Hierarchies can be modeled by a set of exponential functions, from which we can derive a set of power laws indicative of scaling. The solution to a scaling relation equation is always a power law. The scaling laws are followed by many natural and social phenomena such as cities, earthquakes, and rivers. This paper reveals the power law behaviors in systems of natural cities by reconstructing the urban hierarchy with cascade structure. Cities of the U.S.A., Britain, France, and Germany are taken as examples to perform empirical analyses. The hierarchical scaling relations can be well fitted to the data points within the scaling ranges of the number, size and area of the natural cities. The size-number and area-number scaling exponents are close to 1, and the size-area allometric scaling exponent is slightly less than 1. The results show that natural cities follow hierarchical scaling laws very well. The principle of entropy maximization of urban evolution is then employed to explain the hierarchical scaling laws, and differences entropy maximizing processes are used to interpret the scaling exponents. This study is helpful for scientists to understand the power law behavior in the development of cities and systems of cities. Hierarchy is one of the basic characters of complex systems such as cities and networks of cities. A hierarchy can be mathematically described with a power law or a pair of exponential laws. In recent years, many scientists have been interested in the hierarchical structures of natural and social systems . A fractn rule [The hierarchical scaling laws are associated with many mathematical laws of cities. The models are found and reconstructed by Chen , who oncn rule , and Bern rule . Hierarcn rule  who oncen cities, we can organize the cities into a hierarchy comprising M classes according to the generalized 2n rule [m refers to the order number of city class , mN denotes the number of cities of order m, correspondingly, mS and mA represent the average population size and average urban area at the mth class. The parameters are as below: N1 denotes the number of the top-class cities, S1 and A1 are the mean size and mean area of the first-class cities, rn = mN+1/mN refers to the interclass number ratio of cities, rs = mS/mS+1 to the city size ratio, and ra = mA/mA+1 to the urban area ratio. Generally speaking, N1 = 1, but for the three-parameter Zipf’s distribution, N1 > 1 [n principle [n rule that says that if the cities in a geographical region are organized into a hierarchy by means of a fixed size ratio rs = 2, the number ratio of the cities at different levels will be 2, that is, rn = 2 [rn = 2, the size and area ratio of the cities at different levels will range from 1 to 3, respectively, that is, 1 < rs < 3, 1 < ra < 3. The is called generalized 2n rule of hierarchies of cities [The urban hierarchy with cascade structure can be described from two complementary angles of view. The longitudinal distributions can be described with exponential functions, and the latitudinal relationships can be described with power functions ,23. Cons 2n rule ,24. Base, N1 > 1 . Equatiorinciple , which irinciple ,21. Davif cities ,24.number law, the second equation represents the size law, and the third equation represents the area law of urban hierarchies [rn = 2 as given, then it will follow that rs→2, and if rs = 2 as given, then it will follow that rn→2, where the arrow denotes “approach”. If rn = rs = 2, the generalized 2n principle will return to Davis’ standard 2n principle. In this instance, we will have mT = mSmN = N1S1 = S1, where mT denotes the total population at the mth level. This suggests a property of hierarchical conservation of size distributions, which is consistent with the standard 2n rule of cities. If a hierarchy of cities complies with Davis’ 2n rule, the total population size at each level of the hierarchy is theoretically a constant.Among this set of exponential functions, the first equation represents the rarchies . The thrrarchies . For a srn = rs, we can derive a one-parameter Zipf’s model, S(k) = S1/k, where k refers to rank, and S1 is a parameter indicative of the largest city [rn ≠ rs, we can derive a two-parameter Zipf’s model, S(k) = S1/qk, where q is the second parameter indicating scaling exponent. If rn ≠ rs and the largest city cannot influence the whole geographical region, we can derive a three-parameter Zipf’s model, S(k) = C/(k + h)q, where h is the third parameter indicating translational factor, and C denotes proportionality coefficient [vice versa [A set of Zipf’s models is hidden behind the three exponential laws. This suggests that Zipf’s distributions can act as an indication of the self-similar hierarchy. From Equations (1)–(3) we can derive Zipf’s laws of population size distribution and area size distribution. The city size can be measured by both population quantity and urbanized area. The former is termed population size, and the latter is called area size of a city. Where population size distribution is concerned, three types of Zipf’s models can be derived . If rn =est city ,28. The est city . If rn ≠fficient . Where tce versa ,7.μ = N1P1D, D = lnrn/lnrs, η = N1A1d, d = lnrn/lnra, a = A1P1b−, and b = lnra/lnrs. Among these parameters, D denotes the fractal dimension of city population size distributions, d denotes the fractal dimension of urban area size distributions, and b is the allometric scaling exponent of urban hierarchy. In fact, b is the ratio of the fractal dimension D to the dimension d, that is, b = D/d = (lnrn/lnrs)/(lnrn/lnra) = lnra/lnrs. Apparently, from Equations (1) and (2), we can derive Equation (4); from Equations (1) and (3), we can derive Equation (5); from Equations (2) and (3), or from Equations (4) and (5), we can derive Equation (6). This implies that, for the cascade structure of a hierarchy of cities, exponential laws and power laws represent two different sides of the same coin. The exponential laws can be directly derived from the principle of entropy maximization, and thus entropy maximization can be employed to indirectly explain the power laws of cities.The relationships between exponential laws and power laws suggest the relationships between simplicity and complexity. Especially, these relationships suggest the links between characteristic scales and scaling. Exponential laws indicate the conventional growth, distributions, and processes with characteristic scales, while power laws indicate the allometric growth, fractals, and patterns without characteristic scales. The former suggests simplicity, and the latter suggests complexity. The exponential laws and power laws can be integrated into the same framework with the hierarchical scaling concept. The hierarchical scaling in cities performs power law behaviors and can be expressed with the three power functions ,30. Fromsize-number scaling in a hierarchy of cities. It is equivalent to the Pareto law of population size distribution, and D is the fractal dimension of urban hierarchies measured with city size such as population [area-number scaling of cities. It is equivalent to the Pareto law of city-area distribution, and d can be regarded as the fractal dimension of urban hierarchies measured with urbanized area [b is the allometric scaling exponent of urban hierarchy [The above-shown power laws represent three typical aspects of scaling behaviour of cities. Equation 4) suggests the pulation . Equatiozed area . Equatioierarchy . The invierarchy . Hierarc suggestsierarchy . By hierThe power law behaviors of hierarchical scaling in city development can be explained by the principle of entropy maximization. In urban hierarchies, a power law is based on two exponential laws, and the relationships between the power laws and exponential laws can be revealed by the self-similar hierarchy. In fact, exponential distributions can be directly derived by using entropy-maximizing methods ,35,36,37Now, the principle of entropy maximization can be employed to explain the emergence of power law behaviors of cities. In the hierarchy with cascade structure, power laws and exponential laws are compatible with each other. A pairs of exponential laws indicative of longitudinal distributions support a power law reflecting the latitudinal relation between two measurements. In this work, the power laws, Equations (4)–(6), are derivable from the exponential laws, Equations (1)–(3). This suggests that a power law is based on two dual processes of entropy maximization . ConcretD in Equation (4) approaches to 1, i.e., D→1, otherwise, D > 1 or D < 1; (2) if the entropy maximization process of the frequency distribution of cities numbers and that of the urban area size distribution keep in step with each other and fall in the state of balance, the scaling exponent d in Equation (5) approaches to 1, i.e., d→1, otherwise, d > 1 or d < 1; (3) if the entropy maximization process of the city population size distribution and that of the urban area size distribution keep in step with one another and fall in the state of balance, the scaling exponent b in Equation (6) approaches to 1, i.e., b→1, otherwise, b > 1 or b < 1. Using the power law relations and scaling exponents based on the entropy maximization, we can make evaluation on city development in a geographical region.Moreover, the principle of entropy maximization can also be utilized to interpret the scaling exponent values of power laws of cities. If the two dual processes of entropy maximization are of synchronization and in a state of balance, the scaling exponent will be close to 1, or else, the exponent value will departure from 1 (greater than or less than 1). Generally speaking: (1) if the entropy maximization process of the frequency distribution of cities numbers and that of the city population size distribution keep in step with each other and fall in the state of balance, the scaling exponent natural city and developed a new approach to measuring objective city sizes using street nodes or blocks. In urban geography, a city can be defined as a large settlement that has some kind of service functions to the surrounding areas. However, a natural city is the human settlement based on landscape rather than service functions. Natural cities proposed by Jiang and his co-workers [The validity and rationality of the mathematical models can be verified and evaluated through empirical observation data. In fact, the success of natural sciences just rests heavily with their great emphasis on the role of interplay between quantifiable data and models . Four sy-workers ,39 can b-workers . Human a-workers ,6. Thus,-workers ,40. In t-workers ,42, the -workers , and the-workers . In this-workers . Using t-workers ,45.Urban block is an ordinary concept, and the street nodes are defined as street intersections and ends. Using an identification algorithm of urban boundary, Jiang’s research group was able to delineate boundaries of natural cities and yield city areal extents. Thus urban area can be determined by a city’s areal extent containing a large number of street blocks or nodes. The number of street nodes is significantly correlated with the population size of cities. The city data are extracted from massive volunteered geographic information OpenStreetMap databases through some data-intensive computing processes, and four datasets on the cities of the U.S.A., Britain (UK), France, and Germany are available. The process of identifying natural cities is actually an approach of spatial search, and the number of cities is automatically