Already in the 1930s, Otto Warburg observed that tumor cells preferably utilize glycolysis and lactate fermentation for energy production, rather than the mitochondrial oxidative phosphorylation dominating in normal cells, a phenomenon today known as the Warburg effect. Even though many tumor types display a high degree of aerobic glycolysis, they still retain the activity of other energy-producing metabolic pathways. One exception seems to be the clear cell variant of renal cell carcinoma, ccRCC, where the activity of most other pathways than that of glycolysis has been shown to be reduced. This makes ccRCC a promising candidate for the use of glycolytic inhibitors in treatment of the disease. However, few studies have so far addressed this issue. In this report, we show a strikingly reduced mitochondrial respiratory capacity of primary human ccRCC cells, resulting in enhanced sensitivity to glycolytic inhibition by 3-Bromopyruvate (3BrPA). This effect was largely absent in established ccRCC cell lines, a finding that highlights the importance of using biologically relevant models in the search for new candidate cancer therapies. 3BrPA markedly reduced ATP production in primary ccRCC cells, followed by cell death. Our data suggest that glycolytic inhibitors such as 3BrPA, that has been shown to be well tolerated  Increased understanding of this disease that could result in development of new therapies is therefore of great importance.World wide, ~340 000 patients are diagnosed with renal cell carcinoma (RCC) annually.4 The initiating genetic event seems to be loss of function of the tumor suppressor protein von Hippel Lindau, pVHL, found in over 90% of all cases.6 This results in aberrant activation of the hypoxia inducible factors HIF-1α and HIF-2α, transcription factors normally active only at hypoxia, controlling the cellular adaptation to oxygen shortage, such as induction of angiogenesis, cell cycle arrest and pro-survival factors.8 The resulting pseudohypoxic phenotype in ccRCC tumors leads to massive angiogenesis, as well as an extensive metabolic reprogramming, including a shift from mitochondrial oxidative phosphorylation to non-oxygen-requiring glycolysis. In normal cells, when oxygen is abundant, glycolysis is used to metabolize glucose into pyruvate, which enters the citric acid cycle in the mitochondria. ATP is subsequently produced by oxidative phosphorylation, driven by the reducing equivalents formed in the citric acid cycle. During hypoxic conditions, pyruvate is instead fermented to lactate and excreted from the cell. Already in the 1930s the German physiologist Otto Warburg described that tumor cells preferentially utilize the glycolytic pathway to produce lactate also when oxygen is available, a phenomenon today known as the Warburg effect. Although such aerobic glycolysis yields less ATP per molecule of glucose than its full oxidation to CO2 and H2O in mitochondrial metabolism, this pathway provides tumor cells with building blocks needed for growth and proliferation. The current view is that even though increased aerobic glycolysis is one of the characteristics of tumor cells, mitochondrial and other metabolic pathways are usually still maintained alongside with a high glycolytic rate in most cancer forms.9ccRCC derives its name from the histologically empty (clear) appearance of the cancer cells, caused by large deposits of glycogen and lipid droplets in the cytoplasm.13 In this report, we show that the number and respiratory capacity of mitochondria are strongly reduced in ccRCC cells, which instead exhibit high glycolytic activity. We hypothesized that this renders ccRCC cells highly sensitive to inhibition of glycolysis. To test this hypothesis, we exposed primary ccRCC cells to 3-bromopyruvate (3BrPA), a pyruvate analog primarily described as a glycolytic inhibitor. We show that primary human ccRCC cells, in contrast to normal kidney cells and also established ccRCC cell lines, were highly sensitive to inhibition by 3BrPA. The possibility to utilize metabolic inhibitors might be a promising therapeutic alternative to specifically target ccRCC tumors that should be further studied.Recent studies indicate that the metabolism of ccRCC tumors differs from other cancer forms in that most pathways besides glycolysis are downregulated.c oxidase II (MTCO2) revealed high expression in normal epithelial cells of the proximal tubules, whereas the expression in ccRCC cells was strongly reduced  data set. As shown in  reduced . With puThese data urged us to analyze the capacity of ccRCC cells to utilize mitochondrial oxidative phosphorylation for energy production. Measurement of cellular oxygen consumption rate (OCR) by the Seahorse technique can be used to quantify mitochondrial respiration. As shown in 2O3, which inhibits mitochondrial respiration. As2O3 had negligible effect on primary ccRCC cells, while normal primary kidney epithelial cells did not tolerate this treatment , commonly used to study this tumor type in vitro, were included in these studies, in order to determine whether also other ccRCC cell lines were equally resistant to 3BrPA, and also to ensure that the lack of HIF-1α was not a determining factor in the resistance of the established cell lines to 3BrPA treatment. All tested continuous ccRCC cell lines showed an increased resistance to glycolytic inhibition by 3BrPA treatment compared with primary ccRCC cells, although they appeared somewhat more sensitive than primary normal kidney cells ,20 a proton-linked pump that catalyzes the transport of monocarboxylates such as lactate and pyruvate across the cell membrane. The expression of this pump is induced in hypoxic and glycolytic cells. Data obtained from publically available gene expression data sets, together with the TCGA, showed that ccRCC tumors express SLC16A1 at significantly higher levels than normal kidney cells .29 These effects seem to vary depending on concentration, cell type and on the carbon source available for energy metabolism, where the greatest inhibitory effect of 3BrPA on mitochondrial function was found when glucose supply was limited.28 However, in the case of ccRCC cells, their nutrient-rich environment and low mitochondrial content implies that the toxic effect of 3BrPA on these cells most likely acts through glycolytic inhibition. In further support of this theory is our results demonstrating a high tolerance of primary ccRCC cells to known inhibitors of the respiratory chain, as well as the reduction in lactate excretion following 3BrPA treatment.Although 3BrPA has mainly been described as an inhibitor of glycolysis, it has also been reported to target mitochondrial function by inhibiting mitochondrially bound hexokinaseSLC16A1. The expression of this lactate transporter correlates to a more pronounced glycolytic profile.20 In this study we showed that the levels of SLC16A1 was elevated both in ccRCC tissue samples and in cultured primary ccRCC cells compared with normal kidney cells, suggesting an increased uptake of 3BrPA specifically in ccRCC tumor cells. The low SLC16A1 levels found in primary normal kidney cells probably limit their uptake of 3BrPA. Although this may add a level of protection towards any off-target effects of high-dose 3BrPA treatment, we do not believe that the low SLC16A1 levels is the main explanation for the increased resistance of the normal cells to 3BrPA. One of the normal kidney cell samples (R124N) displayed elevated expression of SLC16A1 compared with the others, yet these cells did not show an increased sensitivity to 3BrPA treatment, arguing that the absolute levels of SLC16A1 is not the critical factor determining sensitivity to 3BrPA. Instead, we hypothesize that the cellular metabolic feature is a crucial aspect, where the constrained glycolytic profile of ccRCC cells renders them highly sensitive, whereas normal cells with a broader metabolic repertoire will be spared.Uptake of 3BrPA has been reported to be dependent on the monocarboxylate transporter MCT1, or SLC16A1 at almost the same elevated levels as in the primary ccRCC cells. No correlation between 3BrPA IC50 values and SLC16A1 expression levels was found among the continuous cell lines. Expression levels of SLC16A1 in the SKRC10 cell line was significantly higher compared with the other established cell lines. Again, this was not reflected in any difference in the sensitivity of these cells to 3BrPA.We additionally found that established ccRCC cell lines also were less sensitive to 3BrPA treatment, despite the presence of The tested cell lines displayed higher mitochondrial DNA content and a marked increase in mitochondrial respiration compared with primary ccRCC cells. These findings infer an elevated capacity to utilize other metabolic pathways in established cell lines, a lower dependence on glycolysis for energy production and thus a higher tolerance to glycolytic inhibition. Of note, ccRCC cells isolated from one patient did not respond to 3BrPA treatment (R104T). Seahorse analysis revealed a higher basal OCR and increased maximal respiratory capacity of these cells, suggesting a higher mitochondrial content and lower dependence on glycolysis for energy production. Together, these data imply that cells relying on functional glycolysis for energy production will be selectively sensitive to 3BrPA treatment.in vivo, retained in primary ccRCC cells, that is lost in cell lines during establishment and propagation. These results point to the importance of using relevant model systems, and that established cell lines not always give a correct reflection of the tumor type they are derived from. Hence, screening for metabolic inhibitors using established cell lines might give misleading results and promising candidates might be overlooked.The extreme glycolytic profile of primary ccRCC cells is probably a property of tumor cells In summary, our results demonstrate that primary ccRCC tumor cells are highly sensitive to glycolytic inhibition by 3BrPA, suggesting that the possibility to utilize pharmaceuticals targeting glycolysis, thereby disrupting energy metabolism, should be further studied in ccRCC tumors.30 with ethical approval from the Lund University ethical committee (LU680-08 and LU 289-07). Tumors were classified as ccRCCs by an experienced pathologist. Normal samples were collected from healthy kidney cortex farthest from the tumor. 786-O cells were obtained from ATCC. SKRC10 and SKRC17 were kindly provided by E. Oosterwijk . All cells were grown in DMEM high glucose supplemented with 10% FBS and 1% penicillin/streptavidin , except for SKRC7, SKRC10, SKRC17, SKRC21 and SKRC52, which were kept in RPMI. All cell lines were regularly checked for mycoplasma.Primary human ccRCC cells were isolated and cultured from patient nephrectomies as described previously,3-Bromopyruvate was obtained from Sigma-Aldrich . A fresh stock solution of 20 mM 3BrPA in PBS was prepared for each experiment.