The closeness in the values of the spreading diameter before and after shaking on the table Dsp,1 → Dsp,2 confirms this. The dependences (regression equations) of mobility, coefficients of the Ostwald–Weil equation, and density and strength on the W/C ratio and plasticizer concentration for lightweight concrete with a volume content of hollow microspheres of 46.4% have been established. The limits for homogeneity of lightweight concrete mixtures on hollow microspheres are W/C ≤ 0.6 and CPl ≤ 1.0%. The dispersion of quartz sand (varying the Sp/Sf ratio) in an amount of 8.7% in the composition of lightweight concrete does not have a significant effect on the self-compaction criterion and physical and mechanical properties. Changes in the physical and mechanical properties of lightweight concrete on hollow microspheres in the selected range of varying the W/C ratio and plasticizer concentration are in the following ranges: ρ = 1403–1485 kg/m3, Rfl = 3.34–5.90 MPa, Rcom = 29.6–45.7 MPa. The presence of delamination at W/C ≥ 0.6 does not allow one to correctly establish the influence of variable factors.Producing self-compacting concrete with lightweight aggregates is a difficult task. Mixtures with a high content of expanded aggregate tend to separate. It is possible to evaluate the possibility of producing self-compacting lightweight concrete with low average density. This work presents the results of a study of self-compacting lightweight concrete on hollow microspheres. The ability of a lightweight concrete mixture on hollow microspheres with low density (ρ = 1450 ± 25 kg/m Considerable efforts and investments are being directed toward the search for new technologies for modern methods of construction . The stuCurrently, the process of obtaining and using SCC is very extensive ,5,6,7,8.3 usually meets the requirements for use as a structural material [The most common fillers for reducing the density of concrete are pumice, rice husk, diatomite, sawdust, volcanic cinders, scoria, sintered fly ash, artificial cinders, expanded perlite, bloated clay, coke breeze, and exfoliated vermiculite ,11. Howematerial . The autmaterial  describematerial ,20,21, t0) and plastic viscosity (µ) have been used to describe the rheology of self-compacting concretes. The thixotropy of the mixture is another important property that determines the ability of the system to maintain its uniformity [Rheological and technological characteristics and production features are the main indicators of quality for concrete mixes. An analysis of these key factors is presented in ,24,25,26iformity . The useiformity ,26. Thisiformity .A natural limitation to reducing the density of concrete is the density of the aggregate ,20,28. HA study on the rheological characteristics of a concrete mixture that had partially replaced cement with hollow glass microspheres was carried out in . It was 3) and high strength are effective structural materials. The effectiveness of the physical and mechanical properties of such concrete for the construction of multi-story buildings or long-span structures is substantiated in [It has been shown ,35 that iated in . ViolatiA natural limitation for obtaining self-compacting lightweight concrete mixtures is the presence of a lightweight aggregate, which tends to ascend when critical liquefaction is reached at a certain degree of filling of the dispersion medium . At the 3 [The purpose of this study was to evaluate the possibility of producing self-compacting lightweight concrete with a high content of lightweight aggregate. Also, this study is necessary to establish prescription limits for homogeneous compositions of lightweight concrete with an average density of about 1400 kg/m3 ,33,34.f), quartz powder (Sp), plasticizer (Pl), and water (W). The ratio of dry components (PC:MS:Sf:Sp) was constant and corresponded to the average density of concrete according to the project (1400 kg/m3). The volumetric content of lightweight aggregate was 0.464.Lightweight concretes with hollow microspheres were used as the object of this study ,36. The Ordinary Portland cement—CEM I 42.5 N manufactured by “Maltsevsky Portland Cement”  —was used2 > 95%). The average specific surface area is 20 m2/g, in accordance with [The complex silica additive “FremSilica 2”  consists of small particles of amorphous but spherically shaped silica  were used as a lightweight aggregate to reduce the average density of the concrete. The particle diameter of these microspheres is 10–500 μm. Their main properties are presented in The plasticizer Melflux 2651F  was used to control the technological properties of the concrete mixtures. Melflux 2651F is a second-generation polycarboxylate ester that does not affect the setting of cement.2/kg.Quartz powder is fine sand that is obtained from ground fractional quartz sand  in a ball mill. The specific surface area of the quartz powder we used was 720 mThe properties of the object were studied using the method of mathematical experimental planning. For this purpose, the following standard two-factor model was used:Y on variable factors (X1 and X2), significantly reducing the number of experiments.The search for equation coefficients made it possible to establish the dependence of quality parameters X1—the amount of water (W/C ratio)—and X2—the concentration of the plasticizer —were used to evaluate the technological and rheological properties of the concrete mixture with hollow microspheres. The main levels were 0.5 (X1) and 1.4 (X2), and the variation intervals were 0.1 and 0.4, respectively  sand. Varying the ratio of fine and fractionated sand particles  makes itD × d × h—101.6 × 69.9 × 50.8 mm [The mobility of the concrete mixture was determined in accordance with  using a  50.8 mm  before a−1 for 60 s.The rheological properties were studied based on the shear stress of the concrete mixture. The rotary viscometer MCR 101  was used for testing . The metThe tests were carried out according to the following algorithm: (1) homogenization of dry components of the mixture; (2) addition of water with dissolved plasticizer; (3) mixing of the concrete mixture according to ; (4) filStandard samples were used to determine the average density and flexural and compressive strength after 28 days of hardening. A visual analysis of the concrete samples was carried out before determining their mechanical properties. The purpose of this procedure was to establish the presence or absence of stratification of the mixture and the formation of voids in the concrete sample. Examples of samples with signs of stratification, as well as the presence of voids, and samples without these signs are shown in The Advantest 9 servo-hydraulic system  was used to study the physical and mechanical properties  in accorThe technological and rheological properties and homogeneity of the concrete mixtures, as well as the physical and mechanical properties of the hardened composite, were studied for lightweight concrete compositions with hollow microspheres.Standard methods for determining the mobility of concrete mixtures allow you to establish a quantitative indicator, such as the spreading diameter from a truncated cone. This indicator is a generally accepted fluidity criterion for mixtures based on mineral binders. For the studied concrete, this characteristic determines the ability to spread under shaking.Dsp,1) tends to the spreading diameter after shaking , then, naturally, mobility is taken as the ability of the concrete mixture to self-compact:Thus, the spreading diameter obtained without shaking should be considered a natural criterion for self-compacting mixtures. Consequently, if the spreading diameter without shaking  and the concentration of the plasticizer  at which this possibility is realized. The experiment was mathematically planned , mm; Y2—the spreading diameter of the concrete mixture after shaking , mm.In this case, the intensity of self-compaction will depend on absolute values. A justified prototype for self-compacting lightweight concrete is heavy concrete with an identical composition and target values regarding technological properties but without lightweight aggregate. The determination of the dependence of this criterion on variable factors makes it possible to conclude that it is possible to obtain self-compacting concrete mixtures on hollow microspheres with an average density of 1400 kg/m0 show that the concrete mixture has a close spreading diameter of Dsp,1 ≈ Dsp,2 (differ within 2%) when the variable factors corresponding to the main levels of plan are as follows: W/C = 0.5 and CPl = 1.4%. Each of the coefficients, except B0, has a greater value in modulus in the Y1 model than in the Y2 model. This indicates a more intense influence of the studied factors on spontaneous spreading than on spreading under the influence of shaking. At the same time, the general nature and direction of the influence of factors for both models are naturally identical. The common extreme value for Dsp,1 and Dsp,2 with a spreading diameter of more than 300 mm is the values X1 ≥ 0.25 in the entire range X2  in concrete mixtures on hollow microspheres. This indicates the ability of the studied mixtures to spread independently and obtain values close to the maximum values of the spreading diameter. The dependence of Dsp,2/Dsp,1 = f, as shown in Dsp,2/Dsp,1 ≤ 1.05 at X1 ≥ −0.25 in the entire range of X2. According to the models obtained, this value corresponds to the W/C ratio of 0.475. The parameters that characterize the mobility of the concrete mixture of heavy concrete (prototype) are presented in Dsp,2/Dsp,1. At the same time, the compositions of the lightweight and heavy concrete mixtures, with the same content of water and plasticizer , are different. The Dsp,2 and Dsp,1 of the lightweight concrete mixture are more than those of the heavy concrete mixtures by 8.2 and 2.2%, respectively, and the self-compacting coefficient is lower by 5.6%. An important requirement for self-compacting concrete is uniformity. For example, the separation of water from the mixture was observed in the compositions of prototypes of heavy concrete with a large W/C ratio. The correction of water and plasticizer consumption  eliminates this drawback. The established models make it possible to determine that the spreading diameters of a lightweight concrete mix on hollow microspheres are equal to Dsp,1 = 241 mm and Dsp,2 = 255 mm at W/C = 0.45 and CPl = 1.2%. This means that the self-compacting coefficient  of a lightweight concrete mix on hollow microspheres is 7.0% less than that of Prototype 2 with the same ratio of control factors. This indicates the greater role of water distribution in self-compacting systems on microspheres compared to quartz sand (without hollow particles). Such patterns depend on the structural parameters of dispersed systems  and on the properties of the dispersed phase (hollow microspheres and quartz sand).A comparative analysis of lightweight and heavy concrete mixtures was carried out. The following observations were noted: Firstly, the variation in the W/C ratio and the content of the plasticizer ensures the better mobility of the lightweight concrete mixtures compared to the heavy ones. Secondly, the ability to self-compact in mixtures with hollow microspheres is higher, meaning there is a lower coefficient of f) and fine (Sp) quartz sand. It becomes possible to see how the mineral part of an identical composition affects the distribution of water in the dispersed system. The total volume content of the quartz part in a mixture is constant Sg + Sf = const = 8.7% at a constant consumption of all components of lightweight concrete and W/C = 0.5, CPl = 1.4% . This means that the mobility of the mixture does not demonstrate the role of the dispersion of quartz sand in the studied range. The self-compacting coefficient varies in a statistically insignificant range of 1.03–1.05. The data obtained indicate that the technological criterion for the quality of the mixture does not allow one to make a statistical conclusion about the influence of the dispersion factor of quartz aggregates on