Sex differences in behavioral and neural characteristics can be caused by cultural influences but also by sex-based differences in neurophysiological and sensorimotor features. Since signal-response systems influence decision-making, cooperative and collaborative behaviors, the anatomical or physiological bases for any sex-based difference in sensory mechanisms are important to explore. Here, we use uniform scaling and nonparametric representations of the human cochlea, the main organ of hearing that imprints its adult-like morphology within the petrosal bone from birth. We observe a sex-differentiated torsion along the 3D cochlear curve in samples of 94 adults and 22 juvenile skeletons from cross-cultural contexts. The cochlear sexual dimorphism measured in our study allows sex assessment from the human skeleton with a mean accuracy ranging from 0.91 to 0.93 throughout life. We conclude that the human cochlea is sex-typed from an early post-natal age. This, for the first time, allows nondestructive sex determination of juveniles’ skeletal remains in which the biomolecules are too degraded for study but in which the petrosal is preserved, one of the most common bone within archaeological assemblages. Our observed sex-typed cochlear shape from birth is likely associated with complex evolutionary processes in modern humans for reasons not yet fully understood. Therefore, complex evolutionary processes likely fine-tuned sex-differentiated neurophysiological and sensorimotor features involved in signaling among humans6. For instance, the human voice and hearing sensitivity are known as secondary sexual features10 but with yet unknown possible translations into the morphology of the main organ of hearing, the cochlea. The anatomical basis for sex-based differences in human hearing are therefore important to explore for any biologist and behaviorist interested to track its evolution in the fossil record and, in particular, the origin of the human uniquely “receiver-centered” hearing system that tailors speech content to needs of listeners12. Here, we investigated the shape in three dimensions (3D) of the human cochlea housed in a spiral-shaped cavity within the petrosal part of the temporal. Indeed, from birth, the cochlea is the organ that makes a more crucial contribution than the middle ear in setting the hearing range13. The cochlea also imprints details of its overall adult-like structure within the petrosal, the hardest and most dense bone in the mammal body that is most often preserved in fossil assemblages. Since the inner ear is not notably influenced by postnatal growth and development15, both juveniles and adults can be sampled and compared directly. We used a total of 94 X-ray medical computed-tomographies (CTs) representing adult individuals of African and European descent sampled in France and South Africa. Additional cochlear data were also taken from micro-CTs of 22 dry skulls representing juveniles ranging in age from birth to 10 years .Signaling within a species influences decision-making, maximizes cooperative  and collaborative  behaviors involved in sexual or other forms of social selection18. This was likely caused by the fact that none of these studies18 attempted to measure the allometric effect of the human sex-differentiated body mass on cochlear features. Moreover, previous analyses of cochlear variations among human populations did not filter out the scale and rotational effects to investigate potential differences in pure shape, as defined in detail by Kendall (1984) in his “theory of shape”19. All previously proposed approaches to cochlear morphology used finite-dimensional spaces represented either by a vector of features21 or parametric functions  based on angles and lengths22, leading to strong restrictions when performing statistical studies. As a first step, in order to overcome the drawbacks of the previously proposed approaches of cochlear morphology, we used a uniform scaling, a nonlinear registration and a statistical framework with a nonparametric representation of the 3D cochlear curve. As a second step, we further tested the applicability of our results for sex determination by measuring the accuracy of a method based on our observed sex-differentiated cochlear shapes. Indeed, sex determination from the human juvenile skeleton is an absolute prerequisite in most forensic, bioarchaeological and palaeoanthropological contexts. In the absence of sufficient biomolecule (DNA or peptides) survival, especially when fossil specimens are too old or degraded, this task is cumbersome because of the low degree of prepubertal skeletal sexual dimorphism among humans23, a limiting factor for successful sex determination. In forensic medicine, a minimal and arbitrary value of 0.95 is usually expected24 for accuracy . While some morphological methods show a relatively high accuracy of sex determination on adult skeletons25, this is not the case for the many methods that have been proposed for application to juvenile skeletons, especially for females28. Although some slight sexual dimorphism has been observed from an early post-natal age among humans30, there is still an important uncertainty about the age at which the human skeleton  is quantifiably dimorphic. It is therefore well established that the juvenile skeleton cannot be sexed with any degree of accuracy from morphological observations alone32 without sex-typing from DNA33 or from peptides34. Indeed, any method for sex determination from the juvenile skeleton should be not only based on the description of purported sex-differentiated skeletal features but should also provide repeatable accuracy.Previous studies investigated human sexual dimorphism in cochlear size and gave contradictory results35. We then reduced the dimensionality of the 3D cochlear shape variables through unsupervised learning (clustering) and principal component analysis on the tangent space of shapes  (Methods). We observed a clear separation between the curves representing adult males and females along the first Principal component (PC1) only, independently of the country of origin (France or South Africa) or the continent of origin (Europe or Africa)  representing adult individuals and collected in France  and South Africa . We first tested the presence of sexual dimorphism in the 3D cochlear shapes in this sample of 94 adults by modeling mathematically the 3D cochlear shape by an open curve located along its outer periphery38, we computed the adult female and male Fréchet means for the observed cochlear curves sampled in this study  along the Fréchet means  locations along the normalized cochlear spiral curve, the most significant differences in torsion between the female and male Fréchet means were located between the 8th and 10th deciles and the highest sexual dimorphism appeared between the 8th and 9th deciles  for training while the remaining individuals (n = 54) were retained for sex assessment. In each trial, we computed the female and male Fréchet means. We learned the regression model from the training sample. We then applied it for sex assessment in the test sample. Overall, we obtained an accuracy of 0.93. When we considered the 20 trials of randomly selected text samples, the reliability ranged from 1 to 0.96  for males, and from 1 to 0.66  for females.Finally, in a fourth step, in order to further test our method and determine its applicability to juveniles, we carried out the sex determination of a new sample comprising 22 juvenile skeletons of known sex  ranging in age from birth to 10 years  that optimizes the deformation (transformation from one to another). We also visualize the shape means and covariances enabling a full statistical analysis. We observe that sex is the most dominant source of variation in 3D cochlear shape even when we consider separately each adult sample subset   and for which sex-typing from DNA or from peptides is not (yet) possible. In contrast, the possible absence of sex-typed cochlear shape in other species of great apes would suggest that social structure, mating systems and communication in these species did not affect the morphology of this organ during evolution. Moreover, in the absence of pre-existing knowledge , yet unknown patterns of variation in cochlear shapes within fossil hominin species could be still investigated. In this case, unsupervised learning techniques could use cochlear shapes directly measured in fossil hominin samples for sex assessment of particular specimens.Our approach investigates for the first time 3D cochlear curves directly by their real  shapes with two important advantages over previous methodsca) Fig. . Therefo30. Distinct parts of the human skeleton, especially the cranium, not only display differential degrees of sexual dimorphism at a given stage of growth and development, but also independent growth patterns in size and shape that may also vary with sex39. Little is known about the potential sexual dimorphism of the morphology of the human hearing system. Therefore, we focused our attention on the cochlea because it represents the only sensory system organ that imprints details of its overall adult-like structure within bone by term birth15. It is better amenable to evolutionary studies of communication systems for three main reasons. First, this organ acts as a frequency analyzer of incoming sounds into high and low frequencies along its spiral canal from basal to apical locations respectively40. Second, extensive studies in placental mammals (including primates) demonstrate the possibility