Atomic force microscopy showed that the reconstruction of ice-I surfaces occurs with a short-range (2 × 2) H-atom order. Understanding the precise atomic structure of ice surfaces is critical for revealing the mechanisms of physical and chemical phenomena at the surfaces, such as ice growth, melting, and chemical reactions. Nevertheless, no conclusive structure has been established. In this study, noncontact atomic force microscopy was used to address the characterization of the atomic structures of ice Ih(0001) and Ic(111) surfaces. The topmost hydrogen atoms are arranged with a short-range (2 × 2) order, independent of the ice thickness and growth substrates used. The electrostatic repulsion between non–hydrogen-bonded water molecules at the surface causes a reduction in the number of the topmost hydrogen atoms together with a distortion of the ideal honeycomb arrangement of water molecules, leading to a short-range–ordered surface reconstruction. The atomic structure of the surface of a crystal solid is difficult to determine, even if the bulk structure is perfectly identifiable. The lack of adjacent atoms or molecules at a truncated surface destabilizes the atomic structure of the ideal (bulk-like) surface, which can cause surface reconstruction . Identifying the surface structure at subnanoscale is the first step in understanding the characteristic chemical and physical properties exhibited at the surface. However, the surface structures of ice—a ubiquitous solid—are still not well-understood.The presence or absence of the surface reconstruction of ice Ih, which is a representative phase of crystalline ice that appears below the normal pressure . PreviouTo resolve this serious discrepancy, scanning probe microscopy can provide complementary information to the above-mentioned studies. Although scanning tunneling microscopy (STM) is widely used for the atomic-scale imaging of thin water films .To grow single-crystalline ice layers, we used two types of typical substrates: single-crystalline Rh(111) and Pt(111) (figs. S1 and S2). On Rh(111), ice layers were grown like hexagonal pillars with a difference in height , whereas2, respectively, and their values are approximately half of the density for the ideal surface (2.8 atoms/nm2). The dangling H atoms at these surfaces are more sparsely distributed than those at the ideal surface (1) Reduction in the number of dangling H atoms. According to the atomic-resolution images shown in  surface .2) Short-range (2 × 2) order of dangling H atoms. Although the protrusions in the AFM images seem disordered, the corresponding self-correlation (SC) images exhibit a blunt hexagonal pattern . This in3) Distortion of the O lattice. The protrusions in the images cannot overlap the ideal honeycomb mesh of the O atoms, implying that the ideal lattice of honeycomb O atoms at the surface was distorted. The blunt hexagonal pattern in the SC images was reproduced by a slightly displaced array of spots rather than the perfect (2 × 2)–arranged spots (fig. S7). Note that the (1 × 1) diffraction patterns in the previous diffraction studies (2). This result indicates that surface reconstruction is common between ice-Ih(0001) and Ic(111) and robust against changes in the H-atom orientation in the bulk and all interface effects.As shown in et al.  , we simuet al.  is more We conclude that the reconstruction of the ice-I surfaces originates from the electrostatic repulsion between the dangling H atoms. Several theoretical studies (+ sputtering at 850 (800) K, followed by annealing at 950 (900) K under O2 pressure to remove contaminant C atoms from the surface. The growth of crystalline ice was performed by dosing H2O gas onto the clean substrate. Ultrapure H2O was purified via freeze-and-pump cycles, and the gas was injected into the UHV chamber through a tube nozzle, which is separated from the substrate by 10 cm. For ice on the Pt (Rh) surface, the deposition rate was 3 (2) BL/min, and the substrate temperature was maintained at 145 (141) K. In addition, the samples were postannealed. Although we observed several samples under different postannealing parameters (temperature of 110 to 120 K and duration of 10 min to 10 hours), the results were identical.We used single-crystalline Pt(111) and Rh(111) substrates (MaTecK) to grow crystalline ice under ultrahigh vacuum (UHV) conditions. The Pt (Rh) substrate was cleaned by several cycles of annealing at 1200 K and Ar−10 torr at 85 K (UNISOKU-based custom-built ncAFM/STM). The details of the system were described elsewhere –based ncAFM by two orders of magnitude  An ideal honeycomb O-atom lattice was superimposed on the ncAFM image , green.2) The positions of dangling H atoms were determined from the protrusions in the ncAFM image , red.NN) on the surface BL. Then, ONN for each dangling H was uniquely assigned.3) We assumed that a dangling H atom was located just above the nearest neighboring O atom (ONN became closer to the dangling H atom than in the absence of the defect.4) This process was skipped for the model without heptagonal and pentagonal rings  but was NNN, i.e., three O atoms directly bonded to ONN via hydrogen bonding) were placed around an ONN atom with the ideal O─O length and bonding angle.5) The next-nearest neighboring O atoms for each dangling H  The positions of ONN were fixed. The other O atoms (including ONNN) were displaced from the original positions so that each O atom was located at the weight center of its three neighboring O atoms. This operation resolved the large distortion of the bonding between ONNN and its neighboring atoms. We repeated this process until the position of all the O atoms become stable.7) The positions of OWe varied the lateral positions of the initial honeycomb structure over the ncAFM image  and execNN and ONNN) are as undistorted as possible. We imposed these conditions to express that the distortion was increased in regions where dangling H atoms disappeared due to the rotation of the water molecules (Processes 5 and 6 ensure that oxygen atoms near dangling H atoms (i.e., O


Non-steroidal anti-inflammatory drugs produce antinociceptive effects mainly through peripheral cyclooxygenase inhibition. In opposition to the classical non-steroidal anti-inflammatory drugs, paracetamol and dipyrone exert weak anti-inflammatory activity, their antinociceptive effects appearing to be mostly due to mechanisms other than peripheral cyclooxygenase inhibition. In this review, we classify classical non-steroidal anti-inflammatory drugs, paracetamol and dipyrone as “non-opioid analgesics” and discuss the mechanisms mediating participation of the endocannabinoid system in their antinociceptive effects. Non-opioid analgesics and their metabolites may activate cannabinoid receptors, as well as elevate endocannabinoid levels through different mechanisms: reduction of endocannabinoid degradation via fatty acid amide hydrolase and/or cyclooxygenase-2 inhibition, mobilization of arachidonic acid for the biosynthesis of endocannabinoids due to cyclooxygenase inhibition, inhibition of endocannabinoid cellular uptake directly or through the inhibition of nitric oxide synthase production, and induction of endocannabinoid release. CB1 receptors are mainly expressed in brain structures, but also in peripheral tissues; on the other hand, CB2 receptors are expressed most abundantly in immune system cells in the periphery. Many of the unwanted effects of cannabinoid receptor agonists are caused via CB1 receptors located in the central nervous system . In addition, there are artificially synthetized synthetic cannabinoids, who are functionally similar to Δ9-tetrahydrocannabinol (THC), the main psychoactive and analgesic compound found in the plant ,2. All os system ,6.Endocannabinoid system is comprised of CB1 and CB2 receptors, their endogenous lipid ligands (endocannabinoids) and the enzymes involved in their biosynthesis and inactivation. Endocannabinoids are derived from arachidonic acid (AA); anandamide [arachidonyl ethanolamide (AEA)] and 2-arachydonoyl-glycerol (2-AG) are best characterized and considered to be the main endocannabinoids ,7. AEA aCannabis has been used for the management of pain for centuries, and numerous experimental and clinical research works have found effectiveness of the plant’s constituents, endocannabinoids and synthetic cannabinoids in different pain models ,16,17. HNSAIDs exhibit moderate analgesic, anti-inflammatory and antipyretic properties; they are the most common pain relief medicines in the world. The principal mechanism of action of NSAIDs is inhibiting the activity of COX enzymes, and thereby reducing the production of prostaglandins . Howeverin vivo and in vitro  and prostaglandin-glycerol esters; a reduction in the levels of these proinflammatory and pronociceptive mediators may also contribute for their antinociceptive activity ,13. TherIncrease in endocannabinoid tonus can be reached not only by decreasing their metabolism via inhibition of degradative enzymes, but also by augmenting endocannabinoid biosynthesis. Since AA is also important in endocannabinoid synthesis, COX inhibition probably provides more AA for endocannabinoid synthesis rather than prostaglandin synthesis ,47. IndeRegarding the involvement of the endocannabinoid system in the analgesic effects of NSAIDs, Gühring et al.  proposedParacetamol (acetaminophen) is one of the most widely used drugs as an antipyretic and analgesic. Unlike classical NSAIDs, paracetamol does not exert any anti-inflammatory activity, whereas its analgesic activity is similar to that of NSAIDs. Inhibition of peripheral COX enzymes does not appear to be primarily responsible for the antinociceptive activity of paracetamol; but probably some central mechanisms, including the endocannabinoid system, participate in these effects . InhibitIn most of these studies, pharmacological blockade or genetic deletion of cannabinoid receptors have been performed. Our group measured local endocannabinoid and N-acylethanolamide levels in the brain and spinal cord of rats, in order to observe the interaction of paracetamol and endocannabinoids directly; we observed an increase in 2-AG levels in the PAG and the RVM 12 h after paracetamol administration, but a decrease in AEA levels in the RVM and spinal cord . There aATP channel opening (Dipyrone (metamizole) is another worldwide used antipyretic and analgesic drug. Unlike classical NSAIDs, but similar to paracetamol, it possesses little anti-inflammatory activity. Despite intensive research, the precise mechanism underlying the antinociceptive effect of dipyrone is still unknown. Rather than peripheral COX inhibition, it has been suspected for a long time that dipyrone elicits centrally-mediated antinociceptive action ,75,76. I opening , althoug opening ,85.ATP opening is involved in the antihyperalgesic effect of dipyrone metabolites  by non-opioid analgesics and/or their metabolites ,88,90;2- Increase in endocannabinoid levels by;a) inhibition of degradative enzymes; i) via FAAH inhibition ,41; ii) via COX-2 inhibition ,42,49;b) shifting AA metabolism toward endocannabinoid synthesis due to COX inhibition ,48,49;c) reducing activation of endocannabinoid transporters and thus endocannabinoid degradation due to inhibition of NOS production ,97;d) induction of endocannabinoid release ,94;e) inhibition of cellular uptake of endocannabinoids by the metabolite (paracetamol) ,61.


