The purpose of our study was to utilize a molecular imaging technology based on the retrograde axonal transport mechanism (neurography), to determine if oxaliplatin-induced neurotoxicity affects retrograde axonal transport in an animal model.in vivo fluorescent imaging performed (0–60 min) at baseline, and then weekly for 5 weeks, followed by 2-weekly imaging out to 9 weeks. Tissues were harvested for immunohistochemical analysis.Mice (n = 8/group) were injected with a cumulative dose of 30 mg/kg oxaliplatin (sufficient to induce neurotoxicity) or dextrose control injections. Intramuscular injections of Tetanus Toxin C-fragment (TTc) labeled with Alexa 790 fluorescent dye were done (15 ug/20 uL) in the left calf muscles, and With sham treatment, TTc transport causes fluorescent signal intensity over the thoracic spine to increase from 0 to 60 minutes after injection. On average, fluorescence signal increased 722%+/−117% (Mean+/−SD) from 0 to 60 minutes. Oxaliplatin treated animals had comparable transport at baseline (787%+/−140%), but transport rapidly decreased through the course of the study, falling to 363%+/−88%, 269%+/−96%, 191%+/−58%, 121%+/−39%, 75%+/−21% with each successive week and stabilizing around 57% (+/−15%) at 7 weeks. Statistically significant divergence occurred at approximately 3 weeks . Quantitative immuno-fluorescence histology with a constant cutoff threshold showed reduced TTc in the spinal cord at 7 weeks for treated animals versus controls . There was no significant difference in neural cell mass between the two groups as shown with NeuN staining .in vivo molecular imaging can demonstrate imaging changes in a model of oxaliplatin-induced neuropathy. Impaired retrograde neural transport is suggested to be an important part of the pathophysiology of oxaliplatin-induced neuropathy.We show–for the first time to our knowledge–that neurographic  Oxaliplatin is a platinum-based chemotherapeutic that is widely used in the treatment of colon carcinoma Current modalities of investigation in oxaliplatin-induced neuropathy are the clinical neurologic exam, supplemented by electrophysiologic studies, both of which become abnormal in the majority of patients on oxaliplatin therapy  informative of the basic pathophysiological mechanisms of nerve injury, and b) allows repeated non-invasive assessments allowing the outcomes of interventions to be measured, c) early in the process of nerve injury, while outcomes could perhaps still be altered.There is a great In this paper we address this need by demonstrating the use of a molecular imaging agent based on the retrograde axonal transport mechanism in the setting of oxaliplatin-induced neuropathy. We show that a molecular imaging tracer: a) can demonstrate retrograde neuronal transport impairment in oxaliplatin toxicity, b) using a non-invasive imaging measurement, that can be repeated, allowing longitudinal studies, and c) that imaging changes occur relatively early in the course of the development of neuropathy. Our work suggests impaired retrograde transport to be an important part of the pathophysiology of oxaliplatin-induced neuropathy.This study was approved by our institutional animal care review board. We used two groups of 8 BALB/C mice  for imaging studies: controls and oxaliplatin treatment . An additional two groups of 4 animals each were used for histological studies , euthanized 7 weeks after starting the oxaliplatin regimen. Using a well-characterized model system described in the literature E. coli carrying the expression plasmid pAE-Fc (a kind gift of Dr. Ana L. T. O. Nascimento Recombinant TTc was isolated from −1 cm−2 sr−1)  using bandpass excitation and emission filters for near infrared light  . Animalsr−1) see .Animals were killed for histology at 7 weeks after the start of oxaliplatin treatment. Both control (n = 3) and oxaliplatin (n = 3) treated animals were euthanized 1 hour after the injection of TTc in the calf muscles and the spines with cords were excised and frozen on dried ice. We embedded 1 cm pieces of the complete spinal column ranging from the lower thoracic cord (T12–T13), the complete lumbar area (L1-L6) as well as the higher sacral (S1–S3) area for cryotomy. Serial frozen sections were cut at a thickness of 30 μm using a cryostat. For immunohistochemical detection of TTc and neuron – specific nuclear protein (NeuN), the cryosections were fixed with 4% paraformaldehyde for 5 minutes, sections were blocked in a solution of phosphate-buffered saline (PBS), 1% bovine serum albumin, and 0.03% Triton X-100. The primary detection was done with a polyclonal rabbit anti-TTc antibody  in PBS for 1 hour at room temperature while the secondary immuno reaction included a simultaneous reaction of anti-NeuN  and a secondary goat anti-rabbit Alexa Fluor 546–labeled antibody  at room temperature for 1hour. For detection of fluorescence, sections were mounted in Prolong Gold anti-fade reagent with DAPI . Micrographs were imaged under identical conditions using a10x UPLAPO objective with an inverted Olympus FV1000 scanning confocal microscope . In this study, we used a FV5-LD405 laser diode (Olympus) and both the argon ion 488 nm and green HeNe 543.5 nm lasers  and suitable filter sets (DM405/488/543 nm).We studied 20 cryo-sections per animal and used the Spinal Cord Atlas http://www.r-project.org) version 2.12.0 with package “nlme” was used for statistical analysis. The model accounts for correlation among within animal measurements over time, as well as the quadratic trend over time in mean TTc uptake as a function of week.Student’s paired T-test was done to assess the statistical significance of animal weights, fluorescent histology pixel counts and for simple comparisons of fluorescent values using a spreadsheet . Fluorescent uptake curves over the thoracolumbar spine for oxaliplatin-treated and control animals were evaluated formally by analyzing the observed change from 0 minutes to 60 minutes post TTc injection in fluorescent signal intensity using a linear mixed-effects regression model. The statistical software R  compared to 0.66 g for controls (from baselines of 22.57 g and 23.49 g). By the end of week 5, weights of most animals had again reached pre-injection values. No other phenotypic changes were observed in the animals.The properties of labeled TTc were compared to unaltered TTc  by Western blot and cell uptake studies using PC-12 pheochromocytoma cells  and found to be identical, as shown in prior work by our group Using the methodology demonstrated in The pattern of imaging agent migration was initially similar between the treated and control animals, but rapidly became different . As the Quantitative analysis of Regions of Interest (ROIs) placed over the spinal cord confirmed these findings, as demonstrated by wireplots of the uptake curves , and in In oxaliplatin treated animals, however, there is a rapid loss of uptake from the injection site to the cord, starting from our first imaging time point at 1 week  with fluorescence at 60 minutes has decreased to 4-fold the baseline value. The uptake continues to fall, reaching 2-fold by 3 weeks into the study and stays between 1.5–2 fold for the rest of the study out to 9 weeks , Table 1Control animals also start out at an 8-fold increase of fluorescence over the thoracic spine at 60 minutes after injection. After a brief increase to 8.8-fold at 1 week, the values for control animals gradually decrease to slightly above 4-fold for the rest of the study , Table 1Statistical analysis is given with simple T-tests in the rightmost columns of Analysis by means of a linear mixed statistical model  confirmsAn interesting aside was the observation of a significant trend (p = 0.013) to increase in fluorescence values at 0 minutes in oxaliplatin treated animals  and can be seen in the decreasing slope of values at 60 minutes for both the oxaliplatin and control groups in the wireplot , with oxThe summary statistics for the fixed regression coefficient parameters of interest (in millions) are: intercept 64.4+/−5.22 (estimate +/− standard error), week −6.47+/−1.24, week squared 0.42+/−0.12, oxaliplatin −15.66+/−7.07 and oxaliplatin times week −1.69+/−0.74.th thoracical (T12) to the 3rd sacral-S3 vertebrae illustrate similar histological findings  neurotoxicity impairs neuronal transport, with statistically significant divergence occurring between treated animals and controls by 3 weeks, and b) that retrograde transport impairment appears to be irreversible up to the 9 weeks duration of our study. Our findings are not due to neuronal cell death, as NeuN staining of the cords showed equivalent neuronal cell mass in both groups, with reduced transport of TTc only noted in the Oxaliplatin treated group.We have previously shown that TTc can be used as neurographic imaging agent Tetanus toxin has been inferred, and its effects described from the times of the ancient Egyptians, but it was first isolated in 1967 All platinum based chemotherapeutics can induce sensory neuropathy in cancer patients, with oxaliplatin being especially well known for cold induced allodynia Imaging findings diverge between oxaliplatin treated and control animals almost from the beginning  and 3 wiFuture work will be directed at investigating the very early events in neural transport, and correlating these to behavioral outcomes.The phenomenon of subjective clinical improvement in the face of persistent neuropathy by objective measures (such as electromyographic studies) is well known, and likely reflects neuronal adaptation mechanisms to compensate for a loss of function. Such compensation can occur at brain TTc uptake and transport of control animals did show some changes over time also, but to a much lesser degree than observed in oxaliplatin treated animals . It is pElevations in baseline fluorescence  were observed over the course of the study in oxaliplatin treated animals  to treat or prevent neuropathy. Indeed, prior work has shown some attenuation of neuropathic effects by the administration of nerve growth factors However this linkage may be complex, and other hypotheses, such as selective mitochondrial dysfunction in certain nerve types should also be considered Primary abnormalities of axonal transport may be the underlying mechanism of oxaliplatin-induced neuropathy (and possibly other neuropathies). Molecular imaging offers to make this a testable hypothesis, and offers a measurable imaging parameter that can be targeted by interventions. The success of these interventions can be measured early in the course of the neuropathy and correlated to outcomes. These are the elements needed to develop a cure or preventative strategy for a disease in dire need of such interventions.Our current fluorescent imaging technique could be used today as an intraoperative technique to mark and avoid nerves at risk for injury , but limited photon penetration through tissue makes applications beyond the intra-operative setting difficult with fluorescent imaging. Other imaging modalities will be needed to take this technique beyond the operating room. With additional imaging modalities such as MR, however, neurographic molecular imaging could have wide general applicability as there are many currently incurable nerve conditions  that could benefit from improved diagnosis and assessment of retrograde neuronal transport by means of imaging. It should be noted that recent major developments have been made in using fluorescent imaging (as yet in non-neurographic applications) in humans in the intra-operative setting in vivo molecular imaging tracer in the diagnosis and assessment of oxaliplatin-induced neuropathy, a frequently encountered problem in cancer patients. Our work suggests that molecular imaging is able to provide interesting mechanistic insights into the pathophysiology of neuropathy, specifically suggesting that impaired retrograde axonal transport, perhaps of growth factors, is an important part of this disease.In conclusion, we demonstrate the usefulness of an


