+ and E− plants. All microarray experiments and the first phenotypic response experiment were conducted in Percival growth chambers under the conditions detailed above and the second phenotypic response experiment was conducted in a greenhouse  following previously described methods  between endophyte inoculated and un-inoculated plants. The second phenotypic response experiment was conducted in a greenhouse (using seedlings that were germinated and inoculated in the growth chambers) to confirm photosynthesis results from the first phenotypic response experiment under more natural conditions and to permit an assessment of endophyte induced shifts in carbon and nitrogen isotope signatures without the potential effects of isotope depletion often observed in relatively small closed chambers. Further, this experiment followed a paired leaf E+ and E−, on the same plant, design that permits greater statistical power.The first phenotypic response experiment focused on comparing photosynthesis (AT. cacao 3K microarray) spotted at The Pennsylvania State University (PSU) Core Genomics Facility. This array contained 3347 unigenes (spotted in duplicates) and a set of non-hybridizing oligos as negative and probe spike-in controls . These gene sequences were collated from several different EST sequencing projects and from other cacao genes sequenced at the time of microarray development, prior to the whole genome sequencing. A total of 2154 different T. cacao genes with their respective locus identifiers were represented in this array and 2015 of the genes contained A. thaliana locus identifiers and functional annotation based on e-value cutoff of 1*e-5 using TBLASTX . One μg of total RNA was amplified using mRNA amplification kit  prior to labeling and hybridization. aRNA was dye coupled with Cy3 or Cy5  and subsequently purified according to manufacturer's instructions. The samples were paired and each Cy3 labeled sample (1.5 μg) was combined with a Cy5 labeled sample (with 1.5 μg) in dye-swap design. The paired samples were mixed together, fragmented (using Ambion AM8740) and individually hybridized to a single array at 42°C for 18 h. A total of six arrays were hybridized resulting in a total of 12 measurements per treatment . Arrays were washed to remove non-specifically bound target and were scanned with an Axon 4000A scanner.Hybridizations were performed by the Genomics Core Facility at PSU according to published facility protocols  array, containing four probes of 50–60 mers in length for each of 17,247 unigenes . The microarray was designed based on EST sequences containing 6,853 contigs and 12,959 singlets from mixed cacao tissues  that were used for gene ontology (GO) categorization.The 17,247 unigenes on this microarray were blasted against TAIR9 CDS database and 11,225 unigenes had TcACT (Tc01g01090) and TcUBQ (Tc02g024050) from cacao was also assessed and used to normalize the data. Amplification efficiency of all target and reference genes was calculated from the slopes of the dilution curves for each sample (E = 10(−1/slope)) . Specifically, the expression of eight unigenes (representing six genes) that were highly up-regulated in the first microarray experiment was evaluated in the six E+ and six E− samples  used for first microarray experiment. Gene specific primers  correction for multiple testing correction were considered as significantly differentially expressed. The difference in expression between the controls (E−) and the treatments (E+) is presented as the log2 fold up- or down-regulation. For the second microarray experiment raw data was background corrected and normalized using the RMA algorithm available in the Limma package. Similarly as above, a gene-wise linear model was fitted to normalized data and empirical Bayes used for statistical analysis. A moderated t-test was conducted to identify genes with significant difference (p < 0.05 after BH correction for multiple testing) in gene expression between E+ and E− leaves at 3 dpi. The data from the third microarray experiment were analyzed with software ArrayStar 4 (DNASTAR). Pair files were imported to ArrayStar and processed using the RMA method and quantile normalization. Samples were grouped by time point and an F-test ANOVA was employed for finding genes with significant (p < 0.05 after FDR correction) difference in expression across the time points assessed. Data from the three microarray experiments are available at the NCBI Gene Expression Omnibus .Microarray data analyses of first and second microarray experiment were conducted using the Limma package of the Bioconductor software  were stained with 2% Phloroglucinol-HCL for 20 min. After the staining, 14 E+ and 18 E− sections were immediately assayed for the development of the purple color under an Olympus BX61Epi-Fluorescence Microscope  using a 10× objective. Images were acquired for with a Hamamatsu Orca-ER camera and processed with Olympus SlideBook 4.1 software. The purple color intensity  in the vascular tissue of each sample was measured using ImageJ software (http://rsbweb.nih.gov/ij/). Average intensity of each section was calculated and randomization tests indicated significant differences between the E+ and E− treatments (p < 0.001).Six E− and six E+ cacao plants used for the first microarray analysis were preserved in RNAlater. Three to four approximately 1 cm−1) at each pixel. Brighter regions in the spectral images correspond to higher total intensity within the region of integration.A WITec CRM200 confocal Raman microscope was used to acquire lignin spectral images from mature leaves (preserved in RNAlater) of the six E− and six E+ plants from first microarray experiment. In brief, the spectral images were generated by acquiring one spectrum (1 s integration time) at each pixel. Each pixel spacing was 500 nm. The lignin spectral images were generated using WITec's Witecproject software by integrating over the spectral interval 1495–1560 relative reciprocal centimeters (cm−1) at each pixel. We observed at 100× magnification that the cell corners and cell walls of both treatments had a greater intensity and thus greater lignin concentration than the inner part of the leaf cells. We performed a total of 12,000 individual scans at 40× magnification per E− and E+ treatments using random samples from all 6 biological replicates, including a approximately 240 cells per treatment. We recorded the overall carbohydrate distribution by integrating from 500 to 3000 cm−1. For this analysis Raman bands were assigned to cellulose or lignin components according to published literature and average spectra were calculated for each individual treatment  in E+ plants, full-length protein coding sequences of these genes were compared to α (TUA) and β (TUB) tubulin sequences from Populus tremuloides and A. thaliana. To identify all tubulin genes from cacao, full-length CDS sequences of six A. thaliana (AtTUA) and eight Populus (PoptrTUA) genes were compared against T. cacao ESTs and Genome databases using TBLASTX  genes were used as outgroup. The pairwise deletion option was selected to address alignment gaps and missing data, and branch support was obtained through 2000 bootstrap replicates.A phylogenetic tree was inferred by neighbor-joining analysis of all α-tubulin gene sequences from 2 uptake capacity was measured using a Licor 6400 portable photosynthesis meter . Light saturated rates of photosynthesis (Amax) were obtained from light response curves of net CO2 uptake between 0 and 800 μmol photons m−2 s−1 under ambient CO2 conditions. Cuvete temperature was at 30°C. Air flow through the cuvettes was 500 μmol s−1.Photosynthesis related measurements were conducted in the first phenotypic response experiment at 27 dpi on leaves of developmental stage E  coupled to a continuous flow isotope ratio mass spectrometer . Variation in repeated sample runs of the same material was ±0.01%. All analyses were conducted at the Cornell Stable Isotope Laboratory (COIL).Leaf samples  was up-regulated in host tissues without the presence of C. tropicale or any of its products. The coding sequence of TcChi1 in binary vector pGAM00.0511  under the control of high-level constitutive E12-Ω CaMV-35S promoter  using Takara SYBR® premix EX TaqII kit (Clontech) according to the user's manual. Each reaction was performed in duplicates in Roche Applied Biosystem StepOne Plus Realtime PCR System . The specificity of the primer pairs was examined by visualization on the 2% Agarose Gel and dissociation curve. Analysis was performed using TcACT (Tc01t010900) as reference gene (primer set: TcACT-RTF: AGCTGAGAGATTCCGTTGTCCAGA and TcACT-RTR: CCCACATCAACCAGACTTTGAGTTC).We conducted a series of experiments in which a Phytophthora capsici mycelium as previously described . Three sterile agar plugs were placed at the left side of each leaf segment and used as negative controls. Inoculated leaves were then incubated at 27°C and 12:12 (Light: Dark) light cycle for 3 days before the evaluation of disease symptoms. Images were taken using Nikon D90 digital camera and measurements of each lesion size were performed using ImageJ software. Average lesion sizes were calculated from 18 replicates  and significance was determined by single factor ANOVA.The transient EGFP fluorescence was observed under a microscope as previously described , we used RT-qPCR to measure the ratio of P. capsici actin-coding DNA (PcACT) to cacao actin-coding DNA (TcACT), compared to controls (VC). Four lesions were collected as a 1.4 cm × 1.4 cm square surrounding the inoculation site and genomic DNA was extracted using TissueLyzer and DNeasy plant mini kit . P. capsici actin A (F: GACAACGGCTCCGGTATGTGCAAGG and R: GTCAGCACACCACGCTTGGACTG) and TcACT (Tc01g010900) were used as pathogen and host targets. RT-qPCR was performed as previously described  in E+ leaves relative to E− leaves  compared to un-inoculated (E−) leaves. See Table A. thaliana . We performed RT-qPCR of eight unigenes (representing six genes) identified by this microarray analysis as being highly up-regulated by E+ treatments. Up-regulation was confirmed in all cases , 433 up-regulated and 423 down-regulated genes in E+ relative to E− leaves  correction, Supplementary Table A. thaliana  and found that GO categories of host genes with more genes affected by the endophyte under biological processes and cellular component were similar to the first and second microarray experiment  to view the dynamics of gene expression changes in E+ relative to E− leaves over a period of two weeks. We found 142 genes with significant changes in expression through the entire time course and with fold changes in expression between time points ranging from 1 to 7.4 . Studies of host plant genetic