The spreading of soap bubbles after forming contact with a substrate is experimentally studied. We find for dry glass substrate that the rim of the spreading soap bubble follows the well known scaling law for inertia dominated spreading  The fluid mechanics of their formation through coalescence has recently attracted more attention5. Besides the mutual interaction of soap bubbles to form foams, they are approaching and interacting with wet and dry container walls.Through centuries soap bubbles have fascinated children and adults due to their beauty and fragility alike. Foams which are a dense packing of soap bubbles posses fundamental importance in many industrial processes such as waste water treatmentliquid droplet coalescence and their spreading as a closely connected fluid mechanical problem9. The coalescence of two closely spaced water droplets is initiated with the formation of a liquid bridge between the droplets10. Then the bridge growth et al.6 to follow up to a logarithmic correction factor a linear growth, i. e. 7.Let us first recall et al. suggested the growth of the liquid bridge radius as 8.In the inertia dominated regime , one recognizes that it is rather difficult to observe the viscous regime for water-like fluids 13. It spreads until it reaches a film thickness that is determined by van der Waals forces. On partially wetting surfaces the droplet spreads driven by capillarity as long as it has not reached its equilibrium contact angle12. In the long-time limit the exponent of the spreading for a small, viscous droplet on a surface is 1/10, which was already predicted by Tanner14. Small means in this context smaller than the capillary length g the gravitational acceleration. In small droplets, surface tension plays the dominant role and the exponent can be determined by balancing surface energy and gravitational forces with the dissipation in the fluid14. For low viscosity droplets, the spreading dynamics is governed by the interplay between inertia and surface tension and a exponent of 1/2 was found in the early spreading stage12 and 2/3 for later stages15.The initial stage of droplet spreading on a smooth, flat surface is very similar to the coalescence of two droplets or the coalescence with a wet film. This can be attributed to the existence of a precursor film of typically one molecule in layer height, which is formed by van der Waals forces16. The effect of the latter on the spreading of droplets has been studied: At early stages for partially wetting surfaces and low-viscosity droplets the power law was found to be independent of the surface wettability and only the pre-factor changes. Therefore, drops spread faster on a hydrophilic surface than on hydrophobic 17. In the inertia dominated regime of droplet spreading on partially wetting surfaces Bird et al.11 showed that b decreases monotonously with increasing equilibrium contact angle 13.The cross-over time between the fast and the slow dynamics is determined by the droplet radius, the viscosity of the fluid, and the wettability of the surface3 and numerical18 studies on partial coalescence of a soap bubble with a flat soap films. There the overall time for partial coalescence follows R is the initial radius of the bubble.While spreading and coalescence of droplets is well understood, detailed studies of the contact dynamics of soap bubbles on dry and wet surfaces are scarce. Previous work is limited to experimentalThis work aims to fill the knowledge gap to compare the spreading of soap bubbles on dry and wet substrates while varying film viscosity and their surface properties.Owis LTM 120-300-MiDS). Thereby the surface of a glass substrate is approached with a constant speed of 0.5 mm/s. Upon touching the soap bubble spreads on the surface. The dynamics is illuminated with a collimated green laser , CMC 7–10 mM , a hydrophilic coating either with positive charges or without, and a hydrophobic coating.9. Therefore we report in Table To measure the static contact angle of the soap solutions on the glass slides, a small droplet (0.1 ml) was placed on the glass slide and a photograph was taken of the droplet after 20 s. The results are reported in Table The surface tension was measured with the pendant drop method and analyzed with a software using MATLAB .We report on the spreading of soap bubbles made from the three solutions  as a function of t and in (b) as a function of a, b and c are fitting parameters). The intersection of both fits gives From the space–time plot we extract the spatial coordinate of the rim. Let us first compare the measurements with a power law of for the early spreading of a droplet on a dry surface, i. e. r as a function of time for three different solutions are shown. Additionally, four different glass substrates are used. The soap solutions show different equilibrium contact angles on the different substrates. The values are presented in Table Next the effect of viscosity is investigated by varying the amount of glycerol added to the solution . This coating is obtained by spreading a bubble on the dry surface prior to the present experiment. Solution S1 shows dewetting and is therefore not used as a wet coating. Solutions S2 and S3 form a thin stable fluid layer with an almost uniform thickness of approx. 50 Next we report and discuss the spreading of soap bubbles on wetted surfaces.Note that only the macroscopic wetting is studied here. The wetting according to the molecular-kinetic theorydry surface 22. The contribution of surface active molecules should be included in a model to account their importance.Right within the experimental temporal and spatial resolution of 14.8 The spreading velocities for the three solutions before the cross-over time were determined from the space–time plots (cf. red line in Fig. In Fig. Figure 11 where inertia does not play a role.On wet surfaces we observe marked differences when the bubble contacts and spreads compared to a dry surface. The radius grows linearly right from the beginning and within our experimental resolution. The rim only slows down when it reaches the radius of the soap bubble. We explain this behavior with a purely viscous spreadingAdditionally, the formation of an instability is observed during the spreading on a wetted surface. We find the formation of radial oriented stripes trailing the rim and explain it with height undulations of film downstream of the rim.u is the velocity of the fluid particle in the rim, t is the time, G of 5 can be excluded. Instead we speculate that the wrinkle formation is caused by a Marangoni flow. The wrinkles remind of stripes evolving in evaporating films on an inclined surface, such as wine in a glass23. Similar structures were found by Wodlei et al.24, during evaporation of an oil droplet containing a surfactant, which is placed on a soap solution. The authors connected the number of wrinkles to the relation of a critical layer height, below which wrinkles can occur and the undisturbed film thickness at the edge of the drop. The prediction of such a critical height by Wodlei et al.24 could explain, why in our experiments wrinkles are not present in every experiment, but in only 50% of the cases.To understand if acceleration may cause this instability we estimate its magnitude upon contact from the Laplace pressureIn this work, the spreading of soap bubbles on different substrates is studied. The spreading of the contact line was found to follow the power law


Climate change is strongly impacting the Arctic environment, leading to rapid sea ice loss. In some sectors, the retreating ice edge is perceived as an opportunity to expand and develop economic activities. Previous studies show this development in the Canadian and Russian Arctic. This paper examines mobility patterns of cruise ships and fishing vessels around Svalbard, a major hotspot of maritime activity and retreating sea ice cover, in relation to sea ice variability between August 2012 and September 2019. The results show a slight overall increase in fisheries and cruise activity, as well as remarkable trends of stretching operational seasons and expanding navigational areas in these sectors. Overall increasing activity and changing mobility patterns provoke a discussion about the implications for safe navigation and sustainable management, thus raising issues of high pan-Arctic relevance. This increase in temperatures results in large changes in sea ice conditions, with both thickness and extent following negative trends4. Sea ice is a large impediment for the development of maritime activities in the High Arctic and limits the activity to seasonal operations in most areas. Therefore, the decline of sea ice is opening new opportunities for maritime activities to expand7. The fastest developing sectors are shipping, tourism and recreational craft1, resulting in an increase in the number of ships in the Arctic by 25% between 2013 and 20198. This increase in vessels entails high risks and raises strong concerns about environmental impacts and safety issues as it puts pressure on supporting infrastructures and regulations5.The polar regions are the most climate-sensitive areas in the world, and temperatures in the Arctic are rising more than twice as fast as the global average9. More recently on a national level, the Norwegian Government in its latest Svalbard strategy emphasized the need to asses preparedness and safety challenges arising from increased shipping activity around the archipelago of Svalbard10.From a pan-Arctic perspective, there is a high level of maritime activity in Svalbard waters, together with the rest of the Barents Sea, and notably the Kara Sea12. At the same time, the region experiences the most drastic environmental changes in the Arctic with the northward retreat of the ice edge13, the delay of winter freeze-up14, and the air temperatures rising faster than global or even Arctic average15. Svalbard, therefore, serves as a relevant case study for the development of maritime activities and environmental change, and can provide important lessons applicable to other Arctic regions.Safety concerns regarding high latitude cruise tourism and fishing operations are progressively put on the political agenda, nationally and internationally. In 2017, the International Maritime Organization (IMO) implemented the Polar Code, which sets mandatory requirements for design, construction, equipment, operational, training, search and rescue and environmental protection matters for all vessels sailing in polar watersWhile maritime activity around Svalbard has increased in the past decade, to date the actual developments have not been mapped in a comprehensive way. The objective of this work is to visualize and discuss the relationship between marine economic activities (cruise tourism and fisheries), and sea ice variability around Svalbard between 2012 and 2019, in order to understand the potential implications for safety and sustainable management. The study area is delimited by the Svalbard Fisheries Protection Zone (SFPZ), which reaches out to 200 nm from the coastlines and is roughly located between 72° N and 84° N, and 3° W and 38° E , whereas less sea ice and better accessibility promoted more cod fishing activity into December  to adapt to vessels with growing passenger capacity35  and (2) to access increasingly remote areas, year-round . Increased activity and expanding operational seasons raise concerns for environmental protection and human safety, and pushes the limits of SAR infrastructure in place38. In its recent Svalbard strategy10, the Norwegian government emphasized the need to assess preparedness and safety challenges arising from increased shipping activity in the Arctic, and currently investments are made to improve the material infrastructure. Despite the under-dimensioning in state preparedness around Svalbard, from a pan-Arctic perspective it might be argued that Svalbard has a relatively well-developed safety infrastructure, and it can be assumed that safety preparedness in other Arctic regions is to an even larger extent a major responsibility of the operators39.Supporting infrastructure for safe maritime activity consist of, among others, safety and emergency response capacities, and communication and information systems. While SAR capacity around Svalbard is dimensioned to assist a fishing vessel in distressnges are  to adapt40. In addition, the improvement of Arctic weather and sea ice forecasting services has gained a lot of attention since the Year of Polar Prediction41. The strong seasonal and inter-annual sea ice variability around Svalbard in parallel with increasing maritime activities has a demand-pull effect on achieving the need for accurate and reliable sea ice services43. End-users operating around Svalbard emphasize the need for publicly accessible ice charts to be available daily, even more so when sailing in the north and east of the archipelago. For the moment, ice charts are available on weekdays, while automated charts are published during the weekends, but there is no warranty on their accuracy44. Therefore, it is imperative to put in place opportunities for