Patients then received erlotinib with initial doses based on smoking status. Doses were increased in 75 mg increments every two weeks depending on toxicities until each patient's maximal tolerable dose (MTD) was achieved. Cyclin D1 and D3 biomarkers were measured by immunohistochemistry. The objectives of the study were to evaluate time to progression (TTP) and overall survival (OS) for the entire population and biomarker subgroups. Forty-five patients were enrolled. Intra-patient erlotinib MTD ranged from 0 to 525 mg. Median MTD achieved in smokers was higher than in non-smokers . TTP for the entire cohort was not significantly improved compared to historical controls. Patients with high cyclin D1 expressing tumors demonstrated improved TTP on erlotinib  and improved OS . Intratumoral cyclin D3 expression did not impact clinical outcomes. Current smokers but not former smokers exhibit a higher erlotinib MTD. High cyclin D1 expression was associated with favorable TTP and OS.This phase II study investigated dose-intense erlotinib maintenance after dose-dense chemotherapy for patients with metastatic non-small cell lung cancer and examined two cell cycle biomarkers. Patients with newly diagnosed metastatic non-small cell lung cancer received docetaxel 75 mg/m Dose-dense docetaxel and cisplatin was previously investigated as a first line treatment for patients with metastatic non-small cell lung cancer . The toxErlotinib improves the overall survival of patients with metastatic non-small cell lung cancer in the maintenance, second line and third line settings ,3. The iA phase I/II study investigated the maximal tolerable dose (MTD) of erlotinib in patients who were smoking ≥10 cigarettes daily . This stMolecular genetic properties of individual cancers such as epidermal growth factor receptor (EGFR) mutations impact the likelihood of achieving clinical benefit with treatment. Progression-free survival is dramatically increased in patients with certain EGFR activating mutations ,7–9. PatIn vitro studies have shown that cyclin D3 is not repressed by erlotinib treatment and that high cyclin D3 expression is associated with erlotinib resistance  of 0 or 1 was required. Patients were ineligible if they had received prior chemotherapy, had inadequate organ function, were pregnant or breast feeding, or were currently receiving radiation therapy.ClinicalTrials.gov (NCT00723138). After obtaining written informed consent, patients were treated with cisplatin 75 mg/m2 and docetaxel 75 mg/m2 with both drugs given intravenously on day 1 every two weeks. Prophylactic anti-emetics were given based on investigator's preference and generally included fosaprepitant or aprepitant as well as 5-HT3 antangonists. Prophylactic growth factor support with pegfilgrastim was administered day 2 of every cycle. Treatment was repeated for four cycles or until unacceptable toxicity or disease progression.This study was approved by the Institutional Review Board of Wake Forest University and was registered with Erlotinib was started immediately after completion or discontinuation of chemotherapy for all patients regardless of response or progression on chemotherapy. The starting doses of erlotinib were 300 mg daily for patients who were smoking at ≥10 cigarettes per day and 150 mg daily for all other patients. Doses of erlotinib were increased in 75 mg increments every two weeks until patients developed either grade 2 or 3 toxicities . In the event of grade 3 toxicities, erlotinib was held until resolution to grade 1 and then the dose was reduced by 75 mg daily. If the reduced dose was tolerated with grade 2 or less toxicity, that was determined to be the MTD for that patient. In the event of grade 2 toxicities, medical interventions were added and erlotinib was continued at that dose which was determined to be the MTD for that patient.At the time of enrollment, all patients completed a detailed smoking history questionnaire. Patients were categorized as never smokers (<100 lifetime cigarettes), distant former (>1 year since cessation), former (1 year - 1 month since cessation), recent former (<1 month since cessation), and current smokers. Physical examination and standard laboratory tests were performed prior to each cycle of chemotherapy. Complete blood counts were performed weekly during dose-dense chemotherapy. After initiation of erlotinib, physical examination and laboratory tests were performed every two weeks until the patient's erlotinib MTD was established and then every four weeks. Tumor measurements were performed prior to initiation of treatment, after completion of dose-dense chemotherapy and then every eight weeks until disease progression or unacceptable toxicity. Tumor response was assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) .Formalin-fixed paraffin-embedded biopsy specimens that had been obtained prior to enrollment were analyzed after the completion of the study by a pathologist who was unaware of clinical outcomes (Jennifer Laudadio). Antigen retrieval was performed using the Leica antigen retrieval system according to the manufacturer's protocol for 20 min prior to applying antibodies . Cyclin D1 primary antibody  at a dilution of 1:50 and cyclin D3 primary antibody  at a dilution of 1:20 were independently applied to prepared slides. The percent of cancer cells staining positive for each cyclin was determined. Tumors were then categorized as having high or low expression based on whether the expression was above or below the median percent staining result for each marker.Genomic DNA was extracted from paraffin-embedded biopsy tissues using the DNEasy Tissue kit  according to the manufacturer's protocol. DNA concentrations were measured using spectroscopy. Polymerase chain reaction (PCR) assays were performed using the EGFR PCR Kit Using Scorpions and Amplification Refractory Mutation System (Qiagen) to assess for 28 activating mutations in the EGFR gene. Activating mutations identified with this method were confirmed and tested for the T790M resistance mutation by a second independent analysis using Rotor Gene analysis with the EGFR RGQ PCR kit (Qiagen).2 test except for age which was compared using t-test. OS and TTP were assessed using the Kaplan-Meier method. Comparisons of survival curves were performed using the log-rank test. Cox proportional hazards models were used to assess differences between biomarker expression groups after controlling for chemotherapy response, age and PS. All P-values shown are two-sided.A sample size of 45 evaluable patients was selected to provide 85% power to detect an improvement in time to progression (TTP) by 2.5 months over the historical control of 4 months  using a Forty-five patients were enrolled from August, 2007 to February, 2011. The patient characteristics are displayed in The toxicities during dose-dense chemotherapy and dose-intense erlotinib are displayed in Toxicities during dose-intense erlotinib were primarily rash and diarrhea. However, other toxicities including anorexia, dehydration and fatigue were dose limiting in 14% of patients. No patient on protocol developed grade 5 toxicity.The median erlotinib MTDs for lifelong non-smokers, former smokers, and current smokers were 150, 187.5 and 300 mg daily. The MTD for current smokers was significantly higher as compared to lifelong non-smokers (P=0.019) and former smokers (P<0.001). The MTD for former smokers was not significantly different as compared to lifelong non-smokers (P=0.51). Thirty-four patients had adequate tissue for immunohistochemical biomarker and EGFR mutation studies. Cyclin D1 expression ranged from 1 to 95% with a median of 33%. Cyclin D3 expression ranged from 5 to 85% with a median of 28%.Samples from three patients were initially identified as potentially harboring EGFR mutations. A subsequent analysis confirmed that two of these three patients harbored EGFR activating mutations (one del 19 and one L858R). Neither of these co-expressed the T790M resistance mutation. The biomarker and clinical outcomes for these patients are shown in Radiographic responses following dose-dense chemotherapy were partial response (20%), stable disease (63%) and progressive disease (18%). For the entire study population, the median time to progression was 4.6 months  which was not significantly improved compared to the historical control of 4 months. The median overall survival for the entire study population was 9.5 months .As compared to low cyclin D1 expression, high cyclin D1 expression was associated with longer TTP on erlotinib  and improved OS  as shown in Cox proportional hazards models were used to control for the potential confounding effects of chemotherapy response, age and PS on clinical outcomes. After controlling for these variables, high cyclin D1 continued to predict improvements in TTP on erlotinib  and OS . After controlling for these variables, the effect of cyclin D3 continued to be non-significant for TTP on erlotinib (P=0.71) and OS (P=0.94).Clinical characteristics of patients with high cyclin D1 and low cyclin D1 expressing cancers are compared in In this study, dose-dense chemotherapy was associated with high degree of treatment related toxicities and a response rate lower than that observed in a prior multi-institutional study . This liIn some situations, erlotinib resistance may be related to achieving inadequate drug levels. Intensification of erlotinib dose in the maintenance setting is an attractive approach to prevent this form of resistance. Our findings confirm prior reports indicating that the MTD of erlotinib is higher in patients who continue to smoke. A randomized phase III trial is ongoing to test whether high dose erlotinib treatment will improve the clinical outcomes over standard dose erlotinib for patients who continue to smoke .In the present study, the MTD for former smokers was also examined. The median MTD for former smokers was significantly lower than for current smokers and was not elevated compared to lifelong non-smokers. These findings indicate that the increase in erlotinib metabolism triggered by tobacco exposure is reversible following smoking cessation. Providing smoking cessation interventions to patients after diagnosis of lung cancer improves clinical outcomes and reducing the risk of inadequate drug levels may help to explain this effect ,20.EGFR mutation analysis is a valuable test for identifying highly sensitive tumors in patients who will benefit from first line erlotinib instead of chemotherapy. Consistent with this, the patient in the present study with the longest TTP and OS demonstrated an EGFR activating mutation as well as high cyclin D1 . Some pain vitro models as well as pre- and post-treatment cancer biopsies (Cyclin D1 was identified as a biomarker by studying the mechanism of action of erlotinib using biopsies . Cyclin


Muscle biopsies were obtained from the vastus lateralis muscle at rest, immediately and 3 h after exercise. The participants then underwent 10-d of cycle training which included four high-intensity interval training sessions  and six prolonged moderate-intensity sessions . Participants repeated the pre-training exercise trial at the same absolute work load (64% of pre-training VO2peak). Muscle PGC1-α mRNA expression was attenuated as it increased by 11- and 4- fold (P<0.001) after exercise pre- and post-training, respectively. PGC1-α protein expression increased 1.5 fold (P<0.05) in response to exercise pre-training with no further increases after the post-training exercise bout. RIP140 protein abundance was responsive to acute exercise only (P<0.01). COXIV mRNA  and COXIV protein expression  were increased by training but COXIV protein expression was decreased  by acute exercise pre- and post-training. These findings demonstrate that short-term intensified training promotes increased mitochondrial gene expression and protein abundance. Furthermore, acute indicators of exercise-induced mitochondrial adaptation appear to be blunted in response to exercise at the same absolute intensity following short-term training.Reduced activation of exercise responsive signalling pathways have been reported in response to acute exercise after training; however little is known about the adaptive responses of the mitochondria. Accordingly, we investigated changes in mitochondrial gene expression and protein abundance in response to the same acute exercise before and after 10-d of intensive cycle training. Nine untrained, healthy participants (mean±SD; VO Acute and chronic exercise models have been used to investigate mitochondrial adaptations in skeletal muscle. Traditional studies investigating mitochondrial adaptations were conducted utilising prolonged endurance training protocols in rodents The mitochondrial biogenesis transcriptional programme in skeletal muscle results from the coordinated activation of signalling pathways, transcription co-activators, transcription factors, and repressors initiated in response to muscle contraction Given that skeletal muscle energy flux during contraction is effected by exercise training status, it is not surprising that signal transduction cascades are differentially regulated by acute exercise before and after training At present, there remains a significant gap in the literature on the molecular events (gene expression and changes in protein abundance) that occur in the process of mitochondrial biogenesis and reticulum expansion in human skeletal muscle in response to acute exercise before and after intensified endurance exercise training. The purposes of this study were to firstly examine changes in expression of mitochondrial genes that are involved in the regulation of mitochondrial biogenesis  under exercise conditions that maximally highlighted training adaptions (exercise at the same absolute intensity). Secondly, to investigate changes in protein abundance of mitochondrial biogenesis regulators and mitochondrial function including PGC-1α, RIP140 and the Electron Transport System (ETS) proteins. We hypothesized that with exercise training there would be an attenuation of the increase in key mitochondrial genes expressed in response to the same absolute bout of exercise before compared with after training. Additionally, we hypothesized that there will be an increase in mitochondrial protein abundance after training with no effects of acute exercise both before and after training.The study protocols were approved by the Monash University Standing Committee on Ethics in Research Involving Humans and the Karolinska Institutet Human Research Ethics Committee, and conducted according to the principles expressed in the Declaration of Helsinki. All participants were fully informed of the possible risks involved in the study before providing written informed consent.2peak 3.5±1.8 l/min). The data from this study forms part of a larger study investigating the molecular adaptations to short-term intensified cycle training in humans and have previously been reported Nine healthy untrained male volunteers were recruited for the short-term training study  vastus lateralis. Following the initial biopsy, subjects rode at 71±8% and 64±9% VO2 peak (164±18 W) for 60 min pre- and post-training respectively. The second muscle biopsy was taken immediately (<20 sec) after the completion of the ride. Subjects then rested in a supine position, and were instructed to keep as still as possible for the next 3 h after which the final biopsy was taken from the most proximal incision. Drinking water was provided ad libitum to subjects to consume over the duration of the trial but matched for post training trial. Muscle biopsies were immediately frozen in liquid nitrogen and stored at −80°C until analysis. All metabolic measures, muscle glycogen and selected cell signaling data for these subjects have been previously reported Subjects reported to the laboratory between 0600 or 0700 hours after an overnight fast (∼9 h). Upon arrival, subjects voided and body weight was measured. After 15 min of supine rest the first of three muscle biopsies (120–250 mg each) was obtained, after administration of local anaesthesia, the remaining biopsies were obtained from separate incision sites over the All antibodies were purchased from commercial suppliers. The antibodies use were against total NADH-ubiquinol oxidoreductase (NUO-[complex (C)-I]; Molecular Probes A21344; 1∶5000.) ubiquinol cytochrome c oxidoreductase , Succinate Ubiqinone Oxyreductase ; Cytochrome C Oxidase I  and Cytochrome C Oxidase IV , PGC1α , nuclear co-repressor receptor interfering protein 140 kDa  and GAPDH . All other reagents were of analytical grade , unless otherwise specified.Total RNA was extracted from 30–40 mg of muscle as previously described Reverse transcription was performed on DNase-treated RNA using AMV Reverse Transcriptase  as previously described Primer sequences were obtained from gene sequences from GenBank and are shown in Muscle protein extracts were prepared from freeze-dried muscle and the protein concentration of the resulting supernatant was determined using a commercial kit  as previously described Data is presented as mean ±SD for nine subjects unless otherwise stated. Statistical analysis of gene expression and protein abundance were conducted using two way repeated measures analysis of variance . When significant interactions were observed a LSD Post-hoc tests were conducted to determine where specific differences occurred. The results were considered significant when p<0.05. Analysis was conducted using the statistical package IBM SPSS Statistics 20.0 for Windows .Acute exercise and exercise training altered the gene expression of the PGC1 co-factor family of genes . PGC1 α Transcription factor Tfam mRNA trended to increase by 10% (1.1±0.3 fold) in response to exercise pre-training but remained unchanged in response to exercise after training  similarly before and after training  and remained elevated at this level at 3 h post exercise and before and throughout the post training trial  in COXIV abundance has been demonstrated after a single HIIT session in untrained humans The main limitation of this study was that we were unable to empirically determine mitochondrial biogenesis by protein synthesis In conclusion our study provides evidence that 10-d of intensified cycle training reduced PGC-1α mRNA expression, suggestive of reduced mitochondrial biogenesis processes and adaptation in responses to the same (absolute work done) acute bout of endurance exercise in human skeletal muscle. This alteration in molecular programming is potentially due to reduction of relative exercise intensity, and suggests at a molecular level that maintaining relative exercise intensity is important for continued mitochondrial adaptation after as little as 10 consecutive exercise sessions. Additionally, we provide evidence that exercise acutely reduces the protein abundance of COXI and COXIV before and after training, which may impair post exercise mitochondrial function and explain marked and rapid training induced changes in both COXIV mRNA expression and protein abundance. The practical implications of these findings are that we provide evidence that suggests that maintaining absolute exercise intensity even after a short period of endurance training results in a blunting of the molecular programs for mitochondrial reticulum expansion in human skeletal muscle, and may have implications for health and exercise performance.


Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus reported to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS). While the association of XMRV with CFS and PC has recently been discredited, no studies have been performed in Australian patients to investigate the association between PC and XMRV or related murine leukemia virus (MLV) in matched PC and normal tissue.Genomic DNA (gDNA) was purified from matched normal and cancer formalin-fixed paraffin-embedded (FFPE) prostate tissue from 35 Australian PC patients with Gleason scores ranging from 7 – 10. The presence of the ribonuclease L (RNase L) polymorphism R462Q was determined by allele specific PCR. Samples were screened for XMRV and related murine leukemia virus (MLV) variants by qPCR. Contaminating mouse DNA was detected using qPCR targeting mouse intracisternal A particle long terminal repeat DNA.gDNA was successfully purified from 94% (66/70) of normal and cancer FFPE prostate tissues. RNase L typing revealed 8% were homozygous (QQ), 60% were heterozygous (RQ) and 32% were wild-type (RR) for the RNase L mutation. None of the 66 samples tested were positive for XMRV or related MLV sequences using broad MLV or XMRV specific primers with detection sensitivities of 1 viral copy of MLV/XMRV and XMRV DNA, respectively.Using highly sensitive qPCR we found no evidence of XMRV or related gammaretroviruses in prostate tissues from 35 Australian PC patients. Our findings are consistent with other studies demonstrating that XMRV is a laboratory contaminant that has no role in the aetiology of PC. RNASEL gene (R462Q) was associated with familial PC [in situ hybridization (FISH) and immunohistochemistry assays were observed in either malignant epithelium [Prostate cancer (PC) is one of the most commonly diagnosed cancers in men resulting in approximately 3,300 deaths in Australia per year. While the aetiology of PC remains poorly understood, infection and associated inflammation are risk factors in PC development . The rat gene R46Q was ass gene R46Q was assilial PC . XMRV wa gene R46Q was assGammaretroviruses comprise a group within the larger retrovirus family that reverse-transcribe viral RNA to a cDNA during replication that is inserted into the host cell chromosome. As the name indicates, XMRV is highly related to murine leukemia virus (MLV) sharing 96% sequence identity . In addiIn addition to the original report by Urisman and colleagues 2006) several other groups demonstrated an association of XMRV with PC using nucleic acid detection and immune based assays -5. Howev06 severaDue to public health consequences of potential infection with XMRV or MLVs in humans, we considered it is important to refute or confirm their association with PC in an Australian context. This study describes the first evaluation in Australian PC patients for the presence of XMRV and related gammaretroviruses in matched normal and PC tissue and the elucidation of the RNASEL genotype in Australian PC patients. In addition, patient samples were tested for the presence of mouse DNA contamination using a sensitive quantitative PCR (qPCR) assay that detects mouse intracisternal A-type particle (IAP) sequences. We failed to detect XMRV or related MLVs in Australian PC patients confirming that these gammaretroviruses are highly unlikely to be involved in PC.The patient population comprised individuals with higher-grade PC as determined by Gleason scores . Consistent with PC predominately presenting in older men, the median age of patients was 64 (range 49 – 78). The RNase L genotype was determined using purified DNA recovered from 70 formalin-fixed paraffin-embedded (FFPE) normal and cancer tissue from each of 35 patients. The RNase L genotype of both samples from each patient was 100% concordant and 8% of the patients were homozygous (QQ), 60% were heterozygous (RQ) and 32% were wild-type (RR).7 copies per reaction .DNA derived from FFPE samples is expected to be fragmented due to the fixation process and may contain substances that are inhibitory for PCR amplification. Therefore, to evaluate the suitability of genomic DNA (gDNA) derived from FFPE tissue for qPCR, we determined whether we could amplify a region of the single copy human vesicle-associated membrane protein 2 (HuVAMP2) gene. The size of the amplimer (78 bp) was designed to be similar to the expected amplimer sizes for qPCR detection of XMRV and MLV-related viruses Table . The linn Figure . All sam7 and 1 to 106 copies per reaction, respectively  primers targeting the conserved reverse transcriptase regions of XMRV and Moloney murine leukemia virus (MoMLV)Figure . The lin Figures  and 2C. n Figure . Taken tWe performed qPCR using BMLV primers on gDNA purified from 33 normal prostate and 33 PC FFPE tissue. DNA was subjected to three independent assays except for 10/33 cancer and 16/33 normal tissues where only one qPCR assay was performed due to limited amounts of gDNA. All patient samples were negative using the BMLV primers Table . We alsoTo determine whether patient samples were contaminated with mouse DNA we performed qPCR with primers that detect the mouse IAP retrotransposon ,29. The We assessed mouse DNA contamination in gDNA from FFPE patient samples in qPCR IAP assays and found that 5/33 and 10/33 of normal and cancer tissues, respectively were positive for mouse IAP Table . The levUsing highly sensitive qPCR neither XMRV nor related MLV were detected in any of the prostate tissue samples in our Australian cohort. The BMLV qPCR assay used in this study reproducibly detected as little as one copy of XMRV or MoMLV DNA in a total of one μg of carrier nucleic acid. While several studies have reportedly used primers that simultaneously detect XMRV and related MLV in PC samples ,13,18,31We obtained paired normal and cancer tissue from the same patient to determine whether there was a difference in prevalence of XMRV or related MLV in these tissues to provide clues to their role in PC pathogenesis. This is in contrast to previous studies that have used 10 μm patient tissue slices that are likely to be a mixture of both cancer and normal tissue . Our strgag-leader deletion was challenged with the finding that these primers were able to amplify endogenous MLV sequences present in the gDNA of 12 different mouse strains [bone fide human infection was debunked by Paprotka and colleagues who showed that XMRV was a laboratory virus generated by a rare recombination event between two mouse endogenous retroviruses during passage of the CWR22 PC xenograft in nude mice from which the 22Rv1 cell line was derived [Previous studies have raised serious doubts regarding the role of XMRV in PC and CFS. The first studies to cast doubt on this association demonstrated that PCR reagents and nucleic acid purification columns were contaminated with mouse DNA that harbours MLVs detected by highly sensitive PCR -24. In a strains . These s strains ,28. Howe strains ,26. Furt derived .We used a previously published IAP qPCR assay , to deteWhile mouse DNA contamination was detected in seven cancer samples in the IAP qPCR assay, no signal was observed in normal samples from the corresponding patients. Due to the limited amount of gDNA we were unable to test normal prostate tissue from one of these seven patients. In contrast, both normal and cancer samples from three patients were positive for mouse DNA contamination. The greater number of mouse DNA positive samples in cancer compared to normal tissue is unlikely due to increased processing of the former samples because the samples were all handled a similar number of times. In addition, it is unlikely that PCR reagents used in this study were contaminated with mouse DNA as none of the no template controls (12 wells per plate) or DU145 and LNCaP gDNA negative controls (3 wells of each per plate) were positive for XMRV or related MLV in our qPCR assays. Furthermore, we avoided using a Taq polymerase that relies on a monoclonal antibody to achieve a “hot start” as this has previously been implicated as a source of mouse DNA contamination ,23. The One potential limitation of our study is that we used naked plasmid DNA for determining the analytical range of the qPCR assays used in this study while the samples were formalin-fixed DNA from tissue. This may have led to possible overestimation of the sensitivity of the assays. Regardless, our findings are consistent with previous studies demonstrating no association between XMRV and related gammaretroviruses in prostate cancer patients undertaken in regions geographically distinct from Australia.RNASEL genotype of patients in our cohort was to establish if there was an association with the homozygous (QQ) RNase L variant and XMRV or related MLVs. The RNase L enzyme is an interferon-induced ribonuclease, which has antiviral activity and can also induce apoptosis [RNASEL mutation and infection with these viruses could not be investigated. Regardless, we successfully determined the RNASEL genotype for all DNA samples purified from FFPE cancer and normal prostate tissue using a previously published allele specific PCR assay [RNASEL genotype of Australian PC patients has been determined. The overall allele distribution in our small cohort appears to be similar to non-hereditary PC cases observed in the USA where the heterozygous (RQ) allele has the greatest prevalence (~47%) and the homozygous (QQ) allele the lowest prevalence (9.9 – 15.2%) [RNASEL allele distribution in Australian PC patients are distinct to normal men in the same geographical region, if there are racial differences, and whether there is an association of this allele with disease severity.The original premise for determining the poptosis ,33. Men poptosis . Given tpoptosis . Since nCR assay . The genvalence ~7% and th– 15.2%) . FurtherThe Blood XMRV Scientific Working Group’s findings, along with the discovery that XMRV is a virus generated by a rare recombination event in the laboratory has provided irrefutable evidence that XMRV or related MLV are not associated with CFS ,36,37. TAdded in proof: Following submission of our study a report by Lee and colleagues  demonstrProstate samples used in this study were archival FFPE tissue obtained from patients in the greater Melbourne area who had radical prostatectomies performed with tissues