The androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors.We previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants.This work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients’ serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy.The online version of this article (doi:10.1186/s13059-015-0864-1) contains supplementary material, which is available to authorized users. Advances in prostate cancer (PCa) research have led to the development of novel therapies for the metastatic castration-resistance (CRPC) form of the disease, such as two recent drugs abiraterone , 2 and eIn the majority of cases, CRPC is accompanied by reactivation of the AR signaling axis so that the receptor regulates its numerous target genes including PSA. Not surprisingly, an impressive repertoire of mechanisms has been identified that reactivate the AR signaling axis. These include upregulation of CYP17 , amplifiThe detection of circulating cell-free DNA (cfDNA) has recently emerged as a non-invasive diagnostic tool for a variety of cancers, including CRPC, and as a technology maximizing the efficacy of anticancer therapies , 23. It By improving the sequencing and data analysis processes, we were able to sequence the cfDNA of the 15 CRPC patients that were previously excluded due to low yield of DNA in their plasma samples, to validate all of the mutations reported in our previous work  and to iin vitro characterization of all AR mutations identified in 62 CRPC patients together with seven AR mutants previously reported in the literature , to ascertain the exact mechanisms of resistance to AR pathway inhibitors  on all mutants, as well as investigated their in vitro responses to four different steroids including DHT, progesterone, estradiol, and hydrocortisone. As the result, we present evidence that all identified AR mutations provide evolutionary escape routes from androgen blockade, thus highlighting the need for novel AR inhibitors that bind to the AR outside of the ABS. Finally, we demonstrate that VPC-13566, one of our recently developed class of AR inhibitors bearing a quinolone scaffold . Th. Th46]. Several of these CRPC-associated mutations are well documented in the literature, including hydroxyflutamide- and bicalutamide-resistant AR variants T878A and W742L/C, respectively. Other substitutions, including L702H , V716M , V731M , T878S , and H87in vitro functionalization to determine whether and how the mutations modulate the activity of the AR.In the current work, we employed a modified mutation-identification pipeline that allowed us to establish a detailed cfDNA-based mutation status of all 62 CRPC patients reported in , thus inThe measured responses of 24 single and double AR mutants to four AR antagonists, hydroxyflutamide, bicalutamide, enzalutamide, and ARN509, revealed that all these drugs can behave as AR agonists in the context of certain mutations Table . In partResistance to bicalutamide was also observed for the majority of the AR mutants, with the notable examples of W742L/C, T878A/S, S889G, D891H, and M896V/T mutations conferring strong bicalutamide activating phenotypes , H875Y (7.4 %), and two double mutants F877L/T878A (21.4 %) and T878A/S889G (5.3 %). All four AR mutants demonstrated varying degrees of activation by enzalutamide in our assay  configuration of the protein , 54. UsiOur analysis of the distribution of the 15 mutated residues on the AR 3D structure demonstrated that they are mainly clustered around the AR steroid binding site and adjacent to helix 12 , which makes the agonist conformation improbable with the WT-LBD  - Vancouver Prostate Centre  between August 2013 and March 2014 was described previously . In totaFor this paper we have sequenced blood samples from 15 patients that were reported as not sequenced in the previous manuscript due to either low DNA yield (less than 30 ng as determined by Qubit 2.0 measurement using Qubit dsDNA HS Assay Kit) or repeatedly failed to produce useable sequencing data . DNA was amplified with the Sigma WGA2 kit (Cat No WGA2-10rxn or WGA2-50rxn) as per manufacturer’s instructions. The amplified material was sequenced with the Roche 454 GS FLX+ system, software version 2.9 as described in . The amphttps://github.com/genome/bam-readcount) that allows for direct filtering of reads at each base position based on both sequence phred quality score (only bases with phred score >25 were scored) and mapping score (only bases with mapping score of >18 were scored). The raw base counts for each qualified base were converted to percentage relative to sequence coverage at corresponding position. We defined mutation candidates as non-reference bases with percentage value greater than 4 standard deviations distant to the mean and exceeding 0.1 % and 1 % level in non-amplified samples and amplified samples, respectively, due to increased background of base substitutions in WGA2 data. All calls detected in more than 75 % of sequenced samples were discarded as artifacts. We performed manual curation of all detected calls, in some cases adding calls that were present in other sequenced samples from the same patient, provided that they could be unambiguously identified as outliers on scatter plots of matching samples. Finally, we performed haplotype frequency estimation through manual curation of aligned reads in IGV. Reads with >7 mismatches with the reference sequence were considered uninformative for samples with multiple mutation calls.The biggest challenge in analysis of WGA2 amplified DNA is the significantly higher noise levels introduced by the genomic amplification. Therefore, we modified our pipeline to assure tighter control and more precise filtering of low-quality sequencing data. Raw sequence reads were mapped to the human genome (hg19) using BWA and visualized using the Integrative Genomics Viewer (IGV) . All posFull-length human AR (WT-AR) was encoded on a pcDNA3.1 expression plasmid (Life technologies). The LBD point mutations (single and multiple) were generated using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) as per manufacturer’s instructions using WT-AR as the template. The mutagenic oligonucleotide primers were designed individually with the desired mutation in the middle of the primer with approximately 10 to 15 bases of correct sequence on both sides.PC3 cells lacking the AR and authenticated by IDEXX Laboratories  were maintained in RPMI 1640 media (Life Technologies) and 5 % FBS (Hyclone Thermo Fisher Scientific) at 37 °C and 5 % CO2. Cultures were routinely monitored for mycoplasma contamination. For the steroid activation assay, cells were seeded in 96-well plates  in RPMI 1640 medium with 5 % charcoal-stripped serum (CSS) (Hyclone). After 24 h, cells were co-transfected with 25 ng of wild-type or mutated AR and 25 ng of the reporter plasmid pARR3-tk-luciferase using TransIT20/20 transfection reagent (3 μL/μg of DNA)  in Optimem serum-free media (Life Technologies) for 48 h according to manufacturer’s suggested protocol. Cells were stimulated with increasing concentrations of DHT, estradiol, progesterone, or hydrocortisone in 100 % ethanol (0 to 500 nM). Control cells were treated with 100 % ethanol alone. At 24 h after treatment, the medium was aspirated off and the cells were lysed by adding 60 μL of 1× passive lysis buffer (Promega) followed by shaking at room temperature for 15 min and two freeze/thaw cycles at -80 °C . Twenty microliters of lysate from each well were transferred onto a 96-well white flat bottom plate (Corning) and the luminescence signal was measured after adding 50 μL of luciferase assay reagent (Promega). The chemical oxidation of luciferin into oxyluciferin by the luciferase is accompanied by light production that can be quantified as luminescence by a TECAN M200Pro instrument. Each concentration was assayed in quadruplicate n = 4, with a biological replicate of n = 3. For each steroid, results were averaged and normalized by expressing them as a percentage of WT AR activity.PC3 cells were seeded and transfected as described above. At 48 h after transfection, medium was aspirated and replaced with medium containing 0.1 nM R1881 and either 0.1 % DMSO (control) or serial dilutions of increasing concentrations of AR inhibitors ranging from 0 μM to 50 μM . A non-stimulated/no R1881 control was used. After 24 h, cells were lysed and AR-dependent luciferase activity was quantified. Each concentration was assayed in quadruplicate n = 4, with a biological replicate of n = 3. Results were averaged and normalized by expressing them as a percentage of WT AR activity.Twenty microliters of each of the four replicates of DMSO/control-treated lysate from the luciferase assay were pooled with 20 uL of 5X sample buffer, boiled for 5 min, and 20 uL of the mixture was loaded on a 10 % SDS-PAGE gel and electrophoresed at 120 V for 90 min. Proteins were transferred to PVDF membrane (Millipore) at 100 V for 1.5 h at 4 °C, blocked for 1 h at room temperature with 5 % non-fat skim milk in TBS, followed by incubation with 1/1,000 dilution of AR N20 antibody  overnight at 4 °C. Membranes were incubated with 1/5,000 dilution of goat anti-rabbit IgG HRP  for 1 h at room temperature, washed five times with TBS 0.1 % Tween 20 (Sigma), and bands visualized using Super Signal West Femto (Thermo Scientific) and a digital imager (Syngene G Box).The displacement of a fluorescein isothiocyanate (FITC) labeled BAG1L peptide (FITC-MAQRGGARRPRGDRERLGSR) from the BF3 pocket of the LBD by the compounds VPC13566 and VPC14449 was assessed using a Time-Resolved Fluorescence Energy Transfer (TR-FRET). Compounds were tested in the range of 0.41 to 100 μM in a final concentration of 1.5 % DMSO. The protein AR-LBD, the FITC-BAG1L peptide, and the LanthaScreen® Elite Terbium-labeled anti-His-tag antibody  were used at final concentrations of 100 nM, 500 nM, and 5 nM, respectively. Briefly, the (His)6-tagged AR-LBD was prepared at 4X final concentration in the buffer phosphine) in the presence of 4X Tb-anti-His-antibody (Mix A). Mix B contained 4X FITC-BAG1L peptide in 2 % DMSO. A three-fold serial dilution of the compounds was prepared at 100X final concentration in DMSO. The compounds were then diluted 50-fold in buffer to get a 2X final concentration and 2 % DMSO (Mix C). In a black flat bottom 384-well plate 5 μL of Mix A, 5 μL of Mix B, and 10 μL of Mix C were added.The plate was incubated at room temperature for 2 h and FRET was analyzed on Synergy-4 multi-plate reader with the following settings: excitation, 340 nm; emission, 495 nm and 520 nm. The emission ratio (520:495) was analyzed, normalized to the buffer, and plotted.PC3 cells were plated at 5,000 cells per well in RPMI 1640 containing 5 % CSS in a 96-well plate and treated, after 72 h, with 0.1 nM R1881 and VPC-13566 (0-50 μM). After treatment for 24 h, cell density was measured using the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay according to the manufacturer’s protocol . The percentage of cell survival was normalized to the cell density of control wells treated by vehicle and 0.1 nM R1881.http://www.ebi.ac.uk/ena/data/view/PRJEB12109.All sequence data supporting the results of this article are available in the European Nucleotide Archive (ENA) repository, with the study accession number PRJEB12109 and a direct URL: This study was approved by the University of British Columbia Clinical Research Ethics Board (CREB) with the certificate approval number H09-01628 (renewed on 7 December 2015) and by the Vancouver Coastal Health Research Institute (VCHRI) with the certificate number V09-0320. All patients provided informed written consent. All the experimental methods comply with the Helsinki Declaration.