and phenotypic expression should be designed and interpreted with potential endophyte effects on their host clearly in mind  usually do not control for, or even consider, the potential effects of endophyte colonization. This and other studies have shown that, over the course of even a few days, the effects of foliar endophytes on host genetic and phenotypic expression can be rapid and large [e.g., marked reductions in pathogen and herbivore damage (Craine et al., Recent published and in press work suggests similar patterns and mechanisms are responsible for symbiont-conferred protection against natural enemies across both plants and insects (see Douglas, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Genomic Landscape of Compensatory Evolution Laboratory selection experiment explains how organisms compensate for the loss of genes during evolution, and reveals the deleterious side-effects of this process when adapting to novel environments. Adaptive evolution is generally assumed to progress through the accumulation of beneficial mutations. However, as deleterious mutations are common in natural populations, they generate a strong selection pressure to mitigate their detrimental effects through compensatory genetic changes. This process can potentially influence directions of adaptive evolution by enabling evolutionary routes that are otherwise inaccessible. Therefore, the extent to which compensatory mutations shape genomic evolution is of central importance. Here, we studied the capacity of the baker's yeast genome to compensate the complete loss of genes during evolution, and explored the long-term consequences of this process. We initiated laboratory evolutionary experiments with over 180 haploid baker's yeast genotypes, all of which initially displayed slow growth owing to the deletion of a single gene. Compensatory evolution following gene loss was rapid and pervasive: 68% of the genotypes reached near wild-type fitness through accumulation of adaptive mutations elsewhere in the genome. As compensatory mutations have associated fitness costs, genotypes with especially low fitnesses were more likely to be subjects of compensatory evolution. Genomic analysis revealed that as compensatory mutations were generally specific to the functional defect incurred, convergent evolution at the molecular level was extremely rare. Moreover, the majority of the gene expression changes due to gene deletion remained unrestored. Accordingly, compensatory evolution promoted genomic divergence of parallel evolving populations. However, these different evolutionary outcomes are not phenotypically equivalent, as they generated diverse growth phenotypes across environments. Taken together, these results indicate that gene loss initiates adaptive genomic changes that rapidly restores fitness, but this process has substantial pleiotropic effects on cellular physiology and evolvability upon environmental change. Our work also implies that gene content variation across species could be partly due to the action of compensatory evolution rather than the passive loss of genes. While core cellular processes are generally conserved during evolution, the constituent genes differ somewhat between related species with similar lifestyles. Why should this be so? In this work, we propose that gene loss may initially be deleterious, but organisms can recover fitness by the accumulation of compensatory mutations elsewhere in the genome. To investigate this process in the laboratory, we investigated 180 haploid yeast strains, each of which initially displayed slow growth owing to the deletion of a single gene. Laboratory evolutionary experiments revealed that defects in a broad range of molecular processes can readily be compensated during evolution. Genomic analyses and functional assays demonstrated that compensatory evolution generates hidden genetic and physiological variation across parallel evolving lines, which can be revealed when the environment changes. Strikingly, despite nearly full recovery of fitness, the wild-type genomic expression pattern is generally not restored. Based on these results, we argue that genomes undergo major changes not simply to adapt to external conditions but also to compensate for previously accumulated deleterious mutations. Deleterious, but non-lethal mutations are constantly generated and can hitchhike with adaptive mutations Caenorhabditis elegansCompensatory evolution appears to be common at many levels of molecular interactions. It is involved in the maintenance of RNA and protein secondary structures, it mitigates the costs of antibiotic resistance As regards the potential pleiotropic effects of compensatory evolution, our knowledge is rather limited, not least because it demands detailed exploration of the underlying molecular mechanisms of compensation. Compensatory mutations may enhance fitness either by reducing the need for the gene with the compromised function, or by restoring the efficiency of the affected molecular function Saccharomyces cerevisiae genome to compensate for gene loss through compensatory evolution and to explore the long-term consequences of this process.Compensatory evolution following gene loss is of special interest We initiated laboratory evolutionary experiments with 187 haploid single gene knock-out mutant strains, all of which initially showed slow (but non-zero) growth compared to the wild-type control in a standard laboratory medium . These gFitness may increase during the course of laboratory evolution as a result of general adaptation to the environment and/or accumulation of compensatory mutations that suppress the deleterious effects of gene inactivation. Under the assumption that compensatory evolution was the dominant force in our experiments, fitness should not increase by the same extent in all lineages: genotypes that carry deleterious null mutations are further away from the optimal state and are hence expected to show large fitness gains ; this waS. cerevisiae that influence the rate of mutations Δrad54, Δrad52, Δmre11, and Δrad50). Second, fitness improvements of the corresponding single gene knock-out strains did not differ from the rest of the dataset .The difference in fitness improvement is not due to the elevated mutation rate of mutant genotypes for two reasons. First, a previous study conducted a genome-wide screen with the aim to identify genes in As previously p<10−14)  = 3.9, see also . It has see also . Howeversee also . This pade novo mutations that accumulated during the course of laboratory evolution. Large-scale duplications  were observed in 22% of the laboratory evolved lines. On average, six point mutations and 0.5 small insertions or deletions per clone were detected . Third, genome sequence analysis of the evolved lines revealed only one clear example where evolution proceeded through increasing the dosage of a gene duplicate with redundant function of the deleted gene . This figure is a gross overestimate, as it includes very distant duplicates that most likely diverged functionally . Second,ted gene . All throme XIII . RPL6B iild type .Compensatory evolution may restore wild-type physiology or generate novel alterations with respect to prior physiological states p<10−7), while evolved wild-type populations did not show such a difference . Furthermore, the degree of fitness variation across conditions was especially high for gene deletions that showed large fitness gains during compensatory evolution   . These rΔrpl6b and Δatp11 lines displayed 24%–26% fitness increase compared to that of the wild type in a medium containing sodium chloride . Moreover, the fitnesses of these lines in this medium surpassed all that of the 22 evolved wild-type controls. These results are all the more remarkable, as the corresponding ancestral Δrpl6b and Δatp11 strains showed fitness values significantly lower than wild type under all environmental conditions considered. These preliminary results indicate that gene loss can promote adaptive evolution towards novel environments, a possibility that will be explored further in a future work.Finally, our analysis revealed a few instances where the laboratory evolved lines displayed significantly higher than wild-type fitness in specific environments . Most nochloride , a resulMDM34 deletion with the aim of deciphering the molecular mechanisms and/or potential fitness costs of compensatory mutations . The analysis revealed that the net effect of mutations in three evolved lines were deleterious in the presence of MDM34  into wild type and Δmdm34 resulted in very similar conclusions. mga2-1 and Δmdm34 showed strong sign-epistasis mga2-1 to compensate the loss of MDM34 was restricted to non-acidic conditions   see . Most noheximide . We concOur work addresses one of the most long-standing debates in evolution. Since the early 1920s, Ronald Fisher pioneered the view that adaptation is by and large a hill climbing process: it proceeds through progressive accumulation of beneficial mutations First, compensatory evolution following gene loss was pervasive: 68% of the deleterious, but non-lethal gene disruptions were compensated through the accumulation of adaptive mutations elsewhere in the genome . Furtherp = 0.87), 21 observed point mutations generated in-frame stop codons, most likely yielding proteins with compromised or no activities  evolving wild-type control populations showed only minor changes in fitness, (ii) the rate of adaptation was genotype specific, (ii) convergence at the molecular across genotypes was extremely rare, (iv) evolution preferentially affected genes that are functionally related to that of the disrupted gene, and (v) compensatory mutations had no beneficial impact in a wild-type genetic background.The above results encouraged us to distinguish between two evolutionary scenarios. Organisms may attempt to restore the disrupted molecular function through mutations in genes with redundant functions . Alternatively, they may aim to minimize the cellular damage incurred by gene disruption . While the possibility of full functional restoration cannot be excluded, the rarity of compensation through mutations in gene duplicates and the plasticity of compensatory mutational effects across environments are consistent with the second scenario. Indeed, our work demonstrates that gene loss promotes genetic changes that have a large impact on evolutionary diversification, genomic expression, and viability upon environmental change. An important implication of our study is that the beneficial effects of compensatory mutations should frequently be conditional, and subsequent changes to the environment can reveal the hidden fitness effects . Lack of restoration of fitness across