Background. The prevalence of allergies is steadily increasing worldwide; however, the pathogenesis is still unclear. We hypothesized that Mycobacterium avium subsp. paratuberculosis (MAP) may contribute to allergy development. This organism can be present in dairy foods, it can elicit an immunomodulatory switch from a Th1 to a Th2 response, and it has been speculated that it is linked to several human autoimmune diseases. To determine the contribution, sera from 99 individuals with various atopic disorders and 45 healthy nonallergic controls were assessed for total IgE levels and successively for MAP-specific IgE by ELISA. Results. The mean total serum IgE level in allergic patients was 256 ± 235 IU/mL, and in the healthy controls it was 62 ± 44 IU/mL . Among the patient groups, 50 of the 99 subjects had increased IgE total level ≥ 150 IU/mL, while 49 subjects had IgE ≤ 150 IU/mL . Additionally, 6 out of 50 subjects (12%) with IgE ≥ 150 IU/mL and none (0%) with IgE ≤ 150 IU/mL were positive for specific MAP IgE . Conclusion. The present study revealed that MAP has the ability to induce specific IgE and might contribute to the induction of allergic inflammation in genetically predisposed individuals.  Mycobacterium avium subspecies paratuberculosis (MAP) and its possible contribution to the pathogenesis of these diseases.There is increased prevalence of diseases due to immunological disorders including autoimmune diseases and allergies in developed countries , 2. It hMAP is an intracellular pathogen that causes paratuberculosis (Johne's disease) in ruminants, a chronic granulomatous enteritis causing major economic losses in the dairy industry worldwide .Due to the pathological similarities between paratuberculosis and human Crohn's disease (CD), a chronic inflammatory bowel disease of the gastrointestinal tract, MAP has long been suspected to be the etiological agent of CD due to frequent detection of MAP-specific DNA and its infrequent isolation from the lesion . ExperimThe shedding of MAP into milk is well known in the infected cattle, and there is much evidence of MAP contamination in milk, cheese, powdered milk for infants, and beef meat , 20. TheMoreover, oral exposure of mycobacteria is important in the induction of intestinal tuberculosis in human and paratuberculosis in cattle. Recent vaccination studies for the control of tuberculosis in wild animals showed that oral administration of heat-killed mycobacteria stimulating host immunity , 24.Based on this evidence, we hypothesized that oral exposure to MAP antigens might be able in turn to modulate host immunity enabling the development of allergies. In the present study, we examined the sera of allergic individuals and healthy controls (HCs) for the presence of MAP-specific IgE in the context of total IgE levels. To our knowledge, this is the first report showing greater presence of MAP-specific IgE in the serum of allergic individualswith higher levels of total IgE compared to nonallergic people.A total of 99 subjects with reported allergy  and 45 age- and sex-matched HCs without allergy  were enrolled from healthcare personnel of Sangenjaya hospital, Tokyo, from March to April 2014. All subjects were interviewed using questionnaires and gave their written informed consent. Many of them had a history of sensitivity to allergenic food and/or common aeroallergens. The demographic and clinical characteristics of the patients are reported in The study protocols, the documents for informed consent, and the questionnaire sheets were approved by the Human Ethics Committee of Sangenjaya Hospital  and Tohto College of Health Sciences . Bacillus Calmette-Guérin (BCG) (strain Tokyo 172) lipophilic antigen was performed as previously published [ Mycobacterium avium subsp. hominissuis and Mycobacterium avium subsp. avium [Preparation of MAP (strain ATCC 19698) andublished . MAP lipublished . An analublished . The latp. avium .The commercially available Human IgE ELISA Quantitation Set Kit  was used to detect human IgE in the sera according to the manufacturer's protocol. The test kit provides an affinity purified goat anti-human IgE antibody (Ab), a standard, and a horseradish peroxidase (HRP) conjugated goat anti-human IgE detection Ab . Samples and standards were run in duplicate. Mycobacterium phlei prior to testing was of value in eliminating falsely positive reactions in ELISA for the detection of Abs against MAP [Serum specific IgE to MAP lipophilic antigen were measured through indirect ELISA . The absinst MAP . Indeed inst MAP .μL/well of diluted lipophilic antigens at room temperature overnight. The next day, blocking was performed with 300 μL/well of Blocking One solution  with incubation at room temperature for one hour. The plates were washed three times with 300 μL/well PBST . Duplicates were made on the 96-well plate for each sample. Then 100 μL/well of preabsorbed serum sample at a 1 : 100 dilution in diluting buffer (PBST) was then added and incubated at room temperature for two hours, followed by an additional washing step. HRP-labeled goat anti-human IgE polyclonal Ab  (1 : 1000 dilution) was added and incubated at room temperature for two hours, followed by a washing step. The bound HRP conjugate was detected by adding 100 μL/well of Sure Blue Reserve TMB Microwell Peroxidase Substrate  and the absorbance was measured at a wavelength of 450 nm by using a Bio-Rad iMark Microplate Reader . Data were normalized to a positive control serum included in all assays run, whose Ab reactivity was set at 10,000 arbitrary units (AU)/mL. A negative control  was also included and the mean values were subtracted from all the samples.Sumilon ELISA 96-well plates  were coated with 50  Mycobacterium tuberculosis complex, far from the Mycobacterium avium complex, and concerns other routes of exposition. In any case, all the subjects enrolled in the study were BCG-vaccinated and MAP shares different antigens with BCG [To evaluate the specificity of MAP antigens in stimulating an Abs response and to verify whether cross-recognition between MAP and other mycobacteria occurs, an inhibition ELISA using antigen of BCG was developed. BCG belongs to thewith BCG ; this maAn inhibition assay was performed using two serum pools  as previously reported . BrieflyGraphPad Prism 6.0 statistical software  was used for analysis of the data. Fisher's exact test with Yates' corrections was used to compare the mean total serum IgE levels between subjects with total IgE of ≥ 150 IU/mL  and ≤ 150 IU/mL  and to compare IgE levels between allergic patients and HCs.U test. All statistical tests were two-sided with a significance level of 0.05 and all results are reported as the median ± interquartile range. The optimum cutoff values were calculated by a Receiver Operating Characteristic (ROC) curves analysis, setting the specificity at 95%, with the corresponding sensitivity chosen accordingly. The odds ratios (OR) with a 95% confidence interval (CI) were also calculated.Concerning the specific MAP IgE titer, for comparison between the groups we used Mann–Whitney p < 0.0001) (p < 0.0001).The mean total serum IgE level in the allergic patients was 256 ± 235 IU/mL, whereas in the HCs it was 62 ± 44 IU/mL  . Fifty oTotal IgE serum levels were negatively associated with age/gender. In contrast, total IgE serum levels were associated with sensitivity to different allergens. Of the 50 subjects with high total IgE level of ≥150 IU/mL, 21 (42%) were sensitive at least to one allergenic food  . Twenty-five subjects (50%) were sensitive to aeroallergen, 15 to pollen of Japanese cedar , and 10 to Japanese cypress , while four (8%) had allergy for both food and inhalant allergens . In addition, 40 subjects (80%) habitually consumed imported products .p = 0.03). The mean total serum IgE level in these six patients was 472 ± 237 IU/mL. MAP-specific IgE Abs were not detected (p = 0.01).Regarding the humoral response against MAP-specific antigen, we tested all allergic subjects taking into account their total serum IgE level. As shown in detected  in HCs g (Cryptomeria japonica) and two to Japanese cypress (Chamaecyparis obtusa) . Note that all of the positive subjects preferred to consume imported cheese and milk, and two of them were also allergic to milk.Among the six positive subjects, five were female and one was male . Four were allergic to Japanese cedarConcerning anti-MAP IgG titer (previously reported) , double The interassay coefficient of variation (CV) (with the same serum tested in three separate experiments) for the indirect ELISAs ranged from 5.4% to 8.5%, while the intra-assay CV (with the same serum tested in 20 replicate wells) ranged from 1.9% to 3.3%.Next, we determined the presence or absence of cross-recognition between MAP and BCG by inhibition ELISA using two serum pools. Regarding the specificity of MAP lipophilic antigens, we confirmed by inhibition experiments that anti-MAP and anti-BCG Abs targeting surface lipophilic antigen are not cross-reactive. Indeed, for both serum pools tested, BCG antigen was not capable of significantly blocking the Ab-binding directed to MAP antigens, causing only a small decline in the signal (11–15%), while MAP antigen (positive control) inhibited the signal of all serum pools tested (79–82%) .The present study revealed that Japanese people have anti-MAP-specific IgE and that positive individuals had respiratory allergy with higher levels of total IgE. We detected statistically significant high levels of specific anti-MAP IgE in six subjects with total serum IgE of ≥150 IU/mL and in none of the subjects with IgE of ≤150 IU/mL. This surprising result shows for the first time that MAP antigens are able to elicit a specific IgE response in human. This evidence may provide a new view of pathogenesis of allergy increasing worldwide . ConsideIn our study, all of the IgE positive subjects were affected by allergies caused by aeroallergens and often consumed imported dairy products. Hence, it is possible that these individuals are regularly exposed to allergens to the same degree that they are exposed to MAP antigens orally.The prevalence of allergic disorders is steadily increasing worldwide  includinMAP is reported to have a significantly stronger ability to induce IL-10 in a host macrophage compared with other mycobacteria –37. MAP Another possible link between MAP and allergies is based on a molecular mimicry mechanism. In fact, Blast-p analysis revealed that the major antigenic peptide from the Japanese cedar pollen allergen Cry j 1 (KVTVAFNQF)  shares iIn conclusion, despite the fact that mycobacterial antigen is generally considered to downregulate the IgE response by normalizing the balance of Th1 and Th2 , our datThe present study revealed that MAP has the ability to induce specific IgE and might contribute to the induction of allergies in genetically predisposed individuals.