the distribution of water in the system.Thus, the response of the studied concrete mixtures on hollow microspheres to spontaneous spreading with a volume content of 0.464 was shown. The values of spontaneous spreading are close to the values of mobility during forced spreading (after shaking). The ranges of the main recipe factors are established. It is shown that lightweight concrete mixtures can have better technological properties than heavy concrete mixtures with similar water contents. Therefore, the features of the rheological properties of such mixtures are also superior.For each composition, in accordance with the above experimental plan, rheological curves that describe the dependence of shear stress on shear rate were obtained . The gra−1, it decreases, and this decrease is associated with overcoming the flow resistance threshold. At this moment, a larger volume of the mixture begins to move. Also, for these compositions, areas of opposite changes in shear stress can be observed under increasing shear rates  has lower shear stress, and a uniform change in τ indicates the better fluidity and continuity of the system. This may be due to the influence of the light dispersed phase (hollow microspheres), which has a smaller size and density compared to quartz sand. Thus, fine particles of lightweight aggregate require more water, and as we know, the intensity of the flow decreases in proportion to the gravitational effect, depending on the density. However, the composition of heavy concrete with the specified ratio of W/C and CPl is prone to water separation, which indicates its excess. Reducing the value of the variable factors to W/C = 0.45 and CPl = 1.2% made it possible to obtain a prototype of a homogeneous mixture of heavy concrete. Obviously, the shear stress of such a composition is higher and can act as an approximate boundary for obtaining a homogeneous mixture.It is natural that the shear stress increases with a decrease in the W/C ratio, regardless of the concentration of the plasticizer. A similar pattern can be noted for compositions with varying k—consistency indicator; n—indicator of the flow type . The resulting rheological equations for each composition, according to the experimental plan, were used to establish regression equations Y = f:Y3 and Y4—the coefficient k and n of the Ostwald–Weil equation, respectively.The group of concrete mixtures with shear stress values less than 15 Pa included compositions with W/C = 0.6. The shear stress graphs had no discernible differences when varying the plasticizer concentration. A comparative analysis of the obtained rheological curves was performed using the Ostwald–Weil equationk) has a more significant change from varying the W/C ratio than the additive concentration. At the same time, both factors contribute to a decrease in the value of this criterion. Note the positive sign of the coefficients B12, B11, and B22 in equation Y3. This indicates a change in the nature of both the mutual influence of X1 and X2, as well as a significant increase in their value. That is, both an excess amount of water and plasticizer, and their mutual influence leads to an increase in the density of the concrete mixture. This can be explained by a violation of the homogeneity of dispersed systems when exceeding the threshold number of variable factors leads to an imbalance of water. In this case, water separation is observed. That is, the segregation of the mixture may be associated with a decrease in the thickness of the water layer and an increase in friction between solid particles. Equation Y4 demonstrates the value of the coefficient n < 1 at values of the varied factors corresponding to the main levels of variation (B0 = 0.966). That is, such a dispersed system is characterized by a pseudoplastic type of flow. In this case, the concrete mixture begins to flow at minimal shear loads. The shear stress changes with decreasing intensity as the shear rate increases. At the same time, this rheological behavior is enhanced by the w/c ratio, and its combined effect with the plasticizer enhances this rheological behavior of the mixture. This can be seen by the “–” sign in front of the coefficients B1 and B12 in the regression equation Y4. At the same time, the coefficients of the equation show the possibility of changing Y4 to the region n > 1. That is, the nature of the flow of the concrete mixture may shift to being dilatant depending on the value of the W/C ratio and the concentration of the plasticizer. In such a system, the cement–mineral paste forms a layer between the particles of the dispersed phase and helps reduce friction. As the shear rate increases, the shear stress changes with increasing intensity. From the equation Y4 = f, it is clear that the concentration of the plasticizer (coefficient B2) contributes to the tendency towards such behavior of the dispersed system under study.An analysis of the resulting equations showed that the density coefficient  . The change in the value of the coefficient n indicates a change in the nature of the flow, from pseudoplastic (n < 1) to dilatant (n > 1), with an increase in the proportion of the thin component (Sf). This is due to a decrease in the volume of the dispersed phase (fractionated quartz sand) and an increase in the volume of the dispersed medium .In o 102.9) . This raDsp,2/Dsp,1 = 1.08 and 1.14, respectively, are described by equations with k = 31.2 and 77.7 and n = 0.68 and 0.63, respectively. This shows that mixtures of heavy concrete tend to flow at lower values of shear stress than mixtures of lightweight concrete on hollow microspheres. However, the spread diameter of heavy concrete is smaller , while the movement is directed “down” (towards settling) if ρf > ρm.Let us note the value of the coefficients of the rheological equations for heavy concrete. Compositions Prototype 1 and Prototype 2, with  smaller . This isThe concrete studied mixtures are dispersed systems in which the dispersed phase consists of hollow microspheres (average particle diameter no more than 100 microns) and 0.16–0.63 mm fractionated quartz sand, and the dispersed medium is a cement–mineral system that is mixed with an aqueous solution of a plasticizer. The homogeneity of such a system consists of the condition of ensuring a low speed of movement of phases with a known density and geometric dimensions .3) show that lightweight particles (ρf = 540 kg/m3) with a particle diameter of less than 0.36 mm (rMS < 180 μm) have a slower movement speed than the largest particles of fractionated quartz sand . Thus, with all other things being equal, the movement speeds of particles of different densities can be arranged in the next row of the sequence in accordance with their radius:The theoretical dependences of the movement speed of particles of different radii and densities on the viscosity of the dispersed medium  can be controlled through the viscosity of the cement–mineral paste through the interdependent factors of the W/C ratio and the concentration of plasticizer A key requirement for self-compacting concrete mixtures is their ability to maintain homogeneity. Many factors contribute to maintaining the homogeneity of the structure, including the quality and quantity of mixture components, as well as manufacturing conditions and external influences. In the concrete mixtures under study, an additional factor is the different densities of the components of the dispersed phase (hollow microspheres and quartz sand). Therefore, the tendency for lightweight concrete compositions with high self-compacting abilities to delaminate limits their use. According to the experimental plan, samples of each composition were subjected to a visual analysis that involved recording the presence of heterogeneous layers .CPl < 1.0%, there is no delamination on all compositions of Series 1 and Series 2. That is, the compositions molded immediately after preparation (without compaction), and the compositions molded after mobility tests are homogeneous. However, increasing the content of the plasticizer (composition 4) and the amount of water with the plasticizer (composition 6) is sufficient for the separation of concrete mixtures for these series. For Series 3, only compositions 1, 3, and 5 (W/C ≤ 0.4) did not show visual signs of delamination. That is, for compositions 2, 7, 8, and 9 (W/C ≥ 0.5), a change in the structure of the concrete mixture occurs after external influences due to testing. This can be explained by the thixotropic dilution of these concrete mixtures—associated with a decrease in the viscosity of the cement–mineral matrix (dispersed medium) under the action of vibration forces. At the same time, the external effects of the test conducted to determine of the spread diameter do not have such a dilution. The assumption of the presence of a uniformity threshold is derived from this. The structure of the concrete mixture loses its homogeneity after exposure to extreme energy. The results of our visual analysis of concrete samples with varying sand dispersion values are shown in The data presented in p/Sf ratio was varied, delamination was observed only in samples of Series 3. This means that, regardless of the dispersion of quartz sand (volume content of 8.7%), samples of the composition of the concrete mixture of the series with W/C = 0.5 and CPl = 1.4% have a similar tendency to delaminate under the influence of external influences caused by testing.When the SCPl = 1.4%, the concrete mixtures are characterized by a high tendency to segregate. Compositions with W/C = 0.5 with varying CPl values and ratios of fractional sand and quartz powder tend to stratify after a certain level of external influence. For such compositions, it is advisable to use special organic or mineral viscosity modifiers or stabilizers.Thus, our visual analysis of the stratification of the samples of the concrete under study showed that compositions with W/C = 0.4 have high structural uniformity, including in the presence of external influences. If W/C = 0.6 and The final product when using self-compacting concrete mixtures is a solid product with specified performance properties. Ensuring the homogeneity of the mixture is an important consideration for obtaining a homogeneous structure and properties in the volume of the solidified composite. In this regard, lightweight concretes with hollow microspheres are complex systems with a defined set of rheo-technological and physico-mechanical properties–antagonists: high mobility and homogeneity, low average density, and high strength.Y5 represents the average density of concrete, Y6 represents the flexural strength, and Y7 represents the compressive strength. However, our analysis of the obtained mathematical models was complicated by the presence of segregated compositions at X1, and X2 tends to 1 of the experimental plan  shows the statistically insignificant influence of variable factors on the average densities of the concretes. Only the amount of water is a significant factor, according to the model. In this case, the value of coefficient B1 shows the negative impact of the W/C ratio on the average densities of the concretes. This is explained by the delamination of concrete with an increase in the amount of water (W/C ≥ 0.6). An analysis of equations Y6 and Y7 allows one to conclude that water content has a similar effect on flexural and compressive strength . The role of the plasticizer becomes more significant. This is reflected by a positive influence in quantities close to the boundaries of the variable range and the joint influence of CPl and W/C ratio (coefficients B22 and B12). The established experimental and statistical models highlight the need to optimize the studied compositions in terms of the W/C ratio and plasticizer concentration to obtain concrete with a homogeneous structure and high strength properties. Changes in physical and mechanical properties in the selected range of varying factors are in the following ranges: ρ = 1403–1485 kg/m3, Rfl = 3.34–5.90 MPa, Rcom = 29.6–45.7 MPa.Regression equations describing the relationship between the W/C ratio