Similarly, ultrafiltration also enhanced trypsin inhibition by the alcalase-digested peptides with an IC50 of 21.4 mg/mL for the unfractionated hydrolysate in comparison to 3.1–4.7 mg/mL for the fractions. However, ultrafiltration did not enhance trypsin inhibitory activity of chymotrypsin-digested peptides, while the peptide separation reduced efficacy of pepsin-digested peptides. In contrast, chymotrypsin inhibition by all the enzymatic digests was significantly (p < 0.05) enhanced by ultrafiltration, especially peptide sizes >3 kDa. Kinetics of enzyme inhibition indicate peptides were bound to the enzyme active site in a competitive mode that led to reduced catalysis. We conclude that the pea peptides could function as useful tools to promote human health and as a preservative during food processing and storage. In this work, we report the potency of enzymatic hydrolysates of pea proteins against trypsin and chymotrypsin. Pea protein concentrate was digested with each of alcalase, chymotrypsin, pepsin, and trypsin, followed by membrane separation of the protein hydrolysates into peptide fractions . Peptide size profiling with size-exclusion gel chromatography indicated the narrowest size range (0.85–4.98 kDa) for alcalase. Trypsin activity was strongly ( Food-derived protease inhibitors have essentially been in the form of large proteins that block activities of trypsin and chymotrypsin, the major serine proteases. The initial serine protease inhibitors were isolated from soybean seeds as the Kunitz inhibitor, which predominantly acts against trypsin , while tHowever, inhibition of the enzymes (trypsin and chymotrypsin) involved in protein digestion is also paramount as it is known that excessive protein intake can cause weight gain  as excesIn contrast to whole proteins, food-derived peptides are usually obtained from the enzymatic hydrolysis of animal and plant proteins . Dependip-nitroanilide), BTpNA (N-Benzoyl-l-tyrosine p-nitroanilide), 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), bovine chymotrypsin, porcine pancreatic trypsin, and other analytical grade reagents were purchased from Sigma-Aldrich  and Fisher Scientific Company , respectively.The yellow field pea protein concentrate (PPC) containing 82% protein content (dry wt basis) was purchased from Nutri-Pea Limited . BApNA (N-Benzoyl-D-L arginine w/v) was suspended in double distilled water followed by heating to optimal temperature. After the protein mixtures have been adjusted to the optimal pH of each protease, hydrolysis was initiated by addition of each protease at a ratio of 1:25 enzyme/substrate ratio. Hydrolysis was allowed to proceed for 4 h accompanied by constant adjustment to the set pH value using a 0.5 M NaOH solution for alcalase, trypsin, and chymotrypsin reactions while 1 M HCl was used for the pepsin reaction. The duration of hydrolysis was based on lack of change in pH of the reaction mixture after 4 h, which indicates no further hydrolysis occurred beyond this point. Hydrolysis was then terminated by adjusting to pH 5.0 and the mixtures heated at 95 °C for 15 min to completely inactivate the enzymes. The digestion mixture was cooled to room temperature and then centrifuged . The supernatant was collected, and a portion freeze-dried and stored at −20 °C as the protein hydrolysate. The remaining portion of the supernatant was separated into different peptide fractions using membrane ultrafiltration.PPC was hydrolyzed (in duplicates) using four different proteases  as previously described . The enzDetermination of the peptide size distribution of the four pea protein enzymatic hydrolysates was carried out using the method of Alashi et al. . In summThe membrane ultrafiltration protocols were carried out according to the method of Awosika and Aluko . Brieflyv/v) dimethyl sulfoxide. Following incubation for 10 min at 37 °C, the reaction was terminated by the addition of 100 μL of 30% (v/v) acetic acid. A 200 µL aliquot of the reaction mixture was transferred to a 96-well microplate and the absorbance measured using a microplate reader at 410 nm. Trypsin inhibitory activity was determined by measuring the release of p-nitroanaline from BApNA. AEBSF was used as a positive control. The trypsin inhibitory activity (%) was calculated using the equation:50) values of the protein hydrolysates and membrane fractions were obtained by non-linear regression analysis of a plot of trypsin percentage inhibition versus the sample concentrations using GraphPad Prism version 6.0 .Trypsin inhibitory activity was determined following a previously described method . Briefly2, pH 8.0; final concentration = 20 µg/mL) and 400 μL of samples  were pre-mixed and incubated for 15 min at 37 °C. The reaction was started by the addition of 1 mL of 1 mM BTpNA prepared in 0.01 M Tris-HCL buffer containing 0.02 M CaCl2 and 40% (v/v) ethanol, pH 8.0. Following incubation for 15 min at 37 °C, the reaction was terminated by the addition of 200 μL of 30% (v/v) acetic acid. A 200 µL aliquot of the reaction mixture was transferred to a 96-well microplate and the absorbance measured using a microplate reader at 410 nm. Chymotrypsin inhibitory activity was determined by measuring the release of p-nitroanaline from the substrate BTpNA. AEBSF was used as a positive control. The chymotrypsin inhibitory activity (%) was calculated using the equation:50 values were determined as described above for trypsin.Chymotrypsin-inhibitory activity was determined using previously described protocols . BrieflyThe mode of enzyme inhibition by protein hydrolysates and membrane fractions was determined for trypsin and chymotrypsin by conducting enzyme reactions at various substrate and peptide concentrations . The inhp < 0.05. All analyses were conducted using Statistical Package for the Social Science version 16.0 .All assays were conducted in triplicate and data analyzed as the mean values ± standard deviation. The mean values were examined using analysis of variance (ANOVA) and then compared using Duncan’s multiple range test with significant differences accepted at As shown in 50 values for the trypsin-inhibitory activity of pea protein hydrolysates and fractions are presented in 50 values when compared to the unfractionated hydrolysates, except for pepsin-derived hydrolysate, which had a lower IC50 in comparison to the other pepsin-derived peptide fractions. Overall, trypsin 5–10 kDa peptide fraction had the lowest IC50 value of 2.14 mg/mL. In the alcalase group, alcalase 5–10 kDa fraction had the lowest IC50 value of 3.14 mg/mL, whereas in the chymotrypsin group, the <1 kDa fraction had the lowest IC50 value of 2.63 mg/mL. However, in comparison to the standard trypsin inhibitor drug , the IC50 values of pea protein hydrolysates and peptide fractions were significantly (p < 0.05) higher.Results of the IC50 values could not be determined because percent inhibition did not reach 50% even when sample concentrations exceeded 6 mg/mL. Inhibitory activities of the samples were lower than that of the standard AEBSF (66.39% at 6 μg/mL). Based on the results of the highest concentration tested (6 mg/mL), the pepsin 5–10 kDa fraction had the highest inhibitory activity of 48.13% among all the samples. With regards to each group, the alcalase 3–5 kDa peptide fraction had higher inhibitory activity (29.68%) than the other fractions. In contrast, the 5–10 kDa fraction was the most active within each group of trypsin (36.93 ± 1.04%) and chymotrypsin (38.90%).Percentage chymotrypsin inhibition by the pea protein hydrolysates and peptide fractions was concentration dependent in the 2–6 mg/mL range, as shown in The Lineweaver–Burk plots for the trypsin-catalyzed reaction in the absence and presence of the peptide inhibitors at two concentrations are shown in Inhibition of the chymotrypsin-catalyzed reaction was also competitive as shown by the intersection of the lines at the same point on the y-axis in Determination of the molecular weight of hydrolysate is a key criterion in considering the potential bioactivities of peptides. This is because low molecular weight peptides have better chances of escaping structural degradation within the gastrointestinal tract and being absorbed into blood circulation than larger peptides. In addition, small-sized peptides tend to fit and bind tightly into an enzyme’s active site and produce stronger inhibitory effects than larger peptides. As a result, one of the objectives of protein hydrolysate production is to ensure the abundance of small-sized peptides in order to enhance potency against metabolic targets . The alcp < 0.05) weaker trypsin and chymotrypsin inhibitors when compared to AEBSF, which is considered a standard protease inhibitor. In comparison to this work, an isolated polypeptide from Chinese cabbage seeds inhibited trypsin with an IC50 value of 8.5 µM [Opuntia joconostle Weber seeds, which were inactive against this enzyme [The enzyme inhibition results indicate that fractionation improved the trypsin- and chymotrypsin-inhibitory capacity of pea protein hydrolysates, depending on the hydrolytic enzyme. For example, alcalase and trypsin hydrolysates had weaker trypsin-inhibitory activity than the ultrafiltration fractions. Similarly, the >3 kDa fractions of all the hydrolysates had stronger chymotrypsin inhibition than the unfractionated hydrolysates. The results suggest that there may have been peptide antagonism within the unfractionated hydrolysates, which could have contributed to reduced trypsin and chymotrypsin-inhibitory activities. Ultrafiltration separation could have reduced antagonistic effects in the fractionated peptides, hence the higher inhibitory effects of the peptide fractions. Similar results were reported for cod protein hydrolysates and the ultrafiltration fractions . The benf 8.5 µM . Howevers enzyme .In this present study, the modes of inhibition of trypsin and chymotrypsin activities were investigated via kinetic studies in the absence and presence of the enzymatic hydrolysates or the chosen peptide fractions. Lineweaver–Burk plots were used to determine the possible mode of enzyme inhibition and the results indicate a competitive type of inhibition for both