determined through spatial search technique. By the same technical criterion of spatial search, the numbers of natural cities extracted from different countries may be very different. The reason lies in the different geographical conditions, which result in great differences in the spatial patterns of urban development. In Britain and France, natural cities have better correspondence with the usual cities, while in Germany, natural cities are significantly different from traditional ones.n rule to the above-mentioned datasets, we can create four self-similar hierarchies of European and U.S. cities. Suppose that these systems of cities follow the pure Zipf’s law. Then the cities in each country can be reorganized into a hierarchy with cascade structure. The Zipf’s law cannot be directly derived by using the method of entropy maximizing, but the hierarchical scaling laws can be derived by means of this approach. Curry once tried to derive Zipf’s law using the idea from entropy maximization [The empirical analysis can start from investigating Zipf’s distribution, which, as pointed out above, can be regarded as a signature of the hierarchies with cascade structure. If cities in a region follow Zipf’s law, they can be organized into a self-similar hierarchy ,30. It hmization , but hismization . Table 2Several algorithms can be adopted to evaluate the scaling exponents. The most common ones include the least squares method (LSM) , maximumn principle expressed by Equations (1) and (2), we can organize the cities in each country into a hierarchy with cascade structure. The city number in the mth level is mN = 1, 2, 4, …, 2m−1, … The numbers of levels in the urban hierarchies in the four countries are 15, 11, 11, and 13, respectively. The last levels are lame-duck classes because that city numbers are not big enough. Based on the hierarchical structure, we can calculate the average city size mP and the corresponding average urban area mA at each level (n rule and satisfy Equation (1). It is easy to testify that city size mP and urban area mA follow exponential distribution and meet Equations (2) and (3), respectively, but the lame-duck classes are two outliers due to lack of adequate cities. Strictly speaking, the first class is usually an outlier because the largest city is often an exception [The systems of cities in the U.S.A., U.K., France, and Germany can be well described with hierarchical scaling equations. In light of the generalized 2ch level . The citxception . In factd value (area exponent) is slightly greater than the D values (size exponent). The ratio of D to d can be termed fractal dimension quotient of urban hierarchies. As indicated above, if D approaches 1, the total “population” of the mth level approaches a constant S1. Despite the fact that there are always many smaller cities than larger ones [i, iP, and the rank of class i within all classes when ordered by population size, iR, approaches a constant K, i.e., iRiP = K [mth level of the self-similar hierarchy, mT = mSmN, approaches to a constant, i.e., mT→constant, which suggests a conversation law. The conversation law implies some type of symmetry [The exponential distributions of city size and urban area result in the power-law relations between city number, size, and area. The exceptional values in the exponential laws often manifest themselves on the log-log plots for power laws. In fact, if the scale is too large or too small, a power-law relation always breaks down ,50. Thusger ones ,38,45, tPiRi = K . The difsymmetry ,52. In tSince Zipf’s law is a signature of the self-similar hierarchy of cities, two distributions related to the rank-size distributions should be discussed here. First, the relationship between Zipf’s distribution and the lognormal distribution. Where city-size distributions are concerned, if we do not identify the scaling range, the rank-size relation often satisfy a lognormal distribution rather than a power-law distribution; however, if the scaling range is taken into consideration, the power-law relation is always clearer than the lognormal relation. In order to reveal the power-law relations of urban hierarchies, the data points at the two extremes should be removed as outlier. Second, the rank-size distribution and the primate distribution. Both the city size distributions of Britain and France are regarded as primate distribution. However, according to The relationships between city number and city size or urban area are a pair of fractal dimension relations, from which it follows an allometric scaling relation between city size and urban area. Using the data displayed in b ≤ 1. Second, the allometric scaling exponent is equivalent to the fractal dimension quotient. In theory, the allometric exponent is the ratio of the fractal dimension of urban population size distribution to that of urban area size distribution. Where empirical analysis is concerned, the allometric exponent is close to the fractal dimension ratio. Generally speaking, for the developing systems of cities, the fractal dimension of population size distribution is significantly less than that of area size distribution. The allometric scaling exponent values come between 2/3 and 1, and always approach to 0.85 [q = 1 [b = 1 or 0.85 [q must fall between 1/2 and 2, i.e., 1/2≤ q ≤ 2 [b must come between 2/3 and 1, i.e., 2/3 ≤ b ≤ 1 [The four study areas, U.S.A., U.K., France, and Germany, are all developed countries, and the levels of urbanization are near their respective capacity values, i.e., the upper limit values. The allometric scaling properties of these urban hierarchies are as below: First, the allometric scaling exponent is close to but less than 1. This suggests that the relative growth rate of city size is slightly less than that of urban area. When a city is small, its population density is low, the per capita land use quantity is large, and the city expands fast in the two-dimensional space. With the growth of the city, the population distribution is becoming more and more concentrated, and the urban buildings begin to develop to the higher level, thus the per capita land consumption become smaller and smaller, and intensification of urban land use emerges. As a result, the allometric scaling exponent  to 0.85 ,31,53. H to 0.85 . The sta5 [q = 1 ,27,28, a2≤ q ≤ 2 , and then rule, gravity model, and so on into a logic framework [D, and the area-number scaling analysis yields the fractal dimension of area-size distribution d. From the above-mentioned scaling relations, we can derive the size-area allometric scaling relation, and the allometric exponent b is equal to the ratio of D to d. All these results support the judgment that the evolution of natural cities conforms to the principle of entropy maximization. Entropy maximization means an optimal and coordinated relationships between the efficiency of the whole and the equity among individuals in a self-organized system [The applications and functions of the self-similar hierarchy lie mainly in the following four aspects. First, it can be employed to reveal the physical foundations of power law behaviors of cities using the principle of entropy maximization . Second,ramework ,7,26,30.ramework . Fourth,ramework . The empd system . In a seHierarchical allometry is one of urban allometric scaling relations. Allometric scaling includes longitudinal allometry , transversal allometry , and spab = D/d = Da/Ds, where D and d refer to the fractal dimensions of population and area size distributions, and Ds and Da denote the fractal dimension of the spatial distributions of city population size and land use form [D and d proved to be paradimension according to the relationships between Zipf’s law and hierarchical scaling law [M, where M is a positive integer  direction relations, the first one reflect the size-number scaling relation, the second one reflect the area-number scaling relation, and third one reflect the size-area allometric scaling relation. The self-similar hierarchy indicates a kind of deep structure of systems of cities and latent spatial order in urban evolution, which can be understood from the perspective of entropy maximization. The contribution of this work to the studies on scaling of cities rests with the following aspects. First, the principle of entropy maximization is employed to explain the power law behaviors of urban hierarchies from new angles of view. Three processes of entropy maximizing are used to interpret the emergence of power laws and the related scaling exponents. Second, a series of allometric scaling models are divided into three groups and put in order in a logic framework. The framework includes longitudinal allometry, crosssectional allometry, and hierarchical allometry. Third, an entire case study on hierarchies of natural cities are presented. The case may be helpful for readers to understand power law behaviors in hierarchies from a new angle of view.The main conclusions of this study can be drawn as follows. First, the natural cities provide a new way of understanding the hierarchical scaling laws, which can be represented by a set of power functions. The three hierarchical models can be well fitted to the datasets of natural city size and urban area of the U.S.A., Britain, France, and Germany by taking the scaling range into consideration. In a sense, the natural city is a concept of cities based on geographical landscape, which differs from the traditional city concept based on human administration. Compared with the census data or statistic data of common cities, the observational data of natural cities show better effect of hierarchical scaling analysis. Moreover, the allometric scaling relation comes from a pair of rank-size scaling relations. In theory, the allometric scaling exponent is equal to the ratio of the fractal dimension of population size distribution to that of area size distribution; in practice, the allometric exponent is very close to the quotient of the two fractal dimension values of size distributions (esp. Britain and Germany). Second, the principle of entropy maximization can be employed to explain the emergence of power law behaviors in hierarchies of natural cities. A power law is based on a pair of exponential laws on city number and sizes. An exponential law can be derived by means of the method of entropy maximizing. Thus a power law is determined by two dual entropy maximization processes: the entropy maximization of city frequency distribution and that of city size distribution. An urban hierarchy is involved two types of entropy maximization: the frequency distribution of cities and size distributions of urban population and urbanized area. The fractal models are controlled by an entropy maximization process of frequency distribution and that of size distribution, while an allometric scaling relation is dominated by two entropy maximization processes of size distributions such as population size distribution and area size distribution. Entropy maximization can explain the power law behaviors of traditional city size distribution, but this principle seems to be more suitable for explaining the evolution and power law emergence of natural cities. What is more, the entropy maximization principle can also be used to interpret the scaling exponent values of urban power laws. If the two correlated entropy maximization processes are in step with each other and in a state of balance, the scaling exponent will approach 1, otherwise, the exponent values will deviate from 1 or even exceed the reasonable range.


Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL−1, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL−1). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL−1 with a limit of detection (LOD) of 0.003 ng mL−1. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1–12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody’s primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I–L), two of them in CDR2 , and three of them in CDR3 . Second, the affinity constant of NCA1 was tested as 1.20 × 10 Immunoassays, such as ELISA, are a critical part of mycotoxin detection methods. Based on the reaction between the antigen and the antibody, an immunoassay was used as a quantitative or qualitative analysis of target analytes including protein, enzyme, and some small molecules. Immunoassays not only possess an excellent specificity and sensitivity, but also have advantages in having a relatively short detection time and being cost-effective . Thus, mIn the procedure of most mycotoxin detection methods, researchers were inevitably exposed to toxic synthetic antigens, such as the conjugation of OTA and albumin from bovine serum (OTA-BSA), even if there were effective protection measures. Under long-time exposure, damage to the researchers’ health occurs to some extent. In addition, the common synthesis procedure of these antigens demands a large amount of mycotoxin standards and organic reagents, which are also deleterious to operators and the environment . Thus, t1 monoclonal [1 monoclonal [1 monoclonal [A great idea satisfying this demand is to develop anti-idiotype antibodies to surrogate toxic synthetic antigens used in usual immunome analysis methods. There are many kinds of anti-idiotype antibodies that have been developed. Hsu et al. developed an anti-idiotype polyclonal antibody against aflatoxin Bnoclonal . Guan etnoclonal . Nogami noclonal . Wang etnoclonal . Among anoclonal . In addinoclonal .As is well known, the sensitivity of ELISA decreases with the high affinity of synthetic antigen. The high affinity of antigens inhibits antibodies and detection targets to compete for binding sites of the antigen. However, the relationship between affinity and sensitivity of protein coating antigens remains uncertain. In recent years, the construction and function of nanobodies have been gradually revealed, which provides the opportunity for us to obtain nanobodies with a different affinity constant . Early sAspergillus species and Penicillium species [−1 for wheat, barley, rice, and sorghum [−1 [We aimed to develop an anti-idiotype nanobody for OTA detection to deal with the problem of the serious toxicity and the broad occurrence of OTA. Ochratoxin A, a secondary fungal metabolite, is mainly produced by several  species . OTA is  species ,17. From sorghum . This coghum [−1 . Accordighum [−1 ,23,24,25ghum [−1 . Hence, ghum [−1 . ConsequIn this study, we obtained two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to OTA, that were named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). We are also interested in the question of the relationship between affinity and sensitivity of protein coating antigen. Therefore, we analyzed the prime structure, affinity constant, and sensitivity of the two NCAs. Through using the NCA with higher sensitivity to substitute the routine synthetic toxic antigen, we also established an eco-friendly and green ELISA method for the rapid detection of ochratoxin A in cereals.−1) gradually. The number of phage output per round of panning is shown in The phage-displayed nanobodies were isolated for competitive elution by reducing the concentration of OTA standard  satisfy the standard of valuable antibodies. The affinity constant of NCA2 was about 4.5-fold higher than the affinity constant of NCA1. This result shows NCA2 had a stronger binding ability with mAb 1H2 compared to NCA1.The affinity of antibody reflects its ability to bind with the antigen. Usually, it is regarded as a valuable antibody with high affinity when its affinity constant ranges between 1050 of the eco-friendly ELISA based on NCA1 was nearly 2.7-fold of the eco-friendly ELISA based on NCA2. Therefore, the eco-friendly ELISA based on NCA2 possesses higher sensitivity than eco-friendly ELISA based on NCA1. In combination, these result support the idea that the protein coating antigen with higher affinity constant presents higher sensitivity.As is well known, the high affinity of synthetic antigens inhibits antibodies and detection target to compete for binding sites of antigen, which decreases the sensitivity of ELISA. However, the relationship between affinity and sensitivity of protein coating antigens remains unclear. The sensitivity of the NCA was analyzed through competitive ELISA . As showBecause of the higher sensitivity of NCA2 compared to NCA1, we applied the NCA2 to substitute antigens, such as OTA-BSA, to establish an eco-friendly ELISA for ochratoxin A in cereals. We evaluated the NCA2-based antigen substitute’s ELISA performance in methanol effect, matrix effect, and cross-reactivity.50 value obtained from the ELISA under the methanol concentration ranging from 5–20%, we optimized the methanol content in ELISA. As shown in 50 value increased with a concentration increase. We confirmed the methanol concentration applied for ELISA at 10% with the lowest IC50 value was 0.017 ng mL−1.By comparing the IC50 of this approach was 0.017 ng mL−1 with the LOD of 0.003 ng mL−1. The linear range of standard curve was 0.01–0.51 ng mL−1.By using the OTA standard solution to perform a NCA2-based antigen substitute ELISA method under optimal situation, we obtained the standard curve . FormulaThe sensitivity comparison between the NCA-based eco-friendly ELISA and the previous study are reported in −1. Moreover, there was nearly no difference in the linear range. This indicates there are few matrix effects induced by those sample matrices.In the matrix analysis, we estimated the ELISA method in corn, rice, and wheat matrices. As shown in 1 (AFB1), zearalenone (ZEN) or deoxynivalenol (DON) was used as a competitive standard solution. This phenomenon indicates the specificity of this ELISA was great.The cross-reactivity test was conducted to estimate the specificity of this ELISA using the idiotypic nanobody to substitute the antigen. From −1. These results indicate the ELISA is reliable and accurate.The recovery analysis was performed to estimate the stability and accuracy of the idiotypic nanobody-based antigen substitute ELISA method. The results outlined in 8 L mol−1 and 5.36 × 108 L mol−1, respectively. Moreover, the IC50 of NCA1 and NCA2 were 0.052 ng mL−1 and 0.015 ng mL−1, respectively. These results show the NCA2 possesses a higher affinity constant with better sensitivity compared to NCA1. The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies increasing the affinity constant may enhance the immunoassay sensitivity. Based on NCA2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green ELISA method for the rapid detection of ochratoxin A in cereals. After optimization of NCA2-based ELISA, the IC50 was 0.017 ng mL−1 with a limit of detection of 0.003 ng mL−1. The average recoveries of spiked corn, rice, and wheat were between 80% and 114.8% with a abbreviation of relative standard deviation (RSD) of 3.1–12.3%.In this work, we isolated two kinds of anti-idiotypic nanobodies against mAb 1H2, which is specific to OTA. During the research, we focused on the changes of the nanobody’s primary sequence to improve its immunoassay sensitivity. We were also interested in the relationship of affinity and sensitivity of this anti-idiotypic nanobody as a protein coating antigen. By comparing the difference in the primary structure, we found there were six AAR of changes in the complementarity determining region (CDR), which composed the antigen-binding site. One of them was located in CDR1 (I–L), two of them in CDR2 , and three of them in CDR3 . The changes of amino acid make the CDR of NCA2 a higher polar than the CDR of NCA1. In a further test of the affinity and sensitivity, we obtained the affinity constant of NCA1 and NCA2, which were 1.20 × 10In conclusion, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.1, zearalenone, deoxynivalenol standard, bovine serum albumin (BSA), polyethylene glycol 8000 (PEG8000), Freund’s incomplete adjuvant, 3,3’,5,5’-Tetramethylbenzidine (TMB), and goat anti-mouse monoclonal antibody conjugated to horseradish peroxidase (HRP) were purchased from Sigma . E. coli ER2738 competent cells from the E. coli ER2736 line were purchased from Lucigen Corp. . Top 10F’ competent cells and the Superscript III First-Strand Synthesis system were purchased from Life Technologies . The M13 bacteriophage antibody conjugated to HRP was purchased from GE Healthcare . Helper phage M13KO7 and SfiI were obtained from New England Biolabs . Tween 20 was obtained from J&K Scientific . xTractor buffer for protein extraction and His60 Superflow resin were purchased from Clontech Laboratories, Inc. . The QIAprep Spin MiniPrep Kit, QIAquick Gel Extraction Kit, and QIAquick PCR Purification Kit were all from Qiagen . The LeukoLOCK Total RNA Isolation System was obtained from Applied Biosystems . Costar 96-well ELISA plates were purchased from Corning Incorporated . The pComb3X phagemid vector was a generous gift from Dr. Carlos F. Barbas .All reagents were of analytical grade unless otherwise stated. The monoclonal antibody (mAb) 1H2 against ochratoxin A was produced in our laboratory . Ochrato2HPO4·12H2O, 0.2 g of KH2PO4, and 0.2 g of KCl in 1000 mL of deionized water. Phosphate-buffered saline with Tween 20 (PBST) was prepared by dissolving the Tween 20 with PBS buffer.Phosphate-buffered saline  was prepared by adding 8 g of NaCl, 2.9 g of Na−1 NaCl for 1–2 h before they were discarded or autoclaved.Pure mycotoxin standards were handled in a hood with extreme caution. All items coming in contact with a mycotoxin, phage, and bacterial cultures  were immersed in a solution of 2.5% NaClO and 0.25 mol LA 3-year-old castrated male alpaca was subcutaneously immunized with 200 μL of antigen solution (200 μg mAb 1H2 dissolved with 100 μL 0.01M PBS and mixed with 100 μL Freund’s incomplete adjuvant). This injection was performed every two weeks for a total of eight injections. To isolate lymphocytes, 20 mL of blood was collected one week after the fifth, sixth, seventh, and eighth injections. The lymphocytes were stored at −80 °C until used for total RNA