In recent years, it has been demonstrated that the application of elicitors such as methyl-jasmonate (MeJ) and benzothiadiazole (BTH) to wine grapes can increase their phenolic and aromatic compounds if they are treated at the beginning of ripening (veraison). However, the veraison period is short, and it is not always possible to apply the treatments in a few days. Therefore, it would be of great interest to optimize the moment of elicitor application or extend the treatment period. The aim of this paper was to analyze during two consecutive years (2016–2017) the foliar application of MeJ, BTH, and a combination of both, during two different ripening periods of Monastrell grapes (veraison and mid-ripening), and determine the more appropriate moment to increase the concentration of anthocyanins. To carry out this aim, analysis of anthocyanins by HPLC in grapes and wines was mainly performed. The most suitable period for the application of MeJ, BTH, and MeJ + BTH was at mid-ripening, since the grapes showed a greater accumulation of anthocyanins at harvest. However, the MeJ + BTH treatment applied during veraison also obtained similar results, which would allow extending the application period if necessary. However, the increase in the anthocyanin content of grapes was not reflected in all the wines, which may have been due to reinforcement of the skin cell wall as a result of the application of elicitors. Further analysis is needed to improve the maceration process of the Monastrell grapes and the extraction of the anthocyanins that were increased by the treatments applied in the vineyard. There is growing interest in phenolic compounds as bioactive compounds because of their impact on human well-being, which is mainly influenced by their chemical structures . Grapes Vitis vinifera L. cv Monastrell is the main grapevine in this region (more than 80% of the vineyards), and numerous projects have been undertaken to adapt its cultivation to new scenarios that include climate change [Several studies have been carried out in this respect in wines with the Denominations of Origin “Jumilla”, “Yecla” and “Bullas” from Murcia (south-eastern Spain). e change , new cone change .Other research studies have been developed to obtain new varieties from Monastrell with agronomic and oenological characteristics that are suited to this area , and difGiven the logistical and technological importance of the optimal moment of elicitor application, several studies have focused on the accumulation of anthocyanins, as well as color development; for example, after applying abscisic acid at different times during veraison to table grapes ,26,27, gFor the reasons mentioned above, the aim of this paper was to ascertain which of the two stages of ripening of Monastrell grapes (veraison and mid-ripening) was more advantageous for the foliar application of MeJ and BTH elicitors, or a combination of both, in order to determine the moment of application that increases the concentration of anthocyanins to the greater extent in grapes and consequently in wines.The results obtained for the different parameters measured are shown in On the other hand, the effect of elicitor application on average berry weight during 2016 showed that treatments applied at mid-ripening had a greater effect on this parameter than treatments applied at veraison. Thus, we found that the MeJ + BTH treatment applied at veraison was the only one that managed to increase the average weight of the berries. In relation to this, other studies in which treatments were also carried out at veraison found that BTH applied to Monastrell increased berry weight only in one of the two study seasons ; the samIn the 2017 season, Monastrell berries also responded differently to treatments depending on the time of application. Again, we observed that the response to treatments applied at mid-ripening on berry weight was more notable than treatments applied at veraison. In fact, at veraison, no differences were recorded between the average weight of treated and control grapes. However, at mid-ripening, the MeJ and BTH treatments did lead to increases in the average weight of the berries, thus obtaining results similar to those observed in the mid-ripening treatments in 2016. With regard to the soluble solids analyzed in the two seasons, the average concentration of the grapes in 2016 was 25.7 °Brix, which is a much higher value than the 22.5 °Brix recorded in 2017 . In thisIn addition to the higher rainfall recorded in 2017, there were more days with maximum temperatures above 30 °C in the period between the start of veraison and harvest, which amounted to 33 days in 2017 and 22 days in 2016. In addition, in 2017, the start of veraison was brought forward by 15 days, and the harvest date was 5 days earlier than in 2016, extending the ripening period of the Monastrell grapes by 10 days. However, despite this increase in the ripening period in 2017, the sugar concentration was lower at the time of harvest. In relation to this, it has been described that temperatures above 30 °C negatively influence the accumulation of sugars in the berries ,43, as wIn the 2016 season, the application of treatments did not cause statistically significant changes in the concentration of soluble solids of the treated grapes, if we compare them with the control grapes, both in the treatments that were applied at veraison and those applied at mid-ripening. However, we observed a slight increase in soluble solids, although not statistically significant, in the berries treated with BTH during mid-ripening. In this respect, the studies found regarding the effect of MeJ and BTH application on the concentration of sugars have shown varied results. Thus, it was found that the application of MeJ did not cause changes in the soluble solids of the Tempranillo variety ,40,44 anGrapes treated with MeJ during veraison in the 2017 season also showed a lower soluble solids value than the control grapes. In the case of grapes treated at mid-ripening, BTH treatment produced grapes with higher sugar concentration than the control grapes, as observed in 2016, indicating that BTH treatment might advance the ripening process of Monastrell. However, other researchers observed a lower soluble solids concentration after BTH application in the Syrah variety . Finally, the data for total acidity, tartaric acid, and malic acid showed no inter-annual differences, and no change was observed by the application of the treatments. However, the malic acid analysis of the grapes from the 2016 season revealed that the vines treated with BTH during veraison and MeJ at mid-ripening produced grapes with lower malic acid concentration than the control grapes. Regarding the effect of MeJ and BTH on pH, total acidity, tartaric acid, and malic acid, our results are in agreement with those obtained in previous research ,16,48,49O-glucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin were identified  a few days before the harvest . These eMoreover, the decision on the time of harvest was determined by the technological maturity, and not by the phenolic maturity, so it could be the case that the grapes were not harvested at the point of highest polyphenolic concentration of Monastrell. Related to this, a recent study analyzed the ripening evolution of Monastrell, Merlot, and Syrah grapes treated with MeJ and BTH during the veraison period , where tDuring 2016, the application of all the treatments positively influenced the concentration of total anthocyanins with respect to the control A, and thIn 2017, the treatments applied in mid-ripening were the most effective at increasing the concentration of total anthocyanins compared to treatments applied during veraison. However, the MeJ + BTH treatment applied in veraison that same year was the only one that managed to increase the concentration of total anthocyanins, pointing to a possible synergistic effect due to the combination of inductors. Few research studies have been reported on the synergism or antagonism between MeJ and BTH, although a cooperative interaction has been described in tomato  and in hWith respect to the individual treatments, several studies have shown that the exogenous application of MeJ can increase the concentration of anthocyanins in Monastrell grapes ,16,17; SNon-acylated anthocyanins constituted about 80% of the total anthocyanins in the grape skins C,D. ThisThe elicitors that increased the concentration of these compounds when applied in veraison 2016 were MeJ and BTH, while MeJ and MeJ + BTH increased them when applied at mid-ripening. In the treatments applied in 2017, only MeJ + BTH increased the concentration of non-acylated anthocyanins when applied during veraison, but all the treatments applied mid-ripening positively influenced the activation of these compounds. Additionally, acylated anthocyanins represented the remaining 20% of the total anthocyanins. In the 2016 season, all acylated anthocyanins increased in concentration following the application of the elicitors E both atO-glucoside with pyruvic acid (vitisin A) or with acetaldehyde (vitisin B) [As in grapes, the total anthocyanins analyzed in wines correspond to the sum of non-acylated and acylated anthocyanins . In thistisin B) . The antAs expected, based on the results obtained in grapes, the wines made in the 2016 season showed a higher concentration of total anthocyanins than those of the 2017 season A,B. The concentration of total anthocyanins in wines produced during 2016 was only higher in the wines made from grapes treated with BTH at veraison; while wines from grapes treated with BTH and MeJ + BTH during mid-ripening had lower concentrations than the control wines. The results obtained in 2017 indicated once again that wines from grapes treated with BTH (veraison) had higher concentrations of total anthocyanins, as they did in 2016. Nevertheless, wines from grapes treated during mid-ripening showed slightly higher concentrations of total anthocyanins than control wines, but no statistically significant differences.If total anthocyanins in grapes A,B and tSimilar to total anthocyanins, wines from grapes treated with BTH (veraison) were the only ones that showed a higher concentration of non-acylated anthocyanins with respect to the control, during both the 2016 and 2017 seasons C,D. With regard to the concentration of acylated anthocyanins of wines produced in 2016 E, no staThe wines produced in 2016 had better color parameters than the wines produced in 2017 . In thisTherefore, most of the positive effect observed in the grapes due to the application of elicitors was reflected in the CI of the wines. In this sense, several studies have described that Monastrell grapes treated with MeJ and BTH during the veraison period resulted in wines with a higher CI than wines from control grapes ,17,38,51When L* was measured in the elaborated wines C,D, thosLastly, 2016 wines made from grapes treated with MeJ (Veraison) and BTH (Veraison), as well as those treated with MeJ (Mid-ripening), showed lower hue than the control wines . On the Undoubtedly, the lower concentration of anthocyanins in the grape skins of the 2017 season had a direct impact on the color parameters of the wines produced, resulting in wines with lower CI and therefore with a higher L*.To classify the different individuals into alternative groups based on the analyzed variables (anthocyanins), a discriminant analysis was performed A,B. The O-glucoside.The statistical analysis performed on grapes clearly distinguished between the two years studied A, as welThe statistical analysis performed on wine samples also allowed us to distinguish between the seasons studied and the