Cod muscle has a balanced protein profile that contains potentially bioactive amino acid sequences. However, there is limited information on release of these peptides from the parent proteins and their ability to modulate mammalian blood pressure.in vitro inhibitory effects against angiotensin-converting enzyme (ACE) and renin. The most active peptides were then tested for systolic blood pressure (SBP)-reducing ability in spontaneously hypertensive rats (SHRs).The aim of this study was to generate cod antihypertensive peptides with potent in vitro ACE and renin activities. CPH and the most active RP-HPLC fraction (CF3) were then used for enzyme inhibition kinetics assays followed by oral administration  to SHRs and SBP measurements within 24 h.Cod protein hydrolysate (CPH) was produced by subjecting the muscle proteins to proteolysis first by pepsin and followed by trypsin+chymotrypsin combination. In order to enhance peptide activity, the CPH was subjected to reverse-phase (RP)-HPLC separation to yield four fractions . The CPH and RP-HPLC fractions were each tested at 1 mg/mL for ability to inhibit p<0.05) higher than the values for CF1 (69%) and CF2 (79%). Conversely, the CF3 had the highest (63%) renin-inhibitory activity (p<0.05) when compared to CPH (43%), CF1 (15%), and CF4 (44%). CPH and CF3 exhibited uncompetitive mode of ACE inhibition, whereas renin inhibition was non-competitive. Even at a 6.7-fold lower dosage, the CF3 significantly (p<0.05) reduced SBP (maximum −40.0 mmHg) better than CPH (maximum −19.1 mmHg).The CPH, CF3, and CF4 had similar ACE-inhibitory activities of 84, 85, and 87%, which were significantly (RP-HPLC fractionation led to enhanced antihypertensive effects of cod peptides, which may be due to a stronger renin-inhibitory activity. The aim of this work was to determine the renin- and ACE-inhibitory activities of Atlantic cod fish (Gadus morhua) enzymatic protein hydrolysate in comparison with RP-HPLC peptide fractions. The most active peptide RP-HPLC fraction and unfractionated protein hydrolysate were then used for comparative kinetic studies of renin- and ACE-inhibitory mechanisms as well as systolic blood pressure (SBP)-reducing ability in spontaneously hypertensive rats (SHRs).Hypertension is related to elevated blood pressure (BP) and is an independent yet controllable risk factor for cardiovascular events such as myocardial infarction, atherosclerosis, stroke, and heart failure . It has ® 1:1 was obtained from Enzyme Development Corporation, New York, NY, USA. Pepsin, rabbit lung ACE, and N-[3-(2-furyl) acryloyl]-l-phenylalanyl-glycyl-glycine (FAPGG) were purchased from Sigma Chemicals . Methanol (HPLC grade), trifluoroacetic acid (TFA), 0.45- and 0.2-micron syringe filters, and all other analytical grade chemical reagents were obtained from Fisher Scientific .The Renin Inhibitor Screening Assay Kit was purchased from Cayman Chemicals . Enzeco Trypsin-Chymotrypsing for 20 min at 4°C and the supernatant discarded while the pellet was collected and dispersed in 200 mL of distilled water. The protein dispersion was adjusted to pH 2.0 with 2 mol/L HCl and pepsin (600–1800 units/mg protein) added at 10,000 units/g fish protein. The enzyme-treated protein dispersion was incubated at 37°C for 12 h with continuous stirring and the digest adjusted to pH 7.8 with 2 mol/L NaOH to irreversibly deactivate pepsin. A trypsin-chymotrypsin (Enzeco Trypsin-Chymotrypsin 1:1) mixture was then added at 1,000 units per mg fish protein for both components and digestion allowed to proceed for 4 h at 37°C with constant stirring. The digest was then boiled for 10 min to inactivate the enzymes, cooled to room temperature, centrifuged , and the supernatant filtered through a Whatman #2 qualitative filter paper. The filtrate was passed through a 1 kDa molecular weight cut-off Prep/Scale Tangential Flow Filtration cartridge membrane ultrafiltration setup . The collected membrane permeate (<1 kDa) was lyophilized as the cod protein hydrolysate (CPH) and stored at −20°C.A detailed description of the cod muscle preparation and protein hydrolysis has been previously described . BrieflyCPH was fractionated using RP-HPLC on a Varian 940-LC semi-preparative system according to a previously reported method . Brieflyin vitro ACE activity was determined using a spectrophotometric method with FAPGG as substrate (Ability of cod peptides to inhibit ubstrate . BrieflyA min-1(sample) and ΔA min-1(blank) are ACE activity in the presence and absence of inhibitory peptides, respectively. The concentration of peptide that inhibited ACE activity by 50% (IC50) was calculated by non-linear regression from a plot of percentage ACE inhibition versus four peptide concentrations . The kinetics of ACE inhibition was studied with 0.0625, 0.125, 0.25, and 0.5 mmol/L substrate (FAPGG) concentrations. The mode of ACE inhibition was determined from the Lineweaver–Burk plots while kinetic parameters (Vmax and Km) were estimated from non-linear regression fit of the data to the Michaelis–Menten equation using GraphPad Prism version 5.0 . Inhibition constant (Ki) was calculated as the x-axis intercept from a plot of the slope of the Lineweaver–Burk lines against sample concentrations while catalytic efficiency (CE) was calculated from Vmax/Km ratio.where ΔIn vitro assay of human recombinant renin activity was conducted using the Renin Inhibitor Screening Assay Kit according to the method previously described (escribed . BrieflyFIU min-1(sample) and ΔFIU min-1(blank) are renin activity in the presence and absence of inhibitory peptides, respectively. IC50 was calculated by non-linear regression from a plot of percentage renin inhibition versus peptide concentrations . The renin inhibition kinetics was conducted using 0.625, 1.25, 2.5, 5, and 10 µmol/L substrate concentrations in the absence and presence of samples, while kinetic parameters were calculated as described above for ACE.where ΔAnimal experiments were carried out following the Canadian Council on Animal Care Ethics guidelines with a protocol approved by the University of Manitoba Animal Protocol and Management Review Committee. The male SHRs  with 350–390 g body weight (bw) were kept in the Animal Housing Facility at the Richardson Centre for Functional Foods and Nutraceuticals, under a 12-h day and night cycle at 21°C and fed regular chow diet and tap water. The rats were divided into four groups  with six rats per group. The CPH (200 mg/kg bw), CF3 (30 mg/kg bw), and captopril (10 mg/kg bw) were each dissolved in phosphate-buffered saline and administered to the SHRs by oral gavage followed by measurement of SBP over a 24-h period  using the tail-cuff method in slightly anesthetized rats as previously described . Prior tp<0.05.All assays were conducted in triplicate and analyzed by one-way analysis of variance (ANOVA). The means were compared using Duncan's multiple range test and significant differences accepted at Enzymatic hydrolysis of the cod fish muscles in this study using a combination of pepsin, trypsin, and chymotrypsin enzymes produced the CPH, which was further subjected to RP-HPLC fractionation to obtain four distinct peptide fractions namely CF1, CF2, CF3, and CF4 . The perin vitro testing for ACE- and renin-inhibitory activities and the results obtained are shown in p<0.05) lower than the inhibitory levels exhibited by the most hydrophobic fractions CF3 (85%) and CF4 (87%). These high ACE activities of CF3 and CF4 are similar to the ACE-inhibitory activity of the CPH (84%).CPH and its RP-HPLC fractions  were subjected to in vitro renin-inhibitory activity. CPH (43% renin-inhibitory activity) fractionation significantly (p<0.05) improved renin-inhibitory activity only in CF3 but not in CF1, CF2, and CF4 which exhibited renin-inhibitory activities of 15, 21, and 44%, respectively. Peptide fraction hydrophobic character also seemed to play a role in renin inhibition just as observed for ACE inhibition. This is because the less hydrophobic CF1 and CF2 had lower renin-inhibitory values than the more hydrophobic CF3 and CF4. As expected, the renin inhibition potency of cod peptides (15–63%) was generally lower than that of ACE inhibition (69–87%). Based on the above in vitro bioactivity results, the most active fraction against both ACE and renin (CF3) was chosen for further comparison with CPH in terms of IC50 values, enzyme inhibition kinetics, and SBP-lowering ability in SHRs. The renin IC50 values as shown p<0.05) stronger ACE- and renin-inhibitory properties of CF3 when compared to CPH.Km and Vmax values for both CPH and CF3 were lower in the presence of peptides (Ki), which is a measure of the inhibitor binding strength to ACE enzyme indicates that the CPH peptides (0.263 mg/mL) bind stronger than the CF3 peptides 0.394 mg/mL).In this study, we used Lineweaver–Burk plots to evaluate both the ACE and renin modes of inhibition of the most active CF3 fraction in comparison with the unfractionated hydrolysate (CPH). The double reciprocal plots for ACE-catalyzed reactions in the absence and presence of these cod peptides as shown in peptides . CE was Vmax but with similar Km values  weaker than the 30 mg/kg bw lower dose of CF3. Moreover, persistence of the CPH was also weaker with a −5.0 mmHg effect after 24 h post-administration when compared to −12 mmHg for CF3.In order to confirm the potential BP-reducing effects of cod peptides as observed during the The low protein content of the CPH may be attributed mainly to the presence of non-peptide components, especially salt, that were formed during proteolysis when NaOH was added to neutralize the protons released by the proteases. Therefore, for CF1, the low protein content can be attributed to co-elution of these salts with peptides. This is because the polar salts bind poorly to the hydrophobic reverse-phase column and will co-elute with early eluting peptides in CF1. The CF2–CF4 peptides that eluted later will contain less salt and more protein because most of the salts would have eluted with CF1. Similar results showing that the initial eluted fractions from hydrophobic chromatographic column exhibited low protein contents while later-eluting fractions had higher values have previously been reported for hemp seed protein hydrolysate fractions separated by RP-HPLC . Therefop<0.05) lower ACE and renin-inhibitory values when compared to CPH, CF3, and CF4. However, the reduced ACE and renin-inhibitory activities could also be due to interference from salts because CF1 and CF2 had lower protein contents and hence higher levels of non-peptide components. The results are similar to a previous work showing reduced ACE-inhibitory efficiency of rapeseed peptide fractions that had lower protein contents  and CF2 (79%) because of the significantly  is higher (weaker