Aspergillus fumigatus in patients with cystic fibrosis. To evaluate the results of the tests used to identify the IgE mediated sensitization to A. fumigatus in patients with cystic fibrosis: Total serum IgE, eosinophil count, fungus detection through oropharyngeal swab or sputum culture, serum-specific IgE, and immediate-type hypersensitivity (IgE) skin tests. We compared the results of the different tests performed. This is a cross-sectional descriptive study with a convenience sample of 86 patients diagnosed with cystic fibrosis in the Reference Service in Cystic Fibrosis at a tertiary teaching hospital. The following tests were performed to assess the sensitization to A. fumigatus sensitization test results were: in 16 patients, there was an increase in serum-specific IgE (>0.35 kU/L); in 23, skin tests were positive; and six had sensitization in both tests. We observed two patients with eosinophilia  and seven with increasing total serum IgE , all of whom obtained negative results in skin tests and had no IgE increase specific to A. fumigatus. A. fumigatus was not detected in oropharyngeal swabs and/or sputum culture of any patients. In 33 (38.4%) patients with cystic fibrosis, with ages ranging from 1 to 33 years (median of 8 years), the IgE-mediated A. fumigatus in cystic fibrosis patients, both serum-specific IgE and immediate-type hypersensitivity (IgE) skin tests are required. Serum eosinophilia and respiratory secretion culture were not essential in this study.  We conclude that, among the tests used to assess sensitization to  It mainly affects the respiratory and digestive systems, the sweat glands and the male reproductive system. In CF, mucociliary clearance and bronchial mucous production are defective, and the mucous produced is thick and viscous. Consequently, lung diseases become the major cause of morbidity and mortality for patients with CF. CF is a chronic disease that has seen remarkable progress in prognosis in recent years, besides increased survival rates, especially through early diagnoses using after-birth screening tests and with an improved control of the patients’ pulmonary disease and nutrition.Aspergillus fumigatus may sensitize patients with CF, leading to IgE-mediated hypersensitivity and allergic bronchopulmonary aspergillosis (ABPA) with variable prevalence.,European Epidemiologic Registry of Cystic Fibrosis (ERCF), in comparison to that observed by the Epidemiologic Study of Cystic Fibrosis (ESCF), may be attributed to ethnic variances and the different diagnostic criteria used; the European criteria are more comprehensive than the ones used in the US study.,A. fumigatus, increased total serum IgE and A. fumigatus-specific IgE and/or IgG, blood eosinophilia, oropharyngeal swab or sputum culture for the detection of A. fumigatus.,A. fumigatus, thus generating high costs to health care centers treating CF patients. In this context, the goal of this study was to evaluate the results of tests used to identify the IgE-mediated Aspergillus fumigatus sensitization in patients with cystic fibrosis.The criteria for the diagnosis of ABPA in CF have been constantly reviewed. Currently, these criteria are based on clinical manifestations and complementary tests: a positive result for immediate skin hypersensitivity to The project was approved by the university’s Research Ethics Committee (Project 065/06). An Informed Consent Form was signed by patients and/or guardians prior to any procedure related to the study.A six-month long cross-sectional descriptive study was conducted in 2013, with a convenience sample of CF patients enrolled in outpatient clinics of the Reference Service for the treatment of CF in a tertiary university hospital. Each patient was submitted only once to each test.We evaluated 86 patients with confirmed diagnosis of CF using two sweat chloride tests, by the Gibson and Cooke standardized method:A. fumigatus sensitization:We used the complementary tests and values routinely used to detect Enzyme Linked Immuno Sorbent Assay), considered high when higher than 1,000 IU/mL.Total serum IgE through the enzyme immunoassay method  enzyme immunofluorescence technique. Sensitization was detected for values equal to or higher than 0.35 kUA/L.A. fumigatus immediate hypersensitivity skin test, performed at the Allergy and Immune Disorders division in accordance to standards: either positive for values equal to or higher than 3 cm with positive histamine control, or higher than or equal to positive histamine control with negative saline solution control. We used the International Pharmaceutical Immunology S.A. -  standardized extract3. Automated eosinophil count, conducted at the Clinical Pathology Service. Eosinophilia was diagnosed for values higher than 1,000 eosinophils/mmA. fumigatus, conducted at the Clinical Pathology Service, positive for the presence of fungus.Oropharyngeal swab and/or sputum culture to detect A. fumigatus-specific IgE through RAST (≥0.35 kU/L) and/or positive results on immediate hypersensitivity skin tests. Sixteen showed increase for serum-specific IgE (≥0.35 kU/L) and 23 obtained positive results on skin tests. Eight patients did not undergo skin tests due to difficulties in attendance. Among 33 patients, six were sensitive in two exams: Serum IgE and skin test  was detected in two patients; total serum IgE was high  in seven patients, and patients with eosinophilia were different from those with high total serum IgE. No patients’ oropharyngeal swab and/or sputum culture tested positive for A. fumigatus. Patients with eosinophilia or increased total IgE tested negative for A. fumigatus-specific sensitization skin tests and had no increase in A. fumigatus-specific serum IgE. ABPA was not detected in any of the patients followed.Eosinophilia , thus not meeting the criteria for ABPA.38.4% of patients with CF tested positive in the immediate hypersensitivity skin tests and/or presented an increase in IgE-mediated hypersensitivity develops gradually with age and with the increased exposure to the allergen. Thus, in IgE-mediated reactions, sensitization to the specific allergen starts slow and builds up over time to the point in which one develops the disease. This is the case of well-known IgE-mediated diseases such as allergic rhinitis, allergic conjunctivitis, allergic asthma, food allergy and IgE-mediated latex allergy.A. fumigatus, but negative for ABPA: 42% tested positive in skin tests, and 54% to increased serum-specific IgE levels.,A. fumigatus; therefore, with age, they might develop ABPA.The frequency of sensitization observed in this study is within the range of values reported in the literature for patients with CF sensitized to A. fumigatus in lower airways pulls neutrophils into alveolar spaces, resulting in spore phagocytosis or the release of its granular contents, leading to fungal death. In cases of uncontrolled allergic asthma, research has shown changes in neutrophil activity, contributing with neutrophilia and worse allergy symptoms.A. fumigatus. The prolonged presence of fungi in the lungs results in diseases ranging from hypersensitivity (ABPA) to invasive cavitation secondary to spore germination.The presence of Dermatophagoides pteronyssinus,Dermatophagoides farinae and Blomia tropicalis. Total serum IgE levels may be normal even among patients who are sensitive to all three mites, i.e., patients presenting with increased specific IgE levels corresponding to each of the three most common mites, thus causing respiratory allergies.A. fumigatus sensitization: Even though a specific in vivo IgE is high, this does not imply an increase of total IgE levels. In ABPA cases, sensitization to the fungus is higher, increasing specific IgE levels and a corresponding rise in total serum IgE.In the present study, total serum IgE and peripheral blood eosinophil count increased in a small number of patients, who saw no rise in specific IgE levels and/or tested positive for the skin test. It is known that total serum IgE and eosinophil count may increase or remain normal in cases of IgE-mediated diseases, and that total IgE levels can be high even among individuals without IgE-mediated reactions. Total serum IgE results from the sum of one or more serum-specific IgE.A. fumigatus sensitization:in vitro or in vivo specific IgE. We believe both tests are complementary. A survey on A. fumigatus sensitization in patients with CF had 90% match for both exams.A. fumigatus in CF cases.The present study does not allow us to conclude which of the tests would be more adequate to check for ,A. fumigatus-specific IgE as the lung disease caused by CF progress.The literature further describes that even at an early age children may test positive for the immediate hypersensitivity skin test.A. fumigatus is often isolated in lower airways of patients with CF with negative culture. The lack of effective mucociliary clearance leads to the accumulation of mucus, which is rich in nutrients for microorganisms, facilitating the spread and persistence of bacteria and fungi.,,In the follow-up of patients with CF, respiratory secretions are routinely collected. The results obtained here are likely owed to the short time frame of the study. In studies on the prevalence of ABPA, negative cultures are reported. Studies also highlight the fact that positive cultures have no diagnostic value. In addition, A. Fumigatus are the immediate hypersensitivity skin test and the increase of serum-specific IgE. Assaying the eosinophil count in peripheral blood, total IgE serum levels and cultures to detect A. fumigatus is expensive and often difficult for patients, and may not test positive for A.fumigatus sensitization. The clinical manifestations of CF and ABPA overlap. Pulmonary infiltrates, bronchiectasis and chronic obstructive pulmonary disease are common in CF, resulting from repeated infections and diseases, regardless of the presence of ABPA. ABPA is possible when a clinical condition suffers acute or subacute worsening, with pulmonary exacerbations not attributable to another etiology and/or after failed attempts of treatment with antimicrobial agents against bacteria isolated in cultures. Under these conditions, it is recommended to check for the possibility of concomitant ABPA, considering the criteria proposed by the literature.,,,The results of this study show that the tests indicating sensitization to The survival of patients with CF has increased, as shown by the records.A. fumigatus in patients with CF, we conclude that two tests are necessary: the immediate hypersensitivity skin test and the determination of A.fumigatus-specific IgE levels. Eosinophilia detection, total serum IgE and swabs/cultures were not essential for the detection of A. fumigatus sensitization in young patients with CF. It is worth highlighting the limitations of this research, considering the number of patients and the relatively short time frame of the analysis. Even so, we demonstrated the importance of skin tests and of measuring serum IgE specific A. fumigatus to assess sensitization in cystic fibrosis patients.In order to assess sensitization to in vitro and in vivo tests for IgE specific to A. fumigatus be initially performed and, if sensitization is detected, other tests should be carried out: Total IgE levels, eosinophils count and fungus research, complying with the diagnostic criteria for ABPA in CF.In this context, we suggest that


However, reliable reconstructions are still lacking for many WAIS sectors, particularly for key areas on the outer continental shelf, where the LGM-ice sheet is assumed to have terminated. In many areas of the outer continental shelf around Antarctica, direct geological data for the presence or absence of grounded ice during the LGM is lacking because of post-LGM iceberg scouring. This also applies to most of the outer continental shelf in the Amundsen Sea. Here we present detailed marine geophysical and new geological data documenting a sequence of glaciomarine sediments up to ~12 m thick within the deep outer portion of Abbot Trough, a palaeo-ice stream trough on the outer shelf of the Amundsen Sea Embayment. The upper 2–3 meters of this sediment drape contain calcareous foraminifera of Holocene and (pre-)LGM age and, in combination with palaeomagnetic age constraints, indicate that continuous glaciomarine deposition persisted here since well before the LGM, possibly even since the last interglacial period. Our data therefore indicate that the LGM grounding line, whose exact location was previously uncertain, did not reach the shelf edge everywhere in the Amundsen Sea. The LGM grounding line position coincides with the crest of a distinct grounding-zone wedge ~100 km inland from the continental shelf edge. Thus, an area of ≥6000 km During the Last Glacial Maximum (LGM) both geologic data and numerical models suggest that the Antarctic Ice Sheet advanced towards the continental shelf edge in most sectors of the continent e.g. –4). By i. By i4])Detailed bathymetry data by Klages et al.  revealedHere we present the first direct marine geological and geophysical evidence from the Amundsen Sea Embayment showing that the WAIS did not reach the continental shelf edge everywhere in this critical sector during the last glacial period. This finding is significant as it redefines the shelf limit for the LGM-ice sheet in this area, which is important to help defining its last maximum extent and subsequent retreat history, and also questions about the source of postglacial sea-level rise. Further, this grounded-ice free area may have also served as a glacial refuge for Antarctic shelf benthos, where it could survive the harsh conditions during the last glacial period.The bathymetry presented in detail here  formed porg) content  in Zürich, which is especially suitable for dating very small carbonate sample sizes . Combinin (e.g. ) but prog CaCO3; ). We cor100 yrs; ), which 92-3 see . All 14Csoftware .95 = 8°), matching the present day inclination of -67.15° at the core site according to the International Geomagnetic Reference Field (IGRF-12) )A further significant implication of our data is that our cores penetrated and recovered only the upper one third of a more expanded, thick glaciomarine sequence. We suggest on the basis of our combined radiocarbon-palaeomagnetic chronology from the uppermost part of the postglacial drape that the entire thickness of the drape  documentUltimately, there is ongoing debate surrounding whether parts of the Antarctic shelf acted as refugia for benthic organisms during the LGM (e.g. ), or wheIn conclusion, our data provide new constraints on the LGM ice-sheet position in the Amundsen Sea, and serve as the first geological evidence of a limited GL advance onto the Amundsen Sea shelf. This information will improve regional palaeo-ice sheet reconstructions, and may also provide another piece of evidence for calibrating and evaluating palaeo-ice sheet models, which are used to simulate past and future WAIS configurations. It further adds to the list of potential shelf refugia for Antarctic benthos during the last glacial period.S1 FigThese profiles are perpendicular to profiles x-x’ and y-y’ in Figs (TIF)Click here for additional data file.S2 FigZoomed-in examples for degree of lamination/stratification are included.(TIF)Click here for additional data file.