In this article, we first review the basic mechanisms by which the β2-adrenergic system contributes to the control of airway smooth muscle tone. Then, we go on describing the structural characteristics of β2-AR and the molecular basis of G-protein-coupled receptor signaling and mechanisms of its desensitization/ dysfunction. In particular, phosphorylation mediated by protein kinase A and β-adrenergic receptor kinase are examined in detail. Finally, we discuss the pivotal role of inhaled β2-AR agonists in the treatment of asthma and the concerns about their safety that have been recently raised.Inhaled β In the 1990’s, Jones et al. [2+-activated K+ (BKCa) channels, i.e., charybdotoxin and iberiotoxin, may inhibit the bronchodilator responses to β2-AR agonists and to other agents that can increase cAMP. These effects have been subsequently confirmed by other studies [2-AR agonists in isolated human airways [2-AR may activate BKCa channels in ASM directly via the α-subunit of Gs [Activated βeceptors . Upon bipathways . The respathways . The int protein . In bron protein  and phos protein . Moreoveexchange , thus re+ ATPase . Howevers et al. ,44 showe studies ,46 and a airways . This suit of Gs  . The latter belongs to G-protein-coupled receptor kinases family (GRK) [s subunit by phosphorylating specific sites [s subunit may be a mechanism of allergen-induced β2-AR dysfunction observed in human isolated passively sensitized bronchi [The covalent modification of β-AR represents the earliest event in the desensitization process . Over a ly (GRK) . Both kiic sites . The unc bronchi   activity in mediating the pro-asthmatic changes in ASM responsiveness that accompany its prolonged homologous desensitization. Their findings provide new evidence that prolonged exposure of ASM to β2-AR agonists results in an upregulated expression of PDE4D5 isoform due to PKA-dependent activation of Gi protein signaling. This is consistent with a potential switch in coupling of the β2-AR from Gs to Gi resulting in Gβγ-subunit-mediated activation of ERK1/2 which, in turn, leads to a phosphorylating cascade of transcription factors. Nino et al.  have rec2-AR on the cell are occupied [2-AR fraction is occupied, this is sufficient to increase intracellular cAMP, resulting in activation of PKA and phosphorylation of all β2-AR on the cell. Under these conditions, only the agonist-occupied receptors are phosphorylated by βARK, which contributes minimally to the process under these circumstances. Other G-protein-coupled receptors with PKA sites may also be phosphorylated, resulting in desensitization by β2-AR agonists [During exposure to low levels of agonist, few βoccupied . Such looccupied . Althougagonists .2-AR agonists, as occurring in bronchodilator treatment of asthmatic patients, the majority of β2-AR on the cell may be occupied [2-AR are phosphorylated by this kinase. In addition, all occupied receptors are phosphorylated by βARK. Such phosphorylated receptor binds β-arrestin with subsequent partial uncoupling from Gs [During exposure to high concentrations of βoccupied . Under t from Gs . 2-AR signaling, and is thought to be a mechanism involved with cell and organ homeostasis and tolerance to agonists. However, there is little direct evidence that these events are relevant to β2-AR physiological function, such as ASM relaxation. Recently, Wang et al. [2-AR lacking PKA and/or GRK phosphorylation sites on ASM. They showed that, at low receptor occupancy, β2-PKA(-) had enhanced agonist-promoted relaxation, while β2-GRK(-) airways were unaffected. In contrast, at saturating agonist concentrations, the greatest relaxation enhancement was with β2-GRK(-), with no evidence for additivity when PKA sites were also removed. For the full range of responses, the β2-PKA(-)/GRK(-) airways had the greatest relaxation efficiency, indicating a graded effect of GRKs as agonist concentration increased. Thus, these two mechanisms impact a relevant β2-AR physiologic function, by acting as attenuators of the acute response, and represent specific interfaces where adjunct therapy or biased ligands may improve β2-AR agonist treatment of obstructive lung disease. Phosphorylation by PKA and GRKs leads to desensitization of βg et al.  have she2-AR undergo a process leading to internalization or sequestration of the receptor to a sub-cellular compartment. The proportion of receptors at the surface that undergo sequestration appears to be highly cell-type dependent. In cells where there is little receptor reserve and sequestration results in ~60% loss of cell surface receptor, desensitization by this mechanism would be expected, as shown in lymphocytes [2-AR of human pro-inflammatory cells is exquisitely sensitive to desensitization, whereas β2-AR-mediated relaxation of human ASM is relatively resistant. An explanation for this discrepancy is that a large β2-AR reserve exists in ASM cells for sympathomimetic bronchodilators, which protects against desensitization [2-AR undergoes phosphatases-mediated dephosphorylation [After about 30 min of agonist exposure, cell surface βphocytes . Recent tization . After arylation .down-regulation and reaches a steady state at 18–24 h. The process can result in an loss of as much as 90% of receptors and is presumed to be a major component in long-term agonist-promoted desensitization of β2-AR [2-AR mRNA stability [2-AR mRNA appears to be due to cAMP-promoted binding of a protein to a destabilization sequence (AUUUA) in the 3’ non-coding region of the gene [2-AR pool. One study suggests that PKA-mediated phosphorylation is required for full agonist-promoted down-regulation [After 3–6 h of exposure to agonist, a net loss of receptors occurs independent of compartment . This isof β2-AR . The mectability , althougtability . Destabithe gene . The secgulation .2-AR agonists have been introduced as a therapeutic option in asthma [2-AR agonists [et al. [2-AR agonists was further explored in mice with ablated β-AR genes (β-AR-/-) and transgenic mice overexpressing β2-AR (β2-AR-OE) [-/- mice had markedly decreased bronchoconstrictive responses to MCh and other Gq-coupled receptor agonists such as thromboxane-A2 and 5-hydroxytryptamine (5-HT). In contrast, β2-AR-OE mice had enhanced constrictive responses. Inositol phosphate accumulation by Gq-coupled M3-muscarinic, thromboxane-A2, and 5-HT2 receptors was desensitized in ASM cells from β-AR-/- mice and sensitized in cells from β2-AR-OE mice. Thus, β-AR seem to regulate antithetically constrictive signals, affecting bronchomotor tone/reactivity by additional effects other than direct bronchodilation. Studies of signaling elements in these pathways identified the nodal point of this cross-talk in phospholipase C-β1, whose expression was altered by β-AR in a direction and magnitude consistent with the physiologic and cellular responses. These results establish a possible mechanism of the β-AR paradox and identify a potential asthma modifier gene (phospholipase C-β1), which may also be a therapeutic target in asthma when chronic β-AR agonists are required [2-AR agonists may negatively impact the balance of factors contributing to airway inflammation and remodeling in asthma [Long-acting βn asthma ,6 to impagonists ,93,94. I [et al.  showed t2-AR-OE) . Unexpecrequired . An alten asthma .2-AR, and reduced expression of various spasmogenic proteins [Two elegant studies ,99, in oproteins . In eighproteins ,102.2-AR synthesis, reverse down-regulation, and improve coupling, the last by partially restoring the fraction of receptors in the high-affinity state. A study from our laboratory indicates that short-term (3 h) exposure to beclomethasone dipropionate ablated β2-AR dysfunction induced by allergen challenge in passively sensitized human bronchi. This effect was mediated by an increase in the activity of the α-subunit of Gs protein, suggesting an action independent of gene transcription [As detailed by Davies and Lefkowitz , corticocription . 2-AR is polymorphic within the human population [2-AR agonists and/or receptor dysfunction in asthma [2-AR agonists less than once a day [2-AR agonists plus moderate-dose inhaled corticosteroids irrespective of B16 genotype [A series of studies revealed that the gene encoding for βpulation ,106. Sucn asthma . For exan asthma  or salmen asthma . In contce a day . In asthgenotype . 2-AR function undergoes desensitization during persistent agonist exposure because of receptor phosphorylation by GRKs, which was found to be highly expressed in ASM [2-AR during persistent agonist exposure, thus possibly contributing to β2-AR agonist variability in asthma treatment of African-Americans [βd in ASM . The codmericans .2-AR expression as being constantly regulated by systemic catecholamines or continuosly administered β2-AR agonists, then the glutamic (Glu)27 allele may be expressed to a higher level in the lung, as it is refractory to down-regulation. More than a decade ago, a study by Hall et al. [2-AR agonists in both children and adults with asthma was identified [If one considers βl et al.  addresseentified .2-AR agonists administered by inhalation are the most effective therapy for rapid reversal of airflow obstruction and prompt relief of asthmatic symptoms. The most widely used are salbutamol  and terbutaline; their action occurs in 5 min or less, peaks after 30 to 60 min and lasts 4 to 6 h [1) does not decline, but the duration of action is slightly shortened [2-AR it may have a deleterious effect [2-AR agonists are recommended for use on request to relieve symptoms or before anticipated exposure to known asthmatic triggers, especially exercise. In mild-to-moderate asthmatic patients, a cumulative bronchodilator effect of salbutamol can be seen up to inhaled doses ranging from 700 to 1,500 µg [Short-acting β4 to 6 h . With rehortened . A regulhortened . Moreoves effect ,118. So,1,500 µg . Dose-de1,500 µg .2-AR agonists to use is based largely on cost and patient's or physician's preference. Salbutamol has been made available in a metered-dose inhaler free of chlorofluorocarbons (CFCs), and CFC-containing salbutamol inhalers were taken off the market on 31 December 2008. Like CFCs, the alternative propellant, hydrofluoroalkane (HFA), is inert in the human airway [The decision about which of the various short-acting βn airway . The HFAn airway  and can n airway . Levalbun airway . Howevern airway .2-AR agonists salmeterol and formoterol have a bronchodilator potency similar to that of their short-acting counterpart. Features distinguishing the two long-acting β2-AR agonists are both practical and theoretical. The onset of action of formoterol occurs within 5 min, a period similar to that for short-acting β2-AR agonists, whereas salmeterol has a slower onset of action (15 to 20 min) [2-AR, whereas salmeterol is a partial agonist  [2-AR specificity, have fewer side effects than short-acting β2-AR agonists [2-AR agonists, regular use of long-acting β2-AR agonists results in only mild tachyphylaxis to the maximal bronchodilator effect and the duration of action [2-AR agonists  rapidly wanes with regular use [2-AR agonists is not impeded by regular use of long-acting β2-AR agonists [The inhaled long-acting β 20 min) . For thi 20 min) . Formoteagonist) . The impagonists . As muchf action . In contular use , a contrular use , the quiagonists .1, reduce symptoms and use of rescue medication, and improve exercise capacity and health status [2-AR agonists does not appear to be a problem in clinical practice, concerns have been raised about the side effects, particularly cardiac dysrhythmias, in susceptible elderly populations [In COPD, formoterol and salmeterol are more effective bronchodilators than their short-acting counterpart  and haveh status . There ih status . Though ulations . Neverthulations . Higher ulations .2-AR agonists afford sustained improvement in lung function may tempt clinicians to use them as a long-term controller medication, without concomitant use of anti-inflammatory treatment. However, this strategy results in unsuppressed airway inflammation and an unacceptably high rate of asthmatic exacerbations [2-AR agonists as single treatment for asthma control is not recommended and several formulations combining β2-AR agonist with an inhaled corticosteroid in a single device have been made available. Combinations of salmeterol with inhaled corticosteroid are generally used for regular treatment only, whereas those of salbutamol or formoterol have been suggested for both as needed and regular treatment [The fact that long-acting βrbations ,138,139.reatment .2-AR stimulatory effects were reduced but not eliminated by aerosol inhalation, its widespread use for the treatment of asthma was abandoned. Moreover, up to a five-fold increase in mortality among users of inhaled epinephrine was reported in 1948 [Epinephrine was the first synthetic β-agonist bronchodilator to be used clinically and was introduced in the early 1900’s . As syst in 1948 .2-AR agonist. It gradually replaced epinephrine because of its more potent β-stimulant properties and its virtual absence of α-agonist activity [Isoproterenol was introduced in 1940 into medicine as short-acting βactivity . Howeveractivity . 