potency) than previously reported F3 permeates of skin (IC50=0.003 mg/mL) and bone (IC50=0.013 mg/mL) gelatins of Pangasius catfish (50=0.029 mg/mL) (50=0.021 mg/mL)  (50=0.35 mg/mL) (50=1.53 mg/mL)  and F3-II (IC50=1.44 mg/mL) peptides of the second FPLC purification step of alcalase-derived seaweed pipefish muscle protein hydrolysate  than the 0.00039–0.0085 mg/mL range reported for captopril, the antihypertensive drug  and CF3 (0.16 mg/mL) are lower than the 0.81–1.89 mg/mL and 1.4–2.7 mg/mL values reported for hemp seed  , and cru1 mg/mL) . Howeverctively) , sea cuc5 mg/mL) , whole b5 mg/mL) , and porsh 50=0.0 mg/mL  in the presence of these peptides could be attributed to enzyme conformational rearrangement that reduced ability of the active site to accommodate the substrate. The Km value in absence of an inhibitor (0.449 mmol/L FAPGG) in this study is slightly higher than the previously reported value of 0.306 mmol/L for flaxseed protein hydrolysate  reported for the hemp seed protein hydrolysate fractions (Ki values are higher than the 0.044–0.051 mg/mL reported for chicken skin protein hydrolysate and peptide fractions (It is vital to study the inhibitory kinetic properties of ACE and renin inhibitors because this helps reveal the possible mechanisms by which peptides are able to act as antihypertensive agents. ACE inhibition by CPH and CF3 was through uncompetitive mode, which means that the peptides were bound to the enzyme–substrate complex to form inhibitory complexes with reduced capacity to yield products. As evident in rolysate , but lowrolysate . Ki is ractions . In contractions .Vmax values shown in Contrary to the ACE inhibition pattern, the CPH and CF3 inhibited renin activity mostly through a non-competitive mode. This implies that the inhibitor can achieve its inhibitory effects by binding to both the free renin and renin–substrate complex. In either case the enzyme–peptide interactions reduce enzyme affinity for substrate and hence less conversion to products as evident by the decreased in vivo interactions with ACE and renin. The persistent BP-reducing effect of CF3 suggests that the peptides were perhaps more resistant to in vivo proteases than the CPH peptides. It is difficult to compare the CF3 data with literature values because of the differences in dosage administered to the SHR. For example, 10 and 100 mg/kg bw bovine casein hydrolysate produced −28 and −42 mmHg SBP reductions, respectively, 2 h after oral administration to SHRs (The CF3 peptide demonstrated an excellent potential antihypertensive agent because of the fast-acting BP-lowering effect in SHRs within the first 2 h after oral administration and the persistent effect observed after 24 h. The results are consistent with the higher renin-inhibitory activity of CF3 when compared to CPH b and 2.  to SHRs . In cont to SHRs . Transla to SHRs . Because to SHRs  who repoin vitro assays provide rapid assessment of bioactive potential of peptides, the use of physiologically relevant animal disease models provides for a realistic estimation of applicability to human disease conditions. Hence, the positive relationship between in vitro and in vivo data in this work provides a scientific basis to use these peptides in future human intervention trials to determine potential therapeutic utility. Although the expected human dose will be several times more than that of a drug, the lack of substantial negative side effects during peptide use as reported by several researchers should make the cod peptides attractive antihypertensive options.In conclusion, although


Anisakis spp. can produce allergic reactions after eating raw or undercooked parasitized fish. Specific IgE is detected long after the onset of symptoms, but the changes in specific IgE levels over a long follow-up period are unknown; furthermore, the influence of Anisakis spp. allergen exposure through consumption of fishery products is also unknown.Sensitization to Anisakis spp. allergens over several years of follow-up and the influence of the consumption of fishery products in IgE sensitization.To analyse the changes in IgE sensitization to Anisakis spp.-specific IgE, anti-Ani s 1 and anti-Ani s 4 IgE were repeatedly measured over a median follow-up duration of 49 months in 17 sensitized patients.Total IgE, Anisakis spp.-specific IgE was detected in 16/17 patients throughout the follow-up period. The comparison between baseline and last visit measurements showed significant decreases in both total IgE and specific IgE. The specific IgE values had an exponential or polynomial decay trend in 13/17 patients. In 4/17 patients, an increase in specific IgE level with the introduction of fish to the diet was observed. Three patients reported symptoms after eating aquaculture or previously frozen fish, and in two of those patients, symptom presentation was coincident with an increase in specific IgE level.Anisakis spp. allergens lasts for many years since specific IgE was detectable in some patients after more than 8 years from the allergic episode. Specific IgE monitoring showed that specific IgE titres increase in some allergic patients and that allergen contamination of fishery products can account for the observed increase in Anisakis spp.-specific IgE level.IgE sensitization to Anisakis spp. allergens, the absence of additional exposure to those allergens does not result in the loss of IgE sensitization. Exposure to Anisakis spp. allergens in fishery products can increase the specific IgE level in some sensitized patients.Following sensitization to  Anisakis spp. is a parasite of marine mammals that can parasitize humans when a raw or undercooked fish containing live Anisakis spp. larvae is consumed. As a result, gastrointestinal and/or allergic symptoms are reported. The allergic reaction can be diagnosed by quantifying the serum specific IgE against some parasite allergens. To avoid the appearance of symptoms, sensitized patients are advised to consume frozen or heat-treated fishery products because these treatments kill larvae to prevent new parasitism and allergic reactions. We have analysed the changes in Anisakis spp.-specific IgE levels through repeated measures during a long follow-up period of sensitized individuals whose diets did not include fishery products and subjects who regularly consumed fishery products. We have found that IgE sensitization to Anisakis spp. allergens persists over several years (more than 8 years in some cases) even in subjects who did not include fishery products into their diets. On the other hand, specific IgE titres increase in some allergic patients during the follow-up period and this increase was coincident with the introduction of previously frozen fish into their diet. These findings indicate that Anisakis spp. allergens induce long-lived IgE responses.The nematode  Anisakis spp. is a parasite of marine mammals that can parasitize humans when a raw or undercooked fish containing live Anisakis spp. L3 is consumed. Ingestion of L3 causes an acute and self-limiting infection that can manifest with abdominal pain, nausea, vomiting or diarrhoea. Infection causes a strong polyclonal humoral immune response, and IgM, IgA, and IgG antibodies are detected after one month of infection [The nematode nfection . In somenfection . To avoinfection .Anisakis spp. in sensitized patients have shown the persistence of IgE sensitization up to 38 months after the onset of symptoms [Several groups have investigated the kinetics of specific antibody production in experimental animal models [review in symptoms , 2, 4, 7Anisakis spp.-specific IgE levels through repeated measures during a longer follow-up period than previously reported and to compare IgE sensitization between patients whose diets did not include fishery products and subjects who regularly consumed fishery products.The aim of this study was to analyse the changes in Anisakis spp.-allergic patients with at least 30 months of follow-up after symptom presentation were selected for this study. A total of 17 patients  with a median age of 53 years (IQR = 45–57 years) were diagnosed as being allergic to Anisakis spp. because they reported allergic  and/or gastrointestinal  symptoms within 24 h after eating raw or undercooked fish or seafood. One patient reported symptoms after eating cooked fish (scorpion fish cake). Five patients had grass pollen allergy and one of them had dog dander allergy. The data collected at the first visit are shown in Anisakis spp. and undetectable levels of IgE to shrimp, Ascaris lumbricoides, fish and mites. To assess new sensitization to these allergens, specific IgE levels were quantified at the last visit, and they remained undetectable in all patients. Measurements of the levels of total and specific IgE to Anisakis spp. and clinical evaluations were performed during successive visits. All patients were advised at the first visit to avoid consumption of raw and undercooked fish and to eat farmed fish and deep-frozen fishery products; however, patients with levels of specific IgE to Anisakis spp. higher than 100 kU/L were initially instructed to consume a fish-free diet for six months.To analyse the kinetics of the IgE response, This study was approved by the Ethics Committee of the Hospital Carlos III , and all included subjects were asked to sign an informed consent form.Anisakis spp., and values >0.35 kU/L were considered positive for IgE to the other allergens.The serum total and specific IgE measurements were performed with a Phadia 250 instrument  according to the manufacturer’s instructions. The detection range for total IgE was 2–5000 kU/L. Regarding positivity for specific IgE antibodies, values >0.7 kU/L  were conAnisakis spp. larvae in the third stage of the life cycle (L3) were obtained from parasitized hake (Merluccius merluccius) at local markets in Madrid, Spain. L3 were extracted from fish tissue, washed in PBS and immediately frozen at -20°C until use. Then, L3 were ground in a Potter-ELV homogenizer and sonicated at 18 w for 5 s. Protein extracts were obtained after centrifugation at 16,000 g and 4°C for 10 min.Live Recombinant (r)Ani s 4 and rAni s 1 were obtained as previously reported , 10.In addition to the total and specific IgE measurements, IgE immunoblotting was performed with the parasite crude extract, recombinant (r) Ani s 1 and rAni s 4.Anisakis spp. -allergic patients (1/20) overnight. After washing with PBS, the membranes were incubated for 2 h with 10 mL of a 1:1000 dilution of a monoclonal anti-IgE antiserum . After additional washes, the membranes were incubated with 10 mL of a 1:20,000 dilution of an alkaline phosphatase–labelled goat anti-mouse antiserum . Finally, the membranes were washed and incubated with the BCIP-NBT (Sigma-Aldrich) substrate for 30 min.Proteins extracted from L3 (15 μg), rAni s 1 (3 μg) and r Ani s 4 (3 μg) were subjected to electrophoresis at 120 V on a 4%-20% Tris-glycine gel . Thereafter, proteins were transferred to nitrocellulose membranes by applying a constant current of 1.3 A for 7 min in a Trans-Blot Turbo Instrument (Bio-Rad). Membranes were blocked with PBS, 0.05% Tween 20 and 1% BSA for 1 h at room temperature and then incubated with 10 mL of the sera of Anisakis spp.