Avena sativa) protein isolates and their ability to inhibit the renin, ACE-I, and DPP-IV enzymes. In silico analysis was carried out to predictthe likelihood of bioactive inhibitory peptides occurring from oat protein hydrolysates following in silico hydrolysis with the proteases papain and ficin. Nine peptides, including FFG, IFFFL, PFL, WWK, WCY, FPIL, CPA, FLLA, and FEPL were subsequently chemically synthesised, and their bioactivities were confirmed using in vitro bioassays. The isolated oat proteins derived from seven different oat varieties were found to inhibit the ACE-I enzyme by between 86.5 ± 10.7% and 96.5 ± 25.8%, renin by between 40.5 ± 21.5% and 70.9 ± 7.6%, and DPP-IV by between 3.7 ± 3.9% and 46.2 ± 28.8%. The activity of the synthesised peptides was also determined.The renin-angiotensin-aldosterone system (RAAS) plays an important role in regulating hypertension by controlling vasoconstriction and intravascular fluid volume. RAAS itself is largely regulated by the actions of renin (EC 3.4.23.15) and the angiotensin-I-converting enzyme . The enzyme dipeptidyl peptidase-IV  also plays a role in the development of type-2 diabetes. The inhibition of the renin, ACE-I, and DPP-IV enzymes has therefore become a key therapeutic target for the treatment of hypertension and diabetes. The aim of this study was to assess the bioactivity of different oat ( High blood pressure is the single largest risk factor attributed to deaths worldwide. It is responsible for 12.8% of deaths, and affects all countries and income groups . ACE-I inhibitors have therefore become one of the most commonly studied drugs, with global annual sales exceeding $6 billion USD. ACE-I inhibitory drugs can be considered one of the major protease inhibitor success stories . SynthetGliptins or dipeptidyl peptidase IV (DPP-IV) inhibitors can block the action of the enzyme DPP-IV, and may be used to treat diabetes mellitus type 2. DPP-IV inhibitors increase incretin levels, inhibit glucagon release, increase insulin secretions, and decrease gastric emptying and blood glucose levels.In addition to providing energy and amino acids essential for growth, it has become increasingly recognised that some dietary proteins contain biologically active peptides that can impart a beneficial physiological effect . Such biMilk proteins remain the most common food resource for bioactive peptide generation ,21. The Avena sativa) is a promising source of bioactive peptides, with several peptides already identified, but it has not been fully explored yet [The common oat . The naked, de-husked grains were used to obtain a higher yield, as they are significantly higher in protein and lower in crude fiber . Ammoniuw/v) solution of milled oats in distilled deionised water (ddH2O) was sonicated using an Ultrasonic Bath Branson 3510 at 42 kHz for 1 h. This was followed by stirring overnight at 4 °C. The oats were then centrifuged at 10,000× g for 1 h at 4 °C using a Sorvall™ Lynx 6000 centrifuge . The supernatant was then collected and stored at 4 °C, while the pellet was re-suspended in 4% (w/v) ddH2O. The resuspended oats were sonicated for 1 h, stirred overnight at 4 °C, and centrifuged at 10,000× g for 1 h at 4 °C. The supernatant was then pooled with the first supernatant for further processing. An 80% ammonium sulfate-saturated solution was made with the pooled oat supernatant, and stirred for 1 h at 4 °C, followed by centrifugation at 17,000× g for 1 h at 4 °C. The pellet was resuspended and dialysed overnight at 4 °C. Samples were stored at −20 °C and subsequently freeze-dried using an industrial-scale freeze drier, FD 80 model . A 2%  were synthesised by microwave-assisted solid phase peptide synthesis (MW-SPPS) performed on a Liberty microwave peptide synthesiser . Peptides were synthesised on H-Ala-HMPB-ChemMatrix and H-Ile-HMPB-ChemMatrix resins  and purified using RP-HPLC on a Semi Preparative Jupiter Proteo  column . Fractions containing the desired molecular mass were identified using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) and were pooled and freeze-dried on a Genevac HT 4X lyophilizer .Protein isolates from all seven oat varieties and selected synthesised peptides were tested in vitro for renin inhibition activity. The Renin Inhibition Screening Assay  was carried out as per the manufacturer’s instructions. Briefly, 10 μL of each sample inhibitor at a concentration of 1 mg/mL dimethyl sulfoxide (DMSO) was added to 20 μL renin substrate, 150 μL assay buffer, and 10 μL renin, in triplicate. The samples were incubated at 37 °C for 15 min, and read with excitation wavelengths of 340 nm and emission wavelengths of 500 nm. Fluorescence was read using a FLUOstar Omega microplate reader . The percentage inhibition was calculated using the following equation:2O was added to 20 μL substrate and 20 μL enzyme working solution in triplicate. Samples were incubated at 37 °C for 1 h. Each well then had 200 μL indicator working solution added, followed by a further incubation at room temperature for 10 min. Absorbance at 450 nm was read using a FLUOstar Omega microplate reader . The percentage inhibition was calculated using the following equation:Protein isolates from all seven oat varieties and selected synthesised peptides were tested in vitro for ACE-I inhibition. The bioassay  was carried out according to the manufacturer’s instructions. First, 20 μL of each sample inhibitor at a concentration of 1 mg/mL ddHProtein isolates from all seven oat varieties and selected synthesised peptides were tested in vitro for DPP-IV inhibition. The bioassay  was carried out as per the manufacturer’s instructions. First, 10 μL of each sample inhibitor at a concentration of 1 mg/mL assay buffer was added to 30 μL diluted assay buffer, 10 μL diluted DPP-IV, and 50 μL substrate solution, in triplicate. Samples were incubated at 37 °C for 30 min. Fluorescence was read with excitation wavelengths of 355 nm and emission wavelengths of 460 nm using a FLUOstar Omega microplate reader . The percentage inhibition was calculated using the following equation:In silico analysis of oat protein isolates identified a number of bioactive peptides that had previously been reported in the BIOPEP database . These pBased on the known characteristics of renin, ACE-I and DPP-IV inhibitory peptides , novel pBioactive peptides need to survive degradation by gastrointestinal digestion, reach their target intact, and maintain bioavailability in order to exert a beneficial physiological effect . The novOat protein isolates displayed renin inhibition values ranging between 40.5% (±2.16%) and 70.9% (±7.7%) . Barra oHowever, renin inhibition was not observed with the synthesised peptides. Only the peptide IFFFL was found to inhibit renin by 17.1% (±2.6%) when tested at a concentration of 1 mg/mL compared with the control.The crude oat protein extracts inhibited ACE-I by between 86.6% (±10.7%) and 96.5% (±25.8%). These results were therefore comparable with that of the positive control captopril, which was found to inhibit ACE-I by 97.7% (±23.2%) when tested at a concentration of 1 mg/mL . ProteinC-terminus of an ACE-I inhibiting bioactive peptide [The highest levels of ACE-I inhibition with the synthesised peptides were observed for the peptides WCY (97.8 ± 21.7%), FLLA (97 ± 16.2%), and WWK (95.3 ± 14.2%) . The low peptide . However peptide .The protein isolates of the seven oat varieties did not display significant DPP-IV inhibitory activities when assayed at a concentration of 1 mg/mL, and values ranged from 3.7% (±3.9%) to 46.3% (±8.8%) compared with the positive control sitagliptin (87.4 ± 4%) . The higN-terminus) have been found to be effective DPP-IV inhibitors [Peptides containing the sequence Xaa-Pro  and 70.9% (±7.7%)  in vitrohibition . The oat protein isolates generated in this work were all found to inhibit the enzyme ACE-I by between 86.6% (±10.7%) and 96.5% (±25.8%) . This waN-terminus [The Barra oat protein isolate had the highest activity with 46.3% (±28.8%) inhibition, followed by Rhapsody oat (25.4 ± 1.8%) and Selwyn oat (20.7 ± 3.1%). Similarly, the selected synthesised peptides also had relatively poor DPP-IV inhibition . DPP-IV terminus . Of the In silico methods were used heavily in this study to evaluate the potential of oat protein to generate bioactive peptides, as well as predict generated bioactive peptides following hydrolysis with the food-grade proteases papain and ficin. Similar bioinformatic techniques have already been described in other studies , which hAvena sativa) as a potential source of bioactive peptides. Bioinformatics techniques were used to predict whether oats were indeed a rich source of bioactive peptides following in silico hydrolysis with the proteases papain and ficin on the main storage proteins. Oat protein isolates displayed the highest inhibition bioactivity against the target ACE-I (86.6–96.5%), with lower inhibition levels observed with renin (40.5–70.9%) and DPP-IV (3.7–46.3%). Following the chemical synthesis of nine novel peptides, in vitro bioassays gave mixed results as to their efficacy in inhibiting the injurious enzyme targets ACE-I (48.9–97.8%), renin (0–17.1%), and DPP-IV (0–22.2%). The in silico methods utilised in this study correctly identified ACE-I inhibitory peptides, beyond that of renin and DPP-IV inhibitory peptides. This could reflect a more specific use of these in silico methods until the characteristics of renin and DPP-IV inhibitory peptides are better understood.There has been little work carried out to evaluate the potential of the common oat (