improvements towards higher resolution, accurate charts to better support maritime activity in the Arctic45. It is also essential to provide products with low-bandwidth solutions in order to be easily accessible to a wider range of vessels sailing at higher latitudes. Such sea ice information needs are dependent on the type of use46; while fishermen primarily need information about the location of the ice edge, cruise vessel operators desire more detailed information about the type of sea ice in their navigational areas.With respect to communication and information infrastructure, there are efforts to improve communication in these waters, through prioritizing high frequency radio coverage up to the North Pole9. However, specific regulations are needed to adapt to local development of maritime activities. The HFO ban in protected areas around Svalbard acts as geographical restriction for large ships and could serve as an example for other Arctic regions. Within the IMO and the Arctic Council, there is growing discussion to ban HFOs throughout the Arctic47, which would be an important step forward in sustainable navigation. With regard to the predicted growth in cruise tourism in the Arctic11, in January 2020 the Norwegian Maritime Authority implemented specific regulations for passenger ships sailing in protected territorial waters, which account for 87% of all territorial waters around Svalbard48. These additional regulations include the prohibition of discharge of sewage and gray water within 500 m off land and the prohibition of sea traffic in bird sanctuaries between May 15 and August 1550. Hence, while maritime development around Svalbard will partly be shaped by future ice conditions, its sustainability and trends will largely depend on international and national policy developments51.Several governance regulations have been implemented or expanded in recent years to mitigate risks and limit human impact, such as IMO’s Polar Code, which acts as a baseline for requirements on various levels  for navigation in polar waters52. It will be highly relevant to follow the implementation of the Norwegian government’s recently updated ocean strategy as well as its High North strategy53, and what these imply in terms of safety and sustainable maritime development around Svalbard. Despite ongoing efforts at national and international levels, navigation in these areas will always remain challenging due to persisting seasonal darkness, remoteness and potentially hazardous weather and sea-ice conditions. Still, the development of SAR capacity and communication and information systems are ascending on the political agenda, not only because of the increasing local and destination traffic as illustrated in this paper, but also due to the projections of Svalbard’s strategic future location along a potential Arctic trade route over the North Pole54.Despite ongoing work to enhance supporting infrastructures and sharpen regulations, and thus the growing controllability of maritime traffic around Svalbard, this might invite for a further increase in activities in the decades to come. Indeed, some argue that environmental concerns are unlikely to lead to very strong restrictions and limitations of exploitation of Arctic resourcesThis paper has provided insights into the development of maritime activity around Svalbard, and has presented an approach that will be relevant to understand transformations in other Arctic regions. The combination of vessel tracking data through AIS and sea ice data have enabled us to provide novel regionally contextualized insights about trends in Arctic shipping activity and how these relate to climate change. To conclude, we outline complementary ways to achieve a comprehensive understanding of maritime change and its implications in Arctic regions, serving to guide safety and sustainable development.We approached maritime change by focusing on unique vessel activity per month, which has primarily provided an enhanced understanding of expanding sailing areas and seasons. Naturally, there are several other possible approaches with AIS data, such as measuring the total distance sailed by all vessels per category, which would be crucial information in the dimensioning of safety and response capacity.In the Arctic, investments in satellites recording AIS signals are growing, making higher temporospatial resolution data accessible. This increases the possibilities to analyze mobility patterns in determined areas over shorter time periods (e.g. weekly timeframes), which would elucidate vessel’s behavior in proximity to the ice edge and in the marginal ice zone.55 and is predicted to be an interesting place for maritime activity31, but highly challenging in terms of risk and safety.Developments in the marginal ice zone are very relevant to follow, since this dynamic zone between open ocean and dense pack ice will continue to expand in the summerWe were particularly interested to understand changes in the cruise tourism and fisheries sectors, which provided us with the possibility to reflect upon some of the dynamics that are at play in each of these sectors. Naturally, a more comprehensive picture of maritime change would include several sectors, such as cargo shipping, research, military and surveillance activities. Past trends can be understood by using the wealth of AIS data that have been generated in the past years. However, not all vessels carry AIS transponders, and a mapping of all vessels is vital for understanding the overall safety challenges in the Arctic, especially since more and more pleasure yachts are exploring the area.past trends are analyzed in line with projected maritime change, which might include a focus on foresight and scenario studies and a scrutiny of government and business strategies for industrial innovation and development for any particular region. Such a comprehensive understanding can lead to a better overall picture of maritime transformations in Arctic regions and the risks and uncertainties these entail.A comprehensive study of maritime activity trends increases in value when 56. AMSR2 has been delivering sea ice concentration data since August 201256, which sets the beginning of the study period. AMSR2 has a horizontal resolution of 6.25 km and has a specific grid over the Earth. For each grid point the average sea ice concentration is calculated by ASI and is associated with coordinates representing the center of the grid point. However, this resolution presents some limitations close to land, where coastal reflection was detected as sea ice. This limitation does not affect the study, as ice cover in fjords is not considered in this study.This study used daily sea ice concentration data from ARTIST Sea Ice (ASI) algorithm applied to Advanced Microwave Scanning Radiometer (AMSR2) from the open dataset of the University of Bremen57. Since, AIS has been growing in interest for coastal states to ensure safety and used more frequently as a tool to establish sustainable management of maritime activities58. Several studies have analyzed mobility patterns with AIS data on a global scale59 and in the Arctic60. In this study, AIS data from vessels operating around Svalbard between August 2012 and September 2019 were obtained from the Norwegian Coastal Administration (NCA). Monthly files had the following information; Maritime Mobile Service Identity (MMSI) number, IMO number, name of vessel, vessel length, timestamp (UTC), longitude, latitude, speed over ground, course over ground, true heading , navigation status, message number  and source (satellite/ground). Both commercial and recreational vessels are included in this dataset, which are categorized by class A and class B respectively. AIS signals are recorded every 2 s to 10 minutes61, depending on vessel class and navigational status, and whether signals are detected by satellite or ground receptors. The NCA uses AIS data from four satellites to monitor vessel traffic off Norwegian and Svalbard coasts. As AIS data are publicly available (upon request) not all vessel information is provided. Therefore, to obtain ship type information on vessels sailing in the SFPZ a special request was done; this second dataset excluded vessels under 15 m. In this study, the IMO number was used to merge the AIS and vessel type information data. We processed over 120 million AIS signals and used the IMO number, timestamp (UTC), longitude, latitude, and vessel type to determine mobility patterns around Svalbard.AIS is a precise satellite-based source of data continuously recording spatial ship traffic. Initially established by IMO as a safety measure at sea for large commercial ships to avoid collision, AIS first became effective by the end of 200461. Multiple satellites allow for a more continuous registration of AIS signals, whereas only one satellite results in gaps in the data when orbiting the globe. Furthermore, AIS data are susceptible to problems related to the AIS transponder, issues stemming from modifications by users, and errors in the transmission, reception, storage and decoding of information62. Among these limitations, two in particular affected our analyses: (1) vessel information such as the IMO number filled in by the crew can hold some errors, therefore some vessels were not categorized and therefore excluded from analysis; (2) the poor satellite coverage at high latitudes may limit the position registered and lead to loss of the northernmost positions.AIS data are not homogenous time series due to satellite limitations, technology innovations and transponder problems. Satellites are crucial to receive signals beyond the limits of coastal receptors; however, signals will not be detected if the satellite is out of reach or if the vessel is near a geographical barrier. The investment in new satellites has led to an increase in the frequency of signals between 2012 and 2019. The four AIS satellites in Norway were launched at different periods: AISSat-1 in July 2010, AISSat-2 in July 2014, NorSat-1 and NorSat-2 in July 2017To measure maritime activity in SFPZ we counted the number of unique vessels sailing in the area, using the AIS data where each vessel has a unique IMO number. Each IMO number is counted once per month even if the same vessels sailed multiple times through the SFPZ in that time period. The delimitation of the SFPZ is roughly from 72°N to 84°N and 3°W to 38°E Fig. . For eac63, here the ice colormap was selected from the cmocean gallery. Concerning AIS signals, in order to compare mobility patterns throughout the study period, vessel positions are shown with a frequency of 60 min on the maps. This allowed a better comparison between earlier and later years, due to satellite innovations explained above. In total 86 months were analyzed and ten maps  are presented in this paper. In this study, 2013 is the first complete year of both AMSR2 sea ice concentration and AIS data. 2017 has a particular high sea ice cover over the summer season and is of particular interest to analyze. The selected months in this paper allowed us to give an overview of both seasonal sea ice variability and mobility patterns. All maps between August 2012 and September 2019 can be found in the To visualize the relationship between passenger and fishing vessels with sea ice variability, a series of maps were made. These maps of ice concentration and ship tracks were created with python 3.7, using basemap (1.2.0), matplotlib (3.0.2), numpy (1.15.4) and pandas (0.23.4) libraries, and using a Polar Stereographic projection. Each map shows the monthly mean sea ice extent as well as all passenger and fishing AIS signals detected that same month. The monthly mean sea ice concentrations were calculated from the daily AMSR2 grid. In this study, the ice edge is delimited at 15% ice concentration, and below this limit is defined as open water. On the maps, the boundaries of sea ice concentration are at 15%, 30%, 45%, 60%, 75%, 90% and 100%  over the summer and autumn months of 2018. The participants were captains, sea ice researchers and representatives of fisheries and cruise tourism organizations in the Svalbard and Barents Sea area. These key informants were selected based on their role in decision-making and guidance for the fishing and cruise tourism industries operating around Svalbard. The structure of the interviews allowed to cover important themes around the use of sea ice services around Svalbard, but also gave the participants the freedom to add information they believed important as well. Main themes concerned changes in sea ice variability, vessel traffic, the type of sea ice information used in decision-making and the challenges around operating in waters around Svalbard in the future. All interviews were conducted with the participants’ consent, were all held in person, and most interviews lasted around 30 min. The interviews were conducted in English, which did not result in any language barrier as most Norwegians are fluent in English. Handling and management of the qualitative interview data followed the requirements of the Norwegian Centre for Research Data.Supplementary Information.


