The ice plant is a species that is grown mainly in the dry regions of the American West and contains various minerals and ingredients beneficial for human health, such as inositol and beta-carotene. With the growing trend towards healthy foods, pasta consumption has also increased. Pasta is a convenient and low-glycemic-index food that is composed mainly of carbohydrates, proteins, lipids, dietary fiber, and trace amounts of minerals. The optimal mixing ratio was evaluated to produce pasta of the highest quality in terms of blood sugar elevation and antioxidant efficacy. The components and minerals of the ice plant, including D-pinitol and inositol, were analyzed, and 20 essential amino acids were identified. In this study, we also investigated the quality and characteristics of ice plant paste and eggs, as well as the quality, antioxidant activity, and formulation of raw materials mixed with ice plant at different ratios. Optimal conditions were found to be 46.73 g of ice plant paste in 100 g of durum wheat flour, 20.23 g of egg, and 2 g of salt, providing a way to develop fresh pasta that enhances the health benefits of ice plant paste without excessive moisture and other ingredients. Mesembryanthemum crystallinum) extract ameliorates hyperglycemia [w/w) and utilizing mechanical force to produce fresh pasta, which is then dried. Despite its high carbohydrate content, pasta contains low levels of dietary fiber, vitamins, essential amino acids, and minerals, some of which are lost during the milling process. However, the FDA recognizes the presence of bioactive ingredients in pasta, such as proteins, minerals, phytochemicals, vitamins, etc., with up to 10–15% of these functional ingredients retained without a significant decrease in pasta quality.The ice plant is an annual plant belonging to the genus Aizoaceae. The bladder cells, which look like ice crystals on the surface of stems and leaves, contain ingredients useful for the human body, such as inositol, beta-carotene, as well as various minerals. The ice plant is reported to exhibit various physiological activities, such as antioxidant properties, blood-sugar-lowering activities, and antibacterial effects . It is mglycemia . Other sglycemia ,10,11. Iglycemia ,13. As tglycemia . Made frCistus incanus L. leaves on wheat pasta [Pasta is a typical Italian noodle dish and is divided into raw and dried pasta. In Korea, dried pasta is the most commonly used form, while in the western and northern regions of Italy, fresh raw pasta is preferred due to its mild flavor and the ability to enhance its color, shape, and nutritional value with added ingredients. The trend towards raw noodles is growing, with changes in consumer preferences leading to alterations in dietary habits. Different types of pasta, including yellow lentil pasta ; pasta fat pasta ; add criat pasta ; and pasat pasta , spiruliat pasta , and Morat pasta , have beThe ice plant used in this study was purchased in October 2022 from Yesan ice plant Signal Agricultural Corporation  in Chungcheongnam-do. The general components, inorganic components, inositol, and amine were analyzed in each part of the ice plant. After analysis, durum wheat , salt , and eggs  were purchased and used as ingredients for pasta manufacturing. DW (distilled water) and 70% ethanol were used as the extraction solvents in the antidiabetic and antioxidant experiments. The reagents used in this experiment were products of Sigma-Aldrich Co. .The quantification of raw fat, crude protein ash, and carbohydrates in each part of the ice plant was performed by general component analysis using the AOAC  method. M.crystallinum L.) are calcium, iron, potassium, sodium, magnesium, manganese, zinc, copper, cadmium, and lead, which are approved by the Ministry of Food and Drug Safety (2016) [The raw materials contained in each part of ice plant (y (2016) . A volumM. crystallinum L.), about 1 g was taken, and 50 mL of a 5 mM sodium 1-hexanesulfonate solution was added. Thereafter, the extract was extracted for 30 min with an ultrasonic extractor  at 40 °C, mixed well with the sample, and centrifuged at 15,000 rpm for 15 min. Next, only the supernatant was taken and filtered with a 0.45 μm syringe filter , and HPLC  analysis was performed.After homogenizing the raw sample for each part of the ice plant (v/v%). The column temperature was 40 ℃and the sample temperature was 20 °C. A sample volume of 0.5 uL was injected for sufficient ionization of the material to be analyzed.A Dionex Ultimate 3000 HPLC instrument  and an Agilent 1260 infinity FL detector  were used for analysis. The conditions for use were as follows. The FL detector settings consisted of an emission wavelength of 450 nm and an excitation wavelength of 340 nm (OPA). Additionally, the FL detector had an emission wavelength of 305 nm and an excitation wavelength of 266 nm (FMOC). A UV detector was used at 338 nm, and as a standard, 17 types of 1 nmol/uL amino acids were dissolved in 0.1 N-HC and diluted with tertiary distilled water to test at concentrations of 1000, 500, 100, and 10 pmol/uL. For the analysis conditions, an Inno C18 column  was used, and mobile phase A was 40 mM Sodium phosphate, with a pH value of 7. Mobile phase B was 3DW/acetonitrile/methanol  parts were analyzed using the a Fibertec 1023 System E dietary fiber analyzer . The process involved adding 50 mL of 0.08 M phosphate buffer (pH 6.0) to 1 g of the sample, then adding 0.1 mL of α-amylase solution and performing hydrolysis at 95–100 °C for 15 min. After cooling to room temperature, 10 mL of 0.275 N sodium hydroxide solution was added to adjust the pH to 7.5, followed by the addition of 0.1 mL of protease and hydrolysis in a shaking water bath at 60 °C for 30 min. The solution was cooled again, and 10 mL of 0.325 M HCl solution was added to adjust the pH to 4.0–4.6. Then, 0.1 mL of amyloglucosidase was added, hydrolyzed in a 60 °C shaking water bath for 30 min, and cooled to room temperature. Subsequently, 95% ethanol  was added, and the mixture was stirred and left to stand at room temperature for a day. Finally, celite was added, the mixture was filtered through a crucible to obtain a constant weight; washed with 78% ethanol, 95% ethanol, and acetone in sequence; dried in a hot air dryer at 60 °C for 24 h; and weighed to calculate the results using the following formula:The raw samples of ice plant , durum wheat , salt from CJ , and eggs  were used for experimentation. Fresh ice plant paste was used for raw pasta production. When making pasta, the ice plant was ground using its natural water, then pacotized and freeze-dried to a moisture content of 30% to make a paste. The optimal mixing ratio of ice plant paste and eggs was determined using the Design-Expert program. The experiments were designed using central composite design (CCD) and response surface design (RSM) to evaluate various sensory characteristics, such as chromaticity , texture, and overall quality. The ice plant paste was set at 30–60 g, and the egg was set at 10–30 g, with the mixing ratio adjusted through a preliminary test . All ingTo determine the chromaticity of each part of the ice plant, the values of L (lightness), a (redness), and b (yellowness) were measured using a colorimeter . Each of the three samples for each part was measured three times. The L value of the standard white plate used at this time was 105.61, the a value was −0.18, and the b value was +4.82.The pH measurement for each part of the ice plant was homogenized by adding 50 mL of distilled water to 5 g of the sample. A glass electrode pH meter  was used to measure three samples for each part, with each sample measured three times. The average value was obtained.3 . The analysis was performed using a salinity meter .To measure the salinity of each part of the ice plant, samples were homogenized by adding 50 mL of distilled water to 5 g of the sample. Then, 100 mL of distilled water was used for salinity analysis, and 10 mL of the homogenized mixture was transferred to a test container. The sample was zeroed using a 0.3% NaCl solution before analysis. The titration reagent was titrated with 0.1 N AgNOThe moisture content of fresh noodle pasta with ice plant paste was evaluated using the atmospheric pressure drying method at 105 °C. The evaluation was conducted using a dry oven . Prior to cooking, 0.5 g of raw pasta was weighed and placed in an aluminum dish. The dish was then heated to 105 °C, and the moisture content was measured. After multiple measurements were taken, the average value was calculated and presented.The sample solution was prepared by mixing 70% ethyl alcohol with 5 g of raw ice plant paste noodle pasta before cooking. Then, 50 mL of the solution was added to a stirring device  and homogenized for 90 s. The mixture was then placed in a shaking incubator  for 24 h. The sample was obtained by filtering the mixture twice using Whatman No. 4 filter paper.