The association between psoriasis and carotid intima-media thickness (CIMT) or impaired flow-mediated dilation (FMD) remains controversial. We aimed to evaluate the extent of subclinical atherosclerosis as measured by CIMT and FMD in patients with psoriasis by conducting a meta-analysis.A systematic literature search was performed using PubMed, Embase, Cochrane databases, China National Knowledge Infrastructure, and VIP databases up to February 2015. Observational studies investigating CIMT or FMD in patients with psoriasis and controls were eligible. Psoriatic patients and controls were at least age- and sex-matched. Random-effects analysis was used to estimate the weighted mean difference (WMD) and 95% confidence interval (CI) between psoriatic patients and controls.A total of 20 studies were identified and analyzed. Meta-analysis showed that psoriatic patients had a significantly thicker CIMT  and lower FMD  than those in controls. Subgroup analysis indicated that psoriatic arthritis appeared to have less impaired FMD (WMD −2.45%) and thinner CIMT (WMD 0.10 mm). Psoriatic patients with mean age >45 years had much thicker CIMT (WMD 0.13 mm). The impaired FMD (WMD −3.99%) seemed more pronounced in psoriatic patients with mean age <45 years.This meta-analysis suggests that patients with psoriasis are associated with excessive risk of subclinical atherosclerosis. Screening and monitoring CIMT and brachial artery FMD may be recommended to identify a subgroup of psoriatic patients at higher risk for cardiovascular events. Psoriatic arthritis is defined as inflammatory arthritis associated with psoriasis. Psoriasis not only negatively affects the quality of life, but also increases risk of cardiovascular events5 and cardiovascular mortality.6 Therefore, early detection of subclinical atherosclerosis in psoriatic patients would help to reduce cardiovascular morbidity and mortality.Psoriasis is a chronic inflammatory skin disease characterized by relapsing thick scaling plaques.7 and carotid intima-media thickness (CIMT)8 have been suggested to be an important marker of subclinical atherosclerosis. Assessment of flow-mediated dilation (FMD) with high-resolution ultrasound in the brachial artery is a widely used method to evaluate the endothelial function.9 CIMT is usually determined by using B-mode ultrasound technique in the common carotid artery. Determination of FMD and CIMT is widely used in clinical practice because of their noninvasive technique. Most studies have shown evidence of subclinical atherosclerosis in psoriatic patients as indicated by increased CIMT23 or impaired FMD26 than the matched controls. However, conflict findings regarding the relationship between psoriasis and subclinical atherosclerosis risk still exist.30 These conflicting results might be correlated with the severity or duration of psoriasis and population studied.Endothelial functionThis meta-analysis aims to quantitatively estimate the association between psoriasis and subclinical atherosclerosis as measured by CIMT and FMD in patients with psoriasis by conducting a meta-analysis.31 This meta-analysis was not based on the individual participant data; ethical approval was not applicable. A systematic search of studies published before February 2015 was conducted through PubMed, Embase, Cochrane databases, China National Knowledge Infrastructure, and VIP databases. The following medical subject headings terms were used for the literature search: “psoriasis” OR “psoriatic arthritis” AND “carotid intima-media thickness” OR “carotid atherosclerosis” AND “endothelial function” OR “flow-mediated dilation” AND “subclinical atherosclerosis”. Only fully published articles in peer-reviewed journals were included. The references of retrieved articles were also reviewed to identify any relevant study.This study was conducted according to the recommendations of the Meta-Analysis of Observational Studies in Epidemiology.Inclusion criteria were: observational studies investigating the relationship between psoriasis with or without psoriasis arthritis and endothelial function  or mean CIMT; reporting CIMT or FMD as continuous data for patients with psoriasis and controls; psoriatic patients and controls were at least age- and sex-matched. Exclusion criteria were: lack of an eligible control group; evaluating endothelial function except for FMD; studies did not provide CIMT or FMD as mean values and standard deviation (SD) or standard error.32 with the following 3 items: selection of the study groups, between-group comparability, and the ascertainment of either the exposure or the outcome. Study with NOS score ≥5 was judged to be of higher quality.The following data were extracted from each included study: first author's name, publication year, geographic region, study design, type of psoriasis, psoriasis severity (Psoriasis Area and Severity Index score), characteristics of participants , CIMT (mean and SD), FMD (mean and SD), matched factor, whether exclusion of cardiovascular risk factors in participant selection. The methodological quality of the selected studies was assessed by using the Newcastle–Ottawa Scale (NOS)I2 statistic and Cochran Q statistic. A I2 statistic value <50% or Cochran Q value of P < 0.05 was considered substantial heterogeneity. Random-effects analysis was used to estimate the effect size because of the anticipated clinical heterogeneity among included studies. The presence of publication bias was investigated by the Egger regression33 and Begg correlation test34 with a P value <0.01, which is statistically significant. Subgroups analyses were performed according to the type of disease (psoriasis or psoriatic arthritis), study design , matched factor (matched BMI or not), mean age of psoriatic patients (>45 years or <45 years), and whether participants with cardiovascular risk factors were excluded. All statistical analyses were performed with Stata software 12.0 .CIMT and FMD were expressed as continuous data. The pooled effect size was calculated as the weighted mean difference (WMD) with 95% confidence interval (CI) by the inverse variance optimal approach between the psoriatic patients and control group. The degree of heterogeneity across studies was tested by using the 35 did not provide SD of the outcome; 2 studies37 did not provide average intima-media thickness values; and participants’ selection did not match with age or sex in 2 studies.39 One study40 was further excluded because the reported FMD value was particularly high in both psoriatic patients and controls than other studies. Finally, only 20 studies30 were included in the quantitative synthesis. The flowchart of the studies’ selection process is outlined in Figure The initial literature search yielded a total of 766 potentially relevant articles. After screening on the basis of abstracts or titles, 711 articles were excluded. After full manuscripts assessed for eligibility, 26 articles appeared to satisfy the inclusion criteria. Six articles were excluded for the following reasons: one study30 comprising 1066 psoriatic patients and 962 controls were included in the meta-analysis. The sample size of individual study ranged from 54 to 196. Of the 20 studies, 1630 were case–control studies, 324 were cross-sectional studies, and 126 was cross-sectional plus case–control design. All the studies were published from 2007 to 2014. All studies clearly reported matching of patients and controls by sex and age design. Ten studies excluded participants with preexisting cardiovascular risk factors. The studies enrolled participants with a mean age range from 30 to 57.85 years. The characteristics of the included studies are shown in Table 30 All quality scores of the included studies were >5 according to the NOS. NOS scores of the included studies are presented in Table 30Twenty studies30 assessed the CIMT difference in 934 psoriatic patients and 866 controls. As shown in Figure I2 = 90.7%; P < 0.001). Neither the Begg rank correlation test (P = 0.137) nor the Egger linear regression test (P = 0.634) showed evidence of publication bias.Sixteen studies28 assessed the FMD difference in 424 psoriatic patients and 400 controls. As shown in Figure I2 = 97%; P < 0.001), the pooled random-effect difference in FMD showed that psoriatic patients had a significant decrease in FMD than controls . Neither the Begg rank correlation test (P = 0.348) nor the Egger linear regression test (P = 0.149) showed evidence of publication bias.Eight studiesSubgroup analyses showed that psoriatic arthritis alone appeared to have less CIMT  and less impaired FMD . Cardiovascular risk factors, age, and BMI of participants could influence the results. The detailed results of the subgroup analyses are shown in Table This meta-analysis suggests that patients with psoriasis are associated with an increased CIMT and impaired brachial artery FMD than the healthy controls, which reflects the subclinical atherosclerosis. Compared with controls, psoriatic patients had 2.79% reduction in brachial artery FMD and 0.11 mm thicker CIMT. Particularly, psoriatic arthritis had less impaired brachial artery FMD and thinner CIMT than total psoriatic patients.41 Subgroup analysis indicated that the pooled WMD of CIMT was increased when we pooled studies of matching factor with BMI. Together these findings, psoriatic patients were associated with accelerated subclinical atherosclerosis and might be independent of the classical atherosclerotic risk factors.When we investigated the relationship between psoriasis and subclinical atherosclerosis, the impact of type of disease, BMI, concomitant cardiovascular risk factors, and age should be considered. Subgroup analysis showed that patients with psoriatic arthritis appeared to have less impaired brachial artery FMD and thinner CIMT than the total psoriatic patients. CIMT may be affected by other cardiovascular risk factors. However, WMD of impaired FMD or increased CIMT remained statistically significant for participants without clinical evidence of atherosclerotic risk factors. These findings implied that the clustering of cardiovascular risk factors in psoriatic patients may amplify the effect of psoriasis on subclinical atherosclerosis. Psoriatic patients had a higher prevalence and incidence of obesity.Elder people are usually associated with accelerated atherosclerosis. In this study, psoriatic patients with mean age >45 years appeared to have greater thicker CIMT than those younger than 45 years. On the contrary, the impaired brachial artery FMD was more pronounced in patients with mean age <45 years. Impaired endothelial function might precede any change in CIMT. These findings revealed that determination of FMD may be recommended for psoriatic patients with mean age <45 years, whereas measurement of CIMT might be suitable for the older patients.42 Our meta-analyses indicated that patients with psoriasis were associated with excessive risk of subclinical atherosclerosis. Accordingly, a systematic review summarized that patients with psoriasis and psoriatic arthritis had impaired endothelial function compared with the general population, as measured by pulse wave velocity and aortic stiffness parameters.43 A more recent published meta-analysis44 suggested that patients with psoriatic arthritis appeared significantly associated with markers of subclinical atherosclerosis. However, this meta-analysis mainly focused on patients with psoriatic arthritis but not address total psoriatic patients. These meta-analyses supported that patients with psoriasis may increase future cardiovascular morbidity and mortality.The association between psoriasis and risk of cardiovascular disease is controversial. A well-designed meta-analysis suggested that psoriasis was associated with ischemic heart disease but not cerebrovascular disease and cardiovascular mortality.46 Moreover, many immunological factors involved in psoriasis, such as C-reactive protein and tumor necrosis factor-α, also contribute to atherosclerosis.47The exact mechanisms of psoriasis in promoting atherosclerosis remain unclear. Psoriasis is considered a systemic inflammatory condition. The chronic systemic inflammatory state has been linked to an acceleration of the atherosclerotic lesions. Chronic systemic inflammation induces endothelial dysfunction, altered glucose metabolism, and insulin resistance that play a significant role in the progress of atherosclerosis.I2 = 90.7%) and FMD (I2 = 97%) was observed. The sources of heterogeneity might be correlated with the study design, age of the participant, presence or absence of psoriatic arthritis, and with or without atherosclerotic risk factors. Finally, we were unable to determine the effects of pharmacologic therapy on the progression of atherosclerosis in psoriatic patients.Several limitations should be considered. First, the causal association between subclinical atherosclerosis and psoriasis could not be defined because of the case–control or cross-sectional nature of the included studies. Second, studies using other techniques to evaluate subclinical atherosclerosis were not included in this meta-analysis. Third, as for the included studies did not provide data about the severity or duration of psoriasis on the subclinical atherosclerosis, so we could not determine whether the severity of psoriasis or longer duration of the disease increased the extent of subclinical atherosclerosis. Fourth, significant heterogeneity in pooled CIMT (This meta-analysis suggests that psoriatic patients are associated with excessive risk of subclinical atherosclerosis compared with the healthy controls. Assessment of CIMT and FMD of the brachial artery may be recommended to identify a subgroup of patients at higher risk for cardiovascular events in psoriatic patients. Psoriatic patients with mean age >45 years appeared to have greater thicker CIMTA and need frequent follow-up. Moreover, further studies are needed on whether the treatment of psoriasis will reverse subclinical atherosclerosis.