Enterococcus faecalis from chicken faecal samples in 6 provinces of China to investigate the prevalence and transmission mechanism of the bacitracin resistance locus bcrABDR in E. faecalis. Thirty-seven bcrABDR-positive E. faecalis were detected with 26 different PFGE clusters. The MLST of 14 positive strains belonged to ST16 and we also detected three new sequence types. S1-PFGE analysis indicated that the locus was located on plasmids presenting different sizes, with the most prevalent size being ~50 kb (13/37). Sequence analysis revealed that 17 out of the 37 strains harbored a 5400-bp central region, in which locus bcrABDR was bracketed by two ISEnfa1 of the same orientation. Two types of bcrABDR alleles, differing in around 10% of their sequence were found. In silico analysis showed that bcrABDR is present in a variety of bacteria including the chicken commensal Enterococcus cecorum. Our results indicate that the use of bacitracin at farms might trigger the emergence and spread of the bacitracin resistance determinant bcrABDR among human bacterial pathogens. The finding of bcrABDR in the chicken commensal E. cecorum indicates that farm animals microbiota can be an important reservoir of resistance genes with relevance for human health.In this study we isolated 109  Bacitracin is a peptide antimicrobial that inhibits the cell wall biosynthesis by binding to the lipid undecaprenyl pyrophosphate (UPP) and impeding its dephosphorylation. UPP is the precursor of the undecaprenol monophosphate that serves as a membrane-associated carrier for sugar-peptide units in the peptidoglycan synthesis. After completion of the cycle, UPP is dephosphorylated to form undecaprenol monophosphate (UP) to start another synthetic cycleE. faecalis, a primary resistance mechanism has been reported to be the presence of the bcrABDR cluster, composed by the bcrABD operon and its regulatory gene bcrRbcrABD operon encodes a putative heterodimeric ATP-binding cassette (ABC) transporter BcrAB that has been proposed to mediate the active efflux of bacitracinbcrABDIt has been reported that bacitracin can be an effective antibiotic to treat patients who are infected with vancomycin-resistant enterococci (VRE)25E. faecalis, the bcrABDR cluster has been sporadically detectedbcrABDR cluster in E. faecalis isolates obtained from animal samples from six different provinces in China in the aim of determining whether or not the wide use of bacitracin in farms may render a selection of strains carrying this resistance determinant, which might compromise the use of bacitracin in human therapy. In addition, we have explored the different hierarchical levels (from genes to clones) at which selection of resistance may operate in the aim of defining the elements involved in the allodemicIn E. faecalis from 11 farms in 6 provinces of China    and S1.bcrABDR spread, one of the plasmids (pEF123) was fully sequenced as described in methods. As shown in  were detected in the 7 transconjugants. In the type I genetic organization, the locus bcrABDR was flanked by two copies of the insertion sequence ISEnfa1 located in the same orientation  and the locus bcrABDR was oriented in the opposite direction of ISEnfa1. To determine the stability of this structure, inverse PCR was performed using the primersP13/P14. Sequence analysis of the PCR production revealed that it contained one intact ISEnfa1, supporting that bcrABDR might form a circular intermediate through the recombination of the two copies of ISEnfa1. bcrABDR-positive strains, and 17 of them gave a positive result. Together with previously published worksbcrABDR. In the type II organization, ISEnfa1 was only located upstream the locus bcrABDR  without any ISEnfa1 located downstream. In the type III organization we could not find the insertion sequence ISEnfa1 neither upstream nor downstream bcrABDR (JEF174). Although it is tempting to propose that these results suggest a route for the evolution and spread of bcrABDR from bacteria not harboring ISEnfa1 towards strain carrying one ISEnfa1 copy, ending in those that carry two ISEnfa1 copies, in which the circular intermediate can be found, the opposite situation might also happens if loss of ISEnfa1 occur through abortive recombination events.The regions surrounding the bcrABDR-positive isolates, the four genes in the locus could be amplified just in three of the isolates with the primers previously described. For the other 34 isolates the genes bcrA and bcrD could not be detected using these primers, so new primers for these genes were designed according to the sequence of the plasmid pEF123 determined in this study by using these novel primers , was found and identity of 99% in pTW9 and pJM01, whereas the bcrABDR sequence present in pEF123 (Type II), with a 99% identity was found in the plasmids pXD5 and R17. It has been discussed that the low variability of antibiotic resistance genes found in the mobile gene elements of bacterial pathogens might be the consequence of a founder effectbcrABDR alleles  are present in bacitracin resistant E. faecalis indicating that in this case at least two and likely four, different events might account for the acquisition of resistance. This situation may happen when there exists a close ecological relationship between the donor and the recipient of the resistance element, which allows these transfer events to occur frequently and being efficiently selected, likely as the consequence of bacitracin use in chicken farms.From the 37  primers , a new aent SNPs . IndepenbcrABDR, the sequence of the cluster and the surrounding ISEnfa1 was searched at NCBI DNA database. In addition to the aforementioned plasmids, Type II bcrABDR was found to be present in the genome of Streptocccus pyogenes NGAS322 as well as in the genomes of different strains of E. cecorum  isolates1516Enfa1, in other occasions without IS present around. In addition, the genomes of E. cecorum also contain upstream and downstream sequences nearly identical to those found in pEF123 and other bcrABDR-containing plasmids. All of these will be powerful to improve Enterococcus cecorum can be a reservoir of bacitracin resistance in chicken. Although the whole-genome sequence methods used in this study did not allow distinguishing between chromosomal and plasmidic DNA. Since a route of transmission from E. cecorum towards E. faecalis or vice versa cannot be tracked, we cannot ascertain which is the origin of bcrABDR; however our results indicate that the commensal chicken microbiota can be a reservoir of transferrable bacitracin resistance that may end in relevant human pathogens as E. faecalis.To ascertain the distribution of locus bcrABDR in E. faecalis isolated from 6 provinces in China. Our study showed that resistance to bacitracin, associated to the presence of plasmids containing the bcrABDR cluster was highly prevalent and associated with resistance to other antimicrobials, especially erythromycin, streptomycin, kanamycin, florfenicol and tetracycline. The polyclonal phenomenon result in E. faecalis indicated that different clones are involved in the spread of bacitracin resistant E. faecalis isolates containing the bcrABDR cluster. Persistence and co-selection of the bcrABDR cluster could be promoted under antibiotic selective pressure, when these classes of antimicrobials were used either as feed additives or to cure or prevent bacterial infections in animalsbcrABDR cluster are present in different plasmids, suggesting the existence of multiple events of acquisition (and fixation) of this resistance determinant by E. faecalis from its (still unknown) original host. After these acquisition events, the spread of bcrABDR has been guided by its incorporation in different plasmids, which are also spread among different clonal complexes.In this study, we investigated the prevalence of the bcrABDR flanking regions showed three different organizations, in most cases, two intact ISEnfa1 flanked the bcrABDR cluster and inverse PCR shows that they can form bcrABDR-containing circular intermediates through homologous recombination, which suggests that IS-mediated recombination may account for the transfer of the bcrABDR cluster between different genetic platforms. Interestingly the structure ISEnfa1-bcrABDR-ISEnfa1 is also observed in other bacterial species, which can further suggest that the form of the circular intermediate is the main manner for the transmission of the locus bcrABDR .All these results indicated that multiple levels of selection regulate the spread of E. faecalis were isolated from 181 faecal swabs of healthy chickens. The samples were collected from 11 farms in six provinces of China  in May 2014  were determined by the agar dilution method following the guidelines of the Clinical and Laboratory Standards Institute (CLSI)24E. faecalis ATCC 29212 was used as a quality control strain. Bacterial DNA was extracted using a DNA extraction kit  following the manufacturer’s instructions. The presence of bcrABDR was determined by PCR and further sequencing of the amplicons. Primer walking was performed to analyze more in detail the different bcrABDR and surrounding structures in the selected strains. The primers used in this study were presented in A total of 109 May 2014 . The susE. faecalis strains carrying bcrABDR. PFGE clusters were analyzed with Bio Numerics . Dendrograms were generated using the Dice similarity coefficient, and the analogical values to categorize identical PFGE types cut-off was fixed at 90%. Further determination of clonality was performed by Multi Locus Sequence Typing (MLST), following the protocol described at http://efaecalis.mlst.net.Pulsed-field gel electrophoresis (PFGE) was performed using the CHEF-MAPPER System (Bio-Rad Laboratories) as described previouslybcrABDR positive strains were performed as described previously, using E. faecalis JH2-2 (Rifr) as the recipient strain27bcrABDR positive transconjugants and transformants were detected by PCR, using the primers described in Conjugation and transformation experiments of the bcrABDR in the bcrABDR-positive strains (37 isolates and 7 transconjugants), whole-cell DNA was separated by PFGE after treatment with S1 nuclease (TaKaRa). Afterwards, the plasmids carrying bcrABDR were identified by Southern blot using a bcrB probe.To estimate the size of the plasmids harboring bcrB-positive plasmid from transconjugant JEF123 was extracted using the Qiagen plasmid maxi kit . Sequencing was performed using the Miseq platform  with a 500-bp paired-end library, and assembled by SOAPdenovohttp://blast.ncbi.nlm.nih.gov) and BRIGA bcrABDR, has been deposited in GenBank under the accession number KX579977.The nucleotide sequence of the plasmid pEF123 containing How to cite this article: Chen, M.-Y. et al.Multilevel selection of bcrABDR-mediated bacitracin resistance in Enterococcus faecalis from chicken farms. Sci. Rep.6, 34895; doi: 10.1038/srep34895 (2016).