moment of treatment; however, in 2016, the distinction observed between the control and the moments of application (veraison and mid-ripening) was more pronounced. Five discriminant functions were obtained, the first function explaining 92% of the variance, with coumaryl glucosides  having greater influence as standardized coefficients.Vitis vinifera (syn. Mourvedre) red wine grapevines grafted onto 1103-Paulsen (clone 249) rootstock and trained in a vertical trellis system. Vine rows were arranged N-NW to S-SE with between-row and within-row spacing of 3 × 1.25 m. The soils are brown-limestone and stony, with low percentages of organic matter. The texture is predominantly silty-sandy, with high permeability and good aeration [The experiment, using the Monastrell variety, was carried out over two consecutive years (2016–2017) in Jumilla, Murcia (south-eastern Spain) . Total precipitation and average temperature during the grape ripening period in 2016 were 2.6 mm and 24.9 °C, and 54.6 mm and 25.5 °C in 2017 . The veraeration .v/v). Control plants were sprayed with aqueous solution of Tween 80 alone. The treatments were applied at two different stages of grape maturity: at veraison and at mid-ripening . For all treatments, a second application was performed 7 days after the first in order to maximize anthocyanin accumulation based on the results of previous works with this and other grape cultivars [The experiments were conducted in a randomized block design, in which all treatments were applied to three replicates, using 10 vines for each replication. The protocol used to apply the different treatments, as well as the doses, have been described previously . Plants ultivars ,38. WhenThe traditional flesh parameters were measured: Total soluble solids (°Brix) using an Abbe-type refractometer (Atago RX-5000); titratable acidity using an automatic titrator  with 0.1 N NaOH, and tartaric and malic acids using enzymatic kits from Boehringer 146 Mannheim GmbH . 2/100 kg). Total acidity was corrected to 5.5 g/L with tartaric acid, and selected yeasts were added . A pre-fermentative cold maceration was carried out followed by traditional maceration. Tanks containing crushed grapes were placed in a cold room at 4 °C for 10 days, after which the tanks were returned to the winery to continue the traditional maceration process at 25 ± 1 °C. The fermentative pomace contact period was 10 days. Throughout the fermentation pomace contact period, the cap was punched down twice a day, and the temperature and must density were recorded. At the end of this period, wines were pressed at 1.5 bars in a 75 L tank membrane press. Free-run and press wines were combined and stored at room temperature. The analyses were carried out in triplicate at the end of alcoholic fermentation.All vinifications were made in triplicate in 50-L stainless steel tanks using 50 kg of grapes, which were destemmed, crushed, and sulfited . Grape and wine anthocyanins were determined according to Gil-Muñoz et al. . Twenty Samples were filtered and centrifuged, and a Shimadzu UV/visible spectrophotometer, model 1600PC  was used to analyze color intensity (CI) and CIELAB parameters. CI was calculated as the sum of absorbance at 620 (blue component), 520 (red component), and 420 nm (yellow component) in undiluted wine . The CIEp < 0.05) when the ANOVA test was significant, and a multivariate discriminant analysis was applied to identify the most discriminant variables.Significant differences among wines and grapes and for each variable were assessed by analysis of variance (ANOVA) using Statgraphics 5.0 Plus package . Duncan’s test was used to separate the means  seems to be the most appropriate to increase the concentration of total anthocyanins, whether applied at veraison or mid-ripening since it would give more time to plan treatment, facilitating vineyard management even in unfavorable environmental conditions.However, the increase in the anthocyanin content of grapes achieved by the above-mentioned treatments was not reflected in all the resulting wines, which was probably because the application of elicitors reinforces the skin cell wall, making it difficult to extract anthocyanins. Whatever the reason, it is clear that a more thorough analysis is necessary to improve the maceration process for Monastrell grapes and the extraction of the anthocyanins that were increased by the treatments applied in the vineyard. In this respect, this is the first attempt to optimize the time of application of these elicitors in order to maximize the accumulation of anthocyanins in grapes. The results provide information that will help understand the response of berries to treatments with MeJ and BTH in different circumstances.


Multistable switches are ubiquitous building blocks in both systems and synthetic biology. Given their central role, it is thus imperative to understand how their fundamental properties depend not only on the tunable biophysical properties of the switches themselves, but also on their genetic context. To this end, we reveal in this article how these factors shape the essential characteristics of toggle switches implemented using leaky promoters such as their stability and robustness to noise, both at single-cell and population levels. In particular, our results expose the roles that competition for scarce transcriptional and translational resources, promoter leakiness, and cell-to-cell heterogeneity collectively play. For instance, the interplay between protein expression from leaky promoters and the associated cost of relying on shared cellular resources can give rise to tristable dynamics even in the absence of positive feedback. Similarly, we demonstrate that while promoter leakiness always acts against multistability, resource competition can be leveraged to counteract this undesirable phenomenon. Underpinned by a mechanistic model, our results thus enable the context-aware rational design of multistable genetic switches that are directly translatable to experimental considerations, and can be further leveraged during the synthesis of large-scale genetic systems using computer-aided biodesign automation platforms. Living cells function as microscopic factories, converting energy and building blocks into a large array of products . The visWhile modifying the genetic blueprint of living organisms is now routine practice via genome editing, designing systems of even modest complexity still requires numerous iterative cycles and vast libraries ,9 due toConsidering the central role that multistable switches play in both natural and synthetic gene systems ,38,39, iMotivated by this, our objective here is two-fold: we seek to (i) characterize how tunable biophysical parameters of the toggle switch shape its stability and robustness properties, and to (ii) quantify how burden from the genetic context influences these fundamental characteristics. Significantly extending our prior results ,47 by acTo ensure predictable system-level behavior, the effects of context-dependence must be explicitly accounted for at the part level. To ensure that the stability and robustness of genetic toggle switches can be rationally adjusted by leveraging the explicit design guidelines we uncover, our approach relies on a mechanistic model ,47 that This paper is organized as follows. After presenting the mathematical model and the technical tools underpinning our analysis, we reveal the role that promoter leakiness, scarcity of shared transcriptional and translational resources, and cell-to-cell heterogeneity collectively play to establish the fundamental stability and robustness properties of genetic switches. Following this, we illustrate how these results can be further generalized to account for loading from the genetic context of the switch.y and z) repressing each other’s expression. Assuming that the repressors bind as dimers [The toggle switch represents one of the first rationally engineered genetic devices , compriss dimers ,38,55,56s dimers , the de-eakiness , respecty and z, the widely adopted model of the toggle switch above [y and z; similarly, y and z), this assumption can be relaxed to study the effects of parameter asymmetry [Although cellular resources  are shared among processes responsible for the production of ch above ,57,58 fach above . Consequch above ,16,17,50ion rate ,16,50. Ttal data ,16,17,50ineering , β via doy sites , ν via pineering , and βc proteins ,16. Whilsymmetry ,60.Regarding the typical range of ility in , which o in [α>2 . Since αy number , consideper cell . As β derding to , based oproteins  or decoyproteins  by modifproteins  or by acproteins . To accoFinally, it is important to note that promoter leakiness can be taken into account by considering a different formulation , which lCrucially, these two approaches lead to identical results considering Stability analysis of  when ν=0ried out ,54. The amics in  have a sobian of  are negaimilarly . IncludiIn particular, the dynamics become bistable when therwise  =∂function ,66.With this, we can thus calculate the potential barriers separating metastable fixed points ,67. To toints of  with thexi to xj . Therefoxi to xj .Stochastic simulations were performed by considering the overdamped Langevin dynamics widely used in biomolecular simulations ,45, togeq and To model cellular heterogeneity, assume that Q and With this, and relying on ,71,72,73First, note that the rescalings α←α/+βc and β context . Similar(ν) from  to studyα←α/+βc and βIn this section, we first reveal the role that each parameter plays in determining the stability properties of the toggle switch. Following this, we establish how biophysical parameters affect the robustness of metastable fixed points to noise, subsequently generalizing these results to account for parameter variations to study population-level effects. Finally, we uncover how additional burden from the genetic context of the toggle switch shapes the above relationships, explaining the phenomenon observed in At its core, the toggle switch serves as a memory unit, provided that the underlying dynamics are multistable ,74. Whene if α>2   plane  are sepafined in .These results aid the rational design of multistable toggle switches by providing explicit guidelines to eliminate unwanted behaviors. For instance, decreasing promoter leakiness We next reveal how protein production rate barriers A separatbarriers  defined barriers C, and thq from ..ν=0), fumodality A, as expmodality  in SectiQ when Just as population-level characteristics follow from single-cell properties via fined in  when relAdditionally, to illustrate how our results provide guidelines for eliminating unwanted behaviors, consider the population-level steady-state distributions of oy sites ) is the  grey in B. Robustq from (As the behavior of genetic switches displays strong dependence on their context ,40,41, hf q from  increasetability . In the  crossed A. Thus,  crossed A, additi crossed A.Crucially, our results also expose that mitigating the unwanted effects of the context can be achieved by increasing ld of βc  that cauto noise B. Naturaw red in A, while green in A. More igreen in A.Bioenergetic cost associated with the existence and expression of a gene is not a novel concept . Recent To promote modularity and predictable system-level performance, sources of context-dependence need to be incorporated into the design of genetic modules . ConsideLeveraging this, we not only revealed how tunable macroscopic parameters affect whether the toggle switch displays monostability, bistability, or tristability, but also how they shape the robustness of the metastable states to noise using a potential landscape-based approach . In addiIn this paper, we thus derived explicit guidelines that aid the design of multistable genetic switches. Crucially, these results are directly translatable to experimental considerations due to the mechanistic model underpinning our results: e.g., ineering , β via toy sites , ν via pineering , and βc proteins ,16. Our proteins ,82,83, bproteins ,85,86, aproteins .While competition for shared transcriptional/translational resources represents a major source of context-dependence ,16, it i