(A)Total Phenol Content2CO3 was added, and the mixture was reacted for 2 h in the dark. The absorbance at 725 nm was measured using a UV-visible Spectrophotometer . Gallic acid was used as the standard and analyzed in the same manner as the sample. A calibration curve was established, and the total phenol content of the sample was obtained as milligrams of gallic acid equivalent (GAE) per dry basis.The total phenolic content was determined by modifying the Folin–Denis method (1912) and measured using the Singleton–Rossi method (1965). Volumes of 2400 μL of distilled water and 150 μL of the sample were mixed in a test tube. Then, 150 μL of 2 N Folin–Ciocalteu phenol reagent  was added and stirred. Then, 1 N Na(B)Total Flavonoid ContentThe total flavonoid content was determined by modifying the Davis method . First, (C)DPPH Free Radical Scavenging Activity−4 M, final concentration) was mixed with 0.5 mL of the sample, stirred, and brought to room temperature. After 30 min, the absorbance was measured at 517 nm using a UV-visible spectrophotometer . The DPPH free radical scavenging activity value was obtained using the following formula:The free radical scavenging activity was determined using the method described by . A DPPH For a sensory evaluation study of raw pasta using ice plant paste, we used a 7-point hedonic scale to assess the participants’ preferences, with 1 indicating a strong dislike and 7 indicating a favorable opinion. The panel of 20 participants was composed of individuals with experience in food tasting capable of differentiating various sensory attributes, such as taste, flavor, and texture. The panelists were between the ages of 20 to 50 years old, and the samples were presented in a random order and coded to ensure anonymity. The panelists were each given the opportunity to evaluate the samples, with taste-neutral water provided for rinsing between samples, and all samples were presented at room temperature.The results of the sensory evaluation study were analyzed statistically using the central composite design (CCD) and the response surface methodology (RSM). The data were analyzed using quadratic analysis of variance (ANOVA) to evaluate each response variable and determine the influence of various factors on the outcome. To compare the means between the control and optimized raw pasta of ice plant paste, ANOVA was performed using Microsoft Excel at a significance level of f-value 0.05.In the context of a food products, a general component could refer to the basic ingredients or nutrients that make up the product, such as protein, carbohydrates, fats, vitamins, and minerals. Macro minerals are minerals that are required in relatively large amounts by the human body for normal physiological function. They include calcium, phosphorus, magnesium, sodium, potassium, chloride, and sulfur. These minerals are essential for many bodily processes, such as bone health, muscle and nerve function, fluid balance, and acid–base balance. It is important to maintain a balanced intake of these macro minerals through a healthy diet to support optimal health and prevent deficiencies or imbalances . MicromiPinitol is a cyclic polyol  that occurs naturally in plants and microorganisms. Its chemical structure is similar to that of inositol: a six-carbon cyclic polyol that plays a vital role in several physiological processes. Derived from various plants and microorganisms, pinitol is a cyclitol that is sometimes referred to as 3-O-methyl-chiro-inositol. It shares a structural resemblance to inositol, a cyclic polyol with six carbons that is present in many living organisms and is essential for multiple biological functions . PinitolInositol, a type of cyclic polyol or sugar alcohol, is crucial for numerous physiological processes in living organisms due to its structural similarity to glucose. Inositol exists in many different forms, with myo-inositol being the most common and most widely studied in humans. Inositols occur naturally in many foods, such as fruits, beans, and grains, and are also produced by the body. They serve vital functions in various cellular signaling pathways, including those that regulate cell growth, differentiation, and death, as well as those involved in insulin signaling, lipid metabolism, and neurotransmission . FurtherM. crystallinum L. (as shown in the chromatogram), the respective contents of which are presented in In this study, we present the analysis results of inositol components in the ice plant . InositoOur analysis revealed the presence of a total of 20 amino acids . AspartiAlanine is a non-essential amino acid that is involved in protein synthesis and the regulation of blood sugar levels and is a source of energy for cells, particularly muscle cells. It is also involved in the metabolism of nitrogen .GABA is a neurotransmitter that is involved in the regulation of nerve impulses in the brain. It is synthesized from the amino acid glutamate and has been linked to the regulation of mood and anxiety .Tyrosine is a non-essential amino acid that is involved in protein synthesis and the synthesis of several important neurotransmitters. It is also involved in the regulation of mood and is believed to play a role in the regulation of stress and anxiety .Valine, methionine, tryptophan, phenylalanine, isoleucine, and leucine are amino acids that are considered essential because the body cannot produce them on its own and must obtain them from dietary sources . They arTotal dietary fiber refers to the indigestible components of plant-based foods that resist digestion and absorption in the small intestine. α-Glucosidase inhibitory activity and α-amylase inhibitory activity refer to two methods of measuring how well a substance can hinder the activity of enzymes responsible for carbohydrate digestion. α-Glucosidase is an enzyme that breaks down complex carbohydrates into simple sugars, while α-amylase is an enzyme that breaks down starches into smaller carbohydrate molecules. Limiting the activity of these enzymes can result in slower carbohydrate absorption, potentially aiding in the management of conditions such as diabetes and obesity. Researchers often investigate substances that show α-glucosidase inhibitory activity or α-amylase inhibitory activity to explore their potential as natural remedies or dietary supplements.M. crystallinum L. were investigated at a concentration of 1 mg/mL, as shown in 50 value of 7995 ppm. The cotyledon extract exhibited 5.99 ± 0.53% inhibition, with an IC50 value of 5117 ppm. In the case of shoot extract, inhibition increased significantly from 125 ppm to 5.52 ± 0.59% at 1000 ppm, with an IC50 value of 4592 ppm. At the highest concentration of 1000 ppm, α-glucosidase activity was inhibited in the order of stem > cotyledon > shoot.α-Glucosidase is an enzyme that converts oligosaccharides or disaccharides into glucose. α-Glucosidase inhibitors delay the digestion and absorption of carbohydrates to control postprandial blood sugar without causing hyperinsulinemia or hypoglycemia, which can lead to obesity, and promote insulin secretion. They also promote the secretion of glucagon-like peptide-1, which inhibits glucagon secretion in the small intestine . The α-gM. crystallinum L. was measured in this study. The stem extract showed concentration-dependent inhibition, with 7.44 ± 1.16% inhibition at 62.5 ppm and 31.91 ± 3.90% inhibition at 1000 ppm, with an IC50 of 2451 ppm. The cotyledon extract also showed concentration-dependent inhibition, with 1.58 ± 0.82% inhibition at 62.5 ppm and 17.55 ± 1.69% inhibition at 1000 ppm, with an IC50 of 4748 ppm. The shoot extract showed 2.06 ± 1.10% inhibition at 62.5 ppm and 23.04 ± 1.55% inhibition at 1000 ppm, with an IC50 of 7115 ppm. At the highest concentration of 1000 ppm, the order of inhibition was stem > shoot > cotyledon for all three ethanol extracts -glucan bond in carbohydrates. When α-amylase inhibitors are present, the digestion of starch in the small intestine is slowed down, which delays the absorption of glucose and prevents a rapid increase in blood sugar after meals. Both salivary and pancreatic α-amylase are essential enzymes for carbohydrate metabolism in various living organisms. The α-amylase activity inhibition ability of ethanol extracts from different parts of extracts .p-value of 0.004 and an R2 value of 0.96 was selected , and the R2 value was 0.921, indicating the suitability of the model  and an R2 value of 0.862  and an R2 value of 0.840. The preference for texture ranged from 3.63 to 6.31, and a quadratic model in which each factor interacted was selected, with a p-value of 0.0234 (p < 0.05) and an R2 value of 0.790  and an R2 value of 0.801 (The sensory characteristics of fresh ice plant paste pastas were evaluated for color, flavor, taste, texture, appearance, and overall acceptability using a seven-point preference scale . The accof 0.862 . The preof 0.701 . The tasof 0.790 . The accof 0.801 . The resof 0.801 .The optimization of fresh ice plant paste pasta production yielded significant results as sensory test items including color, taste, flavor, texture, appearance, and overall quality were maximized within the range of independent variables of ice plant paste and eggs. A canonical model was used to predict the optimal point with the highest desirability, which was determined to be 46.73 g of ice plant paste and 20.23 g of egg. The response model was used to generate a graphical optimization and perturbation plot, as shown in Pasta is a staple food that is widely consumed due to its palatability, ease of preparation, low cost, and long shelf life . The incPasta with added vegetables contains a variety of essential nutrients, such as phytochemicals, fiber, vitamins, polyphenols, carotenoids, glucosinolates, and minerals . DespiteVegetable supplementation in pasta has been evaluated, but most studies only use small amounts of vegetables to maintain overall acceptability. While pasta enriched with functional ingredients may have superior nutritional properties compared to traditional pasta, it may negatively impact technical and sensory qualities and be rejected by consumers . The cooIn this study, we examined the components and minerals present in ice plant and analyzed their ability to inhibit the activity of starch-degrading enzymes, specifically focusing on D-pinitol and inositol. Additionally, we conducted an amino acid analysis to identify the presence of 20 essential amino acids. The findings of our study demonstrate that incorporating ice plant into semolina-based fresh pasta had a positive impact on its nutritional quality, particularly in terms of pinitol, inositol, and amino acid contents. Moreover, among the various observed physical and chemical characteristics, color played a significant role in the resulting product.The cooking characteristics of the supplemented fresh pasta were similar to those of regular fresh pasta, except when ice plant paste was used as a replacement. Sensory evaluations indicated that samples containing 45 g of ice plant paste and 20 g of egg received the highest scores among all the supplemented fresh pasta samples. Developing a fortified pasta product with desirable quality poses a challenge, and it is crucial to accurately determine the proportions of individual additives based on consumer preferences. The inclusion of ice plant significantly enhanced the nutritional value of fresh pasta and was well-received by consumers in this study. However, future research should focus on sensory analysis to improve the overall sensory characteristics of pasta when ice plant is added under different conditions, thereby providing a higher nutritional value tailored to consumer acceptance. Furthermore, to explore additional health benefits of incorporating ice plant into food and develop fresh pasta that offers enhanced health advantages, further studies should investigate the elemental composition of ice-plant-incorporated pasta without excess water and other ingredients, particularly those with high levels of antioxidant activity.


