To determine if variations in trial eligibility criteria and patient baseline characteristics could be considered effect modifiers of the treatment response when testing targeted therapies ) for rheumatoid arthritis (RA).th 2013. Eligible trials reported ACR20 data at months 3–6 and used an add-on design. Odds ratios (ORs) were calculated from the response rates and compared among the trial eligibility criteria/patient baseline characteristics of interest. Comparisons are presented as the Ratio of Odds Ratios (ROR).We conducted a meta-epidemiological study of all trials evaluating a targeted therapy approved by regulatory authorities for treating RA. The database search was completed on December 11Sixty-two trials  were included in the primary analyses using ACR20 response. Overall, targeted therapies constituted an effective treatment  3.41 to 4.60). The majority of the trial eligibility criteria and patient baseline characteristics did not modify treatment effect. The added benefit of targeted therapies was lower in trials including "DMARD-naïve" patients compared with trials including "DMARD inadequate responders"  and trials including "targeted therapy inadequate responders" , test for interaction: p = 0.0002. Longer mean disease duration was associated with a higher likelihood of responding to treatment . Analyses conducted using DAS28-remission as the outcome supported the above-mentioned findings.Our results suggest that a highly selective inclusion is not associated with greater treatment effect, as might otherwise be expected. The added benefit of a targeted therapy was lower in trials including patients who were DMARD-naïve and trials including patients with shorter disease durations. Newer drugs, consisting of biological disease modifying antirheumatic drugs (bDMARDs)  as well Randomized controlled trials (RCTs) that have tested targeted therapies vary in several of their trial eligibility criteria and patient baseline characteristics , but litIt is unknown if the difference in benefit from therapy depends on whether the outcome of choice is a measure of change such as the ACR20 response criteria (a 20% reduction in the number of swollen and tender joints and 3/5 other core items)  or the nThe objective of this study was to examine if variations in trial eligibility criteria and patient baseline characteristics can influence the added benefit of targeted therapies compared to the control treatment in RA trials .The protocol describing the study eligibility criteria, data extraction, and analysis was specified in advance and registered at the international prospective register of systematic reviews–PROSPERO (Registration no. CRD42014010322). The study findings are reported according to the Preferred Reporting Items for Systematic reviews and Meta-analyses .clinicaltrials.gov or later publications). Open-label studies were excluded because they have an inherent risk of performance bias [Eligible trials were RCTs of RA ;14. The nce bias .th 2013. The World Health Organization (WHO) Clinical trials Portal (ICTRP), clinicaltrials.gov, FDA, and EMA were searched to identify unpublished data.We searched PubMed, EMBASE, The Cochrane Central Register of Controlled Trials (CENTRAL), and LILACS and used a combination of keywords and text words. Our search strategy has previously been published . The seaFulfillment of the study eligibility criteria was assessed by two independent reviewers. Titles and abstracts were first screened and duplicates were removed. If one reviewer thought a study could potentially be eligible for inclusion the paper was retrieved and read in full text. Disagreements were resolved by discussion until consensus was reached.Data were extracted from trials, using a pre-specified form, by one investigator and subsequently checked by a second investigator. Disagreements were resolved by consensus. Data were extracted from only one active arm (the one using the FDA/EMA approved dosage) and one comparator arm per trial to avoid splitting the comparator group. We extracted outcome data according to the study’s primary outcome time point; if the primary outcome came later than six months into the study, we extracted available data closest to month three.Data on the following trial eligibility criteria were extracted: first author, drug of interest, treatment given in the active and comparator arm, minimum required 66 swollen joint count (SJC), minimum required 68 tender joint count (TJC), minimum required C-reactive protein (CRP), maximum allowed disease duration and rheumatoid factor (RF), and/or anti-cyclic citrullinated peptide (anti-CCP) antibody status. Information regarding the patients’ background DMARD medication was also extracted. For "MTX," "csDMARDs (non-MTX csDMARDs),” and "Targeted therapies," we extracted information about how the medication was handled at randomization and grouped the trials into one of the following categories: Continued , Discontinued , Not using  of interest), Naïve  of interest, and Not reported .In order to stratify trials according to the DMARD history of their included patients, we asked ourselves the question: Had the participants, prior to inclusion, exhausted the treatment potential of at least one DMARD (or targeted therapy). Trials were then grouped into one of the following categories: DMARD-naïve ; DMARD-IR (DMARD inadequate responders), where patients had exhausted at least one csDMARD option and had inadequate response); and TT-IR (patients had experienced an inadequate response to at least one previous targeted therapy).Data on the following patient baseline characteristics were extracted: female%, age, disease duration, CRP, Disease activity score based on 28 joint counts (DAS28), RF%, anti-CCP positive%, SJC, TJC, health assessment questionnaire—disability index (HAQ), MD global assessment of disease activity, patient global assessment of disease activity and patient-reported pain on visual analogue scales of 0–100 mm (VAS). If both median and mean values were given, the former was prioritized. Primary outcome was ACR20 response, as this was most frequently reported. As a secondary outcome, DAS28-remission (DAS28<2.6) was extracted.The risk of bias (RoB) within each trial was assessed using the RoB tool as recommended by The Cochrane Collaboration ;20. EacheOR and SE(logeOR), respectively) based on the intra-trial contrast between active intervention (targeted therapy) and comparator group. Outcome events were presented so that an OR of more than 1 indicated a beneficial effect of the targeted therapy. Heterogeneity statistics were calculated according to a fixed-effect meta-analytic summary and presented as the apparent inconsistency between trials (I2) [For each trial we estimated the odds ratio (OR) and the corresponding standard error  . Howeverals (I2) ; we chosals (I2)  because als (I2) .2 (T2) as a consequence of inclusion in the (mixed effects) statistical model. Comparisons between different trial eligibility criteria or patient baseline characteristics are presented as the Ratio of Odds Ratios (ROR) with a 95% confidence interval (95%CI).[According to our objective, we explored potential effect modification of trial eligibility criteria and patient baseline characteristics, in a number of meta-regression analyses. These analyses were modelled using Restricted Maximum Likelihood (REML)-based models. A priori, we defined a relevant study-level covariate as one that decreased the between-study variance, Tau (95%CI). AnalysesFig 1, from 230 possible RA studies, a total of 62 trials were considered eligible, including 19,923 RA patients in the primary analysis. Of these trials, 35 also reported data on DAS28-remission and were included for the secondary analyses.  presents the characteristics of the included trials, which were overall of a good methodological quality (low RoB).As illustrated in There were only two missing values in the extraction of trial eligibility criteria; one study did not report a minimum required SJC and TJC at inclusion, which later was remedied by using datasets' median values . MissingTable 1presents the analyses of trial eligibility criteria using ACR20 as the outcome. Corresponding analyses of patient baseline characteristics for ACR20 are seen in Table 2.2) was 0.25, and the proportion of variance attributable to heterogeniety (I2) was 77%. The trial eligibility criterion "DMARD History" which grouped trials according to whether their patients were DMARD-naïve, DMARD-IR, or TT-IR, reduced the between-trial heterogeneity by 36% (τ2 = 0.16) with statistically significant interaction among the subgroups (p = 0.0002). The added benefit of targeted therapies was lower in DMARD-naïve trials with a lower ROR compared with DMARD-IR  and TT-IR  trials. Fig 2shows a forest plot of trials stratified by DMARD History.The overall effect-increase of adding a targeted therapy was OR 3.96 (95% CI 3.41 to 4.60). Between-study variance in trials . However, trials that only included patients with early arthritis had significantly lower effect compared with trials that only included patients with established arthritis . The patient baseline characteristic "Baseline Disease Duration" demonstrated increasing effect with longer average disease duration of the trial patients  with 1.05 OR-units per year.The trial eligibility criteria "MTX Handling at Randomization" and "Minimum Required SJC at Inclusion" also demonstrated significant interactions among subgroups .The median absolute response rates for each “DMARD History” group are shown in Table 3presents the analyses conducted using DAS28-remission as the outcome. Corresponding analyses of patient baseline characteristics for DAS28-remission are seen in Table 2.2) was 0.32, and the proportion of variance attributable to heterogeniety (I2) was 65%. The trial eligibility criterion "DMARD History" reduced the between-trial heterogeneity by 50% (τ2 = 0.16) with statistically significant interaction among the subgroups (p = 0.003). The added benefit of targeted therapies was lower in DMARD-naïve trials with a lower likelihood of responding compared with DMARD-IR  and TT-IR trials . Fig 3shows a forest plot of trials stratified by DMARD History.The overall effect-increase of adding a targeted therapy was OR 5.57 . Between-study variance in trials , whereas the patient baseline characteristic "Baseline Disease Duration" demonstrated increasing effect with longer average disease duration of the trial patients (p = 0.005).The trial eligibility criteria "MTX Handling at Randomization," "TT Handling at Randomization," and "Minimum Required CRP at Inclusion" also demonstrated significant interactions among subgroups .). In the ACR20-Modified dataset; "Minimum Required 66 SJC at Inclusion" no longer demonstrated significant interaction among subgroups, which suggests confounding by the included patients' DMARD history. "MTX Handling at Randomization" still showed significant interaction among subgroups (p = 0.02), but inspection of the trials that discontinued MTX showed that the control group received no treatment (these were primarily monotherapy and dose-finding studies). Trials solely including patients with established arthritis had greater added effect of a targeted therapy compared to trials where “Max Disease Duration at Inclusion” was not specified or reported . The patient baseline characteristic “Baseline RF (%)” showed increasing effect with greater proportion of RF positive patients at baseline ) with 1.02 OR-units per percentage point increase.To explore the confounding effect of the trial eligibility criterion "DMARD History" in the remaining trial eligibility criteria and patient baseline characteristics, we omitted trials labelled as DMARD-naïve when conducting post-hoc analyses. Results from these modified datasets  are shown in . Trials including DMARD-IR of TT-IR patients randomize patients to a control treatment that they have previously failed on which results in a large relative effect of the added active treatment and hence a larger odds ratio. However, we found that the added benefit of a targeted therapy was lower even when looking at absolute response rates for trials including DMARD-naïve patients–thus demonstrating the diminished effect . We believe that these findings justify that all RA patients should be exposed to csDMARD treatment before introduction a targeted therapy is