Three novel peptides with ACE inhibitory activity were detected by LC-MS/MS, with sequences LVYP , VYPW and YPWT(Tyr-Pro-Trp-Thr). Molecular docking revealed that the three novel peptides all established hydrogen bonds with the S1 and S2 (His353) pockets of ACE. Among them, LVYP, VYPW and YPWT, respectively, formed eleven hydrogen bonds, six hydrogen bonds and nine hydrogen bonds with ACE. The study revealed that these peptides have the potential for the development of novel ACE inhibitor drugs and provide a new avenue for high-value utilisation of mushrooms scraps.Food-derived hypotensive peptides have attracted attention in the field of active peptide research in recent years. In this study, based on ACE inhibition rate and using the Box–Behnken central combination design principle to optimise the process of ACE inhibitor peptides prepared by double-enzyme hydrolysis. The amino acid sequences of ACE inhibitor peptides were determined by liquid chromatography mass spectrometry (LC-MS/MS), and their binding to ACE was studied by molecular docking. The optimal processing conditions were 1:1 alkaline protease: compound protease, pH was 8.43, enzymolysis temperature was 44.32 °C, and enzymolysis time was 3.52 h. Under these conditions, the ACE inhibition rate reached 65.12%, and the inhibition rate after separation and purification was 80.68% (IC Agaricusbisporus is an edible basidiomycete mushroom that is widely cultivated worldwide, with production accounting for 54% of total edible fungi -L-phenylalanyl-glycyl-glycine) in Tris-HCl (50 mmol·L−1) (purchased from Sigma Aldrich) containing 0.3 mol·L−1 NaCl at pH 7.5 and placed in a 37 °C water bath. A 10 μL sample of enzyme solution was placed in a 96-well plate, 150 μL of substrate solution was added, the plate was immediately placed in an enzyme calibrator, and the absorbance at 340 nm was measured every 30 s for 30 min. A 10 μL volume of buffer solution was used instead of enzyme solution as a blank control, and 10 μL of 0.25 U·mL−1 ACE solution was used instead of enzyme solution as a negative control. The change in absorbance value (ΔA) was plotted against time and the slope was calculated using the following formula:The ACE inhibition rate was determined as described previously  with sli50) was calculated by plotting the ACE inhibition percentage against different peptide concentrations. Experiments were performed in triplicate, and IC50 values of purified peptides were compared with that reported for captopril (used as a positive control).The peptide concentration inhibiting 50% ACE activity (defend as ICw/v) TNBS (purchased from Sigma Aldrich) was added to a small tube containing 1 mL of phosphate buffer , placed in a 50 °C constant temperature water bath in the dark, shaken for 60 min, and cooled at room temperature for 20 min. Then, a 2 mL sample of 0.1 mol/L HCl was added to stop the reaction, and measure the absorbance at 340 nm. The degree of protein hydrolysis was calculated using the following equation:1 is the content of amino nitrogen before protein hydrolysis, AN2 is the content of amino nitrogen after protein hydrolysis, and Npb is the protein content of the protein substrate. The values of AN1 and AN2 can be calculated from the absorption curve of the standard sample at 340 nm .Referring to the method of Adler–Nissen  with a sPowdered mushroom protein was weighed at a substrate concentration of 5% by mass fraction and enzymatically digested using alkaline protease, complex protease, flavour protease, papain, and neutral protease . According to the degree of protein hydrolysis and the ACE inhibition rate, two proteases with superior preparation effects were selected for compounding.−1. The enzymatic digestion time was 2 h, the enzymatic digestion temperature was 55 °C, and the pH of enzymatic digestion was 7.5. The ACE inhibition rate and protein hydrolysis degree were calculated according to Equations (2) and (3), respectively.The substrate concentration of mushroom protein was formulated to 5%. The two enzymes selected were added respectively at the ratios of 2:1, 1:1, 1:2, 1:3, 1:4 and 1:5 for alkaline protease: complex protease, the addition amount of the two enzymes was 6000 u·gAccording to the optimal ratio of the dual enzymes and their optimal addition amounts determined in According to the optimal protease addition ratio determined in According to the optimal addition ratio of protease determined in According to the results of the above single factor experiments, taking the ACE inhibition rate as the response value and using the Box–Behnken central combination experiment design as the principle, a response surface analysis method with three factors and three levels was designed to optimise the process for preparing ACE inhibitors. The setting of factors and levels are shown in Four different macroporous adsorption resins  were tested in static adsorption experiments, and the optimal resin was identified according to the adsorption rate calculated using the following formula: Dynamic adsorption and desorption experiments were performed as described previously  with sli280nm) was measured using an ultraviolet spectrophotometer.Sephadex G-100  was used to fractionate the purified ACE inhibitory peptides, and distilled water was used as the eluent for elution. Eluted fractions were collected in separate tubes (3 mL/tube) and collected based on peaks. The absorbance of fractions at 280 nm (Av/v/v) acetonitrile:water:trifluoroacetic acid, a detection wavelength of 280 nm, a flow rate of 1 mL/min, and a column temperature of 30 °C. Bovine serum albumin (Mw67000), VB12 (Mw 1335) and oxidised glutathione (Mw 614) served as mixed standards. A standard curve was plotted according to the above chromatographic conditions , a TSK(TOSOH) Gel 2000 SWXL 300 × 7.8 mm column was employed, with a mobile phase of 30:70:0.1 (nditions .2 = 0.9987.As shown in www.biotech-pack.com (accessed on 25 April 2021). Beijing, China).Sequence determination of ACE inhibitor peptides was performed by Beijing Biotech Pack Biotechnology Co., Ltd., . A 2 mL sample of 0.5 mg/mL ACE inhibitory peptides were placed in buffers at different pH values, incubated at 4 °C for 12 h, and the method described in ACE inhibitory peptides were preincubated at different temperatures (25~95 °C) for 30 min, and ACE inhibitory activity was tested using the method described in g for 10 min, and the supernatant was used to determine ACE inhibition activity.ACE inhibitory peptides were digested as described previously  with somg for 10 min. The supernatant was used to determine ACE inhibitory activity.A 2 mL sample of the pepsin-digested solution was mixed with 2 mL of 0.05 mg/mL trypsin solution and shaken at 37 °C for 4 h (E/S = 1:200). Subsequently, the reaction was terminated by heating in a water bath at 100 °C for 5 min to inactivate the enzyme and centrifuged at 10,000× A. bisporus proteins, five proteases  were subjected to enzymatic hydrolysis tests, and the results are shown in In order to obtain suitable proteases for hydrolysing All five proteases resulted in ACE inhibitory activity following enzymatic hydrolysis of mushroom protein. Compared with the direct extraction method, the degree of protein hydrolysis and the ACE inhibition rate were significantly altered after hydrolysis with each of the five protein hydrolases. The ACE inhibition rate was lowest after enzymolysis by neutral protease and flavour protease and highest with alkaline protease and complex protease, and the degree of protein hydrolysis followed the same pattern. Therefore, alkaline protease and complex protease were selected for hydrolysis of mushrooms scraps to prepare ACE inhibitory peptides.The ACE inhibition rate and degree of proteolysis first increased, then decreased, with decreasing compound ratio . When thFrom Both the ACE inhibition rate and degree of protein hydrolysis first increased then decreased with increasing enzymatic digestion temperature . Both paFrom Based on the single factor test results, the main factors affecting the preparation of ACE inhibitory peptides from mushrooms were pH, enzymatic digestion duration and enzymatic digestion temperature. Therefore, these three influencing factors were selected to carry out response surface experiments. The response surface results are shown in p < 0.0001). The coefficient of determination of the model (R2) was 0.9977, and the lack of fit term was not significant, indicating that the model fitted well and was relatively reliable. The pH, enzymatic digestion temperature and enzymatic digestion duration were all significant, and the order of significance was pH > enzymatic digestion temperature > enzymatic digestion duration. Thus, pH had the greatest influence on the ACE inhibition rate of mushroom scraps protein, followed by enzymatic digestion temperature, and enzymatic digestion duration has the least influence. Interactions between enzymatic digestion duration and enzymatic digestion temperature, and between enzymatic digestion duration and pH, were significant , a drug prescribed as an ACE inhibitor [Triticum aestivum L. (IC50 was 2.79 mg/mL) [Navodonseptentrionalis (IC50 was 1.96 mg/mL) [The IC peptide . It can nhibitor . However9 mg/mL)  and Navo6 mg/mL) . Therefohttp://bis.zju.edu.cn/biopepdbr/index.php (accessed on 29 April 2021), http://pepbank.mgh.harvard.edu (accessed on 29 April 2021), http://www.uwm.edu.l/biochemia/ (accessed on 29 April 2021)), and found that the three identified peptides were not previously identified as ACE inhibitors, which confirmed that the three tetrapeptides were novel ACE inhibitory peptides.In order to determine the sequences of active peptides, LC-MS/MS was employed . Based oThe sequences of the three ACE inhibitory peptides were LVYP, VYPW and YPWT. Molecular docking showed that both the central six-membered ring and five-membered ring of the overlapping structure of ACE inhibitory peptides engage in Pi-Pi conjugation with His410 of ACE. The specific interaction mode with ACE is shown in In addition, hydrogen bonds also play an important role in the interaction between inhibitors and ACE. The number and length of hydrogen bonds affect the binding force of inhibitors , left. ALVYP, respectively, forms two hydrogen bonds with Ala356 and Asp 358, and one hydrogen bond with Glu403, Arg522, Tyr523, Trp357, Glu384, Ala354 and His387, totalling eleven hydrogen bonds. The molecular docking score is 136 points. VYPW, respectively, forms three hydrogen bonds with His353, and one hydrogen bond with Pro407, Tyr523 and Arg522, totaling six hydrogen bonds. The molecular docking score is 169 points. YPWT, respectively, forms two hydrogen bonds with Ala356 and Tyr360, one hydrogen bond with Ala354, Glu384, Asp358, Tyr523 and Glu403, totalling nine hydrogen bonds. The molecular docking score is 160 points.The active site of ACE is mainly composed of three active pockets; S1 , S2  and S1’ (Glu162) . Some ofMoreover, LVYP and VYPW also interact with Zn2+, and studies have reported that Zn2+ also combines with three other regions  to form a twistable tetrahedral structure, which plays a key role in the stability of the compound . SecondlAmong the three new peptides, we found that both LVYP and VYPW have the same sequence VYP. The two peptides YPWT and VYPW also shared the same sequence YPW. Therefore, we performed molecular docking for these two shared tripeptides respectively, and the results are shown in VYP, respectively, forms one hydrogen bond with Glu123, Arg124, Arg124 and Asn85, and forms a conjugate bond with the six-membered ring of tyrosine, and the molecular docking score is 108 points. YPW, respectively, forms one hydrogen bond with Arg124, Ser517, and Glu123 and, respectively, forms a conjugated bond with the six-membered ring of tyrosine, either of the five-membered ring and six-membered ring of tryptophan, and the five-membered ring of proline. The molecular docking score is 138 points. It shows that the above two tripeptides have certain ACE inhibitory activity.The inhibitory activity of ACE was less affected by pH, and its activity remained above 90% in different pH solutions within the range of 2 to 12 , indicatVery little ACE inhibitory activity was lost at various temperatures, with more than 90% of activity retained after treatment . The resThe results in The research and development of food-derived active peptides is attracting considerable attention ,29,30, ePleurotus eryngii, achieving an ACE inhibitory rate of 67.1%. However, hydrolysis by a single protease is limited by the specificity of the enzyme; hence, the degree of hydrolysis of the target protein was relatively low, and the biological activity of the product was easily affected.Edible fungi are a valuable resource for the preparation of active peptides due to their high protein content . PreparaA. bisporus prepared by single-enzyme and double-enzyme methods was compared. It was found that the inhibition rate of ACE inhibitory peptides obtained by single enzymatic hydrolysis was 46.36%, while the inhibitory rate of ACE inhibitory peptides obtained by double enzymatic hydrolysis was 65.12%, an increase of 18.76%, and the hydrolysis effect was superior, consistent with the results of other related studies [In the present study, the activity of ACE inhibitory peptides derived from  studies . AdditioThe key to preparing ACE inhibitor peptides by enzymatic methods is controlling the protease reaction conditions. Inappropriate temperature, duration and pH can affect enzyme activity , leadingFood-derived bioactive peptides can only exert their effects when they are absorbed by the intestine into the blood circulation . HoweverWhen preparing biologically active peptides, the separation and purification of peptides is very important ,42. CrudMolecular docking is an effective method for predicting the binding mode of ligands to receptors based on calculating the interaction energy and intermolecular forces . In ordeStructure–activity relationship studies have shown that potential ACE-inhibiting peptides typically consist of four to six amino acids . Girgih A. bisporus using dual-enzyme hydrolysis. Under optimal process parameters, the extraction rate of ACE inhibitory peptides was 6.678% . This provides a new path for the high-value utilisation of A. bisporus scraps. The average activity of the three novel ACE inhibitory peptides was 80.68%, and the IC50 value was 0.9 mg/mL. The amino acid sequences of three novel ACE inhibitory peptides were LVYP and VYPW and YPWT. The stability verification results showed good tolerance to temperature, pH and gastrointestinal digestive enzymes, indicating good potential for the development of drugs for lowering blood pressure. Molecular docking revealed their binding mechanism, which provides guidance for the development of antihypertensive peptide drugs.In this study, three novel ACE inhibitory peptides were successfully obtained from scraps of