2-AR agonists and mortality in asthma were the startling rise in asthma deaths in several countries, including the UK, Australia and New Zealand during the early 1960s [et al. [2-AR agonists and restrictions on their over-the-counter sale, the usage of inhaled short-acting β2-AR agonists declined. Simultaneously, the asthma death rate fell sharply, suggesting that the previously noted relationship might have been a causal one. It is noteworthy, however, that this decline in the asthma death rate occurred concomitantly with a greater reliance on corticosteroid therapy, suggesting that the latter might also have contributed to the drop in mortality due to asthma [The most-often-cited examples of the possible relationship between short-acting βly 1960s  and in Nly 1960s ,146. In  [et al. ,147 founo asthma .2-AR agonists as contributing to the second epidemic of asthma deaths that occurred in New Zealand in the late 1970s and early 1980s [2-AR agonists, along with sustained-release theophylline preparations, were prescribed in place of inhaled corticosteroids and cromolyn [2-AR agonists delivered by air-driven home nebulizers for self-treatment of severe asthma. In this case, patients could be exposed to the risk of hypoxic cardiac arrest if large doses of short-acting β2-AR agonists were delivered in the face of uncorrected hypoxaemia [et al. [2-AR agonists. These last included fenoterol , but also salbutamol . The authors concluded that these findings could be due to adverse effects of the β2-AR agonists themselves but raised the alternative possibility that their increased use might simply be a marker of more severe disease. A 1 year clinical trial showed increased airway responsiveness and worsened asthma control during regular treatment with fenoterol added to usual therapy compared with short-acting β2-AR agonists used only as needed for symptom relief [versus as-needed salbutamol did not detect sustained adverse effects on asthma control [2-AR only as needed for symptom relief [Several hypotheses have been advanced incriminating short-acting βly 1980s . One sugcromolyn , possiblpoxaemia . Two caspoxaemia  and frompoxaemia  have led [et al. , by usinm relief . However control ,155. Nevm relief .2-AR agonists salmeterol [2-AR agonist therapy in the situation of severe exacerbations. However, concerns about the possible risks associated with long-acting β2-AR agonists therapy were raised soon after their introduction into clinical practice. This was due to the evidence that their regular use had the potential to reduce bronchodilator sensitivity to β2-AR agonists [2-AR agonists may be at risk of severe exacerbations if the symptom control, achieved with long-acting β2-AR agonists use, led to a discontinuation of inhaled corticosteroid therapy [The introduction of long-acting βlmeterol  and formlmeterol  in the 1lmeterol . There wagonists ,160 and agonists , which magonists . It also therapy .an interim analysis showing a statistically significant, four-fold increase in asthma mortality with salmeterol. By contrast, in a large UK-based case-control study there was no positive association between long-acting β2-AR agonists therapy and asthma death [2-AR agonists therapy were co-prescribed inhaled corticosteroid [There were also concerns about a potential risk of mortality from the Salmeterol Nationwide Surveillance Study . In thisma death . Howeverosteroid . Due to osteroid .2-AR worldwide. Three placebo-controlled trials showed that the higher dose (48 µg/day) of formoterol tended to be associated with more serious asthma exacerbations than a lower one [Formoterol fumarate is currently the most widely used long-acting βower one . Howeverower one . et al. [2-AR agonists in the whole data set, no increased risks of cardiac-related deaths or cardiac-related non-fatal serious adverse events were observed. Similarly, when examined by inhaled corticosteroids use there was no increased risk of cardiac serious adverse events with regimens of formoterol with inhaled corticosteroids vs. inhaled corticosteroids without long-acting β2-AR agonists. Noteworthy, there was a significant reduction in asthma-related serious adverse events with formoterol  speaking against a relationship between formoterol and increased asthma mortality. The main limitation of the study by Sears et al. [Recently, Sears et al.  have reps et al.  was the 2-AR agonists with longer half-lives are currently undergoing development, with the hope of achieving once-daily dosing. Among them, the development of indacaterol is very advanced and it is likely that this drug will be launched by the end of 2010 [A variety of β of 2010 ,172. Ind of 2010 .2-AR instead of doubling or increasing the dose of inhaled corticosteroids [2-AR compared with adding a leukotriene antagonist [2-AR agonists should not be used as monotherapy in asthma but always together with an inhaled corticosteroid. Numerous studies have shown the benefit of adding a long-acting βsteroids ,174,175 tagonist . Howevertagonist  and the tagonist  now emph2-AR agonists have proved effective in achieving a good control of asthma. In particular, they are essential in reducing daytime and especially nighttime symptoms, improving lung function, reducing the risk of exacerbations, and minimizing the required dose of inhaled corticosteroids.In conclusion, either as rescue medications and therapy added to or combined with inhaled corticosteroids, short- and long-acting β


Sodium arsenite can impair male reproductive function by inducing oxidative stress. Silymarin is known as a potent antioxidant. This study was performed to investigate if silymarin can prevent the adverse effect of sodium arsenite on ram sperm viability, motility and mitochondrial membrane potential. Epidydimal spermatozoa obtained from ram were divided into five groups: 1) Spermatozoa at 0 hr, 2) spermatozoa at 180 min (control), 3) spermatozoa treated with sodium arsenite (10 μM) for 180 min, 4) spermatozoa treated with silymarin (20 μM) + sodium arsenite (10 μM) for 180 min and 5) spermatozoa treated with silymarin (20 μM) for 180 min. MTT assay and Rhodamine 123 staining were used to assess sperm viability and mitochondrial membrane potential respectively. Sperm motility was performed according to World Health Organization (WHO) guidelines. Viability (p<0.01), nonprogressive motility (p<0.001) and intact mitochondrial membrane potential (p<0.001) of the spermatozoa were significantly decreased in sodium arsenite treated group compared to control group. In silymarin + sodium arsenite group, silymarin could significantly reverse the adverse effect of sodium arsenite on these sperm parameters compared to sodium arsenite group (p<0.001). In addition, the application of silymarin alone for 180 minutes could significantly increase progressively motile sperm (p<0.001) and decrease non motile sperm (p<0.01) compared to the control.Silymarin could compensate the adverse effect of sodium arsenite on viability, nonprogressive motility and mitochondrial membrane potential of ram sperm. Nowadays infertility has become one of the critical problems for couples. Arsenic as a toxic metal and environmental contaminant could be a risk factor for male fertility. The use of arsenic-containing herbicides, insecticides, rodenticides, and preservatives and by-products of fossil fuels is enough to endanger men fertility . (Silybum marianum) seeds  seeds . This com) seeds -12. ThisViability and motility are considered as the most important parameters of mature sperm which indicate its structural and functional quality to move toward an egg for successful fertilization. Thus a focus on mitochondria integrity, which reflects the metabolic status of sperm to provide energy for survival and movement, can give information about the health of sperm for fertilization process. No doubt, factors that impair mitochondrial integrity and energy production of sperm endanger its health to reach the egg. Due to the toxic effect of arsenic on reproduction and fertility, this study was carried out to investigate if silymarin as a potent antioxidant can prevent the adverse effect of sodium arsenite on viability, motility and mitochondrial membrane potential of ram sperm.Epididimal sperm collection and treatmentsIn this experimental study, Farahani's ram testes were received from Arak slaughterhouse immediately after ram daily slaughter for public consumption and transferred to the research laboratory under standard conditions. The ethical issues in the use of animals in research were observed. The experiment were approved by the ethical committee at Arak University A few incisions were made in the caudal epididymis and spermatozoa were then washed into a sterile Falcon tube by Ham's F10 medium . Firstly sperm number and sperm motility were determined, according to World Health Organization protocol (WHO), to estimate sperm quality .6 spermatozoa and divided into five groups (n=6): 1. Spermatozoa at 0 hr, 2. Control spermatozoa, 3. Spermatozoa treated with sodium arsenite , 4. Spermatozoa treated with silymarin  + sodium arsenite (10 μM) and 5. Spermatozoa treated with silymarin (20 μM). Samples 2-5 were kept at 37oC in a CO2 incubator for 180 min.High quality sperm samples were then used for experiments. The sperm samples were separated in eppendorf tubes as each tube contained 5×10Sperm viabilityoC in CO2 incubator for 1 hr. The tubes centrifuged at 6000 rpm for 6 min and the precipitate was dissolved in 200 µl dimethyl sulfoxide (DMSO). The solution was then centrifuged at 4000 rpm for 4 min. 100 µl of the purple solution were transferred into a 96-well plate and absorbance was measured using ELISA reader  at 505 nm. Optical density of sample was then used for calculating sperm viability percentage .The MTT, 3--2, 5-diphenyl tetrazolium bromide, assay was used to assess viability. This assay was performed based on a method described by Mosmann . In brieSperm motilityEvaluation of sperm motility was done according to WHO guidelines (13). In brief, 10 µl of sperm suspension was placed on semen analysis chamber. Minimum of five microscopic fields was evaluated to estimate sperm motility on at least 200 spermatozoa for each sample. The percentage of sperm motility was evaluated for following motion patterns: progressive motile sperm (PMS), nonprogressive motile sperm (NPMS) and nonmotile sperm (NMS).Sperm mitochondrial membrane potential oC for 10 min in dark place. Tubes were centrifuged at 300 gr for 10 min and 1 ml Phosphate Buffered Saline was then added to remainder precipitate. Thin smears were then prepared and observed under an Olympus fluorescence microscope at 1000× magnification  was measured using Rhodamine 123 which is a cationic, cell permeable and fluorescent dye. It is rapidly absorbed by active mitochondria and has no toxic effect . For stafication . At leasStatistical analysisThe results expressed as mean±SD for six samples per group. One-Way analysis of variance (ANOVA) followed by Tukey's test was used to assess data statistical significance. p<0.05 was considered significant.Sperm viabilityThe percentage of sperm viability in sodium arsenite group was significantly decreased in comparision with control group (spermatozoa at 180 min) (p<0.01). In silymarin+ sodium arsenite group, silymarin could significantly (p<0.001) reverse the adverse effect of sodium arsenite on sperm viability compared to sodium arsenite group .Sperm motilityNo significant differences were found in PMS percentage in the group treated with sodium arsenite compared to the control, while the percentage of NPMS was significantly decreased (p<0.001) and the percentage of NMS was significantly increased (p<0.001) compared to the control . SpermatIn addition, in silymarin group, PMS percentage was significantly increased (p<0.001) while the NMS percentage was decreased significantly (p<0.001) compared to the control .Sperm mitochondrial membrane potential The application of sodium arsenite, significantly decreased (p<0.001) the sperm percentage of with intact MMP compared to the control. In silymarin group+ sodium arsenite, silymarin could significantly (p<0.001) ameliorate the toxic effect of sodium arsenite on MMP compared to sodium arsenite group , 4.In this study the adverse effect of sodium arsenite on viability, motility and mitochondrial membrane potential of ram epididymal sperm was examined. Also the protective effect of silymarin on these parameters in sodium arsenite treated spermatozoa was tested. Evaluation of sperm mitochondrial integrity and metabolic status in the health and pathology could provide information about sperm health as well as sperm motility and viability. Although visual estimation of viable sperm using the traditional methods such as eosin-nigrosin and trypan blue staining are simple and inexpensive, the results can be influenced by experience of analyst . In addition, these methods just assess membrane integrity and provide no information about sperm mitochondrial capacity and metabolic status. MTT reduction assay which evaluates viability in wide variety of cells could be used for the assessment of sperm mitochondrial capacity to estimate sperm fertility , 18-20. Our results showed a significant decrease in viability percentage (p<0.01) and intact MMP (p<0.001) in sodium arsenite treated spermatozoa. In addition, this toxicant was negatively affected the percentage of NPMS and NMS (p<0.001). This result was in agreement with previous findings on sperm viability and motility of rat as well as mice sperm motility under in-vivo conditions. Mitochondria might be the most important target for arsenic toxicity , 25. ArsArsenic has also an ability to complex with protein's SH groups, thus depleting the mitochondrial glutathione (GSH) level which is not only an antioxidant but also is an essential factor for maintenance of mitochondrial proteins thiol groups in the reduced state , 28. UndOn the other hand, a change in mitochondria permeability results in the release of mitochondrial cytochrome C, leading to the activation of caspase-9 and caspase-3 in the mitochondrial pathway . We therBecause of high concentration of polyunsaturated fatty acids and low antioxidant enzymes, mammalian spermatozoa are susceptible to lipid peroxidation mediated by oxidative stress . TherefoThe incubation of spermatozoa for 180 min (control group) caused a significant decrease in PMS percentage as well as significant increase in NPMS and NMS percentage compared to spermatozoa at 0 hr. The prolonged in-vitro incubation of human spermatozoa was shown to induce a time dependent loss of motility and some parameters related to sperm motion . Such deIt is therefore possible that the motion patterns deterioration of ram sperm during 180 min incubation, under aerobic conditions, might be attributed by oxidative attack or an unbalanced between sperm oxidation and antioxidant defense system. To support this idea, we showed that the application of silymarin alone for 180 min significantly increased the PMS percentage (p<0.001) and decreased the NMS percentage (p<0.001) compared to the control. This effective result of silymarin might also be due to its antioxidant role in improving the capacity of sperm antioxidant defense system.Our results indicate that sodium arsenite has a negative influence on sperm viability, motility and intact MMP. In addition, silymarin is able to compensate the adverse effects of sodium arsenite on these parameters.