Protein-protein interactions (PPIs) create the steps in signaling and regulatory networks central to most fundamental biological processes. It is possible to predict these interactions by making use of experimentally determined orthologous interactions in other species.In this study, prediction of PPIs in rice was carried out by the interolog method of mapping deduced orthologous genes to protein interactions supported by experimental evidence from reference organisms. We predicted 37112 interactions for 4567 rice proteins, including 1671 predicted self interactions (homo-interactions) and 35441 predicted interactions between different proteins (hetero-interactions). These matched 168 of 675 experimentally-determined interactions in rice. Interacting proteins were significantly more co-expressed than expected by chance, which is typical of experimentally-determined interactomes. The rice interacting proteins were divided topologically into 981 free ends (proteins with single interactions), 499 pipes (proteins with two interactions) and 3087 hubs of different sizes ranging from three to more than 100 interactions.This predicted rice interactome extends known pathways and improves functional annotation of unknown rice proteins and networks in rice, and is easily explored with software tools presented here.The online version of this article (doi:10.1186/1939-8433-5-15) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae  are essential for many fundamental biological processes. With the advent of high-throughput approaches, genome-wide networks of PPIs have been generated in http://www.ebi.ac.uk/intact; Aranda et al., Arabidopsis thaliana was made consisting 1159 high confidence, 5913 medium confidence and 12907 low confidence interactions. This was established using a confidence scoring based method on the number of different data sets in which the interaction was recorded, the number of different types of experiments supported the interactions, and the number of species in which the interaction was discovered . A confidence value  was calculated to estimate the strength of experimental support for each predicted interaction. We have identified 37112 predicted interactions for 4567 rice proteins  had an ortholog that matched at least one of the eleven reference organisms  database.A gold standard of 675 experimentally confirmed interactions was abstracted from IntAct (Drosophila (9 interacting partners) and Arabidopsis (11 interacting partners) and smaller than in yeast (22 interacting partners). Table The rice interacting proteins were divided into free ends (981 proteins with single interactions), pipes (499 proteins with two interactions) and hubs of different sizes ranging from three to more than 100 interactions. The largest class of proteins is that of medium hubs (between 11 to 50 interactions). The interacting proteins had an average of 14–15 partners, which is comparable to Protein-Protein interactions are often mediated by protein interaction domains (PIDs) which bind to other domains (domain-domain interactions), or short conserved amino acid motifs in the partner protein. PIDs that interact most frequently among the rice protein pairs (interologs) were determined using annotation by PFAM domain models (see methods). In rice, the most enriched domains among the interactors included known interaction domains PF00400 (WD40), PF00069 (protein kinase), PF00270 (DEAD), and PF02985 (HEAT). Looking at domain-domain interactions amongst interacting pairs, domain pairs (with one domain in each interacting protein) with extremely high fold of enrichment (vs. random pairing) were often otherwise rare domains among the protein interactions. Among the twenty most frequent PFAM interacting pairs were 8 self interactions between homodomains and 12 interactions between different PFAM domains. Of these 14 (70%) were enriched more than 2 fold, with the highest fold of enrichment (134 fold) for interaction between the homodomains of the proteasome  gene in flowers . By using the graph definition language GraphML, the color of each protein (node) is painted differently based on its predicted subcellular localization , none of the experimental data sets from reference organisms are complete. The interactions contained in this data set are of evolutionarily conserved proteins and pathways. However as the experimental data are based on high throughput studies, the predicted rice interactome does not represent only well studied interactions like the proteosome, ribosome and base transcriptional machinery: there are still some surprising connections and a number of unknown proteins within the data set as well.A rice interactome has been predicted based on conservation of protein interactions across species over the course of evolution. Each interaction has been assigned a confidence level (CV) as an internal quality control. Assignments for subcellular localization and coexpression can be used to further validate the predicted interaction. Coexpression analysis has shown that interacting proteins tend to have similar expression profiles, and tend to be localized to the same or adjacent compartment. By inputting rice proteins of interest, the Rice Interactions Viewer Figure  can be uNote added in revision: we expanded the number of predictions by incorporating Braun et al.’s Arabidopsis Interactome data set (Arabidopsis Interactome Oryza sativa subsp. japonica), Arabidopsis thaliana, Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Escherichia coli, Bacillus subtilis, Helicobacter pylori were retrieved from TAIR  were retrieved for the prediction of rice interactome.Peptide sequences from rice . A predicted interactome was recorded in an Excel spreadsheet when both interacting proteins in a reference species had orthologs in rice. Duplicates of the same interactions were removed from different interactome data sets and from different reference species to generate unique interacting protein pairs.Interactome databases were obtained from BioGRID  for experimental support of a predicted interaction was calculated using the following formula, CV = N x E x S; where N is the total number of data sets in which the interaction was recorded, E is the number of different experimental methods from which an interaction was inferred, and S is the number of reference species from which the same interaction was recorded. The higher the CV, the more likely an interaction is to be conserved in multiple species and to be more convincing as it is supported by different experimental methods. The interactions were classified according to their CV to three groups: low confidence (CV = 1), medium confidence (CV = 2 to 10) and high confidence (CV > 10).http://geneontology.org). The top 7 enriched GOSlim terms for molecular functions (MF) and cellular component (CC), respectively; were used as node features in the visualization of protein network using Cytoscape 2.6  terms for rice were retrieved from the GO site . Some of the proteins involved in the interactome may possess more than one PFAM domain, thus all possible pairs of PFAM domains of the interacting protein pairs were queried using MySql  of each protein (the number of proteins that interact with it), and the total number of interacting pairs (EE), nab is given byHere Cia = 1 or 0, whether protein i is in compartment a or notCib = 1 or 0, whether protein i is in compartment b or notki,kj = the node degree of protein(node) i,jeij = the probability of protein(node) i and protein(node) j interactsE = total number of interactions(edges)P(multi) = 1 − (1−P)m where for enrichment m equals the number of ab pairs with at least one observed interolog and for depletion m equals the number of ab pairs possible in the ensemble of random networks.The P values are finally subject to a multiple-test correction Coexpression between interacting proteins was determined using the Pearson correlation coefficientith sample, and Y is the expression level for gene Y in the ith sample (−1≤ r ≤1).Where N is the number of expression samples, X is the expression level for gene X in r values indicate a correlation of expression patterns, while low negative r values indicated anti-correlation. A large multi data set compendium consisting of 165 diverse data sets Additional file 1:Table S2. PFAM domains for interacting pairs. (XLSX 3 MB)Additional file 2:Table S3. List of GSM identifiers of microarray expression data. (XLSX 17 KB)Additional file 3:Table S4. Experimental verification of predicted interactions. (XLSX 43 KB)Additional file 4:Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Below are the links to the authors’ original submitted files for images.