and plasticizer concentration with the average density and strength of lightweight concrete on hollow microspheres have been obtained:tal plan . The equp/Sf) of this component on the physical and mechanical properties of lightweight concrete. However, the highest values in terms of flexural and compressive strength were observed in the composition with Sp/Sf = 50/50.p/Sf ratio is important. The flexural strength of the self-compacting lightweight concrete on hollow microspheres is 44–53% of the strength values of Prototype 1 and Prototype 2 and 45–66% of their compressive strength values. Moreover, the average density is 1.5 times less than that of the heavy concretes.At the same time, since the complete absence of a thin or coarse part of the sand does not provide an optimal combination of these properties, optimizing the S3) to self-compact has been established. The close values of the spread diameter before and after shaking on the table  is confirmation of this. The intensity of the influence of the W/C ratio and plasticizer concentration on free flow diameter  is higher than that of the spreading diameter after shaking . The self-compacting ability of the lightweight concrete with hollow microspheres is higher than that of heavy concrete (Prototype 1) at the same W/C ratio and CPl (W/C = 0.5 and CPl = 1.4%).Thus, the ability of a concrete mixture based on hollow microspheres (average density 1450 ± 25 kg/mk) than the concentration of the plasticizer. In this case, a decrease in the thickness of the dispersed systems under study can naturally be observed with an increase in both W/C ratio and CPl. The obtained regression equations for the index n in the Ostwald–Weil equation show the possibility of changing the nature of the flow of mixtures from pseudoplastic (n < 1) to dilatant (n > 1) under varying W/C ratios and CPl values.An assessment of the flow rheology of the concrete mixtures using the coefficients of the Ostwald–Weil equation showed that the W/C ratio is a more significant factor in terms of its influence on the density (thickness) of the concrete mixture  in an amount of 8.7% in the composition of lightweight concrete does not have a significant effect on the self-compaction criterion Dsp,2/Dsp,1. Replacing fractionated sand with fine sand from 100 to 0% contributes to increasing the density by almost three-fold, which is naturally associated with a larger total surface area of the quartz part in the mixture. Concrete mixtures with different ratios of fine and fractionated sand (Sp/Sf) (at W/C = 0.5 and CPl = 1.4%) have similar homogeneity and tend to separate after a certain threshold of external influence. It has been shown that varying the ratio of fine and fractionated sand while maintaining their total volumetric contents (8.7%) in the composition of lightweight concrete does not have a significant effect on the physical and mechanical properties. The highest value of flexural and compression strength was observed in compositions with Sp/Sf = 50/50.The dispersion of quartz sand  to self-compact has been established.The ability of a lightweight concrete mixture with a low density (ρ = 1450 ± 25 kg/mThe dependences (regression equations) of mobility, coefficients of the Ostwald–Weil equation, and density and strength od the W/C ratio and plasticizer concentration for lightweight concrete with a volume content of hollow microspheres of 46.4% have been established.Pl ≤ 1.0%.The limits for homogeneity of lightweight concrete mixtures on hollow microspheres are W/C ≤ 0.6 and Cp/Sf ratio) in an amount of 8.7% in the composition of lightweight concrete does not have a significant effect on the self-compaction criterion and physical and mechanical properties.The dispersion of quartz sand (varying the SThis paper shows the possibility of changing the nature of the flow of lightweight concrete mixtures from pseudoplastic to dilatant under varying W/C ratios and plasticizer concentrations.Carrying out this study allowed us to conclude that it is possible to produce self-compacting lightweight concrete using hollow microspheres. The key contributions and conclusions of this paper are as follows:Establishing the influence of the studied factors on the strength properties in systems without delamination.Establishing the parameters and boundary values of delamination after external influence on the concrete mixture.Studying the time it takes to maintain the mobility and homogeneity of the concrete mixture on hollow microspheres.Future research directions for the development of this research topic include the following:


Antimicrobial, and particularly antibiotic resistance are one of the world’s biggest challenges today, and urgent action is needed to reinvigorate the antibiotic development pipeline. To inform policy discussions during and after the 2023 Swedish Presidency of the Council of the European Union, we critically appraise incentive options recently proposed by the European Commission, and member states, and consider what has been achieved over the last two decades in relation to antibiotic research and development. While several new antibiotics have achieved regulatory approval in recent years, almost none have innovative characteristics such as new chemical classes or novel mechanisms of action. We consider four incentive options to incentivise research and development of new antibiotics, including subscription payments, market entry rewards, transferable exclusivity extensions, and milestone payments. While each option has advantages and drawbacks, a combination of incentives may be required and continued investment is needed by the EU in push incentives, such as direct funding and grants, to incentivise drug discovery and preclinical stages of development. The EU must also coordinate with international initiatives and support access to new and pre-existing antibiotics in LMICs through platforms such as the WHO, and G7 and G20 group of countries. Antimicrobial and particularly antibiotic resistance is one of the most significant challenges facing healthcare systems worldwide. In EU/EEA countries, antibiotic-resistant infections resulted in more than 38,000 deaths and more than 1 million years of life lost because of premature death or years lived with disabilities in 2019 alone.,The COVID-19 pandemic has re-emphasised that infectious diseases do not respect national borders, and there is renewed debate at the EU level on what can be done to tackle growing AMR, including planned revisions to general pharmaceutical legislation,,There are several important policy issues relevant to AMR that are outside the scope of this article. This article does not focus on policies necessary to limit the emergence and dissemination of resistant infections such as stewardship, IPC, public awareness, surveillance, and access to generic antibiotics.,,,,Undoubtedly, a significant success has been raising the profile of AMR among global and national policy makers, evidenced by the 2017 EU One Health Action Plan on AMR,As a result, there has been significant public investment in push incentives for antibiotic research and development over the last decade. The EU established the New Drugs 4 Bad Bugs (ND4BB) programme with EUR 650 million between 2014 and 20 as part of a public-private partnership called the Innovative Medicines Initiative (IMI).Several multilateral organizations have also been established with funding contributions from individual governments and non-governmental organisations (NGOs). A 2020 review of these initiatives,There has been relatively less investment in pull than on push incentives. Several approaches have been tried nationally in Europe . In FranIn England and Sweden, subscription-style payments  have been used for reimbursement of antibiotics at the national level. Subscription payments can replace sales revenues  with upfront payments each year for access to predetermined quantities of antibiotics or complement sales volumes , whereby an annual revenue guarantee is used in return to access to antibiotics. The National Institute for Health and Clinical Excellence (NICE) and NHS England implemented subscription payments to replace sales revenues for access to two new antibiotics as of April 2022, Cefiderocol (Fetcroja) and Ceftazidime/Avibactam (Zavicefta) for a total of £10 million a year for each antibiotic.Beyond Europe, several pull incentives have been implemented in the US. In 2012, the Generating Antibiotic Incentives Now (GAIN) Act was enacted allowing expedited regulatory assessment and an extension of market exclusivity of new antibiotics by five years.A. baumannii, and Carbapenem-resistant P. aeruginosa. The WHO describes the supply of recently approved new antibiotics, and those in clinical development, as “insufficient” to address the growing threat of antibiotic resistance.Internationally, the combined use of push and pull incentives has contributed to several new antibiotics achieving regulatory approval over the last five years. According to 2022 WHO review of the antibiotic pipeline, there have been 12 new antibiotics that have received regulatory approval between July 2017 and November 2021.,This antibiotic pipeline remains insufficient to tackle AMR because of a complex range of scientific, economic, and regulatory factors that can be classified according to different stages of the drug development pathway.Collectively, these scientific barriers mean that the rate of failure during preclinical and clinical phases for antibiotics under development is exceptionally high. The success rate of moving an antibiotic from the preclinical research stage to achieving regulatory approval has been estimated as low as between 1.5 and 3.5% and can take between 10 and 15 years.,,Many antibiotic developers that have launched antibiotics have subsequently experienced significant financial losses or filed for bankruptcy.From the regulatory perspective, procedural differences between national and regional drug regulatory agencies in approving novel antibiotics make global licensing time-consuming and costly, including differences in patient selection criteria, specification of statistical parameters, definitions of clinical endpoints, and rules regarding expedited approvals.There is consensus that no single incentive will be sufficient to stimulate antibiotic research and development, and a combination of push and pull incentives will be required. Each incentive has benefits and disadvantages regarding the expected impact and operational feasibility, and there are a number of principles that policymakers should consider when assessing different options for incentives.,First, a holistic incentive package for antibiotic research and development should reinforce broader public health objectives. In 2019, around 80% of the global health burden attributed to antibiotic resistance globally resulted from respiratory tract infections, bloodstream infections, and intra-abdominal infections.,Second, any package of incentives for antibiotic research and development needs to ensure the expected return on investment increases. As mentioned above, the average NPV for antibiotic drug development is estimated to be negative.Third, it is important to consider the political, regulatory, legislative, industry and financial hurdles potentially faced during implementation of any incentives for antibiotic research and development. In Europe, there are particular challenges in navigating the complexities of EU legislation. For example, the boundaries between the scope of EU- and national-level health policies sometimes create tensions, as pre-existing treaties, such as the Treaty on the Functioning of the European Union, state the EU must respect member states’ autonomy in operating their health systems.,,There is consensus among EU policy makers that increased investment is needed in new and pre-existing incentives to revitalise the antibiotic pipeline.During the development of this article, the Directorate General for Health Emergency Preparedness and Response Authority (HERA) published a report that modelled the expected cost and impact on expected NPV