Phytolacca dodecandra) is presented. The process of saponification, which involves reacting triglycerides with fats and oils in an alkaline solution, produces soap. With the help of WCO and Endod as manufacturing inputs, this study intends to create high-quality, commercially viable eco-friendly soaps. The optimal blend of WCO and Endod with sodium hydroxide solution was used in the current investigation to create laundry soaps. Evaluations were done on the cleansing effects and physico-chemical makeup of prepared soap. As a reference control, the raw oil soaps made without and with frying were employed. The free caustic alkali content, chloride content, moisture content, ethanol-insoluble-matter, total fatty matter, pH, and foam height values of the prepared soap were found to be in the range of 0%, 0%, 16.56–22.52%, 0.1–3.05%, 63.41–75.46%, 9.22–9.82%, and 3.3–8.1 cm respectively. The results obtained by blending fried WCOs and Endod were comparable to the Physico-chemical properties of the Endod-free uncooked/fresh oil soap. The soap made by blending WCO and Endod has higher cleansing power and better lather formation than the prepared soap with WCO without Endod. Moreover, the observed data are comparable with similar data reported in other literature, recommended acceptable standards , and from many countries including the British, Malaysia, and the Philippines. Cooking oils fried at different temperatures do not have much effect on the quality of soap making. This suggested that the blending of WCOs and Endod can be used as raw materials to prepare high-quality and economically feasible soaps by replacing imported oils and fats.A green viewpoint based on the production of soap using waste products such waste cooking oils (WCOs) and Endod ( Now a dPlant-supply oils and lengthy chain animal fat are used nearly in all cleaning soap-making processes ,6. The rIt is vital to observe that once oils are fried at temperatures of 180 °C and above for 5 or greater cycles, it creates a byproduct WCO ,11. EverPhytolacca dodecandra, known locally as Endod in Amharic, Handoodee in Afan-Oromo, and Shibti in Tigrigna, produces a range of saponins that have potent biological properties. These include anti-fungal, anti-bacterial, anti-protozoan, and anti-snails, which cause schistosomiasis, mosquito  control, insecticidal, and spermicidal activities [The African soapberry plant tivities ,28. The tivities . In EthiThus, this study was initiated to use the optimized ratio of WCO and Endod for the production of qualified and cost-effective laundry soap by treating WCO and Endod samples with sodium hydroxide for the first time. Benefiting from the synergistic effect, the prepared soap qualified for the basic quality standards set for grade one laundry soap.22.1Analytical standard chemicals such as carbon tetrachloride , Ethanol , Potassium hydrogen phthalate , potassium hydroxide , sodium hydroxide , hydrochloric acid , phenolphthalein (PubChem), potassium iodide , Nitric acid , Silver nitrate , Acetone , Potassium chromate,  Light petroleum, Sulphuric acid , Sodium hydrogen carbonate , n-Hexane , methyl orange , Chloroform , analytical reagent quality, Glacial acetic acid , and Starch solution were used in this study. In addition, a standard food-grade edible vegetable cooking oil, WCOs, Endod berries powder, Fresh ginger, and double distilled water were also used for the study.2.2Several Materials/Apparatuses were used in this study. The moisture analyzer (Kern DAB 200-2 Weighing range (max.) 200 g, Readability 0.001 g), and pH meter (Seven Excellence pH/Ion meter S500-Std-K) were used during the investigation.2.3To investigate the influence of cooking on the physiochemical properties of pul oil, 10 L of a typical standard food-grade cooking oil was obtained and purposefully burnt at various temperatures over varying time durations. An aliquot of oil (1 L) was removed before to heating and labeled as unheated cooking oil (UHCO). The remaining 9 L oil is then poured into a stainless steel frying pan machine with a capacity of 10 L 1 kg of air dried potatoes were fed to the panning machine for each of the six frying sessions . A 1 L sIn order to remove solid inorganic particles from the collected WCOs, pretreatment procedures from the literature were utilized . Fresh gEndod berries were collected from the trunk of the plant in the Jimma area to be used as filler and the active ingredient in the preparation . Impurit2.4The two crucial parameters, Saponification Values (SV) and acid values (AV) were taken into consideration in this study to evaluate the qualities of the collected WCOs for recycling for soap making. To determine each parameter, the standard titration methods by the American Oil Chemists’ Society (AOCS)  and ISO 2.4.1For the determination of the SV, 2 g of the oil sample was taken into a clean dried 250 mL of a conical flask. Then, 25 mL of 0.5 M KOH solution and some boiling chips were added and a reflux condenser was connected to the round-bottomed flasks. The flask was heated for an hour with frequent shaking. Later, 1 mL of 1% phenolphthalein indicator was added and the hot excess alkali was titrated with 0.5 mol/L of standardized hydrochloric acid solution until the pink color disappeared. At the same time and under the same circumstances, a blank titration had been conducted. Finally, the saponification value was calculated from the recorded data during titration using 0 is the volume in mL of the standardized HCl solution used for the blank test (22.5 mL), V1 is the volume in mL of the standardized HCl solution used for determination, C is the exact concentration of the standardized KOH solution used (0.5 N); m is the mass in g of the oil taken.V2.4.2To determine the AV, 10 g of oil sample were taken and added into the flask, 50 mL of ethanol was added into the second flask, and both were heated in a water bath at the same time at a temperature of 70 °C for 10 min. Next, 0.5 mL phenolphthalein was added to the 50 mL of heated ethanol. A neutralized ethanol was added to the test portion in the first flask, and it was mixed thoroughly. Then, a titration was made using 0.1 N of KOH solution until the definite pink color was formed, and the volume of the titrant was recorded at the endpoint.The AV  and acidN is the exact concentration of standard KOH solution used in mole per litre, V is the volumetric standard KOH solution used in mL; m is the mass in g of the test portion, and M is the molar mass of the KOH (56.1 g/mol).2.5During preparation, keeping the balance between oil and caustic/lye is necessary to maintain the quality of the prepared soap. If it is excessively oily, it is too soft and melts quickly, it will dry slowly and won’t froth well. An overly caustic substance will be unpleasant, dry, or burn your skin. No lye will be remaining in the soap bar if it is prepared properly. First, the oil’s SV was calculated based on Equation 2.6Literature indicated that the physicochemical characteristic of soap depends on several factors such physicochemical characteristics including FCA, Cl, pH, MC, EIM, TFM, and cleaning Power and lather formation ,7,8. The2.6.1For the determination of free caustic alkali, 5 g of soap was weighed followed by pouring 200 mL of ethanol into the flask. The flask was connected to the reflux condenser and boiled gently until the soap had completely dissolved. It was brought to a gentle boil and kept at the boil for 5 min to remove carbon dioxide. After it was cooled to about 70 °C, 4 drops of phenolphthalein indicator were added. Then, it was titrated with the ethanolic solution of hydrochloric acid (0.1 N) until the color is just perceptibly pink. The percentage of free caustic alkali in the soap expressed as sodium hydroxide m is the mass in g of the test portion, V is the volume in mL of the ethanolic hydrochloric acid solution, and T is the normality of the ethanolic hydrochloric acid solution (0.1 N).2.6.23 (1.4 N) until the pH reaches around neutrality. Finally, the resulting solution was titrated against 0.1 M AgNO3 using K2Cr2O7 as an indicator till a brick red color is obtained. The chloride content of the soap, expressed as a percentage by mass of sodium chloride (NaCl), is given by the formula For the determination of chloride content, 5 g of soap samples were dissolved in 50 mL of distilled water and then heated to dissolve the sample. The resulting solution was transferred into a 500 mL volumetric flask, the solution was stirred using a magnetic stirrer and heated at a temperature of 90–95 °C until the solution was completely dissolved. Next, the solution was titrated using HNOm is the mass in g of the test portion (5 g), V is the titrated volume in mL of the silver nitrate solution, and 58.5 is the mass in g of sodium chloride.2.6.3For the determination of the moisture and volatile matter contents, 5 g of samples were precisely weighed into a clean, dried dish. Then, it was heated in a moisture analyzer  at a tem0 is the mass in grams of the dish, m1 is the mass in g of the dish and the test portion before heating, and m2 is the mass in g of the dish and the test portion after heating.m2.6.4Dissolution of the soap in ethanol, filtration, and weighing of the undissolved residue was done. The sample’s test portion, 5 g was put into a conical flask. Next, 200 mL of the ethanol was added to the test portion in the conical flask and connected to the reflux condenser. To avoid as much material sticking to the bottom of the flask as possible, it was heated to a slow boil while being swirled. Then, the filter paper was taken out of the desiccator after 1 h and left in there just long enough for it to cool entirely to room temperature and weighed it.The content of EIM expressed as a percentage by mass is given by 0 is the mass in g of the test portion, and m is the mass in g of the residue .m2.6.5To determine the total fatty matter content of the prepared sample, 5 g of the test samples were added into the beaker. The test portion was dissolved in 100 mL of hot water. The solution was poured into the separating funnels. The beaker was washed with small quantities of water and added the washings to the separating funnel. Then, a few drops of methyl orange solution were added as an indicator, and while violently shaking the separating funnel, a precisely measured known volume of hydrochloric acid solution (1 N) was added until the color of the solution turned pink, indicating that there was an excess of around 5 mL. 100 mL of the light petroleum was then added after the separated funnel’s contents had been cooled to roughly 25 °C. The stopper was inserted and gently inverted the separating funnel whilst maintaining a hold on the stopper. The stop-cock of the separating funnel was opened gradually to release any pressure, then close, gently shaken, and again release the pressure. The shaking was repeated until the aqueous layer had become clear, and then allowed to stand for 5 min. After that, the extracted colorless solution (a mixture of petroleum and oil) was collected in the conical flask and transferred to the pink color solution. The residue was dissolved in 20 mL of the ethanol, a few drops of the phenolphthalein solution was added, and then the ethanolic potassium hydroxide solution (1 N) was titrated to a faint permanent pink color, and the result was recorded. Next to that, the ethanol solution was evaporated into the water bath. When the evaporation was near completion, the flask was rotated to distribute the potassium soap in a thin layer on the sides and bottom of the vessel. The preliminary drying of the potassium soap was Carried out a by adding acetone and evaporating off the acetone on the water bath under a slow stream of cold dry nitrogen or air. Then, it was heated to constant mass in the oven, controlled at 105 ± 2 °C, i.e. until the difference in mass after heating for an additional 15 min does not exceed 3 mg. It was ultimately weighed after cooling in a desiccator.The total fatty matter content is given, as a percentage by mass, by 1 is the mass difference in g between the final flask and initial flask (mass of fat matter), mo is the mass in g of the test portion, V is the volume in mL of the stand and volumetric ethanol potassium hydroxide solution used for the neutralization, T is the exact normality of the standard volumetric ethanolic potassium hydroxide solution.m2.6.6To measure the pH of the aqueous solution, 99 g of distilled water and 1 g of soap sample were weighed out, and the water was heated to 70 °C. Then, soap was added to the distilled water and thoroughly mixed with a magnetic stirrer until the soap was completely dissolved. The pH of the solution was then determined using a pH meter after being chilled in an ice bath at 40 °C [2.6.7The cleaning capacity of the made soap bars was evaluated using a white, extremely dirty cloth that had been dumped on the road and was highly smeared with oil. The capacity of soap to foam allows it to produce lather when used . Its foa33.1In this study, SV and AV were investigated to determine the