enzymes. Therefore, the peptides were bound to the active site of both enzymes, which reduced the rate of substrate binding, hence the lower catalytic activities than when the peptides were not present. Since increases in substrate concentration could overcome the initial inhibition by the peptides, the maximum velocity was the same for inhibited and non-inhibited reactions. However, binding of the peptides to the active site necessitated an increase in substrate concentration required to reach half-maximal velocity, hence the Km values were higher for the inhibited reactions. In other words, binding of the peptide samples affected Km of the reaction catalyzed by both enzymes, proportionately to peptide concentration without affecting maximum velocity (Vmax) of the reaction. The results are similar to our previous report, which also observed that the chymotrypsin pea protein hydrolysate inhibited α-amylase in a competitive manner; in contrast, inhibition of α-glucosidase was non-competitive . TrypsinFor the first time, we report the ability of enzymatic protein hydrolysates to reduce the catalytic rate of trypsin- and chymotrypsin-catalyzed reactions. Peptide size varied, with alcalase producing the smallest peptides, but this factor did not have any consistent effect on potency of enzyme inhibition. Ultrafiltration separation of the peptides yielded fractions with stronger inhibitory activity against chymotrypsin while the effect on trypsin inhibition was dependent on type of enzyme used for protein hydrolysis. Future work should also examine the >10 kDa membrane fraction with respect to its in vitro inhibitory properties against trypsin and chymotrypsin. Inhibition of trypsin and chymotrypsin activities was revealed to be due to competitive binding of the peptides to the enzyme active site, which suggest ability of the peptides to fit into the active sites or interact with active site amino acid residues. Results from this work suggest that the enzymatic pea protein hydrolysates could function to improve human health by modulating metabolic or viral reactions that are dependent on serine protease activities. However, since the hydrolysates could be susceptible to proteolysis in the gastrointestinal tract, animal and human studies will be required to confirm health benefits. However, the hydrolysates may be useful for food preservation by preventing unwanted proteolysis or through toxic effects on pests that are dependent on serine proteases for survival.


Most patients with nonallergic asthma have normal serum immunoglobulin E (IgE) levels. Recent reports suggest that total and aeroallergen-specific IgE levels in induced sputum may be higher in nonallergic asthmatics than in healthy controls. Our objective is to compare total and dust-mite specific (Der p 1) IgE levels in induced sputum in allergic and nonallergic asthmatics and healthy controls.Total and Der p 1-specific IgE were measured in induced sputum (ImmunoCAP immunoassay) from 56 age- and sex-matched asthmatics  and 9 healthy controls. Allergic asthma was defined as asthma with a positive prick test and/or clinically-significant Der p 1-specific serum IgE levels.Patients with allergic asthma presented significantly higher total and Der p 1-specific serum IgE levels. There were no significant between-group differences in total sputum IgE. However, Der p 1-specific sputum IgE levels were significantly higher (p = 0.000) in the allergic asthmatics, but without differences between the controls and nonallergic asthmatics. Serum and sputum IgE levels were significantly correlated, both for total IgE  and Der p 1-specific IgE .Total IgE levels measured in serum and induced sputum are significantly correlated. No significant differences were found between the differents groups in total sputum IgE. Nevertheless, the levels of Der p 1-specific sputum IgE levels were significantly higher in the allergic asthmatics, but without differences between the controls and nonallergic asthmatics. Probably due to the lack of sensitivity of the test used, but with the growing evidence for local allergic reactions better methods are need to explore its presence. The Clinical Trials Identifier for this project is NCT03640936. Although the causes of allergic asthma are well-understood, much less is known about the pathophysiology of nonallergic asthma. Non-allergic asthma is generally defined as nonatopic asthma with or without normal serum levels of immunoglobulin E (IgE) antibodies. Unlike allergic asthma, which is triggered by a specific allergen, nonallergic asthma may be triggered by a wide range of factors, including smoke, viruses, and other nonspecific stimuli.Dermatophagoides pteronyssinus (Der p 1) specific IgE in induced sputum versus healthy controls[Despite their differences, these two types of asthma may share many similarities, including airway inflammation with eosinophilia, increased Th2 cytokine production , hyperreactivity, and airway-induced exacerbations. Although most patients with nonallergic asthma present normal total serum IgE levels, in some cases IgE may be elevated when compared to healthy controls . Mouthuy controls. Those f controls–10. The  controls. Additio controls–16, charIn this context, we hypothesized that patients with nonallergic asthma would present higher levels of IgE in induced sputum than healthy controls. To test this hypothesis, we measured total IgE and Der p 1-specific IgE in serum and induced sputum in three different groups: 1) patients with a confirmed diagnosis of allergic asthma, 2) patients diagnosed with nonallergic asthma, and 3) healthy controls. Secondary aims were to assess the correlation between total and Der p 1-specific IgE levels in serum and induced sputum and to establish a preliminary estimate of total IgE and Der p 1-specific IgE in the induced sputum of healthy individuals.This was a comparative cross-sectional study to measure and compare total IgE and Der p 1-specific IgE levels in the induced sputum of asthmatics and in a group of healthy volunteers. Patients and controls were matched for age, sex, and disease severity; and asthma control for allergic and non-allergic groups. Patients were consecutively enrolled from the outpatient asthma unit of our institution, a tertiary referral university hospital in Spain, between January and December 2013.Total and Der p 1-specific IgE were measured in both serum and induced sputum. The IgE levels in serum and sputum were compared to determine the correlation between IgE levels in these two fluids.We defined asthma as a history of variable respiratory symptoms and evidence of variable expiratory airflow limitation. All patients had a positive bronchodilator test or a document positive methacholine challenge test. Asthma severity was defined according to the Global Initiative for Asthma Management (GINA).D. pteronyssinus was the selected allergen because is the perennial allergen more prevalent in our geographic area.Allergic asthma was defined as asthma with 1) positive skin prick test to aeroallergens and/or 2) clinically-significant Der p 1-specific IgE according to the recommendations of the Committee of Skin Tests of the European Academy of Allergy and Clinical Immunology (EAACI) international task force. PatientInclusion criteria were: 1) age 18–70 years; 2) continuous residence (> 4 years) in the geographic region of the study; 3) diagnosis of stable bronchial asthma according to GINA criteria; 4) non-Exclusion criteria were: 1) pregnancy; 2) moderate to severe active alcohol use; 3) severe atopic dermatitis; 4) presence of any lung disease, autoimmune disease or systemic inflammatory disease, or cancer.The control group consisted of healthy, non-smoking volunteers age 18 to 79 years, without rhinitis, allergic asthma, or other allergic symptoms (GINA criteria) or other lung disease. Controls were recruited from among staff members at our hospital. Participation was completely voluntarily. All controls were required to present a negative prick test for aeroallergens and Der p 1-specific IgE, and negative Phadiatop test.NO ; forced spirometry; inflammatory cell count in induced sputum; eosinophil count in peripheral blood; total serum IgE levels; and skin prick test for common aeroallergens. Patients also completed the validated Spanish-language version of the Asthma Control Test (ACT)[Upon enrolment, demographic and clinical variables were assessed and recorded for all participants. On the same day, the following assessment were performed: Feest (ACT).NO was measured before spirometry using an electrochemical equipment  and an expiratory maneuver providing a sustained 50 mL/s flow from total lung capacity, following the 2005 recommendations of the American Thoracic Society/European Respiratory Society[1 (forced expiratory volume in 1 second) in the reference range when 80% of the predicted value [Fey Society. Spiromeed value ,23.Skin prick testing was performed according to standard procedures, with wheal diameters ≥ 4 mm considered positive. Allergic asthma was defined as the presence of asthma symptoms for one or more allergens, with positive skin prick tests for these allergens. Well-controlled asthma was defined as ACT ≥ 20.6 cells/g) were excluded. Cell viability was determined by light microscopic assessment using trypan blue exclusion staining. The cells underwent centrifugation to obtain a cell pellet and a supernatant. The cell pellet was used for differential cell counts  performed on May-Grünwald-Giemsa-stained preparations. A differential leukocyte analysis of nonsquamous cells (Diff-Quik stained) was performed on a minimum of 400 cells. Differential cell counts are expressed as the percentage of total nonsquamous nucleated cells. Reference values for the cell counts were performed as described in other publications[Induced sputum was evaluated following the procedures previously described by our group and by Plications.Total and specific IgE in induced sputum supernatants were measured by the ImmunoCAP fluoroenzyme immunoassay (Phadia ThermoFisher Scientific) following the manufacturer’s instructions. The test was considered positive at > 2kU/L for total IgE and > 0.35kU/L for specific IgE, according to manufacturer’s recommendation.Categorical variables were expressed as absolute and relative frequencies and quantitative variables as mean and standard deviation (SD). Groups were compared using ANOVA or chi-square test as appropriate. Given that the distribution of total and specific IgE values in serum and sputum were not normal, non-parametric tests  were applied; the values were expressed as medians with interquartile ranges and ranges. The significance values in the case of non-parametric tests were adjusted by the Bonferroni correction. For the correlation analyses, Spearman's Rho test was used. Statistical significance was set at p<0.05. Statistical analysis was performed with Statistical Package for the Social Sciences version 18.0 .The study design complied with the principles of the Declaration of Helsinki and was approved by the Clinical Research Ethics Committee at the Hospital de la Santa Creu i Sant Pau in Barcelona (COD 26/2012). All participants provided written informed consent. All patient-related data were anonymised, with the identity of the participating known only by the treating physicians. The clinicaltrials.gov identifier is NCT03640936.Table 1 shows the clinical, functional, and inflammatory characteristics of the patients and controls.A total of 56 asthmatic patients—21 (37.5%) with allergic asthma and 35 (62.5%) with nonallergic asthma—met all inclusion criteria and completed the study. The control group consisted of nine healthy volunteers. 