environments is broadly consistent with the emerging view that epistatic interactions are plastic across conditions The perspective offered in this work leads to the re-formulation of several fundamental questions. First, it sheds light on an evolutionary paradox: while core cellular processes are generally conserved during evolution S. cerevisiae parental strain. Non-essential single-gene deletion strains from the haploid yeast deletion collection All strains used in this study were derived from the BY4741 The slow-growing yeast deletants used in this study are listed in evolving wild types). The YOR202W deletion strain was used as evolving wild-type control because the fitness of this strain is indistinguishable from the BY4741 parental wild-type strain his3Δ1 allele, thus it was possible to control for the reported mutation-generating effect of the KanMX4 cassette 5 cells (∼0.5 µl sample volume) were transferred every second day to 100 µl of fresh medium in 96-well plates resulting in ∼7.6 generations between transfers. The experiment was run for 104 days  and samples from days 0, 26, 52, 78, and 104 were frozen in 15% glycerol and kept at −80°C until fitness measurement. Cross-contamination events were regularly checked by PCR and visual inspection of empty wells (unpublished data).Compensatory adaptation refers to fitness gains in a gene deletion strain that are greater than fitness gains occurring in an isogenic wild-type strain. We conducted a series of laboratory evolutionary experiments using four independent populations of each of the 187 slow-growing deletants along with 22 independent lineages of an isogenic wild-type strain (referred to as 600) of liquid cultures of each strain using 384-well microtiter plates containing YPD medium (as during the evolutionary experiments). We used relative growth rate as a proxy for relative fitness (see below). Compared to laborious competition based fitness assays, this protocol allows estimating growth rate on a relatively large scale in an environment that is nearly identical to the one used in the evolutionary experiments.We used established protocols specifically designed to measure fitness in yeast populations 600 of the wells was 0.027. Each 384-well plate were inoculated with four different starter plates: one plate having the unevolved wild-type control as a reference strain in all wells in order to estimate various within-plate measurement biases, and three plates containing the same set of mutants from three of the five time points of the evolutionary experiment. The 384-well plates were incubated at 30°C in an STX44 (LiCONiC AG) automated incubator with alternating shaking speed every minute between 1,000 rpm and 1,200 rpm. Plates were transferred by a Microlab Swap 420 robotic arm (Hamilton Bonaduz AG) to Powerwave XS2 plate readers (BioTek Instruments Inc) every 20 minutes and cell growth was followed by recording the optical density at 600 nm. Six technical replicate measurements were executed on all strains sampled from each time-point of the evolutionary experiment. Measurements with growth curve irregularities were automatically removed. Only those strains were further analyzed where at least four technical replicate measurements remained after this quality control step.Starter cultures were inoculated from frozen samples using 96-well plates. The starter plates were grown for 48 hours under identical conditions to the evolutionary experiment. 384-well plates filled with 60 µl rich medium per well were inoculated for growth curve recording from the starter plates using pintool with 1.58 mm floating pins. The pintool was moved by a Microlab Starlet liquid handling workstation (Hamilton Bonaduz AG) to provide uniform inoculum across all samples. The median blank corrected initial ODp-value was corrected with a false discovery rate of 0.05.Growth rate was calculated from the obtained growth curves following an established procedure p-value corrected with a false discovery rate of 0.05). Second, the growth rate increment of the knock-out strain had to be disproportionally larger than that of the evolving wild-type control strains. To test whether fitness gain in a knockout is higher than those occurring in the evolving control lines, we first fitted a normal distribution to the fitness improvement values of the evolving control lines. Next, we defined a fitness improvement cutoff, so that the probability that an evolving control line would show an improvement at least that high is less than 0.05.To determine whether the fitness defect of a given knock-out strain became compensated during the evolutionary experiment two criteria must have been met: First, the growth rate improvement had to be significant , fitness was estimated by colony size on solid agar media. Moreover, it allowed direct comparison of the reliability of our measurements to results of a previous study .We prepared solid agar media of 14 different compositions to expose the strains to fundamentally diverse environments and to obtain sufficient throughput. Our list of 14 growth media was primarily based on a previous study Fitness of the ancestor (day 0) and evolved strains (day 104) was approximated by measuring colony sizes of ordered arrays of strains at 768 density. First, four different 96-well plates of the evolutionary experiment were scaled up to arrays of 384 colonies: one having the unevolved wild-type control in all positions, and three different plates of the mutant set from the same time point. Then pairs of 384 arrays with corresponding strains from day 0 and 104 were combined to reach 768 density. With this set up, all evolving replicate lines derived from the same ancestral genotype from both day 0 and day 104 were grown on the same 768 plate to exclude potential plate-to-plate variations when comparing colony growth of ancestor and evolved lines. Four technical replicates of these 768 arrays were transferred into each of the 14 different media.After acclimatization to the media at 30°C for 48 hours the plates were replicated again onto the same type of media and photographed after 48 hours of incubation at 30°C. Digital images were processed to calculate colony sizes, and potential systematic biases in colony growth were eliminated . For eacp-value corrected for each condition with a false discovery rate of 0.05). The same statistical test was used to determine whether the fitness of an evolved line is different from that of its ancestor in a given environment. See result in To determine whether an ancestor genotype shows a significantly altered fitness compared to the wild-type control in a given environment, we used a Wilcoxon rank sum test  and the CNV-seq p<0.0001) software predicted significant alteration from the read coverage of the reference genome.Genomic DNA was prepared using a glass bead lysis protocol: clones were inoculated into 5 ml YPD+G418 (200 mg/l) and grown to saturation at 30°C. Cells were pelleted and resuspended in 500 µl of lyis buffer . Cells were mechanically disrupted by vortexing for 3 minutes at high speed with 500 µl glass bead . After adding 275 µl 7 M ammonium acetate, samples were incubated at 65°C for 5 minutes, followed by a second incubation on ice for 5 minutes. The samples were extracted with chloroform∶isoamyl alcohol (24∶1) and centrifuged for 10 minutes. The aqueous layer was transferred into a new tube and precipitated with 1 ml isopropanol, pelleted and washed with 70% ethanol, and resuspended in 500 µl RNaseA solution (50 ng/ml). After 30 minutes RNaseA treatment at room temperature, samples were chloroform∶isoamyl alcohol (24∶1) extracted, precipitated with 50 µl sodium acetate (3 M [pH 5.2]) and 1,250 µl ethanol, pelleted and washed with 70% ethanol. Finally, the genomic DNA was dissolved in water. The steps of re-sequencing was done by the UD-GenoMed Medical Genomic Technologies Ltd: amplified genomic shotgun libraries were run on the Illumina HighScan SC with 1×100 bp single read module resulting in an average coverage of about 80×. Reads were aligned to the de novo mutational events. De novo mutations were identified as alterations from the reference genome specifically found in the evolved lines but not present in the ancestral strains. Mutations, which occurred before our evolutionary experiment but after the gene knock-out, are referred to as secondary ancestor mutations. These mutations were identified in the ancestral strains as SNPs and indels present only in the corresponding ancestor strain, not in any other ancestral strains. The rationale behind this consideration is not to classify mutations accumulated in the parental strain of the mutant library prior to the generation of the knock-out strain as a secondary ancestor mutation. The list of identified mutations can be found in Our primary aim was to analyze p = 0.003).Whole-genome re-sequencing revealed that 86% of SNPs in the coding regions were non-synonymous. To statistically test whether the ratio of non-synonymous to synonymous SNPs was higher than expected based on a neutral model of evolution, we employed the method of Barrick and colleagues To test whether the extent of evolutionary compensation is influenced by the disrupted gene's pleiotropy, we used three complementary measures of gene pleiotropy. Environmental pleiotropy of a non-essential gene was defined as the number of unique conditions in which the removal of the gene resulted in a fitness defect according to Dudley and colleagues To investigate whether mutations accumulated during compensatory evolution preferentially affected genes that are functionally related to the disrupted gene, we used different measures of functional relatedness: co-membership within stable protein complexes, shared functional category, genetic interaction profile similarity, co-expression, and paralogy. For protein complexes we used the manually curated dataset based on tandem affinity purification/mass spectrometry studies (YHTP2008) from the Wodak lab −8, (ii) alignment length exceeded 100 residues, (iii) sequence similarity was >30%, and (iv) they were not parts of transposons.Paralog gene pairs were identified by performing all-against-all BLASTP similarity searches of yeast open reading frames. We defined two genes as paralogs if (i) the BLAST score had an expected value E<10Eight evolved lines were selected for microarray analysis, all of them showing high fitness following evolution . The corresponding ancestral strains and the wild-type control were also subjected to gene expression profiling. 