μl media and cells were incubated for 1–6 days. WST-1 (10 μl) solution  was added to cells and absorbance was measured after 4 h incubation.Cells were seeded in triplicates in 96-well plates. The following day 3BrPA or vehicle was added in a total volume of 100 Cells were seeded in 24-well plates and incubated with or without 3BrPA for 24 h. Adherent and floating cells were collected and the number of live and dead cells was determined using the cell count and viability assay on the Nucleocounter NC3000  according to the manufacturer's protocol.2 chamber for 2 h after which the OCR was analyzed according to the manufacturer's instructions. Maximal respiratory capacity was determined after injection of 4 μg/ml oligomycin (Sigma-Aldrich), 4 μM FCCP  and 5 μM Rotenone (Sigma-Aldrich) according to instructions for the Cell Mito Stress Test. OCR was corrected to number of cells. Each sample was run in three to five replicates.Measurement of OCR was performed on a Seahorse XF24 Extracellular Flux Analyzer . Cells were seeded to confluency in a XF24 Seahorse assay plate. The following day media was changed to unbuffered XF Assay medium and the plate was incubated in a non-COTissue samples were fixed in 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M Sorensen phosphate buffer PH 7.4, followed by 1% Osmiumtetroxid in 0.5 M potassium cyanoferrate buffer. After dehydration, embedding and sectioning, samples were analyzed at the Lund University Bioimaging Center using a Tecnai biotwin 120 kV transmission electron microscope .μl and a quantitative PCR was performed using primers for nuclear and mitochondrial encoded genes. The relative mitochondrial DNA content of each sample was determined by dividing the levels of mitochondrial DNA by the nuclear encoded genes. For primer sequences see Total DNA was isolated using the Allprep RNA/DNA kit from Qiagen . DNA samples were diluted to 0.4 ng/Cells were seeded into 96-well plates. The following day, 3BrPA was added to the media and 1 h later lactate concentration in cell medium was determined spectrophotometrically using a Lactate Assay Kit from BioVision  according to the manufacturer's instructions. The assay was performed under serum-free conditions.Intracellular ATP levels were determined using the Cell Viability Kit SL . Cells were seeded in triplicates in a 96-well plate. Twenty-four hours later cells were treated with 3BrPA for 1 h, after which intracellular ATP levels were determined according to the manufacturer's instructions.30 Antibodies used were anti-MTCO2 (Abcam), anti-SLC16A1 (Sigma-Aldrich) and anti-Glut1 .Tissue samples or cultured cells were fixed in 4% neutral buffered formalin and embedded in paraffin according to the standard protocols. Immunohistochemical stainings were performed as described previously.Ct method was used to quantify relative RNA levels and three separate housekeeping genes  were used for normalization. Primer sequences are listed in Total RNA was isolated using the the RNeasy mini kit from Qiagen. cDNA synthesis was performed using M-MuLV reverse transcriptase and random hexamers (Thermo Scientific). Quantitative real-time PCR was performed with the SYBR Green PCR Master Mix (Thermo Scientific) on an Mx 3005 P QPCR system . The comparative Flow cytometry was performed on a FACS Calibur . Percentage of apoptotic double-positive cells was determined after staining with Propidium Iodide (Sigma-Aldrich) and Annexin V-APC (BD Biosciences) according to the manufacturer's protocols. Cell cycle analysis was performed on fixated cells stained with Propidium Iodide. The percentage of cells in each stage of the cell cycle was determined using the Dean/Jett/Fox algorithm in the FlowJo software .http://tcga-data.nci.nih.gov/tcga/dataAccessMatrix.htm) by November 2013. The data comprised 505 ccRCCs and 70 normal kidney specimens analyzed on the Illumina HiSeq platform. RNA-seq data from the following cancer types included in the TCGA data collection were also downloaded for comparisons: BLCA, Bladder Urothelial Carcinoma; BRCA, Breast invasive carcinoma; COAD, Colon adenocarcinoma; GBM, Glioblastoma multiforme; HNSC, Head and Neck squamous cell carcinoma; KICH, Kidney Chromophobe; KIRC, Kidney renal clear cell carcinoma; LAML, Acute Myeloid Leukemia; LGG, Brain Lower Grade Glioma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; OV, Serous Cystadenocarcinoma; PRAD, Prostate adenocarcinoma; READ, Rectum adenocarcinoma; SKCM, Stomach adenocarcinoma; THCA, Thyroid Carcinoma; UCEC, Uterine Corpus Endometrial Carcinoma. RSEM estimate values were multiplied by 106 followed by adding an offset of 10 and subsequently log2 transformed. Two additional data sets  with mRNA expression data of ccRCC and normal kidney were obtained from the Gene Set Omnibus data repository .Level 3 RNA-seq data containing mRNA gene-level RSEM estimates were downloaded from The Cancer Genome Atlas (TCGA) data portal (


Modern flamingos (Phoenicopteridae) occupy a highly specialized ecology unique among birds and represent a potentially powerful model system for informing the mechanisms by which a lineage of birds adapts and radiates. However, despite a rich fossil record and well-studied feeding morphology, molecular investigations of the evolutionary progression among modern flamingos have been limited. Here, using three mitochondrial (mtDNA) markers, we present the first DNA sequence-based study of population genetic variation in the widely distributed Chilean Flamingo and, using two mtDNA and 10 nuclear (nDNA) markers, recover the species tree and divergence time estimates for the six extant species of flamingos. Phylogenetic analyses include likelihood and Bayesian frameworks and account for potential gene tree discordance. Analyses of divergence times are fossil calibrated at the divergence of Mirandornithes (flamingos + grebes) and the divergence of crown grebes.mtDNA sequences confirmed the presence of a single metapopulation represented by two minimally varying mtDNA barcodes in Chilean flamingos. Likelihood and Bayesian methods recovered identical phylogenies with flamingos falling into shallow-keeled  and deep-keeled  sub-clades. The initial divergence among flamingos occurred at or shortly after the Mio-Pliocene boundary (6–3 Ma) followed by quick consecutive divergences throughout the Plio-Pleistocene. There is significant incongruence between the ages recovered by the mtDNA and nDNA datasets, likely due to mutational saturation occurring in the mtDNA loci.Phoeniconaias’ as a junior synonym of ‘Phoenicoparrus’ and redefine the latter genus as containing all flamingos more closely related to Phoenicoparrus andinus than Phoenicopterus roseus.The finding of a single metapopulation in the widespread Chilean Flamingo confirms similar findings in other widespread flamingo species. The robust species phylogeny is congruent with previous classifications of flamingos based on feeding morphology. Modern phoenicopterids likely originated in the New World with each sub-clade dispersing across the Atlantic at least once. Our divergence time estimates place flamingos among the youngest families of birds, counter to the classical notion of flamingos as among the oldest based on biogeography and the fossil record. Finally, we designate ‘ Flamingos are traditionally perceived as among the oldest lineages of living birds with reports of flamingo-like birds appearing in the fossil record as early as the late Cretaceous (e.g.). Howevee.g. and the e.g.-5, suggee.g.. Notablyaleogene .Recent fossil and molecular work have shed new insight into phoenicopterid origins and cast doubt on the classical notion of flamingos as a particularly ancient lineage among the storks, herons and ibises (Ciconiiformes). Most notably, molecular -11 and mPhoenicopterus ruber ruber and P. ruber roseus) that likely form a single genus comprising two distinct sub-clades. This division is congruent with the organization by Jenkin [e.g. blue-green algae and diatoms); the remaining species  have more compact bills in cross-section suited to filtration of larger food items (e.g. mollusks and crustaceans). Further morphological variation within the two sub-clades is not completely known [Sibley and Ahlquist  represeny Jenkin  of flamily known .pers. comm. and Lesser [Intraspecific genomic variation in each flamingo species is also incompletely known but is an important consideration when investigating morphological and geographic origins of species with wide ranges. Recent population genetics studies have identified a lack of genetic structure within the Greater ; Remi Wad Lesser  Flamingod Lesser ,21 on thd Lesser ,23, the The present study has three aims: 1) to assess intraspecific mitochondrial variation within the widespread New World Chilean Flamingo through the use of three mtDNA markers and museum skins collected throughout the range; 2) to test the phylogenetic relationships among modern flamingos through the use of a multi-locus (10 nDNA and two mtDNA markers) and varied analytical  approach; and 3) to estimate the divergence times of living flamingo species from the resulting species tree calibrated with two fossil constraints. How these results inform the taxonomic organization and biogeography of flamingos is discussed.Sequence data for 3 mtDNA loci were obtained and analyzed from 17–26 Chilean Flamingos representing the entire breeding range of the species except Ecuador , though both partitioned and non-partitioned BI analyses recovered the same topology with complete support (posterior probability = 1.0); the ML analysis had >85% bootstrap support for all nodes. A *BEAST analysis including multiple individuals for each flamingo species again recovered the same topology with complete support (posterior probability = 1.0). All jackknife analyses excluding a single locus at a time from the total (nDNA + mtDNA) dataset recovered the complete topology with high (>0.9) posterior probability, suggesting that no single locus was driving the results. The nDNA dataset recovered the same topology, while the mtDNA dataset differed only by recovering a (Andean + (Lesser + James’s)) clade, albeit without significant support.Even though intra-specific variation was discovered in a set of five nDNA loci including two to three individuals per flamingo species, this variation was found to be minimal , American (P. ruber) and Chilean (P. chilensis) Flamingos. Within this clade, the American and Greater Flamingos are often considered sub-species . The 1%ies (e.g.. Althougminor), Andean (andinus) and James’s (jamesi) Flamingos. These species have often been set apart from the classical genus Phoenicopterus with the Lesser placed within Phoeniconaias based on unique characteristics of the bill [Phoenicoparrus based on the shared absence of the hallux (hind toe) [Phoeniconaias does not seem to exclude andinus and jamesi, and the hallux of minor is reduced [Phoenicoparrus. Thus, we suggest that the division of the deep-keeled clade into two genera is arbitrary and uninformative. Instead, we suggest the placement of minor within Phoenicoparrus along with andinus and jamesi based on shared mandibular morphology, ecology and phylogenetic relatedness. We suggest the designation of Phoeniconaias as a junior synonym of Phoenicoparrus based on priority and the redefinition of Phoenicoparrus as all species more closely related to Phoenicoparrus andinus than to Phoenicopterus roseus.The deep-keeled clade comprises those species with bills suited to capturing smaller prey items and includes the Lesser (the bill  and the ind toe) . However reduced , approxie.g.