physicochemical properties of the oils, and the experimental results are shown in .Table 1TDifferent studies demonistrated that repeatedly heating vegetable oil at high temperatures could cause oxidation, which results in colour change, rancid odour, and rancid flavour ,61. WCOsSV reveals the average molecular weight and, consequently, chain length of the fatty acids in the oil or fat . SV is rThe AV, which results from enzymatic activity, indicates the quality of the fatty acids in the oil. Oil with a low AV is long-lasting and resistant to peroxidation and rancidity. The SV of oil is used to assess if it is safe to eat and appropriate to use in the soap industry. Oil samples with higher free fatty acid contents are found to have a higher AV, which donates decreased oil quality . The AV 3.2To determine the effects of over-cooking oil at different temperatures on the quality of the soaps, 6 soaps from frying 1 to 6, and 2 soaps from frying-80 and frying-95 were prepared a–i. The To increase the cleaning action of the prepared soap, the pretreated and crushed Endod berries were mixed frying 6 based on the result from In all soaps, the lye (NaOH) to water ratio  was added. The Physicochemical Properties of the Prepared Soaps were determined as follows.3.33.3.1Free caustic alkali requires the roughness of any soap. The free caustic alkali (FCA) is the amount of alkali-free to counter and avert the soap from becoming oily. The FCA of the prepared soaps were determined and tabulated in 3.3.2The result shows the amount of chloride in all soaps was the same (0.00%) . It is w3.3.3The moisture content (MC) laundry soaps prepared from WCOs obtained from frying 1 up to 6 and collected from a hotel, and a restaurant, and blended with Endod had values 16.41 ± 0.050, 17.17 ± 0.050, 17.54 ± 0.010, 18.15 ± 0.010, 18.51 ± 0.020, 19.15 ± 0.010, 19.37 ± 0.020, 19.70 ± 0.040% respectively. The results clearly showed that the soaps made from blending WCOs and Endod had lower MCs than soap made from non-frying (20.07 ± 0.020%) without Endod. With the use of Endod, oil, lye (NaOH), and distilled water in a consistent ratio, the MCs of the soaps increased when the frying cycle increased a. This iIn this study, the advantage of Endod to control the MC of a soap resulted due to the aforementioned reason. It absorbs excess water generated as a result of overheating making WCO a more suitable raw material for the preparation of a high-grade laundry soap (3.3.4The ethanol-insoluble matter (EIM) values of the prepared laundry soaps were observed to be in the range of 0.104 ± 0.002% to 3.05 ± 0.030%. The result showed that the soap prepared from WCOs and Endod had a greater EIM than soap made from fresh oil (0.104 ± 0.002%) without Endod. This is also another indication that by employing Endod, oil, lye (NaOH), and distilled water in a uniform ratio, the amount of EIM present in the soap increased as the frying cycle or the oil’s temperature increased a. The ef3.3.5The total fatty matter (TFM) content of soap samples involved in this investigation ranged from 63.41 ± 0.010 up to 75.46 ± 0.200%. The TFM values decreased from 75.46 ± 0.200 to 66.46 ± 0.140% as the frying cycle increased a. This iThe effects of Endod in different ratios were determined. In the beginning, the impact of Endod was tested using non-frying oil without Endod or Endod to oil ratio (0:100) as the standard reference and frying 1 oil with Endod ratio Endod to oil (4:100). The results are 74.270 ± 0.200% and 75.460 ± 0.200% respectively a. Simila3.3.6The pH measures the acidity or alkalinity of a substance. The pH results of the prepared soaps ranged from 9.22 ± 0.020 to 9.77 ± 0.010 . This de3.3.7Cleansing power, bubble, hardness, conditioning, creaminess, and lather formation are the principal traits generally used to assess first-class laundry soaps . The detThe comparative physicochemical characterization result of the present work is summarized and given in Table S1. From the table, it can be boldly seen that some of the physicochemical characterization results of the current work are by far better than many reported works in similar areas. The improvement in the present work is attributed to the synergistic effect of the WCO and the Endod powder. The Endod berries power used as the raw material helps to reduce Cl ion content induced by the use of chloride salts alkali metal in the conventional soap preparation. Moreover, the plant contains saponins by nature and improves many of the physicochemical properties of the prepared soap.4Phytolacca dodecandra) for soap preparation has three advantages. First, it minimizes health problems and environmental pollution induced by direct using and discarding WCOs to the environment. Secondly, providing a new, cheap, and effective raw material (Endod plant which grows rapidly and bears fruit twice a year under the right climatic conditions) and is used for soap making instead. Above, all the use of such cheap active material also reduces the number of fillers required in soap production. The third is the blending of WCO and Endod replacing fresh palm oil, animal fats, and alkali metal salts (like NaCl and KCl) in soap making, reducing associated costs to soap production.The effect of blending endod with WCO was carefully investigated. Laundry soaps were prepared by using the proper amount of WCO and Endod powder in the laboratory for the first time. Physicochemical characterizations of the prepared soaps revealed that the products fit into the grade-1/genuine category of laundry bar soaps. Moreover, the result indicated that frying the oil has no significant effect on the quality of oil used as a raw material in soap making. The physicochemical characterization results of the soaps have also shown that all test parameters were within the recommended international standard limits, except for the soap prepared with an Endod to oil ratio of 6:100 (W/W), which exceeded EIM limits set by the EAS, CES (2.5%), and British (2%). Moreover, the result also revealed that a better soap could be prepared by mixing Endod berry powder with WCOs in the appropriate ratio range  of Endod to oil. Soap made by blending WCO and Endod has a comparable quality without Endod and non-fried made soap. Therefore, using WCO and Endod  in the subject matter or materials discussed in this manuscript.


The alterations in the specific IgE levels were analyzed in a subgroup of eleven patients, i.e., five who did not and six who did acquire natural tolerance. Results: The use of MAMA allowed a component-resolved diagnosis of IgE sensitization in each of the children suffering from cow’s-milk-related anaphylaxis according to Sampson grades 1–5 requiring only 20–30 microliters of serum. IgE sensitization to caseins and casein-derived peptides was found in each of the children with Sampson grades of 4–5. Among the grade 1–3 patients, nine patients showed negative reactivity to caseins but showed IgE reactivity to alpha-lactalbumin (n = 7) or beta-lactoglobulin (n = 2). For certain children, an IgE sensitization to cryptic peptide epitopes without detectable allergen-specific IgE was found. Twenty-four children with cow-milk-specific anaphylaxis showed additional IgE sensitizations to BSA, but they were all sensitized to either caseins, alpha-lactalbumin, or beta-lactoglobulin. A total of 17 of the 39 children without anaphylaxis lacked specific IgE reactivity to any of the tested components. The children developing tolerance showed a reduction in allergen and/or peptide-specific IgE levels, whereas those remaining sensitive did not. Conclusions: The use of MAMA allows for the detection, using only a few microliters of serum, of IgE sensitization to multiple cow’s milk allergens and allergen-derived peptides in cow-milk-allergic children with cow-milk-related anaphylaxis.Background: Immunoglobulin-E(IgE)-mediated hypersensitivity to cow’s milk allergens is a frequent cause of severe and life-threatening anaphylactic reactions. Besides case histories and controlled food challenges, the detection of the IgE antibodies specific to cow’s milk allergens is important for the diagnosis of cow-milk-specific IgE sensitization. Cow´s milk allergen molecules provide useful information for the refined detection of cow-milk-specific IgE sensitization. Methods: A micro-array based on ImmunoCAP ISAC technology was developed and designated milk allergen micro-array (MAMA), containing a complete panel of purified natural and recombinant cow’s milk allergens , recombinant BSA fragments, and α-casein-, α-lactalbumin- and β-lactoglobulin-derived synthetic peptides. Sera from 80 children with confirmed symptoms related to cow’s milk intake (without anaphylaxis:  An intolerance to cow’s milk is particularly common. In most cases, it is due to lactose intolerance and can be avoided by the consumption of lactose-free milk products. Immunoglobulin-E-(IgE)-mediated cow’s milk allergy (CMA) is more rare and affects approximately 3% of the population ,2. CMA oTraditionally, the serological detection of CM-allergen-specific IgE antibodies is performed with the use of CM allergen extracts, of which some are able to help quantify CM-allergen-specific IgE levels . InteresIn this study, we present a novel milk allergen micro-array (MAMA) that allows one to measure, with only a few microliters of serum, the IgE levels of to all known CM allergens and certain CM-allergen-derived peptides. In addition, MAMA was also used for the high-resolution mapping of allergen- and peptide-specific IgE reactivity profiles in a cohort of clinically well-defined CM-allergic children. Our study not only provides a multiplex assay that allows a hitherto unmatched detailed survey of CM allergen- and peptide-specific IgE reactivity profiles, but it also appears to be useful for the reliable detection of CM-allergen-specific IgE reactivity in patients with anaphylactic reactions to CM, as well as for monitoring the development of patient tolerance following an elimination diet.Children suffering from CMA  were obsThe CM-related allergic symptoms of patients were ranked as non-anaphylactic and anaphylactic. Anaphylactic reactions were graded as grades 1–5 according to Sampson . The majA subset of 11 children  were subEpisodes of anaphylaxis due to accidental intake of cow’s milk were recorded by the treating physicians.The development of clinical tolerance in the form of baked, fermented, or whole milk when re-introduced into the diet under controlled conditions was monitored also by the treating physicians. No controlled challenges were performed in the clinics.Written informed consent was obtained from the parents or legal guardians of the children in order to obtain blood samples and immunological analysis . The analysis of the pseudonymized samples with MAMA was conducted with approval from the Ethics Committee of the Medical University of Vienna, Austria (EK1641/2014).Allergen-specific IgE antibodies were measured in certain serum samples with ImmunoCAP ISAC micro-arrays containing 112 allergens, and this was conducted according to the manufacturer’s recommendations  with permission from the Ethics Committee of the Medical University of Vienna (EK1641/2014).2HPO4, pH = 8.4) with a SciFlexArrayer S12 .The natural and recombinant milk allergens, recombinant BSA fragments and milk-allergen-derived synthetic peptides are summarized and characterized in IgE reactivity and IgE levels specific for micro-arrayed proteins and peptides were measured as follows: The microarrays were washed for 5 min with phosphate-buffered saline containing 0.5% Tween 20 (PBST) and were dried via centrifugation with a Sigma 2–7 centrifuge and MTP-11113 rotor . Subsequently, 35 µL of undiluted serum sample was added per array and incubated for 2 h at 22 °C. After another washing step, the bound IgE antibodies were detected as described in . The sliImage analysis was performed via MAPIX microarray image acquisition and analysis software version 8.5.0 , and through a conversion of measured fluorescence units to ISAC standardized units (ISU), which was performed as described in .Given the small number of patients in the studied groups, the median and the interquartile range  were used in the description of the variable distributions. The pairwise comparison of groups was performed with a Mann–Whitney U test. Considering the problem of multiple hypothesis testing, the significance threshold was Holm-corrected to 0.000385 .The graphical representation included box and whisker diagrams that demonstrated the median , the first and third quartiles (upper and lower bounds of the “box”) and the minimum and maximum (“whiskers”). Additionally, the distribution of allergen values within the ranges of [0.3; 1), [1; 15) and ≥15 was illustrated using different color codes.The analysis was performed using IBM SPSS Statistics 20.0 .p values of <0.05 were considered as significant.The graphical representation was performed using GraphPad Prism 6 software , RStudio software 2022.11.4-20 and Microsoft Office Excel 2010. Correlations of allergen-specific IgE levels