As inferred from 16S rDNA data, the highly diversified and coupled processes sustained by the monimolimnetic microbiota are essential ecosystem services that enhance mineralization of organic matter and formation of reduced compounds, and also abatement of undesirable greenhouse gasses.In deep lakes, many investigations highlighted the existence of exclusive groups of bacteria adapted to deep oxygenated and hypoxic and anoxic hypolimnia. Nevertheless, the extent of bacterial strain diversity has been much less scrutinized. This aspect is essential for an unbiased estimation of genetic variation, biodiversity, and population structure, which are essential for studying important research questions such as biogeographical patterns, temporal and spatial variability and the environmental factors affecting this variability. This study investigated the bacterioplankton community in the epilimnetic layers and in the oxygenated and hypoxic/anoxic hypolimnia of five large and deep lakes located at the southern border of the Alps using high throughput sequencing (HTS) analyses (16S rDNA) and identification of amplicon sequence variants (ASVs) resolving reads differing by as little as one nucleotide. The study sites, which included two oligomictic (Garda and Como) and three meromictic lakes  with maximum depths spanning from 124 to 410 m, were chosen among large lakes to represent an oxic-hypoxic gradient. The analyses showed the existence of several unique ASVs in the three layers of the five lakes. In the case of cyanobacteria, this confirmed previous analyses made at the level of strains or based on oligotyping methods. As expected, the communities in the hypoxic/anoxic monimolimnia showed a strong differentiation from the oxygenated layer, with the exclusive presence in single lakes of several unique ASVs. In the meromictic lakes, results supported the hypothesis that the formation of isolated monimolimnia sustained the development of highly diversified bacterial communities through ecological selection, leading to the establishment of distinctive biodiversity zones. The genera identified in these layers are well-known to activate a wide range of redox reactions at low O The monimolimnion is often rich in phosphorus and reduced nitrogen compounds. Conversely, while reduced substances accumulate in the monimolimnion, biota in the surface illuminated layers and dark mixolimnion may be deprived of essential nutrients  ]. Owing Planktothrix rubescens and Microcystis aeruginosa) or neurotoxins   in the upper and oxygenated layers, dark oxygenated layers, and deep hypoxic layers of DSL, quantifying the influence of light and oxygen concentrations (as a proxy of redox conditions) on the potential selection of specific bacterial lineages. A specific emphasis will be given to Cyanobacteria. This group of bacteria is widely represented with several adaptations in many aquatic and terrestrial ecosystems . Moreoverrellyi) .m), and volume (V) values set at S 10 km2, zm 50 m, and V 0.5 km3. The two largest lakes included in this work, i.e., Garda and Como, are oligomictic and oligo-mesotrophic; they have surface and maximum depths of 368 and 146 km2, and 350 and 410 m, respectively. The two last complete vertical circulation events were between 2004 (Garda) and 2005–2006 (Garda and Como)  . Since tectively . In lakely, 2005 ; hypoxialy, 2005 . Meromixly, 2005 , eutrophly, 2005 .max = 270 m). The sampling stations in lakes Garda, Como, and Iseo are part of the Italian and European Long Term Ecological Research network . Constant volumes of water were collected in each layer with a 5 L Niskin bottle for successive subsampling. Vertical profiles of temperature and oxygen were carried out using underwater probes . In the upper layers, samples were collected at 0, 10, 20, 40, and 60 m. Additional samples were collected, every 50 m or less, in the hypolimnetic layers , total phosphorus (TP), soluble reactive phosphorus (SRP), reactive silica (Si), alkalinity and major ions  were determined in a single laboratory (FEM) using standard analytical procedures (Samplings and measurements were carried out in 2016 during the stratification period in the deepest zones of the lakes Garda (5 July), Iseo (29 August), Lugano  and Idro (23 August); in Lake Como (14 June), samplings were carried out at the station of Dervio, in the northern basin . DNA concentrations, measured with a NanoDrop ND-8000 , ranged between 5 and 46 ng μL–1. PCR amplification of bacterial sequences was carried out by targeting a ∼ 460-bp fragment of the 16S rRNA gene variable regions V3–V4 using the specific bacterial primer set 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805Rmod (5′-GACTACNVGGGTWTCTAATCC-3′) with overhang Illumina adapters. The final barcoded library was sequenced on an Illumina® MiSeq (PE300) platform. A detailed description of procedures is reported in For HTS analyses, aliquots of water were filtered through 25 mm polycarbonate Isopore membranes (Merck) with 0.2 μm pore size. Filtered volumes ranged between around 500 and 1000 mL, depending on the quantity of suspended particles, and until near-clogging of the filters. Filters were stored at −20°C until DNA extraction with MO BIO PowerWaterSequences were analyzed using the DADA2 package 1.10  in R 3.5The ASVs abundance table, taxonomy, DNA reads and environmental data were imported into the R package phyloseq 1.28.0 . Chlorop2, SRP, NO3-N, NH4-N, Si, Alk, SO4). A second distance matrix was computed including only a subset of variables linked to stratification (water temperature) and major chemical gradients . The bacterial dissimilarity matrix was computed using the same methods used in NMDS. The significance of the statistic was evaluated by 9999 permutations of rows and columns of the dissimilarity matrix.Correlations between environmental variables and the bacterial community in the 5 lakes and selected layers were calculated by computing Mantel tests . The envk species (or higher level taxonomy), the observed optimum environmental levels of oxygen were estimated by computing the average values – weighted by the corresponding abundances values – of the O2 concentrations in the corresponding samples where the k species were identified. Species optima (uk), and species tolerances (tk), were computed using standard approaches  and 20 m (Idro). Below 60 m, O2 concentrations in these three lakes were always below 0.5 mg L–1. Based on the stratification patterns and O2 concentrations, three layers were identified. In the five lakes, the upper oxygenated and productive layer included sampling depths between the surface and 20 m ; the dark oxygenated layers included all the hypolimnetic sampling depths in the oligomictic lakes (Garda and Como), and the depths at 40 and 60 m in lakes Iseo and Lugano ; the third layer included the deep, dark and hypoxic sampling depths in the three meromictic lakes, below 100 m (Iseo and Lugano) and 40 m (Idro) .The five lakes showed steep surface temperature gradients up to 40–50 m  and 20 m  . Dissolv3-N begun to increase after the first 20–30 m, up to around 320 μg L–1 (Garda) and 750 μg L–1 (Como) in the deep layers; conversely, NH4-N was generally below 20 μg L–1. Apart from the increase of nitrate up to 20–50 m, both NO3-N and NH4-N in the three meromictic lakes showed opposite vertical patterns , TP reached the highest concentrations in the meromictic lakes (averages between 88 and 204 μg L–1). All the five lakes showed vertical decreasing values of pH and vertical increasing values of conductivity, Si, Alk, Ca, and Mg. In particular, the hypolimnion of the meromictic lakes showed low-medium pH (range: 7.4–8.3), high conductivity values (229–454 μS cm–1), and high concentrations of calcium (37–71 mg L–1) and alkalinity (117–209 mg L–1) and, especially in Lake Idro, sulfate (7.6–86 mg L–1), magnesium (7.2–20 mg L–1), and silica (0.8–5.4 mg L–1). Corresponding ranges in lakes Garda and Como were: pH, 7.4–8.2; conductivity, 169–240 μS cm–1; calcium, 25–39 mg L–1; alkalinity, 71–134 mg L–1; sulfate, 11–27 mg L–1; magnesium, 5.6–8.5 mg L−1; silica, 0.3–2.3 mg L–1. Differences in the distribution of Cl, Na, and K  in the oligomictic and meromictic lakes were less apparent.The vertical gradients of oxygen were paralleled by similar changes in the vertical distribution of nitrate and ammonium nitrogen . In lakepatterns . In lakeP = 0.32). The median number of ASVs per lake was between 243 (Idro) and 316 (Como). Chao1 values closely followed the observed number of ASVs. Compared to Lake Como, the Shannon diversity in the other lakes showed a larger dispersion and lower values . The number of ASVs was higher in the oxygenated hypolimnion (median 344) than in the other two layers (251–261) (P < 0.01). Shannon diversity was higher in the oxygenated layers (P < 0.01).The number of ASVs in the 5 lakes was generally comparable . Other dominant taxa in the two oligomictic lakes included Proteobacteria , Bacteroidetes , and Planctomycetes (CL500-3), whereas photosynthetic Cyanobacteria were mostly represented by Tychonema bourrellyi. In lakes Garda and Como, the dominant orders almost coincided  and Planctomycetes (family Gemmataceae) (Pseudanabaena) was found in Lake Lugano.The three meromictic lakes showed a strong increase of Proteobacteria in the upper and/or deep hypolimnion. Lake Idro had a peculiar presence of Epsilonbacteraeota  at higher depths. Differences in the bacterial composition in the hypoxic layers were already apparent at the order level . In Lakeataceae) . In the The number of ASVs exclusively present in the single lakes overshadowed those common in two or more lakes . DiffereP < 0.001). A closer inspection of sites in the three layers allowed to identify differences in the positioning of samples belonging to the different lakes , due to, e.g., the position of the samples of Lake Garda in the left area of the configuration, and to the three meromictic lakes, which showed a clear separation of the respective samples in the deep hypoxic hypolimnion  . Samplesolimnion . The sepolimnion , which, olimnion .2, samples in the upper oxygenated layers were characterized by higher temperature values and pH, whereas the deep oxygenated layers had higher concentrations of NO3-N, Cl, Na, and K  or highly significant .The correlations between the environmental variables and the community structure were highly significant considering both the pooled data  was evaluated testing their abundances in one selected layer compared to the remaining layers . The anad change  > 4. As c layers . As expec layers  mostly ik-O2 ranged between 0.05 and 1.4 mg L–1. The taxa identified belonged to genera that are known to be strictly or facultative anaerobic, or microaerobic, and linked to a wide variety of redox reactions at low O2 conditions. Most genera belonging to the class Deltaproteobacteria were associated with sulfate reduction reactions, with Desulfomonile and Desulfurivibrio in lakes Idro and Lugano, Desulfobacca in both lakes, and Desulfatirhabdium in the three meromictic lakes. Along with other members of the Desulfobulbaceae, Desulfurivibrio is known also to perform disproportionation of elemental sulfur to sulfide and sulfate (Candidatus Anammoximicrobium); besides part of the family Rhodocyclaceae, denitrification can be performed by a wide array of microbial groups (including methanotrophs and methylotrophs). Other processes connected with known phenotypic characters included H2-utilization in syntrophic association (Syntrophus) and, particularly in Lake Idro, sulfide (and hydrogen) oxidation at the expense of nitrate (Sulfuricurvum and Sulfurimonas). Sulfurimonas have been shown not only to use sulfide as electron donors but also for example thiosulfate or elemental sulfur. In