Single-atom catalysts have received significant attentionfor theirability to enable highly selective reactions. However, many reactionsrequire more than one adjacent site to align reactants or break specificbonds. For example, breaking a C–O or O–H bond may befacilitated by a dual site containing an oxophilic element and a carbophilicor “hydrogenphilic” element that binds each molecularfragment. However, design of stable and well-defined dual-atom siteswith desirable reactivity is difficult due to the complexity of multicomponentcatalytic surfaces. Here, we describe a new type of dual-atom system,trimetallic dual-atom alloys, which were designed via computationof the alloying energetics. Through a broad computational screeningwe discovered that Pt–Cr dimers embedded in Ag(111) can beformed by virtue of the negative mixing enthalpy of Pt and Cr in Agand the favorable interaction between Pt and Cr. These dual-atom alloysites were then realized experimentally through surface science experimentsthat enabled the active sites to be imaged and their reactivity relatedto their atomic-scale structure. Specifically, Pt–Cr sitesin Ag(111) can convert ethanol, whereas PtAg and CrAg are unreactivetoward ethanol. Calculations show that the oxophilic Cr atom and thehydrogenphilic Pt atom act synergistically to break the O–Hbond. Furthermore, ensembles with more than one Cr atom, present athigher dopant loadings, produce ethylene. Our calculations have identifiedmany other thermodynamically favorable dual-atom alloy sites, andhence this work highlights a new class of materials that should offernew and useful chemical reactivity beyond the single-atom paradigm. Most recently, dual-atom sites have been identifiedas promising active sites for several reactions.10 However, supported dual-atom catalysts are again structurally complex,making them difficult to design, synthesize, and characterize.11 This complexity also impedes understanding thereaction mechanism at a fundamental level. While computational screeningof these materials can identify candidates with high predicted performance,11 synthesizing and characterizing the predictedstructures can be difficult in many cases.The design of well-defined active sitesin heterogeneous catalysisis a long-standing goal of the field, but the structural complexityof supported catalysts and their dynamic changes in response to reactiveatmospheres complicate this goal.13 These dual-atom alloys wouldfeature active sites that are amenable to atomic-scale characterizationwith scanning tunneling microscopy (STM) and accurate modeling withtheory. Furthermore, unlike supported single-atom and dual-atom catalystswhere the dopant is often charged, metal dopants in metal supportshave near 0 valence.14 The dual-atom alloystructure is analogous to single-atom alloys (SAAs), which consistof catalytically active metal atoms atomically dispersed in a second,typically more inert, host metal. However, dual-atom alloys extendbeyond SAAs, as they feature a two-atom active site, which is a significantlylarger and more tunable active site than that of SAAs. SAAs have provento be effective catalysts for many reactions,21 and their well-defined active sites enable them to be highly selectiveand amenable to first-principles design.22 The site isolation in SAAs also provides important catalytic benefitsthat may be shared by dual-atom alloys, such as enabling spilloverof reaction intermediates and deviations from linear scaling relationshipsthat limit the performance of traditional catalysts.25 However, single-atomactive sites may not be able to stabilize larger intermediates ortransition states, or break very strong bonds; therefore, more challengingreactions will require adjacent active atoms.27As an alternativeto oxide-supported dual-atom catalysts, it maybe possible to create stable heterometallic pairs embedded in a thirdmetal host.Dual-atom alloys address these limitations of SAAs by combiningtwo active atoms with different chemical reactivity in one localizedheteroatom dimer active site. A dimer site comprised of two differentdopant metals should allow much more tunability of reactant bindingand activation and hence improved control over chemical reactivitybeyond what can be achieved with SAAs. However, due to the enormousstructure space involved with the design of trimetallic alloy sites,effective computational screening is crucial for a priori identificationof the most stable and chemically reactive combinations.1Ag and Cr1Ag are inert for this reaction. Our calculations on the reactionmechanism reveal that adjacent oxophilic Cr and carbophilic or “hydrogenphilic”Pt atoms are necessary to provide favorable binding and reaction energiesfor the conversion of ethanol to acetaldehyde and that higher dopantloadings lead to the formation of ethylene.Inthis work, we computationally design and experimentally synthesizeand test a dual-atom alloy consisting of a heterometallic dimer, Pt–Cr,embedded in an inert host, Ag. The use of a Ag(111) single-crystalhost enabled us to directly image the different active sites and demonstratethat Pt–Cr dimer sites can dehydrogenate ethanol in temperature-programmeddesorption (TPD) studies, while both Pt12 Specifically,we started with the known Pt1Ag, Pd1Ag, Pd1Au, and Ti1Cu SAAs, which all have positive homometallicdimer formation energies and hence are stable SAAs. We used densityfunctional theory (DFT) to calculate the heterometallic dimer formationenergy for a wide range of elements as possible third metal dopantsto add to these bimetallic SAA systems. These heterometallic dimerformation energies must be negative in order to favor formation ofthe heteroatom dimer site . We also calculatedthe homometallic dimer formation energy of the third metal, whichmust be less negative than the heterometallic dimer so that the heterometallicdimer is energetically favored over the homodimer. From these screeningresults, many heteroatom dual-atom alloy sites are predicted to bestable, and the most promising in terms of active site uniformityare those with a negative heterometallic dimer formation energy buta positive homometallic dimer formation energy seen in the light blueregions of S1.In the search for synthesizabledual-atom alloy materials, we firstcomputationally screened many alloy combinations in order to identifycases where the two different dopant atoms would form a heterometallicdimer as opposed to homometallic dimers 1a.12 Spe28 Cr is relatively oxophilic, while Pt is relatively carbophilic,which suggests it will bind H strongly.29 Our DFT calculations of H and OH adsorbed on the top sites of thePt1Cr1Ag dual-atom alloy confirmed this factand demonstrate that the Pt atom of the dual site binds H more stronglythan OH, while the opposite is true for the Cr atom, as seen in 1Cr1Ag dual-atom alloy is predictedto be both thermodynamically stable and composed of two atoms withcomplementary chemical reactivity.To identify heteroatom dimer sites with desirablechemical reactivityfor O–H activation, the first step of ethanol dehydrogenation,we searched for thermodynamically stable pairs in which one metalatom stabilizes an O-containing intermediate and the other metal atombinds H strongly. Based on previous work,1Cr1Ag dual-atomalloy as having favorable thermodynamicstability and desirable chemical properties for ethanol activation,we synthesized and characterized the trimetallic PtCrAg alloy andits PtAg and CrAg bimetallic counterparts. First, Pt was depositedonto a clean Ag(111) single crystal via electron beam evaporationat an alloying temperature of 380 K. This led to the formation ofa PtAg SAA30 in which the Pt atoms aredispersed in the Ag surface in the form of isolated atoms as seenin S3. These STM images show the appearance of thesingle-atom Pt sites with and without adsorbed CO, which is presentin the ultra-high-vacuum (UHV) chamber background. Next, the alloyingof Cr with Ag(111) was investigated. Given that our goal is to formheteroatom pairs, we used Cr(CO)6 in the gas phase to addthe Cr. Our hypothesis was that Cr(CO)6 is less reactivethan evaporated Cr metal atoms, and hence the longer surface residencetimes before alloying enable the Cr(CO)6 to sample differentsurface sites and decompose next to the Pt atom sites. Specifically,Cr(CO)6 should be more likely to decompose at Pt siteswhich bind CO strongly as opposed to Ag sites that bind CO weakly;hence Pt should facilitate the decomposition of the carbonyl. As wewill describe below, this kinetic trapping of the Cr atom next tothe Pt site, combined with the intrinsic thermodynamic stability ofthe PtCr dimer pair confirmed by our computational studies, providesa new method for synthesizing these dual-atom sites.Based on this computationalscreening that identified the PtS2 show the results ofalloying Cr into Ag(111)by exposure to Cr(CO)6 at a sample temperature of 320 K.Just as with Pt, both bright and dark protrusions are observed, andthe comparison to the DFT-simulated STM images shown in panels c andd reveals that CO-free Cr atoms appear as protrusions and Cr atomswith adsorbed CO appear as depressions. Most interestingly, when bothPt and Cr are alloyed with Ag(111), in addition to isolated Pt andCr atoms, we observe the appearance of oblong asymmetric featuresaligned with the close-packed directions of the Ag(111) surface . These STM images, coupled with the DFT-simulatedimages, indicate that Pt–Cr heteroatom dimers are formed viathe serial deposition of Pt and Cr(CO)6 on Ag(111).35 Our goal was not necessarily to design a better catalyst for thisreaction, as there are existing alloys that can efficiently convertethanol.33 TPD studies do not directly probe catalyticturnover, as the experiment involves a single deposition of ethanolfollowed by heating of the sample to induce reaction. Instead, weuse this reaction as a probe of the reactivity of the Pt–Crheterodimer sites and compare them to Pt and Cr single-atom sites.In order to more accurately detect the desorption of reactively formedhydrogen, we exposed the surface to fully deuterated ethanol and detecteddeuterated acetaldehyde and D2 as products. As seen in S6, and S7, the PtAg and CrAg bimetallic surfaces do not decomposeethanol even at loadings up to 10% of a monolayer. The PtAg resultsare in agreement with previous TPD studies of ethanol on Pt(111),which observe no reaction, merely reversible adsorption/desorptionof intact ethanol.36 In contrast, the presenceof just 3% of both Cr and Pt results in activity toward ethanol dehydrogenationas seen in Figure S7 shows full TPD data for all alloy compositions studied. Interestingly,at low dopant concentrations, ∼3% of a monolayer, the mixedPtCrAg sample is 100% selective to acetaldehyde and hydrogen, andas the concentration of the dopants is increased, the selectivityto ethylene increases, reaching ∼80% at 10% dopant concentrations(see Table S1). Overall, the TPD resultsindicate that varying the dopant loading modifies the observed reactionproducts:After characterizing the atomic-scale structures of the PtAg, CrAg,and PtCrAg alloys, we tested their reactivity for ethanol decompositionto acetaldehyde, hydrogen, and ethylene using TPD experiments. Thisreaction was chosen as a simple but important example where O–Hbond breaking is critical, but complete decomposition of the moleculeis undesirable.1Cr1 dual-atom alloy site are crucial for O–H bond activationin ethanol . Specifically,in the dissociated ethoxy+H state, Pt stabilizes the H atom, as itis quite hydrogenphilic. However, on Pt1Ag, ethoxy is forcedto a Ag site, where it is relatively unstable. Conversely, Cr is oxophilic,such that ethoxy is quite stable on Cr1Ag, but H is forcedto a Ag site, where it is relatively unstable. Hence, there is littleor no thermodynamic driving force for ethanol to dissociate on eitherof the respective SAAs as seen in S9–S11. This indicates that larger Pt–Crn ensembles are the active sites for ethyleneproduction. Specifically, on the Pt–Cr dimer, the C–Obond scission was calculated to have a high barrier of 1.62 eV, whilethe Pt–Cr2 trimer gives a much lower calculatedbarrier, 0.41 eV. On the trimer, the C–O bond scission transitionstate features the O interacting with a Cr, the ethyl fragment interactingwith the other Cr, and both H atoms adsorbed on the Pt. Furthermore,the calculated formation energies of two types of trimers are bothnegative: −0.12 eV for Cr2Pt1Ag formingfrom Cr1Pt1Ag and Cr1Ag and −0.15eV for Cr1Pt2Ag forming from Cr1Pt1Ag and Pt1Ag. This result suggests that the trimersare likely to form in the surface as the concentration of Cr and Ptincreases. This is consistent with the experimental findings thatethylene production dominates the reaction selectivity at higher coveragesof Pt and Cr.Our DFT calculations rationalize these resultsand show that thedifferent reactivities of the two atoms of the Pt37 Our DFT screening highlights many otherdual-atom