extraction.The total RNA in the blood of the alpaca was isolated according to the operation manual of the Life Technology LeukoLOCK Total RNA Extraction Kit. The first strand of cDNA was synthesized using the Superscript III First-Strand Synthesis system. The heavy chain antibody variable region VHH gene fragment from IgG2 and IgG3 was amplified by PCR using the primers designed previously: forward primer VHH-F (CAT GCC ATG ACT GTG GCC CAG GCG GCC CAG KTG CAG CTC GTG GAG TC) targeting framework 1 region; and reverse primers VHH-R1 (CAT GCC ATG ACT CGC GGC CGG CCT GGC CGT CTT GTG GTG TTG GTG TCT TGG G) and VHH-R2 (CAT GCC ATG ACT CGC GGC CGG CCT GGC CAT GGG GGT CTT CGC TGT GGT GCG) correspond to the IgG2 and IgG3 hinge regions, respectively . The PCR−1) in 0.01M PBS in a 96-well microtiter plate, and 100 μL 3 % BSA/PBS in wells of another microtiter plate, were incubated at 4 °C for 12 h. After being washed with 0.05% PBST three times, 100 ul of the freshly prepared phage display nanobody library was added to the wells coated with BSA to reduce BSA-bound phage, while the wells coated with mAb 1H2 was blocked with 3% milk–PBST solution. The microtiter plates were incubated for 1 h at 37 °C. Then, the blocked microtiter plate was washed with 0.05% PBST three times. The phage peptide library supernatant was then transferred to wells coated with an antibody, shaken at room temperature for 2 h, and then washed ten times with 0.05% PBST. The bound phages were competitively eluted in 100 μL of 100, 50, and 10 ng mL−1 OTA standard in 10% methanol/PBS in three rounds of bio-panning and shaken at room temperature for 30 min. The elution solution was then collected and used to infect E. coli ER2738 for the amplification of phage. The amplified phage was used for each subsequent round of panning. The titer of the phage of elution solution in each round represents the level of positive clone enrichment.The solution of 100 μL mAb 1H2  and incubation for 15 min at 37 °C. The optical density (OD) values of those wells were obtained by a microplate reader. Clones that bound to mAb 1H2  but not to BSA  were considered positive and were selected for further characterization. The pComb3X phagemid vector encoding the VHH was extracted from the ER2738 clone and sequenced using primer gback (GCCCCCTTATTAGCGTTTGCCATC).After the last round of panning, 30 individual clones were randomly selected to identify positive clones by performing an indirect competitive phage ELISA. The competitive procedure was the same as the bio-planning. However, after washing ten times with 0.05% PBST, 100 μL of 0.2 μg mL600 value reached 0.6–0.8, a final concentration of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) solution was added, and then continued to incubate at 37 °C for 250 rpm overnight. Protein was isolated using xTractor buffer according to the manufacturer’s instructions. The nanobodies containing 6 × His tag were purified by Ni-NTA metal affinity chromatography according to the manufacturer’s instructions. The purity and size of the nanobodies were identified using 15% reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to standard protocols. The concentration of nanobodies were determined by Bradford Method. For the special use in this paper, we named the nanobody as the nontoxic coating antigen (NCA).The pComb3X phagemid vector encoding VHH having a unique DNA sequence was transformed into and expressed in a non-inhibitor E. coli strain TOP10F’ cell. For expression, 100 mL of SB medium was incubated with an overnight culture of TOP10F’ cells carrying the VHH expression plasmid, and was incubated at 37 °C with shaking at 250 rpm. When the culture ODThe comparison of NCAs was conducted from the primary structure, spatial structure, affinity constant, and sensitivity of the obtained nanobodies.The primary structure of nanobodies was sequenced in the procedure of identification of positive clone. The spatial structure of nanobodies was homology-modeled by the SWISS-MODEL website using the amino acid sequences of nanobodies.The affinity was measured according to Beatty’s method . The proWhen the concentration ratio of coating antigen was 1:250 was used to estimate the sensitivity of those two NCAs.The sensitivity of the nanobody was measured by competitive ELISA under the same condition. The specific steps were the same as ELISA that were employed in the identification of positive clone. The ICThe NCA-based eco-friendly ELISA was developed based on competitive ELISA. The specific steps were roughly same as ELISA that were employed in the identification of positive clone. The only differences of this kind method were using NCA as a new kind of coating antigen and using mAb 1H2 as antibody. The principle of this method was NCA and OTA standard solutions competed the binding site of mAb 1H2.The optimal work concentration of NCA and mAb 1H2 was determined by performing a competitive ELISA in the checkboard test. The concentration groups corresponding to the OD value near to one were employed for further estimates by competitive ELISA.50 values obtained in different concentrations of methanol, we could determine the optimal methanol concentration used in ELISA.To evaluate the effects of methanol on the performance of the NCA-based eco-friendly ELISA, the OTA standard used in ELISA was diluted by 0.01M PBS in 5%, 10%, and 20%. By comparing the IC50 values of different performances when using the OTA standard solution diluted with three kinds of matrices, separately, we could estimate the effect of matrices to the idiotypic nanobody-based antigen substitute ELISA.The matrix effect was analyzed through spiked samples including corn, rice, and wheat. These samples were pretreated as the procedure of sample preparation. By comparing the IC−1. The calculation of LOD and linear range were conducted by previous research [The standard curve of the NCA-based eco-friendly ELISA was obtained through competitive ELISA by using the OTA standard solution. The concentration of OTA ranged from 0 to 10 ng mLresearch .1, Zearalenone, and deoxynivalenol as detecting targets to evaluate the specificity of the NCA-based eco-friendly ELISA.In the cross-reactivity test of the NCA-based eco-friendly ELISA, we employed three standard solutions including aflatoxin B−1) was added into blank sample powders, separately. After extraction, the detection was performed three times on the same day to estimate the accuracy within the assay, and conducted five days later to estimate the accuracy between assay.Recovery analysis of the NCA-based eco-friendly ELISA was also conducted through spiked samples such as corn, rice, and wheat. The OTA standard (10, 20, 50 ng mLFive grams of ground corn, rice, and wheat samples were weighed, extracted with 80% aqueous methanol solution, and incubated at room temperature for 30 min with shaking at 250 rpm. Each sample extract was then filtered twice and centrifuged at 6000 g for 15 min. After dilution, the supernatant was used for ELISA analysis.


Lactic acid bacteria were found in high proportions in Nham, Nam phak and Miang. Bacillus was predominant in Thua nao, in which significant proportions of Lactic acid bacteria of the family Leuconostocaceae were also found. Groups of lactic acid bacteria found varied among different food samples, but three genera were predominant: Lactococcus, Lactobacillus and Leuconostoc, of which many members are recognised as probiotics. The results showed that these traditional Thai fermented food products are rich sources of beneficial bacteria and can potentially be functional/probiotic foods.Many indigenous fermented foods of Northern Thailand and neighbouring regions have traditionally been known for their health benefits. In this study, we explored the communities of bacteria in selected fermented foods which are commonly consumed among ethnic groups around Northern Thailand, for which information on their microbial compositions or their functional properties is still limited. The selected food groups included Thua Nao , Nham (fermented pork sausage/loaf), Nam phak (fermented Chinese cabbage) and Miang (fermented leaves from Miang Tea trees). Bacteria in these fermented foods were isolated and enumerated. Bacterial communities were determined using a culture-independent (pyrosequencing) approach. Lactic acid bacteria were recovered from all of these fermented food samples, with levels ranging from 3.1 to 7.5 log CFU/g throughout the fermentation processes. Analysis of the  Many raw materials, such as fish, meats and vegetables have been used to make various traditional fermented products throughout the world. In Thailand, there are indigenous products such as Nham (fermented pork sausage or fermented meatloaf), Miang (fermented tea leaves from Miang Tea trees), Nam phak (fermented Chinese cabbage) and Thua nao . These products, although have some common features with other fermented foods of similar types in South China and South East Asia, are unique to Thai culture, particularly Lanna or Northern Thai culture. However, some of these products have also been popularly consumed and increasingly recognised as health foods in many other parts of Thailand.i.e. natural fermentation processes. A prominent example of this is Miang. This is a fermented product named after the Miang tea tree (Camellia sinensis var. Assamica) from which leaves are used for fermentation and is traditionally consumed as snacks in the northern part of Thailand [Sus scrofa domesticus) [Brassica rapa ssp. chinensis). It is usually consumed as a side dish. Nam phak is similar to Korean kimchi, which has the cabbage as a main ingredient. Thua nao is made from soybean (Glycine max (L.) Merrill) [Most of the traditional Thai fermented products are manufactured using raw materials available locally or, in some cases, raw materials indigenous to the geographical area and natural starter cultures, Thailand . To makeThailand . The ferThailand . Anotheresticus) . It is uMerrill) . It is uMerrill) . The appLactobacillus plantarum, Lactobacillus camelliae, Lactobacillus thailandensis and Pediococcus siamensis [Pediococcus spp., Lactobacillus spp. and Micrococcus spp. [Leuconostoc mesenteroides, Lactobacillus brevis and Lactobacillus plantarum were among the predominant species reported [Bacillus, especially B. subtilis [Bacillus species, such as B. pumilus, B. megaterium and B. licheniformis, were also found in this or similar products [Beneficial microorganisms, especially lactic acid bacteria (LAB), are involved in the fermentation process of Miang, Nham and Nam phak –7. LAB tiamensis ,8. In Nhcus spp. ,10. Therreported . As for subtilis ,12. Seveproducts ,14. ThesBacillus groups have been recognised for their function as probiotics for humans. Because of the production of lactic acid and other antimicrobial substances, LAB can also inhibit undesirable pathogenic bacteria in the fermented products themselves, thus promoting natural preservation. In addition, some Lactobacilli can also inhibit microbial pathogens in the gastrointestinal tract. Therefore, these traditional fermented foods, which are associated with LAB and Bacillus, could be considered potential foods for promoting human health [Many members of LAB and n health . In fact16S rRNA gene sequencing (454-pyrosequencing), respectively. The results obtained from both approaches were expected to reveal predominant bacterial taxa and a broader range of bacterial communities; some of which can potentially be probiotics and may contribute to the probiotic and functional properties of these traditional fermented foods.Most of the studies on the microorganisms involved in fermentation of these products or their varieties have been carried out using cultured methods. However, for traditional fermented products such as these, many types of microorganisms usually exist throughout the fermentation process and contribute to the unique characteristics of the products, although not all of them can be recovered or distinguishably isolated in the laboratory. Recently, next-generation DNA sequencing has been used to demonstrate a broader range of bacterial communities in food . HoweverIn this study, four Northern Thai fermented foods  and Thua nao) were sampled from selected locations in different districts , where the products are produced in the traditional ways. Briefly for the fermentation process of Miang, Miang tea leaves are steamed for 1–2 hours, left to cool, then packed and fermented with or without filamentous fungi and kept under an anaerobic condition . Miang sA ten-gram portion of each sample was homogenised in 90 mL of 0.85% (w/v) sodium chloride and ten-fold serial dilutions were prepared until the desired concentrations were obtained. The suspensions from different dilutions were spread onto Tryptic Soy Agar (TSA), de Man Rogosa Sharp (MRS) agar with Bromocresol green added as a pH indicator and Yeast Extract Malt Extract (YM) agar. All agar plates were incubated at 37°C for 24–48 hours. Colonies grown on TSA, MRS agar and YM agar were counted as total bacteria, presumptive lactic bacteria and fungi, respectively. The numbers of colonies were calculated into colony forming units per gram (CFU/gram) of sample.2 production and protease production. The LAB colonies were preserved in 20% (v/v) glycerol at -20°C for further analysis.Presumptive LAB isolates obtained from MRS agar were analysed for their morphology, Gram-stain reaction, catalase production, CO16S rRNA gene were amplified by polymerase chain reaction using the universal bacterial primers 27F (5´-AGAGTTTGATCMTGGCTCAG-3´) and 1492R (5´-TACGGYTACCTTGTTACGACT-3´). A 25 μL PCR reaction mixture contained 1×buffer, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.8 μM of each primer, 1 unit of Tag DNA polymerase (Invitrogen), 1 μL of DNA template and ddH2O. The PCR was carried out in a thermal cycler (BIO RAD), using the following condition: an initial denaturation for 3 min at 94°C; 30 cycles of denaturation for 45 sec at 94°C, annealing for 30 sec at 55°C and extension for 1.5 min at 72°C; and a final extension for 10 min at 72°C. The PCR products were analysed using gel electrophoresis on a 1% (w/v) agarose gel, stained with ethidium bromide and visualised under a UV transilluminator.The fermented foods with the highest proportions of LAB were selected for high-throughput sequencing analysis. All fermented samples were homogenised in sterile distilled water and 1.8 mL portions of the food homogenates were centrifuged at 13,000 g for 3 min. Genomic DNA was extracted from the cell pellets using the MO BIO Power Food Microbial DNA isolation kit and QIAGEN DNeasy Blood and Tissue kit according to the manufacturer’s instructions. The fragments of 16S rRNA fragment was amplified using primers 27F (5´-GAGTTTGATCMTGGCTCAG-3´) and 518R (5´-WTTACCGCGGCTGCTGG-3´). The sequence data were processed using Quantitative Insights Into Microbial Ecology (QIIME) version 1.9.1 [The genomic DNA samples prepared as above were subjected to GS-FLX Titanium 454 pyrosequencing (Roche) ). The total bacterial on 1.9.1 . The lowon 1.9.1 , the lonon 1.9.1  and RDP on 1.9.1 . Non-meton 1.9.1 .The numbers of total bacteria, LAB and fungi in Thai fermented foods , as recovered from Tryptic Soy Agar (TSA), de Man Rogosa Sharp with Bromocresol green (MRS-bromocresol green) and Yeast Extract Malt Extract (YM) agar, at different fermentation times are shown in From ca. 5 log CFU/g after 2 days of fermentation, which was in accordance with the gradual decrease of pH from 6.24 to 3.96  sequences. From all of the fermented foods samples, 152,805 sequences were detected. A total number of 113,844 sequences were retained after discarding low-quality sequences. Based on the 97% identity reads, OTUs were generated and subjected to taxonomic classification.The abundance of bacterial taxa in the traditional fermented foods was investigated based on Among the traditional fermented Thai foods analysed in this study, LAB were most abundant in Nham (with the proportion of 87.12% or higher) . In NhamLactococcus, Lactobacillus and Leuconostoc. The majority of LAB in Nham belonged to Streptococcaceae and Lactobacillaceae families  similar to those of the lactic-fermentation types of food. Such high numbers of lactic acid bacteria were previously reported in alkaline-fermented foods such as in African alkaline products  and in TMiso in Japan, Sufu in China and Tempeh in Indonesia [In Miang and Thua nao, fungal counts were high, especially in the last stages of fermentation. Although some filamentous fungi or mycotoxic fungi could be contaminants , many fundonesia . In MianIn Nham, the number of LAB decreased gradually after reaching the peak at 2 days of fermentation. For Miang, the numbers of LAB after five months of fermentation slightly decreased from those after one month of fermentation. This observation was in accordance with a previous study that reported the change in microbial population during the traditional fermentation processes of fermented tea (Miang) .Bacillus spp. This finding was in accordance with other studies, which reported that the bacteria in the genus Bacillus played the most crucial role in soybean fermentation [Kinema of India, Natto of Japan and Chungkok-jang of Korea, in which B. subtilis plays an important role in fermentation [From the analysis of bacterial communities, the bacterial group that was predominant in Thua nao was entation ,12,14. Ventation .Lactococcus was found as a predominant genus. This had also been observed by other researchers [Lactococcus species that had been identified were Lc. garvieae and Lc. lactis [et al. [Lactococcus lactis strain WNC 20, which originated from Nham, had inhibitory effects against some foodborne pathogens. Moreover, this strain could produce bacteriocin and was recommended for use in fermented meat products. As for Lactobacilli, the other major LAB group found in Nham in our study, some of its members are known for their potential probiotic properties [Leuconostoc has been found among fermented foods similar to Nam phak, such as kimchi (fermented Chinese cabbage of Korea) [paocai (pickled cabbages of China) [Lactobacillus and Weissella were also found in the former product and Lactobacillus, while Enterococcus and Lactococcus were found in the latter [Lactobacillus plantarum was associated with Miang samples collected from many provinces in Northern Thailand [In Nham, earchers ,39. Lact. lactis . Interes [et al.  showed toperties . Leuconof Korea)  and paocf China) . In addie latter ,43. AnotThailand .Lactococcus, Lactobacillus and Leuconostoc, have been known to have probiotic potential [Lactobacillus species have been reported to have cholesterol lowering properties [Helicobacter pylori [Lactococcus species have been described for therapeutic treatments for various diseases, e.g. atopic dermatitis and inflammatory bowel diseases (IBDs) [Leuconostoc sp. was reported to have an inhibitory effect against pathogens [Some members of LAB are recognised as probiotics, which are beneficial to human’s health, particularly through the ability to survive and colonise gastrointestinal tracts of humans . Some geotential ,47 and hoperties  and redur pylori ,50. Lacts (IBDs) ,52. Leucathogens .It can be seen that the traditional fermented foods, such as those from Thai culture as presented in this study, which have both economic and cultural values, can be natural carriers or sources of beneficial LAB or potential probiotic bacteria. This fact makes them potential probiotic or functional foods and the beneficial bacteria contained in these traditional food products also have great potential health benefits.ca. 5–7 log CFU/g) in the later stages of fermentation of Miang (fermented tea leaves), Nham (fermented pork sausage) and Nam Phak (fermented cabbage), which belong to the lactic acid fermentation class of fermented foods. The culture-independent pyrosequencing analysis revealed that LAB formed the majority of a large part of the bacterial populations in these traditional fermented foods. In Thua nao, an alkaline fermented soybean product, although LAB was not the most abundant group, it reached a significantly high level at the later stage of fermentation also. Although the presence of LAB was a common characteristic in these fermented products, each of these, however, had unique bacterial communities. Many members of LAB genera found in these products are well-known as probiotics. Some members of the genera have the capacity to produce substances that contribute to products’ unique flavour, and some have potential nutraceutical or pharmaceutical properties. The indigenous foods of Northern Thailand are potential sources of beneficial LAB and could be utilised for diverse applications.This study demonstrated that lactic acid bacteria (LAB) were associated with traditional fermented foods in Lanna or Northern Thailand region. The numbers of LAB increased to significantly high levels Click here for additional data file.