1 values in the allergic (83.4%) and nonallergic (80%) patient groups were similar, but substantially higher (100.2%) in the healthy controls (p = 0.013) and the need for more than one round of oral corticosteroids was significantly higher (p = 0.022) in the nonallergic group. As for other not clinically relevant sensitizations detected by prick test in the group of patients with allergic asthma, the most frequent were polysensitized patients (17), sensitized to D. farinae (8) and pet epithelia (4). A single patient had a positive prick test for molds.There were no significant differences between the three groups with regard to age, sex, or body mass index (BMI). There were significant differences between the groups in: FEV1 and need for more than one round of oral corticosteroids last 12 months. FEVTable 2 shows the total and Der p 1-specific levels of IgE in serum and sputum for all three groups. Significant between-group differences in serum IgE levels were observed for both total and Der p 1-specific IgE, with the allergic asthma group presenting significantly higher levels of total IgE  and Der p1-specific IgE levels (15.5 KU/L) than the nonallergic group and healthy controls. In the sputum samples, no significant between-group differences in total IgE were observed. However, Der p 1-specific IgE levels were significantly higher (p<0.0001) in the allergic asthma group compared to the other two groups. Fig 1 shows the total sputum IgE levels in the three groups, with no significant differences between the groups.Fig 2 shows the specific Der p 1-specific IgE levels in sputum for each group. The allergic asthma group presented significantly higher Der p 1-specific IgE levels than both the nonallergic patients (p<0.0001) and the healthy controls (p = 0.006). There were no significant differences between the nonallergic patients and the healthy controls. In the group of patients with non-allergic asthma, a subgroup analysis was performed among those with total sputum IgE >2 or <2, with no statistically significant differences either at clinical characteristics or regarding the other IgE measurements.Table 3 shows a matrix with all significant correlations (p<0.0001) in descending order from strongest to weakest. As that table makes clear, the strongest correlation was between total IgE and Der p 1-specific IgE in serum.Using data from the whole sample (patients and controls), we calculated the correlations between total and Der p 1-specific IgE levels in sputum and serum. In the present study, we sought to test the hypothesis that the airways of patients with nonallergic asthma exhibit a local inflammatory response by determining total and dust mite-specific IgE antibody levels in induced sputum. Our results showed that patients with allergic asthma presented significantly higher total and Der p 1-specific IgE levels in serum compared to both nonallergic asthmatics and healthy controls. However, contrary to our expectations, there were no significant differences among the three groups in total sputum IgE levels. Moreover, there were no differences in Der p 1-specific sputum IgE levels between healthy controls and nonallergic asthma patients. Overall, the lack of significant differences in total sputum IgE levels between the three groups was surprising. However, diverse factors could explain this unexpected finding, as we discuss in detail below.7. Moreover, the findings from multiple studies that the anti-IgE treatment omalizumab provides symptom relief in nonatopic patients implies that an inflammatory reaction does, in fact, play a role in these patients[The hypothesis that patients with nonallergic asthma may present a local allergic response in the airways derives from the mounting evidence for a new phenotype of local allergic rhinitis in nonatopic rhinitis–15. Stud patients,9,10. Mo patients, who—in There are several reasons that could explain the discrepancy between our results and those reported by Mouthuy et al. First, the lack of any significant differences in IgE levels between the nonallergic asthmatics and healthy controls in our study could potentially be attributed to the size of the control group in our study (n = 9) versus the large control group (n = 25) in the study by Mouthy and colleagues. Another explanation could be related to the ImmunoCAP method, which may not be sufficiently sensitive to detect very small differences in IgE levels. In our sample, although the test did detect higher Der p 1-specific IgE levels in the sputum of allergic asthmatics verus both nonallergic asthmatics and healthy controls, this is probably because there were large differences in Der p 1-specific IgE levels in the allergic asthmatics versus the nonallergic asthmatics and healthy controls given that the study inclusion criteria for those latter two groups specifically required that they have a negative skin prick and negative serum Der p 1. By contrast, the differences in total IgE may have been less marked, making it more difficult to detect using the ImmunoCap technique.Local IgE production has been documented previously in nonallergic asthma patients using bronchial biopsy,29,30. IIn the present study, we used the sputum induction and analysis methods described by Araujo et al., who valData from a recent study conducted by Pillai et al. could heOverall, the findings reported in studies which use more invasive but more sensitive methods  strongly suggest the presence of elevated total IgE production in the airways of both allergic and nonallergic asthma patients,34–36. TA secondary aim of this study was to determine, on a pilot basis, the standard levels of total and Der p 1-specific IgE in the induced sputum of healthy controls. We found the following values: median (IQR) total IgE in sputum in the controls was 3.16 (1.41) KUA/L. The Der p 1-specific values were 0.06 (0.02). Evidently, the small sample (n = 9) of healthy controls are insufficient to define standard levels, but these data provide an initial estimate. Nevertheless, more data from larger studies will be needed to confirm these initial levels, especially considering the small sample and the potential limitations in the assay technique used to measure IgE in the sputum supernatant.The main limitation of the present study is the limited number of healthy controls. Another limitation may be related to the lack of significant differences between the groups in total IgE in induced sputum, which points to limitations in the measurement technique that may have influenced our findings. An important strength of the study is that this is, to our knowledge, only the second study conducted to date to specifically determine total and Der p-specific IgE in induced sputum of allergic, nonallergic, and healthy controls. Given the conflicting results of our study and those reported by Mouthuy et al., additional studies are needed. Finally, another important strength is the well-selected and well-defined sample of patients and controls; we used very strict diagnostic criteria  to define both allergic and nonallergic asthma, as well as for the healthy controls.The findings of this study show that total IgE levels measured in serum and induced sputum are significantly correlated. The significantly higher levels of Der p 1-specific IgE detected in the induced sputum of the allergic asthmatics underscores the potential value of measuring aeroallergen-specific IgE in induced sputum. Nevertheless, the lack of significant between-group differences in total sputum IgE levels suggests that the ImmunoCAP immunoassay technique used in this study may not be sufficiently sensitive to detect small differences in total sputum IgE. To support the results of this work, a larger sample size would be necessary for future studies.A growing body of evidence indicates that both allergic and nonallergic asthmatics present elevated airway inflammation. Measuring IgE levels in induced sputum is a non-invasive, cost-effective approach that could provide valuable clinical data to help individualize the treatment of nonallergic asthma. However, more sensitive methods are needed to measure IgE levels in induced sputum. Nonetheless, there exists a clear potential to use total and/or aeroallergen-specific IgE levels measured in induced sputum as a marker of treatment efficacy. 28 Nov 2019PONE-D-19-27179Total and specific immunoglobulin E in induced sputum in allergic and non-allergic asthmaPLOS ONEDear Dr. Crespo,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by Jan 12 2020 11:59PM. When you are ready to submit your revision, log on to If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.We look forward to receiving your revised manuscript.Kind regards,Aleksandra BaracAcademic EditorPLOS ONEJournal Requirements:1. When submitting your revision, we need you to address these additional requirements.