different stages of drug development for subscription payments, MERs, and milestone payments.Subscription payments  for antibiotics either pay developers an annual fixed price to access antibiotics upfront (for example in the UK) or guarantee annual revenues (for example in Sweden).As subscription payment models are typically agreed upon towards the end of antibiotic development, the impact of this incentive on stimulating the antibiotic pipeline would depend on the size of incentive and whether the institution responsible for assessing antibiotic candidates provides reliable signals to antibiotic developers during the early stages of development that they may be eligible for subscription payments upon market approval . The same applies to other incentives implemented towards the end of antibiotic development, including MERs and TEEs. The HERA study modelled the impact of two options for revenue guarantees of €150 million and €100 million per year, that would complement revenues generated from sales, and expected to cost €750 million and €421 million over 10 years for each new antibiotic.Implementation feasibility at the EU level may depend upon achieving consensus among member states regarding the size of payments, the public health value of the new antibiotic, and sales volumes required to meet demand. The HERA study conducted a survey of member states regarding their interest in participating in the mechanism for subscription payments proposed by JAMRA, and 80% of member states responded stating they needed more information before taking a position.MERs are a financial reward for antibiotic developers that successfully achieve regulatory approval for a new antibiotic. They could be a one-off reward, or payments could be spread over several years. They may also be granted in exchange for intellectual property rights to allow earlier entry of generic manufacturers and diversification of supply. The size of MER could vary according to the extent to which a new antibiotic targets high-priority medical needs. MERs can include stewardship, access, and environmental health agreements, although these may be challenging to enforce if the MER is a one-off payment. There is also a risk that antibiotic developers may later become bankrupt if the size of MER does not reflect research and development costs or if funds are mismanaged. MERs would likely benefit both large pharmaceutical companies and SMEs since they would make attracting investment by SMEs at early stages of development more feasible if appropriately sized.MERs can be used to fund post-approval costs related to pharmacovigilance or commercialisation,,,,Transferable exclusivity extensions (TEEs) are a reward granted to antibiotic developers that successfully develop and launch a new antibiotic that can be used to extend the intellectual property rights of another medicine for a fixed period .Milestone payments involves granting funding to developers at specific stages of antibiotic development. Milestone payments could target high-priority medical needs by establishing eligibility criteria, but this would be operationally challenging and require substantial technical expertise. This function could be undertaken by an “antibiotic pipeline coordinator”, who should have oversight of the antibiotic pipeline internationally and facilitate cooperation and exchange of technical expertise among relevant stakeholders across the antibiotic pipeline. Such an antibiotic coordinator would also be tasked with interpreting the reliability, validity, and transparency of clinical trial data. Some institutions already undertake this role at different stages of development, including CARB-X and GARDP.While the policy discussion at the EU level is predominantly focused on pull incentives,Over the last two decades, the EU has demonstrated global leadership in combating AMR through interventions such as enhanced surveillance, strengthened legislation of antimicrobial use, advocating for a “One-Health” approach, and investment in push incentives for antibiotic development.MA drafted the manuscript. MA and EM designed the analysis. All other authors provided comments and edits to iterative versions of the manuscript.The authors have no relevant conflicts of interest to declare.


The scientific community widely acknowledges that the gut microbiota plays a critical role in maintaining host health and can be altered by a range of factors, such as antibiotic use, diet, stress, and infections. Therefore, this study utilized bibliometric analysis to thoroughly investigate research trends in the microbiota and antibiotics. Scopus was used to extract papers linked to microbiota and antibiotics published between 2002 and 2021, and both Microsoft Excel and VOSviewer were used to conduct the analysis of the data. A total of 2,816 publications discussed the connection between the microbiota and antibiotics. Growth occurred in two stages: the first (2002–2015) was characterized by fairly slow publication production, while the second (2016–2021) saw a rapid increase in publishing progress. The United States has the most publications, 654, representing 23.22% of the total. China came second with 372 publications (13.21%), followed by the United Kingdom with 161 publications (5.72%) and India with 157 publications (5.58%). In addition, publications on ‘altered intestinal microbiota composition with antibiotic treatment’ were introduced after 2017, while ‘gut microbiota and antimicrobial resistance’ and ‘probiotics as an alternative antimicrobial therapy’ were introduced before 2017. Based on these results, this study provides an in-depth look at key moments in the history of microbiota and antibiotic research, as well as possible directions for future research in different areas of microbiota and antibiotic research. Therefore, it is suggested that more attention should be given to the latest promising hotspots, such as how antibiotic treatment changes the composition of the gut microbiota. Research in recent decades has focused on and linked the gastrointestinal microbiome to many diseases and human health2.Many microbes, including viruses, bacteria, fungi, protozoa, and archaea, colonize the human gastrointestinal tract (GI), termed the gut microbiota3. Colonization resistance may occur directly through competitive bacterial interaction or host defense against pathogens, which is triggered indirectly by bacteria. Many studies have shown not only the association but also the causality of many diseases with gut microbiomes. Furthermore, studies have revealed that intrinsic and extrinsic factors, mainly drugs, can affect gut microbiome function and/or composition, ultimately affecting human health2.The gut microbiota protects the GI tract against infection using different mechanisms that contribute to resistance against colonization by exogenous microorganisms and protection against fatal pathogens6, has been brought to light by research conducted in recent years. Because research output plays an important function in science development by providing a key association between the production of knowledge and its application, the volume of research that encompasses almost all of the world’s regions that are interested in the production of health sciences is growing. This is one of the reasons why there is a growing interest in the production of health sciences. Despite the fact that only a few bibliometric studies have been conducted in the field of gut microbiota11, none of them have investigated the links between the microbiota and antibiotics. To fill this gap, the authors of this study used bibliometric analysis to thoroughly investigate the key research trends related to microbiota and antibiotics. Bibliometric analysis refers to the quantitative and qualitative examination of the literature in a specific field utilizing statistical and mathematical methods. Over the past decade, numerous studies have been conducted to explore bibliometrics in various scientific disciplines14. Unlike systematic reviews, which aim to answer a particular research question based on a limited number of publications, bibliometric analysis aims to provide a comprehensive overview of the literature in a particular field15. Similarly, it differs from scoping reviews, which focus on identifying the nature and scope of research evidence16. Despite these differences, bibliometric analysis remains a valuable tool for obtaining a snapshot of both national and international contributions to the literature in a particular field, as well as identifying research gaps that future studies may address14. By investigating the global links between the research dynamics and hotspots of the microbiota and antibiotics, this paper aims to gain insight into the research trends of the microbiota and antibiotics to provide references for future research. Furthermore, by gaining insights into the research trends and hotspots in the field, policymakers and funding agencies can make informed decisions about where to allocate research resources and prioritize future research efforts.The importance of the gut microbiome, as well as its connection to health, the immune system, and the use of antibiotics17. Typically, either SciVerse Scopus or Web of Knowledge is used as the single database, and no gray literature is included in the analysis. For this particular study, publications relevant to microbiota and antibiotics from 2002 to 2021 were retrieved using SciVerse Scopus, which was chosen due to its many advantages over other databases, such as Web of Science, Medline, and Google Scholar19. One of the key features of Scopus is its ability to provide bibliometric indicators in a direct and simple way. Additionally, Scopus includes 100% of Medline's publications, so by using Scopus, publications in Medline are automatically included as well. Many academics have extensively used the Scopus database, one of the most complete, systematic, and reliable databases, for bibliometric analysis and visualization of the scientific literature. Because the Scopus database is always accessible, we decided to perform the literature retrieval from it on a single day, December 1, 2022, to eliminate any potential for bias that daily additions to the database could have caused.In bibliometric analysis, documents are retrieved from a single database and then analyzed both quantitatively and qualitatively22 and antibiotics26). These terms were then included in the Scopus Engine. Each of the chosen “keywords” was used as an entry for the “Article Title”. The keywords used in the study, ‘antibiotics’ or ‘antimicrobials’, ‘antibiotics’ or ‘antimicrobials’, were those related to antibiotics per se rather than other related terminology, such as specific name or class of antibiotics.The terms related to the microbiota and antibiotics were identified using PubMed Medical Subject Headings (MeSH) and relevant publications on the topics  were relevant to the topic being looked up. The author contacted two bibliometrics professionals and requested that they verify false-positive results by reviewing the titles and abstracts of the most-cited documents that were supplied to them. When experts established that there were no false positive results, the search query was deemed to have reached its conclusion. The correlation test was implemented between the information retrieved by the search query and the real findings for the top ten active researchers to validate that there are no false-negative results. The fact that the correlation test yielded a strong correlation (r = 0.952) and a significant result (p < 0.01) provides evidence that the search query was accurate. This particular validation method has been used in bibliometric investigations carried out in the past and published in the literaturehttps://www.vosviewer.com/), which is a free online program that can be used for mapping purposes30. The VOSviewer application was used to create network visualization maps that presented international research collaboration and research hotspots.The data retrieved were transferred to Microsoft Excel for analysis and tabulation. Export