Individuals with autism spectrum disorder (ASD) reportedly possess preserved or superior music-processing skills compared to their typically developing counterparts. We examined auditory imagery and earworms (tunes that get “stuck” in the head) in adults with ASD and controls. Both groups completed a short earworm questionnaire together with the Bucknell Auditory Imagery Scale. Results showed poorer auditory imagery in the ASD group for all types of auditory imagery. However, the ASD group did not report fewer earworms than matched controls. These data suggest a possible basis in poor auditory imagery for poor prosody in ASD, but also highlight a separability between auditory imagery and control of musical memories. The separability is present in the ASD group but not in typically developing individuals. Musical ability is dependent upon a group of auditory perceptual and cognitive skills which may be affected by autism in ways that are poorly understood. The relationship between language and music is itself complex. In 1871, Darwin speculated that they might share a common evolutionary origin Darwin . Both arEarly studies suggested that the processing of music may be preserved in autism even when language is impaired  studies of pitch processing in ASD. A recent meta-analysis of results using these techniques suggests that infants and children (but not adults) with ASD have impaired auditory brainstem response to sound  predicts vocal pitch imitation accuracy  subscale predicts performance on musical imagery related tasks  processing and control, an area which has been hitherto neglected in studies of musicality and ASD.The study was approved by the University of Reading ethics committee and performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments. Participants were recruited through a variety of ways, such as word-of-mouth, contacting local organisations associated with autism, and online advertising.t-tests confirmed that the two groups did not differ in age, self-reported years of musical training, non-verbal IQ as measured by Raven’s progressive matrices, or receptive vocabulary as measured by the fourth edition of the Receptive One Word Picture Vocabulary Test , (t (32) = 5.1, p < .001, Cohen’s d = 1.75; BF10 = 1115.205, median = − 1.59, 95% CI − 2.42, − .77) and Systemizing Quotient (SQ) (t(32) = 4.7, p < .001, Cohen’s d = 1.61, BF10 = 400.729, median = 1.46, 95% CI .66, 2.25).The sample consisted of 34 participants: 17 ASD participants , aged 16–56, with an average of 36.6 years old matched with 17 controls on gender. All ASD participants had an official clinical diagnosis, which is also confirmed by their AQ scores. Bayesian https://osf.io/q7k9n/. Three ASD participants answered all questions with only 1 (one participant) or 7 (two participants), and these participants (and their matched controls) were excluded from subsequent analyses on the presumption that they had failed to understand the task. Importantly, including these individuals does not affect the results except on the one occasion noted in the text.The full data-set, including all participants, are available via the Open Science Framework at The materials used were two online questionnaires, which were provided through the JISC (formerly BOS) survey platform. First, the Bucknell Auditory Imagery Scale (BAIS) was given to participants online. The second questionnaire used was an earworms questionnaire, initially used by Beaman and Williams  and showLinks to both questionnaires were sent by email, for participants to complete in their own time. The AQ was sent first, then the BAIS and finally the earworms questionnaire. Time pressure was removed for participants as there was no time limit given and no supervision was given to ensure that they necessarily completed the questionnaires in this order. As part of a general recruitment procedure all individuals also subsequently completed a number of other tests, as indicated in Table Bayesian analyses were run on JASP software using default priors because there were no strong a priori commitments to any particular effect size  = 8.13, p = .008, eta squared = .238), a main effect of type of vividness  = 207.04, p < .001, partial eta squared = .89), and an interaction between group and vividness type  = 3.31, p = .04, partial eta squared = .11), as shown in Fig. p value of .052 for the interaction, but the results are otherwise unaffected. Bayesian model comparisons lead to similar conclusions: Using JASP to calculate BFinclusion values to indicate the extent to which Bayes the data support inclusion of the factor of interest, a Bayesian model with an effect of group was revealed to be more likely than the model of no group differences (BF10 = 9.106). A model with an effect of soundtype was also more likely than the null model of no difference in soundtype . Finally, a model in which these factors interact was more likely than a model with no interaction (BF10 = 4.95).1The three factors identified by factor analysis Halpern  as contrF,26 = 8.1t-tests and a Holm’s sequential Bonferroni correction (Holm t(26) = 3.55, p = .001, Cohen’s d = 1.34, BF10 = 22.37, median = − 1.1.35, 95% CI − 1.976, − .328) for environmental sounds (t(26) = 2.86, p = .008, Cohen’s d = 1.08, BF10 = 5.93, median = − .878, 95% CI − 1.693, − .137) and for music (t(26) = 1.69, p = .104, Cohen’s d = .64, BF10 = .997, median = − .48, 95% CI − 1.204, .178). Thus, the evidence favours the hypothesis that ASD individuals differ from controls in vividness of auditory imagery in two cases , in the third case (music) the observed effect size was smaller and the results inconclusive.Follow-up analyses (2-tailed) using Bayesian ion Holm  for the F = 11.16, p = .003, eta squared = .3), a main effect of type of control  = 4.74, p < .001, partial eta squared = .82) and a group by control type interaction  = 118.3, p = .013, partial eta squared = .15), as shown in Fig. inclusion values revealed that a model with an effect of group was more likely than the null model, (BF10 = 34.34), one with an effect of soundtype was also more likely than the null model of no effect (BF10 = 6.005 × 1015), and a model in which both effects interact was more likely than a model with no interaction (BF10 = 11.58).A similar pattern was apparent with Control scores of the BAIS where once again there was strong evidence for an effect of group  = 3.99, p < .001, Cohen’s d = 1.51, and t(26) = 3.34, p < .001, p = .003, Cohen’s d = 1.26, t(26) = 2.36, p = .026, Cohen’s d = .89, respectively) and in all cases a difference between groups is more likely than a null difference . Note however that the evidence in support of the alternate hypothesis is much weaker in the case of verbal/environmental sounds than in the other instances and also that the frequentist t test would not have survived a stricter Bonferroni correction.For BAIS-C, the three factors contributing to control scores were identifiable as arising from questions concerning verbal/environmental sounds, music, and verbal sounds Halpern . Follow-fewer earworms in the ASD group relative to the control group but this hypothesis is decisively rejected by a 1-tailed Bayesian t-test . Figure 10 = 1.48, median = .462, 95% CI .037, 1.17 when including only the individuals who successfully completed the BAIS scale; BF10 = 2.67, median = .551, 95% CI .06, 1.239 when all individual are included).Given that BAIS scores were lower amongst the ASD group than in controls, the positive correlation between BAIS scores and earworms in typically developing individuals reported by Beaman  would sumore prevalent in an ASD population—the current data-set is insufficient to settle this point. It is clear, however, that any hypothesis about earworms predicated upon superior auditory imagery is not supported. It was also plausible that either or both of the vividness and control subscales would show differential effects of ASD upon the types of auditory imagery such that, for example, musical imagery might be preserved or even enhanced whereas vocal or verbal imagery might not be. Analyses of variance showed interaction effects, but Figs. As far as we are aware, these results represent the first attempt to examine the phenomenology of auditory imagery in an ASD population and directly compare this to a matched control group. The results of this study show that both self-reported vividness and control of auditory imagery are reduced in our ASD group relative to the typically developing control group. Contrary to predictions, however, this did not result in fewer earworms and it remains possible that Ockelford’s  speculatSignificantly, the Bucknell Auditory Imagery Scale was previously validated in a nonclinical population and shown to possess some interesting properties with respect to that population. Most notably, BAIS-V not only predicted individual differences in gray matter density in auditory and sensory-motor regions of the brain regions (Lima et al. The interpretation outlined above raises the question of why, if auditory imagery is deficient the ASD group examined here did not also show fewer earworms. Indeed, the data are roughly equally consistent with the ASD participants being equivalent to the matched controls or showing more earworms as speculated by Ockelford . The mosA clear limitation of the current study is the relatively small sample size, which requires us to be cautious not to over-interpret these results until such time they may be replicated with a larger sample and, perhaps, a subgroup analyses of ASD individuals. Notably, the sample size was too small for meaningful correlation coefficients to be calculated so that, for example, the correlations between BAIS scores and self-reports of earworms previously observed Beaman  were notImportantly, the groups were matched on a number of cognitive and demographic variables; including verbal and non-verbal ability, and age and musical training. It is not clear what results might be obtained with a less cognitively capable ASD group, but the data are clear in indicating that the differences in auditory imagery reported here cannot be explained in terms of poorer overall cognitive capability or even just a simple failure to understand the task. This is important because the literature on musical ability in ASD is mixed, with some suggestions that preserved or superior musical capability is the provenance of a subgroup of ASD individuals who show little or no cognitive impairment (Bonnel et al. There are also strengths and weaknesses associated with the auditory imagery scales employed within the current study. The vividness of a mental image, being the property of introspection, is intrinsically subjective and can only be directly assessed by self-evaluation. The advantages and disadvantages attendant to a questionnaire design are discussed by Hubbard . StrengtAuditory imagery scores are shown to be lower in an ASD group than in a matched control group in terms of both the self-rated vividness and mental control experienced over imagery. This contrasts with the case of visual imagery, in which poorer mental imagery in ASD is only associated with “unreal” or impossible objects. Contrary to predictions, this poorer auditory imagery was not associated with fewer earworms amongst individuals with ASD. There was limited evidence that individuals with ASD might in fact experience more earworms than the control group, consistent with clinical observations by Ockelford .


