Lentivirus-mediated knockdown of hypoxia-inducible factor 1α (HIF1α) or of Notch signaling components had a similar effect, and pharmacologic inhibition of HIF or Notch reduced the number of proliferating Pax7+ cells. In contrast, a prolonged hypoxic response induced either by uncoupling of respiration from oxidative phosphorylation or through HIF stabilization by dimethyloxalylglycine (DMOG) had an immediate anti-inflammatory effect. Although significant satellite cell proliferation occurred in presence of DMOG, expression of differentiation markers was affected. These results emphasize the importance of transient hypoxia as opposed to prolonged hypoxia for myogenesis.The microenvironment of the injury site can have profound effects on wound healing. Muscle injury results in ischemia leading to short-term local hypoxia, but there are conflicting reports on the role of hypoxia on the myogenic program in vivo and in vitro. In our rat model of mitochondrial restoration (MR), temporary upregulation of mitochondrial activity by a cocktail of organelle-encoded RNAs results in satellite cell proliferation and initiation of myogenesis. We now report that MR leads to a transient hypoxic response in situ. Inhibition of hypoxia by lowering mitochondrial O Regeneration of adult mammalian muscle postinjury is a complex process involving tightly regulated molecular events that follow an initial inflammatory response, including infiltration of immune cells and clearance of cellular debris at the injury site, activation and proliferation of satellite cells (SCs) to form myoblasts, and their subsequent differentiation and fusion to form myofibers . In para2 concentration. Local hypoxia has been observed at the injury site after incision, with gradual rise of O2 levels after 4–10 days [+MyoD− cells and decline of Pax7+MyoD+ cells, without an overall effect on myoblast proliferation under hypoxia, have been reported [α in muscle cells did not affect cellular proliferation, but KD of HIF1α in myeloid cells resulted in reduced macrophage infiltration and numbers of proliferating cells [One of the consequences of muscle injury caused by burn, laceration, or freezing is the rupture of blood vessels resulting in ischemia and lowering of local O–10 days . Prolong–10 days ; accumulreported . Tissue-ng cells , suggestWe have previously reported that restoration of mitochondrial activity at the injury site of rat muscle by administration of a cocktail of mitochondrion-targeted RNAs remarkably accelerates satellite cell activation and initiation of the myogenic program . Since, Sprague-Dawley rats received needle injury in the hind limb quadriceps muscle. Lesion circumference was measured daily. At the height of inflammation (6 d after injury), a cocktail of three polycistronic RNAs encoding various portions of the rat mitochondrial genome, or control D arm oligonucleotide, was administered at the injury site as ribonucleoprotein (RNP) complexes with RNA import complex (RIC), as described .Following administration of pcRNAs, 0.9% Hypoxyprobe-1  in PBS was injected intraperitoneally at a dosage of 60 mg/kg body weight. After 60 min, the animals were sacrificed and the muscle was excised and immediately fixed with paraformaldehyde. Sections were stained with FITC-conjugated monoclonal antibody against pimonidazole (1 : 500) and DAPI or anti-COII antibody, for confocal microscopy.+ satellite cells by FACS as described [Mononuclear cells isolated from regenerating muscle were analyzed for Pax7escribed .α protein [γ-Secretase inhibitor XX  inhibits processing of Notch protein to active NICD [μL of 2 mM HIF-In, 2 mM DMOG, 100 μM γ-secretase inhibitor, or 1 mM DETA-NO) was injected into the wound site 4 h after pcRNA administration. Chloramphenicol (100 μg/mL), m-chlorocarbonylcyanide phenylhydrazone , or antimycin A  was injected in 0.1 mL at the wound site 3 h following administration of pcRNA and the muscle was harvested after a further 0.5–1 h.HIF inhibitor -acetylamino)-4-hydroxybenzoic acid methyl ester) specifically prevents hypoxia-induced upregulation of HIF1 protein . Dimethy protein . γ-Secreive NICD . Diethyl6 TU/mL; Sigma Aldrich; thrice daily) expressing nontargeted or gene-targeted short hairpin (sh) RNA (see Supplementary Table S1 available online at http://dx.doi.org/10.1155/2015/961307).At 3 d and 4 d after injury, the injury site was infected with lentivirus pLKO.1-CMV-tGFP .The rate of Oμm) were fixed, incubated with the appropriate primary (Supplementary Table S2) and Alexa Fluor (AF) 395 (blue), AF 488 (green), or AF 633 (red) labeled secondary antibody (1 : 1000), and imaged with the Nikon A1R confocal microscope using the corresponding laser lines.Muscle sections (10 Blots of muscle protein were probed with the appropriate antibody (Supplementary Table S2) and signals detected by enhanced chemiluminescence.t−ΔΔC, where ΔCt = Ct (target gene) − Ct (β-actin), Ct is the critical threshold of the SYBR Green signal, and β-actin is the internal control.Muscle RNA was reverse-transcribed using antisense cyclin D1 (Ccnd1) primer, and the cDNA subjected to real-time PCR in presence of SYBR Green in the StepOnePlus (Applied Biosystems) thermal cycler, for 40 cycles with a 45 s annealing step at 55°C, using sense and antisense rat Ccnd1 primers (Supplementary Table S3). The use of gene-specific primers for cDNA synthesis was preferred over synthesis of total cDNA by random priming since the latter yields cDNAs of variable length and sequence coverage from any particular mRNA, only a fraction of which would be amplified by gene-specific primers in the PCR step. mRNA was quantified as fold-change between pcRNA-treated samples and controls; fold-change = 22 uptake rate of injured muscle of 12–14-month-old rats was reduced to less than 5% of untreated controls . Admini2 tensions below 10 mm Hg [+ SCs with intense cytoplasmic Hypoxyprobe stain were observed within hypoxic areas between or close to myofibers, while SCs at some distance from these areas were not stained (Hypoxia in pcRNA-treated injured muscle in situ was detected as intracellular adducts of pimonidazole (Hypoxyprobe), the formation of which is detectable only at O10 mm Hg . In quad stained , suggestTyr [α and 2α) remained low for at least 24 h  [Western blot analysis of control injured muscle treated with a mitochondrion-targeted RNA that has no effect on mitochondrial translation (the D arm of tRNATyr ) showed ast 24 h . Upon MRast 24 h . In normrds 24 h . In indichondria . These dse (PHD) .2 consumption by the electron transport chain, thereby increasing availability of cytosolic O2 (the oxygen redistribution hypothesis). We enquired if changes in the local O2 concentration by respiratory inhibitors and uncouplers had any effect on wound resolution. Needle-injured muscle was treated with pcRNAs for 3 h  and then locally injected with mitochondrial inhibitors that directly or indirectly influence O2 uptake. HIFα induction was inhibited by (1) chloramphenicol, showing the requirement of mitochondrial protein synthesis to stimulate respiration (2 (2 consumption ( m-chlorocarbonylcyanide phenylhydrazone (CCCP), which stimulates respiration through uncoupling of electron transport from oxidative phosphorylation, significantly stimulated HIFα levels at early times  antation  DETsumption ; and (4)sumption . In contly times . Furtherast 24 h .The respiratory inhibitors had distinct effects on regeneration. In presence of the hypoxia-inhibiting NO, the inflammation was prolonged, delaying the resolution phase, which, however, occurred at approximately the same rate as in pcRNA-treated controls . In pres+PCNA+) cells in the mononuclear fraction 24 h after pcRNA treatment was reduced from 70% to 15% in presence of CCCP (data not shown).In presence of CCCP, which induces prolonged hypoxia, the activation of SCs was affected, as evidenced by reduced expression of cyclin D1, a G1 phase marker, and of PCNA, which is expressed in the S phase . The fraα in the nucleus and Dll1 on the plasma membrane were observed . Conversely, in injured muscle treated with recombinant Dll1 (in the absence of pcRNA), cyclin D1 transcript levels were upregulated ~5-fold after 12 h (data not shown). These results provide evidence for hypoxia-driven activation of the HIF-Delta-Notch pathway influencing SC proliferation after pcRNA-induced mitochondrial restoration.KD of HIF1r effect . After 2yofibers . The mRN Figures , while t Figures . In presα with persistently high levels up to at least 24 h . These results indicate a high level of satellite cell proliferation in presence of DMOG. However, in contrast to pcRNA-treated muscle, there was no activation of the myogenic regulators Myf5 and Myog in DMOG-treated muscle  should effectively replace pcRNA-induced respiratory hypoxia as a wound-healing agent. In rats injected at the wound site with the PHD inhibitor dimethyloxalylglycine (DMOG ), there ast 24 h . High least 24 h . This waast 24 h . FACS an2 concentration with inhibitors or uncouplers has specific effects on wound resolution. Our results demonstrate the importance of the microenvironment of the injury site in myogenesis.We show here that MR produces acute hypoxia at the injury site and that manipulating the local O2 levels to rise quickly after a short respiratory burst. On the other hand, in intact muscle, MR induced only a gradual hypoxic response , and delayed myogenesis, whereas KO of HIF1α in muscle cells had no such effects [α at the injury site affects SC proliferation  of the HIF1 effects . In contferation . The reaIn our rat quadriceps injury model, specific effects of environmental insults on the lesion size profile, with well-defined inflammatory and resolution phases (see Figures Hypoxia is essential for myogenesis and changes in the hypoxic condition of the microenvironment of the wound site affect wound healing in specific ways. Hypoxia stimulates SC proliferation but inhibits myoblast differentiation in vivo. While transient hypoxia is beneficial, prolonged hypoxia has a detrimental effect.The Supplementary Material contains sequences of lentivirus shRNA inserts, primer sequences and description of antibodies used in this study.