-specific IgE >100 kU/L were assigned a value of 101 kU/L [Statistical analyses were performed using SPSS 20.0 software . Quantitative variables are described as medians and interquartile ranges (IQR). The Mann-Whitney U test was used to compare the values of total IgE and specific IgE quantified at the first and last visits. A paired-samples comparison between the baseline and last visit values was performed using the Wilcoxon signed-rank test. Regression was used to analyse the trends in the changes in specific IgE values. Changes in specific IgE values over time were estimated with linear and non-linear regression models, and the best fit was selected. A p–value of <0.05 was considered statistically significant. For the statistical analysis, all values of 101 kU/L .Anisakis spp.-specific IgE, specific IgE to rAni s 1 was detected in all patients, and specific IgE to rAni s 4 was detected in six patients  or allergic and gastrointestinal symptoms (n = 8) after eating raw or undercooked fish, except P14 who reported urticaria after eating cooked fish (scorpion fish cake). Raw anchovies in vinegar sauce were the most frequently consumed fish related to the onset of symptoms . The medpatients . AlthougAnisakis spp. values (>0.7 kU/L) were detected in all patients throughout the follow-up period, except for P14, who had a specific IgE to Anisakis spp. value of 0.6 kU/L at the end of a 112-month follow-up period  were significantly higher than the final total IgE value  and the Anisakis spp.-specific IgE titre  (Anisakis spp.-specific IgE titres (p < 0.01). No significant differences were found between the patients who were and were not re-exposed to Anisakis spp. in the decrease in the Anisakis spp.-specific IgE level over the follow-up period  or in the follow-up duration .Positive specific IgE to p period . Baselinp< 0.01) . The medAnisakis spp.-specific IgE values. The Anisakis spp.-specific IgE values underwent an exponential or polynomial decay trend in 13/17 patients, including the patients who had not included fish in their diet during the follow-up period  did not fit a regression model  and, then, increased up to 37 kU/L at one year later. The changes in the Anisakis spp.-specific IgE level in P6 were similar to those in P1, with a decrease of specific IgE titre until reaching 3 kU/L at 12 months and an obvious increase at the next visit (50 kU/L). P15 showed a moderate level of Anisakis spp.-specific IgE (10 kU/L) at the first visit; it decreased at one month (6 kU/L) and increased at the following visit. The Anisakis spp.-specific IgE increases in these four patients were coincident with the introduction of aquaculture and frozen fish into their diet  and last visit Anisakis spp.-specific IgE . When the total and specific IgE titres and the duration of follow-up for the patients whose data fit a regression model were compared to those of these four patients, only total IgE at last visit was found to be higher in the latter group .Regression was used to analyse the trend in the changes in p period . Howeveron model . The Anieir diet . The paiAnisakis spp.-specific IgE level. P3 and P5 did not report new episodes in the subsequent visits. P13 showed generalized acute urticaria after eating commercial frozen hake  than previous studies (6–38 months) [Anisakis spp. L3 can produce an acute and self-limiting infection in humans with allergic and gastrointestinal symptoms. To avoid the appearance of symptoms, patients are advised to consume frozen or heat-treated fishery products because these treatments kill larvae and thus prevent new parasitism [Anisakis spp. larvae in farmed fish has been shown to be minimal, because no Anisakis spp. larvae have been found in their viscera or flesh [Anisakis spp.-specific IgE levels in these patients, who were considered to have not been re-exposed to parasite material, was higher than those found previously in patients on a fish-free diet (57%) with a follow-up period of 13 months, and this discrepancy was probably due to our longer follow-up period [Anisakis spp.-specific IgE values between the patients who did and did not consume fish during the follow-up period [Anisakis spp.-specific IgE decay does not seem to be influenced by the consumption of previously frozen fish.Our study analysed the changes in the values of total IgE and specific IgE to  months) , 4, 7. R months)  and Ani  months) . Exposurrasitism . An alteor flesh , 13. TweAnisakis spp. allergens persists over several years since specific IgE was detectable in some patients after more than 8 years from the allergic episode. Similar results were observed in a nine-year follow–up study of adult subjects sensitized to food allergens [Anisakis spp. infection cases. Therefore, the persistent IgE sensitization observed in Anisakis spp. allergic patients who were not exposed to parasite allergens for several years indicates that Anisakis spp. allergens induce long-lived IgE responses.Our results show that IgE sensitization to llergens . In our llergens  and suggAnisakis spp. allergens and in some patients who regularly consume aquaculture and frozen fish shows that the decrease in specific IgE titres fit a non-linear regression model. However, we observed that the Anisakis spp.-specific IgE level in four patients  increased at some time points during follow–up. This increase in Anisakis spp.-specific IgE could be due to sensitization to other allergen sources that have been reported to cross-react with Anisakis spp. allergens, i.e., other parasite nematodes, mites and crustaceans [Anisakis spp. allergens at baseline, and no new onset hypersensitivity to those allergens was found during the follow-up period. In addition, the increase in Anisakis spp.-specific IgE was parallel to the increase in Ani s 1 detected by immunoblotting, which supports the hypothesis that the observed changes in Anisakis spp.-specific IgE were actually due to exposure to Anisakis spp. allergens. Another explanation for this observation is re-infection with live L3. However, we do not believe this is the case because the patients reported symptoms after eating frozen or aquaculture fish, and it is unlikely that an acute parasitism episode went unnoticed by these patients, as they had experienced one previously. On the other hand, previously unrecognized allergens have been detected in the course of gastro-allergic anisakiasis [Anisakis spp.-specific IgE level. Some Anisakis spp. antigens have been shown to be stable and to maintain their capacity to bind IgE after different freezing and heat treatments [Anisakis spp. antigens have been detected in farmed salmon and processed fish products [The analysis of the changes over time in specific IgE level in the patients that were not re-exposed to staceans . Howeversakiasis . Becauseeatments , 20. Furproducts , which cAnisakis spp. parasitism of fish in our region would result in frequent contact with parasite material, which would cause symptoms in sensitized subjects exposed to it when previously frozen or heat-treated fish is included in their diet [Anisakis spp. antigens present in fishery products may contribute to the persistence and even to the increase in Anisakis spp.-specific IgE level. The increase in the Anisakis spp.-specific IgE level associated with the appearance of symptoms indicates that exposure to Anisakis spp. material could be involved in the onset of symptoms in some Anisakis spp. allergic patients [Anisakis spp. in sensitized patients.The clinical significance of these results is difficult to determine because very few patients (n = 3) reported symptoms after the initial acute parasitism episode in our study. It has been proposed that the high rate of eir diet . Howeverpatients . HoweverAnisakis spp. allergens can last more than 8 years. Specific IgE monitoring showed that specific IgE titres increase in some allergic patients and that allergen contamination of fishery products can account for the observed increase in Anisakis spp.-specific IgE level.In conclusion, we have shown that IgE sensitization to S1 Checklist(DOCX)Click here for additional data file.S1 FigAnisakis spp. crude extract; lane 2: rAni s 1; lane 3: rAni s 4. The shadow box indicates the follow-up period during which the patients were consuming fish. sIgE: specific IgE to Anisakis spp.Letters indicate the time points during follow-up when IgE immunoblotting was performed. Lane 1: (PDF)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


The fundamental reason for this is the activation and high engagement of the pentose phosphate pathway (PPP) in response to the production of reactive oxygen species (ROS) H2O2 by mitochondria and the high concentration of H2O2 (produced by mitochondria and other sources). This makes oxidative phosphorylation an inefficient source of energy since it leads (despite high levels of ATP production) to a concomitant high energy consumption in order to respond to the hazardous waste products resulting from cellular processes associated with this metabolic pathway. We also demonstrate that the high concentration of H2O2 results in an increased glucose consumption, and also increases the lactate production in the case of glycolysis.Since the original observation of the Warburg Effect in cancer cells, over 8 decades ago, the major question of why aerobic glycolysis is favored over oxidative phosphorylation has remained unresolved. An understanding of this phenomenon may well be the key to the development of more effective cancer therapies. In this paper, we use a semi-empirical method to throw light on this puzzle. We show that aerobic glycolysis is in fact energetically more favorable than oxidative phosphorylation for concentrations of peroxide (H Increased aerobic glycolysis (the Warburg Effect) in proliferating cancer cells has been a perplexing puzzle that has remained unresolved for more than 80 years Warburg, . The obs2O2 in non-cancerous and cancerous cells during oxidative phosphorylation  HTurrens, . Moreove2O2 are the major players in the cell metabolism dynamics and the three chemical reactions involved are therefore  level. For concentrations of H2O2 much lower than CSC the production rate of GSH is very low. As the concentration of H2O2 becomes comparable to CSC, cells start to activate the PPP to produce more GSH. The produced H2O2 by cell mitochondria is a major player