considered.We found that absolute response rates differed for the control arms across the three “DMARD History” groups, which was expected (S2 Table), trials solely including patients with established arthritis had greater added effect of a targeted therapy compared to trials where maximum disease duration at inclusion was not specified (i.e. patients were not selected on the basis of their disease duration). On the other hand, the patient baseline characteristic “Baseline Disease Duration” was no longer statistically significant, which suggests confounding by the “DMARD-History”  (S2 Table). As these two variables (both reflecting disease duration) give different results in the ancillary analyses we are unable to conclude whether disease duration is an independent effect modifier or whether it is confounded by the fact that DMARD-naïve trials also included patients with the shortest disease duration.Both "Maximum Disease Duration at Inclusion" and "Baseline Disease Duration" reflect the same construct and our analyses support previous findings of greater added effect of targeted therapies in patients with longer disease duration ;7. HowevAll trial eligibility criteria and patient baseline characteristics were analyzed using both ACR20 and DAS28-remission as the outcome of interest. Although these two outcomes are very different in nature, the results for “DMARD History” and disease duration were very similar for both outcomes. Some trial eligibility criteria were significant in the primary or secondary analyses, but not in the ancillary analyses. They were believed to be confounded by the trial characteristic “DMARD History” and not considered further , but not in the primary analysis (ACR20). In the DAS28-remission-Modified dataset , this trial eligibility criterion showed significant interaction among subgroups and it was borderline significant in the ACR20-Modified dataset. This indicates that baseline CRP had different effects in different sub-groups; it could indicate a positive prognostic value of patients' having an elevated baseline CRP when looking solely at patients who have failed previous treatment with csDMARDs and/or bDMARDs/ tsDMARDs.This study is the most comprehensive analysis to date of the effect of csDMARDs and bDMARDs/ tsDMARDs for RA. Only FDA/EMA-recommended dosages were included, which adds to the external validity of the study. Previous meta-epidemiological studies in this field have focused mainly on patient baseline characteristics whereas we also looked into the trial eligibility criteria. We focused on short-term (3–6 months) follow-up, which is also the time frame where it is relevant for a primary evaluation of the initiated treatments efficacy.This study has several limitations. All extracted variables were limited to publicly available information. Outcome data were limited to clinical endpoints . Missing data could limit our findings for some variables, since this was handled by imputing the median value for that variable . This conservative approach could mean overlooking potential effect modifiers concerning variables with many missing data Finally, all data were obtained at trial level; use of aggregated data to draw conclusions on individuals should be done with care. Because confounders and effect modification cannot be controlled with aggregated data, using aggregated data can give rise to wrongful conclusions—a phenomenon known as ecological fallacy . It is tWe have demonstrated that the majority of trial eligibility criteria and patient baseline characteristics cannot be considered effect modifiers in trials testing a targeted therapy for rheumatoid arthritis. This suggests that trials with a highly selective inclusion do not show better effect of a targeted therapy as might otherwise be expected. Therefore, a selective inclusion might also not be necessary when designing trials to test new bDMARDs or tsDMARDs.We found that the medication history and baseline disease duration of the included participants modified the added effect of the targeted therapy. The added benefit of a bDMARD or tsDMARD was lower in trials including patients who were DMARD-naïve.Researchers conducting meta-analyses, assessing the efficacy of targeted therapies for RA, should include the patients medication history as a covariate in order to improve the precision of estimates. We recommended that future trials report adequately on the patients previously failed number of csDMARDs and bDMARDs/ tsDMARDs. Also the trial definition of treatment failure for each class of drugs should be given.S4 Table).The dataset has been published as supporting information  and shows how much added effect there was in the arm receiving the targeted therapy (grey).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Atherosclerosis is a chronic inflammatory disease characterized by the infiltration of pro-inflammatory macrophages into a lipid-laden plaque. ITCH is an E3 ubiquitin ligase that has been shown to polarize macrophages to an anti-inflammatory phenotype. We therefore investigated the effect of ITCH deficiency on the development of atherosclerosis. ApoE−/−ITCH−/− mice fed a western diet for 12 weeks showed increased circulating M2 macrophages together with a reduction in plaque formation. Bone marrow transplantation recreated the haemopoietic phenotype of increased circulating M2 macrophages but failed to affect plaque development. Intriguingly, the loss of ITCH lead to a reduction in circulating cholesterol levels through interference with nuclear SREBP2 clearance. This resulted in increased LDL reuptake through upregulation of LDL receptor expression. Furthermore, ApoE−/−ITCH−/− mice exhibit reduced hepatic steatosis, increased mitochondrial oxidative capacity and an increased reliance on fatty acids as energy source. We found that ITCH ubiquitinates SIRT6, leading to its breakdown, and thus promoting hepatic lipid infiltration through reduced fatty acid oxidation. The E3 Ubiquitin Ligase ITCH modulates lipid metabolism impacting on atherosclerosis progression independently from effects on myeloid cells polarization through control of SIRT6 and SREBP2 ubiquitination. Thus, modulation of ITCH may provide a target for the treatment of hypercholesterolemia and hyperlipidemia. Atherosclerosis is a vascular disease in which cholesterol accumulation within the arterial wall leads to a chronic low grade state of inflammation4ITCH is an E3 ubiquitin ligase initially discovered in the agouti locus responsible for the coat color of miceHere we investigate the role of ITCH deficiency on the development of atherosclerosis.After 12 weeks of Western Diet (WD), ApoE−/−ITCH−/− male mice showed reduced weight gain and improved glycaemia with no differences in systolic and diastolic blood pressures  coupled MOMA-2 staining of the aortic root showed a reduction in macrophage infiltration in the aortic root  while FATo differentiate between the effect of ITCH loss on the immune system versus its effect on the stromal fraction we performed Bone Marrow Transplantation (BMT) experiments .In the BMT animals we found no differences in either the development of atherosclerosis , weight,As the liver is the main site of triglyceride and cholesterol synthesis we next examined the hepatic tissue. Histological analysis showed conserved liver architecture with reduced accumulation of neutral lipids in ApoE−/−ITCH−/− animals . InteresTo determine the underlying mechanism behind the reduced triglyceride accumulation in the liver we performed indirect calorimetry measurements. These revealed an increased production of VO2 and VCO2 coupled with a mild but significant decrease in the Respiratory Exchange Ratio (RER). Together these values suggest that ApoE−/−ITCH−/− have a higher metabolic rate couple to an increased reliance on fatty acids as an energy source . A limitThe metabolic phenotype of reduced weight gain, improved glycaemia, reduced liver triglycerides and increased mitochondrial oxidation strongly recalls the phenotype of Sirt6 transgenic mice. Indeed, western blotting revealed an upregulation of SIRT6 in the liver while there were no changes in SIRT1 . InteresSiRNA knockdown of ITCH in HepG2 cells confirmed the link by increasing SIRT6 in a dose dependent manner .To investigate in more depth the reduction of circulating cholesterol we measured the expression of several genes involved in the synthesis, excretion and re-uptake of cholesterol. Unexpectedly, in the ApoE−/−ITCH−/−, we observed an upregulation of several genes involved in cholesterol synthesis, while genes involved in cholesterol excretion were minimally modulated .With a gene expression profile indicative of increased cholesterol production we next analyzed cholesterol re-uptake and found that the LDL-receptor is upregulated at a transcriptional as well as protein content level . HoweverITCH SiRNA knockdown in HepG2 cells resulted in increased nuclear localization of SREBP2 .We herein show for the first time that the loss of ITCH results in a reduced development of atherosclerosis coupled with reduced hepatic fatty infiltration in the hypercholesterolemic ApoE−/− mouse model through reduced clearing of SREBP2 and SIRT6 respectively.While the loss of ITCH also results in a shift towards an anti-inflammatory M2 phenotype, this appears to be uncoupled, in our model, from the development of atherosclerosis, suggesting that the main effect is driven by the reduction in the concentrations of circulating lipoproteins. Indeed this effect is lost when performing bone marrow transplantation. It has recently been suggested by Ferrante et al. that, in the adipose tissue, it may not be the shift towards the classical M1 macrophage that is responsible for the deleterious effects of increased cytokine production, but rather the overall expansion of the monocyte population conferring insulin resistanceWe therefore postulate that the loss of ITCH in the liver hampers the breakdown of SREBP2 thus leading to an increased expression of the LDLr, which is enough to compensate for the increased cholesterol synthesis and levels of circulating PCSK9. As a matter of fact the BMT animals showed increased levels of cholesterol as the bone marrow transplantation did not effectively reduce liver levels of ITCH resulting in a selective upregulation of PCSK9, a mechanism that remains to be explored.The main regulation of SREBP2 is through the intracellular content of cholesterol. Increased intracellular cholesterol usually inhibits the cleavage of the inactive form of SREBP2 to its active, nuclear form. The active nuclear form of SREBP2 is mainly regulated by ubiquitination dependent degradationThe clinical importance of SREBP2 has been clearly demonstrated in patients with hypercholesterolemia in whom the treatment with a statin results in the upregulation of the LDL receptor through increased activation of SREBP2. While there has been some discussion regarding the concomitant upregulation of PCSK9 there are currently promising agents under development, which specifically target PCSK9 thus allowing, further decreases in LDL levels.2 consumption and CO2 production shown in our calorimetric studies and the upregulation of several mitochondrial markers of oxidative metabolism in the liver of ApoE−/−ITCH−/− mice, ITCH emerges as a direct regulator of lipid metabolism in the liver. Since the bone marrow transplant experiments de-emphasized the metabolic role of a Th2 bias, at least in this model, we consequently looked for targets at a hepatocyte level. Despite several putative targets, such as the P73/PML/PPARδ pathway  guidelines and in accordance with the “Guide for the Care and Use of Laboratory Animals” (1996) by the Institute of Laboratory Animal Research Commission on Life Sciences .Itch−/− mice were crossbred with ApoE−/− mice to generate heterozygotes. These were then interbred to generate a pure lineage, which was then backcrossed into ApoE−/− for 5 generations. ApoE−/−Itch−/− mice and ApoE−/− littermates were maintained on 12 hrs light and dark cycles under controlled environmental conditions, with free access to water and food. Studies were performed only in male mice. Where described, after weaning, animals were fed a western diet  for 8 or 12 weeks. Metabolic testing procedures and ITCH−/− fed high fat diet (HFD) were previously describedBlood was collected after overnight fast, allowed to clot at room temperature