to estimate hearing capacities for gross dimensions and shape of the cochlear cavity within bone41. Finally, the cochlear overall structure is also accessible in juvenile and adult fossil hominins42. Given the sexual differences in the human fundamental and resonance frequencies8, the unique human sexual dimorphism caused by a male disproportionate glottal-size43 , it was important to investigate whether these secondary sexual features translated into the morphology of the cochlea. Little emphasis has been laid on human sexually selected communication systems, their relationships with signal reliability (“honesty”) and sociocultural behaviors, particularly on how women and men perceive others in gendered societies through signals emitted to represent the sender’s characteristics46. In non-human primates, male vocal signals mediate mate choice48 and voice pitch is perceived as an indicator of mate quality49 while vocal attractiveness is correlated with both body and facial attractiveness in humans51. In multilingual and cross-cultural contexts, women find men with a more masculine voice more attractive49. Our results may suggest that sexual selection has played an as yet unrealized role in the evolution of a sex-differentiated human hearing system from birth.The ontogeny of the human skeletal sexual dimorphism in size, structure and shape is still the subject of ongoing debate52 and structure-function relationships within the cochlea should be considered with caution. When compared to other extant and extinct apes, humans have a longer cochlea than expected for their body mass, a proxy indicating a major auditory change that likely occurred in early representatives of the genus Homo approximately 2 million years ago20. This finding is consistent with the unique human sensitivity at low-frequencies53. If we posit a sender-receiver co-evolution, the hearing organ may reveal important evolutionary trends in the human sound communication system, including speech. Should our proposed evidence for a sex-differentiated cochlea among humans be associated with neurobiological and social underpinnings of our unique sexual dimorphism in the production and the reception of sounds, it would facilitate the understanding of key features of human communication systems.The differences highlighted in our study may induce sex-based hearing properties. However, cochlear mechanics in humans are still debatedOur sampling strategy aimed to investigate potential differences in 3D cochlear shapes caused by (i) sex, (ii) country of sampling (France or South Africa) and (iii) continent of origin (African and European descent) for the sample from South Africa only. For both samples (France and South Africa), all the patients were adults  and their identity was made anonymous to remain strictly confidential. Given the legal frameworks relating to medical data protection issues in France and in South Africa, no information about “ethnicity” was available for either samples. “Ethnicity” is a self-ascribed category by which individuals seek to assert their identity. It is here considered as highly imprecise, inconsistent and defined differently in different places and at different times. However, in South Africa, the public and private registrations require South Africans to indicate their “race”  by ticking one of four boxes . Since this information was available, we could consider the “continent of origin” (either Europe for “Whites” or Africa for “Blacks”) as a third additional stratification variable (in addition to sex and country of sampling) for the sample from South Africa only . We nevertheless considered this information with caution since it was self-ascribed by the patient. Moreover, the language(s) spoken by the patients were not known because the medical data were collected retrospectively. However, the sample from South Africa was collected from the Steve Biko Academic Hospital in Pretoria by one of us (AO), one of its faculty members at the time of sampling. This hospital is a public health and training institution that provides tertiary healthcare to the multicultural population living in Pretoria. It is therefore representative of the pattern of diverse linguistic groupings in this city. Indeed, the official languages in South Africa include English and Afrikaans (derived from Dutch) and nine Southern Bantu languages; Zulu being the most spoken as a first language, followed by Xhosa. Afrikaans and English are respectively the third and fourth most common first language in South Africa. South Africa, children usually learn in their mother-tongue at school for the first three years (a Southern Bantu language more than 60% of children). They then switch to either English or Afrikaans and continue with that language for the rest of their schooling career.https://www.cnil.fr/sites/default/files/atoms/files/mr-003.pdf). For this study, we complied with the reference methodology MR-003. In South Africa, we obtained ethical clearance in 2017 from the Research Ethics committee of the Faculty of Health Sciences, University of Pretoria. Ethics Reference No: 465/2017.All the steps of the present study were performed in accordance with relevant guidelines and regulations. No data used in this study involved experimentation, intervention, risk or constraint added by the research. For both samples (France and South Africa), the information regarding sex and continent of origin was tabulated and used for statistical purposes only. Our research was non-interventional and was based on anonymous medical data collected retrospectively. In France, the processing of personal data in the healthcare sector for research must be authorized by the National Commission of Informatics and Civil Liberties (CNIL). Moreover, since our data were only digital, anonymized and collected retrospectively, the approval of the committee for the protection of persons (a French ethics committee) was not necessary. In order to simplify the authorization procedure, the CNIL has adopted the reference methodology MR-003 for data collections of healthrelated data for non-interventional research purposes , and the Steve Biko Academic Hospital . For the sample from France, the CT scans were performed between 2012 and 2014, mainly to visualize the maxillary sinuses. For the sample from South Africa, the CT scans were performed in 2016. The pixel size ranged from 0.46 to 0.52 mm with a maximum slice thickness of 2 mm.Institut d’Anatomie Normale et Pathologique » of the University of Strasbourg. These skeletons correspond to individuals of known sex and age at death  and G Schwalbe (1844–1916) before 191854 (Rampont 1994). We obtained permissions to access these skeletons that have already been used in several published studies16. The micro-ct scans (pixel size of 0.041 mm) were obtained from the XtremeCT  system at the Institut de Médecine et de Physiologie Spatiales, Toulouse, France (http://www.medes.fr/).The juvenile sample is represented by micro-ct scans of crania curated in the « www.vsg3d.com/avizo) for the segmentation and the 3D reconstruction of the air-filled cochlea. We decided arbitrarily to only use the left cochlea and we assumed that sexual dimorphism also occurred on the right side. We used a threshold of 550–650 Hounsfield Units to extract the bone structures and for further reconstructions. The cochlear size and shape were modelled following a method previously used in Braga et al.20 by a series of landmarks placed at small intervals along the outer periphery of the cochlea, between the apex and the point marking the origin of the basal turn (where there is a saddle between the cochlear part and the vestibule) very close to the inferior margin of the round window. We subsequently analyzed the cochlear 3D shape by imposing appropriate geometric invariances including rigid motions, uniform scaling, and re-parametrization. Here, we point out that there is a difference between methods that perform registration based on features vectors and those that perform intrinsic statistical shape analysis. For both registration methods, the main goal is to establish correspondences between pairs of observations. Comparisons in a Euclidean space for methods based on features vectors lead to statistical analyses where the template is either selected as a predetermined observation from the sample, or constructed using Euclidean averages. In this case, a major drawback consists in ignoring the intrinsic complexity and nonlinearity caused by the structure of the data. The aim of the intrinsic shape analysis conducted in our study was to (i) achieve accurate registration with uniform scaling and correspondences between shapes, (ii) focus on the representation of the shape, (iii) analyze shape on a nonlinear manifold. Thus, the objectives of such intrinsic shape analysis encompassed correspondence-based registration methods while simultaneously integrating the tools for statistical analysis into a nonlinear registration framework.We first imported the CT scans into the Avizo software package  from the 94 adult cochlear curves sampled in this study. We evaluated the classification performance depending on the average estimates. We repeated these experiments 20 times to avoid a biased sampling (and we reported the mean and the standard deviation of these repeated experiments). In the Supplementary Figure Fig. , we giveThe dimensionality reduction of the population shape variables through tangent principal component analysis (TPCA) allowed local geometric shape analysis to characterize infinitesimal