in this response, since they are at the center of H2O2 production and they, if functional, can activate TNF. At high concentrations of H2O2, the H2O2 produced by mitochondria does not diffuse into the cell cytoplasm and accumulates instead around the mitochondria which, as a result, activate TNF.To understand the mechanism behind aerobic glycolysis and the role of ROS, we consider the following major metabolisms and detoxification pathway: oxidative phosphorylation, glycolysis, and the PPP. We assume that ATP, GSH, and Hherefore ,(1)Glucces GPxr . The conces GPxr  and 10 nces GPxr , hence iPGSH, 2O2 by mitochondria and H2O2 by external sources, and β and γ are functions of the concentration of GSH and, the difference between CSC (C0) and the concentration of H2O2 (CROS). We choose β and γ to be different since, in general, the response of mitochondria to the accumulation of H2O2 differs from that of the rest of the cell. β and γ are very small for low concentrations of H2O2 and start to increase as the concentration of H2O2 increases. Close to C0 they become very large in order to drive most of the consumed glucose to the PPP pathway. Since mitochondria can activate TNF in their oxidative phosphorylation state . We obtain the production and consumption as functions of the oxygen and glucose consumption and GSH production (qO) converted to H2O2 by mitochondria [about 1/100−2/100 Turrens, qG is the total consumption of glucose and r is the ratio between oxygen and metabolic glucose consumptions. We now consider the case when the net production rate of ATP exactly balances the energy requirements of the cell and derive the following result for the consumption of glucose in terms of the concentration and production rate of H2O2 (r = 0) reads:2O2 . In this case, and for a purely glycolytic metabolism (r = 0), PATP = 2qG which implies that 2ATP are produced for each molecule of glucose—this is the well-known case of pure glycolysis. In the case of dominant respiration (r = 6) PATP = 36qG, which is again the net energy production rate for a cell under oxidative phosphorylation. A simple comparison of 2–36 ATP production leads to the superficial, albeit prominent conclusion that, for the case of low H2O2 concentrations, oxidative phosphorylation is the most efficient metabolism.We assume that both oxidative phosphorylation and glycolysis are activated to produce energy for cell needs and for the PPP and investigate the production rate of ATP in the presence of H of H2O2 :(6)qGSS2O2 by finding the maximum production rate of ATP (PATP) as a function of r. For αβ < αT, where αT = 68/9 the maximum production of ATP arises at r = 6. This is in agreement with the well-known fact that oxidative phosphorylation is the most efficient way for ATP production. However, rather interestingly, for αβ > αT the maximum production rate of ATP occurs at r = 0 and the transition point is independent of 2O2, glycolysis is in fact energetically more efficient than oxidative phosphorylation, which directly contradicts current prevailing explanations. To illustrate this metabolic transition in terms of the concentration of H2O2 we choose two functions 2O2 increases. The coefficient β is chosen to be larger because (a) TNF is activated by mitochondria and (b) at high concentrations of H2O2, the produced H2O2 cannot diffuse into the cell and accumulates in the vicinity of the mitochondria which occupy a much smaller space within the cell. In Figure PATP  as a function of CROS and r. For CROS < 0.87C0, which corresponds to β < αT, the maximum production rate arises at r = 6 and for CROS > 0.87C0, which corresponds to αβ > αT, it transitions to pure glycolysis (r = 0).We now search for the most efficient metabolism in the presence of H2O2, a cell must expend most of its produced ATP in detoxifying its self-generated H2O2 (by mitochondria). This is supported by the fact that for α = 0 , it is respiration that is the more efficient metabolism for any concentration of H2O2. In contrast, for α≠0, no matter how small α is, the shift in metabolism (from respiration to glycolysis) occurs when concentrations of H2O2 exceed some critical threshold value. At these concentration levels of H2O2, the net produced energy by oxidative phosphorylation for one molecule of glucose is less than that produced through glycolysis. To gain a more quantitative understanding of this phenomenon, we first observe that α is a property of mitochondrial efficiency. It can safely be assumed that this remains constant independent of H2O2. Meanwhile β changes through either an increase in the concentration levels of H2O2 or a decrease in the concentration levels of GSH and thus results in a crossing of the transition point to glycolysis. In non-cancerous cells and under normal conditions, H2O2 is mainly produced by mitochondria and diffuses through the cell. In this case, the low production of GSH and other antioxidants are sufficient to detoxify the H2O2, hence normal cells function in the αβ < αT regime. In this case, oxidative phosphorylation is the most efficient mechanism for ATP production. However, in proliferating cancer cells, H2O2 is produced by growth factors and mitochondria that work at higher rates to compensate for the increased energy needs of proliferating cells. Hence, the concentrations of H2O2 are much higher and this can push the cell into the regime αβ > αT in which glycolysis is the more efficient metabolism.As a first step to better understand the nature of the transition from oxidative phosphorylation to glycolysis, we first take note that the inefficiency observed in oxidative phosphorylation is fundamentally due to the fact that for some concentrations of HCROS for the two cases r = 0 and r = 6 under constant cell needs. These plots show the significant increase in glucose consumption as functions of the concentration of H2O2. Therefore, the generated H2O2 by growth factors and other sources is one of the major reasons for the increase in glucose consumption. Also, the two plots intersect for CROS = 0.87C0, which means that for a given cell glucose requirement, the energy expended for the consumption of glucose through oxidative phosphorylation exceeds the corresponding expenditure when the cell has shifted to a glycolysis metabolism and this is due to the production of H2O2 by mitochondria.In Figure r = 0 as a function of CROS assuming that 2O2 leads to enhanced lactate production. This suggests that the observed high lactate production in cancer cells does not occur solely because of the cell needs, but may also be related to the increase in the concentration levels of H2O2. We plot in the inset of Figure In Figure 2O2. These functions could be measured in vitro by putting different cell lines in a steady state flow of H2O2 and measuring the production rate of GSH for different concentrations of GSH. We also note that, at the same concentration of H2O2, glycolytic cells produce less GSH than cells that use oxidative phosphorylation.Notice that the presented results are qualitatively independent of the form of β and γ and we can derive the obtained results for any β and γ as long as they increase with increasing H2O2 in hepatoma cells, glycolysis activity increases, and by reducing H2O2 levels, this activity decreases. These observations are consistent with our prediction of a concomitant increase in glycolysis activity with increase in H2O2 and vice versa. We also note that Brand and Hermfisse  and CROS less than the critical value for transition from oxidative phosphorylation to glycolysis (COG), the metabolism is anaerobic glycolysis. When the concentration of oxygen passes the hypoxic concentration levels, cells transit to oxidative phosphorylation or aerobic glycolysis depending on whether CROS is less or greater than COG. As the concentration of H2O2 increases and exceeds CGP, cells close all their metabolic pathways to drive the whole consumption of glucose toward PPP in order to reduce cell damage by H2O2. However, this process cannot continue indefinitely because changing glucose to G6P is ATP-dependent. Thus, cells need to keep their glycolytic metabolism active in order to continue the process of generating GSH and for detoxification of H2O2. When the concentrations of H2O2 exceed the critical concentration C0 tumor cells undergo apoptosis. Notice that this diagram is based on the most efficient mechanism of producing ATP and the availability of oxygen. It is possible that mutated cells activate less dominant metabolic pathways in any of these regions.In Figure 2O2 stress and are more resistant to ROS. Hence, at high concentrations of H2O2, they have a higher survival advantage as compared to cells that rely on respiration. As a result, the glycolytic phenotypic population becomes the dominant population under ROS stress. Switching to the glycolytic phenotype may be realized through overexpression of glycolytic agents. In fact, experimental results report that PKM2 is activated in cancer cells which serves to shift the metabolism from oxidative phosphorylation to aerobic glycolysis , PKM2 is inhibited to drive the whole glucose flux to the PPP pathway and thus minimize the adverse effects of ROS (Anastasiou et al., When some cells adopt the glycolytic phenotype, they have chosen the most efficient way of generating energy under HWe anticipate that there are other physiological and pathological situations in which our results might be pertinent and might help to explain certain biological behaviors. Two such examples are the observation of the glycolysis metabolism in embryos (Kondoh et al., 2O2 exceed a certain critical value. This is because the energy generated by mitochondria is consumed by PPP to respond to the production and high concentrations of H2O2, generated by mitochondria. This makes oxidative phosphorylation an inefficient source of energy since it results in high energy consumption in order to respond to the production of H2O2 by mitochondria under high concentrations of H2O2. We have also shown that by increasing H2O2 levels, cells need to increase their glucose consumption via the glycolysis metabolism and PPP in order to satisfy their nutritional needs and for the purposes of removing H2O2. Thus, we propose that H2O2 is the major player behind the observed shift toward aerobic glycolysis in proliferating cancer cells.In conclusion, we have demonstrated that aerobic glycolysis is energetically more favorable than oxidative phosphorylation when the concentration levels of HHM, MK and SS conceived and designed the research project. HM carried out the calculations and simulations. HM, MK and SS analyzed the results and wrote the manuscript.SS and MK acknowledge NSERC funding through individual Discovery Grants.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.