PLOS Genetics publications that exemplify recent progress in domestic animal genetics. Rather than grouping by species, we assigned the publications to 5 thematically related topics, which we will briefly introduce in the following sections.Domestication of wild animal and plant species may be considered the largest genetic experiment in human history. Human selection for specific traits and targeted breeding over hundreds or thousands of generations has shaped very special populations that provide unique opportunities for research. Domestic animal genetics contributes to technological advances facilitating a more sustainable, environment and climate friendly food production that respects changing societal expectations related to ethical and animal welfare issues. Domestic animal genetics also enables fundamental insights into gene functions and provides animal models for biomedical research. Under this theme, we present a selection of The 7 articles in this group provide either new methods or resources for research in domestic animal genetics. This ranges from an improved cat reference genome assembly over the generation of a comprehensive gene expression atlas in sheep to the presentation of a comprehensive database on breed-specific disease allele frequencies in dogs.The genetic changes associated with domestication show many parallels across different animal species, such as specific changes in behavior or the loss of seasonality of reproduction. In our collection, we present 8 articles that investigate specific genetic changes that are the consequence of breeding in closed populations or that investigate admixture and subsequent selection mechanisms in domestic animals.Nine articles in this section nicely reflect different levels of genetic complexity. They range from oligogenic traits to very complex traits such as the influence of the host genome on rumen microbiome diversity. In many livestock populations, a wealth of environmental and phenotypic data is recorded. This enables powerful studies with respect to many complex traits and genotype x environment interactions. With the intensive use of artificial insemination, elite livestock sires can have many thousands of offspring, which is a unique situation in mammalian species and predestines livestock populations for genetic research. When it comes to disease genetics, dogs with their many breed-specific predispositions for specific diseases provide excellent opportunities to identify genetic risk factors for complex diseases.With 23 articles, this is by far the largest group in our collection. The special population structure of domestic animals greatly facilitates the identification of the causative genetic variants for hereditary traits. At the same time, sophisticated veterinary diagnostics approaching the standards in human medicine is widely available for cats, dogs, and also other domestic animals. Purebred animals are maintained in strictly closed populations with a substantial amount of inbreeding, which favors the emergence of new recessive traits and diseases. The elucidation of the causative genetic variants in spontaneous domestic animal mutants may assign physiological functions to uncharacterized genes, provides candidate genes for human medical genetics, and helps to obtain more comprehensive genotype–phenotype correlations.Dogs and also cats may develop very similar tumors as humans. The shorter life span and striking breed predispositions for certain cancer types facilitate the identification of genetic risk factors. With 5 articles, this is the smallest group in our collection. However, the potential of domestic animals and especially dogs for cancer research in general and cancer genetics in particular has by far not yet been fully utilized.PLOS Genetics continues to publish articles with some of the best research in domestic animal genetics. This is a field with many facets enabling fascinating new fundamental insights as well as laying the basis for applications in animal breeding that are absolutely vital for a sustainable future of mankind.


With social and economic environment changes occurring in the world and deepening of the urbanization process, China’s urban development exhibits a new phenomenon of growth and shrinkage fluctuations. The resource-based city shrinkage phenomenon is particularly prominent. Research on the commonalities and patterns of similar groups should be enhanced. We constructed an urban shrinkage evaluation index system from the three dimensions of population, economy and space. Accordingly, we explored the spatiotemporal evolution characteristics of 175 resource-based cities in China from a multidimensional perspective with the entropy method, shrinkage model and transfer matrix method. The results indicated that most resource-based cities in China occurred in the non-shrinking state, but their development speed gradually decreased or even presented stagflation. The shrinkage measure-related results in the different dimensions revealed that the number of shrinking cities is increasing. The population, economic and comprehensive shrinkage levels were mainly slight and remained stable. The number of cities experiencing moderate and severe shrinkage was relatively small and mostly encompassed short-term shrinkage. Spatial shrinkage demonstrated a clear administrative hierarchy difference. Moreover, the spatial distribution range of shrinking cities in each dimension expanded and exhibited obviously similar characteristics, i.e., shrinking cities were relatively concentrated in Northeast China, while they were more scattered in other regions. Furthermore, the geodetector technique was applied to reveal the influencing factors of resource-based city growth and shrinkage. Based on the results, the change in the secondary industry output value share at the start of the study was the primary factor. The impact of each employment structure indicator from 2014 to 2018 was particularly significant. Comprehensive exploration of the shrinkage characteristics of this particular group of cities and their development behavior from a multidimensional perspective can provide an important reference for the transformation and high-quality development of resource-based cities. Since the reform and opening up, the economy and cities in China have experienced continued growth and long-term rapid expansion. As economic development transitions into the new normal state and urbanization enters the second half of its course, the economic and urbanization growth rates gradually decrease, and urban development in China joins the postgrowth era . Urban gUrban shrinkage is not a new phenomenon. It initially originated from the notable population loss experienced by developed countries, but no clear shrinkage direction was provided in early studies. In the 1990s, Häußermann formally proposed the concept of shrinking cities in empirical research on the Ruhr area in Germany from a sociological perspective, which represented the demographic and economic decline conditions in cities in Germany due to deindustrialization . Since tUnder the background of globalization, the spatial distribution of shrinking cities is no longer limited to a few old industrial cities in developed countries represented by Britain and Germany , but graTo explore the evolution of shrinking resource-based cities, we considered 116 prefecture-level and 59 county-level resource-based cities as the research area. A comprehensive analysis of resource-based city shrinkage in China from 2010 to 2018 was conducted, and spatiotemporal evolution characteristics were analyzed. Then, with the application of the geodetector method, we explored the influencing factors within the context of the development characteristics of resource-based cities. This study aims to provide a reference for the transformation and development of shrinking resource-based cities, as well as other similar shrinking cities in China and abroad.According to the list of resource-based cities in China’s Sustainable Development Plan of National Resource-based Cities, 2013–2020 (SDPNRBC), which was issued by the State Council in November 2013, there are 262 resource-based cities nationwide. According to this list, there are 126 prefecture-level administrative regions, 62 county-level cities, 58 counties and 16 municipal districts. Since the research target encompasses the territory of urban entities, based on the existing data conditions, a total of 175 resource-based cities in 116 prefecture-level cities and 59 county-level cities were selected to minimize the differences in research unit data level. The study area is shown in Two types of data are mainly considered in this research: NTL data and socioeconomic statistics. NTL data comprise important monitoring data of the intensity of human activities, which reflect changes in urban expansion, social and economic activities, etc. These data facilitate the identification of urban spatial shrinkage and have been widely applied , 28–30. Against the new normal background, a single urban population index hardly fully characterizes the urban shrinkage phenomenon. However, it is easy to downplay its core features if numerous indicators, such as population, economy, society and spatial aspects, are all included in the conceptual system of shrinking cities . TherefoSa is the shrinkage degree of city a. a in t1 and t2 respectively. That is, Sa is the change rate of the development index of city a from t1 to t2. Sa < 0 indicates that the city is shrinking, and the lower the value, the more serious the decline in the urban development level during the reporting period is over the initial period level, i.e., the higher the urban shrinkage level is. Conversely, city a occurs in a state of stagnation or growth, and the higher the Sa value, the greater the development level of city a is improved over the initial stage level.Based on the obtained evaluation index data pertaining to the study period, comprehensive and fractal development index values for each city were calculated by using the weight of each index, and the growth and shrinkage levels of each city were calculated with a shrinkage model. The specific equation is as follows:Moreover, the classification of growth and shrinkage levels obviously cannot adopt a unified standard in different spatiotemporal scales. Therefore, the natural break point method is applied to our classification , 34. BasL1 to Ln denote urban growth or shrinkage types. Aij indicates the number of cities that changed from type i into type j during the period from T1 to T2. i-type cities during the T1 period, while j-type development state during the T2 period. Moreover, i-type cities converted from type i into type j.In this paper, based on the classification of urban growth and shrinkage, the calculation idea of the land use type transfer matrix is adopted for reference , 37, theq denotes the influence of the detection factor, and the value occurs in . The larger the q value is, the greater the influence of a given detection factor on the dependent variable. h is the number of layers of the dependent or independent variable, and its value range is . Nh and N are the number of units