and then centrifuged at 2000 G for 20 mins at 4C to obtain serum. Total cholesterol, HDL and the VLDL/LDL fraction were measured by colorimetric assay according to the manufacturers instructions (Abcam).To determine atherosclerosis in the outflow tract and valve area the top half of the heart was embedded in Optimized Tissue Compound (Tissue Tek) and stored at −80 until sectioning. Serial 8 micrometer sections where all 3 valve leaflets were visible were stained with Oil Red O and mounted with gelatin.Cholesterol and triglycerides were extracted from liver tissues and analyzed using the Total cholesterol Assay Kit and Triglyceride Quantification Kit  according to the manufacturer's instruction.6 cells bone marrow cells were then injected in the retro-orbital venous plexus of the recipient ApoE−/− mice. Feeding with western diet started 3 weeks after engraftment. PCR for the Itch−/− genotype was performed on DNA extracted from blood of recipient mice 8 weeks after BMT to confirm the efficiency of this BMT technique.Bone marrow transplantation (BMT) studies were performed as previously describedFACS analysis was performed as previously describedFor labeling of T-regulatory cells we used a staining kit according to the manufacturers instructions (Milteny Biotec). T-Regs were defined as CD4+, CD25+ and FoxP3+.Samples were analyzed using a FACScalibur (BD bioscience) running BD Cellquest Pro and analyzed with Flow JO .Itch siRNA using the Nucleofecteor AMAXA transfection kit according to the manufacturer's instructions with the use of a Nucleofector machine (Amaxa/Lonza). In short, cells 1 × 106 were resuspended in 100 μL of KIT V nucleofection solution (Amaxa/Lonza) with stated concentrations of Itch siRNA or control scramble sequences , permeabilized and then plated and incubated at 37°C for 2 days before harvesting for Protein and RNA studies.HepG2, human hepatoma cells , were maintained in RPMI containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin . Cells were transfected with Nuclear protein was extracted by using the NE-PER kit according to the manufacturers instructions (Thermo scientific).Western blotting and immunoprecipitation were performed with the following antibodies: SIRT1 (ab28170), SIRT6 (ab62739), SREBP2 (ab30682), LDL receptor (ab52818), ITCH (BD pharmigen 611198), PCSK9 (R&D biosystems AF3985). Actin and Lamin A/C (Santa Cruz Biotechnology) and AKT (CST 9272) were used for control.Immunoprecipitation with SIRT6 was performed as previously describedt test with GraphPAD Prism 5.1. Values of p < 0.05 were considered statistically significant.Results of the experimental studies are mean ± SD or SEM as specified. Statistical analyses were performed using the one-way ANOVA or unpaired Student's R.S. carried out the experimental work and data analysis; M.M., A.M., B.K. and V.C. helped with the animal experiments; J.M. performed the histological stainings; R.M. helped with in-vitro experiments; G.M. provided the ITCH −/− mice and contributed to discussion; R.S. and M.F. wrote the manuscript; M.F. designed the study and the experiments. All authors reviewed the manuscript.Supplementary Figures


For this, sperm are cryopreserved prior to therapy and in younger children testicular biopsies are cryopreserved with a hope to mature the germ cells into sperm later on for assisted reproduction. In addition, lot of hope was bestowed on pluripotent embryonic and induced pluripotent stem cells to differentiate into sperm and oocytes. However, obtaining functional gametes from pluripotent stem cells still remains a distant dream and major bottle-neck appears to be their inefficient differentiation into primordial germ cells (PGCs). There exists yet another population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) in adult body organs including gonads. We have earlier reported that busulphan (25 mg/Kg) treatment to 4 weeks old mice destroys actively dividing cells and sperm but VSELs survive and differentiate into sperm when a healthy niche is provided Mouse testicular VSELs that survived busulphan treatment were cultured for 3 weeks. A mix of surviving cells in seminiferous tubules  were cultured using Sertoli cells conditioned medium containing fetal bovine serum, follicle stimulating hormone and with no additional growth factors.Sca1) and SSCs (Gfra) proliferate (Pcna), undergo spermatogenesis (spermatocyte specific marker prohibitin), meiosis (Scp3) and differentiate into sperm (post-meiotic marker protamine).Stem cells underwent proliferation and clonal expansion in culture and spontaneously differentiated into sperm whereas Sertoli cells attached and provided a somatic support. Transcripts specific for various stages of spermatogenesis were up-regulated by qRT-PCR studies on day 7 suggesting VSELs  contains supplementary material, which is available to authorized users. By genetic modifications, human ES and iPS cells were differentiated into germ cells that underwent complete meiosis [Pluripotent stem cells (PSCs) including embryonic stem (ES) and induced pluripotent stem (iPS) cells have been studied for differentiation into gametes but the goal to achieve normal gametes to treat infertility still remains a distant dream . The pro meiosis ,9. Eguiz meiosis  achievedAgainst this background, there is yet another novel PSCs population which exists in various adult organs  includinLIN-/CD45-/SCA1+ VSELs comprise 0.03 ± 0.002% of cells in adult mouse testis. Immuno-phenotyping studies reveal that unlike bigger spermatogonial stem cells (SSCs) which are more than 10 μm in size and GFRA positive, VSELs are smaller in size, negative for GFRA and express pluripotent transcripts including nuclear OCT-4. VSELs are possibly the pluripotent primordial germ cells which survive in adult testis in small numbers. They undergo asymmetric cell division to self-renew themselves and also give rise to SSCs which express cytoplasmic OCT-4, divide rapidly and further differentiate into sperm . VSELs cin vivo on inter-tubular transplantation of healthy Sertoli or mesenchymal cells [see Additional file in vitro into oocyte-like structures [in vitro culture of VSELs enriched from adult mammalian  ovary surface epithelium spontaneously differentiate into oocyte-like structures in a very simple culture medium  within three weeks; whereas the epithelial cells differentiate into mesenchymal-like fibroblasts and act essentially as a source of growth factors and cytokines required for the differentiation of oocyte-like structures [Of the four different doses of busulphan used by us to treat mice in an earlier study, the dose of 25 mg/Kg resulted in nearly complete testicular ablation associated with significant loss of Dazl and Gfra at both mRNA and protein level . Howeverructures . Our groructures ,28.in vitro differentiation potential of surviving stem cells collected from busulphan treated mouse testis. For this, cells collected by enzymatic digestion of seminiferous tubules  were used to establish primary cultures. Results show that the whole process of spermatogenesis gets replicated in vitro in basic culture medium.The aim of the present study was to study the The study was approved by the Institute’s Animal Ethics Committee (IAEC). Adult male Swiss mice maintained in the Institute experimental animal facility were used for the study. They were housed in a temperature and humidity controlled room on a 12 hour light/12 hour darkness cycle with free access to food and water. Eight weeks old Swiss mice were treated with busulphan . One month after the treatment, mice were sacrificed by cervical dislocation; testes were collected and further processed for the study.Dazl ) and also at protein level for DAZL. Besides the Sertoli cells, relatively quiescent VSELs were found to persist and increase in numbers as confirmed by flow cytometry and higher expression of specific transcripts Oct-4A and Sca-1 by qRT-PCR studies [As reported earlier by our group, this dose of busulphan caused effective loss of SSCs and germ cell aplasia evidenced by histology, significant reduction of transcripts specific for SSCs (Gfra) and germ cells  supplemented with penicillin 100 U/ml and streptomycin 100 mg/ml. All reagents were from Invitrogen (USA) unless otherwise specified. Single cell suspension of testicular cells was obtained by a two-step enzymatic process as described earlier . Brieflyin vitro culture.The cells suspension obtained by digesting the testicular tissue was heterogeneous in nature and different strategies were used to further enrich the surviving VSELs including (i) differential plating i.e. cells suspension was plated in 35 mm culture dish, Sertoli cells attached overnight and unattached floating cells were collected next day for the study (ii) immuno-magnetic enrichment of SCA-1 sorted cells (described below) (iii) differential centrifugation, which involved initial centrifugation at 500 rpm for 10 minutes that allowed larger cells to settle down and VSELs in supernatant were later pelleted down at a higher speed of 1000 g and used for in vitro culture.SCA-1 positive cells were magnetically separated using SCA-1-FITC antibody  and Easysep kit  according to manufacturer’s instructions. Briefly, the mouse testicular cell suspension was sequentially incubated with blocking solution (provided in the kit), SCA-1 antibody, selection cocktail and magnetic nanoparticles. Then, the cell suspension was placed on a magnet and the positive cells were selected. Magnetically enriched SCA-1 positive cells were used for °C till further use.Sertoli cells are known to provide various growth factors in a paracrine manner for germ cells to undergo spermatogenesis . Hence a2 atmosphere. Cells for initial seeding, obtained and enriched for VSELs as described above, were cultured using Sertoli cells conditioned media supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin and follicle stimulating hormone (5 IU/ml). During culture, the media was changed partially every second day and care was taken not to disturb the cells in culture too much. The cultures were monitored over time and images were captured under inverted microscope . The cultures were established five times.All cultures were carried out in 35 mm dish and maintained at 37°C in humidified 5% COTesticular cells were fixed in Trizol (Invitrogen) for RNA extraction at the start of culture (D0) and on day 7 (D7) after culture. Total RNA was extracted using standard protocol and was treated with DNase I  to remove any genomic DNA contamination. First-strand cDNA was synthesized using the iScript cDNA synthesis Kit  according to the manufacturer’s instructions. Briefly, 1 μg of total RNA was incubated with 5X iScript reaction mix and reverse transcriptase mix. The reaction was carried out in G-STORM thermocycler . The reaction mix was first incubated at 25°C for 5minutes, then at 42°C for 30 minutes and finally at 85°C for 5 minutes.Dazl (germ cell), Gfra , prohibitin (meiotic) and protamine (post-meiotic) markers using specific primers and PCR conditions  and SYBR Green chemistry (Bio-Rad). The amplification conditions included initial denaturation at 94°C for 3 minutes followed by 40 cycles comprising of denaturation at 94°C for 10 seconds, primer annealing for 20 seconds, and extension at 72°C for 30 seconds followed by melt curve analysis step from 55°C to 95°C. The fluorescence emitted at each cycle was collected during the extension step of each cycle. The homogeneity of the PCR amplicons was verified by running the products on 1.5% agarose gels and also by studying the melt curve. All PCR amplifications were carried out in duplicate. Mean Ct values generated in each experiment using the CFX Manager software (Bio-Rad) were used to calculate the mRNA expression levels. The fold change was calculated using ΔΔCt method. The Q-PCR studies were done on two independent cultures and results analyzed separately.The expression levels of various gene transcripts were studied on day 7 compared to initial cells that were put in culture using specific primers Table  by qRT-PThe culture experiments were repeated five times using cells suspension from chemoablated testis with VSELs enriched using three different approaches as described in the Methods section. We obtained similar results however initial presence of few spermatogonial cells which may also have survived busulphan treatment cannot be ruled out