alloy materials that are predicted to be stable and hencesynthesizable. Compared to the relatively small structure space ofSAAs, these dual-atom alloys offer many stable and potentially usefulheteroatom active sites and feature a larger active site in whichthe two different elements can stabilize distinctly different chemicalintermediates. These mixed-metal active sites also offer the intriguingpossibility of performing selective cross-coupling reactions betweentwo different species by virtue of the reactants’ preferredbinding to each of the two different atoms of the dual site.In summary, we have computationally designedand experimentallytested a new type of active site, the dual-atom alloy, in which heterodimersof two reactive atoms (Pt and Cr) are embedded in a more inert host(Ag). This trimetallic was identified based on computational examinationof both the stability and reactivity of the dual site. STM confirmedthe presence of Pt–Cr sites in the experimentally synthesizedtrimetallic, and TPD demonstrated that these dual sites are activefor ethanol dehydrogenation, while the individual component PtAg andCrAg bimetallics are not. DFT calculations revealed that the PtCrmixed site is needed in order for O–H bond breaking to be boththermodynamically and kinetically favorable, as Pt binds the dissociatedH while Cr stabilizes the ethoxy intermediate. Moreover, the largerPtCr ensembles present at higher dopant loadings changed the reactionselectivity to favor the production of ethylene, demonstrating howsensitive the reaction pathway is to the atomic-scale structure ofthe heteroatom active site. More generally, this work opens up a newclass of dual-atom alloy materials that can be computationally designedusing the methods we developed here. These materials extend beyondSAAs to a larger design space and larger active site. The well-definednature of the dual-atom active site provides a strong synergy betweencomputational modeling and experiment, which is critical in the questto design catalytic sites from first principles. For example, thisapproach involving fundamental studies of the active site structureand associated reactivity has proven very successful in the recentdevelopment of a variety of SAA catalysts.–10 mbar. The Ag(111) single crystal  was cleaned via Ar+ sputtering followed by750 and 1000 K anneals in the TPD and STM chambers, respectively.The PtAg alloy was synthesized following a previously reported procedure,in which Pt was deposited using an electron beam evaporator onto theAg(111) surface held at 380 K.30 The Cr(CO)6 was deposited using the vapor pressure above solid Cr(CO)6 purchased from Sigma-Aldrich (98% purity). The compound wasplaced into a glass sample vial and connected via a turbo-pumped gaslineto a precision leak valve on the UHV chambers. The evacuated pressurein the gas line was <1 × 10–4 mbar, andonce the Cr(CO)6 vial was opened to the gas line, the pressurerose to 5 × 10–1 mbar. During Cr(CO)6 deposition the Ag(111) crystal was held at 320 K, and the pressurein the chamber was 1 × 10–6 mbar during thedeposition. Liquid ethanol-d6 (Sigma-Aldrich99.5%) was purified via freeze–pump thaw cycles prior to usein order to remove any dissolved gases. Ethanol doses are given inLangmuirs (1 L = 1 × 10–6 Torr·s). TheTPD experiments were collected with a heating rate of 1.5 K/s. Quantitativemass spectrometry was used to determine the surface coverage of thePt and Cr dopants using CO TPDs following a literature procedure.38 The TPD traces in m/z = 34) from the acetaldehyde (m/z = 48) signal. Product selectivity was calculatedby correcting the desorption peaks for ionization cross section, fragmentationpattern, and the mass spectrometer quadrupole sensitivity.39 In the TPD experiments the surface temperatureis ramped up to 600 K, which leads to the dopants diffusing beneaththe surface, which is a common phenomenon in TPD studies of dilutealloys.The surface science experimentswere performed on an Omicron low-temperature STM and a separate home-builtTPD chamber. Both chambers have a base pressure < 1 × 1040 exchange–correlationfunctional with the Tkatchenko–Scheffler method41 for van der Waals interactions and were performedwith the Vienna Ab initio Simulation Package (VASP).43 The cutoff energy for all the calculations was set to 400 eV. Thesurfaces calculated in this work are all fcc(111) structures, andthe relaxation calculations were done on a 7 × 7 × 1 k-point mesh with 3 × 3 × 4 unit cells with thebottom two layers fixed. The lattice constants and the convergencetests were performed in our previous work.44 The transition states were optimized by the improved dimer method45 and were confirmed by frequency calculations.The harmonic approximation was used to calculate free energy correctionsand CO vibrational frequencies (Table S3). For STM image simulation, a bias voltage of −200 mV wasapplied; Cr1Ag, Cr1(CO)Ag, Pt1Ag,and Pt1(CO)Ag structures were modeled with a 5 × 5× 4 unit cell, and Cr1Pt1(CO)Ag was modeledwith a 6 × 6 × 4 unit cell. Spin-polarized calculationswith different initial magnetism were performed to determine the moststable spin state of each dopant–host system.All periodic plane wave DFT calculationswere carried out by using the Perdew–Burke–Ernzerhofgeneralized gradient (GGA-PBE)


Horseshoes are placed on the hooves of horses to prevent excessive wear on the hoof when horses are ridden or used for work. Typically, horseshoes are attached to the bottom of the hoof with nails. Although nails have been used for centuries, we sought to understand whether nailing a shoe to the hoof would adversely affect hoof expansion that normally occurs when weight is borne on the limb. Horseshoes were attached to cadaveric hooves by nails in three different sets of positions. Weight bearing was simulated while hoof expansion and compression and tension on the surface of the hoof were measured. We found that placing nails closer to the back of the hoof limited hoof expansion and caused changes in compressive and tensile forces on the surface of the hoof. Limiting hoof expansion interferes with the hoof’s ability to absorb concussion. Over time, the observed changes in compression and tension may cause abnormal changes in hoof shape that can affect soundness and performance.p < 0.05). Nails placed palmar to the quarters of the hoof decreased heel expansion (p < 0.001). Heel nails resulted in the largest changes in hoof wall principal strain directions distally. The application of nails palmar to the hoof quarters alters hoof wall deformation during limb loading. The continued loading of the hoof with palmer nails could alter hoof conformation over time.Racehorses are susceptible to underrun heel hoof conformation. Racehorses are often shod with nails placed toward the heel. It is unknown if palmar nails restrict or alter hoof deformation in a manner that could promote the development of underrun heel conformation over time with repeated loading. To determine how the addition of palmar nails affects heel deformation during limb loading, hoof expansion and hoof wall deformations were quantified using rosette strain gauges and kinematic markers during in the vitro limb loading of cadaveric limbs that simulated midstance for walk, trot, and canter loads. Nail treatments used to attach a horseshoe to the hoof included: toe nails (T), toe and quarter nails (TQ), and toe, quarter, and heel nails (TQH). The effects of nail treatment on heel expansion and hoof wall deformations were assessed using repeated measures analysis of variance ( Heel expansion and elastic structures within the foot provide some mechanisms for attenuating ground impact forces along with bones and joints in the distal part of the limb ,2. HorseAbnormal hoof conformations have been associated with injuries and lameness. One type of abnormal hoof conformation is the underrun heel, defined as a heel angle at least 5° lower than the toe angle . A studyHoof wall expansions are influenced by the application of a shoe and by the sites of attachment of the shoe to the hoof wall. The placement of shoes decreases normal hoof expansion when compared to an unshod foot . In a stThe study objectives were to determine the effects of nail placement on hoof wall surface strains, deformation, and expansion during the loading of cadaveric limbs in vitro and to determine if changes are consistent with the development of the underrun heel conformation. We hypothesize that greater constraint of the hoof by a horseshoe attached with nails placed progressively more palmar would increase hoof wall strains, change principal strain directions, and alter hoof deformations in a manner consistent with a mechanism that may lead to the development of underrun heel conformation.The effects of nail placement for horseshoe attachment on heel and quarter expansions, hoof wall  deformations, hoof wall  surface strains, and fetlock extension were assessed during the loading of cadaveric equine limbs in vitro to simulate the middle of the stance for walk, trot, and canter loads. Treatments included no shoe (NS) and horseshoe with toe nails (T), toe plus quarter nails (TQ), or toe plus quarter plus heel nails (TQH). The order of the three nail treatments was different among individual limbs so that the variance associated with the effect of the order of treatment  could be partitioned out from the variance associated with the effect of nail placement to minimize the effect of the test order on statistical nail treatment outcomes. In addition, NS treatments were included before and after the assigned nail treatments for comparison with the nail treatments and to determine if repeated limb loading affected outcome measures . Treatment effects were assessed using a repeated measures analysis of variance (ANOVA).A sample of nine unilateral cadaver forelimbs from nine horses (mean age 14 years ± 4.1 (SD); 5 left, 4 right; 3 female, 3 male, 3 unknown; 3 Quarter Horse, 1 Paint, 1 Andalusian, 2 Thoroughbred, 2 unknown breed) were removed by transection at the level of the middle of the radius to maintain the passive components of the fetlock stay apparatus . The lim6 N/m) filled the solar surface of the hoof to the level of the weight bearing surface of the unshod or shod hoof. Nails were placed in the pre-existing holes for respective treatments. Due to the location of the hoof markers and strain gauges, the need to maintain the shoe in the same position on the hoof for all treatments, and the likelihood that driving nails in an instrumented hoof would detach the strain gauges from the hoof, the same nail holes were used from when the shoe was initially fitted for all nail treatments. Since the nails were removed and replaced according to the treatment order assignment for each limb, the nails were not clenched.The hooves were trimmed and shod by one Certified Journeyman Farrier to ensure medial and lateral balance was achieved. The shoe was attached to the hoof by the toe, quarter, and heel nails A. Then, The fetlock angle was quantified using kinematic markers on mediolateral transfixation pins located through the centers of the proximal and distal aspects of the third metacarpal bone (MC3) and proximal phalanx (P1) D. Dorso-Global hoof wall deformations were measured with kinematic markers applied to the medial and lateral aspects of the heels and quarters, at approximately 33% and 66% of the distance from the hairline to the solar margin of the hoof B,C. SpheLocal hoof wall deformations were measured  on the lateral side of the hoof wall at six locations: three proximal-distal locations  on the quarter (widest part of the hoof) and heel . Locations are defined as the proximal heel (PH), middle heel (MH), distal heel (DH), proximal quarter (PQ), middle quarter (MQ), and distal quarter (DQ) B. GaugesMechanical loading was performed in a servohydraulic material testing system  equipped with an axial-torsional load transducer  and software . The proximal end of the radius was potted in an aluminum cylinder with polymethylmethacrylate , which was secured to the mechanical testing system. The foot was secured to the system actuator via a translation table on linear bearings to allow the hoof to translate dorsally during limb loading, which maintained the radius and metacarpal bones parallel to the axis of loading. The initial positioning was standardized by obtaining a physiologic (mean 210°) palmar fetlock angle at a load of ~700 N. The limb was preconditioned by 200 sinusoidal cycles (700–1800 N) at 0.25 Hz under axial displacement control with rotation displacement fixed. Subsequently, the limb was similarly loaded (700–6700 N) for three sinusoidal cycles at 0.25 Hz (~3350 N/s) to capture the peak vertical forces reported for stance (1800 N), walk (3600 N), trot (4600 N), and canter (6700 N) ,14, whilData were analyzed for 3600, 4600, and 6200 N loads. Since one limb did not achieve 6700 N load, 6200 N