Lake Lugano, Gallionella included iron-oxidizing bacteria requiring at least a certain amount of O2. Further genera belonging to the less abundant phyla were linked to the mineralization of a wide variety of organic matter, including complex carbohydrates, cellulose, proteinaceous C, and organic acids  the optimum range of O2 of “monimolimnetic” families was between 0.06 and 1.45 mg L–1.The deep and hypoxic layers showed a number of classifiable genera typically present in the three meromictic lakes . Consist sulfate . A varieic acids . Though Flavobacterium, the variability in ASVs was very high (61) and, considering the high number of species potentially included in this genus, not straightforward to interpret (mean sequence similarity in the 61 ASVs was 94%). For this genus, it was actually possible to assign a species name to only a low fraction of ASVs, i.e., F. pectinovorum, F. succinicans, F. paronense, F. chungnamense/koreense, and F. terrigena, each one present with only one sequence variant. Most of the different variants were mostly present in 2 or 3 lakes, only rarely 5 (figure not shown).The reads identified at the genus level were characterized by a very variable number of ASVs, due to variability in how many ASVs were nested within each genus. In a number of genera, such as Cyanobium PCC-6307. The similarity between each pair of aligned sequences assigned to this taxon ranged between 94.8 and 99.8%. Of these, only one ASV was present in the 5 lakes, whereas the majority (8) was found in one or several samples in one unique lake , with a number of reads greater in Lake Garda. Conversely, the second more abundant strain present in four lakes did not show apparent differences . At the species level, the two Planktothrix NIVA CYA 15 were assigned to the complex P. agardhii/rubescens. These two ASVs were distinguished by two sequence variants (AG). After a BLAST analysis, the more abundant ASV, (A), which was identified in all the lakes with the exclusion of Lake Idro, showed 100% similarity with many P. rubescens/P. agardhii sequences, including those previously associated to P. rubescens in the largest lakes south of the Alps (P < 0.001), with higher abundances in Lake Lugano. The second variant  was previously identified in Lake Garda using HTS approaches (Aphanizomenon NIES 81), fully coincided  with that of Dolichospermum lemmermannii ; the identification was confirmed also by previous observations carried out in all the DSL using a polyphasic approach . Tychonema sequences were identified, with one unique abundant oligotype, in lakes Como, Garda, and Iseo.A similar distribution characterized other genera, such as those included in Cyanobacteria . The larque lake . The disthe Alps . The disproaches . The seqch e.g., , and by ch e.g.,  and SILVIn this work, the new established group of non-photosynthetic Cyanobacteria was identified only in the three meromictic lakes, both in the oxygenated and hypoxic layers  and in the hypoxic layers (Class Sericytochromatia). The identity of the ASV attributed to Sericytochromatia was confirmed using a further downstream classification in SILVA 132, and evaluation of its position in the general phylogenetic tree. Considering the overall means, the relative abundances of NCY on the total of Cyanobacteria was however low, with maximum contributions < 6% in Melainabacteria and < 1% in Sericytocromatia.Methylotenera and Crenothrix, not shared with the meromictic lakes, in the two oligomictic lakes Garda and Como.The most impressive differences in the ASVs composition characterized the populations inhabiting the deep hypoxic/anoxic layers. Focusing on the most abundant taxa of This work demonstrated the existence of a clear link between the vertical partition of water layers and the ecological selection of different bacterial populations and groups adapted to different local habitats. The BCC showed a large and significant divergence in the three main water layers that were identified on the basis of the oxygen concentrations and the main vertical physical and chemical gradients, namely the upper and oxygenated layers, dark oxygenated layers, and deep hypoxic layers. The differentiation was detected at different taxonomic levels and was linked to the ecological selection of different taxa adapted to perform a variety of metabolic functions constrained and driven by the availability of oxygen (as a proxy of redox conditions) and chemical compounds. Further, the higher sequence resolution to marker gene surveys provided by the oligotyping approach allowed to identify several exclusive ASVs present in single lakes and/or specific water layers and depths. After a brief examination of the elements that should be taken into account when analyzing ASVs, these aspects will be addressed in the next sections.Microcystis 16S rDNA oligotypes were not monophyletic and that they could not be used to infer toxicity  for oxygen, which, in the hypoxic/anoxic layers, and in the oxygenated layers, were between 0.05 and 1.4 mg L–1, and 7.1 and 11.9 mg L–1, respectively. These optima values allowed to quantify the O2 concentration requirements of distinct taxa developing in distinct strata. At the same time, these values can contribute to compare, on a quantitative basis, the oxygen niche of the same group of taxa in other aquatic systems.The identification of genera typical of the three water layers was fully consistent with the corresponding optima , as well as aerobic, anoxygenic  . Methanogenesis represents the largest biogenic source of methane on Earth   . Selecteen cycle . A furthon Earth . Though on Earth , methanopolimnia . Candida nitrite  was deteacceptor . The phy nitrate ; their h species . Overall 5–22 m) .zm < 30 m) stratified lakes, which showed a higher number of unique OTUs in the hypolimnia than epilimnia (m = 92 m) and dimictic  perialpine lakes , the most represented genera in the anoxic zone were Syntrophus, Methylotenera, Gallionella, Sulfurimonas, Desulfurivibrio, and Sulfuritalea. These genera were mostly present in the meromictic lakes included in this work, further confirming how predominant vertical environmental gradients can affect the environmental filtering of bacterial communities.Besides large and deep lakes , the higpilimnia . A few one lakes . BesidesThe identification of genera in P < 0.001), highlighting the general relationship between sequencing depth and ASVs numbers. This was exemplified by the high number of ASVs in the genera identified with higher abundances in the hypoxic and anoxic hypolimnia (Cyanobium), i.e., the fraction roughly corresponding to the picocyanobacteria  with study accession PRJEB33405.NS was involved in all aspects of the manuscript; see Acknowledgments for technical support.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: To determine the effects of social capital on harmful drinking (HD) among Chinese community residents using a multilevel study. Methods: A cross-sectional study conducted from 2017–2018. In total, 13,610 participants were randomly interviewed from 29 districts of 3 cities in China with a multi-stage sampling procedure. Social capital, including social cohesion, membership in social organizations, and frequency of social participation, were assessed using validated scales. HD was assessed using the CAGE four-item questionnaire. Multilevel models were developed to determine whether social capital was related to HD when socioeconomic and demographic covariates were controlled. Results: In general, the prevalence of HD was 8.18%, and more specifically, 13.77% for men and 2.74% for women. After controlling for covariates and stratifying by gender, compared to residents in the low individual-level membership of social organizations, we found that the odds ratio (OR) for HD was 1.30 with a 95% confidence interval (CI) of 1.07–1.56 among men and 1.95 (95% CI: 1.29–2.97) among women. Compared to residents in the low individual-level frequency of social participation groups, the odds ratio of HD among women was 1.58 (95% CI: 1.10–2.26). There was no association between district-level social capital and HD. Conclusions: A high level of social capital may promote HD among the residents of Chinese neighborhoods. Intervention to modify social capital under the Chinese drinking culture may help reduce HD. Harmful drinking (HD) is the most significant public health challenge because of its serious individual and collective consequences, including poor health outcomes, loss of productivity, increased mortality, association with more than 60 diseases, and increased propensity for violence and crime ,2,3,4. ISocial capital can affect health as a social determinant and studies have shown that social capital was related to some health outcomes, such as physical health, mental health, violent crime, and even mortality ,11,12,13Although plenty of studies showed that social capital is negatively associated to HD ,17,18,19Our previous study found that workplace social capital was a protective factor for HD , but it This study was conducted in Shanghai, Zhengzhou, and Xinzheng in Henan province, China from June 2017 to March 2018. Neighborhood residents from 29 districts were randomly selected using multi-staged sampling. All 16 districts in Shanghai, all 7 districts in Zhengzhou, and 6 districts randomly selected from Xinzheng were selected as clusters, then 5% of the population aged 15 to 75 years old were randomly selected from each district. Data was collected using a self-administered “health city needs assessment questionnaire” This study has been approved by the Institutional Review Board of Fudan University .The CAGE  four-item questionnaire was used to measure HD see . The fouBecause there was no uniform social capital concept and measure, we used social cohesion to measure the cognitive component and membership of social organizations and frequency of social participation to measure the structural component based on previous studies .(1) Social cohesion: It was assessed using the Chinese version of the Neighborhood Scales see  13,28].,28.13,28(2) Membership in social organizations: We assessed it by asking participants whether they were a member of the following six social organizations in their respective district see . Partici(3) Frequency of social participation: We assessed this by asking participants how often they participated in eight different activities see  27]. Ea. Ea27]. To analyze the three dimensions of social capital, we assessed them at the individual and aggregate levels. Both levels of social capital scores were dichotomized into high versus low level using the median ,13.Base on a literature review ,30,31,32(1) Smoking statusSmoking status was measured using two questions: (1) have you smoked more than 100 cigarettes in your entire life? (2) Have you smoked, even one puff, in the past 30 days? Current smokers responded “yes” to both questions; otherwise, they were regarded as nonsmokers .