in our generic cultures. This was anyways not an issue of great concern to us as we have earlier reported that VSELs are the quiescent stem cells which undergo asymmetric cell division to give rise to the SSCs which further undergo symmetric cell divisions, proliferate rapidly, differentiate, undergo meiosis to produce sperm . The undThe cells  were studied after overnight seeding and two distinct populations of cells comprising the bigger Sertoli cells and distinctly spherical stem cells could be observed at lower magnification Figure A. SertolGfra, prohibitin and protamine were found to be expressed on day 7 , proliferation (Pcna), meiosis (Scp3), pre-meiotic stage (prohibitin) and post-meiotic sperm (protamine) were repeated twice and results are represented individually for both the experiments  which survive busulphan treatment in adult mice testis undergo spontaneous proliferation, clonal expansion, differentiation and meiosis resulting in sperm formation during  in 2011  and rece in 2011  to diffein vitro is further supported by more than 40-60 folds up-regulation of Sca-1 and also around 8 fold increase in Gfra transcripts by qRT-PCR. Elevated levels of Sca-1 in both the culture samples analyzed by qRT-PCR was associated with low level of Gfra expression in one of the sample and this perhaps represents different state of proliferation/differentiation of the cells in culture taken for RNA extraction.Although VSELs exist as a small sub-population comprising approximately 0.03% cells in normal testis, the numbers almost double after busulphan treatment . In cultin vitro because VSELs invariably will give rise to SSCs which will further differentiate into sperm. The concept that VSELs give rise to SSCs was proposed by us [Whether a small population of SSCs survives after treatment depends on the dose of busulphan used. They may survive because this could only explain the spontaneous and endogenous recovery later on in certain cases. The concept that VSELs exist in testes, survive because of their quiescent status and may be responsible for endogenous recovery of spermatogonial cells are novel concepts in testicular biology put forth for the first time by our group. Similarly VSELs survive in adult mouse ovary after chemotherapy and are responsible for endogenous recovery of oocytes . Choi eted by us  based oned by us . Similared by us . Being pin vitro has remained the major bottle-neck and various groups have used chemicals like retinoic acid for meiotic entry of germ cells [in vitro was reported earlier by our group [Correct meiotic progression rm cells . The sucur group -28. Thusin vitro and was recently reviewed [in vitro since the support provided by Sertoli cells and peritubular cells in vivo could not be replicated in vitro and also even though several combinations of culture media were tried, none was competent enough to support complete spermatogenesis.Considerable progress has been made by various groups to demonstrate spermatogenesis reviewed ,4,34. Sareviewed  producedreviewed  reportedreviewed  and alsoreviewed . Howeverin vitro, unlike the ES/iPS cells lies in their source of origin during development and was recently discussed by us in details. It is the distinct epigenetic status of the VSELs and their close similarity to PGCs confers on them the superior ability to spontaneously differentiate into sperm compared to ES/iPS cells [in vitro (present study) and in vivo [in vitro. Similar experiments need to be urgently undertaken using human testicular biopsies from azoospermic cancer survivors. Differences exist between mice and men and the question remains whether our work in mice has relevance to humans. Our clinical collaborators have observed similar VSELs in adult azoospermic survivors of childhood cancer patients . We are not advocating obtaining sperm in vitro for clinical use. We will rather take advantage of Mother Nature and prefer to restore spermatogenesis by providing a healthy niche in situ as described in our recent study [in vitro will always have associated epigenetic concerns and results of the present study only demonstrate the differentiation potential of VSELs to the scientific community.The Views and Reviews section in Fertility Sterility published in January 2014 concludes that the field of obtaining gametes from pluripotent stem cells remains a distant dream and much more remains to be achieved on the bench before moving to the clinic [PS cells . RecentlPS cells . But VSE in vivo  potentia in vivo , traversnt study . Obtainiin vitro in the present study. Importance of the niche was highlighted in a focus issue on Regenerative Medicine recently published in the journal Nature Medicine [The scientific community needs to take note of this novel population of pluripotent stem cells that exist in adult testis and exploit their potential. Importance of a healthy niche/ microenvironment also needs to be appreciated. Several decades of research targeted the ‘seeds’ for fertility preservation; little realizing that it is the soil that needs to be rejuvenated. The results of present study and our recently published studies ,26 implyMedicine .in vitro. This better potential of VSELs compared to ES/iPS cells is because they are the PGCs surviving in adult testis. PGCs are more mature and pre-programmed to easily differentiate into gametes compared to inner cell mass cells in a blastocyst from which ES cells are derived. However, for clinical applications we advocate to restore spermatogenesis by providing a healthy niche in vivo as reported earlier [in vitro maturation. The in vivo re-initiation of spermatogenesis by injecting healthy niche cells may provide normal fertility and biological parenthood to cancer survivors with minimal safety, ethical, regulatory and societal concerns.To conclude, it is possible to coax VSELs that survive busulphan treatment in mice testis to undergo spermatogenesis  earlier  rather t


In skeletal muscle, the mutant allele was highly expressed leading to an abnormal transcript consisting of a 41-bp inclusion and premature termination codons and to residual levels of a correctly spliced transcript. This expression pattern, caused by a leaky intronic mutation with regard to spliceosome activity and its apparent stability with regard to surveillance mechanisms, could contribute to the moderate muscle hypertrophy in this cattle breed. This finding is of importance for genetic counseling for meat quantity and quality in livestock production and possibly to manipulate myostatin pre-mRNA in human muscle diseases.Myostatin is essential for proper regulation of myogenesis, and inactivation of Myostatin results in muscle hypertrophy. Here, we identified an unexpected mutation in the  Myostatin is expressed almost exclusively in cells of the skeletal muscle lineage, from the embryonic myotome to striated muscle in adults myostatin gene has been extensively investigated considering the potential benefits of enhancing muscle growth in clinical and agricultural settings. Loss-of-function mutations which impair Myostatin function or those which knockdown myostatin gene expression, result in muscle hypertrophy often referred to as “double-muscling” myostatin overexpression induces profound muscle loss myostatin mutation has been identified in a young boy myostatin pre-mRNA splicing are being developed for the treatment of muscle-wasting disorders such as Duchenne muscular dystrophy Since its discovery in mice in 1997 myostatin mRNA levels, the myostatin gene does not seem to be responsible for muscle phenotype in BAB In cattle, several mutations that cause different degrees of hypermuscularity have been reported. The most extreme phenotype is seen in the Belgian Blue breed (BBB) myostatin gene. In skeletal muscle, the mutant allele was highly expressed leading to an abnormal transcript with a premature termination codon and to residual levels of a correctly spliced transcript.Here, we identified an unexpected mutation in the myostatin cDNA obtained by RT-PCR in Blonde d'Aquitaine longissimus dorsi, a hypertrophied skeletal muscle in cattle. The amplification products . Remarkably, the mutation created a new 5′ donor splice site. Nine animals were found G/G homozygous  in the ten animals. Direct sequencing of PCR products revealed a mozygous  and the expected . We did ice site . This as animals  whereas T3811>G3811) leading to an aberrant myostatin mRNA. This likely results in a truncated Myostatin, lacking the entire bioactive region and hence reduction in functional Myostatin concentrations contributing to muscular hypertrophy in animals belonging to Blonde d'Aquitaine breed.We genotyped 445 animals from several European cattle breeds by RFLP-PCR  for the The most striking result revealed by our work was the apparent stability of the aberrant transcript despite the presence of premature termination codons as assessed by RT-PCR and direct sequencing . Indeed,G/G or G/T animals) or both to wild-type and abnormal transcripts . As expected, the commonly used TaqMan probe detected myostatin mRNA in all animals regardless of their genotype  animals used as control  animals at variable levels ranging from undetectable  to detectable levels  as compared to the G/T heterozygous animal (line 10). Aberrant transcripts were more expressed in G/G animals than in the G/T heterozygous animal (line 10). This result was confirmed by cloning and sequencing. Unfortunately, we were unable to quantify the residual correctly spliced transcript by either semi-quantitative RT-PCR or TaqMan real-time quantitative RT-PCR.The genotyping studies above showed that the deep intronic mutation was almost fixed in Blonde d'Aquitaine cattle . Howevermyostatin gene also lead to hypermuscular phenotype in cattle breeds such as the Belgian Blue and Piedmontese Myostatin is a TGF-β superfamily member that acts as a negative regulator of skeletal muscle growth. Mice lacking Myostatin have a dramatic and widespread increase in skeletal muscle mass as a result of a combination of muscle fiber hypertrophy and hyperplasia T3811>G3811 mutation within intron 2 of the myostatin gene  and activin type I receptors (ALK4 and ALK5) to phosphorylate responsive Smad proteins , which enables the Smad proteins to form a transcriptional complex with Smad4 to transcribe target genes myostatin gene that most likely contributes to muscular hypertrophy of the Blonde d'Aquitaine breed. Taken together, it is attempting to speculate that this leaky mutation, with regard to spliceosome activity, appears to buffer the nonsense mutation and prevents extreme muscle overgrowth making it biologically significant in the context of mutations causing hypermuscularity. Indeed, extreme muscle overgrowth in cattle has disadvantages including reduction in female fertility, lower viability of offspring, delay in sexual maturation and systematic use of cesarean delivery due to large calf size. This finding is of importance for genetic counseling for meat quantity and quality in livestock production and possibly opens a new therapeutic option using myostatin pre-mRNA to treat human muscle diseases.In conclusion, we identified a mutation in the Longissimus dorsi samples were collected within 15 minutes after slaughter , cut into small pieces and immediately frozen in liquid nitrogen, transported to the laboratory and stored at −80°C until use. Muscle samples (used as controls) from two animals belonging to the Limousine breed were obtained from Limoges slaughterhouse (France).Ten 12 months old Blonde d'Aquitaine bulls were used in this study. They were reared in the same conditions of feeding, housing and health status at the beef progeny test station of MIDATEST . DNA was extracted from the ten muscle samples using the QIAamp DNA midi kit (Qiagen) according to the manufacturer's instructions and stored at −20°C. Total RNA was extracted from muscle samples using Tri reagent (Sigma-Aldrich) and treated with DNase I (Sigma-Aldrich) following manufacturer's instructions. The quality and quantity of RNA were evaluated by the Agilent 2000 bioanalyzer and conserved at −80°C until use.myostatin transcript (RT-PCR assay) that may escape surveillance mechanisms . 2 µg of total RNA from the ten Blonde d'Aquitaine bulls and two Limousine animals were reverse-transcribed (RT) into cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems). 100 ng  of cDNA for each sample were used as template (RT-PCR assay) to amplify the entire coding region of the myostatin transcript  and a specific TaqMan probe  respectively. The positions of TaqMan probe hybridization targets are indicated in CASC3  and SF3A1  were used as internal control for more accurate normalization of expression data. cDNAs were amplified on an ABI PRISM©7900 system (Applied Biosystems) according to manufacturer's instructions. Relative mRNA expression values were calculated by the ΔΔCt method with normalization of each sample to the average change in cycle thresholds of controls.Quantitative real-time RT-PCR assays were performed in triplicate for each sample using 100 ng cDNAs prepared as described above. Relative amounts either of wild-type and aberrant myostatin intron 2 was amplified from DNA from the ten Blonde d'Aquitaine animals using Taq DNApolymerase Expand Long Template (Roche Applied Science) according to manufacturer's instructions; intron2seq-F and intron2seq-R primers used are indicated in The 2.033-kb of T3811>G3811). A 763-bp fragment encompassing the SNP was amplified by PCR from genomic DNA using Intron2-F and Intron2-R primers (AflII restriction enzyme (Biolabs) and size fractionated by agarose gel electrophoresis  into 565-bp and 198-bp fragments  was used as a cutting control.We genotyped 445 animals from several European cattle breeds  by RFLP- primers , digestephoresis . The T38ragments . DigestiAflII and HindIII were made using 10 µl unpurified PCR products, 2 µl enzyme buffer and 1 µl restriction enzyme in 20 µl final volume. 0,2 µl BSA were added for the AflII digestion. Samples were incubated overnight at 37°C.Digestions with