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttp://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2. We noticed you have some minor occurrence(s) of overlapping text with the following previous publication(s), which needs to be addressed:https://doi.org/10.1159/000431259https://doi.org/10.2147/JAA.S142200http://www.jiaci.org/issues/vol25issue6/6.pdfIn your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the Methods section. Further consideration is dependent on these concerns being addressed.3. We note that you have reported significance probabilities of 0 in places. Since p=0 is not strictly possible, please correct this to a more appropriate limit, eg 'p<0.0001'.http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.4. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see We will update your Data Availability statement on your behalf to reflect the information you provide.5. Your ethics statement must appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please also ensure that your ethics statement is included in your manuscript, as the ethics section of your online submission will not be published alongside your manuscript.6. Thank you for stating the following in the Competing Interests section:AC. in the last three years received honoraria for speaking at sponsored meetings from AstraZeneca, Chiesi, Esteve Laboratories, Faes Farma, Ferrer, GlaxoSmithKline, Novartis, Teva, Zambon.  Received help assistance to meeting travel from Bial, Novartis. Act as a consultant for AstraZeneca, Boehringer, GlaxoSmithKline, Novartis. And received funding/grant support for research projects from a variety of Government agencies and not-for-profit foundations, as well as AstraZeneca.EC has received funding to travel to and attend training activities from ALK, Menarini, Teva, AstraZeneca, Chiesi, Boehringer, and Novartis.LS, EM and declare no conflict of interests.JG has received funding to travel and attend to training activities from Menarini, Teva, AstraZeneca, Chiesi, GSK, Mundipharma, Boehringer.In the last three years, VP has received honoraria for speaking at sponsored meetings from AstraZeneca, Boehringer-Ingelheim, Chiesi, GSK, and Novartis. VP has also received financial support to travel to meetings organized by Chiesi and Novartis. VP is a consultant for ALK, AstraZeneca, Boehringer, MundiPharma, and Sanofi. VP has also received funding/grant support for research projects from a variety of governmental agencies and not-for-profit foundations, as well as from AstraZeneca, Chiesi and Menarini.http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” . The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Dear Editor,The article entitled as “Total and specific immunoglobulin E in induced sputum in allergic and non-allergic Asthma” was reviewed for PLOS ONE on 20 November 2019. It is a valuable study in means of using and evaluating induced sputum and it has a control group. However there are the following points that needs to be solved;- The size of the study group seems to be too small, this may be increased, or the power of the study may be calculated and added.- The subject of the study that comparing total IgE between allergic and nonallergic asthma is not new even tough using induced sputum and spesicifc IgE.- Local allergy or entopy was showed in nonallergic rhinitis or asthma in mucosa biopsy or lavage in 2000’s. However, it has lost its popularity because of the difficulty to diagnose these patients, and not changing the treatment. Furthermore, it is not evaluated as an asthma phenotype. The new point may be investigating entopy between allergic, non-allergic eosinophilic, and non-allergic non-eosinophilic phenotypes.- Subgroup analysis as high and low total ıge may be performed.- There are other allergens such as pollens that may be present in nonallergic astmatics sputum. This question effects the results unconfident.- Mold sensitivity has a great impact on total IgE, and should have been explored.Reviewer #2: The detection limit was 0.35 kU/L for specific IgE and yet the authors report levels in sputum markedly below this level, e.g. for Der p-specific IgE 0.055 kU/L median and 0.04 kU/L average for non-allergic asthma. (See line 173-4 and Table 2.) Thus unfortunately, the data appear to be invalid.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentDear Editor.docxSubmitted filename: Click here for additional data file. 3 Jan 20201. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: NoAuthor’s response: According to the recommendations of the reviewer, we have better drafted the manuscript in key points to make it more understandable, especially in the conclusions (see line 41)2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: NoAuthor’s response: The statistical analysis was carried out under the support of a statistician who guided us at all times on the most appropriate analyzes for this study. If you suggest doing some analysis of greater interest in our study, using the database that we have attached to the study, we would be delighted to perform it.3. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: YesAuthor’s response: As required by the internal policy of Plos one, we have attached our data to a public repository. 4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: YesAuthor’s response: Thanks for your feedback5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Dear Editor, The article entitled as “Total and specific immunoglobulin E in induced sputum in allergic and non-allergic Asthma” was reviewed for PLOS ONE on 20 November 2019. It is a valuable study in means of using and evaluating induced sputum and it has a control group. However there are the following points that needs to be solved; - The size of the study group seems to be too small, this may be increased, or the power of the study may be calculated and added.Author’s response: Due to the limited literature and great variability in the results that exist of the IgE values in the induced sputum, for the calculation of the sample size, the reference values of the IgE in the induced sputum will be considered as binding variable reports in literature (references 31 and 32). This sample size is calculated with the Granmo V7.10 program, setting the type I error at the usual 5% (α = 0.05), with a bilateral approximation and with a minimum difference required for a power of 80% or higher (ß = 0.2). Also our study had as a secondary objective to establish a preliminary estimate of total IgE and Der p 1-specific IgE in the induced sputum of healthy individuals. To support the results of this work, a larger sample size would be necessary for future studies. A comment has been added in the manuscript (see line 327) - The subject of the study that comparing total IgE between allergic and nonallergic asthma is not new even tough using induced sputum and specific IgE.Author’s response: The levels of total and specific IgE in blood are extensively studied comparatively in asthmatic and healthy patients, and also in different types of asthmatic patients. However, sputum-induced levels of this immunoglobulin have been poorly reported. Our particular interest was to assess its potential applicability as a diagnostic test in the detection of local allergic reactions at the bronchial level. - Local allergy or entopy was showed in nonallergic rhinitis or asthma in mucosa biopsy or lavage in 2000’s. However, it has lost its popularity because of the difficulty to diagnose these patients, and not changing the treatment. Furthermore, it is not evaluated as an asthma phenotype. The new point may be investigating entopy between allergic, non-allergic eosinophilic, and non-allergic non-eosinophilic phenotypes.Author’s response: We totally agree with the reviewer's opinion. Local allergy has been a controversial issue in the last decade, and in our view further research is needed to define this entity properly. Its loss of popularity has been due, on the one hand, to diagnostic difficulty, so this study has been, in fact, a first approach to a technique that could allow diagnosing it in a simple way. On the other hand, until now it did not imply differences in treatment, but we believe that with the availability of new monoclonal drugs this may change. We also consider that it could help to better understand a group of patients, who because a priori they do not present signs of atopy or eosinophilia, remain limited to conventional treatments. - Subgroup analysis as high and low total IgE may be performed.Author’s response: The main focus of our work was:1) To measure the levels of total and Dermatophagoides pteronyssinus (d1) specific IgE in the induced sputum (IS) of asthmatic patients and healthy volunteers2) To correlate the levels of total local IgE and specific IgE to d1  in patients with allergic and non-allergic asthmaSince there are few published papers in the literature that have measured levels of total IgE in sputum and since there are no established values of normal sputum total IgE levels, we cannot make this classification as there is no established cut-off point.- There are other allergens such as pollens that may be present in nonallergic asthmatics sputum. This question effects the results unconfident.Author’s response: Our inclusion criteria were particularly strict and detailed in this study. We only selected patients with allergic asthma in whom D. pteronyssinus was the only clinically relevant allergen, in addition to presenting positive prick test, and/or previous high specific IgE for this allergen. If they presented a positive prick test for other allergens and there was any suspicion that their symptoms were related to other allergens to which they were sensitized, they were excluded from the study. This has been clarified in the manuscript (see line 140) - Mold sensitivity has a great impact on total IgE, and should have been explored.Author’s response: This is an interesting point of view, but in our area, dust mites are the most common perennial allergens, and mold sensitization is not particularly common. In fact, in this group of patients, only one of them had a positive prick test for Aspergillus, and it was not clinically relevant (see line 219)Reviewer #2: The detection limit was 0.35 kU/L for specific IgE and yet the authors report levels in sputum markedly below this level, e.g. for Der p-specific IgE 0.055 kU/L median and 0.04 kU/L average for non-allergic asthma. (See line 173-4 and Table 2.) Thus unfortunately, the data appear to be invalid.Author’s response: According to manufacturer’s recommendation, > 2kU/L for total IgE and > 0.35kU/L for specific IgE, are the values to consider a positive test, but as we previously stated, the levels in induced sputum are not described yet. We understand that the previous phrasing could be confusing, so we have modified the corresponding paragraph to better understand this aspect.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 7 Jan 2020Total and specific immunoglobulin E in induced sputum in allergic and non-allergic asthmaPONE-D-19-27179R1Dear Dr. Crespo-Lessman,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Aleksandra BaracAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 8 Jan 2020PONE-D-19-27179R1 Total and specific immunoglobulin E in induced sputum in allergic and non-allergic asthma Dear Dr. Crespo-Lessman:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Aleksandra Barac Academic EditorPLOS ONE