A major determinant in the efficiency of ribosome loading onto mRNAs is the 5′ TL (transcript leader or 5′ UTR). In addition, elements within this region also impact on start site selection demonstrating that it can modulate the protein readout at both quantitative and qualitative levels. With the increasing wealth of data generated by the mining of the mammalian transcriptome, it has become evident that a genes 5′ TL is not homogeneous but actually exhibits significant heterogeneity. This arises due to the utilization of alternative promoters, and is further compounded by significant variability with regards to the precise transcriptional start sites of each . Consequently, the transcript for a protein coding gene is not a unique mRNA, but in-fact a complexed quasi-species of variants whose composition may respond to the changing physiological environment of the cell. Here we examine the potential impact of these events with regards to the protein readout. The cellular phenotype is determined mainly by the cells protein composition. Events such as cell growth, differentiation, the stress response, apoptosis and even circadian regulation involve a re-seeding of the polysome with specific mRNA populations . The dynWith the advent of high-throughput RNA sequencing (RNA-seq) it has become increasingly popular to define the genetic expression profile of a cell by its transcriptome . An apprTable 1 and Figure 1). The ternary complex (TC) composed of Met-tRNAi-eIF2-GTP is first loaded onto the 40S ribosomal subunit in combination with a series of eIFs. The Met-tRNAi is located in the P site in association with eIF2-GTP. This initiation factor is composed of tree subunits . Its GTP loading is catalyzed by the Guanine Exchange Factor (GEF) eIF2B whose activity is also tightly regulated. The additional factors constituting the pre-initiation complex (PIC) include eIF1, eIF1A, eIF3, and eIF5. eIF1/1A are involved in the fidelity of start codon recognition with eIF1A positioned in the A-site and eIF1 near the P-site , which form a trimolecular complex referred to as eIF4F. The smallest of these, eIF4E, binds directly to the cap and is conserved across the eukaryotic kingdom ,b. It isOnce recruited to the cap, the PIC moves forward (5′–3′) along the mRNA, scanning for the first AUG. Generally, this movement is facilitated by the RNA helicase eIF4A in an ATP dependent manner assisted by the single stranded RNA binding proteins eIF4B/eIF4H. They serve both to stimulate helicase activity and to limit RNA re-association . StudiesThe nucleotides immediately surrounding an AUG codon influence the efficiency of its recognition. The sequence 5′-ACCAUGG-3′ is referred to as the optimal Loading of the PIC onto an mRNA and the subsequent identification of the initiation start site are the key events that dictate the translational readout both quantitatively and qualitatively. Central in both these events is the 5′ TL which carries features that will modulate both PIC recruitment and start site selection.Approximately 50% of human 5′ TLs contain one or multiple uORFs . DespiteGCN4  and the proteins that continue to be expressed during this phase will ultimately determine cell fate, i.e., recovery or apoptosis. The translational brake reflects increased eIF2α phosphorylation, an inhibition in TC regeneration and a subsequent reduction in the pool of 43S ribosomes. Until recently, it was widely accepted that the inhibitory and reinitiation phenotypes associated with the ISR were mechanistically coupled in that both arose due to a simple reduction in TC levels. However, recent studies from our lab suggest that it may not be so simple . Using tHighly structured 5′ TLs are frequently observed in the transcripts of genes whose protein products impact on the regulation of cellular proliferations and differentiation. As such it is a characteristic signature associated with tight translational control. Structure impacts on the protein readout at multiple levels. When positioned close to the 5′ it can render the cap less accessible, consequently these mRNAs compete poorly for the limiting amounts of eIF4E . MoreoveXIAP gene, alternative splicing within the 5′ TL generates two variants one of which recruits via the cap whilst the second has IRES activity. This splicing event is coupled to cellular stress and ensures continued XIAP expression under these conditions  modulate activity in large part by serving as chaperones that ensure the correct RNA folding . Many cenditions . [NOTE: These transcripts harbor a terminal oligopyrimidine (TOP) tract at their 5′ end consisting of a cytosine at the penultimate nucleotide position followed by a stretch of 4–14 pyrimidines . TOP mRNAUG codons close to the 5′ end (<20 nts) are generally considered to be silent or very leaky . HoweverFigure 1). Furthermore, we know absolutely nothing about how the mRNA sits on this 43S ribosome and what actually occurs to release the 43S from the 5′ end to permit scanning, if indeed it occurs. This has made it difficult to interpret the initiation events that one observes on short 5′ TLs. However, thanks to high resolution cross-linking studies we know considerably more about the path of the mRNA on the 48S positioned over a start codon that is 5′ distal  . Indeed, 3→5′ scanning has recently been evoked . In another model direct loading is via the mRNA 5′ end on empty 80S particles free of eIFs . The mRNA would then be treaded through the ribosome until the AUG enters the P site. Translation initiation with bacterial 70S monosomes on leaderless mRNAs has been known for some time  . The crogure 2A) . This won evoked . Furthern evoked . Howeverome time . It is aome time . Nonetheome time .At another level, the translational readout can also be modulated by trans-acting cellular proteins binding to specific RNA features within the 5′ TL. This includes the IRPs (iron-regulatory proteins), which bind to stem–loop structures called the IRE , but it In vitro studies indicated that this TSS variability impacted on translation efficiency and abundance of the different GR N-terminal protein isoform levels , a collection of promoter mapping experiments from multiple species generated by NGS . An interesting group of these cis-acting peptides are responsive to environmental signals and have been coined “peptoswitches”  with the potential to encode biologically active peptides . One souwitches” . The sigATXN1) transcript a small ioORF starting 30 nts downstream of the AUGATXN1and in the -1 reading frame is expressed by leaky scanning .Detecting the products of smORFs, which may be numerous and small (<100 aas), is technically not straightforward . Howevercis-regulatory elements residing within the mammalian 5′ TL modulate the protein readout we are only beginning to understand the impact of transcriptional heterogeneity on this process. In this review we have focused mainly on the promoter. However, another twist on this story is that promoter usage can impact on splicing patterns and transcription TTSs, events that can in-turn also modulate the protein readout (Despite major advances in our understanding of how the  readout . For exaThe text was in large part written by JC after numerous discussions with BW. All graphic art was prepared by BW.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 (long-term) and transplanted (short-term) plant incubations of the seagrass Cymodocea nodosa were performed near and away the influence of volcanic CO2 vents at Vulcano Island to test the hypothesis of beneficial effects of CO2 on plant productivity. We relate, for the first time, the expression of photosynthetic, antioxidant and metal detoxification-related genes to net plant productivity (NPP). Results revealed a consistent pattern between gene expression and productivity indicating water origin as the main source of variability. However, the hypothesised beneficial effect of high CO2 around vents was not supported. We observed a consistent long- and short-term pattern of gene down-regulation and 2.5-fold NPP decrease in plants incubated in water from the vents and a generalized up-regulation and NPP increase in plants from the vent site incubated with water from the Reference site. Contrastingly, NPP of specimens experimentally exposed to a CO2 range significantly correlated with CO2 availability. The down-regulation of metal-related genes in C. nodosa leaves exposed to water from the venting site suggests that other factors than heavy metals, may be at play at Vulcano confounding the CO2 effects.Ocean acidification is a major threat for marine life but seagrasses are expected to benefit from high CO Projections forecast that ocean acidification will continue and that by the end of the century the mean global ocean surface pH will decrease up to 0.4 units1Rising atmospheric carbon dioxide CO is incre2 associated with ocean acidification may favour carbon-limited species, such as seagrasses, by increasing both the passive diffusion of CO2 and the efficiency of RuBisCO carboxylation over photorespiration22 levels and ocean acidification is inconclusive, and particularly scarce over long-time scale. Indeed, recent meta-analysis did not identify significant effects of ocean acidification on seagrass photosynthesisThe increase in seawater DIC and CO2 play in altering specific metabolic pathways related with the physiological response, but also the fast acclimation and reversibility capacity to short-term acute disturbances , single generation experiments8112 enriched sites, such as submarine volcanic CO2 vents, have been used to study the species and communities potential to face ocean acidification. The use of CO2 vents provides the enormous advantage of testing organisms and communities that have been exposed for long-term over multiple generations to high CO22 and varies among vents2, such as heavy metals or sulphides, have been disregarded due to the low concentrations observed at the seagrass meadows2 vents revealed that seagrasses have successfully adapted to live under permanently high CO2 levels31Cymodocea nodosa, there is evidence of an increase in chlorophyll a content and maximum electron transport rate but low biomass production in meadows growing in acidified areas near CO2 ventsNaturally COCymodocea nodosa to high CO2 to test the hypothesis of beneficial effects of ocean acidification on seagrass productivity, taking advantage of the high CO2 environment existing near the submerged volcanic vents at Vulcano Island .The aim of this study was to evaluate the long- and short-term responses of the seagrass A combination of molecular and physiological techniques was used to relate, for the first time, the expression of target genes involved in plant metabolism  to the net plant productivity (NPP) to investigate if molecular and physiological responses are coordinated. Antioxidant and metal-related genes were also targeted to evaluate the existence of a gene activation to deal with oxidative stress and heavy metal toxicity of plants near the vents in relation to plants away from the venting influence.C. nodosa specimens and water from sites under (CO2 site) and away (Reference site) the influence of CO2 vents at Vulcano Island was performed at the same depth. Differences in the gene expression and productivity between plants exposed for a long-time to high CO2 (CO2 plants) and present-day levels of CO2 (Reference plants) were investigated (in situ incubations) to assess if plasticity and long-term adaptation to high CO2 affected specific metabolic pathways and productivity response of C. nodosa. The short-term acclimation responses to CO2 and reversibility capacity of C. nodosa were assessed investigating the gene expression and productivity of plants from one site (CO2 or Reference) incubated with water from the other site (transplant incubations).A set of plant incubations with 2 on productivity and evaluate the adequacy of volcanic CO2 vents as natural laboratories, a set of controlled experimental incubations of C. nodosa plants were conducted in Ria Formosa  under a wide range of CO2 concentrations.To validate the observed effects of high CO2 concentrations (and associated parameters of the DIC system) were significantly higher in the CO2 site than in Reference site . In accordance with descriptions from previous studies29302 site) was higher than in the Reference site, particularly regarding the inorganic carbon system. Indeed, occasional extreme high values of