This work in strawberry fruit predicts new genes relevant to a key metabolic pathway using correlation analysis, and then demonstrates their function in a rapid and effective transient assay. LEUCOANTHOCYANIDIN REDUCTASE (LAR) and FLAVONOL 3’-HYDROXYLASE (F3’H) transcript accumulation upon overexpression. Another affects LAR and FLAVONOL SYNTHASE (FLS) after overexpression. The third putative transcription factor appears to be a universal regulator of flavonoid-pathway genes, as many pathway transcripts decrease in abundance when this gene is silenced. This report demonstrates that such systems-level approaches may be especially powerful when connected to an effective transient expression system, helping to provide rapid and strong evidence of gene function in key fruit-ripening processes.New modulators of the strawberry flavonoid pathway were identified through correlation network analysis. The transcriptomes of red, ripe fruit from two parental lines and 14 of their progeny were compared, and uncharacterized transcripts matching the expression patterns of known flavonoid-pathway genes were identified. Fifteen transcripts corresponded with putative transcription factors, and several of these were examined experimentally using transient expression in developing fruits. The results suggest that two of the newly-identified regulators likely contribute to discrete nodes of the flavonoid pathway. One increases only  Fragaria × ananassa) is a high-value crop, recognized for its sweet flavours, nutrient content and attractive appearance. Consumer analysis has shown that rich red colour and a promise of flavour and nutrition can affect desire to purchase the product  plants. The data revealed co-expression relationships between common flavonoid pathway genes and allowed the identification of coincident candidates, focusing on transcription factors potentially playing a role in ripening-related processes. A subset of the candidates was either silenced or overexpresssed in transient fruit assays, and revealed specific mechanisms of action in the regulation of discrete pathway functions. This work couples bioinformatics-based network analysis with a robust transient assay to functionally identify new regulators of flavonoid pathway genes.This report uses a TCNA approach to identify new candidates that affect specific nodes of the flavonoid pathway, using a segregating population of octoploid strawberry , while silencing was performed in ‘Elyana’. These cultivars have been noted for robust overexpression or silencing responses in the agroinfiltration method . For the developmental series, ‘Festival’ fruits were harvested at the green, white, turning and red stages. Achenes were removed from frozen fruits by grinding them off with a frozen steel file. The resulting powder was collected and analysed separately.Two different cultivars were used for transient fruit assays. Overexpression was performed in r) with a two-tailed test and the P-value was established for each comparison with 11 degrees of freedom. Correlations were considered positive when Pearson’s r>0.65 and P<0.05.Correlation tests were performed using Pearson product-moment correlation coefficient (Pearson’s FaTCP11 (gene09614) and FaPCL1-like (gene17673) overexpression constructs, full-length sequences were amplified from strawberry cv. ‘Elyana’ cDNA using specific primers (see Supplementary Table S1) and cloned into the gateway vector pK7WG2D.1. Silencing of FaSCL8 (gene13212) was accomplished by amplifying a 330bp sequence from cv. ‘Elyana’ cDNA and inserting it into pHELLSGATE12 using gateway technology. Primers used are listed in Supplementary Table S1.For Agrobacterium tumefaciens strain AGL0 (Agrobacterium cells were harvested and resuspended in a modified MacConkey (MMA) medium . The Agrobacterium suspension was then injected into multiple sites over the entire fruit while it was still attached to the plant ~14 d after pollination using a sterile 1ml hypodermic syringe. The fruits were harvested 3–7 d after injection, the day they turned fully red. Fruits at the same developmental stage were used as control. Control for the agroinfiltration effect on the flavonoid pathway was tested using a control plasmid containing the GUS gene and a comparable injection protocol .Transient expression in strawberry fruit by agroinfiltration was performed according to FaCHP1 as a stable reference transcript . Reaction mixes (20 μl) were prepared, which included 10 μl of EvaGreen 2X qPCR MasterMix-ROX (ABM), 0.2mM of each primer and 1 μl of diluted cDNA. Gene transcripts were quantified with normalization to anscript . Specifihttp://www.ncbi.nlm.nih.gov/sra/?term=SRP039356). Data represent all reads from parental lines (‘Mara des Bois’ and ‘Elyana’) as well as reads from individual F1 plants.The RNAseq reads have been deposited into the NCBI Short Read Archive and are accessible under project SRP039356 . Global analysis of these variations showed the absence of general trends between the samples and confirmed the apparent homogeneity of the dataset. This allowed investigation of possible correlations among genes expressed in specific biosynthetic pathways.‘Mara des Bois’, a 1990’s cultivar with a unique aromatic profile, and ‘Elyana’, a Florida cultivar that produces large, flavourful fruits, were crossed and fruits from 14 F1 individuals were harvested at a uniform fully-expanded, red-ripe stage. An average of 11.7 million clean reads were produced per genotype with a maximum of 20.3 and 19 million reads for the parents ‘Mara des Bois’ and ‘Elyana’, respectively . The reaPHENYLALANINE AMMONIA LYASE (PAL) and some family-specific genes like the UDP-GLUCOSE FLAVONOID GLUCOSYL TRANSFERASE (UFGT) that leads to anthocyanin synthesis. Two isoforms of PAL and two of CHALCHONE SYNTHASE (CHS) were also included.The phenylpropanoid pathway, and more specifically the flavonoid pathway, is a well-studied biosynthetic pathway that includes several enzymes that catalyse production of different families of molecules such as the flavonols, the condensed tannins and the anthocyanidins . The corr>0.65, P<0.05) were identified  and CHI (CHALCONE ISOMERASE). These transcripts share six, seven and six positive correlations with other genes, respectively. Even when positive correlations were found between either PAL1 and PAL2 and the same other genes, no correlation between the two PAL isoforms was detected, suggesting different roles or, more likely, different regulation processes for the two isoforms. Furthermore, the global correlation pattern is in agreement with the metabolic flux occurring in the ripe fruit. Indeed, positive correlations are only found between genes that are involved in the anthocyanin pathway and no correlation were established with genes like LAR and ANR that are specific for the synthesis of tannins or FLS for the flavonols. No correlation was found for the F3’H gene suggesting the implication of this enzyme in processes other than anthocyanin synthesis in ripening strawberry fruit.Correlations in the expression profiles within this set of genes were calculated, and positive correlations between genes  with transcripts from the flavonoid pathway  homologues were selected, and their individual transcript accumulation patterns were compared to the expression profiles of the flavonoid pathway transcripts. Only 15 TF transcripts showed four or more positive correlations , gene17673 (a MYB TF) and gene18718  and another yet uncharacterized MYB TF (gene18815) also show strong correlations. Another interesting candidate is gene13212 due to its relatively high expression (RPKM between 379 and 1074). This transcript is most similar to Arabidopsis SCARECROW-LIKE 8 (SCL8). Three candidate genes were selected for comprehensive expression analysis. The first was gene17673, predicted to encode a putative MYB TF. This gene shared positive correlations with PAL1/2, C4H, 4CL and CHS1/2  were selected based on potential inferred roles in ripening or due to their expression levels. Based on sequence and domain similarity compared to ke genes . ClosestFaTCP11 and FaPCL1-like expression is stable during development and decreases during the red stage . Next, FaTCP11 and FaPCL1-like were overexpressed in ‘Strawberry Festival’. Overexpression assays were performed because these transcripts were expressed at low levels in developing fruit. FaTCP11 was overexpressed 10-fold higher in three independent fruits  and more than 70-fold higher in one fruit (OEX 4) compared to control fruits  in three independent fruits  and >15-fold lower in HP 4  1 from maize, CYCLOIDEA (CYC) from Antirrhinum majus, and PROLIFERATING CELL FACTORS (PCFs) from rice  and the Late-regulated Biosynthesis Genes (LBGs) genes in anthocyanin production, including those functioning in ripened fruit receptacles  but the induction of F3’H could lead to the synthesis of a different sub-family of anthocyanins. In strawberry the two major classes of anthocyanins are cyanidin and pelargonidin derivatives (F3’H in FaSCL8-silenced fruit suggests a modification in the cyanidin-pelargonidin balance that will be the subject of further investigation. Again the action of FaSCL8 on the pathway seems to be related to another transcription factor. Indeed, the induction of F3’H by FaSCL8 silencing could be due to the induction of FaMYB9/11. In conclusion, FaSCL8 seems to be able to modify the flavonoid genes expression in an indirect way by modulating the expression of known flavonoid regulating TFs. Future work will also include further characterization of these transcripts in stable transgenic lines.Suppression of  of F3’H  transcriTaken together, the results presented here confirm the applicability of using TCNA to identify transcription factors associated with a given biological process. The strength of using a segregating F1 population in order to identify new candidate genes for a metabolic pathway was clearly demonstrated. Roles for candidate genes were functionally assessed, and raise indications of complex connections between different sub-pathways that will be examined more precisely in transgenic lines. A well-studied pathway was investigated in this report, but the real power in this analysis could be proven by elucidating less well-characterized pathways of interest. This approach could provide a technical solution for resolving pathway components in diverse species of interest.JXB online.Supplementary data are available at Supplementary Table S1. A list of the primer sequences used in this study.Supplementary Fig. S1. The effect of the agroinfiltration process on the induction of flavonoid pathway genes.Supplementary Fig. S2. Hierarchical clustering of the ‘Mara des Bois’ × ‘Elyana’ F1 population.


