Endoscopic submucosal dissection (ESD) is challenging in the right colon. Traction devices can make it technically easier. In this study, we evaluated a flexible grasper with articulating tip and elbow-like bending (IgE) through a double-balloon surgical platform (DESP), compared with an earlier generation grasper without elbow-like bending (Ig). The reach of Ig/IgE was investigated at eight locations using a synthetic colon within a 3D model. Using a fresh porcine colorectum, 4 cm pseudo-polyps were created at the posterior wall of the ascending colon. Fifty-four ESD procedures were performed using three techniques: standard ESD (STD), ESD using Ig (DESP + Ig), and ESD using IgE (DESP + IgE). IgE was able to reach the full circumference at all the locations, whereas the medial walls proximal to the descending colon were out of Ig’s reach. Compared with the STD, both DESP + Ig and DESP + IgE showed significantly shorter procedure time (STD vs. DESP + Ig vs. DESP + IgE = median 48.9 min vs. 38.6 vs. 29.9) and fewer injuries (1.5 vs. 0 vs. 0). Moreover, the DESP + IgE had a shorter procedure time than the DESP + Ig (p = 0.0025). The IgE with DESP increased instrument reach compared to Ig, and likely represented a traction tool for excision of large pseudo-polyps in the right colon. However, ESD has not been adopted widely due to technical difficulties, longer procedure times, higher complication rates, and longer learning curves, especially for lesions located in the right colon9. As a result, more than 25,000 colectomies for non-malignant polyps are performed annually in the United States10.Endoscopic submucosal dissection (ESD) has been established as an acceptable technique to treat premalignant colorectal lesions11. Various traction devices have been proposed to reduce procedure times and complication rates13. However, most devices are limited in their ability to navigate to the right colon or maintain dexterity of the instruments once situated in the right colon14.A traction device can increase the efficiency and safety of ESD procedures by maintaining visualization of the submucosal plane2, Lumendi LLC, Westport CT USA) is one example of a platform used to advance the endoscope to the lesion, improve stability of the colon, and maintain visualization of the lumen throughout the colon15. The endoluminal platform also allows the use of dedicated instruments  through two external tool channels. The endolumenal interventional grasper with an articulating distal tip  provides a good traction during endoluminal procedures16, however, Ig is not necessarily able to reach the lesion or provide traction in all directions due to the limitation of its movement in clinical cases. To resolve these issues, the endolumenal interventional grasper with Elbow  adds an elbow-like bending section more proximally. In this study, we evaluated the efficacy of a novel flexible grasper with an articulating distal tip and elbow-like bending section (IgE) in a preclinical model, compared with Ig.A double-balloon endolumenal surgical platform , which is fixed behind the endoscope tip, provides stability for the endoscope. A “therapeutic zone (TZ)” is created by extending the Fore Balloon (FB) beyond the endoscope tip. This adjustable FB improves luminal visualization by flattening folds and straightening flexures. Both balloons are inflated by a pump on the inflation handle16. Ig and IgE instruments consist of a pistol style handle and flexible shaft with an articulating jaw at the distal tip  with two independently inflatable balloons and bilateral external tool channels , equipped with DESP, was navigated through a 3-D synthetic colon model  Fig. . Eight t16. The pseudo-polyp was placed on the posterior wall of the ascending colon. Three different ESD techniques were evaluated and compared:Standard Cap-assisted ESD technique (STD group)ESD with DESP and Ig (DESP + Ig group)ESD with DESP and IgE (DESP + IgE group)In this study, we used ex-vivo porcine colons and no live animals were used. Using electrocautery, 4 cm pseudo-polyp was created in a fresh porcine colorectum and the intestine was positioned within an established 3D colon model  and one endoscopist performs EMR (> 100 procedures); these endoscopists were defined as the experienced group. The other three endoscopists only had ESD experience in ex-vivo models (> 10) and were defined as the novice group.A straight transparent cap  was attached to a pediatric colonoscope. The colonoscope, equipped with/without DESP, was advanced to the cecum. Abdominal pressure and use of the balloon technique were applied to reduce loops and shorten the colon at the endoscopist’s discretion.After navigation to the cecum, the FB and AB were deployed proximal and distal to the lesion. The FB was docked during the procedure at the endoscopist’s discretion. Normal saline with 0.04% methylene blue was used for submucosal injection by injection needle . The same settings (Endo Cut Q mode) of the ERBE electrosurgical unit  was used for all parts of the ESD procedures. Using a DualKnife , a circumferential mucosal incision was made around the lesion with a 5 mm margin Fig. .The primary outcomes were the reach of the Ig/IgE and the total procedure time for ESD procedures. Total procedure time was defined as the time from submucosal injection to the removal of the specimen. Secondary outcomes included the cecal intubation time, resected specimen size, grasping time (for DESP + Ig and DESP + IgE groups), submucosal dissection time/speed, the number of muscularis propria injuries, and occurrence of perforations. Submucosal dissection speed was calculated as follows: (major radius) x (minor radius) π/ (dissection time). Grasping time was defined as the time from Ig/IgE insertion through the tool channel to grasping of the lesion.Sample-size calculation of ESD procedures in this study was as follows; at least 16 procedures were required to detect a decrease in total procedure time from 42 min in the Ig group to 35 min in the IgE group, with 80% power to show a significant difference between groups and 5% type I error. Each endoscopist performed three ESD procedures using each technique.p values < 0.05 were considered to be significant.All variables are expressed as a median. The associations between continuous parameters were evaluated by the Mann–Whitney U test to compare two groups and by the Kruskal–Wallis test to compare more than two groups. Variables with p value < 0.05 in the Kruskal–Wallis test were subjected to multiple comparisons using the Steel–Dwass method. All analyses were performed using JMP Pro 14 statistical DiscoveryTM . Ig was able to reach the full circumference of the synthetic colon at the rectum and the sigmoid colon via both the right and left channels Fig. . Howeverp = 0.60, Table. A total of 36 colonoscopies were performed (18 without DESP and 18 with DESP). The cecal intubation rate was 100% with no perforation, regardless of the use of DESP. There was no difference in the navigation time ; this reduction was driven by significantly reduced submucosal dissection time , DESP + Ig (n = 18), and DESP + IgE (n = 18)). All lesions were successfully resected p = 0.025), shorter submucosal dissection times (p = 0.022), and smaller volumes of submucosal injection fluid (p = 0.0069) (Table. p = 0.33). However, the IgE showed a smaller number of times the lesion was re-grasped compared to Ig .Regarding the difference between DESP + Ig and DESP + IgE groups, the DESP + IgE group showed significantly shorter total procedure times p = 0.05, shortep = 0.030, Fig. p = 0.63), total procedure time , and the number of muscularis propria injuries .On subgroup analysis of ESD procedures using DESP + IgE performed by the experienced (n = 9) and the novice (n = 9) endoscopists, grasping time was significantly longer in the experienced group than the novice group  allow for the removal of large polyps in the right colon with a significantly shorter dissection time, total procedure time, and fewer injuries than standard ESD technique in an ex vivo porcine model. Moreover, IgE provided a broader reach of the grasper and decreased ESD procedural times and submucosal injection volumes. Finally, this surgical platform and these tools may help bridge the gap of skills in ESD procedures between the expert and the novice.17. In this study, the Ig instrument could not reach behind each flexure. Moreover, the same difficulty also occurred in the ascending colon, the transverse colon, and the descending colon since the overtube device itself made the wide intestinal arch. The elbow bending function can resolve this issue, allowing the IgE to reach everywhere in the large intestine.In order to access the more proximal colon, endoscopes and devices pass along the outer side of the intestinal curves and loops, leading to limited access to inner lesions, especially lesions located just beyond the splenic flexure and the hepatic flexure18. Similarly, the advantages of the double-balloon platform to improve the endoscope operability in the right colon were not demonstrated in this simplified model19.In this study, each procedure was able to successfully reach the cecum. Navigation of the colonoscope equipped with DESP may be more difficult as it required assistance with abdominal pressure, unlike standard colonoscopy. However, the simplified colon model does not allow demonstration of the advantages of the balloon technique that assists navigation under more challenging situations21. The clip-nylon method and the suture/clip method utilizing the FB of the double-balloon interventional platform (DEIP) can provide real-time traction, but only in a single dimension, proximal and distal/back and forth23. Robotic platforms have not been applied to the right colon yet, though new technology might resolve this limitation24.Traction devices have been introduced to provide tension and counter-tension to facilitate resection of larger lesions, more complex lesions, and lesions located in difficult locations. However, few traction devices can be used anywhere in the large intestine, provide real-time dynamic traction in all directions, and move independently from the endoscope, allowing for continuous direct visualization of the target plane of submucosa. The S–O clip , traction wire  and elastic band  can be used in the right colon and provide traction in all directions, but it cannot be repositioned during the procedure25.The dexterity of IgE led to significantly shorter dissection time and shorter total procedure time compared to using Ig. First, IgE was able to reach the targeted site, even the inner side at the ascending colon which was out of the Ig’s reach Fig. c. Second24. Using the grasper during mucosal incision may further emphasize the benefits of this instrument. Moreover, the use of an additional knife/scissors in combination with IgE might also be helpful. These multi-instrument techniques should be investigated further. After tumor resection, closure of large mucosal defects (> 20 mm) in the right colon is recommended3. We believe the ease of mucosal closure would also greatly facilitated using the grasper to approximate the two edges of the defect26.This flexible grasper was not used during mucosal incision; in fact, there was no significant difference in mucosal incision time between the three techniques. This grasper has the potential to make mucosal incision easier by providing stability and tension, as observed with endoscopic models of robotic surgeryIn the United States, ESD has been less widely adopted, compared to East Asian countries, since the lower incidence of gastric cancer forces endoscopists to start with the more challenging colorectal tumors, leading to the increased risk and a longer time to acquire skills in ESD. In our study, there was no difference in ESD procedural outcomes using DESP + IgE between the experienced and the novice groups. The novice could perform ESD efficiently and safely as well as the expert. This surgical platform has the potential to bridge the gap between the expert-novice differences, and shorten the learning curve of ESD.A synthetic colon, which keeps the shape compared to human colons, was used to investigate the reach of Ig/IgE. The function of Ig/IgE might be underestimated since we could collapse the colon clinically in grasping the tissue. Nevertheless, we experienced Ig wasn’t able to reach the lesion in certain locations.(2) The ex-vivo model in this study has no peristalsis or bleeding, so performing ESD may be more difficult clinically. On the other hand, the simplicity of the model may underestimate the benefits of DESP and IgE grasper. A similar double-balloon platform, DEIP, is clinically used in the right colon and has demonstrated the benefits of the double-balloon platform in navigation. Traction devices have also been used for ESD and demonstrated clinical advantages. However, DESP, which has two more external channels, creates a larger device and its application may be limited in patients who have a narrowed colon due to diverticulitis and inflammatory bowel diseases.(3) All lesions were 4 cm in diameter and placed at the posterior wall of the ascending colon to limit variability. The use of this traction device will need to be evaluated for resection of other challenging lesions and lesions at other challenging locations, such as circumferential lesions or lesions at the hepatic flexure or under a fold. Furthermore, this platform cannot be rotated in the proximal colon; therefore, it may be more difficult to excise the lesions located at the anterior or lateral walls.(4) IgE may require additional time to obtain a good traction since it is necessary to articulate both distal tip and elbow-like bending section. This also highlights the need for multiple endoscopists to perform ESD and to articulate the grasper, and an assistant to secure the platform and inflate/ deflate the balloons.27.(5) The procedure time may be exaggerated by the use of normal saline as the submucosal injection fluid, not a long-lasting gel. The need for repeated injections with longer procedures is exaggerated with the use of short-acting injection fluid. Additionally, the need for repeated injections further increases the total procedure time9. Our previous study showed the experience in five ESD procedures might be enough in an ex-vivo model16.All participants are advanced endoscopists and participants familiar with the ex-vivo research model and using this endolumenal platform. Therefore, this study didn’t investigate the learning curve of this platformThis was a preclinical study. No other traction comparator was done. Larger human feasibility studies are warranted to validate these results.There are several limitations in this study.In conclusion, we demonstrated a novel flexible grasper with DESP likely represents an important traction tool for removal of large pseudo-polyps in the right colon. This new technology has the potential to expand the capacity for endoluminal resection, leading to lower colectomy rates for complex polyps.Supplementary Video 1.Supplementary Video 2.