was used as the experimental load for all limbs. Principal strain magnitudes and directions were calculated based on equations by Schajer . The hoop > 0.05) for any variable and was therefore removed from the ANOVA model. To determine whether the order influenced the outcomes, ANOVA was performed on the data without NS treatment. If the order was found to be significant, and the residuals were normally distributed (Shapiro–Wilks test), the model assumptions were assumed to be valid. If the order was not found to be significant, the order effect was removed from the model, the initial NS treatment was added back in, and the model was re-run. An analysis of variance  that accounted for repeated measures was used to assess the effects of being barefoot (NS) and shod with shoes of different nail positions  on the hoof wall strains, deformations, heel expansion, and fetlock angle using a commercial statistical program . Differences between the initial and final NS treatment were not detected, so the initial NS measurements were used for the NS data points. The treatment, load, order, and treatment*load interaction were treated as fixed effects. The horse was treated as a random effect. The treatment*load interaction was not statistically significant  were examined for normality using a Shapiro–Wilks test . The residuals were considered normally distributed if W ≥ 0.9 and the residuals were aligned on the QQ plot. For variables that had ANOVA residuals that were not normally distributed, a box plot  was used to determine outliers (observations that fell below Q1 − 1.5 IQR or above Q3 + 1.5 IQR). The ANOVA was performed after outlier removal, and the residuals were assessed for normality. For variables with non-normally distributed ANOVA residuals, the Box Cox procedure was used to determine a transformation appropriate for each variable . For variables that had any negative values, a constant  was added to all values for the application of the Box Cox procedure. The transformed data were again run through the ANOVA model, and the residuals were assessed for normality. For variables that were still non-normally distributed, raw data were transformed using the Rank procedure (Proc Rank), and ranked data were analyzed using the analysis of variance. A Fetlock extension increased significantly with an increasing load  but was The largest differences in expansion were observed in the heel location. Proximal and distal heel expansions increased with increasing loads . ExpansiThe distances between proximal and distal heel markers decreased with the addition of more palmar nails , indicatThe Proximal, Distal, and PrPa-DiDo segment lengths, parallel and oblique to the solar aspect of the hoof, respectively, increased with increasing loads . The DorNail treatment differences were observed for the Distal and Palmar segments . The DisThe principal tensile strains decreased for the PH, MH, and PQ gauges, while they increased for the DH, MQ, and DQ gauges as the load was increased; however, only the heel gauges were found to have significant differences between loads . The priTensile principal strains for distal heel (DH) and all quarter gauges differed among nail treatments . TensileThe PH, MQ, and DQ compressive principal strains differed among nail treatments . The MQ Maximum shear strains in the MQ and DQ gauges increased with the addition of nails placed more palmar; however, maximum shear strains varied inconsistently among treatments for PH and PQ gauges . Principal strain directions did not differ significantly among loads but differed among treatments . The priPrincipal compressive strain directions tended to roughly align perpendicular to the hoof wall tubules . The largest changes in direction occurred with the TQH treatment and were most pronounced in the MH and DQ locations  at the coronary region. Hoof growth both responds to and reflects hoof loading conditions. In hooves where there is a medio-lateral imbalance, the side where the hoof bears more loads have slower growth compared to the side that experiences less of a load . While hSeveral findings associated with the addition of heel nails could contribute to the development of underrun heels. The direction of the principal tensile strain in the distal heel and quarter regions changed the orientation to exert dorsal traction on the distal aspect of the heel when the hoof is fixed to the ground A. Simila6 N/m) and was less stiff than typical surfaces on which horses are generally worked . The effect of the nail position  could not be separated from the effect of the number of nails applied  because the addition of nails was only performed in the palmar direction. Finally, while there have been no studies, to our knowledge, on the effects of freezing on hoof tissue and its mechanical behavior compared to that of fresh tissue, we acknowledge that there is a potential for freezing and thawing to affect the results. However, since some of our results in strain and hoof expansion measurements do match those that were performed in live horse hooves, we are confident that the effects of freezing on the hoof are minimal.Limitations of the current study include the in vitro use of cadaver limbs. Pins placed within the bones and hoof wall precluded the use of live horses and restricted the assessment of deformations to pin locations. The potential effects of pins on the strain results were minimized by placing pins distant to strain gauges, considering Saint-Venant’s principle . SpeckleIn conclusion, shoeing with the addition of nails palmar to the widest portion of the hoof (quarters) reduces hoof expansion and hoof wall deformation at the heels and alters hoof wall principal strain directions in a manner consistent with mechanisms leading to the development of underrun heels and thus injury risk. The practice of placing nails more palmarly in relation to the quarters has been observed in racing horses , and inj


Corrosion constitutes a significant issue in industries that handle metals. Corrosion inhibitors with a low impact on the environment provide a significant economic benefit in various engineering applications. In this work, the effectiveness of olive leaves extract is evaluated as a cost-effective and environmentally-friendly corrosion inhibitor. The corrosion of carbon steel in different concentrations of hydrochloric acid  when protected by an aqueous solution of olive leaf extract of concentrations ranging from 10 to 60 ppm is investigated. A green extraction process based upon water extraction is used to ensure minimum impact on the environment. Results show that the corrosion inhibition efficiency increased as the concentration of the olive leaf extract increased. An analysis of variance showed a significant effect of acidic molarity, temperature, and inhibitor concentration on the corrosion rate. A significant statistical model indicates that the inhibitor exhibits higher efficiencies at higher acidic molarity. Results of SEM and EDX also demonstrated that a protective film of the inhibitor on the specimen surface plays a role in corrosion inhibition, suggesting that the inhibitor molecules are adsorbed at the interface between the carbon steel and the acid solution. The study provides an insight on the corrosion mechanism and highlights the potential of olive oil extract as an eco-friendly alternative to traditional corrosion inhibitors. This process is a significant challenge in applications involving steel structures3, leading to equipment breakdown, reduced production, and safety concerns4. For instance, corrosion in pipelines used for oil and gas transportation is a major issue and has been observed to arise within pipelines, with an estimated annual cost of $1.372 billion5. In some estimates, the cumulative annual cost of corrosion is valued at $1.372 billion, consisting of the overall expenses associated with the surface pipeline and facility costs ($589 million), down-hole tubing costs ($463 million), and capital expenses ($320 million). In addition, the cumulative yearly expenditure on corrosion amounts to a staggering $70 billion; with a potential saving of 14% achievable through the direct application of currently available anti-corrosion methods6.Corrosion is a characteristic phenomenon that reveals itself in metals, where they tend to revert to their original state, metal oxide due to environmental factors7. The utilization of natural resources, known as green inhibitors, has gained momentum due to the imperative to minimize environmental impacts. Green corrosion inhibitors are used in many industries, including oil and gas, automotive, and construction to protect metal equipment and infrastructure from rust and corrosion caused by exposure to harsh environments such as saltwater, hydrogen sulfide gases, and acidic conditions, these inhibitors help extend the life of pipelines, storage tanks, and other equipment, minimizing maintenance and replacement costs8.To mitigate corrosion, chemical inhibitors are commonly used, but their negative effects on human health and the environment have led to a growing interest in exploring natural substances as eco-friendly corrosion inhibitors10. Among the array of natural products explored for their potential as corrosion inhibitors for various metals are peels, alum sludge, and olive leaves14.Plant extracts have been extensively investigated as environmentally friendly corrosion inhibitors since the 1960s15 because olive waste products are found in abundance in both countries as olive-growing and exporting countries. It is known that olive leaves contain antioxidants and tannins, which strongly contribute to its use as an inhibitor12.Olive leaf extract has garnered significant attention in Turkey and EgyptExtensive research has been conducted to address the environmental concerns associated with synthetic corrosion inhibitors by exploring natural resources as innovative and environmentally-friendly alternatives. Among countries with a rich agricultural tradition, such as Egypt and Turkey, the cultivation of olive trees has been prominent for centuries. Olive trees thrive in elevated temperate and arid regions, resulting in significant agricultural waste in the form of olive leaves. The potential utilization of olive leaves as a corrosion inhibitor offers a promising opportunity to transform this waste into a valuable resource while effectively addressing environmental concerns.16.Olive leaves are rich in various phenolic compounds, including oleuropein, hydroxytyrosol, and tyrosol, which have been extensively studied for their notable antioxidant, antimicrobial, and anti-inflammatory properties17 studied the inhibiting action of olive leaf extract on corrosion of copper in a NaCl solution, observing an inhibition efficiency of up to 90%. They suggested that oleuropein was the major compound of the leaf extract and thus more likely the main inhibiting species. However, the authors did not provide sufficient details on the mechanism of inhibition and used a single acid molarity. Elabbasy18 studied the inhibiting action of olive leaf extract on corrosion of C-steel in Sulfamic acid solution, observing an inhibition efficiency of up to 90%. Their observations using Scanning Electron Microscopy (SEM) suggested that the inhibition mechanism involves physisorption. These studies imply that olive leaf extract could potentially serve as a sustainable corrosion inhibitor. Nevertheless, further research is required to fully comprehend the mechanism of action of olive leaf extract and optimize its use as a corrosion inhibitor.Recently, olive leaf extract has been proposed as a corrosion inhibition agent. Refait et al.19. The inhibitor molecules undergo adsorption onto the metal surface, leading to the creation of a protective layer that serves to impede the reaction of the metal with the corrosive environment20. The efficacy of the inhibitor is predicated on various factors, including the concentration of the inhibitor, temperature, the nature of the metal and PH. The effectiveness of OLE as a corrosion inhibitor is significantly influenced by the pH of the solution in which it is employed21. At specific pH values, the OLE may exhibit greater potential in generating a protective film on the metal surface. many studies revealed that when decreasing the pH from 8 to 3.9, different changes were observed in the corrosion potential after adding the inhibitor and that the efficiency of the inhibitor decreases with increasing acidity, and also the weight loss decreases with increasing the pH from 3.15 to 8.023 It was noticed in the practical results that as the acidity of the solution increased and the pH decreased, the effectiveness of the extract decreased. Therefore, comprehending the correlation between pH and its impact on the efficacy of OLE as a corrosion inhibitor is of utmost importance in optimizing its usage.The process of inhibiting corrosion