However, it is not known whether this is related to an intrinsic property of this subclass or only the allergen specificity. We tested the hypothesis that allergen specificity is the critical determinant for this activity using a panel of antibodies with identical specificity but different subclasses. The different antibodies were all able to inhibit the activity of IgE to the same extent. We demonstrate that specificity is the dominant factor determining the ability of an antibody to block allergen‐dependent IgE activity.IgG BATbasophil activation testBSAbovine serum albuminEDTAethylenediaminetetraacetic acidELISAenzyme‐linked immunosorbent assayFAPfacilitated antigen presentationHBSHepes‐buffered salineHPLChigh‐performance liquid chromotographyPBSphosphate‐buffered salinePIPEpolymerase incomplete primer extensionSCITsubcutaneous immunotherapySPRsurface plasmon resonanceTMBtetramethybenzidine1, IgG2, IgG3 and IgG4, differ mainly in the length and rigidity of the hinge region. These differences impart different functional roles, based on their ability to activate the immune system. Similarly, IgA is represented by two subclasses: IgA1, which predominates within the serum, and IgA2, with a shorter hinge region and more compact structure, which predominates at mucosal surfaces Allergy is associated with the excessive production of allergen‐specific IgE. However, allergen‐specific antibodies of other isotypes are produced both in allergic disease and in states of tolerance, for example in hyperimmune beekeepers and in patients treated by allergen immunotherapy 4 fraction 4 from this serum correlates with a decrease in IgE inhibitory activity 4 subclasses or whether other subclasses that recognize the same epitope are equally effective in blocking IgE‐mediated activity. To test this, we generated a set of recombinant monoclonal antibodies of the same specificity for the grass pollen allergen Phl p 7, with different constant region domains, representing all of the IgG and IgA subclasses. We then measured the affinity of antigen binding and ability of these antibodies to inhibit IgE‐mediated activities in in vitro assays.Serum from patients receiving allergen immunotherapy blocks the activity of IgE, and this inhibitory activity co‐elutes with the IgGDetailed methods are available in the supporting information.1, IgG2, IgG3, and IgA1 and IgA2 expression vectors were subsequently cloned and expressed using the PIPE method Matched heavy‐ and light‐chain variable antibody sequences specific to Phl p 7 allergen were previously isolated from a single B cell derived from a patient undergoing grass pollen immunotherapy 1 and IgG2, and IgG3 and IgG4 were purified by affinity chromatography with a 5‐ml HiTrap Protein‐G HP column . Human 102.1F10 IgA1 and IgA2 were purified by affinity chromatography with immobilized SSL7/Agarose . The purified antibodies were analysed by size exclusion chromatography Human 102.1F10 IgGIgE‐facilitated allergen binding to B cells was performed as previously described −, CD303−, CD294+) activation (upregulation of CD63) was measured by flow cytometry following incubation of blood from a Phl p 7‐sensitized donor with recombinant Phl p 7 in the additional presence of Phl p 7‐specific antibodies (10 μg/ml), postsubcutaneous immunotherapy serum (SCIT) (patient 102) or control serum .Basophil .Size exclusion chromatography and ELISA confirmed the purified Phl p 7‐specific IgG4 has been previously shown to be an effective blocking antibody for IgE‐mediated activity, we wished to determine whether this blocking activity was specific to the IgG4 subclass. We therefore tested the IgG and IgA subclasses in two independent in vitro assays of IgE activity. Similar to IgG4, IgG1, IgG2, IgG3, IgA1 and IgA2 were able to inhibit binding of IgE‐Phl p 7 complexes to the IgE receptor CD23 (FcεRII) on the surface of B cells  subclass of the antibody, we compared the ability of other subclasses to inhibit IgE. Although the affinities for Phl p 7 were similar in all of the recombinant antibodies we generated, subtle but significant differences in binding rates were observed , which tended to cancel out to give very similar overall affinities (ranging from 250–570 pm). These differences may reflect subtle conformational changes that constant region domains impart on the variable region, which have been reported to influence the fine specificity and affinity of isotype‐swapped antibodies in vitro assays. Of course, we cannot exclude the possibility that more subtle effects might be observed by titration of the different antibodies. However, under the conditions used here, blocking activities were dependent only on the ability to bind allergen and not on the constant region effector function.The inhibitory activity of non‐IgE antibodies in an allergic reaction is thought to be due to their competition with IgE, by masking the epitopes on the allergen. These so‐called blocking antibodies represent a potentially valuable but as yet untested therapeutic commodity for use in passive allergen immunotherapy 4‐expressing B cells secrete the most efficacious blocking antibodies in vivo after specific allergen immunotherapy 4 has properties that may favour its use over other IgG subclasses, such as its inability to bind complement, and also the unique property of exchanging one heavy‐/light‐chain pair with an IgG4 antibody of a different specificity to generate a bispecific antibody unable to form immune complexes It is almost certain that the blocking activity of an antibody is dependent on several factors such as epitope specificity, concentration and affinity for antigen in vivo or whether other mechanisms as mentioned above come into play. Phl p 7 is a relatively small allergen, and while our study provides a proof‐of‐concept for passive immunotherapy, it is likely that combinations of allergen‐specific monoclonal antibodies directed against multiple allergens, and possibly multiple epitopes, will be required to ameliorate symptoms depending on the sensitization pattern of each individual In summary, our results demonstrate that all IgG and IgA subclasses are capable of inhibiting the activity of IgE in an allergen‐specific manner. Further experiments will be required to examine whether antibodies with the same specificity but different isotype are similarly inhibitory TSD, HB and HJB produced and characterized antibodies; MHS performed functional assays; JMM helped to design experiments and analyse data; LKJ, AJB, SRD and BJS helped to design experiments and write the manuscript; and LKJ performed and coordinated experiments. HJG proposed the study and helped to write the manuscript.None of the authors have any conflict of interest to declare.Figure S1. ELISA plates were coated with Phl p 7 and antibody binding was confirmed using subclass‐specific monoclonal detection antibodies.Click here for additional data file.Appendix S1. Methods.Table SI. SPR analysis of the interaction between Phl p 7 and recombinant antibodies (±SE).Click here for additional data file.