changes along the cochlear curve, but also highlighted global changes in shape covariates using the dominant modes of variation. The key advantage of this approach was that it allowed the use of 3D cochlear shape as a geometric phenotype in more sophisticated multivariate analyses. While the choice of TPCA for representing the tangent space was made for computational convenience, it is important to note that PCA yields a basis that maximizes the feature variance in the sample along each projection. The study of shape variance shows discriminatory rules that help in classifying groups. Additionally, even if the 3D shape analysis was performed locally on the curves, the principal projections captured global shape properties in the data, and hence helped to interpret appropriately the variability in 3D cochlear shapes. The application of our analysis to samples of 3D shapes demonstrated both global and local effects of sex for 3D cochlear shape variation.37 was then used to capture the characteristic of the 3D cochlear curves for a given sample. It was also used as a common reference template for registering the individual shapes to perform a group analysis. Instead of being a subject-specific template, the Fréchet mean represents a statistical template that minimizes the shape variance. The main advantage of this approach is that it also allowed us to explore the covariance structure of the 3D cochlear shapes in an intrinsic manner. All the computations were made using programs written in C/C++ and Matlab 2016a. We used a regression model on the shape space. It is known that these models depend on unknown re-parametrization functions (or domain deformations)55 which remain a challenging task. For instance, in a regression case where the shape is available as a finite number of observed data, we are interested in studying their re-parametrizations for describing the underlying shape of any newly observed input. For shape regression, each of the observed shapes comes with its own reparametrization. We were interested in computing the Fréchet mean of re-parametrization and in learning a Gaussian process on the space of cumulative distributions as a subspace of the unit sphere. Furthermore, we used a Bayesian inference for tuning the regression model, one among the most effective existing solutions particularly for regression and classification of shapes.The Fréchet mean Example of misclassified males and females during one trial.


Staphylococcus aureus and Bacillus subtilis) and two Gram-negative bacteria . The best activity was displayed by compound 19 against E. coli with an inhibition zone of 16.0 ± 0.82 mm and 14.67 ± 0.94 mm at 500 and 250 μg/mL, respectively. This is close to ciprofloxacin which is used as a positive control. The results of in silico molecular docking evaluation of the compounds against E. coli DNA gyraseB were in good agreement with the in vitro antibacterial analysis. Compounds 19  and 24  showed the maximum binding affinity close to ciprofloxacin  used as positive control. Therefore, the antibacterial activity displayed by these compounds is encouraging for further investigation to improve the activities of quinoline-stilbenes by incorporating various bioisosteric groups in one or more positions of the phenyl nuclei for their potential pharmacological use. Findings of the DPPH radical scavenging assay indicated that some of the quinolone stilbenes and pinacol possess moderate antioxidant properties compared to ascorbic acid used as a natural antioxidant.Emergence of antimicrobial resistance to standard commercial drugs has become a critical public health concern worldwide. Hence, novel antimicrobials with improved biological activities are urgently needed. In this regard, a series of quinoline-stilbene derivatives were synthesized from substituted quinoline and benzyltriphenylphosphonium chloride using Wittig reaction. Furthermore, a novel pinacol of quinoline was synthesized by pinacolinazation of 2-methoxyquinoline-3-carbaldehyde which was achieved by aluminum powder-potassium hydroxide reagent combination at ambient temperature in methanol. The structures of the synthesized compounds were established based on their spectral data. The antibacterial activities of the synthesized compounds were evaluated in vitro by the paper disc diffusion method against two Gram-positive bacteria ( Staphylococcus aureus (MRSA), vancomycin- resistant Staphylococcus aureus (VRSA), vancomycin-resistant enterococcus (VRE), and fluconazol-resistant Candida have become a serious medical problem worldwide . Re. Retranstilbenes . StilbenSeveral studies have shown that the introduction of different functional groups into quinolines and stilbenes can improve their biological activities. Thus, due to their useful applications in various fields, scientists pay much attention to the modification of quinolines and stilbenes. Especially noteworthy in this regard is the emergence of new drugs with hybrid molecular architectures possessing better pharmacological properties than small molecules. Hence, in the present work, the synthesis of quinoline-stilbene conjugates and pinacols of quinoline was reported. The result of the synthesis of novel pinacols of quinoline was also reported here for the first time. Herein, we also report the results of the antibacterial and radical scavenging activities of synthetic derivatives of quinoline-stilbenes and pinacols of quinoline. It is rational to anticipate better bioactivity from a bigger scaffold developed by conjugating the phenyl ring to the quinoline nucleus which has wider sites for suspending different bioisosteres.3 as a solvent, and chemical shifts (δ) were reported in ppm and the coupling constants (J) are reported in Hertz. The infrared spectra of the compounds were recorded using KBr pellets on a PerkinElmer BX IR Spectrometer (400–4000 cm−1) at Addis Ababa University. UV-Vis spectra were determined using a double beam UV-VIS Spectrophotometer (SM-1600 Spectrophotometer)) using methanol as a solvent. Analytical thin-layer chromatography was conducted on a 0.2 mm-thick layer of silicagel GF254 (Merck) on an aluminum plate, and the spots were visualized with 254 nm and 366 nm wavelength UV light. Silica gel gravity column chromatography was carried out using 100-mesh silica gel.All solvents and organic reagents were purchased from LOVA CHEMIE PVT LTD. Melting points were determined using capillary tubes with the Japson analytical melting point apparatus and are uncorrected. The NMR spectra of the compounds were obtained using the NMR Bruker Avance 400 spectrometer operating at 400 MHz using DMSO-d6 and CDCl2-Chloroquinoline-3-carbaldehyde and 2-chloro-8-methylquinoline-3-carbaldehyde were prepared according to the literature report . The var1The structures of the synthetic compounds synthesized by the procedure have been established using spectroscopic methods including UV-Vis, FT-IR, and NMR, and the result is summarized below.Rf = 0.22 (n-hexane : EtOAc = 9 : 1). UV-Vis (MeOH) λmax = 280 nm; IR : 3035 (CH-arom.), 1693 , 1621 (quinoline C=N str.), and 599 (aromatic C=C str.); 1H NMR : δH 7.65 , 7.88 , 7.97 , 8.07 , 8.74 , and 10.54 ; 13C NMR : δC 126.3 (C-1), 126.5 (C-8), 128.2 (C-6), 128.6 (C-4a), 129.7 (C-5), 133.6 (C-7), 140.3 (C-4), 149.6 (C-8a), 150.1 (C-2), and 189.1 (C-9).The crude product of 4 was purified by recrystallization from ethyl acetate. It was yellow crystal; mp 146–148°C; yield 49%; Rf = 0.63 (n-hexane : EtOAc = 9 : 1). UV-Vis λmax (MeOH) = 375 nm; IR : 3035 (CH-arom.), 1635 , 1621 (quinoline C=N str.), and 599 : δH 6.72 , 6.91 , 7.16 , 7.44 , 7.56 , 7.66 , 7.78 , 7.94 , and 8.14 ; 13C NMR : δC 125.7 (C-9), 127.2 (C-4), 127.4 (C-5), 127.9 (C-6), 128.0 (C-3), 128.3 (C-4′), 128.4 (C-8), 129.0 , 129.1 , 131.2 (C-10), 133.7 (C-4), 135.9 (C-7), 138.9 (C-11), 146.6 (C-2), and 149.9 (C-8a).The crude product of 5 was purified by silica gel column chromatography using n-hexane : acetate (9 : 1) as an eluent. The yield was 35.5%, a white powder; mp 58–60°C; n-hexane : ethyl acetate (8 : 1) as an eluent. The yield was 40.5%, a dull yellow powder; mp 178–180°C; Rf = 0.29 (n-hexane : EtOAc = 6 : 1). UV-Vis λmax (MeOH) = 400 nm; IR : 3429 (N-H str.), 3023 (aromatic C-H), 1673 , 1610 (quinoline C=N str.), and 1558 and 1465 (aromatic C=C str.); 1H NMR : δH 6.56 , 6.75 , 7.09 , 7.23 , 7.32 , 7.44 , 7.59 . 