Populus trichocarpa to determine this species capacity for climate adaptation. We measured bud burst, leaf coloration, and leaf senescence traits across two years (2017–2018) and combine these observations with measures of lifetime growth to determine how genetic correlations between phenology and growth may facilitate or constrain adaptation. Timing of transitions differed between years, although we found strong cross year genetic correlations in all traits, suggesting that genotypes respond in consistent ways to seasonal cues. Spring and autumn phenology were correlated with lifetime growth, where genotypes that burst leaves early and shed them late had the highest lifetime growth. We also identified substantial heritable variation in the timing of all phenological transitions (h2 = 0.5–0.8) and in lifetime growth (h2 = 0.8). The combination of additive variation and favourable genetic correlations in phenology traits suggests that populations of cultivated varieties of P. Trichocarpa may have the capability to adapt their phenology to climatic changes without negative impacts on growth.In a warming climate, the ability to accurately predict and track shifting environmental conditions will be fundamental for plant survival. Environmental cues define the transitions between growth and dormancy as plants synchronise development with favourable environmental conditions, however these cues are predicted to change under future climate projections which may have profound impacts on tree survival and growth. Here, we use a quantitative genetic approach to estimate the genetic basis of spring and autumn phenology in  Perennial plants transition between periods of growth and dormancy in response to seasonal changes. Patterns of growth cessation and dormancy minimise the risk of tissue damage from freezing over winter and are primarily induced by a change in photoperiod  is one of several large, deciduous, tree species within the genus Populus. Populus species are generally fast-growing and as such have great ecological significance as pioneer species  aim to characterise heritable genetic variation for phenology traits in this population, and (2) test whether genetic correlations that exist between phenology traits may facilitate or constrain adaptation to novel climatic conditions.Here, we analyse phenotypic variation in a clonally replicated population of P. Trichocarpa trees in a plantation at Krusenberg (59°44'44.2“N 17°40'31.5“E) in central Sweden. Material in this plantation was originally generated from 9 female and 10 male trees collected over a latitudinal range from 44 to 60o in North America  to col8 (100% yellow). Leaf senescence (ls) was measured on a 3-point scale where ls1 = full foliage, ls2 = half leaves remaining and stage ls3 full defoliated. We estimated growing season length by the duration of photosynthetically active leaf canopy. Canopy duration (CD) was defined as the period between the beginning of budburst (bb2) and beginning of leaf yellowing (co3). Phenology measurements were taken every 2–5 days during the spring and autumn seasons in 2017 and 2018. Lifetime growth was determined by measuring diameter at breast height (DBH) in 2017To assess variation in phenology we included the traits that best define the major milestones of phenology during the annual growth cycle. For spring development, bud burst (bb) was scored on a scale from 1 to 5, with stage bb2 representing initial shoot emergence, bb3 leaf primordia exposed, bb4 leaves half shed with bud scales dropped and bb5 leaves completely shed. Autumn phenology was scored on a scale of 1–8 based on a continuous range of crown colouring from While late season growth cessation is best described by bud set, this trait is difficult to accurately measure in mature trees. Due to the difficulty of determining the exact transitions between beginning of growth, cessation of growth and bud development in grown trees, we split these seasonal transitions into 5 biologically relevant proxy stages which describe the start and end of seasonal transition; (bb2) the first bud emergence, (bb5) full leaf emergence, (col2) first stage of yellowing, (col5) complete yellowing, (ls3) day of full leaf shed. These phenology traits are combined with growth of the tree at 14 years of age as measured by cross calliper measurement (DBH) in 2017.R = 0.98-1).Screening was conducted at intervals of 2–5 days meaning that individual trees occasionally passed through developmental stages between screenings. To account for these missing estimates of the day of transition we estimated the transition days for each developmental stage using local regression models (LOESS) fit to each individual tree. Models were fit through the data point of the first day an individual tree was observed at a stage transition and day estimates were calculated for any stage transitions for which there was no direct observation Fig. . This meh2) for each genotype, and to determine the additive genetic co-variance between measurement years, we implemented the following bivariate models, where measures of each transition trait  taken in subsequent years 2017 (Y1) and 2018 (Y2) formed a bivariate response variable, μ is a fixed intercept, g is the random additive genetic component drawn from pedigree information and Va , and implemented the following model:μ is a fixed intercept, Ti is the fixed effect of year, gj is the random term describing the additive genetic variance drawn from pedigree information and R , leaf expansion (bb5), start of leaf yellowing (col2), end of leaf yellowing, (col5) leaf shed (ls3), canopy duration (CD) and lifetime growth (DBH) as a 7-trait multivariate response (Hadfield . We implWe extracted the 7 × 7 genetic variance-(co)variance matrix from this model, and calculated genetic correlations as the genetic covariance between traits divided by the square root of the product of the variances. We extract the genetic correlation between the 6 phenology traits and fitness  to determine the genetic relationship between phenology and growth. This correlation between phenology and growth ultimately determines whether adaptive shifts in phenology in response to climate change will be associated with increased growth, or be constrained by associated negative effects on tree growth.All phenology transitions occurred earlier in 2018 than 2017 . Lifetime growth was the highest and most precise of the heritability estimates (h2 = 0.76), which reflects a substantial genetic basis for differences in lifetime growth between genotypes.Phenology traits had a strong genetic basis, with spring heritability in the range of 0.48–0.72 (bb2) and 0.51–0.62 (bb4), respectively. Autumn phenology was also highly heritable, with highest and most consistent estimates for leaf shed (ls3) , onset of colour change (col3), and leaf shed (ls3) all overlap 1, showing complete correlation between years. The contribution of genetic variation to leaf out (bb5) and canopy duration also very consistent with correlations above R = 0.77), but showed little to no link to autumn phenology characteristics (Table R = 0.80–0.87) suggesting a developmental cascade brought on by pleiotropic effects of genes involved in sensing and responding to the end of favourable growing conditions. Spring phenology was negatively correlated with both canopy duration and lifetime growth, where clones bursting leaves early in the season hold their leaf canopy longer and grow more. Leaf colouring was positively correlated with growth reflecting that clones with late onset of leaf colouration and senescence conferred higher lifetime growth (Table Spring traits of budburst (bb2) and leaf out (bb5) displayed highly positive genetic correlations (cs Table . Similarth Table .Table 1GP. Trichocarpa has a heritable genetic basis, but also that these traits can vary between years in response to climatic variation. With heritable genetic variation underlying both spring and autumn phenology, and an absence of genetic constraint between these transitions, our results suggest that these traits have potential to evolve independently in order to track future climate changes. As projections suggest climate change may provide conditions for a longer growing season, the absence of constraint may allow trees to have a longer canopy duration, which will in turn lead to higher growth. Together these findings provide insight into the genetic architecture of phenology traits, and identify the capacity of P. Trichocarpa to adapt its phenology to novel environmental contexts as they colonise areas outside their original range, or as environmental conditions shift via global climate change.The ability of plants to survive the novel environmental conditions brought on by climate change is dependent on populations harbouring sufficient genetic variation to adapt to changes in seasonality. Here, we show that timing of phenology in an experimental population of There is a considerable contribution of genetics to all phenology traits surveyed in this field experiment and similar to other studies (e.g., Olson et al. Growing season in deciduous forest trees is defined by the leaf phenology traits, and we show with this experiment that canopy duration has a negative genetic correlation with spring phenology, and a positive correlation with autumn phenology. Perhaps unsurprisingly, we identify a genetic association where early bud burst and late leaf drop leads to longer growing season and greater lifetime growth. These patterns have been shown in other studies of this species (McKown et al. Populus species for estimates of bud flush however, it is important to note that the majority of these studies have been conducted on juvenile trees (e.g., McKown et al. P. Trichocarpa (Marron et al. A number of studies have found moderate heritability for phenology traits in this, and other P. Trichocarpa. This is consistent with the observation of conserved patterns of environmental responses across the plant kingdom where bud burst is largely regulated by local environment not local adaptation (MacKenzie et al. While we find evidence for a heritable basis to spring and autumn phenology, there is only a weak relationship between leaf unfurling and senescence. This finding adds to existing evidence that different genetic architectures are controlling phenological responses of spring and autumn environmental cues in P. Trichocarpa, however the genetic links between phenology and growth suggest that shifts in the timing of spring and autumn transitions have the potential to affect growth if shifts lead to a shortening of the growth period. Previous work has shown that autumn phenology is primarily initiated as trees respond to predictable changes in photoperiod, but data presented here suggests that large interannual shifts in autumn timing are possible, which suggest that leaf colour and senescence traits may also be responding to multiple environmental cues either during the summer growing season, or during the onset of autumn (Rohde et al. Variability and uncertainty in relation to the future timing of seasonal transitions and the potential for extreme or unseasonal weather events such as out of season frost pose a significant risk of damage to deciduous trees. Our findings here suggest that adaptive shifts in phenology may be possible in Multiple studies have shown that temperature is not a trigger for senescence (Bhalerao et al. It is important to note that a substantial shift in autumn phenology occurred between the two years of observation. In 2018, there was a faster increase in temperature and more sunny days during early spring, which is reflected in the faster spring development and earlier leaf out in that year. Autumn was also drier, and leaf colouration earlier in 2018, which is consistent with the presence of drought induced bud set, so it is likely that although light characteristics are the primary cue for growth cessation and senescence (Michelson et al. This study presents data from a collection of source genotypes sampled over ~15 degrees of latitude in north-western north America. This sampling design may inflate estimates of heritability by sampling across locally adapted populations, although this does not undermine the key findings that phenology transitions have a strong genetic basis. Also, we are unable to explore the contribution of dominance and epistatic interactions to our estimates of additive genetic variance due to limitations of the pedigree, however we note that these are common limitations of quantitative genetic analyses of experimental systems, and we acknowledge that some of the phenotypic variance attributed to additive genetic variation may be the result of dominance and epistatic interactions.P. Trichocarpa populations have the ability to adjust their phenology in response to a changing climate. The wide and environmentally variable native range of P. Trichocarpa is indicative of this species ability to persist across variable and unpredictable climates. We show here that a genetic variation underlies a large part of variation in phenology traits, but that trees also have the capacity for significant phenology shifts in response to year to year weather variation across the two years investigated here. This suggests this species has capacity to adapt its phenology to rapid climatic shifts, and that there is sufficient genetic variation underlying these traits that either natural or artificial selection could lead to evolutionary changes in adaptive phenology traits which may facilitate colonisation outside its natural rangeOverall, the substantial heritability found in phenology traits and the ability of trees to shift the timing of phenology transitions drastically between years suggests that Supplementary material