Concerning in vitro validation, 31 SSR primers among those used in the in silico PCR were synthesized and tested for their ability to detect polymorphism among six Egyptian date palm cultivars. All tested primers have successfully amplified products, but only 18 primers detected polymorphic amplicons among the studied date palm cultivars.The present investigation was carried out aiming to use the bioinformatics tools in order to identify and characterize, simple sequence repeats within the third Version of the date palm genome and develop a new SSR primers database. In addition single nucleotide polymorphisms (SNPs) that are located within the SSR flanking regions were recognized. Moreover, the pathways for the sequences assigned by SSR primers, the biological functions and gene interaction were determined. A total of 172,075 SSR motifs was identified on date palm genome sequence with a frequency of 450.97 SSRs per Mb. Out of these, 130,014 SSRs (75.6%) were located within the intergenic regions with a frequency of 499 SSRs per Mb. While, only 42,061 SSRs (24.4%) were located within the genic regions with a frequency of 347.5 SSRs per Mb. A total of 111,403 of SSR primer pairs were designed, that represents 291.9 SSR primers per Mb. Out of the 111,403, only 31,380 SSR primers were in the genic regions, while 80,023 primers were in the intergenic regions. A number of 250,507 SNPs were recognized in 84,172 SSR flanking regions, which represents 75.55% of the total SSR flanking regions. Out of 12,274 genes only 463 genes comprising 896 SSR primers were mapped onto 111 pathways using KEGG data base. The most abundant enzymes were identified in the pathway related to the biosynthesis of antibiotics. We tested 1031 SSR primers using both publicly available date palm genome sequences as templates in the  Phoenix dactylifera L., 2n = 36) [Date palm 2n = 36)  is one on = 36  i2n = 36) . Despite2n = 36) . Morphol2n = 36) , randoml2n = 36) . Compare2n = 36) .In recent years date palm has been subjected to intensive genome sequencing studies. Yang et al.  reportedNowadays, the commonly published genome data and the availability of bioinformatics tools provide the scientists with tools to identify simple sequence repeats that could be used to develop useful SSR markers. Hamwieh et al.  surveyedThe present investigation was carried out aiming to: 1) identify and characterize, simple sequence repeats within the third Version (VS 3.0) of the date palm genome; 2) comparatively analyze genic and intergenic SSR motifs and develop a new SSR primers database; 3) recognize all single nucleotide polymorphisms (SNPs) that are located within the SSR flanking regions and 4) annotate the pathways for the sequences assigned by SSR primers and consequently determine the biological functions and gene interactionhttp://qatar-weill.cornell.edu/research/datepalmGenome/download.html.In this study, we used the Version 3.0 of the date palm draft genome sequence of Khalas female variety published by Al-Dous et al. . It conthttp://pgrc.ipk-gatersleben.de/misa) and primer3_core [The Perl script MISA  and designated as (ATBV01 sequence) [Two methods were used to validate our designed SSR primers. The first method was s et al.  and Al-Ms et al. . The firs et al. . While, equence) .In vitro validation was used as the second method. This was performed through designing and testing 31 SSR primer pairs, randomly selected from the 1031 primers previously tested in silico. In vitro test was applied to investigate the polymorphism among six Egyptian date palm cultivars . Total genomic DNA was extracted from date palm leaves, using the DNeasy Plant Kit (QIAGEN). The PCR amplification reaction was prepared in 25 μl containing 50 ng of DNA, 2.5 mM MgCl2, 5 μl 5X PCR buffer, 0.5 mM each SSR primer, 0.5U of GoTaq Flexi DNA Polymerase, and 2.5 mM dNTPs. The PCR temperature profile was 95°C for 5 min, followed by 35 cycles of 95°C for 50 s, the annealing temperature for each SSR primer  were located within the intergenic regions with a frequency of 499 SSRs per Mb. While, 42,061 SSRs (24.4%) were located within the genic regions with a frequency of 347.5 SSRs per Mb. Interestingly, Zhang et al. [Among the genic regions 1,785 SSRs were located in exons. This represents about 4.24% of the total genic regions (121018751 bp). However, 40,276 SSRs (95.76%) were located within the introns. The details of the identified SSRs in date palm are summarized in the .Our investigation of the different SSR motifs present in the intergenic region showed that the most abundant type was the mono- (63.6%), followed by the di- (27.8%), tri- (6.2%), tetra-(2%), penta- (0.3%) and hexa- (0.1%). However, in the introns of the genic regions the percentages of mono-, di-, tri-, tetra-, penta- and hexa- repeats were 62.7%, 29.3%, 5.8%, 1.9%, 0.2% and 0.1% respectively. While in the exons, the most frequent type was the tri-, followed by the mono-, di-, hexa- and tetra- repeats . The penta- repeat was not found in the exon regions Similarly, Zhao et al.  reportedThe total population of the detected SSRs across the genome was classified as non-triplet repeats  and triplet repeats (tri and hexa). The results are summarized in Tables As shown in Fig 2 illustrates the mono, di and tri repeat sequences, number, and locations of the repeats whether in the genic or intergenic regions using the Circos software developed by Krzywinski et al. [The high appearance of tri repeats in coding regions was consistent with previous studies in plant genomes –24. Fig i et al. .. While, 48 repeats were discovered in the genic regions only and comprised 11 tetra, 9 penta and 28 hexa repeats. In addition, 236 repeats were unique to the intergenic regions. The latter comprised 63 tetra, 84 penta and 88 hexa repeats. The present study represents the first report concerning the comparison of these specific SSR motifs  in both genic and intergenic regions in the date palm genome.Concerning the other types of SSR repeats, 134 SSRs  repeats were present in both genic and intergenic regions Among the tri repeats, 60 different repeat sequences were determined In this context, Morgante et al. , Li et aIn non-coding regions the rate of base substitution is high , however.A total of 111,403 SSR primer pairs were designed, that represents 291.9 SSR primers per Mb and covers the amplification of 22,652,015 bp (5.9%) of the total genome. Out of the 111,403, only 31,380 SSR primers were developed in the genic regions. These primers could amplify about 7,727,736 bp (6.38%) of the total genic regions. However, 80,023 primers covered the amplification of about 14924279 bp (5.72%) of the total intergenic regions Phoenix dactylifera Genic SSR primers, and Pd_IG_SSR was refers to Phoenix dactylifera Intergenic SSR primers and x refers to serial number).Using the Version 1.0 date palm genome, Hamwieh et al.  designedhttp://qatarweill.cornell.edu/research/datepalmGenome/download.html, and were subjected to Perl script developed by our research team to recognize all SNPs located in the SSR flanking regions. A total of 250,507 SNPs were determined in 84,172 SSR regions which represent 75.55% of the total SSR flanking regions. Therefore, the frequency of SNPs per SSR flanking region was 2.97. The distribution of these SNPs among the genome revealed that a number of 186,953 SNPs was located in 60,764 intergenic SSR flanking regions, while 63,554 SNPs were located in 23,408 genic SSR flanking regions . Thus, the high range of variation among the date palm genotypes could be attributed to the high record of SNPs in the genic regions.SNPs data were downloaded from The newly designed SSR primer pairs and related SNPs within the genic and intergenic regions were listed in the  sequence [in silico PCR reactions. When using the date palm genome PDK30 sequence[To validate our designed SSR primers, the first test was sequence  and ATBVsequence were used sequence, all the sequence with an in vitro validation, 31 SSR primers among those used in the in silico PCR were synthesized and tested for their ability to detect polymorphism among six Egyptian date palm cultivars. All tested primers have successfully amplified products, but only 18 primers detected polymorphic amplicons among the studied date palm cultivars .Concerning the in silico and in vitro PCR are summarized in in silico and in vitro PCR were consistent in the number and length of alleles for some primers, while other primers recorded polymorphism in fragments sequence and differences in some alleles length. Moreover, some alleles could be detected in one genome and absent in other genome sequence. As shown in in vitro only 23 were consistent in both in silico and in vitro PCR.The comparisons between the results of the The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database is a useful tool for understanding genes biological functions and their molecular interactions. We usedBased on the basic parameters of the Blast2GO program, the blast results revealed that 3787 sequences were partial genes, thus, this number was excluded from the next steps of analysis. The remaining 8487 sequences showed blast result path to gene ontology (GO) annotation step. This step indicated 3201 sequences with no GO terms, while, 5286 sequences were assigned to 13554 GO terms, ranging from 1 to 45 GO terms per sequence. Sequences with GO terms were introduced into the EC number detect step. This analysis revealed only 463 unique sequences (genes), which encode an actual number of 258 enzymes distributed among 111 different pathways. These genes comprised 896 SSR primers because each gene sequence might contain more than one SSR primer. The sequences with blast results, GO terms and EC numbers were listed in .The data for the pathways name, name of each enzyme and the position of their coding genes within the genome, in addition to the EC numbers for each enzyme were listed in Figs  respectively.To confirm the pathway annotation results, two pathways, i.e., the starch and sucrose metabolism and the amino sugar and nucleotide sugar metabolism pathways were selected to apply the Blast2GO program. This program highlighted the identified Enzyme codes on the respective KEGG map as shown in in silico PCR for testing the primers before their use in vitro, thus saving time, effort and cost.This study extends the basic research on date palm in an attempt to promote applying genomics and bioinformatics to enhance breeding and improvement programs of this important crop. The comparative studies depending on SSR motifs are useful since they represent co-dominant markers, highly polymorphic regions and could be used in genetic diversity analysis and breeding programs through designing specific primers for these regions. Genic SSR primers are helpful to detect the polymorphism within functional regions. Therefore, the breeder can choose the appropriate primers according to their localization in the biological pathways related to the trait of interest. Moreover, SNPs in the SSR flanking regions could provide an additional advantage to the breeders. Furthermore, this investigation highlights the usefulness of the S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(XLSX)Click here for additional data file.S6 File(XLSX)Click here for additional data file.S7 File(XLSX)Click here for additional data file.