Biologists have long been fascinated by the striking diversity of complex color patterns in tropical reef fishes. However, the origins and evolution of this diversity are still poorly understood. Disentangling the evolution of simple color patterns offers the opportunity to dissect both ultimate and proximate causes underlying color diversity.Amphiprion frenatus) have supplementary stripes when compared to their respective adults. These stripes disappear caudo-rostrally during the juvenile phase leading to the definitive color pattern. Remarkably, the reduction of stripe number over ontogeny matches the sequences of stripe losses during evolution, showing that color pattern diversification among clownfish lineages results from changes in developmental processes. Finally, we reveal that the diversity of striped patterns plays a key role for species recognition.Here, we study clownfishes, a tribe of 30 species within the Pomacentridae that displays a relatively simple color pattern made of zero to three vertical white stripes on a dark body background. Mapping the number of white stripes on the evolutionary tree of clownfishes reveals that their color pattern diversification results from successive caudal to rostral losses of stripes. Moreover, we demonstrate that stripes always appear with a rostral to caudal stereotyped sequence during larval to juvenile transition. Drug treatments (TAE 684) during this period leads to a dose-dependent loss of stripes, demonstrating that white stripes are made of iridophores and that these cells initiate the stripe formation. Surprisingly, juveniles of several species  contains supplementary material, which is available to authorized users. Understanding the diversification of phenotypes requires to integrate developmental and evolutionary analysis in an ecological context . Having Pomacentrus amboinensis have been suggested to serve as a subordinate signal directed to dominant males [Pigmentation, in particular color patterns, provides an incredible number of cases that allow the exploration of the interplay between ecology, evolution, and development that are at the basis of trait diversification –6. Amongnt males . To datent males . Howevernt males , 16.Chrysiptera leucopoma may retain its juvenile color  or shift to the adult phenotype (a dark brown body) depending on habitat type and/or population densities [It is now well known that phenotypic diversification between lineages may be achieved by changes in developmental processes , 17. Theensities . HoweverAmphiprion and the monotypic Premnas) are iconic coral reef fishes [Amphiprion offers a good opportunity to better delineate the patterns and processes allowing the diversification of such ornamental diversity. The clownfish evolutionary radiation has recently received much attention, providing a suitable phylogenetic framework for testing new evolutionary hypotheses on the rise of color diversity in coral reef fishes [Clownfishes (f fishes . This trf fishes ) within f fishes . Their lf fishes , followef fishes . The funf fishes , 25. Thef fishes .Amphiprion. We first map their occurrence and pattern on the clownfish evolutionary tree and reconstruct the ancestral state in terms of white stripe presence/absence. Our results provide evidences that the diversification of clownfish color pattern results from successive caudal to rostral losses of stripes during evolution. Using specific drugs , we reveal that the white stripes are formed by iridophores and are essential for the patterning of the neighboring black stripes. Then, by an ontogenetic approach, we show that either the juvenile has the same number of stripes than adults or the juvenile has supplementary stripes that disappear caudo-rostrally later on. The reduction of stripe number over ontogeny totally matches the sequences of stripe losses across evolution, demonstrating that the diversification in color pattern among clownfish lineages results from changes in developmental processes. Finally, we determine the links between the number of stripes and other external morphological traits, and we provide some evidence that the various striped patterns have evolved for species recognition. This approach allows us to consider the relationships among striped patterns, fish morphology, and their ecology to suggest that both developmental and ecological processes have shaped the diversity of color patterns in clownfishes.In this study, we focus on the vertical white stripes present in most species of Clownfishes can be classified into four categories according to their striped pattern at the adult stage: species without vertical stripe (group A) or species having one white vertical stripe (on the head—group B), two vertical stripes (on the head and the trunk—group C), or three vertical stripes  Fig. . White sAmphiprion chrysogaster—Fig. To understand the evolution of color pattern in clownfishes, we performed a stochastic mapping of striped patterns on the most complete time-calibrated phylogeny of Amphiprionini . The anaThis evolutionary analysis therefore highlights that the diversification of white stripe pattern in clownfishes is a history of loss from an ancestral lineage having three stripes and that these losses always occurred in a progressive and sequential fashion from caudal to rostral regions. For example, in all two striped species, the peduncle stripe has been lost and the head and the trunk stripes are retained. All one-stripe species retained the head stripe and have lost the peduncle and trunk stripes.Amphiprion ocellaris and Amphiprion frenatus that display three stripes  or a single head stripe at the adult stage, respectively.The fact that stripes always appear at the same location in clownfishes and that the losses of stripes during evolution occurred in a sequential manner from caudal to rostral suggests that this loss may be constrained by a developmental mechanism. We thus tested whether a variation in the number of stripes could occur during clownfish ontogeny by studying the development of A. ocellaris larvae acquire the head and trunk stripes simultaneously , larvae of both species do not harbor any vertical stripe  an evolutionary pattern of stripe loss, with a caudo-rostral progression that is observed in adult fish, and (ii) a developmental pattern of rostro to caudal stripe gain during the larval to juvenile transition exemplified in A. ocellaris, we observed at least three types of cells: xanthophores, melanophores, and white cells , known as an inhibitor of leukocyte tyrosine kinase (Ltk) and anaplastic lymphoma kinase (Alk) and leading to a decrease of iridophore number in zebrafish [A. ocellaris larvae  induced a dose-response effect into the formation of white stripes  are described according to their ultrastructure and their pigment type . Xanthoplls Fig. . From julls Fig. . To deteebrafish , could dpes Fig. –d. Whilepes Fig. , fish trpes Fig. , e. Fishpes Fig. , e and 5pes Fig. , e. Simipes Fig. –c, thosepes Fig. . TogetheA. ocellaris larvae allow the understanding of how iridophores contribute to color pattern development. White vertical stripes are surrounded by a thin stripe of melanophores over an orange body. Interestingly, we observe that when no white vertical stripe is formed over the body  one in A. chrysopterus, (iii) one in A. latifasciatus, (iv) one in A. allardi, and (v) one in the clade grouping A. mccullochi and A. akindynos  and body elongation  whereas the co-evolution of posterior lobe morphology and the number of white stripes is weaker . Overall, these data provide evidence that the distribution of striped patterns is not random in clownfish communities, suggesting that stripes help for species recognition.