There are concerns that diminished prostaglandin action in fetal life could increase the risk of congenital malformations. Many endocrine-disrupting chemicals have been found to suppress prostaglandin synthesis, but to our knowledge, pesticides have never been tested for these effects.We assessed the ability of pesticides that are commonly used in the European Union to suppress prostaglandin D2 (PGD2) synthesis.Changes in PGD2 secretion in juvenile mouse Sertoli cells (SC5 cells) were measured using an ELISA. Coincubation with arachidonic acid (AA) was conducted to determine the site of action in the PGD2 synthetic pathway. Molecular modeling studies were performed to assess whether pesticides identified as PGD2-active could serve as ligands of the cyclooxygenase-2 (COX-2) binding pocket.The pesticides boscalid, chlorpropham, cypermethrin, cyprodinil, fenhexamid, fludioxonil, imazalil (enilconazole), imidacloprid, iprodione, linuron, methiocarb, o-phenylphenol, pirimiphos-methyl, pyrimethanil, and tebuconazole suppressed PGD2 production. Strikingly, some of these substances—o-phenylphenol, cypermethrin, cyprodinil, linuron, and imazalil (enilconazole)—showed potencies (IC50) in the range between 175 and 1,500 nM, similar to those of analgesics intended to block COX enzymes. Supplementation with AA failed to reverse this effect, suggesting that the sites of action of these pesticides are COX enzymes. The molecular modeling studies revealed that the COX-2 binding pocket can accommodate most of the pesticides shown to suppress PGD2 synthesis. Some of these pesticides are also capable of antagonizing the androgen receptor.Chemicals with structural features more varied than previously thought can suppress PGD2 synthesis. Our findings signal a need for in vivo studies to establish the extent of endocrine-disrupting effects that might arise from simultaneous interference with PGD2 signaling and androgen action.Kugathas S, Audouze K, Ermler S, Orton F, Rosivatz E, Scholze M, Kortenkamp A. 2016. Effects of common pesticides on prostaglandin D2 (PGD2) inhibition in SC5 mouse Sertoli cells, evidence of binding at the COX-2 active site, and implications for endocrine disruption. Environ Health Perspect 124:452–459; http://dx.doi.org/10.1289/ehp.1409544 Although the importance of androgens as drivers of male sexual differentiation in fetal life is widely recognized , the invSox9 gene). As first suggested by Sox9 gene, which drives the differentiation of Sertoli cells in the genital ridge of the mouse. Only fully differentiated Sertoli cells can coordinate the differentiation of all other testicular cell types, including the androgen-producing Leydig cells. Sry and Sox9 upregulate prostaglandin D2 synthase, thereby promoting prostaglandin D2 (PGD2) synthesis and secretion. In turn, PGD2 can act via its prostaglandin D receptor (DP) to upregulate Sox9 expression in an autocrine and paracrine manner -2,2-dichloroethene (DDE)–induced eggshell thinning in raptors was linked to direct inhibition of prostaglandin synthesis in the shell gland mucosa, thereby disturbing calcium metabolism  is associated with an increased risk of testis maldescent (cryptorchidism) in their sons . COX inhin vitro and that these chemicals can act in concert when present as mixtures as well as in combination with other antiandrogenic chemicals , imidacloprid, malathion, mancozeb, o-phenylphenol (OPP), pirimiphos-methyl, prochloraz, propamocarb, pyrimethanil, and thiabendazole) for in-depth study. Owing to evidence of their antiandrogenicity ; and all other pesticides  were purchased from Sigma Aldrich. Ethanol (> 99.7% purity) was obtained from VWR International Ltd. All test compounds were dissolved in ethanol to prepare stock solutions (20 mM) for use in the SC5 assay. Solubilization of mancozeb and imidacloprid was achieved via sonication.SC5 assay for PGD2 inhibition. SC5 mouse juvenile Sertoli cells  and 24-well plates  were used. Before each experiment, SC5 cells were seeded in 24-well plates . On the following day, all of the medium was removed and 400 μL fresh medium with test compound was added. Each plate contained a solvent control (0.5% ethanol in medium) and a positive control  that served as a reference for cytotoxicity potentially induced by the test compounds. All pesticides were tested in three independent experiments and at eight different concentrations; ibuprofen was included in each experiment as a positive control for PGD2 suppression. Plates were incubated at 37°C for 24 hr in an atmosphere of 5% CO2. At the end of the exposure period, 100 μL medium was removed for PGD2 measurements and kept on ice. PGD2 levels were determined using a prostaglandin D2-MOX enzyme immunoassay  according to the manufacturer’s instructions. Plates were read at a wavelength of 405 nm with a reference wavelength of 620 nm (Spectramax 340 PC). PGD2 concentrations in samples from treated cell cultures were determined using the linear range of a standard curve and are presented as percentages of the PGD2 levels measured in the solvent controls. To correct for inter-experiment data variability and to allow for regression analysis of pooled data, the PGD2 readings for the tested compounds on each EIA plate were expressed as percentages of the average of the values obtained from the solvent controls.li cells  were kinCytotoxicity determination. After aliquots of supernatant medium were removed for PGD2 determination, the plated SC5 cells were subjected to cytotoxicity testing with a modified version of the 3--2,5-diphenyltetrazolium bromide (MTT) assay (T) assay , as descStudies of the mode of PGD2 inhibitory action. Time series studies were conducted in which SC5 cells were exposed to ibuprofen (130 nM), aspirin , and OPP (175 nM) for 5 min, 15 min, 30 min, 1 hr, 2 hr, 3 hr, 6 hr, and 24 hr (three experiments performed in duplicate). The selected concentrations corresponded to half-maximal suppression of PGD2 levels (IC50) as determined in concentration–response analyses for the full duration of exposure (24 hr) in the SC5 assay.2 (PLA2) inhibitor MJ33 in terms of the reversibility of PGD2 inhibition and the influence of arachidonic acid (AA) on PGD2 levels. SC5 cells were exposed to ibuprofen (10 μM), aspirin (100 μM), OPP (10 μM), or MJ33  for 4 hr. After 4 hr of exposure, the medium was processed for PGD2 measurements as described previously. Some cultures received one of the following treatments for a further 2 hr: a) the treatment medium was removed and the cells were washed with Hanks’ Balanced Salt Solution (HBSS), after which 400 μL fresh culture medium  was added; b) the same as in a), but the added culture medium (400 μL) contained 10 μM AA; or c) a small volume of AA solution was added to the treatment medium containing the test chemicals to a final concentration of 10 μM AA. At the end of the experiments, PGD2 concentrations were measured as described previously.We assessed the mode of action of OPP by comparing its effects with those of ibuprofen, aspirin, and the phospholipase AThe influence of AA supplementation on PGD2 levels was also studied using a range of pesticides. After exposing SC5 cells to increasing concentrations of AA (between 0.3 and 100 μM) for 24 hr, we chose to use 10 μM AA for experiments with test chemicals. We exposed SC5 cells to varying concentrations of ibuprofen or OPP for 22 hr without AA and then added AA (10 μM) for another 2 hr before terminating the experiment. PGD2 levels were determined as described above. The same protocol was used for nine pesticides , where each pesticide was tested at levels producing 80% (IC80) PGD2 suppression relative to the solvent controls. These (highly effective) concentrations were chosen to provide unambiguous evidence of the effects of AA supplementation.Molecular modeling studies of binding to the COX-2 active site. We conducted computer modeling studies using Molecular Operating Environment (MOE) software . The crystal structure of murine COX-2, which is very similar to human COX-2 (Ki score as an estimate of how well the selected pose(s) of the test chemical fit to an average binding site. The quality of the fit was computed from the test pesticide’s structure alone and was based on the physicochemical properties  without considering features of the COX-2 binding pocket. We also calculated a predicted pKi score based on computed positions that the ligand can assume in the COX-2 active site. The predicted pKi score takes into account the structural conformation of the test chemical and the properties of the binding site of COX-2. The best poses were selected based on energy minima and interactions within the binding site. By comparing the Andrews pKi score and the predicted pKi, we judged whether the pesticide used all of its potential energy for binding in a particular position. If the predicted pKi was substantially greater than the Andrews pKi score, the ligand was specific to COX-2.an COX-2 , was obtStatistical analysis. For all concentration–response analyses, experiments were repeated three times; the data from the same test compound were pooled, and nonlinear regression analyses were performed using the best-fit approach  to be tested in the present study. After converting the PGD2 readings for the tested compounds on each EIA plate to percentages of the average values for solvents, 15 of the initial 24  were found to inhibit PGD2 synthesis in a concentration-dependent manner in the SC5 assay, as shown in With an IC50 of 175 nM for PGD2, the fungicide OPP was the most potent of the selected pesticides and was nearly as potent as the analgesic ibuprofen (IC50: 128 nM), which we used as a positive control . CypermeStudies of the mode of PGD2-inhibitory action. To obtain more information about the mode of action by which pesticides inhibited PGD2 synthesis, we initially conducted detailed experiments only with OPP, the most potent of the pesticides studied herein. We compared its effects with those of ibuprofen, aspirin, and the PLA2 inhibitor MJ33. Aspirin suppresses PGD2 synthesis by irreversibly inactivating COX-1 and COX-2 through relatively rapid, covalent modification of the COX active site (acetylation of Ser530), thereby blocking AA binding. Ibuprofen inhibits PGD2 synthesis through competitive enzyme inhibition by reversibly binding to the AA binding site of both COX isoforms (reviewed by 2 inhibitor MJ33. We treated SC5 cells for 4 hr with OPP (10 μM), ibuprofen (10 μM), aspirin (100 μM), or MJ33 (100 μM), removed the medium, briefly washed the cells, and incubated them with fresh medium  for another 2 hr. For OPP and ibuprofen, the replenishment with fresh medium led to increases in PGD2 levels relative to cultures that had not received media changes at the end of the 4-hr exposure period (We sought to further corroborate the mode of action of OPP by comparing it with the PLAe period .2 (as with the PLA2 inhibitor MJ33) or by inhibition of COX isoforms (as with ibuprofen and aspirin). To distinguish between these possibilities, we studied the effects of AA supplementation on PGD2 levels. In cells exposed to OPP, ibuprofen, or MJ33, removal of the treatment medium followed by washing and adding fresh medium with AA (10 μM) but no test chemical gave rise to PGD2 levels of a magnitude similar to those observed before exposure, indicating that AA could once again be used for PGD2 production after changes of medium  from phospholipids through inhibition of PLA2) because they have degraded and thus lost their ability to inhibit COX isoforms, or because COX expression increased during the long incubation period. To establish an optimal AA concentration for these experiments, we studied the influence of increasing concentrations of AA on PGD2 levels. After 24 hr, 10 μM AA produced approximately 300% increases in PGD2 levels relative to solvent controls . This concentration of AA was selected for coincubation with PGD2-active pesticides. When cells were incubated with ibuprofen and OPP for 22 hr followed by AA (10 μM) supplementation for another 2 hr, no changes in PGD2 levels were observed , again suggesting that OPP suppresses PGD2 synthesis by inhibiting COX.Next, we tailored our AA supplementation studies to the treatment regimen used to record the concentration–response relationships with pesticides over a 24-hr exposure period. Accordingly, we treated SC5 cells with test chemicals for 22 hr and then added AA for an additional 2 hr. Under these conditions, AA was expected to stimulate PGD2 levels only if COX enzymes were active, either because the test chemicals act upstream of COX  as well as at its base (Tyr385); another possible target for hydrogen bonding is the acetyl salicylic acid acetylation site (Ser530) positioned below Tyr385 at the base of the channel  of the binding of the PGD2-active pesticides were higher than the Andrews mean pKi scores, as with ibuprofen, these compounds could be fitted in the ligand-binding pocket of COX-2. As shown in Ki scores were higher than the predicted pKi scores. channel . We perfLack of correlation between the ability to suppress PGD2 and antagonism at the androgen receptor. Of the 15 pesticides identified as capable of suppressing PGD2 synthesis, 11 were also found to antagonize the androgen receptor in vitro ] showed potencies (IC50s) ranging between 175 and 2,300 nM  may provide useful tools for disentangling the relative importance of prostaglandin synthesis inhibition in producing adverse male reproductive effects. The lack of correlation between potency in suppressing PGD2 and antagonizing the androgen receptor  PDFClick here for additional data file.