by olive extract entails the interplay between the molecules of the inhibitor and the surface of the metalWhile it is evident that olive leaf extract is an environmentally-friendly and cheap extraction solution and can potentially offer a sustainable solution for corrosion management, the discussion above reveals gaps in the data available in literature on the mechanism of the corrosion inhibition. In addition, there is no detailed information regarding the water heating extraction process. In this article, the use of olive leaf extract as a corrosion inhibition agent for carbon steel in acidic media is experimentally investigated. The efficacy of an extract obtained from waste olive leaf is evaluated as a potent inhibitor for X70 carbon steel alloy submerged in different concentrations of hydrochloric acid and at different temperatures. Measurements of metal loss, along with Scanning Electron Microscopy and Energy-Dispersive X-ray spectroscopy are used to investigate the mechanism of corrosion inhibition.Samples of X70 Carbon Steel (CS)with dimensions of 2 × 1 × 0.02 cm (± 1%), were prepared. The samples experienced a thorough cleaning process and were subsequently polished with emery paper until their surface exhibited a glossy and smooth appearance. Following this, the samples were strictly rinsed with distilled water before being subjected to treatment with acetone.24. Subsequently, the extraction solution was filtered to remove the grounded olive leaves as shown in Fig. Olive leaves collected as wastes from the factory presented the olive oil in Egypt and Turkey. Olive leaf extract (OLE) was produced through the incorporation of 10 g of pulverized olive leaves with 100 ml of distilled water. Two sources for olive leaves were used, originating from Egypt and Turkey. Extraction was carried on at 100 °C during 60 min with a 500 rpm mixing speedThe chemical composition of the samples was performed using Trace GC1310-ISQ mass spectrometer  with a direct capillary column TG–5MS (30 m × 0.25 mm × 0.25 µm film thickness). The column oven temperature was initially held at 50 °C and then increased by 5 °C/min to 230 °C hold for 2 min. Increased to the final temperature 290 °C by 30 °C/min and hold for 2 min. The injector and MS transfer line temperatures were kept at 250, 260 °C respectively; Helium was used as a carrier gas at a constant flow rate of 1 ml/min. The solvent delay was 3 min and diluted samples of 1 µl were injected automatically using Autosampler AS1300 coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 40–1000 in full scan mode. The ion source temperature was set at 200 °C. The components were identified by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 11 mass spectral database.1 and W2 express weight loss of the samples before and after submerging the treated metal samples in the corrosive medium.Samples of carbon steel were employed to measure weight loss. The samples were first smoothed with emery paper, then weighed, and subsequently exposed to varying concentrations of hydrochloric acid  in the absence and presence of a range of concentrations of olive leaf extract for eight hours at ambient temperature. The carbon steel samples were subsequently extracted, washed with water, cleaned with acetone, dried, and then strictly weighed on a lab scale with an accuracy of 0.01 g. The inhibitory effectiveness percentage (IE%) was determined according to Eq. (CR was determined from Eq. (And the corrosion rate from Eq. :2\documeg to Eq. :3\documeThe utilization of the scanning electron microscopy (SEM) technique allowed for the demonstration of OLE inhibitor adsorption by surface characterization of carbon steel with and without the corrosion inhibiting treatment. Surface morphology investigations were conducted on CS immersed in 0.5 M HCl solution for 3 h before and after the addition of inhibitor, using an analytical scanning electron microscope (Quanta FEG 250) at the EDRC, DRC, Cairo.25. The statistical technique known as analysis of variance (ANOVA) can be used. This technique is based on variance ratios to significant differences exist among the means of several groups of observation. ANOVA analysis involves the consideration of inputs, which belong to the independent variable or the factor that is being tested. In this research, the independent variables consist of the inhibitor type, inhibitor concentration, concentration of corrosive media and the temperature at which the inhibitor is subjected to testing. The response, which is the dependent variable, is the corrosion rate that is being measured.The response surface methodology refers to a collection of statistical and mathematical techniques employed in the modelling and analysis of problems where the response of interest is contingent on diverse variablesTwo-way ANOVA has been used to determine the effect of temperature and inhibitor concentration on corrosion rate at different corrosive solution,  HCL. The analysis determines whether variations in temperature affects and inhibitor concentration result in significant differences in inhibitor effectiveness.26.The null hypothesis is rejected if the computed F values (F) are too largeThe collection and handling of plants leaves were in accordance with all the relevant guidelines.Several parameters, such as temperature, concentration of corrosion inhibitors, composition of the corrosive medium, surface features, and environmental conditions, have an effect on the rate of corrosion. By controlling and changing these input factors, it is possible to change the rate of corrosion and possibly slow it down. In this section, the effect of selected parameters on the effectiveness of oil leaf extract as a corrosion inhibition agent is analysed.27.The results obtained from gas chromatography-mass spectrometry (GC–MS), as presented in Table Weight loss and corrosion rates of carbon steel samples treated with OLE solutions obtained from samples originating in Egypt and Turkey for 8 h, under the effect of considering different temperatures and HCl molarities. The inhibition efficiencies values were calculated and compared for the OLE inhibitor, at various concentrations for 8 h of immersion at different temperature, and the obtained results are summarized in Table 28. This effect is particularly noticeable at a concentration of 60 ppm, where the inhibitor demonstrates superior effectiveness compared to a lower concentration.The data in Table Scanning Electron Microscopy (SEM) images provide important insights into the surface characteristics and microstructure of materials, allowing to characterize and evaluate their properties at the micro- or nanoscale. In this section, Fig. The severe corrosion is evident through the presence of large and deep vacuoles on the steel surface. However, the SEM image in Fig. 29. These observations indicate a reduction in the corrosion of metal after the addition OLE inhibitor. This surface characterization is consistent with the results obtained by gravimetric measurements and statistical calculations.Additionally, in order to recognize the elemental composition of the metal surface samples before and after implementation of the studied inhibitor, Energy-Dispersive X-ray spectroscopy (EDX) was carried out and examined. It is evident that the surface of metal which immersed in uninhibited solution composed of a significant proportion of atoms consisting of iron, oxygen, and carbon as shown in Fig. (RSM) is a statistical technique utilized for the purpose of enhancing and optimizing processes through the examination of the correlation between input variables and response variables. The identification of optimal input variable settings is crucial for achieving the desired response and providing effective and productive process optimization.An analysis of variance (ANOVA) was performed to investigate the influence of OLE concentration on corrosion inhibition. The study utilized a Taguchi Orthogonal Array (OA) design, which involved the integration of three levels of temperature, three levels of OLE concentration, three levels of corrosive media molarity, and two distinct sources of olive leaves. Table A screening test was carried out and the origin of OLE did not have any significant effect on corrosion inhibition. Subsequently, the effect of inhibitor concentration, temperature and time as independent variables along with their interactions up to the third degree on the inhibition efficiency as a response variable was screened via a full factorial model.The results of the screening model illustrated a strongly significant effect of OLE concentration and the molarity of the solution. The temperature effect on the inhibition efficiency was marginally significant. The interaction between OLE concentration and the Molarity was the only second or third order effect to have an effect on the inhibition efficiency. The detailed parameters of the screening model are shown in Table To analyze the effect of the significant parameters on the corrosion inhibition efficiency, an Analysis of Variance was performed considering only the first order parameters along with the interaction between Molarity and OLE concentration. The parameters of this due to the reduction of the noise error by disregarding the contribution of insignificant variables, the F ratios are improved compared to the full factorial model.The second-order model predicts that the corrosion inhibition efficiency is related to the independent effects by the form in Eq. , with hiThe model predictions agree well with the measurements as shown in Fig. Interaction plots of the three parameters are presented in Fig. The increase in OLE concentration leads to a significant increase in inhibition efficiency at all temperatures and all solution molarity values. On the other hand, the interaction between molarity and OLE concentration results in a higher inhibition effect at higher solution molarities, resulting in enhanced protection when required.Figure From the previous discussion, results indicate that the corrosion inhibition activity of OLE may be due to the presence of various organic compounds in its composition such as phenolic compounds, tannins, flavonoids and oleuropein. These organic compounds contain polar function groups with hetero atom such as O in addition to conjugated double bonds or aromatic rings, which are the major adsorption centers.30. The corrosion medium generates hydrogen ions on the outer layer of metal, which combine with other ions to form hydrogen gas. However, inhibitory compounds can generate neutral molecules that attach to the metal surface, forming a protective layer instead of hydrogen ions, thereby inhibiting corrosion31. Consequently, it may be suggested that the protective films may be formed on the metal surface through the adsorption process to inhibit the corrosion of CS in HCl medium.The inhibitory process involves the absorption of compounds by the metallic exterior, resulting in the formation of a protective layer that shields the alloy from corrosionIn this study, the use of an environmentally friendly extract from olive leaf as a corrosion inhibition agent is considered to address economic and environmental challenges while also providing additional revenue opportunities through the utilization of an underappreciated material. A process to produce an aqueous extract from oil leave waste is proposed. The effect of treating carbon steel surfaces with the olive oil extract is analyzed through metal-loss measurements, SEM, chromatography-mass spectrometry, and EDX to quantify corrosion inhibition efficiency and give an insight on its mechanism. Results showed that olive leaf extract exhibits corrosion inhibition properties on carbon steel surfaces, with an efficiency of 53% at an inhibitor concentration of 60 ppm compared with the reference case. Statistical analysis confirms that corrosion rate is affected by temperature, inhibitor concentration, and their synergistic impact. SEM analysis further reveal that the olive leaf extract acts as an inhibitor through adsorption, effectively blocking the active sites on the carbon-steel surface. This inhibitory process is facilitated by the interaction between the organic component in the olive leaf extract and the metal surface. Olive leaf extract offers promising potential as a green corrosion inhibitor in various applications, such as oil and gas, construction, and transportation. By promoting the use of olive leaf extract as a green corrosion inhibitor, we can reduce the dependence on toxic chemicals and contribute to a more sustainable future, while reducing the environmental footprint of heavy industries. Given that it is a natural and biodegradable product, it could also contribute to cost-efficiency as it represents a cost-effective alternative to traditional corrosion inhibitors (Supplementary Figure Supplementary Figures.Supplementary Information 2.