In the present study, the mechanism of mercury chloride (HgCl2) induced inactivation of tyrosinase was investigated for the first time. The IC50 values were 29.97 and 77.93 μmol/L for monophenolase and diphenolase, respectively. A kinetic analysis revealed that HgCl2 inhibited tyrosinase activity in an irreversible non-competitive manner. The strong intrinsic fluorescence quenching suggested that the formation of the HgCl2-tyrosinase complex induced conformational changes of the enzyme, and HgCl2 had only one single binding site or a single class of binding site on tyrosinase. The molecular docking and further experiments demonstrated that HgCl2 bound to the amino residuals (His) in the catalytic center of tyrosinase. To our knowledge, these findings presented in this paper were the first evidence of the direct interactions between HgCl2 and tyrosinase, which provided a deep understanding of the inhibition mechanism of mercury on tyrosinase.Inorganic mercury compounds have been used in skin-lightening products since ancient times. Although a previous study demonstrated that mercury impeded the transfer of Cu Tyrosinase (EC 1.14.18.1), a type III copper protein, is ubiquitously distributed in bacteria, fungi, plants, insects, crustaceans, and mammals . It is aTo decrease the abnormal accumulation of melanin, the use of skin-lightening  products has been prevalent among women all over the world for decades . The actThe failure in the prohibition of adding inorganic mercury to skin-lightening products is an interesting question worthy of consideration. We believe that the main reason may be the good performance of inorganic mercury compounds in skin-lightening and anti-freckle effects. So now comes the question, why do these compounds exhibit such strong inhibition ability of melanin production. It is well known that mercury can replace the copper needed for tyrosinase activity, thereby deactivating enzymes and producing skin-lightening effects . Specifi2, 99.5%) and kojic acid (99%) was obtained from Aladdin Bio-technology . Histidine (His), valine , glutamate (Glu), and alanine (Ala) were of analytical grade and bought from Xiya Chemical Industry . All other chemicals and reagents used were of analytical grade or pharmaceutical grade. A Milli-Q-Plus ultra-pure water system from Millipore  was used throughout the study to obtain water used during the experiments.Tyrosine (98%), dopa (98%), and mushroom tyrosinase  were purchased from Sigma-Aldrich . Mercuric chloride  at 305 K. A 100 μL aliquot of HgCl2 solutions with various concentrations  was added to the reaction system to give concentrations ranged from 0 to 333.33 μmol/L. The final concentration of tyrosinase was 33.33 U/mL. The optical densities (OD) at wavelength of 475 nm were recorded using UV spectrophotometer  every 30 s in the first 2 mins and then every 1 min until 30 mins. Kojic acid was used as a positive control. The appropriate blanks (containing both tyrosine and HgCl2 but without tyrosinase) were tested and subtracted to correct the absorbance.The method was modified based on the previous description . In brie2 solutions with various concentrations  was added to the reaction system to give concentrations ranged from 10.42 to 666.67 μmol/L. The OD values of the reaction system in the presence of HgCl2 solutions (A1) were recorded at the wavelength of 475 nm using the UV spectrophotometer. The OD values of the reaction system in the absence of HgCl2 and tyrosinase were used as a positive (A2) and a negative (A3) control, respectively. Relative enzymatic activity (%) = (A1-A3)/A2×100%. The experiment was repeated three times. The IC50 value was calculated as the concentration (leading to achieve 50% inhibition) according to the equation, which is obtained by curve fitting of the enzyme activity versus the concentrations of inhibitor.The method was performed as previously described . The ass2 on tyrosinase furtherly by using immobilized tyrosinase. In brief, we added 200 µL of diluted tyrosinase (2500 U/mL) to each well of a 24-well polystyrene plate and then covered the plate and incubated it at 4°C overnight to produce immobilized tyrosinase. The plate was brought to room temperature, and we thoroughly decanted the solution from wells and washed the wells two times with PBS to remove the unfixed enzyme. We added 3 mL of inhibitor solution to each well and incubated them at room temperature for 30 mins to assure the fully reaction between the inhibitor and the enzyme, discarding the liquid following by washing the wells two times with PBS to remove the excess inhibitor. Then, 3 mL of PBS was added to each well, and they stood for 10 mins to make the dissociation of inhibitor-tyrosinase complex. WE thoroughly decanted the solution from wells to remove the dissociated inhibitor (the procedure was repeated three times). Finally, 3 mL of substrate (dopa) was added to each well and react at room temperature for 30 mins to test the residual activity of tyrosinase. The tested samples included PBS (negative control), 95% ethanol (positive control of irreversible inhibitor), kojic acid (positive control of reversible inhibitor), and HgCl2, and each sample was tested three times. The concentration of Kojic acid and HgCl2 was both 666.67 μmol/L.Experiments were conducted to confirm the inhibitory mechanism of HgClThe non-competitive inhibition type of tyrosinase was assayed by a Lineweaver–Burk plot, which can be described as the following equation: and a secondary plot is presented below2. Km and Ki are the Michaelis–Menten constant and inhibition constant, respectively. The values of Km can be calculated from the equation (1). [I] and [S] are the concentrations of inhibitor and substrate, respectively. In. is the intercept value of the Lineweaver–Burk curve. Ki should be calculated by the secondary plot of In. vs. [I].where ν is velocity of the enzyme reaction calculated by a change in absorbance at the wavelength of 475 nm per minute in the absence and presence of HgCl2 to give concentrations ranged from 0 to 400 μmol/L. After five-minute incubation periods, fluorescence in the range of 290–500 nm was measured while the excitation wavelength was fixed at 280 nm. All the fluorescence data collected from these experiments were corrected for absorption of excitation light and re-absorption of emitted light according to the following equation:  equipped with a 150 W xenon lamp and a thermostat bath. We added a 2.0 mL aliquot of tyrosinase solution (400 U/mL) to the quartz cuvette (1 cm path length) and gradually added 2 mmol/L solution of HgClquation: Fc and Fm represent the corrected and measured fluorescence, respectively. A1 and A2 are the absorbance of HgCl2 at excitation and emission wavelengths, respectively.where Agaricus bisporus tyrosinase (PDB ID: 2Y9W) and the 3D structure of HgCl2 were both retrieved from the RCSB Protein Data Bank . The docking results generated 100 conformations of HgCl2, which were grouped into clusters by a root mean square (RMS) deviation tolerance of 2.0. The lowest energy  and the largest number cluster  were chosen for further analyses with the PyMOL molecular graphics system.Molecular docking study was performed by using AutoDock 4.2.6. The X-ray crystal structure of ata Bank . All inp2 and amino acid. We assumed that if HgCl2 indeed binds to the amino acid residues of the enzyme, such as His, Val, Glu, and Ala, premixing the amino acid with HgCl2 would affect the inhibitory effects. Thus, the monophenolase and diphenolase activity of tyrosinase both were determined by adding the mixture of HgCl2 and amino acid. The mixture was prepared just before use by adding an aliquot of 200 μL HgCl2 solution to the equal volume of amino acid solution, and which was allowed to react for 10 mins at room temperature. Furtherly, to prove the interaction between His and HgCl2, the UV-Vis spectra of His, HgCl2, and the mixture of both were measured. In brief, 5 and 10 mmol/L solutions of His, HgCl2 were prepared beforehand, and the mixture was produced by mixing the His and HgCl2 solutions (10 mmol/L) in the volume ratio of 1:1. The spectra of the samples at the wavelengths between 200 and 800 nm were scanned by using UV spectrophotometer .The experiment was repeated three times.According to the results of docking study, we furtherly checked the interactions between HgCl2, was repeated at least three times. The data are presented as the mean ± SD (standard deviation). The statistical significance was determined at the level of P-value (< 0.05) by one-way analysis of variance.Each data point of the experimental results, including tyrosinase activity assay, kinetic analysis for non-competitive type inhibition, fluorescence quenching test, and interactions between amino acid and HgCl2, respectively. With the increase of kojic acid concentration, the lag time was prolonged from 1.44 min to approximately 10.33 min. Similarly, HgCl2 also exhibited a marked inhibitory effect with significant prolongation of the lag period from 2.00 to 13.4 mins. The inhibitory effects of HgCl2 on monophenolase were activity dependent on the concentrations because the steady rate (the slope of linear part of the kinetic equation) decreased with the increasing concentration of the inhibitors  was investigated. A series of parallel  lines with the same slope and different abscissa intercepts  and its concentration  in the presence of different concentrations of HgClversible . The inc2 on tyrosinase, the results of which were shown in 2 returned to 28.32%, indicating HgCl2 indeed inhibits the tyrosinase in an irreversible way compared to kojic acid. However, the enzyme activity is much higher than the one treated by 95% ethanol, which may be due to the fact that the amount of the inhibitor (HgCl2) is not enough to inactivate the whole enzyme molecules coated on the plate.In order to prove the irreversible inhibition furtherly, immobilized tyrosinase was used to study the effects of HgCl2 on the diphenolase activity was studied by the Lineweaver–Burk double reciprocal plots. As shown in Km) keeps the value unchanged and the vertical axis intercept (2 induced a non-competitive inhibition (2 could bind both free enzyme and the enzyme-substrate complex with the same inhibition constants (Ki). The value of Ki (29.41 μmol/L) was obtained from a plot of the vertical axis intercept  versus the inhibitor concentration  were 0, 95.24, 181.82, 260.87, 333.33, and 400.00 μmol/L, respectively. Although the decrease in the fluorescence intensity were caused by quenching, there were no significant wavelength shift with the accumulation of HgCl2, indicating that tyrosinase does not undergo conspicuous overall structural changes. Moreover, the fluorescence intensity decreased with the increasing concentration of the inhibitor because of the tryptophan-masking properties of HgCl2, which implied that the tyrosinase became disagglomerated, and its structure was loosened , respectively, [Q] denotes the concentration of HgCl2, Kq is the quenching rate constant of the biomolecule (Kq = Ksv/τ0), Ksv is the Stern–Volmer dynamic quenching constant, and τ0 is the average lifetime of the fluorophore in the absence of quencher (τ0 = 10-8 s). The value of Ksv determined by the linear regression plot of F0/F vs. [Q] at 298 and 304 K were (2.14 ± 0.05) × 104 and 1.83 ± 0.03 × 104 L/mol, respectively. The plot  × 1012 and (1.83 ± 0.03) × 1012 L/mol/s at 298 and 304 K were much higher than the maximum scattering collision quenching constant (2.0×1010 L/mol/s)  , suggesteraction .Ka) and the number of binding sites (n) can be obtained from the following equation:For static quenching interactions, if there are similar and independent sites in biological molecules, the apparent binding constants . The Ka value of (2.87 ± 0.02) × 104 L/mol achieved the order of magnitude of 104 L/mol, indicating the moderate binding of HgCl2 to tyrosinase (2 ON TYROSINASE) showed that HgCl2 had a significant inhibitory effect on tyrosinase that was irreversible. We suggested that HgCl2 might be irreversibly coordinated with the amino acid residues of tyrosinase to form the HgCl2-tyrosinase complexes, resulting in the conformation change of the catalytic center of