(2) Mental healthWHO-5 Wellbeing Index was used to assess mental health see Table S5(3) Self-rated health Self-rated health (SRH) is a commonly-used health indicator for the association between social capital and health . In our Several sociodemographic and socioeconomic factors were included as covariates: gender, age categorized in 10-year increments (ranging from ≤29 to ≥70 years old), occupation status , marital status , region of origin (native vs immigrant). In addition, participants came from three cities: Shanghai, Zhengzhou, and Xinzheng.2 test was used for univariate analysis, and data from this study had a multilevel structure comprised of neighborhood residents (at level 1) nested within districts (at level 2). The analysis proceeded as follows [p-value was set at <0.05 (two tailed). The STATA version 13.0 program  was used for the whole analysis [At the first step, a χ follows : (1) Emp follows , and theanalysis .In total, 13,610 residents (48.43% men and 51.57% women) with a mean ± standard deviation (SD) age of 46.89 ± 15.01 years responded. Of these, 30.96% had enrolled in the university, 64.95% were employed, 86.64% were married, and 81.22% were native. In general, the prevalence of HD was 8.18%, more specifically, 13.77% for men and 2.74% for women.Descriptive results of sociodemographic characteristics and the prevalence of HD are given in 2 = 422.26, p < 0.001) and women . The intraclass correlation coefficients (ICC) were 0.169 for men and 0.178 for women, indicating that 16.9% (men) and 17.8% (women) of the variation was explained by a random effect by the different districts.Since the prevalence of HD was much higher among men, we modeled the relationships between social capital and HD among men and women. The multilevel logistic model results are shown in The results of the model showed that the adjusted OR of social capital and HD were greater among those who were employed , current smokers , and residents with good mental health . Additionally, among women, the prevalence of HD was lower at higher education levels, where university or higher levels of education had the lowest odds ratio of HD . Of particular interest, associations between three dimensions of individual-level social capital and the prevalence of HD were different after controlling for covariates and stratifying by gender. At the individual-level, the adjusted OR were greater among those who had a higher level of membership in social organizations , and the adjusted OR were greater among women who had a higher level of social cohesion .In model 2, we found that none of three dimensions of district-level social capital were related to HD, but some individual-level covariates were significantly associated with HD: current smokers  and residents with higher mental health scores . Among women, the prevalence of HD was lower with higher educational levels and lowest with a university or higher educational level . The adjusted OR of HD were lower among men that were local residents .In model 3, after controlling for district-level social capital, the results of model 3 indicated that the associations between the covariates and the HD were comparable between model 1 and 2, and among women, mental health status was not significantly associated with HD. Furthermore, the associations between the three dimensions of social capital and HD were comparable between model 1 and 2. Neither the individual-level frequency nor district-level frequency of social participation was associated with HD.To our best knowledge, it is the first time that the “dark side” effects of social capital impacting HD were found in the Chinese context. We found that a high membership rate in social organizations was associated with a high prevalence of HD among men, and high social cohesion and membership in social organizations were associated with a high prevalence of HD among women; the results were consistent with those of previous studies ,21,24,35There are several plausible explanations for social capital positively associated with HD, but there are differences between men and women. First, for both men and women, high-level membership in social organizations was positively associated with a high prevalence of HD. This is similar to the drinking culture in Denmark ,23, and The different measurements of HD and social capital may cause the inconsistency of our findings with other “negative relation” studies . Gao et Nevertheless, no relationships between district-level social capital and HD were found in the study. In China, social capital mainly exists within the family or other social organizations, which means that people trust those belonging to the same group , so the In the multilevel models, other covariates were associated with HD. For example, native men had a lower rate of HD than non-local men who have a rural “Hukou” . These results were similar to those of a previous study indicating that rural–urban migrants were more likely to engage in HD than urban residents due to the greater number of work-related stressors they experience . MoreoveThere are some limitations in our study. First, the data from this cross-sectional study prohibited us from drawing conclusions about causality. In other words, we cannot tell social capital caused HD or vice versa. Further longitudinal studies, such as cohort studies or nested case control studies, that investigate the relationship between social capital and HD from various industries is imperative. Additionally, the data were from a self-administered questionnaire, so the possibility of a lack of attention or non-professional investigators should be considered . Second,Our study found positive associations between three dimensions of social capital and HD, and for the first time, we revealed the “dark side” of social capital impacting HD in China and the Chinese drinking culture that may influence drinking behaviors. After stratification by gender, we discovered that individual- and district-level social cohesion were positively related to HD only among women. In contrast, there was no association between district-level social capital and HD. In light of these findings, we should administer some longitudinal studies to examine the relationship between additional aspects of social capital and HD. Interventions regarding creating a healthy drinking environment and a rational usage of social capital may contribute to reduce the rate of harmful drinking.


The mammalian CIP/KIP family proteins are intrinsically disordered proteins (IDPs) that can regulate various cellular processes. However, many reports have shown that IDPs generally evolve more rapidly than ordered proteins. Here, to elucidate the functional adaptability of CIP/KIP proteins in vertebrate, we analysed the rates of evolution in relation to their structural and sequence properties and predicted the post-translational modification based on the sequence data. The results showed that CIP/KIP proteins generally could maintain their function through evolution in the vertebrate. Basically, the disordered region that acts as a flexible linker or spacer has a conserved propensity for structural disorder and a persistent, fast rate of amino acid substitution, which could result in a significantly faster rate of evolution compared to the ordered proteins. Describing the pattern of structural order-disorder evolution, this study may give an insight into the well-known characteristics of IDPs in the evolution of CIP/KIP proteins. Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2(refs6). The family members can be distinguished into another CDI protein family by a conserved N-terminal domain called a CDI domain or kinase inhibitory domain (KID), which allows them to regulate cell cycle progression by binding to both cyclin and CDK subunits of mitogens, whereas INK4 members bind to cyclin only7. Although CIP/KIP members have similar specificities on their CDI/KID, they have shown to have distinct functions and regulatory mechanisms related to the divergence in their remaining sequence7. Correspondingly, CIP/KIP members have a different motif near their C-terminal, in which p21, p27, and p57 have proliferating cell nuclear antigen (PCNA)-binding motif, a stretch of conserved glutamine and threonine residues namely QT-box motif, and both PCNA-binding motif and QT-box motif, respectively9. Initially recognised as inhibitors of cell cycle progression, CIP/KIP proteins have also been reported to be involved in many other regulatory mechanisms, including transcriptional regulation7, cell migration10, and apoptosis11. Furthermore, recent studies have indicated that all CIP/KIP proteins also act as tumour suppressors and may be oncogenic under certain conditions1. The multifaceted abilities of these proteins may originate from their partial or complete lack of a cooperative, folded structure under native conditions, namely intrinsically disordered proteins (IDPs)18. However, it was reported that IDPs generally evolve more rapidly than ordered proteins20. Therefore, to understand how these proteins get along with the IDP fast rates of evolution, it is critical to examine their evolution in relation to structural features.The CIP/KIP family is one of two cyclin-dependent kinases (CDKs) inhibitor (CDI) protein families and it includes p2122. These characteristics may result in a flexible conformation of disordered regions, which are unable to fold spontaneously into three-dimensional structures and tend to adopt specific tertiary conformations, at least locally, only after binding to their partners23. The molecular interactions of IDPs can be transient and dynamic, such as to function as switches fine-tuned by post-translational modifications (PTMs) and to function as chaperones. On the other hand, it also can be permanent, such as those that enable them to modify the activity of other proteins, assemble multiple binding partners to promote protein complexes, and function as scavengers, which store and neutralize small ligands26. In addition, disordered regions can also retain their structure without ever becoming structured or showing the so-called “fuzziness” phenomenon and may function as flexible linkers or spacers27. Accordingly, these characteristics have consequences for IDP ability to exhibit functional promiscuity under different conditions23.Generally, the primary sequence of IDPs is composed of a high proportion of charged and polar amino acids and is deficient of bulky hydrophobic amino acids20. These differences may be due to differences in amino acid compositions, intramolecular contacts, and functional differences28. Additionally, recent assessments have shown that disordered proteins have different accepted point mutations and higher insertion and deletion rates compared with ordered proteins29. Disordered regions are also associated with more modification sites than ordered regions, particularly phosphorylation30. Given the ability to mediate multiple biological processes, CIP/KIP proteins also have a higher tendency to evolve over time. Thus, the evolutionary mechanism of CIP/KIP proteins may provide important information about genome evolution, in general. Here, to elucidate the functional adaptability of CIP/KIP proteins, we calculated the rates of evolution per site and the rates of structural properties transitions, and predicted their structural disorder and secondary structure propensities. In addition, we also predicted and examined the phosphorylation and the conserved phosphorylation sites, respectively.Because conformationally flexible proteins tend to have functional promiscuity, studies on IDPs evolution have attracted a lot of attention, particularly with regards to the rates of amino acid substitution compared with ordered proteins. Many reports have shown that IDPs generally evolve more rapidly than ordered proteinsTo investigate the evolutionary patterns of structural properties of the CIP/KIP members in vertebrate, we predicted the propensity of structural order-disorder and secondary structures and mapped the results of which to the taxon position in the phylogenetic tree and multiple sequence alignment Figs  and 2. WThe CDI/KID domain was encountered through pfam for all CIP/KIP members in all species, and there were two CDI/KID domains found for p21 of amphibian. This domain of CIP/KIP members which is responsible to control cell-cycle progression in mammals generally had a similar and conserved secondary structure propensity, with a little distinction in the transition patterns and rates Fig. . On the Based on the distribution of the