Inhibitory neurons in the midbrain spatial attention network, called isthmi pars magnocellularis (Imc), control stimulus selection by the sensorimotor and attentional hub, the optic tectum (OT). Here, we investigate in the barn owl how classical as well as extraclassical  inhibitory surrounds of Imc receptive fields (RFs), fundamental units of Imc computational function, are constructed. We find that focal, reversible blockade of GABAergic input onto Imc neurons disconnects their extraclassical inhibitory surrounds, but leaves intact their classical inhibitory surrounds. Subsequently, with paired recordings and iontophoresis, first at spatially aligned site-pairs in Imc and OT, and then, at mutually distant site-pairs within Imc, we demonstrate that classical inhibitory surrounds of Imc RFs are inherited from OT, but their extraclassical inhibitory surrounds are constructed within Imc. These results reveal key design principles of the midbrain spatial attention circuit and highlight the critical importance of competitive interactions within Imc for its operation. Imc is a midbrain inhibitory nucleus essential for stimulus selection across space. Here, the authors show in the barn owl that global inhibitory surrounds of Imc neurons are constructed within Imc, but their classical inhibitory surrounds are inherited from inputs. Equally essential is the processing and representation of the stimulus at the selected location. For neurons involved in spatial selection, a core characteristic that impacts both these functions is their spatial receptive field (RF), defined as the subset of the spatial locations that a neuron responds to selectively. The excitatory center and classical inhibitory surround of the RF together control the responses of neurons to a stimulus inside the RF, whereas the extraclassical surround controls the modulation of the neuron’s responses by a competing stimulus outside the RF. Thus, understanding how classical and extraclassical surrounds are constructed is essential for understanding how neurons involved in selection achieve both competitive selection among multiple competing stimuli as well as the processing and representation of the selected target.Animals behave in complex environments and are constantly faced with multiple competing stimuli. Selecting the location with the most “important” or highest priority stimulus to guide behavior at any instant is an essential part of adaptive behavior, and operates upon the foundation of a spatial map of relative stimulus priority7. The SC/OT is required for the control of spatial selection and selective attention when a target is present amidst distracters10, with OT neurons signaling the highest priority stimulus among competing stimuli categorically13. Notably, these competitive interactions within the OT are controlled by long-range inhibition generated by GABAergic neurons in the nearby isthmi pars magnocellularis  is a major sensorimotor hub in the midbrain Fig. . SC/OT n18, exhibit both classical inhibitory surrounds and extraclassical competitive surrounds20 Specifically, responses of Imc neurons to a bar stimulus of increasing length (or a circular stimulus of increasing size) have been shown to drop to low values at large lengths (sizes), demonstrating the presence of classical inhibitory surrounds18. In parallel, responses of Imc neurons to a stimulus inside the RF are divisively suppressed by a second stimulus anywhere outside the RF, demonstrating a global, extraclassical inhibitory surround. Whereas the source of the excitatory drive for Imc receptive fields is known to be neurons in layer 10 of the OT (OT10)21, how the inhibitory surrounds of Imc receptive fields are constructed is not understood. Addressing this question is key to understanding how the map of relative stimulus priority is constructed and stimulus selection orchestrated in the OT.In turn, Imc neurons, which have well-defined spatial receptive fields10 neurons – the sole source of excitatory input to Imc neurons. Finally, with paired recording experiments at spatially misaligned sites within Imc, we demonstrate that global competitive surrounds of Imc RFs are controlled by long-range inhibition from distant Imc neurons. Our results reveal that distinct mechanisms are involved in the construction of the inhibitory surrounds of Imc neurons: classical, local inhibitory surrounds are conferred onto Imc neurons by OT10 neurons, while global, competitive surrounds are constructed within the Imc using inhibition from distant Imc neurons.Here, we systematically dissect the mechanisms by which the inhibitory surrounds of Imc neurons in the barn owl are constructed. We do so in a series of experiments involving extracellular recordings in the Imc coupled with iontophoretic silencing of GABAergic input onto Imc neurons, silencing of GABAergic input onto spatially aligned OT sites, or silencing of excitatory (glutamatergic) input onto other/distant Imc sites. First, with iontophoretic blockade of GABAergic input on Imc neurons, we show that whereas global competitive surrounds of Imc neurons are abolished, the classical inhibitory surrounds are not affected. Then, with paired recording experiments at spatially aligned sites in the Imc and OT, we demonstrate that classical inhibitory surrounds of Imc RFs are controlled by inhibition onto OT20 was due to a comparison occurring at the Imc site itself, or if this response reduction reflected computations occurring elsewhere.We first investigated mechanisms underlying the global, extraclassical inhibitory surrounds exhibited by Imc neurons. Specifically, we asked if the reduction of stimulus-evoked Imc responses by a distant competitor presented outside the RFp = 0.044, permutation test (GABA blockade vs. baseline slopes)).To this end, we conducted extracellular recordings in the Imc using multibarrel glass iontophoresis electrodes filled with a bicuculline methiodide solution Fig. , Methodsn = 40, following the removal of four outliers; Methods), we found that bicuculline iontophoresis at Imc consistently weakened this competitor-dependent response reduction . We verified that these results were specifically due to drug iontophoresis by measuring responses following recovery from bicuculline iontophoresis at a subset of Imc sites . We found that competitor-dependent response reduction returned to strong, near-baseline levels . This recovery occurred despite a small, but progressive reduction in the maximum evoked firing rates to S1 alone over time—from the baseline through recovery conditions, consistent with typical time-dependent run-down effects during extended electrophysiological recordings  surrounds are thus constructed locally at the Imc neurons themselves.; Fig. 2A)23. This protocol is well-established to produce response profiles as a function of bar length in which firing rates rise to a peak before decreasing to an asymptotic value  without and with bicuculline iontophoresis Fig.  at Imc sp = 0.438; permutation test).We found that the responses of Imc sites to the bar-length protocol exhibited the classic shape . Notably, nearly all these sites (n = 24/28) were the same as those at which we also obtained the tuning curve data for Fig. n = 8) of the sites .Across a population of Imc sites at which we presented bars of different lengths, we found no systematic effect of bicuculline iontophoresis on SI  provide focal input to Imc neurons, and are the only known source of excitatory drive to them21. We, therefore, tested if Imc classical surrounds reflect those of OT10, by performing paired recordings of classical inhibitory surrounds at spatially aligned Imc and OT10 sites, without and with iontophoresis of bicuculline onto OT10 neurons , p = 0.002; permutation test), indicating weakening of the classical inhibitory surround in OT10. Across a population of OT10 sites, we found that GABA blockade consistently reduced SI . Notably, SI values returned to near-baseline following recovery from bicuculline iontophoresis . Thus, GABA blockade in OT10 weakened the classical inhibitory surrounds of OT10 neurons.To this end, we recorded bar length response profiles at paired Imc-OTtes Fig. . We firsT10 Fig. , Methods10 GABA blockade on bar-length response profiles at paired  Imc sites , we also found a significant decrease in the SI value following GABA blockade at the paired OT10 site, indicating weakening of Imc’s classical inhibitory surround , p = 0; permutation test). Across a population of paired  Imc-OT10 sites, we found that GABA blockade at OT10 consistently reduced SI at Imc sites . Indeed, SI values returned to near-baseline values following recovery from OT10 bicuculline iontophoresis, demonstrating that the observed effects on SI were due to the drug . Thus, GABA blockade in OT10 also weakened the classical inhibitory surrounds of Imc neurons.Next, we examined the effect of this OTtes Fig. ; Methods10 sites increased upon GABA blockade at OT10 and decreased after recovery from the drug  surrounds in Imc, we conducted paired recordings at two mutually distant sites within the Imc . This effect was consistent across the population of tested site pairs : inactivation of Imc site B significantly weakened S2-induced suppression of S1 responses at Imc site A .We found that in the baseline condition Fig. , S2 effep = 0.004, baseline vs. site B inactivation; p = 0.004, site B inactivation vs. recovery; p = 0.82 baseline vs. recovery, paired sign rank tests corrected for multiple comparisons).The observed effects were due, specifically, to drug iontophoresis. First, evoked responses at site B, which were inactivated by kynurenic acid Fig. , returne16, and for this reason, in our experiments here, A and B sites were chosen to be much further apart (average distance = 44.7° ± 5.17°). To test any effects of potential spread directly, we performed separate experiments in which A and B sites in Imc were similarly far apart , unlike at B sites at which they were suppressed significantly .We were able to rule out the possibility that the observed effects were due to potential spread of kynurenic acid from Imc site B to Imc site A. We have shown previously that iontophoresis of kynurenic acid at an Imc site does not block evoked responses at distant Imc sites whose RFs are located more than 25° awayTogether, these results established that extraclassical inhibitory surrounds of Imc neurons are constructed at the Imc using long-range inhibition from distant Imc neurons, i.e., intra-Imc inhibition is the source of Imc’s extraclassical inhibitory surrounds.8. In the parallel thalamo-cortical pathway of visual processing, a related debate on whether the classical surrounds of simple cells in V128 are constructed within V1 by the action of local inhibitory neurons, or by modulation of excitatory inputs from upstream LGN cells, was only recently resolved29. Selective manipulation of inhibitory neurons within V1 in mice showed that cortical inhibition played a key role in the construction of the classical surround30, thereby establishing a clear mechanism for a basic function of V1 neurons.This study uncovers the mechanistic implementation of classical as well as global inhibitory surrounds of neurons in the Imc, a key midbrain inhibitory nucleus in the tecto-fugal pathway for visuomotor  processing in vertebrates31. In turn, neurons in the inhibitory Imc control the competitive interactions within OT16. This inhibitory output of Imc neurons reflects computations occurring within Imc rather than being just a sign-flipped version of excitatory inputs into Imc20. Indeed, the Imc itself encodes a map of relative stimulus priority20, much like the SC/OT – the first reported in inhibitory neurons, to the best of our knowledge. In this context, then, how the responses of Imc neurons to (single and multiple competing) stimuli are constructed, and specifically, how Imc receptive fields are constructed, is a critical, but unanswered, question.In the tecto-fugal pathway, the sensorimotor hub, SC/OT, is not only involved in the processing of individual sensory stimuli, but also plays a critical role in the selection of the highest priority stimulus  among competing distracters10 neurons have clearly identified OT10 as the source of Imc’s excitatory drive21. However, although that study also examined the effect of iontophoretic blockade of GABAergic input in Imc on Imc responses21, the results are difficult to interpret in the context of the construction of Imc’s inhibitory surrounds. In that study, data obtained from the presentation of a single stimulus inside the RF, and from the presentation of two competing stimuli (one inside and one outside the RF), were combined and reported together as a single