The involvement of PKG-Iα was further studied using cultured endothelial cells subjected to hypoxia reoxygenation (HR) injury in vitro. In rat MIR model, SCU  significantly reduced ischemic size and restored the endothelium-dependent vasodilation of isolated CA rings. PKG inhibitor Rp-8-Br-cGMP  could ameliorate the protective effects of SCU. Increase in phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a main substrate of PKG, at Ser 239 was observed in both heart tissue and serum of SCU-treated animals. In cultured human cardiac microvascular endothelial cells (HCMECs), SCU (1 and 10 μM) dose-dependently protected cell viability and increased the mRNA and protein level of PKG-Iα against HR injury. The activity of PKG was also increased by SCU treatment. The activation of PKG–1α was then studied using targeted proteomic analysis (MRM-MS) checking the phosphorylation state of the autophosphorylation domain (aa42-94). Significant decrease in phosphorylation of PKG-Iα at Ser50, Ser72, Ser89 was induced by HR injury while SCU treatment significantly increased the phosphorylation of PKG-Iα, not only at Ser50, Ser72 and Ser89, but also at Ser44 and Thr58 (two novel phosphorylation domains). Our results demonstrate PKG-Iα might play an important role in the protective effects of SCU on ED against MIR injury.Scutellarin (SCU), a flavonoid glycoside compound, has been successfully used in clinic for treatment of ischemic diseases in China. In this report, we checked the effects of SCU on endothelium dysfunction (ED) of coronary artery (CA) against myocardial ischemia reperfusion (MIR) injury  Erigeron breviscapus (Vant.) Hand. Mazz , triphenyl tetrazolium chloride (TTC) and ACH were purchased from Sigma-Aldrich .in vivo and in HR cells of HCMECs in vitro. In the animal studies, the protective effect of SCU on MIR injury was tested by evaluating myocardial ischemia area, and then the hypothesis that SCU improves endothelium dependent vasodilation reduced by MIR was proved in the animal model with ED induced by MIR, while PKG inhibitor was used to reveal the influence of PKG. In vivo animal studies, there were four independent experimental design series. 1) To evaluate the effects of SCU on myocardial ischemia area induced by MIR injury, SD rats were divided into four groups: sham, MIR model and two SCU groups . 2) In another experiment, the influence of PKG inhibitor and SCU on myocardial ischemia area was assessed. The rats were divided into four groups: MIR model, SCU , PKG inhibitor , and SCU  + PKG inhibitor  treated group. 3) In another independent experiment of MIR, endothelial-dependent vasodilation in CA rings was assayed. Rats were divided into five groups: sham, MIR model, SCU groups , PKG inhibitor  and SCU  + PKG inhibitor  treated groups. 4) To evaluate the effects of SCU on PKG-I and p-VASP Ser 239 changes induced by MIR injury, SD rats were divided into five groups: sham, MIR model and two SCU groups , nitroglycerin  group (used as positive control).In order to study cardiovascular endothelium protective mechanism of SCU and the role of PKG, the investigations were carried out in both MIR rats vitro cell studies, HR treatment was used to simulate vivo IR injury. To investigate whether SCU has direct cytoprotective effects and the role of PKG, cell viability and protein, activity and mRNA of PKG were determined. In addition, the phosphorylation site of PKG–1α was further studied using MRM-MS to clarify how SCU affects the phosphorylation of PKG activation. In vitro studies, cells of HCMECs were incubated with SCU (0.1–10 μM) under normal culture and simulated HR conditions. In each assay, three independent experiments (each conducted in triplicate) were used.In Adult Sprague-Dawley (SD) rats (180–220 g) were provided by the animal center of Kunming Medical University. The rats were housed under controlled temperature (23–25°C) and lighting  with free access to food and drinking water. The study and the protocol were approved by the Animal Care and Use Committee of Kunming Medical University and conformed to the standards set by the Yunnan Experimental Animal Management Board. The animal procedures were performed conform the NIH guidelines . For anesthesia, 10% urethane (1mL/100 g with dose of 1g/kg) in saline solution was intraperitoneally injected to the rats. Before initiating surgery, to render the animal areflexic to strong ear- or tail-pinch, if needed, additional doses of ≤0.4 ml 10% urethane were used. SCU and other treated drug infusion was initiated intravenously 15 min prior to MIR surgery via the tail vein (2 mL/h) until the ending of reperfusion. The sham and MIR groups were given normal saline (NS).The MIR model was induced according to the previously described procedure with minor modifications . The aneThe hearts were frozen and stored in a −20°C freezer for 5 min, and then sliced into 1-mm-thick transverse sections across the long axis. Slices were incubated in 1% TTC in a phosphate buffer (pH 7.4) for 15 min at 37°C. After staining, the slices were immersed in 4% formalin. The tissues that stained brick red were considered viable, whereas pale or white tissues were considered necrotic. The infarct areas were analyzed by Image-Pro Plus 6.0 (IPP) software, and the ischemic area was expressed as a percentage of the area at risk: infarct size = 100×infarct area (white area)/risk area (white area + red area). Infarct size was expressed as the percentage of the total cross-sectional area of the ventricles. In addition, to measure PKG and p-VASP in heart tissue and serum, the infarct section of heart and serum sample were collected and wrapped in foil and stored in liquid nitrogen for Western blotting analysis.2, 1.6; MgSO4, 1.2; 3-[N-morpholino]-propanesulfonic acid (MOPS), 1.2; Na2HPO4, 1.4; EDTA, 0.02; D-glucose, 5.6; pH adjusted to 7.4 at 37°C. KCl (60 mmol/L)-PSS is a substituted PSS in which NaCl has been substituted by KCl (60 mmol/L) in an equal molar fashion.The treated rats were sacrificed and the left heart placed in a dissection dish filled with ice-cold (4°C) PSS solution (in mmol/L): NaCl, 140.0; KCl, 4.7; CaCl2; washout was performed by draining and replacing the bathing solution using a syringe.An integrated wire myograph system  was applied with a Motic SMZ168-TL stereomicroscope to dissect the blood vessels. The CA of LAD was dissected free, adhering connective tissue cleaned off, and cut the segments following the point of occlusion into 1-mm-long ring. The rings were mounted with 60-μm steel wires in separated tissue baths of the wire myograph system for tension recording. The tissue baths were filled with PSS solution (pH 7.4) at 37±1°C and aerated with O50 and Emax values.Isometric tension signals were recorded and data collected by a Powerlab data acquisition system . Each ring was stretched to an optimal tension of 1 mN and permitted to equilibrate for 90 minutes before the experiment started. Thirty minutes after setting up the wire myograph, each ring was initially contracted by KCl (60 mmol/L)-PSS and washed and the viability of the rings measured. Rings with twice amplitude of contraction less than 10% were used for the experiment. The rings were contracted by U46619 (1 μmol/L) and relaxed using cumulative addition of ACH (0.001–100 μmol/L) to test the endothelial-dependent vasodilation by calculating EC2+ 2% O2 + 93% N2) with medium free of glucose and serum at 37°C, followed by 12 h of re-oxygenation (5% CO2+ 95% air) in complete medium containing glucose and serum. At the end of the experiment, cell viability was examined using MTT assay as described below. Furthermore, cells of each group were also collected for real-time PCR assay, western blotting and ELISA assay as described in following sections.HCMECs were obtained from the Shanghai Yansheng Biochemical Technology Company  and grown in 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin antibiotics. Tightly confluent monolayers of HCMECs from 4th-15th passage were used in all experiments. In experiments checking the effects of SCU under normal condition, cells were treated with vehicle control (NS) or SCU at different concentrations or PKG activator  for 26 h. In experiments checking the effects of SCU under HR condition, cells were incubated with SCU at different concentrations or PKG activator  for 2 h prior to HR injury and during HR injury (24 h). Control cells were cultured in parallel and kept in normal culture condition for the entire time period (26 h). Simulated HR injury was induced according to previously described procedures  with min4 cells/ml and 90μl/well. Cells were cultured in normal culture condition or treated with simulated HR injury as described above. After the treatments, 20 μl of MTT (5 mg/ml) was added to each well and the plates were incubated for 4 h at 37°C. Then, 100 μl of lysis buffer  was added to each well and incubated overnight. Cell viability was determined by measuring the ability of metabolically active cells to convert the yellow tetrazolium salt MTT into purple formazan crystals with a Microplate Reader at 570 nm. Results of three independent experiments (each conducted in triplicate) were used for statistical analysis. Cell counting and trypan blue exclusion viability was performed on Countstar IC 1000 (Inno-Alliance Biotech) automated cell counter and the percentage of viable cells was obtained.For MTT assay, cells were plated in 96-well flat-bottomed plates at a density of 3×10Total RNAs were extracted using the TRIzol® reagent (Invitrogen) and reverse transcribed using PrimeScript® RT reagent Kit with gDNA Eraser (TaKaRa) according to the manufacturer’s instructions. Real-time PCR amplifications was performed using SYBR® Premix Ex TaqTM II (TaKaRa) and the thermal profile was 95°C for 2 min followed by 40 cycles at 95°C for 15 sec and 61°C for 20 sec. Each sample was measured in triplicate and the mean threshold cycle (Ct) value was calculated. Relative expression was calculated using the ΔΔCt method. The mRNA expression of GAPDH was used as an internal control. Primer sequences used for PKG-Iα analysis were 5’- GGCTGTCAGAGAAGGAGGAAG–3’ and 5’-GGAAGGACCTGTACGTCTGC–3’. Primer sequences used for GAPDH analysis were 5’-GGAGCGAGATCCCTCCAAAAT–3’ and 5’-GGCTGTTGTCATACTTCTCATGG–3’.For rat heart tissue homogenate samples, the preparation comprises following steps: after removal from liquid nitrogen, tissue wrapped in foil were hammered and transferred into a precooled glass homogenizer containing 200 μL precooled RIPA buffer and homogenized for 10 min, transferred to Eppendorf tube, and centrifuged at 18,000 rpm for 10 min at 4°C. The supernatants were collected and stored at −80°C until Western blotting analysis. For cell samples, cells were lyzed in RIPA buffer containing 50 mM Tris HCl (pH 7.4) with 150 mM sodium chloride, 1.0% Nonidet P–40, 1% sodium deoxycholate, 1.0% Triton