7.66 , and 11.97 ; 13C NMR : δC 115.3 (C-8), 119.3 (C-4a), 122.4 (C-3), 125.4 (C-4), 127.0 (C-6), 127.9 (C-9), 128.9 , 129.0 , 129.3 (C-4′), 130.6 (C-7), 132.2 (C-5), 136.9 (C-1′), 137.5 (C-10), 138.7 (C-8a), and 161.8 (C-2).The crude product of 7 was purified by silica gel column chromatography using Rf = 0.32 (n-hexane : EtOAc = 9 : 1). UV-Vis λmax (MeOH) = 295 nm; IR : 3065.4 (aromatic C-H str.), 2919.3 , 2847 , 1673 , 1620 (quinoline C=N str.), and 1599 and 1579 (aromatic C=C str.); 1H NMR : δH 4.22 , 7.45 , 7.76 , 7.85 , 8.60 , and 10.49 ; 13C NMR : δC 55.9 (C-10), 120.0 (C-3), 124.4 (C-6), 125.1 (C-4a), 127.1 (C-8), 129.8 (C-5), 132.6 (C-7), 140.0 (C-3), 149.0 (C-8a), 161.2 (C-2), and 189.4 (C-9).Compound 8 was a gray powder; the yield was 90%; mp 106–108°C; n-hexane : ethylacetate (9 : 1) as an eluent. The yield was 28.3%; it was a gray powder; mp 138–140°C; Rf = 0.52 (n-hexane : EtOAc = 9 : 1). UV-Vis λmax (MeOH) = 322 nm; IR : 3065.4 (aromatic C-H str.), 2919.3 , 2847 , 1630 , 1620 (quinoline C=N str.), and 1599 and 1579 (aromatic C=C str.); 1H NMR : δH 3.97 , 6.65 , 6.78 , 7.21 , 7.34 , 7.42 , 7.61 , 7.78 , and 7.91 ; 13C NMR : δC 54.1 (-OCH3), 122.1 (C-3), 124.6 (C-4a), 124.7 (C-9), 126.9 (C-2′), 127.1 (C-8), 127.9 (C-5), 128.0 (C-6′), 128.8 , 129.29 (C-4′), 130.0 (C-10), 132.7 (C-4), 136.7 (C-7), 137.7 (C-1′), 145.6 (C-8a), and 160.2 (C-2).The crude product of 9 was purified by silica gel column chromatography using Rf = 0.25 (n-hexane : Methanol = 7 : 3). UV-Vis λmax (MeOH) = 390 nm; IR : 3525–3510 (O-H and N-H), 3065.4 (aromatic C-H str.), 2919.3 , 2847 , 1623 (imine C=N str.), 1620 (quinoline C=N str.), and 1599 and 1579 (aromatic C=C str.) 1H NMR : δH 3.65 , 4.72 , 4.92 , 7.19 , 7.55 , 7.72 , 8.21 , 8.5 , and 9.55 ; 13C NMR : δC 43.4 (C-14), 60.5 (C-12), 61.2 (C-15), 63.7 (C-11), 117.2 (C-3), 121.9 (C-8), 122.4 (C-4a), 125.7 (C-5), 128.9 (C-6), 131.5 (C-7), 143.0 (C-4), 148.3 (C-8a), 155.4 (C-2), and 163.8 (C-9); Dept-135 δC 43.4 (C-14 down), 60.5 (C-12 down), 61.2 (C-15 down), 63.7 ( C-11 down), 121.9 (C-8), 125.7 (C-5), 128.9 (C-6), 131.5 (C-7), 143.0 (C-4), and 163.8 (C-9).Compound 10 was a yellow powder, and the overall yield was 84%; mp 80–82°C; Rf = 0.30 (n-hexane : EtOAc = 9 : 1). UV-Vis λmax (MeOH) = 295 nm; IR  3223, 3025 (arom-C-H.), 2956 , 2837 , 1683.3 (C=O str.), 1621 (quinoline C=N str.), and 1579 (aromatic C=C str.); 1H NMR : δH 2.80 , 7.53 , 7.70 , 7.78 , 8.70 , and 10.6 ; 13C NMR : δC 17.8 (C-10), 126.0 (C-3), 126.5 (C-6), 127.5 (C-5), 127.8 (C-4a), 133.6 (C-7), 136.9 (C-8), 140.4 (C-4), 148.7 (C-2), 149.4 (C-8a), and 189.5 (C-1).The crude product 14 was recrystallized from EtOAc. Yield after recrystallization was 38.75%, pale yellow crystal; mp 139 140°C; n-hexane : ethyl acetate 9 : 1). The yield was a yellow gum; Rf = 0.7 (n-hexane : EtOAc = 9 : 1). UV-Vis λmax (MeOH) = 375 nm; IR  3025 (arom-C-H.), 2956 , 2837 , 1630 , 1621 (quinoline C=N str.), and 1579 (aromatic C=C str.); 1H NMR : δH 7.36 , 7.44 , 7.52 , 7.61 , 7.66 , 7.85 , and 7 and 8.78 ; 13C NMR : δC 17.5 (C-11), 123.1 (C-6), 126.4 (C-6), 126.4 (C-9), 127.4 , 127.7 (C-9), 127.8 (C-4′), 128.1 (C-6′), 129.4 , 129.7 (C-4a), 131.1 (C-2′), 133.9 (C-7), 135.4 (C-4), 135.8 (C-8), 136.8 (C-1′), 145.7 (C-2), and 148.7 (C-8a).The crude product of 14 was purified by silica gel column chromatography (solvent Rf = 0.41 (n-hexane : EtOAc = 1 : 1). UV-Vis λmax (MeOH) = 375 nm, IR  3429 (O-H str.), 3179.5 (NH str.), 3023 (aromatic C-H), 2919.8 , 2837.2  1673 (C=O str.), 1610 (quinoline C=N str.), and 1558 and 1465 (aromatic C=C str.); 1H NMR : δH 2.56 , 7.23 , 7.51 , 7.61 , 8.49 , and 10.48 ; 13C NMR : δC 16.7 (C-10), 117.9 (C-6), 123.3 , 129.1 , 135.0 (C-5), 139.1 (C-8a), 144.2 (C-4), 162.7 (C-2), 190.00 (C-9).Compound 15 was yellow powder; mp 176–178°C; 16 was purified by column chromatography with solvent n-hexane: ethylacetate (8 : 1). The pure product was a dull yellow powder; mp 60–62°C; Rf = 0.38 (n-hexane : EtOAc = 6 : 1).The crude product of λmax (MeOH) = 375 nm; IR : 3429 (N-H str.), 3023 (aromatic C-H), 2919.8 , 2837.2 , 1630 , 1610 (quinoline C=N str.), and 1558 and 1465 (aromatic C=C str.); 1H NMR : δH 2.56 , 7.11 , 7.24–7.42 , 7.69 , 8.23 , and 11.10 ; 13C NMR : δC 17.7 (C-11), 119.9 (C-8), 122.37 (C-6), 123.4 (C-4), 123.6 (C-5), 123.7 (C-9), 126.5 (C-4′), 127.0 , 128.3 (C-4a), 128.4 , 131.3 (C-7), 133.7 (C-3), 136.1 (C-10), 137.7 (C-1′), 136.7 (C-8a), and 162.1 (C-2).UV-Vis n-hexane : ethylacetate (7 : 1). The pure product was a pink powder; mp 150–152°C; Rf = 0.61 (n-hexane : EtOAc = 7 : 3). UV-Vis λmax (MeOH) 315 nm, 319 nm; IR : 3055 (aromatic C-H str.), 1620 , 1615 (quinoline C=N str.), 1577 (aromatic C=C str.), and 1512 and 1340 (O=N-O str.); 1H NMR : δH 2.36 , 6.78 , 6.88 , 7-21-7.78 , 8.14 , 8.26 , 8.33 , and 8.72 , 13C NMR : δC 17.4 (-CH3), 121.1 (C-9), 122.3 , 122.7 (C-6), 123.6 (C-5), 127.3 , 128.1 (C-14), 128.8 , 129.3 , 130.5 (C-10), 133.8 (C-7), 135.0 , 136.9 , 137.1 (C-20), 140.0 (C-4), 143.6 (C-17), 157.2 (C-2), and 159.2 (C-8a), and peaks due to their stereoisomers were also seen.The crude product of 19 was purified by silica gel column chromatography using Rf = 0.42 (n-hexane : EtOAc = 7 : 5). UV-Vis λmax (MeOH); IR  3281.8 (N-H str), 3055 (aromatic C-H str.), 1645.7 (NHC=O) 1620 , 1615 (quinoline C=N str.), and 1577 (aromatic C=C str.); 1H NMR : δH 2.3 , 2.33 , 6.81 , 6.89 , 7.08 , 7.25 , 7.42 , 7.63 , 7.68 , 8.06 , and 8.66 (NH); 13C NMR : δC 17.4 (C-25), 24.4 (C-24), 120.0 , 122.3 , 124.2 (C-6), 125.1 (C-5), 125.5 , 127.2 (C-9), 128.0 (C-28), 128.9 , 129.3 (C-13), 130.3 (C-15), 133.3 (C-7), 134.7 (C-20) 136.4 (C-8), 137.0 (C-11), 139.2 (C-10), 143.9 (C-17), 148.9 (C-8a), 158.6 (C-23), and 168.6 (C-2).The crude product 22 was purified by silica gel column chromatography with n-hexane : ethyl acetate (7 : 4) as eluent. mp 178–180°C; Rf = 0.4 (n-hexane : EtOAc = 7 : 3); UV-Vis λmax (MeOH) 325 nm; IR : 3470 , 3013 (aromatic C-H str.), 2930.9 , 1625 (quinoline C=N str.), 1620 , and 1589.6 and 1475.5 (aromatic C=C str.); 1H NMR : δH 2.65 , 3.99 , 5.20 , 5.25 , 7.31 , 7.51 , 7.72 , and 8.28 ; 13C NMR : δC 17.3 (C-10), 52.9 (C-11), 69.2 (C-9), 117.2 (C-6), 124.79 (C-3), 125.33 (C-5), 127.1 (C-4a), 128.9 (C-7), 133.8 (C-8), 136.7 (C-4), 143.6 (C-8a), and 158.26 (C-2).Compound 24 was purified by silica gel column chromatography using solvent n-hexane : ethyl acetate (8 : 1). The final product was a white powder; mp 202–204°C; Staphylococcus aureus (ATCC25923) and Bacillus subtilis (ATCC6633)), and two Gram-negative bacteria  and Salmonella typhimurium (ATCC 13311)) were obtained from the Adama Public Health Research & Referral Laboratory Center to evaluate the antibacterial activity of the synthetic compounds. The identity of the bacterial strains were recognized and confirmed by morphology of colony and Gram staining and by standard biochemical tests following the methods of Bergey's Manual of Determinative Bacteriology (1994) [μg/mL. Sterile filter paper discs of 6 mm diameter were soaked in 1 mL DMSO solution of the compounds at 250 and 500 μg/mL concentrations. Then, the saturated paper discs were placed on the centre of each MHA plate. Ciprofloxacin was the standard drug used as positive control, and DMSO was used as negative control. The plates were then inverted and incubated for 24 hours at 37°C, and the zone of inhibition was recorded. The results were expressed as the mean of three measurements . The DPPH radical scavenging rate of each sample was calculated as follows [Ao is the absorbance of the control reaction and A1 is the absorbance in the presence of the test or standard sample.DPPH radical scavenging activity was used to evaluate the antioxidant activity of the synthetic compounds and compared with ascorbic acid . All syn follows , 22:(1)%The control DPPH solution was prepared by mixing 2 mL 0.004% DPPH with 2 mL methanol to afford 0.002% DPPH methanolic solution. The results were presented in percent radical scavenging activity , and theE. coli gyrase A and synthetic compounds in a 3D fashion, the compounds were docked within the binding site of the protein. AutoDock Vina with our previously reported protocol was used to dock the proteins  and compounds (5–24) into the active site of proteins [E. coli DNA gyrase A (PDB ID 1ZI0) and S. aureus Gyrase complex with ciprofloxacin and DNA (PDB ID: 2XCT) was downloaded from protein data bank. The protein preparation was carried out using the reported [To investigate the mode of interaction between the proteins , 23. Theproteins , 23. Thepounds 5– into thepounds 5– into thehttp://www.graphpad.com). Groups were analyzed for significant differences using a linear model of variance analysis (ANOVA) test for comparisons, with significance accepted for p < 0.05.Experiments were conducted in triplicates. The data presented are mean ± SD of the three independent experiments. GraphPad Prism version 5.00 for Windows was used to perform the Analysis  and 2-methylacetanilide (11) were synthesized by acetylation of aniline and o-toluidine in acetic anhydride-acetic acid mixtures (3) and 2-chloro-8-methylquinoline-3-carbaldehyde (13) were developed by the application of Vilsmeier–Haack reaction  as a solvent and K2CO3 as a base. The latter was selected due to its weak basicity and poor nucleophilic character which does not interfere with nucleophiles in most nucleophilic substitution reactions. The attractive features of DMF including high dielectric constant, its aprotic nature, wide liquid range, low volatility, dissolving all reactants, and supplying sufficient activation energy for the reactions allow us to use this reagent as a solvent. Furthermore, it is also miscible in water which eases the isolation of the desired product by adding the reaction mixture into cold ice water which causes the water-insoluble product to precipitate out [The hybridization of biologically active molecules is a nice tool for drug development used to treat a variety of diseases. It provides a means to improve the bioactivity of bioactive molecules through synergetic effect. Hybrid drugs can provide combination therapies in a single multifunctional agent , 24, 25.mixtures  and 2. Treaction  and 2 us3 in DMF , 24. Subtate out . Compouncis isomers which were confirmed by the coupling constant of the olefinic double bonds in the stilbenes. This agreed very well with previous products of Wittig reactions carried out at ambient temperature in DMF/KOH which gives the cis-stilbenes in good yield [cis and trans stereoisomers. A further attempt to purify 19 using silica gel column chromatography was unsuccessful since the spots of the two stereoisomers overlapped in its TLC profile. Pinacolization of carbonyls has been conducted by using a number of reagents such as