In vivo studies were performed to evaluate the activities of two thiazolidinedione PPARγ agonists, rosiglitazone and pioglitazone, as inhibitors of oral carcinogenesis in rats. Oral squamous cell carcinomas (OSCC) were induced in male F344 rats by 4-nitroquinoline-1-oxide . In each study, groups of 30 NQO-treated rats were exposed to a PPARγ agonist beginning at week 10 (one day after completion of NQO administration) or at week 17 (7 weeks post-NQO); chemopreventive agent exposure was continued until study termination at week 22 (rosiglitazone study) or week 24 (pioglitazone study). Administration of rosiglitazone (800 mg/kg diet) beginning at week 10 increased survival, reduced oral cancer incidence, and reduced oral cancer invasion score in comparison to dietary controls; however, chemopreventive activity was largely lost when rosiglitazone administration was delayed until week 17. Administration of pioglitazone (500 mg/kg diet beginning at week 10 or 1000 mg/kg diet beginning at week 17) induced significant reductions in oral cancer incidence without significant effects on OSCC invasion scores. Transcript levels of PPARγ and its three transcriptional variants  were not significantly different in OSCC versus age- and site-matched phenotypically normal oral tissues from rats treated with NQO. These data suggest that PPARγ provides a useful molecular target for oral cancer chemoprevention, and that overexpression of PPARγ at the transcriptional level in neoplastic lesions is not essential for chemopreventive efficacy.Peroxisome-proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor that regulates cell proliferation, differentiation, and apoptosis.  In spite of continuing improvements in cancer therapy, oral squamous cell carcinoma (OSCC) remains a significant problem in the United States and around the world. The American Cancer Society projects that approximately 39,500 new cases of oral or oropharyngeal cancer will be diagnosed in the United States in 2015, and that approximately 7500 people will die of these cancers [The oral cancer problem is even more significant outside of the United States, as approximately 2/3 of new oral cancer cases are diagnosed in developing countries . SubstanMuch of the variation in international rates of oral cancer appears to reflect differences in lifestyle factors that underlie disease etiology. The most important risk factors for human oral carcinogenesis are the use of tobacco and alcohol –9. RecenClearly, differences in smoking behavior are responsible for much of the variation in OSCC incidences seen in different countries. In addition, the use of smokeless tobacco products (chewing tobacco and snuff) is clearly linked to increased oral cancer risk –12, as iExposure to human papillomavirus (HPV) is an emerging and potentially major etiologic factor for oral cancer. Individuals infected with HPV demonstrate an increased risk of OSCC ,14, and e.g., tobacco, alcohol, HPV) and secondary prevention efforts involving cancer chemoprevention are necessary to reduce mortality from this disease.Data collected in the United States for the period of 2005 to 2011 demonstrate a 5-year survival rate of 63.2% for patients with oral or pharyngeal cancer; this compares to a 5-year survival rate of 52.7% reported in 1975 . The modPeroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor that plays key roles in the regulation of cell proliferation, differentiation, and apoptosis . Three dAn expanding body of evidence supports the hypothesis that activation of signaling pathways regulated by PPARγ may provide an effective strategy to prevent and/or treat cancer . Activation of PPARγ modulates numerous signaling pathways and associated cellular responses that are clearly linked to carcinogenesis. On this basis, PPARγ presents an attractive molecular target for cancer chemoprevention. Furthermore, the ubiquitous expression of PPARγ1 suggests that its activation by pharmacologic agents may provide a useful strategy for cancer prevention in several organ sites. To test this hypothesis, the present studies were performed to evaluate the activity of rosiglitazone and pioglitazone, two thiazolidinedione agonists of PPARγ, as inhibitors of the induction of OSCC. Studies were performed in a model for oral carcinogenesis that has been used in chemoprevention efficacy evaluations of numerous other classes of agents (reviewed in ).in vivo studies, study protocols were reviewed and approved by the IIT Research Institute Animal Care and Use Committee. All aspects of the program involving animal care, use, and welfare were performed in compliance with United States Public Health Service Policy on Humane Care and Use of Laboratory Animals, and in compliance with the guidelines stated in the National Research Council Guide for the Care and Use of Laboratory Animals. During twice daily mortality/moribundity checks, sixteen criteria for animal euthanasia are used to identify animals that should be considered for euthanasia. These criteria are consistent with the American Veterinary Medical Association Guidelines for the Euthanasia of Animals (2013 Edition). Specific criteria for euthanasia that were used in the present studies included: loss of > 15% of body weight in one week; gradual but continuous decline in body weight; adequate indication that a study animal may not survive until the next scheduled observation; and prolonged unhealthy appearance such as rough coat or hunched posture.Prior to the initiation of in vivo carcinogenesis studies were performed to evaluate the chemopreventive efficacy of rosiglitazone and pioglitazone. The dose levels of each agent used in chemoprevention studies were selected on the basis of the results of a preliminary toxicity/dose selection study of that agent. The goals of dose selection studies were to identify dietary levels of each agent that induced no mortality, body weight suppression, or other evidence of limiting toxicity.Separate Male F344 rats were received at approximately seven weeks of age from virus-free barrier colonies either: (a) maintained under contract to the National Cancer Institute, Frederick, MD (rosiglitazone studies); or (b) at Charles River Laboratories, Raleigh NC (pioglitazone studies). After receipt from the vendor, rats were held in quarantine for a minimum of one week prior to randomization into a dose selection or oral cancer chemoprevention study. Prior to release from quarantine and randomization into a study, each rat underwent a hand-held clinical and physical examination to ensure its suitability for use as a test subject.Rats were housed in pairs or trios on hardwood bedding in polycarbonate shoebox cages in a windowless room that was illuminated for 12 hours each day and maintained at 22 ± 1°C and within the range of 30% to 70% relative humidity. Throughout all studies, rats were permitted free access to Purina 5001 Laboratory Diet  and City of Chicago drinking water (provided in water bottles).2 and subjected to a limited gross necropsy to identify any gross pathology associated with agent exposure.A preliminary toxicity/dose selection study was performed for each agent to select dose levels for use in the chemoprevention study. In each dose selection study, groups of 10 male F344 rats received diets supplemented with rosiglitazone or pioglitazone at doses of 0, 200, 400, 600, 800, or 1000 mg/kg diet for 6 weeks. Rats were observed twice daily to identify any gross clinical evidence of toxicity; body weight was measured individually for each rat once per week. At the end of the exposure period, each rat was humanely euthanized with COHyperplastic and neoplastic lesions were induced on the tongue of male F344 rats by drinking water administration of 4-nitroquinoline-1-oxide (NQO), as described by Tanaka (reviewed in ) and useAfter release from quarantine, rats were assorted into groups of 30 using a constrained randomization procedure that blocks for body weight. Rosiglitazone and pioglitazone were obtained from the Chemopreventive Agent Repository maintained by the Division of Cancer Prevention, National Cancer Institute. Dietary administration of each chemopreventive agent was initiated either: (a) one day after completion of NQO exposure (study week 10); or (b) 7 weeks after completion of NQO exposure (study week 17). Once initiated, administration of chemopreventive agents was continued until study termination at week 22 (rosiglitazone study) or week 24 (pioglitazone study).Cage-side observations were performed a minimum of twice daily to evaluate animal health and identify possible toxicities resulting from administration of NQO or PPARγ agonists. Rats were weighed weekly throughout the studies. Body weight is a key metric in oral cancer chemoprevention studies in the NQO model, since body weight loss provides a useful indication of the clinical progression of OSCC . Hand-he2 asphyxiation. All study animals  received a limited gross necropsy focused on the tongue and oral cavity. The tongue from each rat was carefully excised, all gross lesions were charted, and the tongue was bisected longitudinally. At necropsy, half of each tongue was fixed in 10% neutral buffered formalin for histopathologic evaluation, and the other half of each tongue was snap-frozen in liquid nitrogen and stored at -80°C for use in molecular analyses. To avoid possible artifacts due to post-mortem changes, only tissues from freshly euthanized animals were used for molecular studies.At the termination of each chemoprevention study; surviving rats were humanely euthanized by COEach bisected tongue was processed by routine histologic methods, and replicate sections were cut at 5 μm and stained with hematoxylin and eosin. The histopathologic classification of each lesion and its depth of invasion were determined in order to (a) quantify tumor incidence and (b) determine if a shift to a less invasive lesion may have occurred as a result of chemopreventive agent administration. Cancer invasion was classified using a semi-quantitative grading system . In thisMicroarray analysis was performed on 11 pairs of histologically confirmed oral cancers and adjacent phenotypically normal oral tissues from dietary control rats treated with NQO. After tissues were removed from storage at -80°C, RNA was isolated, and the quantity and quality of each RNA sample was determined using an Agilent Bioanalyzer . Gene expression in each tissue pair was then analyzed using Agilent Rat GE 4x44K v3 arrays . First and second strand cDNAs were prepared, cRNA target was prepared from the DNA template, verified, fragmented to uniform size, and hybridized