pCO2 and DIC were recorded in the CO2 site  and Ria Formosa  is given in CO2 site .Expression stability of reference genes (RGs): Four RGs (Eukaryotic initiation factor 4A (eIF4A), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S) and Ubiquitin (UBI)), belonging to different functional classes, were tested for stability in the different experimental conditions . The thrC. nodosa plants from both Reference and CO2 sites after in situ and transplanted incubations (Genes of interest (GOIs) expression level: Relative expression levels of 20 GOIs involved in photosynthesis, carbon dioxide fixation and metabolic carbon assimilation pathways, antioxidant and metal responses were analysed in ubations .in situ incubations in CO2 site) with respect to plants from the Reference site (in situ incubations in Reference site) (2 site compared to plants from Reference site (P(H1): 0.000), as well as the large subunit of RuBisCO (rbcL) (P(H1): 0.037). The transcripts for the Ferredoxin (FD) and Phosphoenolpyruvate carboxylase (PEPC) were close to the significance level (P(H1): 0.058 for both) (P(H1): 0.000). Down-regulation of metal tolerance and metal detoxification proteins (MTP and MT) was also significant (P(H1): 0.000). The general down-regulation pattern observed points to an overall reduction of metabolic processes related with photosynthesis, oxidative (ROS) and metal detoxification response in plants growing near the CO2 vents with respect to plants from the Reference site.Major changes in gene expression patterns were observed in plants long-term growing near the vents (ce site) , as confce site) . Overallnce site . Accordior both) . The samor both) . The expC. nodosa from the Reference site were incubated in water from the CO2 site (transplant incubation), expression of almost all the GOIs analysed was reduced (in situ conditions (i.e. plants from the Reference site incubated in water from the Reference site). Significant down-regulation was also detected for the enzymes Phosphoenolpyruvate carboxylase (PEPC) and Glutathione reductase (GR) (P(H1): 0.000)  .2 site were incubated in water from the Reference site, a clear gene up-regulation pattern was observed on the metabolic paths linked to photosynthesis as well as oxidative (ROS) and metal detoxification to acclimate to the new conditions (P(H1): 0.000) (P(H1) < 0.1)  . Several) < 0.1) .C. nodosa was significantly affected by the origin of the water where plants were incubated (p < 0.01). NPP was significantly lower when plants were incubated with water from the CO2 site, both in plants long-term growing near the CO2 vents (i.e. CO2 plants in situ incubations) or away from them but incubated with water from the CO2 site   of bations) .2 concentrations in water  (in situ incubations) and plants from CO2 site incubated in water from the Reference site (transplant incubations). Reversely, plants long-term growing in the CO2 site (in situ incubations) and plants from the Reference site incubated in water from the CO2 site (transplant incubations) showed a generalized gene down-regulation and lower NPP. The biplot in the PCA identify GR, MT, PEPC, SOD, GSH-S, LHCA1 and psbA as the genes most contributing to this separation  defined by the origin of the water where plants were incubated (i.e. water from Reference O2 site) . These tDirect correlations were found between gene expression and productivity (Spearman’s rank correlation). Significant correlations were detected between NPP and expression of photosynthetic , and carbon metabolism-related genes  as well as antioxidant-related genes  .in situ incubations of C. nodosa plants were performed under the same physico-chemical conditions with CO2 levels experimentally controlled   .C. nodosa were significantly lower in plants incubated in CO2–rich water from Vulcano CO2 vents when compared to Reference water. Plants living in the venting site showed a general reduction in the expression of genes involved in various metabolic processes, particularly those related with the light-dependent reactions of photosynthesis, carbon fixation and metabolic carbon assimilation when compared to plants from the Reference site. The same pattern was observed for genes involved in the oxidative stress antioxidant response and metal detoxification. This generalized gene down-regulation was paralleled by a reduction in plant metabolism, which resulted in 2.5 fold decrease of NPP in plants naturally growing under acidified water near the vents. Broad-scale decreases in the expression of genes related to key cellular processes, including metabolism and stress response, affecting physiological processes have been already described in different marine taxa in response to ocean acidification C. nodosa exposed to volcanic CO2 vents and the expression level of the genes of interest (GOIs). The pioneer information reported here can represent a baseline for understanding adaptive features and stress responses in this species and in the Cymodoceaceae family in general.Our results showed that both the net plant productivity (NPP) and the gene expression of C. nodosa to high CO2, evaluated through the transplant incubations, agreed with the molecular and productivity long-term responses measured in situ. Reference plants incubated in water from the CO2 site experienced a generalized, drastic down-regulation in the gene expression of several photosynthetic, antioxidant and metal-related genes that globally reduced plant metabolism and resulted in a significant NPP decrease. On the contrary, plants from CO2 site incubated in Reference water significantly up-regulated the expression of those genes and moderately increased NPP.The short-term acclimation responses of C. nodosa leading to significant differences in plant productivity when incubated in water with different CO2 concentrations. Significant positive correlations were detected between gene expression and productivity for specific genes encoding proteins related with the efficiency of the photosynthetic process, such as the Photosystem I reaction center (psaJ) and the Photosystem II protein D1 (psbA), the Photosystem I light harvesting complex (LHCA1), and the carbon fixation . Significant correlations were also detected between oxidative stress-related genes  and productivity. These results support the use of molecular tools as early indicators of potential stress or physiological readjustments in response to environmental changeGlobally, we identified a coordinated gene expression pattern among photosynthetic antioxidant and metal-detoxification genes in all incubations conducted in Vulcano. We also detected a fast response of gene expression over physiology and global metabolism in in situ incubations) could be attributed to local adaptation and differential selection of specific genotypes under acidified conditions2 and Reference sites . Consequently, results point to a low contribution of local adaptation to the observed differences between plants from the two sites. Organisms may also be able to cope with conditions found within the CO2 vents through plastic responses at different levels, from molecular (i.e. gene expression) to physiological, morphological and behavioural, without genotypic changes occurring through selectionC. nodosa, in terms of photosynthetic response capacity42The contrasting response pattern recorded in Vulcano between plants long-term growing at each site  in Ria Formosa) when exposed to high CO2. However, the sharp NPP decrease observed in C. nodosa growing at present day CO2 levels in Vulcano (Reference plants) when incubated in CO2-rich seawater (i.e. transplants) is difficult to justify, particularly when considering the photosynthetic carbon-limited physiology of this species442 showed by Ria Formosa plants short-term exposed to a CO2 range.The observed decrease of 2 in Vulcano2 and Reference sites in Vulcano were growing and incubated under similar environmental conditions  with sulphide (H2S) concentrations of about 270 μM2S and heavy metals on the photosynthetic apparatus, metabolism and survival of marine organisms, including seagrasses, have been previously described49C. nodosa leaves when incubated in water from CO2 site suggests no significant damage associated with heavy metals. However, attenuation, and even inhibition, of cytochromes functioning has been reported at concentrations of H2S as low as 100 μM compromising the photosynthetic output of photosystems51C. nodosa plants incubated with water from the venting site in Vulcano. Unfortunately, no data on seawater sulphide or metal concentration during the experimental period are available. At present, a major knowledge gap exists regarding the combined effects of high CO2 and other drivers associated with Global Climate Change on marine benthic communities, including those dominated by seagrasses. The particular chemical composition of seeping fluids at each venting site may recreate different foreseen multi-drivers scenarios associated with Climate Change Submarine COCymodocea nodosa, exposed to high CO2 in volcanic CO2 vents. We found that the gene expression and productivity responses of C. nodosa to high CO2 at Vulcano vents were coupled indicating water origin as the main source of variability among all the incubations. However, the hypothesis that high CO2 environment near the vents is beneficial to plant productivity was not supported. The consistent and unexpected long- and short-term pattern of gene down-regulation and NPP decrease observed in C. nodosa when incubated with water from the vent site suggests that environmental factors, other than increased inorganic carbon availability, may be at a play affecting photosynthetic metabolism and decreasing productivity. The down-regulation of metal detoxification genes suggests no significant damage associated with heavy metals. Further research is needed to clarify if sulphide or other components of seeping fluids may explain our observations. Natural submarine CO2 vents provide unique experimental conditions to evaluate the acclimation and adaptive potential of species and communities to scenarios where ocean acidification occurs in combination with other environmental factors.In conclusion, we related, for the first time, gene expression with net plant productivity in the seagrass species, th centuryThis study was conducted in May 2013 in Vulcano Island  where submarine gas emissions date from late 18Cymodocea nodosa grows30342 site (CO2) is located approximately 200 m from the vent at 2 m depth and 50 m from the shore. The Reference site (REF), away from the vent influence, is located approximately 450 m from the vent at 2.5 m deep and 40 m from the shore.We selected two sites, characterized by different pH regimes, where the seagrass C. nodosa growing in Ria Formosa , a lagoon system with no venting influence, were incubated following the same methodological approach used in Vulcano, i.e. in situ NPP estimates through O2 determination in the same incubation chambers used in Vulcano.In order to test the productivity results obtained in Vulcano Island, we conducted a set of incubations at an independent control site. Specimens of −1) was checked using CRM batch #126 . DIC system parameters were calculated with the CO2SYS programet al. (1973) refitted by Dickson and Millero (1987) (references included in the CO2SYS).During the incubations, underwater irradiance (Licor Li-192SA) above the incubation chambers, salinity (VWR CO310), temperature , and pH (Thermo Scientific Orion) were monitored in both Vulcano  and Ria Formosa . Alkalinity, for characterization of the DIC system, was measured by final point potentiometric titration and accuracy  and quickly transported (less than 5 minutes) to an intermediate incubation location, halfway of donor sites, where all incubations took place at 2 m depth. Plants were gently cleaned from sediment and epiphytes and placed into the incubation chambers with water from their own collection site (in situ incubations) or from the other site (transplant incubations).in situ or transplants) two runs were conducted, one at midday (around 12:00 h) and one in the afternoon (around 16:00 h). At each run, four incubation chambers from each site were incubated together and thus they were exposed to the same irradiance. Light measurements (Licor sensor) were done at the incubation location along the incubation time. No significant differences were found between the midday  or the afternoon (p = 0.2) irradiances