structure propensity score in all regions, p27 tended to be more disordered than p21 and p57, with scores of 0.523 ± 0.179, 0.685 ± 0.177, and 0.601 ± 0.241 (means and standard deviations) for p21, p27, and p57, respectively . In addition, for the CDI/KID domain, the means and standard deviations were 0.386 ± 0.101, 0.535 ± 0.106, and 0.474 ± 0.113 for p21, p27, and p57, respectively . Thus, on an average, the frequency of the sequence that tends to be ordered is greater in the CDI domain than in the entire sequence for all CIP/KIP members. Each human CIP/KIP protein exhibits a different genetic distance pattern relative to the vertebrate class. As shown in the p-distance value analysis, compared with the human sequence, chondrichthyes had the most diverse sequence identity for p21 protein (59.8%), whereas p27 was the most diverse in amphibia (54.3%) and p57 was the most diverse in ray-finned fish (51.4%). p27 had the lowest genetic distance value for each class, except amphibia . Thus, p27 had the slowest rate of evolution of all CIP/KIP proteins.To investigate the relationship between rates of amino acid substitution and the structural properties and function, we calculated the rates of amino acid substitution. The results are Z-scores, which mean the scores of more than zero is evolving faster than average, and the scores of less than zero is evolving slower than average. In addition, we also predicted the sites of post-translational modification (PTM), i.e., phosphorylation, in the vertebrate and examined the modification site conservation through species evolution following the 50% majority rule. This approach was used to investigate the contributions of protein modifications to the rates of amino acid substitution.In general, each protein had its own specific distribution of the rates of evolution. However, similar patterns were observed in relation to the functional region. Generally, but not always, conserved modification sites and the adjacent region have a lower rate of amino acid substitution Fig. . HoweverRegarding the modification site prediction results, 14 phosphorylation sites had been predicted to occur in the human p27 sequence, whereas 10 and 11 sites had been found for p21 and p57, respectively. Most modification sites were found in the disordered region outside of the CDI/KID domain . Only two sites located in the CDI/KID domain were found, one from p21 and another from p57. In contrast, p27 possessed a higher number of predicted phosphorylation sites than other protein members, and it also possessed the highest number of conserved phosphorylation sites as well. In human p21, there were T145, S146, and S153 that showed to be conserved in propensity and sequence to a vertebrate; on the other hand, there were S7, S10, S12, T42, S110, S138, S140, T157, S161, S175, S178, S183, T187, and T198 in human p27; while there were only S28 in human p57. Except for S7, S161, S175, S178, and S183 in p27 and S28 in p57  analysis, respectively17. Consistent with this, the prediction of order-disorder propensity of CIP/KIP proteins in this study also showed that these proteins are highly disordered in structure. Further, their rates of amino acid substitution showed a relatively similar distribution, and some disordered regions involved in intermolecular interactions and post-translational modifications were constrained. Describing the pattern of structural order-disorder evolution, this study may give an insight into the well-known characteristics of IDPs in the evolution of CIP/KIP proteins.The structural studies of p21 using proteolytic mapping, circular dichroism (CD) spectropolarimetry, and nuclear magnetic resonance (NMR) revealed that the protein and CDI/KID domain are lack of folded structure17. Even though this region, particularly in p21 and p57, were often predicted to be ordered in this study, the structural studies reported that this domain is lack of folded structure. The characteristics of these fragments that could pre-formed structural elements are likely to result in the order propensity in the prediction method. On the other hand, even though the order-disorder transitions were commonly centered in this region based on the prediction, the CDI/KID domain of all Cip/Kip proteins generally had lower rates of evolution than average, despite there were still some other sites that showed faster rates than average. The CDI/KID domain itself is the binding site for regulating cyclin and CDK complexes1; thus its structure could be altered through the balance between the specificity and affinity of cyclins and CDKs throughout evolution. Evolutionarily, cyclins show great diversification and complexity in various organisms ranging from protists to animals. Likewise, the numbers of CDKs have also increased throughout species evolution as well as cyclins31. The faster rate of amino acid substitution found in the CDI/KID domain is likely to be an important site for adapting the affinity to the binding partner. However, the generally conserved sequences found here suggested that the CDI/KID domain still maintain their specific structures in the vertebrate.Interesting results were shown in the CDI/KID domain, in which all CIP/KIP proteins noted to have a conserved secondary structure in all vertebrate sample, and both p21 and p57 have several transitions of order-disorder propensities in the evolution of vertebrate. The structural studies of these proteins revealed that the CDI/KID domain is likely to have a transiently populated secondary structure which may exhibit a pre-molten globule-like of structure Fig. 16,17. Ev33. In addition, p57 was also found to interact with PCNA (via aa 271–275), with a lower affinity than p21 (ref.33). PCNA itself appears to be conserved throughout evolution, and most plant PCNAs exhibit features similar to those found in PCNAs of other eukaryotes34. Thus, the PCNA-binding motif of p21 and p57 might be constrained to maintain the binding affinity. The short linear motif (SLiM) displays a common functional module in the disordered region. It can bind to the globular domains helped by flexible structure around them26. Accordingly, both proteins have a similar critical function in their interaction with PCNA; both proteins have an ability to inhibit PCNA activity and ability in DNA replication, thereby facilitating the role of the cell repair machinery throughout the cell cycle33. Further, the PCNA-binding motifs of both p21 and p57 have the constrained disordered structures in vertebrates, even though the structural secondary structure prediction showed that p21 PCNA-binding motif commonly have a strand propensity. These supported the well-known characteristics of IDP that essential intermolecular interactions and conserved binding partners could constrain disordered regions through evolution.Another important region of the CIP/KIP proteins is located near the C-terminal, of which p21 has proliferating nuclear antigen (PCNA)-binding motif Fig.  and p27 6. Predicted to be conserved in disordered structure, the fragments that have lower rates of evolution are relevant to mediate the protein-protein interaction. For p27, many conserved and functional phosphorylation sites, which determine various fates including cytoplasmic localization, stability, and degradation, lie in the QT-box motif36. Likewise, for p57, besides the existence of a PCNA-binding motif inside the QT-box motif, an important phosphorylation site, T310, for protein degradation is also located in this domain37. Since there are many important phosphosites in the QT-box motif, flexibility may be required in the structure of this domain to keep those sites exposed. In addition, functional phosphosites and binding sites need to be constrained in order to maintain the function.The QT-box motif showed to have both lower and faster rate of amino acid substitution than average in both p27 and p57, even though its structure was constrained to be disordered. The heterogeneity of the rates of amino acid substitution here likely displays that this region contains multiple short linear motifs (SLiMs). The QT-box is a structural motif that previously reported to function in protein-protein interaction39. Thus, it suggested that the substitution of amino acid in this region of CIP/KIP proteins is constrained to a specific residue. The conservation of disordered structure having rapid amino acid substitution rates may be related to their functions as flexible linkers that allow movement of the important region positioned on either end or spacers which regulate the distances between important region26. Indeed, this mechanism of evolution seems to only maintain the flexible properties of the region. Here, the constrained sequences may not be necessary, as the mutation yields a sequence that is flexible in structure. Accordingly, there may still be a selection for faster rates of substitution in disordered regions. This characteristic may be the main reason why disordered proteins generally have faster rates of evolution than ordered proteins.All human CIP/KIP proteins have important regions near the N-terminal and C-terminal that are separated by regions whose rate of amino acid substitution, for each sequence, is commonly faster than average for all vertebrate species. However, despite their faster rate of substitution, these regions were constrained to have a disordered structure for all vertebrate species, except for the more primitive mammalian species with p57, which does not have these regions. Based on the sequence composition, disordered regions generally have more charged and fewer aromatic amino acids as well as faster rates of amino acid substitution than ordered regions29. Frequent repeat expansions, insertions, and deletions could create an obvious divergence in the sequence length over evolution. However, only p57 showed a clearly different sequence length over evolution in its disordered region. In contrast, p21 and p27 exhibited high rates of amino acid substitutions without any obvious differences in sequence length. In addition, the PCNA-binding motif and QT-box motif of p57 also could only be detected from aves to mammals and primates based on the multiple sequence alignment. What are the factors that make p57 to occupy similar motifs to other CIP/KIP members in the evolution process of vertebrates? the elucidation of the actual factors responsible for the distinct frequency of insertions and deletions between these proteins clearly needs further studies.Interestingly, the CIP/KIP family has two different mechanisms of evolution in maintaining the length of the sequence, particularly in disordered regions from after the CDI/KID domain to the C-terminal. Generally, disordered regions also tend to occupy repeat expansions and to have more insertions and deletions than ordered regions despite the faster rate of amino acid substitution40. In addition, for example in yeast, disordered proteins are associated with twice as many kinases as structured proteins on an average30. Phosphorylation plays important roles in protein function and is critical for biological processes and subcellular protein localisation41. Therefore, the evolution rates of phosphosites are assumed to be different from those of nonphosphorylated residues in the disordered region. Based on the predictions here, several of the predicted phosphosites of human CIP/KIP proteins were shown to have a lower rate of amino acid substitution. Some of them are well-studied phosphosites whose functions are known and have a conserved propensity to be phosphorylated; however, some phosphosites showed a different pattern; in particular, T157 in p27 that showed to have a higher rate of amino acid substitution. On the other hand, some well-known functional phosphosites