X–100, 1 mM EDTA, and protease inhibitor cocktail . After centrifugation, the supernatant were collected and stored at −80°C until Western blotting analysis.Protein concentrations were determined using a BCA Protein Assay Kit . The protein samples were denatured by mixing with equal volume of 2 × sample loading buffer and then boiling at 100°C for 5 min. An aliquot (20 μg as protein) of the supernatant was loaded onto a 10% SDS gel, separated electrophoretically, and transferred to a polyvinylidene fluoride membranes . After the membrane was incubated with 10 mM TBS with 20% Tween 20 and 5% dehydrated skim milk to block nonspecific protein binding, the membrane was incubated with primary antibodies overnight at 4°C. The primary antibodies used were rabbit anti-PKG-I polyclonal antibody , rabbit anti-vasodilator-stimulated phosphoprotein (VASP) , rabbit anti-phosphorylated-VASP (p-VASP)  and rabbit anti-GAPDH. After TBS washes, blots were then incubated with secondary antibody for 1 h at room temperature at a 1:5000 dilution and then visualized using enhanced chemiluminescence (ECL) kit . The secondary antibodies used were horseradish peroxidase (HRP)-coupled anti-rabbit secondary antibody (Santa Cruz Biotechnology). The density values of bands were quantified by densitometric analysis of scanned images (Scion image 4.03). The relative protein ratio was calculated by determining the integrated intensity of the bands of each treated group as a ratio of the control condition.. The kit is primarily designed to measure the activities of PKG (including PKG-I and–II), and in which the phosphor-specific monoclonal antibody used has been demonstrated to recognize the phosphor-threonine 68/119 residues on substrate of PKG. Collecting HCMECs under normal and HR condition, determined the activities of PKG in the cells following the kit procedures.An enzyme-linked immunosorbent assay (ELISA) kit for PKG activity was supplied by CycLex Co., Ltd Cells were divided into four groups including control, HR model, SCU 1 μM, HR+SCU 1 μM group and underwent different treatments as described above. After treatments, cells were washed with ice-cold PBS and then scraped off with a cell scraper. After centrifugation for 5 min at 1500 g, the supernatant was discarded and the cell pellet was resuspended in lysis buffer containing 7 M urea, 2 M thiourea, 2% CHAPS and 1% DTT, 0.8% Pharmalyte, a mixture of Protease inhibitors  and incubated 30 min on ice. The lysed cells were centrifuged at 15000 g for 30 min at 4°C and the supernatants were collected for analysis. Protein sample (1mg) was reduced by incubating with 50 μl 100 mM DTT in 100mM ammonium bicarbonate for 45 min at 56°C. After cooling to room temperature, alkylation was induced by adding 250 μl 200mM IAA and incubating in darkness for 30 min at room temperature. After alkylation, equal volume of acetone was added to precipitate protein at -20°C for 30 min. After centrifugation at 15000 g at 4°C for 15 min, the supernatant was discarded and the protein pellet was washed twice by 75% alcohol. The protein sample was dried down by evaporation at room temperature in a vacuum concentrator/centrifugal evaporator and then dissolved in 1 ml 50 mM ammonium bicarbonate buffer. Digestion was conducted by adding 20 μg trypsin (1:50 enzyme:protein ratio) at 37°C overnight. After drying by evaporation at room temperature in a vacuum concentrator/centrifugal evaporator, the sample was redissolved with 900 μl 0.1% formic acid and the desalted by the Waters sep-pak Vac C18 Spin Column . The sample was dried by evaporation at room temperature in a vacuum concentrator/centrifugal evaporator and then stored at -80°C until mass spectrography (MS) analysis.The nanoHPLC-ESI MS/MS analysis was performed with a 4000 Q TRAP system  operated in the positive mode using the Nanospray® III Source and heated interface. Briefly, each protein sample (5 μl) was eluted onto a C18 reversed phase column  at a flow rate of 300 nl/min. Peptides were separated using a 60 min gradient ranging from 5% to 50% mobile phase B . By using the MRM Pilot software  to predict the mass signals which might be produced by the hydrolysates, a MRM initiated detection and sequencing scan (MIDAS) method was developed for detection of PKG-I α phosphopeptides in sequence of aa42-94. The MS analysis was performed on the 4000 Q TRAP system in information dependent acquisition (IDA) mode and the setting parameters were: ion spray voltage: 2300 V; curtain gas: 20; ion source gas: 15; and heated interface: 150°C. Protein samples from three independent experiments were analyzed. And, for each experiment of protein samples, triplicate technological repeat were performed to ensure reproducibility. The detected amounts of PKG I α phosphopeptides (peak areas) were further normalized using internal standard. The two un-phosphorylated peptides of PKG I alpha used as internal standards were ILMLKEER (516.3/2+) and EEEIQELKR (587.3/2+), aa10-17 and aa29-37, respectively. Briefly, the raw peaks were processed, and extracted ion chromatograms (XIC) were generated from the full-scan MS using MultiQuant 2.0.2 . Peak areas of PKG Iα phosphopeptides were normalized by peak areas of tryptic digested peptides of internal standards. The normalized phosphorylation rates were calculated as ratio of peak area of PKG I alpha phosphopeptides to peak area of the average of two un-phosphorylated peptides of PKG I alpha.50” value to be calculated. The Emax value represents the maximal vasodilative response . Comparisons were made using one-way ANOVA, two-way ANOVA analysis or unpaired t-test. P< 0.05 was considered statistically significant.Values were expressed as mean ± SEM. Statistical analysis was performed using statistical software Sigma Stat 3.1. Nonlinear regression analysis for individual concentration-response curves was performed using a Hill algorithm in Sigma Plot 10.0, allowing for an individual geometric ‘‘ECTo evaluate the effects of SCU on MIR injury and the role of PKG, myocardial ischemia area and endothelial-dependent vasodilation (as an ED index) were assessed. As shown in 50 of ACH . Therefore, SCU at doses of 1 and 10 μM was used in the present study. As shown in As shown in Since there is no specific antibody for PKG-Iα or PKG-Iβ, Western blotting assay was conducted using antibody against PKG-I. As shown in The effects of SCU on protein expression of PKG-I and phosphorylation of VASP under HR injury were shown in In addition, ELISA assay proved that SCU (1 and 10 μM) incubations significantly raised PKG activity in normal cultured HCMECs while PKG activator  used as positive control also increased PKG activity . HR treaTo clarify the role of PKG-Iα in IR injury and the protective mechanism of SCU, the phosphorylation states of PKG-Iα under normal and HR treatments were analyzed using MRM-MS . PKG-Iα phosphopeptides with phosphorylation at Ser44, Ser50, Thr58, Ser64, Thr69, Ser72, Ser89 were detected. The PKG-Iα phosphopeptides were listed in in vivo. In rats, MIR injury caused impairment in endothelium-dependent vasodilation, as assessed by ACH-induced dilation in isolated CA. A significant blunting of endothelium-mediated dilation was observed in MIR model group. The results were consistent to other reports supporting that IR injures endothelium and causes vascular ED VLPVPSTHIGPRTTR, fragment ions b3-b18 suggested that the phosphorylation site might be on the S44,S50,T51,T57 or T58 while a loss of −98 Da at b3 confirmed the phosphorylation site to be S44. (B) CQSVLPVPS[Pho]THIGPRTTR, the CID-MS/MS product ion spectrum consisted of both b- and y-type ions, of which the fragment ions suggested that the phosphorylation site might be on the S44,S50,T51,T57 or T58 while a loss of −98 Da at b9 and y10 confirmed the phosphorylation site to be S50. (C) CQS[Pho]VLPVPS[Pho]THIGPRTTR, the CID-MS/MS product ion spectrum consisted of both b- and y-type ions, of which the fragment ions suggested that the phosphorylation site might be on the S44 and S50, while a loss of −98 Da at b3 and y10 confirmed the phosphorylation site to be S44 and S50. (D) CQSVLPVPSTHIGPRTT[Pho]R, the CID-MS/MS product ion spectrum consisted of both b- and y-type ions, of which the fragment ions suggested that the phosphorylation site might be on the S44,S50,T51,T57 or T58 while a loss of −98 Da at y2 confirmed the phosphorylation site to be T58. (E) AQGIS[Pho]AEPQTYR, fragment ions b5-b12 suggested that the phosphorylation site might be on the S64,T69 or Y70, while a loss of −98 Da at b5 confirmed the phosphorylation site to be S64. (F) AQGIS[Pho]AEPQT[Pho]YR, the CID-MS/MS product ion spectrum consisted of both b- and y-type ions, of which the fragment ions suggested that the phosphorylation site might be on the S64,T69 or Y70, while a loss of −98 Da at y3 and b5 confirmed the phosphorylation site to be S64,T69. (G) S[Pho]FHDLR, fragment ions b1–b4 or y1–y4 suggested that the phosphorylation site might be on the S72, while a neutral loss of −98 Da at b2 confirmed the phosphorylation site to be S89,the b- and y-type ions which were detected in the CID-MS/MS product ion spectrum were highlighted in red. (H) S[Pho]KDLIK, fragment ions b1–b5 or y6 suggested that the phosphorylation site might be on the S89, while a neutral loss of −98 Da at b5 confirmed the phosphorylation site to be S89, the b- and y-type ions which were detected in the CID-MS/MS product ion spectrum were highlighted in red.Highlighted in red circle are the site ions that lost 98 Da. ((TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Jatropha curcas (physic nut), an inedible oilseed, is a potential source of bioenergy. The seeds, however, contain allergens such as Jat c 1, a 2S albumin that can induce hypersensitivity reactions in humans and result in allergic diseases. Recent advances in identifying and characterizing plant allergens and, in particular, their immunoglobulin E (IgE)-binding epitopes have produced a wealth of information. We identified IgE-binding regions and the critical amino acids involved in the degranulation of mast cells and the release of histamine, preliminary steps for the prevention and treatment of this allergy. Four IgE-binding regions were identified in the sequence of Jat c 1. We identified and demonstrated the fundamental role of two glutamic acid residues in IgE binding. The sequence LEKQLEEGEVGS produces a random loop on the most exposed part of Jat c 1. This region is important to the stimulation of the allergic response. The possibility of using this information to produce vaccines and other pharmacological agents for allergy treatment is discussed.Increasing energy demand has spurred interest in the use of biofuels. The online version of this article (doi:10.1186/s40064-016-2036-5) contains supplementary material, which is available to authorized users. The increasing demand for food