The importance of different allergens in association with IgE production and canine atopic dermatitis (CAD) has been poorly studied and few studies exist on factors influencing allergen-specific IgE antibodies in serum. The aim of this cross-sectional study was to investigate the prevalence of elevated IgE levels to different environmental allergens in Norwegian dogs with a suspicion of CAD. The secondary aim was to identify risk factors associated with elevated serum levels of allergen-specific IgE.Acarus siro 84.0%, Dermatophagoides farinae 80.2%, Tyrophagus putrescentiae 79.9%). Sheep sorrel was the most commonly encountered outdoor allergen (40.0%). Only 2.6% of the dogs with elevated IgE levels were positive to flea saliva.The study sample consisted of serum from 1313 dogs of 161 different breeds. All samples were submitted for serologic IgE-testing  based on suspicion of CAD. Overall, 84.3% of the dogs had elevated IgE levels to one or more of the allergen(s). The predominant allergens amongst the positive results were the indoor allergens (The test results varied significantly depending on when the serum samples were taken. Samples taken during summer and autumn more often came out positive than samples taken during winter and spring. Geographical variations were also demonstrated. A greater proportion of females than males had positive test results, and more females than males tested positive to outdoor allergens. The mean age was significantly higher in the dogs testing positive than amongst the dogs testing negative. The allergen-specific IgE levels varied with breed. The boxer was the only breed with a significantly higher proportion of positive test results compared to the other breeds. Boxers also had a higher prevalence of elevated IgE levels to outdoor allergens, whereas the Rottweiler had a higher prevalence of elevated IgE levels to indoor allergens compared to the other breeds.IgE hypersensitivity was most often associated with indoor allergens. Outdoor allergens were of minor importance and IgE reactivity to flea saliva was rare. Breed differences in allergen-specific IgE levels were identified. Season of sampling, and the dogs’ geographical localisation, sex and age also affected the results of the IgE analysis. Canine atopic dermatitis (CAD) is a commonly encountered disease in small animal practice. It is defined as “a genetically predisposed inflammatory and pruritic allergic skin disease with characteristic clinical features, most commonly associated with IgE antibodies to environmental allergens” . EpidermInteraction between allergen-specific IgE antibodies and the relevant allergens is widely accepted as an important part of the pathogenesis of CAD, although it is known that CAD is a multifactorial disease that cannot be explained by IgE-mediated hypersensitivity alone ,4. Cutanet al. [The initial diagnosis of CAD should be based on a set of clinical criteria; the currently favoured set was proposed by Favrot et al. . It is aet al. ,13. Afteet al. .The influence of potential risk factors on serum levels of allergen-specific IgE has been poorly studied in the dog. It seems likely that geographical factors have an impact on the results of serologic IgE tests, as dogs in different regions are exposed to varying amounts of different allergens. Also, seasonal differences in allergen exposure presumably affect the serum levels of allergen-specific IgE . WhetherThe prevalence of elevated IgE levels to specific allergens has been poorly studied in Norwegian dogs. Worldwide, only a few studies exist on factors that may increase the risk of developing elevated allergen-specific IgE levels in canine serum. Hence, the aim of the present study was to investigate the prevalence of elevated serum levels of IgE to different environmental allergens in serum samples from Norwegian dogs that underwent serologic allergen testing. A secondary aim was to identify risk factors associated with elevated serum levels of allergen-specific IgE.In this cross-sectional study, the material was obtained from Dr. Baddaky AS , which is the only Norwegian enterprise to analyze allergen-specific IgE in animals. The full dataset consisted of serum samples from 1313 dogs, submitted to Dr. Baddaky AS for measurement of allergen-specific IgE (Heska Allercept® Serum IgE Test Nordic Panel) between March 2009 and September 2012. The serum samples were taken by veterinarians working in various geographic regions of Norway and were presumably submitted for IgE analysis based on suspicion of CAD.Forms containing information from the referring veterinarians followed the serum samples and were registered in Dr. Baddaky's database. Information regarding when the samples were taken (the sample date), the postal codes of the submitting veterinarians, each dog's breed, sex and date of birth as well as the results from the IgE analysis were obtained from the database.Acarus siro, the storage mite Tyrophagus putrescentiae and the house dust mite Dermatophagoides farinae. The outdoor allergens included are timothy grass (Phleum pratense), cocksfoot (Dactylis glomerata), blue grass annual (Poa annua), perennial rye grass (Lolium perenne), sheep sorrel (Rumex sp.), english plantain (Plantago lanceolatum), nettle (Urtica dioica), lambs quarter (Chenopodium sp.), mugwort (Artemisia vulgaris), alder (Alnus sp.), oak (Quercus sp.), birch (Betula sp.), meadow fescue (Festuca elatior), velvet grass (Holcos lanatus), redtop grass (Agrostis sp.), elm (Ulmus sp.), beech (Fagus sylvatica), hazel (Corylus sp.), sycamore (Acer pseudoplatanus) and ragweed (Ambrosia sp.). Flea saliva allergen is also included in the analysis.Heska Allercept® Serum IgE Test is an ELISA-based IgE analysis using the alpha chain of the human high affinity IgE receptor FcεRI as the IgE-specific reactant. The Nordic Panel includes indoor and outdoor allergens commonly occurring in Norway. The indoor allergens included in the Nordic Panel are the flour mite The test results are given as a number on a continuous scale, ranging from 0 to 3000  enzyme linked immunosorbent assay (ELISA) absorbance units (EA units). This represents the amount of allergen-specific IgE measured in a serum sample. More information about the test procedure is available in the references -22.A test result of 150 EA units or higher was considered positive. The dogs comprising the study sample were sorted according to their individual test results, providing the distribution of test results in the study sample.Based on the international breed standards , the dogs were categorized by their respective breed, giving a total of 161 different breeds. All mixed breeds were collectively called mixed breed.Each dog was categorized as either male or female, based on the information from the submitting veterinarians. Neutered individuals were not separated into isolated categories, because of uncertainty regarding the completeness of this information. Routine spay/neutering of healthy dogs is not commonly practiced in Norway.Each dog's age in months was calculated by subtracting the birth date from the sample date. The dogs were categorized into ten age categories: 0–6 months, >6 months −1 year, >1-2 years, >2-3 years, >3-4 years, >4-5 years, >5-6 years, >6-7 years, >7-8 years and >8 years.Each dog's birth season was defined based on the birth date: Spring , summer , autumn  and winter . Season of sampling was categorized in the same manner, depending on the sample date.The postal codes of the submitting veterinarians were used to identify the geographical distribution of the study sample. Counties were generated from the postal codes . The 19 The data was transferred into a database created in Excel® . The dataset was sorted in pivot tables, and all tables and graphs were created using a filtering function in Excel®.The mean age of the dogs in the study sample was estimated with a 95% confidence interval (CI) using Epitools . Non-oveP-value of <0.05 was considered statistically significant.The relationship between the risk factors sex, birth season, sample season and geographical region and the results from the allergen-specific IgE test (+/−) were tabulated and analyzed using a chi-square test. The chi-square test was performed with a 95% confidence limit using Epitools . A P-valStatistical analysis of breed as a risk factor was performed using the software package Stata SE 12 . The 95% confidence limit for the proportion of dogs with positive test results within each breed was calculated. Deviation from the “average breed”  was evaluated based on non-overlapping confidence intervals. The breeds represented with less than 10 individuals in the study sample were excluded from the analysis of breed effect.The study sample consisted of serum from 1313 dogs. A total of 161 breeds were represented. Besides mixed breed , the German shepherd dog was the most numerous breed . The sex distribution was similar, with 673 males (51.3%) and 640 females (48.7%). The age of 106 of the 1313 individuals could not be calculated due to lack of information. The remaining 1207 dogs had a mean age of 3.3 years (39.2 months). As with age, the birth season of 106 dogs could not be defined due to lack of information. Amongst the remaining 1207 dogs, a slight majority were born in the spring  and slightly fewer were born in the winter . 285 dogs (23.6%) were born in the autumn and 291 dogs (24.1%) were born in the summer. The season of IgE analysis showed more variation. Most serum samples were taken in the summer  and autumn , while only 181 samples (13.8%) were taken during the winter months. 270 samples (20.6%) were taken in the spring. A majority of the 1313 samples were sent from the southern parts of Norway, with a total of 1174 (89.4%) coming from regions south of Trøndelag.-).1107 of the 1313 dogs (84.3%) had IgE levels of 150 EA units or higher (i.e. positive results) for one or more of the allergen(s) included in the test (T+). 595 dogs (45.3%) had a positive reaction against at least one outdoor allergen (O+), while 1018 dogs (77.5%) reacted positive against at least one indoor allergen (I+). Most dogs that tested positive had elevated IgE levels for both outdoor and indoor allergens  or indoor allergens alone . Only 89 of the dogs with positive results (8.0%) were positive to outdoor allergens alone , D. farinae  and T. putrescentiae . Sheep sorrel was the most commonly encountered outdoor allergen, with 443 of the 1107 dogs (40.0%) testing positive. Elevated IgE levels to flea saliva was by far the least common result in the study sample, with 29 of the 1107 dogs (2.6%) testing positive. Only two dogs tested positive to flea saliva alone.The distribution of positive results to each allergen is presented in Table P = 0.035), and more females tested positive to outdoor allergens than did males . No statistically significant difference between sexes was detected for indoor allergens.The association between sex and results of the IgE analysis are shown in Table The age was known for 1022 of the 1107 dogs testing positive (T+). Only one of the positive individuals (0.1%) was younger than six months, while 42 dogs (4.1%) were between six and twelve months of age. A majority of the dogs with positive results were between one and four years old . The age category >1-2 years was the most numerous . The number of positive dogs then decreased with advancing age.I-; mean age 38.1 months; 37.7-38.5).The mean age was significantly higher in the dogs testing positive  than amongst the dogs with negative results . The same significant variation was seen for outdoor allergens , while the mean age of the dogs testing positive and negative to indoor allergens, respectively, was not significantly different , while 56 dogs (15.0%) were negative to all allergens (T-). 