of CIP/KIP proteins in humans were shown to have a higher rate of evolution but were not conserved to be phosphorylated in vertebrates. Therefore, in CIP/KIP proteins, the existence of phosphosites in the disordered region may constrain the rate of evolution.The promiscuity of IDPs is highly affected by post-translational modifications (PTMs), particularly phosphorylation, which influences the binding affinity of disordered proteins44. In human p27, the number of conserved phosphosites was higher than that in the other Cip/Kip proteins; however, some of the conserved phosphosites, including S7, S161, S175, S178, and S183, have not yet been well-studied. Interestingly, the well-studied conserved phosphosites in this protein, S10, T157, T187, and T198, have a similar function to the human p21 conserved phosphosites. The activities of p21 and p27 are controlled by their concentration and subcellular localisation, and modulating these factors may expose the proteins to a different range of potential binding partners. Thus, phosphosites modulating essential functions may be conserved. However, identifying other phosphosites that have overlapping functions must also be a concern, since some of the non-conserved human p21 and p27 phosphosites were also shown to have a similar function to the conserved phosphosites of each protein, even though they are modulated by different signals; for example, T57 and T80 in human p21 that modulate degradation45.In human p21, T145, S146, and S153 showed a conserved propensity to be phosphorylated in the vertebrate. Each of these phosphosites has an important function in modulating either cytoplasmic relocalisation, protein degradation, or protein stability of p2146. However, only T310 was shown to have a lower rate of amino acid substitution than the whole site on average. Since p57 has a frequent insertion in vertebrates throughout evolution, the functional modification sites may have been shifted via the unstable length of the linkers, and thus, making it difficult to trace the conserved function through evolution. Therefore, an extensive amount of insertions and deletions in disordered regions could affect the perceived rate of evolution of modification sites, particularly at greater divergence times29.On the other hand, based on the phosphorylation propensity, the single conserved phosphosite of p57 found here, S28, has not yet been well-studied. Currently, only S282 and T310 of human p57 have been well-studiedIt is important to remember that the properties of structural disorder and phosphorylation sites of CIP/KIP proteins in this study have been inferred using linear sequence predictors. Therefore, the results in this study cannot be interpreted as a perfect or true depiction of the nature of IDPs. However, since experimental data is not available, these results can still be used and considered as the viewpoint of computational biology.In conclusion, the disordered region in CIP/KIP proteins that exposes many functional phosphosites and binds to various regulators is generally essential for the multifaceted biological roles of CIP/KIP proteins. These regions of CIP/KIP proteins frequently have lower rates of evolution and could conserve the structural properties. The CIP/KIP disordered region that is placed between the important regions in the N-terminal and C-terminal has a conserved disorder propensity with high rates of evolution. The presence of this type of region could result in a significantly faster rate of evolution compared to the ordered proteins. Considering this study was based on prediction, it cannot be given as a true depiction. Therefore, further study related to the dynamic nature of IDPs is required. However, this study could give an insight into the well-known characteristics of IDPs in the evolution of CIP/KIP proteins.47) under the blastp (protein-protein BLAST) algorithm (Supplementary Dataset 1). For each CIP/KIP protein, the human protein sequence  was used as the reference to obtain vertebrate sequences in the RefSeq database (ref.48). Only one sequence with the highest Max score of BLAST for each species was used for each CIP/KIP protein to minimise redundancy. For alignment, whole vertebrate sequences and representative organism sequences were constructed for each dataset of CIP/KIP proteins. The species included in the representative dataset were Homo sapiens, Rhinopithecus roxellana, Mus musculus, Sarcophilus harrisii (for p21 and p27), Plethodon cinereus (for p57), Chrysemys picta bellii, Gallus gallus, Latimeria chalumnae, Danio rerio, Xenopus laevis (for p21 and p27), Nanorana parkeri (for p57), Callorhinchus milii (for p21 and p27), and Rhincodon typus (for p57). All datasets were aligned using MAFFT version 7 (ref.49). The iterative refinement method L-INS-I was used for the alignment strategy with maximum of 1000 iterations. Pairwise distance was calculated for representative data alignment of each CIP/KIP protein using Mega7 (ref.50).Vertebrate sequences were collected for each CIP/KIP protein, including p21, p27, and p57, from the NCBI database using NCBI BLAST (ref.51) in the CIPRES Science Gateway (ref.52), with Jones, Taylor, and Thornton53 amino acid substitutions with + F method and gamma shape parameter (JTT + F + G) model which was selected as the best fit model under Akaike Information Criterion (AIC) by Aminosan (ref.54) for all vertebrate datasets. The analysis used four simultaneous metropolis-coupled Markov chain Monte Carlo for 5,000,000 generations with a sampling frequency of 1,000 generations. The consensus topology trees were calculated by discarding the first 25% of trees using the 50% majority rule.Phylogenetic trees were constructed using Bayesian inference from each dataset of vertebrate CIP/KIP protein alignment. The trees were estimated using MrBayes 3.2 (ref.55) with the option for long disordered regions. This prediction has values ranging from 0 (strong propensity for an ordered structure) to 1 (strong propensity for a disordered structure), with 0.5 as the cut-off between order and disorder. However, we used 0.4 as the order-disorder propensity cut-off instead of 0.5 in this study because the former value is considered more accurate in predicting the experimentally verified disordered structure in the DisProt database than the original cut-off 56. The order-disorder propensity scores were mapped onto the multiple sequence alignment and taxon position in the tree, and visualised in a heat map using iTOL (ref.57). Further, the order and disorder propensity results were also converted into binary matrix, 0 and 1, respectively, to analyse the evolutionary dynamics of structural order-disorder.The structural disorder propensity of all unaligned vertebrate datasets was predicted using IUPred (ref.58) for all sequences in the datasets. A site having a value of greater than or equal to 0.75 was predicted to be phosphorylated, whereas a value of less than 0.75 was predicted to be not phosphorylated. The prediction results for each sequence were plotted following the multiple sequence alignment and phylogenetic tree of each dataset. Furthermore, the predicted phosphorylated site of the human sequence was considered to be conserved through evolution if it was predicted to be phosphorylated in the vertebrate sequence following the 50% majority rule of the amount of sequence in the alignment.Phosphorylation site was predicted using NetPhos (ref.59). This tool incorporates the Jnet algorithm and has 82.0% accuracy in predicting secondary structure59. The prediction was performed for all sequences in each dataset. The results of the prediction were mapped and visualised in a heat map. In addition, the results of secondary structure propensity were also converted into binary matrix, 1 for helix and strand, and 0 for loop, to examine the evolutionary dynamics of secondary structure-loop transitions.Secondary structure was predicted using Jpred 4 (ref.60). The analysis was performed using the empirical Bayesian principle with the Jones, Taylor, and Thornton amino acid substitution model and 16 discrete categories of the prior gamma distribution. The Bayesian inference tree of each dataset was used for the input tree, and the branch lengths were not optimised. Human CIP/KIP protein was used as the reference sequence. Gap was treated as missing data based on sequence reference.The substitution rates of amino acids for each vertebrate dataset alignment were analysed using Rate4Site (ref.61. Gain Loss Mapping Engine (GLOOME) was used with equal gain and loss and 6 categories of gamma rate distribution62. The Bayesian inference tree and human sequence for each dataset were used for the input tree and the reference sequence, respectively. The gap was treated as missing data, and the outputs of evolution rates were standardized as Z-score.The evolutionary dynamics was investigated by calculating the evolution rates based on the phylogenetic trees and binary matrices for each secondary structure prediction and structural order-disorder prediction. This analysis was used to study the gain/loss transitions of the structural property by adopting the phyletic patternsSupplementary figure 1, Figure 2, Table 1, Table 2, and Table 3Dataset 1


However, the concentrations of these metals were not correlated with traffic density, probably due to extensive road maintenance and expansion. The contamination factor demonstrated that the road edge soils were moderately- to highly-polluted with Cd, Cu, Pb, and Zn. The safest distance to minimize metal pollution for agricultural production is proposed to be greater than 10 m away from the road edge.Vehicle emissions have been known to cause trace metal contamination in soils. The extent of such contaminations in soils, and of the effects of traffic density and distance from highways on the concentration of trace metals in roadside agricultural soils is largely unknown. This study examined the total concentrations of common trace metals  in roadside agricultural soils from Thailand with diverse traffic densities (approximately 30–200 million vehicles/kilometer/year), roadside distances , and crops . Cadmium, Cu, Pb, and Zn concentrations significantly decreased with increasing distance away from the roads ( Traffic is a primary source of trace metal pollution in roadside soils, and plays a pivotal role in the biogeochemical cycling of trace metals, and in turn substantially affects human health ,2,3. Trap < 0.05) correlated with vehicle volume [The distribution patterns of trace metals in soils have been found to be associated with the distance from the road. A number of studies have demonstrated that trace metal concentrations typically decrease with increasing distance from the road ,8,9,18. e volume .Thailand is an agricultural country, and roadside soils are often being used for economic crop production, such as rice, maize, and sugarcane. However, little research has been done in relation to trace metal concentrations in roadside agricultural soils. Therefore, the objectives of this study were to evaluate the concentrations of trace metals in roadside agricultural soils and to examine the effects of vehicle density and the distance from the road on the concentrations of trace metals contamination in roadside agricultural soils. The results from this research could provide a suitable distance from the road for crop production to help reduce the pollution risk of trace metals from traffic.Samples of roadside agricultural soils in this study were obtained in 2016, and the sampling sites are shown in 4OAc (pH 7.0). Organic carbon content was determined using the dichromate oxidation procedure, then multiplied by the Van Bemmelen factor of 1.724 to calculate organic matter [Three transects were sampled next