to the microarrays. Slides were washed and scanned using an Agilent G2565 Microarray Scanner. Microarray data were analyzed using Agilent Feature Extraction and GeneSpring GX v7.3.1 software packages. Microarray data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO accession GSE51125).RT-PCR analyses were performed to compare the relative abundance of total PPARγ and its variants  in fifteen pairs of age- and site matched normal and neoplastic oral tissues. Tissues used for RT-PCR analyses were harvested from NQO-treated dietary control rats from a different study than tissues used for microarray analysis. Total RNA was isolated from these tissues by homogenization in 0.2 ml TRIzol reagent , followed by extraction using an additional 0.8 ml TRIzol. RNA integrity was determined by 1% agarose gel electrophoresis; purity was assessed by spectrophotometry.http://bioinfo.ut.ee/primer3-0.4.0/); primer sequences are provided in -(∆Cq), where ∆Cq is Cq(sample)—Cq(HSP90ab1). HSP90ab1 was used as a common reference gene for normalization; we have recently identified HSP90ab1 as a reliable reference gene in this model. To ensure the specificity of PCR for each primer pair, melting curve analysis was performed after PCR reactions.Gene-specific primers were designed using Primer 3 software (p < 0.05) reduction in oral cancer incidence, reduction in oral cancer invasion score, or increase in survival in a carcinogen-treated group receiving a PPARγ agonist when compared to the carcinogen-treated dietary control group. Comparisons of OSCC incidence were performed using X2 analysis. Because oral cancer invasiveness was evaluated using a semi-quantitative scoring system, comparisons of invasion scores were performed using non-parametric statistics . Animal survival was evaluated using X2 analysis. Body weights and other continuous data were compared by analysis of variance, with post-hoc comparisons made using Dunnett's test.Evidence of chemopreventive activity was defined as a statistically significant , diet supplemented with rosiglitazone (800 mg/kg diet) from weeks 10 to 22 , or diet supplemented with rosiglitazone (800 mg/kg diet) during weeks 17 to 22 (delayed intervention schedule). The 800 mg/kg diet dose level of rosiglitazone was selected on the basis of the results of a preliminary toxicity/dose selection study.Administration of rosiglitazone beginning at week 10 conferred significant protection against the induction of OSCC by NQO . Chemoprp < 0.05). The decrease in the incidence of OSCC in rats receiving rosiglitazone by the normal intervention schedule (weeks 10 to 22) was accompanied by a statistically significant increase in the incidence of squamous cell papillomas, a putative preneoplastic lesion, and a non- significant increase in the incidence of squamous epithelial hyperplasia  in the dietary control group, the incidence of OSCC in rats receiving rosiglitazone from weeks 10 to 22 was 63%  versus 73% (22/30) in the dietary control group; p < 0.05).Administration of rosiglitazone beginning at week 10 also induced a statistically significant shift in OSCC invasion scores . WhereasAlthough administration of rosiglitazone beginning at study week 10 conferred significant protection against oral carcinogenesis, delaying the start of rosiglitazone exposure until week 17 resulted in the loss of much of its chemopreventive activity. When compared to dietary controls, the group receiving delayed administration of rosiglitazone demonstrated a small reduction in oral cancer incidence  and a mop < 0.05; Changes in body weight provide a useful marker for oral cancer incidence and chemopreventive efficacy in this model. Actively growing NQO-induced oral cancers are commonly associated with body weight loss during later weeks of chemoprevention studies ; these cAdministration of rosiglitazone beginning at week 17 had no statistically significant effect on group mean body weight, but did prevent the apparent tumor-associated body weight loss seen in dietary controls between weeks 19 and 22 of the study .In the chemoprevention efficacy evaluation of pioglitazone, groups of 30 NQO-treated rats received either unsupplemented basal diet (control); diet supplemented with the high dose of pioglitazone (1000 mg/kg diet) from weeks 10 to 24 ; diet supplemented with the low dose of pioglitazone (500 mg/kg diet) from weeks 10 to 24; or diet supplemented with the high dose of pioglitazone from weeks 17 to 24 (delayed intervention schedule). The high dose of pioglitazone (1000 mg/kg diet) was selected on the basis of the results of a preliminary toxicity/dose selection study.p < 0.05 for both comparisons). In both groups, the lower incidence of invasive oral squamous cell carcinomas was accompanied by an apparently compensatory increase in the incidence of squamous epithelial hyperplasia, a putative precursor lesion in this experimental model (0.05 < p < 0.10 for both comparisons). Unexpectedly, when administered by the standard intervention protocol, the high dose of pioglitazone had no significant effect on oral carcinogenesis, and also did not increase the incidence of oral squamous epithelial hyperplasia. When compared to dietary controls, pioglitazone had no statistically significant effect on oral cancer invasion scores in any group  of pioglitazone and delayed administration of the high dose (1000 mg/kg diet) of pioglitazone .A similar lack of dose-relatedness was seen in animal survival . At studMicroarray analyses of 11 pairs of OSCC and histologically normal oral tissues collected from NQO-treated rats failed to demonstrate any differential expression of PPARγ. In these tissue pairs, the mean level of PPARγ transcripts in OSCC was 101.4 ± 4.4% of the mean level of PPARγ transcripts in phenotypically normal oral tissues . Most of the genes that were identified as differentially expressed in OSCC are involved in lipid metabolism: significantly down-regulated genes included CD36 (thrombospondin receptor); acyl-CoA dehydrogenases (C-4 to C-12 straight chain and long chain); acyl-coA synthetases (long chain family members 1 and 6); acyl co-A oxidases (branched chain); and carnitine palmitoyltransferases 1b and 2. Significantly up-regulated genes in OSCC included angiopoietin-like 4, adiponectin, and fatty acid desaturase 2. The roles, if any, of these genes in the etiology and biology of OSCC remain to be defined.Although microarray data did not demonstrate significant differential expression of PPARγ in rat oral cancers, KEGG analysis of microarray data from these samples did identify statistically significant differential expression of PPARγ-associated signaling pathways in OSCC. Analysis of microarray data using Genespring software identified 19 PPARγ-associated genes whose expression was significantly different in OSCC versus paired histologically normal oral tissues  that can be used to characterize disease progression and agent efficacy. Convenient access to preneoplastic and neoplastic oral lesions also facilitates collection of tissues for histopathology, biochemical testing, or molecular analyses.in vivo studies were performed to determine the chemopreventive activity of rosiglitazone and pioglitazone, two thiazolidinedione compounds that are well-studied agonists of PPARγ. We report that rosiglitazone and pioglitazone both confer statistically significant protection against carcinogenesis in a well-studied rat model for OSCC. Chemopreventive efficacy was expressed as a statistically significant reduction in oral cancer incidence (rosiglitazone and pioglitazone); a statistically significant reduction in oral cancer invasiveness (rosiglitazone); and a statistically significant reduction in oral cancer-related mortality (rosiglitazone). Although a substantial number of PPARγ-associated molecular pathways were differentially expressed in OSCC in comparison to site-matched phenotypically normal oral tissues, neither total PPARγ nor any PPARγ transcript variant was significantly overexpressed at the RNA level in OSCC. On this basis, it is concluded that the chemopreventive efficacy of the two PPARγ agonists evaluated in these studies is not dependent on overexpression of PPARγ or any PPARγ transcript variant in OSCC.The present studies were performed to evaluate the hypothesis that neoplastic development in the oral cavity can be inhibited or delayed by pharmacologic activation of PPARγ action. To address this hypothesis, N-butyl-N-(4-hydroxybutyl)nitrosamine [Although rosiglitazone and pioglitazone both demonstrate significant activity as chemopreventive agents for oral cancer in the NQO/F344 rat model, the toxicity of these agents in humans makes them unacceptable for possible clinical use in cancer prevention. Clinical administration of thiazolidinediones has been linked to several serious toxicities in humans , includirosamine .N-acetylfarnesylcysteine [e.g., dual PPARα/γ agonists such the glitizars [e.g., N-(4-hydroxyphenethyl)-3-mercapto-2-methylpropanamide [Although rosiglitazone and pioglitazone are not appropriate candidates for use in human cancer chemoprevention, it has been suggested that the clinical toxicities reported for these and other PPARγ modulators may be compound-specific rather than the result of the mechanism of action of the drug class . For thicysteine  or isoqucysteine  that arelitizars ,35); andpanamide ) that repanamide .in vitro [in vitro [The chemopreventive activity of PPARγ agonists in the oral cavity and other epithelial tissues may be mediated by effects on a substantial number of signaling pathways, including several that play key regulatory roles in inflammation, cell proliferation, and carcinogenesis. Treatment with a PPARγ agonist has been reported to reduce COX-2 expression in human prostate cancer cells in vitro ; we havein vitro . Similarin vitro . The chein vitro . It shouin vitro . ClearlyThe results of the present studies suggest that additional agonists or partial agonists of PPARγ merit consideration for efficacy evaluation in preclinical models of oral carcinogenesis. In consideration of the clinical toxicity of rosiglitazone and pioglitazone, emphasis should be placed on natural products and new molecular entities whose structures are unrelated to thiazolidinediones. Should a novel agent or natural product demonstrate both significant chemopreventive activity in preclinical models and a toxicity profile that is consistent with use in healthy or at-risk individuals , clinical studies to evaluate the efficacy of that agent in the prevention of human oral cancer could provide a valuable addition to clinical strategies to control this malignancy.