Mg-MgI2 [2, transition metals, actinides, and lanthanides [II and TiIII reagents have also received considerable attention, although olefination is a competing reaction with these reagents [After securing crucial intermediates 3 and 13, attention was given to the synthesis of a series of stilbenes quinolines and their substituted analogs using Wittig reaction. In this regard, the desired products were made by reacting derivatives of compounds 3 and 13 with benzyltriphenylphosphonium chloride using DMF as a solvent and KOH as a base. The products produced are mainly od yield . However Mg-MgI2 , Zn-ZnC1thanides . TiII anreagents . Among treagents –28. In treagents . Methanoreagents  (Scheme S.aureus (ATCC25923), B.subtilis (ATCC6633), E. coli , and S. typhimurium (ATCC 13311)) using the paper disc diffusion method. The results of this evaluation are presented in Tables E. coli varies from 8.0 ± 0.82 to 16.0 ± 0.82 mm, whereas for standard drug (ciprofloxacin) at the same concentration, it was 18.67 ± 0.47 diameter. Compounds 14 (with a mean inhibition zone of 12.67 ± 0.82 mm) and 19 (with a mean inhibition zone of 16.0 ± 0.82 mm) were the ones with maximum activity against E. coli.Quinolines and stilbenes and their analogs have been associated with various biological activities. Compounds containing the quinoline moiety have shown good amoebicidal, bactericidal, fungicidal, and antimalarial activities , 32. ExpZ-factor is a reflective of both the assay signal dynamic range and the data variation associated with signal measurements and, therefore, is suitable for assay quality assessment [Z-factor provides a useful tool for comparison and evaluation of the quality of assay and can be utilized in assay optimization and validation [Z-factor is defined in terms of four parameters: the means (μ) and standard deviations (σ) of the sample and the positive and negative controls as shown below [σs and σc are the standard deviation of the sample and control and μs and μc are the mean of the sample and control.sessment . Z-factolidation . The Z-fwn below , 31.(2)ZZ′-factor can be calculated using only control data as follows:σc+ and σc− are the standard deviation of the positive and negative and μc+ and μc− are the mean of the positive and negative control, respectively.A Z-factor of the synthetic compounds and standards was calculated, and the results are presented in Z′-factor for the controls ranges from 0.82 to 0.92, a reflective of excellent quality assay. It was also found out that the Z-factor for six samples was greater than 0.5, while for another six samples, the Z-value was between 0.0 to 0.5. For four and two samples, the values were between −0.5 to 0 and between −0.5 to −1.0, respectively. Hence, the data obtained showed a good overall assay.The S. typhimurium. Within the series, compound 16 with a mean inhibition zone of 9.67 ± 0.47 mm diameter was the maximum while a 13.56 ± 0.82 mm mean inhibition zone was observed for the standard drug at the same concentration. In addition, 24 showed the strongest activity against S. aureus with a 16.0 ± 1.63 mm mean inhibition zone compared to 11.67 ± 0.47 mm for the standard drug. However, none of the compounds showed activity against B. subtilis. Overall, among the compounds reported herein, only five of them displayed potent bioactivity.Compounds 5, 16, and 24 have moderate activity against ortho position relative to the amino group) to afford sufficient yields. However, most important bioisosters such as F, Cl, carbonyl, carboxylic acid, and sulfonyl group are electron withdrawers and deactivators and cannot be incorporated in the quinoline nucleus. But, the current hybrid scaffold overcomes this limitation since the phenyl nucleus can bear various types of functional groups (electron withdrawer or donors) in one or more of the five positions. Thus, the current strategy provides a manageable way to optimize the bioactivity through structural modification. Owing to Wittig reaction tolerance of various functional groups, phenyl ring can comprise one or more bioisosters which would be used to improve the bioactivity of quinoline-stilbenes. Regarding compound 24, which was generated by pinacolization of 2-chloro-8-methylquinoline-3-carbaldehyde, a huge improvement in the bacterial activity was observed relative to the monoalcohol  methanol of the same reactant. Previously, we reported [E. coli (with mean inhibition zone of 9.33 ± 0.89 mm diameter), whereas the correspondent diol (24) was active against both S. aureus (with a mean inhibition zone of 16.0 ± 1.63 mm diameter) and S. typhimurium (with a mean inhibition zone of 8.67 ± 0.47 mm diameter), respectively. Furthermore, the study revealed that there is an opportunity of developing stronger bioactive molecules by pinacolization of quionoline-3-carbaldehyde derivatives bearing various bioisosteric substituents.In the current synthetic compound, the phenyl nucleus did not bear any substituents and the substituents in the quinoline nucleus were limited to the second and eight position. As a limitation, quinoline analogs synthesized by application of Vilsmeier–Hack reaction require activated aniline derivatives  used as a natural antioxidant. This indicates that the activities shown by the synthetic compounds are moderate in antioxidant activity. This is in good agreement with the literature reported for closely related compounds [Most of the synthetic quinolone stilbenes and their analogs possess moderate antioxidant activity relative to ascorbic acid . Compounompounds , 33.Z-factors for the radical scavenging activity of the synthetic compounds lie between 0.88 and 0.99 . The compounds (5–24) were displayed a minimum binding energy ranging from −5.6 to −7.1 kcal/mol , 14 , and 22  have shown hydrogen bond interaction with different amino-acid residues. The in silico interaction results are agreeing with in vitro antimicrobial analysis of the synthesized compounds against E.coli.DNA gyrase is an enzyme belonging to a member of bacterial topoisomerase which controls the topology of DNA –37. Henckcal/mol , with thE.coli, and among them, compound 24 revealed better activity [5 and 9 do not show any hydrogen bond interaction with any amino-acid residues within the active site. Compounds 5, 7, 9, 10, and 22 docking results were partially matching the clinical drug ciprofloxacin interactions with amino-acid residues. Based on the in silico molecular docking analysis result, compound 24  showed the highest binding affinity compared to ciprofloxacin . Therefore, compound 24 might be a better antibacterial agent than the other synthesized compounds reported herein. The binding affinity, H-bond, and residual interaction of nine compounds are summarized in E.coli DNA gyrase A are depicted in Figures Compounds 14 , 16 , 19 , and 24  showed good activities against activity . CompounE. coli with an inhibition zone of 16.0 ± 0.82 mm and 14.67 ± 0.94 mm at 500 and 250 μg/mL, respectively. This is close to ciprofloxacin which is used as a positive control. The results of in silico molecular docking evaluation of the compounds against E. coli DNA gyrase A were also in good agreement with in vitro antibacterial analysis. Two of the compounds, 19 and 24, exhibited the highest binding affinity comparable to ciprofloxacin. Some of these compounds  possess potent antibacterial properties. The antioxidant properties of these compounds revealed that they were active only to a half extent of ascorbic acid at the same concentration. Finally, we recommended that both the antibacterial and antioxidant activities can be further optimized by incorporating active bioisostere groups on the phenyl ring of the hybrid scaffold.In conclusion, quinolone-stilbenes-merged hybrid molecules were reported by utilizing Wittig reaction between 2-chloroquinoline-3-carbaldehyde derivatives and benzyltriphenylphosphonium chloride. The 2-chloroquinoline-3- carbldehyde derivatives were synthesized by Vilsmeier–Haack reaction, and the chlorine atom was replaced by various nucleophiles via a nucleophilic substitution reaction. A novel pinacol of quinoline was also synthesized by pinacolization of 2-methoxyquinoline-3-carbaldehyde with Al/KOH reagent system. The antibacterial activities of the compounds were evaluated with paper-disc diffusion against two Gram-negative and two Gram- positive bacterial strains with the best activity displayed by compound 19 against