determined by MAMA and ImmunoCAP ISAC were assessed by Spearman´s rank correlation coefficient. In this study, we investigated 80 subjects  with allergic symptoms that could be clearly attributed to the consumption of CM . In totan = 11) who were prescribed a CM elimination or modification diet. The duration of the prescribed diet was from 8 to 18 months. Two children suffered from CM-related anaphylaxis at grade 4–5, five from anaphylaxis grade 1–3 and four had mild symptoms  contained six milk allergen micro-arrays (MAMAs), which were surrounded by a frame to avoid an overflow of serum A. The MAn = 41), showed an IgE reactivity to at least one of the CM-allergen components. The heat map indicates already that patients with Sampson grades of 4–5 exhibited higher specific IgE levels than patients with anaphylaxis grades of 1–3 . By con = 7) or beta-lactoglobulin (n = 2). Of note, all seven alpha-lactalbumin-positive patients were negative to caseins and beta-lactoglobulin but showed grade 1–3 reactions. However, BSA was also recognized in casein-negative patients, but these patients were positive to alpha-lactalbumin and/or beta-lactoglobulin  developed by us is unique and different from published and currently available multi-allergen tests for measuring CM-specific IgE antibodies  responses [In our study, we were not able to investigate patients who underwent AIT or OIT with CM allergens and therefore we do not have data regarding the development of allergen-specific IgG. However, the technology used by us for generating the MAMA is based on the well-established printing of allergens or peptides on pre-activated glass slides, which has been shown to also be useful for measuring allergen-specific IgG (IgGesponses . Althougesponses ,37,38.Although we were able to test a relatively large number of sera from CM-allergic children from different centers, it may be considered a limitation of our study that no double-blind, placebo-controlled food challenges had been performed in our patients. However, clinicians were able to provide a meticulous clinical characterization of the milk-allergic children, and this corresponded with the requirements set by international guidelines , includiIn conclusion, we developed MAMA, containing milk allergens and allergen-derived peptides, as a useful research tool for the detection of IgE sensitization to multiple allergen molecules and allergen-derived peptides in CM-allergic patients, particularly for children that requires only small amounts of serum samples.


Furthermore, the nanogenerator shows a power density of 556.72 μW·m−2 with a load resistance of 76.89 MΩ and under 4g acceleration at 15 Hz. The output voltage of the NOP-TENG depicts a stable output performance even after 27,000 operation cycles. This nanogenerator can light eighteen green commercial LEDs and power a digital calculator. The proposed NOP-TENG has a simple structure, easy manufacturing process, stable electric behavior, and cost-effective output performance. This portable nanogenerator may power electronic devices using different vibration energy sources.Triboelectric nanogenerators (TENGs) based on organic materials can harvest green energy to convert it into electrical energy. These nanogenerators could be used for Internet-of-Things (IoT) devices, substituting solid-state chemical batteries that have toxic materials and limited-service time. Herein, we develop a portable triboelectric nanogenerator based on dehydrated nopal powder (NOP-TENG) as novel triboelectric material. In addition, this nanogenerator uses a polyimide film tape adhered to two copper-coated Bakelite plates. The NOP-TENG generates a power density of 2309.98 μW·m Due to the growth of the Internet of Things (IoT), the number of devices will increase by over 200 billion by 2025 . These dThe kinetic energy of the vibrations in the environment can be converted into electrical energy using triboelectric nanogenerators (TENGs) . The worBy considering organic materials, Shaukat et al.  fabricatNopal (opuntia ficus-indica) is a cactus that grows in semi-arid and arid environments in Mexico, South Africa, Latin America, and Mediterranean countries ,38. HereOur nanogenerator used dehydrated nopal powder and polyimide film tape as triboelectric materials. These materials were adhered onto two copper-coated bakelite electrode plates (52.2 mm × 52.2 mm × 1.5 mm). The dehydrated nopal powder was obtained from Natura Bio Foods (NBF)  ,40. ThisThe working mechanism of NOP-TENG uses the vertical contact-separation mode , which iIn the fabrication process of the NOP-TENG, the two bakelite plates have their top surface covered with copper a,d. In aA nopal powder sample a was useFurthermore, p-pV) of 14.56 V. Instead, the second TENG has a maximum p-pV of 9.92 V. Based on these results, the maximum p-pV of the TENG with NOP increases approximately by 46.7% compared to the maximum p-pV of the TENG without NOP. Thus, the dehydrated nopal powder acts as a triboelectric material that can increase the output performance of the proposed TENG. The output performance of both TENGs is shown in To study the triboelectric effect of the dehydrated nopal powder (NOP), the output voltages of a TENG without NOP and another with NOP were measured. For both TENGs, a polyimide film tape adheres to the copper electrode of the top plate. The first TENG included dehydrated nopal powder adhered on the surface of the bottom plate, and the second TENG had no NOP but only glue-coating on the surface of the copper electrode of the bottom plate. This glue-coating was deposited using a spin coating at 1550 RPM for 15 s. The two TENGs were electrically connected in parallel to an oscilloscope a, and ang, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, and 4g) at the frequency of 15 Hz. The maximum peak-to-peak open-circuit voltages  is shown in −2 with a load resistance of 76.89 MΩ. The output performance of the NOP-TENG was measured using the setup of voltages a of the g cycles b. Accord−2 with a load resistance of 76.89 MΩ. The output performance of the NOP-TENG was tested by applying a hand force on its outer surface. Based on this test, the nanogenerator has a power density of 2309.98 μW·mg at 15 Hz. The rectified signal was employed to charge four different capacitors. The NOP-TENG generates an AC signal that can be rectified with a diode bridge and stored in a capacitor. For this test, the output of the NOP-TENG was connected to a commercial diode bridge. In addition, electrolytic capacitors were connected to a diode bridge a. Afterwg at 15 Hz (Finally, we applied the NOP-TENG to power small electronic devices. For this, a 22 μF capacitor was charged for a period of 900 s with an acceleration of 4at 15 Hz c. FigureThe proposed nanogenerator has a portable structure for its easy application by green energy harvesting from different vibration sources. This nanogenerator can be employed on different environment surfaces under random vibrations. For instance, an array of portable nanogenerators can be implemented on different sources of mechanical vibrations such as bridges, buildings, machine tools, and automobiles. In addition, rectifier circuits can be used to convert the AC output signal of the nanogenerators into DC signals. These DC signals can be stored in a capacitors array. Thus, these capacitors can be charged and discharged to power other electronic device types such as pressure, temperature, and humidity sensors. The proposed nanogenerator has a low-cost and simple manufacturing process as well as a stable output performance to power future IoT electronic devices. Moreover, the use of organic or recycled materials is suitable for the circular economy by decreasing waste and environmental pollution. For instance, dehydrated nopal powder can be adhered to some recycled material surfaces to develop cost-effective triboelectric nanogenerators.Due to the friction and impact between both triboelectric layers of the NOP-TENG, the dehydrated nopal powder film suffered wear as shown in The proposed nanogenerator has a biodegradable triboelectric material that suffers wear due to friction. This wear of the dehydrated nopal powder is a weakness of our nanogenerator. To increase the lifetime of the organic triboelectric film, the dehydrated nopal powder must have strong adhesion to the copper electrode, obtaining a better homogenous surface layer. Nevertheless, more investigations on the lifetime of the proposed organic triboelectric material are required by considering the effect of the size and thickness of the dehydrated nopal powder as well as the influence of the environmental humidity.Our nanogenerator needs a rectifier circuit to convert its AC output signal into a DC signal. In addition, this DC signal can be stored by employing capacitors to power electronic devices. An important challenge of triboelectric nanogenerators is their coupling with cost-effective energy storage systems. Energy management strategies of the proposed nanogenerator can be considered by adding electrical interfaces of high efficiency and minimum power consumption ,43. Anot−2 with a load resistance of 76.89 MΩ under 2 g acceleration at 15 Hz. This nanogenerator depicted a cost-effective output performance with the ability to power a digital calculator and light 18 LEDs.This paper reported a novel triboelectric nanogenerator based on dehydrated nopal powder and polyimide film tape to harvest green energy from vibrations. This nanogenerator was developed using a simple portable structure with a low-cost manufacturing process. The output performance of the nanogenerator was measured by considering various accelerations and load resistances. The output voltage of the nanogenerator showed a stable behavior during a period of 27,000 operation cycles. The output power density of the nanogenerator reaches a maximum value of 556.72 μW·m


Six hundred and three patients with cleft-related human phenotype ontology terms were identified within the Deciphering Developmental Disorders study. Genes carrying pathogenic/likely pathogenic variants were identified and reviewed enabling a diagnostic yield of 36.5%. In total, 124 candidate genes for syndromic OC were identified, including 34 new genes that should be considered for inclusion in clinical clefting panels. Functional enrichment and gene expression analyses identified three key processes that were significantly overrepresented in syndromic OC gene lists: embryonic morphogenesis, protein stability and chromatin organization. Comparison with non-syndromic OC gene networks led us to propose that chromatin remodelling specifically contributes to the aetiology of syndromic OC. Disease-driven gene discovery is a valid approach to gene identification and curation of gene panels. Through this approach, we have started to unravel common molecular pathways contributing to syndromic orofacial clefting.Orofacial cleft (OC) is a common congenital anomaly in humans, which has lifelong implications for affected individuals. This disorder can be classified as syndromic or non-syndromic depending on the presence or absence of additional physical or neurodevelopmental abnormalities, respectively. Non-syndromic cleft is often non-familial in nature and has a complex aetiology, whereas syndromic forms tend to be monogenic. Although individual OC-related syndromes have been frequently described in the medical literature, there has not been a comprehensive review  Orofacial cleft (OC) is a common congenital anomaly in humans, which has lifelong implications for affected individuals. The incidence of OC in England, Wales and Northern Ireland is 15 per 10 000 live births . Cleft phttps://panelapp.genomicsengland.co.uk) is a reputable, publicly available database of regularly updated and curated virtual gene panels that is a valuable reference tool for genes associated with syndromic OC  ,7. Accorand DLG1  but thesand DLG1 ,13. Metaand DLG1 . These fCDH1, CTNND1, FGFR1, GRHL3 and SATB2)  or associated phenotypes. Instead, it seems likely that both rare and common variants contribute across all OCs and that some genes contribute across multiple clinical subgroupings (e.g. d SATB2) ,15. The Genetic testing through exome and genome sequencing has become increasingly available in clinical and research settings creating an opportunity to enhance our knowledge of the diagnoses, molecular pathways and networks associated with syndromic OC. The Deciphering Developmental Disorders (DDD) is a national collaborative project in the United Kingdom established to facilitate the translation of genomic sequencing technologies into the National Health Service. It includes over 13 000 patients with congenital anomalies and/or undiagnosed developmental disorders, all of whom have broad clinical phenotypes and available exome data ,17. Pati2 = 15.75(1), P = 7.2 ×10−5] but not compared with the DDD study from which the samples were drawn . The male excess was observed across cases with and without ID.Approximately 5% of the patients with data available through the DDD study, which includes individuals with undiagnosed developmental syndromes (DataFreeze 2017-12-15: 13 612 probands), had the human phenotype ontology (HPO) terms ‘cleft’ and/or ‘bifid uvula’   16). Af. Af16). 