Critically, ROCK inhibition in normal hearts of NTLs without α1A-AR overexpression caused hypophosphorylation of both MYPT1 and cMLC2, and rapidly reduced basal contractility.Hypercontractility without hypertrophy with α1A-AR signaling and the physiological role of RhoA/ROCK signaling in maintaining contractility in the normal heart.We report for the first time pleiotropic α Sympathetic regulation of contractility reflects catecholamine stimulation of CM adrenergic receptors (ARs). β1-AR effects are normally dominant. In heart failure, however, β-ARs are downregulated and uncoupled from G proteins, and α1-ARs may act to maintain contractility.Heart failure is a major cause of death, disability and escalating health costs worldwide as populations age. Inotropic drugs are useful short term, but their long term use may increase mortality, in part due to increased calcium (Ca1A-AR overexpression (α1A-TG) display hypercontractility that is proportional to receptor number, is inhibited by selective α1A-AR blockade, and is not due to β-AR cross-talk 2+ overload We reported previously that transgenic mice with cardiac-restricted α1A-TG mice is indeed an agonist-induced increase in intracellular Ca2+ release. In the absence of agonist, however, contractility is reduced. This unexpected finding was not due to reduced intracellular Ca2+ concentration [Ca2+]i but to reduced Ca2+ sensitivity of the myofilaments resulting from inhibition of the RhoA/Rho kinase (ROCK) signaling pathway. This inhibition results in hypophosphorylation of myosin phosphatase target subunit 1 (MYPT1), relieving its inhibition of myosin light chain phosphatase (MLCP) and leading to inactivation of cardiac myosin light chain 2 (cMLC2), a key myofilament protein mediating contraction. The inhibition of RhoA/ROCK signaling was mediated by the α1A-AR in the absence of ligand, consistent with spontaneous receptor isomerization to a conformation distinct from that which activates increased Ca2+ release in the presence of ligand, indicating pleiotropic receptor signaling.Here, we demonstrate that the mechanism underlying the enhanced cardiac contractility of α2+]i, the RhoA/ROCK signaling pathway may be a suitable target for development of novel inotropic interventions.Critically, the regulation of basal contractility by the RhoA/ROCK pathway is shown to be physiologically relevant because its inhibition in non-transgenic mice with normal receptor expression caused a significant reduction in basal contractile function. Because modulation of cMLC activity can increase contractility without altering i indicator Indo-1/AM (1.2 µM) were field-stimulated at 0.5 Hz, and [Ca2+]i obtained from photomultipliers. Simultaneously, shortening was measured by edge detection . The concentration-response to phenylephrine  was recorded.CMs were isolated by enzymatic retrograde infusion 2 and 5% CO2 at 37°C and pH 7.4. A fluid-filled balloon was inserted via the mitral valve, and inflated to a diastolic pressure of ∼5 mmHg. Hearts were maintained at 37±0.1°C in a water-jacketed bath. Experiments, performed in separate groups, were: 1) perfusion with successively increasing concentrations of A61603 , a selective α1A-AR agonist; 2) before and after perfusion with angiotensin II  or with one of two selective α1A-AR antagonists, RS100329  or KMD3213 dihydrobromide ; 3) for contractile protein measurements, perfusion with saline or RS100329 , then snap-frozen (liquid nitrogen); 4) for RhoA/ROCK pathway, perfusion with saline or Y-27632, a selective ROCK inhibitor , then snap-frozen; 5) for RhoA/ROCK signaling in agonist-induced responses, A61603 (0.1 nM−1.0 µM) in absence or presence of Y-27632.Hearts were excised into ice-cold modified Krebs-Henseleit perfusion buffer. The aorta was perfused at 80 mmHg with perfusion buffer equilibrated with 95% O2+] Skinned left ventricular fiber strips were prepared as described previously Steady state levels of the following were determined by Western blot analysis: cardiac troponin I (cTnI) and its Ser23/24 and Ser43 phosphorylated forms (p-cTnI); cTnC; cTnT; cMLC2 and its Ser20 phosphorylated form (p-cMLC2); MYPT1 and its Thr696 phosphorylated form (p-MYPT1); myosin light chain kinase (MLCK); protein kinase Cα (PKCα) and PKCε. Left ventricular tissue was lysed in a buffer , homogenized, and proteins quantified using the Pierce BCA Protein Assay Kit. Protein (40 µg) was separated by SDS-PAGE, and transferred to PVDF membranes (Bio-Rad Laboratories) blocked for 2 hours at room temperature with 5% bovine serum albumin (Sigma) dissolved in Tris-buffered saline with 0.1% Tween.Primary mouse monoclonal antibodies were: cTnC ; cTnT ; smooth muscle MLCK . Primary rabbit polyclonal antibodies were from Abcam: p-cTnI (Ser43) ; p-cMLC2 (Ser20) ; cMLC2 ; or Cell Signalling: p-cTnI (Ser23/24) ; cTnI ; p-MYPT1 (Thr696) ; MYPT1; PKCα ; PKCε ; RhoA(67B9) . GAPDH  was used to standardize for loading. Horseradish peroxidase-conjugated goat anti-mouse (1∶5000) or anti-rabbit (1∶10000) secondary antibodies  were used at room temperature for 1 hour. Immunologic detection was accomplished using Amersham ECL Western blotting detection reagents . Protein levels were quantified by densitometry using NIH ImageJ analysis software.A G-LISA kit was used . After lysis in a buffer containing 50 mM NaF (Sigma Aldrich), 20 mM sodium pyrophosphate (Sigma Aldrich) and 1 mM p-nitrophenyl phosphate  to block phosphatase activity, homogenization, and protein concentration quantification, aliquots of lysate (0.5 mg protein/ml) were added to wells linked to a Rho-GTP-binding protein. Active (GTP-bound) RhoA attached to the wells. Inactive (GDP-bound) RhoA was eluted. Active RhoA was detected with an anti-RhoA antibody, and absorbance read at 490 nm with a PHERAStar FS microplate reader .10 [Ca2+]) and fitted with Hill plots to determine pCa50  and Hill coefficient values max) was scaled as a % of baseline, and data from individual experiments fitted to a non-linear regression equation  to determine EC25, EC50 and EC75, the agonist concentrations generating 25%, 50% and 75% of the maximum dP/dtmax response, respectively, and analyzed using one-way ANOVA. Data points from different dose-response curves were compared using two-way ANOVA for repeated measures, with Newman-Keuls post-hoc test when indicated. For protein expression quantification, unpaired t tests were used for comparisons between two groups, and two-way ANOVA with multiple comparisons was used among groups . Results are mean ±1 SEM.All experiments and analyses were blinded. In skinned fibers, relative force was plotted against pCa .In the absence of αortening P<0.01. eticulum . Kinetic1A-TG CMs observed in the absence of agonist stimulation, concentration-response curves to incremental doses of the α1-AR agonist, PE, demonstrated greater shortening of α1A-TG than NTL CMs . Heart rate (α1A-TG 381±17 vs. NTL 371±14 bpm) and coronary flow (2.5±0.2 vs. 2.2±0.1 ml/min) were not different.The isolated CM experiments suggested that the hypercontractility of αesponses  that cloactility , evidenc1A-AR agonist, A61603, the α1A-TG hearts demonstrated marked hypercontractility, evidenced by higher peak pressure and higher dP/dtmax for any given concentration of A61603, with parallel increments in dP/dtmin . A61603 concentrations above 3 nM caused rapid decompensation in α1A-TG hearts, corresponding to the sudden death phenotype documented previously in vivoWith the selective αdP/dtmin . EC50 wa1A-TG hypocontractility is that sustained overstimulation of the contractile apparatus, due to activation of the greatly increased number of α1A-ARs by endogenous catecholamines, results in heterologous downregulation of contractility at a sub-receptor level . If so, stimulation of the same sub-receptor pathway via an alternate Gαq-linked receptor would be expected to elicit a reduced contractile response in α1A-TG hearts. To test this hypothesis, isolated perfused hearts were treated with AngII to activate the Gαq/11-coupled AT1 receptor. AngII produced a transient negative, followed by a large sustained positive inotropic response : pCa50 5.86±0.12 α1A-TG vs. 5.88±0.11 NTL; Hill coefficient 3.03±0.12 nCa α1A-TG vs. 2.82±0.19 nCa NTL. The pCa50 is a measure of the sensitivity of the contractile apparatus to Ca2+. The Hill coefficient gives an indication of the affinity of the functional unit of the contractile apparatus for Ca2+.Next we evaluated myofilament Ca2+ sensitivity was not different in α1A-TG skinned cardiac fibers, a modulating effect of myofilament protein phosphorylation on Ca2+ sensitivity in vivo, where the cell membrane is intact, could not be excluded. To address this issue, we assessed the phosphorylation status of the key contractile proteins, cTnI and cMLC2, in isolated hearts from α1A-TG and NTL mice perfused with vehicle  or RS100329. There were no differences in total cTnI or p-cTnI, or in cTnC or cTnT, between α1A-TG and NTL hearts (data not shown), but α1A-TG mice exhibited significant hypophosphorylation of cMLC2  than NTL hearts , but was not altered by RS100329 treatment. PKCε was unaffected by genotype or treatment (data not shown). MLCK phosphorylates MLC in response to changes in [Ca2+]i, but was unaffected by genotype or RS100329 treatment (data not shown), consistent with the fact that baseline hypocontractility in α1A-TG hearts was not associated with changes in [Ca2+]i.PKC, MLCK and MLCP regulate MLC phosphorylation in smooth muscle. PKCα was significantly higher in α1A-TG mice  was directly correlated with RhoA activity  RhoA binds to the C-terminal region of MYPT1, and that activated ROCK inhibits MLCP by phosphorylating MYPT1 at Thr696, we next examined RhoA/ROCK signaling. RhoA activity was significantly reduced in αG hearts , a reducG hearts , or of Rmax and dP/dtmin in NTL hearts .To further evaluate