253 of the dogs born in the summer (86.9%), 239 of the dogs born in the autumn (83.9%) and 212 of the dogs born in the winter (82.5%) had positive test results (T+). The corresponding P-value, as calculated by chi-squared test, was 0.5166. The relationship between birth season and results to outdoor and indoor allergens (O+/O- and I+/I-) also was insignificant .No statistically significant association could be found between the dog's birth season and results of the IgE analysis. Amongst the dogs born during spring, 318 (85.0%) had positive test results (T+) than samples taken during winter and spring. The same pattern was identified in terms of serum IgE levels to outdoor and indoor allergens, respectively.The test results varied significantly depending on when the serum samples were taken Table . SamplesP-value for the geographical distribution of positive and negative test results (T+/T-), as calculated by chi-squared test, was 0.0008.The relationship between the dog’s geographical region and results of the IgE analysis were statistically significant. The region North had the lowest proportion of positive results , while the southern regions Agder/Rogaland and Southeast had the highest proportion of positive results . The proportion of positive test results amongst the remaining regions varied between 80.2% (Northwest) and 84.4% (Trøndelag). The P-value for the geographical distribution of positive and negative results to outdoor allergens (O+/O-), was <0.0001.Oslo/Akershus had the highest proportion of dogs testing positive to outdoor allergens , with the southern regions Agder/Rogaland and Southeast having the second highest proportion of positive results to outdoor allergens . Northwest had the lowest proportion of positive results to outdoor allergens (32.6%). The remaining regions had the following distribution of positive results to outdoor allergens (O+): North 40.0%, Hedmark/Oppland 42.6%, Trøndelag 45.3%. The P-value (<0.0001) was calculated for the geographical distribution of positive and negative results to indoor allergens (I+/I-). Agder/Rogaland had the highest proportion of dogs testing positive to indoor allergens . The region North had the lowest proportion of positive results to indoor allergens (60.0%). The proportion of positive results to indoor allergens amongst the remaining regions varied between 74.1% (Oslo/Akershus) and 83.7% (Southeast).The same Out of the 161 breeds registered in the study sample, 30 breeds were represented with at least 10 individuals. A total of 915 dogs were represented amongst these 30 breeds. The distribution of positive test results within these breeds is shown in the Tables A large majority (84.3%) of the dogs in the present study had positive results to one or more of the allergens included in the Nordic panel serum IgE test. The actual prevalence of elevated IgE levels in the general dog population is not known , althougA. siro (84.0%), D. farinae (80.2%) and T. putrescentiae (79.9%). It should be noted that the Nordic Panel only includes a subset of selected indoor allergens, and some of these might be false positive results as former studies have documented cross-reactivity amongst the mites [D. farinae was found to be the most important allergen amongst Norwegian dogs with CAD [D. farinae and/or D. pteronyssinus). Only 16.1% were positive to pollen allergens in the same study, and none of the dogs reacted to pollen allergens alone. This study was largely based on intradermal testing [Dermatophagoides spp. seems to be a major allergen in many parts of the world, including Europe. Pollen allergens on the other hand, appear to be important in the USA, but of less importance in Europe [The predominant allergens amongst the positive results were the indoor allergens: he mites . Most dowith CAD . In a st testing . Unfortu testing . Severalrinae 80.% and T. n Europe . The prerinae 80.% and T. Elevated IgE levels to flea saliva was by far the least common result in the study sample (2.6%). This is not surprising, because the flea burden in Norway is relatively low and dogs in areas with low numbers of fleas have a low prevalence of hypersensitivity to flea allergens .In this study, the dogs’ sex, age, breed and geographical region influenced serum levels of allergen-specific IgE. Sampling season also affected the test results, whereas birth season had no significant influence on the IgE levels.The results from previous studies are ambiguous regarding whether or not there is a sex predilection to elevated serum levels of allergen-specific IgE in dogs -19. In tIn the current study, the group of dogs with elevated IgE levels to one or more of the allergens had a higher mean age (39.6 months) than the group of dogs with non-elevated IgE levels (37.3 months). Dogs with positive results to outdoor allergens had a higher mean age (40.6 months) than dogs testing negative to the same group of allergens (38.1 months). The age differences were statistically significant, as documented by non-overlapping confidence intervals. This implies that the production of allergen-specific IgE, particularly to outdoor allergens, varies with age. On the other hand, no significant variation was found in mean age between dogs testing positive and negative to indoor allergens . Another interesting finding, however, is that dogs with positive results to outdoor allergens had a higher mean age than dogs testing positive to indoor allergens. This could imply that the production of IgE to outdoor allergens is dependent on allergen exposure over a prolonged period of time, compared to the production of IgE to indoor allergens. One could also speculate that Norwegian dogs are more exposed to indoor allergens than outdoor allergens, making elevated IgE levels to indoor allergens appear earlier. The relationship between dogs’ age and serum levels of IgE has been poorly studied, and the existing reports are conflicting. A study on healthy beagles found that total serum IgE levels varied with age , whereasThe present study documents statistically significant geographical variations in canine serum levels of allergen-specific IgE. Several underlying factors could explain this variation. Climate differences might be a factor involved. The region with the lowest proportion of positive results, North, has a lower level of average annual rainfall and lower humidity compared to the other regions . This leSignificantly higher proportions of the serum samples taken during summer and autumn were positive on the IgE test than samples taken during winter and spring in the present study. This pattern was found for the test overall (T+), as well as for the outdoor (O+) and indoor allergens (I+). The literature is limited in terms of seasonal variations in allergen-specific IgE levels in dogs, but season of the year has been found to influence the results of serum IgE tests in people . One couThe dogs' birth season did not affect the results of the IgE analysis in this study. To the authors’ knowledge, no other published studies have investigated the association between birth season and allergen-specific IgE levels, and results from studies on CAD are conflicting ,15,16.The present study documents breed differences in serum levels of allergen-specific IgE. There was a significantly higher proportion of elevated IgE levels (T+) amongst the boxers than amongst the other breeds in the study sample, indicating that boxers may be more prone to produce IgE antibodies in response to environmental allergens than other breeds. Pugs and Jack Russell terriers seem to be at a lower risk to develop IgE hypersensitivity, as these breeds had a significantly lower proportion of positive results on the IgE test compared to the other breeds represented. Provided that the history of the dogs in the study sample matches the clinical diagnosis of CAD, one could speculate whether pugs and Jack Russell terriers are overrepresented with ALD compared to other breeds.Further, the present study shows that the propensity to produce IgE antibodies to different allergen groups varies with breed. On the basis of the current results, it seems like the boxer are predisposed to producing IgE antibodies to outdoor allergens (O+), while the Rottweiler are predisposed to developing IgE hypersensitivity towards indoor allergens (I+). This is an interesting finding, as breed differences in allergen-specific IgE reactivity previously have been poorly studied in the dog; most studies on the subject have evaluated breed differences in total serum IgE levels. Also, several studies exist on breed predispositions to the disease CAD. Both total IgE levels and the occurrence of CAD have been documented to vary with breed ,11,17,18IgE hypersensitivity among Norwegian dogs with suspected CAD was most often associated with indoor allergens. Outdoor allergens were of minor importance and IgE reactivity to flea saliva was rare. Serum levels of allergen-specific IgE varied with sex, age, geographical localisation, and sampling season. Breed differences in allergen-specific IgE levels were documented. Relative to the other breeds represented in the study sample, the boxer had a higher prevalence of elevated IgE levels to outdoor allergens, while the Rottweiler had a higher prevalence of elevated IgE levels to indoor allergens.


Outlet glaciers grounded on a bed that deepens inland and extends below sea level are potentially vulnerable to ‘marine ice sheet instability'. This instability, which may lead to runaway ice loss, has been simulated in models, but its consequences have not been directly observed in geological records. Here we provide new surface-exposure ages from an outlet of the East Antarctic Ice Sheet that reveal rapid glacier thinning occurred approximately 7,000 years ago, in the absence of large environmental changes. Glacier thinning persisted for more than two and a half centuries, resulting in hundreds of metres of ice loss. Numerical simulations indicate that ice surface drawdown accelerated when the otherwise steadily retreating glacier encountered a bedrock trough. Together, the geological reconstruction and numerical simulations suggest that centennial-scale glacier thinning arose from unstable grounding line retreat. Capturing these instability processes in ice sheet models is important for predicting Antarctica's future contribution to sea level change. Irreversible ice loss from East Antarctic outlet glaciers during periods of ice sheet instability is yet to be observed in the geological record. Here, Jones et al. combine surface-exposure ages and model simulations to show the centennial-scale glacier thinning of Mackay Glacier during the mid-Holocene. The East Antarctic Ice Sheet (EAIS) contains more than two-thirds of the total volume of Antarctic ice grounded below sea level23455791157813Here we report a new terrestrial record of ice sheet thinning from Mackay Glacier, an outlet of the EAIS . Mackay Dating of glacial erratic clasts in altitudinal transects has the potential to record the past surface lowering of a glacier from a previously thicker configurationCosmogenic nuclide surface-exposure dating allowed us to establish a chronology of ice surface lowering (see Methods). Across all 4 transects, 44 exposure ages ranged from 22.3±2 thousand years to 245±75 years BP ; Fig. 2.The resulting chronology provides a near-complete record of outlet glacier thinning, with the Mt Suess/Gondola Ridge (upper) transect revealing ice surface lowering from ∼22 ka BP into the late Holocene . Undatedn=6), we were able to use the Bayesian-modelled ages to estimate thinning rates, which we consider to provide the best estimates of the surface lowering history. Here regression analysis indicates thinning of 33.1–80.2 cm per year (2 σ) and, as the rate likely varied during this period, we suggest that the upper end of this range better represents the peak of thinning  and Low Ridge transects. While the peak rate of surface lowering during this episode and the corresponding duration are not possible to determine, this analysis provides the most probable time-averaged estimates for the whole episode of rapid thinning. Regression was applied randomly to exposure ages through a Monte Carlo simulation . First, we simulated the modern ice surface at the sampling transects with a grounding line located upstream of the large overdeepening, and an advanced glacier that extended beyond the most distal GZW immediately offshore from Mackay Glacier and which honoured our empirically derived estimates of ice thickening along its longitudinal profile.To assess whether the change in surface elevation at our thinning transects could be explained simply by recession of the grounding line, we forced the grounding line from the outer trough, through the zone of GZWs and across the reverse bed slope of the inner trough. This was achieved using a range of individual ocean temperature and calving rate scenarios as the forcing. We also investigated the effects of pulses of sea level rise on the glacier, for example, if the rate of eustatic sea level rise exceeded that of isostatic rebound. In all experiments, we were able to simulate retreat