“Species recognition” refers to the behavior whereby individuals identify and keep track of conspecifics for group coherence or identify a suitable sexual partner . AccordiOur study reveals an unexpected link between the variation in the number of stripes occurring during ontogeny and the same type of variation occurring during evolution of clownfishes. Strikingly, the sequence of events observed during evolution mirror the one seen during ontogeny. An identical sequence of white stripe loss than the one observed during evolution was observed during late juvenile stages. These observations strongly suggest that the diversification of stripe patterns observed during clownfish radiation is the product of modifications of an ancestral stereotyped ontogenetic trajectory. From a mechanistic point of view, such a successive gain of stripes from the head to the caudal region during ontogeny support the hypothesis that loss of stripes during evolution is likely constrained by ontogenesis.A. ocellaris and A. frenatus, is remarkable. This highly suggests a conserved mechanism of color pattern ontogeny across clownfishes. Here, we provide a first analysis into the mechanistic underpinnings of the patterns seen. In a first part, using an inhibitor of two receptor tyrosine kinases (Ltk and Alk) that are instrumental in iridophore formations in zebrafish, we show that the white coloration of stripes is produced by iridophores and not leucophores. After TAE treatments, we show that stripes are sometimes absent, incomplete, and/or less white. Disrupting the white stripe formation reveals that the presence of iridophores is instrumental for the distribution of the two other chromatophore types. In TAE-treated fish, xanthophores and melanophores are scattered on the flank and not properly organized as in wild type. This suggests that, as in zebrafish [Pomacanthus imperator [A. frenatus and A. chrysopterus) does not fit with Turing predictions and thus allows a clear refutation of the “Turing pattern hypothesis.” This suggests that, in clownfishes, when and where the stripes must be formed are controlled by specific patterning mechanisms that remain to be analyzed.The sequential appearance of white stripes from the anterior to the posterior region during the development of two distantly related species, ebrafish , cell-ceebrafish  as in zeebrafish ). Howeveebrafish . This moebrafish . Such Tuebrafish  and durimperator . The casVarious clownfish species exhibit extra stripes during the juvenile phase when compared to the adult stage, and this convergent loss of stripes occurred at least five times across the evolution of clownfishes Fig. . Such a The correlated evolution between the numbers of vertical white stripes and dorsal fin shape suggests that both phenotypic traits may depend on the same or linked developmental processes. An ecomorphological interpretation of these results would be that patterns of stripes are somehow linked to differences in macro-habitats, but this hypothesis certainly needs further analyses. On the other hand, the fact that the white stripe is present mostly in species with an indentation in the dorsal fin may suggest that it may be linked to a disruptive effect , the banBeyond ecological adaptation, we provide evidence that the striped patterns play a role for species recognition in clownfishes. Indeed, the number of species pairs showing the same striped pattern is exceptionally low in natural communities compared with random ones. Non-significant results from Keppels and Komodo Table  are probOur study highlights a strong link between the diversification of pigmentation pattern in clownfish and the developmental trajectories underlying white stripe formation. This sets up exciting perspectives for the study of color pattern diversity in coral reef fishes, for which an Evo/Devo approach was clearly underused until now. As suggested by our integrated study of clownfishes, the diversification of color patterns results from interplay among developmental, ecological, and behavioral processes.A. akallopisos, A. sandaracinos, A. perideraion, and A. pacificus) show an atypical pattern with a white line browsing the dorsal region [A. clarkii, A. melanopus, and A. polymnus) are polymorphic with two different patterns observed in natural populations  for R [ 0.5.38; . We thenkage ape . The likq14 = q41), (iii) a model in which the loss or the acquisition of two stripes by evolutionary color shift is not allowed , and (iv) a model combining the constrains of model (ii) and model (iii). In order to reduce the number of parameters, every model assumed that the speciation rates (λ) and the extinction rates (μ) are equal among stripe morphs. Moreover, we corrected for incompletely resolved phylogeny without specifying the state of missing species and we assumed that the missing species are randomly distributed on the phylogenetic tree. The fit of models was compared using sample-size-corrected Akaike’s Information Criterion (AICc) scores and weights [To test whether the evolution of striped patterns is not random in clownfishes, we compared transition rates between stripe morphs using the “multiple state speciation extinction” (MuSSE) method. MuSSE is an extension of the BiSSE maximum likelihood-based test, which is described in , 29. To  weights . A ΔAICc weights .Amphiprion sp. individuals found in Fishbase [http://pbs.bishopmuseum.org/images/JER/) and J. Williams. Others were studied in the marine vertebrate collections of S. Planes (CRIOBE) and Museum of Natural National History (MNHN). This analysis includes 22 species over the 30 described species , the length of the most posterior spine (l2), the length of the longest soft ray (lr), and the length of the dorsal fin (L). Anterior and posterior lobe morphology indexes correspond to (l1-l2)/L and (lr-l2)/L, respectively . VariatA. ocellaris and A. frenatus were maintained at 26 °C in separate 60-L aquaria. Breeding pairs laid egg clutches on the underside of a terracotta pot placed in their aquarium. On the night of hatching , egg clutches were transferred from the parental aquarium to a 30-L larval rearing aquarium. Larvae were fed rotifers (Brachionus plicatilis) at 10 individuals per milliliter three times a day for the first 7 days. The ratio of Artemia nauplii to rotifers was increased each day until larvae were fed only five individuals of Artemia nauplii per milliliter from day 7. From day 7 until day 20, larvae are euthanized or anesthetized in MS222 at 200 mg/L and 100 mg/L, respectively, in filtered aquarium water and photographed under a stereomicroscope. At least three larvae per species per day of development are studied.TAE684 (NVP-TAE684) , a specific inhibitor of Ltk and Alk , was dilAdditional file 1:Table S1. Dataset list of studied species. Dataset list of studied species, with relative coding based on their color pattern [http://pbs.bishopmuseum.org/images/JER/), or nicely provided by J. Williams. Others were studied in the marine vertebrate collections of S. Planes (CRIOBE) and the Museum of Natural National History (MNHN). Excel document 19 ko. (XLSX 18 kb) pattern , color p pattern –72, and  pattern  and withAdditional file 2:Figure S1. Successive caudo-rostral loss of stripes during evolution is independent of clownfish color polymorphism. Phylogenetic trees of clownfishes from Litsios et al. (2014) [. (2014)  with a sAdditional file 3:Table S2. Summary statistics of the stochastic mapping. Summary statistics of the stochastic mapping , assuming that all transition rates (q) among stripe morphs are free to vary. Morphs are coded as followed: species without vertical stripe (A), species having one white vertical stripe on the head (B), species having two white vertical stripes (head and trunk) (C), species having three white vertical stripes  (D). Results are provided for every combination of coding. Word document 13 ko. (DOCX 12 kb)Additional file 4:Table S3. Model fitting of the four striped pattern evolutionary models. Model fitting of the four striped pattern evolutionary models, using the different coding of color patterns detailed in Additional file Additional file 5:Table S4. Relationship between the number of vertical white stripes and eco-morphological variables. Results from PGLS analyses. X and Y refer to the variables in the linear regression model. See Additional file Additional file 6:Figure S2. Lobe morphology index of dorsal fin of clownfish species. Graphs representing the quantifications of posterior (a-b) and anterior (c-d) lobe morphology index in each species. In each graph, we presented the mean from the smallest to the biggest for each stripe morph. (a-c) Graphs represent the mean of posterior (a) and anterior (b) lobe morphology index (dots). Error bars indicate the standard deviation. (b-d) Boxplots of the mean of posterior (c) and anterior (d) lobe morphology index. Boxes extend from the 25th to 75th percentiles, whereas whiskers go down to the smallest value and up to the largest. Median data are indicated by horizontal line within each box. PDF document 67 ko. (PDF 66 kb)


Fourteen well-defined ribozyme classes have been identified to date, among which nine are site-specific self-cleaving ribozymes. Very recently, small self-cleaving ribozymes have attracted renewed interest in their structure, biochemistry, and biological function since the discovery, during the last three years, of four novel ribozymes, termed twister, twister sister, pistol, and hatchet. In this review, we mainly address the structure, biochemistry, and catalytic mechanism of the novel ribozymes. They are characterized by distinct active site architectures and divergent, but similar, biochemical properties. The cleavage activities of the ribozymes are highly dependent upon divalent cations, pH, and base-specific mutations, which can cause changes in the nucleotide arrangement and/or electrostatic potential around the cleavage site. It is most likely that a guanine and adenine in close proximity of the cleavage site are involved in general acid-base catalysis. In addition, metal ions appear to play a structural rather than catalytic role although some of their crystal structures have shown a direct metal ion coordination to a non-bridging phosphate oxygen at the cleavage site. Collectively, the structural and biochemical data of the four newest ribozymes could contribute to advance our mechanistic understanding of how self-cleaving ribozymes accomplish their efficient site-specific RNA cleavages. The N2-reaction mechanism for RNA phosphodiester backbone cleavage. There is general agreement that ribozymes employ an internal phosphoester transfer mechanism in which the 2′ oxygen nucleophile of a nucleotide at position +1 attacks its adjacent phosphorus atom, accompanied by the protonation and departure of the 5′ oxygen of a nucleotide at position −1  [The theoretical rate limit (approximately two per minute) for a nuclease employing the α and γ catalysis is much smaller than the rate constant (approximately 1000 per minute) of twister ribozymes . Therefontation) . The sol2+ ion concentrations: their maximum apparent rate constants are approximately 5 min−1 and 1000 min−1, respectively. The pH profile and maximum rate constant for TS-1 are highly