600 of 0.15 in 60 ml YPD and grown to early mid-log phase (OD600 0.6±0.05) in 250 ml Erlenmeyer flasks with 220 rpm shaking at 30°C. Cells were harvested by centrifugation  and immediately frozen in liquid nitrogen after removal of supernatant. Total RNA was prepared by hot acidic phenol extraction and cleaned up using the QIAGEN's RNAeasy kit.Two independent colonies of the wild-type control, evolved, and corresponding ancestor knock-out strains were inoculated into 15 ml YPD and grown overnight at 30°C. The saturated populations were diluted to an ODp-value<0.05 when comparing two strains. The raw dataset is available online at ArrayExpress .All steps after RNA isolation were automated using robotic liquid handlers as described previously rpl43a), which was not sequenced. In the case of Δrpl43a, whole chromosome duplication was predicted on the basis of visual inspection of expression profiles. The position of partial chromosome duplication was predicted by the Charm algorithm All transcriptome comparisons of the wild-type, knockout, and evolved strains were repeated on a dataset where CNVs, genes showing expression response to aneuploidy, and growth rate related genes were excluded. CNVs were identified on the basis of the read coverage of the genome sequence data  with themdm34 were chosen for in-depth genetic analysis. The fitness cost of the set of compensatory mutations accumulated in the evolved Δmdm34 lineages was measured in wild-type genetic background. To this end, the MDM34 gene was re-introduced into the ancestor and evolved Δmdm34 lineages according to the delitto perfetto method mdm34 lineages was swapped with the CORE-UH cassette, containing the KlURA3 and hyg markers. Then the MDM34 open reading frame with longer than 0.3 kb flanking regions on both sides was amplified from the unevolved wild-type control strain and transformed into the cells to replace the CORE-UH cassette. The replacement of the KlURA3 marker was counter-selected using 5-FOA containing medium. The loss of hygr was confirmed, the site and orientation of gene replacement was verified by PCR and the sequence of the MDM34 gene was determined by capillary sequencing.The evolved lines of ΔMGA2 gene in one of the evolved Δmdm34 lineages was reinserted into both the wild-type and ancestor Δmdm34 background. This specific point mutation changes the 750th codon of MGA2 from GAT to TAT resulting in the incorporation of tyrosine instead of aspartic acid. We refer to the mutant allele as mga2-1. Using the delitto perfetto method KlURA3 marker with the missense SNP was counter-selected using 5-FOA containing medium, loss of hygr was confirmed, and the result of the site-directed mutagenesis was verified by capillary sequencing. Attempts to introduce the mga2-1 mutation into the ancestor Δmdm34 strain in this way were not successful, presumably due to the severe slow growth of the intermediate strain that lacks both MDM34 and MGA2 gene in a functional form. To complement this, a helper plasmid with MDM34 gene . The RNA samples were concentrated by the NucleoSpin RNA Plant Kit (Macherey-Nagel), according to the manufacturer's instructions. A total of 500 ng RNA was used as a template to prepare cDNA using the Maxima First Strand cDNA Synthesis kit (Thermo Scientific). Reactions without template were set up to detect contaminations of the reagents used in the cDNA synthesis. qPCR reactions were set up in 20 µl volume, using the following templates: no template control, 10 ng non-transcribed RNA and cDNA transcribed from 10 ng RNA. The qPCR reactions were run in a Bioer LineK Gene device, using 2× Maxima SYBR Green qPCR Master Mix (Thermo Scientific). All samples had three technical replicates. Gene expression was determined in arbitrary units using a standard curve fitted on triplicates of a four-step 10-fold dilution series. OLE1 expression level was determined relative to TUB1 expression level as an internal control. All control reactions, not treated with reverse transcriptase or not having template, gave Ct values at least 10 cycles higher than the corresponding samples.Yeast samples were grown in 20 ml YPD medium to mid-log phase . RNA was extracted from 10Figure S1Fitness trajectories often show a saturating trend by day 104 of the evolution experiment. Fitness was measured at five time points during laboratory evolution , and fitness improvements were tested for each line and time interval , indicating saturating compensatory evolution. (B) Representative examples of fitness trajectories showing a saturating trend (replicate lines of six genotypes are depicted).(TIF)Click here for additional data file.Figure S2The extent of compensatory evolution in knock-outs is genotype-specific. Here, we tested whether there are inherent differences in the propensity for compensation across genotypes . We defined compensatory evolution as a fitness increase that is disproportionally large relative to that in the evolving wild-type lines Click here for additional data file.Figure S3Global transcriptome changes following compensatory evolution. (A and B) were prepared by reproducing the main results of Δmdm34), only a small fraction of the expression changes were restored in the evolved lines Click here for additional data file.Figure S4Pleiotropic effects and mechanism of compensation of Δmdm34. (A) Diversity of pleiotropic effects in independently evolved lines. Relative fitness across environments of isolated clones of independently evolving lines founded from the same Δmdm34 genotype were measured as colony sizes grown on different media (Δmdm34 strain in the corresponding environment. In (A) and (D) log2 values are shown according to the color coding. (B) Quantitative PCR confirmation of upregulation of OLE1 in both the evolved line carrying the mga2-1 mutation and in the Δmdm34 mga2-1 double mutant strain  Addition of oleic acid to the medium suppresses the fitness defect of Δmdm34, but does not affect the fitness of the evolved line carrying the mga2-1 mutation or the strain carrying both Δmdm34 and mga2-1 mutations. Fitness was measured as colony sizes relative to unevolved wild-type control on rich media supplemented with DMSO as solvent control (non-treated), 0.1 mM oleic acid and 0.1 mM stearic acid Click here for additional data file.Figure S5Validation of the phenotypic profiling experiment. We compared our colony size measurements .urements  of the a(TIF)Click here for additional data file.Table S1Fitness of strains in various environments. The table includes fitness values of ancestor and evolved strains as measured in liquid YPD and in different agar media. Pleiotropy measures and GO process terms of the deleted genes are also presented.(XLSX)Click here for additional data file.Table S2Mutations identified by Illumina next generation sequencing. The table contains all the identified de novo and ancestral mutations in the sequenced genomes.(XLS)Click here for additional data file.Table S3Microarray analysis results. The table contains microarray data on all ancestral and evolved lines subjected to microarray analysis.(XLS)Click here for additional data file.Table S4Composition of media used for phenotypic profiling. Each media contained 1% yeast extract, 2% pepton, 2% agar, and different carbon sources. Some media also contained growth inhibitors as indicated. Concentration of drug inhibitors were set to have a minor but detectable growth inhibitory effect on the evolving wild-type control (unpublished data). The list of 14 growth media was primarily based on a previous study (XLSX)Click here for additional data file.Table S5Data supporting . The table contains fitness measurements supporting dosage compensation of Δrpl6b by increased copy number of RPL6A.(XLSX)Click here for additional data file.Table S6Data supporting . (4B) Euclidean distances between pairs of wild-type evolved and ancestor knock-out strains, and also between the corresponding biological replicates. (4C) Categories of expression changes for each gene in the eight evolved knockout strains. Genes, which show initial expression change in the knockout can be categorized as restored, if expression during evolution goes in the opposite direction or unrestored if not. The category “other” includes genes not showing initial expression change.(XLSX)Click here for additional data file.Table S7Data supporting . The table contains fitness (colony size) data employed for epistasis analysis between the mdm34 gene deletion and the mutations accumulated in the evolving strains ( strains  and betw strains  in both (XLSX)Click here for additional data file.Table S8Data supporting . The table contains colony size measurement data on the environment-dependent compensation of the deletion of rpb9 by a loss-of-function mutation of whi2.(XLSX)Click here for additional data file.Table S9Data supporting . Table includes single-gene knockout fitness and relative frequency of suppressing mutations for 3880 non-essential yeast genes.(XLSX)Click here for additional data file.Table S10Data supporting  The table contains data on the fitness trajectories of the evolving strains. Fitness was measured at day 0, 26, 52, 78, and 104. The columns “improved day x-y” show whether there is a statistically significant fitness improvement between day x and y, as assessed by one-sided Wilcoxon tests .(XLSX)Click here for additional data file.Table S11Data supporting  (S3A) Euclidean distances between pairs of wild-type, evolved, and ancestor knock-outs, after excluding genes from the transcriptome profiles that (i) show copy number changes in the evolved lines, (ii) change expression level in aneuploid lines, or (iii) whose expression level depends on cellular growth rate. (S3B) Table includes categories of expression changes for each gene in the eight evolved knockout strains, excluding genes from the transcriptome profiles that (i) show copy number changes in the evolved lines, (ii) change expression level in aneuploid lines, or (iii) whose expression level depends on cellular growth rate. Genes displaying an initial expression change in the knockout can be categorized as restored, if its expression level changes in the opposite direction during evolution, or unrestored. The category “other” includes genes that did not display an initial expression change.(XLSX)Click here for additional data file.Table S12Data supporting  The table contains data on the pleiotropic effects and mechanism of compensation of the deletion strain Δmdm34.(XLSX)Click here for additional data file.Text S1Additional analyses supporting the prevalence of and mechanisms underlying compensatory evolution following gene loss. The text includes a bioinformatic analyses of deleterious loss-of-function variants in natural yeast populations, a case study on compensatory mutations, and a brief description of image analysis used for measuring the extent of compensatory evolution.(DOC)Click here for additional data file.