Certain mutations are represented with more than one line, which allows for studies of variable genetic backgrounds. In addition, these iPSCs can be successfully differentiated to astroglia, a cell type known to play a critical role in ALS disease progression. This library represents a comprehensive resource that can be used for ALS disease modeling and the development of novel therapeutics.Amyotrophic lateral sclerosis is a progressive disease characterized by the loss of upper and lower motor neurons, leading to paralysis of voluntary muscles. About 10% of all ALS cases are familial (fALS), among which 15–20% are linked to Cu/Zn superoxide dismutase (SOD1) mutations, usually inherited in an autosomal dominant manner. To date only one FDA approved drug is available which increases survival moderately. Our understanding of ALS disease mechanisms is largely derived from rodent model studies, however due to the differences between rodents and humans, it is necessary to have humanized models for studies of disease pathogenesis as well as drug development. Therefore, we generated a comprehensive library of a total 22 of fALS patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized before being deposited into the library. The library of cells includes a variety of C9orf72 mutations,  Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a fatal disease characterized by the loss of upper and lower motor neurons, leading to paralysis of voluntary muscles . The mecTo date, only one drug, riluzole, is FDA approved for delaying disease progression for ALS patients with only modest efficacy in increasing survival . The vasFortunately, significant progress in human induced pluripotent stem cell (iPSC) research provides a novel valuable tool for ALS research. Soon after the first reports on human iPSC generation ,16, neurHere we report that we generated a library of familial ALS patient-specific iPSCs (fALS-iPSCs) carrying a large variety of ALS mutations. These fALS-iPSCs can be differentiated to astroglia, a critical cell type that plays a role in ALS progression. In order to be widely useful, this “library” is based on the more common different ALS mutations, including SOD1 and FUS, and several lines of the same mutation are collected from different families. The fALS-iPSCs and their parental fibroblasts were generated to be publicly available to academic and commercial entities alike. The fibroblast lines have been reported separately . We beliThe use of human tissues and associated demographic information was approved by the Johns Hopkins University Institutional Review Board and Ethics Committee . All skin biopsies were collected following written informed consent from the donor. The informed consent was approved by the Johns Hopkins University Institutional Review Board and Ethics Committee of Johns Hopkins Hospital (IRB protocol: NA-00021979). The consent allowed for use by all parties, commercial and academic, for research purposes only but not for human therapy.GAPDH and plotted relative to the expression level in the control H9 and/or H7 Human ES cells. All iPS cell lines and fibroblasts were deposited with Coriell (http://www.coriell.org) for widespread public distribution.The Sox2, Oct4, Klf4 and c-Myc encoding vectors were transduced into ALS-fibroblasts by using retrovirus based delivery. All iPS cell lines were created and initially characterized with an NIH-sponsored commercial agreement with iPierian (USA). Promising colonies were picked and evaluated for the expression of multiple pluripotent markers by quantitative PCR (qPCR) and/or immunocytochemistry. Their in vitro pluripotency was further determined by three germ layer differentiation via embryoid body formation. The transgene expression was determined by qPCR. Individual PCR reactions were normalized against The iPSCs were maintained in mTeSR1 (StemCell Technology) and passed once a week using dispase (StemCell Technology) following the manufacturer’s instructions. Partially differentiated colonies were removed manually before differentiation analyses. The iPSCs were differentiated to neuroprogenitor cells (NPCs) via embryoid body (EB) formation by following the methods described previously . To incrThe cells were fixed with 4% paraformaldehyde at room temperature (RT) for 10min, and then permeabilized with 0.1% Triton X-100 in PBS at RT for another 10min. After incubation with 10% donkey serum or 3% BSA for 20min at room temperature, the primary antibody was applied and incubated at RT for 1hr and Alexa Flour 488 or 555 labeled secondary antibody for another hour. Nuclei were stained with DAPI. Images were randomly taken using a Nikon microscope with SPOT camera. Primary antibodies used in the study are listed in Statistic analysis was performed by Prism (GraphPad Software). Student’s t test was used for the percentage of rosette positive colony comparison between control and SOD1 groups. One-way ANOVA with Tukey’s post analysis was carried out to compare maker expression at different time points. P<0.05 was considered significant. Data are summarized from more than three independent experiments and shown as mean±SEM.sod1 mutations, three FUS mutations, one angiogenin (ANG) and one FIG4 mutation. All iPSCs have been generated by using Sox2, Oct4, Klf4 and c-Myc factors delivered by retroviruses with a programming efficiency of ~0.1%. Putative iPSCs underwent a validation profile that included: 1) morphological analysis, compact colonies with clear boundary and are comprised of cells with big nucleus and large nucleoli and scant cytoplasm  dermal fibroblasts were collected through a nationwide National Institutes of Health supported collaboration . The fibroblast cultures were established at Johns Hopkins School of Medicine for iPSC generation. A total of four healthy control iPSC lines, twenty-two fALS-iPSC lines and two sporadic ALS-iPSC lines with c9of72 repeat mutation were generated  plurytoplasm .; 3) pluytoplasm , Tra1–60ytoplasm .; 4) in ytoplasm .. The exytoplasm .. Only csod1 mutations , and controls . We first followed the well-established method to differentiate the cells to NPCs via EB formation. Both control and SOD1-iPSC generated rosettes structure after about ten days of induction and developed to neural tube like rosettes at around 12–14 days. The cells in the neural tube like structures expressed NPC marker Pax6 and Sox1  at week 5–6 (not shown). We further examined the cells for any evidence of mutation altering biology. None of the GFAP+ astroglia with SOD1 mutations had evidence of aberrant morphology compared to the control cells. In addition, no evidence for intracellular, either cytoplasmic or nuclear, inclusions with ubiquitin immunoreactivity has been seen (not shown).In 2009, through the American Recovery and Reinvestment Act (ARRA), the National Institutes of Health (NINDS) funded three consortia to develop well-characterized and publicly available iPSCs from familial forms of ALS, Parkinson’s disease (PD) and Huntington’s disease (HD). As a member of this consortium, we were tasked with generating a comprehensive library of fALS-iPSCs, which includes 22 fALS-iPSC and 4 control lines. This consortium was assembled to collect fALS biopsies and fibroblasts, including relevant clinical information, generating initial iPSC lines, employing a commercial collaborator (iPierian) specialized in rapid reliable development of iPSC lines, and validating their basal biology to differentiate into astroglia and motor neurons. In this report we outline this large collection of diverse fALS-iPSC lines and their reliable differentiation to astroglia. Although sporadic ALS iPS cell lines could be useful as well, early review of the program provided support for only the generation of cell lines from available fALS patients. Subsequently, our labs have collected over 100 fibroblast lines from sporadic ALS as well (unpublished). Most recently, we generated several c9orf72 ALS/FTD iPS cell lines from sporadic ALS patients (see above) .iPSCs can be generated from different tissue resources –45 by ussod1 primarily into rudimentary GFAP expressing-astroglia and some biologically relevant mature astroglia expressing EAAT1, EAAT2 or aquaporin 4. In the previous report, hESC and healthy human iPSC lines were differentiated to functional astroglia [We were able to successfully differentiate fALS-iPSCs with different mutant stroglia . By usinThe astroglia derived from this fALS-iPSC library represent the natural disease in terms of allelic copies of the mutant gene, which would provide a faithful model to study cell-cell interactions responsible for disease pathophysiology in ALS and astroglia pathophysiology. From the mutations we tested, there was no gross evidence of aberrant cellular morphology or intracellular inclusions under basal conditions. It is possible that the majority of the cells were immature astroglia, or the cells were not purified or enriched, or extrinsic signals (such as from neurons) are necessary. In vivo studies from both human and mice showed there is dramatic dysregulation of EAAT2 expression in disease and EAAT2 expression is tightly coupled to the presence of neurons , future sod1 mutations of the known >105 sod1 mutations [In addition to its application in disease modeling, the fALS-iPS derived cells offer a valuable tool for screening and validating CNS compounds. Candidate compounds for treating CNS defects fail in clinical trials in over 90% of cases due to poor targeting, lack of efficacy, and unacceptable side effects . Recent utations , includiutations  as so cautations .During astroglia differentiation, most cells expressed markers of astroglial progenitor fate compared to GFAP and mature astroglial markers. The generation and maturation of iPS derived-astroglia takes a long time in vitro, which is consistent with human embryonic astroglial development. However, this lengthy time to differentiate provides a platform to screen drugs which may facilitate astroglia development and maturation. For example, as EAAT2 expression is downregulated in ALS , maintenS1 FigTaqMan assay was used to determine gene expression. The log ratio is the difference in Ct between gene of interest and the housekeeping gene GAPDH.(PDF)Click here for additional data file.S2 FigEmbryoid Body culture was used to examine the differentiation potential of the iPSC lines. These aggregates are allowed to grow for several days and then assessed by antibody staining for their ability to differentiate into cell types representing three germ layers.(PDF)Click here for additional data file.S3 FigThe iPS cells were analyzed between passage number 4 to 10 (P4—P10). Line 010 showed balanced translocation due to reciprocal exchange between the long-arm of chromosome 1 and the short-arm of chromosome 17.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Highlights from ASCO 2015 demonstrate the impasse we face in solid tumour oncology: the compelling novel immune and targeted therapies are often associated with cost–benefit ratios significantly above the thresholds for reimbursement. This is at least in part a consequence of our incomplete understanding of the mechanisms of response and resistance to these agents. For example, ipilimumab is associated with durable clinical benefit in 15%–20% of unselected advanced melanoma patients (∼£75 000 per patient treated), and while the responses to single-agent targeted therapies such as vemurafenib are higher, they are often relatively short-lived (∼£42 000 per median PFS of 6–7 months). New trial design strategies such as basket and umbrella studies have improved upon patient selection, but have not yielded detailed biological understanding of the drug targets, nor polygenic mechanisms of resistance within or between patients. Academically led studies have the opportunity and the responsibility to prioritize biological insights as trial end points, maximising research gain, increasing patient benefit/safety and ultimately, improving cost-effectiveness. Collection of tumour material is fundamental to these aims but the timing, handling and sample analysis are of critical importance Kα inhibition [BRCA2 mutant carrier who progressed on olaparib and carboplatin [Resistance to targeted therapies can be mediated by pre-existing rather than  de novo . Thus, cure of PIKα inhibiboplatin . Distincboplatin  and coloboplatin , respectEGFR mutation in lung cancer [The benefit of combination strategies can be limited by excess toxicity (combined targeting of the PI3K and MAPK pathways ), cross-g cancer ). Informg cancer , academig cancer , providiPD-L1 expression, a putative predictive marker for PD1/PDL1 inhibition, is also spatially heterogeneous . GenomicNon-genetic causes of treatment resistance have been largely overlooked but studies that incorporate longitudinal biological sample collection and novel imaging techniques are well placed to examine tumour drug exposure (including heterogeneity of drug distribution ) and indThere clearly are challenges to implementation of such complex studies but they can be overcome through close interdisciplinary work of academic/clinical consortia as illustrated by the Lung TRACERx programme , the useThe authors have declared no conflicts of interest.