tyrosinase. Furthermore, the n values (0.96 ± 0.01) were close to one, suggesting that there was a single binding site or a single class of binding sites in tyrosinase for HgCl2.According to the intercept and slope value of the regression curve  with ADT by a root mean square (RMS) deviation tolerance of 2.0, resulting in four clusters, as shown in 2 is surrounded by amino acid residues , whilst the suboptimal conformation is located adjacent to amino acid residues . The detailed information of interaction between the amino acid residues and HgCl2 is summarized in 2 may change the morphology of catalytic center by binding with the amino acid residues, such as His 94, His 296, and His 263. Some experiments (in next section) were furtherly designed to support this point.Molecular docking was furtherly performed to explore the interaction between HgCl2 on tyrosinase. It was found that only His exhibited the significant effect on protecting the diphenolase activity in the presence of HgCl2, as shown in 2 were shown in 2 preferred to bind with His rather than other amino acid residues, such as Val, Glu, and Ala. Once HgCl2 bound to free His, it was difficult to bind it to the residual His, which resulted in the protective effects. In order to further prove the interaction between His and HgCl2, the UV-Vis spectra of His, HgCl2, and the mixture of both is presented in 2), suggesting the formation of His-HgCl2 complexes. Thus, based on this indirect and direct evidence (results of docking study), we believe that HgCl2 exhibits inhibitory effects on tyrosinase by binding to the amino residual (His) of the catalytic center of tyrosinase.Amino acids, including His, Val, Glu, and Ala, were checked to see whether they have any impact on the inhibitory effect of HgCl2 on tyrosinase. For monophenolase, HgCl2 could obviously prolong its lag time and decrease its steady-state rate with the IC50 value of 29.97 μmol/L. A kinetic study showed that HgCl2 could also significantly inhibit the diphenolase activity with the IC50 value of 77.93 μmol/L in an irreversible non-competitive manner. HgCl2 quenched the fluorescence of tyrosinase by a static procedure and formed an irreversible complex with Ki value of 29.41 μmol/L. The molecular docking study suggested that HgCl2 bound to the His residual of the catalytic center of tyrosinase and might change the morphology, leading to the inhibitory effects. Our studies firstly demonstrated that HgCl2 has the capability to inhibit tyrosinase directly, which is helpful to further our understanding of the inhibition mechanism of mercury on tyrosinase and to expand the scientific understanding of the toxicity of mercury in organisms.In this paper, kinetics studies, different spectroscopic measurements, and molecular docking studies were employed to explore the inhibitory effects and mechanism of HgClThe datasets generated for this study are available on request to the corresponding author.2 on tyrosinase. All authors contributed to manuscript revisions and read and approved the submitted version.JC, ZR, and NJ contributed conception and design of the study and performed the docking study. YY, MR, and QL designed and performed the inhibitory effects of HgClThis work was supported by the Natural Science Foundation of Fujian Province (grant number 2019J01806), the Education Department of Fujian Province (grant number JZ160470), the Putian Science and Technology Bureau (grant number 2018SP3004), and the Training Program of Innovation and Entrepreneurship for Undergraduates .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition, the electrochemical impedance spectroscopy (EIS) was investigated for the detection of IgE. The limit of detection (LOD), limit of quantification (LOQ), and dynamic range of the electrochemical sensor were 3 IU/mL, 10 IU/mL, and from 10 IU/mL to 100 IU/mL, respectively.The immunoglobulin E (IgE) level in serum is an important factor in the examination of allergy. Ferrocene (Fc)-modified self-assembled monolayers (SAMs) were placed on an indium tin oxide (ITO) electrode as a sensing layer for the detection of human IgE. The Fc moiety in the SAMs facilitated the electron transfer through the organic SAMs layer and electrocatalytic signal amplification. The electrochemical measurement was accomplished after the sandwich type immobilization of the receptor antibody, target human IgE, and enzyme conjugated secondary antibody. The enzyme product,  Allergy  is a major cause of various allergic diseases, such as atopic dermatitis, allergic asthma, allergic rhinitis, urticaria, and food/animal/insect/plant allergies and has a significant effect on a person’s health ,2,3,4. TIgE is synthesized by plasma cells like other immunoglobulin and is typically the least abundant isotype in blood, usually less than 1 IU/mL (1 IU = 2.4 ng) . HoweverThe majority of allergy tests nowadays are based on the antibody receptor (immunoassay). Common methods include radioimmunoassay , enzyme-In this paper, we investigated the electrochemical immunosensing of human IgE using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Even though Indium tin oxide (ITO), one of the most widely utilized conductive oxide thin films, presents an advantage in spectroelectrochemistry due to its transparent property, the ITO electrode is a still promising material for developing commercial electrochemical biosensors instead of noble metal electrodes such as Au electrodes, owing to its low electrical resistivity, low background current, wide potential window, applicability to self-assembled monolayers (SAMs), and cheaper price than Au electrodes . TherefoFor the cyclic voltammetric detection, the electron transfer mediator Fc-modified SAMs were constructed on the ITO electrode. Next, streptavidin, biotin conjugated receptor antibodies, target human IgE, and alkaline phosphatase (ALP) conjugated secondary antibodies were immobilized in turn. Consequently, the electrochemical signal amplification by enzymatic reaction and redox cycling via Fc was successfully observed. Not only the CV, but also the EIS, was applied to investigate the IgE concentration. The EIS is a label-free and convenient tools for monitoring the charge transfer processes of immunosensors ,24. Ther4), streptavidin, 4-aminophenol (p-AP), 4-aminophenyl phosphate monosodium salt hydrate (p-APP), thrombin for human plasma, ferri/ferrocyanide, bovine serum albumin (BSA), human IgG, hemoglobin, horse serum, and all buffer salts were purchased from Sigma Aldrich , unless otherwise stated. NH4OH (30% diluted) and H2O2 (35% diluted) were obtained from Samchun . Immunoglobulin E (IgE) human serum (Target IgE) was obtained from the National Institute of Biological Standards . Biotin-conjugated rabbit anti-human IgE (heavy chain) antibody (receptor antibody), alkaline phosphatase (ALP)-conjugated goat (or mouse) anti-human IgE antibody (secondary antibody), and goat immunoglobulin G (IgG), mouse IgG, and ALP-conjugated streptavidin were obtained from Thermo Fischer Scientific . All chemicals were used as received. Ultrapure water  was used in all experiments.(3-Aminopropyl)triethoxysilane (APTES), ferrocenecarboxaldehyde, sodium borohydride  5:1:1] at 70 °C for 2 h, rinsed with distilled water, and dried with N2 gas [ITO was obtained from Samsung Corning . A cut of the ITO electrode was cleaned with ethanol and dried with Nh N2 gas .4) for 1 h. Then, the Fc-modified SAMs was obtained on the ITO electrode [For the formation of APTES SAMs, the cleaned electrode was incubated in a diluted APTES solution (APTES:ethanol = (v/v) 1:50) for 2 h, and washed with pure ethanol ,27. Nextlectrode .w/w) BSA solution to prevent the non-specific binding of proteins for 0.5 h [The SAMs modified electrode was incubated in phosphate buffered saline (PBS)  solution containing 10 μg/mL of streptavidin for the immobilization of the receptor antibody. The streptavidin is addressed on the electrode surface through non-specific binding. After rinsing with PBS  buffer, the electrode was incubated in PBS  containing 10 μg/mL of biotin-conjugated receptor antibody for 1 h, after which the unreacted reagent was washed by PBS . The electrode was incubated in a PBS  containing 0.1% , mouse IgG (100 μg/mL), thrombin (100 μg/mL), human IgG (1 mg/mL), and hemoglobin (5 mg/mL) were used instead of target IgE. Target IgE and the other reference materials were prepared using PBS  buffer.The electrochemical experiments, both CV and EIS, were performed by using a CHI 750 potentiostat  with three electrode cells placed in a Faraday cage. The electrochemical cell consisted of a prepared ITO working electrode, a Pt wire counter electrode, and an Ag/AgCl (1 M KCl) reference electrode. All the potentials are reported vs. Ag/AgCl when not indicated differently.2 (pH 9.0) with/without 1 mM p-AP or 1 mM p-APP. The solutions of p-AP or p-APP were prepared daily before use. All the electrochemical measurements were obtained after having 10 min of quiet time for the enzymatic reaction.CV was performed in an electrolyte solution consisting of 50 mM tris(hydroxymethyl)aminomethane (Tris), 10 mM KCl, and 1 g/L MgClEIS was performed using ferri/ferrocyanide (2.5/2.5 mM) as a redox probe in 1X PBS (pH = 7.4). A potential of 0.18 V was applied with a perturbation amplitude of 5 mV between frequencies of 1 MHz and 0.01 Hz. Although the Fc moiety was placed on the surface of the biosensor electrode, it was difficult to observe the apparent signal changes caused by the Fc reporting (the data is not shown here). This is because the surface concentration of Fc was too small, and the internal Fc was not sensitive to the externally attached target IgE. Therefore, the redox probe, ferri/ferrocyanide, was added into the solution for the EIS measurement.In the electrochemical immunosensor, the use of self-assembled monolayers (SAMs) can provide a reproducible and robust path to functionalize electrode surfaces. Various organic molecules containing anchor groups such as thiols, amines, or silanes are used for the formation of SAMs depending on the electrode materials ,31. The Generally, in electrochemical biosensors, because the additional organic layers of SAMs and biomolecules make the electron transfer difficult, redox-mediating functional groups that enable electron transfer between electroactive species and the SAMs-modified electrodes are required for the successive electrochemical detection. Therefore, the redox mediator-introduced SAMs can be an excellent platform for electrochemical biosensors.In this research, we introduced the electron transfer mediator, Fc, as a functionality in the APTES SAMs-modified ITO electrode. In previous studies ,34,35, tp-AP is employed as an enzyme label and a signal-reporting molecule for the electrochemical measurement [p-AP. Here, the electroactive species, p-AP, is generated by the enzyme label, ALP, from its substrate, p-APP.Then, the redox-active Fc SAMs-modified electrode was investigated as an immunosensor for the detection of human IgE. The combination of ALP and surement . The Fc First, the redox reaction of the Fc moiety on the electrode surface was investigated to characterize the formation of the Fc-functionalized SAM onto the ITO electrodes. As shown in Γs, were 4.1 (±1.0) × 10−7 mol/cm2.The full width at half-maximums (FWHM) was ~160 mV, relatively larger than the ideal value for reversible responses, 90.6 mV . The oxip-AP or p-APP to characterize the electrocatalytic redox cycling performance of the Fc-functionalized SAMs layer. Even if the p-APP existed, the electrocatalytic reaction between p-APP and Fc did not appear. The background current of p-APP was obtained only at the potential region of 0.0 V to 0.5 V. However, in the presence of p-AP, the oxidation part of the current was amplified by the electrocatalytic redox-mediated oxidation of p-AP, even though the reduction part of the current stayed intact. The mechanism is as follows: the Fc moiety in the SAMs is oxidized to the ferrocenium ion (Fc+) by the electrochemical oxidation reaction. Then, the Fc+ is reduced back to Fc by the chemical redox reaction with p-AP. As discussed in a previous report, this can be explained by the following mechanism [p-AP that has been