from the outermost GZW to upstream of the overdeepened Mackay Sea Valley in ∼300–400 years, in good agreement with our chronology .In all retreat scenarios, the grounding line fluctuates in the outer trough, retreats more rapidly over the reverse bed slope and then re-establishes stability on the normal slope of the bedrock ridge upstream of the overdeepening . A highe1133A rapid episode of dynamic thinning in our model may have occurred due to a process other than that provided by the reverse bed slope. Therefore, we also carried out an idealized experiment in which Mackay Sea Valley was replaced by a flat bed . Under tIn summary, simulated accelerated thinning corresponds with increased ice flux as the grounding line retreats over the reverse bed slope, irrespective of the forcing applied. Together, the modelling experiments and regional geological constraints suggest that the substantial thinning recorded at our nunatak sites most likely resulted from marine ice sheet instability245Rapid and protracted ice loss of marine-terminating glaciers that retreat into deeper water has been observed in temperate and sub-polar settings7134344833848Most tidewater glacier cycles have been observed to last for up to several decades as a result of small variations in the bed topography71344505152Glacial troughs similar to Mackay Sea Valley (excavated to ∼1 km below sea level or greater) and their associated reverse bed slopes are common features on the inner continental shelf surrounding Antarctica15417195657Our new data from East Antarctica reveals that accelerated ice sheet thinning as a consequence of marine ice sheet instability can lead to hundreds of metres of ice surface lowering. We highlight that periods of rapid, topographically driven ice loss are likely typical of marine ice sheet margins, even in East Antarctica. These findings provide confidence for models that are able to simulate such ice-dynamic retreat1459Forty-four samples were collected for surface-exposure dating from two nunataks in the lower reaches of Mackay Glacier. The upstream nunatak comprises Gondola Ridge, an elongate granitic ridge, and Mt Suess, a basaltic dome that peaks at 1,127 m a.s.l. Low Ridge is an area of exposed granitic bedrock (∼205 m above the ice surface) that occurs downstream of the present-day grounding line. Geomorphological evidence shows that Mackay Glacier was larger in the geologically recent pastWe carried out altitudinal sampling transects at Mt Suess/Gondola Ridge to constrain thickness at the LGM and subsequent initial thinning, and at Low Ridge to record more recent thinning near the present-day terminus . To miniin situ) beryllium extraction.Preparation of samples for surface-exposure dating was conducted at the sedimentology, cosmogenic nuclide and geochemistry laboratories of Victoria University of Wellington and GNS Science, New Zealand. The upper ∼3–7 cm of each sample was extracted for processing using a large circular saw. This material was then crushed with a fine jaw-crusher and sieved to retain sand-sized grains (250–500 μm). Each sample was initially cleaned using a Frantz Isodynamic separator (at ∼0.5 A and a 10° tilt) to remove the magnetic component. Further cleaning was required to remove non-quartz minerals and etch any meteoric beryllium from the outer surface of the grains. This was achieved for each sample (up to 150 g) with a 1-day leach in hydrochloric acid and then three 2-day leaches in a 5% mixture of weak hydrofluoric acid and nitric acid, warmed and rotated on hotdog rollers. A final 1-h hydrofluoric acid (7 M) leach and concentrated aqua regia cleaning was carried out to remove any remaining meteoric beryllium before sample dissolution and (9Be spike (∼0.15–0.18 mg per sample) was added to the quartz samples, which were then dissolved in concentrated hydrofluoric acid. Anion exchange columns were first used to remove iron, and then cation exchange columns were used to isolate beryllium from iron, aluminium, titanium, sodium and magnesium. Procedural blanks yielded mean 10Be/9Be ratios of 1.38 × 10−15 with a s.d. of 5.91 × 10−16. Analytical measurements of 10Be/9Be were undertaken at ETH Zurich mass spectrometry facilities using both Tandem and Tandy accelerator mass spectrometers10Be/9Be ratio of 28.1±0.8 × 10−12), which in turn was calibrated relative to the ICN 01-5-1 standard (10Be/9Be ratio of 27.09±0.3 × 10−12)  ref. , and wer10Be, corrected for topographic shielding, sample thickness, quartz density (2.7 g cm3) and an Antarctic atmospheric pressure gradient, using CRONUS-Earth online calculator10Be is currently unconstrained in Antarctica, and production rates from both a global data set17Surface-exposure ages were calculated from the measured concentrations of Four sample outliers were identified in the data set. On Gondola Ridge (mid-lower), two samples were erroneously old or young (GR38 and GR40), representing micro-inheritance and some post-depositional effects, respectively. At Gondola Ridge (lower), the oldest sample  is not consistent with the thinning trends recorded in adjacent transects; if the surface elevation here was <50 m above present before ∼13 ka, then an unfeasible ice surface profile would exist with sites upstream and downstream, which record glacier surfaces >200 m higher at this time. For this reason, sample GR83 most likely contains a small amount of inheritance. A single sample on Low Ridge (CC93) is not consistent with exposure ages above and below its elevation. This minor outlier has probably been affected by post-depositional rotation or spalling.We applied Bayesian modellingTo estimate the rate and duration of rapid thinning at Mackay Glacier, linear regression analysis was carried out on data from the Mt Suess/Gondola Ridge (upper) and Low Ridge transects for the period of ∼6.8–6.0 ka. Error-weighted least-squares regression was applied randomly to normally distributed exposure ages (2 σ) through a 4,000-iteration Monte Carlo simulation. Rates and durations of rapid thinning were estimated from the distribution of feasible, positive-sloping linear regressions, with uncertainty generally reflective of the number of samples contributing to each transect and their respective uncertainties. Linear rates of thinning were calculated using the raw exposure ages at Low Ridge and the Bayesian-modelled ages at Mt Suess/Gondola Ridge (upper). The duration of this thinning episode was additionally determined, based on the start and end points of the modelled regressions.We employed a one-dimensional finite difference flowline model to investigate whether retreat of Mackay Glacier as a consequence of grounding line retreat through an overdeepening could result in the magnitude of thinning observed at our exposure dating transects. The flowline model has previously been applied to Transantarctic Mountain outlet glaciers and is fully described in Golledge and LevyNet mass balance is determined using a positive degree-day scheme, with mean surface air temperature, annual temperature range and precipitation rate initially prescribed. Surface melt is calculated if mean surface temperatures are above the freezing point. Although ice shelves are not directly incorporated into the model, negative mass balance is imposed in areas where the glacier bed is below sea level. Sub-shelf melting is calculated using an ocean temperature-based melt scheme when the ice thickness at the grounding line is less than floatation thicknessThe model was initialized with bed topography and ice thickness obtained from multiple radar data profiles. Glacier surface elevations were obtained from airborne altimetry measurements Investigating the Cryospheric Evolution of the Central Antarctic Plate (ICECAP), while the bed topography was interpolated from BEDMAP2 dataModel experiments used climate and glaciological parameterisations from field measurements and commonly used physical values . First, Transient simulations were then carried out by forcing retreat of the grounding line from the outer trough. Glacier retreat was achieved for a range of scenarios by enhancing the calving coefficient and summer ocean temperature. No retreat scenarios simulated ice thicker than floatation with a grounding line positioned within the trough in the model domain, and therefore all negative changes in mass balance were achieved through basal melt at the grounding line. Further model experiments used increased sea level to force retreat of the glacier; however, a similar pattern of retreat and thinning was observed irrespective of environmental forcing. In all scenarios, the grounding line initially fluctuates in the outer trough, but then accelerates over the reverse bed slope, corresponding to increased ice flux and rapid glacier thinning. In some cases , temporary grounding line stability that is implied by the presence of GZWs is not simulated in any retreat scenarios. This could be because of reduced trough width and complex lateral drag in this location , a stabiHow to cite this article: Jones, R. S. et al. Rapid Holocene thinning of an East Antarctic outlet glacier driven by marine ice sheet instability. Nat. Commun. 6:8910 doi: 10.1038/ncomms9910 (2015).Supplementary Figures 1-5, Supplementary Tables 1-2, Supplementary Note 1 and Supplementary ReferencesSurface-exposure dating sample details and ages from Mackay Glacier. Ages from each transect have been calculated using a global production rate9 with a range of production rate scaling schemes, as well as using a New Zealand (NZ) calibration site production rate10 with the time-dependent (t-d) scheme. Best estimates are derived from Bayesian age-elevation modelling, which includes weighted mean ages for samples at equal elevations (see Methods). Samples identified as outliers are shown in grey italics.


Here, we elucidate the mechanism of cilostazol-induced PGI2 stimulation in endothelial cells.Cilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is known as an anti-platelet drug and acts directly on platelets. Cilostazol has been shown to exhibit vascular protection in ischemic diseases. Although vascular endothelium-derived prostaglandin I2 accumulation in the culture media was measured. Cilostazol increased PGI2 synthesis via the arachidonic acid pathway. Cilostazol-induced intracellular calcium also promoted PGI2 synthesis via the inositol 1,4,5-trisphosphate receptor. Using RNAi, silencing of PDE3B abolished the induction effect of cilostazol on PGI2 synthesis and intracellular cAMP accumulation. Inhibition of the exchange protein, which was directly activated by cyclic AMP 1 (Epac-1) and its downstream signal the Ras-like small GTPase (Rap-1), abolished cilostazol-induced PGI2 synthesis, but this did not take place via protein kinase A (PKA). Inhibition of downstream signaling, such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) γ, and phospholipase C (PLC) ε, suppressed cilostazol-induced PGI2 synthesis.Human aortic endothelial cells (HAECs) were stimulated with cilostazol and PGI2 synthesis. Namely, stimulation of HAECs with cilostazol induces intracellular calcium elevation via the Rap-1/PLCε/IP3 pathway, along with MAPK activation via direct activation by Epac-1/Rap-1 and indirect activation by Epac-1/Rap-1/PI3Kγ, resulting in synergistically induced PGI2 synthesis.The PDE3/Epac-1/Rap-1 signaling pathway plays an important role in cilostazol-induced PGI H-tetrazol-5-yl) butyloxy]-3,4-dihydroquinolin-2-(1H)-one] is a selective phosphodiesterase 3 (PDE3) inhibitor, which has been shown to prevent platelet aggregation and peripheral vasodilation . C. C2 prodon et al . discoveon et al . The preal cells . Milrinoal cells , slightlat aorta  and rat at aorta , 29]. F. F2 prodS1 FigTop, Effect of Epac-1-targeting siRNAs (iEpac-1) or non-targeting siRNAs (iCt) on phosphorylation of MAPK and Akt in HAECs. Bottom, Effect of Epac-1-targeting siRNAs (iEpac-1) or non-targeting siRNAs (iCt) on cilostazol-induced phosphorylation of ERK and Akt in HAECs. HAECs were transfected with iEPAC-1, or with iCT. Post-transfection HAECs were treated with vehicle or 30 μM cilostazol . Phosphorylation of both proteins was normalized with their total proteins.(A) (PPTX)Click here for additional data file.S2 FigDirect bindings of 007 to Epac-1-binding PDE3B peptide. Relative responses of Epac-1-binding PDE3B peptide to 007 at concentrations of 1.25, 2.5, and 5μM. Cilostazol  was used as a positive control (n = 4).(PPTX)Click here for additional data file.