in the h-th layer and whole region, respectively, and σ2 are the variances in the dependent variables in the h-th layer and whole region, respectively.The geodetector technique can reveal the internal and external influencing factors of geographical spatial differentiation through spatial variance analysis of statistical principles and has been widely applied in economic, social and ecological fields . In thisY) as dependent variables and selected 13 indicators from 7 aspects, such as population growth, employment structure, and differences in people’s living conditions and regional economy, to constitute an index system of influencing factors regarding the shrinkage of resource-based cities  cities were more stable than were type IV and V cities, and the number and proportion of cities whose population dimensions slightly shrank were the highest in all dimensions. From the perspective of the transformation direction, the cities experiencing shrinkage in the population and comprehensive dimensions mainly transformed from type III into type II cities, revealing the characteristics of the coexistence of growth and shrinkage fluctuations. Cities subject to slight economic shrinkage were mainly transformed into type I cities. In contrast, the spatial dimension showed the reverse transformation characteristics, and the number of cities transformed from type III into type IV cities accounted for 58.33% of all cities, indicating that the spatial shrinkage level of resource-based cities greatly increased compared with the initial stage level. A final point to address here is, cities experiencing rapid growth (I) in all dimensions indicated an obvious deceleration or stagnation tendency, and their transformation direction was mainly from type I into type II cities. The proportion of cities with this transformation pattern in the population, economy and spatial dimensions reached 76.48%, 58.18% and 60.00%, respectively.The spatial distribution of comprehensive urban shrinkage  indicateAs the core feature and the most intuitive expression of urban shrinkage, population shrinkage is more significant than are economic and spatial dimension shrinkage aspects . From 20The spatial distribution of resource-based cities with economic shrinkage is shown in The spatial pattern of urban spatial shrinkage is shown in We applied Geodetector software to identify the influencing factors and corresponding action intensity of the shrinkage phenomenon of China’s resource-based cities from 2010 to 2018. And the action directions of significant influencing factors were further analyzed in combination with the Pearson correlation coefficient. The results are shown in q value, the impact of indicators from large to small is X9, X12, X10, X5, X2, X8, X7, X4, X13, X3. According to correlation analysis, X2, X5, X7, X9, X12 and X13 have positive effects on the development of resource-based cities, while X3, X8 and X10 yield negative effects. However, X3 and X4 fail the correlation test. During this period, the change in the output value of the secondary industry was the primary factor influencing the growth and shrinkage levels of resource-based cities, i.e., the lack of development of resource-based industries was closely related to the emergence of the shrinkage phenomenon of resource-based cities.From 2010 to 2014, there were 10 indicators that impose significant effects on the spatial difference in growth and shrinkage levels between prefecture-level resource-based cities. According to the X5, X4, X13, X3, X9, X8, X12. Overall, the factors of the employment structure imposed a particularly prominent influence on the shrinkage level of resource-based cities. During this period, except for a negative correlation between X8 and the dependent variable, all other indicators revealed positive effects. The influence of various factors and corresponding indicators on the shrinkage level of resource-based cities is examined in the fourth section.With increasing regional differences in the growth and shrinkage levels of resource-based cities and with increasing scattered distribution degree, the index with a notable influence greatly decreased from 2014 to 2018, and only 7 elements such as the change in the number of employees in the mining and manufacturing industries exhibited significant detection results, but no new significant influence factors were observed. The influencing factors are sorted from large values as follows: The economic development has entered a new normal in China. With an increasing demand for industrial structure transformation and upgrading and intensification of the contradictions between production cities, the shrinking characteristics of resource-based cities are gradually obvious, which is subject to their unique development path. Although growth remains the mainstream trend, the overall development speed is obviously decreasing, while the number and the shrinkage level of shrinking cities are increasing daily, and the situation is relatively pessimistic. It should be noted that type II cities can maintain low growth or stagnation for a long period, and these cities do not experience large-scale factor loss at the moment. However, in the urban development network, the regional growth advantage is reduced, and the region faces the risk of marginalization. Therefore, this kind of potential shrinking city should be given more attention over shrinking cities.In additions, the results showed that relatively few cities could maintain shrinkage in both periods. This means, compared with the overall recession that is sometimes difficult to reverse in some developed countries, the shrinkage of resource-based cities in all dimensions exhibited significant instability. Then, from the long-term continuous series, are these cities still in continuous shrinkage, or do expansion and shrinkage exist at the same time? Urban shrinkage in this paper is calculated by the shrinkage model, which is lack of research on its sustainability. So, it should be further improved in the follow-up research.The foregoing shows that there are many factors that significantly impact the development of China’s resource-based cities in different periods. Hence, in order to explore the sustainable development path of resource-based cities, we further explained the influence mechanism of main factors and put forward its countermeasures based on the existing research and our empirical results. In addition, we focus on the agglomeration area of shrinking cities, that is, Northeast China, and qualitatively explore the shrinkage incentives combined with the shrinkage characteristics of resource-based cities.X1 failed the significance test. Therefore, research on the influence mechanisms of the different dimensions and various scales should be strengthened. In contrast, population migration exerted a significant positive impact on the spatial difference between the growth and shrinkage levels of resource-based cities. This is similar to the conclusions of other studies [Among the resource-based cities, the proportion of cities experiencing a negative natural population growth represented by Northeast China was relatively small. It may be the main reason why  studies , 40. It  studies .Based on the empirical results of the INS and EMS, it is found that resolution of the contradiction between industrial structure optimization and labor employment transfer is increasingly becoming a major problem faced by resource-based cities during critical periods. At the initial research stage, the scale of the emerging industries in resource-based cities was still small. As a result, regional resource elements were concentrated in traditional industries with a relatively high development level. Therefore, in a considerable number of cities, expansion of the traditional industrial scale and number of employees facilitates urban economic and social development to a certain extent, while the high-end manufacturing industry and tertiary industry are difficult to reap their economic benefits in the short term. At the latter period, further implementation of development planning of resource-based cities has effectively promoted the orderly development of alternative industries and consequent reduction in traditional resource-based industries. But the poor matching between the traditional industrial labor force and replacement industry  may leadWhen exploring the causes of the decline of American urban centers, it was found that the location relative to larger urban centers was one of the basic factors that contributed to urban shrinkage . It is sAs a concentration area of urban shrinkage, Northeast China has attracted considerable attention. It is also impacted by many factors, experiencing both historical reasons and realistic causes. With reference to relevant literature, the following analysis can be seen here. First of all, earlier urbanization and the implementation of family planning have led to a lower overall fertility level from the perspective of population . The resResource-based cities are already located along the outer edges of metropolitan areas. In the new era of the overall economic slowdown, China’s resource-based cities, especially resource-exhausted cities, are experiencing or will soon face urban shrinkage represented by population loss and economic recession under the influence of multiple factors. This paper analyzed the spatiotemporal evolution of shrinkage and its influencing factors in 175 resource-based cities in China from 2010 to 2018. The study found that the development of China’s resource-based cities was still dominated by growth. However, the number of shrinking cities in the population, economy, and spatial dimensions increased. The spatial distribution range continued to expand. Certain areas in Northeast China became small clusters of shrinking cities. The industry and employment structures were the key factors influencing the growth and shrinkage levels of the resource-based cities in China during the two research periods. In summary, this study was conducted to explore the commonalities and differences between China’s resource-based cities from a multidimensional perspective, which perfected the framework for different types of urban shrinkage research to a certain extent. Drawing lessons from the idea of the land use transfer matrix, we employed the transfer matrix to describe the transformation characteristics of urban development. This method better highlights the direction of data change over other methods. Additionally, the combination of the geodetector technique and Pearson correlation coefficient analysis complemented the quantitative study on the factors of urban shrinkage.It also should be pointed out that some deficiencies also exist in our research, which is worth studying in the future. Firstly, as mentioned earlier, our research on the sustainability of resource-based cities is relatively lacking. Secondly, the purpose of this study is to explore the law of the shrinkage of resource-based cities. But there are actually a few cities in the state of shrinkage according to the results. And the spatial distribution is very scattered, so it is relatively difficult to comprehensively summarize their distribution characteristics. From the comparison between resource-based cities and non-resource-based cities, we may get more reasonable results. Finally, we explored the influencing factors of the shrinkage of prefecture level resource-based cities from a macro perspective. However, quantitative research on local influencing factors of significant shrinking cities is still insufficient. It is also the focus of future research.S1 Data(ZIP)Click here for additional data file.