The scientific assessment of regional ecosystem service value (ESV) is helpful in developing scientific ecological protection plans and compensation policies. However, an ESV evaluation method that can adapt to the complex and diverse characteristics of the ecological environment has not been established. This study takes Gansu Province in China as an example, fully considering the regional differences in ecosystem service function. Five correction indices for the value equivalent factor per unit area were constructed on a provincial scale, and a regional difference adjustment index for 11 categories of ecosystem services was constructed on a regional scale. In this way, a value evaluation model based on regional differences was established. The results show that in 2015, the total ESV reached 2,239.56 billion yuan in Gansu Province, with ESV gradually increasing from the northeast to the southwest, and the high-value areas of service function being located in Qilian and Longnan Mountains. The forest and grassland ecosystems contributed the most to the ESV. From the perspective of value composition, local climate regulation and biodiversity maintenance functions are the main service functions of Gansu Province. From 2000 to 2015, ESV increased by 3.43 billion yuan in the province. The value of forest and urban ecosystems continued to increase, whereas the value of cultivated land ecosystem continued to decrease. In terms of spatial characteristics of the service value change, the area that experienced value reduction gradually moved from the central part of Gansu Province to the surrounding areas. The evaluation method proposed in this paper provides a relatively comprehensive evaluation scheme for the spatiotemporal dynamic evaluation of ESV in complex ecological environments. Ecosystems not only provide various raw materials or products directly for human survival, but also have other functions such as regulating climate, reducing pollution, conserving water sources, maintaining soil quality, preventing wind and sand erosion, reducing disasters such as floods and fires, and protecting biodiversity. All ecosystem products and services are collectively referred to as ecosystem services (ES) ,2. The eAt present, research on the evaluation method of ESV can be roughly divided into two categories. The fist is a method based on the service function price per unit area. This method evaluates some key service functions by means of a series of ecological equations, such as food production, soil and water conservation, carbon and oxygen production, and habitat quality –14. The However, scholars have found that the evaluation results of the equivalent factor method are valid and reliable only when the equivalent factor accurately reflects the ecological background in the study area ,20,21. TBased on the research framework of value equivalent factor per unit area, we adopted the method of meta-analysis and fully used the evaluation results based on the physical quantity method to determine the average unit area equivalent factor in different ecosystems in Gansu Province. This method avoids or reduces the subjective conjecture easily caused by relying on the experience of experts. Additionally, abundant ecological environment data are used to correct the equivalent factor, thus completing the evaluation of ESV in complex ecological environments. Compared with previous studies ,19,27,28The types of ecosystems are complex and diverse in Gansu Province. The diversity of ecosystem types has caused significant regional differences. However, current research mostly focuses on single or several ecosystems, and only investigate certain ES functions in the ESV in Gansu Province, such as forests –31, grasThis study considers the regional differences and the simplicity of the equivalent factor method. In view of the application of the equivalent factors of various ecosystems on a large scale, it is necessary to closely relate the equivalent factors to the national (large) scale and to the actual situation in Gansu Province. Based on a more refined classification of the ecosystem types in Gansu Province, the study added some ecosystem equivalent factors, constructed a revised index, and revised the equivalent factors studied by Xie et al.  to form 2, with complex and diverse geological landforms and climate types. In addition to the marine ecosystem, there are six main land use or cover types, including forests, grasslands, deserts, wetlands, farmland, and urban areas. The Gansu region forms part of China’s “two screens and three belts” strategic ecological security barrier policy, which aims to maintain and protect the survival and reproduction of organisms, maintain the natural ecological balance, and guarantee people’s livelihoods on the Qinghai Tibet Plateau, the Sichuan–Yunnan Loess Plateau and the north sand belt. It is an important water conservation and supply area in the upper reaches of the Yangtze River and the Yellow River.The case study region is located in northwest China , at the We used national ecosystem type datasets from the Satellite Application Center of the Ministry of Ecology and Environment and the Chinese Academy of Sciences, for the periods 2000, 2005, 2010, and 2015, for the classification of ecosystems. The resolution of Landsat TM/ETM images was 30 m, SPOT-5 images was 5 or 2.5 m, Envisat image was 30 m, and HJ-1 images was 30 m. From this data, and according to the study requirements, the ecosystem types were divided into 7 primary types and 21 secondary types in the research area, and a corresponding database was established.We then used data from 2,508 ground verification points, including 38 different ecosystem types, and integrated these ecosystem types with the ecosystem types obtained through the satellite images, into a final database with 21 ecosystem types, as shown in http://cdc.nmic.cn).In this study, the monthly average temperature, precipitation, and sunshine hours from 1981 to 2012 in Gansu Province and its surrounding meteorological stations were used. The data was obtained from Gansu Meteorological Bureau and China Meteorological Science Data Sharing Service Network , and the grain output of each county is from Gansu Province Rural Yearbook (2000–2014). The monitoring data of atmospheric environmental quality status comes from Gansu Environmental Monitoring Center Station for the period 2015–2018, and the monitoring data of surface water comes from the Bulletin of Environmental Conditions in Gansu Province and the Bulletin of Environmental Conditions in China, for the period 2000–2015.Based on the research results of Costanza et al. , de GrooD represents the ESV of a standard equivalent factor (yuan · hm-1); SW, St, SV, and SX represe the sowing area proportion of wheat, corn, potato, and oil to the sowing area of the four crops; and FW, FT, FV, and FX represent the average net profit of wheat, corn, potato, and oil crops per unit area in Gansu Province (yuan · hm-1).The research period in this study is 2000–2015, and the net profit of agricultural products differed each year due to changes in social and economic conditions, and agricultural production technology. The ESV calculated only by the net profit of agricultural products during the study period is not representative. Therefore, in addition to the statistical information obtained , the sowing area and net profit of wheat, corn, potato, and oil crops per unit area were obtained, and the average sowing proportion and average net profit were calculated. Based on this, the value of a standard equivalent factor was calculated. The calculation method was as follows:The basic value equivalent of ecosystem service function per unit area  refers to the annual average value equivalent of various service functions of different ecosystem types per unit area. Previous studies on equivalence factors ,18,19 arFor the types of ecosystems and the corresponding ecosystem service types in Gansu Province , the naRelevant international literature, such as publications by Elsevier, Springer Nature, Wiley, and the Chinese How Net database, was searched. We inputted retrieval words such as Gansu Province, the names of each basin and city in Gansu Province, Qilian Mountain, and Gannan Plateau, to obtain research results on ecosystem service value calculated by ecosystem service function quantity in Gansu Province. If, in the future, there are many more papers on the evaluation of ESV, the journals with the highest influence should be selected for average calculation, and the proportion with standard equivalent should be calculated as the basic equivalent of ecosystem service functions, such as the local climate regulation and soil conservation functions of shrubs.We prioritized the collection and sorting of domestic published research results of ecosystem service value calculated by ecosystem service function quantity. The average of selected ESV, and thereafter the proportion with standard equivalents, should be calculated, so as to convert them into the average value equivalent factor of ecosystem service function, as the basic equivalent of the ecosystem service function, which is used to determine the value equivalent of ecosystem service functions, such as garden land, shrub land, forest land, and swamp wetland.If no relevant research results for Gansu Province can be found, relevant research results for other regions in China should be collected. ESV per unit area of ecosystem should be calculated, and then compared with the standard equivalent value. It can be converted into an average value equivalent factor of ecosystem service function by constructing a correction coefficient, as the basic equivalent of the ecosystem service function, which is used to determine the value equivalent of some ecosystem service functions, such as lakes, reservoirs, saline alkali land, and urban green space.There are great differences between the ecosystem service functions in Gansu Province and those of the entire country. Therefore, in this study, the value equivalence factor was localized and calibrated by calculating the ecosystem service function quantity per unit area, such as the biodiversity maintenance value of forest land, grassland, wetlands, and desert ecosystems, and the crop supply service of paddy fields and dry land.If there is no ecosystem service function value listed in directly corresponding documents in the secondary classification of ecosystem, and it is therefore difficult to calculate the ESV, refer to the equivalent factors listed by Xie et al. , as theyThrough the above six steps, the value of the main ecosystem types for a certain ecosystem service function per unit area in