submitted to TissuPath Specialist Pathology  for diagnostic pathology between 2007 and 2011. For this study, the samples were prepared by TissuPath scientists from normal and cancer affected regions of the prostate, guided by the associated haematoxylin and eosin (H&E) stained tissue sections which had been evaluated by light microscopy. Samples were received as 2–3 FFPE punch biopsies of 2 mm × 2 mm (diameter × depth). Specimens, coded to mask whether they were normal or cancer tissue and to maintain patient confidentiality, were provided to investigators at the Burnet Institute. Samples were unblinded following completion of the assays at which time the age and Gleason scores for each patient was provided. The study was approved by the Alfred Health Human Ethics Committee (Project Number 32/11).The human prostate carcinoma cell line 22Rv1  and the The plasmid, pVP62, encodes the full length molecular clone of XMRV inserted in the mammalian expression vector pcDNA3.1(−) and was obtained from the NIH AIDS Research & Reference Reagent Program . pHuVAMPTo avoid cross contamination each tissue sample was obtained using a separate sterile 2 mm punch biopsy. To minimize the exposure of patient samples to potential sources of mouse, XMRV and MLV DNA, standard laboratory procedures for sterile DNA extraction were practiced when handling and processing specimens for purification of gDNA and PCR. These measures included the use of sterile UV irradiated microcentrifuge tubes, filter-barrier pipette tips and dedicated micropipettes. All tissue processing was performed in a biosafety class II cabinet and equipment was exposed to UV light for 15–20 min prior to use.gDNA was purified from FFPE patient tissue using the QIAamp® DNA FFPE Tissue Kit and the QIAGEN Deparafinisation solution . Since the QIAamp kit recommends extracting gDNA from tissue sections of 10 μm in thickness, we introduced modifications to optimise DNA recovery from the thicker core biopsies used in this study. These modifications included trimming excess paraffin from the core biopsies, dissecting the tissues into a maximum of five pieces prior to the addition of DPS, and an overnight incubation of tissues with proteinase K. gDNA was extracted from a total of 70 prostate tissue punch biopsies. gDNA was purified from DU145, LNCaP, MRC-5, 22Rv1 cells and PBMCs using the DNeasy Blood and Tissue Kit (QIAGEN) according to manufacturer’s instructions.The RNase L R462Q polymorphism in patient samples was determined using the ARMS assay as previously published . ARMS is7 to 100 copies in the presence of 1 μg Saccharomyces cerevisiae transfer RNA (tRNA) carrier nucleic acid per reaction (Sigma-Aldrich). PCR was performed using 10 μl of diluted pHuVAMP2 containing the required plasmid copy numbers or 1 μg of sample gDNA in a final volume of 25 μl. Threshold cycle (Ct) values that were not within the average Ct ± SD (8.9×105 ± 5.4×105 copies/μg) for gDNA from 22Rv1, LNCaP, DU145 and human PBMCs were considered unsuitable for further analysis by qPCR.To verify the quality of gDNA purified from FFPE tissue and to rule out the presence of PCR inhibitors, patient samples were subjected to a qPCR assay targeting the human VAMP2 gene as described previously . Quantitpol  and the TaqMan probe (BMLV-Probe) targeting conserved regions in the reverse transcriptase region of XMRV and MoMLV l Figure   from the average Ct of the lowest standard tested  were considered negative. Samples where a signal was detected within the linear range of the assay and in the majority of independent assays performed, were considered positive.XMRV: Xenotropic murine leukemia virus-related virus XMRV; PC: Prostate cancer; CFS: Chronic fatigue syndrome; gDNA: Genomic DNA; FFPE: Formalin-fixed paraffin-embedded; RNase L: Ribonuclease L; MLV: Murine leukemia virus; qPCR: Quantitative PCR; X-MLV: Xenotropic-MLV; IAP: Intracisternal A-type particle; PBMCs: Peripheral blood mononuclear cells; VAMP2: Human vesicle-associated membrane protein 2; DPS: Deparafinisation solution; ARMS: Amplification refractory mutation system; Ct: Threshold cycle; tRNA: Transfer RNA; SD: Standard deviation; MoMLV: Moloney murine leukemia virus; BMLV: Broad MLV.The authors declare that they have no competing interests.The project was conceived and funding obtained by GT. Project protocol and ethics was prepared by GT and ACH. Tissue was supplied by JP and JM. Experiments were performed by SDR. Data analysis was performed by SDR and ACH. Statistical analysis was performed by GT. The manuscript was drafted by GT and SDR and all authors were involved in critical revision of the manuscript and approved it for submission.


With the completion of genome sequences belonging to some of the major crop plants, new challenges arise to utilize this data for crop improvement and increased food security. The field of genetical genomics has the potential to identify genes displaying heritable differential expression associated to important phenotypic traits. Here we describe the identification of expression QTLs (eQTLs) in two different potato tissues of a segregating potato population and query the potato genome sequence to differentiate between cis- and trans-acting eQTLs in relation to gene subfunctionalization.Leaf and tuber samples were analysed and screened for the presence of conserved and tissue dependent eQTLs. Expression QTLs present in both tissues are predominantly cis-acting whilst for tissue specific QTLs, the percentage of trans-acting QTLs increases. Tissue dependent eQTLs were assigned to functional classes and visualized in metabolic pathways. We identified a potential regulatory network on chromosome 10 involving genes crucial for maintaining circadian rhythms and controlling clock output genes. In addition, we show that the type of genetic material screened and sampling strategy applied, can have a high impact on the output of genetical genomics studies.Identification of tissue dependent regulatory networks based on mapped differential expression not only gives us insight in tissue dependent gene subfunctionalization but brings new insights into key biological processes and delivers targets for future haplotyping and genetic marker development. The field of associative genomics or genetical genomics attempts to combine the heritability of generated ~ omics data with phenotypic variation through genetic marker associations . The aimDespite the high potential of genetical genomics studies in plants, the association of differential gene expression or metabolite concentration with the phenotypic variation does not always disclose the responsible genetic factors, due to genetic linkage of additional genes with the genomic region. Identification of causative polymorphisms is important for the development of applicable genetic markers. For genetical genomics experiments, factors like population size, marker density, sampling strategy, environmental factors and screening platform used, greatly influence the outcome and the ability to detect small additive biological effects . ThePotato cultivars are highly heterozygous tetraploid outcrossing plants, which complicates genetic analysis aimed at understanding the molecular mechanisms underlying trait variation. Genetic studies therefore are often performed in diploid genotypes and populations. Precision breeding relies on the identification of causative polymorphisms in genes responsible for trait variation. Once a gene has been assigned a regulatory function for a targeted trait, the allelic diversity of the gene in tetraploid cultivars and wild type accessions can be determined and potentially exploited in marker assisted breeding .The genome sequence of a dihaploid potato clone (DM) has recently been completed  and thisExpression profiles of leaf and tuber RNA extracts were obtained from two independent experiments. For tuber profiling, each sample was assayed twice in a two-colour dye experiment, allowing cross-validation of the observed variation and subsequent filtering based on correlation scores (Methods). The total number of array features exhibiting significant expression that could be used for QTL mapping was higher for leaf  than tuber samples . A wide overlap of expressed genes  was found between both organs, which was somewhat surprising considering their highly contrasting functions in terms of architecture and morphology, environmental exposure and developmental stage. However, the organs harvested consist of a multitude of different cell types thereby reducing the overall transcriptomic complexity and no distinction between basal or high gene expression was made. Similarly, the potato microarray is based on available EST sequences where temporal and spatially restricted transcripts have a lower change of being captured and thus not profiled when using the microarray.QTL analysis was performed based on the R/qtl package that allows high-throughput QTL mapping in an outbreeding species using a single integrated genetic map (Methods). A total of 17,764 QTLs were identified as significant for leaf and tuber data combined, corrected for gene redundancy present on the array where possible (i.e. similar blast scores for independent array features against the potato genome gene prediction sequences v3.2).The distribution of QTLs over the different linkage groups is presented as a sliding window graph using 5 cM stepping size for tuber and leaf  [For the majority of features on the array 12,331; 70.1%), only a single QTL was detected while for 2,503 features (29.9%), two or more QTLs were found for both tissues combined (Table 29M16TH)  involved29M16TH) . For bot31; 70.1%29M16TH) . The numOne of the most exciting outputs from large-scale eQTL studies in species with a full genome sequence available is the ability to distinguish cis- and trans-acting regulation of gene expression, which holds the potential to reveal regulatory networks. With the availability of the potato draft genome sequence we were able to distinguish cis- and trans-acting QTLs for most genes. EST based unigenes representing the features on the array were blasted against the genome scaffolds as described in Methods. Putative chromosome locations could be assigned to the vast majority of genes (92%). Genes that could not be assigned a chromosome