Lacticaseibacillus casei LUHS210] almond, coconut, and oat drinks preparation BPs were used in wheat bread (WB) formulations. Microbiological and other quality parameters  as well as bread texture hardness during the storage and acrylamide content in the WB were evaluated. Among the fermented samples, 12-h-fermented almond and oat, as well as 24-h-fermented coconut drinks preparation BPs  were selected for WB production. In most cases, the dough and bread quality parameters were significantly (p ≤ 0.05) influenced by the BPs used, the treatment of the BPs, and the quantity of the BPs. The highest overall acceptability of the WB prepared with 20% fermented almond drink preparation by-product (AP), 15% fermented oat drink preparation by-product (OP), and 15% ultrasonicated OP was established. After 96 h of storage, the lowest hardness  of the breads prepared with 5% fermented AP, coconut drink preparation by-product (CP), and OP and ultrasonicated CP was found. The lowest content of acrylamide in the WB prepared with OP was found . Finally, 15% fermented OP could be safely used for WB preparation because the prepared bread showed high overall acceptability, as well as low acrylamide content.During plant-based drinks production a significant amount of valuable by-products (BPs) is obtained. The valorization of BPs is beneficial for both the environment and the food industry. The direct incorporation of the fermented and/or ultrasonicated almond, coconut, and oat drinks production BPs in other food products, such as wheat bread (WB) could lead to the better nutritional value as well as quality of WB. Therefore, in this study, various quantities  of differently treated [ultrasonicated (37 kHz) or fermented with  Nowadays, food processing BPs are considered a source of valuable compounds that could be recovered to fortify different products . The nonre found . Oxalatere found . Howeverre found . ConsideLacticaseibacillus casei LUHS210 strain) and ultrasound treatment of almond, coconut, and oat drink preparation BPs could be useful for the BPs biosafety improving without negative changes in the chemical composition  could be one of these methods because it not only enhances the organoleptic properties and nutritional value of fermented substrates, but it also improves their microbiological stability and possesses a detoxifying effect . In addiposition . Moreoveresearch .The most effective valorization of by-products can be done without expensive intermediate treatment steps , and we hypothesize that such direct incorporation of the fermented and/or ultrasonicated almond, coconut, and oat drink preparation BPs in other food products, such as wheat bread, is possible. Bread continues to be a significant part of daily human nutrition. Wheat bread has a high glycaemic index and contains gluten. Due to this, frequently consumed wheat bread may elicit some health-related issues such as insulin resistance or celiac disease . To miniLacticaseibacillus casei LUHS210 strain and ultrasonication at 37 kHz. Furthermore, the wheat bread samples were prepared with the addition of different quantities  of fermented and ultrasonicated by-products, and quality as well as safety parameters of the wheat bread were evaluated.In this study, the challenges associated with the application of almond, coconut, and oat drinks preparation BPs in wheat bread technology were analyzed. To reduce microbial contamination before the use as wheat bread ingredients, two treatments for the by-products were applied: fermentation with the Moisture content was determined by drying the samples to constant weight  with hexane technical grade   in 100 g of sample].Plant-based drinks production BPs  were obtained from a European company in 2019, producing plant-based drinks. The chemical composition of AP, CP, and OP (not dehydrated) is given in t weight . Ash conat 900°C . Dietary3 method . Kjeldahproteins . The tot States) . Carbohy2 intensity) for 2 min at 60°C at the Fraunhofer Institute UMSICHT (Germany). The BPs were stored in an airtight container at −18°C until further analysis.To disintegrate the by-products, all AP, CP, and OP samples were treated with a low-frequency (18 kHz) ultrasonic device  where it was stored at −80°C  until the use. From previous studies, it was known that the LUS210 strain possesses antimicrobial activities against various pathogenic and opportunistic strains (L. casei LUHS210 was grown in Man-Rogosa-Sharpe (MRS) broth  at 30 ± 2°C for 48 h. Two percent of the MRS solution (v/v) in which the strain was multiplied was inoculated into fresh medium and propagated for 18 h. The multiplied LAB strain was further used for the fermentation of BPs. strains . Before 10 CFU/mL were fermented at 30 ± 2°C for 48 h. For 100 g of processed by-product, 60 mL of water was used. The final moisture content of the processed by-products was, on average, 60%. The moisture content was determined according to method described in The BPs, water, and a suspension of the LUHS210 strain (3% from dry matter of the by-product mass) containing 8.9 logUltrasonication of BPs was performed at a frequency of 37 kHz using a 100 W power level. The equipment employed was a ultrasonication processor . Each 250-g sample of by-product was processed for 30 min. Before ultrasonication, for 100 g of processed by-product, 60 mL of water was used.Total titratable acidity (TTA) and pH were measured only in not dehydrated fermented BPs.By-products sample (5 g) was homogenized with 50 mL of distilled water and the pH was measured using a pH electrode . TTA was determined in a10 mL of sample homogenized with 90 mL of distilled water, titrated with a volume (mL) of 0.1 mol/L NaOH to obtain a final pH value of 8.2, and expressed as Neiman degrees (°N). For the concentration evaluation of L-(+) and D-(−) lactic acid isomers, a specific Megazyme Assay Kit  was used.The determination of LAB, total bacteria (TBC), enterobacteria (TEC), and mold/yeast (M/Y) counts in the by-products was performed according to The wheat bread recipe consisted of 1 kg of flour (100%), 2% salt, 3% fresh compressed yeast, and 56% water (control bread). The dough was mixed (3 min at a low-speed regime and 8 min at a high-speed regime) in a mixer , shaped, and proofed at 30°C and at 80% relative humidity for 45 min. Dough loaves of 350 g were formed and baked in a deck oven  at 220°C for 25 min. The wheat flour was substituted with 5, 10, 15, and 20% of fermented and ultrasonicated by-products. In total, 25 different wheat bread formulations were prepared and tested .∗, a∗, b∗), and texture hardness were determined in dough with the addition of fermented BPs or ultrasonicated BPs. The samples were taken after the dough was mixed.pH, TTA, color coordinates , overall acceptability, bread shape coefficient, mass loss after baking, and acrylamide content. The analysis was performed 12 h after baking. In addition, bread crumb firmness during storage was evaluated.Bread samples were analyzed for TTA, moisture content, specific volume, crumb porosity, color coordinates  after dissolving 5 g of dough in 10 ml distilled water.∗a∗b∗  system .The color characteristics of dough and bread were evaluated using a CIE LDough texture hardness was determined using texture profile analysis (TPA) . Dough samples (50 g) were placed in a plastic measurement vessel (diameter of 25 mm and height of 50 mm) and hardness was fixed as a maximum compression force . The samples were taken after the dough was mixed.The moisture content of bread was established by drying the sample at 103 ± 2°C to constant weight.Bread crumb porosity was ascertained according to the Lithuanian standard method .Bread volume was measured by the rapeseed displacement method .Bread specific volume was calculated by measuring and dividing the volume and weight of the bread loaf.The bread shape coefficient was calculated by measuring and dividing the height and width of a bread slice.The overall acceptability of each bread loaf was evaluated according to Bread crumb firmness during storage was determined as the maximum compression force using texture profile analysis (TPA) . Bread was immediately sliced (thickness 6 mm), and crumb hardness was fixed as a maximum compression force .18 column . The conditions for detection by MS/MS were as follows: ionization was performed using electrospray in the positive mode . The acrylamide concentrations in the selective samples were quantified by the internal standard (13C3-acrylamide) method.The quantitative analysis of acrylamide was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a Waters Alliance HPLC system 2695 coupled to a Micromass Premier XE mass spectrometer , as described by post-hoc tests. A linear Pearson’s correlation was used to quantify the strength of the relationship between the variables . The correlation coefficients were calculated using the statistical package SPSS for Windows . The results were recognized as statistically significant at p ≤ 0.05.Analyses are based on three independent fermentation and baking trials. Results were expressed as the mean ± standard deviation (SD). To evaluate the effects of the different types of by-products, their different treatments, and the quantity of additives used on bread quality parameters, the data were analyzed by multivariate analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) procedure as p = 0.0001) affected by the fermentable substrate, the duration of fermentation, and the interaction of the analyzed factors (substrate × duration). When comparing the L-(+)/D-(−) lactic acid ratio, the highest ratio (12.4) in AP samples was found.The pH, TTA, L-(+) and D-(−)-lactic acid concentration, and microbiological parameters of AP, CP, and OP by-products are shown in 10 CFU/g). At the same time, samples that were ultrasonicated at 37 kHz did not contain enterobacteria, but the presence of M/Y was established in AP samples (4.61 log10 CFU/g). A comparison of fermented samples showed that none of these samples contained enterobacteria and M/Y, while the LAB count after 12 h of fermentation ranged from 7.90 to 8.95 log10 CFU/g , the LAB count after 24 h of fermentation ranged from 8.38 to 8.75 log10 CFU/g , and the LAB count after 48 h of fermentation ranged from 8.32 to 8.67 log10 CFU/g . A moderate negative correlation between the pH values of the by-products and TBC was established (r = −0.4103), as well as a weak negative correlation (r = −0.1082) between the pH values of the by-products and the LAB count.Total bacteria, TEC, LAB, and M/Y counts in samples after 12, 24, and 48 h of fermentation and ultrasonication are shown in Finally, after summarizing the acidity and microbiological parameters of the fermented samples, for the wheat bread preparation, 12-h-fermented AP and OP samples  and 24-h-fermented CP samples  were selected.p = 0.043), the treatment applied for the by-product (p = 0.007), the quantity of by-product used (p ≤ 0.0001), and the interaction of the analyzed factors  was significant, except