2 = 5.915(1), P = 0.015, χ2 = 11.717(1), P = 0.0006, χ2 = 11.032(1), P = 0.0009, respectively] (2 = 7.317(1), P = 0.0068]  , abnormalities of the musculoskeletal system   and abnormalities of the nervous system  . These three systems were more commonly affected in patients with syndromic CPO [χctively] . Abnorma 0.0068] . The sec 0.0068] . The mos 0.0068] .Consanguinity was recorded in 4.7% of OC cases , as compThrough examination of exome sequence and array-CGH data, DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources (DECIPHER) listed 836 ‘plausibly diagnostic’ sequence variants in 413 of the 603 (68.5%) patients On average, each patient had 7.85 non-OC HPO terms  and ‘plausibly diagnostic’ copy number variants (CNVs) were recorded in 45 cases (7.5%), 20 of whom also had ‘plausibly diagnostic’ sequence variants. Each of these variants were assessed according to standard American College of Medical Genetics (ACMG) and Association for Molecular Pathology (AMP) guidelines  identifyde novo. The parental phenotype data did not indicate the presence of a cleft in these cases. Twenty variants were inherited in a heterozygous form , 20 in a biallelic form  and 4 were hemizygous variants. All variants and inheritance are shown in Eighty percent (192/238) of P/LP variants were present in a heterozygous form, consistent with a dominant pattern of inheritance. Twelve variants (5.0%) were identified in a homozygous state and 12 compound heterozygotes were present, consistent with a recessive pattern of inheritance. Inheritance information was available for 86.1% (205/238) of P/LP variants and 72.7% (149/205) of these variants were SATB2 was the most commonly identified gene and harboured potentially pathogenic variants in 2.7% (16/603) of the total patients investigated, all of whom had CPO (KMT2D variants accounted for a further 1.0% (6/603) of patients  and seven further genes  are currently classified as red or amber, thereby increasing the number of green genes in the panel by 34 , which included 34 genes (41%) connected by 66 edges (average node degree of 3.88), in addition to 5 smaller connections with 2–4 nodes (CHD4 and TRRAP (each with 10 edges)  functioned in chromatin remodelling, a process that is essential to cellular differentiation and maintenance and has been implicated across a range of neurological disorders  enrichment, –4 nodes . The mos0 edges)  and S9. isorders ,21. The isorders . Of note−5) and limb bud formation .Seventeen Gene Ontology (GO) terms (3 GO components and 14 GO processes) were overrepresented  < 0.0001 and ≥ 1.2-fold) in the OxSOC green gene list . These tN = 82) was combined with the green genes in the existing PA Clefting panel (N = 142) to generate an updated list of 176 green cleft genes   were not included in the PA Clefting panel as green genes (The OxSOC green gene list (× 10−16) . Eighty-en genes . Again, N = 176) revealed a significant overrepresentation across 54 GO terms (1 GO component and 53 GO processes) with FDR < 0.0001 and ≥ 1.2-fold enrichment  and included chromatid cohesion  and neuronal cell fate . Of these three GO categories, GO:0060173 was primarily associated with CL/P in OxSOC patients . In contrast, genes in GO:0007062 and GO:0048663 categories were found to carry P/LP variants primarily in OxSOC patients with CPO  at a level consistent with presentation in the OxSOC cohort as a whole (77.2% of classified patients CPO).Enrichment analyses of the combined green gene list , 2 of 4 classified patients (50.0%) and 10 of 16 classified patients (62.5%), respectively]. These were at a level that reflected the CPO enrichment in OxSOC as a whole (77.2% of classified patients).ClueGO analysis allowed clustering of related gene functions across the combined green gene list and highlighted 28 functional groups with FDR < 0.5 . The funhttps://bioinfo.uth.edu/CleftGeneDB/). It is generally considered that non-syndromic forms of OC tend to be polygenic. As such, there is no gold-standard or complete gene list. We chose this list because it provides an overview of current literature in relation to non-syndromic genes and is manually curated by developmental biologists. From the combined green gene list, 53% of genes were associated with syndromic CPO and 44% with syndromic CL/P in the OxSOC patients and/or current literature (ARHGAP29) were associated with syndromic OC  and non-syndromic (N = 106) gene lists, and syndromic CL/P (N = 77) and syndromic CPO (N = 93) gene lists in samples from human brain and dental pulp and from mouse mandibular and maxillary arches and palate tissue. Comparisons were also made across the three identified functional clusters .To further investigate the possibility that clinical subgroups map onto distinct functional networks, developmental gene expression patterns were examined between syndromic . Genes related to syndromic CPO showed a higher level of expression than syndromic CL/P  as did the chromatin organization and protein stability gene clusters   .P = 2.07 × 10−4, Trimester 1), whereas non-syndromic genes were expressed at a lower level than other protein-coding genes . The relative increased expression of the syndromic genes appeared to be driven by the CPO subset of genes (min adjusted-P = 1.91 × 10−4) with no contribution from the CL/P genes and specifically, came from the gene networks related to chromatin organization and protein stability (min adjusted-P = 9.66 × 10−6 and 1.05 × 10−4)  . All P-vThis report represents one of the largest genetic studies of syndromic OC to date involving 603 patients (the OxSOC cohort) and, importantly, is one of only a few studies focussing on monogenic causes of syndromic OC across syndromes. Our genomic analyses demonstrate the utility of exome sequencing and array-CGH as a diagnostic tool for these patients and add 34 genes to current diagnostic panels. Network analyses revealed systematic differences in the molecular mechanisms underlying not only syndromic and non-syndromic OC, but also syndromic CPO and syndromic CLP. Together, these findings provide a primary overview of the genetic architecture of syndromic OC allowing a comparison to that described in non-syndromic forms of cleft.This cohort represents syndromic OC alone and hence unsurprisingly there was an excess of patients with CPO (74%) compared with CLP 22%). Contrary to the previously reported male to female ratios of 2:1 for CLP and 1:1.77 for CPO, which includes both syndromic and non-syndromic cleft types, our cohort showed an excess of males for both syndromic CLP and syndromic CPO %. Contra. In addiANKRD11, CHD7, CTCF, CTNND1, EFTUD2, KMT2D, PGAP3, PIEZO2 and SATB2) accounted for approximately one-quarter  of the patients with P/LP SNVs, including 42 of 167 patients with CPO (25.1%) and 8 of 38 patients with CL/P (21.1%)  carried P/LP variants across two or more OxSOC CL/P patients, whereas 37 genes carried P/LP variants across two or more OxSOC CPO patients. In particular, SATB2, PGAP3, KMT2D, EFTUD2 and PIEZO2, all of which were reported in four or more OxSOC CPO cases. The most commonly reported gene across all OxSOC cases was SATB2. P/LP variants (both CNVs and SNVs) in this gene accounted for 2.65% (16/603) of patients investigated and 7.27% (16/220) of patients diagnosed. Disruptions of this gene are recognized to cause cleft (SATB2-associated syndrome (Pathogenic or Likely Pathogenic variants were identified in 220 of the 603 probands (36.5%) investigated in this study, all of whom were selected on the presence of OC and had exome and CNV data available. The diagnostic yield was similar across all types of cleft investigated and in keeping with that reported in the literature ,17. Nine (21.1%) . Six gen 51 of 21, 23.8% osyndrome .MID1 in three patients  of patients had a P/LP CNV demonstrating the utility of CNV analysis for patients with syndromic OC . Two sinpatients  supportisyndrome . Microdesyndrome  supportisyndrome . MAPK3, clefting . It is, Our study identified 34 genes that now meet criteria to be listed as green in the PA Clefting panel, extending the size of the panel by > 20%. PA applies established gene panels, databases and crowdsourcing to compile phenotype- and disease-specific gene panels . In the EFTUD2, PIEZO2, CHD7, TP63 and PTCH1)    ,29 and n, TBX22)  OC, as wnd KAT6B . These ohttp://www.bristol.ac.uk/dental/cleft-collective/about/), would enable a more detailed genotype–phenotype correlation analysis and a study of larger sample sets, for example the 100 000 Genomes Project, are required for the validation of our findings. Together, such studies will allow us to address questions of heterogeneity within a clinically selected population.The current study aimed to specifically investigate syndromic forms of cleft and to explore genetic heterogeneity across cleft types but this retrospective approach has its limitations. Prospective clinical studies, for example the Cleft Collective  or CHD (chromodomain-helicase-DNA binding) classes of ATP-dependent chromatin remodellers , highligWNT , SHH  and this was driven by the genes that function in chromatin remodelling and protein stability . In contIRF6). This likely represents the ascertainment of the DDD cohort in which individuals with a genetic diagnosis fully explaining the phenotype were not enrolled. Our conclusions were also limited by the variable phenotypic information available in DECIPHER and a lack of parental segregation testing in some cases. It is likely that the diagnostic rate for the study cohort would have been higher if these limitations were resolved. We applied network and expression analyses to compare genes that contribute to syndromic and non-syndromic forms of OC but differences in the underlying genetic mechanisms make direct comparisons difficult. Syndromic OC is usually monogenic making it relatively simple by comparison to pinpoint a single causative variant, even if functional studies may be needed to demonstrate causality. In comparison, non-familial non-syndromic OCs are multifactorial meaning that gene lists are derived from linkage and association studies and often have little supporting functional evidence. In this study, we chose to work with a non-syndromic gene list derived from a systematic literature review with a manual curation step (CleftDB). Other lists are available and may have given different results but this reflects the field as a whole—there is no gold-standard gene list for either of the OC types investigated here. However, studies such as the one presented here generate a fuller picture of the genetic aetiology and heterogeneity of OC and allow the assessment of candidate genes within this wider picture; a first step in more comprehensive gene lists for future studies.Our paper adds to the current literature in which studies focussing on the genetic architecture of syndromic OC are limited. We apply standardized and accepted guidelines to define pathogenicity in a representative cohort and compare the genes identified with those currently included in clinical panels and in the literature. Nonetheless, some limitations should be noted. As discussed earlier, our study did not discover variants in some genes that are known to be causative of syndromic OC  arise from disruption of embryonic morphogenesis. In our analyses, the MAP kinase and WNT cascades were particularly highlighted in both syndromic and non-syndromic forms of OC. Interestingly, our exploratory network and gene expression analyses indicate that the point of disruption differs between syndromic and non-syndromic OC. Syndromic CPO in particular is related to genes involved in ATP-dependent chromatin remodelling. These are highly expressed across both brain and palatal tissue and are critical to the establishment and maintenance of lineage-specific open chromatin regions. Non-syndromic OC is more likely to involve genes that control lip/palate-specific gene expression, perhaps by directing transcription factors to existing regions of open chromatin. These findings support the observation that Genome Wide Association Study (GWAS) significant non-syndromic loci are enriched in enhancer regions ,13. ThisFull details of all analyses are available in Supplementary methods, but are presented briefly here.https://hpo.jax.org/app/) terms of ‘cleft’ and ‘bifid uvula’ were identified for this Complementary Analysis Project (CAP#5). All patients have undergone exon-array-CGH and exome sequencing , Clinvar (https://www.ncbi.nlm.nih.gov/clinvar/) or in peer-reviewed medical journals] and CNVs recorded in DECIPHER (https://deciphergenomics.org) (All SNVs within known disease-causing genes  and Fac1E-1JE2) .Supplementary_Materials_and_Methods_R1_ddad023Click here for additional data file.References_for_Supplementary_Tables_ddad023Click here for additional data file.SuppTables_HMG-2022-CE-00520-R1_Wilson_without_ID_ddad023Click here for additional data file.