Amplification, overexpression, and somatic mutation of the HER2 gene have been reported to play a critical role in tumorigenesis of various cancers. The HER2 H878Y mutation was recently reported in 11% of hepatocellular carcinoma (HCC) patients. However, its functional impact on the HER2 protein and its role in tumorigenesis has not been determined. Here, we show that HER2 H878Y is a gain-of-function mutation. Y878 represents a phosphorylation site, and phospho-Y878 interacts with R898 residue to stabilize the active conformation of HER2, thereby enhancing its kinase activity. H878Y mutant is transforming and the transformed cells are sensitive to HER2 kinase inhibitors. Thus, our study reveals the following novel mechanism underlying the tumorigenic function of the HER2 H878Y mutation: the introduction of a tyrosine residue into the kinase activation loop via mutagenesis modulates the conformation of the kinase, thereby enhancing its activity. ErbB2 belongs to the ErbB family of receptor tyrosine kinases, which consists of ErbB1, ErbB2, ErbB3 and ErbB4, also known as EGFR, HER2, HER3 and HER4, respectively in humans. Members of the ErbB family play critical roles in normal cellular function and organismal development, as evidenced by the embryonic lethality exhibited by ErbB2 knockout mice [Although HER2 has no known ligand, it is a preferred dimerization partner for other ErbB family members. The activation of the ErbB receptor results in the autophosphorylation of its C-terminal tyrosine residues, which recruits signaling partners, including members of the Ras-Raf-MEK-MAPK pathway, PLC-γ1, phosphatidylinositol-3 kinase (PI3K)-AKT-S6 kinase (S6K), SRC, stress-activated protein kinases (SAPKs), members of the PAK-JNKK-JNK pathway and the signal transducers and activators of transcription (STATs)  patients . However2 incubator.NIH-3T3 cells were purchased from the ATCC and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FCS (Hyclone). Beas2b (ATCC) cells were cultured in DMEM medium supplemented with 10% FBS (Gibco). AML12 (ATCC) cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium with 10% FBS, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone. WEHI-3B cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco). Ba/F3 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 10% WEHI-3B conditioned medium (filtered supernatant). Ba/f3 and WEHI-3B cell lines are gifts from Prof. Pasi A. Jänne (Harvard University) . All of 3) were incubated with the indicated inhibitors or DMSO (as a vehicle control) for 3 days. Viability was assayed using the CellTiter-Glo luminescent cell viability assay (Promega). The resulting luminescent signals were recorded using a multimode plate reader (PerkinElmer). All of the inhibitors were purchased from Selleck Chemicals. Cell culture grade dimethyl sulfoxide (DMSO) was purchased from Sigma.HER2-transformed Ba/F3 cells (2 x 104) were seeded in 0.4% low melting point agarose (invitrogen) with 10% FCS or FBS and DMEM. Bottom agar consisted of DMEM with 0.6% agarose, and 10% FCS or FBS. Colonies was counted 1 month after seeding in soft-agar. Assays were carried in 6-replicate for each isoform. When necessary, single colony was transferred to 24-well plate to establish a stable cell line. The transfected Ba/F3 cell were deprived of IL-3 after selected with G418 for 12 days. The transformed cells were then seeded at one cell/well in a 96-well plate to establish a stable clone.For mammalian HER2 expression, the DNA fragment encoding wild-type hHER2 was inserted into pCAG-IN (CAG promoter-intron-IRES-Neomycin ) multiplAll cells were lysed in RIPA buffer (Beyotime) supplemented with protease and phosphatase inhibitors (Roche). Following lysis, the cells were sonicated. Western blotting was performed using standard methods. The blots were probed with the following antibodies: phospho-HER2-Tyr1221/1222 (Epitomics), β-actin (Sigma), phospho-HER2-Tyr877, phospho- HER2-Tyr1248(p-HER2), HER2, PLCγ1, phospho-PLCγ1-Tyr783, PARP, STAT5, phospho- STAT5-Tyr694, AKT, phospho-AKT-Ser473 (pAKT), ERK1/2, phospho-ERK1/2-Thr202/Tyr204, S6, phospho-S6-Ser235/236 (pS6) and phosphor-4E-BP1-Thr37/46 .H878Y mutant HER2 was identified in 11% of HCC patients and H878 locates in kinase domain , suggestHER2 mutations were reported in a portion of lung cancer patients  and H878To investigate the HER2 protein elicited signaling underlying gain-of-function phenotype for H878Y mutant, we assessed the phosphorylation status of HER2, Erk and Akt, two typical downstream molecules . Both thWe next sought to determine the molecular mechanism underlying the increased kinase activity of the H878Y isoform. Autophosphorylation of ErbB receptor family members is crucial for the activation of their tyrosine kinase activity. We therefore ask whether the H878Y isoform increases the autophosphorylation ability as compared to the WT isoform. To this end, the cytoplasmic domains of WT and H878Y HER2 were expressed in Sf21 cells using a baculovirus system and were subsequently purified for kinase assays. After onehour treatment with ATP using equal amounts of the purified kinases, the proteins were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the levels of autophosphorylation were assessed by western blot analysis. H878Y isoform exhibited increased levels of autophosphorylation on the Y1221/Y1222 residues. We also detected increased levels of phosphorylated Y877 . These dAs a receptor tyrosine kinase, kinase activity affects many aspects of HER2 function. We asked whether H878Y has enhanced kinase activity. Indeed, we found that the H878Y mutant exhibited significantly increased kinase activity compared to the WT enzyme . To furtOur data therefore demonstrated that H878Y mutation conferred higher kinase activity in comparison to WT HER2.Phosphorylation of Y877 confers higher kinase activity of HER2 and correlates with activated states of HER2 . SimilarWe further investigated how phosphorylated Y878 (pY878) increases the kinase activity of HER2. The crystal structure of the HER2 kinase domain has been previously resolved . We thusP > 0.05, To validate that pY878 enhances the kinase activity of HER2 by interacting with R898, we substituted R898 with alanine (R898A) to abrogate the formation of this potential salt bridge and assessed the resulting mutant kinase activity, as reflected by the colony formation ability of HER2-expressing cells in a soft-agar assay. The R898A HER2 mutant exhibited increased transforming ability, indicating that the R898A mutation by itself does not impair HER2 activity . Interesin vivo. HKI-272 efficiently eliminated phosphorylation of HER2 proteins and induced cleaved PARP  are able to efficiently block H878Y mediated transformation capability. We first overexpressed the H878Y HER2 mutant in Ba/F3 cells and found that the transformants were no longer dependent on IL-3 for survival. Interestingly, the second-generation irreversible pan-ErbB inhibitors exhibited significant toxicity against the HER2-driven Ba/F3 cells . We nextved PARP  in theseved PARP , clearlyWe next studied how HKI-272 inhibited the signal transduction elicited by HER2. We found that HKI-272 efficiently eliminated phosphorylation of ectopically expressed WT and H878Y HER2 protein in all 3 cell background. Interestingly, although H878Y elicited stronger PLC-γ1, STAT5 and Akt than WT isoform, these downstream mediators were more sensitive to HKI-272 inhibition in H878Y and WT isoform expressing cells, suggesting that H878Y is a sensitizing mutation. We also observed inhibition of Erk phophorylation in response to HKI-272 administration and found that Erk phosphorylation in H878Y overexpressing-cells were more sensitive in all 3 cell background. Similarly, Akt was sensitive to HKI-272 inhibition in all 3T3 and Beas-2B cell background. To study signals further downstream, we checked S6 phosphoryation as surrogate for S6K activity and 4E-BP1, both being mTOR substrates that are controlled by Akt. Interestingly, we found that S6 phosphorylation was sensitive to HKI-272 inhibition in 3T3 and AML12 background, but not Beas-2B background. Instead, 4E-BP1 was sensitive to HKI-272 inhibition in 3T3, but not in AML12 background . Our datHCC therapy in clinic is limited by paucity of valid druggable targets. We here show that H878Y, a HER2 mutation recently identified in 11% of HCC patients, represents a gain-of-function mutation and that Y878 enhances the kinase activity of HER2 through a salt bridge interaction between pY878 and R898 that stabilizes the kinase in an active conformation. H878Y is transforming and transformed cell lines are sensitive to treatment with HKI-272.http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/). However, the functional impact of these mutations remains to be characterized. The PDGFRa D842Y and D846Y mutations were reported to sensitize PDGFRa to imatinib [Our work on the H878Y mutation has revealed a novel mechanism by which mutations can impact kinase activity. We demonstrate that the Y878 in HER2 mutant is phosphorylated and interacts with R898 to stabilize the active conformation of the kinase, thereby enhancing its activity. This mode of function mimics, to a certain extent, the Y1007 and Y1008 residues within the activation loop of the JAK2 kinase . Tyrosinimatinib . Furtherimatinib , KIT D82imatinib  and FLT3imatinib  mutationH878Y mutation is a gain-of-function mutation that is capable of transforming NIH-3T3, Ba/F3 Beas-2B, and AML12 cells. Interestingly, we found that H878Y is a sensitizing mutation for HER2 kinase inhibitors, consistent with a previous report . TherefoIn summary, our work clearly shows that H878Y, a gain-of-function mutation found in a portion of HCC patients, positively impact on kinase activity of HER2 through phosphorylation of this mutationally introduced tyrosine to impact on structure of HER2 kinase domain.S1 Fig(A) Mass spectrometry detection of Y878 phosphorylation. A single phosphorylation site on Y878, on the background of unphosphorylated Y877 is detected by mass spectrometry. (B) The last snapshot of HER2-WT in 10 ns MD simulation, where pY877 coordinating with residues K762 and R844. (C) Comparison of the RMSDs in 10 ns simulations of HER2-WT (black), HER2-pY878 (red). The RMSDs were calculated over the Cα atoms of kinase domain with respect to the crystal structure.(TIF)Click here for additional data file.S2 Fig3 cells were treated with HER2 inhibitors for 3 days, cell viability were determined by CellTiter-Glo luminescent cell viability assay. n = 8. (D) WT and H878Y transformed Ba/f3 cells were treated with 50nM of various HER2 inhibitors for 12 hours, immunoblots of HER2 signaling were shown. D,DMSO; H,HKI-272; B, BIBW2992; C, CI1033; CP, CP724714. (E) Colony formation assay. Vector, WT or H878Y transfected AML12 cells (1×105 cells) were treated with 500nmM of HKI-272 for 4 days, cells were fixed and stained with 0.5% crystal violet.(A), (B), WT and H878Y transformed Ba/f3 and parental cells were treated with 200nM of various HER2 inhibitors for 3 days to determine the viability, n = 6 (A); or for 4 hours to probe HER2 downstream signaling (B). D,DMSO; H,HKI-272; B, BIBW2992; C, CI1033. (C) Viability of Ba/F3 cells transformed by WT or H878Y mutant HER2. 2×10(TIF)Click here for additional data file.S1 Protocol(DOC)Click here for additional data file.