interaction of by-product quantity × treatment. No correlation was established between the dough pH and the TTA; the lowest TTA values of the dough samples prepared with CP and OP (fermented and ultrasonicated) were found (81.5% lower on average compared with the control dough). Dough samples with 5% fermented AP and with 15% and 20% ultrasonicated AP by-products had, on average, 17.3% higher TTA compared with the control dough. The dough TTA was significantly influenced by the by-product used (p ≤ 0.0001), and the interaction of the analyzed factors  was significant.The quality parameters  of dough, prepared with 5, 10, 15, and 20% of fermented and ultrasonicated AP, CP, and OP, are shown in ∗, lightness; a∗, redness; b∗, yellowness), in most of the cases, by-product addition increased the a∗ coordinate of the dough compared with the control dough, which was prepared without by-products. In compare all the groups, the highest a∗ of dough samples prepared with OP was found .In the comparison of the color coordinates of the doughs  and fermented CP , the hardness of the doughs was reduced . Conversely, by the addition of ultrasonicated CP additives at concentrations of 15% and 20%, the hardness of the doughs increased significantly (by 32.5% on average compared with the control).r = 0.7747). By increasing by-products content in bread formulations, moisture content of the breads was increased, and between breads TTA and moisture content moderate positive correlation was found (r = 0.4948). The TTA of the breads was increased, by increasing content of the fermented by-products in bread formulations. Also, by increasing bread specific volume and porosity, moisture content of the samples showed tendency to reduce, and negative moderate correlations between the bread specific volume and porosity with the moisture content were found . In compare bread samples mass loss after baking, 5% of the fermented and ultrasonicated AP, 5 and 10% of the ultrasonicated AP, and 15% of the ultrasonicated CP reduces breads mass loss after baking, compare with control breads, and between the moisture content and mass loss after baking negative moderate correlation was found (r = −0.4323). The highest overall acceptability of the breads, prepared with 20% of fermented AP, 15% of fermented OP, as well as 15% of ultrasonicated OP was established. However, it should be mentioned that the specific volume and porosity has a low influence on overall acceptability of the breads, as above mentioned parameters showed very weak correlation with overall acceptability . In all the cases OP (fermented and ultrasonicated) reduce bread shape coefficient, compare with control breads. Also, lower bread shape coefficient of the samples, prepared with 5 and 10% of fermented and 5% of ultrasonicated AP by-products, as well as with 5% of fermented and 15% of ultrasonicated CP was found. Finally, 20% of fermented AP, 15% of fermented OP, and 15% of ultrasonicated OP could increase overall acceptability of the wheat bread; however, further analyzed characteristics  are very important, as it can lead to future consumers choice of the proposed product.The quality parameters  of wheat bread prepared with fermented and ultrasonicated BPs are shown in r = 0.3399), however, with other analyzed bread parameters correlations with texture hardness were not established. Also, a weak correlation between the dough pH and bread texture hardness after 96 h of storage was found (r = 0.3849).The changes of wheat bread texture hardness during the storage are shown in ∗) of all breads prepared with the addition of by-products was found . In compare acrylamide concentration in wheat bread samples, in all the bread groups the same tendency was established that by the increasing the tested by-products concentration in wheat bread formula, acrylamide concentration was increased, and the highest acrylamide concentration in breads, prepared with the addition of fermented AP and CP was found . HoweveL. casei LUHS210 had a good microbial viability at pH 2.5 – the concentration of viable cells after 2 h incubation at pH 2.5 was 8.36 ± 0.2 (CFU/mL) (+) ion, and the amino acid becomes positively charged  or base (OH–) from solution. Moreover, the alkaline-producing activities also occur during fermentation. The increase in pH during fermentation could be related with the higher protein content in substrate, proteolytic activities, and the release of ammonia by microorganisms involved in fermentation. Protein content in OP was the highest  . The act(CFU/mL) . Differe(CFU/mL) . The pH (CFU/mL) . Also, p charged . If base highest  between  highest . During As the main acidic nature metabolites of lacto fermentation, L(+) and D(−)-lactic acid isomers were also evaluated in this study. L-(+)-lactic acid was produced in a significantly higher amount than D-(−)-lactic acid among all fermented by-products. L- and D-lactate dehydrogenases are responsible for the production of lactic acid optical isomers. L-(+)-lactic acid is also synthesized in the human body, while D-(−)-lactic acid is a toxic compound and may cause acidosis when accumulated in the body .Lacticaseibacillus casei group, the growth of pathogenic and opportunistic microorganisms could be limited in fermented substrates  produced by LAB, as well as species of the bstrates .When high intensity ultrasound is applied, the cavitation phenomenon increases cell membrane penetrability and induces the formation of free radicals in the cells of microorganisms . This leAfter summarizing the acidity and microbiological parameters of the fermented samples, for the wheat bread preparation, 12-h-fermented AP and OP samples  and 24-h-fermented CP samples  were selected.In general, the addition of different fermented plant drink preparation BPs with lower pH could reduce the dough pH and affect the solubility of the dough components and the activity of the endogenous enzymes . However∗ coordinate of the doughs could be related to the redness-brownness color of AP, CP, and OP. However, a significant influence of the by-products on the b∗ and L∗ coordinates of the doughs was not established. Changes in the texture of the wheat dough, supplemented with plant drink preparation by-products, could be related to the higher content of fat, non-gluten proteins, and dietary fibers in AP, OP, and CP, which have an impact on gluten dilution and increased water absorption , and the most acceptable breads were prepared with 20% fermented AP, 15% fermented OP, and 15% ultrasonicated OP. Also, the tested by-products influenced the bread staling process, and the lowest hardness (lower than that control bread) after 96 h of storage of the breads prepared with 5% fermented AP, CP, and OP  and with 5% ultrasonicated CP (1.2 mJ) was found. The addition of the selected by-products to the main wheat bread formula could reduce acrylamide formation; a significantly lower acrylamide content was found in breads prepared with 5% and 10% ultrasonicated AP, compared with the control breads. However, the lowest acrylamide content in bread prepared with OP was found (except for breads prepared with 15% and 20% ultrasonicated OP). Finally, 15% fermented OP can be safely used for wheat bread preparation because the prepared bread showed high OA, as well as lower acrylamide concentrations than the control bread.This study showed that ultrasonication at 37 kHz and fermentation are both effective for improving the biosafety of by-products. The tested by-products had a different influence on the dough and bread quality parameters; however, the specific volume and porosity of the breads showed a very weak correlation with overall acceptability (The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.EB planned the experiment, performed the statistical analyses, and wrote the original draft. VB, IP, AB, EZ, VL, VS, and PZ carried out the experiment and performed all analyses. DK wrote the original draft and revised the manuscript. DZ and GJ revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Creole animals are an important genetic resource due to their adaptation to adverse conditions, however, many of them have disappeared or have been reduced in number; therefore, their identification and preservation should be promoted. In the Mixteca zone of Oaxaca, México, there is a group of creole sheep with physical differences with respect to the other known breeds. The objective of the present study was to determine the degree of differentiation among the individuals of the population of creole sheep and the similarities with other breeds, as well as to measure their level of conservation and consanguinity. It was found that there is a group of sheep which is different from the presently known breeds and that they do not present high degree of consanguinity, and thus may be considered a new breed. Therefore, it is proposed that they are identified as the Chocholteca Creole breed, in honor of the ethnic group which inhabits this region. The present study is very important because it discovers a new genotype of sheep, which amplifies the genetic diversity. Therefore, further studies are needed, given that they are a potential alternative of meat and wool.locus and an effective number of 3.33. The intra-racial analysis showed a moderate genetic diversity with values of expected heterozygosity of 0.686 and observed of 0.756, a mean polymorphic information content of 0.609, and a mean coefficient of consanguinity of −0.002. In interracial genetic diversity for the coefficients of consanguinity, the values were FIS = 0.0774, FIT = 0.16993, and FST = 0.10028, showing an elevated genetic distance with other creole breeds, but close to Argentine Creole, to another Creole of México and the Spanish Merino. Its genetic structure showed that it does not have any populational subdivision nor mixes with the others analyzed. It is concluded that it is a distinct and isolated population and is proposed as the creole breed “Chocholteca” for its conservation.Creole sheep in México have undergone crossbreeding, provoking the loss of genetic variability. The objective of the present study is to determine the intra-racial genetic diversity, the genetic relationship with other genotypes, and the populational substructure of the Oaxacan Creole sheep. Twenty-nine blood samples were obtained of Creole sheep of the Oaxaca Mixteca region in México. A genetic analysis was made with 41 microsatellites recommended for studies of genetic diversity in sheep. An analysis was made of genetic diversity, populational structure, and genetic distance with 27 other sheep populations. The study found 205 alleles with a range of 2 to 9 by  Sheep farming is one of the principal agricultural activities in the state of Oaxaca, Mexico. The Mixteca region has the highest number of production units ,2, princThe High Mixteca zone, of the state of Oaxaca, inhabited by the Chocholteca ethnic group, still conserves a sheep population with no more than 100 animals that present morphological