Analysis of enzyme kinetics revealed that 5c is a competitive and reversible inhibitor with dissociation constant (Ki) value 0.0072 μM. In silico docking studies with mushroom tyrosinase (PDB ID 2Y9X) predicted possible binding modes in the enzymatic pocket for these compounds. The orthohydroxyl of the cinnamic acid moiety of 5c is predicted to form hydrogen bond with the active site side chain carbonyl of Asn 260 (2.16 Å) closer to the catalytic site Cu ions. The acetyl carbonyl is picking up another hydrogen bond with Asn 81 (1.90 Å). The inhibitor 5c passed the panassay interference (PAINS) alerts. This study presents the potential of hydroxyl-substituted benzoic and cinnamic acids and could be beneficial for various cosmetic formulations.Tyrosinase and its related proteins are responsible for pigmentation disorders, and inhibiting tyrosinase is an established strategy to treat hyperpigmentation. The carbonyl scaffolds can be effective inhibitors of tyrosinase activity, and the fact that both benzoic and cinnamic acids are safe natural substances with such a scaffolded structure, it was speculated that hydroxyl-substituted benzoic and cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. These moieties were incorporated into new chemotypes that displayed in vitro inhibitory effect against mushroom tyrosinase with a view to explore antimelanogenic ingredients. The most active compound, 2-((3-acetylphenyl)amino)-2-oxoethyl(E)-3-acrylate (5c), inhibited mushroom tyrosinase with an IC VariChemDraw Professional 15.2 was used to sketch all the ligands and LigPrep (Schrödinger) to prepare in their neutral form. The conformation of the prepared ligands was optimized in the OPLS-3 force field for further docking analysis.For this purpose, the crystal structure of target protein (PDB ID 2Y9X) was retrieved from RCSB and prepared in “Protein Preparation Wizard” workflow in Maestro Schrödinger for adding hydrogens, adjusting protonation and bond orders appropriate for pH 7. The water molecules beyond 5 Å from het groups were removed and minimized the prepared protein structure for further grid generation and docking analysis.For grid generation, the catalytic pocket is selected from its cocrystallized ligand and literature . The griin vitro tyrosinase inhibition activity. para position. The hydroxyl substitution at the second and fourth positions of the cinnamic acid phenyl ring in 5c resulted in a considerable rise in the enzyme inhibitory activity (50 46.43 μM), 5b (IC50 309.20 μM), and 5d (IC50 61.62 μM) displayed lower inhibitory activity than 5c (IC50 0.0020 μM) compared to reference inhibitor kojic acid (IC50 16.69 μM) and arbutin (IC50 191.17 μM). It is reported here that the ortho- and parahydroxyls of the phenyl ring in 5c appear to show an increased tyrosinase inhibitory activity  and 5(a–e) were designed and synthesized to evaluate activity . The pheactivity .max vs. 1/[S] at different doses of 5c reveal that the Michaelis-Menten constant (Km) changes while that of 1/Vmax remained the same representing the competitive nature of the most potent inhibitor (5c)  and 5(a–e) with the binding site of mushroom tyrosinase was determined using Michaelis-Menten kinetic studies. The inhibitors exhibit a dose-dependent inhibition of the enzyme tyrosinase. A striking reduction in the reaction rate in the presence of inhibitors refers to a decrease in the final absorbance in comparison to control without inhibitors. The inhibitory index of all these tested compounds varied depending upon the position of various substituents and classes of compounds . Inhibittor (5c) . The disμg/mL) of enzyme vs. various doses  of the inhibitor 5c for L-DOPA catalysis showed a group of straight lines with varying slopes and intersecting on the same point on the y-axis preferring the reversible effect of 5c on tyrosinase  was visualized to determine its interactions in the catalytic site of the protein tyrosinase (π-π stacking with side chain His 259 and His 263. The keto carbonyl oxygen from the tail moiety of this compound is interacting through another hydrogen bond with neighboring Asn 81 (1.90 Å) predicting a competitive type of inhibition for 5c. In the tyrosinase signature, His 61, His 85, His 94 (Cu 400), His 259, His 263, and His 296 (Cu 401) are conserved residues in the core region. Previous studies exposed that Asn 81, Asn 260, His 259, His 263, and Met 280 are the crucial residues responsible for stabilizing the protein inhibitor complex [A computational molecular docking study was also conducted to examine the binding conformations of all the synthesized compounds within the catalytic pocket of enzyme tyrosinase. The docked ligand-protein complexes were investigated based on docking score  and the hydrophobic/philic bonding interaction pattern. The docking scores had little fluctuations, and the comparison depicted no significant energy difference among all docked molecules due to similar basic skeleton of the ligands. Therefore, majority of the ligands showed efficient docking energy values. From docking results and rosinase . Two str complex .2 PSA, and the molar volume of 355.34 A3 proved to have a drug-like behavior within the standard range , polarizability ≤ 5, and solubility ≤ 4 has been reported in literature . The comrd range . Furtherα,β-unsaturation present in the 5(a–e). Among all the synthesized compounds, 2-(3-acetyl phenylamino)-2-oxoethyl(E)-3-acrylate (5c) exhibited the most promising inhibitory activity against tyrosinase (IC50 0.0020 μM) better than the standard kojic acid (IC50 16.69 μM) and arbutin (IC50 191.17 μM). Moreover, in the cinnamate amide 5(a–e) scaffold, the presence of -OH groups is of great interest for antityrosinase activity. In addition, from benzoic acid analogues, 3c (2-((3-Acetylphenylamino)-2-oxoethyl 2,4-dihydroxy benzoate) also displayed considerable activity against tyrosinase (IC50 27.35 μM) and has potential to be explored further for antityrosinase and cosmetic drug discovery and its clinical exploitation.The findings of the current study have provided a comprehensive overview of tyrosinase inhibitory activity of acetophenone analogues. According to the results presented, cinnamic acid derivatives 5(a–e) are more potent than benzoic acids 3(a–e), likely due to the additional


For both the ethanol and isopropanol HS, the active ingredient was the greatest overall contributing factor to the environmental impact (83.24% and 68.68% respectively). For HW with liquid soap and bar soap, there were additional contributing factors other than the soap itself: for example tap water use (28.12% and 48.68% respectively) and the laundering of a hand towel to dry the hands (10.17% and 17.92% respectively). All forms of hand hygiene have an environmental cost, and this needs to be weighed up against the health benefits of preventing disease transmission. When comparing hand sanitizers to handwashing with soap and water, this study found that using isopropanol based hand sanitizer is better for planetary health. However, no method of hand hygiene was ideal; isopropanol had a greater fossil fuel resource use than ethanol based hand sanitizer. More research is needed to find hand hygiene sources which do not diminish planetary health, and environmental impact is a consideration for public health campaigns around hand hygiene.In order to reduce the transmission of pathogens, and COVID-19, WHO and NHS England recommend hand washing (HW) and/or the use of hand sanitizer (HS). The planetary health consequences of these different methods of hand hygiene have not been quantified. A comparative life cycle assessment (LCA) was carried out to compare the environmental impact of the UK population practising increased levels of hand hygiene during the COVID-19 pandemic for 1 year. Washing hands with soap and water was compared to using hand sanitizer (both ethanol and isopropanol based sanitizers were studied). The isopropanol-based HS had the lowest environmental impact in 14 out of the 16 impact categories used in this study. For climate change, hand hygiene using isopropanol HS produced the equivalent of 1060 million kg COThe online version contains supplementary material available at 10.1007/s11356-022-18918-4. Hand hygiene is one of the most important ways of avoiding pathogen transmission. WHO .st 2019, originating in Wuhan, China.  on December 31ina. WHO . Currentina. WHO .To help prevent pathogen spread and in particular of Covid-19, the WHO recommends hand washing with soap and water (HW) or cleaning hands with alcohol hand sanitizer (HS) whenever hands are visibly dirty, as well as if hands are not visibly dirty. The WHO and Centre for Disease Control (CDC) both state that it is necessary to clean hands at key times with soap and water or HS with at least 60% alcohol. The WHO provides guidance for the contents of HS sanitizer based on ethanol or isopropanol  can also be used to consider the entire life cycle of a product from ‘cradle to grave’. LCA data can be used to quantify multiple environmental impacts as well as other useful public health information such as Disability Adjusted Life Years (DALYs). DALYs consider both the time lost to premature death and reduced quality of life due to illness. DALYs .Although LCAs have been used to compare different types of hand drying Isopropanol-based HS: individual disinfects hands with 0.4 ml of hand sanitiser, made from WHO formulation II (WHO 2009)Liquid soap HW: individual washes hands with 0.75 ml of liquid soap and 3.33L of tap water, drying hands with a laundered hand towel.Bar soap HW: individual washes hands with 0.15 ml of bar soap and 3.33L of tap water, drying hands with a laundered hand towel.A comparative attributional LCA of 4 different hand hygiene techniques was undertaken at the Eastman Dental Hospital, London, in partnership with the Dublin Dental University Hospital . The four hand hygiene techniques were:The functional unit was defined as the UK population practicing COVID-19-related hand hygiene for 1 year. This time period was chosen as it reflects the time that the COVID-19 pandemic is currently expected to be at least as prevalent. . The weight of each constituent of the sanitiser was based on the density and volume needed to produce 1 L of product. Fragrances and pigments were excluded from all products as they were not consistent product by product, and were assumed to be present in equal quantities across all types of products assessed. It was assumed that components for hand sanitiser were mixed in a pressure-protected mixing machine and immediately packaged in screw top bottles to prevent evaporation, as per WHO guidelines . It was assumed that all liquid soap and sanitizer products were packaged by machine assembly in 1 L cylindrical plastic bottles  with a plastic lid (polyethylene) and a printed paper label that wraps around the bottle. The weight of the bottle, lid, and label were based on a tear down of a sample 1L sanitizer bottle. The constituents of the liquid and bar soap were assumed to include palm oil, cottonseed oil, and soybean oil as per the Ecoinvent  dataset Assumptions for transport of the products from the factory to retailer, and then from retailer to consumer, were