the involvement of RhoA/ROCK signaling in basal contractility, hearts were treated with Y-27632, a selective ROCK inhibitor. Selective ROCK inhibition caused significant falls in peak pressure, dP/dtL hearts  within 5L hearts , but cau1A-AR overexpression was increased intracellular Ca2+ release in response to agonist stimulation. This was not unexpected because other Gαq/11-coupled receptors, including the AT1 and endothelin receptors, increase Ca2+ release by activating phospholipase Cβ, and this would account for the increased PKCα expression we observed. In addition, α1A-AR coupled Ca2+ entry depends on a novel mechanism involving redirection and activation of the transient receptor potential canonical 6 (TRPC6) channel from the cytoplasm to the plasma membrane via interaction with Snapin, but α1A-AR activation of Gαq/11 also produces diacylglycerol that independently activates TRPC6 in the plasma membrane 1A-ARs by endogenous catecholamines could thus account for the hypercontractility seen in vivo2+]i might be expected to stimulate cardiac hypertrophy also. Marked hypertrophy is observed, for example, in mice with cardiac overexpression of Gαqq/11-coupled receptors, such as the AT1 receptor 1A-TG CMs or in mouse or rat hearts in vivo1A-AR activation in vivo is not sustained but intermittent, fluctuating with endogenous catecholamine levels. This is consistent with the propensity of α1A-TG mice to stress-related sudden cardiac death suggestive of Ca2+ overload 1A-AR activation would be expected to cause heterologous desensitization of the contractile response amplitude of the [Ca2+]i transient with agonist stimulation of α1A-TG CMs, we observed no change in resting [Ca2+]i with repeated but non-sustained α1A-AR activation  We propose a model of pleiotropic receptor signaling  in which1A-AR raises the question of physiological relevance. Although contractility is reduced in mice with a non-phosphorylatable form of cMLC2, and reduced phosphorylation of cMLC2 has been found in failing human and mouse hearts The link between cardiac contractility and RhoA/ROCK signaling in animals with 170-fold overexpression of the α1A-AR expression, demonstrating a significant reduction in baseline contractility in association with reduced phosphorylation of MYPT1 and cMLC2. These findings indicate that the RhoA/ROCK pathway plays an important physiological role in maintaining normal baseline contractility. This normal role may be amplified in heart failure, when the β-ARs are downregulated and uncoupled from G proteins, and with the increasing therapeutic use of β-AR blockers. Moreover, increased contractility with RhoA/ROCK pathway activation does not depend on increased Ca2+ release, suggesting it as a promising target for development of novel inotropic agents that might not increase mortality with long term use.To address this issue more directly, we examined ROCK inhibition in NTL hearts with normal α1A-AR is based on a model with 170-fold overexpression of the receptor. Nevertheless, this model has allowed us to identify pleiotropic signaling by the receptor that may have broader significance for receptor physiology. As noted above, promiscuous coupling due to receptor overexpression can be excluded because the model is based on the wild type α1A-AR. This model also allowed us to identify the importance of RhoA/ROCK signaling and its control of MLC2 phosphorylation in modulating cardiac contractility, and we have demonstrated that this mechanism supports baseline contractility even in the setting of normal α1A-AR expression. The mechanism by which the α1A-AR inhibits RhoA activity in the absence of ligand remains to be determined in future experiments.Our novel finding of depressed cardiac contractility due to agonist-independent activity of the αFigure S11AHypocontractility and impaired relaxation in α-1ATG hearts are reversed with the selective α-AR antagtonist, KMD3213. A, representative recordings of left ventricular pressure (LVP) and dP/dt at baseline and during KMD3213 infusion (100 nM) in isolated perfused contracting hearts. B, composite data at baseline (Control) and after KMD3213 infusion (100 nM) for 10 min in NTL (n = 4) and α1A-TG (n = 4) hearts. Data are shown as the mean ± SEM. *P<0.05, **P<0.01.(TIF)Click here for additional data file.


Chrysanthemum morifolium, is a well-known flowering plant worldwide, and has a high commercial, floricultural, and medicinal value. In this study, simple-sequence repeat (SSR) markers were generated from EST datasets and were applied to assess the genetic diversity among 32 cultivars. A total of 218 in silico SSR loci were identified from 7300 C. morifolium ESTs retrieved from GenBank. Of all SSR loci, 61.47% of them (134) were hexa-nucleotide repeats, followed by tri-nucleotide repeats (17.89%), di-nucleotide repeats (12.39%), tetra-nucleotide repeats (4.13%), and penta-nucleotide repeats (4.13%). In this study, 17 novel EST-SSR markers were verified. Along with 38 SSR markers reported previously, 55 C. morifolium SSR markers were selected for further genetic diversity analysis. PCR amplification of these EST-SSRs produced 1319 fragments, 1306 of which showed polymorphism. The average polymorphism information content of the SSR primer pairs was 0.972 (0.938–0.993), which showed high genetic diversity among C. morifolium cultivars. Based on SSR markers, 32 C. morifolium cultivars were separated into two main groups by partitioning of the clusters using the unweighted pair group method with arithmetic mean dendrogram, which was further supported by a principal coordinate analysis plot. Phylogenetic relationship among C. morifolium cultivars as revealed by SSR markers was highly consistent with the classification of medicinal C. morifolium populations according to their origin and ecological distribution. Our results demonstrated that SSR markers were highly reproducible and informative, and could be used to evaluate genetic diversity and relationships among medicinal C. morifolium cultivars. Chrysanthemum morifolium (Ramat.) Kitam is an important member of the family Asteraceae. A large number of C. morifolium cultivars are ornamental and medicinally important plants that are planted all over the world , also known as microsatellites, are short tandem repeated motifs that may vary in the number of repeats at a given locus Tautz, . SSR marC. morifolium cultivars, with the aim to provide new information that could be used to improve the utilization and conservation of C. morifolium genetic resources.In this study, SSR markers were developed and were applied to investigate genetic diversity and phylogenetic relationships among medicinal C. morifolium were collected from the main distribution areas in China. The sampled germplasms and voucher specimens are shown in Table http://www.nhpe.org). Voucher samples were deposited in the Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou Normal University, China.A total of 32 cultivars of Fresh, young leaf tissues from 10 individuals of each cultivar were randomly collected for genomic DNA isolation. The genomic DNA was isolated as described previously  were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/), with a mean length of 531 bp. These ESTs were analyzed to identify the perfect SSR loci using the MIcroSAtellite (MISA) software (http://pgrc.ipk-gatersleben.de/misa/), following the set to detect tandem repeats of hexa-, penta-, tetra-, tri-, and di-nucleotides, with a minimum number of 4, 4, 5, 7, and 10 tandem arrays of the core repeat, respectively. SSR loci embedded the ESTs with appropriate flanking sequences were selected for primer design using software Primer 3.0  EST-SSRs . After a trial run of 136 pairs of SSR primers, 55 of them with clearly separated bands, stable amplification, and rich polymorphism were chosen for further analysis.A total of 136 SSR primer pairs, targeting at 86 4)2SO4, 100 mM KCl, 1% Triton X-100, pH 8.8), 2 μL MgCl2 (20 mM), 0.4 μL dNTPs (10 mM), 1 μL of each primer (10 μM) (forward and reverse), 1 U Taq DNA polymerase , and 50 ng genomic DNA templates. PCR amplification was run using a GeneAmp PCR System 9700  with the following program: 94°C for 5 min, followed by 35 cycles of 94°C for 40 s, then 40 s at the annealing temperature of each primer pair, 72°C for 1 min 30 s, and a final extension at 72°C for 10 min. PCR products were separated on 1.5% agarose gel, stained with ethidium bromide and photographed under UV light. Sanger sequencing was used to confirm SSRs in amplified genomic DNA fragments as described previously  or absent (0) for each of the SSR markers. The polymorphism information content (PIC) of each pair of SSR primers was calculated using the formula:n is the number of alleles (marker), qi is the ith allele frequency, and qj is the jth allele frequency  to 38 (primer pair ID. 1774) Table . The PICA total of 1319 loci were accounted to calculate the genetic diversity among the 32 Chrysanthemum cultivars. Binary data matrices produced by SSRs were used to estimate the genetic similarity of the genotyped Chrysanthemum samples. The pairwise similarity coefficient among the 32 cultivars ranged from a maximum of 0.809 (between Huaiju “Hdbj” and Huaiju “Hxbj”) to a minimum of 0.533 (between Huangju “Jj3” and Gongju “Wgj”).C. morifolium populations , which means that the genetic relationship between Hangju cultivars and Machengju “Mcj” is very close, consistant with a previous study . In our study, all the Boju, Chuju and Gongju cultivars were grouped together within subgroup II-1, which confirmed the inference above  ; the National science and technology support Programme project (2011BAI04B02); Zhejiang Provincial New Agricultural Varieties Breeding Of Traditional Chinese Medicinal Materials Major R&D Projects (2012C12912).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.