[e.g. within grebes) may have introduced saturation artifact. It is likely that homoplasy within the relatively longer and more quickly-evolving mtDNA sequences, with more mutational sites, conflicted with the signal of the shorter and more slowly evolving nDNA sequences, resulting in artificially old age estimates. Thus, the true ages of the component flamingo divergences are probably somewhere between those predicted by the total dataset and by the nDNA dataset alone.Even though the various subsets of data recovered incongruent divergence times Table , all agre.g.) but is e.g., which ie.g.. Though Phoenicopterus suggests a New World origin for the shallow-keeled flamingos. The origin of Phoenicoparrus is not so straightforward, as the basal divergence within the clade spans the Atlantic. Plio-Pleistocene fossil flamingos are known only from the New World [Phoenicoparrus, and crown Phoenicopteridae, in the Western Hemisphere, followed by trans-Atlantic dispersal. Better phylogenetic resolution for the Australian forms will further inform phoenicopterid origins.The two Old World species (Greater and Lesser) are each recovered in clades with otherwise exclusively New World distributions. The recovery of the Greater Flamingo as derived within ew World -37 and Aew World ,38 and tew World . The NewJuncitarsus[Elornis and Agnopterus[The phoenicopteriform fossil record is rich but poorly understood and revision will greatly advise divergence time estimation and paleo-biogeographical reconstruction for crown flamingos. The stem phoenicopteriform record is characterized by fragmentary material with only tentative assignment to the flamingos. While the earliest palaelodids (early Oligocene) are the earliest to be reliably placed in Phoenicopteriformes, ncitarsus,40 (but ncitarsus), Elornignopterus from theHarrisonavis croizeti from the Oligo-Miocene of Europe displayed a mandibular morphology approximating that of modern flamingos [Finally, while we present the first information on the timing of diversification and specialization within crown flamingos, better understanding of interspecific variation (particularly with respect to the filter feeding mandibular morphology) is necessary to interpret the ecological significance of these results. Likewise, revision of the stem phoenicopteriform fossil record is necessary to place these ages in a more meaningful context. Most notably, the primitive flamingo lamingos ,43. ReasPhoenicopterus while the Lesser is moved to genus Phoenicoparrus (= ‘Phoeniconaias’) along with the James’s and Andean Flamingos. The living species diverged from each other ~4.37 million years ago, followed by Phoenicoparrus (~2.56 mya) and Phoenicopterus (~2.29 mya).Chilean Flamingos show no evidence of population structure across their entire range, a condition similar to other widely-distributed flamingo species. The six living species of flamingos fall into two clades reflecting differences in bill morphology and feeding ecology. We propose slight reorganization of flamingo taxonomy to indicate these differences: the Greater, American and Chilean Flamingos comprise genus Field sampling of all flamingo populations remains impractical for logistic reasons, including geographic inaccessibility and conservation protection. Sampling from captive birds alone is not guaranteed to capture species-wide variation due to a persistent lack of information regarding the geographic origins of the ancestors of captive populations. To assess phylogeographic structure, we thus set out to sample museum specimens of Chilean Flamingos with known locality. Available DNA barcodes from Chilean Flamingos suggest the presence of at least two haplotypes in Argentina  but no dPodilymbus podiceps and Aechmophorus clarkii were included to represent the basal split within grebes [eg.[eg.[Tissue samples were obtained for each flamingo species  an [eg.[eg.). Sample2O, 5 µl 10× detergent- and Mg+-free reaction buffer, 5 µl MgCl2 (25 mM), 1 µl dNTP (10 mM each), 2.6 µl each primer, 0.2 µl Hot MultiTaq (US DNA) polymerase (5 u/µl) and 2.5 µl DNA template (20 mM). Thermocycling comprised an initial denaturation at 95°C for 5 min followed by 35–40 cycles of annealing at 95°C for 30 sec, extension at 50°C for 30 sec and denaturation at 95°C for 40 sec, followed by a final annealing/extension at 72°C for 5 min. Excess dNTPs and primers were removed from PCR product by addition of 1.45 µl Exonuclease 1 , 2.85 µl FastAP (Thermo Scientific) shrimp alkaline phosphatase (1 u/µl) and 2.85 µl dH20 and held at 37°C for 30 min and then 80°C for 15 min. Product was sequenced offsite at MacroGen USA (Maryland).Genomic DNA was extracted from tissue samples using the DNeasy Tissue Kit (Qiagen) and was amplified for 10 nuclear and two mitochondrial loci from one individual per species, which comprised the primary dataset Table . Two addSequence data for one to two additional flamingo individuals per species for five loci . Genbank accession numbers are provided in Additional files All supporting data are included as additional files. Newly obtained DNA sequence reads have been uploaded to Genbank (The authors declare that they have no competing interests.CRT carried out DNA extraction, amplification, alignment, performed all phylogenetic and divergence time analysis, and drafted the manuscript. LMO and MAFG carried out DNA extraction, amplification, and alignment, and helped draft the manuscript. BF carried out ancient DNA extraction, amplification and alignment. MVT conceived of the study, participated in its design and coordination, assisted with alignment and analysis, and helped to draft the manuscript. All authors read and approved the final manuscript.Specimen and locality information for the Chilean Flamingo individuals included in the population genetics analysis.Click here for filePairwise genetic distances between multiple individuals of each flamingo species for five nDNA loci.Click here for filePairwise genetic distances between Phoenicopterus ruber and P. roseus for 10 nDNA and two mtDNA loci.Click here for filePrimer sequences for each locus used in this study.Click here for fileAncient DNA extraction and amplification methods.Click here for fileSample information for individuals used in the primary phylogenetic analyses.Click here for fileGenBank accession numbers for sequence data used in the primary phylogenetic analyses.Click here for fileSample information for the additional flamingo individuals used in the pairwise genetic distance analyses and GenBank accession numbers for associated sequence data.Click here for file


Different cancer cells exhibit altered sensitivity to metformin treatment. Recent studies suggest these findings may be due in part to the common cell culture practice of utilizing high glucose, and when glucose is lowered, metformin becomes increasingly cytotoxic to cancer cells. In low glucose conditions ranging from 0 to 5 mM, metformin was cytotoxic to breast cancer cell lines MCF7, MDAMB231 and SKBR3, and ovarian cancer cell lines OVCAR3, and PA-1. MDAMB231 and SKBR3 were previously shown to be resistant to metformin in normal high glucose medium. When glucose was increased to 10 mM or above, all of these cell lines become less responsive to metformin treatment. Metformin treatment significantly reduced ATP levels in cells incubated in media with low glucose (2.5 mM), high fructose (25 mM) or galactose (25 mM). Reductions in ATP levels were not observed with high glucose (25 mM). This was compensated by enhanced glycolysis through activation of AMPK when oxidative phosphorylation was inhibited by metformin. However, enhanced glycolysis was either diminished or abolished by replacing 25 mM glucose with 2.5 mM glucose, 25 mM fructose or 25 mM galactose. These findings suggest that lowering glucose potentiates metformin induced cell death by reducing metformin stimulated glycolysis. Additionally, under low glucose conditions metformin significantly decreased phosphorylation of AKT and various targets of mTOR, while phospho-AMPK was not significantly altered. Thus inhibition of mTOR signaling appears to be independent of AMPK activation. Further in vivo studies using the 4T1 breast cancer mouse model confirmed that metformin inhibition of tumor growth was enhanced when serum glucose levels were reduced via low carbohydrate ketogenic diets. The data support a model in which metformin treatment of cancer cells in low glucose medium leads to cell death by decreasing ATP production and inhibition of survival signaling pathways. The enhanced cytotoxicity of metformin against cancer cells was observed both in vitro and in vivo. In the transformation to cancer, cells undergo reprogramming of their ordinary metabolic functions to facilitate rapid growth potential. Otto Warburg reported high rates of glycolysis in cancer cells even in aerobic conditions. This paradoxical change is one mechanism by which cancer cells have adapted for rapid proliferation. As a result of this altered metabolism, cancer cells use large amounts of glucose and generate high amounts of lactate. Glucose metabolism through glycolysis contributes to ATP synthesis and provides intermediates for other biosynthetic processes. Thus, cancer cells are dependent on the high rates of glucose uptake and metabolism for survival in vitro cancer cell culture commonly use high glucose, 25 mM (450 mg/dL), in the growth medium. While high glucose medium creates an optimal environment for cancer cell proliferation, these glucose levels may complicate the interpretation of drug efficacy studies. High glucose alone has the ability to activate proliferation pathways in a cancer cell in vivo. In pancreatic cancer cells, Sinnett-Smith et al. Current methods of Normal serum glucose is usually maintained between 4 and 6 mM (approximately 72–108 mg/dL). In cases of low nutrient availability, serum glucose levels may drop to 2.5 mM (45 mg/dL), with tissue levels of glucose commonly lower. Reducing glucose availability has also been attempted as a cancer treatment by a variety of methods. Modifying diet by direct caloric restriction or fasting has been investigated as a method of reducing cancer growth with some promising results in vitro in a variety of cancer types in vitro that are observed in vivo, high concentrations, commonly 5–20 mM, of metformin are