A 45-year-old female was referred for endocrine evaluation of an incidental mass (31×24 mm in diameter) on the right adrenal gland. The patient was normotensive and nondiabetic, and had no history of generalised obesity ; however, her waist-to-hip ratio was 0.97. Elevated urinary free cortisol levels (112–118 μg/day) and other findings indicated adrenocorticotrophic hormone-independent Cushing's syndrome due to right adrenocortical adenoma. Echocardiography before adrenalectomy revealed concentric left ventricular (LV) hypertrophy with a particular increase in interventricular septum thickness leading to impaired systolic and diastolic functions. Upon surgical remission of hypercortisolism, the asymmetric hypertrophy disappeared and the cardiac dysfunctions were considerably ameliorated. Although the mechanism(s) by which excessive cortisol contributes to LV wall thickness remain(s) unclear, serial echocardiography and cardiac multidetector-row computed tomography may support the notion that abnormal fat deposition in the myocardium owing to hypercortisolism appears to be an important factor for the reversible change in the cardiac morphology.Patients with Cushing's syndrome occasionally exhibit severe LV hypertrophy related to systolic and diastolic dysfunctions although they have neither hypertension nor diabetes mellitus.Biological remission of hypercortisolism can normalise structural and functional cardiac parameters and help in differentiating the cardiac alterations induced by excessive cortisol from those induced by other diseases.Excessive lipid accumulation within the heart before myocardial fibrosis may be implicated in reversible alterations in the cardiac morphology by Cushing's syndrome.Early diagnosis and treatment of Cushing's syndrome appear to be pivotal in preventing irreversible cardiac dysfunctions subsequent to cardiovascular events and heart failure. Cushing's syndrome, resulting from excessive autonomous cortisol secretion by the adrenal gland, ectopic adrenocorticotrophic hormone (ACTH) or corticotrophin-releasing hormone secretion by a pituitary or nonpituitary tumour and chronic glucocorticoid therapy, leads to a constellation of complications, including hypertension, diabetes mellitus, cardiovascular disease, stroke and thromboembolism Left ventricular (LV) concentric remodelling with an increased interventricular septum thickness, termed asymmetric septal hypertrophy, is typically a finding of hypertrophic cardiomyopathy due to mutations in the genes encoding sarcomeric proteins; however, it may appear to be a sign of other cardiac and systemic diseases This report describes a patient with active Cushing's syndrome due to adrenocortical adenoma accompanied by unexplained cardiomegaly over the past 3 years. The patient was not diagnosed with hypertension or diabetes but displayed severe LV concentric hypertrophy with asymmetric septal thickness concomitant to systolic and diastolic dysfunctions. Serial echocardiography revealed that surgical remission of hypercortisolism led to normalisation of parameters for the LV structure along with the recovery of cardiac functions, suggesting the pivotal role of excessive cortisol in the cardiac alterations.A 45-year-old female was referred for evaluation of a right adrenal incidental mass on abdominal computed tomography (CT) that was ordered to assess intermittent abdominal discomfort. Three years before the first visit to our department, the patient underwent health check-up at a hospital including electrocardiogram and chest X-ray examination. LV hypertrophy and cardiac enlargement were noted; she was immediately referred to the cardiology department of the same hospital. A cardiologist performed echocardiography and confirmed LV concentric remodelling without specifying its aetiology and recommended regular electrocardiogram, echocardiography and chest X-ray examinations. The patient underwent these examinations every year but took no further medical treatment before adrenalectomy. She also noticed a menstrual disorder (oligomenorrhoea) 2 years before admission; however, emotional liability, sleep disorders and depression were not observed.The patient had no history of generalised obesity and her body weight on admission was 51.2 kg , although she gradually gained weight by 4 kg for the past 5 years and her waist-to-hip ratio was 0.97  at the time of diagnosis. During admission, her blood pressure was measured several times during the day  and was found to be between 112/64 and 128/78 mmHg without antihypertensive drugs and salt restriction. Physical examination revealed bilateral malar erythaema, bruising and thin skin. The patient showed no other features, such as apparent accumulation of fat in the face (a moon face) or around the neck , hirsutism, acne, prominent purple striae on the abdomen and proximal muscle weakness. Neither crackles nor murmurs were heard from the chest, although Valsalva manoeuvre was positive. Other physical findings were unremarkable.4–6, inverted T-wave in leads aVL and V1–6 and ST segment elevations in leads aVR and V1–2 (Z-score of −0.35 and a T-score of −0.35).On admission, chest X-ray revealed cardiac enlargement defined as a 58.0% cardiothoracic ratio. Electrocardiogram revealed marked high-voltage QRS complexes, ST segment depressions in leads Vand V1–2 A. Bone m131I]adosterol imaging; however, the left adrenal gland was negative  without administration of any medication such as angiotensin-converting enzyme inhibitor, angiotensin receptor type 1 blocker, aldosterone antagonist, calcium channel blocker and sodium-wasting diuretics. Other laboratory findings revealed a loss of the circadian rhythm of cortisol secretion  and elevated urinary free cortisol levels  with no suppression after low (1 mg)- and high (8 mg)-dose dexamethasone tests (20.2 and 18.4 μg/dl respectively). ACTH levels were consistently suppressed (<2.0 pg/ml). Plasma renin activity  and plasma aldosterone concentration  were within the normal limits and the PAC-to-PRA ratio (80.8) was <200. Diabetes mellitus was ruled out according to the current Japan Diabetes Society's guideline using a 75 g oral glucose tolerance test. CT of the abdomen also revealed a 31×24 mm low-density mass within the right adrenal gland with a regular border and mild enhancement post-contrast A. The rinegative B, sugges2) and the relative wall thickness (0.75) were high and the interventricular septum was remarkably thick (20.1 mm)  B, indicaThe patient underwent laparoscopic right adrenalectomy C. HistolThe surgical treatment was effective on features of Cushing's syndrome, with a relief of the physical signs. Regular menstrual cycles (25 days/cycle) resumed 3 months after adrenalectomy. The patient had gradually lost 10.5% of the initial body weight  up to 17 months after adrenalectomy and maintained the loss for >3 years A, suggesThe abnormal electrocardiogram findings promptly disappeared at 8 months after adrenalectomy C. The caTo investigate whether surgical remission of hypercortisolism affects myocardial characteristics, ultrasound reflectivity of the LV anteroseptal and posterior walls was quantified by densitometry. Serial echocardiography revealed that echogenicity in the LV anteroseptal and posterior walls at 8 months after adrenalectomy was higher than that during the untreated active state by 36% and that the level of intensity was sustained for 43 months after the treatment A. ContraThis report describes a patient with Cushing's syndrome, who presented with a specific cardiac morphology characterised by disproportional hypertrophy of LV with an impaired systolic performance and relaxation, as shown by the mid-wall FS, E/A ratio and E-wave deceleration time. These findings are predictive of the occurrence of subsequent cardiovascular events and heart failure Approximately 80% of patients with Cushing's syndrome have high blood pressure and present with a more severe alteration of cardiac mass parameters compared with normotensive patients with Cushing's syndrome In five previous case reports, severe congestive heart failure associated with Cushing's syndrome was improved by medical and surgical treatment for hypercortisolism In contrast to the present patient's manifestation of severe concentric LV hypertrophy with asymmetric septal thickness, the patients in the above five cases presented dilated cardiomyopathy without apparent septal hypertrophy (IVSd, 8.6 and 13 mm in the two reports Assessment using ultrasonic integrated backscatter has demonstrated that myocardial fibrosis is significantly increased in active, untreated patients with Cushing's syndrome and is related to LV systolic and diastolic dysfunctions Cushing's syndrome is characterised by abnormal fat deposition in subcutaneous sites on the face, neck, abdomen and skeletal muscle et al. et al. Sugihara Written informed consent was obtained from the patient for publication of this case report.H Iwasaki was the patient's physician, was responsible for case description and literature review and wrote the manuscript.