Homobutein had kinh = (2.8 ± 0.9) × 103 M−1s−1, pairing the relative BDEOH measured by radical equilibration EPR as 78.4 ± 0.2 kcal/mol for BU and estimated as 82.6 kcal/mol for HB. The inhibition of mushroom tyrosinase (mTYR) by HB and BU was also investigated. BU gives a reversible uncompetitive inhibition of monophenolase reaction with KI′ = 9.95 ± 2.69 µM and mixed-type diphenolase inhibition with KI = 3.30 ± 0.75 µM and KI′ = 18.75 ± 5.15 µM, while HB was nearly competitive toward both mono- and diphenolase with respective KI of 2.76 ± 0.70 µM and 2.50 ± 1.56 µM. IC50 values (monophenolase/diphenolase at 1 mM substrate) were 10.88 ± 2.19 µM/15.20 ± 1.25 µM, 14.78 ± 1.05 µM/12.36 ± 2.00 µM, and 33.14 ± 5.03 µM/18.27 ± 3.42 µM, respectively, for BU, HB, and reference kojic acid. Molecular docking studies confirmed the mechanism. Results indicate very potent antioxidant activity for BU and potent anti-tyrosinase activity for both chalcones, which is discussed in relation to bioactivity toward protection from skin disorders and food oxidative spoilage.Butein (BU) and homobutein (HB) are bioactive polyhydroxylated chalcones widespread in dietary plants, whose antioxidant properties require mechanistic definition. They were investigated by inhibited autoxidation kinetic studies of methyl linoleate in Triton™ X-100 micelles at pH 7.4, 37 °C. Butein had  Asteraceae , Asparagaceae , Anacardiaceae , Fabaceae , Pinaceae , Solanaceae , and others  is the initial substrate concentration, while Vmax and Km are, respectively, the maximum reaction rate (at saturating substrate concentration) and the M-M constant, with the substrate concentration yielding half-maximum rate . Results in 50 of butein decreases on increasing the concentration of l-tyrosine for monophenolase inhibition, owing to the uncompetitive mechanism that requires pre-formation of the E-S complex, values variably increase with the concentration of substrate for butein diphenolase inhibition and for homobutein and kojic acid, all showing a mixed-type inhibition with the (variable) prevalence of competitive behavior. This further highlights the limits of IC50 in quantifying inhibitors’ performance.Determination of M-M inhibition constants trations ,35. None50 for butein and homobutein, against both mono- and diphenolase reactions, were sensibly lower, confirming their higher inhibiting potency. Concerning butein, our values are lower but compatible with those of Khatib et al.  = 1 mM as “standard” settings . Resultsb et al.  for monob et al. . While wTo help rationalize the inhibiting mechanism of butein and homobutein, molecular docking simulations were performed against mTYR (PDBID: 2Y9X) to determine binding structures and affinities ,50,51 usl-tyrosine and l-dopa; setting flexibility in the ligand and rigid structure in the protein; and were repeated using AutoDock 4.2, AutoDock GPU, and AutoDock Vina to identify consistent binding poses and the protein residues which are mainly involved in interaction with the ligands. Results guided the setting of the flexible residues in AutoDock Vina, which was used for re-docking all the ligands so as to obtain optimized interaction at a low computational cost [Computations were first performed by individually docking butein, homobutein, and substrates nal cost . This reKI value for homobutein compared to butein measured for diphenolase inhibition , in the absence of other substrates, i.e., without l-tyrosine and l-dopa. Their full UV–Vis spectrum was monitored for 60 min. Results showed a rapid spectral variation attributable to the complexation with the enzyme for butein, followed by a very slow decay of its concentration, which was identical in the presence or absence of the enzyme . The lower BDEOH of catechol butein  compared to homobutein  justifies its higher reactivity. However, it explains the outstanding antioxidant performance in the protection of methyl linoleate micelles only in part. Indeed, butein was able to outperform nature’s premiere antioxidant, α-tocopherol, despite its BDEOH being higher by 1.3 kcal/mol, and, most interestingly, butein was able to trap almost twice as many peroxyl radicals per molecule of antioxidant compared to α-tocopherol. This behavior is reminiscent of the behavior previously found for catechol/quinone antioxidant systems [•) radicals. It is based on the very fast reduction by HOO• of the semiquinone and quinone from the antioxidant to re-generate fresh catechol ready to carry on the inhibition. However, it was observed here without the deliberate addition of a source of hydroperoxyl radicals. We suggest that butein is able to exploit the HOO• radical formed as a side event during the autoxidation of methyl linoleate [2-•/HOO• is produced under both physiologic and pathologic conditions, which lends support to the redox bioactivity reported for butein in previous studies [Both butein and homobutein are effective antioxidants working with a chain-breaking mechanism consisting of the fast trapping of chain-carrying peroxyl radicals , with KI of 3.30 and 2.50 µM, respectively, both outperforming the reference kojic acid. This mechanism, which is supported by molecular docking studies, is investigated for the first time for homobutein and is at variance from previous knowledge for butein. Both chalcones were also active against the l-tyrosine substrate (monophenolase), albeit with a different mechanism, which was uncompetitive for butein (KI′ = 9.95 µM), and it was nearly competitive for homobutein (KI = 2.76 µM). While the mechanism of homobutein is nicely supported by molecular docking, that of butein appears to require further efforts for full rationalization.Along with their antioxidant activity, both butein and homobutein have major efficacy in the inhibition of tyrosinase, showing a prevailing competitive mechanism when Of interest, the combined antioxidant and anti-tyrosinase activities open previously unexplored applications for the two chalcones, particularly in dermatology, to contrast photoaging, melasma, and age-related pigmentation disorders  and in f


In calcaneal fractures, the percutaneous screw fixation (PSF) is currently considered to be the better choice, but it is difficult to accurately place the screw into the sustentaculum tali (ST) during the operation. In this study, the ideal entry point, angle, diameter and length of the screw were calculated by simulating the operation process.We retrospectively collected the calcaneus computed tomography (CT) scans of 180 adults, DICOM-formatted CT-scan images of each patient were imported into Mimics software to establish calcaneus model. Virtual screws were placed on the lateral of the posterior talar articular surface (PTAS), the lateral edge of the anterior process of calcaneus (APC), and the calcaneal tuberosity, respectively, the trajectory and size of the screws were calculated.The mean maximum diameter of the PTAS screw was 42.20 ± 3.71 mm. The vertical distance between the midpoint of the APC optimal screw trajectory and the lowest point of the tarsal sinus was 10.67 ± 1.84 mm, and the distance between the midpoint of the APC optimal screw trajectory and the calcaneocuboid joint was 5 mm ~ 19.81 ± 2.08 mm. The mean maximum lengths of APC screws was 44.69 ± 4.81 mm, and the Angle between the screw and the coronal plane of the calcaneus from proximal to distal was 4.72°±2.15° to 20.52°±3.77°. The optimal point of the maximum diameter of the calcaneal tuberosity screw was located at the lateral border of the achilles tendon endpoint. The mean maximum diameters of calcaneal tuberosity screws was 4.46 ± 0.85 mm, the mean maximum lengths of screws was 65.31 ± 4.76 mm. We found gender-dependent differences for the mean maximum diameter and the maximum length of the three screws.The study provides effective positioning for percutaneous screw fixation of calcaneal fractures. For safer and more efficient screw placement, we suggest individualised preoperative 3D reconstruction simulations. Further biomechanical studies are needed to verify the function of the screw. Calcaneal fractures are the most common traumatic injuries of the tarsal bones and approximately 75% of calcaneal fractures have a displaced intra-articular component . SurgicaThe major challenge of PSF for displaced intra-articular calcaneus fractures is placing screw that will maintain reduction by achieving fixation in the center of the ST without passing through the subtalar joint or the medial calcaneal cortex or affecting important medial structures. We simulated the placement of three screws into the ST on a calcaneal specimen from the lateral edge of PTAS(4 mm), the lateral edge of the anterior process of calcaneus (APC)(4 mm), and the calcaneal tuberosity(6 mm) Fig. . There hThe purpose of the study is to specify the ideal insertion point, the largest secure diameter, length and the accurate angle of the calcaneal screw through the method of 3D simulation.We retrospectively collected the calcaneus CT scans of 180 adults who had undergone continuous slice CT scanning at the imaging research center of our hospital during January 2017 and July 2022, including 80 females and 100 males. Patients were excluded if they had deformity, fracture, arthritis, or tumor in the foot. This study was conducted in accordance with the World Medical Association Declaration of Helsinki and approved by the Ethical Committee of Liaocheng People’s Hospital. Informed consent was obtained from the patient who participated in this study. All methods were carried out in accordance with relevant guidelines. The mean age of the patients on whom the models were based was 45.08 ± 10.32 years (range18–88years).DICOM-formatted CT-scan images of each patient were imported into Mimics software . We removed the soft tissue and other tarsal bones by the function of image segmentation, region growth and multiple slice editing of Mimics software, respectively. A total of 180 virtual calcaneus models were created.1 was 10.67 ± 1.84 mm, L2 was 19.81 ± 2.08 mm, the distance between the midpoint of the APC optimal screw trajectory and the calcaneocuboid joint was 5 mm ~ 19.81 ± 2.08 mm. For the data captured above, the intersex difference was significant (P < 0.05). As shown in Table Then as shown in Table Through the axial perspective, we found that the center point of the largest translucent area was located at the lateral border of the achilles tendon endpoint Fig. B. From TThe safety tracks of PTAS and APC allowed 3.5 and 4 mm screws to be inserted into the ST, and the maximum safe diameter of the calcaneal tuberosity screw is 4 mm.In PSF, ST screw can maintain a firm and stable fixation, but it is difficult for us to accurately place the screw into the ST , 10. LiaIn addition, Gitajn et al. used axial Harris heel views and Geerling et al. used 3D imaging techniques to evaluate the accuracy of PTAS screw placement . HoweverAPC can involve various fracture types, and its fixation affects foot function . Most ofLong et al. provided a reliable and reproducible protocol for PSF treatment of displaced intra-articular calcaneal fractures, but calcaneal tuberosity screws positioning and dimensions were not studied . We founAfter screw placement, multi-directional perspective is required to ensure that the screw is as close to the articular surface as possible without penetrating. If screw penetrates the articular surface, the insertion point and appropriate Angle need to be re-selected. If there is a navigation system, the effect is better, and it is closer to the articular surface to obtain optimal holding force, so as to prevent the fracture fragment from shifting.The parameters of the three screws may provide the surgeon with appropriate information on safe screw placement for PSF treatment of calcaneal fractures, so as to complete the operation more quickly and effectively. The large standard deviation of our results indicates great differences among individuals. As a result, preoperative planning should be implemented in detail for each patient. 