to each highway, and for each transect soil samples were obtained at five distances—0, 10, 20, 50, and 100 m—from the road edge. The soil samples were taken from the surface to the base of the Ap horizon, except for the road edge sample (0 m distance) that was taken from 0–10 cm. The road edge samples were considered as representative of the trace metal concentrations released from motor vehicles. A total of 165 soil samples were collected and stored in plastic bags for laboratory analysis. Soil samples were air-dried, gently disaggregated, and sieved through a 2 mm sieve to remove gravel, plant debris, and plant litter. The sieved soil samples were used for particle size distribution using the pipette method and general chemical analysis, except for the determination of organic carbon and total metal concentrations, where the sieved soil was finely ground to <0.5 mm. The methods used for the soil analyses are described in Soil Survey Staff . Soil pHc matter .3:HClO4 (5:2). One gram of each sample (0.5 mm sieve) was weighed into a digestion tube, then 10 mL of the mixed acids was added to the digestion tube, and the sample was open digested at 150 °C for 3 h, or until a clear solution was obtained. The final volume was made up to 25 mL. The clear solution was preserved, and subsequently diluted before the elemental analysis. Total concentrations of Cd, Co, Cr, Cu, Ni, Pb, V, and Zn were determined by inductively coupled plasma-optical emission spectrometer (ICP-OES)  using an axial view of the plasma. Standard solution (PerkinElmer Number N9300281) for the ICP was used for preparing calibration curves and used as the internal standard. Metal concentrations in the digests were checked against stream sediment reference material (STSD-3)  for quality assurance. The deviation between standard replicates (n = 3) for the metal concentrations was within 0.98–3.97% RSD (mean = 2.02%).Total trace metal concentrations of soil samples were determined by a wet digestion procedure using a mixture of HNOi is the contamination factor for a trace metal , Ci is the concentration of the trace metal in the sample, and Si is the background concentration of the trace metal in the soil sample at a distance of 100 m from the road edge [The contamination factor (CF) was used to assess the enrichment of trace metals in the soils and to measure the pollution levels ,27. The oad edge ,20,21,22oad edge ,27:CFip < 0.05. A Pearson correlation was also performed at p < 0.05 and p < 0.01. A principal component analysis (PCA) was performed to demonstrate the association of metal concentrations with relevant soil properties and related factors.The trace metal concentrations were analyzed using a one-way analysis of variance (ANOVA), and the mean values were compared among sampling distances for each highway using Duncan’s Multiple Range Test (DMRT) at −1). The organic matter (OM) content varied from low to moderately high (7–61 g kg−1), while the cation exchange capacity ranged from low to high (3.5–28.0 cmolc kg−1) , and non-saline (ECe 0.2–2.4 dS mlc kg−1) . Most ofp < 0.05), as shown in The total concentrations of trace metals in the roadside agricultural soils  are presented in In this study, the Co, Cr, Cu, Ni, V, and Zn concentrations in some roadside agricultural soils were found to be within, or higher than, the critical values for total trace metal concentrations in soils that may be toxic to plants . Most ofr = −0.176 *, −0.220 **, −0.323 **, and −0.359 **, respectively), as shown in The results showed the negative correlation of Cd, Cu, Pb, and Zn concentrations with increasing distance from the road edge .Although several studies reported that trace metal distributions in roadsides soils were related to traffic density ,18,23, tThe first two factors from the PCA accounted for 69.38% of the data variability, which signified the heterogeneous nature of the studied soils, as shown in The second group of elemental affinity was related to Ni, Co, V, and Cr, and their concentrations were elevated in the maize growing soils taken from highways 2090 and 2273 at the sampling distances of 10–100 m. These metal groups are typically substituted in the structure of natural clay minerals and iron oxides ,37. HighRoadside agricultural soils under paddy rice showed CFs of trace metals  ranging from 0.9 to 3.8, indicating a range from no pollution (CF < 1) to considerable pollution (3 ≤ CF ≤ 6). However, in the road edge soils the Cd, Pb, and Zn levels (CF) were clearly higher than in the 10–50 m soils. The CFs of maize growing soils varied from 0.5 to 1.9, indicating no pollution to moderate pollution (1 ≤ CF < 3). For the sugarcane growing soils, the CFs (0.9–10.2) indicated a range from no pollution to very high pollution (CF > 6); the levels of trace metals at the road edge were clearly higher than those in the 10–50 m soils, as shown in Rice: Zn > V > Cr > Cu > Pb ≈ Ni > Co > Cd;Maize: V ≈ Zn > Cr > Cu ≈ Ni > Co > Pb > Cd;Sugarcane: Zn > V > Cr ≈ Cu > Pb > Co > Ni > Cd.The results showed that roadside agricultural soils contained Cd, Co, Cr, Cu, Ni, Pb, V, and Zn. The levels of trace metals in roadside agricultural soils for rice, maize, and sugarcane were as follows:p < 0.05). Furthermore, Cd, Cu, Pb, and Zn showed a negative correlation with the distance from the highway, with the concentrations of Cd, Cu, Pb, and Zn decreasing with increasing distance from the road edge. However, the traffic density failed to make a clear contribution to the accumulation of trace metals in the roadside agricultural soils in this study, probably due to road expansion as a part of the urban development process. Moreover, mean concentrations of Cd, Cu, Pb, and Zn in road edge soils indicated that the soils were moderately to considerably polluted. This study proposes that the optimum spacing for plantations is more than 10 m away from the road edge to reduce the pollution risk of Cd, Cu, Pb, and Zn in roadside soils. Large accumulations of Cd, Cu, Pb, and Zn in roadside agricultural soils should be taken into consideration, as they have a high potential to enter into the food chain, which can cause adverse effects on biota and terrestrial environments.The Cd, Cu, Pb, and Zn concentrations in most road edge soils were statistically significantly higher than those in the soils at a distance of 10–100 m (


ReIn) from multiple experiments in a large-scale metabolomic profiling has become a widely used strategy for enhancing the reliability and robustness of analytical results, and the strategy of direct data merging (DiMe) among experiments is also proposed to increase statistical power, reduce experimental bias, enhance reproducibility and improve overall biological understanding. However, compared with the ReIn, the DiMe has not yet been widely adopted in current metabolomics studies, due to the difficulty in removing unwanted variations and the inexistence of prior knowledges on the performance of the available merging methods. It is therefore urgently needed to clarify whether DiMe can enhance the performance of metabolic profiling or not. Herein, the performance of DiMe on 4 pairs of benchmark datasets was comprehensively assessed by multiple criteria . As a result, integration/merging-based strategies (ReIn and DiMe) were found to perform better under all criteria than those strategies based on single experiment. Moreover, DiMe was discovered to outperform ReIn in classification capacity and robustness, while the ReIn showed superior capacity in controlling false discovery rate. In conclusion, these findings provided valuable guidance to the selection of suitable analytical strategy for current metabolomics.Because of the extended period of clinic data collection and huge size of analyzed samples, the long-term and large-scale pharmacometabonomics profiling is frequently encountered in the discovery of drug/target and the guidance of personalized medicine. So far, integration of the results ( ReIn of multiple experiments in large-scale metabolomics has been applied to enhance the reliability and robustness in cancer-related metabolites profiling  together with the strategies based on single experiment were collectively evaluated by multiple criteria. In conclusion, these findings provided a valuable guidance to the selection of suitable analytical strategy in a given metabolomics study.In the study, comprehensive evaluation of different analytical strategies was conducted by assessing their MetaboLights database . Second, several criteria were used to ensure the availability and processability of raw metabolomics data, which included (a) complete set of raw data files, (b) well-defined parameters , (c) enough samples (>10) in each experiment, (d) same classes of both cases and controls in different experiments, and (e) clear description on the sample groups. The application of the above criteria to those 339 projects resulted in eight benchmark metabolomics datasets of varied sample sizes. In particular, these eight datasets included (1) a UPLC-QTOF MS dataset based on the serums of 59 patients of HCC and 129 CIR patients collected at Georgetown University Hospital (GUH) and run in positive mode from an experiment conducted in May 2010  a mMay 2010 , (3) a UMay 2010 , (4) theMay 2010 , (5) theMay 2010 , (6) theMay 2010 , (7) UPLMay 2010 , and (8)May 2010 .DiMe strategy applied in this work was systematically illustrated in DiMe strategy, which included the pair of experimental dataset (1) and dataset (2) from MTBLS17 ESI+ . Furthermore, another 10 datasets were generated by the random sampling of half of the merged dataset for 10 times, which were further used for evaluating the robustness of DiMe strategy .Prior to the biomarker identification, the datasets were frequently pretreated in current metabolomics study . Herein,p-value < 0.05 . Meanwhile, another 10 datasets were generated by the random sampling of half of the pretreated experimental dataset for 10 times, which were applied for the evaluation of robustness of the ReIn strategy . Fourth, PLSDA was used to identify the differential metabolic peaks between distinct sample groups within each dataset . The resulting metabolites annotated based on HMDB by setting the m/z tolerance equal to 20 ppm were the metabolic biomarkers finally identified. Finally, the metabolites annotated from two experimental datasets were collectively considered for assessing identification precision of the ReIn strategy, the classification models constructed based on experimental datasets were integrated for evaluating ReIn’s classification capacity, and the robustness of the ReIn strategy was also collectively determined by the average overlap values between two experiments. All in all, the workflow of ReIn strategy applied in this study was systematically illustrated in The workflow of the  package  by settiw to ten . Second,identification precision, classification capacity and robustness. As reported, these three criteria were independent from each other . Then, independent test set was used to assess the classification capacity of constructed model, which was evaluated by the ROC analysis together with the measurement of AUC  for each dataset, and ten lists of biomarkers were discovered. Third, for any 2 marker lists, the fraction of shared marker appearing on both lists were used to measure the similarity of these two lists. Particularly, overlap value was calculated (shown in Eq. 6) based on marker lists a and b. The closer the overlap value equal to 1, the more robust the markers discovered in that study (102 ) overlap values denoting all possible combinations between any two sub-datasets were thus calculated and analyzed here.First, ten sub-datasets were generated by the random sampling of half of the pretreated