necessary. Recent studies suggest these findings may be due in part to the common cell culture practice of utilizing high glucose, and when glucose is lowered, metformin becomes increasingly cytotoxic to cancer cells Recently metformin has gained renewed interest as a potential cancer therapeutic and chemotherapy adjuvant. Metformin’s potential anti-cancer activity was implicated by the lower incidence of cancer in type II diabetics versus other glucose controlling drugs While the precise mechanism for metformin’s cancer cytotoxicity remains unidentified, metformin’s systemic actions likely combine with the direct cellular action to enhance its effect on cancer cell growth inhibition and cancer cell death. Here we show that low to normal glucose levels, as opposed to high glucose conditions, potentiate the effect of metformin in breast and ovarian cancer cell lines. The possible mechanisms for this activity are explored. These observations may reveal both more relevant cell culture techniques for studying metformin in cancer as well as provide insight into clinical use of metformin as an adjuvant cancer therapy.The following chemicals were used in this study: metformin , D-(−)-fructose, D-(+)-galactose, 2-deoxy-D-glucose, oligomycin . Dulbecco’s modified Eagle’s Medium (DMEM) with high glucose (Hyclone), Gibco DMEM without glucose (Life Technology), SYTOX Green Nucleic Acid Stain (Invitrogen), and ATP Assay kit (Invitrogen).2. All the cell lines were used within passage ten after receiving from ATCC to ensure cell line authentication.Human cancer cell lines MCF7, MDAMB231, OVCAR3, PA-1, SKRB3, and human mammary epithelial MCF10A cells were all purchased from ATCC and maintained in DMEM containing 10% fetal bovine serum with varying glucose levels and 1% penicillin-streptomycin at 37°C under a humidified atmosphere containing 5% COCells were plated into 96 well plates and treated with the indicated treatment for one or two days. Sytox Green nucleic acid stain (10 µM) was added directly to cells in 96 well plates and incubated for 10 minutes. The plate was read at excitation/emission of 485 nm and 530 nm, respectively, with a 515 nm cutoff using a fluorescence plate reader . Fluorescence was measured to obtain number of dead cells. Subsequently, to determine total cell number, 0.4% Triton-X100 was added to each sample and it was incubated for 30 minutes at room temperature to permeabilize all the cells, and fluorescence at 485/530 nm was measured again to obtain the total cell number.Cells were seeded into 6 well plates followed by incubation for 24 hours. Treatment was given with fresh medium for 24 hours. Equal numbers of cells were lysed in each treatment group and 10 µl was used from each sample following the manufacturer’s protocol for ATP assays . Briefly, 100 µl of the standard reaction solution was measured in a luminometer for background luminescence. Then 10 µl of the lysate supernatant was added to the reaction solution and the luminescence was again measured. Background luminescence was subtracted from sample luminescence and results were plotted as fold change from control samples.Cells in 35 mm dishes were rinsed once with PBS and lysed by addition of sodium dodecylsulfate (SDS) sample buffer . Equal amounts of protein from each treatment group were separated on 10% or 15% SDS-polyacrylamide gels. Proteins were transferred to Immobilon P membranes (Millipore) using a semi-dry Bio-Rad Trans-blot apparatus with a transfer buffer of 48 mM Tris-HCl and 39 mM glycine. The membranes were blocked with 5% non-fat dry milk or 5% BSA in Tris-buffered saline  containing 0.1% Tween-20 (TBS-T) for one hour at room temperature. The membrane was then incubated with the appropriate antibody in TBS-T containing 5% non-fat dry milk or 5% BSA for 1 hour at room temperature or overnight at 4°C. After washing in TBS-T the membrane was incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. Proteins were detected using the Super Signal West Pico chemiluminescent substrate . Anti-β-actin monoclonal antibody  was purchased from Sigma. Antibodies against phosphorylated AKT at threonine 473 (#05-669 used at 1∶1000) was purchased from Upstate Biotechnologies. Antibodies against AKT phosphorylated at threonine 308 , ATK , phosphorylated S6K , S6K , PARP , cleaved PARP , AMPK phosphorylated at threonine 172 , and AMPK , Cleaved Caspase 7  were purchased from Cell Signalling Technology. Secondary horseradish peroxidase-linked anti-mouse  and anti-rabbit  IgG antibodies were purchased from Pierce Biochemical.MCF7 were plated in 96 well plates and treated as indicated for 15 hours. Medium from each treatment was collected and tested for lactate concentration using an L-Lactate Assay Kit (Eton Biosciences Inc.). Cells were counted using Sytox Green staining method as described previously. Relative lactate levels were obtained after normalizing by total cell number.2-free incubator for one hour. Initial measurements of ECAR and OCR were obtained followed by addition of different concentrations of glucose (2.5 mM or 25 mM), fructose (25 mM) or galactose (25 mM). ECAR and OCR were further measured following injection of oligomycin and 2-deoxyglucose. Cell number was obtained by trypsinizing cells and counting using a hemocytometer. ECAR and OCR were plotted after normalizing by total cell number.ECAR and OCR were measured using a Seahorse XF24 analyzer according to manufacturer’s instructions (Seahorse Bioscience). Briefly, MCF7 cells were plated at 40,000 cells/well in XF24-well plates. Cells were treated as indicated the next day. Before being processed with the Seahorse XF24 analyzer, cells were washed and equilibrated with buffer free medium  at 37°C in a CO5 cells in the flank (10 mice per treatment condition). Four days after tumor cell injection, the mice were switched to a calorie restricted low carbohydrate ketogenic diet (BioServ P3666) for the KD group (see below for details of diets). The control groups remained on normal mouse chow  and were fed as libitum. Metformin (2 mg/mouse) was administered intraperitoneally daily starting 7 days after tumor injection. Tumor volume was estimated using A2×B where A is the larger diameter and B is the smaller diameter. Animals were euthanized when the tumor size was greater than 15 mm in its greatest dimension, or tumor volumes reach 3000 mm3, or when the animal was substantially emaciated.All experiments were performed in accord with institutional and national guidelines and regulations; the protocol was approved by the institutional animal care and use committee at Sanford Research. Briefly, using a 25-gauge needle, Balb/C mice were subcutaneously injected with 1×10Serum glucose levels were measured using tail vein blood after puncturing the tail with a 25 gauge needle to produce one drop of blood for each test. Glucose was measured using a Bayer Contour Glucometer and glucose test strips.th day after tumor cell injection. Calorie restriction was determined by measuring the weight of food consumed each day. The appropriate amount of the ketogenic diet was provided every day on a petri dish within the cage. Calories were increased to 7.5 kCal/day/mouse (25% restriction) at day 7 after initiation of the ketogenic diet to prevent weight loss. Calories were further increased to 8 kCal/day/mouse at day 11 after the initiation of ketogenic diet and maintained at this level until the end of the experiment. The control diet groups were fed ad libitum on standard mouse chow  which provides calories from protein, fat and carbohydrates at approximately 24%, 18%, and 58%, respectively.The low carbohydrate ketogenic diet was purchased from BioServ (#F3666). This diet provides calories from protein, fat and carbohydrates at approximately 4.6%, 93.4%, and 2%, respectively. All mice being fed this diet were subjected to 30% calorie restriction  starting the 4t tests were used to compare two groups. P values less than or equal to 0.05 were considered to have significance.Error bars shown are standard deviations from the mean of at least three replicates. Two-tailed pairwise Student’s Our previous research has demonstrated that metformin is cytotoxic to many breast and ovarian cancer cell lines. However, some cell lines such as the breast cell lines MDAMB231 and SKBR3 exhibited resistance to metformin cytotoxicity Concurrently, the effects of metformin at different concentrations of glucose were examined in the non-cancer human mammary epithelial cell line MCF10A . In contIn order to test whether these effects on metformin cytotoxicity applied to other cancer cell types, we also examined ovarian cancer cells. In a similar fashion, lowering the glucose was found to increase the cytotoxicity of metformin in the ovarian cancer cell lines OVCAR3 and PA-1 . This suOther known cellular actions of metformin include inhibition of complex I of the electron transport chain and oxidative phosphorylation In high glucose conditions, metformin treated cells potentially maintain ATP levels by activation of AMPK and enhancing glycolysis AMPK is known to regulate glycolysis by enhancing glucose uptake and regulation of several key enzymes in the pathway To better understand the intracellular changes that occur with metformin treatment in high and low glucose media, protein levels of several kinases were examined. Both MCF7 and MDAMB231 cells were treated with metformin (8 mM) for one day in medium containing either 25 mM or 2.5 mM glucose. Western blotting was performed to test phosphorylation of AKT and targets of mTOR signaling. The response to metformin was greatly considerably altered in low glucose conditions. In low glucose conditions metformin was found to substantially reduce the phosphorylation of AKT and decrease the phosphorylation levels of targets of mTOR (S6K and 4EBP1), compared to high glucose conditions . Since tIn order to further investigate the role of glucose in protecting cells from metformin-induced death, the effects of glucose were compared to those of related sugars, including the hexoses fructose and galactose. It has previously been reported that fructose and galactose do not contribute significantly to glycolytic metabolism in cancer cells To further examine glucose as a specific carbon source for maintaining glycolysis and the production of ATP in the presence of metformin, we next tested the effects of the drug on oxidative phosphorylation and glycolysis by measuring OCR and ECAR in 25 mM glucose, 25 mM fructose, or 25 mM galactose media. Metformin inhibited OCR in all three conditions , consistThe cell culture experiments described above suggest that limiting the availability of glucose to tumors would enhance the anti-cancer effects of metformin in vivo. To test this a calorie restricted low carbohydrate ketogenic diet was used to lower serum glucose in Balb/c mice. Tumors were established from mouse mammary cancer 4T1 cells and tumor growth was monitored. The low carbohydrate ketogenic