The primary measurement was the subjective estimation of quality of the visibility of the operating field provided by the 3D exoscope—VITOM-3D  in comparison to the operating microscope. Results: All procedures, except for two (3.3%) converted to the microscope, were successfully completed using a 3D exoscope. In both stapedotomy and tympanoplasty, the exoscope was superior to the microscope during more superficial portions of the procedures. By contrast, in deeper areas of the middle ear, the exoscope provided significantly worse visibility, but usually not suboptimal. Both intraoperative bleeding and the narrow surgical field substantially reduced the visibility with the 3D exoscope in comparison to the microscope. Conclusions: Overall, our study shows that the 3D exoscope offers excellent, highly magnified, and well-illuminated high-definition images of the surgical field. However, our experience revealed several important limitations of this system, including decreased depth perception in deep areas of the tympanic cavity and reduced visibility in a difficult surgical field, with subsequent need to switch to an operating microscope in select cases. Background: The aim of the study was to describe our initial experience with the high-definition three-dimensional (3D) exoscope for middle ear surgery versus the operating microscope. Methods: The study included 60 randomly chosen patients diagnosed with otosclerosis ( Advances in intraoperative optics and new surgical visualization systems such as the three-dimensional 3D) exoscope system have recently been reported as a viable alternative to surgical microscopes and endoscopes . The 3D D exoscopIn this context, we carried a cross-sectional analysis to assess the feasibility of using the 3D exoscope to perform stapedotomy in otosclerosis and tympanoplasty in chronic suppurative otitis media. In this observational study, we evaluated the quality of subjective visibility with the 3D exoscope perceived by an operating surgeon and compared it to the operating microscope at all the stages of both surgical procedures.n = 30) or chronic suppurative otitis media with cholesteatoma or granulation (n = 30) with a clinical indication for surgery. All the patients underwent video-assisted surgery with the 3D exoscope, which was performed between January 2018 and December 2018. All stapedotomy procedures were performed by a single surgeon . The tympanoplasty procedures were performed by two surgeons . Both surgeons are right-handed, and they have more than 20 years of experience in otosurgery. An endaural approach (with an incision of skin between tragus and helix and with use of retractors) was used in all the patients undergoing stapedotomy. In all the tympanoplasty cases, an incision was made behind the auricle. All stapedotomies were performed under local anesthesia and neuroleptoanalgesia. All tympanoplasties were performed under general anesthesia.This was a prospective cross-sectional analysis conducted in an academic, tertiary referral center. The regional ethics committee deemed this study to be exempt. The study included 60 randomly chosen patients diagnosed with otosclerosis  and 7 men (23.3%), with a mean age of 37.3 years . Of these, 22 patients (73.3%) underwent an initial stapedotomy while the remaining eight underwent stapedotomy in a second ear (26.7%). The tympanoplasty group included 18 women (60.0%) and 12 men (40.0%), with a mean age of 42.8 years (range from 23 to 59). In 24 patients, tympanoplasty was performed for the first time (80.0%), and in 6 (20.0%) it was a second-look procedure. Among the 30 patients with chronic suppurative otitis media, 26 were with cholesteatoma and 4 with granulation tissue. According to the European Academy of Otology and Neurotology (EAONO)/Japanese Otological Society (JOS) Joint Consensus Statements on the Definitions, Classification, and Staging of Middle Ear Cholesteatoma , we had All the analyzed patients underwent video-assisted surgery with the VITOM 3D, a high-definition exoscope  . The imaThe operating surgeons compared the quality of visibility with the exoscope to their prior recollection of visibility through the microscope from thousands of other middle ear surgeries. In order to make their subjective assessment, the surgeons filled in a form at each stage (stages 1–10) intraoperatively, and the subjective comparison was based on the following scale: (0) both instruments are considered comparable; (−1), the microscope is considered superior, and (+1) the exoscope is considered superior. During all the surgeries, the microscope was also prepared to be used at any single moment in case a surgeon needed to confirm his/her subjective assessment or to convert from the 3D exoscope to the operating microscope.In stapedotomy, the various surgical stages are relatively consistent across most cases. For the purposes of this study, we defined and assessed the following stages: the initial image of the operating field (stage S1); ease of tympanomeatal flap formation (S2); ease of opening the tympanic cavity (S3); ease of widening the external auditory canal (S4); visualization of the incudostapedial joint (S5); assessment of the oval window area (S6); ease of removal of the stapes superstructure (S7); facial nerve assessment (S8); footplate assessment and perforation (S9); and prosthesis placement maneuver (S10).The surgical stages for tympanoplasty, by contrast, can vary from patient to patient. Therefore, we assessed only the main stages of tympanoplasty, which had to be assessable for all operations. All cases included cholesteatoma or granulation tissue removal. The following stages were assessed: initial view of the operating field—planum mastoideum (stage T1); posterior meatal wall skin incision + tympanic membrane assessment (T2); antromastoidectomy (T3); cholesteatoma or granulation removal form mastoid process (T4); lateral semicircular canal visualization (T5); cholesteatoma or granulation removal form tympanic cavity (T6); cholesteatoma or granulation removal form hidden areas (protympanum and/or sinus tympani) (T7); temporal fascia harvesting (T8); ossiculoplasty (T9); and myringoplasty (T10).The primary measurement was the comparison of the surgeons’ subjective assessments of the quality of the visibility of the operating field provided by the 3D exoscope and the operating microscope at each stage and for the whole procedure in both types of surgery. Furthermore, we evaluated the impact of intraoperative conditions on the visibility with the exoscope. What is more, we analyzed the necessity for the conversion from the 3D exoscope to the microscope. The conversion was defined as the moment when the visibility with the exoscope was suboptimal due to bleeding, difficult anatomy, or a narrow operating field, and an experienced surgeon did not have an operational sense of surgical confidence and decided to switch to the microscope, which he/she had known for decades. Additionally, we assessed the duration of the procedure (minutes).p < 0.05.Statistica software  was used to perform the statistical analysis. The test of proportion was used to compare the visibility provided by the exoscope with the one provided by the microscope throughout the procedure and in the particular steps of the surgery. Due to the lack of a normal distribution for the total visibility variable, the Mann–Whitney test was used for comparison of normal and difficult operating conditions (intraoperative bleeding and narrow operating field). The level of statistical significance was set at The mean duration of the procedure was 41.3 min , and it did not differ significantly from the average time it took for this procedure to be performed with the microscope in our department by the same surgeon (40 min). In all the cases, surgery and healing were uneventful. All 30 procedures were completed with the 3D exoscope without requiring a switch to the operating microscope. As shown in p = 0.4884). The sum of the obtained points for all the stages of all the surgeries was 47, with a mean of 1.57 and a median 3.00 for one complete procedure. Furthermore, there were some evident differences concerning the influence of intraoperative conditions. Both intraoperative bleeding (observed in four cases) and the narrow surgical field (found in another four cases) significantly reduced the visibility with the 3D exoscope in comparison to the microscope. The details are presented in In comparison to the microscope, the 3D exoscope was evaluated as better in 48%, as comparable in 19.7%, and as worse in 32.3% for the whole procedure . The differences were not statistically significant  and it did not differ significantly from the average time it took for this procedure to be performed with the microscope in our department by the same surgeons (76 min for cholesteatoma stage 2). In all the cases, surgery and healing were uneventful. Most  of the tympanoplasty procedures were completed with the 3D exoscope. However, the conversion to the operating microscope was necessary in two cases, both due to bleeding during cholesteatoma and granulation removal from the tympanic cavity and hidden areas of the middle ear (protympanum and/or sinus tympani) (stages T6/T7). As shown in p = 0.0283). The sum of the obtained points for all the stages of all the surgeries was 116 with a mean of 3.87 and a median 5.00 for one complete procedure. There were no significant differences in the surgical field visibility for the first and second cholesteatoma surgeries. However, we found some evident differences concerning the influence of intraoperative conditions. Both intraoperative bleeding (observed in six cases) and the narrow surgical field (found in another four cases) significantly reduced the visibility with the 3D exoscope in comparison to the microscope. The details are presented in In comparison to the microscope, the 3D exoscope was evaluated as better in 56.3%, as comparable in 26.0%, and as worse in 17.7% for the whole procedure. Differences were statistically significant  using the 3D exoscope, which provided sufficient visualization of crucial anatomical structures. Conversion to the operating microscope was necessary in only two cases (3.3%), both during cholesteatoma or granulation removal from tympanic cavity and hidden areas of the middle ear (protympanum and/or sinus tympani). These findings support the use of the 3D exoscope for middle ear surgery. Based on our findings, the 3D exoscope appears to represent a valid 3D stereoscopic visualization tool for use in middle ear surgery. Our study showed that exoscope is an excellent tool for the more lateral part of the middle ear but has some disadvantages for surgery medial to the tympanic membrane.The main advantages of the exoscope include high-resolution 3D images, a greater degree of freedom for the exoscope adjustment, and a comfortable surgical posture ,13. WhatHowever, based on our subjective observations, the 3D exoscope has some important limitations, both during stapedotomy and tympanoplasty. In both procedures, the superiority of the exoscope over the microscope was only observed during more superficial stages. By contrast, in deeper areas of the middle ear, as the surgical field narrows and bigger magnification is required, the exoscope provided statistically significantly worse visibility. During stapedotomy, all the steps in the oval window region (S7–S10) were visible better with the microscope. Similarly, during tympanoplasty, we found that the microscope was more comfortable when removing the cholesteatoma or granulation tissue from the tympanic cavity and hidden areas of the middle ear (stages T6 and T7). Similar observations in microvascular bypass procedures have been reported . As thosAnother limitation of visualizing the surgical field with the 3D exoscope is the narrow operating field and bleeding during the surgery, cases in which a microscope is preferable. The presence of these unfavorable factors in the operative field had a significant and negative impact on the quality of the visibility provided by the 3D exoscope in comparison to the microscope. Thus, in such cases, we strongly advise that a microscope be chosen rather than the 3D exoscope.This study has several limitations. Firstly, the operating surgeons compared the quality of visibility with the exoscope to their prior recollection of visibility from other surgeries with the microscope. Study design relies heavily on a subjective assessment of a surgeon in real time. Undoubtedly, achieving an unbiased fashion is very difficult in such a comparison. However, from a technical point of view, conducting surgery on the same patient by means of two instruments simultaneously (an exoscope and a microscope) was not possible. That is why a surgeon’s recollection, knowledge, and experience seemed to be an invaluable reference point. Secondly, we were unable to unify all steps of tympanoplasty. For this reason, to facilitate the comparison, we selected main surgical milestones for tympanoplasty. A third limitation is that we did not include data concerning body habitus , even though such data are important for the 3D exoscope application. One of the challenges of using a microscope is that visualization may be limited, and a surgeon’s operative comfort decreases when the patient’s body habitus or anatomy is unfavorable. The next limitation of our research is the lack of postoperative hearing test results. This is due to the initial boundary assumption that the results must be as good as in cases of the classical method. Therefore, in cases of difficulties in visualization with the exoscope and suboptimal insight, we were immediately ready to convert the operation to the microscope to avoid complications or a worse audiological result. Finally, the last limitation is our inability to include 3D photographs in this manuscript, as one of the major aspects of the comparison between the use of the 3D exoscope and a microscope involved the comparison of the quality of 3D images. By contrast, the main strength of this study is the fact that, to our knowledge, it was the first study to assess the value of the 3D exoscope in stapedotomy and tympanoplasty. In addition, the analysis was carried out by a reference center with a 40-year tradition in otosurgery that performs approximately 900 procedures per year.Overall, our study shows that the 3D exoscope offers excellent, highly magnified, and well-illuminated high-definition images of the surgical field. However, our experience revealed several important limitations of this system, including decreased depth perception in deep areas of the tympanic cavity and reduced visibility in a difficult surgical field, with the subsequent need to switch to an operating microscope in select cases.