based on the PEF recommendations for modelling transport  and would launder this hand towel every other day Sturt .For the bottled products, it was assumed that individuals would dispose of the plastic bottle and lid in plastic recycling, and the paper label in paper recycling in the UK. For the bar soap, it was assumed the cardboard box of soap was recycled.For the ethanol HS, the amount of ethanol absorbed through the skin is negligible, and the amount of ethanol inhaled by the user is only a couple of percent  evaporates quite rapidly. The first-order half-life was found to be approximately 8 min, which means that half of the amount present at any time will evaporate in the next 8 min Pinhas . TherefoFor the isopropanol HS, the boiling point isopropyl alcohol is 82 degrees C and its vapor pressure at 35 degrees C is 95 mm Hg NLM . Unlike Data from the life cycle inventory was modelled and analysed in OpenLCA  v11, aloThe results of the LCIA are shown in Table The normalised results compare the impact of using HS or HW against one global person’s annual share of all emission and resource use in the world (the impact that the “average Joe” would be expected to make from living their daily lives for 1 year). Figure As per PEF recommendations, the toxicity categories have been removed . Similarly, doubling the pump volume, to 3 ml, increased the LCIA results by an average of 61% (range from 26% in carcinogenic effects to 94% in freshwater eutrophication).Another sensitivity analysis for liquid soap HW was performed to examine the effect of increasing or decreasing the volume of water used to wash the hands. The original assumption was that 3.33L of water were used per episode of handwashing (tap running for 40 s at 5L/min). Halving the water use, to 1.67L, reduced the LCIA impact by an average of 86% (range from 66% in carcinogenic effects to 98% in land use); and doubling the water use, to 6.66L, increased the LCIA impact by an average of 28% (range from 3% in land use to 69% in carcinogenic effects).The results of the DALY impact calculations are shown in Fig. Hoekman et al.  providesThe overall human health impact was measured using DALYs. The biggest contributor to the DALY impact, for all methods of hand hygiene, was ozone depletion, which damages the protective ozone layer surrounding our planet. The next highest contributor was for the use of water in each product. Water was required at different stages of the pathway. The first was in packaging and manufacture, water is required to grow and convert the fruit for the ethanol gel, manufacture the isopropanol and also to mould the plastic associated with the packaging. It is also required to manufacture the soap. It is not possible to eliminate water from the manufacturing process, however it may be possible for industry to consider more sustainable packaging which doesn’t have such a need for this high water consumption. The other major use of water was in the actual handwashing process. For HW with any type of soap, water is used to wash hands, and undertake laundry processes. Water is also required to treat the waste water that is produced from this process. This is discussed further below.There is some evidence that one type of HS might be more clinically effective than another. One study showed that 80% ethanol HS, was more effective than isopropanol  but using additional plain soap and water reduced the presence of bacteria to a lower 8% (comparison of both handwashing arms: p < 0.001)  had a contribution of between 24% (toward the ionizing radiation impact) and 93.60% (towards the freshwater eutrophication aspect) of the environmental footprint. Freshwater eutrophication impact arises from a number of sources, including growing the oils to produce the soap  as well as water required to manufacture the soap. It is acknowledged that there is wide variability within the constituents of soap. We know that palm oil is associated with high negative impacts on the environment, but there is no evidence to suggest alternatives would be any better Standard . Palm oiFor both liquid and bar soap, the process of handwashing requires using tap water (tap water use contributed 28%). There are a number of ways to reduce this footprint. Washing your hands for less time, or with less soap  would be one factor, but might also reduce the effectiveness of the process. The NHS  suggestsThe laundry of the hand towel contributed an average of 10.17% for liquid soap and 17.92% for hand soap. If the consumer used a smaller hand towel, or washed their towels weekly instead of every second day, this contribution would lower. The Ecoinvent dataset for laundry includes the cleaning, drying and ironing of laundry therefore removing the machine drying and ironing of your towels could also reduce the overall environmental impact (Benjamen 2020).Our study reinforces the environmental benefits of using bar soap compared with liquid soap. This is for a number of reasons including less plastic packaging with the latter, but also as less soap is released when using a bar. It is unsure as to whether this reduction in soap may also reduce pathogenic efficacy of the bar.Rubbing most commercial hand sanitizers on one’s hands for as little as 15 s reduces the amount of many viruses by a factor of 1000 and reduces the amount of a wide range of bacteria by 100,000 . When the results were normalised, fossil fuel use was found to be the least important of the 16 impact categories measured in this LCA . The fact that the impact of ethanol via fermentation was worse for the environment than a fossil fuel based isopropanol is not as surprising as it seems, and is in line with fuel studies showing the problems with using ethanol as a fossil fuel substitute. Northern American studies are mostly based on ethanol produced from corn. A mono-culture crop such as corn can inevitably reduce plant and animal biodiversity, and contribute to erosion, nutrient run off and other adverse environmental impacts. Corn ethanol production uses a modest amount of water which can be a significant resource problem where there are constraints of water Supplementary file2 (PDF 0.99 MB)Below is the link to the electronic supplementary material.


People experiencing homelessness are at increased risk of SARS-CoV-2 infection. This study reports the point prevalence of SARS-CoV-2 infection during testing conducted at sites serving people experiencing homelessness in Toronto during the first wave of the COVID-19 pandemic. We also explored the association between site characteristics and prevalence rates.The study included individuals who were staying at shelters, encampments, COVID-19 physical distancing sites, and drop-in and respite sites and completed outreach-based testing for SARS-CoV-2 during the period April 17 to July 31, 2020. We examined test positivity rates over time and compared them to rates in the general population of Toronto. Negative binomial regression was used to examine the relationship between each shelter-level characteristic and SARS-CoV-2 positivity rates. We also compared the rates across 3 time periods .The overall prevalence of SARS-CoV-2 infection was 8.5% (394/4657). Site-specific rates showed great heterogeneity with infection rates ranging from 0% to 70.6%. Compared to T1, positivity rates were 0.21 times lower (95% CI: 0.06–0.75) during T2 and 0.14 times lower (95% CI: 0.04–0.44) during T3. Most cases were detected during outbreak testing (384/394 [97.5%]) rather than active case finding.During the first wave of the pandemic, rates of SARS-CoV-2 infection at sites for people experiencing homelessness in Toronto varied significantly over time. The observation of lower rates at certain sites may be attributable to overall time trends, expansion of outreach-based testing to include sites without known outbreaks, and/or individual site characteristics.The online version contains supplementary material available at 10.17269/s41997-021-00591-8. The COVID-19 pandemic has disproportionately impacted and continued to pose extraordinary challenges for people experiencing homelessness  an active case-finding program involving testing in shelters without identified cases throughout the study period, and (2) an outbreak management program involving testing in response to positive cases that were identified through community testing or symptoms. Testing was conducted across various types of sites, including shelter-based programs, drop-in and respite programs, encampments, and COVID-19 physical distancing sites, which are sites that were opened during the pandemic to allow safe physical distancing for shelter residents. The study included data on testing performed on residents at these sites between April 17 and July 31, 2020, which included the first wave of Ontario’s COVID-19 pandemic. Each on-site testing was done either for active case finding or for outbreak management purposes. Outbreak was defined as having at least one laboratory-confirmed case in residents or staff. This is in accordance with the Ontario Ministry of Health’s outbreak guidance for congregate settings , and the City of Toronto Shelter, Support and Housing Administration (SSHA) to ascertain outbreak status, COVID-19 test results, and shelter characteristics. Data from Ontario Health Toronto contained information on test dates, total number of residents tested, the number tested positive for each date, and the location of each site. The outreach shelter-based testing was conducted by various community agencies and healthcare organizations. TPH data were used to determine outbreak status for each testing date. We also used two separate TPH datasets to ascertain weekly laboratory positivity rates  of the Toronto general population, and the number of sporadic cases by neighbourhood regions  and gender . We further categorized site type as to whether or not it served exclusively refugee claimants. For each site, we defined its layout as one of the following: single room, shared room (2–5 beds), dorm or open style (more than 6 beds), and encampment (outdoor). If a site had a mixed layout (both single rooms and shared rooms), we classified the layout as shared rooms to reflect COVD-19 transmission risk in shared spaces.As a proxy measure of COVID-19 transmission in the community that could potentially affect site positivity rates, data were obtained on the number of sporadic cases of COVID-19  in the neighbourhood where the sites were located.Prevalence of SARS-CoV-2 infection was expressed as positivity rates per 100 tested residents between April 17 and July 31, 2020, and it is described by site and by site characteristics. The overall prevalence of SARS-CoV-2 infection was calculated by dividing the total number of confirmed cases across all sites by the total number of tested individuals during the study period with 95% confidence interval. We also compared the weekly COVID-19 positivity rates among individuals screened at sites for people experiencing homelessness in relationship to the positivity rates in the general Toronto population.The association between site characteristics and site prevalence rates was evaluated using a negative binomial regression. The model’s outcome was the total number of positive cases in each site and included one site characteristic and the log of the total number of residents tested as the offset. Only univariate models were fit, since most the site characteristics were highly correlated. For our regression analysis, we focused on data between April 17 and June 25, 2020, since this period constituted most of the first wave of the pandemic. In addition, on June 25, 2020, Toronto entered stage 2 wherein businesses and services were allowed to reopen , 8 had missing test results, 23 were not at a site for people experiencing homelessness, and 18 were repeated testing.The final sample included 97 unique sites and 111 testing dates. Eight sites had to perform testing on multiple days because of high resident volume, which led to purposefully testing over more than 1 day, or because not all residents were available on the first day of testing. Among the excluded testing dates . For the period of April 17 to June 25, the prevalence was 11.5% . Site-specific rates ranged from 0 to 70.6% and declined over time  in the prevalence of SARS-CoV-2 infection across shelters in Toronto during the period April 17 to July 31, 2020. We found significantly higher positivity rates during the T1 (April 17–April 25) compared to later testing periods. We also found that site-based active case finding (testing in the absence of an outbreak) identified few cases and likely provided minimal benefits in terms of reducing the transmission of SARS-CoV-2. This finding is consistent with modeling studies that indicated that mass testing has to occur every 7 days to be a useful part of shelter COVID management  is higher compared to previous Ontario-based studies that used administrative health records to ascertain positivity rates (2.3–6.4%) in homeless populations Below is the link to the electronic supplementary material.