diet (KD) significantly reduced serum glucose levels  from ∼6 in vivo. While previous groups have shown that metformin is concentrated within cells, especially the mitochondria in vivo. While commonly used glucose concentration is 25 mM in cell culture growth media, plasma levels of glucose are usually maintained in a range of 5–7 mM. Using low concentrations of glucose in vitro may also be more relevant to metformin’s mechanism in vivo. Using low glucose concentrations can mimic the systemic glucose modulation effects of metformin and potentially provide a more accurate view of the pathways activated within cancer cells.A common criticism of current metformin research is that in vitro concentrations are unattainable et al. Our results support and extend previous observations of enhanced effects of glucose deprivation on metformin activity against tumor cells. Several groups have found synergism between metformin or other biguanides and 2-deoxyglucose, a compound that blocks glycolysis and has similar effects as glucose deprivation Based on our results and the discussion above we propose that high glucose protects against metformin cytotoxicity by providing a fuel source for glycolytic metabolism, which maintains cellular ATP levels even when metformin blocks mitochondrial oxidative metabolism. Enhanced glycolytic metabolism induced by metformin requires activation of AMPK and the availability of glucose allows glycolytic metabolism to run at high efficiency. When glucose is limiting, AMPK is not effectively activated by metformin and cancer cells lack sufficient fuel to maintain glycolytic metabolism. Also, mTOR signalling is blocked in an AMPK-independent manner, further enhancing the metabolic deficiency. Cellular ATP becomes depleted, leading to energy collapse and cell death. This is consistent with previous reports of the ability of AMPK signalling to maintain ATP levels in response to hypoxia, fuel deprivation, and other stressors in vitro may provide a more accurate model of studying cancer, and further highlight the importance of reducing glucose availability to cancer cells. Despite a long history of use as a diabetes medication, the precise cellular actions of metformin are still largely a mystery. Furthermore, the mechanism of action for metformin’s cancer inhibition and killing effects remains unidentified. The results reported in this study contribute to evidence that both the systemic glucose lowering effects and direct cancer cellular effects contribute to metformin’s mechanism of action. The increased cancer cytotoxicity when glucose concentrations were lowered may also be evidence that the multiple pathways affected by metformin enhance one another to promote cancer cell death. As discussed above, alternative carbon energy sources such as fructose and galactose did not alter metformin’s cancer inhibitory and cell death effects in the same manner as increasing glucose concentrations. This suggests that the altered cancer cell metabolism has sacrificed flexibility in carbon source in order to maintain rapid proliferation Notably, when glucose concentration is lowered, the pH change in media induced by metformin treatment no longer occurs. This is likely a result of less lactate production when normal to low glucose concentrations are used. High lactate production has recently been proposed as a protective factor to glucose deprivation Oleksyszyn proposed using a ketogenic diet with metformin as anti-cancer therapy to control glucose levels These results may also impact clinical use of metformin in cancer patients, as ketogenic diets may be utilized to promote low glucose states while still providing necessary energy for many normal cell types. Ketotic states are common in diets focused on carbohydrate reduction, and fasting states do not appear to be harmful to normal cells. Moreover, metformin and caloric restriction have both been shown to increase lifespan in mice With metformin’s extensive safety data from decades of use as a diabetic medication, another role for the drug may be as an adjuvant between other courses of therapy such as surgery, radiation, or traditional chemotherapy. In cancers that are difficult to screen and follow such as ovarian cancer, metformin could potentially have use as a relatively low side effect adjuvant therapy following surgical debulking. Because of metformin’s low side effect profile, therapy could potentially be initiated much sooner following surgery and would generally not interfere with the standard chemotherapy schedule. In patients with lower risk stratification, where chemotherapy is generally not indicated, adjuvant therapies such as metformin may be a useful addition to routine surveillance. Metformin could potentially play a similar role in cancers as tamoxifen maintenance therapy in estrogen receptor positive breast cancers. While metformin’s effect may not be as strong as tamoxifen’s, metformin is also not limited by estrogen receptor status. Past metformin research has shown many benefits: from promising epidemiological data for cancer reduction


Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabilize microtubules in neuronal axons at all stages of development. In Alzheimer's and other tauopathies, tau forms intracellular insoluble amyloid aggregates known as neurofibrillary tangles, a process that appears in many cases to be preceded by hyperphosphorylation of tau monomers. Understanding the shift in conformational bias induced by hyperphosphorylation is key to elucidating the structural factors that drive tau pathology, however, as an IDP, tau is not amenable to conventional structural characterization. In this work, we employ a straightforward technique based on Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) and Hydrogen/Deuterium Exchange (HDX) to provide a detailed picture of residual structure in tau, and the shifts in conformational bias induced by hyperphosphorylation. By comparing the native and hyperphosphorylated ensembles, we are able to define specific conformational biases that can easily be rationalized as enhancing amyloidogenic propensity. Representative structures for the native and hyperphosphorylated tau ensembles were generated by refinement of a broad sample of conformations generated by low-computational complexity modeling, based on agreement with the TRESI-HDX profiles. Full length tau protein htau40) and it’s splice variant isoforms  were ori0 and it’via chemical shift index analysis) . In. In26]. The process of analyzing raw TRESI-HDX data to yield protection factors is summarized in kobs for each peptide. The primary-sequence dependent ‘random coil’ rate constants krc  . . 23]. A decrease in deuterium uptake upon hyperphosphorylation is bounded by G326 and S352, corresponding to the microtubule-associated R3 and R4 regions in the repeat domain. This nucleus of new residual structure could be linked to the decreased affinity of hyperphosphorylated tau for microtubules [114LEDEAAGHVTQ124, corresponding exactly to a region identified as having significant (18%) helical structure in the native ensemble [One region that shows a marked otubules . The othnificant % helicalR between an individual FRODAN model’s solvent accessibility profile and the experimentally-derived HDX profile. A similar approach has been shown to be highly predictive of HDX in weakly structured regions of proteins [In order to characterize the structural ensembles that are predicted from our HDX profiles, we have generated theoretical ensembles for native and hyperphosphorylated tau using Framework Rigidity Optimized Dynamics Algorithm New (FRODAN), a low computational complexity alternative to MD . This approteins .Rmax = 0.3). Nonetheless, the ‘most representative’ structures exhibit all of the features predicted from a qualitative evaluation of the TRESI-HDX profiles. High-ranking structures for the native protein (corresponding to R > 0.275 or the top 10%) are universally compact with many exhibiting precisely the expected global ‘S’-fold (R > 0.16 or the top 10%) are all extended with a few remaining structural nodes surrounding regions of relatively low deuterium uptake  IDP tau. Many of the global and local structural changes are easily rationalized as increasing amyloidogenic propensity in the GSK-3β hyperphosphorylated tau monomer. Specifically, hyperphosphorylation results in release of the global fold, development of new intramolecular interactions in the microtubule binding region and greatly increased exposure of H2, however, H1, which has also been implicated in amyloidogenesis, remains largely sequestered. These data provide structural evidence for a causative role for hyperphosphorylation in tau amyloidogenesis S1 Table(DOCX)Click here for additional data file.S1 FigIntrinsic rates are shown as dashed lines and single exponential fits to the experimental data are shown as a solid line. Residue numbers and protection factor of are provided at the top of each plot.(DOCX)Click here for additional data file.S2 FigNo additional phosphorylation was observed beyond 30 hours.(DOCX)Click here for additional data file.S3 FigSmaller paired helical filaments were observed as early as 48 hours after completion of phosphorylation.(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.


Juvenile Idiopathic Arthritis (JIA) pathology is characterized by a disregulated adaptive immune system. This fact is exemplified by reduced regulatory T cell (Treg) functionality and increased T helper (Th)17 cell activity.In order to further understand the relation between Treg and Th17 development, we investigated the induction of these cells from naïve T cells in early human life.For this, we compared cells from umbilical cord blood (CB) with cells from adult volunteers, as well as cells from schisis patients who undergo several corrective operations in early life. We cultured these cells with antiCD3 for 5 days and subsequently investigated T cell subtype induction by flow cytometry.We show that upon activation, CB cells easily adopt a Treg phenotype, whereas Th17 cells can not be induced. Although production of inflammatory cytokines is dramatically lower in CB cells, addition of Th17 inducing cytokines IL-1b or IL-6 did not influence this phenomenon. Instead, an increased programmed death (PD)-1 signaling and decreased Th17 defining transcription factor RORC activation underlie the prevention of inflammatory T cell activity. The propensity for Treg development is a phenomenon which we still observed in samples from 12 month old children, whereas we found Th17 cell development as early as three months after birth. This development in the first year of life is likely related to prevention of aberrant T cell responses towards self and the developing microbiome.These data give more insight into the development of the immune system and inflammation and can lead to novel targets in JIA therapy, aiming at the balance between Treg and Th17 cells.None declared.


Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair.8,5′ cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases  were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases , DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal  Oxidative DNA damage represented by a spectrum of bases or sugar modifications is incurred by reactive oxygen species that arise from endogenous biochemical processes and can also be induced exogenously by environmental agents such as chemical compounds  or ionizing radiation. Oxidative DNA lesions in nuclear and/or mitochondrial genomes result in perturbations to cellular DNA replication and transcription; furthermore, their accumulation predisposes individuals to accelerated tissue aging, neurodegeneration, and cancer. A variety of oxidative DNA lesions exist, and recent efforts have focused on establishing meaningful relationships between the accumulation of a particular oxidative lesion and aberrant cellular and organismal phenotypes as well as the pathways for repairing and tolerating the spectrum of oxidative DNA lesions in vivo is supported by observations that under controlled environmental conditions, cPu lesions accumulate with age in wild-type mice compared to young mice, and also in congenic progeroid Ercc1−/Δ mice deficient in the XPF-ERCC1 endonuclease implicated in NER A class of endogenous oxidative DNA lesions that has attracted considerable attention for potential roles in human disease and mutagenesis is 8,5′-cyclopurine-2′-deoxynucleoside (cPu) While the effects of cPu lesions on the functions of DNA polymerases E. coli (Ec) EcDnaB Thermoplasma acidophilum (ta) XPD Recombinant human FANCJ 32P-end-labeled partial duplex forked substrates labeled and annealed as described previously Table S1.Cyclo dA and Cyclo dG phosphoramidites were purchased from Berry & Associates . Synthesis and purification by polyacrylamide gel electrophoresis (PAGE) of oligonucleotides including those which used cyclo dA or cyclo dG phosphoramidites for synthesis was performed by Loftstrand Labs . The DNA substrates used were 5′-Helicase reactions were carried out in 20 µl volumes which contained 10 fmol of the forked duplex DNA substrate carrying the cyclopurine lesion either in the top, bottom, or neither strand. The reactions were performed using previously described conditions FANCJ . Unless For helicase sequestration experiments, FANCJ (9.6 nM) or RECQ1 (8.8 nM) was preincubated for 3 min at 30°C or 37°C, respectively, with ATP (2 mM) and the indicated amounts of unlabeled forked duplex competitor DNA substrates containing the cdA lesion in the top strand, bottom strand, or neither strand. Subsequently, 10 fmol of radiolabeled 19 bp forked duplex, also known as the tracker substrate 200 (to serve as protein trap) was added simultaneously to the reaction mixture and incubated at 30°C or 37°C. Aliquots (20 µl) of the reaction mixture were quenched at 10-sec intervals with 2X Stop Buffer containing a 10-fold excess of unlabeled oligonucleotide with the same sequence as the labeled strand. Products of helicase reaction mixtures were then resolved on 12% polyacrylamide gels and visualized as described under “Standard helicase assays”.For protein trap kinetic helicase assays, FANCJ (0.6 nM), DDX11 (1 nM), EcUvrD (1 nM), and RECQ1 (7 nM) were preincubated for 3 min at 24°C with 5 nM of the radiolabeled forked duplex DNA substrate carrying the cyclo dA lesion in the top, bottom, or neither strand. After 3 min, ATP (2 mM) and 500 nM oligo dTTable S1). The control substrate consisted of the same oligonucleotides except there was no cPu lesion present in either strand.Up to this point, there has been no assessment of the effect of cPu base damage on DNA unwinding by any helicase. Given that cPu lesions arising from oxidative stress are believed to be fairly abundant We began with the human RecQ DNA helicase BLM implicated in the hereditary chromosomal instability disorder Bloom Syndrome, and also the human RECQ1 DNA helicase which is not yet reported to be genetically linked to a human disease but thought to play a role in cancer suppression RECQ1 was inhibited by the cyclo dA in the translocating strand, showing nearly a four-fold reduced level of helicase activity at the 9 nM RECQ1 concentration where 80% of the control substrate as well as the substrate with the lesion in the non-translocating strand was unwound, compared to only 20% of the substrate with the cyclo dA in the translocating strand , whose human homologue is linked to Xeroderma Pigmentosum We next tested the The modest effects of a cyclopurine seen with EcDinG and DDX11 led us to investigate how taXPD might behave when it encounters the cycloadduct damage. This question was particularly interesting to us because taXPD is believed to play a critical role in DNA damage verification during a relatively early step of NER A series of experiments were also performed with FANCJ helicase. FANCJ unwound the DNA substrates in a protein concentration-dependent manner in the 15-min incubation. However, in this case there was markedly less unwinding by FANCJ of the DNA substrate harboring the cyclo dA  Previously, we observed that certain forms of DNA damage to the base  or top (non-translocating) strand had no discernible effect on the ability of RECQ1 to unwind the tracker substrate compared to preincubation with the control undamaged forked duplex  In order to further examine the effect that cPu had on helicase activity, rates of unwinding for select helicases were conducted under single-turnover conditions. The SF2 Fe-S cluster helicase FANCJ is strongly inhibited by the cyclopurine lesion, a result that is consistent with the strand-specific inhibition observed under the multi-turnover conditions. In contrast, the rate of DNA unwinding by the Fe-S cluster helicase DDX11 is only modestly affected by the cyclopurine damage, a result that is also consistent with what was observed under multi-turnover conditions. Thus, DDX11 possesses an intrinsic ability to unwind and bypass the cyclopurine, even in the translocating strand, without invoking the requirement for additional helicase molecules to load on the same substrate and help stalled DDX11 helicase molecule translocate forward. In the case of 3′ to 5′ helicases, SF1 helicase EcUvrD is detectably affected by the translocating strand cyclopurine under single-turnover conditions, whereas the enzyme fully tolerated the lesion under multi-turnover conditions. These results suggest that the ability of EcUvrD to efficiently unwind the DNA substrate with the translocating strand cyclopurine lesion in a protein concentration manner under multi-turnover conditions may be in part due to the loading of more than a single functional helicase molecule on the DNA substrate during the reaction incubation period. In contrast, the 3′ to 5′ helicase RECQ1 is more negatively affected in its ability to unwind the DNA substrate with the translocating strand cyclopurine lesion under either single-turnover or multi-turnover conditions. Thus, inhibition of both RECQ1 and FANCJ stalling by the cyclopurine lesion in the translocating strand is not efficiently overcome by increasing the number of helicase molecules in solution.The selective deterrence of DNA unwinding by the RecQ helicases when the cPu lesion resided in the translocating strand is interesting in light of recent experimental findings that BLM . In contrast, the sequence-related ChlR1 and EcDinG helicases fully tolerated the cPu lesion in either the translocating or non-translocating strands. Therefore, the Fe-S cluster helicases ChlR1 and EcDinG are likely to unwind damaged DNA by a mechanism that is distinguishable from FANCJ.FANCJ was the sole Fe-S cluster helicase tested that displayed a sensitivity similar to that of the RecQ helicases in which inhibition was observed only when the cPu adduct resided in the helicase translocating strand. To our knowledge, strand-switching by FANCJ or any Fe-S helicase for that matter has not been examined, leaving in question the mechanistic basis for inhibition of FANCJ helicase by the cPu lesion. Unlike most, if not all RecQ helicases, FANCJ does not efficiently catalyze strand annealing of pre-existing complementary single-stranded DNA molecules Ferroplasma acidarmanus XPD (faXPD) utilizes its conserved Fe-S cluster domain as a damage sensor pocket, which scans for lesions when translocating on a DNA substrate Previously, the effects of more classic NER lesions such as the UV photoproduct CPD have been examined for their effects on DNA binding and unwinding by various archaeal XPD helicases Sulfolobus acidocaldarius XPD and Endonuclease III) with redox active Fe-S clusters can utilize charge transport along the DNA double helical molecules as a means for detecting DNA lesions and recruiting other repair proteins It is also possible that the differences in sensitivity of Fe-S cluster helicases to DNA damage such as a cPu adduct could be due to other mechanisms. Recent experimental evidence from the Barton lab suggested that DNA repair proteins Click here for additional data file.


The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors.TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations.We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.The online version of this article (doi:10.1186/s13059-014-0530-z) contains supplementary material, which is available to authorized users. EGFR and PDGFRA in glioblastoma multiforme (GBM) using fluorescence in situ hybridization (FISH) and array-comparative genomic hybridization on multiple regions within primary tumors [Regional heterogeneity of mutations has been observed in a variety of tumor types ,2. This y tumors ,4. Despiy tumors . This isWe recently developed a method to identify low frequency mutations across known cancer genes  using thTo assess heterogeneity within gliomas, we dissected each of 14 tumors into 3 to 5 regions per tumor  secondary to the contamination of a subset of control tissues with tumor cells. After applying our filters . We used DNACopy  to segmePDGFRA and KIT, and three tumors had amplification of EGFR. We validated copy number estimates for a subset of calls using a variety of different methods, including Taqman quantitative PCR , as well as whole genome sequencing (in tumor BI15 for EGFR). MIP copy number estimates of EGFR were highly correlated (R2 = 0.90) with delta Ct obtained by Taqman qPCR when compared across all 62 regions sequenced  inactivating mutation (R248H) in TP53 in only two regions of the tumor (A and B). This tumor had high allele fraction mutations in RB1 exclusively in two other regions (D and E) within the same tumor. Both TP53 and RB1 mutations were present at trace levels (<1%) within region C. As clinical workup indicated that BI09 had an IDH1 mutation, we investigated all regions of this tumor by Sanger sequencing and found that regions A to E shared the IDH1 R132H mutation. Sanger sequencing also validated the TP53 mutation in regions A and B as well as the RB1 mutation in regions D and E  in region D of the glioblastoma BI12 and was not detected in at least one other region , we observed heterogeneity for clinically relevant mutations in 3 of 14 (21%) glial tumors. These include point mutations in TP53 were found to stratify outcomes, with median survival of 71 versus 16 months in patients with mutant versus wild-type TP53, respectively [TP53 status from different regions within an individual tumor nevertheless show the potential of genetic heterogeneity to confound the assignment of prognostication based on the detection of specific molecular markers.Historically, in anaplastic oligodendroglioma with intact 1p, mutations in ectively . While nPDGFRA. Our finding of regional heterogeneity of PDGFRA within tumors BI05 and BI06 confirms recent work by Sottoriva et al. and others and suggests that a single biopsy may not be sufficient to allow for informed application of targeted therapies against these presumed oncogenic drivers [et al. [PIK3CA and PTEN mutation was detected at the first recurrence, and heterogeneity of PIK3CA, TP53 and EGFR mutation was detected at the second recurrence.In addition, decision-making regarding the use of receptor tyrosine kinase inhibitors could be influenced by the status of amplifications/mutations in  drivers ,4,13. ClThis study used tumor-adjacent brain to serve as a ‘control tissue’ to help identify somatic mutations. However, after extracting DNA and investigating mutations, we observed that in several cases the grossly normal brain tissue actually contained a large fraction of infiltrating tumor cells. This aspect of our study ultimately complicated the process of calling somatic point mutations (requiring filtering against databases of germline variation) and copy number . While blood would be the most appropriate material to use as a control we did not have institutional review board approval to collect blood at the time these experiments were conducted .EGFR VIII deletion commonly found in GBM. However, one can imagine expanding this assay to consider amplifications and deletions with smMIPs by tiling probes at higher density and incorporating known SNP positions to aid in identifying cases of loss of heterozygosity. One could also capture additional glioma-relevant genes like IDH1 and IDH2 by adding probes targeting these genomic regions.In this study we were able to identify amplification of only a subset of genes of interest, as some genes had too few probes to accurately determine copy number. Our study also did not detect genomic rearrangements and deletions such as the Our investigation focused on regional heterogeneity within a tumor, instead of the microscopic heterogeneity that is likely present within a given tumor biopsy. As we performed the smMIP assay on DNA extracted from tissue pieces that likely contained millions of cells, we would likely miss cases of heterogeneity where only a small population of cells within a biopsy contained a mutation (such as an amplification). Use of techniques such as immunohistochemistry, FISH and, more recently, single cell sequencing remain necessary to characterize the extent of microscopic heterogeneity in tumors.These results validate the smMIP approach as a scalable and cost-effective platform for deep sequencing of cancer genomes to examine subclonal variation. Despite deeply sequencing multiple sections of 14 tumors, our survey required only one lane of sequencing on the Illumina HiSeq because we focused on well-known gene targets of mutation in cancer. In contrast to the technique used by a similar investigation , our metWe find multiple instances of regional heterogeneity in clinically relevant cancer genes within malignant gliomas at the time of diagnosis. We also demonstrate a scalable technique that can be used to efficiently characterize regional genetic heterogeneity for both point mutations and copy number alterations in tumors. Future challenges will include how best to interpret cases of intratumoral heterogeneity and test its impact in the context of clinical trials using targeted therapy approaches.Freshly resected brain tumor specimens from adult patients were obtained with informed consent as part of the Genomics Big Idea pilot program (UW/FHCRC). Tissue, patient demographics and final diagnosis were obtained in accordance with protocols approved by the institutional review board at the University of Washington. Tumors were divided into three to five regions, depending on size. Tissue from each region was then subdivided into four pieces for use in next generation sequencing, histology, cell culture and xenotransplantation Figure . In 10 cet al. [The smMIP assay was used to genotype candidate genes. Probes were previously designed by Hiatt et al.  against et al. . After sInitial analysis steps through to read mapping were performed as previously described , except phred base quality ≥30, ≥30× coverage and the absence of a neighboring homopolymer run of four bases or more  that had fewer than 15 probes with sufficient coverage in the control tissue (BI12).We compared read depth of smMIP-targeted regions in each tumor against that of the control tissue BI12 to identify high level gene amplifications in tumors. We restricted the copy number analysis to targets with greater than 30× coverage in control tissue and a GC content ranging from 30 to 60%. To reduce the number of potential artifacts remaining, we removed from consideration  12 genes  against positions within EGFR and PDGFRA detected using smMIP sequencing were confirmed using Taqman qPCR analysis. DNA from each region was analyzed in quadruplicate using commercially available probes against PDGFRA  and EGFR . Reference primers amplified a fragment from TERT . Finally, to compare sensitivity of the smMIP approach, all regions from all tumors were assayed in duplicate for EGFR copy number.Tumors with regional heterogeneity in EGFR FISH was performed using commercially available probes  with DAPI counterstain using standard methods. Slides were imaged using an Olympus DP72 digital camera mounted on a Nikon E400 microscope. Fifty nuclei were scored for each region. EGFR amplification was called if more than 10% of nuclei either contained many EGFR signals or exhibited a EGFR:CEP7 ratio greater than 2. 1p19q deletion FISH was performed using commercially available probes  using standard methods.Immunohistochemistry for IDH1 and p53 was performed on 4-micron paraffin sections using mouse anti-human p53 clone  and mouse anti-human IDH1 R132H . All tumors were investigated for IDH1 mutation by neuropathology, while only a subset of tumors was investigated for p53 expression by immunohistochemistry  under accession number SRP049298.