Sound localization relies on minute differences in the timing and intensity of sound arriving at both ears. Neurons of the lateral superior olive (LSO) in the brainstem process these interaural disparities by precisely detecting excitatory and inhibitory synaptic inputs. Aging generally induces selective loss of inhibitory synaptic transmission along the entire auditory pathways, including the reduction of inhibitory afferents to LSO. Electrophysiological recordings in animals, however, reported only minor functional changes in aged LSO. The perplexing discrepancy between anatomical and physiological observations suggests a role for activity-dependent plasticity that would help neurons retain their binaural tuning function despite loss of inhibitory inputs. To explore this hypothesis, we use a computational model of LSO to investigate mechanisms underlying the observed functional robustness against age-related loss of inhibitory inputs. The LSO model is an integrate-and-fire type enhanced with a small amount of low-voltage activated potassium conductance and driven with (in)homogeneous Poissonian inputs. Without synaptic input loss, model spike rates varied smoothly with interaural time and level differences, replicating empirical tuning properties of LSO. By reducing the number of inhibitory afferents to mimic age-related loss of inhibition, overall spike rates increased, which negatively impacted binaural tuning performance, measured as modulation depth and neuronal discriminability. To simulate a recovery process compensating for the loss of inhibitory fibers, the strength of remaining inhibitory inputs was increased. By this modification, effects of inhibition loss on binaural tuning were considerably weakened, leading to an improvement of functional performance. These neuron-level observations were further confirmed by population modeling, in which binaural tuning properties of multiple LSO neurons were varied according to empirical measurements. These results demonstrate the plausibility that homeostatic plasticity could effectively counteract known age-dependent loss of inhibitory fibers in LSO and suggest that behavioral degradation of sound localization might originate from changes occurring more centrally. Accurately locating a sound source is one of the most fundamental functions of the auditory system. Behavioral studies report that sound localization ability generally degrades with aging in both laboratory animals and humans. Anatomical studies in aged animals have observed considerable reductions of inhibitory inputs into the neuronal circuit responsible for sound localization. Paradoxically, physiological studies at the brainstem level, however, find only minor age-related changes in the function of the sound localization circuit. In order to bridge the puzzling discrepancy between these observations, we developed a model of the corresponding neuronal circuit and simulate its function by varying the number of inhibitory afferents. To simply mimic the activity-dependent adjustment of synaptic inputs, we also varied the strength of remaining inhibition. Consistent with the empirical findings, our simulations demonstrate that functional changes of the sound localization circuit remain relatively small, compared to the reduced number of inhibitory inputs. This functional robustness is further enhanced by adjusting the synaptic strength. These results suggest that the age-related degradation of sound localization behavior might be caused by altered functions of higher stages in the auditory pathways above the level of the brainstem. The ability to accurately locate the source of a sound, or sound localization, is a fundamental property of the auditory system, required for exploring the acoustic environment, performing effective communication, avoiding danger, and finding pray or mates ,2. MammaBehavioral studies have consistently showed that binaural hearing, including sound localization, generally degrades with age both in humans   model of LSO. The model was tuned to replicate known physiological response properties of LSO neurons . The terV as well as the activation of the KLVA conductance are described each by a first-order differential equation. When the membrane potential reaches or exceeds the threshold Vθ, an output spike is counted and the model is in an absolutely refractory period of Tref, in which no more spikes are counted. In addition, a spike-associated current Ispike(t -Tθ) is initiated at each threshold crossing to simulate a spike-like trajectory of the membrane potential.The model equations and the corresponding parameter values are summarized in V reaches the threshold Vθ, a spike is counted and then the membrane potential V is fixed to the reset potential V0 for a refractory period of Tref. The passive IF model was used only in the specific subsection titled "Binaural tuning simulated with passive IF model" in the Results. In all other sections, we used the active IF model described above.In addition to the active IF model, we also used a passive IF model to confirm the generalizability of our results. The parameters of this model  were takαex or αinh), and is then summed into the total excitatory (gex) and inhibitory (ginh) synaptic conductances. The spiking activity of the excitatory and inhibitory input fibers  and the degree of phase-locking VS(fm), with fm being the modulation frequency. We assumed that all the inputs are mutually independent and that, in phase-locked cases, all excitatory inputs are locked to one single phase and the inhibitory inputs are locked to another. See [We adopted the same configurations for the excitatory and inhibitory synaptic inputs to the modeled LSO neuron as in our previous study . The modher. See  for the her. See .ILD-tuning of the LSO model for non-modulated tones was simulated by varying the contralateral sound intensity level that determines the inhibitory synaptic input intensity . The ipsEnvelope phase-tuning of the LSO model for AM tones was simulated by varying the relative phase between the ipsilateral (excitatory) and contralateral (inhibitory) inputs. Namely, the difference between the locking phases of both inputs was varied from -180 to +180 degrees. A positive phase means that inhibitory inputs arrive earlier than excitatory inputs. Unless otherwise noted, the modulation frequency was fixed to 300 Hz, at which the effect of amplitude-modulation is highly prominent .D is defined as the spike rate difference between two stimulus conditions normalized by the mean standard deviation of the spike rates [μ and σ are the mean and standard deviation of the spike rate for each stimulus value, respectively. The discriminability can be theoretically related to the Fisher information IF(S) as: S denotes the stimulus value and ΔS is the difference between two neighboring stimulus values . In othThe Fano factor, which is the spike count variance in a fixed period divided by the average number of spikes, is often used to characterize the variation of spiking response. LSO neurons typically have a Fano factor near or slightly below 1, indicating somewhat more regular spiking activity than the Poisson process . Our siminh of inhibitory input to the model LSO neuron was fixed to 8  condition, the number Mxed to 8 , which iinh = 8 to 4, for example, the unitary amplitude was doubled from Ainh = 12 to 24 nS to compensate the loss. In the overcompensated case, the amplitude of the unitary inhibitory input was multiplied by a factor (2—Minh/8), so that the total inhibitory input conductance linearly decreased with the number of inputs. For instance, the total inhibitory input became 50% larger than the default value when the number of inputs was 4. Even in these compensated and overcompensate cases, all model parameters other than the number and amplitude of inhibitory inputs were unchanged throughout the simulations.In order to examine the effects of inhibition loss, we considered three cases named "uncompensated", "compensated", and "overcompensated". In the uncompensated case, only the number of inhibitory inputs was changed, while all the other parameters were kept unchanged. In the compensated case, the unitary amplitude magnitude of inhibitory conductance was varied according to the number of remaining inhibitory inputs so that the total amount of inhibitory conductance remained unchanged. In this second case, when the number of inhibitory inputs was halved from MWhile the spike rate of an LSO neuron monotonically decreases with ILD e.g., , the locFor each of the LSO neurons, we next assumed a "mirrored neuron" whose ILD-tuning function is a flipped image of the original tuning curve about the midline (zero ILD). This mirrored neuron is the delegate of a similarly-tuned neuron in the contralateral LSO. We then calculated the spike rate difference between the original and mirrored neuron to obtain the bilateral rate difference . We note that the number of neuron pairs used would not affect our conclusion, since the calculated discriminability was all normalized to eliminate the influence of the population size.LSO neurons change their spike rates according to binaural sound level differences and to binaural envelope phase differences. As demonstrated in our previous study , these fIn response to binaural AM sounds, the model LSO generates many spikes when phase-locked excitatory inputs precede inhibitory inputs . In suchTo simulate age-related loss of inhibition, we changed the number of inhibitory inputs to the model LSO and examined how binaural level- and phase-tuning curves are altered. First, we considered an "uncompensated" case, in which only the number of inhibitory inputs was varied from the default value of 8, while all other model parameters remained unchanged . This siinh = 8 inputs to -17.3 dB for Minh = 4 inputs). This shift of midpoints qualitatively agrees with previous experimental findings [D was averaged over the entire ILD range tested and normalized to its value for Minh = 8 inputs. The curve of this normalized discriminability monotonically increased with the number of inhibitory inputs and saturated  and only marginal for larger numbers of inputs (Minh ≥ 4). As a result, the simulated modulation depth showed a prominent change for 0–4 inputs but was almost constant for the range above 4 inputs (inh = 8), the curves for the modulation depth and the discriminability in the compensated case were even shallower than in the uncompensated case (gray dash-dotted lines). These observations indicate that loss of a small to moderate number of inhibitory fibers does not impair binaural tuning in LSO.In comparison to the uncompensated case shown in , effects4 inputs . Around D , however, (over)compensation did not improve the performance, because the overall rate increase in the uncompensated case counteracted the decrease of the modulation depth. In sum, our simulation results suggest that activity-dependent plasticity may effectively counteract the age-dependent loss of inhibitory fibers projecting to the LSO, and that this robustness of binaural tuning does not strongly depend on the exact amount of compensation.inh = 8 inputs , and slTo test if the existence of this KLVA conductance affects our conclusion of the robustness of binaural tuning in LSO, we repeated the same simulations using a passive IF model without any active conductances see . DespiteD of each LSO pair  of the ABR, which is presumed to reflect the reduced efficacy of inhibition in LSO ,32. Our Previous behavioral studies demonstrated that aged human subjects show more difficulties in sound lateralization tasks relying on interaural time disparities than in those using level differences ,75. Our Measurements of monaural and binaural ABRs suggest a general reduction of synchronized neuronal activity along the auditory pathways in aged animals . ComparaBased on anatomical measurements in cats , we assuWe used modulation depths and neuronal discriminability functions to estimate the performance of binaural processing in the model LSO. While these measures are supposed to reflect the encoding properties of LSO neurons, it is still unknown how the outputs of LSO neurons and other brainstem nuclei are combined in higher auditory areas that "decode" the binaural information to form the perception of sounds . In our comparisons across different number of inhibitory inputs, we took a population average of the discriminability and normalized it to the default condition . This avIn combination with prior physiological observations ,26, our