oxidized by the loss of two electrons. This redox cycling would continue until all the p-AP is spent. As a result, an amplified oxidation current proportional to the concentration of p-AP was obtained. In the absence of the electron transfer mediator, Fc, the electrochemical oxidation of p-AP was obtained at a higher potential region (~0.6 V), as shown in p-APP occurs at this potential region, it is difficult to distinguish the signal current by the p-AP from the background current by the reactant p-APP. Therefore, the electron transfer mediator, Fc, is required.As shown in echanism ,38:(1)2 p-APP, the electrocatalytic amplified current appeared at the same potential region as in the case of p-AP, indicating that p-APP was converted to p-AP by the enzymatic reaction. Therefore, in the presence of both p-APP and ALP, as in the case of p-AP, the electrocatalytic amplified current of Fc occurred at a ~0.3 V potential region. Consequently, the electron transfer mediator, Fc, plays an important role in the reduction of the reaction overpotential and in the amplification of the catalytic current. This is the expected operation of the Fc SAMs layer in the biosensor.To investigate the electron transfer mediation of Fc with the enzymatic reaction, the Fc SAM-modified electrode was incubated in a PBS containing 10 μg/mL of ALP-conjugated streptavidin for 1 h. As shown in Recently, a direct introduction of avidin on an electrode surface using non-specific binding was used as a simple and effective immobilization for biotin-labeled (bio)molecules on ITO ,40. Therp-APP after 10 min of enzymatic reaction time. The electrochemistry of the sensing mechanism is depicted in p-APP was dephosphorylated to p-AP by the enzyme label, ALP, after which the p-AP was diffused to the nearby electrode surface by a concentration gradient. At the electrode surface, the Fc moiety of the SAMs was electrochemically oxidized to Fc+ by the electrode, and it reacted with the nearby p-AP. The redox cycling, as mentioned above, will be maintained until all the p-AP is consumed. Consequently, the amplified electrocatalytic current is proportional to the concentration of p-AP.For the electrochemical measurement, the prepared immunosensor was investigated by CV in the electrochemical buffer containing The amplified electrocatalytic current was obtained in relation to the concentration of target human IgE, as shown in p-APP, the oxidation current was similar to the background current level of the target IgE case, as shown in In order to confirm the selectivity of the immunosensor for the target IgE, we assembled electrochemical sensors detecting other biomolecule targets such as goat IgG, mouse IgG, thrombin, human IgG, and hemoglobin. Then, when each sensor was measured in a solution containing 1 mM The shelf life of the biosensor was investigated. The Fc-SAM-modified ITO electrodes were prepared on the same day. A few days later, the Fc-SAM-modified ITO electrodes were constructed by bio-reagents, and the electrochemical measurement were carried out. The biosensor layer consists of the organic SAM-modified inorganic material electrode; therefore, the electrode surface is expected to be very stable even for long-term storage. As shown in The redox-marker and label are usually required for the electrochemical measurement. The requirement of this specific marker and label makes the electrochemical system more complicated than the other methods, resulting in the electrochemical sensor being uncompetitive. The electrochemical biosensor has advantages in terms of price and miniaturization, and therefore there have been many studies conducted to solve this marker and label problem. The EIS, a label-free electrochemical method, is one of the alternatives ,24.EIS was tried with the exact same sensor after target immobilization, without the secondary antibody incubation step. Depending on the amount of target IgE immobilized on the sensor surface, the thickness of the insulating layer by the biomolecule changed. The redox reaction of a redox probe, ferri/ferrocyanide, was perturbed by the insulating layer on the electrode surface, resulting in a change of charge transfer resistance. As shown in The EIS method is time- and cost-effective for the detection of IgE because it is label-free. However, the results were less reproducible than the CV measurements because all kinds of non-specific binding can affect the charge transfer resistance in EIS methods. To improve the selectivity and the sensitivity of EIS measurement, a probe molecule with a high binding affinity should be used, and a special pre-treatment or method to reduce the non-specific binding should be introduced.We also investigated both electrochemical methods in a condition using a serum matrix. Human serum and horse serum were used as a matrix for the dilution of target human IgE. However, the noise background signals at 0 IU/mL of the target concentration were, in both serums, considerably high. Consequently, the analytical properties, such as the LOD or dynamic range, will be poor in serum media (the data is not shown here). Therefore, pre-treatments such as filtration or separation are required for application in real serum.Interestingly, the detection limit of the EIS method was also ~3 IU/mL, close to the CV measurement results, despite the absence of an electrocatalytic amplification process in the EIS measurements. Because the same receptor antibody and target IgE were used, the observed similar analytical characteristics may indicate that the binding constant between the receptor antibody and the target IgE is the dominant variable for determining the selectivity of the immunosensor, rather than other factors such as the signal amplification. The sensing ability of the immunosensor actually varied greatly depending on the type of receptor antibody. Further studies may be needed on the competitive effects between the receptor’s binding affinity and the signal amplification scheme in relation to the sensitivity of the biosensor.We introduced the electron transfer mediator-functionalized layer on the ITO electrode for the electrochemical immunosensing of human IgE. The electron-transfer mediator, Fc, was directly introduced on the ITO electrode by an aldehyde-amine reaction. The formation of Fc SAMs-modified ITO electrodes is easier and cheaper than that of a similar Fc modification on Au electrodes using mixed SAMs.p-AP oxidation reaction. The electrocatalytic current amplification was achieved firstly with enzymatic production and secondly with redox cycling by Fc. The LOQ of the immunosensor was 10 IU/mL of the target IgE. This is good enough to determine the allergy based on the total IgE level. In addition to this, a label-free EIS measurement was also applied, obtaining a similar level of sensitivity. Therefore, the electrochemical detection of human IgE was successfully obtained via both CV and EIS.The electron-transfer mediation of the Fc SAMs-modified ITO electrode was measured by using a


However, we erroneously introduced the mutation based on the sequence numbering of NL3 containing the 20-residue spliced sequence A1 (UniProt entry Q9NZ94-1), which, by chance, also has an Arg at position 451 (sequence motif RETIKF). This latter mutation locates to the Site II interface and resembles the other ΔSite II mutants we described in Figure 6A. The correct R451C mutation, however, is not situated in either the Site I or Site II NL–MDGA interfaces. Therefore, our conclusion that R451C prevents NL–MDGA complex formation is wrong, and any speculation based on it in the Discussion section may be disregarded. We note that this error has no impact on the validity of any of the crystal structures or other results.Furthermore, we noticed two unrelated minor errors within Figures 2, 4, and S3.In Figures 2A and S3A, a N-linked glycosylation site is incorrectly shown in the intracellular (IC) tail of NL1.−1 instead of 1.02 kcal mol−1. Therefore, ΔG = −8.66 kcal mol−1 instead of −6.62 kcal mol−1. The value for the KD is correct, however.In Figure 4C, there is a sign error in the bottom left panel: it reads −TΔS = −1.02 kcal molThe authors apologize for any confusion the errors may have caused.


Capsosiphon fulvescens glycoproteins (Cf-hGP) enhanced probiotic-induced improvement of memory in aged rats and the underlying mechanism in the dorsal hippocampus. Cf-hGP were isolated using lectin resin. Cf-hGP (15 mg/kg/day) and/or Lactobacillus plantarum (L. plantarum) (109 CFU/rat/day) were orally administered once a day for 4 weeks. Co-treatment with Cf-hGP and L. plantarum synergistically improved spatial memory in aged rats, which was overturned by functional blocks of brain-derived neurotrophic factor (BDNF) signaling. Increases in BDNF expression and nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation were accompanied by mono- and/or co-administration in the dorsal hippocampus, while c-Jun N-terminal kinase (JNK) phosphorylation and glucose-regulated protein 78 expression were decreased. These synergistic effects were downregulated by blocks of BDNF/Nrf2-mediated signaling. In particular, co-treatment, not mono-treatment, reduced phosphorylation of eukaryotic elongation factor 2 (eEF2) regulated by eEF2 kinase and protein phosphatase 2A. Additionally, co-treatment downregulated the interaction between eEF2 kinase and JNK. These data demonstrated that cognitive impairment in aged rats was synergistically diminished by co-treatment with Cf-hGP and L. plantarum through BDNF-mediated regulation of Nrf2 and eEF2 signaling pathways in the dorsal hippocampus.Aging-induced cognitive dysfunction can be regulated by probiotics through bidirectional communication with the brain. This study aimed to investigate whether  Aging is a natural process associated with functional and cognitive decline, which is closely associated with neurobiological changes in the hippocampus, including a decrease in the number of newborn neurons in the subgranular zone of the hippocampus ,2,3. FurProbiotics have been proposed as a key player in mental health. They are live microorganisms that confer a health benefit on the host when administered in adequate amounts . The micBrain-derived neurotrophic factor (BDNF) plays an essential role in neuronal survival and growth, differentiation of new neurons, and hippocampal-dependent neuronal plasticity and long-term potentiation ,15,16. ANeuronal proteostasis is essential for controlling synaptic plasticity and memory formation by regulating protein turnover in the ER, which plays a pivotal role in the correct synthesis, folding, and modification of proteins . Aging-iCapsosiphon fulvescens (C. fulvescens), is widely consumed as a nutrient source or functional food due to its beneficial health effects , while the frequency of platform crossings was increased , for both treatment with only Cf-hGP and L. plantarum and co-treatment  and/or ter maze A. After ter maze B. Furthereatment C,D. Integed rats , we furtL. plantarum regulated BDNF-mediated signaling linked to plasticity-related proteins, specifically, alterations of BDNF, Nrf2, eEF2, JNK, and GRP78, and their interactions in the dorsal hippocampus. We first examined expression of mature BDNF and phosphorylation of Nrf2 and JNK. Following oral administration of Cf-hGP (15 mg/kg) and/or L. plantarum (109 CFU/rat/day) for 4 weeks F = 32.00; p < 0.05; n = 5 rats per group] and expression of mature BDNF  in the dorsal hippocampus, while JNK phosphorylation was downregulated compared to the aged untreated group . In particular, when Cf-hGP and L. plantarum were used for co-treatment for 4 weeks, Nrf2 phosphorylation and mature BDNF expression were synergistically increased, while JNK phosphorylation was decreased compared to mono-treatment  as a positive control was unilaterally injected into the dorsal hippocampus, which significantly increased Nrf2 phosphorylation , but JNK phosphorylation was decreased . The co-treatment-induced increase in Nrf2 phosphorylation was downregulated by functional blocks with the anti-BDNF antibody (1 µg/µL)   F = 27.85; p < 0.05; n = 6 rats per group] and GRP78 expression  in the dorsal hippocampus.Next, we investigated whether BDNF signaling activated by co-treatment with Cf-hGP and 1 µg/µL) A. We furr group] B. In addL. plantarum downregulated eEF2 phosphorylation at threonine 56 ; however, there were no significant differences in the mono-treatment with Cf-hGP or L. plantarum F = 42.67; p < 0.05; n = 6 rats per group], while it reduced eEF2 phosphorylation , which was overturned by functional blocks with the anti-BDNF antibody (1 µg/µL) F = 25.29; p < 0.05; n = 6 rats per group]  B. The cor group] C.L. plantarum significantly increased eEF2K phosphorylation  and induced its inactivation, which downregulated the interaction between eEF2K and JNK and reduced JNK phosphorylation  in the dorsal hippocampus . Next, to explore these synergistic effects caused by Cf-hGP, we further determined that BDNF-mediated signaling was differentially regulated by the co-treatment in the dorsal hippocampus, which is a pivotal region in spatial memory [L. plantarum synergistically upregulated the alterations caused by mono-treatment , not any mono-treatment (L. plantarum significantly increased eEF2K phosphorylation and inactivated eEF2K, which reduced its interaction with JNK and JNK phosphorylation in the dorsal hippocampus (In this study, only the co-treatment with Cf-hGP and reatment A. The dereatment B. Furthereatment C. In addpocampus B,C. ThesL. plantarum could synergistically improve the cognitive function in aged rats compared to any mono-treatment through activation of BDNF-mediated Nrf2 and eEF2 signaling in the dorsal hippocampus (These data demonstrate that chronic co-administration of Cf-hGP with pocampus . Therefo