and fuel as lifestyle expectations rise in a population expected to reach nine billion by 2050 will soon become global challenges , Brazil, and Jat c 1 was isolated and identified by SDS-PAGE and immunoblotting as described by Maciel et al. .2OH3 and 5 mg of nigrosin. This mixture was injected into the four foot pads of the animals (250 µL/foot pad). Five rats were anesthetized after 15 days and bled by cardiac puncture, and the titer was determined by indirect competitive ELISA. The sera were pooled, lyophilized and stored at −20 °C until used in the mast-cell degranulation assay.Jat c 1 (10 mg) in 500 µL of sterile 0.01 M phosphate-buffered saline (PBS) was emulsified with an equal volume of an adjuvant containing 5 mg of Al2O2, 6.5 ml of 0.1 M citric acid, 7.0 ml of 0.2 M sodium phosphate and 9.0 mL of distilled H2O. The reaction was stopped by adding 50 µL of H2SO4 per well. The plate was read at 492 nm.Microtiter-plate wells were each coated with 100 µL of 1 mg/mL Jat c 1 in 0.2 M sodium carbonate buffer, pH 9.6, and incubated for 18 h at 37 °C. The subsequent steps were performed at 37 °C. A microtiter plate was washed twice with PBS containing 0.05 % TWEEN 20 (300 µL/well for 1 h), 300 µL of blocking buffer (1 % gelatin in PBS containing TWEEN 20) were added to each well and the plate was incubated for 1 h. The plate was again washed with PBS containing 0.05 % TWEEN (300 µL/well for 1 h). After coating with Jat c c1, each well received 50 µL of antiserum diluted 1:500 in PBS containing 0.05 % TWEEN 20. IgE was detected by a 1:2000 dilution of goat anti-rat immunoglobulin (anti IgE) conjugated to peroxidase (Sigma) developed in 50 µL of a solution containing 10 mg OPD, 10 µL of 30 % HTwo blanks were included in the experiment. Blank 1 contained protein and the development solution to detect any intrinsic peroxidase activity in the samples, and Blank 2 contained carbonate/bicarbonate buffer, antibody and the development solution.Wistar rats were bred at the animal house of the Universidade Estadual do Norte Fluminense. All experimental procedures were approved by the university’s animal research ethics board (Protocol 112).2. The abdomens were gently massaged for approximately 90 s, and then an incision approximately 5 cm in length was cut in the peritoneal cavities, which were carefully opened. The cavities were washed prior to the injection of 20 mL of DMEM (Gibco) containing 12 U/mL of heparin, as described by Deus-de-Oliveira et al. (5 mast cells/mL) was separated into aliquots of 100 µL in Eppendorf tubes.For obtaining peritoneal mast cells, three rats (250 ± 20 g) were individually euthanized with COa et al. . The fluThe mast cells (100 µL) were incubated with 1 μL of R/A Tor rat pre-immune serum as a control or were initially sensitized for 1 h at 37 °C using 1 μL of anti-Jat c 1 polyclonal antibodies. After sensitization, the mast cells were washed twice with Dulbecco’s Modified Eagle Medium (DMEM) (Sigma) and were then incubated with 100 ng of synthetic peptides or 100 ng of native Jat c 1 or its isolated chains (100 ng) for 1 h at 37 °C. To evaluate the mast-cell degranulation, we mixed and incubated an aliquot of 10 µL of the cell suspension, after the various incubations, for 15 min with 10 µL of an aqueous solution containing 0.1 % toluidine blue, 10 % formaldehyde and 1 % acetic acid, pH 2.8. The intact and degranulated mast cells were counted in a Neubauer chamber. The four quadrants were microscopically viewed using Nomarski differential interference contrast with a Zeiss Axioplan optical microscope. Untreated mast cells were also incubated as a negative control with the dye under the above conditions and were observed with the optical microscope. The counting of intact and degranulated cells allowed an assessment of the acquisition procedure. Each experiment was performed in triplicate using mast cells from the same animals. Graphs of the results of the degranulation assays were constructed using GraphPad Prism.P < 0.001). This analysis was performed by GraphPad Prism.The data obtained in the degranulation assays were analyzed by one-way ANOVAs and multiple comparisons using Tukey’s tests  (Sigma) strategy , and mast-cell degranulation was evaluated as described below. A peptide was considered to be an IgE-binding region at levels of mast-cell degranulation >40 % , and substitution of strategic amino acid residues in the characterized IgE-binding-regions.N-ethyl-5-phenylisoxazolium-3′-sulfonate) (Sigma) is a specific reagent for the modification of dicarboxylic amino acids. The methodology proposed by Dunn et al. . We determined if these residues were critical in these epitopes by constructing a library of peptides substituting glutamic acid by leucine using the Fmoc strategy described above. All peptides were purified by C18 reverse-phase HPLC. The IgE binding of mutated peptides was analyzed by a mast-cell degranulation assay as previously described.IgE-epitope candidates were localized using JHg, modeled by Nair et al. . The modJ. curcas seeds. We also identified IgE binding-regions of Jat c 1 and searched for homologous sequences in allergenic proteins from other plants that trigger allergenic cross-reactions.We purified and fully characterized Jat c 1, a known allergenic protein from J. curcas seeds was obtained by saline extraction and chromatography on Sephadex G-50. Jat c 1 was then isolated by reverse-phase chromatography, as previously reported , two polypeptide chains were isolated by reverse-phase chromatography on a C18 column . The small chain aligned at position 33–61, and the large chain  aligned at position 72–103. The molecular weight determined by mass spectrometry suggested that the large chain continued to position 133  and second (large chain) Jac c 1 peaks were VRDICKKEAERRVLSSCENTITQRRGRSE and PRQQVPRQCCNQAKELSAICRCESIHYLLEKQ, respectively. Alignment and analysis of these sequences were obtained from the Jatropha Genome Database  showed approximately 60 % degranulation. Mast cells challenged with isolated Jat c 1 polypeptide chains produced similar results. The small and large chains produced 58 and 64 % degranulation, respectively, indicating the presence of continuous IgE epitopes in both chains  contained at least two glutamic acid residues. To verify the possible involvement of these residues in IgE binding, we treated P3 and the synthetic peptides that degranulated mast cells in the previous experiment  with WRK, a reagent known to modify glutamic acid. After WRK treatment, all peptides except P4 exhibited reduced mast-cell degranulation, with levels similar to those of the negative controls (~30 %). Mast-cell degranulation induced by P4 was 42 %, a value near a negative mast-cell degranulation reported by Deus-de-Oliveira et al. .To confirm the critical role of glutamic acid in IgE binding, we synthesized a series of related peptides substituting the strategic glutamic acid residues by leucine. The IgE-binding abilities of these modified peptides were evaluated by mast-cell degranulation assays. The substitution of glutamic acid residues by leucine in P5, P6 and P8 significantly reduced mast-cell degranulation to approximately 30 %. Substitution of glutamic acid residues in P1 and P4 decreased mast-cell degranulation to 40 %, similar to that of the negative control (Table J. curcas hemagglutinin (JcCA0234191), a protein homologous to Jat c 1, that was modeled by Nair et al.  within the three-dimensional structure of r et al. . P5 and r et al. .Fig. 7MaR. communis . Indeed, knowing the identity of IgE-binding epitopes is essential for the synthesis of hypoallergens employed in the development of such vaccines. Our evidence suggests that, in the case of Jat c 1, the potential IgE-binding epitopes should have their glutamic acid residues substituted by leucine to produce a hypoallergenic protein. The production of minitopes  from modified Jat c 1 IgE-binding peptides, where the strategic glutamic acid residues could be replaced by leucine residues, is a further ASIT possibility. A similar approach, the modification of critical amino acids in the epitope, was used successfully for the major birch pollen allergen Bet v 1 (Valenta and Niederberger Jatrophacurcas, Jat c1 was characterized. The IgE-binding peptides and the critical amino acids involved in mast cells degranulation and histamine release were also identified. Since characterization of allergen/IgE-binding is a fundamental step to produce proteins mutant hypoallergenic and vaccine based on peptides, our study contribute to improved efficacy/safety profiles for safer allergy vaccination.In this study an allergenic protein from


Combined BRAF and MEK inhibition may delay the onset of resistance compared with BRAF inhibition alone. The safety and tolerability of the BRAF and MEK inhibitors, vemurafenib and cobimetinib were evaluated in a phase 1B trial, BRIM7 (NCT01271803). Modulation of signaling pathways, transcriptional outputs and T-cell dynamics were assessed in tumor samples obtained from BRIM7.+ T-cells in tumor biopsies were assessed by immunohistochemistry. Changes in transcription profiles upon treatment were measured by qRT-PCR using the Fluidigm/Nanostring platforms.Tumor tissues were collected at baseline prior to treatment, on-treatment at cycle 1 day 14 (C1D14) and at disease progression (PD). Modulation of the MAPK and PI3K pathways, cell proliferation, and CD8133 tumor samples have been collected representing 82 unique patients. There are six paired biopsies (baseline and C1D14) from the same patients, and seven evaluable PD samples. In the six paired biopsies (baseline and C1D14), robust inhibition of the MAPK pathway was observed in tumors following vemurafenib + cobimetinib treatment as measured by reduction of pERK levels compared to baseline (mean H-score inhibition 88 +/- 12%). This was observed in samples from BRAF inhibitor-naïve patients (n=2) and also from patients who had progressed while on vemurafenib (n=4). The inhibition of pERK correlated with the reduction of the proliferation marker Ki67 in C1D14 tumor samples (77 +/- 10%), while inhibition of the PI3K pathway marker pS6 showed greater variability (68 +/- 28%). At PD, varying levels of MAPK pathway activity and moderate-to-high expression of Ki67 were seen in tumors. Ongoing analyses of combination treatment effects on the MAPK pathway, immune regulatory gene transcription and CD8+T-cell status will be presented.The combination of vemurafenib + cobimetinib results in MAPK pathway inhibition in both BRAF inhibitor naïve patients and patients who have progressed on vemurafenib. Increased levels of pERK and Ki67 at PD suggest the renewal of proliferation as tumors may have escaped the inhibitory effects of the combination therapy. Effects of the combined treatment on the MAPK pathway and immune gene signatures will also be discussed.