data included types of retrieved documents, the annual growth of publications, prolific countries, prolific institutions, prolific funding agencies, the most cited publications, and journals involved in publishing the retrieved documents. The retrieved data were also exported to VOSviewer v.1.6.18  original articles, 292 reviews, and 299 (10.62) other documents, including letters, conference papers, and editorials.Figure Table Ministry of Education of China and the Chinese Academy of Sciences published the highest number of articles, with 35 publications each. Next came Wageningen University & Research with 33 publications and INSERM with 30 publications.The top 10 institutions in terms of the amount of research they have conducted in this area are listed in Table The National Natural Science Foundation of China supported the highest number of articles, with 178 publications, followed by the National Institutes of Health with 162 publications and the National Institute of Allergy and Infectious Diseases with 83 publications.Table Plos One , followed by Frontiers in Microbiology , Scientific Reports , and Microorganisms . With an IF of 16.837, Microbiome was the most influential journal of the top 10 prolific journals.Table 40. The work that received the most citations in this area was written by Dethlefsen et al.35 and published in PLoS Biology. The works by Dethlefsen and Relman37 and Cho et al.34 received the second and third most citations, respectively.The 10 most cited publications on the microbiota and antibiotics are shown in Table By mapping the cooccurrences of terms in articles on microbiota and antibiotics, research areas were grouped into 3 clusters. There were 49,251 terms, and 111 of those terms appeared at least 100 times. The visual network map reveals that three clusters can be formed from all of these terms , followed by "ampicillin" (n = 258), "ciprofloxacin" (n = 244), and "metronidazole" (n = 233).Table 43. However, no bibliometric research has been conducted on the links between antibiotic use and the microbiota. In the current study, we analyzed global trends and research horizons on the microbiota and antibiotics through bibliometric analysis to look back over the past 20 years. Researchers were shown to have a significant interest in microbiota and antibiotics, as evidenced by the increasing number of annual publications. The United States held the leadership position in microbiota and antibiotics with regard to publications and collaboration. However, this position cannot be separated from its substantial support from financial institutions.To date, studies have revealed a link between the microbiota and several neurological and digestive diseases. The gut microbiota has become a focus of intense investigation in various diseases during the past two decades due to the growing interest in the function of intestinal microbe alterations in the pathogenesis of diseases. The status and tendencies of the gut microbiota in particular subjects are increasingly being investigated using bibliometric analysis44. This is likely due to several factors, including the availability of research funding, a strong infrastructure for scientific research, and a robust pharmaceutical industry. The United States is also home to several prestigious universities and research institutions that attract talented researchers and scientists from all over the world.The results of this study indicate that the United States, China, the United Kingdom, and India have made the most progress in microbiota-related antibiotic research in the past two decades, with the United States leading the way. This research is critical to combating antibiotic resistance, which is a global concern. It is essential that countries continue to invest in scientific research to develop new strategies for the use of antibiotics and combat this issue. According to the results, the United States has been at the forefront of antibiotic research trends44, making this research a priority. In addition, the country has invested heavily in scientific research in recent years and has a growing pharmaceutical industry.China ranked second in microbiota-related antibiotic research trends, indicating that the country has made significant strides in this field in recent years. China has a large population and has been hard hit by antibiotic-resistant infections46. The UK has a long history of scientific research and innovation and is home to several renowned universities and research institutions.The United Kingdom ranked third in the results, indicating that the country has also been active in microbiota-related research26, making this research a priority. However, India faces several challenges in the field of scientific research, including limited funding, a lack of infrastructure, and brain drain, where talented scientists leave the country for better opportunities elsewhere.India ranked fourth in the results, indicating that the country has progressed in microbiota-related antibiotic research. India is home to a large population with a high burden of infectious diseasesaltered composition of the gut microbiota with antibiotic treatment was particularly interesting in the current study. Treatment with antibiotics has been shown to decrease the diversity of bacteria in the intestinal microbiome, causing metabolic changes that increase the potential subsequent susceptibility of the intestinal tract to colonization. This allows exogenous pathogens to invade the GI tract and cause infection, in addition to the development of antibiotic resistance48. Antibiotics may also disrupt the normal balance between the gut microbiota and various species. For example, Clostridium difficile may result from the antibiotic effect of antibiotics on decreasing species diversity49. Administration of a combination of gentamicin, meropenem, and vancomycin in adults resulted in a decrease in Bifidobacterium and butyrate-producing species and an increase in the prevalence of Enterobacteriaceae during the first treatment, which was then partially restored within the next month, while some bacterial species remained undetectable for longer periods in the gut50.The current study identified three research themes related to antibiotic use and microbiota. These research themes were closely related. The research theme that focused on the 51. Intestinal bacteria resistant to antibiotics can be passed from mother to child during birth and may remain for weeks. Twelve percent of the commensal E. coli bacteria in the Swedish study were positive for tetracycline resistance despite never being exposed to antibiotics52. As bacteria sensitive to the antibiotic are eliminated, resistant bacteria for the same antibiotic start to take their place by multiplying. After antibiotic treatment, the diversity of the bacterial species is reduced, while the total microbial load may increase. For example, evidence showed that patients treated with a week of b-lactams doubled the fecal microbial load in fecal samples47. Even if transient, colonization with multidrug-resistant bacteria can lead to the transfer of antibiotic resistance genes into the microbiota, which can eventually result in increased resistance to a variety of effective antibiotics and an increased risk of fatal infection53.In the current study, ‘gut microbiota and antimicrobial resistance’ was among the most popular hot topics in research on the links between antibiotic use and the gut microbiota. Antibiotic resistance genes are present in the human gut microbiome. The number of resistance genes in the stomach increases rapidly during antibiotic therapy and gradually decreases once treatment ends54. Although probiotic treatment also alters the luminal microbiota, this luminal alteration of the microbiota by probiotics could significantly influence systemic metabolism, including insulin resistance55. Multidrug-resistant bacteria, such as carbapenemase-producing enterobacteria, extended-spectrum beta-lactamase strains and vancomycin-resistant enterococci, are all serious pathogens that cause a high mortality rate and are considered an important public health problem56. Therefore, strategies to prevent the colonization of the luminal microbiota, especially in the colon, could be achieved using probiotics. However, little is known about their biological action despite substantial research on probiotics in recent years57.Another topic that has received much attention is ‘probiotics as an alternative antimicrobial therapy’. There is evidence that probiotics may help treat and prevent infectious diseases40. Furthermore, these results demonstrate the increased emphasis and attention given to this area of research in recent years. For example, the most frequently cited article on the subject is published in PLoS Biology and has been cited 1695 times. This investigation used pyrosequencing technology to identify changes that occur in the microbiota population of three individuals before and after ciprofloxacin treatment35. The study clearly showed how ciprofloxacin impacted the diversity of the bacterial population in the intestine and its variability and balance. Furthermore, numerous taxa were unable to return to normal after six months of treatment35. The paper by Dethlefsen and Relman37, which was published in Proceedings of the National Academy of Sciences of the United States of America, was the second most cited article. This article was conducted to test the influence of the use of ciprofloxacin antibiotic on the communities of normal intestinal flora by studying approximately 1.7 million hypervariable 16S rRNA tags of 16S rRNA of the bacteria37. Ciprofloxacin greatly affected the bacterial population within 3–4 days of treatment. However, these populations began to recover after one week of stopping antibiotics, and although the recovery was imperfect, they did not return to their former status37.The results of our research demonstrate that several subtopics closely related to study hotspots were highlighted in the most cited publications on the links between antibiotics and the microbiotaNature34. Again, the investigators adopted an animal model to examine the effect of administering a low-dose antibiotic to a young murine on the composition of the gut microbiome and its metabolic pathway. As a result, several changes were documented. An example of these changes is the alteration of essential genes responsible for converting carbohydrates to short-chain fatty acids34.The third most cited paper was published in 40, published in Science, was the fourth most cited article. This study showed how antibacterial lectin, called RegIIIγ, maintains mutualism between the gut microbiota and the host intestine. This material forms a thin layer as a physical barrier between the microbiota and the intestinal epithelial lining. However, losing this zone was associated with an increase in colonic bacteria and, consequently, activation of the immune response40. The paper by Sartor31, published in Gastroenterology, was the fifth most cited article. This review article highlighted a key topic of rationale for manipulating gut microbiota in the treatment of inflammatory bowel disease (IBD). Although this therapeutic strategy is uncontested, clinical studies do not follow standard criteria based on rigorous evidence for the use of these therapeutic modalities. However, some experts have proposed the use of ciprofloxacin and/or metronidazole or certain types of probiotics to treat IBD31.The paper by Vaishnava et al.The study highlights that research on altered intestinal microbiota composition with antibiotic treatment was introduced after 2017. Therefore, it is necessary to explore the implications of such alterations in the gut microbiota for host health and how these changes can be mitigated.The study also shows that research on gut microbiota and antimicrobial resistance has been ongoing for several years. However, further research is needed to investigate the mechanisms by which the microbiota contribute to antimicrobial resistance and how this knowledge can be utilized to develop new strategies to