The conjugated planar rings of tyrosine and tryptophan may interact with histidine within the active site to provide stability upon enzyme-peptide binding. This postulation was later confirmed as the Lineweaver–Burk analysis had identified DIP1 as a competitive inhibitor and DIP1 also showed 36.27 ± 1.17% of copper chelating activity. In addition, DIP1 provided the highest SPF value (11.9 ± 0.04) as well as ferric reducing antioxidant power (FRAP) (5.09 ± 0.13 mM FeSO4), 2,2′–azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) (11.34 ± 0.90%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (29.14 ± 1.36%) free radical scavenging activities compared to other peptides. These results demonstrated that DIP1 could be a multifunctional anti-tyrosinase agent with pharmaceutical and cosmeceutical applications.Nature-derived tyrosinase inhibitors are of great industrial interest. Three monophenolase inhibitor peptides (MIPs) and three diphenolase inhibitor peptides (DIPs) from a previous study were investigated for their in vitro tyrosinase inhibitory effects, mode of inhibition, copper-chelating activity, sun protection factor (SPF) and antioxidant activities. DIP1 was found to be the most potent tyrosinase inhibitor (IC The subsequent oxidation and polymerization of quinones leads to the formation of melanin, which is responsible for hair, skin and eye pigmentation in human and animals. This ubiquitous pigment plays a crucial role in photoprotection by forming supranuclear melanin caps in keratinocytes to protect the cells against ultraviolet (UV)-induced DNA damages and mutations  ranging from 0.125 to 0.5 mM. The initial velocity, V of the reaction was determined using the Beer–Lambert law:The mode of inhibition of peptides was subsequently determined using the Lineweaver–Burk plot of 4∙5H2O and lastly 6 µL pyrocatechol violet were added to a 96-well plate. The sample solution was mixed well and the absorbance was measured at 632 nm using a spectrophotometer . Ethylenediaminetetraacetic acid (EDTA) was used as the positive control. The copper chelating activity is calculated as follows:controlA denotes the absorbance of the system containing sodium acetate buffer, pyrocatechol violet and CuSO4∙5H2O whereas sampleA denotes the absorbance of the system containing peptide, sodium acetate buffer, pyrocatechol violet and CuSO4∙5H2O.The copper chelating activity was determined according to Kubglomsong et al. . BrieflyThe PepFold3 web server  was usedThe X-ray crystal structure of mushroom tyrosinase (PDB ID: 2Y9X) was obtained from the Protein Data Bank (PDB) . AdditioCF denotes correction factor of 10 (for the short pathlength used); EE denotes erythemal effect spectrum; I denotes solar intensity spectrum and Abs denotes the absorbance of sample.The ability of peptides to absorb light in the UVB region (290–320 nm) was measured according to the methods described by Dutra, Kedor-Hackmann and Santoro . The UV–The values of EE×I were normalized to 1 by Sayre, Agin, LeVee and Marlowe  i.e. the3⸱6H2O in the ratio of 10:1:1 was incubated at 37 °C for 30 min. Then, 200 µL of FRAP reagent was added to 2.7 µL peptide sample (10 mg/mL) in a 96-well plate and mixed well before incubating at 37 °C for 1 h. The absorbance of the sample was then measured spectrophotometrically at 593 nm. Freshly prepared FRAP reagent was used for each run. A standard curve was prepared using FeSO4⸱7H2O at 0–3 mM. The peptide FRAP was expressed in mM FeSO4. Gallic acid was used as the positive control.The reducing potential of peptides was measured according to Benzie and Strain . ferric controlA denotes the absorbance of the system containing ABTS and water whereas sampleA denotes the absorbance of the system containing ABTS and peptide sample.The free radical scavenging potential of peptides was measured according to Cai, Luo, Sun and Corke . BrieflycontrolA denotes the absorbance of the system containing DPPH and ethanol whereas sampleA denotes the absorbance of the system containing DPPH and peptide sample.The free radical scavenging potential of peptides was measured according to Blois . Brieflyp-value < 0.05 implies a significant difference between the sample means. Three independent experiments were performed in triplicates each and the results were reported as mean ± standard deviation.Statistical analysis was conducted using SPSS version 20.0 . The results were analysed using one-way ANOVA followed by Duncan’s test. 50 < 10 µM were found to comprise of only 2–3 residues [50 values < 0.36 mM and 14 µM, respectively [The effects of MIPs and DIPs on the monophenolase and diphenolase activities of tyrosinase were shown in residues ,24. Thusectively ,26.50 determined was 3.04 ± 0.39 mM. Results from kinetic studies suggested this peptide as a competitive inhibitor  b and thenhibitor . Howevernhibitor b. In connhibitor . DIP3, onhibitor b. The panhibitor . A simile enzyme ,28.o-dopaquinone functional group. The acceptance of hydrogen reduces o-dopaquinone back to ʟ-DOPA hence hindering the subsequent melanogenesis steps. On the other hand, the high copper chelating activity of DIP1 was accompanied by high diphenolase inhibition  lower monophenolase inhibition and copper chelating activities than MIP1. The large molecular weight and bulky side chains of MIP2 may reduce its diffusion towards the active site due to large steric hindrance. On the other hand, MIP3 did not interact with any of the tyrosinase hotspots and was bound to a groove at the back of the enzyme active site with a binding energy of −7.0 kcal/mol  or single electron transfer (SET) mechanisms [The antioxidant activities of MIPs and DIPs were shown in chanisms ,44. Nonechanisms . Howeverchanisms .Based on the results, MIPs and DIPs showed various degrees of tyrosinase inhibition using different mechanism of actions. The peptides also showed copper chelating activity, antioxidant activity and provided sunscreen effects. Notably, the strong copper chelating activity of MIP1 and MIP3 resulted in only the modest inhibition of monophenolase activity, suggesting an alternative mechanism of inhibition. The large molecular weight peptide (such as MIP2) may not be a favourable characteristic for the inhibition of monophenolase reaction and copper chelation. Similar results were observed for diphenolase peptide inhibitors where DIP2 and DIP3 showed lower inhibitory effects on tyrosinase. DIP1 was found to be the most potent diphenolase reaction peptide inhibitor in which it showed a competitive mode of inhibition towards tyrosinase. The strong inhibitory effect of DIP1 may be attributed to its ability to chelate copper ion within the enzyme catalytic site. In addition, DIP1 also showed the highest UV absorption and antioxidant activities compared to other peptides. The strong activities of DIP1 could be due to the presence of aromatic amino acid residues such as tyrosine and tryptophan within the peptide sequence. The planar ring structures of the peptide side chains may effectively absorb UV or even interact with those from the histidine residue in the tyrosinase catalytic site to confer stability upon the enzyme-peptide binding. Therefore, the enrichment of peptides with aromatic residues such as Tyr, Phe, Pro, His and Trp may provide positive effects to their tyrosinase inhibitory and antioxidant activities ,48.The strong tyrosinase inhibitory activity of DIP1 may find potential applications in the cosmeceutical industry as a natural-derived multifunctional anti-tyrosinase agent. However, the transdermal absorption of peptide represents the major challenge for commercialization. The molecular weight of < 500 Da is ideal for skin delivery , however


Emerging ice-sheet modeling suggests once initiated, retreat of the Antarctic Ice Sheet (AIS) can continue for centuries. Unfortunately, the short observational record cannot resolve the tipping points, rate of change, and timescale of responses. Iceberg-rafted debris data from Iceberg Alley identify eight retreat phases after the Last Glacial Maximum that each destabilized the AIS within a decade, contributing to global sea-level rise for centuries to a millennium, which subsequently re-stabilized equally rapidly. This dynamic response of the AIS is supported by (i) a West Antarctic blue ice record of ice-elevation drawdown >600 m during three such retreat events related to globally recognized deglacial meltwater pulses, (ii) step-wise retreat up to 400 km across the Ross Sea shelf, (iii) independent ice sheet modeling, and (iv) tipping point analysis. Our findings are consistent with a growing body of evidence suggesting the recent acceleration of AIS mass loss may mark the beginning of a prolonged period of ice sheet retreat and substantial global sea level rise. It may have taken only a decade to repeatedly destabilize the Antarctic Ice Sheet after the last Ice Age as shown by a new data-model study. The ice sheet lost ice for centuries each time before it abruptly re-stabilized again. The future evolution of the AIS represents the largest uncertainty in sea-level projections of this and upcoming centuries2. The West Antarctic Ice Sheet (WAIS) holds an equivalent of 4.3 m global mean sea-level rise (GMSLR), of which 3.4 m rests on a bed below present sea level. The marine-based Wilkes Land basin of the East Antarctic Ice Sheet (EAIS), which holds an equivalent of 3–4 m GMSLR, also has sectors exposed to marine ice-sheet instability that have the potential to discharge ice for centuries to millennia3.Deciphering past sea-level changes during times of natural climate warming can be useful for understanding the consequences of current and future human-induced climate warming. Here, the role of the Antarctic Ice Sheet (AIS) as the largest remaining ice sheet on Earth is crucial, yet remains poorly understood4. Invoking additional processes such as hydrofracturing and ice-cliff failure, AIS sea-level contributions could be up to ~1 m by the year 2100, and up to ~16 m by the year 2500 in higher CO2 emission scenarios5. However, recent reassessment shows that these mechanisms are not necessarily needed to explain geological and historical mass loss. Models that do not include them have a more modest GSLR projection for the 21st century with an AIS contribution of just 10–20 cm, under representative concentration pathway 8.58.Basal ice-shelf melting induced by a warming ocean has been identified as a key trigger of marine ice sheet instability, leading to enhanced ice flow across the grounding line9. The natural warming following the last Ice Age—the deglaciation—lasted from ~19,000 to ~8000 years ago and was accompanied by a GMSLR up to 134 m10. Sea level did not rise uniformly but in distinct Meltwater Pulses (MWP) such as MWP-19K, MWP-1A—when sea level rose ~16 m over ~360 years11—and MWP-1B. Since the AIS most likely contributed to these periods of rapid sea level rise, and since future AIS collapse could be substantial, approaching deglacial rates, studying processes of ice decay during the last deglaciation in pristine data archives can be extremely valuable to better understand the present state and future