CAPN3 gene. It is a rare disease and there is no cure or treatment for the disease while the pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. However, key proteins implicated in Wnt and mTOR signaling pathways, which regulate muscle homeostasis, showed a considerable reduction in their expression and in their phosphorylation in LGMDR1 patients’ muscles. Finally, the administration of tideglusib and VP0.7, ATP non-competitive inhibitors of glycogen synthase kinase 3β (GSK-3β), restore the expression and phosphorylation of these proteins in LGMDR1 cells, opening the possibility of their use as therapeutic options.Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the  CAPN3 gene that causes progressive degeneration of the proximal muscles of the pelvic and shoulder girdle. The disease begins in the second decade of life and muscle degeneration leads to muscle weakness and atrophy that confines patients to a wheelchair in around 20 years of disease progression [Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the 2+ flow between the sarcoplasmic reticulum/cytoplasm [Calpain 3 is a muscle-specific protease that may participate in several functions, such as muscle contraction due to its link to titin ,5,6,7, cytoplasm .Balanced homeostasis between the synthesis and degradation of proteins in the muscle fiber is key to maintain the muscle and thus to avoid muscle atrophy and weakness . For thaWhen the Wnt signaling pathway is active, Wnt ligands induce the inactivation of GSK-3β preventing β-catenin phosphorylation, allowing its accumulation in the cytoplasm and translocating it to the nucleus. Then, β-catenin binds to T-Cell Factor/Lymphoid Enhancer Factor (TCF/LEF) and activates downstream target genes ,15. On tAmong the drugs that inhibit GSK-3β, lithium is a widely used drug. Due to its activator role in the Wnt signaling pathway, certain studies showed beneficial results in vitro ,22,23,24Among the molecules that are able to inhibit GSK-3, the ATP-competitive ones have often presented important adverse side effects in long-term treatments. On the other hand, those that inhibit GSK-3β in a non-competitive or allosteric way are more selective ,28,29, wTideglusib is an irreversible drug designed for the treatment of Alzheimer’s disease and whose safety for human treatment has been demonstrated . VP0.7, The pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. Loss of calpain 3 leads to the deregulation of the expression of several genes/proteins and to abnormal sarcomere formation in the muscles ,32,33. CIn this work, we report expression alterations in proteins implicated in signaling pathways that regulate muscle homeostasis, such as Wnt and mTOR pathways. LGMDR1 patients’ muscles showed a severe reduction in the expression of the proteins involved in these pathways. Finally, our study showed that tideglusib and VP0.7, ATP non-competitive GSK-3β inhibitors, restore the expression and phosphorylation of key proteins in Wnt and mTOR pathways, opening up the possibility of their use as therapeutic options in LGMDR1.Previous studies had described the overexpression of FRZB, a Wnt1, 5a, 8 and 9a antagonist, in the muscle of LGMDR1 patients . Due to n = 2) has been observed in pseudo-asymptomatic cases with respect to the controls . On the contrary, the active form of β-catenin showed a drastic decrease of the protein in patients, except in a benign patient (B09-26). This reduction was not observed in the pseudo-asymptomatic cases (05 and 07-109). These pseudo-asymptomatic cases were 13- and 10-year-old patients who only presented hyperCKemia and eosinophilia. In most of the analyzed proteins, an increase in expression .Subsequently, the expression of kinases downstream of this pathway was analyzed. p70S6K is a kinase participating in the mTOR pathway that is activated by sequential phosphorylations ,40,41. Op = 0.0381)  .As a summary, a schematic representation of the altered proteins in Wnt and mTOR pathways in LGMDR1 muscle are shown .Lithium and the small molecule drugs tideglusib and VP0.7 were used in this study. While lithium is a known and widely used GSK-3 inhibitor, its unwanted adverse effects entailed in clinical trials led us to the search of selective GSK-3 inhibitors with an optimal safety profile, including tideglusib and VP0.7. These drugs were administered in different cell types: primary myotubes, CD56- cells and skin fibroblasts. The effect on the response for the administered drugs was different depending on the cell type.We proceeded to analyze the expression of GSK-3β, as well as its downstream effect on the Wnt pathway in myotubes from controls and patients after drug administration. Inhibition by tideglusib and VP0.7 slightly increases the amount of total GSK-3β and its phosphorylation (Ser9) in patients. The activation of the Wnt pathway was confirmed by effective inhibition of GSK-3β, since an increase in total β-catenin expression as well as active β-catenin was observed with tideglusib and VP0.7. As a control assay, lithium was administered, and an increase in active β-catenin was also verified A.FRZB gene or with treatment with lithium, the expression of some altered genes in the muscle of patients was recovered [FRZB gene silencing, that is, an increase in the expression of the CAPN3, FOS and ANOS1, and an increase in ITGB1BP2 was also observed  increased slightly. For mTOR, hardly any changes were observed in total mTOR, but there was a substantial increase in phosphorylation of mTOR in two of its residues (Ser2448 and Ser2481) A.Downstream of the mTOR pathway, the p70S6K and RPS6 proteins were analyzed. After administration of the drugs, the expression of total p70S6K, its phosphorylations (Thr421/Ser424 and Thr389), as well as the levels of phosphorylation of the RPS6 protein (Ser235/Ser236) increased in the patient’s sample. In the control, the effect observed after the treatments is very mild A.Finally, AMPK was analyzed after drug administration, and a slight increase in Thr172 residue phosphorylation was observed in the patient and a very marked increase in the control A.FRZB gene had shown recovery in the expression of various altered genes in patients, we proceeded to analyze its possible effect beyond the Wnt pathway, specifically in the mTOR pathway. The silencing of the FRZB gene carried out in the myotubes did not show any effect on the regulation of the expression or on the phosphorylation of mTOR (data not shown). In addition, the activations of the downstream kinases of this pathway, P-p70S6K (Thr389), P-p70S6K (Thr421/Ser424) and P-RPS6 (Ser235/Ser236) were analyzed, but little effect or a slight decrease was observed  occurred similarly in other cell types, we proceeded to study skin fibroblasts and CD56- cells obtained from muscle patients. We used two non-myogenic cell lines  to show that these drugs may not affect other cell lineages.FibroblastsTreatment with lithium in fibroblasts showed increased phosphorylation of GSK-3β (Ser9), confirming that the treatment produces the desired kinase inhibition . SubsequTreatment with tideglusib and VP0.7 did not show an increase in the phosphorylation of GSK-3β (Ser9) . Since tCD56- cellsAs expected, the administration of lithium to CD56- cells showed that inhibition of GSK-3β through phosphorylation of the Ser9 residue occurred, since the amount of phosphorylated GSK-3β was increased . On the In the same manner, after treatment with tideglusib and VP0.7 in CD56- cells, no substantial alteration was observed in the amount of total β-catenin or in its active form A. FinallIt is known that as a consequence of the primary deficiency of a specific protein, there are alterations in different signaling pathways, as occurs in various forms of muscular dystrophy, where the signaling pathways of NF-κB, MAPK, PI3K/Akt and Cn/NFAT are altered . It had In this work, two signaling pathways that regulate homeostasis in muscle fiber in LGMDR1 patients have been analyzed to establish, first, the pathways with the greatest impact on the pathophysiology of the disease, and second, the effect of the treatment with GSK-3β inhibitor drugs to regulate signaling pathways in patients.CAPN3-KO model, after generating muscle damage, the altered expression of proteins that are involved in the Akt/mTOR signaling pathway. In muscle tissue from LGMDR1 patients, we also observed alterations in this signaling pathway, since they present a severe decrease in mTOR expression, its phosphorylation, as well as a reduction in the activation of downstream proteins of the pathway, which leads to the dysregulation of the growth of muscle fiber. This signaling pathway, like many others, receives different stimuli through growth factors, cellular energy levels, etc. that must be correctly integrated to stimulate protein synthesis and cell growth according to physiological requirements [Our study has allowed to establish that in the muscle of patients, there is a reduction of active β-catenin, which confirms that the Wnt pathway is altered. Additionally, Yalvac and colleagues  already irements ,51,52. Airements  demonstrOn one hand, it has been shown that GSK-3β inhibits the mTOR pathway by phosphorylating TSC2, and thus, inhibits the downstream phosphorylation of S6K. Stimulation of the mTOR pathway by Wnt is independent of β-catenin-mediated transcription. The Wnt pathway bifurcates at the level of GSK-3β to regulate, on the one hand, translation by means of mTOR and, on the other hand, transcription by means of β-catenin . InhibitRegarding the energy requirement of the cell, AMPK, AMP-activated protein kinase, is a cellular energy sensor that plays an important role in cell homeostasis . Like GSRelated to protein expression in pseudo-asymptomatic patients, we observed an increase in most of the analyzed proteins. This fact could be due to the expression profile derived from their young age, since pseudo-asymptomatic patients are 13 and 14 years old and it is known that age is a factor for regulating the expression of genes. Finally, the fact that the muscle samples from pseudo-asymptomatic patients present levels of phosphorylation of p70S6K and RPS6 similar to that of muscles of controls highlights the relevance of the activity of this pathway for the correct maintaining of the muscle function.With the administration of tideglusib and VP0.7 in myotubes, there is a total inhibition of GSK-3β that increases the amount of β-catenin and activates the mTOR pathway, confirming the participation of GSK-3β in both processes. Since the in vitro data shown here have been obtained from a patient and a control, these should be treated with caution. However, this finding supports the suitability of these drugs to be used as a treatment in LGMDR1, given that it could suggest that the undesirable side effects would be low.FRZB gene silencing, it has been established that the GSK-3β pool involved in the Wnt pathway  does not participate in the regulation of the mTOR pathway. This is because Wnt does not affect the phosphorylation state of GSK-3β [Finally, thanks to the results obtained with the f GSK-3β  and becaf GSK-3β .In the case of Tideglusib and VP0.7, their inhibition is not occurring by the phosphorylation of Ser9 and it cannot be confirmed this way. However, the effect of these drugs at the used concentrations have been confirmed in other experiments ,61. MoreAlthough the results seem promising, they should be treated with