We also demonstrate that serially-reaggregated engineered kidneys can transport organic anions and cations through specific uptake and efflux channels. These results support the physiological relevance of kidneys grown in culture, a commonly used model for kidney development and research, and suggest that serially-reaggregated kidneys self-assembled from separated cells have some functional characteristics of intact kidneys.Recent advances in renal tissue engineering have shown that dissociated, early renogenic tissue from the developing embryo can self-assemble into morphologically accurate kidney-like organs arranged around a central collecting duct tree. In order for such self-assembled kidneys to be useful therapeutically or as models for drug screening, it is necessary to demonstrate that they are functional. One of the main functional characteristics of mature kidneys is transport of organic anions and cations into and out of the proximal tubule. Here, we show that the transport function of embryonic kidneys allowed to develop in culture follows a developmental time-course that is comparable to embryonic kidney development  The ability of separated kidney progenitor cells to self-assemble into functional kidney-like rudiments centred around a single collecting duct would be of great importance both to regenerative medicine and for use of cultured or engineered kidneys as a drug screening platform.The mature vertebrate kidney is a highly specialised and complex excretory organ, consisting of many different cell types, with numerous physiological functions. Murine kidney development begins with formation of a ureteric bud as a branch from the nephric duct at the caudal end of the embryo. At embryonic day 11.5 (E11.5), the rudimentary kidney is composed of two distinct tissue types, the epithelial ureteric bud surrounded by condensed mesenchyme. Previous work in this laboratory has shown that kidney rudiments at this stage can be dissociated into a single cell suspension, and these cells can self-assemble into organotypic renal structuresThe nephrons of mature kidneys in vertebrates represent one of the major excretory routes in the body. Specialised transporters and efflux pumps at the basolateral and apical sides of the proximal tubules mediate the transport of organic anions and cations, providing a mechanism for excretion of xenobiotics and endogenous compounds  and organic cation transporters (Octs) have been shown to be detectable in embryonic stages both in vivoin vivo and that efflux of anions through the Mrp family of efflux pumps occurs at a later stage than anion uptake. We also find that cultured kidneys dissected at E11.5 and placed in culture continue to develop functional characteristics at roughly the same rate as kidneys allowed to develop in vivo, supporting their validity as a relevant model for kidney research. Furthermore, we find that the tubules of serially-reaggregated engineered kidneys are able to transport anions and cations in a manner similar to the mature kidney, supporting their possible use therapeutically or for pharmacological testing.Using the Sebinger culture system, which encourages cultures to grow flat whilst preserving development of renal structures, we have optimised and developed a panel of live fluorescence-based assays to test tubular function in cultured embryonic kidneys and in serially-reaggregated, engineered kidneys. We find that, in agreement with transcript data from rat embryonic kidneys In order to investigate the emergence of tubular function in renal development, we developed and optimised a series of assays to test tubular transport of organic anions and cations in live, cultured, developing murine kidneys. These assays could then be used to test transport function in the serially-reaggregated, engineered kidneys. Specific inhibitors of the different transport channels were employed to demonstrate specificity of fluorescent cation uptake and fluorescent anion uptake or efflux. The assays  are summarised in The fluorescent anion 6-carboxyfluorescein (6-CF) was chosen as a tracer dye to investigate organic anion transport in cultured embryonic kidneys and in serially-reaggregated engineered kidneys. The 376 Dalton fluorescein derivative 6-CF has been shown to be transported by organic anion transporters (Oats)91011516Probenecid is a potent and broad inhibitor of anion transporters, including the murine basolateral renal tubular Oat family of anion transporters2023Anion uptake was assessed by incubating live, cultured kidneys growing in the Sebinger culture system with the fluorescent anion 6-CF, either with or without probenecid (2.5 mM)Efflux of anions into the lumen of the proximal tubules is mediated by the Mrp family and Breast Cancer Resistance Protein (Bcrp), and to some extent by P-glycoprotein . Although the basolateral Oatp4c1 is thought to mediate uptake of anions into the cellFluorescein efflux has been shown to be mediated by the Mrp family of luminal efflux pumps25Digoxin is known to be transported by Oatp4c118Anion efflux at the apical side was again assessed using the fluorescent anion 6-CF. For the efflux assay, both conditions were allowed to take up 6-CF through the organic anion transporters . After aIn addition to anion uptake and efflux, we investigated the ability of the proximal tubules of cultured embryonic kidneys to take up organic cations using the fluorescent cationic molecule 4',6-diamidino-2-phenylindole (DAPI) . DAPI is3233These assays were used as the basis for analysing tubular function in cultured kidneys and serially-reaggregated engineered kidneys.We first assessed qualitatively the ability of embryonic kidneys, cultured from E11.5, to take up the fluorescent anion 6-CF specifically in the presence or absence of probenecid, a well-characterised Oat inhibitor. We found that cultured embryonic kidneys are able to transport organic anions early in development . The spein vivo, we analysed transcripts for the main renal anion transporters at different embryonic time-points, using semi-quantitative multiplex RT-PCR , either with or without the Mrp inhibitor MK-571No efflux of anions by cultured embryonic kidneys was observed  before 3in vivo and dissected at different embryonic time-points indicated that the transcript for Mrp2 can be clearly detected at E15.5 . DAPI does not normally penetrate intact membranes of live cells, but can be transported into cells expressing Oct1in vivo and dissected at different embryonic time-points indicated that the transcripts for Oct1 and Oct2 are detectable at E15.5 . Specific uptake of the fluorescent anion 6-CF by organic anion transporters was observed from 3 days in culture, with no detectable uptake in serially reaggregated, engineered kidneys treated with the Oat inhibitor probenecid . Specifiin vivo with respect to tubular function. In addition, we have shown that serially-reaggregated engineered kidneys also develop this transport function. Whilst basolateral to luminal transport is an important component of tubular function, tubules also transport molecules from the tubule lumen that have passed through the glomerular filtration barrier, and we have not yet determined if this process is active in cultured or serially-reaggregated engineered kidneys.Our results demonstrate that tubular transport function begins early in kidney development in agreement with previous microarray analysesWe have shown that transport of organic anions and cations is not dependent on development of vasculature and glomerular filtration. There are other examples of the uncoupling of tubular transport and blood filtration in development, for example in insects, where the functions take place in different locations in the body39Previous work has suggested that anatomically-correct, reaggregated kidneys are able to develop vasculature and to develop some degree of glomerular filtrationEmbryonic kidneys were dissected from E11.5 mouse embryos and placed in Sebinger cultureSerial reaggregates were engineered according to the method described in refs. The following reagents were purchased from Sigma Aldrich; Metformin hydrochloride (PHR1084), Cimetidine (C4522), Probenecid (P8761), MK-571 (M7571), Digoxin (D6003). 6-carboxyfluorescein was purchased from Invitrogen (C1360). Glycyl-H 1152 dihydrochloride was purchased from Tocris (2485). Metformin, cimetidine, and 6-carboxyfluorescein were solubilised in water to make stock concentrations as follows: metformin 100 mM, cimetidine 25 mM, 6-carboxyfluorescein 1 mM. Probenecid was solubilised in 500 mM NaOH, to make a stock concentration of 250 mM. MK-571 and digoxin were solubilised in DMSO to make stock concentrations as follows: MK-571 10 mM, digoxin 93.5 mM. Reagents for live assays were diluted in PBS.All assays were carried out on live cultures grown in the Sebinger culture system. Only cultures that were healthy at the time of assay were used; for intact, cultured kidneys this was approximately 8 in 10. For serially reaggregated engineered kidneys this number dropped to approximately 6 in 10. Live staining to outline morphological structure was done in all cases by incubating cultures, at same time as the transporter assay, with rhodamine-conjugated peanut aggulutinin . Prior to assay all cultures were washed once in PBS. Specific uptake of anions in the tubules of cultured kidneys was assayed by incubating live cultures with the fluorescent anion 6-carboxyfluorecein (6-CF) (1 μM) either with probenecid (2.5 mM), or with vehicle only  for one hour at 37°C. Cultures were washed once in PBS and then incubated with probenecid (10 mM) for 15 min at 37°C to trap intracellular fluorophore before imaging. Anion uptake assays in cultured intact kidneys were repeated 3 times (1 d), twice (2 d) or 7 times (6 d). Uptake in untreated kidneys and inhibition of uptake in inhibitor-treated kidneys was evident in every case. Specific efflux of anions by the Mrp family of efflux pumps was assayed by pre-loading live cultures with 6-CF for one hour at 37°C (1 μM), washing once in PBS and imaging briefly to confirm anion uptake, and then subsequently incubating with either digoxin (500 uM) and vehicle (0.33% DMSO), or digoxin (500 μM) and MK-571 (30 uM) , for 30–60 min at 37°C. Anion efflux assays in cultured intact kidneys were repeated twice  or 4 times  with efflux in kidneys untreated with MK571, and inhibition of efflux in MK571-treated kidneys evident in every case. Specific uptake of cations in the tubules of cultured kidneys was assayed by incubating live cultures with the fluorescent cation DAPI (1 μM) alone or in the presence of metformin (5 mM) and cimetidine (200 μM) for 30 minutes at 37°C. Cultures were washed 3x in PBS before imaging. Because