course of the AIS.Geological evidence can provide the pace, timing and source of previous contributions from the AIS and thus offer a context for these predicted future changes. For the mid to late Holocene, sea level can be considered more or less stable, with decimeter-scale fluctuations13 and the past15. In this model, freshwater induced variations in deep-water formation regulate the Atlantic meridional overturning circulation and related heat transport into the Southern Hemisphere. As a consequence of reduced vertical mixing in the circum-Antarctic Southern Ocean, upwelling Circumpolar Deep Water does not lose its heat to the atmosphere as effectively, thereby intensifying the subsurface heat transport and intrusion beneath Antarctic ice shelves16, where it contributes to melting the AIS.During deglaciation the AIS might have lost ice mass in response to three global environmental drivers or a combination thereof: subsurface ocean warming, atmospheric warming due to teleconnections to the equatorial Pacific, or sea-level forcing through Northern Hemisphere ice sheet mass loss. A warming subsurface ocean (in ~500–1500 m water depth) has been accepted as an important causal mechanism to destabilize the AIS in both the present17 and the El Nino-Southern Oscillation18 today. Although these relations are not well understood for the deglaciation, changes in tropical Pacific sea-surface temperatures could have induced changes in the surface mass balance of Northern Hemisphere ice sheets and thus were likely important for the growth to and retreat from their Last Glacial Maximum (LGM) extent19. For the AIS, however, such teleconnections would not lead to substantial changes in surface mass balance for the end of the LGM because of the different ice-sheet configuration and the colder climate, leading to a slightly positive ice-mass balance during ENSO-driven temperature increase because the threshold to thawing would not be crossed20.The surface mass balance of the AIS is influenced by atmospheric teleconnections such as the Southern Annular Mode22, and conversely that a sea-level fall or bedrock uplift re-stabilizes the grounding line, reduces ice loss or promotes grounding line advance23. New ice-sheet models that are coupled to a global sea-level model show that AIS dynamics are amplified by Northern Hemisphere sea-level forcing24, suggesting a higher AIS volume during the LGM relative to models without this interaction, and a dynamic response of the AIS during deglaciation that is well in line with periods of rapid sea-level rise (MWPs) and the geological data sets presented here.It is well established that bedrock subsidence beneath the AIS and/or sea-level rise at its edge play a critical role in driving grounding-line retreat or prohibit AIS growth14, Methods). These events, termed Antarctic Ice Sheet Discharge (AID) events, lasted for centuries to millennia and are documented in deep-ocean sediment in the Scotia Sea’s Iceberg Alley14.Despite the mounting evidence for present-day and future AIS instability, and increasing concerns about GMSLR, until recently there has been no individual record combining the pace, timing, and magnitude of past AIS mass loss to provide essential constraints for projections of the future AIS evolution. However, multiple AIS mass loss events for the deglaciation have recently been dated precisely using dust-climate synchronizations between marine cores and ice core cores  ice core, the newer AICC 2012 age scale29, which relies on interhemispheric methane correlation, was applied24. This age scale allows for direct comparison of climate events documented in ice cores from both hemisphere and has recently also been used to compare Northern Hemisphere ice-sheet events to our AID events24. Also, we carefully re-counted all IBRD transitions leading into, and out of, an AID event using the new chronology. For details concerning the age model, the stratigraphic resolution of the sampling, and the underlying uncertainties and IBRD counting procedures see ref. 14 and Methods.In this study, we primarily rely on published IBRD records from Iceberg Alley sites MD07-3133 and MD07-3134 and use the timing and magnitude of the deglacial AID events31, and to a novel, shallow-marine core transect across the Ross Sea shelf32 to infer the deglacial grounding line retreat history. We then use new model output from previous ice-sheet model experiments15 to examine the role and timing of calving in relation to total ice-mass loss. Finally, we analyze the IBRD records and ice-sheet model outputs for early signs of destabilization and re-stabilization using tipping point analyses.We compare this offshore ice discharge record to published isotopic and chronological data from a blue ice field in the Patriot Hills, an area sensitive to ice-sheet thinning14. Three out of eight AID events occurred at known times of global MWP events  constituting the peak of AIS deglaciation around MWP-1A11; and AID2 (~11.3 ka) at the time of MWP-1B33.The sediment record from Iceberg Alley indicates multiple phases of enhanced iceberg routing during the last deglaciation that commenced and terminated abruptly, and lasted from centuries to a millenniumnts Fig. : AID8  is about the same as the duration of deglacial ice-sheet re-stabilization .The age model34. In most cases the flux of debris increased from low background levels to high values within a single sample and the decrease occurred equally rapidly. These rapid transitions from low to high and high to low IBRD contents form the basis of our manually assigned AID event classification. The counting interval was 1 cm, which translates into 8–17 years resolution for AID1–8, depending on the time interval and core. For core MD07-3133, the resolution was ~8 years/cm for most parts of the deglaciation, whereas it was mostly ~14 years/cm for core MD3134. Considering AID1–8 of the stacked IBRD record, which represents a larger-scale, regional response to ice-sheet changes, it took, on average, 17 years to initiate AIS mass loss, and 22 years to re-stabilize the AIS again , the Antarctic Coastal Current (ACoC), and the Antarctic Divergence as well as the Coriolis Force. Also, important mechanisms such as ocean thermal forcing or sea-ice variability do not affect iceberg transport significantly. Hence, these present-day processes likely operated in a more or less similar manner during deglaciation. For further information see ref. 36). However, our recognition of a highly dynamic AIS that deglaciated early and substantially starting with AID 7 at ~17 ka, is in line with an Antarctic-wide deglacial warming and retreat of sea-ice inferred from ice cores37 as well as enhanced upwelling in the Southern Ocean38 and a general southward displacement of the Southern Hemisphere Westerly winds39. In addition, we find clear evidence for early deglaciation in novel and pristine archives on the Antarctic shelf and continent.Knowledge of the response of the AIS to past climate changes was previously based on only a few near-field, short sediment sequences, which limited our understanding of the dominant feedbacks between the AIS, Southern Hemisphere and Northern Hemisphere climate, and global sea level. Most shallow marine and terrestrial Antarctic sequences cannot be dated adequately, reveal only the final stage of ice retreat, or resolve only local responses to climate forcing, therefore leaving much room for speculation. Such shallow marine and terrestrial archives pointed to a rather stable AIS that mainly deglaciated late, in post-MWP-1A to late Holocene times with minor contributions to the deglacial GMSLR  suggest ice elevation drawdown of 300–400 m for AID8  that is buttressed by the Institute Ice Stream via the Horseshoe Valley Trough. Ice-sheet modeling demonstrates that this area is highly sensitive to dynamic ice-sheet changes across the broader Weddell Sea Embayment and provides an independent measure of AIS surface elevation change32. The Ross Sea records suggest ice-margin changes across the Antarctic during AID events, most noticeably for AID6 and AID2 and associated MWP-1A and MWP-1B, respectively.Second, the first well-constrained chronology for deglacial grounding line retreat for the eastern Ross Sea shelf was developed from sediment wedges in Whales Deep Basin. Carbonate shell dating on a core transect revealed a step-wise retreat of the WAIS largely in-phase with AID7 to AID2 Fig. , exhibit15 simulate AIS ice mass change in response to changes in air and ocean temperatures, precipitation, and global mean sea-level. Although no specific iceberg calving mechanism was included, the temporal phasing of AIS mass loss arising from the combination of applied environmental forcings and independent age determination bears extremely close correspondence to the phasing, shape, and relative magnitudes of IBRD fluxes recorded in Iceberg Alley14 41. More recent estimates report calving rates of ~1300 Gt/yr, with equal mass loss from melting26, or 1100 Gt/yr with slightly higher relative melt rates42. In a steady-state system, this annual ice mass loss will be compensated by snowfall on land or basal freezing, a condition that can be assumed for the AIS during the mid to late Holocene and during the LGM; both rather stable ice-sheet periods when the IBRD flux rates remained relatively low. However, during deglaciation, the IBRD flux increased distinctly—up to nine times above the low average during MWP14 . These are maximum numbers since the actual contribution to GMSLR will be due only to the fraction of ice that is above flotation thickness and therefore sea-level relevant. Based on ref. 15, ~32% of ice mass delivered to the ocean corresponds to ice above the flotation thickness. Hence, the global mean sea-level contribution of the AIS to MWP-1A could be at least 2.5–3.5 m. Due to uncertainties these are rough estimates only; however, they indicate that a notable fraction of MWP-1A’s 16 m GMSLR could have originated from Antarctica.Since an annual ice mass loss of an additional 1000 Gt/yr would lead to ~2.6 mm of GMSL/yr, the IBRD flux record implies that, over the course of MWP-1A for instance, which lasted a little less than 400 years, ~9 m of GMSL could have originated from the AIS at average annual calving rates of 1300 Gt/yr .The abruptness of the initiation and termination of the AID events suggests that there may be thresholds that, once passed, could trigger a destabilization or re-stabilization of the ice sheet. If these tipping points are irreversible in the sense that there is hysteresis—that is a direct reversal of the forcing does not allow a return to the previous state—certain ‘early warning indicators’ may identify some characteristic fluctuation patterns present in a timeseries preceding an abrupt change. These patterns form due to the increasingly slow return to equilibrium from perturbations as a critical threshold is approached, termed ‘critical slowing down’45, which can mask the patterns of critical slowing down. Alternatively, the signals may not have been recorded due to their absence, either because there is no critical transition, or if the system ‘tipped’ before reaching the critical threshold. To try to resolve this we undertook the same analysis on the model simulations for sea-level equivalent (SLE) mass loss , AID2 (MWP-1B), and AID1 were identified in our simulations. We found an increase in autocorrelation and variance, leading indicators of critical slowing down48, prior to both the destabilization and re-stabilization phases of the simulated AID events  and during an AID event (for re-stabilization). Although some AID events displayed increases in autocorrelation and variance prior to the abrupt change, most did not  ice-shelf thinning or collapse60. According to our combined reconstruction-modeling study of past extreme