caution since they are results that were obtained in vitro and in a single patient. However, this work could help to shed some light on the knowledge of the underlying mechanisms of the LGMDR1 disease. The rescue of signaling pathways responsible of the synthesis of proteins that conform the costamere could help to improve its altered structure. Thus, it could also lead to a better functioning of the muscle fiber, improving the contraction and preventing the degeneration and, consequently, the death of the fiber.FRZB gene silencing, through the inhibition exerted on the GSK-3β pool of the Wnt pathway, cannot recover the mTOR pathway. Finally, (d) tideglusib and VP0.7, through efficient inhibition of GSK-3β, rescue the Wnt and mTOR pathways in vitro in the myotubes of LGMDR1 patients.In summary, we established the following relevant events that could orient towards possible therapeutic targets: (a) Wnt and mTOR signaling pathways are altered in the muscles of LGMDR1 patients. (b) Maintaining active the mTOR pathway, pseudo-asymptomatic patients could escape the dystrophic phenotype. (c) There are no other approaches in progress for this disease and these treatments could help correct some of the deficits observed in patients. Moreover, tideglusib has shown to be a safe compound for chronic treatments in phase 2 trials ,63,64,65All participants gave informed consent, using forms approved by the Ethics Committee on the Use of Human Subjects in Research at Donostia University Hospital. Muscle biopsy specimens were obtained from adult and pseudo-asymptomatic  patients with LGMDR1, in whom the diagnosis had been confirmed genetically by the identification of both mutations in the calpain 3 gene, and from healthy adult controls. These controls were otherwise healthy individuals that underwent surgery for bone fractures and the muscle biopsies were obtained during the surgery . The pseHuman proximal muscle biopsies were minced and cultured in a monolayer according to the method described by Askanas . To obta2 in culture medium for the myoblast stage. When the myoblasts started to fuse, the medium was replaced by a medium containing 2% of fetal bovine serum (FBS)  and without growth factors to obtain myotubes. CD56- cells were seeded in collagen-coated plates.CD56+ cells were seeded at 2500–3000 cells/cm2. Subsequently, fibroblasts were cultivated in DMEM, 10% FBS and 2% penicillin/streptomycin.On collection, skin samples were immersed in RPMI 1640 medium and 2% penicillin (1000 U/mL)/streptomycin  . Then, skin fragments were placed on a moistened surface with Modified Eagle Medium (MEM) , 13% Newborn Calf Serum , 0.4% penicillin/streptomycin and 2 mM L-Glutamine  and incubated at 37 °C, with 5% COFRZB (s5369) knockdown was purchased from Thermo Fisher Scientific. A scrambled siRNA was used as a negative control . Cells plated at 24,000 cells/cm2 were transfected with the siRNA at a concentration of 5 nM using RiboCellin transfection reagent  following the manufacturer’s instructions. After 8 days of differentiation, human primary myotubes were incubated with the corresponding siRNA and the transfection agent. Finally, the RNA obtained from these cultures was analyzed 48 h post-transfection by quantitative real-time PCR. Likewise, proteins from these cultures were analyzed 72 h post-transfection.The siRNA for Tideglusib and VP0.7 were synthetized in or laboratories followed previously described procedures ,29. CompRNA extraction from primary myoblast/myotubes was performed with a RNeasy Mini Kit . In the case of the muscle biopsies, which were snap frozen and stored at −80 °C until use, total RNA was obtained using an RNA-Plus Kit .CAPN3, FOS, ITGB1BP2, ANOS1 and GAPDH, FRZB gene and the lithium treatment in a previous study [The isolated RNA was reverse-transcribed to first-strand complementary DNA (cDNA) in a final volume of 50 μL using a High-Capacity cDNA Reverse Transcription Kit , according to the manufacturer’s instructions. To investigate the levels of expression of the differentially expressed genes /p70S6K could not be represented because available samples did not match to obtain the ratio. When a single sample was available, statistical analysis was not performed.


Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the  Human cysteine proteases participate in several physiological processes, such as the degradation of peptides and proteins , and conIxodes scapularis tick. Sialostatin L inhibits the protective proteolytic activity of host cells at infestation sites, thus promoting tick survival  and cultivated under the influence of 350 rpm orbital shaking at 28 °C for 24 h. Cells were harvested by centrifugation at 450× g for 5 min at 4 °C and resuspended in 50 mL of BMMY medium  to absorbance at 600 nm of 5.0. Incubation was carried out at 30 °C and 350 rpm orbital shaking for 44 h, with further additions of methanol to a final concentration of 0.5% every 12 h, approximately. Samples (1 mL) were taken during the assay, submitted to protein precipitation with methanol/chloroform ) ) , using t


The interaction of ERL with BSA at Site II is ruled out based on the site marker studies. The suggested Site on BSA for interaction with ERL is Site I. Stability of the BSA-ERL system was established with molecular dynamic simulation studies for both Site I and Site III interaction. In addition, the analysis can significantly help evaluate the effect of various quercetin-containing foods and supplements during the ERL-treatment regimen. In vitro binding evaluation provides a cheaper alternative approach to investigate ligand-protein interaction before clinical studies.The interaction between erlotinib (ERL) and bovine serum albumin (BSA) was studied in the presence of quercetin (QUR), a flavonoid with antioxidant properties. Ligands bind to the transport protein BSA resulting in competition between different ligands and displacing a bound ligand, resulting in higher plasma concentrations. Therefore, various spectroscopic experiments were conducted in addition to in silico studies to evaluate the interaction behavior of the BSA-ERL system in the presence and absence of QUR. The quenching curve and binding constants values suggest competition between QUR and ERL to bind to BSA. The binding constant for the BSA-ERL system decreased from 2.07 × 10 The most affluent blood protein present in vertebrates is serum albumin (S.A.) and it is mainly responsible for transporting both endogenous and exogenous molecules to their target sites. It is the principal constituent of the plasma proteins ≈60%)  and play0%  and pSimultaneous administration of two or more drugs might lead to a competition between the two drugs to bind to a similar site present on the BSA. The conformation of the protein binding pockets can be altered on interaction with ligands, thereby influencing the binding of other ligands to the same binding pockets ,9. The tErlotinib (ERL), a tyrosine kinase inhibitor, has anticancer properties and is approved to treat non-small cell lung cancer. There is a high epidermal growth factor receptor (EGRF) expression in certain cancers, and ERL inhibits EGFR . One of Fresh fruits and vegetables contain phenolic compounds such as flavonoids and tannins, and one such flavonoid is quercetin . QuercetQuercetin is highly metabolized, and the amount of QUR in systemic circulation varies based on inter-individual pharmacokinetics and the source of QUR. In addition, although different organs metabolize quercetin, its transport to these organs is dependent on systemic circulation ,22. ThusThe flavonoid QUR binds to Site I, subdomain IIA of BSA. Further, the interaction of QUR with other concomitantly used drugs has also been investigated ,29,30,31The chemicals used in the study ERL and QUR were procured from Weihua Pharma Co., Limited  and fatty acid-free BSA from Sigma-Aldrich . The phosphate buffer saline (PBS) pH-7 was prepared afresh to prepare the stock solution. QUR and ERL were dissolved in DMSO (dimethyl sulfoxide) followed by dilution with PBS. Millipore water (type-I) was used to prepare the stock solutions.The spectrofluorometer FP-8200  was used to measure the fluorescence spectra. The instrument had a quartz cell, and the slit width was 5 nm. The inner filter effects were corrected for the reabsorption of emitted light using the equation :Fcor=FobcorF and obsF are the corrected and observed fluorescence in the above equation, respectively, and exA and emA are excitation and emission wavelengths absorbance values, respectively. The BSA spectra were recorded for the binary system, which consisted of BSA-ERL and BSA-QUR, and the ternary system consisted of BSA-QUR-ERL and BSA-ERL-QUR systems. For the measurement of spectra in the binary system, the concentration of BSA was held constant at 1.50 μM and the concentration of ERL was between 0.00–27.5 μM and QUR was between (0.00–35 μM) The spectra were recorded at three temperatures of 298, 303 and 310 K for BSA-ERL system and 298 K for BSA-QUR system. The concentration of BSA was 1.50 μM in the presence of ERL, and the spectra were recorded with ERL 0.00–27.50 μM in the presence of QUR 5.5 μM in the ternary system.The site marker studies were undertaken to establish the binding Site for ERL with phenylbutazone for Site I and Ibuprofen for Site II as markers. The spectra for BSA were recorded with ERL in the presence of site markers phenylbutazone and ibuprofen.The UV-Vis absorbance spectra were recorded with Shimadzu 1800 . The spectra for BSA were recorded in the presence of ERL (0.00–15.00 μM) and a fixed concentration of BSA μM.The microenvironmental changes in the fluorophore residues were identified with the SFS technique. The changes in the amino acid residues Tyr (tyrosine) and Trp (tryptophan) can be observed at ∆λ = 15 nm and ∆λ = 60 nm. A shift in the emission spectra indicates changes in the polarity of amino acid residues around the fluorophore molecules.Structural changes and conformational changes in the protein are identified with 3D fluorescence spectra. In addition, microenvironmental fluctuations in the vicinity of fluorophore residues lead to conformational or structural changes in the protein molecules. The BSA-ERL 3D spectra were matched to the 3D spectra of BSA to identify any such variations in the fluorophore microenvironment.http://www.ks.uiuc.edu/Research/namd (accessed on 20 September 2021)) [+ or Cl− with their maximum concentration of 0.15 M. The minimization of the system was carried out in MOE. The default MOE parameters were used for the minimization, which included Amber10: EHT force field. Partial Charge on LL toms was calculated using Amber10: EHT force field. The system’s energy was minimized to an RMS gradient of 0.1 kcal/mol/A2. As per the MOE manual, an RMS of 0.1 is sufficient for structure minimization for molecular dynamic simulation. The minimized system was gradually heated. The temperature raised from 0 K to 310 K at an incremental level of 50 K for 100 ps and equilibrated at each temperature step followed by an unrestrained run of 20 ns. Constrained light bond with 2 fs time step. Long-range electrostatics were treated via the particle mesh Ewald method. The grid box formed had following parameters and dimensions: solvent molecules—26,824, 1.009 g/cm3; Space group—P1, Triclinic, 1, 1×; size—; cell shape—. The parameters RMSD and RMSF were obtained for the Protein BSA and the protein in the