3D reconstruction and simulated screw placement techniques with digital software before surgery are valuable.There are still some limitations to this study. We only analyzed the data based on genders, not different age, body size and type of fracture groups. These factors may also affect screw placement and fracture block stability. We only studied the calcaneus of Chinese people, who have different skeletal shapes with American and European populations. In addition, the study was performed on intact calcaneus, but the normal anatomy is distorted in the case of calcaneal fractures. It is necessary to improve the quality of fracture reduction by preoperative 3D reconstruction and intraoperative use of reduction forceps. Thus, the effect of ST screw and other calcaneal screws needs to be verified by more biomechanical research and related clinical research.We provided guidelines for PSF of calcaneal fractures in a 3D simulation. In the 3D model, we positioned and measured the dimensions of the three screws. Data from simulated screws need to be combined with clinical experience for safer placement of screws into the ST. Further biomechanical tests and clinical studies are needed to verify its effects.


Spheroids mimic 3-D tissue niches better than standard cell cultures. Cryopreserving spheroids, however, remains challenging as conventional cryoprotectants do not mitigate all damage mechanisms. Here chemically-programmed extracellular ice nucleation is used to prevent supercooling, alongside proline pre-conditioning, which are found to synergystically improve post-thaw recovery of spheroids. This validates the need to identify compounds and materials to address both biochemical and biophysical damage pathways beyond standard cryoprotectants. Addressing both biochemical and biophysical mechanisms of cryo-damage through proline preconditioning and induced ice nucleation rescues spheroids from cryopreservation damage. Cryopreservation is successful for cells in suspension but 2-D monolayers and 3-D models  are more challenging to store. 3-D Models are more predictive of in vivo fate . In 2022 the US FDA removed some requirements for animal testing prior to human studies, where advanced cellular models are available.6 There is a clear need to develop and discover freezing strategies to protect 3-D tissue models to allow their wider uptake and to improve reproducibility.5Cell culture is a crucial tool to explore biochemical pathways, to discover new drugs and to produce biologics. Continuous cell culture leads to phenotype drift,7 including ice growth8 and delayed onset apoptosis.9 Polyampholytes can protect 2-D and 3-D cellular models,10–13 potentially through membrane stabilisation and cellular dehydration. During cryopreservation, water tends to supercool, meaning nucleation of ice does not occur until ∼−20 °C in microliter droplets14 and −15 °C in multiwell plates.15,16 Induced nucleation within 96 well plates is known to increase recovery of adherent (2D) cell monolayers post-thaw.15,16 Induced nucleation at warmer temperatures reduces intracellular ice formation (IIF) and leads to less ice propagation at cell–cell contacts17 which is particularly relevant for spheroids.Conventional cryopreservation methods use ∼10% DMSO, but there is growing interest in a chemistry-driven approach to discovering compounds and materials which mitigate other mechanisms of cryopreservation-induced damage,18 Minerals such as feldspar19,20 can nucleate but require specialised devices to segregate the insoluble powders from the cells.21 Physical nucleation requires external stimuli, at a precise point in the freezing process, requiring thermal monitoring. Soluble polysaccharides extracted from pollen have emerged as potent ice nucleators22 and we have shown they improved post-thaw recovery of cryopreserved spheroids by inducing nucleation at a pre-determined and reproducible temperature.23There are few useful ice nucleators for cellular cryopreservation. Ice nucleating proteins from bacteria are not isolated pure, only as lyophilized powders.24,25 (Rho-associated kinase) and caspase26,27 inhibitors improve post-thaw yield. l-Proline, as well as being a protective osmolyte, can promote stress tolerance in several species.28,29 Application of proline to cell monolayers pre-conditions them for freezing and can improve cell recovery30,31 although the mechanism and cell scope of this is not understood.In addition to the physical modes of damage/protection, a ‘medicinal chemistry’ approach to cryopreservation, targeting specific deleterious pathways, can mitigate cold damage. ROCKHere we explore the synergy of proline preconditioning  and induced extracellular nucleation  for the cryopreservation of spheroids. Each approach alone gives benefit, but together lead to substantial improvements in cell recovery. There is evidence for reduced reactive oxygen species and actin depolymerization, as a consequence of this approach, highlighting the importance of discovering new cryoprotectants for emerging cellular technologies.10,11,32 Spheroids were prepared using an agar-mould33,34 to ensure homogeneity, seeded with 8000 cells per spheroid and allowed to grow for 5 days before use, A549 adenocarcinomic human alveolar basal epithelial cells, were cultured and assembled into spheroids,.30,35 beyond its impact on osmolarity, which is not seen for other amino acids, although some mitigate ice recrystallisation.36 The spheroids were thawed, and allowed to recover for 24 hours . The recovery rate of the spheroids was then measured by ATP content, Fig. S3B  and replaced with 10% DMSO in cell culture medium and cryopreserved. Proline has a unique cryoprotective mechanism,. S3B ESI. In thistions ESI.37 or mechanical shock.38 Soluble ice nucleating macromolecules were extracted from Carpinus betulus pollen, following reported procedures.16,23 These extracellular macromolecules increase the nucleation temperature of water from −15 °C to as high as −6 °C in microwell plates.16 Fig. S8  and osmolytes . To probe the impact of these strategies on spheroid function A549 and HepG2 spheroids were investigated using confocal microscopy. Live (green)/dead (red) staining was used showing that for both A549 , which might lead to more inter cellular ice propagation or slow DMSO removal post-thaw, which will be investigated in the future.To increase recovery, spheroids were transferred from moulds to u-bottom 96-well plates, which have been used for spheroid freezing with induced nucleation.oth A549  and HepGoth A549  the propining ESI showed rbicin ESI confirmeIn conclusion, we have demonstrated the synergy between mitigating both biochemical and biophysical modes of damage during spheroid cryopreservation. Initial screening of spheroids in agar moulds confirmed that 24 hours pre-incubation with proline (which was removed prior to freezing) increased post-thaw recovery levels of A549 spheroids. Addition of soluble ice nucleators (from pollen) to prevent super-cooling and reduce intracellular ice formation, also increased recovery. Combining these two strategies in microwell-plate based freezing enabled an overall increase in spheroid recovery from 40 to 70%, which was shown to be associated with reduced reactive oxygen species and increased F-actin polymerization. HepG2 spheroids cryopreserved by this strategy showed improved function in a toxicology assay. Initial observations that spheroid density impairs function were also made. This work shows that the chemistry-led discovery and application of specific biochemical inhibitors and extracellular nucleators can advance the banking and deployment of 3D models for basic and applied research.MG, YG and TW devised the experiments. YG and AB undertook the cell culture experimental work. NLHK performed ice nucleation experiments. MG and QG supervised the project. All authors contributed to data analysis and manuscript writing.This project has received funding from the European Research Council under the European Union's Horizon 2020 research and innovation program grant agreements 866056 and 899872. MIG thanks the Royal Society for an Industry Fellowship (191037) with Cytiva. AB thanks BBSRC/UoW for a Flexible Talent Mobility Award (BB/W510907/1). NLHK thanks NERC (NE/S007350/1) and YG thanks SUSTECH/UoW for PhD scholarships. TFW thanks the Leverhulme Trust/UoW for a Fellowship (ECF-2018-127). Q. G. thanks the National Natural Science Foundation of China (81971764) and the Innovation Committee of Shenzhen Municipality (JCYJ 20200109141638004). For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) license to any author-accepted manuscript version arising from this submission.TFW, NLHK and MIG are named inventors of a patent application relating to this work.CC-059-D3CC02252H-s001


Physical catalysts often have multiple sites where reactionscantake place. One prominent example is single-atom alloys, where thereactive dopant atoms can preferentially locate in the bulk or atdifferent sites on the surface of the nanoparticle. However, ab initiomodeling of catalysts usually only considers one site of the catalyst,neglecting the effects of multiple sites. Here, nanoparticles of copperdoped with single-atom rhodium or palladium are modeled for the dehydrogenationof propane. Single-atom alloy nanoparticles are simulated at 400–600K, using machine learning potentials trained on density functionaltheory calculations, and then the occupation of different single-atomactive sites is identified using a similarity kernel. Further, theturnover frequency for all possible sites is calculated for propanedehydrogenation to propene through microkinetic modeling using densityfunctional theory calculations. The total turnover frequencies ofthe whole nanoparticle are then described from both the populationand the individual turnover frequency of each site. Under operatingconditions, rhodium as a dopant is found to almost exclusively occupy(111) surface sites while palladium as a dopant occupies a greatervariety of facets. Undercoordinated dopant surface sites are foundto tend to be more reactive for propane dehydrogenation compared tothe (111) surface. It is found that considering the dynamics of thesingle-atom alloy nanoparticle has a profound effect on the calculatedcatalytic activity of single-atom alloys by several orders of magnitude. Density functional theory (DFT) is themost extensively used method to study surface science from an ab initioperspective, offering a good balance between computational cost andchemical accuracy.3 Slab models are thestandard within heterogeneous catalysis, where a certain cleave/surfaceof the bulk phase is considered. Often, the most stable facet is consideredfor