events, the negative mass balance of the AIS in recent decades61 and especially the rapid retreat of parts of the WAIS62 might very well constitute a novel AID event, marking the onset of self-sustaining and irreversible ice-sheet retreat for centuries.Local destabilization could cause a complete disintegration of the marine ice in West AntarcticaSample-based investigations concentrated on deep-sea cores retrieved in the Scotia Sea during Marion Dufresne II cruise 160 in March 2007. Sites MD07-3133  and MD07-3134  originate from the northern end of Dove Basin and Pirie Bank, respectively.63. The age model of the Scotia Sea sites is based on low-resolution ground-truth data sets associated with Marine Isotopic Stage (MIS) boundaries and a high-resolution age model relying on distinct dust-climate couplings between Southern Ocean sediment and the Antarctic EPICA Dronning Maud Land (EDML) ice core14. The Scotia Sea sites lie downwind from the major dust source (Patagonia) but in closer proximity than Antarctic ice cores  providing clear evidence for coherent and synchronous changes in dust deposition across the Southern Ocean and the AIS14.The original chronology and AID records of sites MD07-3133 and MD07-3134 have been published before2+ (nssCa)14. All in all, the published tie points were sufficient to achieve synchronicity between the marine and ice-core record during deglaciation, especially during the time ~19–13 ka, when the main decline in atmospheric dust occurred.The age model of Site MD07-3134 consisted of 48 control points based on dust proxy tie points. For the last deglaciation, five confident tie points relative to EDML exist for the period ~20 to ~8 ka, which covers 10 m and 7 m core length for Sites MD07-3133 and MD07-3134, respectively. The five tie points correspond to consistent breaks in slopes and characteristic and reproducible peaks in the dust proxy records of MS and Ca relative to ice-core dust proxy non-sea salt Ca29 was developed based on methane correlation. Accordingly, all ages of the IBRD record have been transferred from EDML1 to AICC 2012 (ref. 24). This new age scale makes ages progressively older further back in time. The difference is minimal for the mid to late Holocene relative to EDML1. At the time of MWP-1B the difference is on the order of 150 years, whereas it is 350 years during MWP-1A and ~500 years at the LGM, before which the differences become smaller again  happened syn-sedimentary with minor effect on the preservation of signals.63 built upon a bootstrap algorithm that simulates possibly wrong TPs and therefore provides conservative error estimates for each AID . Resulting dating errors (2-sigma) of the tie point correlation were used to calculate uncertainties for the start, end, and duration of AIDs. All uncertainties are incorporated into the absolute dating uncertainty (2-sigma error) of the AIDs. The bootstrap algorithm allows for the derivation of relative dating uncertainties for the AIDs. Since interpolation errors of nearby layers are highly correlated, these uncertainties are naturally smaller than the absolute dating uncertainties. Hence, significantly more precise estimates were made on the duration of and the distance between AID events. These durations are at least several centuries with relative uncertainties of only a few decades . However, the absolute age uncertainties of EDML1 age model range from 400 to 1000 years between 8 and 21 ka.The original age uncertainty estimates of the Scotia Sea sites24). The results reiterate that AID events 8, 6, and 2 occurred, within uncertainties, during the timing of MWP-19ka, 1A and 1B, respectively, and that no neighboring AID event would overlap enough to indicate otherwise. Any leads or lags between MWPs and AID events can, however, not be established due to the uncertainties.For the AICC 2012 age scale, we considered full uncertainty estimates by propagating the square roots of the uncertainties from the tie point correlation and the uncertainties of the AICC 2012 age scale and marked those for the upper and lower bounds of each AID event . Such loose IBRD packing is markedly different from Northern Hemisphere Heinrich Events, where IBRD is usually packed in discrete layers that consist almost entirely of lithic grains 0.1–3 mm in size (ref. 64).The sediment matrix of Sites MD07-31333 and MD07-3134 ranges from fine-grained mud  to diatom-bearing mud to diatom oozes . Individual small IBRD  are loosely embedded in this continuously deposited, fine-grained matrix. IBRD counting was conducted every cm on x-radiographs taken from 1-cm thick and 10-cm wide slices that were cut out from the center of each core segment and exposed to an X-ray system. 0–2 IBRD grains within a 1-cm interval would be considered low background values. However, slightly higher counts  can already translate into an AID event if neighboring counts are elevated, too. Even high counts typical for peak iceberg discharge during an AID event rarely exceed 10 grains per 1-cm interval, translating into 1 debris grain only that is loosely embedded in a muddy sediment matrix of 1 cm14.The classification into AID events has been made by eye. In a first step, we defined events using the transitions from low to high IBRD content for both sites independently. In a second step, the distinction has been repeated for IBRD flux for both sites independently to find the best compromise between content and flux records. In a third step, the classification has been applied to the stacked record to find the best compromise for a regional record consisting of both sites rather than local records31.A new ‘horizontal’ ice core (1600 m) has been extracted from exposed ancient ice from within Horseshoe Valley . The site is located 50 km inland from the modern grounding line of the Filchner-Ronne Ice Shelf in the Weddell Sea Embayment (WSE), making the area sensitive to dynamic ice-sheet changes across the broader region30, and utilizes Bayesian modeling to integrate the chronological control points through the record. The trace gas measurements provide a range of possible age solutions, which together with the absolute constraint provided by the tephra horizons, allows the development of a robust chronological framework that can be tied directly to the isotopic profile through high-resolution ground-penetrating radar survey65. This can be tested against the ages of independent geochemically typed tephras identified in the BIA profile30. Geochemical analysis confirms the interpretation of WCM-93-25 (ref. 66) (18.2 ± 5.8 ka) as PH279, and further analyses suggests the Trachyte PH282b correlates to TD822a (17.61 ± 0.73 ka)67 with a similarity coefficient of >0.953.The chronological framework for the Patriot Hills Blue Ice Area (BIA) has been developed using a combination of volcanic (tephra) horizons and trace gas samples. The record presented here includes new data that builds upon that presented in ref. 69 to integrate the above accepted gas measurements with the tephra ages. Using Bayes theorem, the algorithms employed sample possible solutions with a probability that is the product of the prior and likelihood probabilities. Crucially, Bayesian modeling enables the relative stratigraphic information from the BIA transect to be incorporated along with the ‘calibrated likelihoods’ or ‘calibrated probability distributions’.We undertook Bayesian age modeling using the software package OxCal v.4.2.4 using a Poisson process deposition model (P_sequence)70 with 20-year resolution. Each gas species was given a Delta_R term to allow for uncertainty between the measurements at Patriot Hills and those reported in the calibration curves70. The Delta_R term was calculated using the ‘f’ and ‘t’ terms. The ‘f’ variable sets the default error on the measurements as a factor of the standard deviation in reported calibration curves; the ‘t’ value sets how tight the tails are on the Delta_R. Our age model used a ‘f’ value of 0.3 and a ‘t’ value of 2 degrees of freedom, allowing the value to be further from the mean than a Normal distribution. Taking into account the deposition model, the ages provided by the tephras and the common age solutions offered by the trace gas measurements, the posterior probability densities quantify the most probable age distributions. To account for the gas-ice age difference (Δage), we used a Delta_R term for the different sections of the BIA, guided by recently reported values from the WAIS ice core71. For the glacial age ice, we used the prior U  and the Holocene, U . For the glacial age ice, we found a mean Δage of 350 ± 87 year; for the Holocene we determined a Δage of 225 ± 43 year.‘Calibration curves’ were developed for the three trace gas species using the same datasets used above18O isotopic measurements were performed between 1 and 3 m resolution at James Cook University (JCU) using Diffusion Sampling - Cavity Ring-down Spectrometry (DS-CRDS)  (ref. 31). This system continuously converts liquid water into water vapor for real-time stable isotope analysis by laser spectroscopy . To ensure reproducibility, a subset of samples was rerun at UNSW ICELAB for δD and δ18O using a Los Gatos Research Liquid Water Isotope Analyser 24d . Reported overall analytical precision on long term ice core standards is <0.32‰ for δD and <0.13 for δ18O values. All isotopic values are expressed relative to the Vienna Standard Mean Ocean Water 2 (VSMOW2). Given the isolated nature of Horseshoe Valley both during contemporary times and deglaciation, and the buttressing effect of the AIS on ice flow from the valley, we interpret the isotopic trend captured in the Patriot Hills BIA as the result of ice-sheet elevation changes due to mass loss across the broader WSE. Thus, increasing δD and δ18O water isotope values across the ACR and the apparent local warming can only reflect regional ice-sheet draw down.δD and δ40 within the Antarctic Coastal Current (ACoC)  Fig. . Althoug75). This is specifically true for the Drake Passage, which is a major bottle neck for the ACC east of our core sites. On the other hand, winds blow west and feed counterclockwise into the ACoC around Antarctica south of the Antarctic Divergence. These currents are large planetary geostrophic currents that mainly operate independent of climate. The ACC and ACoC will thus likely exist under different climates of the past76, and although individual fronts connected with them likely shifted north or south in response to changing sea-ice coverage and climate, these shifts can be considered minor directly east of the Drake Passage.There are several factors supporting the notion that iceberg routing during the deglaciation was not much different from today. First, the Antarctic Divergence is the major driver of the ocean-atmospheric circulation in the Southern Ocean. It forces winds north of it to blow east and to feed clockwise into the ACC, which is geographically constrained to 50–55°S ; although smaller icebergs and crawlers may have escaped the ACoC during times of thicker sea-ice coverage farther away from the continent.Second, the Coriolis force is important for the routing of icebergs after calving from the AIS. This force is also independent from the state of climate because it depends on the rotation of earth. It causes all Antarctic icebergs to be deflected to the left, move counterclockwise and maintain a course very close to the edge of the Antarctic continent. In addition, the Coriolis force exerts a greater force on larger icebergs78. This further implies a vigorous ACoC also during glacial and deglacial times.Third, several studies have reconstructed extensive coastal polynyas immediately in front of the AIS during colder periods when sea-ice coverage was dense and reached farther away from the continent79. However, the requisite temperature increase would be