The etiology of persistent lung inflammation in preterm infants with chronic lung disease of prematurity (CLD) is poorly characterized, hampering efforts to stratify prognosis and treatment. Airway macrophages are important innate immune cells with roles in both the induction and resolution of tissue inflammation.To investigate airway innate immune cellular phenotypes in preterm infants with respiratory distress syndrome (RDS) or CLD.Bronchoalveolar lavage (BAL) fluid was obtained from term and preterm infants requiring mechanical ventilation. BAL cells were phenotyped by flow cytometry.+ macrophages compared with the CLD group . At day 3, infants born at a low gestational age are more likely to have greater numbers of CD14+ mononuclear phagocytes in the airway (p = 0.03), but fewer of these cells are functionally polarized as assessed by HLA-DR (p = 0.05) or CD36 (p = 0.05) positivity, suggesting increased recruitment of monocytes or a failure to mature these cells in the lung.Preterm birth was associated with an increase in the proportion of non-classical CD14+/CD16+ monocytes on the day of delivery . Infants with RDS were born with significantly more CD36These findings suggest that macrophage polarization may be affected by gestational maturity, that more immature macrophage phenotypes may be associated with the progression of RDS to CLD and that phenotyping mononuclear cells in BAL could predict disease outcome. Chronic lung disease (CLD) of prematurity, often also called bronchopulmonary dysplasia (BPD), is a significant complication of preterm birth, leading to increased respiratory symptoms, repeated hospital admissions and abnormal long term lung physiology resulting in great economic cost and markedly increased parental burden. The pathogenesis of CLD is linked to a number of clinical factors, including prematurity, mechanical ventilation, oxygen therapy and post- and ante-natal infection all of which help to initiate or sustain an inflammatory process in the preterm lung. Persistent airway neutrophilia and elevated levels of neutrophil chemoattractants, including CXCL8, in broncho-alveolar lavage (BAL) fluid, are associated with the development of CLD in preterm infants Our previous results have demonstrated increased numbers of macrophages in infants with respiratory distress syndrome (RDS), a condition associated with more effective resolution of inflammation and better clinical outcome than CLD, which is characterized by chronic distal airway inflammation and poor lung function. This observation, together with the known roles of macrophages, suggests macrophages may regulate inflammatory responses in the preterm lung These data led us to hypothesise that the relative abundance of macrophages in the preterm lung, and their differentiation status and activation phenotypes, may be associated with either the resolution of RDS or the progression to CLD. In this study, macrophages in BAL fluid samples from preterm infants retrospectively diagnosed with RDS or CLD, and from infants born at term, were phenotyped by flow cytometry, and the relationships between macrophage phenotype, disease severity and gestational age were examined.Patients were recruited in the regional neonatal intensive care unit at the University Hospital of Wales, Cardiff. The study was approved by the Cardiff and Vale NHS Trust Research and Development Committee and the South East Wales Research and Ethics Committee (05/WSE04/85). Written informed consent was obtained from the parents.Two groups of infants were recruited: 1) Preterm infants (≤32 weeks gestation) who required mechanical ventilation for RDS defined by clinical and radiological features, were recruited within the first 12 hours of life (n = 18). This group was later subdivided into those infants who went on to develop CLD  and those infants where RDS resolved .2) Term control infants  who required mechanical ventilation for non-respiratory reasons (for example infants ventilated peri-operatively for gastroschisis).Bronchoalveolar lavage (BAL) was performed within 24 hours of birth on ventilated infants as previously described Cellular pellets were separated by centrifugation and mucus disrupted by treatment with dithiothreitol (DTT). DTT had no effect on antigen detection by any antibody used (data not shown). A cell count was performed and cytospins prepared  for morphological analysis. Slides were air dried, fixed in methanol and stained with Giemsa based stains .Cells were washed to remove DTT and stained with the indicated antibodies: HLA-DR-APC , CD16-PE, CD36-PE, CD15-APC  and CD14-biotin . Staining combinations were as follows: CD15, CD14/CD16, CD14/HLA-DR/CD36. Streptavidin-PE-Cy5.5 tandem conjugate (eBioscience) was used as a secondary reagent to detect biotinylated primary antibodies. Following staining, samples were immediately analysed by flow cytometry .+ events. Cells of the monocyte/macrophage lineage were identified as CD14+. CD15+ neutrophils were CD14 negative, allowing distinction of these two cell types +/16++ non-classical monocytes were identified as highly CD16 positive CD14+ cells. Forward scatter (FSC) and side scatter (SSC) profiles were also examined. Flow cytometry data were analysed using FlowJo .Neutrophils were identified as CD15Cytokines were measured in cell-free BAL using a CBA custom flex set multiplexed assay (BD Biosciences). Antibodies to IL-1β, CXCL8, CCL3, CCL4 (pro-inflammatory indicators) and IL-10 (anti-inflammatory indicator) were used and samples analyzed by FACS array (BD Biosciences).Data were analysed by Mann Whitney test and regression analysis by linear regression  as defined in the figure legend.6) recovered/ml were 1.02±0.29 (term), 4.17±1.78 (RDS) and 1.42±0.2.86 (CLD). The numbers of neutrophils (CD15+) recovered were significantly higher in preterm infants compared with term infants within 24 hours of birth, both as a percentage of all cells and as absolute numbers  while HLA-DR+/CD36+ events were characterized as mid FSC and mid-high SSC .Characteristics of the 22 infants included in this study are shown in  numbers . CD14, ae number  in some egy used . Since Cegy used . Cells segy used  appear ir HLA-DR  or CD36 r HLA-DR . Since b+/16++) are associated with inflammation and injury + cells, the proportion of CD14+/16++ cells was significantly greater in preterm infants compared with term infants at on the within 24 hours of birth  and can lead to acute lung injury (e.g ARDS). It may also be beneficial to have immune defences already in place in anticipation of the potentially threatening event of birth.Macrophages containing apoptotic neutrophils have been visualized in BAL from preterm infants, supporting a functional role for these cells in the preterm infant lung in vivoThe origins of mature airway macrophages at birth are unclear. They may be rapidly released from the pulmonary interstitial compartment at the time of birth + cells in the lung, yet infants of a low gestational age have, as a proportion of these CD14+ cells, fewer mature macrophages. These data may indicate a greater stimulus for monocyte recruitment into the lung and/or a possible failure of maturation in the more preterm individuals. This idea is in accordance with studies that show decreased HLA-DR expression on monocytes isolated from cord or venous blood from infants of a low gestational age compared with less preterm infants In adults following acute pulmonary inflammatory stress, numbers of alveolar macrophages decrease and are replaced by macrophages of a more immature phenotype, possibly by rapid recruitment of monocytes from the vascular compartment in vitro and in vivoThe importance of the mononuclear cell phenotype in the context of inflammation has also been demonstrated in the form of CD14+/CD16+ populations, also referred to as ‘non-classical’ monocytes + macrophages are associated with IL-1β, CXCL-8, CCL4 and IL-10 levels, suggesting these cell types may be an important source of these cytokines + and HLA-DR+ macrophages represent mature phenotypes, they may differ in their cytokine output in the tissue. CD36+ macrophages are able to take part in efferocytosis, which is known to be associated with the production of anti-inflammatory cytokines by macrophages + macrophages does not correlate with pro-inflammatory cytokines such as IL-1β and CXCL8. Unexpectedly, CD36+ macrophage numbers are not associated with IL-10 in this study, a key macrophage anti-inflammatory cytokine.We also examined the relationship between neutrophils, and macrophages expressing HLA-DR or CD36, which as a significant source of cytokines and chemokines may co-ordinate the recruitment of other cells. A strong correlation between mature macrophage and neutrophil numbers is seen. This could reflect a role for one of these cell types in the recruitment of the other, or simply that individual infants are more likely to globally recruit cells than others. Our preliminary study of relationships between BAL cytokine levels and different cell phenotypes suggests the former explanation, where The former explanation is more probable since CBA analysis of BAL also indicates a strong correlation between individual cell populations and cytokine levels at preterm birth, where in particular, HLA-DRWe show that infants with progressive inflammatory disease (CLD) have fewer mature airway macrophages in the immediate peri-natal period compared to infants with resolving disease (RDS) suggesting phenotyping macrophages in ventilated infants may be a predictive marker of disease outcome allowing the stratification of treatment based on risk. Based on these findings, we speculate that the presence of mature airway macrophages may drive inflammation resolution, or indeed that the process of inflammation resolution may drive macrophage maturity. In support of this, we have previously shown an increase in total macrophages in BAL from infants with RDS compared to those with CLD, as determined morphologically + macrophages in RDS infants compared to CLD infants may reflect the greater need for efferocytosis due to the increased neutrophil burden in the RDS lung + macrophages is associated with Wilson-Mikity syndrome in preterm infants CD36 facilitates efferocytosis by macrophages, driving the resolution of inflammation In summary, we have demonstrated an association between macrophage phenotypes and disease progression in the context of chronic lung disease of prematurity. Not only do these findings associate macrophage phenotype with disease outcome in the context of neonatal lung disease, but also suggest new therapeutic possibilities focusing on delivering treatments to mature macrophage populations in the lung in preterm infants at risk of CLD. Furthermore, ex vivo phenotyping studies of ventilated preterm babies at birth may allow the stratification of risk in this patient group and enable treatment strategies to be tailored according to likely disease prognosis. These findings may help develop new therapeutic strategies focused on macrophage maturation in this clinically important disease.