result. Classical versus global  surrounds have fundamentally different properties in terms of their function, strength profiles, spatial scope, and requirements of the underlying circuitry27. As a result, the properties of one cannot predict those of the other27, highlighting the critical need for considering results from single stimulus versus two-stimulus (competition) protocols separately in order to disambiguate the mechanistic underpinnings of the respective inhibitory surrounds.Past work involving recordings in the Imc paired with focal iontophoretic silencing of OT26 create global inhibitory surrounds of Imc RFs 32. Due to this specialized anatomical and functional feature, Imc does not participate in shaping OTid’s responses to single stimuli. Consequently, not having a dedicated mechanism within the Imc for generating classical inhibitory surrounds  avoids potentially ‘wasteful’ circuitry that would not aid the core functional role of Imc.By contrast, Imc neurons have been shown to not send inhibition to the portion of the OT space map from which they receive input , and as well, on competitive surrounds .The goal of this study was to determine the mechanisms underlying the construction of classical and extraclassical inhibitory surrounds of Imc neurons. This was done by measuring (i) spatial tuning curves at Imc neurons using a visual stimulus , (ii) with or without a second visual stimulus present outside of the RF (S2), and separately, measuring (iii) responses to bar stimuli of increasing lengths. All these measurements were made in baseline conditions, and while microiontophoretically applying bicuculline methiodide (Sigma-Aldrich), a GABA10 neurons in the construction of classical inhibitory surrounds of Imc neurons by applying bicuculline methiodide at OT10 sites while simultaneously recording tuning curves and bar length response profiles at both the OT10 site and a paired Imc site encoding for the same area of sensory space . Further, to examine the role of Imc neurons in mediating the extraclassical surrounds of other (distant) Imc neurons, we recorded from one Imc site encoding for S1 (site A) without and with S2 presented far outside of the site’s RF (>30° away). A second electrode, placed at the distant Imc site encoding for S2 , was then used to inactivate Imc site B (using kynurenic acid). We compared the responses of Imc sites A in the intact versus site B-inactivated cases. Additionally, in control experiments to assess potential spread of kynurenic acid iontophoresis at site B to site A, we compared the responses of Imc sites A in the intact versus site B-inactivated cases while recording spatial receptive fields at both sites.Additionally, we examined the role of OTEight adult barn owls  were used for electrophysiological recordings. All protocols and animal care were in accordance with NIH guidelines for care and use of laboratory animals and approved by the Johns Hopkins University Institutional Animal Care and Use Committee. Birds were shared across different studies and group housed in an aviary with a 12 h/12 h light/dark cycle. Before electrophysiological experiments, head bolts were affixed to the skull under anesthesia . Birds were administered intramuscular injections of 0.1 mL of meloxicam and 0.1 mL of butorphanol, incision areas were disinfected with betadine, and locally anesthetized with subcutaneously injected bupivacaine. Bilateral craniotomies were performed and small plastic cylinders with removable caps were placed on the skull to allow access to midbrain structures over multiple experiments. Polysporin antibiotic ointment was applied to any exposed brain surface and incisions. Owls were returned to the aviary following recovery from surgery, and experiments were performed starting after a week of recovery.34.On experiment days, owls were initially anesthetized with isoflurane (1–2%) and a mixture of nitrous oxide and oxygen (45:55), and administered with intramuscular injections of 0.1 mL of meloxicam and 0.1 mL of butorphanol. Birds were then secured in a sound-attenuating booth, and head-fixation was calibrated for each owl as follows. The pupils were dilated with atropine eye drops, and the pectin structures in the eye were sighted using an ophthalmoscope. The position of the head of the owl was adjusted in roll, pitch and yaw directions such that the pectin structures in the two eyes were positioned symmetrically on either side of the midline , and positioned approximately 7 degrees above horizon11.Once the bird was calibrated, isoflurane was turned off after the bird was secured, and owls were maintained on oxygen and nitrous oxide for the duration of the experiment. As recovery from isoflurane occurs well under 30 min after it is turned off, recordings were made in animals that were not anesthetized. When possible, nitrous oxide as well was turned off 5 min before data collection . Notably, previous work has demonstrated that neural responses in the midbrain network do not differ under nitrous oxide tranquilization from non-tranquilized conditions16 and with electrolytic lesions17, and established its location as approximately 500 μm medial to the medial-most part of the OT. Recording sites in the Imc were targeted by either navigating first to the optic tectum and then to the Imc using the OT’s topographic space map as ref. 20, or by referencing reliable stereotaxic coordinates from prior experiments and verified on the basis of established distinct neural responses27. For recording in OT10, OT layers were identified by their distinctive neuronal responses34.The Imc is an oblong structure in the avian midbrain that is elongated along the rostrocaudal axis, parallel to the OT. Previous work has confirmed in vivo targeting of the Imc with fluorescent dye injection10-Imc recordings, the OT10 targeting electrode was also angled at a 5° medial-leading angle to sterically accommodate both OT10 and Imc electrodes. Extracellular activity in Imc was recorded primarily using multi-barreled glass electrodes with the central barrel containing a carbon fiber electrode for recording neural activity . Paired recordings with two electrodes utilized a multi-barreled glass electrode to administer drug and record responses at one brain site, and an epoxy-coated tungsten microelectrode  to record responses at the paired site. All data in this paper represent a combination of both well-isolated single as well as multi-unit sites. Spike times were recorded using Tucker-Davis hardware and analyzed using MATLAB.For Imc recordings, an electrode was positioned to enter the brain at a medial-leading angle of 5° to avoid a major blood vessel. During some paired Imc-Imc recordings, electrodes were additionally angled in the caudal-leading direction (2°−5°) to accommodate space for two electrodes. For paired OT34. The drug of interest was filled in one barrel of a multi-barreled electrode and was microiontophoretically applied to the recording site. Electrophysiological responses were recorded using a carbon fiber electrode in one of the other barrels.Microiontophoresis was performed using a 1-channel iontophoresis box (DAGAN Corp PS-100). This is an established technique for focal delivery of drugs, and has been used extensively for this purpose in the literatureA antagonist bicuculline methiodide  was used. For bicuculline iontophoresis in OT10, we used ejection currents based on published work that has used bicuculline iontophoresis in owl OT . This was guided by the ranges in published work: 50–100 nA in Imc21, 30–120 nA in OT36, 20–80 nA in other regions of the central nervous system of several species41, and pilot experiments. Bicuculline was ejected at Imc or OT sites for 15 min before data collection in the drug condition and ceased for 25–35 min before data collection in the recovery condition. When not being ejected, bicuculline was retained at a current of −15nA.To achieve blockade of inhibitory synaptic input, the GABA16. Kynurenic acid was ejected for 15 min before data collection in the drug condition and ceased for 15 min before data collection in the recovery condition16. When not being ejected, Kynurenic acid was retained at +15 nA.To achieve focal blockade of excitatory synaptic input in Imc, the pan-glutamate receptor antagonist kynurenic acid  was used. For kynurenic acid iontophoresis (in Imc), we used ejection currents established in published work (−500nA)Microiontophoretic experiments typically involved three conditions: baseline, drug delivery (GABA or Kynurenic acid) and recovery. By necessity, they are performed serially. The time between the start of “baseline” data collection and the start of “drug” data collection is ~ 45 min . Similarly, the time between the start of “drug” data collection and the start of “recovery” data collection is ~ 45–60 (30 min data collection + ~15–30 min wait time for drug’s effect to wear off). Such extended electrophysiological recording sessions are known to often result in a progressive run-down of responses over time, which we also observed in our experiments Figs. , S2.27. Bar stimuli were of fixed height (3°), but varying lengths. Stimuli were presented using MATLAB and Psychtoolbox (PTB-343). The spatial locations of visual stimuli were defined by double pole coordinates relative to the midsagittal plane for azimuth or the visual plane for elevation34.Stimuli were black dots (looming stimuli) or black horizontal bars (stationary stimuli) on a gray background presented on a 65” monitor placed tangentially at a distance of 12.5” in front of the owl. The looming dot stimuli expanded linearly over a duration of 250 ms to mimic approaching objects; previous work has estasblished that looming dot stimuli evoke reliably strong responses in OT and Imc with relatively low response habituation10), we collected two-dimensional RFs (azimuth × elevation) by presenting a looming dot stimulus at various azimuthal and elevational locations. Spatial locations at which the stimulus elicited higher firing rates compared to baseline were defined as the site’s spatial RF. This was used for determining the placement of S1 (within the RF), of S2 (outside the RF), and for determining the alignment of Imc and OT10 sites.To determine the extent of space encoded by a recorded site (in Imc or OTTuning curves were measured by presenting S1 of a fixed strength (6°/s) at multiple azimuthal locations spanning the extent of a site’s RF. For examining extraclassical surrounds, S2 was presented far outside the RF, at a distant location from S1 (mean distance from RF center = 33.3 ± 0.91°). S2 loom speed (10°/s), S2 was stronger than of S1. Presentations of S1 alone, and S1 with S2, were interleaved pseudo randomly. Stimuli were presented for 15 repetitions for collecting the data in Figs. 19, and 100 ms to 250 ms (for OT1033). For bar length response curves, the count window was from 65 ms to 175 ms (for both OT10 and Imc sites27). Average rates were calculated across all presentation repetitions.All analyses were done with MATLAB scripts. Response firing rates were determined by counting the number of spikes over a time window following stimulus onset and converting this count to firing rate (sp/s) after subtracting the baseline firing rate. This window was chosen to capture well the evoked neural responses: for tuning curves, the count window was from 125 ms to 275 ms with respect to stimulus onset (for Imckstest), non-parametric tests  were used otherwise. Correction for multiple comparisons was performed using the Holm-Bonferroni method  and Dunn-Sidak for Kruskal-Wallis, if needed. All tests were two-sided. Data shown as a ± b refer to mean ± s.e.m, unless specified otherwise. For bar length response functions, the suppression index (SI), was defined as the difference between the peak response and the asymptotic response (estimated as the average response to three widest bars) divided by the peak response.All statistical analyses were performed in MATLAB. Outliers were identified as data points that lay outside the range of median ± 1.5*interquartile range of the distribution. Parametric tests  were used if data were normally distributed (tested using a Kolmogorov-Smirnov test, The permutation test comparing the SI values of bar length response profiles between baseline and GABA blockade conditions (at the example sites in Figs. The permutation test comparing the slopes of competitive suppression between baseline and GABA blockade conditions (at the example sites in Figs. For paired Imc-Imc recordings, site-pairs were included only when kynurenic acid iontophoresis caused significant reduction of responses at site B.Further information on research design is available in the Supplementary InformationReporting Summary