combat antibiotic resistance.The study highlights the importance of probiotics as an alternative antimicrobial therapy, and it is necessary to further explore their potential to reduce antibiotic use and mitigate the negative effects of antibiotics on the gut microbiota.The study emphasizes that various factors, including diet and stress, can disrupt the gut microbiota. Therefore, it is necessary to explore how these factors influence the composition of the gut microbiota and antibiotic efficacy and how this knowledge can be used to optimize antibiotic therapy.The study of the links between the microbiota and antibiotic use is a rapidly evolving field and is expected to continue to be an area of intense research in the coming years. Based on the current trajectory of research related to the impact of antibiotics on the human microbiome and its consequences on health, there are several future perspectives to consider:This study is the first comprehensive bibliometric investigation of the links between antibiotics and microbiota research conducted worldwide. The study of the connection between antibiotics and the microbiota is still in its infancy, but it is expected to develop further soon. Therefore, scientific researchers, clinicians, and medical educators use the aforementioned development hotspots as a foundation and guide for developing new projects in their respective fields.59.The current study has some limitations. First, we only extracted data from the Scopus database where it was necessary to do so to conform to the data formatting requirements of visualization tools such as VOSviewer. Nevertheless, the Scopus database, one of the most generally accessible and well-known resources globally, has been utilized in several previously conducted bibliometric research projects of a particularly high standard. Second, however, we are concerned with ‘antibiotics’ or ‘antimicrobials’, ‘antibiotics’ or ‘antimicrobials’, were those related to antibiotics per se rather than other related terminology, such as specific name or class of antibiotics. It is important to note that we must acknowledge the potential for bias in our sample selection due to the exclusion of certain publications that use specific antibiotic names in their titles. We are committed to ensuring that our research is as representative and unbiased as possible, and we urge caution in drawing conclusions from studies that may not have taken this into account. Third, the bibliometric analysis cannot directly assess the quality of the evidence presented in the publications. It can only provide a quantitative assessment of the impact of the publication, such as how many times it has been cited or the number of publications in a particular field. The quality of evidence presented in a publication depends on many factors, including study design, the methods used, and the validity of the results. Bibliometric analysis cannot assess these factors. Finally, the scope of the present study was limited to the title search and only contained search terms related to the microbiota and antibiotics. Therefore, it is possible that this study missed some articles that used the terms ‘microbiota’ and “antibiotics” or closely related terms as keywords or appeared anywhere in the text of the publication. However, if these false negative outcomes occur, their impact on overall results will be minimalNational Natural Science Foundation of China and the National Institutes of Health provide significant support for this type of research. Therefore, it is recommended that researchers pay more attention to the latest promising hotspots, including altered intestinal microbiota composition with antibiotic treatment. Further research in these areas could provide new insights and strategies to maintain host health and combat antibiotic resistance.This study is the first bibliometric analysis that objectively and thoroughly examines global microbiota trends related to antibiotic research over the past 20 years. In conclusion, this study provides a comprehensive summary of the current trends in global publications and helps researchers determine the current hot topics in this area. Currently, the number of publications in this field is increasing every year, with the United States and China making the greatest contributions. Furthermore, the


Concrete is among the most widely used structural materials in buildings and bridges all over the world. During their service life, concrete structures may inevitably display cracks due to long-term fatigue loads, leading to the degradation of structural integrity. Thus, it is very important to detect cracks and their growth in concrete structures using an automated structural health monitoring system. In this paper, experimental research on crack detection and imaging of concrete structures by using sensing skin and electrical resistance tomography (ERT) is presented. Carbon ink is screen-printed on the surface of concrete as a conductive material to form sensing skins. With these sensing skins, when cracks occur on or near the surface, it breaks the continuity of the sensing skins and significantly reduces conductivity in cracking areas. Then, after exciting small currents in sensing skins and measuring related voltage data, an inverse analysis based on total variation (TV) regularization is adopted to reconstruct tomographic images showing conductivity changes in sensing skins, to detect the occurrence and growth of cracks. The effectiveness of conductive sensing skins and our related crack detection method is validated in experimental studies on a concrete beam subjected to fatigue tests. Concrete is among the most widely used structural materials in infrastructures, such as buildings and bridges, all over the world. During their service life, concrete structures may inevitably display different types of damage, leading to the degradation of structural integrity. Among the different types of damage, cracking is a major factor in the premature deterioration of concrete structures, which seriously affects the service life of structures. Cracks provide erosion paths for water and corrosive agents to enter the interior of the concrete structures, accelerating the degradation of concrete structure performance ,2,3. TheDuring the past several decades, non-destructive inspection/evaluation (NDI/E) techniques, like ultrasound , acoustiIn addition to the aforementioned studies, considering the fact that the observation of concrete cracks is mainly conducted on the surface, researchers have proposed different types of conductive sensing skins, which can be integrated into the concrete surface to detect surface damage by monitoring changes in conductivity or resistivity. For example, Loh et al.  carried In this paper, a kind of carbon ink is proposed to screen print onto concrete surfaces as smart sensing skins to detect and identify cracks with ERT. Upon excitation of small currents in smart sensing skins and the generation of related voltage data, a total variation (TV) regularization-based algorithm is employed to generate tomographic images of conductivity changes in sensing skins caused by cracking, so as to determine the existence and development of concrete cracks in the whole process. Experimental studies on a concrete beam subjected to a fatigue test were performed to validate the effectiveness of the conductive sensing skin and the related crack detection method.Using voltage measurements from the boundary with a flowing electric current, ERT is a soft-field tomography method that can reconstruct the distribution of internal electrical properties of a conductive object ,27. In oDerived from Maxwell’s equations, the forward problem is governed by a Laplacian equation as follows:To apply boundary conditions to Equation (1), the complete electrode model (CEM) is employed. It is difficult to analytically solve Equation (1), so the most widely used numerical method, the finite element method (FEM), is usually adopted to solve a discrete approximation of Equation (1).Then, the inverse problem can be formulated to minimize Equation (2) in the least-squares sense, i.e.,Generally speaking, reconstructing the change in conductivity by directly inversing the matrix from Equation (3) is not straightforward, because Nonetheless, the Tikhonov regularization is an L2 norm-based regularization method; it usually overly smooths the solution and blurs the boundary of the damage, reducing the accuracy of crack identification. Thus, in this study, total variation (TV) regularization that protects edges and details from loss is employed to solve the ERT inverse problem . Its uncBy introducing an auxiliary variable According to the Bregman formula, Equation (8) can be solved iteratively in this way:Solving the first optimization problem in Equation (9) needs to be carried out in two steps. First, fix Then, fix Algorithm 1: Steps of TV-SB Algorithm.Step 11.2 Initialization: 1.1 Set iteration number Step 22.2 If the condition for termination is met, go to Step 52.1 If the condition for termination is not met, go to Step 3Step 33.2 Use Equation (11) to obtain 3.3 Set 3.1 Use Equation (10) to obtain Step 44.1 Set iteration number Step 55.1 End algorithm, output The above method for solving the TV regularization problem using the split Bregman method is called the TV-SB algorithm ,30. Its In the experimental studies, a reinforced concrete beam with dimensions of 200 mm × 300 mm × 2700 mm was considered. As illustrated in 2. According to the producer, a very small amount of graphene is doped to the ink to increase the conductivity. The carbon ink has a black paste appearance and was uniformly integrated on the surface of the concrete through silk printing. It was cured at ambient temperature for 8 h. After the solvent in the carbon ink evaporated, the conductive fillers came close to each other to form a conductive network. For each sensing skin, 24 copper foil electrodes were then evenly placed at designated positions on their boundaries using Type 3703 conductive silver paste produced by Xinwei New Materials Co., Ltd. . The surface resistivity of the conductive silver paste was about 2.5 × 10−3 Ω/mm2, and it was cured at ambient temperature for 24 h.To detect the occurrence of cracks and monitor their growth, carbon ink was coated on the surface of the concrete beam as sensing skins using a manual screen-printing process. As shown in For ERT testing, the electrodes of the sensing skins were connected to a set of data acquisition systems developed based on the PXI platform, as shown in To accelerate the occurrence of cracks, before fatigue tests, a static load of 90 kN was applied, and the first initial crack appeared in the middle, with a maximum crack width of 0.10 mm and crack length of 61 mm. Following this initial crack, fatigue loads were applied to the concrete beam. When the fatigue test was loaded to 20,000 cycles, three new cracks appeared. In the subsequent cyclic loading process, there were a few new cracks, mainly due to the increase in the length and width of existing cracks. When loaded to 100,000 cycles, the maximum crack length reached 191 mm; when loaded to 200,000 cycles, two new cracks appeared, one of which was a diagonal shear crack with a length of 184 mm. The original cracks had evolved, and the maximum crack width reached 0.62 mm; when loaded to 290,100 cycles, two new oblique shear cracks appeared, each with a length exceeding 200 mm. In addition, two cracks intersected near the mid-span in the tensile zone of the lower part of the test beam, forming a “herringbone”-shaped crack. The maximum crack width quickly exceeded 1.50 mm, and the crack width on its symmetrical plane also exceeded 1.25 mm, basically forming a vertical crack that ran through the cross-section. At this moment, the concrete beam was considered as failed under the applied fatigue load, and the test stopped.The evolution of cracks on the lateral surface of the concrete beam is summarized and depicted in Referring to the process of crack evolution in fatigue tests of the reinforced concrete beam, screen-printed sensing skins