Gansu Province can be obtained, by referring to relevant literature or doing calculations, as shown in 1N)Crop supply correction index  × sowing proportion of wheat + (average yield per unit area of corn/average yield per unit area of corn) × sowing proportion of corn + (average yield per unit area of potato/average yield per unit area of potato) × sowing proportion of potato + (average yield per unit area of oil/average yield per unit area of oil) × sowing proportion of oil.The calculation of y is based on the following formula: Y is the same as that of y.The calculation method of 2D)Fresh water supply correction index , and W is the average water supply per unit area in China . The water supply data comes from the Water Resources Bulletins (2000–2015) for Gansu Province and China.The fresh water supply correction index is calculated as follows:3Air quality regulation correction index (K)a is the average proportion of air quality standards of prefecture level cities in Gansu Province, and A is the average proportion of air quality standards of prefecture level cities in China. The air quality standards data come from the Environmental Quality Bulletins (2000–2015) for Gansu Province and China.The air quality regulation correction index is calculated as follows:4S)Water purification correction index  for Gansu Province and China.The calculation method of water purification correction index is as follows:5Y)Entertainment and aesthetic value correction index  for Gansu Province and China.The calculation method of entertainment and aesthetics value is calculated as follows:1A1)Crop supply regulation index Livestock supply adjustment index Fresh water supply regulation index di is the average NPP of each partition; d1 is the NPP value of the high adjustment area; d2 is the mean value of NPP in the middle regulation area; and d3 is the NPP value of the low regulation area.The local climate regulation index was calculated as follows:A large amount of observation data analysis shows that a change in surface vegetation may have a significant impact on local and regional climate by changing surface attributes such as surface albedo, roughness, and soil moisture –76. The 5A5)Air quality regulation index Groundwater recharge regulation index Soil conservation regulation index Regulation index of windbreak and sand fixation Water purification regulation index Entertainment aesthetics value adjustment index  is generally regarded as the tourism area, with the remaining areas having the lowest entertainment and aesthetic value. The adjustment indexes of the different regions are assigned by expert judgment.11a11)Biodiversity maintenance value regulation index  was calculated using the following equation ; and n is the grid number.Different geomorphic types will affect the distribution of light, heat, water, and soil types in the region . Similarequation :V=∑i=1cDesert is the largest ecosystem type in Gansu Province , followeIn general, ESV decreases from south to north, and from east to west in Gansu Province , which iThe value of local climate regulation and biodiversity maintenance  is much The value of the grassland and forest ecosystems is the highest, accounting for more than 75% of the total value , whereasOver the past 15 y, the townships with increased ESV are mainly distributed in the east and south of Gansu Province, and west of the Hexi Corridor. The townships which had a decrease in ESV are located in Qilian Mountain and Gannan Plateau .Over the past 15 y, the number of townships with increased ESV has decreased. There were 959 townships with an increased ESV in 2000–2005, 758 townships in 2005–2010, and only 391 townships increased in value in 2010–2015. From 2000 to 2015, there were 818 townships with increased ESV.From 2000 to 2005, townships with increased ESV were mainly distributed in the Qilian Mountain, and the eastern and southern parts of Gansu . From 2005 to 2010, the ESV of most townships decreased in the Gannan Plateau and Qilian Mountain, whereas the increased townships were mainly located in Tianshui, Longnan, and the western section of the Hexi Corridor. From 2010 to 2015, the ESV of most townships declined in Lanzhou, Baiyin, Dingxi, Gannan Plateau, and Hexi Corridor, and the townships where the value increased were concentrated in the north and south.In this study, ESV was evaluated by improving the value equivalent factor per unit area and constructing a regional differential value assessment model in Gansu Province. Different ecosystem types provide different ES types to humans. In the current research on ESV, scholars mostly refer to the classification of ecosystem types by Costanza et al. , which iIn different regions, the same ecosystem type provides different ES and their value to humans are quite different. Therefore, the value equivalent factor of Costanza et al.  and Xie The accurate construction of the equivalent factor table is the core of the equivalent factor method. In the process of improving the equivalent factor table of Xie et al. , this stESV assessment has been studied extensively in other regions of China, whereas there are few related studies on ESV in Gansu Province. The existing research focuses on the value of a single ecosystem service on a provincial scale ,34,81, aBased on the characteristics of ecosystems in Gansu Province, this study developed a revised index based on an increase in some ecosystem equivalent factors, and revised the equivalent factors studied by Xie et al.  to form The desert ecosystem type covers the largest area in Gansu Province, followed by grassland, arable land, and forest ecosystems, and the remaining ecosystems account for only a small part. Desert is mostly located in northern Gansu. Grasslands are mainly located in the Gannan Plateau, Longzhong, and Longdong. Cultivated land is mainly located in Longzhong and Hexi Corridor, and forests are mainly located in Longnan, Ziwuling, and the Qilian Mountains.From 2000 to 2015, the grassland ecosystem area increased the most, whereas the cultivated land ecosystem area decreased the most. Forest, grassland, and urban ecosystems in Gansu Province continue to increase. Cultivated land ecosystems continue to decrease. Although wetlands and glacial ecosystems have increased over the past 15 y, they have decreased over the long term. The number of desert ecosystems has increased.In 2015, the total ESV for Gansu Province reached 2,239.56 billion yuan, to which the forest and grassland ecosystems contributed the most. In terms of the value of each service function of the ecosystem, the local climate regulation and biodiversity maintenance functions are the main service functions in the province. Regarding the spatial distribution of service values, ESV gradually increases from northeast to southwest, and the areas with high ESV concentrations are Qilian and Longnan Mountains.From 2000 to 2015, ESV increased by 3.43 billion yuan in Gansu Province. The value of forest ecosystems increased the most, whereas the value of grassland ecosystems decreased the most, showing a trend of increasing first, then decreasing, and then slowly increasing again. The value of forest and urban ecosystems continues to increase, and the value of cultivated land ecosystems continues to decrease. From the spatial characteristics of ESV changes, areas with reduced values gradually move from central Gansu to the surrounding areas.S1 Data(ZIP)Click here for additional data file. 27 Oct 2020PONE-D-20-28811Evaluation method of ecosystem service value under complex ecological environment: A case study of Gansu Province, ChinaPLOS ONEDear Dr. zhao,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. 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Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: N/A**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this paper, xiaojiong Zhao et al. has taken Gansu Province as an example, the regional division method of ecosystem service function is proposed based on the characteristics of differences among different regions, and the variation trend of ESV in Gansu province from 2000 to 2015 is analyzed, which expands the thinking of regional ecological evaluation. The subject matter of this paper is novel, the Angle is clear, the data analysis process is accurate and careful. However, the manuscript needs revision before it is acceptable for publication. The writing of the paper should be improved, I found some of the text is repetition and some parts are not very fluid. The specific sections are questions that should be answered before accepting and proceeding to publications.1. In introduction, It is recommended that authors add comparisons with other ecosystem service value studies and introduce the advantages of the approach used in this article;2. In the discussion section, I suggest comparing with other ecological service value assessment methods in Gansu Province;3.There are still some problems in English grammar and sentences. Please check and correct them carefully.Reviewer #2: comments to the Author:The first time I looked at the title " Evaluation method of ecosystem service value under complex ecological environment: A case study of Gansu Province, China ", I was just wondering to know how would the authors deal with this topic. I was a little impressed and felt that this might be a very interesting manuscript. When I reviewed the manuscript, I didn't feel disappointed. In the manuscript, On the one hand, the average value equivalent factor per unit area of different ecosystems is determined