Cytomegalovirus (CMV) is the most common congenital infection and is the leading non-genetic cause of neurological defects. CMV infection in early life is also associated with intense and prolonged viral excretion, indicating limited control of viral replication. This review summarizes our current understanding of the innate and adaptive immune responses to CMV infection during fetal life and infancy. It illustrates the fact that studies of congenital CMV infection have provided a proof of principle that the human fetus can develop anti-viral innate and adaptive immune responses, indicating that such responses should be inducible by vaccination in early life. The review also emphasizes the fact that our understanding of the mechanisms involved in symptomatic congenital CMV infection remains limited. Cytomegaloviruses (CMV) belong to the betaherpesvirus family and establish a lifelong infection in their host. A number of species can be infected by CMV but each virus has adapted to its host. Specifically, the virus infecting humans is unable to establish a productive infection in animals . This reHuman CMV is the most common congenital infection, affecting 0.2–2.2% of all live births. Congenital CMV infection is the leading non-genetic cause of neurological defects, including mental retardation, cerebral palsy, and hearing impairment , 4. ApprPost-natal CMV infection is usually asymptomatic but severe disease, including respiratory distress syndrome and sepsis, can develop in very premature babies . Howeverγδ T lymphocytes and natural killer (NK) cells are innate lymphocytes that develop and differentiate early during fetal life (reviewed by Vermijlen and Prinz in this Research Topic). γδ T cells express a receptor formed of a γ and a δ chain on their cell surface and are a prototype of unconventional T cells: they can react rapidly during the course of an immune response and they are major histocompatibility complex (MHC)-unrestricted. NK cells are innate lymphocytes that do not possess Rag recombinase-dependent rearranged antigen receptors and that are activated by the balance between activating and inhibitory invariant receptors.−) γδ T cells. Expansions of Vδ2− cells are also observed in CMV-infected healthy adults and hematopoietic stem cell (HSC) transplanted patients  as a model of mDCs, CD34+ bone marrow progenitor cell-derived Langerhans cell-type DCs (LDCs) as well as differentiated mDCs and pDCs purified from peripheral blood. In the presence of CMV, adult mDCs, moDCs, and pDCs produce high levels of pro-inflammatory cytokines and type I IFNs  can be classified into myeloïd DCs (mDCs), whose main function is to prime and functionally polarize naive T lymphocytes, and plasmacytoïd DCs (pDCs) that produce high levels of type I interferons (IFNs). The interactions between CMV and human DCs have been studied e I IFNs –41. Thise I IFNs , 43. Howe I IFNs . Inhibit in Ref. ]. These he virus .In vitro studies have shown that cord blood-derived moDCs and adult moDCs are equally susceptible to CMV infection . Most of our current understanding of the biology of DCs in early life comes from studies of the interactions between pathogen-associated molecular patterns (PAMPs) with specific innate receptors. In contrast, how DCs interact with complex pathogens remains poorly understood. nfection . As obsenfection . Similarwith CMV . In contwith CMV . The redwith CMV . As discwith CMV –50. AlsoMore than 20 years ago, studies of mouse CMV infection demonstrated the central role played by αβ CD4 and CD8 T lymphocytes in the control of CMV infection –53. Clinin vitro stimulation with viral antigens and both subsets express high levels of perforin and granzyme and are cytolytic  equivalent to the one acquired by adult T cells . These results suggest that the increased frequency of cytokine-producing CD8 T cells observed during the first year after congenital CMV infection is related to an increase in their functional capacity , 78. IntStudies of mouse CMV indicate that neutralizing antibodies protect against primary infection and reactivation by limiting viral dissemination in tissues and that transfer of B lymphocytes from immune animals protects immunocompromised hosts against lethal infection , 95. Ant+CD20+CD21low) and atypical (CD27−CD20+CD21low) memory B cells are detected in pregnant women diagnosed with primary CMV infection, particularly in those who were viremic at the time of analysis . Study of the B cell response to CMV in early life should help us understand the potential contribution of this subset in viral control and may also provide important information on the ontogeny of B cell responses during fetal life.Very little is known about the B cell response to CMV infection in early life. Studies have shown that CMV-specific IgM are commonly detected in congenitally infected newborns –111. As Trypanosoma cruzi, and Toxoplasma gondii infections, indicating the capacity of the fetal immune system to respond to a range of different intracellular pathogens , the enzyme responsible for template-independent nucleotide additions, is detected around 1 week of life and this activity correlates with the acquisition of an increased diversity of CDR3 sequences . In humaThe development of effector and memory B cell responses in early life remains poorly understood. This gap of knowledge remains a major limitation to the improvement of neonatal immunization strategies , 129. ThAs discussed above, our recent observations indicate that effector CD8 and CD4 T cells are functionally exhausted during primary CMV infection and suggest that this phenomenon is particularly intense in early life. This intense degree of functional exhaustion may be related to a more intense CMV replication in the fetus and young infant than in the adult. A similar association between functional exhaustion and intense viral excretion was observed following primary CMV infection of juvenile rhesus macaques . As descin utero and after birth. Ganciclovir is recommended for the prevention of long-term neurological sequelae in symptomatic infants (Finally, our understanding of the pathogenesis of symptomatic congenital CMV infection in humans remains very limited. However, mouse studies have provided important insights in the potential mechanisms involved and suggest that the immune response to CMV may play a dual role in the neurological pathology of neonatal mice by controlling CMV replication but also by inducing inflammatory responses promoting developmental abnormalities [reviewed in Ref. ]. The fe infants . If the  infants . HoweverThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, the role of the EP1 receptor is complex and depends on the type of brain injury. This study is focused on the investigation of the role of the EP1 receptor in a controlled cortical impact (CCI) model, a preclinical model of traumatic brain injury (TBI). The therapeutic effects of post-treatments with a widely studied EP1 receptor antagonist, SC-51089, were examined in wildtype and EP1 receptor knockout C57BL/6 mice. Neurological deficit scores (NDS) were assessed 24 and 48 h following CCI or sham surgery, and brain immunohistochemical pathology was assessed 48 h after surgery. In wildtype mice, CCI resulted in an obvious cortical lesion and localized hippocampal edema with an associated significant increase in NDS compared to sham-operated animals. Post-treatments with the selective EP1 receptor antagonist SC-51089 or genetic knockout of EP1 receptor had no significant effects on cortical lesions and hippocampal swelling or on the NDS 24 and 48 h after CCI. Immunohistochemistry studies revealed CCI-induced gliosis and microglial activation in selected ipsilateral brain regions that were not affected by SC-51089 or in the EP1 receptor-deleted mice. This study provides further clarification on the respective contribution of the EP1 receptor in TBI and suggests that, under this experimental paradigm, the EP1 receptor would have limited effects in modulating acute neurological and anatomical pathologies following contusive brain trauma. Findings from this protocol, in combination with previous studies demonstrating differential roles of EP1 receptor in ischemic, neurotoxic, and hemorrhagic conditions, provide scientific background and further clarification of potential therapeutic application of prospective prostaglandin G-protein-coupled receptor drugs in the clinic for treatment of TBI and other acute brain injuries.Brain injuries promote upregulation of so-called proinflammatory prostaglandins, notably prostaglandin E TBI is a complex disorder resulting from coexisting primary and secondary mechanisms such as mechanical brain damage, parenchymal subarachnoid hemorrhages, excitotoxicity, brain edema, and activation of neuroinflammatory pathways −/− mice) in a preclinical controlled cortical impact (CCI) model of TBI, demonstrated that a single treatment with its selective antagonist administered after experimental TBI would be beneficial in reducing acute neurological deficits, hippocampal edema, and inflammatory reactions such as gliosis and secondary microglial activation. With the known molecular mechanisms of EP1 and FP receptors in intracellular Ca2+-signaling −/−).Our previous study concerning the role of the FP receptor in TBI, which is structurally and functionally related to the EP1 receptor, using pharmacological tools and genetic deletion of this receptor , with access to food and water ad libitum. All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize the pain and distress of the experimental animals.Age-matched adult (2–4 months) wildtype (WT) and EP1−/− C57BL/6 genetic backgrounds. Physiological parameters , cerebral vessel diameters, and cerebral arterial vasculature morphology are not significantly different between EP1−/− and WT mice All mice used in the study were obtained from the in-house breeding colony and the WT and EP1Pharmacological treatments with the selective EP1 receptor antagonist SC-51089 were performed with minor modifications Neurobehavioral deficits were assessed using a 24-point NDS scale in all mice 24 and 48 h after CCI or sham surgeries, as we described in detail elsewhere All subjects were survived for 48 h (2 days) post-injury and were then sacrificed. A series of eight 30-µm-thick coronal sections were obtained throughout the entire brain and were processed for histological analysis as described previously For quantitative histopathological and immunohistochemical data, Student’s t-test was used to compare the drug-treated group with the matching vehicle-treated control group or between genotypes. Statistical comparisons among multiple groups were done using one-way ANOVA. For non-parametric data , the Mann-Whitney rank sum test was used. Data are presented as the mean ± standard error, and P values <0.05 were considered significant 2+-modulating EP1 receptor −/− mice in