presence of ligand (BSA-ERL) systems. The trajectory was visualized and analyzed with VMD 1.9.3 tool.The molecular docking was performed with bioinformatics tools MGL Tools , Auto Dor 2021)) . The ligF0 is BSA’s fluorescence intensity, and F is BSA’s fluorescence intensity in the presence of quencher. Stern Volmer constant is given as svK, and (Q) is quencher concentration. The qk biomolecular quenching constant and τ0 the lifetime of the fluorophore in the absence of quencher and is valued at 10−8 s for biopolymers.The fluorescence spectra for the protein BSA for the binary and the ternary system were recorded at 280 nm (excitation wavelength) and an emission wavelength of 300–500 nm a–c. BothsvK values was observed with a rise in temperature for the BSA-ERL system . The ternary system (BSA-QUR)-ERL had binding constants of the order of ≈102 .In the equation above, ΔH°, ΔS° and ΔG°, were calculated from the van’t Hoff plot for ln(bK) vs. 1/T and are given in The thermodynamic parameters Δ−1 whereas entropy was −0.79 kJ·mol−1, the BSA and ERL interaction indicate the interaction being enthalpy-driven, and the interaction had an unfavorable entropy.All the three parameters, enthalpy change, entropy change, and Gibbs free energy, given in In the BSA-ERL binary system, the quenching constants  decrease4 suggests moderate binding between the ERL and BSA. Our results corroborated earlier studies for the interaction between BSA and ERL [4 to 0.2 × 102. -ERL was also studied, and it was different from both the binary system spectra b.The microenvironment alterations in the fluorophore residues can be identified with synchronous fluorescence spectroscopy. The changes in emission wavelength indicate polarity change in the fluorophore residue microenvironment . The synex = λem) and IInd order scattering peak (λem = 2ex). Higher intensity and scattering were observed for Peak a in the BSA-ERL system compared to BSA since the BSA-ERL complex formed a bigger macromolecule than BSA. The other peaks, Peak I representing Trp and Tyr fluorophore residues and Peak II (polypeptide backbone structures), were evaluated in the 3D spectra. The intensity of both peaks, Peak I and Peak II, declined in the BSA-ERL protein–ligand system compared to the protein BSA. Hence, it is concluded that the interaction of ERL with BSA altered the microenvironment of the fluorophore residues, which led to these changes in the 3D spectra. Hence it was concluded that structural changes might have occurred in BSA on interaction with ERL.The 3D fluorescence spectroscopy provides information about protein structural alteration on interaction with a ligand . The 3D Molecular docking studies confirmed the experimental results for the BSA-ERL interaction. The site probe experiments with phenylbutazone suggest that the ERL interacted with BSA’s subdomain IIA (Site I). However, an earlier study using molecular docking suggested that ERL binds to subdomain IB (Site III) and subdomain IIIA (site II) of BSA . Further−1 was lower than the binding energy −29.91 kJ·mol−1 for Site III of BSA. The binding pocket of Site I of BSA  and root mean square fluctuation (RMSF) studies. The RMSD studies for BSA and BSA Site I and Site III with ERL are provided in Further, in the simulation of the complex and comparison to the native protein residue Cys168 and Ala78, the N-terminus seems to be responsible for the higher RMSD values from 14–20 ns. Additional minor peaks can also be observed in the remaining loops 299, 309, and 504 and the C-terminus.In the simulations between 2 to 8 ns, the resulting RMSD plots seem to be responsible for the higher RMSD values beyond residue Pro110 on the N-terminus for the native protein. No fluctuation was observed in the RMSD or RMSF plots of the BSA-ERL system at either of the binding sites, Site I or Site III. The RMSD averages were 1.98 and 1.82 Å, respectively, and the variation in the RMSD for the BSA-ERL system was between 0.258–3.013 Å for Site I and 0.214–2.318 Å for Site III. Therefore, a stable complex between BSA and ERL is concluded as the RMSD values did not fluctuate too high. Further, the residual flexibility is interpreted from the RMSF studies. The RMSF plot for both the studied sites (Site I and Site III) of the BSA-ERL system suggests that the complex formed between BSA and ERL was stable with a fluctuation of less than 3 Å .This study examined the influence of flavonoid QUR on the BSA-ERL interaction by multispectroscopic and computational methods. Quercetin presence in the BSA-ERL system reduced the binding constant of the BSA-ERL system to almost half of what was observed in its absence. Thus, it can be concluded that there will be a higher free drug fraction of ERL in the system in the presence of QUR. However, the use of ERL in a therapeutic regimen leads to several adverse events, which in turn are associated with ERL plasma concentrations. Thus, co-administration of QUR and ERL might influence the pharmacokinetics of ERL and needs to be investigated by in vivo studies. Further, QUR is highly metabolized in the human body, necessitating studying the effect of the QUR on co-administered drugs in future studies. Hence, the information gained from such studies can benefit from dose optimization where the two drugs are intended to be co-administered.


Fram Strait is the major gateway connecting the Arctic Ocean and North Atlantic Ocean, where nearly 90% of the sea ice export from the Arctic Ocean takes place. The exported sea ice is a large source of freshwater to the Nordic Seas and Subpolar North Atlantic, thereby preconditioning European climate and deep water formation in the North Atlantic Ocean. Here we show that in 2018, the ice export through Fram Strait showed an unprecedented decline since the early 1990s. The 2018 ice export was reduced to less than 40% relative to that between 2000 and 2017. The minimum export is attributed to regional sea ice-ocean processes driven by an anomalous atmospheric circulation over the Atlantic sector of the Arctic. The result indicates that a drastic change of the Arctic sea ice outflow and its environmental consequences happen not only through Arctic-wide ice thinning, but also by regional scale atmospheric anomalies. Fram Strait is the major gateway connecting the Arctic Ocean and North Atlantic Ocean, where nearly 90% of the sea ice export from the Arctic Ocean takes place. Here, the authors show that in 2018, ice export showed an unprecedented decline since at least the 1990s, attributed to ongoing Arctic-wide ice thinning and regional-scale atmospheric anomalies. Since the Arctic Ocean gathers 11% of the global river discharge into 3% area of the global ocean3, sea ice export, together with low salinity seawater carried by ocean currents, returns excess of the freshwater inputs toward lower latitudes4. The freshwater export mainly occurs in the Atlantic sector of the Arctic, thereby setting up surface stratification of the neighboring Nordic Seas and further downstream. The freshwater delivered to the Nordic Seas, either in liquid form or as sea ice, preconditions the physical environment for marine ecosystems6, European weather, and climate8, and modulates the rate of deep water formation in the downstream North Atlantic Ocean with implications for climate on a global scale11.Sea ice export through the Fram Strait plays a central role in the freshwater cycle connecting the Arctic and subarctic regions in the Northern Hemisphere13. In the 20th century, the amount of ice export was equivalent to the liquid freshwater transport in the East Greenland Current (EGC)13. The sea ice export, however, has shown a reduction in the last decades, mainly attributed to a reduction of mean ice thickness in Fram Strait over the last two decades at a rate of ~15% per decade14. Climate simulations point to a continued decline in the coming decades with direct consequences for the hydrological cycle in the Arctic and Subarctic regions16.Sea ice export through Fram Strait accounts for ~90% of the total sea ice outflow from the Arctic18  gradient between Greenland and the Barents Sea25. A perennial difference of surface temperature between northern Greenland and the Barents Sea maintains this pressure gradient, setting up northerly winds and resulting in southward sea ice motion through Fram Strait year-round, though most prominent in winter26.Sea ice in Fram Strait is originally formed upstream in the Arctic Ocean. The major part of this ice originates from the Siberian shelves  and the southern perimeter of the perennial sea ice cover in the central ArcticHere we report a strong decline in sea ice export through Fram Strait in 2018, unprecedented since observations started in the 1990s, and evidenced by ice thickness measurements from Upward Looking Sonar (ULS) combined with satellite remote sensing observations. The cause of this decline is found through an analysis combining backward trajectories of ice floes, remote sensing products, and atmospheric reanalysis data.28, whereas ice thickness estimates from satellites are still limited to the freezing season due to properties of the ice surface in the melting season29. Upward Looking Sonar (ULS) installed underneath sea ice, however, can provide accurate, near-continuous measurements of ice thickness covering all seasons26. The Fram Strait Arctic Outflow Observatory (AOO) maintained by the Norwegian Polar Institute has been monitoring ice thickness and ocean properties in the EGC at about 79°N in the western Fram Strait since the early 1990s. An array of four ocean moorings at ~3.25°W, 4°W, 5°W, and 6.5°W has been equipped with ULSs covering the main tongue of sea ice in the EGC. The observatory has documented long-term estimates, variability, and changes of the freshwater, and sea ice export from the Arctic Ocean to the Nordic Seas30.Sea ice volume export is calculated from ice thickness, an areal fraction of sea ice (sea ice concentration), and ice drift speed. Passive microwave satellite observations have provided the fraction of sea ice and ice drift speed covering the entire Arctic and Subarctic for all seasons since 1979In 2018, the ULS array recorded an unprecedented decline of ice draft  since the comprehensive measurements started in 1990. A sudden drop of ice thickness was recorded by all four ULSs starting in August 2017 and lasting until the end of the record in August 2018 . Figure Associated with the 2018 trajectories, we find anomalously warm atmospheric conditions during the preceding winter months (Oct. 2017–Feb. 2018 or preceding months −8 to −3) at the locations where the ice pack were then, before arriving to Fram Strait Fig. . The war33. The trajectory analysis shows that the ice floes were thinning rapidly between 82°N (~300 km north of Fram Strait) and the mooring array at ~79°N  and 1.7 cm day−1 in early spring (March–April)  Table , which i018 Fig. –S6.36. The spatial pattern of these dipole anomalies is characterized by a high-pressure center in the Barents Sea and a low-pressure center over the Canadian Archipelago and Greenland.The longer residence time of the ice floes in the northern vicinity of Fram Strait was a consequence of slow and stagnant ice motion from autumn 2017 to summer 2018. The stagnation was caused by a repeated reversal of the SLP gradient across the strait and associated changes in prevailing wind direction during this period Fig. . An east37. The Arctic dipole