Taurine (2-aminoethanesulfonic acid) is found in milimolar concentrations in mammalian tissues. One of its main functions is osmoregulation; however, it also exhibits cytoprotective activity by diminishing injury caused by stress and disease. Taurine depletion is associated with several defects, many of which are found in the aging animal, suggesting that taurine might exert anti-aging actions. Therefore, in the present study, we examined the hypothesis that taurine depletion accelerates aging by reducing longevity and accelerating aging-associated tissue damage. Tissue taurine depletion in taurine transporter knockout (TauTKO) mouse was found to shorten lifespan and accelerate skeletal muscle histological and functional defects, including an increase in central nuclei containing myotubes, a reduction in mitochondrial complex 1 activity and an induction in an aging biomarker, Cyclin-dependent kinase 4 inhibitor A (p16INK4a). Tissue taurine depletion also enhances unfolded protein response (UPR), which may be associated with an improvement in protein folding by taurine. Our data reveal that tissue taurine depletion affects longevity and cellular senescence; an effect possibly linked to a disturbance in protein folding. Senescence is one of the major contributors to the development and progression of diseases, suggesting that regulation of senescence could significantly benefit general health. To date, diet restriction is the most reliable means of slowing the aging process Taurine (2-aminoethanesulfonic acid) is a candidate for use against aging. Taurine is widely distributed in vertebrate animals, whose tissue taurine concentration averages 1–20 micromol/g tissue. These large levels of taurine are maintained through both the diet and hepatic biosynthesis in human beings. The main source of dietary taurine is meat, with seafood being particularly rich in taurine Taurine, a zwitter ion, possesses many biological actions, one of the important ones being its role as an organic osmolyte In this study, we evaluated the role of taurine on longevity and tissue aging using a tissue taurine deficient mouse model generated by knocking out the taurine transporter (TauT) In vivo wound healing assay was performed under anesthesia with 2% isoflurane inhalation. Mice were euthanized by CO2 inhalation or cervical dislocation, and all efforts were made to minimize suffering of the animals.Animal studies were carried out in accordance with an animal protocol approved by the Institutional Animal Care and Use Committee of Hyogo University of Health Sciences. ad libitum and maintained on a 12-h light/dark cycle. Mice ), n = 20 (heterozygous (hetero)) and n = 58 (TauTKO)) were monitored for survival once per one or two days by our veterinary staff. During monitoring survival, apparent sick and injury were not observed. Young  and old , n = 10 (hetero), n = 16 (WT)) mice were euthanized to isolate tissues for each experiments.TauTKO and littermate mice (C57BL/6 background) were obtained by breeding heterozygous male and female. Mice were housed in Specific pathogen-free (SPF) environment, fed a standard chow , had access to water Total RNA was isolated from skeletal muscles of TauTKO and WT mice by using Sepazol , and cDNA was generated from total RNA by the reverse transcription with Rever Tra Ace . Quantitative RT-PCR analyses were performed by using Applied Biosystems Step One Plus (Applied Biosystems) with THUNDERBIRD SYBR qPCR Mix . The primers used are shown in Sections from frozen tissues were cut by cryostat (Carl Zeiss). Sections we stained by Hematoxylin&Eosin method Mitochondria were isolated from skeletal muscle as described elsewhere with slight modifications Tissues were homogenized in RIPA buffer  by Potter homogenizer and then sonicated on ice for 15 s by using a microtip sonicator. After centrifusion, protein concentration of supernatant was determined by bicinchoninic acid assay . SDS sample buffer  was added into lysates. The membrane transfer was performed by semi-dry method. Western blot was performed as described previously RNA from skeletal muscle was isolated by using Sepazol-RNA super G, and cleaned by using RNeasy mini kit (Qiagen). A microarray analysis was performed by using SurePrint G3 Mouse Gene Expression 8×60K arrays (Agilent Technologies) according to the manufacturer’s instructions. The microarray data have been deposited in Gene Expression Omnibus (GSE57373). Data analysis was performed with GeneSpring software (version GX 10.3). All comparisons of expression levels between the groups were performed using un-paired t-tests. Genes were identified as differentially expressed if they showed a fold-change of at least 1.8 with a p value lower than 0.05. Pathway analysis was performed by using Ingenuity Pathway Analysis (IPA) software (Agilent technology). The Fisher’s exact test was used to determine the statistical significance of association with the recorded knowledge concerning molecular networks, upstream regulators and biological functions in IPA.Student’s t-test or Tukey-Kramer test (for multiple comparisons) was used to determine statistical significance between groups. Log lank test was used in the longevity study. Differences were considered statistically significant when the calculated P value was less than 0.05.Knocking out TauT causes a 99% decrease in taurine content in muscle and heart and ∼90% decline in brain, adipose tissue and liver as well as about 75% decline in plasma taurine concentration 2 = 7.83, P = 0.020, 2 = 5.99, P = 0.00054, 2 = 6.11, P = 0.013, TauT in female mice had little effect  and 794 days (TauTKO)) ), n = 10 (heterozygous (hetero)) and n = 41 (TauTKO). Log-rank test analysis showed a significant difference in the survival curves of TauTKO, hetero and wild-type mice. The median life span of the mice varied from 795 days for WT, 723 days for hetero and 591 days for TauTKO mice  mice the expression of p16INK4a, which is a robust biomarker of cellular aging One of the features of skeletal muscle aging is sarcopenia, which is characterized by a decrease in the size of the muscle fibers. Yet, muscle size of young TauTKO mice is already reduced and associated with consistently lower body weight  and dimiAnother characteristic feature of aging muscle is the decline in respiratory chain complex I activity Moreover, we analyzed red-ragged fibers, which are markers of abnormal subsarcolemmal aggregates of mitochondria characteristically present in one of the mitochondrial diseases Our data suggest that tissue taurine depletion of skeletal muscle accelerates the aging process. To uncover the mechanism involved in acceleration of tissue aging in the TauTKO mouse, transcriptome assays and pathway analysis were carried out on skeletal muscle samples isolated from both young and older TauTKO and WT mice ,S3. To eSince organic osmolytes contribute to protein folding, we tested the hypothesis that tissue taurine depletion leads to the accumulation of misfolded and unfolded proteins in the ER, thereby triggering ER stress. We initially examined the signal pathways involved in UPR to assess potential involvement of ER stress in the pathology of tissue taurine depletion . The proThis study demonstrated that tissue taurine depletion shortens lifespan concomitant with acceleration in tissue aging. In TauTKO skeletal muscle, enhanced expression of p16 is associated with advanced aging. A recent study revealed that p16-positive senescent cells play a key role in the progression of whole body aging, which is a determinant of lifespan. Baker et al. demonstrated that clearance of p16-positive senescent cells by a transgenic technique extends the lifespan of BubR1 progeroid mice Lys, a reaction implicated in the development of the mitochondrial disease, myoclonic epilepsy and ragged-red fiber syndrome (MERRF), which leads to the formation of ragged red fibers In this study, we showed that tissue taurine depletion increases the aging process in central nuclei containing myotubes. While we have previously reported the histological change in young TauTKO muscle et al. has demonstrated that different chemical chaperones, such as TMAO, proline and glycerol, differentially influence the folding pathways unique to mutant variants, suggesting that osmolytes play a specific role in protein folding In this study, we demonstrated that ER stress may be increased by tissue taurine depletion in skeletal muscle, suggesting that endogenous taurine contributes to the stabilization of protein folding. Moreover, we found inductions in genes involved in amino acid metabolism , protein synthesis  and protein folding  in taurine-depleted muscles, suggesting that tissue taurine depletion affects protein homeostasis. These are consistent with the theory that organic osmolytes act as chemical chaperones. The effect of taurine treatment on ER stress has been previously investigated Cells possess various mechanisms to prevent and eliminate protein misfolding or damage, termed proteostasis (protein homeostasis). However, the proteostasis machinery declines with aging, and damaged proteins accumulate in cells In this study, we found that lifespan is significantly shorter in male TauTKO mice but not in female. Moreover, acceleration of skeletal muscle senescence is less severe in female TauTKO than male. The reason for these gender differences is unclear, so far. Gender effects for lifespan have been reported in some experimental geriatric studies. Treatment of metoformin, an anti-diabetic drug, extends lifespan in female mice but not male Since control of calcium handling is also an important action of taurine in skeletal muscle, alteration of calcium homeostasis by taurine depletion may also contribute to impairment of muscle function In conclusion, tissue taurine depletion accelerates muscle aging and shortens lifespan; effects may be related to the enhancement of UPR. This study provides the evidence that taurine acts as an anti-aging molecule and that prevention of chronic taurine depletion may serve as a strategy for delaying skeletal muscle senescence.Figure S1A) Significant induction of p16INK4A in aged female TauTKO muscle. n = 4 (WT), 3 (TauTKO) B) Significant increase in central nuclei-myotubes is detected in aged female TauTKO muscle. n = 4 (WT), 5 (TauTKO). C) Gomori trichrome stain shows no red-ragged fibers in aged TauTKO muscle. Similar results were obtained from 3 independent experiments.(PDF)Click here for additional data file.Table S1Top 50 genes increased or decreased in old TauTKO muscles than old WT muscles.(PDF)Click here for additional data file.Table S2Top 50 genes increased or decreased in young TauTKO muscles than young WT muscles.(PDF)Click here for additional data file.Table S3Biological function identified by IPA of increased or decreased genes of old TauTKO muscle.(PDF)Click here for additional data file.Table S4Biological function identified by IPA in overlap genes which are significantly changed in both young and old TauTKO muscle.(PDF)Click here for additional data file.Table S5(PDF)Click here for additional data file.Checklist S1ARRIVE Checklist.(PDF)Click here for additional data file.