The sequence and structure of the substrate near-site context are not sufficient alone to model the in vivo phosphorylation rules, and they should be integrated with orthogonal information in all possible applications. Here we provide an overview of the different contexts that contribute to protein phosphorylation, discussing their potential impact in phosphorylation site annotation and in predicting kinase-substrate specificity.Phosphate plays a chemically unique role in shaping cellular signaling of all current living systems, especially eukaryotes. Protein phosphorylation has been studied at several levels, from the near-site context, both in sequence and structure, to the crowded cellular environment, and ultimately to the systems-level perspective. Despite the tremendous advances in mass spectrometry and efforts dedicated to the development of  This mechanism of Post-Translational Modification (PTM) is strikingly common throughout evolution and in particular for eukaryotes where it is involved in a myriad of cellular processes  information and multi-organisms prediction. For a complete list of currently available methods, see Table The first method to predict the specific kinases that are responsible for the phosphorylations is Scansite , biological systems have evolved efficient strategies to increase the signal to noise ratio and more importantly to minimize those off target phosphorylations leading to detrimental consequences.The mechanisms of kinase-substrate specificity can be explored at several levels. A major separation is usually operated between peptide specificity and recruitment. Peptide specificity arises from the interactions between the catalytic kinase domain and the substrate peptide, while recruitment is based on interactions between kinase and substrate that do not involve surfaces localized at the catalytic center. During the phosphorylation reaction, the substrate is located together with the ATP in the structural region between the two kinase domain lobes, so that the gamma phosphate of the ATP can be transferred to the substrate site. The binding site differs between Ser/Thr and Tyr kinases, allowing the enzymes to discriminate between the three residues. In general each kinase shows a preference for one of these residues. Not only the site, but also its surrounding sequence provides information that is used by kinases to recognize their target sites Figure . The geoconsensus sequences, in Position Specific Scoring Matrices or more complex classifiers  , that can increase locally the concentration of kinases and substrates. Data is obviously critical to all this, therefore more OPL screenings on kinases with unknown specificity, combined with more comprehensive studies to explore scaffold-mediated kinase-substrate interactions and also more efforts dedicated to assess the impact of mutations in domain-domain interactions involved in signaling could contribute to the development of more refined models of kinase-substrate specificity. Probably the most difficult challenge is to convert results from in vitro studies to approaches that make reliable in vivo predictions. Different kinases vary in their dependence on contextual information. Holistic, i.e., highly integrative, approaches, that allow the modeling of many contexts at the same time, will be able in the future to dissect for each kinase (or kinase group) the different contributions that shape the logic of the signaling system, like for instance in the remarkable case of mitotic kinases, studied by Yaffe's group (Alexander et al., The two main challenges in developing computational approaches to kinase-substrate specificity are: modeling kinase-peptide specificity and substrate recruitment preferences. Bioinformatics solutions have extensively explored the motif-context, and as pointed out by Joughin et al. the construction of higher order mathematical models might have limited, if any, advantage, at the high cost of overfitted models (Joughin et al., Function-dependent classifiers, like those considering the identity of the domain where the phosphorylation is located, can be an alternative way to boost performances in phosphorylation prediction and they might be considered also for biomedical applications. Lastly, from the computational integration of different PTMs models (Creixell and Linding, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to compare the outcomes after phacotrabeculectomy at 1 or 2 sites in the small adult eyes with concomitant cataract and glaucoma.Patients who had 1-site (n = 26) or 2-site (n = 14) phacotrabeculectomy over a 4-year period at an eye surgery center were included. Eighteen eyes of 18 patients with glaucoma using any 1 prostaglandin analogue  were compared with 8 normal control patients. The records of patients were reviewed, and intraocular pressure, best-corrected visual acuity, axial length, anterior chamber depth, corneal endothelial cell (CEC) density, Diopter were measured. The outcome was compared with postoperative and preoperative measurements for 3-month follow-ups.The follow-up time was 3 months. There was no difference between the operations in improving best-corrected visual acuity, lowering intraocular pressure, shortening axial length, and deepening anterior chamber depth. However, 2-site surgery was associated with significantly more CEC loss and refractive error. Postoperative complications were not different between the 2 groups.The CEC loss and the refractive error in 2-site group were higher than that of 1-site group. One-site surgery seems to cause less CEC damage and refractive error than the 2-site operation during the follow-up time of 3 months. The estimated prevalence for microphthalmos is 0.002% to 0.017% worldwide and 0.009% in China.5 It was suggested that patients with simple microphthalmos consist of 0.05% to 0.11% of ophthalmic patients.6 Up to now, there is still no consensus on the definition of nanophthalmos and microphthalmos, and axial length (AL) <21 mm is generally accepted as the definition of nanophthalmos/microphthalmos. Diagnostic features include a small eye, short AL, hypermetropic, microcornea, shallow anterior chamber, narrow angle, large crystalline lenses, high incidence of angle closure glaucoma, thickened sclera, choroidal congestion, uveal effusion, and serous retinal detachment.10The small adult eye phenotypes include anophthalmos, nanophthalmos, and microphthalmos, and the latter 2 are synonymous and could be divided into simplex and complex microphthalmos according to ocular or systemic abnormalities.13 Nevertheless, phacotrabeculectomy in nanophthalmic/microphthalmos eyes has high incidences of intraoperative and postoperative complications. Surgical manipulation in a narrow and crowded anterior chamber with increased vitreous pressure is challenging, which always tends to cause pupillary blockage. During surgery, a sudden decrease in intraocular pressure (IOP) may result in uveal effusions, secondary retinal detachment, vitreous hemorrhage or malignant glaucoma, and even loss of the affected eye. Despite several potential complications, surgical outcomes for small eyes have been encouraging with the advances in phacotrabeculectomy and intraocular lens.18Angle closure glaucoma and cataract are both common comorbidities among the small adult eye. Nanophthalmos/microphthalmos is associated with significant visual morbidity owing to angle closure glaucoma and lens opacity, and needs treatment by phacotrabeculectomy.In this retrospective, consecutive, case–control series study, IOP, keratometry, corneal endothelial cells (CECs), best-corrected visual acuity (BCVA), and intraoperative and postoperative complications were reviewed in patients with small adult eye and concomitant cataract and glaucoma who underwent 1-site and 2-site phacotrabeculectomy. We aimed to evaluate 2 surgery techniques in patients who have had cataract extraction with foldable intraocular lens implant (IOL) and trabeculectomy to better aid in the process of selecting appropriate preoperative therapy approaches.8 Exclusion criteria included corneal opacities, corneal endothelial abnormalities, posterior segment disorders, cataracts not suitable for phacoemulsification, and increased risk of endophthalmitis such as active adnexal and ocular surface infection. All enrolled patients received simultaneous phacoemulsification and trabeculectomies at either 1-site  or 2-site , and pre- and postoperation assessments of the eyes were performed as described previously.19Forty small adult eyes of 24 patients that met the eligibility criteria were enrolled from patients at our hospital between March 1, 2010, and March 31, 2014. Inclusion criteria were based on clinical diagnosis of nanophthalmos/microphthalmos and ocular surgery for cataract and angle closure glaucoma. The inclusion criteria were defined as corneal diameter (CD) <11 mm, anterior chamber depth (ACD) <2.2 mm, and AL <21 mm.Phacotrabeculectomy and posterior chamber foldable IOL implantation were performed by the same experienced surgeon. Before the operation, the pupil was dilated using 1.0% tropicamide and 2.5% phenylephrine , and the eyes received topical anesthesia by using 0.5% proparacaine hydrochloride and 4% lidocaine. One-site group had a trabeculectomy in a superior scleral flap  combined with phacoemulsification through the same incision; 2-site group had a separate, superior scleral flap  for trabeculectomy and clear corneal incision for phacoemulsification. The incision for phacoemulsification was made by stab knife , and 1% sodium hyaluronate  was injected into the anterior chamber. Next, phacoemulsification was performed and the residual cortex was cleared by using an irrigation/aspiration system. The lens capsule was inflated with Healon, and the foldable IOLs  were implanted in the capsular bag. The scleral flap was sutured 2-pin with 10-0 Nylons  and conjunctiva wound was sutured with 8-0 absorbable catgut .P values of less than 0.05 indicated significant differences.Statistical analyses were performed using SPSS 17.0 software . Differences among 1-site and 2-site phacotrabeculectomy were assessed by Kruskal–Wallis test for continuous parameters and Fisher exact test for numeric parameters. Forty small adult eyes of 24 patients  who were enrolled into this study between March 1, 2010, and March 31, 2014, met the inclusion criteria for the definition of nanophthalmos/microphthalmos and diagnostic criteria of angle closure glaucoma and cataract. Thirty-two eyes of 16 patients were diagnosed as bilateral angle closure glaucoma accompanied by cataracts. Preoperatively, 8 eyes of 8 patients were unilateral angle closure glaucoma and cataract. IOP of all nanophthalmic/microphthalmos eyes were controlled below 25 mm Hg with medicines preoperatively. Ten patients have undergone bilateral 1-site phacotrabeculectomy, and bilateral 2-site phacotrabeculectomy was performed in 6 patients. Phacotrabeculectomy and bag Posterior chamber intraocular lens (PCIOL) implantation were performed using the above-mentioned technique.P = 0.02). These results showed that the prevalence of female was higher than that of male.The demographics of the eyes with 1-site group and 2-site group are summarized in Table 2; 2-site group: 2389.12/mm2). The mean lens thickness was 5.34 mm (range 5.16–5.58 mm) in 1-site group and 5.26 mm (range 5.12–5.74 mm) in 2-site group, compared with less than 5 mm in normal eyes. The CD of the nanophthalmos/microphthalmos was usually less than 10 mm. The minimum and maximum CD in 2 groups was 8.00 and 9.80 mm, respectively, and the average values were 8.90 mm. According to the keratometric reading (KR) and AL in 2 groups , the mean PCIOL power was 27.78 D (range 24.50–29.50 D) versus 27.64 D (range 25.50–29.00 D) and mean predicted spherical equivalent was +0.34 D (range +0.012 to +0.48 D) versus +0.28 D (range +0.016 to +0.35 D) in 1-site group and 2-site group, respectively. There were no significant differences in these data between 1-site group and 2-site group.All of the eyes in 2 groups underwent antiglaucomatic medical treatment. Preoperatively, the mean IOP was 28.24 mm Hg (range 23–38 mm Hg) in 1-site group and 27.26 mm Hg (range 22–41 mm Hg) in 2-site group. The mean AL and mean ACD was 19.46 mm (range 16.22–19.90 mm) and 2.01 mm (range 1.76–2.24 mm), respectively, in 1-site