anomaly was reported to influence the sea ice export through Fram Strait in winter38. The SLP anomalies that occurred in the 2017–2018 period had a more direct impact on the ice export compared to the Arctic dipole anomaly pattern, since they were situated such that Fram Strait was sandwiched between the anomaly centers. The reversals of zonal SLP gradient across Fram Strait led to concurrent southerly winds that reduced, and even halted, southward sea ice motion in Fram Strait  mode of daily SLP in the Atlantic sector ). Though the EOF mode was derived from daily SLP analysis, the 2018 dipolar anomalies emerged repeatedly on a monthly time scale with a slightly different spatial pattern  to quantify the potential effects of significantly reduced sea ice export.The difference of the fate of sea ice export versus oceanic transport results in different consequences depending on the phase of the freshwater passing through the Fram Strait. A significant portion of sea ice passing through the Fram Strait can drift into the Nordic Seas48. Another study pointed out that the anomalous ocean stratification in 2018 affected local biogeochemical cycles north of the Fram Strait49. The anomalous ice melt reported here can have contributed to the reduction of sea ice cover in this area, while the southerly winds can have advected the meltwater eastward by Ekman transport and contributed to the observed fresher surface layer and shallower warm AW layer observed in the western Eurasian basin in winter 201721. The anomalous thickness reduction, together with the observed hydrographic anomalies in the northeast of Fram Strait, might be a leading signal of further retreat of marginal ice zones and concurrent northward shift of water mass modification area in the Atlantic sector of the Arctic in the coming years to decades. Sustained monitoring efforts of both sea ice and upper ocean properties in the Atlantic sector of the Arctic Ocean are necessary to clarify both short-term and long-term impacts of the 2018 event for the Arctic Ocean itself.It is also important to note the consequences of the anomalous thickness reduction and the southerly wind events on the local hydrography, water mass modification, and biological processes north of the Fram Strait. A recent study reported that anomalous reduction of sea ice concentration and extreme deep mixed layers occurred northeast of Fram Strait in the 2017/2018 winter51 might result in an increase of ocean heat content available for melting sea ice, a decline of basin-wide ice thickness52 making ice floes more vulnerable to environmental changes, an increase of mobility of sea ice in a number of regions53 making sea ice more sensitive to local wind forcing; all these might have preconditioned the 2018 ice export decline. Climate model simulations suggest that the freshwater cycle of the Arctic may shift into a completely different regime in the next decades, with a consequence of reduced ice export and increased liquid fraction of freshwater transport16. Hence, a repeated appearance and the cause of the dipole SLP anomaly like in 2018, and the likelihood of recurrences in the future also need further investigation in this context. A relation between the most prominent anomaly in February 2018 and sudden stratospheric warming has been pointed out35, whereas the cause of the repeated occurrence of similar patterns in 2017–2018, its relation to dominant atmospheric patterns such as North Atlantic Oscillation or North Atlantic blocking activity, and potential occurrence in a changing climate is still unclear. Interactions and feedbacks between these components in the freshwater cycle and resulting impacts on the ocean ecosystem and European climate are ongoing challenges.The effect of a preconditioned environment and potential recurrences of such southerly wind events in a changing climate also need to be considered. More heat transport into the Arctic Ocean14. After 2014, four IPSs were continuously operated except for two data gaps (missing F13 in 2014 and F12 in 2015). Although the zonal locations of the moorings have slightly changed from year to year, the changes are taken into account in the zonal interpolation procedure of the ice volume transport calculation.Ice thickness data were obtained from Upward Looking Sonars (ULSs) and Ice Profiling Sonars (IPSs) moored in the East Greenland Current in Fram Strait. Four ULS/IPSs were zonally aligned at 79°N from 1990 to 2001 and at 78.8°N from 2002 to 2018 with two temporal gaps in 1996 and 2008 and with several spatial gaps in between. Zonal positions (names) of the moorings are 3°W (F11), 4°W (F12), 5°W (F13), and 6.5–7.0°W (F14), respectively Fig. . The moo54. The raw data were processed to ice draft data by ASL Environmental Science Inc., following standard procedures55 including screening of erroneous records  and sound speed corrections56. The ice draft is converted to ice thickness by an average ratio of thickness to draft in Fram Strait, 1.136, derived from on-site drilling measurements57. Ice draft measurement interval varies with the instruments used and their respective settings. From 1990 to 2005, ULSs (ES300) were used to obtain the draft data, measurement intervals of which vary from 60 to 600 seconds, though in most cases an interval of 240 seconds was employed. After 2006, IPSs (IPS4 and IPS5) were used to measure the ice draft. The measurement interval of IPS4 and IPS5 was set to two seconds, though an interval of one second was applied in some IPSs from 2014 to 2016.The ULS and IPS measure the underwater fraction of sea ice (sea ice draft) by recording travel times of sound reflected at the bottom of floating sea iceDaily mean ice draft and thickness are calculated by averaging the original data except for the open water fraction. Hence the daily mean thickness exhibits a spiky time series when the area is largely occupied by open water Fig. . Tempora59. To take into account the uncertainty of the thickness estimates from remote sensing, we apply three different thickness products for the trajectory analysis independently and use the mean and standard deviation of the results of the three applications as a measure of thickness estimates along the trajectories. The used products are, the weekly Arctic sea ice thickness derived from CryoSat and SMOS using an optimal interpolation scheme (hereafter AWI CS2smos product)29, Arctic sea ice and physical oceanography derived from CryoSat-2 Baseline-C Level 1b waveform observations (hereafter Univ. Bristol thickness product)60, and CryoSat Operational Polar Monitoring Product (hereafter NERC-CPOM thickness product)61. These three products provide ice thickness estimates based on CryoSat-2 radar altimeter measurements62 combined with different thickness retrieval algorithms59. Two of the products (AWI CS2smos and Univ. Bristol) in addition merged ice thickness estimates from Soil Moisture and Ocean Salinity satellite (SMOS) to exploit its advantage in thin sea ice area63. The time period of the products used in this study is from 2010 to 2018. Ice thickness estimates by Envisat64 are also used for validation of interannual variation of the ULS/IPS time series in the period 2002–2010 for winter months  is used for both the volume flux and the trajectory calculation. The product serves the longest temporal coverage (1979–2018) among available drift products by a combination of ice motion estimates derived from various sensors and tracks of ice tethered buoys, which suits both the volume flux and trajectory calculation for a long-range , and the Polar Pathfinder Daily 25 km EASE-Grid Sea Ice Motion Vectors, version 268 (hereafter NSIDCv2 ice drift). The three products are combined to take their full advantage: OSI-405 is employed if data are available, KIMURA is used for deriving ice drift for summer months, and NSIDCv2 ice drift is used to fill temporal gaps of the two products. Due to the combination of the products in different quality, the merged drift has artificial temporal variations in basin-averaged drift speed69. Hence we carefully apply the merged ice drift for partial validation of NSIDCv4 ice drift time series after 2002 . The products contain robust uncertainty estimates as well as sea ice concentration estimates. The sea ice concentration and its uncertainty are used to calculate sea ice volume flux at the Fram Strait section and to provide a termination condition of the backward trajectory calculation.Sea ice concentration is taken from the Global Sea Ice Concentration Climate Data Records of the EUMETSAT OSI SAF70 (ERA5). Daily and monthly mean 2 m air temperature, sea-level pressure, and 10 m wind are used in the analyses.Atmospheric data are taken from European Center for Medium-Range Weather Forecasts Reanalysis v5Salinity data in the western part of Fram Strait in April–May are obtained from shipboard CTD (conductivity temperature depth) measurements during the following cruises: Arctic Ocean 2002 (Oden), Fram2005 , iAOOS 2007 , iAOOS 2008 , and JR17005 .T, A, and V are sea ice thickness, ice concentration, and ice drift velocity perpendicular to the section at location x, respectively. Sea ice concentration and ice drift speed at each segment are linearly interpolated from monthly mean values at the nearest data points. Monthly mean sea ice concentration is calculated from the daily sea ice concentration of OSI-409 and OSI-430, while monthly mean sea ice drift speed is calculated from daily values of NSIDCv4. Sea ice thickness at each segment is calculated from a linear interpolation of thickness between nearest mooring sites if the segment is located in the range of the mooring array (6.5° W < x < 3.0° W). The validity of the interpolation is supported by high correlations of monthly mean values of ice thickness (0.64–0.94) and effective ice thickness (0.81–0.95) between the mooring sites , an extrapolation is applied. The extrapolated thickness is calculated from ice thickness at the outermost mooring site and a mean zonal gradient of ice thickness in the Fram Strait section. Monthly mean values of the zonal  gradient, α are estimated from the ice thickness gradient between the four mooring sites for all observations between 2010 and 2018  zonal segments for the sea ice volume transport calculation. The volume transport at a segment is given bytes Fig. . If the 018 Fig. .F, is given by a zonal integration of (1),The total sea ice volume transport in Fram Strait, xW, is set to 13° W, where sea ice does not move throughout year17, while the eastern edge xE varies in time depending on sea ice extent. The time series of the monthly mean sea ice volume transport estimate is shown in Fig. The western edge of the integration, f, is calculated by the following formula,T, ΔA, and ΔV, are uncertainty estimates of monthly mean values of ice thickness, ice concentration, and ice drift speed, respectively. The formula assumes that ΔT, ΔA, and ΔV are small compared to T, A, and V, respectively, and they are independent variables. Equation . The uncertainty of sea ice drift speed is given by an empirical error function formula72, which takes into account an increase of uncertainty in higher drift speed and lower sea ice concentration. The uncertainty of the monthly mean ice thickness ΔT is estimated based on the accuracies of respective instruments54, with an assumption of no systematic bias and negligible influence of snow depth variation  