Fluoxetine is still one of the most widely used antidepressants in the world. The drug is extensively metabolized by several cytochrome P450 (CYP450) enzymes and subjected to a myriad of CYP450-mediated drug interactions. In a multidrug regimen, preemptive mitigation of drug–drug interactions requires knowledge of fluoxetine actions on these CYP450 enzymes. The major metabolic pathway of fluoxetine leading to the formation of its active metabolite, norfluoxetine, is mediated by CYP2D6. Fluoxetine and norfluoxetine are strong affinity substrates of CYP2D6 and can inhibit, potentially through various mechanisms, the metabolism of other sensitive CYP2D6 substrates. Remarkably, fluoxetine-mediated CYP2D6 inhibition subsides long after fluoxetine first passes through the liver and even remains long after the discontinuation of the drug. Herein, we review pharmacokinetic and pharmacogenetic information to help us understand the mechanisms underlying the prolonged inhibition of CYP2D6 following fluoxetine administration. We propose that long-term inhibition of CYP2D6 is likely a result of competitive inhibition. This is due to strong affinity binding of fluoxetine and norfluoxetine to the enzyme and unbound fluoxetine and norfluoxetine levels circulating in the blood for a long period of time because of their long elimination half-life. Additionally, we describe that fluoxetine is a CYP2C9 substrate and a mechanism-based inhibitor of CYP2C19. Patients presenting with several chronic conditions such as psychiatric disorders, cardiovascular disorders, diabetes, and rheumatoid arthritis often require multiple therapeutic approaches, including the use of numerous drugs, to achieve clinical benefits and experience favorable outcomes ,2,3,4. TIn recent years, an increased incidence of depression with an associated increase in the use of antidepressants has been noted ,14,15,16Drug interactions associated with CYP450 inhibition are classified as reversible  or irreversible  . FirstlyN-demethylation of fluoxetine produces norfluoxetine, an active metabolite with potent serotonin selective reuptake inhibition (SSRI) activity [O-dealkylation converting fluoxetine into p-trifluoromethylphenol which subsequently is transformed into hippuric acid, a glycine conjugate [Fluoxetine is extensively metabolized by various CYP450s such that only ~2.5% of the administered dose is excreted unchanged in urine . The N-dactivity . Other monjugate ,34,35. Fonjugate ,36. SincCYP2D6 is mainly responsible for metabolizing both S- and R-fluoxetine to norfluoxetine corresponding enantiomers  . HoweverS-Fluoxetine and S-norfluoxetine are both substrates and exhibit a high binding affinity to CYP2D6  . Thus, iThe first possibility is that prolonged CYP2D6 inhibition by fluoxetine might be due to noncompetitive inhibition. As a CYP2D6 substrate, fluoxetine must bind to the active site to be metabolized into norfluoxetine. By definition, pure noncompetitive inhibitors are not metabolized by the enzyme they inhibit . AdditioN-demethylation of fluoxetine with a magnitude similar to that observed with anti-CYP2D6 antibodies [Second, fluoxetine could be a mixed CYP2D6 inhibitor, i.e., exhibiting both competitive and noncompetitive inhibition. This mechanism of inhibition gives rise to very potent enzyme inhibitors, often acting as perpetrator drugs with other sensitive substrates, while their pharmacokinetics are not affected by other inhibitors of the same enzyme . In vitrtibodies ,42. Thes50 for CYP2D6 inhibition by fluoxetine did not decrease with various times of pre-incubation periods to allow a potential reactive metabolite to be formed. These observations led to the conclusion that fluoxetine does not exhibit a time-dependent increase in its inhibitory potency and thus is not a mechanism-based inhibitor. Further evidence for the lack of noncompetitive and mechanism-based inhibition of CYP2D6 by fluoxetine is also obtained looking at results from pharmacogenetic studies. It is well known that poor metabolizers of CYP2D6  exhibit decreased clearance characteristics of CYP2D6 probe substrates compared to normal CYP2D6 metabolizers  [CYP2D6 genes) was also reported in other clinical studies [Third, since fluoxetine is a CYP2D6 substrate, studies have been conducted to determine whether fluoxetine produces the time-dependent, mechanism-based inactivation of CYP2D6 . Bertelsalleles) . If noncalleles) . The plaalleles) . Higher alleles) . Finally studies ,46,47,48Conceptually, separating the time of administration between two competing substrates, such that their maximum concentrations in the liver or plasma do not overlap, is a meaningful way to mitigate DDIs caused by competitive inhibition. For most drugs, CYP2D6 inhibition is observed following drug absorption during the hepatic first pass phase  since drug concentrations in the portal vein (which irrigates perilobular hepatocytes) are much higher than concentrations observed after peak plasma concentrations when drugs return to the liver via the hepatic artery. Generally, the ratio of drug concentrations observed in the portal vein during the absorption phase to that measured in the hepatic artery after the peak plasma concentration  is very large (likely in the range of 100 to 1000). This is why separating the time of administration of two substrates that compete for the same enzyme circumvents competitive inhibition between these two substrates. However, as mentioned previously, in the case of fluoxetine, a prolonged CYP2D6 inhibition over two to three weeks was observed when dextromethorphan was used as a probe substrate ,27. OtheN-demethylation pathway, fluoxetine and possibly norfluoxetine undergo CYP2C19-mediated O-dealkylation to form the p-trifluoromethylphenol metabolite [Fluoxetine is a substrate  and a mechanism-based inhibitor of CYP2C19, as CYP2C19 is marginally involved in metabolizing fluoxetine into norfluoxetine ,48. In atabolite ,35. A 30tabolite . Additiotabolite . In conttabolite . In agretabolite . Findingtabolite . Howevertabolite . In vitro studies suggest an important role for CYP2C9 in the metabolism of fluoxetine with stereoselectivity toward the R-enantiomer ,46. PharN-demethylation pathway, in vitro studies suggested that CYP3A4 could contribute to the O-dealkylation of fluoxetine and perhaps norfluoxetine [O-dealkylation can be revealed; however, the incongruity between in vivo and in vitro evidence will require further studies to provide any definitive conclusions.In vitro experiments have suggested a role for CYP3A4 in the metabolism of fluoxetine  . Furtheruoxetine . Based oBased on the information presented above, the strong CYP2D6 binding affinity of both S-fluoxetine and S-norfluoxetine, their unbound steady-state plasma concentrations above their respective Ki values, and their long elimination half-life all contribute to the prolonged inhibitory effects of fluoxetine and norfluoxetine on CYP2D6 activity. The mechanism of CYP2D6 inhibition by fluoxetine and norfluoxetine appears to be principally explained by competitive reversible inhibition. Clinically, separating the time of administration between fluoxetine and other CYP2D6 substrates will not be effective in mitigating DDIs, and the dose reduction of sensitive CYP2D6 substrates co-administered with fluoxetine is warranted to avoid adverse drug events.


Zizyphus jujuba fruits. Proteins from Zizyphus jujuba were lysed by trypsin, papain and their combination. Acquired peptides were purified and evaluated for ACE inhibitory activity. Peptide fractions with inhibitory activity were sequenced using tandem mass spectrometry. To elucidate the mode of peptide binding to ACE, homology modeling, molecular docking and molecular dynamics simulation were performed. Amino acid sequence of F2 and F4 peptides, which were the most active hydrolysates, were determined to be IER and IGK with the IC50 values of 0.060 and 0.072 mg/ml, respectively. Results obtained by computational analysis revealed that similar to the common ACE competitive inhibitors such as captopril, IER tripeptide binds to the enzyme active site, in vicinity of the zinc binding site, and occupies the S1 and S2’ subsites. Binding occurs through hydrogen bonding with Gln293, Lys522, His524, Tyr531 and also several hydrophobic interactions. Collectively, these findings indicate that IER tripeptide inhibits the rabbit ACE enzyme through a competitive mechanism of inhibition with IC50 values in the millimolar range.The synthetic Angiotensin Converting Enzyme (ACE) inhibitors have side effects and hence demands for natural ACE inhibitors have been rising. The aim of this study is to purify and introduce natural ACE inhibitors extracted from  Although the synthetic drugs are often prescribed due to their well-known role and convenience of access, they have undesirable effects, such as extreme low blood pressure, cough, bad tastes and allergic reactions. Contrariwise, natural inhibitors regulate hypertension under mild conditions without significant side effects; hence, they can be considered as worthy alternatives to synthetic drugs2.Angiotensin converting enzyme (ACE) inhibitors are a new class of drugs which are critical in the treatment of hypertension and heart failure3. ACE is a Zn-metallopeptidase that removes a dipeptide from the decapeptide angiotensin-I to produce the potent vasoconstriction octapeptide angiotensin-II4. Several peptides originated from animal or plant proteins exhibit significant therapeutic/regulatory performances like antihypertensive, antimicrobial, antioxidant, immunoregulatory, anticancer activities, etc3.ACE inhibitory peptides are the best known amongst various classes of regulatory bioactive peptides due to the development of functional foods with homeostasis role in the human body5. So far, several ACE inhibitory peptides have been reported from hydrolysis of different food proteins, such as hen egg white lysozyme4, fish6, corn7, soybean8, milk9, casein10, etc. These peptides are good candidates to regulate blood pressure and also to balance fluid and electrolytes in mammals.Investigations to find more bioactive peptides, especially multifunctional ones have attracted the attention of researchers. The bioactive peptides are often inactive in their natural forms and when released by enzymatic hydrolysis, display their biological activityZizyphus jujuba is a well-known medicinal plant with several bioactivities, such as antimicrobial11, antidiabetic13, hypoglycemic13, and analgesic14. Likewise, we recently reported some antioxidant peptides extracted from Z. jujuba5. Although there are several reports on the presence of different