and ERT were used to image and identify the crack situation at fatigue intervals of 20,000, 100,000, and 200,000 cycles, respectively. The distribution of cracks in fatigue tests was relatively complex, with multiple cracks occurring and developing in the same monitoring area. If traditional dynamic ERT algorithms are used, that is, damage identification at different times is based on the initial “intact state” for damage image reconstruction, due to the overlapping interference of cracks, the limited number of tested boundary voltage data is insufficient to provide feedback on all the cracks. Therefore, for each sensing skin, the method of “phased identification and superposition” is adopted, which means that by subtracting different states, the cracks of each time period are identified separately and then stacked and superposed together. Specifically, before static and fatigue loads were applied, small currents were injected into the sensing skins and corresponding voltage measurements were collected according to the selected adjacent electrode mode to obtain a baseline dataset from the pristine state; then, at fatigue intervals of 20,000 cycles, the same testing procedure was performed to collect a voltage dataset, and by comparing with the baseline dataset, the TV-SB algorithm was used to reconstruct changes in conductivities for sensing skins to generate information about cracks at this interval. At the fatigue intervals of 100,000 and 200,000 cycles, the testing procedure was still the same; however, in the TV-SB algorithm, the voltage dataset was obtained at the last time period, i.e., fatigue intervals of 20,000 and 100,000 cycles were used as the “baseline” dataset, respectively. Since ERT images were only used to indicate the locations and sizes of the cracks, for convenience, the reconstructed tomographic images were first binarized into black and white images and then superimposed together. As shown in From From From the above experimental results, it can be seen that sensing skins fabricated by using carbon ink can detect cracks at an early stage during the fatigue process, and the growth of cracks can be monitored by using the ERT method to some extent. According to the authors’ knowledge, this is the first time that carbon ink has been applied to the health monitoring of concrete structures through screen printing. Carbon ink can be produced on an industrial scale, which greatly enriches the sensing materials required for monitoring large-scale structures such as concrete, and provides an important reference for the application of other similar low-cost materials in civil engineering.However, there were also some shortcomings in our work. It can be observed from the results that although the dynamic ERT algorithm based on TV regularization can detect crack growth at different fatigue stages, generally, it is only qualitative. It cannot provide very accurate information about the width and length of the cracks. In particular, it is necessary to develop quantifiable indicators to quantitatively determine the error of crack identification and provide a basis for decision-making. We will strengthen our work in this area in the future.In this paper, an experimental work in crack detection and imaging for concrete structures using screen-printed sensing skins and ERT is presented. Carbon ink is employed as a conductive material to be screen-printed on the surface of concrete structures to form sensing skins, and an ERT testing system is used to excite small currents in sensing skins and measure related voltage data from their boundaries. By processing voltage datasets with a TV regularization-based algorithm, changes in conductivity of the sensing skins caused by cracks and their growth are reconstructed to provide information about the cracks. Experimental studies on a concrete beam subjected to fatigue tests have demonstrated that carbon ink is a good sensing material for the fabrication of printed sensing skins on the surface of concrete structures, and it is feasible and effective to use a combination of printed sensing skins and ERT to detect cracks and their growth. This TV-SB algorithm can reconstruct the distribution of conductivity changes in printed sensing skins to capture the occurrence of cracks in a timely manner and provide information about the location and direction of the cracks, achieving crack identification in concrete structures. However, quantitatively identifying the widths and lengths of cracks is still challenging; the currently identified lengths of cracks from tomographic images show a certain deviation from the true lengths of the cracks. This requires further investigation for damage identification in concrete structures using screen-printed sensing skins and ERT.


Melanoma is a cancer of cells that produce melanin pigment, and it can develop in dogs as well as in humans. Canine melanoma has a high risk of metastasis and is resistant to systemic chemotherapy. Therefore, alternative effective systemic treatment is urgently needed. Abnormal expression and activation of the receptor tyrosine kinase MET is reported in many human cancers, including melanoma. However, the role of MET in canine melanoma is unknown. The purpose of this study is to confirm MET expression in canine melanomas and determine if the levels of expression correlate with prognosis. A review of 30 canine melanoma cases confirmed mild MET expression in more than 50% of these samples. While the levels of MET expression were not significantly associated with key histologic features or survival, a difference in time to metastasis to lymph nodes versus other organs was noted. This may suggest a role for MET expression in the pattern of metastasis, which may in turn help with patient stratification and treatment recommendation.The overexpression and activation of the MET receptor tyrosine kinase has been identified in many human malignancies, but its role in canine cancer has only been minimally investigated. In this study we evaluated the expression of MET in two canine malignant melanoma (CMM) cell lines as well as in 30 CMM tissue samples that were collected from the clinical service at our institution. We were able to confirm the expression of the MET protein in both melanoma cell lines, and we demonstrated MET activation by its ligand, HGF, through phosphorylation, in Western blot analysis. We were also able to demonstrate, by immunohistochemistry, the expression of MET in 63% of the tumor tissue samples analyzed, with the majority demonstrating a relatively low expression profile. We then evaluated the association of MET expression scores with histologic parameters, metastasis, and survival. While statistically significant associations were not found across these parameters, an inverse relationship between MET expression levels and time to lymph node versus distant metastasis was suggested in our cohort. These findings may require assessment in a larger group of specimens to further evaluate the role of MET expression in the homing of metastasis in lymph nodes versus that in distant organs. The reported one-year survival rate for dogs treated with surgery alone is about 20% . PositivHuman and canine melanoma, particularly mucosal and oral subtypes , share similar clinical and molecular similarities and are generally resistant to traditional chemotherapy ,5,6,7. MIn dogs, oral melanoma is the most common oral malignancy with higher reported incidences in breeds such as cocker spaniel, miniature poodle, Anatolian sheepdog, Gordon setter, chow chow, and golden retriever . While sThe MET protooncogene is a surface receptor tyrosine kinase that is expressed on epithelial cells of many organs in physiologic conditions. After binding to the hepatocyte growth factor (HGF), its native and selective ligand, MET activates multiple downstream signaling pathways that result in cell proliferation, motility, and survival. Aberrant activation of the MET/HGF signaling pathway, commonly in both a ligand-dependent autocrine and paracrine mechanism or in a ligand-independent fashion, has been found in many human malignancies, such as papillary renal carcinoma, small cell lung cancer, and cutaneous melanoma ,13,14,15Given that the genetic landscape and mutations that have been identified in canine mucosal melanoma resemble that of human mucosal and acral melanoma, spontaneously occurring oral melanoma in dogs is a potential comparative oncology model for humans ,8,18,19.Here we investigate the expression of MET in melanoma cell lines and tissue samples of melanoma from our clinical service and correlate the expression scores with histologic parameters, metastatic status, and survival outcomes.A search of the University of Minnesota Veterinary Medical Center (VMC) patient database from 2004 to 2019 for client-owned canine patients with documented diagnoses of melanoma yielded 517 cases, of which 175 cases had histopathology reports available for review. Cases were excluded for further analysis if the patient did not have a malignant melanoma as the final diagnosis, underwent any form of treatment before surgery, was considered to have a recurrent tumor based on the medical records, or if the surgery was performed at the referring veterinarian where tissue samples were not available for further evaluation. After reviewing the medical records and histopathology reports, 79 cases were identified as naïve canine malignant melanoma but only 37 cases had matching tissue samples available from Marshfield Laboratory, now a part of Idexx Laboratories. Recuts of tumor tissues were independently evaluated by a board-certified dermatopathologist (A.G.) and a veterinary anatomic pathologist (A.W.), and seven of those cases did not have evidence of tumor on recut so they were excluded from the final analysis. A total of 30 cases were included in the study which consisted of 20 oral malignant melanomas, 5 acral, and 5 cutaneous malignant melanomas.Clinical data collected for each case included breed, gender, weight, age at diagnosis, date of diagnosis, tumor type , treatment pursued, status and date of lymph node and/or distant metastasis, last contact date, and date and cause of death if available. Pertinent data that are presented in this study are available in Informed consent was obtained from all of the University of Minnesota VMC clients for use of patients’ tissues for scientific study and teaching purposes. Only de-identified, archival, diagnostic material was used for this study so institutional review board or ethics committee approval were not applicable.To confirm the expression of MET protein and MET phosphorylation after HGF stimulation, Western blots were performed using two CMM cell lines (TLM-1 and CMGD-2). The canine osteosarcoma cell line OSCA-8 (previously reported to express MET) was used as the positive control . Sample g for 5 min at room temperature (RT). Formalin supernatant was then removed, and the pellets were washed with PBS. Finally, the cell pellets were re-suspended in 80% ethanol and paraffin embedded in cell blocks. To validate the MET antibody (Met (D1C2) XP® Rabbit mAb #8198, Cell Signaling Technology, Danvers, MA, USA) to be used in immunohistochemistry (IHC), cell pellets were generated from the three canine cell lines and processed by BioNet according to standard laboratory protocols.To prepare cell pellet blocks, cells were grown in T-75 flasks to near confluence, washed three times with PBS and fixed with 10% neutral-buffered formalin (NBF) in flasks overnight. After fixation, the cells were gently scraped and transferred to a conical tube and centrifuged to ~300× Unstained tissue sections (4 µm) of the 30 identified cases were de-paraffinized and rehydrated using standard methods. For antigen retrieval, the slides were incubated in 6.0 pH buffer  in a steamer for 30 min at 95–98 °C, followed by a 20-min cool down period. Subsequent steps were carried out manually. Endogenous peroxidase activity was quenched by slide immersion in 3% hydrogen peroxide solution  for 10 min followed by TBST rinse. A serum-free blocking solution  was placed on sections