experimental/merged dataset for ten times. Second, the biomarkers were identified using Student’s at study . For eacwhere a and b indicated two maker lists, and Na and Nb denoted the number of markers in each list.ACC, SEN, SPE, MCC, ROC, and the area under ROC curve  . Similar to ACCs, the MCCs of DiMe (0.44∼0.60) were discovered to be robustly higher than that of the other strategies (0.06∼0.32), and the majority (75%) of DiMe’s MCCs were larger than 0.50.Classification model was frequently constructed in current metabolomics research to predict samples of different disease states  or assesC value) . As illurectness  and stabrectness  of constACC and MCC, the ROC and AUC were two other popular metrics widely used to assess classification ability, which were acknowledged to achieve a comprehensive performance evaluation. As illustrated in ROC curves and the AUC values of 4 benchmark datasets  were compared. Two benchmark sets (MTBLS17-NEG and MTBLS17-POS) contained 503 samples , and the other datasets MTBLS19-NEG and MTBLS19-POS consisted of 180 samples (100 patients with liver cirrhosis and 80 patients with HCC). The gray diagonals represented an invalid model with the corresponding AUC value equaled to 0.5. As shown in AUC values of DiMe among different datasets (0.82∼0.93) were substantially and consistently higher than that of the other 3 strategies (0.57∼0.80), which were similar to the results assessed by ROC curves. In conclusion, this finding indicated that classification correctness  and prediction stability  of the direct merge strategy (DiMe) were found consistently better across multiple benchmark datasets compared with the SiE1 and SiE2 strategies and the one of results integration (ReIn).Apart from overlap value had been recognized as the quantitative measure of the robustness of the identified markers , and the value of overlap between any two sub-datasets was calculated using their corresponding lists of markers identified. In total, there were 45 . Moreover, although there was great difference among the median overlap values (from 0.15 to 0.40), the median overlap of DiMe was found consistently larger than that of the other three strategies.Apart from prediction capacity evaluated simultaneously by classification correctness and prediction stability, the robustness of identified metabolic markers was widely accepted to be another important metric with underlying theory distinct from that of prediction capacity . So far, markers . The higoverlap, the statistical difference of 45 overlap values between different analytical strategies was more meaningful to reveal the level of robustness for each strategy. Thus, comprehensive statistical comparison of robustness among different strategies was conducted and illustrated in overlap values of SiE1, SiE2, ReIn, and DiMe were colored in light green, dark green, blue, and orange, respectively. Apart from the enhanced median values of overlap by DiMe, all overlap values of DiMe were found statistically higher  compared with that of the other strategies. In particular, as illustrated in DiMe and other strategies  were always lower than 0.05 within the range from 4.25E-16 to 1.81E-02. Moreover, the majority of the overlap values of DiMe were larger than 0.3, while that of the other strategies were lower than 0.3. These findings indicated that the DiMe strategy performed better than others in the robustness of the identified markers. Additionally, N ≥ 6, ≥ 7, ≥ 8, ≥ 9, = 10) sub-datasets, which included the number and percentage of markers co-identified by these N datasets. It was very clearly to see that the robustness of metabolic markers identified by DiMe was much better than other three strategies in terms of both the number and the percentage of co-identified markers. Particularly, the percentages of markers identified by over five sub-datasets using DiMe were within 3.25%∼5.07%, while that using SiE1 and SiE2 were 0.87%∼2.74% and 0.93%∼2.06%, respectively. Moreover, the percentages of markers identified by all sub-datasets using DiMe were within 0.00%∼0.41%, while that using SiE1 and SiE2 were 0.00%∼0.25% and 0.00%∼0.21%, respectively.Compared with the median value of glycochenodeoxycholic acid) were identified from serum samples combining TOF MS/MS with UPLC-SRM-MS/MS based on the internal standard isotope dilution  were detected by the targeted analysis based on UPLC-ESI-TQMS , the EF values of ReIn in three experimental datasets  were found to be obviously higher than those of DiMe strategy, which reflected the superior ability of ReIn strategy in controlling false discovery rate. However, in one extreme case (MTBLS17-NEG), the EF of ReIn was lower than that of DiMe. Careful investigation of ReIn, which led to a huge decline in its EF values. Therefore, although ReIn demonstrated superior ability to control false discovery rate, its application could be limited by its relatively small number of true markers identified.MetaboLights, a comprehensive evaluation of different analytical strategies was conducted by assessing the classification capacity, robustness and false discovery rate. As a result, the integration/merging-based strategies (ReIn & DiMe) performed better than strategies based on single experiment (SiE1 & SiE2). Moreover, DiMe strategy was found to outperform ReIn in classification capacity and robustness, while ReIn demonstrated superior capacity in controlling false discovery rate. In summary, these findings may facilitate current metabolomics study in classification capacity, identification precision, and robustness.Based on the systematic review of FZ conceived the idea and supervised the work. XC, QY, and BL performed the research. XC, QY, BL, JT, XZ, SL, FL, JH, YL, YQ, and WX prepared the program and analyzed the data. FZ and JH wrote the manuscript. All authors have read and approved this manuscript.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.


Quality indicators and possible ecological risks for metals in surface sediments were evaluated at 60 sampling sites of these three gulfs using the contamination factor (CF), the contamination degree (CD), the pollution load index (PLI), the geoaccumulation index (Igeo), the potential risk factor (PRFi), and the potential ecological risk index (PERI). Based on Igeo, the Ierissos Gulf sampling sites IER 2, 3, 7, and 9 exhibit moderate Pb pollution, whereas the sampling sites IER 6 and 8 show moderate to strong and strong Pb pollution, respectively. Based on the PRFi and PERI, the studied heavy metals did not pose any significant environmental risks for most of the investigated sites except IER 6 and 8 sampling sites, which may pose considerable environmental risk for Pb. To evaluate potential sources for each metal, multivariate techniques including hierarchical cluster analysis and ANOVA were used.Pollution with copper (Cu), lead (Pb), zinc (Zn), chromium (Cr), and nickel (Ni) heavy metals of the surface sediments collected from three semi-closed East Mediterranean Gulfs, namely Kavala, Strymonikos, and Ierissos Gulfs, North Aegean Sea, Greece, was investigated to evaluate potential benthic ecological risks. The mean concentrations of the studied metals decrease according to the order: Zn > Pb > Cr > Ni > Cu (176.50, 166.23, 127.41, 43.12, and 33.64 mg kg Benthic habitats such as coastal zone sediments play an important role in the marine environment. Surface sediment contamination is one of the most important quality indicators for the assessment of potential ecological risks in coastal marine ecosystems. Therefore, the sediment may be regarded as a repository of pollutants, since after their entry in the water column, adsorption to finer particles and complexation may lead to their accumulation through the settling process into sediment ,2,3,4,5.Some common anthropogenic activities that contribute to the release of Cu, Pb, Zn, Cr, and Ni species into the coastal marine environment consist in the following: mining operations, effluent discharges from fertilizer factories, oil refineries, and municipal sewage, spills during shipping and terminal transfers, intense agricultural activities, leaks of oil from offshore platforms, and usage of anti-corrosive and anti-fouling paints. Furthermore, river discharges, containing both natural and anthropogenic Cu, Pb, Zn, Cr, and Ni species contribute to the increase in metal contents of the coastal environment ,17,18,19The Gulf of Kavala is a shallow marine ecosystem in the North Aegean Sea, flanked by the Symvolon Mountains to the west, the island of Thassos to the southeast, and the Nestos delta plain to the east and north . The gulThe Strymon River originates from the Vitosha Mountain in Bulgaria, flows through the central part of the metamorphic basement of the Rhodope Mountains, passes through the dam lake Kerkini, and outflows into Strymonikos Gulf in the North Aegean Sea . Its draThe Gulf of Ierissos, located to the south of Strymonikos Gulf, has an elliptical shape and NW-SE orientation with a 10 km opening in length towards the Strymonikos Gulf . In its geo, PRFi, and PERI, and (3) to identify potential sources of these metals, using statistical methods. This research approach could be helpful to understand the sediment quality distribution under the effects of the existed anthropogenic and/or natural activities, and thus support the strategic management of pollution with heavy metals in this specific coastal area.The water and the sediments in the above three gulfs contaminated in toxic pollution including the studied heavy metals, could have adverse effects to phytoplankton and other organisms or benthic communities, most of which form the basis for the marine food chain. Thus, the objective of the present study is (1) to assess the associated possible ecological risks of the existent concentration of five  heavy metals in the surface sediments of the three neighboring gulfs by analyzing data from 60 sampling sites, (2) to assess possible ecological risks associated to sediment contamination in heavy metals using various indexes, such as CF, CD, PR, PLI, IThe sampling strategy adopted aimed at detecting pollution sources as local point and non-point pollution sources, such as effluent discharges from mining operations or fertilizer factories, oil desulfurization plant and municipal sewage outflows, intense agricultural activities, possible spills during shipping transfers, leaks of oil from offshore drilling platforms, and usage of anti-corrosive and anti-fowling paints from the local shipping repair yards, as shown in 3 for 1 h and rinsed thoroughly with double distilled deionized water.The sediment samples were carefully collected to avoid disturbance with a Van Veen stainless-steel grab (dimensions 40 × 40 cm) from the upper 5 cm, during four sampling cruises . The upper 5 cm layer was collected because it is more chemically active than the deeper layers, and substance exchanges between sediment and water occur in this layer. After collection, sediment samples were immediately placed at 0 °C, on board. Subsequently, until metal analyses, the samples were stored at −28 °C in plastic vessels, cleansed with 1N HNO3 and rinsed with double distilled deionized water to avoid any metal contamination. Acid washed glassware, analytical grade reagents, and double distilled deionized water were used for the sediment analysis. The analyses were carried out in triplicate, and the significance level was set at 0.05. Detection limits (in μg kg−1) were 0.014, 0.05, 0.02, 0.004, and 0.07 for Cu, Pb, Zn, Cr, and Ni, respectively. Analytical quality control was achieved using certified reference material for marine sediment . The recovery percentage of Cu, Pb, Zn, Cr, and Ni concentrations from the reference material was (%) 98.77, 98.33, 98.15, 