DAPI is bound stably to DNA once inside a cell, the cultured kidneys could be imaged directly without needing to trap the fluorophore intracellularly. Cation uptake assays in cultured intact kidneys were repeated twice  or 10 times (6 d). Uptake in untreated kidneys and inhibition of uptake in inhibitor-treated kidneys was evident in every case. All assays (uptake and efflux) in serially-reaggregated kidneys were performed once  or twice (6 d). Inhibition of uptake or efflux in inhibitor-treated cultures, and no inhibition of uptake or efflux in untreated cultures, was evident in every case.An AnnexinV assay kit was purchased from Biovision (K103-25). After carrying out the cation transporter assay, live cultures were washed 3x in PBS and then incubated with AnnexinV binding buffer for 5 min at room temperature. Cultures were then incubated with AnnexinV solution diluted 1:100 in binding buffer at 37°C. Cultures were imaged immediately by confocal microscopy.Live cultures were imaged using a Zeiss Axiovert epifluorescence microscope with Axiovision software, or using a Nikon A1R confocal microscope with NIS elements software. When applicable all images were taken with identical exposures for comparison. All images were analysed using ImageJ.Total mRNA was isolated from 25–30 pooled embryonic kidneys, pooled cultured kidneys, or adult kidney using the RNeasy kit from Qiagen and according to kit instructions. After isolation, purity of RNA and absence of genomic DNA was confirmed by PCR for β-actin using primers that span an intron and using RNA preparations made with and without addition of reverse transcriptase. PCRs for genes of interest were carried out in multiplex reactions with primers for β-actin as loading controls. A table of primers used can be found in the This project involved no experiments conducted in living animals. Normal embryonic tissues used for post-mortem staining and culture were obtained from healthy CD1 mice killed by trained staff of the UK Home Office-licenced animal house under Schedule 1 of the UK Animals (Scientific Procedures) Act 1986.M.L. performed all experiments, drafted the manuscript and prepared the figures. C.C. prepared RNA from cultured kidneys and helped to make the serial reaggregates. J.D. contributed to experimental design and data analysis, and edited the manuscript. All authors reviewed the manuscript.Supplementary info


Perifosine did reduce primary breast cancer orthotopic xenograft tumor size, but did not impact metastatic burden in a statistically significant manner. However, Perifosine reduced telomerase activity in four of six CLL patients evaluated. Two of the patients were treated for four to six months and shortening of the shortest telomeres occurred in both patients' cells. These results indicate that it may be possible to repurpose Perifosine or other AKT pathway inhibitors as a novel approach to targeting telomerase.Most tumors circumvent telomere-length imposed replicative limits through expression of telomerase, the reverse transcriptase that maintains telomere length. Substantial evidence that AKT activity is required for telomerase activity exists, indicating that AKT inhibitors may also function as telomerase inhibitors. This possibility has not been investigated in a clinical context despite many clinical trials evaluating AKT inhibitors. We tested if Perifosine, an AKT inhibitor in clinical trials, inhibits telomerase activity and telomere maintenance in tissue culture and orthotopic xenograft models as well as in purified CLL samples from a phase II Perifosine clinical trial. We demonstrate that Perifosine inhibits telomerase activity and induces telomere shortening in a wide variety of cell lines  The ends of chromosomes are protected from recognition as double strand DNA breaks by telomeres, nucleoprotein structures on the ends of the chromosomes which consist of telomeric TTAGGG repeats and telomere-binding proteins known collectively as the Shelterin complex  . This prActivation of telomerase is one of the most common perturbations observed in malignancy, present in roughly 90% of tumors . Given tAKT, also known as Protein Kinase B, is a serine/threonine kinase downstream of PI3K, important in a wide variety of signaling events . The proin vivo.Perifosine is an orally available alkylphospholipid in phase III clinical trials for multiple myeloma. Perifosine inhibits AKT activity by interfering with the pleckstrin homology domain of AKT, preventing its membrane localization and phosphorylation . Perifosth and 1/2 of the Hela LD50, respectively) because the blood concentrations from orally-available doses of Perifosine are reported to be the same order of magnitude as the LD50 for a number of cell lines including Hela cells [To evaluate the effects of Perifosine on telomere length and telomerase activity, we cultured a panel of cell lines exposed to clinically relevant Perifosine concentrations for extended periods of time. Due to the heterogeneity in signaling dependence in cancer cell lines , we decila cells , 16. We p = 0.04) reduced the number of colonies per well, while transient treatment with Perifosine did not induce a statistically significant reduction in colony number , Hela cells treated with Perifosine during the soft agar colony formation assay (transient treated cells), and Hela cells treated with Perifosine for 70 population doublings prior to the assay and during the assay (long-term treated cells). Long-term Perifosine treatment significantly  were available from before therapy, cycle 1 day 8, cycle 4 day 1 (day 84), and at discontinuation of treatment, while four other patients had samples from before initiation of therapy and at cycle 1, day 8. Telomerase enzymatic activity was reduced at cycle 1 day 8 in four of the six patients tested. Telomerase activity was lower than before initiation of therapy at discontinuation in both patients treated for 4 or more months Figure . Howeverin vitro do not lead to a reduction in mean TRF in spite of the reduced telomerase activity measured. To test if the shortest telomeres in the population were getting shorter, we performed universal Single TElomere Length Analysis (Uni-STELA) in order to quantify the length and dynamics of the shortest telomeres in these samples.CLL is composed of varying proportions of proliferative cells depending on a number of poorly-understood factors; for most patients, the majority of the tumor is both non-proliferative and telomerase negative, though periodic reversion to a telomerase positive proliferative state is believed to occur . Due to There was a significant reduction in total Uni-STELA product length in patient 5, while there was no change in patient 11 Figure  and 4B. in vitro, in xenograft models and in CLL patients continuously treated with Perifosine. Perifosine did not increase the normal rate of telomere length reduction in telomerase negative cells, indicating that the reduction in telomere length was telomerase-dependent in cancer cells. HCC4017 did not have telomere shortening in response to Perifosine, and because of its very low baseline activity we evaluated it for the presence of c-circles, a marker for Alternative Lengthening of Telomeres (ALT) [in vitro and that a number of cell lines exhibited telomere shortening with continuous Perifosine treatment without a reduction in telomerase activity.This work has shown that Perifosine, an AKT inhibitor, can prevent telomere maintenance es (ALT) . HCC4017Inhibition of telomerase activity occurred in some lines that exhibited telomere shortening in response to Perifosine, though a change in telomerase activity did not universally predict an alteration in telomere length. Both H2009 and H1819 exhibited telomere shortening without a reduction in telomerase activity, suggesting that Perifosine may impair telomere maintenance in some backgrounds in a telomerase enzymatic activity independent manner, such as by regulating telomerase nuclear localization. AKT may modulate telomerase activity through a number of mechanisms, and Perifosine impacts pathways other than AKT, such as the p38 pathway , which mSomatic telomerase activity in mouse cells coupled with the fact that laboratory mouse telomeres are an order of magnitude larger than human telomeres  have comin vitro  with the addition of 10% calf serum . Cells were treated with the indicated concentrations of Perifosine with media changes and fresh Perifosine added every 3 days; cells were passaged 1:32 when they approached 100% confluence.The other AKT inhibitors, AKT inhibitor I, AKT inhibitor IV, and the PDK1/AKT/Flt Dual Pathway Inhibitor (EMD4 Biosciences), were tested under the same conditions as Perifosine using the concentrations indicated, replaced every three days.HCC38 cells expressing a Luciferase (HCC38 + Luc) construct were obtained from the Brekken lab (UT Southwestern). HCC38 + Luc cells were infected with a lentiviral vector bearing an empty pGipZ plasmid containing GFP and puromycin resistance genes (HCC38+Luc+GFP).The soft agar colony formation was performed as in  with a nThe second mammary fat pads of 30 (15 control and 15 treated) 6-8 week old female NOD/SCID IL2G mice (obtained from an on-campus supplier) were injected with 3*10^6 HCC38 breast cancer cells, and tumors were allowed to grow for 2 weeks before Perifosine was administered to the treated group. Primary tumors were measured once weekly in weeks 4 to 6 by digital calipers. Primary tumors were removed after measurement in week 6. Bioluminescent measurements of recurrent primary tumor burden were obtained in week 12. The mice were subsequently sacrificed and lung tumor burden was measured by luciferase activity. Tumor samples were disassociated and cultured for telomere analysis at primary removal in week 6 as well as from both recurrent primary and lung metastatic tumors in week 12. All xenograft experiments were conducted under UT Southwestern IACUC approval.The treated group was given an initial loading dose of 62.6 mg/kg Perifosine suspended in sterile PBS by oral gavage in the first week of treatment followed by weekly maintenance doses of 35.8 mg/kg Perifosine which are reported to maintain Perifosine blood concentrations between 12 uM immediately following a dose and 5 uM immediately prior to a dose based on previous studies on Perifosine pharmacokinetics in mice .Following surgical excision, tumor samples were placed into 750 uL of Gibco Minimum Essential Media  on ice and transported to the tissue culture facility. Tumor samples were then chopped into small pieces with autoclaved surgical scissors and resuspended in 750 uL MEM containing 0.5 mg/mL Liberase DL research grade (Roche). Tissue was incubated at 37° C for 60 minutes