a key elementary step of a process to describe the reactivityof a catalyst. This does not give a full description of the catalyticsystem outside of single-crystal catalysts.4Catalysis contributes to a third of theglobal economy and is crucialacross several fields of science.5 Besides the most stablefacet, these metal nanoparticles can have a variety of available facetsand sites for reactions to take place. Each distinct site has itsown reactivity and contributes to the overall reactivity of a nanoparticle.6 Desirably, a theoretical model would offer acomplete understanding of the catalyst as a whole, where all siteson the surface are modeled. However, theoretical models that employDFT are limited by the system size, with typically cubic scaling ofthe computational cost with respect to the number of electrons.3 In real terms, metal slab models in catalysisare limited to ∼100 atoms; metal nanoparticles can range insize from ∼200 to 100,000+ atoms. New methods are requiredto capture the reactivity of an experimentally representative nanoparticlecatalyst.One of the most widely used classes of catalysts are metal-basedcatalysts. Most metal-based catalysts are dispersed nanoparticlesof varying sizes.7 ML potentials cantherefore overcome the technical limitations of DFT calculations,enabling experimentally sized nanoparticles to be considered overextended timescales. ML potentials have effectively been used to studyhydrogen coupling,8 CO oxidation,9 CO2 reduction,10 and ethanol reforming.11Machine learning (ML) potentials have been demonstratedas effectivetools to describe very large atomic systems with equivalent accuracyto ab initio methods.13 The dopant is dilute and dispersedenough throughout the host metal lattice so as to not interact withother dopant atoms. For traditional alloys, dopants usually influencethe properties of the host metal, changing the reactivity of the surface.This is counter to SAAs, where the chemical reaction takes place effectivelyexclusively at the dopant site; SAAs have localized active centerson the surface of the catalyst.14 Localizedreactions offer unique selectivities specific to this special classof metal catalysts, such as the punctured molecular cork effect.15 The key advantage of SAAs is their ability tobreak linear scaling relationships, increasing both reactivity andselectivity.17 The potential of this effecthas been shown for selective methane activation,18 dehydrogenation of ethanol,19 selective acetylene hydrogenation,20 andselective propane dehydrogenation.21 Propanedehydrogenation to propene is a challenging process due to selectivityrequirements.24 Excess dehydrogenation of the propene product isundesirable from not only a yield perspective but also from a catalystdeactivation perspective, as it can lead to carbon coking of the surfacewhich will deactivate the surface. Rh–Cu and Pt–Cu SAAshave shown promise of enhanced dehydrogenation selectivity which minimizescoke formation.26 In addition to their selectivity, considerable design space is availablewith the mixture of different host metals and dopant metals.13Single-atom alloys (SAAs) are a unique class of metal–alloy-basedcatalysts, where the dopant constitutes a small amount (∼1%)of the alloy.21 Thiscontributes to the difficulty of modeling the reactivity of a single-atomalloy, as not only the chemical reactivity of certain sites needsto be considered but also the dopant occupation of sites on the surface.The dynamics of the catalyst are crucial in the overall reactivityof the catalyst.Dopant metals can diffuse to a varietyof sites on the surfaceor into the bulk of the nanoparticle, with different SAA dopant–hostcombinations having varying propensities for the dopant to be morestable in the bulk or surface sites.Herein, the reactivity of experimentally representativeSAA nanoparticlesis considered using ML methods and DFT microkinetic modeling. In thiswork, we train an equivariant neural network potential for Rh–Cuand Pd–Cu SAAs using DFT training data; classify sites on thesurface of a nanoparticle; discern the probability of finding thedopant atom in each type of site by performing molecular dynamics;model the reactivity of each possible site through microkinetic modelingusing DFT; and, ultimately, determine the total reactivity of bothRh–Cu and Pd–Cu SAA nanoparticle catalysts for the dehydrogenationof propane as a function of temperature.27 functional within the generalizedgradient approximation (GGA) in the Vienna Ab initio Simulation Package(VASP).30 The D3 dispersion correction with Becke–Johnsondamping was also applied to consider van der Waals forces.31Density functionaltheory calculations were performed with the Perdew–Burke–Ernzerhof(PBE)33 Structures were optimized until the forces on all atoms were below0.05 eV Å–1. Transition states were searchedwith the constrained minimization technique and verified with a vibrationalfrequency calculation, where no imaginary frequencies were identifiedas an initial state and one imaginary frequency as a transition state.35The projector-augmented wave (PAW) methodwas used to represent the core–valence electron interaction.k-point mesh for all surfaces, a 5 × 5 × 5 gridfor bulk metal, and to the gamma point for molecules.36 A vacuum layer of at least 12 Å was also used forall surfaces. Gas-phase molecules were calculated in a 25 × 25× 25 Å3 box to prevent periodic interactions.For the DFT calculations of the metal surfaces, the cutoffenergyof the plane-wave basis set was 450 eV. The Brillouin zone was sampledusing the Monkhorst–Pack scheme with a 5 × 5 × 1 p(4 × 4) slab was used for the (100) surface; a p(3 × 4) slab for the (110) surface; an orthorhombic p(4 × 3) slab for the (111) surface; and a p(2 × 4) slab for the stepped (211) surface. For thesurfaces, all slabs were 5 layers thick. A larger supercell was usedfor the ML potential dataset, so structures with a ∼1% dopantloading could be considered.For the dataset DFT calculations, the following models were used:A 3 × 3 × 3 supercell was used for bulk metal; a p(3 × 3) slab was used for the (100)surface; a p(2 × 3) slab for the (110) surface;an orthorhombic p(3 × 2) slab for the (111)surface; and a p(1 × 3) slab for the (211) surface.Single-atom alloys were modeled as one surface copper atom replacedin the unit cell with the defined dopant atom. All unique sites weremodeled on these surfaces through adding adatoms or making surfacevacancies.For the microkinetic modeling DFTcalculations, the following modelswere used: all slabs were 4 layers thick with the bottom 2 layersfixed; a 37 Transmutation Monte Carlo swap simulations wereperformed for 100,000 swaps at each temperature and equilibrated for10,000 swaps. Molecular dynamics calculations were performed withinthe NVT ensemble with the Nosé–Hoover thermostat.38 A step size of 1 fs was used. For the moleculardynamics with transmutation Monte Carlo swap,39 simulations were run for 1,000,000 time steps, with atom swappingbeing performed every 100 steps for 15 atoms, and equilibrated for10,000 steps. Each time step was structurally optimized to a minimumwith a force criterion of 0.05 eV Å–1 beforesurface site classification was performed. The more occupied a siteis, the more favorable its formation is.All molecular dynamicscalculations were performed within the LAMMPS package.40 for bulk copper and the various slabs. Snapshots were taken of thesesimulations, and 2–3 copper atoms were swapped with respectivedopant atoms dependent on the SAA considered. Single-point-energyDFT calculations were performed, and an ML potential was trained foreach SAA.An initial datasetwas generated with molecular dynamics calculations within the NVTensemble using an embedded atom modelThe final dataset was generated through moleculardynamics withtransmutation Monte Carlo swap simulations with the initial ML potentials.Bulk metal, 5 layer thick relaxed slab models ((100)/(110)/(111)/(211)surfaces) were considered. A range of 1–3 atoms in the variousstructures were replaced with dopants in the dataset. Calculationswere performed at 200, 500, and 1200 K.For bulk metal and surfaces,1800 structures were generated foreach category. Within this, 600 are at each defined temperature, andat each temperature, 200 are with 1 dopant, 200 with 2 dopants, and200 with 3 dopants. In total, there are 9000 structures in each datasetfor each SAA.41 The architecture of the network uses 3 interaction blocks. 32 featuresare considered with a maximum rotation order of l = 1. The radial network uses 8 basis functions with a polynomialcutoff p-exponent of 6. The radial network architecturehas 2 layers with 64 neurons. A cutoff radius of 4 Å was used.8640 randomly selected structures were used for the training set (90%),and the other 960 structures (10%) are used for the validation set.The ML potentialwas trained using the NequIP package.42 A cutoff distance of 5.2 Å was used with4 spherical Gaussian-type orbitals as radial basis functions and sphericalharmonics with lmax = 3 for mapping theGaussian-type density functions with σ = 1 around the atom positions.Upon comparing SOAP descriptors, the radial basis function pairwisecomparison kernel implemented in the DScribe package used σ= 1.All smooth overlap of atomic positions (SOAP)descriptors used were generated with the DScribe package.43 Adsorbed surfacespecies were modeled as having restricted translational and rotationalentropic contributions on the surface. Only vibrational entropic contributionswere considered within the harmonic approximation.43 Free energy barriers for the forward and reverse reactionsof each elementary step were calculated for the temperatures of 400,500, and 600 K under standard pressure. Energy barriers were set to0.05 eV if a lower barrier than this value is calculated to removenumerical issues. Barriers of adsorption/desorption steps were alsoset to be 0.05 eV.Forthe free energy calculations and microkinetic modeling, zero-pointenergy corrections were applied to all surface species and gas molecules.The entropic contributions of gas molecules were considered with theideal gas approximation.1.3H8(g) + * →C3H8*C2.3H8* →C*H2CH2CH3 + H*C3.3H8* →CH3C*HCH3 + H*C4.2CH2CH3 + H* →C3H6* + 2H*C*H5.3C*HCH3 + H*→ C3H6* + 2H*CH6.3H6* + 2H* →C3H6(g) + 2H*C7.2*2H* → H8.2* → H2(g) + *HFor the microkinetic modeling, the consideredsteps are:44The diffusion of species between sites was assumed tobe negligible.Standard pressures of 0.014 bar of propane and 0.007 bar of hydrogenwere selected for the conditions. The system was considered to beunder a steady state for the calculated rates. The steady state wasfound by solving the series of differential equations correlatingthe rates of each elementary step. A variable-coefficient ordinarydifferential equation solver, implemented in the SciPy package, wasused to solve the concentration of surface species.21 Pd is selected as a dopant as Pd–Cu SAA catalystshave successfully been used in a variety of reactions.49 Single point energies are calculated for the dataset with DFT, usingthe PBE functional with the D3(BJ) dispersion correction.51 This dataset is used for the training of an equivariant neural networkinteratomic potential using the NequIP package.41The training datasets of the ML potentials are made from snapshotsof classical molecular dynamics with transmutation Monte Carlo swapsimulations of bulk doped copper and slab models of the doped Cu(100),Cu(110), Cu(111), and Cu(211) surfaces. For this, an initially generatedML potential is used. The dopant is either Rh or Pd, dependent onthe SAA that is modeled. Rh is selected as a dopant due to a Rh–CuSAA catalyst being used for propane dehydrogenation.