subsurface warming of Circumpolar Deepwater in ~500–1500 m water depths, i.e., lies deeper than the typical draughts of drifting icebergs. Therefore, ocean thermal forcing could induce enhanced ice melt at the grounding line at times of increased resumption of the Atlantic Meridional Overturning Circulation but this would not affect drifting icebergs.Fourth, ocean thermal forcing has been proposed as primary mechanism to explain distinct phases of AIS mass loss during deglaciation15. Our deglacial ice-sheet model15 employed the Parallel Ice Sheet Model (PISM) at 15-km resolution for transient ice-sheet/ice-shelf simulations from the LGM to present. The age models in these simulations are completely independent from the IBRD age models. Full methodological details are provided elsewhere, but in summary, PISM calculates evolution of the three-dimensional thermal and flow characteristics of the ice-sheet using a combination of the shallow-ice/shallow-shelf approximations of the Stokes equations80, and a novel enthalpy scheme81. Fast-flowing grounded ice evolves in areas where modeled basal porewater pressure increases and thereby reduces basal substrate shear strength, following the plastic till failure approach82.For this study, we re-analyzed outputs from previous ice-sheet simulations16. We apply this timeseries forcing to all floating ice in the domain using sub-ice shelf mass flux rates that are scaled to allow both LGM and present-day grounded ice extents to be reproduced15. Optimization experiments revealed that different scalings of effective ‘melt rate’ were required for each of the four ocean thermal forcings used, but all resulted in rates in the range 0–20 m/yr. During episodes of rapid ocean warming such as MWP-1A, thermal forcing of 0.5–1 °C translates to an equivalent change in sub-ice shelf mass flux of -10 m/yr. Bedrock deformation due to ice thickness changes are accounted for using the native elastic lithosphere, relaxing asthenosphere model of PISM (e.g. ref. 83). This model allows the long-term viscous effects of ice loading to be calculated and bedrock elevation adjustments to take place through the model simulations in a way that in previous experiments allowed present-day uplift rates measured from in situ Antarctic global positioning system receivers to be well captured84. Due to limitations of the 1-dimensional earth model, however, we are only able to implement single values for parameters that affect earth deformation across the entire domain. We use a lithospheric density of 3300 kg/m3, a flexural rigidity of 5 × 1024 Nm, and a mantle viscosity of 1 × 1021 Pa s. Our model does not capture changes arising from self-gravitational or earth rotational effects.Ice-mass loss is assumed to take place through calving of icebergs and sub-ice shelf melting. Although no transiently evolving calving margin is accommodated in our simulations, we nonetheless impose a fixed calving front at the continental shelf break by removing ice seaward of the −2000 m bathymetric contour. This is justified on the grounds that contiguous floating ice of a thickness less than the depth of the continental shelf (approximately 1000 m) is unlikely to survive in the open Southern Ocean. Ice-mass lost through this mechanism changes through time as a consequence of changes in ice thickness reaching the calving front, but the margin itself does not migrate. Given that the change in the length of the calving front during deglaciation is small relative to its total length, we anticipate that this limitation of our model should not substantially affect the results presented here. Sub-shelf mass flux in our model is prescribed according to a timeseries scalar of zonal mean subsurface (approximately −400 m) ocean temperature evolution from transient deglacial ocean-atmosphere simulations using the LOVECLIM intermediate complexity modelEnvironmental boundary conditions such as global mean sea level, air temperature, and subsurface ocean temperature are set using proxy-based and model-derived time series perturbations to present-day gridded datasets. Our ensemble of experiments combines three different sea-level reconstructions with four different ocean temperature curves, yielding twelve discrete ice sheet simulations. Parameter optimization and iterative adjustment of the scaling of the applied ocean forcing allow the thickness evolution of the ice-sheet from 25–0 ka to reproduce the spatially variable pattern of elevation changes indicated by twelve Antarctic ice core records. Marine geological data are used to constrain the horizontal extent of the simulated LGM ice sheet. The age scales used for each of these forcings, as well as their respective temporal uncertainties, differ, however. In presenting the ensemble mean pattern of Antarctic mass loss, therefore, we are unfortunately not able to reproduce the precise absolute timing of ice mass change revealed by the IBRD record. For this reason, we focus primarily on noting the correspondence between the pattern and rate of changes in each dataset, rather than the chronological synchroneity.6. The verified mass loss partitioning in those experiments thus allows reliable inferences to be made regarding the link between calving (leading to IBRD deposition) and mass loss from the ice sheet.Ice-sheet modeling experiments used to investigate the temporal relationship between calving and grounded mass loss rates Fig.  make use85. These patterns, manifested as an increase in autocorrelation and variance over a sliding window, form due to the increasingly slow response of the system to perturbations as a critical threshold is approached, termed ‘critical slowing down’. Procedures involving measuring the indicators of critical slowing down are described in detail in refs. 86. The data is pre-processed by removing long-term trends using a loess filter and interpolated (if necessary) to create an equidistant timeseries. Autocorrelation and variance  are then measured over a sliding window of size 50% of the data length. To provide a quantitative measure of the strength of the trends, the nonparametric Kendall’s tau rank correlation coefficient is applied87 to each individual time series. Significance tests provide a quantitative assessment of whether indicator trends are robust, by varying certain parameters such as the loess smoothing span and size of the sliding window. A null model is created by randomization of the dataset over several thousand permutations, which guarantees the same amplitude distribution as the original time series but destroys the linear correlation and structures. The statistical significance of the original analysis is obtained by comparing against the probability distribution of the null model. Specific sections of data from the Scotia Sea IBRD stack were selected to investigate potential thresholds for destabilization and re-stabilization. Sections of data for analysis were chosen to include only data prior to the re-stabilization/destabilization event. To investigate whether the stacking procedure had an effect on the results, we also applied the same analysis on data from the individual sites MD07-3133 and MD07-3134. Selected data from the deglacial ice-sheet model simulations were also analyzed; these data have a higher data resolution, lower noise level, and constitute a more direct proxy to the stability of the ice sheet.Generic rules have been developed that can identify certain ‘early warning indicators’ to identify the characteristic pattern of fluctuations present in data preceding a non-linear changeSupplementary Information


Infectious bronchitis virus (IBV) poses significant financial and biosecurity challenges to the commercial poultry farming industry. IBV is the causative agent of multi-systemic infection in the respiratory, reproductive and renal systems, which is similar to the symptoms of various viral and bacterial diseases reported in chickens. The avian immune system manifests the ability to respond to subsequent exposure with an antigen by stimulating mucosal, humoral and cell-mediated immunity. However, the immune response against IBV presents a dilemma due to the similarities between the different serotypes that infect poultry. Currently, the live attenuated and killed vaccines are applied for the control of IBV infection; however, the continual emergence of IB variants with rapidly evolving genetic variants increases the risk of outbreaks in intensive poultry farms. This review aims to focus on IBV challenge–infection, route and delivery of vaccines and vaccine-induced immune responses to IBV. Various commercial vaccines currently have been developed against IBV protection for accurate evaluation depending on the local situation. This review also highlights and updates the limitations in controlling IBV infection in poultry with issues pertaining to antiviral therapy and good biosecurity practices, which may aid in establishing good biorisk management protocols for its control and which will, in turn, result in a reduction in economic losses attributed to IBV infection. Infectious Bronchitis Virus (IBV) is an acute and highly contagious respiratory pathogen of chicken that has a major economic impact on poultry stakeholders. The mutable tissue tropism and continuous emergence in various serotypes or genotypes of IBV are prevalent across various geographic regions. As a consequence of the severity and highly contagious nature of IBV infection, it has been implicated in higher economic loss in breeders and layer chickens attributed to reproductive disorders and renal dysfunction. Long-term impacts on the reduction in egg production up to 20–70%) and loss of egg quality for trade and hatching are observed . IBV inf0–70% andNidovirales, family Coronaviridae) with a positive-sense single-stranded RNA, (+) ssRNA and genome of approximately 27 kb with gene organization: 5′UTR-1a/1ab-S-3a-3b-E-M-5a-5b-N-3′UTR [Coronaviridae is further divided into two subfamilies: Coronavirinae and Torovirinae. The Coronavirinae subfamily comprises four genera of viruses such as Alphacoronavirus (αCoV), Betacoronavirus (βCoV), γCoV and Deltacoronavirus (δCoV) based on morphology and genome structure [IBV belongs to gammacoronavirus (γCoV) or Group-3 coronavirus  vaccine immunity must be long-term, otherwise re-vaccination is necessary; (2) the selection of the correct antigenic type of vaccination that is specified for wide antigenic variation in order to cover the maximum virulent serotypes; and (3) timing, technique and applications of vaccine according to flock’s status. Expression and delivery systems of various kinds of IBV vaccine with their existing features are shown in Attenuation of virus or live vaccine is carried out by serial passages of IBV strains in embryonated chicken eggs (ECE) for reducing the required level of virulence. IBV vaccines can either be mild or virulent depending on the level of attenuation. Live vaccines should be covered if those serotypes or strains are circulating in the surrounding region or the predominant antigenic nature refers to strains of serotype-specific vaccines ,46. LiveInactivated vaccines have provided safety and stimulation for long-lasting, prolonged immunity without vaccination reactions or post-vaccine challenge. The killed IB vaccine is relatively cost-effective compared to attenuated live vaccines and applies either single or two or more combined inactivated antigens or two or more serotypes in bivalent vaccines . InactivThe foreign genes are expressed by viral vectors, which is an attractive method of gene distribution for vaccine manufacturers. Nowadays, vaccine producers have advanced since they can be exposed to genetic manipulation of the coronavirus genome. Coronaviruses have been exposed to accept and express foreign gene delivery by gene therapy vectors followed by vaccine production ,61. For Lactococcus lactis) has been used to deliver peptide vaccines orally; consequently, this technique has been described to be a strong elicitor of mucosal immune response [Subunit vaccines comprise a portion of the virus or antigenic principles of the virus, which is cloned and expressed in either bacterial cells, yeast cells or cell lines with the objective of providing a protective immune response without the risk posed by vaccination with the complete pathogen. Subunit vaccines have been prepared from purified viral antigens but are constructed from viral peptides or are a part of the genome encoding for immunogenic epitopes for designing multi-epitope vaccines . Epitoperesponse . For exaresponse . The curresponse . These wresponse .p < 0.01) with strong concurrent immunity against viral infection. Bande et al. [Plasmid DNA vaccines consist of a DNA backbone, which carries the antigenic principle that can be transcribed and translated in the host, under the regulation of the appropriate host promoter and protein translational machinery. Plasmid DNA vaccines can be delivered as a monovalent construct, carrying a single antigen regulated by a single promoter, or a multivalent construct, containing one or more antigen coding genes regulated by either a single or multiple promoters . Currente et al.  conductee et al. . Alternae et al. . Intramue et al.  