In situ underwater microsensor measurements of pH, temperature, and irradiance in the porous ikaite crystal matrix, revealed an extreme microenvironment characterized by low temperatures, strong light attenuation, and gradients of pH changing from pH 9 at the outer column surface to above pH 10 over the first 1–2 cm of the ikaite. This outer layer of the freshly deposited ikaite matrix contained densely pigmented yellow and green zones harboring a diverse phototrophic community dominated by diatoms and cyanobacteria, respectively, as shown by amplicon sequencing. In situ O2 measurements, as well as underwater variable chlorophyll fluorescence measurements of photosynthetic activity, demonstrated high levels of oxygenic photosynthesis in this extreme gradient environment with strong irradiance-driven O2 dynamics ranging from anoxia to hyperoxic conditions in the ikaite matrix, albeit the local formation of gas bubbles buffered the day-night dynamics of O2 in the tufa columns. The microbial phototrophs in the ikaite matrix are embedded in exopolymers forming endolithic biofilms that may interact with mineral formation and cementing of ikaite crystals.The Ikka Fjord (SW Greenland) harbors a unique microbial habitat in the form of several hundred submarine tufa columns composed of ikaite, a special hexahydrate form of calcium carbonate that precipitates when alkaline phosphate- and carbonate-enriched spring water seeping out of the sea floor meets cold seawater. While several unique heterotrophic microbes have been isolated from the tufa columns, the microbial activity, and the boundary conditions for microbial growth in ikaite have remained unexplored. We present the first detailed  The Ikka Fjord in southwestern Greenland harbors a unique assemblage of tufa columns found nowhere else on the planet . The mineral has also been found as minor deposits in other cold habitats e.g., in sea-ice, precipitating during the freezing process of seawater . The Ikka Fjord is long and narrow with a maximum depth of 30 m oriented from NE-SW in a glacial valley bordered by steep, ~500 m high Precambrian mountains mainly composed of carbonatite, syenite, and gneiss. Tufa columns are only found in the shallow inner part of the fjord, “Ikka Bund,” that is protected by a geological land barrier forming a narrow entrance to the inner fjord  connected to an underwater microsensor meter . Light measurements were done in parallel with, and in close proximity to, the site of O2 and pH profile measurements (see below). Photon irradiance (PAR) at the water surface was monitored by a universal light meter .Light measurements of photon scalar irradiance  at the ikaite surface were performed www.dmi.dk) for the same period as temperature recording was performed.Temperature loggers  were placed on the ikaite column at three different depths in August 2013 recording temperature at an interval of 5 min for 7 days. The temperature loggers were placed at the top platform of the column of interest, at ~7 m mean sea level, and 1.5 m above and below this depth, Tidal data of the neighboring fjord Kangilinnguit , were obtained from the Danish Meteorological Institute .The chemical conditions in the ikaite matrix were measured by detailed mapping of OThe needle sensor measurements were carried out handheld by a diver, who could carefully insert the needle sensors into the porous ikaite matrix in steps of 5 mm, following markings on the needle tips Figure . Profili2 were measured at varying orientations on the tufa column relative to the incident sun light, to map any differences in O2 in relation to changes in the prevailing incident light conditions. The measured concentration profiles thus represent O2 levels at different light environments from the surface of the column to a depth of 20–30 mm within the matrix. All measuring points were sorted according to their orientation on the column, yielding profiles at different sun angles relative to the column apex, i.e., east, south, west, north, and exposed (top). The definition of N, S, E, and W was done by the divers compass. Measurements within an orientation zone were obtained in a line going down the column with a few cm between measurements on the side of the column, whereas top measurements were obtained more randomly on the top of the column. For each profile measurement, the microsensor was inserted normal to the ikaite surface. The measured O2 profiles were further separated according to time of day; when the sun was at its highest around noon, and in darkness 1–3 h after sunset, and between 2 years (August 2012 and 2013).Concentration profiles of Oin-situ dynamics of O2 concentration and photon scalar irradiance were measured during two 24 h cycles; one diel cycle with 100% cloud cover (completely overcast), and one diel cycle with 0% cloud cover (blue sky). During these measurements, the tip of the O2 needle sensor was fixed at a depth of ~15 mm inside the ikaite matrix, yielding a relatively high O2 reading at the beginning of a cycle. The photon scalar irradiance sensor was positioned on the ikaite surface in close proximity to the insertion point of the O2 electrode. About 4–5% of the incident irradiance remained at a depth of 15 mm, as calculated from the ikaite surface light measurements obtained in this study and light attenuation coefficients of ikaite measured in the laboratory .Diel in-situ under natural light conditions by a submersible, diver-operated pulse-amplitude modulated (PAM) fluorometer , equivalent to the fraction of energy that is photochemically converted in PSII, was determined as  is the incident photon irradiance of ambient photosynthetically active radiation (400–700 nm) and the constant 0.5 is from the assumption that the photon energy absorbed is equally divided between PSII and PSI. The ETR rates are relative since we did not measure the absorption cross-section of PSII.The effective quantum yield of photochemical energy conversion via photosystem (PS)II activity in the ikaite matrix was assessed in situ light acclimation of the phototrophs in the ikaite matrix. The light saturation coefficient, i. e., the irradiance at onset of light saturation of photosynthesis was calculated as Ek = rETRmax∕α, where rETRmax is the maximum activity and α is the initial slope of the rETR vs. irradiance curve; both parameters were obtained by curve fitting of rETR vs. irradiance curves with an exponential function  exposure to each irradiance level before a measurement pulse, so-called rapid light curves (RLC) of PSII activity vs. photon irradiance  list of photon irradiance levels ranging from 0 to 258 μmol photons m−2 s−1.Before  Figures . The fib Figures , and ϕPSIkaite samples for molecular analysis of the microbial diversity were retrieved by collecting ikaite material from distinct colored zones in a larger cross section taken from the column apex. The microbial diversity in the samples was analyzed by pyrosequencing of 16S rRNA genes, and sequence analysis was carried out as previously described . OTUs were clustered at 97% sequence identity using USEARCH . OTUs containing only one sequence (singletons) were discarded. Phylogenetic analysis was performed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline version 1.9.1 (www.qiime.org) . In this study, we mainly focused on analyzing the microbial phototrophs  in ikaite. The complete dataset of 92,266 quality-filtered sequences in QIIME-compatible format is available from the MG-RAST server .Trimming and quality-filtering of 16S rRNA gene sequences was performed using the software suite Biopieces  at various times of day, and thus shifting irradiance levels (data not shown). Thus, all pH measurements were pooled and plotted as the mean pH Figure . The pH 2 concentration profiles measured in the outermost 20–30 mm of the ikaite column apex displayed a great variability, not only from the column surface to deeper layers of the column, but also at different orientations on the column toward incident sunlight, as well as time of day .The Om Figure . From ab2 concentration were recorded at a depth of 15 mm inside the ikaite matrix of the column apex with accompanying irradiance measurements on the ikaite surface during two distinct 24 h cycles; one with clear skies and one with 100% cloud cover  reaching 20–25 μmol photons m−2 s−1 at a depth of 15 mm inside the column during midday. With decreasing irradiance, we observed a steep drop in O2 concentration rapidly reaching ~0% air saturation just after sunset. In the morning, O2 levels in the ikaite matrix increased again with a short delay after sunrise  that could be assigned to cyanobacteria and microalgal plastids. At the phylum level, cyanobacterial OTUs represented about 0.2–33%, and microalgal plastids about 0.1–27% of all sequences from the ikaite samples, with microalgal plastids dominating in the outer yellowish ikaite zones and cyanobacteria dominating in the green zones found deeper in the ikaite, with only little overlap between the two zones Figure . MicroscSynechococcales and Chroococcales and the filamentous orders Nostocales, Oscillatoriales, and Pseudanabaenales. In the microalgal zone 0–1 cm inside the ikaite, we identified plastids of microalgae belonging to the phylum Chlorophyta, and orders Cryptophyta, Haptophyceae, and Stramenopiles. The green layers were dominated by cyanobacteria, whereas the white-yellowish, yellow, yellow-green and white-green layers were dominated by plastid-bearing organisms. Nostocales predominated in one of the two sampled green zones, and Chroococcales in the other. Cryptophyta dominated in the white-yellowish layer, and Stramenopiles dominated in the two sampled yellow and yellow-green layers.A more detailed analysis of the distribution of different phototrophs across differently colored zones of ikaite revealed distinct community changes in composition of oxygenic phototrophs Figure , Table 1in situ studies of ikaite tufa columns have mostly focused on their geological characteristics  respiration, photosynthesis and bubble formation in the EPS bound crystal matrix, where the phototrophs reside, and (ii) the slow perfusion and mixing of the spring water through the outer ikaite crystal matrix that may also slowly push gas bubbles toward the exterior. We did not quantify or map the distribution of gas bubbles, but we speculate that such bubble formation might be induced in the pigmented zones, where phototrophs and ikaite crystals were bound together in EPS , and such measurements might reveal more details on the zonation and dynamics of anaerobic processes in the ikaite matrix.Our measurements were performed during August, and are presumably representative of photosynthesis in the tufa columns under maximum light availability. How the community of phototrophic microbes and other microbes in the tufa columns survive under very low light levels or even endure extended periods in darkness during the winter months remains to be studied. However, The microbial community within the ikaite tufa columns has previously been shown to be relatively diverse (including anaerobic phylotypes) and with a clear difference in the overall taxonomic composition between the interior and surface parts of the column (Stougaard et al., While the deeper green zone harbored a phototrophic community predominated by unicellular and filamentous cyanobacteria, the outer yellowish zones mainly harbored a diversity of microalgae Figure . Prelimi2 buffering properties mentioned above. As in photosynthetic biofilms (e.g., Kühl et al., 2 gradients and dynamics in the ikaite matrix further support the notion that phototrophic communities in the ikaite tufa columns have characteristics of photosynthetic endolithic biofilms. We speculate that EPS secreted by the microbial biofilms within the ikaite columns may play an important role, affecting the microenvironmental conditions, not only in bubble formation and oxygen buffering by slowing down diffusion of O2 within the matrix, but also playing a role in column formation. We speculate that ikaite crystals are cemented together by biogenic EPS into a porous matrix strengthening and shaping the cementing processes involved in tufa column growth. It has been shown that ikaite crystals can easily be chemically precipitated in the laboratory (Bischoff et al., Microscopic observation of fresh ikaite samples as well as macroscopic observations of ikaite cross-sections showed that ikaite crystals and cells in the pigmented zones were embedded in exopolymers (Supplementary Video In situ microenvironmental analysis revealed the presence of distinct O2 and pH gradients across the outermost 2 cm of the ikaite crystal matrix. pH increased from pH 8.2 in seawater to ~pH 9 at the tufa column surface and further increased to ~pH 10 1–2 cm below the surface, irrespective of ambient irradiance levels. Measurements of O2 concentrations in the ikaite showed pronounced dynamics with irradiance ranging from anoxia to hyperoxic conditions, albeit the porous ikaite matrix of crystals and phototrophs embedded in EPS sometimes exhibited accumulation of gas bubbles buffering such dynamics. Variable chlorophyll fluorescence measurements confirmed active photosynthesis in the pigmented layers of ikaite, which displayed low-light acclimation and high quantum yields in situ. The presence of dense pigmented layers of phototrophs and the observed chemical dynamics thus indicate the presence of endolithic biofilms inside the ikaite crystal matrix that may affect ikaite deposition and other biogeochemical processes in the tufa columns.The uppermost parts of ikaite tufa columns in Ikka Fjord, Greenland, harbor active populations of microalgae and cyanobacteria embedded in exopolymers and stratified into yellowish and green zones, respectively, in the outermost ~2 cm of the porous ikaite matrix. ET and MK designed the study. Field work was carried out by ET, MK, and JL. All analyses were completed by ET, MK, MG, and PS. All authors contributed to the discussion of the results. Manuscript was written by ET and MK with inputs from MG, PS, and JL.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.