–1 for all force components.For the Pd–Cu SAA ML potential, the error is 0.46 meV per atomfor energy and 12.7 meV Å–1 for all force components.To validate the ML potentials, static DFT calculations are performed and compared to static calculations using the ML potentials.The bulk lattice constant of copper is equivalent for both DFT andthe ML potentials (3.56 Å). Additionally, the total energy changeof the dopant atom moving from the bulk of the nanoparticle to the(100), (110), or (111) surface with the ML potential matches closelywithin 0.05 eV with respect to slab calculations using DFT (SI.1). This discrepancy can be explained by thechange of surface stress going from a slab to a nanoparticle model.The final trained ML potential for the Rh–CuSAA has a root-mean-squared training error of 0.53 meV per atom forenergy and 14.5 meV Å52 ((111), (100), (110)), as seen in 21 Many distinct single-atom sites are present on the surface.If only the first nearest neighbors of the single-atom sites are considered,there are 5 surface sites. These correspond to the (100) surface site,(110) surface site, (111) surface site, (211) surface site, and (100)–(110)interface site. There are also two non-surface sites, equating tothe (110) subsurface and bulk lattice sites. However, this only describesthe sites available on the ordered most stable nanoparticle. Whenall possible local environments are considered, derived from the cleavingof the face-centered cubic (FCC) crystal structure, 13 possible surfacesites are found, as listed and shown in A copper nanoparticleis made using a Wulff construction based on the surface energies ofthe experimentally most stable surfacesClassification of all possible sites is requiredto designate whichsite the SAA dopant atom occupies during a simulation. An initialclassification is done by counting the number of neighbors the single-atomsite has. From 10 to 12 neighbors, the single-atom site is eitherpart of the subsurface (10 and 11 neighbors) or bulk lattice (12 neighbors).These are classified as non-surface sites. For 9 neighbors, the siteis either the (111) surface or two possible subsurface (non-surface)sites. Between 3 and 8 neighbors ,a variety of surface sites are available and must be classified.53 descriptor as implementedin the DScribe package.42 SOAP descriptorsare capable of describing the similarity of different atomic environments.53 Local structures (to the first neighbor) ofall atoms are made from neighbor lists as implemented in the atomicsimulation environment (ASE) package.43 SOAP descriptors are then made for the central atom of all atomiclocal structures within the nanoparticle that have 3–9 neighbors.SOAP descriptors of the local atomic environments are compared tothe SOAP descriptors of the dictionary. The sites are classified usinga radial basis function pairwise comparison kernel between two setsof SOAP descriptors x1 and x2 defined as2 isthe Euclidean distance and σ is the width of the Gaussian distribution. 42 is used to attain similaritykernel values between a local environment and all of the dictionaryentries of the nanoparticle sites. The comparison is made for alldictionary entries with the same number of nearest neighbors as theselected site. The dictionary entry with the highest similarity kernelvalue is chosen as the classification for that site.A dictionary of the possible structures for 3–9 neighborsingle-atom sites is constructed manually, as shown in 54 is builtfrom all SOAP descriptors originating from the local environmentsof every single atom in the ML potential optimized Wulff-constructedcopper nanoparticle  mapparticle 1c and thparticle 1d. The fThe clustering of sites on the PCA map in 55 To consider the effects of finite temperature, as in the local structuralrelaxation, thermal vibrations, and expansion of the nanoparticlewith respect to temperature, along with the formation of adatoms andother defects, molecular dynamics with transmutation Monte Carlo swapcalculations are performed. Atom swapping of dopant atoms with copperis performed to sample the slow diffusion process of dopant atomsmoving throughout the lattice. Snapshots from the simulations arefirst optimized to a local minimum and then classified to minimizemisclassification. The results of the classification are shown in SI.2).With all sites on a nanoparticle being classifiable, the dynamicsof the sites on the SAA nanoparticles can be quantified. Initial structuresfor SAA nanoparticles for Rh–Cu and Pd–Cu are made byrandomly replacing Cu atoms within the Wulff-constructed nanoparticlewith the typical experimental percentage of dopant atoms  for SAAs. While the dopant loading is kept constantto allow comparison with experimental work, changing the dopant loadingcould influence the surface sites that form, including aggregationof dopant atoms, as seen in other work.49Both systemsmodeled are described by the simulations as SAAs, matching experimentalwork.SI.1). Some sites are considerablyless stable than the dominant bulk or surface sites, such as the (110)site for Rh and the (100)–(110) interface site for Pd. Forboth dopants, when temperature increases, more unstable sites becomeavailable. The stability of the surface sites can generally be orderedby their number of neighbors, observed by the population of differentsites with increasing temperature. At higher temperatures, less stablesites become populated, such as the (110) terminal site (7 neighbors),the (211) step site (7 neighbors), and the (100)–(110) interfacestep site (6 neighbors).Across all temperatures for simulations, for both Rh–Cuand Pd–Cu, the most populated surface site for the dopant isthe (111) terminal site 1b. The  is the overall rate constant for a surfacesite and ρi(T)is the corresponding probability that the dopant atom occupies thatsite in the nanoparticle. The probability of each site being occupiedis found from the performed calculations in The rate constantfor the dehydrogenation of propane can be calculated for each site,for both Rh–Cu and Pd–Cu, through microkinetic modeling.The turnover frequency (TOF) of an SAA catalyst with SI.3). With these energies, microkinetic modelingis subsequently performed. Our methodology enables ML methods andDFT methods to complement each other to effectively describe experimentallyrepresentative catalytic systems.The free energy pathway is calculated2a for thTwo pathways are considered for the dehydrogenationof propane:a terminal carbon or the central carbon of propane  arecalculated from the free energy barriers for the forward and reversereactions using harmonic transition state theory, usingG† is the free energy change from the initial state to the transitionstate, kB is the Boltzmann constant, h is Planck constant, and T is the temperature.Rate constants During the course of the reaction, the concentration of each surfacespecies/intermediate equilibrates, where a steady state is achieved.Under steady-state conditions, the overall rate of propene formationis calculated. The overall rate is solved at a typical experimentalconcentration of reactants, at constant pressures of 0.014 and 0.007bar for propane and hydrogen, respectively. The overall rate of thesite is taken at the initial rate of reaction, i.e., the pressureof the product, propene, is considered to be constantly 0 bar.SI.3). Atomic hydrogen couplingto form molecular hydrogen is found to be effectively barrier-lessand not significant to the overall pathway in comparison to the C–Hactivation steps. This is in agreement with other theoretical work.21 Rh as a dopant atom gives higher reactivity compared to Pd, shownby the higher rate of reaction if a site-per-site comparison is made(2 more reactive than the (111) site; for Pd, it is the orderof ∼107 more reactive. Comparing the most stablefacet, (111), with the second most stable facet, (100): for Rh asa dopant, the (100) site is ∼30× more reactive, whilefor Pd it is ∼1000× more reactive. The (111) site is theleast reactive site for both SAAs.For both Rh–Cu and Pd–Cu, the rate-determining stepsfor all sites are the C–H activation steps if it is not propenedesorption-limited  microkinetic model at 400K, Rh–Cuhas a calculated rate of 9 × 1010–9 s–1 3. With aThere are two key reasons for the failure of a model that onlyconsiders all of the dopants occupying the (111) surface site. Thefirst reason is that not all of the dopant occupies a site on thesurface. At 400 K, only ∼1% of rhodium dopant atoms are atsurface sites, while this is ∼86% for palladium as a dopantatom 1b. This–6) for thetemperature range of 443–488 K.21 This overestimation of rate could be caused by underestimation ofenergy barriers at the DFT level, which is typical of GGA functionals.58 It could also be caused by the breakdown ofthe harmonic approximation at higher temperatures.59 The influence of surface species on surface site formationwas not considered, yet it does have the potential to have an effectfor some single-atom alloys, as seen in other work with the stronglyadsorbing molecule, CO.60 There is alsoa needed simplification of the surface site classification by onlyconsidering the first nearest neighbors. This causes similar facets,such as the stepped (211) and (311) sites, to be modeled as beingequivalent. A marginal effect from the next nearest neighbors forthe chemical reactivity of a site would be expected.61In comparison to experimental work, the calculatedrate for Rh–Cuat 400 K with the full nanoparticle model is within the same orderof the recorded experimental result  site. If a cube-shaped Rh–Cunanoparticle with only (100) facets is synthesized, less surface dopantsites may form.For metal surfaces, edge sites and other facets mustinevitablyexist. It has been demonstrated experimentally that the majority ofreaction events will take place at an edge site of a surface.We demonstrate the ability that machinelearning techniques haveto give a deeper understanding of the assembly of catalytic materials.The combination of DFT calculations with ML potential calculationscovers the respective weaknesses of the methods; DFT calculationsallow consideration of complex potential energy surfaces for reactionsacross a variety of different surfaces, while ML potentials allowconsideration of the catalyst at the experimental scale. Our resultsstress the importance that the changing configuration of SAA catalystsand the availability of sites must be considered to better model thereactivity of SAAs.Beyond modeling the reactivity of catalysts,greater insight intothe unique character of SAAs has been given. The special site selectivitythat SAAs possess could be exploited in different ways to possiblyoffer enhanced chemical selectivity if certain surface sites are selectivefor specific reactants or for forming certain products. This insightcould offer new approaches to selecting dopant atoms and host metalsfor building new single-atom alloy catalysts.We hope our methodswill be used in other catalytic systems ofinterest, such as formation of catalytic clusters on a support, sitelocations of supported catalytic single atoms, and surface defectformation.