similar to those of artificial ribozymes and deoxyribozymes that might employ only two catalytic strategies (α and γ). However, two additional constructs, called TS-3 and TS-4, exhibited much higher rate constants under suboptimal pH and Mg2+ ion concentrations; TS-3 is predicted to have a rate constant of >100 min−1 under optimal reaction conditions. Taken together, the large gap between the rate constants for TS-1 and TS-3 can cause controversy about the general catalytic strategy used by twister sister ribozymes.Twister sister ribozymes can be autonomously cleaved at nucleotides C13 and A14 on the loop connecting P1 and P2 . This clLysinibacillus sphaericus was assessed [2 showed that the rate constant increases as the pH increases: a gradient of approximately 2 between pH 5.0 and 6.25, and a gradient of approximately 1 above pH 6.25. Based on the profiles, it could be predicted that the apparent rate constant is >10 min−1 under physiological conditions (1 mM Mg2+ and pH 7.5) and >100 min−1 under optimal reaction conditions (Mg2+ concentration above 50 mM and pH 7.5–9.0) [−1) that has been shown for enzymes that exclusively exploit the α and γ catalytic strategies mentioned above, suggesting that pistol ribozyme employs additional β and/or δ strategies to enhance cleavage activity.The pH dependence on the cleavage rate of a pistol ribozyme from assessed . The pH-The cleavage site of the hatchet ribozyme was found to be positioned on C-U nucleotides at the base of P1 by analysing the mass peaks for the cleavage products including a 5′ product with a terminal 2′,3′-cyclic phosphate group and a 3′ product with a 5′-hydroxyl group . This reKa close to neutrality, which is shown for histidine residue of protein. This functional group can be directly involved in acid- and base-catalyzed cleavage reactions at the active site. In this regard, an early assumption was that most ribozymes in nature act as obligate metalloenzymes [glmS ribozyme, which is also a riboswitch, uses the amine of a bound glucosamine-6-phosphate ligand as a general acid [2+) in catalysis are also found in other ribozymes such as self-splicing group I and II introns [Ribozymes appear to have a remarkable disadvantage in their ability to catalyze cleavage reactions when compared to protein nucleases, because RNA is deficient of functional group that has a poenzymes . Howeveroenzymes ,42,43,44oenzymes ,46. Furtoenzymes ,42,45,47ral acid ,26,48. Mral acid ,50,51. Tral acid ,53. The  introns ,56,57,58 introns . It is gThe mechanistic investigations into nucleolytic ribozymes must necessarily be supported by their extensive biochemical data. To conduct in vitro ribozyme activity assays under variable conditions, a ribozyme can be divided into two RNA strands, one of which serves as the substrate that carries the cleavage site, and the other as the enzyme that carries the catalytic RNA sequence and necessary structural elements. In particular, these constructs can be used for studying the effects of pH, divalent metal ions, and specific atomic mutations on the cleavage activity of ribozymes. The use of wild-type self-cleavage ribozyme has been limited owing to significant self-cleavage during large-scale ribozyme production that used in vitro transcription.2+ ion concentrations (50 µM), and rate constants plateaued above pH 6.5. If the two factors are independent of each other, the rate constant for this twister ribozyme in physiological conditions  would be predicted to approach approximately 1000 per minute, which is the fastest cleavage rate of those of the four recently identified ribozymes. Furthermore, significant reductions in ribozyme activity were shown for various twister mutations of highly conserved nucleotides and base-pairing nucleotides in pseudoknots and stems P1, P2, and P4. Another P3-type twister ribozyme derived from the ES ribozyme also exhibited pH-dependence on cleavage activity [Ka values of 6.9 and 9.5. The simplest explanation for this profile is that two ionizations of the ribozyme are related to the transfer of two protons in the transition state of a concerted, general acid-base catalysis. However, reliable interpretation of such profiles can be hampered by coupling of catalytic proton transfer events with a conformational change and acid and alkaline denaturation in a variable pH [O. sativa twister suggest that the higher pKa is derived primarily from residue G45, and G45 N1, which is close to the U6 O2′, acts as general base for activation/deprotonation of the nucleophile for attack at the phosphorus center during the cleavage reaction  [O. sativa, env22, and env9) suggest a crucial catalytic role of the same conserved guanines (N1) as general base , although they adopt slightly different conformations of the active site. However, the guanine N1 is expected to be fully protonated at neutral pH with a pKa of around 10, leading to uncertainties about the catalytic role of the guanines in the proximity of the 2′-OH nucleophile. It remains possible that an energetically unfavorable enol tautomer of the guanine is transiently formed and allows the N1 to accept a proton. For the O. sativa and env22 twisters, the guanine could also be involved in neutralization of the negative charge of a non-bridging phosphate oxygen at the cleavage site. The two catalytic roles of the same guanine are also found in G8 and G638 in the hairpin [A P3 type of the twister ribozyme, derived from an environmental DNA sequence, was used for evaluating pH-dependent activities . The resactivity . Notablyiable pH , which ctalysis) ,30. It i hairpin ,68,69 an hairpin ,70,71, rKa ~6.5) is achieved by the N3 group of the adenine located immediately 3′ of the cleavage site [Ka values of 3.63, 2.15, and 1.5, respectively [Ka of its N3 group toward neutrality, which is accompanied by increased fractional protonation of the N3. Indeed, pH-dependent NMR experiments with a 13C2-A6 labeled, non-cleavable substrate determined the pKa value of the adenine as 5.1, which shows a shift of 1.4 from the pKa value of the substrate strand alone [Ka is the exocyclic N6 of the adenine that forms hydrogen bonds with the non-bridging oxygen atoms of two successive phosphates (C25 and C26), which could stabilize the protonation state of the adenosine N3. This possibility was supported by the significantly reduced activities caused by the replacement of the adenine with guanine or 2-aminopurine, both of which do not have the exocyclic N6 [Ka of active site functional groups can be caused by metal ion-mediated electrostatic modulation, as demonstrated by the pH-activity profile of the VS ribozyme showing differential modulation of nucleobase pKa by different cations [Ka of serine and cysteine residues is lowered by its neighboring positive charges [In addition, there is evidence suggesting that the second ionization event  spectra of the non-cleavable pistol with 13C2-labelled A32 obtained under varying pH conditions indicated that the pKa value of the A32 N3 atom is approximately 4.7 [Ka shifts by one pH unit toward neutrality, compared to the reference pKa of adenine in the duplex and single-strand contexts. The shifted pKa increases a chance of the protonation of the A32 N3, which could donate a proton to the 5′-oxygen leaving group at the cleavage site.The crystal structures of pistol ribozymes have proposed that the A32 N3 (or G32 N3) and G40 N1 atoms are properly located to serve as a general acid and base for cleavage activity, respectively. This was well consistent with the mutagenesis studies from the env25 pistol ribozyme that single mutations of the conserved residues  that are adjacent to the G53-U54 cleavage site caused considerably reduced cleavage activities, compared to the wild-type control . The restely 4.7 . The pKa2+ ion concentration of 10 mM as the pH value increases up to 7.5, above which ribozyme activity plateaus. Notably, the pH-activity profile is different from the bell-shape profile of the twister ribozyme that indicates two apparent pKa values, as described above. The simplest explanation is that the key deprotonatable group of the hatchet ribozyme is the 2′ oxygen nucleophile at the cleavage site that have a pKa near 7, which might be shifted from its normal pKa value of ~13.7 by unique folding of the active site. The shifted pKa would activate the 2′-hydroxyl nucleophile, rendering it almost fully deprotonanted at neutral pH. This process is related to the γ catalytic strategy.The hatchet activity increases at a constant MgKa value of a water molecule is lowered from ~16.0 in a free state to ~11.4 in a Mg2+ ion-bound state, which facilitates a proton transfer of the water molecule [2+ ion is one of the most common cofactors for cleavage reactions of nucleases, because of its abundance, solubility, redox stability, small size and stringent coordination geometry, compared with those of other divalent cations [It has been suggested that divalent metal ions serve as cofactors to promote ribozyme activities, possibly due to their abilities to compensate for the limited chemical versatility of ribonucleotide functional groups. In particular, it is possible for metal ions to neutralize the abundant negative charge of the phosphodiester backbone of ribozymes at near neutral pH, and this counterbalance could enable ribozymes to fold into their functional compact conformation . The elemolecule . Mg2+ io cations ,80.2+ ion, displaying an increased ribozyme activity as Mg2+ ion concentration increased up to 1 mM, at pH 5.5. Furthermore, several other divalent metal ions also induced RNA cleavages to a similar extent as that of Mg2+ ion, indicating a low selectivity of metal ions. This observation could support the idea that Mg2+ ion plays a structural rather than catalytic role in the cleavage activity, which is consistent with the crystal structure of the O. sativa twister that is devoid of metal ions at the cleavage site. Real-time fluorescence experiments using fluorescent 2-aminopurine (AP) to replace U3 in stem P1 were carried out to obtain kinetic profiles for cleavage activity of the env22 twister [2+ ion, suggesting that it promotes instantaneous, catalytically active folding of the ribozyme or