97.61, and 96.32, respectively. In order to determine the precision of the analytical procedure, samples from 5 sediments were analyzed four times. The standard deviation for these samples was calculated to (%) 1.9, 2.5, 2.2, 2.7, and 3.1 for Cu, Pb, Zn, Cr, and Ni, respectively, reaching the conclusion that precision of analysis is at satisfactory levels. In the chemistry laboratory, sediment samples, after defrosting, were passed through a 1 mm plastic sieve, dried at 50 °C up to a constant weight, and homogenized in a porcelain mortar with a pestle. A sediment sub-sample of 0.25 g was digested in a mixture of 4 mL of aqua regia and 1 mL of concentrated HF for 30 min using a Q-6000 microwave digestion system . The resultant solutions were then diluted to a 25 mL volume (Erlenmeyer flask) with double distilled deionized water. Quantitative determinations of Cu, Pb, Zn, Cr, and Ni species were accomplished in triplicate using an Analyst 800 atomic absorption spectrometer  equipped with a transversely heated graphite furnace (THGA) and longitudinal Zeeman-effect background corrector, AS-800 autosampler, 8-lamp turret, PE AA accessory cooling system, and the WinLab32 PC work station. The sediment material was always handled using plastic materials that were washed with 1N HNO−1 for Ni.As described below, several different methods were used to express heavy metal pollution in the studied sediments. Due to the lack of relevant background data for the unpolluted sediment in the area under investigation, the background heavy metal concentrations  from the closely located Alexandroupolis Gulf, Northeast Aegean Sea, provided by Kanelopoulos et al.  were usem denotes the content of the specific heavy metal investigated, and Cb is the local uncontaminated background level for the same metal. Generally, CF values are categorized at four classes, e.g. CF < 1; 1 ≤ CF < 3; 3 ≤ CF < 6 and CF ≥ 6, indicating low, moderate, considerable, and very high contamination, respectively [CF and CD are widely used proxies for the estimation of the anthropogenic impact on sediment chemistry, referring to the enrichment degree of metal concentrations in sediments investigated relative to uncontaminated background levels (baseline) ,28,29,30ectively . Likewisectively ,31.n is the number of metals and CF the contamination factor of metals studied. PLI values close to 0 or 1 indicate perfection or baseline levels of pollutants present, respectively; on the contrary, PLI values >1 indicate progressive deterioration of the area [The overall contamination sediments in heavy metals content of sediments at the studied sites was evaluated using the PLI, calculated with the following equation .PLI = (the area . geo) was applied using the following equation [n is the concentration of the examined metal in the studied surface sediment sample, Cbn is the geochemical background concentration of a given metal [geo  [mi, the concentration of metal i in the sediment; and Cbi, the regional background of the metal i in sediments. The following thresholds were used to interpret the PERI and PRFi values [A potential ecological risk index (PERI) was used to evaluate the potential ecological risk of heavy metals in the sediments ,31. The  Ni = 2) ; Cmi, thi values :Low risk:PRFi < 30; PERI < 100Moderate risk:30 < PRFi < 50; 100 < PERI < 150Considerable risk:50 < PRFi < 100; 150 < PERI < 200Very high risk:i < 150; 200 < PERI < 300100 < PRFDisastrous risk:i > 150; PERI > 300PRFp < 0.05 was considered to indicate statistical significance; (2) cluster analysis among sampling sites and among metals were applied to the standardized through z-scale - transformed data [r) were also calculated. For the interpretation of the data of the chemical analysis the following methodology was used: (1) descriptive statistics  and correlations between elements and sampling areas (gulfs) were performed using the Stat soft Statistica 10 software . The results obtained were expressed as the mean ± standard deviation (SD) and were analyzed by means of analysis of variance (ANOVA). A value of med data  in ordermed data . For themed data ; (3) PolSummary statistics for metal concentrations in the surface sediments of each gulf of the studied area and each metal are presented in p < 0.05), whereas those of all metals exhibited higher mean concentrations in Ierissos Gulf compared to Kavala and Strymonikos Gulf samples. Mean values of Cr and Ni were also significantly higher in Strymonikos compared to Kavala Gulf samples.Mean values of Cu, Pb, and Zn in Kavala and Strymonikos Gulf show no significant differences between samples  and some urban activities (KAV 8–12), as well as a single station positioned at the north of the gulf (close to oil desulfurization plant). This subarea also includes three inshore Strymonikos Gulf stations and two stations from Ierissos Gulf: IER 5 located close to the shore and IER 10 positioned at the gulf’s entrance. Subarea II showed intermediate mean metal concentrations and includes all but one (KAV 13), the remaining eastern (KAV 16–18) and the offshore Kavala Gulf sites , the rest of Strymonikos sites regardless of their position , showing gulf’s homogeneity, and two Ierissos Gulf sites, the southernmost IER 4 and IER 1-site located at the boundary between Strymonikos and Ierissos Gulfs. Subarea III demonstrates elevated mean values for all metals, containing five sites in total: KAV 13 located at the industrial part of Kavala Gulf (close to phosphoric fertilizer plant) and four Ierissos sites  positioned at the gulf’s intermitter-depth transect. Finally, subarea IV includes the most polluted sites of Ierissos Gulf, mostly influenced by mining activities. r = 0.98, p < 0.05) and the second by Cu, Pb, and Zn . The cluster analysis applied for the five investigated metals revealed two individual groups : the firAccording to mean and range CF values , all theThe highest metal pollution load indexes (PLI) were recorded for IER 6 and IER 8 sites  followed by IER 7 (6.68), IER 3 (5.65), IER 9 (5.22), IER 2 (4.98) and KAV 13 (3.41); while the lowest PLI was recorded for KAV 7 site (0.20). According to mean PLI values, the pollution in the study area follows a descending order of: Ierissos Gulf > Strymonikos Gulf > Kavala Gulf . In genegeo values for the three studied gulf areas were represented in geo values for Cu, Pb, Zn, Cr, and Ni in all studied sediments from Kavala and Strymonikos Gulfs belong to class zero, indicating that sediments in all stations are unpolluted by these metals. On the other hand, sediments of Ierissos Gulf sites fluctuated from unpolluted  to moderately , moderately/strongly , and strongly polluted  by Pb (The I7) by Pb . i and PERI for metals in the sediments of the three studied gulf areas. Among the studied heavy metals, Pb reported the highest ecological risk, because of its considerable high toxicity factor (PRFi) in some sampling sites of Ierissos Gulf. Moreover, the PRFi Pb mean value is calculated by 5.77 ± 12.24, which indicated that Pb content varied greatly through the sampling sites of the three gulfs. About 97% of the sampling sites recorded PRFi Pb values lower than 30 indicating a low risk from Pb, while 3% of the sites recorded PRFi Pb higher than 50, which indicates a considerable risk. All sampling sites in the studied three gulfs recorded PRFi << 30 for the other four metals Cu, Zn, Cr, and Ni  located in the vicinity of the ore unloading terminal of the mining operations (‘flotation’) in Ierissos Gulf (Stratoni Bay) and sediments of the site KAV 13, adjacent to phosphoric fertilizer plant in Kavala Gulf exhibit the highest contamination in the examined metals.. RegardiMost of the sediments in Strymonikos Gulf show moderate to high and low values for the CD, PLI, and PERI indexes. Strymon River is the important anthropogenic source of metal pollution for the gulf as, it carries effluents from the industrial activities located along its banks, in Greece and Bulgaria, agricultural wastes originating from Serres valley, as well as domestic sewage. Moreover, drainage basin of the gulf contains maphic and ultramaphic rocks (ophiolites) rich in Cr and Ni, thus, the weathering products contain elevated concentrations of natural origin of such metals . Furtheri << 30) except in IER 6 and IER 8 sampling sites PRF indicates a considerable risk, mostly due to Pb. However, the overall potential ecological risk (PERI) of the studied heavy metals in the surface sediments for the study area exhibited a low risk (PERI << 100). The highest number that PERI scored is at station IER 8. Further research is needed in terms of investigations of various mechanisms influencing metal impacts on organisms living in sediment or overlying water layers of the studied gulfs, like speciation, aging and weathering, limitations to uptake by organisms, as influenced by absorption and eliminator kinetics, etc. The Igeo factor is not readily comparable to the other metal enrichment indexes in sediments due to the nature of the Igeo calculation, which involves a background multiplication of 1.5 and a log function. Based on the classification system proposed for Igeo indexes, Kavala Gulf and Strymonikos Gulf sediments are grouped on average as “unpolluted”. On the contrary, most of the sediments of Ierissos Gulf sites showed moderate to strong pollution in Pb. The Igeo “unpolluted” designation is clearly inappropriate as part of an overall description of the heavy metal results for the sediments from our study area.Overall, the potential risk factor of heavy metals in the surface sediments for the studied gulfs recorded a low risk , the potential risk factor (PRFi), and the potential ecological risk index (PERI) per sampling site for Cu, Pb, Zn, Cr, and Ni in 60 surface sediment samples. The impact of anthropogenic and/or natural heavy metal pollution on three neighboring gulfs in the North Aegean Sea, Greece was evaluated using the contamination factor (CF), the contamination degree (CD), the pollution load index (PLI), the geoaccumulation index  and in the central and deeper parts of Kavala Gulf  showed increased values of the above indexes. Based on the PRFi index, the studied heavy metals did not pose significant environmental risks for the most of the investigated sampling sites, except IER 6 and IER 8, which may pose considerable environmental risk for Pb.IER 6 and IER 8 surface sediments from the Ierissos Gulf, as well as KAV 13 sediment from the Kavala Gulf showed the maximum values for CF, CD, PLI, PRFgeo) are distinctly variable and suggest that sediments in the various Ierissos gulf samples range from unpolluted to moderately, moderately/strongly, and strongly polluted with respect to the Müller’s scale and to the analyzed Pb. Sediments from the other two gulfs as well as from Ierissos Gulf are unpolluted by all investigated metals and by Cu, Zn, Cr, and Ni, respectively. This finding, for uncontaminated sampling sites by using Igeo index, did not correspond with the other indexes findings about metal pollution impact in the study area.The geoaccumulation indexes  source, while Cu, Zn, and Pb were largely contributed to by the anthropogenic sources, such as mining activities in Ierissos Gulf and other industrial  and urban activities in Kavala Gulf. The pollution level of heavy metals at the surface layer of sediments in the three gulfs decreased in the order of Pb ~ Zn > Cr > Ni ~ Cu.In terms of an ecological risk assessment in these three ecosystems, further research is needed, such as studying of various mechanisms which could influence metal impacts on local organisms living on sediment or overlying water layers, like speciation, aging and weathering, limitations to uptake by organisms, as influenced by absorption and eliminator kinetics, etc.