during the two consecutive days of incubations.For each type of incubation  with one sampling port to withdraw water samples . Chamberin situ incubations (n = 64) were conducted at 2 m depth to measure the productivity of C. nodosa to varying CO2 concentrations. Seawater used in the incubations was collected from the field and bubbled with air at different concentrations of CO2, ranging from ~250 up to ~1500 μatm (pCO2) ), thus covering the range found in Vulcano and the predicted CO2 scenarios for the end of the centuryIn Ria Formosa, in situ and transplants incubations. Apical (youngest) shoots were avoided and only middle parts of the youngest fully mature leaves (2° or 3° rank leaves) of intermediate shoots were selected to standardize leaf developmental stages. Immediately after collection, leaves were blotted dry with tissue paper and preserved in RNAlater©  to protect RNA from degradation. The whole procedure, from collection to fixing, lasted less than 5 minutes. After one night at 4 °C, samples were stored at −20 °C until RNA extraction. RNA was extracted from C. nodosa leaves with the Aurum™ Total RNA Mini Kit (BIO-RAD) following manufacturer’s instructions. The full protocol for RNA extraction and cDNA preparation is described in Appendix 2, One shoot from three randomly chosen incubation chambers was collected at the end of both 2 availability  that can be directly correlated with plant productivity . Additionally, some studies have related high-CO2 environments with antioxidant and oxidative stress responses in land plants, and more recently in other seagrass species growing close to natural volcanic CO2 ventsReverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) analysis was performed to investigate differences in expression levels of genes belonging to major pathways affected by changes in CO2) was determined spectrophotometrically by the modified Winkler method according to the protocol described in ref. 2 g DW−1 h−1) rates were calculated by the difference between final and initial oxygen concentration normalized by the incubation time, the volume of water and the biomass incubated.Net plant productivity (NPP) was estimated following the methodology described in ref. t-tests.Normality (Shapiro) and homoscedasticity (Levene) tests were run to test parametric assumptions. Differences in seawater parameters between sites in Vulcano were tested with paired in situ incubations and transplants) relative to controls, we used the method developed in REST 2009 Software version 2.0.13For gene expression, multivariate differences in gene expression among different treatments were tested by an analysis of similarity (ANOSIM), with Primer 6 (v.6.1.12). To inspect the significant gene regulation in the different conditions (2 on productivity (NPP) of C. nodosa plants long- (in situ incubations) and short-term (transplants incubations) exposed to high CO2 water from CO2 vents, an ANCOVA analysis considering two factors, the “origin” of the plants (plants from CO2 or Reference site) and the “water” used in the incubation (water from CO2 or Reference site), and “irradiance” (as a continuous covariate) was performed in order to account for any potential effect of irradiance on NPP. ANCOVA analysis indicated a highly significant main effect of the “water” (p = 0.002) whereas the effects of “irradiance” or “origin” were not significant. A significant three-way  interaction was also revealed (p = 0.03), but the post-hoc tests did not provide a clear interpretation of the occurrence of any other relevant processes, i.e. it was not clear what explanatory factors needed to be retained in the model. We then tested for a model simplification using an Akaike’s information criterion (AIC) test among 3-way and 2-way factorial and additive possible ANCOVA and ANOVA models. The 2-way ANOVA (two fixed crossed factors) with “origin” and “water” as explanatory factors produced the lowest AIC value, that is, the best fit and explanatory power indicating that a simplification of the ANCOVA model was justifiedTo investigate the effect of CO2 on productivity (NPP) of C. nodosa plants. The explanatory power of DIC was highly significant (p = 0.01) as opposed to irradiance (p = 0.2) and the best AIC fit was obtained with DIC as single explanatory variable. For this reason we performed a linear regression with DIC as single explanatory variable.Likewise, at Ria Formosa the incubations were carried out along the day with changing irradiances. A multiple regression analysis including irradiance and DIC as explanatory variables was performed to investigate the effect of COin situ and transplant incubations, a Principal Component Analysis (PCA) and a Cluster analysis were also performed with the software PAST (v.3.03). To link gene expression to productivity a Spearman’s Rank correlation analysis was conducted between the NPP and expression of each gene measured (n = 20 genes) considering the mean gene expression of each incubation type . Unless otherwise indicated, data were analysed using RTo analyse the combined response of gene expression patterns and NPP on plants from How to cite this article: Olivé, I. et al. Linking gene expression to productivity to unravel long- and short-term responses of seagrasses exposed to CO2 in volcanic vents. Sci. Rep.7, 42278; doi: 10.1038/srep42278 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Water capture is not achieved through strategic positioning of a water molecule in the active site, but through a coordinated series of loop movements that allow bulk water access to the final carbocation in the active site prior to product release.δ‐Cadinene synthase is a sesquiterpene cyclase that utilises the universal achiral precursor farnesyl diphosphate (FDP) to generate predominantly the bicyclic sesquiterpene δ‐cadinene and about 2 % germacradien‐4‐ol, which is also generated from FDP by the cyclase germacradien‐4‐ol synthase. Herein, the mechanism by which sesquiterpene synthases discriminate between deprotonation and reaction with a nucleophilic water molecule was investigated by site‐directed mutagenesis of δ‐cadinene synthase. If W279 in δ‐cadinene synthase was replaced with various smaller amino acids, the ratio of alcohol versus hydrocarbon product was directly proportional to the van der Waals volume of the amino acid side chain. DCS‐W279A is a catalytically highly efficient germacradien‐4‐ol synthase ( Present in all kingdoms of life, terpene synthases catalyse highly complex biosynthetic reactions, in which achiral, linear isoprenyl diphosphates are converted into complex often cyclic or polycyclic structures.2+‐binding motifs (DDXXD and NSE/DTE) on opposite sides of the active site.i)–carbocation pair.E,E)‐farnesyl diphosphate , several analogues of 1, Mg2+ and PPi, together with molecular dynamics simulations, have provided strong support that loop movements and conformational changes are required to form the closed form of the enzyme, in which substrate 1 is in a reaction‐ready conformation.i carbocation interactions and general acid–base catalysis by PPi and/or the enzyme.Examination of the structures of terpene synthases and the mechanisms of the catalysed reactions has revealed common structural features and distinct phases of activity.ate FDP, , severalate FDP, , severali, little information is available for terpene alcohol synthases, such as epicubenol,6, so as not to produce δ‐cadinene ,7(11)‐diene synthase from Gossypium arboreum produces 7 in the first committed step of the biosynthetic pathway to the phytoalexin gossypol ‐2F‐farnesyl diphosphate (2F‐FDP) revealed an unusual Mg2+‐binding motif, in which the NSE/DTE motif is replaced by a second DDXXD motif.1.8.DCS from l Scheme .22 Despi1 into 8 (93 %) and an additional unidentified cyclic sesquiterpene alcohol.The double‐mutant protein DCS‐N403P/L405H was shown to convert 7, but also to form hydrophobic interactions that may help to control the active‐site conformation of 1 and mediate active‐site closure and opening. Hence, to test the hypothesis that alteration of W279 can disrupt hydrophobic interactions with Y410 and 1 and allow increased water access to the active site, the contribution of W279 to catalysis was explored by sitedirected mutagenesis. Herein, we show that single amino acid changes can convert DCS into GdolS that produce up to 90 % 8 with high catalytic efficiency. The results suggest that W279 plays a key role in shielding the active site of DCS from solvent.In DCS‐N403P/L405H, the active site is exposed to solvent through a potential water channel created by alteration of the G‐helix residues.6 was produced in Escherichia coli and its catalytic properties determined. Steady‐state kinetic experiments with radiolabelled [1‐3H]1kcat, of 1.26×10−3 s−1; a Michaelis constant, KM, of 0.58 μm ×10−4 s−1 μm−1, which was identical to the value previously measured for WT‐DCS with no His‐tag (kcat/KM=(3.1±0.2)×10−3 s−1 μm−1).DCS‐Hislabelled ‐3H1kca1 by GC‐MS showed that, in addition to 7, 11 % of 8 was produced by the His‐tagged enzyme. The products were identified by comparison with the GC retention times and mass spectra of authentic products generated through the incubation of 1 with WT‐DCS and GdolS from Streptomyces citricolor.Analysis of the pentane‐extractable products arising from incubations with 1 were analysed by GC‐MS. Remarkably, DCS‐His6‐W279A produced only 11 % 7 and 81 % 8  and KM (2.23 μm) were similar to the values measured for DCS‐His6; this indicated that replacement of the hydrophobic and bulky indole group with hydrogen allowed water access to the active site to efficiently quench the intermediate ‐germacryl cation (5) without loss of the catalytic efficiency. If W279 was replaced by tyrosine, the quantity of alcohol 8 formed was only slightly increased relative to that of DCS‐His6.To address the role of W279 during DCS catalysis, tryptophan was replaced by Glu, Gln, Asp, Leu, Met, Als and Tyr and the pentane‐extractable products generated from 6‐W279M and DCS‐His6‐W279L, in which residues with similar hydrophobicity, but with reduced van der Waals volume, replaced tryptophan,6 (7 and 8 in approximately 2:1 ratio). These results show that the relative amounts of 7 and 8 are dependent on the volume of the side chain of residue 279. The values of KM for DCS‐His6‐W279M and DCS‐His6‐W279L were slightly increased, relative to that of the DCS‐His6  medium (10 mL) containing ampicillin (100 μg mL−1)) by using the miniprep kit, as described by the manufacturer. Deletion was confirmed by DNA sequencing.Expression and purification: DCS‐His6 was produced in E. coli BL21(DE3) cells that harboured the cDNA for DCS‐His6 under control of the T7 promoter. E. coli BL21(DE3) cells were gently defrosted on ice before plasmid  was added to the cell suspension. The resulting mixture was stored on ice (30 min), heat‐shocked in a water bath  and then returned to the ice (2 min). LB medium (1 mL) was added and the solution was incubated for 1 h at 37 °C with shaking (150 rpm). The cells were harvested by centrifugation , resuspended in a minimum amount of LB medium and spread on an agar plate containing ampicillin (100 μg mL−1). The plate was then incubated overnight at 37 °C. A single colony from the agar plate harbouring the transformed cells was used to inoculate LB medium (100 mL) containing ampicillin (100 μg mL−1) and the culture was incubated at 37 °C with shaking (150 rpm) overnight. The overnight culture (10 mL) was transferred to each of 6×500 mL of LB medium containing the same concentration of ampicillin as before. Cells were incubated at 37 °C with shaking (150 rpm). When an optical density (OD600) of 0.6 was reached, isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) was added  and the cultures were incubated for 24 h with shaking (250 rpm), at 20 °C. Cells were harvested by centrifugation at 5 °C . The supernatant solution was discarded and the pellets were stored at −20 °C.m Tris‐Base, 5 mm β‐mercaptoethanol (βME), pH 8) and stirred gently for 1 h at 0 °C. Cells were then disrupted by sonication at 5 °C (40 % amplitude for 3 min with 5 s on/10 s off cycles) and the resulting suspension was centrifuged at 5 °C . SDS‐PAGE analysis showed that protein was in the soluble fraction and the pellets were discarded. The supernatant solution was then loaded onto a 2 cm Amintra nitrilotriacetic acid (NTA) Ni2+ column  and eluted under gravity‐controlled drip flow. After 40 min, the column was washed with four column volumes (CV) of binding buffer . The column was then washed with a gradient of imidazole  in binding buffer. DCS‐His6 eluted in the range 60–100 mm imidazole; column fractions were analysed by SDS‐PAGE. The fractions containing pure protein corresponding to a molecular weight of 64 000 (DCS‐His6) were pooled, dialysed overnight  30000) and then concentrated to a final volume of about 5 mL . The solution was aliquoted and stored at 0 °C. The concentration of protein was estimated by using the method of Bradford.Pellets were allowed to thaw at 5 °C and resuspended in cell lysis buffer  was added to assay buffer  followed by addition of enzyme . The aqueous solution was overlaid with HPLC‐grade pentane (0.5 mL) and the resulting mixture was incubated with gentle agitation (18–24 h) at 25 °C. The incubations were repeated without enzyme as negative controls. The pentane extracts were then analysed by GC‐MS as described in the Supporting Information.6 and mutantsSteady‐state kinetics of DCS‐His: Kinetic assays were performed according to the standard, linear range, micro‐assay procedure previously developed for DCS (see the Supporting Information).As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