TERT promoter mutations in gliobastomas (80%) followed by oligodendrogliomas (70%) and astrocytomas (39%). We observed positive association between TERT promoter and IDH mutations in oligodendroglial tumors  and inverse association in primary glioblastomas . Tumors with TERT promoter mutations compared to those without showed increased TERT transcription; we also showed difference in the transcription levels due to the two main mutations. Tumors with TERT promoter mutations had shorter telomeres than those without. The patients with only TERT promoter mutations showed worst survival  and patients with both IDH and TERT promoter mutations showed best survival (246.5 months). In patients with astrocytoma, the TERT promoter mutations only associated with poor survival (P < 0.0001); IDH mutations and 1p/19q deletions associated with increased survival (P = 0.0004). TERT promoter mutations in low grade gliomas associated with reduced progression free survival . While our data affirm the role of TERT promoter mutations in glial tumors, effects on transcription and telomere length emphasise the importance of telomere biology in disease genesis and outcome.In this report on 303 gliomas we show the highest frequency of  Gliomas are the most common primary malignant brain tumors in adults that mainly arise in glial tissue of the brain. Those tumors are either astrocytic, oligodendrocytic or a mixture of the two cell types and are typically categorized according to the International Classification of Diseases – Oncology, version 3 (ICD-O-3) and World Health Organization (WHO) grade , 2. The IDH1) and - less common - Isocitrate dehydrogenase 2 (IDH2) are examples for genetic alterations with prognostic value and potential as novel therapeutic targets [IDH (IDH1 and IDH2) mutations are associated with prolonged progression-free survival and seem to indicate patients that might benefit from chemoradiotherapy [IDH mutations, representing a typical feature of the oligodendroglial subtype [Challenging histopathological features of glial tumors and new possibilities in treatment add to the need of genetic markers with prognostic as well as predictive potential –10. Muta targets , 11. As otherapy . Those motherapy , 14. Co- subtype .TERT) gene in different cancers have consistently shown occurrence at highest frequencies in adult gliomas [TERT encodes the rate-limiting catalytic subunit of telomerase, which is involved in de novo addition of telomere repeats at chromosomal ends. The discovery of the TERT promoter mutations provides a possibility for (i) gaining insight into mechanistic questions in glioma development and (ii) a new biomarker and eventual therapeutic target [TERT promoter mutations exert influence through increased expression due to creation of new binding motifs for Ets/TCF transcription factors [TERT promoter mutation in different gliomas, which in combination with IDH mutations allows classification according to their histological subgroups [Reports on mutations within the core promoter of the telomerase reverse transcriptase  gliomas. The –124C > T mutation was present in 147 tumors (74%), whereas –146C > T was found in 51 cases (26%) and one tumor carried the –124_125CC > TT tandem mutation. Primary glioblastomas had the highest frequency of TERT promoter mutations  followed by oligodendrogliomas ; astrocytomas showed the lowest frequency, with 22 out of 56 (39%) samples showing mutations . Also, TERT promoter mutations were found associated with high grade (grade III + IV) tumors when compared to lower grade (grade II) lesions  were present in 100 of 303 (33%) glioma tumors ; two gliomas harbored the IDH1 c.394C > T mutation (R132H). IDH2 mutations were found in four tumors (4%), with c.515G > A (R172K) in three tumors and c.515G > T (R172M) in one tumor. The highest frequency of IDH mutations was detected in oligoastrocytomas , followed by astrocytomas  and oligodendrogliomas  (Table Besides CDKN2C (p18INK4C) locus at chromosome 1p showed focal deletions in nine gliomas, of which eight were primary glioblastomas and one was a grade II astrocytoma. Of these nine, six tumors were also deleted at the CDKN2A/B and eight tumors carried TERT promoter mutations , followed by oligoastrocytomas 19/47 (40%). The IDH and deletions at 1p and/or 19q were associated with an odds ratio (OR) of 9.97 . TERT promoter mutations and deletions at 1p/19q when present tended to occur together  . However, in oligodendroglial tumors, TERT promoter and IDH mutations occurred together . In primary glioblastomas the frequency of IDH mutations was low  and showed an inverse correlation  with TERT promoter mutations . In astrocytomas, where both, TERT promoter and IDH mutations, were frequent, the OR of 0.60 suggested an inverse association; however this observation was not statistically significant (In the entire set of tumors mutations in 3) Table . In contnificant .CDKN2A/B locus was deleted in 119 of 257 (46%) gliomas. The highest frequency of deletions was observed in primary glioblastomas   (The  0.0001) .TERT promoter mutations  and 23 were without the mutations. Analysis of quantitative real-time PCR data showed statistically significant higher levels of mRNA in gliomas with TERT promoter mutations than in wildtype tumors  compared to tumors without mutations . Stratification of the data according to the combined IDH and TERT promoter status revealed that only in the two groups that carry TERT promoter mutations (TERT promoter mutated and either IDH mutated or wildtype) telomere lengths is statistically significant shorter than in tumors that carry neither of the mutations ; further, patients aged younger than 20 years at diagnosis (n = 7) were excluded from the analysis.To assess the effect of the investigated alterations, we conducted a survival analysis excluding those patients with incomplete mutation data , followed by secondary glioblastomas ; diagnoses of oligoastrocytomas, oligodendrogliomas and astrocytomas were associated with better survival than primary and secondary glioblastomas , followed by the two groups without TERT promoter mutations that were either wildtype for IDH  or carried IDH mutations . Best overall survival was associated with the presence of both TERT promoter and IDH mutations , which resembles oligodendroglial progression. A multivariate model that included age, grade, treatment and loss of CDKN2A/B showed that the combined presence of TERT promoter and IDH mutations was the only independent factor  .Stratification of patients based on the mutational status of the  = 0.03) . The advTERT promoter mutations. Both, patients with and without TERT promoter mutations, had a median survival of 14.0 months , followed by the group with neither TERT promoter nor IDH mutations  and patients carrying only IDH mutations . TERT promoter mutations in combination with IDH mutations associated with best survival   whereas TERT promoter mutations alone associated with poor survival   . A multi = 0.02) .IDH mutations and TERT promoter mutations in astrocytomas. Patients with 1p/19q codeletions were associated with better survival than patients without codeletions  as well as poor survival for patients with TERT promoter mutations  and patients with CDKN2A deletions  (A multivariate model that included  = 0.01) .P = 0.03, IDH mutations showed a median survival of 245.5 months compared to 230.3 months in patients wildtype for IDH; however, this difference was not statistically significant  and 1p/19q codeletions have a protective effect   compared to patients without mutations , IDH mutations  and 1p/19q codeletions .In high grade gliomas erations . HoweverTERT promoter mutations but revealed a protective effect for IDH mutations as well as for 1p/19q deletions  did not receive any treatment, group 2) received radiotherapy alone or in combination with procarbazine or temozolomide and group 3) received temozolomide and radiotherapy combined with a third adjuvant. Patients who did not receive any treatment showed poor overall survival, followed by patients in group 2). In both groups the presence of TERT promoter mutations showed no effect. However, for patients in group 3) we observed that those without TERT promoter mutations  showed better overall survival than patients with the mutations  benefitted from the combination therapy (temozolomide + radiotherapy + adjuvant) in comparison to patients wildtype for the TERT promoter .We further investigated the effect of TERT promoter, IDH1/2, CDKN2A/B deletions, 1p/19q codeletions) we included data on rs2853669, a common SNP within the investigated fragment of the TERT promoter, in order to serve as an internal control that tissues were acquired from the same patients. In 15 cases, glioma tumors and their matched recurrences were consistent in all of the investigated alterations. In contrast, in another 5 pairs, tumors varied in one or more alterations, when compared to their recurrences. In four pairs, an initial CDKN2A/B deletion could not be detected in the recurrences. For two pairs the initial TERT promoter mutations and one IDH1 mutation were not found in the recurrences and those also showed differences in their histology  that had TERT promoter mutations in the initial tumors but not in the recurrence had median progression free survival of 65.3 months compared to 