under agitation at 225 RPM. Fragments were further dissociated by repeat pipetting through 10, 5, and 1mL pipettes (5× each), and then filtered through a 200 micrometer nylon mesh. Samples were centrifuged at 1500 × g for 5 minutes, and then resuspended in 10mL Media X + 10% fetal calf serum + 5 ug/mL puromycin and plated into 10cm dishes for cell culture to remove mouse cells. Fresh Media X + 10% fetal calf serum + 5ug/mL puromycin was replaced 3 days following cell culture to remove dead cells. On day 7 of puromycin selection (to remove mouse contaminating cells), culture dishes were washed 2× with 10 mL phosphate buffered saline, and then harvested for TRF analysis by trypsinization (5 minutes).6 HCC+Luc+GFP cells suspended in 50uL autoclaved PBS+EDTA were injected into the fat pad using a 30-gauge needle. The incision was closed with wound clips, and mice were placed in a recovery cage on a heating pad. Mice were given 0.1 mg/kg intraperitoneal buprenorphine analgesia following breathing stabilization postoperatively and 24 hours later. All surgical instruments were autoclaved prior to operating and a hot bead sterilizer was used to sterilize tools between subjects.For orthotopic mammary fat pad xenograft injections, mice were anesthetized with 2% continuous isoflurane and the area of the incision was thoroughly cleaned with 70% ethanol and betadine. A small incision was made over the right axillary fat pad and 3 × 10in situ. Incisions were closed with wound clips, and mice were placed in a recovery cage on a heating pad, given buprenorphine analgesia, and monitored until ambulatory.The primary tumors were removed using the same anesthesia and pre-surgical conditions as the xenograft injection procedure. A small incision was made in the skin directly overlying the tumor, and the skin/subcutaneous tissue surrounding the tumor was carefully dissected free from the tumor surface circumferentially using both blunt and sharp dissection. The entire macroscopic primary tumor was removed when possible; however in cases where the primary tumor was involving surrounding structures  a small amount of tumor was left 40 mg/mL D Luciferin  suspended in autoclaved PBS was injected subcutaneously in mice anesthetized with 2% continuous isoflurane. Mice were placed on a heated imaging stage for ten minutes, and then imaged using the IVIS Lumina imaging system . Luciferase signals were quantified using the manufacturer's software. Mice were sacrificed immediately following acquisition of the recurrent primary tumor image in week 12; lungs were removed and imaged under the same conditions.Animals were housed in a pathogen-free facility and the animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Southwestern Medical Center.Terminal restriction fragment assays were performed as in . The droUniversal STELA data was quantified using the manufacturer's software (GeneSnap) for the Syngene G-Box , using the quantitative methods for determining the molecular weight of western blot data. The TRF ladders run on the gel were used to establish areas of known molecular weight, and the molecular weight of each STELA product was established from the ladders as determined by the software. The automated band identification algorithm provided with the software was used to identify peaks, with minimal human curation to remove obvious errors .CLL samples (10 million cells/pellet) were purified as part of the previous IRB approved clinical study at Duke University Medical Center . PatientTwo-sided t-tests performed with Graphpad Prism 7 software were used to compare group means.The human and animal studies were conducted with approval from the relevant institutional review boards. Human subjects gave written informed consent prior to their inclusion in the phase II trial.


ETFA and ETFB) and ETFDH encoding gene (ETFDH) in the rice blast fungus Magnaporthe oryzae. It was demonstrated that, by impacting fatty acid metabolism, ETF and ETFDH mutations led to severe growth and conidiation defects, which could be largely rescued by exogenous acetate or carbonate. Furthermore, although conidium germination and appressorium formation appeared to be normal in ETF and ETFDH mutants, most appressoria failed to penetrate the host epidermis due to low turgor pressure. The few appressoria that succeeded in penetration were severely restricted in invasive growth and consequently failed to cause disease. Moreover, ETF mutant etfb− induced ROS accumulation in infected host cells and exogenous antioxidant GSH accelerated mutant invading growth without increasing the penetration rate. In addition, mutant etfb− displayed elevated lipid body accumulation and reduced ATP synthesis. Taken together, ETF and ETFDH play an important role in fungal development and plant infection in M. oryzae by regulation of fatty acid metabolism, turgor establishment and induction of host ROS accumulation.Electron-transferring flavoprotein (ETF) and its dehydrogenase (ETFDH) are highly conserved electron carriers which mainly function in mitochondrial fatty acid β oxidation. Here, we report the identification and characterization of ETF α and β subunit encoding genes ( Magnaporthe oryzea (M. oryzae) is a destructive disease in rice-planting areas worldwide, and poses a severe threat to global food security, especially in the current changing climate1M. oryzae has emerged as a model organism for the study of plant fungal pathogens and their interaction with hosts34M. oryzae infection starts with a three-celled conidium contacting the host surface, which subsequently germinates and develops into a specialized structure called the appressorium7Rice blast caused by The initial infection processes at the host surface from conidia adhesion to appressorial development do not require nutrient acquisition by the fungus, but does require the mobilization and metabolism of the storage reserves in conidiaIn addition to glycerol, lipolysis also generates fatty acids that enter the β oxidation pathway for breakdown in either mitochondria or peroxisomes depending on the carbon chain length of the fatty acids15192119ETFA and ETFB) and one ETFDH gene in the rice blast fungus M. oryzae by targeted gene deletion and/or T-DNA insertion mutation. Our data demonstrate that both ETF and ETFDH play an important role in vegetative growth, conidiation and infection-related development of M. oryzae through regulation of fatty acid metabolism, turgor establishment and host ROS accumulation.The roles of ETF and ETFDH are not well understood in filamentous fungi. In the current study, we first identify and functionally characterize two ETF genes  as described previouslyETFB  on supercontig 8 of chromosome II  and an ETFDH gene  in the M. oryzae genome  and the ETFDH gene (ETFDH) in M. oryzae, we conducted targeted gene deletion mutagenesis and obtained at least two mutants for each gene. The gene deletions were confirmed by Southern blot analysis  completely lacked pigmentation in comparison to the grey colonies of Guy11 and the complemention strains  and minimal medium (MM). On the CM, the colonies of ETF and ETFDH mutants ( strains . In addi strains . These r strains . Growth  strains .7/plate) produced by Guy11 and the complemention strains after incubation for 12 days  to rescue the mutant defects. Our results showed that this treatment almost fully recovered the colony color and growth rate of the mutants , we quantified the ROS accumulation in infected host cells using DAB staining. Our results showed that mutant etfb− displayed a darker redish color at the infection site than Guy11 , and found large quantities of dark grey lipid bodies in the conidia and appressoria of mutants, in comparison to Guy11  and the T-DNA insertion mutant library construction were performed as described previouslyAll fungal mutants and transformants described in this study were generated from trpC-hph cassette with KpnІ and XbaІ ends was cloned to pGEM-T Easy vector (A1360 Promega USA). Then, approximately 1 kb up- and down-stream regions of each gene were amplified from M. oryzae genomic DNA and subsequently cloned into the sites flanking the trpC-hph cassette. For building gene complementation vectors, the full length of each gene with about 1.5-kb native promoter regions was cloned into pCB1532 vector. The 1.5kb GFP sequence with a TrpC-terminator was fused to the C-terminus of the targeted gene for protein localization assays. The mitochondrial marker ATP1-RFP was constructed by fusing RFP to the C-terminus of ATP1 (MGG_07752) driven by the RP27 promoter and loading into pTE11 vector harboring HPH resistance. Peroxisomal targeting protein of M. oryzae (MGG_10840)For constructing gene deletion vectors, 1.5-kb HPH and targeted gene probe were prepared respectively for labeling digested genomes of all fungal strains. Complementation assay was performed by introducing corresponding vector DNA into mutant protoplasts and selecting transformants for chlorimuron-ethyl resistance from pCB1532. Co-localization assays were conducted by co-transformation of ETF-GFP with ATP-RFP and PTS1-RFP into ETF mutant protoplasts.Fungal protoplasts were prepared as described previouslyLive rice and barley infection assays were performed as described previously. Conidial suspensions were prepared and sprayed on 15-day-old dwarf Indica rice cv. CO-39 and Golden Promise barley using an airbrush. Post spray-inoculation, plants were placed in a sealed chamber and incubated in a controlled environment with an alternation of 12 h- light and darkness and 90% relative humidity at 25 °C. By 48 hpi, the inoculated plants were moved out and continually incubated for 2–3 days for disease symptom development. Wounded infection was conducted by scratching live barley leaves with a toothpick and inoculated with 20 μl conidial droplets. Infection assays for microscopy were conducted on rice sheaths, barley leaf abaxial epidermis and onion bulb epidermis38Mobilization of Lipid bodies was visualized by staining with Bodipy 493/503 (Invitrogen) as described previouslyTEM samples were prepared as describedHost ROS accumulation during infection was detected by staining with DAB as described previouslyhttp://www.cominbio.com/). ATP amount was tested by high performance liquid chromatography (HPLC) according to its absorption peak at 254 nm as describedIn the presence of alkaline copper sulfide, fatty acids generate copper soaps and react with 1,5-diphenylcarbazide producing a cerise color in solution. The solution has a characteristic absorption peak at 550 nm, and the degree of color has a linear relation to the amount of fatty acid. The mycelia sample was prepared and fatty acids extracted and measured by Suzhou Comin Biotechnology Co., Ltd .