directly participates in cleavage reaction. The latter corresponds well with the crystal structure of the env22 twister wherein a Mg2+ ion is directly bound to the scissile phosphate backbone of RNA. However, there has been no clear biochemical data that support the direct participation of Mg2+ ion in catalytic processes when compared to the data suggesting an indirect structural role of Mg2+ ion in catalysis.A P3 type of the twister ribozyme, derived from an environmental DNA sequence, displayed metal ion-dependent activities . Cleavag twister . In pringlmS and twister ribozymes but not twister sister and hatchet ribozymes [2+ ion has a relatively large propensity for outer-sphere interactions due to its small ionic radius and high charge density with a large hydration energy, compared to other divalent ions such as Ca2+, Mn2+, and Zn2+ [2+ ion-bound, well-ordered water molecules in ribozyme activity [The binding mode of divalent metal ions to ribozymes can be probed by using cobalt hexammine, which is highly analogous to fully hydrated magnesium ion in terms of size and hydrogen bonding potential ,82. If aibozymes ,11,83. Tibozymes . Types oand Zn2+ . This faactivity .2+ ion commonly binds to oxygen approximately 30,000-fold more strongly than to sulphur, whereas Mn2+ binds to the two atoms with an equal affinity [2+ ion and a non-bridging oxygen at the active site, the Mg2+ ion would cause a substantial loss of cleavage activity for a phosphorothioate substrate. The phosphorothioate substrates were used for cleavage assays of twister ribozyme in the presence of Mg2+ or Mn2+ ions [pro-R and pro-S, at the cleavage site. The ribozyme activity is considerably decreased when the pro-R non-bridging scissile phosphate oxygen at the cleavage site is replaced with sulphur, but this reduced activity is not effectively rescued by thiophilic metal ions such as Mn2+ or Cd2+ [pro-R oxygen, but this contact does not involve inner-sphere coordination of a divalent metal ion. Indeed, the structural models of the active site have proposed that the pro-R oxygen forms a direct hydrogen bond either with the highly conserved guanine located at the bottom of stem P2 in the env22 and O. sativa twisters [env9 twister [pro-R oxygen and the exocyclic N2 of the guanine is the most important component to accelerate β catalysis [pro-R phosphorothioate substrate is most likely due to the impairment of the proper positioning of the exocyclic N2 with regard to the scissile phosphate. Turning the attention to the pro-S oxygen, the phosphorothioate substrate that replaces the pro-S oxygen with sulphur was used to examine the effect of the substitution on the ribozyme activity in the presence of Mg2+ ion [pro-S oxygen does not serve as a ligand for Mg2+ ion. However, this finding is not in agreement with two crystal structures of the env22 twister ribozyme in which a Mg2+ ion forms an inner-sphere interaction with the pro-S oxygen at the cleavage site [2+ ion to bind to pro-S oxygen at the cleavage site.Another approach for examining the nature of a metal binding site is to use a comparison of reactivities of substrates that have either a phosphate group or a phosphorothioate group at the cleavage site; the latter has a sulphur atom substituted for one of the non-bridging oxygen atoms. This experimental design is based on the fact that Mgaffinity ,87. Ther or Cd2+ ,72. Thestwisters ,42,44,72 twister . SpecifiMg2+ ion ,72. As aage site ,31. To a2+ ions, which have the potential to affect the overall conformation of the ribozyme or be coordinated with its catalytic site. In particular, it shows different cleavage activities in the presence of Ni2+ and Sr2+ ions, compared to twister ribozyme; twister sister prefers Ni2+ rather than Sr2+ as a cofactor for cleavage activity, whereas twister shows the opposite preference for the two ions. Among five Group 1 monovalent cations  tested, Li+ ion induces the largest cleavage activity, which is similar to that of the twister ribozyme. The twister sister ribozyme can also employ divalent metal ions as cofactors for its nucleolytic activity, which is similar to many other self-cleaving ribozymes . The cleL. sphaericus were examined at different Mg2+ ion concentrations [2+ ion and a near constant cleavage rate at Mg2+ ion concentrations above ~50 mM. It could be deduced from the profile obtained at a concentration above 1 mM that pistol ribozyme can be almost fully activated at a physiological concentration of Mg2+ ion (>1 mM). Eight other divalent cations were also used as cofactors for pistol cleavage assays. Of these, six cations, Mn2+, Ca2+, Co2+, Ni2+, Cd2+, and Ba2+, support the cleavage activity, suggesting that pistol activity non-specifically requires divalent cations. Two other cations, Zn2+ and Cu2+, do not promote the cleavage activity. In assays with monovalent cations, modest nucleolytic activities of pistol were shown in the presence of Na+ and Li+ ions, which have small ionic radii. Three monovalent ions, K+, Cs+, and Rb+, having larger ionic radii could not induce ribozyme activity. In addition, cleavage assays using phosphorothioate substitution at the non-bridging oxygen of the scissile phosphate showed that Mn2+ ion cannot rescued the reduced activity caused by the substitution, suggesting that a metal ion does not make inner-sphere contact with the non-bridging phosphate oxygen [Self-cleavage activities of a pistol ribozyme from trations . The mete oxygen . This ise oxygen ,35. Coll2+ ions up to approximately 10 mM, above which ribozyme activity plateaus [2+ ion might be due to one or a combination of structural contribution and direct participation in catalysis. Furthermore, the ribozyme activity is promoted with different levels of efficiency in the presence of some other divalent metal ions such as Mn2+, Co2+, Zn2+, and Cd2+, but is inhibited in the presence of the three metal ions, Ba2+, Ni2+, and Cu2+. The inhibitory effects could be explained by the displacement of Mg2+ ions or by distortion of cleavage-competent RNA folding. Compared to twister, twister sister, and pistol ribozymes, hatchet ribozyme appears relatively restrictive in the use of the divalent cations to enhance cleavage activity, suggesting that the hatchet ribozyme has specialized sites for metal binding by tertiary RNA folding, which could accommodate a limited number of divalent cations. The enzyme strand of the hatchet ribozyme efficiently cleaves the normal substrate in the presence of either Mg2+ or Mn2+ ions, for which rate constants are 1.5 min−1 and 4.6 min−1, respectively [2+ ion cannot rescue a decreased ribozyme activity for a phosphorothioate substrate. This is possibly due to a lack of direct, inner-sphere metal contact to a non-bridging phosphate oxygen at the cleavage site. The self-cleavage activity of hatchet was also shown to rapidly increase at pH 7.5 in the presence of increasing concentrations of Mgplateaus . The larectively . HoweverOver the last three years, four small self-cleaving ribozyme classes recently discovered has sparked much of the renewed interest in the structure, biochemistry, and catalytic mechanism of self-cleaving ribozymes, and related papers are already beginning to be published. Moreover, novel catalytic RNA motifs are still expected to be discovered by the ongoing development of high-throughput bioinformatics tools. The four brand new ribozymes, named twister, twister sister, pistol, and hatchet, show distinct active site architectures and divergent, but similar, biochemical properties. However, there have been some arguments about the mechanistic details underlying the self-cleavage activity of the four ribozymes although their structural and biochemical data have accumulated. This is primarily due to slight, but significant, structural differences in the active sites and to the absence of high-resolution tertiary structures. The former could be explained in terms of the spatial arrangement of key residues and the presence/absence of a divalent metal ion near the cleavage site. These differences are found in the crystal structures of the twister  and pistol ribozymes (PDB codes: 5KTJ and 5K7C), respectively. The active site structures provide the information on the orientation of the 2′-oxygen nucleophile, phosphorus electrophile, and 5′-oxygen leaving group at the cleavage site. The appropriate in-line orientations responsible for α catalysis are adopted by the two pistol structures and only one of the four twister structures. Notably, the structural inconsistency of the twister and pistol ribozymes provokes thought on its plausible causes such as the influences of different P1 stem base-parings, inner-sphere metal binding, crystal contacts, and non-attacking 2′-H substitution on the active site conformation. For the twister sister and hatchet ribozymes, only the consensus sequence and secondary structure model are known ,11. TherglmS, and among these ribozymes, hairpin and VS use adenine as a general acid (2+ ion in ribozyme activity because the crystal structures of the same ribozyme have shown different coordinates of a metal ion in the active site. Two of the four twister structures show a direct binding of a divalent cation to a non-bridging phosphate oxygen at the cleavage site, as does one of the two pistol structures. Despite these structural discrepancies, comprehensive structural and biochemical data on the four novel ribozyme classes could support the idea that they use divalent cations, especially Mg2+ ion, to participate in structural integrity rather than being directly involved in catalysis. Among other self-cleaving ribozymes, only HDV ribozyme uses a Mg2+ ion as a direct contributor to β and γ catalysis [A combination of pH-dependent activity profiles, atomic mutagenesis data, and active site structures of the twister and pistol ribozymes can provide their mechanistic insights into general acid-base catalysis, possibly involving an adenine and guanine in close proximity of the cleavage site, as a general acid and base, respectively. Notably, the use of guanine as a general base is well conserved in other self-cleaving ribozymes such as hammerhead, hairpin, VS, and ral acid . In addiatalysis ,61. Furt