characteristics similar to those introduced over 500 years ago, as well as from other regions of México , which oAt present, one of the tools most widely used is the characterization of production animals through the employment of molecular markers, such as microsatellites, in order to determine their purity or degree of crossbreeding with other breeds, the genetic diversity within the same population, the level of endogamy, etc. ,12,13,14Twenty-nine creole wool sheep  of the High Mixteca, Oaxaca, México were included. The region is located in the coordinates 17°39′19” longitude west, 91°16′51” latitude north, at an altitude between 2100 and 2500 masl. The climate is humid temperate with rains in summer and an annual temperature of 14 to 20 °C . A samplOne-hundred microliters were taken from each sample and placed in filter paper with the individual dates. These were left to dry at room temperature and thus conserved until their analysis in the Applied Molecular Genetics Laboratory of the University of Córdoba, Spain. The DNA of each sample was obtained employing the Walsh technique , using t® 400 HD ROX size Standard”  to corroborate the sizes of the amplified microsatellites. The analysis of fragments and allelic typification were made using the programs Genescan Analysis® 3.1.2  and Genotyper® 2.5.2 (Fischer Scientific), respectively.In the analysis, 41 microsatellites were used , which have been utilized for the identification of other breeds of sheep and are recommended by the Food and Agriculture Organization and the International Society of Animal Genetics (FAO/ISAG). Each microsatellite was amplified by PCR and the obtained fragments were identified by means of 4-capillary electrophoresis in an automatic sequencer ABI 3130XL , using the marker “GenescanThe average number of alleles was determined per locus (MNA), the allelic frequency (Ae), the expected and observed heterozygosis (He), (Ho), along with polymorphic information contained (PIC) using the programs MICROSATELLITE TOOLKIT software of excel  and CERVhttps://biovis.jimdo.com/breeds/ accessed on 21 October 2019). The intellectual property of the data of these breeds belongs to investigators of the network CONBIAND (https://biovis.jimdo.com/investigadores/ accessed on 21 October 2019). The F statistics of Wright [IT), as well as the coefficient of genetic differentiation (FST) and the endogamy coefficient (FIS), and a Factorial Analysis of Correspondence was also made using the program GENETIX v 4.05 [A) [For the analysis of genetic differentiation of the Oaxacan Creole sheep, it was compared with 27 breeds , includif Wright  were cal4.05 [A)  in the p4.05 [A) . With th4.05 [A)  was madeFor the analysis of the genetic substructure among individuals of the Oaxacan Creole sheep, a Bayesian algorithm was made with the program STRUCTURE v 2.1  that useThe population analyzed of the Oaxacan Creole sheep presented a polymorphic character with respect to the allelic frequencies corresponding to the 41 microsatellites analyzed. A total of 205 alleles were found, with a minimum of two in the markers ETH010 and ILSTS008, and a maximum of nine in the marker OarCP49, showing a moderate allelic diversity in the population .In the analysis of genetic variability , the OaxThe values of Polymorphic Information Content (PIC) in the population presented 8 markers with a value less than 0.5 and 33 with a value above 0.5 considered very informative for the characterization of genetic variability. The marker ETH010 is the only uninformative marker showing a PIC of < 0.25.IS) had a range of −0.301 (BM6526) to 0.332 (SPS115) with a mean of −0.022 (−0.087–0.002), utilizing 10,000 re-samplings and with a confidence interval of 95%, manifesting that this population does not have a significant deviation of Hardy–Weinberg equilibrium. When the Bonferroni correction was made, it was found that the markers BM6506, BM6526, CD5, ETH010, INRA063, TGLA053, and TGLA122 have a significant defect (p < 0.05) of homozygotes with negative values of FIS, presenting a deviation of the Hardy–Weinberg equilibrium.The value for the frequency of the coefficient of consanguinity (FIS = 0.0774 (0.05353–0.10389), FIT = 0.16993 (0.14598–0.19770), and FST = 0.10028 (0.09159–0.10917). In the Factorial Correspondence Analysis  and the coefficients of genetic differentiation with respect to other populations (FST) , Africa , and the highest values with the creole of Brazil  and Palmera of Spain .Analyzing the genetic distance (Dns (FST) , the OaxA genetic distances through a neighbor-net graphic shows that the Oaxacan Creole sheep are grouped in the same branch as the Argentine Creole and share the same trunk of origin with the Creole of Mexico, Spanish Merino, and Chilean Merino  . This anWhen two populations are analyzed (K2) (African vs. Merino), the Oaxacan Creole is grouped with those that descend from the Merino. The analysis of K3 shows the difference from the Spanish Merino and those that carry a greater genetic load of their ancestors (dark blue). When utilizing 10 populations (K10), more than 85% of the individuals of the Oaxacan Creole were assigned to the same group (data not shown). When compared with 16 populations, it is separated as an independent group, even from the other Mexican creoles. In the analysis of 25 (K25)  and 28 (K28) populations, 86.8% of the individuals are assigned to the same group, where no subdivision or substructure of the population is observed. Therefore, it can be considered a homogeneous flock without a populational substructure or genetic mixing with the rest of the breeds used.Sheep breeding in Mexico, in order to satisfy the productive, economic, and social needs of the producers and the population, has opted for improving flocks through the introduction of improved breeds and has carried out crossbreeding with no established control . Howeverloci are used. Furthermore, it is considered that the optimum sample size is 30, thus this study has a high degree of reliability due to the number of individuals and markers employed.In the present study, 29 sheep of the Oaxacan Creoles population, corresponding to one-third of the entire population, were genetically characterized, utilizing 41 microsatellites recommended by the FAO/ISAG for sheep . These oAM) of 5 and an effective number (Ae) of 3.327, parameters which in conjunction with heterozygosity, which indicate the genetic variability within a population. The value of NAM is low compared with that of the sheep breeds of Colombia (14.27) [It was found that the population has a mean of alleles (N (14.27) , of Chin (14.27) , but it  (14.27) , to the  (14.27) , to Ital (14.27) . The val (14.27)  in 4 pop (14.27) .IS = 0.022, showing polymorphic values of moderate to high and a moderate genetic diversity [IS = 0.09) [IS = 0.026–0.076) [IS = 0.030 [IS = 0.074). Therefore, it can be concluded that the Oaxacan Creole is a population with a moderate intra-racial diversity without a significant deviation from the Hardy–Weinberg equilibrium, similar to other creole breeds of America and the Spanish Merino. Furthermore, the presence of markers with values of FIS very far from zero (reduction of heterozygosity) in the Oaxacan Creole sheep may be due to the low number of individuals in the population, which favors endogamy, added to the lack of planning of crosses between unrelated individuals.The heterozygosity in the Oaxacan Creole is considered from medium to high according to what was reported by Martínez , given tiversity , lower t = 0.09) , but sim = 0.030 , and theST) and the measurement of the genetic distance among them (DA) [Genetic diversity among populations can be analyzed using two variables, the statistic of the coefficient of genetic differentiation (Fhem (DA) . EstimatST−0.1003. This is similar to what was reported by Quiroz et al. [The Oaxacan Creole sheep, in the analysis of genetic differentiation with the 27 populations utilized, showed a mean value of Fz et al.  for the z et al. . Accordiz et al. , this vaz et al. , it is cz et al.  for Spanz et al. , and to z et al. .A genetic distances showed the separation of 4 groups: grouping the Oaxacan Creole with another creole of Mexico, the Chilean Merino, the Spanish Merino, and with the lowest DA, with the Argentinian creole. This analysis makes it possible to suggest that the Oaxacan Creole conserves certain ancestry to the breeds introduced during the colonization [A with the Spanish Merino and the Chilean Merino. On the other hand, it is grouped in the same phylogenic trunk as the Argentinian Creole and to the other creole of Mexico, suggesting the same origin, although with elevated genetic differences, and an origin different from that of the creole of Chiapas. This diversity or genetic differentiation of the Oaxacan Creole could be due to its geographic localization and to the management established by the producer, resulting in reproductive isolation, thus allowing the conservation of its genetic characteristics.The analysis of the genetic relationships among the 28 populations in a neighbor-net built with Dnization , and by This analysis provides information for estimating the proportion that each individual or population carries the genome of its parents or ancestors, making it possible to distinguish if there is a mixing of breeds or not in order to assign individuals or homogeneous populations. This analysis helped to confirm what was observed in the dendrogram of the union of neighbors, revealing that the Oaxacan Creole sheep is a homogeneous population, with the ancestral influence of the Merino; without a populational substructure or mixing of its genome with any of the 27 populations analyzed, even with the other populations of the country, thus separating them as an independent breed.Finally, the data obtained show that the sheep flock subjected to study in the region of the High Mixteca of Oaxaca is a creole breed different from those analyzed, maintaining its genetic merit. Therefore, its recognition is proposed as Chocholteca Creole breed for the zone in which it has been conserved and in honor of the ethnic group which inhabits this region. Furthermore, this recognition will help to promote its conservation and to implement populational recovery programs in conjunction with the producer and his cultural system.The Chocholteca Creole sheep has been maintained as an isolated breed, conserving genetic characteristics since it was introduced to America, and does not present features of crossbreeding with the new lines of improved sheep or other creoles of America, or breeds of Spain and Africa.This creole sheep has in its population a moderate–high genetic diversity, with a homogeneous structure, and does not present a subdivision or genetic substructure.It can be considered as a creole breed different from the others of the American continent and Spain, establishing itself as an important new zoogenic resource. Therefore, it is necessary to establish reproductive management for its conservation.