We describe the detailed microscopic changes in a peptide monolayer following kinase-mediated phosphorylation. A reversible electrochemical transformation was observed using square wave voltammetry (SWV) in the reversible cycle of peptide phosphorylation by ERK2 followed by dephosphorylation by alkaline phosphatase. A newly developed method for analyzing local roughness, measured by atomic force microscope (AFM), showed a bimodal distribution. This may indicate either a hole-formation mechanism and/or regions on the surface in which the peptide changed its conformation upon phosphorylation, resulting in increased roughness and current. Our results provide the mechanistic basis for developing biosensors for detecting kinase-mediated phosphorylation in disease. Kinases play a central role in cellular signalling234567910111213To understand the microscopic changes in the monolayer and the mechanism of how the phosphorylation affects the monolayer, we developed a new analysis method and used it for characterizing the topography of a peptide monolayer. The monolayer was composed of the Hepatoma Derived Growth Factor (HDGF) 160–174 peptide, derived from the HDGF protein that is strongly related to several types of cancer1617Here we studied the phosphorylation and dephosphorylation of the peptide monolayer using square wave voltammetry (SWV) and local roughness measurements with AFM. Previously we have shown that the EIS can be used as a sensitive method for characterizing the capacitance vs. resistance changes in the peptide monolayerSurface roughness is commonly calculated by averaging over the whole scanned area to provide a single value of the global rms roughness2021To overcome these disadvantages, we divided the scanned areas to many smaller sub-areas and created an overall distribution of the rms roughness of each one of them. This method provides a much better sensitivity and enables to observe and distinguish between few roughness values on the same surface. It is also much less sensitive to large contaminants, such as aggregates of peptides, impurities in the solution including residues from preparation processes and atmospheric particles of various types and sizes that adsorb to the surface. With the current approach, these do not affect the distribution as they do in global rms roughness analysis, where they up-shift and widen the overall roughness distribution. Here, we measured the roughness changes of the peptide monolayer on ultra-flat gold surface2 glass was attached using epoxy glue  and dried for 3 hours at 80 °C, then the mica sheet was removed by submerging in pure tetrahydrofuran (THF) for 3 minutes.Ultra-flat gold substrates were prepared as follows2, 0.2 mg/mL BSA and 100 μM ATP) at 37 °C. The reaction was initiated by adding freshly prepared protein kinase solution and stopped by rinsing, after 30 min.The surface was submerged overnight in a peptide containing solution  immediately after mica removal. Phosphorylation with GST – tagged ERK2 (New England Biolabs) was done by immersing the slides in 300 μl kinase reaction medium  AP is defined as the amount of enzyme that can hydrolyze 1 μmol of phosphorylated peptide in a total reaction volume of 1 ml in 1 min at 37 °C). After each step the surface was thoroughly washed in tri-distilled water and dried in N2 flow.For dephosphorylation, AP, 0.1 Units in a final reaction volume of 300 μl, was added for 30 minutes at 37 °C. 1 Unit AP was dissolved in a 1 ml of Tris borate buffer, (pH 8.5), containing 10 mM MgCl6]3− solution additionally containing 100 mM KNO3 as supporting electrolyte was used. In the current study the distance between the working and counter electrodes was invariable.Square Wave Voltammetry (SWV) measurements were performed using an Autolab PGSTAT12 digital potentiostat  connected to NOVA software. A conventional three-electrode cell was employed; a Platinum electrode as a counter electrode, a 2 mm gold electrode (CH instruments) as a working electrode, and a standard Ag/AgCl reference electrode (Metrohm) with a KCl concentration of 3.0 M. Peptide immobilization on the gold electrode was achieved by chemisorption via cysteine thiol functional-group anchoring to the Au surface. The peptide monolayer formation, phosphorylation by ERK2 and dephosphorylation by AP on the working electrode were performed in the same conditions and with the same solutions as on the gold surfaces (see above). The volume of the enzyme solutions used on each electrode was 40 μl. The electrodes were characterized by SWV after each modification step. The voltammograms were obtained by scanning from −0.3 to −0.6 V with a step potential of 25 mV, amplitude of 10 mV and frequency 25 Hz at a scan rate of 625 mV/s. To provide redox species, a 1 mM [Fe(CN)Topographic characterization was done in non-contact AFM  with soft cantilevers  to minimize tip impact on the surface. Initial image processing was carried out with WSxM 5.0 develop 7.0. Global roughness was calculated using WSxM on the whole image2. The matrix is divided to 100 small sub-matrices, 10,000 pixels each. RMS roughness is calculated for every sub-matrix of 100 × 100 pixels, as shown in The roughness of each scanned area was analysed in MATLAB as a matrix of 1000 × 1000 elements, each element representing the height of one pixel. i.e., 1 × 1 nmhere Z is the height value measured by AFM and n is the number of pixels in calculation. This process was repeated on four different samples with 5–10 macroscopically distant areas that were analysed on each sample after each step of the experiment. Histograms of the roughness distributions of the whole samples were made and fitted with two-term Gaussian. Areas of low and high roughness were calculated by integration of the Gaussian fit within the standard deviation range.The process of phosphorylation and dephosphorylation was performed on gold macro electrodes and SWV measurements were taken after each step of the process . The ads2 area was less than 2 nm and the RMS roughness was 0.18 ± 0.02 nm. Therefore, a flat substrate may be assumed for the examined 1 × 1 μm2 areas, with respect to the monolayer thickness and roughness.We characterized using AFM the roughness of HDGF 160–174 peptide monolayer on ultra-flat gold substrate after deposition, after phosphorylation and after dephosphorylation. These measurements are linking the macroscopic electrochemical observations to the microscopic interpretation. The roughness of the ultra-flat gold substrate was checked before the peptide deposition. The maximum peak-to-peak height in a 5 × 5 μm2 peptide monolayer areas after monolayer formation . After phosphorylation the roughness increased to 0.61 ± 0.17 nm (10 scans of 1 × 1 μm2) and after dephosphorylation it decreased to 0.50 ± 0.14 nm (11 scans of 1 × 1 μm2). These results show a small change in the average roughness, which we term “global roughness”, during the experiment. The error margins are smaller relative to the global roughness analysis method and therefore the scanned areas had close values. Thus, at the micron scale the surface is quite uniform.RMS roughness was measured and calculated at various 1 × 1 μmOur results show visually and indicate numerically that phosphorylation of a peptide substrate by a kinase induces structural changes in the monolayer and increases the surface roughness while dephosphorylation reverses the process to its initial values. However, the observed changes in the average surface roughness in each stage are rather small and fall within each other’s margins of error.2 were examined and a semi-automated process was implemented (see Methods for details). This enabled a much larger statistical base – up to 1000 areas at each step versus 5–14 areas previouslyTo address this problem, extract finer information and get a deeper insight into the surface changes during the phosphorylation-dephosphorylation cycle, a higher resolution analysis was performed. Smaller areas of 100 × 100 nmEach distribution was fit to a two-terms Gaussian, represented by the solid lines in 15The local roughness analysis presented here shows that the peptide monolayers consist of low and high roughness domains, which could not be observed by the previously performed global analysis. Initially the peptide monolayer consists mostly of low roughness domains  that is generated in the monolayer upon phosphorylation, thus damaging the insulating capacity of the peptides layer. This is supported by the morphological observation that the smooth HDGF 160–174 peptide monolayer behaves as a well packed self-assembled monolayer having dominant local roughness of ~0.3 nm  and is pThe second contribution is changes in the orientation and packing of peptides in the monolayer. The increased roughness upon phosphorylation could be related to the substitution of an alcohol group by a phosphate group in the monolayer. When the peptides in the monolayer are phosphorylated, two main parameters may contribute to order-disorder transition: a) steric effect of the bulky phosphate group containing larger hydration layer than that of the alcohol group, and b) the electrostatic repulsion between negatively charged neighbouring phosphates. Both parameters may disrupt the orientation and packing of the peptides and therefore the initial ordering of the native peptides monolayer. Although cis-trans isomerisation has been shown to take place in the XSPX phosphorylation motifIn summary, a dense and uniform layer of the HDGF 160–174 peptide was formed on gold electrodes and ultra-flat gold surfaces. It was characterized by SWV and AFM before phosphorylation by ERK2, after phosphorylation and after dephosphorylation by AP. We observed a reversible electrochemical transformation of the peptide monolayer upon reversible phosphorylation and dephosphorylation and further confirmed our model of surface reaction by using a general local roughness analysis. While the experiment was performed on a very specific surface, the analysis method does not take into account any properties of the monolayer or the substrate. Therefore it can be applied to any kind of smooth surface to reveal additional information that is hidden in regular roughness analysis. While the average roughness of the peptides monolayer at 1 μm scale shows slight increase after phosphorylation, the changes are very small. Roughness calculations at 0.1 μm scale reveal a much more complicated picture – the surface is composed of two kinds of domains with different roughness. Phosphorylation changes the ratio between these two kinds and slightly increases the roughness of the smoother domains, presumably by introducing defects and disturbing the ordering of the densely packed domains. This process seems completely reversible as upon dephosphorylation the surface recovers to its initial morphology. The microscopic morphological analysis supports the model that provides a mechanistic insight into the behaviour of the monolayer in the electrochemical assays.How to cite this article: Zhuravel, R. et al. Atomic force microscopy characterization of kinase-mediated phosphorylation of a peptide monolayer. Sci. Rep.6, 36793; doi: 10.1038/srep36793 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.