introduces an additional uncertainty associated with the uncertainty of the zonal gradient of thickness,xF14 and xF11 are locations of the westernmost and the easternmost mooring site F14 and F11, respectively. The standard error of the zonal gradient, Δα, is given by the standard deviation of the zonal gradient between the mooring sites in the 2010–2018 period , while ice concentration (drift speed) contributes to 13–24 (13–26) % of the total uncertainty. Uncertainties of respective monthly mean volume flux are shown in Fig. Backward trajectories of sea ice floes are calculated by the daily sea ice motion vectors from NSIDCv4 Fig. . Pseudo 73. We used 83 buoy tracks that arrived in Fram Strait in the period from 2000 to 2018 and calculated corresponding pseudo buoy tracks backward in time. The comparisons show that the backward trajectory calculation has ~100 km (230 km) error after 3 months (1 year) calculation back in time and the error can be reasonably approximated by a linear function of backtracking days ays Fig. . The errCS2/CS2smos ice thickness products from three different institutes  are independently applied to estimate temporal evolutions of ice thickness along the trajectories. Mean positions of the ice floes in each month are calculated by averaging positions of surviving floes on the 15th of a month, and the Gaussian kernel is applied to estimate ice thickness for the position. The temporal evolutions of ice thickness are obtained at monthly interval except for the melting season (May–September) Fig. .A salinity section in the western part of Fram Strait is analyzed to examine the effect of the ice thickness reduction on the liquid freshwater content in Fram Strait. The salinity anomaly in the EGC in May 2018 Fig. S is calcuSupplementary InformationPeer Review File


In vitro-in vivo extrapolation (IVIVE) showed that the threshold concentration of 2,4,6-TBP to induce inhibition of SULT1A3 was 1.628 µM. In silico docking, the method utilized indicated that more hydrogen bonds formation contributed to the stronger inhibition of 3,5-DBP than 3-BP. In conclusion, our study gave the full description of the inhibition of BPs towards four SULT isoforms, which may provide a new perspective on the toxicity mechanism of BPs and further explain the interference of BPs on endocrine hormone metabolism.Bromophenols (BPs), known as an important environmental contaminant, can cause endocrine disruption and other chronic toxicity. The study aimed to investigate the potential inhibitory capability of BPs on four human sulfotransferase isoforms  and interpret how to interfere with endocrine hormone metabolism. P-nitrophenol(PNP) was utilized as a nonselective probe substrate, and recombinant SULT isoforms were utilized as the enzyme resources. PNP and its metabolite PNP-sulfate were analyzed using a UPLC-UV detecting system. SULT1A1 and SULT1B1 were demonstrated to be the most vulnerable SULT isoforms towards BPs’ inhibition. To determine the inhibition kinetics, 2,4,6-TBP and SULT1A3 were selected as the representative BPs and SULT isoform respectively. The competitive inhibition of 2,4,6-TBP on SULT1A3. The fitting equation was y=90.065x+1466.7, and the inhibition kinetic parameter (K Bromophenols (BPs), a group of brominated compounds, could come from both synthetic and natural sources. With the development of science and technology, BPs are widely used in herbicides, pesticides, wood protectants, and flame retardants, thus leading to frequent contact with such chemical products in daily life , and detvia interfering with the function of the Ca2+ channel in neuroendocrine cells, or it can inhibit the activity of thyroid hormone SULT in human hepatocytes  substance, has significant thyroid hormone activity and estrogen effect. 2,4,6-TBP can disrupt the endocrine system  is regarded as a more vital role in protecting against xenobiotics and in regulating hormone functions during the development of fetal, neonatal, and infant –7. In moin vitro enzymatic reactions, which can further interpret environmental pollutants to interfere with endocrine hormone metabolism.Several xenobiotics, e.g. polychlorinated biphenyls, halogenated phenol, and dietary polyphenol, have been considered as typical environmental endocrine disruptors, which exist in persistent organic pollutants. However, there is currently no systematic and comprehensive study of BPs on endocrine hormone metabolism. In this study, the inhibitory properties of BPs on SULT were investigated through 2 were from Sigma-Aldrich , and so is PAPS with a purity of 60%. Acetonitrile was purchased from Tianjin Saifurui Technology Ltd. ultra-pure water was prepared by Millipore Elix 5 UV and Milli-Q Gradient Ultra-Pure Water System. The other reagents were high-performance liquid chromatography (HPLC) grade or the highest grade commercially available.Eight BPs  were purchased from J&K Chemical Ltd. , with purity over 98%. Recombinant human SULT isoforms were obtained from BD Gentest Corp. . P-nitrophenol (PNP) and its sulfate PNP-S, Tris-HCl, and MgCl2, 40 μM of PAPS, SULTs, and PNP. The concentrations of SULT1A1, SULT1A3, SULT1B1 and SULT1E1 were 10 μg/mL, 10 μg/mL, 10 μg/mL and 20 μg/mL, respectively. After 3-min pre-incubation, PAPS was added to initiate the metabolic reaction. The reaction temperature was set to be 37°C, and the reaction time was 30-60 min. 100 μL of ice-cold acetonitrile was added to terminate the reaction. The incubation mixture without BPs was used as a control. After centrifugation at 12,000 rpm, 10 μL of supernatants were analyzed using ultra-performance liquid chromatography (UPLC)-UV instrument. UPLC separation was achieved using a C18 column  at a flow rate of 0.4 mL/min, and the column temperature was 25°C. The mobile phases contained phase A (H2O containing 0.5% (v/v) formic acid) and phase B (acetonitrile). Gradient condition was used as followed: initiated at 5% B, increased to 95% over 2 minute after 6 minute, held constant for 6 minutes. The UV detection wavelength was 280 nm. In order to determine the Michaelis-constant (Km) of PNP metabolites, incubation mixture  contained BPs (100 μM), Tris-HCl buffer , MgCl2 (5 mM), PAPS (4 mM), SULTs and PNP. The corresponding PNP concentrations for SULT1A1, SULT1A3, SULT1B1 and SULT1E1 were 1-400 μM, 0.1-7 mM, 0.1-1 mM and 0.1-1.2 mM, respectively. Incubation and analysis conditions were conducted as described above. The kinetic parameters Vmax and Km were determined by fitting data to the Michaelis–Menten equation or substrate inhibition equation.The incubation mixture  with a t2, 40 μM of PAPS, SULTs, and PNP. The concentrations of SULT1A1, SULT1A3, SULT1B1 and SULT1E1 are 10 μg/mL, 10 μg/mL, 10 μg/mL and 20 μg/mL. The concentrations of PNP were 40, 1200, 25, 120 μM for SULT1A1, SULT1A3, SULT1B1, SULT1E1 . Incubation mixture without BPs was used as negative controls. All incubations were carried out in duplicate.The incubation mixture  contained 100 μM of BPs, 100 mM of Tris-HCl buffer (pH=7.4), 5 mM of MgCl50) was conducted by adding 0 μM to 100 μM BPs. The inhibition kinetics were determined with BPs and PNP covering the Km (for PNP) and IC50 values (for BPs). Lineweaver-Burk (L-B) plot was drawn using 1/reaction velocity (v) versus 1/the concentration of PNP ([PNP]) to determine the inhibition kinetic type. In the second plot, inhibition kinetics (Ki) were calculated by the slopes of the lines in the Lineweaver-Burk plots.Half inhibition concentration (ICIn vitro-in vivo extrapolation (IVIVE) was employed to predict in vivo inhibition possibility for BPs towards SULTs by using the following equation:i/AUC was the predicted ratio of in vivo exposure of xenobiotics or endogenous substances with or without the co-exposure of BPs. [I] was the in vivo exposure concentration of BPs, and the Ki value was in vitro inhibition constant. The AUCi/AUC cutoffs of < 1.25 were considered as an inhibition for a significant in vivo (AUC in vivo .In silico docking was employed to understand the molecular interaction between BPs and SULTs. We constructed the structure of SULT isoforms by homology modeling method with the MODELLER9v14 program. Autodock software (version 4.2) was utilized to dock BPs into the activity cavity of SULT isoforms, respectively. The non-polar hydrogen atoms of SULTs were merged. The grid box was generated with 60 ×60×60 in X, Y, and Z coordinate to cover the entire ligand-binding site. Lamarckian Genetic Algorithm (LGA) method was utilized to study the molecular docking of active domain between BPs and SULTs. The LGA runs were set to 50 runs for each BPs. The best conformation with the lowest docked energy was analyzed for the interactions between BPs and SULTs including hydrogen bonds and hydrophobic contacts.The experimental data were presented as the mean value plus standard deviation (SD). Statistical analysis was carried out using GraphPad Prism 5.0. Comparisons between two groups were performed using a two-tailed unpaired Student’s t-test. Multiple groups were compared using a one-way ANOVA.m, Ki) of PNP catalyzed by SULTs were summarized  were determined for SULT1A3 by 2,4,6-TBP. As shown in i) was calculated to be 16.28 µM.2,4,6-TBP was selected as the representative BPs to determine the inhibition kinetics. As shown in in silico docking showed the influence of the introduction of substituted bromine on the inhibition capability. The binding free energy of 3-BP and 3,5-DBP with SULT1A3 were -5.77 kcal/mol and -6.41kcal/mol, respectively, which is consistent with the experimental results that 3,5-DBP exerted stronger inhibition than 3-BP towards SULT1A3. 3-BP formed one hydrogen bond with SULT1A3 (Results of i). Based on the IVIVE equation, the threshold value of 2,4,6-TBP was 1.628 µM. 2,4,6-TBP was regarded as the most abundant BP in the human body. The concentrations of 2,4,6-TBP ranged from 27 ng/g lipid to 81 ng/g (0.08 - 0.24 µM) lipid in serum  and SULTs, the most important phase II metabolic enzymes, are the main endogenous substances that undergo the metabolism catalyzed. For example, both UGTs and SULTs are involved in the metabolic elimination of thyroid hormones , 19. TheIn silico docking, we get the conclusion that more hydrogen bonds formation contributed to the stronger inhibition effect of 3,5-DBP than 3-BP towards SULT1A3 and SULT1E1. In vitro-in vivo extrapolation (IVIVE) was performed to predict the threshold value of 2,4,6-TBP to induce in vivo inhibition of SULTs. These results may interpret BPs to interfere with endocrine hormone metabolism.This study demonstrated the inhibition of BPs towards SULT1. Our results showed that SULT1A1 and SULT1B1 were demonstrated to be the most vulnerable SULT isoforms towards BPs’ inhibition. Structure-inhibition activity relationship analysis showed that 2-substituted bromophenol exhibited severe broad inhibition potential towards four SULTs, and the addition of 5-position bromide in BPs can increase the inhibition capability. The original contributions presented in the study are included in the article/ZL contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by ZD and FZ. The first draft of the manuscript was written by ZD and YL. JX commented on previous versions of the manuscript. All authors read and approved the final manuscript.This work was supported by the Educational Department of Liaoning Province (No. JYTZD2020003).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.