group, and was 19.18 mm (range 16.46–19.68 mm) and 2.03 mm (range 1.64–2.12 mm), respectively, in 2-site group. Although the cornea diameter of the nanophthalmos/microphthalmos was small, CEC density was not different form the normal eyes  in 1-site group was switched to extracapsular cataract extraction because of crowded and shallow anterior chamber. Clear corneal incision was sutured because of the leakage in 3 eyes of 2-site group (21.43%) and corneal stroma edema occurred at incision in 2-site group . There were no other complications in 2 groups.All patients received examination with slit lamp, IOP, and VA on the day after surgery. Five eyes of 1-site group (19.23%) and 2 eyes of 2-site group (14.29%) had mild corneal edema and the remaining were completely transparent. In 2 groups, exudation and Tyndall phenomenon was negative or weakly positive in anterior chamber, and each IOL was properly located in the capsular bag of posterior chamber. Twenty-four eyes (92.31%) had increased uncorrected visual acuity from 2 to 8 lines on the Snellen chart in 1-site group, and the remaining 2 eyes (7.69%) in 1-site group had no change with uncorrected visual acuity. In 2-site group, 9 of 14 eyes (64.29%) had increased uncorrected visual acuity from 3 to 6 lines on the Snellen chart; 2 of 14 eyes (14.29%) had decreased uncorrected visual acuity from 1 to 2 lines, and the remaining 3 eyes (21.43%) had no change. IOP was detected with Goldmann tonometer from 6 to 16 mm Hg in 2 groups.P = 0.005 and 0.01; respectively). These results showed that BCVA was improved to the same extent by 2 different surgical methods.Table P = 0.001). Postoperatively, mean AL was shortened 0.54 mm (1-site group) and 0.93 mm (2-site group), and significant difference between 1-site group and 2-site group was detected. AL was shortened much more in 2-site incision than 1-site incision. Mean ACD was deepened 1.99 mm in 1-site group and 1.92 mm in 2-site group, with no significant differences between 1-site group and 2-site group. The mean CEC density was decreased from 2415.12 to 2387.34/mm2 in 1-site group and from 2389.12 to 1867.13/mm2 in 2-site group. The results showed that phacotrabeculectomy using 2-site technique led to more CEC loss than 1-site technique (P = 0.001). In addition, there was a significant difference in refractive error between 1-site group and 2-site group postoperatively, and 2-site phacotrabeculectomy led to more refractive errors and astigmatism than 1-site technique.As summarized in Table 18 Nanophthalmos is known to be related to a high risk of angle closure glaucoma, and nanophthalmos and angle closure glaucoma exhibit similarity such as short AL, small radius of corneal curvature, shallow anterior chamber, and thick crystalline lens.6 Cataract of small eyes is formed gradually with the increase of age, and angle closure glaucoma could increase the incidence of cataract. Therefore, phacotrabeculectomy has to be performed.Currently, birth prevalence of microphthalmos is 0.002% to 0.017%, and the prevalence of simple microphthalmos in ophthalmic patients is 0.05% to 0.11%.19 Nevertheless, phacotrabeculectomy in small eyes could cause high incidence of complications such as uveal effusions, secondary retinal detachment, vitreous hemorrhage, or malignant glaucoma, perhaps due to sudden decrease of IOP.9 Furthermore, trabeculectomy and cataract incision could impact keratometry readings.20.21Phacotrabeculectomy can be performed via 2 approaches: using 1-site for trabeculectomy and phacoemulsification; or using 2 sites with a prelimbal filtering incision for trabeculectomy and a clear cornea incision for phacoemulsification, respectively.24 In addition, we compared the effects of 1-site versus 2-site phacotrabeculectomy on CEC density, and found that 1-site procedure led to less central CEC loss and less central CEC area expansion, perhaps due to a more posterior approach employed in 1-site phacotrabeculectomy. A previous study compared phacoemulsification through a scleral tunnel and phacoemulsification through a clear corneal incision and showed that the former led to less postoperative endothelial damage.25In this retrospective study, we found that both 1-site and 2-site techniques led to significantly shortened AL, consistent with previous reports that AL was significantly decreased following trabeculectomy.2, which has been reported as the threshold for endothelial decompensation.26 It was suggested that CEC density would be stabilized 3 months after cataract extraction.27 In current study with 3 months follow-up, the mean CEC loss rate was 9.44% in 1-site group and 20.97% in 2-site group, indicating that 1-site surgery seems to cause less CEC damage than 2-site surgery. One-site surgery may be a better option for managing coexisting visually significant cataract and uncontrolled glaucoma in patients with small eyes and higher risk of endothelial damage. Our data are consistent with a previous study showing the correlation between shorter AL and higher risk of CEC loss.28The overall mean CEC loss was 15.7%, which may result in a CEC density of less than 500 cells/mmIn summary, when small adult eyes with concomitant cataract and glaucoma underwent phacotrabeculectomy, CEC loss and the refractive error in 2-site group were higher than in 1-site group. Therefore, nanophthalmos/microphthalmos patients who have had cataract extraction with foldable IOL and trabeculectomy should be performed with 1-site surgery approach preoperatively.


Thus, for equivalent ATP turnover (cellular ATP demand), coupling efficiency is even higher in aged muscle mitochondria, due to the down-regulation of inner membrane proton leak caused by the decrease in membrane potential. In the framework of the radical theory of aging, these modifications in ANT function may be the result of oxidative damage caused by intra mitochondrial ROS and may appear like a virtuous circle where ROS induce a mechanism that reduces their production, without causing uncoupling, and even leading in improved efficiency. Because of the importance of ROS as therapeutic targets, this new mechanism deserves further studies.Mitochondrial membrane potential is the major regulator of mitochondrial functions, including coupling efficiency and production of reactive oxygen species (ROS). Both functions are crucial for cell bioenergetics. We previously presented evidences for a specific modulation of adenine nucleotide translocase (ANT) appearing during aging that results in a decrease in membrane potential - and therefore ROS production—but surprisingly increases coupling efficiency under conditions of low ATP turnover. Careful study of the bioenergetic parameters  of isolated mitochondria from skeletal muscles (gastrocnemius) of aged and young rats revealed a remodeling at the level of the phosphorylation system, in the absence of alteration of the inner mitochondrial membrane (uncoupling) or respiratory chain complexes regulation. We further observed a decrease in mitochondrial affinity for ADP in aged isolated mitochondria, and higher sensitivity of ANT to its specific inhibitor atractyloside. This age-induced modification of ANT results in an increase in the ADP concentration required to sustain the same ATP turnover as compared to young muscle, and therefore in a lower membrane potential under phosphorylating— Mitochondria, because of their central role in ATP supply through oxidative phosphorylation, but also in oxygen radical production and ion homeostasis, play a crucial role in skeletal muscle function. During last decades, a growing interest has been focused on the mechanisms linking dysfunctions of the mitochondrial oxidative phosphorylation and the aging process. One of the current leading hypothesis to explain the decrease in muscle energetics with aging is the mitochondrial theory of aging. The now accepted view hypothesizes that the reactive oxygen species (ROS) production linked to the activity of respiratory chain is responsible for the damages accumulated with aging to several components of the oxidative phosphorylation machinery and mitochondrial DNA  and young adult (6 months) rats with modular control analysis and 31P magnetic resonance spectroscopy of energetic metabolites  of oxidative phosphorylation to the increase in ATP demand induced by contraction is significantly reduced in skeletal muscle from aged rats, a dysfunction especially marked for low contractile activity  with aging. The analysis of the flow-force relationships under variable phosphorylation rates (mimicking variable cellular ATP demand) revealed that for equivalent ATP demand the energetic intermediate of oxidative phosphorylation  is lower in aged mitochondria as compared to young ones. Decrease in mitochondrial membrane potential is known to reduce proton leak .Experiments were conducted on male Wistar rats aged 6 and 21 months. Rats were housed in an environmentally controlled facility  and received water and food −1). The GAS muscle of the right leg was then dissected and washed in the isolation medium containing 100 mM sucrose, 180 mM KCl, 50 mM Tris, 5 mM MgCl2, 10 mM EDTA, and 0.1% (w/v) BSA (pH 7.2). Before homogeinization, muscles were minced and exposed for 5 min to protease . Mitochondria were extracted as previously described , glucose-6-phosphatase dehydrogenase , NADP+ (1.6 mM) allowed the determination of phosphorylation rate since the NADPH production by this system is stoichiometrically linked to mitochondrial ATP synthesis rate. Changes in the rate of NADPH production were monitored at 340 nm by placing an optic fiber connected to a spectrophotometer in the oxygraphic vessel . Conversion of NADPH absorbance into NADPH concentration was performed using a molar extinction coefficient of 6.22 mM−1 cm−1. Bioluminescent assays were used to measure both ADP and ATP concentration during each experiment . The use of a combined enzymatic system composed of glucose (5 mM), hexokinase  in a glass vessel  in a medium containing 140 mM sucrose, 100 mM KCl, 1 mM EGTA, 20 mM MgCl2, 10 mM KH2PO4, and 0.1% (wt./vol.) BSA (pH 7.2). Mitochondrial protein concentration used in the measurement vessel was approximately 0.3 mg ml−1 unless for the proton leak flux determination where the protein concentration used was approximately 0.6 mg ml−1. Succinate (5 mM+rotenone 2 μg ml−1) was used as a substrate for the monitoring of oxidative phosphorylation parameters  and top-down control analysis experiments. To inhibit residual adenylate kinase activity, excess of P1,P5-Di(adenosine-5) pentaphosphate  was added to the measurement medium in all experiments. State III oxidation rates were obtained by adding ADP (250 μM) and state IV oxidation rate was measured after complete ADP phosphorylation. Oxidation rates were determined polarographically with a Clark electrode (Rank Brothers) at 25°C. Concentration in air-equilibrated medium was taken as 240 μM  electrode coupled to an Ag/AgCl- saturated reference electrode . Any change in the regulation of the module under consideration will be evidenced by a change in the elasticity curve of this very module. The complete description of the experimental protocol used to determine the kinetic response of each module to membrane potential and the calculation of elasticity coefficients for each module has been described elsewhere . ATP to O ratio was determined under conditions of constant ADP concentration across the whole range of mitochondrial oxidative phosphorylation activity  the responsiveness (elasticity) of energy production is effectively reduced in the GAS of aged rats  is unlikely to explain the decrease in the elasticity of the energy-supply module found in aged rats . In fact, a decreased affinity for ADP indicates that for a given rate of mitochondrial oxidative phosphorylation to match ATP demand, a higher increase in the ADP concentration is necessary. This lower ADP sensitivity of mitochondria in aged rats fits well the regulation of phosphorylation (elasticity) measured in vivo in aged rat muscles. We also showed that mitochondria from gastrocnemius muscle of aged rat manifest an interesting increase in atractyloside-sensitivity, as compared to young ones  and IHU-LIRYC  (ANR-10-IAHU-04), INSERM U1045 and the “Région Aquitaine.” GG is funded by a NSERC discovery grant (RGPIN2014-04668).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.