bioactive compounds in this plant, there is no evidence indicating the presence of ACE inhibitory peptides. In the present study, from protein hydrolysate of Z. jujuba fruit, two ACE inhibitory peptides were purified and characterized. Investigating the inhibition pattern of these peptides showed that they inhibited the enzyme action by competitive mechanism. As a complement to our experiment, homology modeling, molecular docking and 100 nanoseconds (ns) molecular dynamics (MD) simulation were performed to provide an atomistic insight into the interaction of IER peptide (due to the higher affinity) with the ACE.TFA (trifluoroacetic acid), acetonitrile, trypsin (from bovine pancreas), papain (from pawpaw sap), FAPGG (N-(3-[2-furylacryloyl-Phe-Gly-Gly])), ACE (angiotensin-I converting enzyme) from rabbit lung and captopril (≥98% purity), were purchased from Sigma-Aldrich Co. . Ultrafiltration membranes with a 3 kDa cut-off were purchased from Millipore . Analytical and semi-preparative columns were purchased from Macherey Nagel GmbH Co. . All other chemicals used were of analytical grades.Z. jujuba fruit according to the method described by Memarpoor-Yazdi et al.5. Briefly, jujuba fruits were soaked in water and then crushed in a porcelain mortar to attain a homogeneous semi-solid mixture. Excess salt was removed by centrifugation  and the supernatant was filtered using a filter paper. The extract from jujuba fruit was brought from zero to 85% concentration using the standard ammonium sulphate precipitation procedure, and then saturated with salt using gentle agitation at 4 °C. Extracted proteins were suspended in phosphate buffer  forming colloidal particles. Deposits including small and large proteins in the solution were collected by centrifugation , dissolved in distilled deionized H2O (ddH2O) and dialyzed against water at 4 °C for 24 hours. Samples separated by dialysis were lyophilized and their proteins were quantified by Bradford method15, using bovine serum albumin as the control. A total of 500 mg lyophilized powder was prepared from 10 g jujuba fruit. After ammonium sulphate precipitation, the fruit powder yielded 6 mg protein.Proteins were extracted from Enzymatic hydrolysis is essential for releasing bioactive peptides. Accordingly, the extracted proteins were incubated with two commercial enzymes, papain and trypsin, and a combination of the two. Using these enzymes and a protein/enzyme ratio of 50:1, the protein solution (4 mg/ml) in Tris-HCl buffer  was hydrolyzed. Each enzyme was dissolved separately (0.08 mg/ml) in the same buffer. The hydrolysis reaction was conducted at 37 °C for 4 h. Finally, hydrolysis reaction was stopped by heating the solution in boiling water for 15 min. The hydrolysates were centrifuged at 7000 g for 10 min and the supernatants were collected and transferred to test tubes for further investigations.18 semi-preparative RP-HPLC column . Elution was performed using 0.1% trifluoroacetic acid (TFA) in ddH2O (v/v) as solution A and a 50% gradient of 0.098% TFA in acetonitrile as solution B for 55 min at 1 ml/min flow rate. Finally, the absorbance of the eluted peaks was recorded at 220 nm and major peaks were lyophilized and subjected to enzymatic assay. Among active fractions, two fractions with the maximum ACE inhibitory potential were purified more using the same RP-HPLC column with a gradient of 0.5% eluent B/min.The prepared hydrolysates were filtered using a 3 kDa cut-off ultramembrane and subsequently were subjected to peptide purification using a C16. Samples contained hydrolysate or peptide , ACE , FAPGG  and ACE buffer . The control was the same as the samples, except that ACE buffer (50 µl) was used instead of inhibitor sample. Absorbance changes were measured for 30 min at 340 nm. ACE inhibition percentage was obtained based on the following equation:Activity of the peptide fractions for ACE inhibition was measured according to our previous study50) was calculated by plotting the ACE inhibition percentage against different peptide concentrations. Experiments were performed in triplicates. The IC50 values of the purified peptides were compared with that reported for captopril (used as a positive control).Peptide concentration to inhibit 50% ACE activity  for each peptide was calculated using the secondary plot of m at different concentrations of the inhibitor) as a function of the inhibitor concentration. The value of the inhibition constant Ki was obtained from the negative x intercept of the secondary plot.The inhibition patterns of the target peptides (F2 and F4) were determined by Lineweaver-Burk plots. Enzymatic assay was carried out using different concentrations  of FAPGG as substrate at 37 °C. The inhibition patterns of the peptides were determined in the absence and presence of the inhibitory peptide at two concentrations of 0.06 and 0.12 mg/ml using Lineweaver-Burk plots. Inhibition constant (K17) as template using MODELLER 9.2018. Except for Zn and Cl ions, all other heteroatoms were removed from the original PDB file of 2C6N, and the resulting structure was subjected to restraint-based homology modeling as implemented in MODELLER 9.2018. The best model was selected based on the lowest DOPE score and evaluated by PROCHECK19, ERRAT20 and Verify3D21 servers.No crystal structure for rabbit ACE enzyme has been reported in Protein Data Bank (PDB). So, the structure of the enzyme was constructed based on the amino acid sequence of rabbit ACE (UniProt ID: P12822) and the crystal structure of human somatic ACE N-domain  with Amber parameters for standard residues, and 500 steepest descent minimization steps with a step size of 0.02 Å. The molecular structure files were prepared in pdbqt format for both IER and the modeled rabbit ACE using AutoDock Tools. The grid box dimensions were set to 60, 60 and 60 grid points centered on the zinc-binding site and a grid spacing of 0.375 Å was applied.In order to predict the binding conformation of IER to ACE enzyme, molecular docking simulation was performed using AutoDock 4.2.5.124 with CHARMM36 forcefiled25. The protein-tripeptide complex was solvated in TIP3P water molecules inside a cubic box. Thirteen water molecules were replaced by Na+ ions to neutralize the system charge. The neutralized system was then subjected to energy minimization until the maximum force became less than 500 kJ/mol.nm. After energy minimization, two separate position-restrained MD simulations were carried out to equilibrate the system. First, a 200 picoseconds (ps) NVT MD simulation was carried out at 300 K and constant volume using the V-rescale thermostat26. Then, Parrinello-Rahman barostat was used for 500 ps to control the system at 1 bar and 300 K27. Finally, 100 ns molecular dynamics simulation without position restraint was performed under similar condition. The LINCS algorithm was used to constrain the bonds connecting hydrogen atoms28. Long range electrostatic interactions were computed using particle mesh by Ewald method29. Both short-range and Lennard-Jones interactions were calculated with a 1.2 nm cut-off distance. A time step of 2 fs was used to integrate the motion equation. The resulting trajectories were analysed using the built-in tools of GROMACS 5.0.5. The minimum energy conformation of the ACE-IER complex was extracted from the trajectory using free energy landscape (FEL) analysis and considered as a representative structure of the complex. The FEL was generated using the gmx sham tool of GROMACS by projecting radius of gyration (Rg) and RMSD to the average structure along with free energy change and visualization of the 2D and 3D diagrams of FEL was done using the trial version of Mathematica 9 . The 3D structures were prepared using UCSF Chimera 1.1123 and 2D diagrams of ACE-inhibitors were drawn using PoseView (http://proteins.plus)30.Molecular dynamics simulation procedure was conducted using GROMACS 5.0.5Z. jujuba fruits were used to purify ACE inhibitory peptides. Bioactive peptides were released by enzymatic digestion using trypsin, papain and a combination of the two. Primary assay showed that all hydrolysates had inhibitory effects on ACE activity. The percentages of ACE inhibitory activity were calculated to be 26.1% (±2.0), 14.2% (±1.2), and 20.8% (±1.8) for trypsin, papain and their combination at a concentration of 0.15 mg/ml, respectively. According to the results, tryptic hydrolysate with the highest ACE inhibitory potential was used for peptide purification.Proteins extracted from 50 values of 0.060 mg/ml (0.144 mM) and 0.072 mg/ml (0.228 mM), respectively , RADFY (IC50 = 1.35 mM), and RVFDGAV (IC50 = 1.00 mM) purified from Ginkgo biloba seeds31, while they are less active than some other peptides such as KAQYPYV (IC50 = 0.0370 mM), KIIIYN (IC50 = 0.058 mM), and KILIYG (IC50 = 0.053 mM) purified from coconut cake albumin hydrolysates32. The IC50 value of captopril was calculated to be 2.2 ng/ml which is lower than those of F2 and F4 peptides.Bioactive peptide fractions were purified from the tryptic hydrolysate using RP-HPLC Fig. . After Aely Fig. . Accordimax was found to be unchanged, but Km value was altered, which is an indication of competitive inhibitory pattern , while it increased to 0.255 and 0.156 mM in the presence of IER and IGK peptides at 0.060 mg/ml of inhibitory peptide, respectively. Furthermore, at 0.12 mg/ml of inhibitory peptide, Km values increased to 0.4 and 0.272 mM for IER and IGK peptides, respectively, which is indicative of reduced affinity of the enzyme for its substrate in the presence of these peptides. Collectively, results imply that these peptides inhibited the enzyme by binding to the free enzyme, but not to the ACE-substrate complex. Since the inhibition pattern of the ACE inhibitory peptides is related to their inhibitory structures, we hypothesize that these peptides may have similar structures to the substrate; hence, they might compete with the substrate for binding to the same interaction site on the enzyme. Most of the ACE inhibitory peptides show competitive inhibition patterns31, as is also seen in this study. By using secondary plots  as the template using MODELLER 9.2018. The best modeled structure was selected based on the lowest DOPE score, and the reliability of the predicted structure was validated with PROCHECK, ERRAT and Verify3D servers21  with human ACE (UniProt ID: P12821). Hence, the three-dimensional structure of the rabbit ACE was constructed based on the human somatic ACE structure  which was used as template for homology modeling. Comparison of the structures revealed that the position and orientation of IER peptide in the docked structure were very similar to those of lisinopril in the template crystal structure  for the alpha carbon (Cα) atoms and gyration radius of the protein are shown in Fig. The hydrogen bonds with existence probability greater than 5% are depicted in Fig. 35. A more detailed look at the IER binding site revealed that the arginine residue of IER peptide makes hydrogen bonds with Gln293, Lys522 and Tyr531 and also hydrophobic interactions with Phe468 and Tyr534 of ACE. The glutamate residue of IER, which is coordinated to Zn2+, makes a hydrogen bond with His524 via its backbone oxygen and also interacts with Glu396 and His395 through hydrophobic contacts (Fig. 34 (Fig. Structural comparison of the interaction networks of IER, captopril, enalaprilat and lisinopril with ACE active site indicates that the results of this study are in good agreement with those obtained for known competitive inhibitors of testicular ACEcts Fig. . The res 34 Fig. , while b 34 Fig. . In addi 34 Fig. . The oth50 values equal to 0.060 and 0.072 mg/ml, respectively. Analysing the inhibition mechanism by Lineweaver-Burk plots revealed that these peptides compete with the substrate in binding to the enzyme active site. MD simulation results implied that the IER peptide, similar to common inhibitors of ACE such as captopril, binds to the active site and occupies the S1 and S2’ subsites of ACE, confirming that the IER peptide inhibits the rabbit ACE enzyme through a competitive mechanism.In this study, we purified and characterized two tripeptides, IER and IGK, that have the ability to inhibit ACE with the IC