location were either similar to multiple regions on the genome, had similarity scores below the threshold, or represented significant allelic diversity.Identified QTLs on the same linkage group as their physical map position are identified as cis-acting while QTLs on different linkage groups are defined as trans-acting. Our results show that almost twice as many cis-eQTLs were identified in comparison to trans-acting eQTLs for leaf and tuber data  in the same genomic region as the eQTL hotspot found for the tuber data on chromosome 5 [El locus on chromosome 5, shows that regulation of gene expression in the tuber persists even after tuber harvest and throughout storage. These observations emphasize the impact of sampling strategies on the outcome of genetical genomics experiments.One explanation for the relative high percentage of cis-eQTLs in potato could be its highly heterozygous nature and therefore possibly a tolerance to higher levels of sequence polymorphisms (SNPs and indels). Local (cis) control of transcriptional regulation may consequently be the preferred mechanism for transcriptional control in heterozygous plant species such as potato. Alternatively, the ability to detect trans-eQTLs with minor phenotypic effects is largely depending on the statistical power of the QTL study. Applying conservative filtering may result in many false negatives biasing detection towards cis-eQTLs as suggested by Petretto et al. . One strmosome 5 . The C ×In addition to the major QTL peak on chromosome 5, further comparison of trans-acting QTL distribution between leaf and tuber data reveals few other overlapping regions of high QTL frequency in comparison to the distribution plots containing all QTLs. Overlapping trans-eQTL hotspots for both tissues can be found on LG 5 and 10 and to a lesser extent on LG 3, 9 and 11.A total of 5,818 overlapping QTLs (spanning the same genomic region) were found in leaf and tuber, with only 401 features (6.9%) exhibiting trans-acting transcriptional control  that under favourable conditions are able to develop tubers acting as sink organs. The genome-wide RNAseq data  demonstrtop right corner).Co-localization of trans-acting eQTLs may indicate a similar mechanism of transcriptional control. We initially looked for over-representation of GO categories in those regions in the genome with a high number of trans-acting eQTLs from either leaf or tuber. On chromosome 10 35-40 cM), we found a disproportionally large number of tuber-specific eQTLs 42.3%) associated with photosystem I, II, and tetrapyrrole metabolism  and PRR9  were identified. Both genes code for integral components of the central clock [LHY and PRR9 genes, suggesting that LHY and/or PRR9 may be involved in regulation of GIGANTEA, FKF1, PRR5 genes. LHY expression is negatively correlated with downstream effectors GI, FKF1 and PRR5, while PRR9 expression is positively correlated  [CCA1 and LHY [LHY) can be identified possibly indicating a different mechanism of clock control. Similar to Arabidopsis, expression patterns of GI and FKF1 in potato follow a diurnal rhythm with peak expression in the afternoon, while LHY expression peaks at dawn in agreement with the observed negative correlation (data not shown). In Arabidopsis, GI and FKF1 form a complex that controls CDF1 stability [CO expression depending on time of day [GI and FKF1 within a population could therefore cause a phase shift or a change in phase amplitude effecting downstream effectors such as CO and FT orthologs. Here, we propose a tentative role of LHY or PPR9 in controlling the clock output by targeting expression of GI, FKF1 and PRR5 . In the genomic region spanning the QTL interval, cis-eQTLs for genes/array features with high homology to al clock -39. Inteal clock  and contANS (CO)  controllANS (CO) . In Arab and LHY ,43. In ttability . CDF1 ine of day . VariatiThe strategy of deductive reasoning as used in the above examples requires a priori knowledge of the pathways and the potential interactors. For a model species such as Arabidopsis there is a wealth of scientific evidence on gene function, expression patterns and protein interactions that can be integrated in genetical genomics studies. Still, many gene functions are not yet resolved. For the majority of other plant species the annotation of biological functions of genes is often based on sequence similarity alone, which can generate various assumptions lacking scientific support. Genetical genomics studies can provide evidence supporting proposed functions of genes through newly found interactions and co-regulation of genes. This approach will lead to better understanding of transcriptional regulation, and may lead to identification of key regulatory genes underlying trait QTLs.Based on sequence homology of the representative unigenes to the recently published potato genome sequence, physical map positions can be attributed to a large portion of the features present on the array. However, due to the heterozygous nature of potato, the sequences of genotypes are often relatively divergent from the published genome sequence, which makes distinguishing between allelic variation, different gene family members or multi-copy genes distributed throughout the genome not straightforward. SNP frequency estimations in potato range from 1 SNP per 29 bp, found in a single phase comparison targeting 6.6 Mb of sequence , to 1 SNIn this paper we describe the identification of expression QTLs (eQTLs) in two different tissues of one of the most important food crops in the world; potato. With the completed potato genome sequence, population-wide differential gene expression can be queried to differentiate between cis- and trans-acting eQTLs. Overlapping QTLs present in both tissues are predominantly cis-acting while for tissue specific QTLs, the percentage of trans-acting QTLs increases. The type of genetic material screened and sampling strategy applied in genetical genomics studies has a high impact on the output of a genetical genomics study. Interesting regulatory networks have been identified on chromosome 10 associated to the photosystem and circadian clock control. Identification of key regulatory genes and networks unique to either source or sink tissues not only gives us insight in tissue dependent gene subfunctionalization but will also greatly enhance the identification of the causative polymorphism(s) underlying important trait QTLs.Ninety-six individuals, including the parental clones, of a diploid backcross population (C × E) were used in this study. This population is derived from an original cross between potato clones C (USW533.7) and E (77.2102.37) and is described in detail in . Tuber s® 11.1. As tuber samples were measured twice, only genes with a Pearson correlation coefficient higher than 0.8 across the 94 individuals between the Cy3 and Cy5 datasets were included resulting in 19,956 features. For calculation of genome distribution in tubers the Cy3 data was used. All raw and normalized data files are available as Additional files All tuber samples were labelled with both Cy3 and Cy5-dye using the Low RNA Input Linear Amplification Kit, PLUS, Two-colour (Agilent technologies) according to the manufacturer's protocol starting with 2 μg of purified total RNA. Leaf samples were labelled with Cy3-dye following the same protocol as the tuber samples but starting amounts were 200 ng of purified total RNA. Hybridization and washing was performed according to the Agilent's two-colour hybridization protocol with the following change: 1 μg of labelled Cy5 and/or Cy3 cRNA was used as input in the hybridization mixture. Slides were scanned on the Agilent DNA Microarray Scanner and data extracted using the feature extraction software package v9.1.3.1) using a standard two-colour protocol. Genes which show consistent low expression were removed and data sets were independently normalized using the quantile normalization procedure (mean) available in Genstat.1.3.1 us® labelling kit (Invitrogen) with a modified dNTPmix  and incubated for 16 h at 37°. Labelled samples were purified using PureLink™ PCR purification System (Invitrogen) and heat fragmented for 30 min resulting in fragments ranging from ~150 to 600 bp. 1 ug of labelled samples (cy3 and cy5) were hybridized at 65° for 17 h in a standard swop-dye experiment using the independently labelled samples. Washing and scanning of the slides was carried out as described for the gene expression experiments. Feature normalization and ratio calculations were carried out using default method available in Agilent Feature extraction software package (v9.1.3.1). Features showing consistent differential expression (> 33%) between both parents are presented in Additional file Genomic DNA hybridizations were performed after labelling 800 ng of genomic DNA from both parents (C and E) in duplo, using the BioPrime® [The genetic map used in all QTL studies was generated using mapping software Joinmap 4.0®  and is a® , includi® . The pot® . To allo®  and resumakeqtl and the fitqtl functions. The QTL interval is computed using LOD interval method. The default analysis performs a QTL mapping using the Haley-Knott regression with a mapping step size of 5 cM. The LOD threshold used to determine the QTLs is calculated using Li & Ji algorithm [We have used the main module of the R/qtl package, and optimized it for high-throughput QTL mapping in outcrossing species such as potato. The R-script checks the ratio of missing values before automatically converting JoinMap segregation scores to R/qtl scores after which the QTL mapping is performed . The genlgorithm . The numhttps://earray.chem.agilent.com/earray/). The number of array features having eQTLs (cis or trans) were counted per GO group and totals are listed. To reduce the number of classes, GO identifiers targeting the same metabolic routes were in some cases merged (Table http://mapman.gabipd.org/web/guest/mapman). Instead of visualizing gene expression data we display the presence of tissue specific eQTLs . In this manner a quick overview can be obtained for each pre-designed metabolic route and genes can be identified that are differentially expressed within a segregating population  assignment for all the potato micro-array features has been described previously . GO