separate cohorts. At the 48-h time point, CCI produced consistently significant cortical lesions characterized by morphological alternations in cellular morphology and density, including cell and tissue loss  and parenchymal ICH, whereas in sham animals, only marginal changes were observed in the cortical tissue due to craniotomy surgery. The significant hippocampal swelling (edema) previously reported in this model Based on the previous data indicating that blockade or ablation of Ca3 and the median NDS values were 5.5 (ranged between 3 and 8) and 6 (ranged between 3 and 8) at 24 and 48 h after CCI, respectively. These results revealed no significant differences between lesion volumes in the groups with different SC-51089 treatment regimens and vehicle .In the first set of experiments, we tested whether post-treatment with SC-51089 could limit anatomical deficits after CCI in WT mice using a dose and treatment regimen reported to improve outcomes in a mouse model of ischemia induced by transient middle cerebral artery occlusion −/− mice. The CCI procedures performed with the same parameters resulted in similar lesion size and hippocampal swelling with values not significantly different compared to WT mice from the corresponding control group. Similar to WT mice, no considerably significant edema or cortical alternations were observed in EP1−/− mice 48 h following sham procedures  −/− mice exhibit worsened outcomes of brain lesion volumes and neurological and sensorimotor deficits compared with WT mice, possibly affecting microglial phagocytosis The data suggest that the prostaglandin receptors, which mediate most of their actions through mobilization of intracellular Ca2α FP receptor, which exerts Ca2+-modulating effects similar to the EP1 receptor in ischemic stroke and TBI, and these data indicate that despite considerable differences between the mechanisms of primary injury in these models, beneficial effects were obtained with similar treatment regimens et. al.Our hypothesis of the involvement of the EP1 receptor cascade in the pathologies following TBI was based on the wealth of literature data showing COX-2 upregulation following brain injuries such as stroke and trauma, and the data demonstrating the neuroprotective role of pharmacological and genetic ablation of the EP1 receptor in the in vitro models of excitotoxicity and hemin toxicity −/− mice −/− mice with and without drug treatment, suggesting appropriate SC-51089 bioavailability with the treatment regimen used in this study. The effective doses of SC-51089 in published studies focusing on ischemia and excitotoxicity are between 5 and 100 µg/kg −/− mice, and all drug treatment experiments performed in WT mice were replicated in EP−/− mice. The lack of effects of the EP1 receptor pharmacological blockade or knockout on either neurobehavioral and anatomical outcomes or Iba1 and GFAP immunohistochemistry in our in vivo CCI model of TBI, which is in contrast with the neuroprotective effects observed in the related in vitro studies −/− mice in contrast to those observed in an ICH model of hemorrhagic stroke The main goal of this study was to determine whether pharmacological interventions targeting COX-2 downstream cascades involving the EP1 receptor could be beneficial for the management of TBI and the development of novel therapeutic strategies for its effective treatment. The experimental design of drug treatment was based on the published study performed in models of excitotoxicity and ischemia in the WT and EP12-mediated vasoconstriction and that male EP1−/− mice have significantly reduced blood pressure compared to WT mice −/− mice have enhanced cerebral blood flow at reperfusion in a ischemic stroke model −/− and WT mice or between FP−/− and WT mice in our related studies 2+ signaling such as extensive glutamate release and calpain-dependent cortical necrosis The differential roles of the EP1 receptor in acute brain injuries might be attributed to its well-known vascular effects. The reports indicate that activation of the EP1 receptor is involved in PGENeuroinflammatory processes involving activation of reactive microglia and gliosis are well-established hallmarks of TBI and the data show that neuroinflammation following brain injuries is associated with COX-2 expression In conclusion, the data obtained in this study extend our understanding of pathophysiological downstream COX-2 pathways in acute brain injuries. Based on the literature data demonstrating that COX-2 upregulation and increased levels of prostaglandin precursor start within hours


Arabidopsis thaliana and many other plants from temperate climates increase in freezing tolerance in a process termed cold acclimation. However, the correct timing and rate of deacclimation, resulting in loss of freezing tolerance and initiation of growth is equally important for plant fitness and survival. While the molecular basis of cold acclimation has been investigated in detail, much less information is available about deacclimation. We have characterized the responses of 10 natural accessions of Arabidopsis thaliana that vary widely in their freezing tolerance, to deacclimation conditions. Sugar, proline and transcript levels declined sharply over three days in all accessions after transfer of cold acclimated plants to ambient temperatures, while freezing tolerance only declined in tolerant accessions. Correlations between freezing tolerance and the expression levels of COR genes and the content of glucose, fructose and sucrose, as well as many correlations among transcript and solute levels, that were highly significant in cold acclimated plants, were lost during deacclimation. Other correlations persisted, indicating that after three days of deacclimation, plant metabolism had not completely reverted back to the non-acclimated state. These data provide the basis for further molecular and genetic studies to unravel the regulation of deacclimation.During low temperature exposure,  Cold acclimation, the increase in freezing tolerance during exposure to low, but non-freezing temperatures, is a common response of plants from temperate climate zones to seasonal low temperature234However, cold acclimation is only one, albeit the most thoroughly investigated, aspect of successful winter survival. Of equal importance for plant fitness and survival is the correct timing and rate of the loss of freezing tolerance (deacclimation) in spring when plants undergo the transition to growth and development , after 14 d of cold acclimation (ACC) and after 1 d, 2 d and 3 d of deacclimation (DEACC1-3). In agreement with previous resultsCOR6.6 and COR15A and the lowest for the transcription factor genes CBF1-3, ZAT6 and ZAT12. As expected for this selection of genes, they all showed strong cold induction in all accessions. During deacclimation, the transcript changes varied strongly among both the genes and the accessions. In general, the less freezing tolerant accessions showed stronger reductions in transcript abundance than the tolerant accessions  correlations between LT50 NA and the content of the investigated solutes or transcripts for the 10 accessions investigated here Hydrangea spec.26Trifolium repens L.)Vaccinium spec.)3031While it is widely assumed that a shift of plants from cold to warm conditions results in a rapid loss of freezing tolerance12Hydrangea26Metabolite profiling of Arabidopsis Col-0 revealed that the global composition of the metabolome after 24 h of deacclimation approached that of non-acclimated plantsCOR gene transcripts and sugar content were lost during deacclimation. Interestingly, most of the correlations between CBF and COR transcripts that were evident in the cold acclimated plants also persisted during deacclimation, indicating that the CBF regulon remained tightly coordinated during this process. On the metabolic level, this coordination extended from CBF1 and CBF2 through GolS3 expression to the final product Raf. In addition, all steps of this pathway remained correlated with leaf freezing tolerance, indicating its central importance in the regulated decline in freezing tolerance during deacclimation. In striking contrast, transcript levels of COR6.6 and COR15A were no longer correlated with LT50 after 3 d of deacclimation, while transcript levels of COR47 and COR78 remained correlated. A strongly reduced expression of the cold induced genes COR15A, COR15B, COR47 and GolS3 during deacclimation was also reported for Col-0 from microarray hybridization experimentsChanges in transcript levels of cold induced genes during deacclimation were more variable both among genes and among accessions. Most transcripts were more strongly reduced in abundance during deacclimation in sensitive than in tolerant accessions. A strong reduction in the abundance of transcripts of almost all cold induced genes during deacclimation has previously been shown for the accession Col-017Deschampsia antarctica) decreased strongly during deacclimation, in parallel with the corresponding transcript levelsWhile gene expression data during deacclimation have rarely been reported, the content of cold induced dehydrin proteins during deacclimation has been reported in several studies. All three dehydrins investigated in blueberryIn conclusion, the present study is the first to provide a systematic investigation into the natural variation of plant deacclimation on the phenotype, metabolite and transcript levels. It shows that deacclimation is a tightly regulated process with a strong genetic basis and thereby indicates the way for further genetic and molecular studies in this important and largely neglected area of plant-environment interactions.Arabidopsis thaliana accessions that were previously characterized for their acclimated and non-acclimated freezing tolerance as part of a larger population of 54 accessions−2 s−1 until 42 days after sowing (non-acclimated plants). For cold acclimation, plants were transferred for an additional 14 days to a 4 °C growth cabinet at 16 h day length at 90 μE cm−2 s−1 . Soluble sugars were extracted and quantified by high performance anion exchange chromatography as described previously2341Total RNA was extracted from rosette material pooled from five plants in each of three replicate experiments using either Trizol reagent (Invitrogen) or a lysis buffer containing 100 mM Tris-HCl (pH 8.5–9.0), 25 mM EDTA, 25 mM EGTA, 2% SDS, 100 mM ß-mercaptoethanol supplemented with phenol, chloroform and isoamylalcohol. RNA samples were DNase treated . RNA quality controls, first strand cDNA synthesis and cDNA quality controls were performed as described recentlyrcorr from the package Hmisc.Spearman rank order correlation analysis was performed in R using the command How to cite this article: Zuther, E. et al. Time-dependent deacclimation after cold acclimation in Arabidopsis thaliana accessions. Sci. Rep.5, 12199; doi: 10.1038/srep12199 (2015).