for 30 min. The blocking solution was removed and the slides were incubated in anti-MET (clone D1C2 XP(R))  diluted in 10% Sniper blocking solution/90% TBST overnight at 4 °C. On the following day, the slides were rinsed in TBST and biotinylated anti-rabbit (Vector Labs 1:200) was applied. The slides were incubated for 30 min at room temperature. Again, the slides were rinsed with TBST and 4+Streptavidin AP Label  was applied for 30 min. All the slides proceeded with TBST rinse and detection with Warp Red Chromogen  using the manufacturer’s specification for 7 min at room temperature. The slides were then rinsed for 5 min in running tap water and counterstained with CAT Hematoxylin diluted 1:2  for 5 min, air-dried, dipped in xylene, and cover slipped.Hematoxylin and eosin (H&E) and MET-stained canine melanoma samples were reviewed independently by a board-certified dermatopathologist (A.G.) and a veterinary anatomic pathologist (A.W.). The histologic data that were evaluated and collected included mitotic index (defined as mitotic count per 10 high-power fields), degree of pigmentation (<50% or >50%), presence of ulceration (Y or N), presence of necrosis (Y or N), presence of deep inflammation (Y or N), and Breslow thickness in millimeters. In human cutaneous melanocytic tumors, Breslow thickness is the measurement from the top of the granular layer of the epidermis or base of the ulceration to the deepest invasive cell across the base of the tumor. It is one of the most important independent prognostic indicators with thinner melanomas conferring a higher disease-free survival. While Breslow thickness has not been widely utilized in veterinary medicine, Silvestri et al. reported that tumor thickness was a prognostic indicator in canine melanocytic neoplasms, with cutoffs of 9.5 mm and 7.5 mm conferring a higher hazard for shorter overall survival and disease-free intervals, respectively .The MET score was assigned to each sample based on the intensity of cell membrane staining and was independently evaluated by each of the two reviewers. The scores ranged from 0 to 2, with 0 for negative immunopositivity, 1 for mild/moderate, and 2 for marked immunopositivity. All of the slides were processed together to eliminate variations in staining between batches. A consensus was reached when there were discrepancies in the assigned scores. Normal canine liver tissue was used as a positive control. Representative histologic pictures after H&E and MET staining are shown in Several analyses were performed to assess the MET prognostic value. For binary values , the Cochran–Armitage trend test was used; for numerical values (Mitotic index), Spearman’s rank correlation was used; and for censored data (Breslow) and time to event data, the log-rank test for trend was used. Analyses were also performed to assess differences between tumor types. For binary values, Fisher’s exact test was used; for numerical values, the Kruskal–Wallis test was used; and for censored data and time to event data the log-rank test was used. Time to lymph node metastasis and distant metastasis were considered censored at either time to last follow-up or time of death; therefore, these should be interpreted as the time to these events if they had not died first. Time to death was censored if they were lost to follow-up while still alive. For binary variables, counts and percentages are reported; for continuous data, medians and ranges; for censored data (Breslow), the estimated percent >5 from the Kaplan–Meier curve and the number of uncensored data; and for time to event data, Kaplan–Meier curves are shown. All calculations were performed in R version 4.0.2 (22 June 2020).n = 6, 20.0%), German shepherd , and golden retriever . A total of 15 dogs (50.0%) had reported lymph node and/or distant metastasis, of which nine dogs (30.0%) had distant metastasis only, three dogs (10.0%) had both confirmed or suspected lymph node and distant metastasis, and two dogs (6.7%) had lymph node involvement only. Additional clinical characteristics of these cases can be found in A total of 30 cases of CMM were selected from our database, including 18 female dogs (60.0%) with an average weight of 30.1 kg (range 3.2–68.8 kg) and an average age of 10 years at the time of diagnosis, and 12 male dogs (40.0%) with an average weight of 35.5 kg (range 12.3–47.2 kg) and an average age of 9.5 years at time of diagnosis. The three most common breeds were Labrador retriever  received some form of treatment after diagnosis. Of these, 11 (36.7%) and 8 (26.7%) of these dogs underwent surgery plus adjuvant melanoma vaccine or radiation therapy, respectively. The breakdown of the treatment options elected can be found in To confirm the expression of MET protein and MET phosphorylation after HGF stimulation, Western blots were performed using two melanomas and one osteosarcoma cell lines. The results of the Western blots confirmed MET protein expression in all three cell lines. Critical tyrosine residues in the kinase domain and a multifunctional docking site of the MET receptors, namely tyrosine 1234/1235 and 1349 which modulate enzyme activity and recruit transducers and adaptors after MET activation, respectively, were also found to be phosphorylated appropriately after stimulation with recombinant human HGF . These fTo validate the use of the same antibody for immunohistochemistry, we created formalin-fixed and paraffin-embedded cell blocks from pellets of the same three cell lines. Immunohistochemistry with the monoclonal MET-specific antibody was positive in the three cell lines, effectively validating this antibody for immunoprofiling of tissue sections from our cohort of canine melanoma samples .The parameters that were evaluated during histopathologic review of the 30 CMM cases included the degree of pigmentation, presence of ulceration, presence of necrosis, presence of deep inflammation, mitotic index (MI), and Breslow thickness. A summary of the results by tumor type is available in Survival outcome data were available for 17 dogs  while 11 of the dogs were lost to follow-up (36.7%). There were two dogs that were alive at the end of data collection period (6.6%). The median survival time for all dogs with oral melanoma was 457 days (range 41–1023 days). The two dogs with acral melanoma survived 829 day and 290 days, respectively. There was one dog with cutaneous melanoma that had a survival time of 329 days.We then performed immunohistochemical assessment of MET expression on the CMM clinical tissue samples. Of all 30 cases of CMM that were reviewed, 16 (53.3%) had an MET score of 1, and 3 of the cases (10.0%) had an MET score of 2. The rest of the 11 cases (36.7%) were negative for MET expression. When the MET scoring was broken down by tumor types, an MET score of 1 was the most common among all the subtypes, including 10 oral melanomas  and 3 of each acral and cutaneous melanomas (60.0% of each subtype of melanoma) .We then evaluated the relationship between histopathologic features and MET expression for all canine melanomas. Using univariate analysis, we did not find statistical significance for any of the parameters evaluated ; similarFinally, we investigated the relationship between MET expression and the time to metastasis or death . The association of MET score with time to disease metastasis, including lymph node metastasis or distant spread, and death for all types of melanomas was not found to be statistically significant a–d. SimiWhile no statistically significant associations were identified across these parameters, a possible inverse relationship between the MET score and time to lymph node metastasis was noted a,e. On tIn this study, we aimed to investigate MET protein expression and activation in canine melanoma cell lines as well as investigate MET expression in tissue samples of canine melanoma from our clinical practice in a tertiary care academic veterinary practice. Canine melanoma, unlike human melanoma, is most commonly a mucosal or acral disease, rather than cutaneous. Interestingly, there are several similarities between oral and acral melanoma in humans and the corresponding disease in dogs, making the latter an attractive comparative model ,8,18,19.Our current knowledge of the molecular underpinning of melanoma in dogs is piecemeal at best; this lack of knowledge is reflected by the limited therapeutic options in canine patients, accompanied by a lack of robust markers for disease progression and prognosis. Thus, development of novel predictive and prognostic biomarkers is a priority.Receptor tyrosine kinases (RTKs) are attractive targets for oncologic therapy. Several selective inhibitors have been developed over the past two decades. Among these RTKs, the MET receptor appears to play a role in the pathogenesis and progression of melanoma in humans ,16,26. TIn this study, we confirmed MET protein expression in canine melanoma cell lines as well as tumor tissues. We also demonstrated the activation through phosphorylation of MET in key tyrosine kinase domains in canine melanoma cell lines through Western blots. This includes the phosphorylation of tyrosine residues 1234/1235 in the kinase domain of MET with consequent activation of the catalytic activity of the receptor and activation through phosphorylation of tyrosine residue 1349 in the multifunctional docking site in the c-terminus of the receptor. This domain is particularly important for connecting the activated MET receptor to downstream signaling through the use of adaptor proteins and enzymatic reactions, which will ultimately lead to relevant gene transcriptions in the nucleus. Thus, we have demonstrated that the MET receptor is present and can be activated in canine melanoma cell lines.The expression of MET in canine melanoma tissue was confirmed through immunohistochemistry with a validated antibody. We confirmed MET immunopositivity in canine malignant melanoma tissues in 63% of the specimens. This demonstrates that MET is present in primary melanoma tumors in dogs, although the expression appears to occur at low levels, as demonstrated by the presence of +1/mild immunopositivity in the majority of the specimens.MET expression has been reported in the literature in other canine tumors, although reports are sporadic. In 2005, Liao et al. evaluated the expression and function of MET in 25 canine cancer cell lines including osteosarcoma and melanoma . All celOverall, we did not observe statistical significance in the associations of MET expression with histopathologic parameters that are commonly investigated and recorded during histopathologic evaluation. However, we made some interesting observations that are important to outline here. By comparing the time to event curves of all canine melanoma patients with the three levels of MET expression, we noted that the line representing negative MET staining (“CMET = 0”) is consistently lower than the one for mild immunopositivity (“CMET = 1”) for time to lymph node metastasis a. This sBased on a comprehensive review of prognostic factors in canine melanoma, the mitotic index has been previously identified as an independent prognostic factor . An MI >Similar to other retrospective studies, a small sample size and incomplete data from loss to follow-up are among the limitations of the current study. Of the 30 cases with histologic diagnosis of melanoma and tissue samples available, 13 were censored with 11 lost to follow-up and 2 dogs alive at the end of data collection. The inconsistency in the follow-up of these cases also affected the reported time to metastasis and death—biases that a larger scale prospective study with set recheck schedules and re-staging requirements can overcome. Despite the limitations in the data collected, we were able to retrieve clinical and histopathologic information as reported in In conclusion, this is the first study to evaluate the level of MET expression with prognosis and survival in canine melanoma tissue samples. These findings may require assessment in a larger group of specimens to confirm the clinical implications of MET expression assessment in CMM.