Saccharomyces cerevisiae crosses on different culture conditions, we show environment-specific genetic incompatibility segregates readily within yeast and contributes to reproductive isolation. Over 24% (117 out of 481) of cases tested show potential epistasis, among which 6.7% (32 out of 481) are severe, with at least 20% of progeny loss on tested conditions. Based on the segregation patterns, we further characterize a two-locus Dobzhansky–Müller incompatibility case leading to offspring respiratory deficiency caused by nonsense mutation in a nuclear-encoding mitochondrial gene and tRNA suppressor. We provide evidence that this precise configuration could be adaptive in fluctuating environments, highlighting the role of ecological selection in the onset of genetic incompatibility and reproductive isolation in yeast.Genetic variation within a species could cause negative epistasis leading to reduced hybrid fitness and post-zygotic reproductive isolation. Recent studies in yeasts revealed chromosomal rearrangements as a major mechanism dampening intraspecific hybrid fertility on rich media. Here, by analysing a large number of  Chromosomal rearrangements may hamper intraspecific hybrid fertility. Here the authors show that environment-specific genetic incompatibility segregates readily within intermating populations and leads to intrinsic reproductive isolation within a yeast species. Genetic variation accumulated in different natural populations could occasionally cause deleterious epistatic effects leading to reduced hybrid fitness and intrinsic post-zygotic reproductive isolation. The Dobzhansky–Müller model described such negative epistasis as genetic incompatibility, where independently fixed mutations in allopatric populations could not properly function together when combined in hybrids2345678101112131415Caenorhabditis elegans, the specific allelic combination of a paternal-delivered toxin peel-1 and a zygotic-acting antidote zeel-1 was incompatible, which led to F2 offspring inviability in a cross between Hawaii and Bristol isolates10Arabidopsis thaliana emphasized the role of genetic drift, where reciprocal inactivation of a duplicated essential gene histidinol-phosphate aminotransferase (HPA) in different parental genomes led to F2 segregation distortion1819A. thaliana further confirmed the essential role of plant autoimmunity and identified several hot spots leading to hybrid necrosisIn different model organisms, multiple molecular mechanisms have been identified to explain the observed genetic incompatibility within populations. For example, in Yeasts are free-living unicellular eukaryotes, which can be isolated from various ecological  and geographical  niches14Saccharomyces cerevisiae. We measure the offspring viability of 27 crosses on a large number of conditions . We show condition-specific incompatibilities are frequent: over one-fourth of the tested cases show loss of offspring viability to varied degrees (from 1 to 62%), possibly indicating the presence of negative epistasis. We analyse the segregation patterns of identified cases and focus on one case that demonstrated a potential recessive two loci Dobzhansky–Müller incompatibility related to the loss of offspring viability on media containing non-fermentable carbon sources . By further analysing the genes and mutations involved, we show that the incompatibility is due to the presence of a nonsense mutation in a nuclear-encoding mitochondrial gene and a tRNA nonsense suppressor. This is the first identified case of classic two loci Dobzhansky–Müller genetic incompatibility within a yeast species. To identify the evolutionary origin of the onset and maintenance of this particular configuration, we evaluate the effect of the suppressor in different genetic backgrounds and show that the presence of this suppressor confers a surprising level of phenotypic variation, which could potentially offer selective advantages across various environmental stresses. Interestingly, despite frequent observation of tRNA suppressors in laboratory selections22232425S. cerevisiae.Here, we perform a global evaluation of the onset of genetic incompatibility across various environments using the yeast In total, 27 natural isolates previously shown to be compatible with the reference strain S288c (offspring viability >90% on YPD) were selectedTo assess the genetic complexity of the observed cases, we analysed the segregation patterns of the lethal phenotype . Most ofTo map the loci involved, we used a bulk segregant analysis strategy followed by whole-genome sequencing (BSA-seq)http://www.yeastgenome.org/), among which three were found in the region on chromosome V  and two in the region on chromosome X (TIM54 and AIM22). We examined the DNA sequences of these genes in the YJM421 background and found a nonsense mutation at the position +115 in the open reading frame of COX15 . COX15 encodes an inner membrane cargo protein in the mitochondria, the function of which is essential for respirationCOX15 and lead to respiratory deficiency; whereas the strain YJM421 carrying the mutation was respiratory competent.To identify the causative genes for the observed respiratory deficiency, we closely examined the mapped regions for potential candidates involved in respiration. In total, five genes in these regions were potentially involved in respiration according to the SGD annotations ( COX15 (cox15stop) was functional, we deleted the cox15stop in YJM421, and the resulting mutant was unable to grow on media containing non-fermentable carbon sources . The presence of this suppressor would effectively read-through the premature stop codon in cox15stop, which leads to a functional protein product in YJM421. However, this configuration of cox15stop/SUP7 in YJM421 renders the strain incompatible when crossed with S288c, as one-fourth of the segregants would inherent only the non-functional cox15stop allele but not the suppressor, leading to respiratory deficiency. To confirm this hypothesis, we transformed segregants that are non-viable on 2% YP glycerol with a yeast centromeric plasmid containing the suppressor SUP7 (CEN_SUP7), and confirmed that the presence of this suppressor restored their respiration capacity J1 , which in turn transformed this tRNA into a TAA nonsense suppressor  or an empty control plasmid (CEN_Ctrl) and measured their growth rate in a non-stressful condition (YPD at 30 °C) using microcultures , with the most severe case of 2.53-folds lower growth in the presence of the suppressor compared with the strain carrying the control plasmid . Interestingly, despite an overall deleterious effect of SUP7, several isolates, including YJM421, YJM320 and T7, showed similar or higher growth rates in the presence of the suppressor  on solid media for various stress conditions  with an overall gain of fitness across all conditions. In addition, for most of the conditions tested (7 out of 11), significantly increased phenotypic variance was observed in the presence of SUP7 compared with the controls across all strains  and FY5 (MATα) were also used. A uracil auxotrophic mutant of YJM421 (YJM421 ura3Δ0) was generated by deleting the URA3 gene using 5-FOA selection.A collection of 27 strains isolated from diverse ecological  and geographical  origins was used in this study for crossing, phenotyping and survey of nonsense mutations . Laborat−1 hygromycin (Euromedex) was supplemented to maintain the plasmid CEN_SUP7 or CEN_Ctrl carrying a resistance marker gene hygroMX. Sporulation was induced on potassium acetate plates . The 5-FOA selection plate was made by supplementing 25 μg ml−1 uracil and 1 mg ml−1 5-FOA in a synthetic complete uracil dropout media (SC-URA). Detailed media composition for the screening of offspring viability and stress conditions are listed in Standard media conditions were used  for growth and maintenance of the strains. A final concentration of 200 μg mlMATα and were previously shown to be compatible when crossed with FY4 The 27 strains are all For the cross between YJM421 and S288c, 25% of the segregants were respiratory deficient and not viable on 2% YP glycerol. In total, 80 independent respiratory-deficient segregants were separately cultured then pooled by equal OD readings at 600 nm and the DNA was sequenced. Causative genes for the observed incompatibility are presumably united in these segregants and genomic regions involved are mapped by analysing the allele frequency variation.Genomic DNA was extracted using the Qiagen Genomic-tip kit. DNA of the pooled segregants was sequenced using Illumina Hiseq 2000. Detailed procedures of DNA preparation were described previouslySUP7 was amplified with its original regulatory regions from the genomic DNA of YJM421, tagged with attB1/attB2 sites compatible with the Gateway cloning system (Invitrogen). Cloning of the fragment was performed according to instruction. The resulting plasmid (CEN_SUP7) was verified using restriction enzymes as well as Sanger sequencing. Transformation of yeast strains was performed using EZ transformation kit . Single colonies of transformants were maintained on YPD media containing 200 μg ml−1 hygromycin.Empty centromeric Gateway plasmid with hygroMX as a resistance marker was kindly provided by Dr S. Treusch (CEN_Ctrl)−1 hygromycin and then transferred into 150 μl of the same media in flat bottom 96-well plates  using a long pin replicator. Cultures were grown in six replicates for 48 h and the absorbance of each well was read at 595 nm in 10 min intervals  using a TECAN plate reader (Infinite series) with horizontal and orbital shaking. Growth curves were retrieved using the programme GATHODETo measure the growth of strains in the presence or absence of the suppressor in non-stressful condition, strains tranformed with the corresponding plasmids (CEN_Ctrl or CEN_SUP) were pregrown for 24 h in YPD containing 200 μg mlSUP7 in stress conditions, 23 isolates carrying different plasmids (CEN_Ctrl or CEN_SUP) were pregrown in liquid YPD with 200 μg ml−1 hygromycin and pinned onto a solid YPD plate with hygromycin to a 384 format using a replicating robot RoToR (Singer instruments). The resulting matrix plate was allowed to grow for 48 h at 30 °C, and was subsequently replicated on 12 different conditions including YPD as a pinning control. All media conditions tested were supplemented with 200 μg ml−1 hygromycin to maintain the plasmids, detailed compositions are listed in To measure growth variation due to the presence of S. cerevisiae  database under the accession codes PRJNA280047 and SRP056771.How to cite this article: Hou, J. et al. Comprehensive survey of condition-specific reproductive isolation reveals genetic incompatibility in yeast. Nat. Commun. 6:7214 doi: 10.1038/ncomms8214 (2015).Supplementary Figures 1-3, Supplementary Tables 1-3 and Supplementary Reference