information was downloaded through Agilent E-array published designs (n Figure . AdditioSNP: Single nucleotide polymorphism; QTL: Quantitative trait loci; LOD: Logarithm of oddsBK designed the tuber expression study, performed QTL analysis, data interpretation and wrote the manuscript. AMA designed and performed the leaf expression study and carried out hybridizations together with MO. PC optimized the R/qtl program and performed QTL and statistical analysis. GL designed the leaf expression study and helped draft the manuscript. RGFV and CWBB helped design the tuber expression experiment and help draft the manuscript. All authors read and approved the final manuscript.Frequency distribution of eQTLs over the different potato chromosomes.Click here for fileTissue specific and Tissue dependent eQTLs of tuber and leaf tissues belonging to the major carbohydrate metabolism groups.Click here for fileSchematic overview of tuber and leaf eQTL specificity for major CHO metabolism genes.Click here for fileHybridization specificity of genomic DNA from C and E parental clones.Click here for fileTuber Cy3 and Cy5 raw data + genome info partI.Click here for fileTuber Cy3 and Cy5 raw data + genome info partII.Click here for fileTuber Cy3 and Cy5 raw data + genome info partIII.Click here for fileTuber Cy3 and Cy5 qnormalized data + genome info partI.Click here for fileTuber Cy3 and Cy5 qnormalized data + genome info partII.Click here for fileTuber Cy3 and Cy5 qnormalized data + genome info partIII.Click here for fileLeaf Cy3 Leaf raw data + genome info partI.Click here for fileLeaf Cy3 Leaf raw data + genome info partII.Click here for fileLeaf Cy3 qnormalized + genome info partI.Click here for fileLeaf Cy3 qnormalized + genome info partII.Click here for fileMarker to Genome.Click here for file4x44k 015425 POCI annotation.Click here for fileGenotypic scores CxE population.Click here for file


Parrya nudicaulis spanning Alaska. The frequency of white-flowered individuals increases with increasing growing-season temperature, consistent with the role of anthocyanin pigments in stress tolerance. White petals fail to produce the stress responsive flavonoid intermediates in the anthocyanin biosynthetic pathway (ABP), suggesting an early pathway blockage. Petal cDNA sequences did not reveal blockages in any of the eight enzyme-coding genes in white-flowered individuals, nor any color differentiating SNPs. A qRT-PCR analysis of white petals identified a 24-fold reduction in chalcone synthase (CHS) at the threshold of the ABP, but no change in CHS expression in leaves and sepals. This arctic species has avoided the deleterious effects associated with the loss of flavonoid intermediates in vegetative tissues by decoupling CHS expression in petals and leaves, yet the correlation of flower color and climate suggests that the loss of flavonoids in the petals alone may affect the tolerance of white-flowered individuals to colder environments.Intra- and interspecific variation in flower color is a hallmark of angiosperm diversity. The evolutionary forces underlying the variety of flower colors can be nearly as diverse as the colors themselves. In addition to pollinator preferences, non-pollinator agents of selection can have a major influence on the evolution of flower color polymorphisms, especially when the pigments in question are also expressed in vegetative tissues. In such cases, identifying the target(s) of selection starts with determining the biochemical and molecular basis for the flower color variation and examining any pleiotropic effects manifested in vegetative tissues. Herein, we describe a widespread purple-white flower color polymorphism in the mustard  Linanthus parryae provided the motivating example for Wright's shifting balance theory that relied on the absence of natural selection The spectacular diversity of angiosperm flower colors is often attributed to the preferences of their pollinators The most common and conspicuous flower color polymorphism among angiosperms is the loss of floral anthocyanins causing pigmented flowers to become white. Although subtle flower color variation can be caused by changes in epidermal cell shape, vacuolar pH, or the presence of cofactors CHS duplicates in Ipomoea purpurea that are confined to different portions of the corolla cis- or trans-regulatory control of a single locus. For example, the promoter of CHS contains binding sites for petal-specific regulatory elements such as bZIP proteins and mybs The genes that are targeted during the loss of floral anthocyanins may be constrained by the stress-related functions of anthocyanins and their intermediates. First, anthocyanins are not the only products of the ABP - the early half of the pathway produces several flavonoid intermediates, such as flavonols and flavones, known to function during the plant's stress response Parrya nudicaulis. This species exhibits a purple-white flower color polymorphism across its range in Alaska in tundra habitats where pollinators are rare P. nudicaulis, pollinator visitation rates were not detectably influenced by flower color . Furthermore, the frequency of white-flowered individuals is positively correlated with temperature during the growing season  and (2) establish if the loss of petal anthocyanins also affects leaf pigmentation and gene expression.Herein, we have examined the biochemical and molecular basis of a flower color polymorphism in the arctic mustard, g season . Our goaP. nudicaulis in Alaska. Populations were chosen based on their geographical distinctiveness and accessibility. All populations were greater than 10 km distant from one another and approximately 250 km apart on average. In order to correlate flower color frequency with climate, we used mean July temperature which approximates the midpoint of the three month growing season for these plants. We also used growing degree days (GDDs) - the annual accumulation of degrees above a minimum baseline temperature below which physiological function is assumed to be inhibited [GDD  =  (maximum temperature – minimum temperature)/2 – baseline temperature]. We compare GDDs calculated using a 4°C baseline with GDDs calculated with a 0°C baseline. All three measures of climate were based on monthly mean temperatures using historic Climate Research Unit data from 1961–1990 and downscaled to a 2 km grid . Because of uncertainty in the accuracy of climate predictions at particular geographic localities, we used a Spearman rank-based correlation of climate and flower color.The proportion of white- versus purple-flowered individuals was estimated from 14 populations spanning the geographic range of Although this species shows a wide range of pigmentation levels among purple morphs in all populations, we have focused on only purple- and white-flowered individuals for the following biochemical and molecular analyses. Petal tissue from purple-flowered plants (n = 5) and white-flowered plants (n = 4) was collected from the 12 Mile Summit population  and immeArabidopsis thaliana) and total sepal pigmentation (dark vs. light). Data from both sites were pooled and differences in petal, leaf and sepal tissue pigmentation levels were analyzed using a chi-square test.A phenotypic survey was conducted at 12 Mile Summit (n = 45) and Savage River (n = 50) populations in June 2008 . Each pl20 primers following the manufacturer's suggested protocol.Tissue from purple- and white-flowered plants was collected from the 12 Mile Summit and Savage River populations . Within PAL). All primer sequences are available on request from the authors. The seven genes were amplified with degenerate primers via reverse-transcriptase polymerase chain reaction (RT-PCR). After gel purification, RT-PCR products were cloned into the pCR 4-TOPO TA vector (Invitrogen). Inserts were confirmed with PCR of the colonies using M13 forward and reverse primers, then sequenced with T3 and T7 primers on an ABI 3730xl DNA Analyzer  following the suggested BigDye protocol . We sequenced an average of 25 clones per gene (range 21–31 clones). Sequences were assembled with Sequencher software  and aligned with ClustalW We isolated and characterized the six core genes involved in anthocyanin biosynthesis using previously published degenerate primers for angiosperms from Whittall et al. CHS.To extend the coverage of the ABP genes and recover loci for which there were no degenerate primers available, we compared one early bud sample from each color morph using mRNA-Seq and massively parallel sequencing by synthesis following the manufacturer's protocol (Illumina). On average, we gained 300 bps in the open reading frame (ORF) and 50 bps of UTR sequence (when present) for genes previously characterized with degenerate primers – including 93% of the ORF for PAL, CHS, CHI, F3H, DFR and ANS using the manufacturer's suggested protocol . New primers were developed from the genome walking sequences to obtain as much of each gene as possible in cDNA. cDNA from one to two purple-flowered and one to four white-flowered plants was amplified following the methods of Whittall et al. In order to obtain even greater ORF coverage of ABP genes, we designed genome walking primers and amplified 5′ and/or 3′ ends of P. nudicaulis  TaqMan assays were designed using Primer Express 3.0 software (Applied Biosystems). Probes were labeled with the 5′ reporter dye 6-FAM and the 3′ quencher Iowa Black FQ. qRT-PCR was performed on the StepOne Real Time PCR System (Applied Biosystems) in 20 µl reaction volumes using the following reagents : 1X TaqMan Gene Expression Master Mix, 900 nM forward and reverse primers, 250 nM probe, and 25 ng of cDNA. In addition, 6.25 ng of raw RNA extract was included for five samples and was amplified with each gene to detect the presence of any genomic DNA contamination. Thermal cycling conditions were: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. Each sample was run in duplicate and the specificity of primers and probes was verified by running the qRT-PCR products on an agarose gel.T method described in Livak and Schmittgen Ts for each sample and target were obtained by normalizing the threshold cycle number (CT) to the constitutively expressed gene glyceraldehyde-3 phosphate dehydrogenase . Relative quantification (RQ) values were calculated