AR induction of hHSC proliferation and matrix production was estimated in the presence of antagonists. AR involvement in fibrogenesis was also assessed in a mouse model of NASH and in humans with NASH. hHSC time dependently expressed AR and TACE. AR increased hHSC proliferation through several mitogenic signaling pathways such as EGFR, PI3K and p38. AR also induced marked upregulation of hHSC fibrogenic markers and reduced hHSC death. AR expression was enhanced in the HSC of a murine model of NASH and of severe human NASH. In conclusion, AR induces hHSC fibrogenic activity via multiple mitogenic signaling pathways, and is upregulated in murine and human NASH, suggesting that AR antagonists may be clinically useful anti-fibrotics in NAFLD.Amphiregulin (AR) involvement in liver fibrogenesis and hepatic stellate cells (HSC) regulation is under study. Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular cancer (HCC). Our aim was to investigate  Non-alcoholic fatty liver disease (NAFLD), the commonest cause of chronic liver disease in affluent countries, is a spectrum of liver diseases ranging from hepatic steatosis (simple intrahepatic accumulation of lipid droplets) through steatosis with inflammation and fibrosis  to cirrhosis and hepatocellular cancer (HCC)14The epidermal growth factor receptor (EGFR) ligand amphiregulin (AR) plays a central role in branching morphogenesis in organs and is expressed both in healthy and in cancer tissues. It is an autocrine growth factor as well as a mitogen for fibroblasts and regulatory T-cells9The association of AR expression with liver disease has been demonstrated in different animal models of liver damage and in human samples. AR is induced in the fibrotic liver of mice chronically treated with CCl4 and in models of acute liver damage induced by CCl4, activation of Fas and LPS administration, AR induction was also demonstrated in the liver of cirrhotic patients and rats, as well as in human HCC, suggesting that AR is also implicated in hepatic carcinogenesis9111213ex vivo role of AR signaling mechanisms in the fibrogenic physiology of primary hHSC, and secondarily to determine the in vivo significance and disease relevance of these findings by assaying AR in the HSC compartment of a mouse model of NASH and of human livers with NAFLD/NASH at different stages of severity. We show for the first time that AR is expressed in hHSC; further and importantly, we describe AR up-regulation in the HSC of mice and patients with NASH.In this study, our aims were primarily to study the AR has been detected in the whole liver but has not previously been reported specifically in HSC. To determine if AR is expressed by freshly isolated (quiescent) HSC and activated HSC we analysed hHSC for AR mRNA and protein expression, as shown in AR has been suggested to be pro-fibrogenic because mice lacking AR develop less hepatic fibrosis after carbon tetrachloride challenge compared to controlsAR is thought to act through the EGFR in non-liver cellsdata not shown). The effect is comparable to the effect of the pro-fibrogenic cytokine TGF-β used here as a positive controlBesides proliferation, a characteristic of activated hHSC is the production of matrix proteins such as collagen, and over-exuberant matrix over-production results in fibrosis and cirrhosisin vivo relevance of these cellular studies, C57/B6 mice were fed for 4 weeks either normal chow or a methionine choline-deficient (MCD) diet, a well-known regimen inducing NASH23To confirm the in vivo relevance of these ex vivo and animal studies, we analyzed specimens obtained from healthy liver segments of patients undergoing resection of metastases and compared them to samples from patients with histologically proven NASH scored for fibrosis according to both Brunt and NAFLD Activity Score (NAS) of fibrosis severity25To study in humans the These results were corroborated by immunohistochemical analyses of liver biopsies from patients with increased fibrosis scores , as visualized by Masson trichrome stainings . ImmunopHSCs have previously been identified as the primary subtype responsible for remodeling liver tissue following injury271510via the matrix-metalloproteinase TACE , allowing “shedding” of the ligand from its plasma membrane anchoring and subsequent interaction with cognate receptors3132339data not shown). Thus, AR can act as a survival factor when expressed in liver. The survival pathway by which AR operates has only been partially described. AR has been previously shown to interact with EGFR in various cell types and EGFR expression may in fact constitute a requirement for liver recoveryAR is expressed as a 252 amino acid glycoprotein that exists stably in an uncleaved pro-form31data not shown). Collagen 1α is important for recovery of damaged liver tissues, but its over-production is a clinical hallmark of fibrosis36Our work and others have suggested that AR is capable of inducing proliferation in multiple cell types. Additionally, our investigations revealed that collagen 1α, ASMA and positive control TGF-β mRNA are increased upon stimulation of AR in hHSCs  that supports repopulating hepatocytes. The data compiled here supports the hypothesis, as collagen and ASMA production is concurrent with AR-induced proliferation of hHSC. The observation is complemented in both a mouse model of NASH and patient biopsies with NASH. The human data lends added importance to this study as we suggest that AR expression may be a component of the liver's survival response to NAFLD. However, overproduction of AR may lead to increased levels of fibrosis and lead to cirrhosis and potentially HCC. Thus, regulation of AR may be used to control the severity of the disease, using a regime that will promote liver recovery but control overstimulation and the potential formation of fibrosis.2 and cervical dislocation.Adult C57/BL6 animals  8 to 10 weeks old, were used. All animal studies were approved by the University Hospitals of Leuven, Belgium, and all the procedures described have been carried out in accordance with EU Directive 2010/63/EU for animal experiments. The methionine-choline deficient (MCD) model is an established dietary model of murine NASHLiver specimens were obtained from patients undergoing clinically indicated hepatic biopsies, with the approval of the University of Palermo (Department of Pathology), patient consent and in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. Liver biopsy specimens were scored for the presence of NAFLD according to the Brunt and NAFLD Activity Score (NAS) of fibrosis severity26Immunostainings were performed by iVIEW DAB Detection Kit for Ventana BenchMark XT automated slide stainer on human biopsieshHSC were obtained, with appropriate local Ethical approval and patient consent, from healthy liver segments of patients undergoing metastases resection. hHSCs were isolated by collagenase (type IV) perfusion, followed by pronase treatment, as previously described21Mouse HSCs (mHSCs) were isolated from the livers of wild type and MCD male mice by in situ perfusion using collagenase and pronase4141hHSC were harvested at days 0, 2, 4, and 10. Subsequently, the cells were fixed in 50% ethanol/acetic acid and cooled for 10 min at −20°C. Fixing buffer was then removed and the cells washed with PBS and processed for antigen unmasking using 10 μM sodium citrate and 0.2% Triton X. After blocking with 10% goat serum  at room temperature for 20 min, the protein of interest was labeled by incubating the cells overnight at 4°C with a mouse monoclonal primary antibody  at a 1:400 dilution. The primary antibody binding was later detected using the appropriate anti-mouse secondary antibody  conjugated with FITC. DAPI was used in the mounting medium to stain the nuclei and samples examined by confocal microscopy.EGFR inhibitor PD 153035, pertussis toxin (PT), wortmannin (WT), PD98059 (PD), SB202190 (SB), and Ro-32-0432 (RO) were obtained from Sigma and were used on hHSC at concentration previously describedBasal and induced hHSC proliferation was determined using a colorimetric assayEqual numbers of hHSC were plated on 6 mm petri dishes with or without AR. At harvest, apoptotic activity was assessed with the Vybrant (annexin V) apoptosis assay kit 2 . Flow cytometric analysis was performed using a Becton-Dickinson flow cytometer.Collagen 1α2: sense 5′-ATA TTG CAC CTT TGG ACA TC 3′, antisense 5′-TGC TCT GAT CAA TCC TTC TT-3′ ; TGF-β1: sense 5′-AAC CCA CAA CGA AAT CTA TG and antisense 5′-GTG CTG CTC CAC TTT TAA CT-3′ .RNA was isolated from activated hHSCs using Trizol reagent (Invitrogen) and DNA generated with Superscript III one-step RT-PCR with platinum Taq kit . Relative quantification of the mRNAs was performed on a Rotorgene RG-3000 instrument (Corbett Research). Collagen 1-α2, TGF-β1, ASMA, AR and GAPDH primers were custom made (InVitrogen) while classic II 18S internal standard kit was from Ambion . Primers sequences, product sizes and Tm were as follows: AR: sense 5′-GAC ACC TAC TCC GGG AAA GCG TG-3′ and antisense 5′-AGC CAG GTA TTT GTG GTT CG-3′ ; TACE: sense 5′-GAA GTG CCA GGA GGC GAT TA-3′ and antisense 5′-CGG GCA CTC ACT GCT ATT ACC-3′ ; GAPDH: sense 5′-ACA GTC CAT GCC ATC ACT GCC-3′ and antisense 5′-GCC TGC TTC ACC ACC TTC TTG-3′ . To monitor specificity, final PCR products were analyzed both by melting curves and agarose gel (1%) electrophoresis. The amount of transcript was calculated and expressed as the difference relative to the control gene GAPDH .Total caspase-3 was evaluated by western blot analysis, as previously describedTACE activity was assessed in cultured hHSC using the commercial fluorimetric SensoLyte® 520 TACE (α-Secretase) Activity Assay Kit , according to manufacturer's instructions.All data are expressed as mean (±SEM) of at least 3 separate experiments. Statistical analyses were performed using the Mann-Whitney test. A p value was considered statistically significant if p < 0.05.Study concept, design and supervision: J.A.O.; Acquisition of data: C.M., B.S., J.S., A.M., M.M., F.R. and V.P.; Analysis and interpretation of data: F.C., V.P., C.M., B.S., J.S., A.M., M.M., M.V. and J.A.O.; Drafting of the manuscript: G.M., M.V. and J.A.O.; Statistical analysis: G.M.; Obtained funding: M.V. and J.A.O.; Technical support: D.C., C.S. and T.R.