12.3 months for those patients (n = 3) that did not have the mutations in both initial tumors and recurrences. The median progression free survival for patients (n = 15) that had TERT promoter mutations in both initial tumors and recurrences was 8.5 months. The difference in progression free survival in three categories was, however, not statistically significant  and Chr5:1, 295, 250 (hg19 co-ordinate), which led to the erroneous labeling of the alterations as C228T and C250T [TERT promoter including the causal –57 C > A germline single nucleotide change reported in a melanoma family are joined by a common feature of de novo creation of CCGGAA/T sequence, which, besides being a recognition motif for Ets transcription factors, is a specific binding motif for ternary complex factors like Elk1 and Elk4 [TERT up-regulation, which conceivably would lead to increased telomerase levels [TERT promoter, in most other malignancies –124 C > T has been reported to be the most common alteration [TERT transcription, the increase in the expression in tumors with the –124 C > T mutation was significantly higher than in tumors with the –146 C > T mutations. One of the plausible reasons for the novel observation of differences in TERT transcription due to two mutations with apparently same phenotype could be context specific variations in accessibility to transcription factors through chromatin remodeling.The nd C250T , 43. Altand Elk4 . The mute levels , 44, 45.TERT transcription, we also for the first time in glioma observed influence on relative telomere lengths. The tumors with TERT promoter mutations in accordance with earlier observations in thyroid cancer had shorter telomeres than tumors without the mutations [TERT promoter mutations at the point of telomere crisis in the course of cellular transformation has been cited as a reason for an association with telomere length. The critically shortened telomeres in oncogene-driven proliferating tumor cells, in the absence of active telomerase, are thought to be causal in cell cycle arrest through activation of signaling cascade involving check point inhibitors [TERT promoter mutations and regeneration of telomerase to overcome telomeric crisis, probably provides the context for shorter telomeres in tumors with mutations [The promoter mutations not only affected utations . Presumehibitors , 47. Byphibitors . Selectiutations .IDH mutations in the absence of TERT promoter mutations had telomere lengths similar to those without mutations. Biologically IDH mutations are known to deregulate metabolic process. Mutant IDH proteins prevent normal conversion of isocitrate and lead to formation of the R-enantiomer of 2-hydroxylglutarate (R-2-HG) instead of a-ketoglutarate (a-KG) [TERT promoter mutations independently associate with poor in survival in many cancers, the mutations also associate with reduced telomere length. Here we show that IDH mutations that are in general markers of better prognosis in glioma do not associate with changed telomere length in tumors.Tumors with e (a-KG) . Increase (a-KG) . While TTERT promoter mutations than without mutations as an increased TERT expression is associated with the presence of mutations.An inverse correlation between relative telomere length and TERT expression in this study was in accorandance with an earlier report that showed shorter telomere length in gliobastoma that were positive for TERT expression and telomerase activity than those glioblastoma that were negative for both . ConversTERT promoter mutations add further complexity to the scenario but at the same time hold clear potential as prognostic markers. A combination of TERT promoter and IDH mutations support placement of gliomas into the subgroups, which is also reflected in survival and can be of considerable use in making appropriate treatment decisions. Data suggest that patients with TERT promoter mutations in tumors probably require more aggressive treatment than their wildtype counterparts. Further studies will help in elucidating the value of TERT promoter mutations as biomarkers in clinical practice and eventual therapeutic targets. Expression data and an association with shorter telomeres already strongly indicate the role of the TERT promoter mutations not only in glioma, but many other cancer types, and future functional studies will aid placing the TERT promoter mutations into the right context.Development of glioma subtypes along discrete pathways is characterized by the different genetic alterations that emerge along the way , 24, 52.Informed consent was obtained from all patients included in the study. The study was approved by the appropriate ethical committee. 303 primary tumors and additional 22 recurrences were collected between March 2010 and August 2013. Fresh tumor tissue was obtained directly from the operating theatre, snap-frozen and processed for further analysis as described below. In the majority of cases tumor samples were taken based on the MRI image using neuronavigation. Neuropathological diagnosis was made from paraffin-embedded tissue by a board-certified neuropathologist. Clinical data was collected in a database which included parameters such as patient age at first diagnosis, sex, histology at primary diagnosis and at recurrence, progression-free survival and overall survival or date of last patient contact. Progression-free survival was assessed on MRI imaging according to Macdonald criteria. Follow-up data was obtained from the family physicians and from the German Cancer Registry. The patients were treated according to the appropriate guidelines or in clinical trials. The date and number of surgeries, type and number of radiation and chemotherapy were documented.From fresh frozen glioma samples, DNA and RNA were extracted using the QIAGEN AllPrep DNA/RNA MiniKit. Tissues were homogenized in a Tissuelyser LT  with 5 mm stainless steel beads in 600 ml RLT buffer and were further processed. Concentrations of total DNA and RNA in all samples were measured using an ultraviolet–visible spectrophotometer  and absorption ratio at 260/280 nm was determined. RNA consistency was examined for a representative number of samples using the Bioanalyzer 2100 System  with the corresponding RNA nanochips.TERT promoter region (from position −27 to −286 from ATG start site) were identified by PCR and Sanger sequencing. Mutational status of IDH1 was cross-validated by sequencing for all available samples. Mutations in IDH2 were confirmed by sequencing if the respective probes indicated a signal in MLPA. PCR was carried out in a 10 μl volume containing 10 ng DNA, 50 mM KCl, 0.11 mM dNTP and 0.11 mM of each primer. Concentrations of MgCl2 and other additives and temperature conditions were adjusted according to the primer sequences , IDH1 (NC_000002.12) and IDH2 (NC_000015.).Mutations in the equences . AmplifiTo detect deletions at various relevant sites we used the P088-C1 probemix . This probemix can be used to detect loss of chromosome arms 1p and 19q, as well as copy number changes in CDKN2A and CDKN2B genes. Furthermore, it contains mutation-specific probes for IDH1 (R132H and R132C) and IDH2 (R172K and R172M). In addition, 14 reference probes are included, detecting different chromosomal locations known to be silent in glioma. Since probes for hot spot mutations in IDH1 (R132H and R132C) and IDH2 (R172K and R172M) were not established for the use in tumor tissue by the supplier, results were cross-validated by Sanger sequencing and we can confirm consistent results for both methods. Fifteen glioma samples were analyzed with a second kit also covering the 9p21 locus to compare results . The method was performed following the suggested protocol. In short, 50 ng of DNA per sample were subjected to 16 h of incubation with the probe mix and afterwards processed in a ligation reaction, followed by a multiplex PCR. Fragment analysis was performed on a capillary sequencer . The results were evaluated using Coffalyser software (MRC-Holland); threshold to define deletions was set at the suggested delta value of 0.3.TERT expression, reverse transcription reaction was performed using between 0.75 and 1.0 mg RNA and random hexamer primers using a cDNA synthesis kit . TERT expression levels were then determined by quantitative real-time PCR using a Sybr Green kit (Qiagen). The real-time PCR was carried out in triplicates on a 384-well layout using primers specific for TERT (GUSB gene (Qiagen), a housekeeping gene used as an internal standard. TERT expression levels were calculated using GUSB expression as a reference and relative quantification was performed using the ΔΔCT method and log2 transformation.For measurement of for TERT  and primRelative telomere length in tumor DNA was measured using the monochrome multiplex PCR assay as described previously including minor modifications , 54. DesTERT promoter mutations and other parameters were determined by chi2-test. A difference was considered statistically significant if the P-value was 0.05 or smaller. Odds ratios were calculated to assess the effect size. Overall survival was studied using univariate and multivariate Cox proportional hazards modeling, where appropriate. Survival endpoints were analyzed by Kaplan-Meier estimates and differences between the curves were analyzed with log-rank test.Associations between the