IBV N gee et al.  or chicke et al. . The stue et al. ,83. The e et al. . The lime et al. . Currente et al. . A nanopA reverse genetic vaccine is frequently employed to manipulate the full-length genomic cDNAs of viral genomes from RNA virions with the following synthesis of infectious RNA to produce recombinant viruses. This novel technology for operating one or more viral genes is given the potential to develop various modified IBV vaccine candidates . These rCurrently, IBV vaccination programs have gained more attention with respect to the use of low-virulent, live or inactivated killed vaccines with the aim of booster shots at certain times to increase immunity and reduce the antagonistic response of epithelial cells in the respiratory region . HoweverVarious defense mechanisms have shown to neutralize a virus for the sake of building up the immune system in the chicken body against IBV. Primarily, the virus enters the body system and is detected and neutralized by the non-specific immune response . VariousA vaccine requires a certain period of time in order to elicit a protective immune response in avian hosts. Moreover, passive immunization can induce immunity from maternal derivative’s antibody (MDA), which is particularly supportive during the early stages of life . The strPassive immunity typically refers to IgG antibodies or maternally derived antibodies (MDAs) that play a significant role in the immediate local protection of chicks for short-term defense against IBV. During the third part of embryonation, IgG is secreted from the yolk and entrance to the bloodstream in order to prevent the virus replication at a point in time. Moreover, the newly hatched chick can receive the IgG level from 5 days of post-hatch, and the continuous circulation of IgG levels in chicken serves as humoral immunity for progeny and improves performance and survival ,109. In The innate immune system is the first-line defense of IBV infection that can be directly activated during the initial exposure of the virus into the chicken body. The inflammatory response is increased by the flow of blood to assist carrying immune cells to the site of infection . VariousThe immune system is activated by the physical and functional barriers offered by the skin and mucous membranes, which is intended to avoid infection and attack the invading pathogens. Moreover, leukocytes include heterophils, macrophages, NK cells, mast cells, acute phase proteins, basophils and eosinophils that participate in innate immune responses by killing organisms or destroying enzymes and free radicals . The immToll-like receptors (TLRs) are a part of pattern recognition receptors (PRRs), which are activated by the innate immune response through sensing-conserved molecular patterns . VariousThe Type I interferon is observed in the mucosal lining of trachea, lungs and kidney when an innate immune response is activated during infection; however, the response of stimulation depends on the virulence and host adaptability of the viral strain ,127. TheThe humoral immune response is triggered by the activation of IBV-specific antibodies to inhibit viral replication and virus circulation in the blood steam or hinders viremia from the trachea, kidneys and oviduct ,136. TheThe antibodies’ response was confirmed in serum samples or tracheal swabs, and lacrimal secretion after successful IBV vaccination . The stuIn chickens, CMI is one of the essential immunoregulatory weapons during IBV infection, especially for aiding the clearance of viruses, decline of infection and reducing virus shedding and vaccine development . The evaThe mucosal immune responses are a more advanced technique for respiratory viruses and show ongoing progress in the development of mucosal vaccines . Mucosal−3 to 10−5 errors per replicating cycle) and recombination are the modern instruments of coronavirus that may be responsible for the outbreaks of different strains of IBV [The limitations of controlling IBV are multifactorial, including age of birds, breed, nutrition and management status; and variant or potential use of a live vaccine of IBV against virulence strain. Moreover, mutation (10s of IBV . For this of IBV ,169. As s of IBV . The bess of IBV ,171.The methods of inhibiting coronavirus replication included targeting the main proteinase of coronaviruses that is responsible for virion replication by using antiviral drug therapy. The use of cysteine–proteinase inhibitors in cell cultures has inhibited the replication of many coronavirus species, including IBV . AnotherThe basic requirement of biosecurity is to improve primary and secondary containment by implementing control measures such as limited or controlled site access, providing boots and uniform, farm equipment and litter sanitation, foot-baths and wheel baths, which may all need to be set up at all entrances inside/outside where the maximum truck enters the farm. Setting up all biosecurity plans routinely during an emergency outbreak in order to reduce the greater risk of IBV spreading is enforced. Cleaning is the most hygienic measurement and is a significant part of biosecurity for reducing the level of virus infection and transmission. Cleaning should be categorized as dry or wet, which can be applied with an accurate concentration of proper sanitization and disinfected for reducing residual virus contamination. It is highly recommended that similar disinfectants should not be used more than the last 6–12 months, resulting in the development of resistance. Moreover, the downtime between consecutive bird houses is recommended to be at least 10–14 days, except for the multi-age flocks that are challenging and may need to follow strict biosecurity measures for movement and equipment among the flock. Several factors aggravate IBV infection, such as the bird’s strain, age of birds, nutritional aspect and other environmental stress, e.g., ammonia levels, ventilation, temperature and humidity ,155. AddIBV has circulated in the world almost 90 years apart as it was first isolated in early 1931. Currently, the live and killed vaccines followed by booster immunization have been the ideal approaches in IBV vaccination programs; however, the outbreaks of IBV in poultry farms have become persistent, which poses a significant problem to commercial farming. The emergence of new serotypes and variants has necessitated the development of new strategies for the management of IBV and the engineering of new vaccines to address novel serotypes. The innate immunity against IBV virus is generally weak and needs an advanced understanding of the immune responses to IBV. The vaccine technologies have been frequently expanded based on genetic experience and attempts to apply a broad-spectrum or new generation of genetic vaccines based on virulent viruses initiating the field challenge, which is the best method for the control of IBV. This review outlines the present situation of IBV control, including vaccination, immune status of birds and current developments in vaccine technology, for inducing cross protection against multiple IBV serotypes in different regions of IBV that help effective management in the future.


Hypertension causes many deaths worldwide and has shown an increasing trend as a severe non-communicable disease. Conventional antihypertensive drugs inevitably cause side effects, and great efforts have been made to exploit healthier and more-available substitutes. Microalgae have shown great potential in this regard and have been applied in the food and pharmaceutical industries. Some compounds in microalgae have been proven to have antihypertensive effects. Among these natural compounds, peptides from microalgae are promising angiotensin-converting enzyme (ACE) inhibitors because an increasing number of peptides show hypertensive effects and ACE inhibitory-like activity. In addition to acting as ACE inhibitors for the treatment of hypertension, these peptides have other probiotic properties, such as antioxidant and anti-inflammatory properties, that are important for the prevention and treatment of hypertension. Numerous studies have revealed the important bioactivities of ACE inhibitors and their mechanisms. This review discusses the antihypertensive effects, structure-activity relationships, molecular docking studies, interaction mechanisms, and other probiotic properties of microalgal ACE inhibitory peptides according to the current research related to microalgae as potential antihypertensive drugs. Possible research directions are proposed. This review contributes to a more comprehensive understanding of microalgal antihypertensive peptides. Microalgae are the main contributors to primary production in aquatic ecosystems. They are widespread, fast-growing, and resilient in different harsh environments, such as those with high salinity and temperature, as well as wastewater. In recent years, microalgae and their derived natural compounds have been regarded as important and sustainable food supplements and biodiesel sources for the purpose of overcoming food shortages and energy crises associated with population growth and limited land resources ,2,3. FurHypertension, a chronic disease in humans, is a multifactorial disorder that is considered one of the major causes of premature death worldwide. As a “silent killer”, this chronic disease causes over nine million deaths annually and affects approximately one billion people . HyperteAngiotensin-converting enzyme (ACE) is a ubiquitous enzyme in mammalian tissues that is involved in the renin-angiotensin and kinin nitric oxide systems, and blood pressure in humans is regulated by the renin-angiotensin-aldosterone system (RAAS) through two main proteases, renin and ACE  33]. Br. Br33]. Chlorella decreased blood pressure by modulating the RAAS [Chlorella was also shown to decrease human blood pressure after ingestion [Chlorella vulgaris (C. vulgaris) and Spirulina platensis (S. platensis) showed marked antihypertensive effects on spontaneously hypertensive rats (SHRs) [Chlorella, Spirulina, Isochrysis, and Nannochloropsis were found to show ACE inhibitory effects with functions similar to those of conventional antihypertensive drugs [The first study of the antihypertensive effects of microalgae was conducted in the 1980s, when Miyakoshi et al., reported that the RAAS ,47. Chlongestion . Suetsuns (SHRs) . After tve drugs ,51,52,53ve drugs . Since tve drugs ,56,57. Tve drugs . Such a ve drugs . The simve drugs ,61,62. C50), which indicates the ACE inhibitor concentration that leads to 50% inhibition of ACE activity. Moreover, a few studies have demonstrated that the composition of the peptide and the amino acid sequence impacts the inhibitory activity of hypertensive peptides [50 values in ACE inhibitor and ACE inhibitor-like peptides could inhibit ACE through two strategies, namely, competitive and noncompetitive, which are determined by Lineweaver–Burk plots. Competitive inhibitors compete with the substrate and bind to the active site of ACE, and noncompetitive inhibitors change the conformation of ACE, which prevents the substrate from binding to the active site of ACE. When the Lineweaver-Burk plot cannot demonstrate the inhibitory strategy of the peptide, a mixed-noncompetitive pattern appears, which has been observed for some peptides ,64,65. Npeptides . Thus, wDaskaya-Dikmen et al., noted that potent ACE inhibitory peptides are generally short-chain peptides consisting of 2–12 amino acids . To the 50 values of microalgal ACE inhibitory peptides ranging from 0.5 to 474.36 μM, and most of these microalgal peptides were clearly noncompetitive ACE inhibitors  from Spirulina sp. . Pujiastuti et al., pointed out that there is no correlation between ACE inhibitory activity and the inhibitory pattern in marine organisms  [Isochrysis, Spirulina, and Nannochloropsis have shown only noncompetitive properties in studies that have analyzed this pattern to date, and Chlorella shows both inhibition patterns  . When fopatterns .The primary structure of the peptide  is also an important factor associated with ACE inhibitory activity. The N-terminus and C-terminus both strongly affect the ACE inhibitory activity of biopeptides . Many sh1–5) showed low IC50 values (0.5–1.11 μM), indicating a strong inhibitory effect, while W at the N-terminus of a dipeptide (number 4) showed a relatively high IC50 value (307.61 μM). In addition, Y as the C-terminal residue of the microalgal peptides did not have strong inhibitory effects [To better understand the correlation between the structural ACE inhibitory activity from the C-terminal residues in microalgal biopeptides, the structural formulas of these amino acids are shown in  effects ,75.1, 2, 3, 4, 5, and 8. It may be inferred that the bioactivities of peptides 1, 2, and 5 are influenced by their crystal structures, which showed short-chain alkyl groups or a certain number of carbon atoms, whereby the isobutyl group showed the strongest inhibitory activity and n-butyl showed the lowest inhibitory activity. The different bioactivities among peptides with similar termini indicated that the other amino acid residues also affect the biofunctions and efficacy against ACE. For example, some residues or atoms can form hydrogen bonds  [In addition to the amino acids at the N- and C-termini, the type of functional group at these positions affects the ACE inhibitory effects of microalgal biopeptides. Hydrophobic and aromatic residues at the N- and C-termini usually show strong antihypertensive activity . Notablyesidues) ,81.50 value with the formula LibDock score = 10.063 lg (1/IC50) + 68.08 [Great efforts have been made in recent years in the molecular docking of biopeptides from conventional foods to ACE ,83. Howe + 68.08 , and a lHuman tACE includes three active site pockets named S1, S2, and S1′ . These pC. vulgaris forms five short hydrogen bonds within the active sites of ACE (ranging from 2.08 to 2.48 Å), which include hydrogen bonds with H353 and H513 in the S2 pocket and A354 in the S1 pocket  is much higher than that of TTW (0.61 μM) and VHW (0.91 μM), even though FEIHCC formed more hydrogen bonds with ACE  from B. malleus increased the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and metal chelation ability at a concentration of 2 mg/mL [S. platensis at a concentration of 6.23 mg/mL exhibited a 75.72% radical scavenging activity (RSA) percentage, which reflects the DPPH radical scavenging effects [C. vulgaris could effectively scavenge superoxide radicals (IC50 of 7.5 ± 0.12 μM); the IC50 value of the hydroxyl radical scavenging effect was 8.3 ± 0.15 μM; and the DPPH radical scavenging activity could reach more than 40% at a concentration of 60 μM [Tetradesmus obliquus, such as Ala-Asp-Val-Pro-Phe-Arg (ADVPFR), Ser-Gly-Ser-Trp-Asp-Gly-Thr-Leu-Arg (SGSWDGTLR), Gly-Pro-Lys-Asp-Asp-Pro-Ala-Ala-Trp (GPKDDPAAW), Ser-Trp-Asp-Gly-Thr-Leu-Arg (SWDGTLR), and Ser-Trp-Ile-Ala-Arg (SWIAR), showed antioxidant properties according to their DPPH scavenging activity [Oxidative stress has been identified as a key etiological factor in the development of hypertension . Some bi 2 mg/mL . The ACE effects . The pepof 60 μM . A peptiactivity .I. zhanjiangensis inhibits expression of the NF-κB, MAPK, and Akt signaling pathways to block inflammation and endothelial cell apoptosis after Ang II treatment and activates the Nrf2 signaling pathway [The inflammatory processes are associated with hypertension and participate in its development and maintenance . Inflamm pathway . In deta pathway , and FEI pathway . The act pathway ,100. It C. vulgaris significantly reduced the systolic blood pressure of SHRs from 1 to 4 h, and the highest antihypertensive effect appeared at 1 h (a decrease of 49.9 mmHg). This antihypertensive effect continued for 4 h after oral administration, and the highest effect was comparable to that of the captopril group (10 mg/kg) [Chlamydomonas reinhardtii (C. reinhardtii) significantly reduced the systolic blood pressure of SHRs after intragastric administration , which contained more than 29% of the peptide Val-Leu-Pro-Val-Pro (VLPVP) [C. reinhardtii were demonstrated after intragastric administration of the genetically modified strain of SHRs at a dose of 10 mg of recombinant AHP3  protein per kg of body weight; the maximal decrease in blood pressure was observed 6 h post-administration [p > 0.05). Both TTW and VHW from C. vulgaris also influenced diastolic blood pressure (DBP), with TTW leading to a significant reduction in DBP from 180 to 140 mmHg at 2 h (p < 0.05) and VHW showing a decrease in DBP from 174 to 153 mmHg at 1 h (p > 0.05) in SHRs at a dose of 5 mg/kg body weight, where these peptides were digested in vitro and administered by gavage [Bellerochea malleus  were found to reduce blood pressure in SHRs by 17 mmHg after 5 days of oral administration [Microalgal biopeptides could be potential sources of ACE inhibitors . The ant (VLPVP) . The antstration . The pepy gavage . Proteinstration . These fMicroalgae have great potential for the prevention and treatment of hypertension due to excellent biological properties; they are easily cultured, possess low/no toxicity, and do not require land use. Some have found applications in many health-related industries. Microalgal peptides are effective ACE inhibitor peptides both in vivo and in vitro. The ACE inhibitory effects of these peptides are becoming a popular research topic, as are ACE inhibitor peptides from other food sources. Microalgal peptide activity may be determined by many factors, such as the species of origin, inhibitory pattern, sequence of amino acids, functional groups present, crystal structure, and molecular docking sites. In addition, these peptides protect cells by improving their antioxidant and anti-inflammatory properties through the regulation of physiological signaling pathways such as NF-κB, Nrf2, MAPK, and Akt, which also contribute to the prevention, diagnosis, and treatment of hypertension and its syndrome. However, knowledge of the structure-activity relationships of microalgal peptides is still deficient, especially regarding contributions from other functional groups. Future studies should focus on the bioactivity of microalgal peptides, the diversity of microalgal biopeptides, the relationship between their higher-order structure and activity, and additional mechanisms of molecular docking and interactions to achieve a better understanding of their use in healthy food supplemental sources and drug design.


It is caused by IgE antibodies to the carbohydrate galactose‐α‐1,3‐galactose  found on glycoproteins and glycolipids of non‐primate mammals. Patients develop symptoms after ingestion of mammalian meat and products  or pharmaceuticals of mammalian origin .We investigated the IgE levels against α‐Gal and beef  as well as total IgE in 50 well‐characterized Swedish patients with a doctor's diagnosis of AGSIxodes ricinus) at both visits were evaluated as previously.p value < 0.05 was considered significant.The IgE levels were determined both at the time of diagnosis (visit 1) and at a follow‐up visit (visit 2) at least 3 weeks (median 2.5‐month range <1–102 months) after diagnosis. All patients reported a history of several tick bites at the time of diagnosis.n = 24) compared to the ANA group (n = 26) . We noted that at visit 2, the α‐Gal IgE levels in the ANA group had not changed significantly , whereas the IgE levels in the non‐ANA group had decreased significantly . This was observed regardless of season (winter/summer) for blood collection . The fold change of beef IgE levels were similar between the ANA and non‐ANA groups , reflecting that they had been avoiding mammalian meat to the same extent. In the group of patients with visit 2 ≤5 months after the diagnosis, we observed that in 20/30 the IgE levels decreased (Table p = 0.07). Likewise, the IgE levels to beef decreased significantly in the non‐ANA group only .We found that neither the anti‐α‐Gal IgE levels nor the ratio between anti‐α‐Gal IgE and total IgE correlated with the severity of the allergic reaction (data not shown), which is in line with other studies.n Figure . There wed Table , probablp = 0.42). Of five patients who did not report tick bites, three had at least a three‐fold decrease in α‐Gal IgE levels . Moreover, one of them had not experienced symptoms after the diagnosis (No. 38). Our IgE data are firmly in line with previous reports showing that anti‐α‐Gal and anti‐tick IgE antibodies are maintained by continuous tick bites,When the patients whose visit 2 was ≥5 months after the diagnosis (18/20) were interviewed, all except one had avoided mammalian foods, and 17/18 reported consuming milk and dairy products. The remaining patient (No.44) reported consuming meat 2–5 times per year. The majority (15/18) had experienced symptoms after diagnosis, 72% (13/18) gastro‐intestinal discomfort, and 55.6% (10/18) urticaria, probably due to non‐awareness of consuming α‐Gal‐containing food. Only three patients (16.6%) reported an absence of allergic symptoms. With respect to tick bites, 72% of the interviewed patients (13/18) reported that they had been tick bitten after the diagnosis Table . Their In = 10, non‐ANA n = 5). Basophils from 10 patients were activated at different concentrations of HSA‐α‐Gal (Figure Furthermore, 15 patients with a second visit ≥5 months after the diagnosis were tested in BAT against HSA‐α‐Gal (ANA l Figure , showingIn conclusion, we found that the IgE levels to α‐Gal in AGS patients experiencing anaphylaxis are maintained over time. Although the patients avoided mammalian meat, most had been tick bitten and experienced symptoms, probably caused by the consumption of α‐Gal‐containing food. This is in concordance with still having basophils that are activated by α‐Gal. Thus, AGS patients with severe reactions should strictly be avoiding α‐Gal containing food as well as tick bites.Prof. M. van Hage reports personal fees from Thermo Fisher Scientific, outside the submitted work. Dr. M. Starkhammar reports personal fees from ALK, personal fees from CHIESI, personal fees from MEDA, personal fees from AstraZeneca, outside the submitted work. The rest of the authors have nothing to disclose.Table S1Click here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.