using the equation RQ  =  (1+E)−ΔΔCT, where E is the amplification efficiency of each target determined by a standard curve and ΔΔCT  =  ΔCTpurple reference – ΔCTsample. The five dilutions used in the standard curve to calculate amplification efficiency were run in triplicate and were serially diluted two-fold from a starting quantity of 20 ng/µl.Threshold levels for each gene were manually adjusted in order to control for inter-assay variation and relative expression of samples was determined with the comparative ΔΔCT values were first converted to a linear form as described in Livak and Schmittgen −ΔCT. We compared linear ΔCT values of the two color morphs in each tissue type using a two-sample Student's t-test with unequal sample sizes and unequal variance. To assess significance, we compared our observed t-statistic to t-statistics from 104 bootstrapped datasets of comparable sizes (sampled with replacement).Due to limited sample sizes within each developmental stage, samples for each color morph were pooled across developmental stages for statistical analyses. ΔCWe tested for correlations among all genes within each tissue type using a Pearson's correlation coefficient. Expression levels for the comparisons also were converted to a linear form as explained above. Data were natural-log transformed to meet assumptions of normality, when necessary, and statistical analyses were completed in Jmp 4.0 . In each tissue type, 28 tests were done on all combinations of the eight ABP genes . To control for multiple comparisons we used the Bonferroni correction as described in Abdi's equation 8 Cis-regulation in CHS has been well characterized in the Brassicaceae CHS promoter are highly conserved P. nudicaulis-specific primers for a survey extending 444 bp upstream from the start codon for 10 purple alleles and six white alleles to look for color differentiating SNPs.trans-regulation of CHS often co-regulate flavonol synthase (FLS), we also examined this locus at the sequence and expression levels. Using Illumina transcriptome sequencing, we were able to assemble large portions of FLS expressed in petals (841 bps). We amplified FLS with cDNA and genomic DNA and designed qRT-PCR assays as for the other ABP genes.Since several candidates for petal specific P = 0.021). The correlation between climate and flower color was equally strong using 0°C as a baseline  or using average July temperature, the midpoint of most populations' three month growing season .The percentage of white-flowered individuals among 14 populations ranged from zero to 24% . Five po3× lower flavonol concentrations than purple-petal extracts , and an unidentified phenolic compound . Petal eextracts . The abs2 = 0.008; df = 93; P = 0.93). However, when we compared leaf pigmentation levels with sepal pigmentation, there was a significant correlation . Seventy-eight percent of the leaves without visible anthocyanin pigmentation on the petioles had light colored sepals, yet plants with pigmented leaf petioles had an equal frequency of light and dark colored sepals. Although sepals and leaves show correlated pigmentation levels, anthocyanin pigmentation levels in petal and vegetative tissues are decoupled.We conducted a phenotypic survey to determine if petal color correlated with vegetative pigmentation levels. Ninety-five individuals across two populations were visually scored for flower color, leaf petiole pigmentation and sepal pigmentation. Over 70% of plants had pigmentation on the leaf petiole, regardless of their flower color. There was no significant correlation between flower color and vegetative pigmentation level . The recombination survey was conducted on cDNA sequences within 300 bp of the start and stop codons on average, well within the range of linkage disequilibrium reported from the closely related Brassica napus and even Oryza sativaPortions of all seven ABP enzyme-coding genes were examined for DNA sequence changes that could lead to loss-of-function phenotype using RNA samples preserved in the field. A degenerate primer reverse-transcriptase PCR approach To test whether the same ABP genes were expressed in leaves, we compared petal-derived cDNA sequences to those obtained from leaf cDNAs from purple- and white-flowered plants. Identical alleles were recovered from leaves as were found in the petals for all seven ABP genes, indicating that the same copy was at least weakly expressed in both tissues of both color morphs (RT-PCR conditions optimized for relatively low levels of gene expression).F3H and DFR, which produced mixed sequence due to poly-T repeats in the introns , intron sequences had higher variation (7.51% variable sites), yet we did not find any consistent differences between color morphs. The lack of distinguishing SNPs in these introns suggests that the immediately adjacent regions of the ORF are unlikely responsible for the purple-to-white transition due to the low frequency of recombination (average distance from intron to start and stop codon is 640 bps). In addition, there were no significant departures from neutrality (Tajima's D) in the coding and noncoding regions, when present  (P = 0.0006). The largest difference was at the opening stage, where purple petals had 66× higher expression than white petals. No other genes involved in anthocyanin biosynthesis had significant petal expression differences between color morphs , consistent with the uncorrelated pigmentation levels of petal and vegetative tissues found in the phenotypic survey described previously. The only gene with a weak, yet non-significant trend in expression differences in leaves of purple and white flowers was CHI .There were no significant differences in expression of ABP genes in leaves from purple- and white-flowered individuals ; Table 3CHS expression was significantly different between purple and white petals, but not between the leaves of those plants, we also examined expression of CHS in sepal tissue – the adjacent floral whorl to the petals. Sepals from purple-flowered plants did not have significantly different CHS expression than sepals from white-flowered plants (P = 0.74). In fact, mean CHS expression in sepals from white-flowered plants was ∼1.5× higher than that of purple-flowered plants, the opposite pattern detected in the adjacent petals.Since CHS was the only locus with differential expression, we looked for correlated expression of ABP loci to test for potential trans-acting regulatory elements. We detected significant expression correlations among several of the late genes in the ABP, yet no genes showed a correlation in expression with petal CHS , as was expression of ANS and UF3GT . In leaves, expression of DFR was correlated with both ANS  and UF3GT , yet again, no correlated expression with CHS in leaves. Correlated expression of these late genes in the ABP of other angiosperms has often been attributed to one of three conserved regulatory elements, R2R3 MYB, WD40 and bHLH CHS expression does not correlate with any other ABP gene surveyed, the CHS downregulation is unlikely to be caused by one of the common ABP regulatory genes.Although petal etal CHS . In petaCHS, the 5′ UTR is very short (∼100bp) and we were able to identify the TATA box based on conserved sequence across the Brassicaceae (TTATATA) at 111 bp from the start codon 7CT] starting 171 and 154 bp upstream from the start codon, comparable to the location of the H-box in Koch et al. CHS promoter.Primers from genome walking produced 444 bp upstream from the start codon. In Arabidopsis thaliana seedlings, CHS expression was correlated with expression of the ABP side-branch enzyme FLS via three closely related R2R3-type MYBs Petunia hybrida (Baccara Rose Picotee), CHS downregulation was also positively correlated with FLS expression FLS for any color-differentiating SNPs and expression differences between purple- and white-flowered individuals. Primers created from Illumina transcriptome sequencing results produced 91.1% of the ORF for FLS, yet we did not detect any consistent differences among color morphs in a cDNA survey of two purple- and two white-flowered individuals. Expression analyses of FLS showed no significant differences among the color morphs in petal and leaf tissue  causing the change in CHS expression.The decreased trans-regulation through changes in the amino acids of the regulatory element or their expression could lead to the decrease in CHS expression in white flowers. Trans-regulation of the ABP almost always involves multiple ABP genes P. nudicaulis, we find no other enzyme-coding genes in the ABP that have correlated expression with CHS, although we are able to detect the predicted co-regulation of late genes in the ABP trans-regulated CHS expression in petals [Atmyb111;CHI, F3H, and FLS, yet we find no correlation between CHS and these three other genes in P. nudicaulis.Alternatively, CHS). Further examination of pigmentation within the flower  could also provide additional clues to the target(s) of natural selection on the flower color polymorphism in P. nudicaulis. Although the widespread nature of this flower color polymorphism and lack of population structure at neutral loci could facilitate further association mapping, the ability of making controlled crosses has been stymied by the difficulty growing plants under controlled conditions and the length of time it takes them to flower (likely >10 years).In focusing on the loss-of-function phenotypes, in which we expected pleiotropic effects to be most pronounced (pigmented vs. white), we have ignored the variation within the pigmented morphs. Most populations in Alaska exhibit a wide range of variation within the pigmented category ranging from dark purple to light pink. The next step in characterizing this color variation will be to quantify the petal color with UV-Vis spectra, then compare the petal phenotype with their flavonoid profiles and the levels of gene expression across the ABP . Although “thermal rewards” can attract pollinators, since we see no differential visitation to purple and white morphs , we predict the increased floral temperature functions independently of pollinators through expediting fruit maturation by warming the gynoecium - a limiting factor in arctic plant reproduction We conclude that white-flowered