Cities are the innovation centers of the US economy, but technological disruptions can exclude workers and inhibit a middle class. Therefore, urban policy must promote the jobs and skills that increase worker pay, create employment, and foster economic resilience. In this paper, we model labor market resilience with an ecologically-inspired job network constructed from the similarity of occupations’ skill requirements. This framework reveals that the economic resilience of cities is universally and uniquely determined by the connectivity within a city’s job network. US cities with greater job connectivity experienced lower unemployment during the Great Recession. Further, cities that increase their job connectivity see increasing wage bills, and workers of embedded occupations enjoy higher wages than their peers elsewhere. Finally, we show how job connectivity may clarify the augmenting and deleterious impact of automation in US cities. Policies that promote labor connectivity may grow labor markets and promote economic resilience. Recent technological, social, and educational changes are profoundly impacting our work, but what makes labour markets resilient to those labour shocks? Here, the authors show that labour markets resemble ecological systems whose resilience depends critically on the network of skill similarities between different jobs. So what makes cities adaptable? This question remains open because the typical study of urban labor at equilibrium obfuscates responses to out-of-equilibrium disruptions. For example, most automation research relies on occupation-level estimates of technological exposure4. But, labor markets are heterogeneous systems in which skills5, jobs, geographies7, and sectors all interact8. Accordingly, models built on empirical skills data may better capture worker mobility and better identify the sources of urban adaptability through the interdependencies of workplace skills10. Analogous strategies predict resilience to shocks in ecological systems  from the density of mutualistic interdependencies between species—independent of population dynamics at equilibrium12. Similar network models have been applied in a variety of economic systems including global socioeconomic systems13, regional economies14, banking systems15, and interfirm worker mobility16 through an analogy connecting ecological mutualism to economic complementarity. In these studies, the interdependencies between cities, banks, and firms undergird systemic resilience to shocks even though dynamics in those systems are very different. Despite their success in other economic domains, studies of interdependencies between skills and occupations remain absent from urban labor studies on economic resilience.Like ecosystems12. Thus, we ignore the different equilibrium dynamics that govern a labor market or an ecosystem, and we apply analogous measures to occupation networks in US cities constructed from employment distributions and skills data provided by the US Department of Labor. While existing foundational studies have identified similar structures in urban labor markets20, we are not aware of any study empirically relating these economic structures to economic resilience following a labor shock. Thus, we expand this body of work by applying our theoretical framework to measure labor market resilience compared to the unemployment experienced across US cities following the Great Recession. Finally, having empirically validated our measure following an actual labor disruption, we apply our framework to understand the impact of another labor disruption: automation via computerization.How strong is the analogy between labor market resilience and the resilience of ecosystems? To study this question, we first expand existing labor theory using an analogy between the overlapping skill requirements of occupations within a labor market and the mutualistic interactions between species within an ecosystem. Since labor markets and ecosystems may be governed by different dynamics, how can ecological methods apply to labor market resilience? Recent studies demonstrate the prominent role of structure in determining ecological resilience independent of the ecosystem’s population-level dynamics21) lack the granularity required by this ecological view of labor. In general, the job matching function M describes the dynamics of employment, E, as a function of unemployed workers, U, filling job vacancies, V, according toλ represents the rate of job match dissolution. Employment is increased as job seekers fill job vacancies through the matching function22 between unemployed workers and available jobs; most research models this process as M ∝ UγVγ1− where γ ∈ . However, the abstract nature of this model obfuscates the frictions that limit workers’ career mobility23. In particular, skill mismatch21 is difficult to identify without refining the model with empirical skill interdependencies.Traditional labor market models  that respects frictions in the flow of workers between occupations i and j. To this end, recent studies using the O*NET database from the US Bureau of Labor Statistics (BLS) have revealed skill polarization26, the impact of automation in cities3, and demonstrated how similar skill requirements are predictive of worker transitions between occupations28.To describe career mobility between occupations, the model must be extended to treat each occupation i and j according to wij = 1 − ∣∣Oi − Oj∣∣2 where Oj denotes the skill vector of occupation j (see Methods to learn more about this skills database from the BLS). We normalize skill similarity scores so that wij ∈ . Our results throughout are robust to alternative skill similarity calculations . We assume that a worker’s skills are approximated by the skills required by their most recent employment  and 11We test this hypothesis by applying eq. mentclass2pt{minimmentclass2pt{minimThese effective variables capture the expected long-term dynamics in each city given our model and rely heavily on each city’s job network. In particular, λ), the variation in simulated long-term employment levels  followsentclass1pt{minimaentclass1pt{minimaλ clarifies this relationship and shows how cities can have different stationary solutions under the same exogenous forces , some cities, such as New York, NY, are able to resist changes to net employment, but other cities, such as Yuma, AZ, experience significant decreases in their fraction of employed workers according to simulations. The results in Fig. λ  and occupational diversity . Employment diversity is given by j’s fraction of employment in city c. Even after controlling for employment diversity and occupational diversity, job connectivity significantly improves predictions of peak unemployment rates during the Great Recession  using Local Area Employment Statistics see Fig. , Model 1Since job connectivity may promote economic resilience to labor disruptions, how can policy makers and individuals leverage connectivity and job network projections? One way is to compare the embeddedness . With Chicago, IL as an example  from automation. The gray points in Fig. 3. However, simulating long-term employment dynamics using eq. 34), this type of insight sheds light on the growing economic and labor disparity between large US cities and smaller rural areas35 and offers a potential pathway to forecast labor dynamics and resilience given the uncertain nature of future technologies.In general, these simulations offer a new tool to policy makers for distinguishing between potentially substitutive technological impact and augmentation. First, consistent with previous studies of automation and cities36 and identify specific policy instruments for promoting job connectivity and occupational embeddedness, and quantify the most impactful use of these policy options.As complex interdependent systems, urban labor markets are more than the sum of individual occupations or sectors. Specifically, this study maps the dependencies between occupations in urban labor markets based on occupational skill requirements and demonstrates how the topology of these connections between occupations relates economic resilience to job network connectivity and relates workers’ wages to occupational embeddedness. Job connectivity and occupational embeddedness are topological features that rely entirely on the job network in each city, and insights based on these features would not be calculable in the absence of the job network . Further, our analysis of job connectivity and occupation embeddedness is careful to control for potential confounders from traditional network-independent variables that might limit the salience of our conclusions  the New England cities and town areas (NECTA) included in the US Census. By definition the CBSAS or NECTA are geographical areas that consist of one of more counties  anchored by an urban center of at least 10,000 people plus adjacent counties that are socioeconomical tied to the urban center. Some of the cities/areas span over different states, like the ones in New York or Boston, for example. The BLS data contains information about the number of workers and wage estimates of 772 occupations and 422 cities. Occupations are described by the Standard Occupational Classification 6 digits38.Number of jobs and wage estimates by sector and city were obtained from the US BLS for the 2017 year39. The O*NET program is the “nation’s primary source of occupational information,” and is continually updated through surveys of various workers from each occupation in the Standard Occupation Classification (SOC) taxonomy. The O*NET database identifies the distinguishing characteristics of each occupation.The O*NET Database is a data product of the US BLSs has some real-valued importance to occupation j denoted by O ∈  such that O = 1 indicates that s is essential to j and O = 0 indicates that s is irrelevant to j. Letting S denote the set of 232 O*NET variables, we also use Oj = s∈S to represent the skill vector associated with occupation j.Every occupation requires a different mix of knowledge, skills, and abilities, and is performed using a variety of activities and tasks. In this study, we focus on the abilities, interests, knowledge, skills, work activities, work contexts, education, training, and experience O*NET variables to identify the importance of 232 different workplace skills for 775 different occupations. These O*NET variables result from a composition of surveys each conducted with varying Likert scales; thus, we normalize the responses from each survey so that each O*NET variable 40. Specifically we used the seasonally adjusted metropolitan area estimates by month.Unemployment rates were obtained from the US BLS Local Area Unemployment StatisticsEeff = 0 which indicates a systemic collapse where employment in that city has disappeared. However, the instability of this fixed point suggests it is difficult for real-world labor systems to realize this abysmal future. Rather, the second—and more preferable—fixed point describes the long-term fraction of workers with employment (denoted c’s job connectivity Equation  describeEeff and Ueff, we begin withTo derive this second fixed point associated with Supplementary Information