Drosophila and identified the Chitinases (Chts) and imaginal-disc-growth-factors (Idgfs) that are essential for larval and adult molting. We demonstrate that Cht and idgf depletion results in deformed cuticles, larval and adult molting defects, and insufficient protection against wounding and bacterial infection, which altogether leads to early lethality. We show that Cht2/Cht5/Cht7/Cht9/Cht12 and idgf1/idgf3/idgf4/idgf5/idgf6 are needed for organizing proteins and chitin-matrix at the apical cell surface. Our data indicate that normal ECM formation requires Chts, which potentially hydrolyze chitin-polymers. We further suggest that the non-enzymatic idgfs act as structural proteins to maintain the ECM scaffold against chitinolytic degradation. Conservation of Chts and Idgfs proposes analogous roles in ECM dynamics across the insect taxa, indicating that Chts/Idgfs are new targets for species specific pest control.The cuticle forms an apical extracellular-matrix (ECM) that covers exposed organs, such as epidermis, trachea and gut, for organizing morphogenesis and protection of insects. Recently, we reported that cuticle proteins and chitin are involved in ECM formation. However, molecular mechanisms that control assembly, maturation and replacement of the ECM and its components are not well known. Here we investigated the poorly described glyco-18-domain hydrolase family in  The epithelial apical extracellular matrix (ECM) controls development and structural maintenance of organisms, organ morphogenesis, wound healing, cell signaling, and the local defense barrier against a hostile environment1234In mice and humans mutations of the extracellular collagen and the cornified envelope have been linked to skin disorder diseases, characterized by skin fragility, painful wounding and accompanied skin infections. The skin layers establish stability and an essential protective barrier by crosslinking of extracellular components5719Drosophila4121618Tribolium and a similar role in Drosophila has been discussed recently20Genes involved in the biosynthesis and secretion of chitin-ECM components have been studied intensively in family18 due to the characteristic glyco18 domain. The family18 comprises numerous Chitinases (Chts) and Imaginal-disc-growth-factors (Idgfs)26Drosophila imaginal disc cells263031family18 members in ECM organization and accompanied dynamics of the cuticle remained largely unknown.Chitin-polymers may spontaneously assemble into microfibrils of various lengths and diameter232425262728family18 members in ECM formation in Drosophila. By individually knocking down genes in the cuticle secreting tissues, we show that a large number of the Chts and idgfs are involved in cuticle molting during larval as well as in pupal stages. This is supported by gene specific spatial-temporal expression profiles and by developmental lethality profiles upon gene knockdowns. Moreover, we demonstrate that many Chts and idgfs genes are required also during the intermolt stages for forming the epidermal cuticle in order to organize the outermost protective barrier, since knockdown mutants are highly susceptible to mechanical stresses and bacterial infections. Investigating the molecular network in Cht2 and idgf6 RNAi induced mutants, which showed strongest lethality and most severe cuticle defects, we propose that certain Chts are involved in organizing the chitin-matrix scaffold, which is essential for further enzyme-mediated ECM maturation and cuticle thickness. In contrast to Chts, the non-enzymatic Idgfs play an essential role in the protection of the newly synthesized cuticle matrix against degradation in order to stabilize and expand the ECM size in the larvae.We systematically investigated Drosophila family18 comprises sixteen members consisting of ten Chitinase  and six idgf genes 252627Tribolium castaneum that Cht5, Cht10 and idgf4 genes are involved in the molting processChts and idgfs in cuticle molting remains elusive.The Cht and idgf genes systematically in cuticle forming tissues, we performed individual gene specific transgenic RNA interference (RNAi)-mediated knockdown in Drosophila using lines of the very efficient (see below) Vienna stock collection (www.vdrc.at)RNAi was driven in cuticle-forming organs with the help of the 69B-Gal4 driver line (Wild-type (wt) and 69B-Gal4 animals were taken as control. Our knockdown studies identified a large subset of genes required in larval cuticle molting and a distinct set essential in pupal-adult molting  were not penetrated by Pseudomonas at the epidermal cuticle, as GFP spots and the accompanied melanization darkening were not observed. In contrast, most of the Cht2 and idgf6 RNAi induced mutants (n = 45 for each genotype) showed melanization-like darkening spots at the epidermal wounding sites where GFP-expressing bacteria accumulated  RNAi-induced knockdown showed lethality and severe cuticle molting defects in larvae and pupa of the red flour beetlesChts in Drosophila. The observation that Drosophila Cht11 did not cause any molting or barrier defects is consistent with its recently proposed function in the mitochondriaCht4 knockdown did not show any cuticle phenotypes in Drosophila and other insects, but Cht4 is rather involved in reproductionCht genes for insect moltingDrosophila extends initial observations and shows that a large number of Cht and idgf genes are required to control the repetitive reorganization of the chitin-ECM at the epidermis.We investigated the role of otection . Our datCht and idgf genes are essential for forming the epidermal barrier-ECM. We show that the Cht2,5,7,9,12 and idgf1,3,4,5,6 genes are required for chitin-ECM formation and barrier function throughout larval development. These morphological data and in vivo barrier analysis were supported by analysis of the cuticle structure and the involved molecular network. The cuticle assembly zone is associated with the apical cell surfaceCht2 is required for cuticle assembly zone function. In the Cht2 knockdown the assembly zone was absent and consequently Obst-A, Serp and Verm were not enriched at the apical cell surface but mislocalized or reduced. This finally led to reduced number and defective morphology of chitin-lamellae when normally the number of lamellae should increase in third instar and the cuticle becomes thickened. There are different molecular scenarios how the glycosylhydrolase Cht2 could determine procuticle size. It could act in the cuticle assembly zone in order to modify for example length of chitin-fibrils, which supports Obst-A-mediated chitin-scaffold formation and maturation by the deacetylases Serp and Verm. A distinct scenario could be that Cht2 inhibits premature assembly, or acts in later stages for elongation and ordering the filaments. Defects in each of these processes could lead to pushing the cuticle proteins towards the membrane, and the disappearance of cuticle assembly zone. Indeed, preliminary data for Cht2 protein localization within the epidermal cuticle support both scenarios Tc-Knk downregulation at the body wall and elytra cuticleRecently, it has been shown in Cht genes : w1118 (referred to as wild-type (wt)), 69-BGal4 line, UAS-RNAi-Cht and UAS-RNAi-idgf lines (Vienna stock center). For knockdown analysis, 69B-Gal4 flies were mated with UAS-RNAi flies. For negative controls we used Wild-type (wt) animals and animals from the 69B-Gal4 and the different UAS-RNAi lines but without inducing the RNAi system. In general, RNAi induced phenotypes can be variable to some extent. We therefore show representative images of observed phenotypes and indicate the number of analyzed animals.Vienna (Austria) /Bloomington (USA) stock centers  17 embryos (n ≥ 100 for Chts n ≥ 75 for idgfs) and were transferred onto new agar plates and incubated at 29 °C. The number of dead and living animals were counted every 24 hours with Stemi stereomicroscopes  and characterized from first instar larvae to adult flies. Every genetic experiment was repeated at least three times. For cuticle preparation first, second and third instar larvae were washed with water and incubated in a 1:1 lactate and Hoyer’s reagent mixture overnight at 65 °C. Larval stages were determined by the morphology of mouth hooks. Larvae were analyzed with 60x and 100x objectives in Olympus immersion oil and by Normarski microscopy . Pupae and adult flies were analyzed using an Olympus Binocular microscope (SZX12). All images were taken with Cell’F software. In general for all assays (see below) images were cropped in Adobe Photoshop CS6 and figures designed with Adobe Illustrator CS6. For statistical analysis Excel 2010 was used.Embryos from First instar larvae (n > 50) were gently laterally wounded with a standard glass needle for micro injections as recently described16N-acetylneuraminacid and N-acetylglucosamine sugar residues and can be used as a marker for plasma membrane surfaces and cuticle layers. Used antibodies: Knk Third instar larvae were fixed overnight in 4% paraformaldehyde and embedded in JB-4 resin  as described by the manufacturer. Slices of 7 μm were cut using an Ultracut E . For each genotype n ≥ 15 individuals were analyzed. The immunofluorescent labeling was performed as recently describedFor immunofluorescent analysis LSM710 and LSM780  confocal microscopes and 63x LCI Plan Neofluar objectives were used. For scanning procedure pinhole “airy unit 1” ZEN software standard settings were used and cross-talk was avoided by using according filter settings and lasers. Images were taken with the ZEN 2010 and ZEN 2011, cropped with Adobe Photoshop CS6 and figures were designed with Adobe Illustrator CS6.Drosophila second and third instar larvae were placed on a 150 μm flat embedding specimen holder  and frozen using 1-hexadecene in a Leica HBM 100 high pressure freezer  according to the described methodPseudomonas entomophila (PE) was cultured in LB-medium with 50 μg/ml rifampicin at 29 °C. For infection experiments, second instar larvae were placed on filter papers moistened with PBS. For each genotype and condition, three dishes were prepared. Uninfected control larvae were fed with 500 μl saccharose solution. For infection of larvae, 500 μl of 10% saccharose solution containing 0.5 g resuspended bacteria was added to the filter paper. Larvae were incubated at 29 °C for 24 h and afterwards imaged with a Zeiss Apotome Axiovert 200 M . Pseudomonas positive larvae reveal the GFP signal. Multidimensional photographs were taken with AxioVision software (Zeiss). For each genotype n = 45 animals were analyzed and the infection experiment was repeated three times. To prove infection by Pseudomonas and to rule out contamination of the control, homogenized larval material was streaked out on Rifampicin-agar plates with milk, on which pathogenic Pseudomonas grows colonies. After overnight culturing at 29 °C only plates with infected animals showed Pseudomonas colonies, while non-infected did not.rp49  as a reference. The specificity of all primers was tested by melt curve analysis. Standard control PCR reactions  were performed and showed no signal. Expression data were calculated according to the delta-delta-CT method and analyzed using Bio-Rad iQ5 Optical System Software and Microsoft Excel 2010. All primer sequences and efficiencies are provided for Chts  and lysed in a Precellys homogenizer using a 1.25–1.65 diameter glass bead mix . The lysed samples were frozen in liquid nitrogen and stored at −80 °C. RNA was extracted within two weeks. The NucleoSpin RNA II Kit  was used following the manual instructions including DNase treatment. RNA quantification was performed by the use of NanoDrop . A second genomic DNA digestion and the first strand cDNA synthesis was carried out with 500 ng of total RNA using the QuantiTect Reverse Transcription Kit  following the manual instructions and using the provided oligo-dT and random primer mix. All RNA reactions were performed on ice, cDNA was stored at −20 °C. qPCR experiments were done as recently describedfor Chts , for idgs .