in Gansu Province, with reference to the six calculation process. This method of meta-analysis will avoid or reduce the subjective conjecture which is easy to be caused by relying on experts' experience; On the other, through abundant eco-environmental data, this study established a value evaluation model and completed the evaluation of ecosystem service value under complex ecological environment. In particular, one thing is affirmed that the human activity factors that affect ecosystem service value are fully considered by Author to the evaluation of ecosystem service value, such as carrying capacity, air pollution, over exploitation of groundwater and water pollution etc. The methods and data used in the study are new. The manuscript meets the requirement for acceptation for publication.My main concerns are as follows:Introduction1. The description of Gansu Province may be suitable for the part of study area.2. There lacks enough literature review on the topic in the study area. The authors may check some references on this topic in last decades.Materials and Methods1. What is the basis of the ecosystem type classification?2. Please supplement the distribution map of ecosystem types3. from 2000-2015, what does this mean? The authors have to state clearly which year was used.4. Classification of ecosystem service functions. Please explain the specific problem of repeatability. The description is not fully understood due to the ecological integrity has been evaluated in previous research.Result1. In the Table3, the unit of area is km2, while in formula 19, the unit of area is ha.2. Biodiversity maintenance value regulation index (a11), How the habitat quality index is measured?3. "invest" should be "InVEST"Other detail questionEnglish needs proofreading and editing, especially those sentences related to comparison. In addition, there exists many formatting problems in this manuscript, please verify the full text.1.Please unify the number of decimal points in the whole text.2. Table1. Please note that the black space.Reviewer #3: (1)The paper takes Gansu Province as an example, on the basis of fully considering the 6 regional differences of ecosystem service function. But it don’t explain why Gansu was taken as an example? Does Gansu have a typical ecological service system?(2)making dialogue with the ESV evaluation and calculation literatures by taking the role of your specific context.(3)This paper did a poor job in readability, with many typo errors and unstructured sentences. The authors need a copy editor before next submission.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Attachmentreviewers comments.docxSubmitted filename: Click here for additional data file. 12 Dec 2020Reply to Reviewer #1Comment1:“In introduction, It is recommended that authors add comparisons with other ecosystem service value studies and introduce the advantages of the approach used in this article.”Response1: We added comparisons with other ecosystem service value studies and introduce the advantages of the approach used in this article, line108-118 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment2:“In the discussion section, I suggest comparing with other ecological service value assessment methods in Gansu Province.”Response2: There are few methods to evaluate the value of ecosystem services in Gansu Province, through literature retrieval, we compared with the study by Wang et al; line735-736 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment3:“There are still some problems in English grammar and sentences. Please check and correct them carefully.”Response3: We used Editage for English language editing that edited my manuscript, and we have carefully and thoroughly proofread the manuscript to correct all the grammar and typos. For details of manuscript editing, please refer to marked-up copy of my manuscript.Reply to Reviewer #2Comment1: “In introduction, the description of Gansu Province may be suitable for the part of study area.”Response1:In introduction,“Gansu Province is located in the northwestern inland in China. …..Its special geographical location and natural conditions form a more distinct ecological structure.”, this part is repeated with the content in the study area, so this description of Gansu Province has been deleted in introduction. line 119 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment2: “There lacks enough literature review on the topic in the study area. The authors may check some references on this topic in last decades. ”Response2: We checked some references about method of ecosystem services value, and added other ecosystem service value studies. line 108-118 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment3: “What is the basis of the ecosystem type classification? ”Response3: The basis of the ecosystem type classification was added in Ecosystem type data, line 160-184 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment4: “Please supplement the distribution map of ecosystem types.”Response4:The distribution map of ecosystem types was added in line 185 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment5: “from 2000-2015, what does this mean? The authors have to state clearly which year was used. ”Response5: The mean is “from 2000, 2005, 2010, and 2015”. line 194-195 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment6: “Classification of ecosystem service functions. Please explain the specific problem of repeatability. The description is not fully understood due to the ecological integrity has been evaluated in previous research. ”Response6: “The specific problem of repeatability” means is “the double- counting problem between ecological integrity and other ecosystem services”. line 217-219 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment7: “In the Table3, the unit of area is km2, while in formula 19, the unit of area is ha. ”Response7: The unit of area is unified as km2. line 593 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment8: “Biodiversity maintenance value regulation index (a11), How the habitat quality index is measured? ”Response8: The habitat quality index was measured in detail in another article of the author, so we added the reference. line 562-563 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment9:“"invest" should be "InVEST"”Response9: We modified the invest to InVEST. line 562 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment10:“English needs proofreading and editing, especially those sentences related to comparison. In addition, there exists many formatting problems in this manuscript, please verify the full text. ”Response10: We used Editage for English language editing that edited my manuscript, and we have carefully and thoroughly proofread the manuscript to correct all the grammar and typos. For details of manuscript editing, please refer to marked-up copy of my manuscript.Comment11:“Please unify the number of decimal points in the whole text. ”Response11: We unified the number of decimal points in the whole text.line 37, 42,567,612,632,646 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment 12:“Table1. Please note that the black space. ”Response12: We revised the black space. line 314 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Reply to Reviewer #3Comment1: “The paper takes Gansu Province as an example, on the basis of fully considering the 6 regional differences of ecosystem service function. But it don’t explain why Gansu was taken as an example? Does Gansu have a typical ecological service system? ”Response1: We stated the reasons that the types of ecosystems are complex and diverse in Gansu Province, line 119-127 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment2: “making dialogue with the ESV evaluation and calculation literatures by taking the role of your specific context. ”Response2: We have supplemented the relevant references on the other ecosystem service value studies, line108-118, 858-863 in marked-up copy of my manuscript. The modified part is highlighted in yellow.Comment3: “This paper did a poor job in readability, with many typo errors and unstructured sentences. The authors need a copy editor before next submission. ”Response3: We used Editage for English language editing that edited my manuscript, and we have carefully and thoroughly proofread the manuscript to correct all the grammar and typos. For details of manuscript editing, please refer to marked-up copy of my manuscript.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 18 Jan 2021Ecosystem service value evaluation method in a complex ecological environment: A case study of Gansu Province, ChinaPONE-D-20-28811R1Dear Dr. zhao,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Bing Xue, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #3: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The author has added comparisons with other ecosystem service value studies and introduce the advantages of the approach used in the introduction and grammar problems also have been solved.The overall structure of the article is clear and has strong scientific and practical significance.Reviewer #3: I really appreciate the revision made by the author, which can be seen to improve the readability of the paper.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #3: No 21 Jan 2021PONE-D-20-28811R1 Ecosystem service value evaluation method in a complex ecological environment: A case study of Gansu Province, China Dear Dr. Zhao:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProfessor Bing Xue Academic EditorPLOS ONE