Synechococcus, and haptophytes) across the Pacific Ocean . At the basin scale, heterogeneous selection  dominated the assembly processes of all phytoplankton groups; however, its relative importance varied greatly at the climatic zonal scale, explaining the distinct latitudinal α- and β-diversity among phytoplankton groups. Assembly processes in Synechococcus and haptophyte communities were mainly controlled by physical and nutrient factors, respectively. High temperature drove Synechococcus communities to be more deterministic with higher diversity, while haptophyte communities were less environmentally selected at low latitudes due to their wide niche breadth and mixotrophic lifestyle. Diatom communities were overwhelmingly dominated by the selection process but with low correlation of measured environmental factors to their community compositions. This could be attributed to the high growth rate of diatoms, as indicated by their lower site occupation frequency than predicted in the neutral community model. Our study showed that heterogeneous selection is the main force that shaped the biogeography of three key phytoplankton groups in the Pacific Ocean, with a latitudinal variation of relative importance due to the distinct traits among phytoplankton.Phytoplankton diversity and community compositions vary across spaces and are fundamentally affected by several deterministic  and stochastic  processes. How this suite of different processes regulates the biogeography of phytoplankton remains to be comprehensively explored. Using high-throughput sequencing data and null model analysis, we revealed the ecological processes shaping the latitudinal community structure of three major phytoplankton groups  regulate their global biogeography remains to be comprehensively explored. In this study, we disentangled the ecological processes of three key phytoplankton groups  along the same latitudinal gradients in the Pacific Ocean. Heterogeneous selection, by promoting species richness and reducing similarity between communities, was the dominant process shaping the communities of each phytoplankton group at the basin scale. However, its relative importance varied greatly among different phytoplankton groups in different climate zones, explaining the uneven latitudinal α- and β-diversity. We also highlight the importance of identifying key factors mediating the relative importance of assembly processes in phytoplankton communities, which will enhance our understanding of their biogeography in the ocean and future patterns under climate changes. Phytoplankton, including both prokaryotes  and eukaryotes , are diverse primary producers in the ocean. The dynamics of their community structure may have great consequences for global biogeochemical cycles . Due to 2–6–8–According to the framework proposed by Vellend, ecological processes that shape the community assemblages  can be classified into four fundamental types: selection, dispersal, ecological drift, and speciation , 15. Sel16–Vellend’s conceptual framework has gained popularity in the study of microbial community, as it defines the process at a finer level and systematically takes both deterministic processes  and stochastic processes  into consideration, both of which are crucial for modeling microbial ecology . Followi21–24–In this study, we characterized the phytoplankton community structures in the pelagic Pacific Ocean (PO) along a latitudinal transect  using high-throughput sequencing data, with the aim of attributing their biogeography to different ecological processes using Vellend’s framework . As the Synechococcus, diatoms, and haptophytes) because they are diverse, abundant, widespread, and ecologically important phytoplankton in the ocean, with distinct traits that may influence the contribution of different ecological processes in determining their communities (Synechococcus are pico-sized (<2 μm) prokaryotes, while diatoms and haptophytes are larger eukaryotes, with sizes ranging from nanophytoplankton (2 to 20 μm) to microphytoplankton (>20 μm); together they cover the full size spectrum of phytoplankton. Diatoms are autotrophic r strategists with high growth rate, while most haptophytes are K strategists with low growth rate and the ability to shift between autotroph and heterotroph   , 31. Prexotroph) , 33. We Synechococcus, and haptophytes, respectively (Synechococcus (0.084) and diatoms (0.068)  . All Syn10.1128/mSystems.01203-21.2FIG S2Synechococcus; TR, tropic; ST, subtropic; SA, subarctic; SUR, surface. Download FIG S2, TIF file, 0.8 MB.Rarefaction curves of phytoplankton communities. Rarefied species richness  was estimated for the three groups of phytoplankton at different scales: local individual samples, combined samples within a climatic zone, and combined basin samples . Syn, Copyright © 2022 Xu et al.2022Xu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01203-21.3FIG S3FIG S3, TIF file, 0.2 MB.Site occupation frequency of OTUs in phytoplankton communities. Each dot represents an OTU in a community assembly of phytoplankton. Site occupation frequency refers to the ratio of number of occupied sites  divided by total number of sites in a community assembly. An OTU with a site occupation frequency of 1 occurs at all sites in the community assembly. Mean relative abundance is the average value of relative abundance of a certain OTU in each local sample. Download Copyright © 2022 Xu et al.2022Xu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01203-21.9TABLE S2H) and (Gini-) Simpson (1 − classic Simpson index) values, with standard deviation. Download Table S2, DOCX file, 0.02 MB.Sequence, OTU, and alpha diversity information of samples. Syn, Synechococcus; Hapto., haptophytes; Size, number of samples. Total seqs, total number of sequences of all samples. Av. seqs, average number of sequences within one sample; OTUs were clustered at 97% sequence identity. Total OTUs, total number of observed OTUs of all samples; Av. OTUs, average number of observed OTUs within one sample. Alpha diversity indices include richness , Chao1, Shannon-Wiener   between surface and deep chlorophyll maximum (DCM) layers and (ii) among climatic zones  using Bray-Curtis dissimilarity. We found significant zonal differences in the OTU structure in all three types of phytoplankton, as shown in the nonmetric multidimensional scaling (NMDS) plots  . Zonal d< 0.001) . NotablySynechococcus . A similar pattern was found in the DCM samples. We found significant phylogenetic signal (positive correlation) at relatively shorter phylogenetic distances  for all the phytoplankton groups (We found a significant distance decay relationship (DDR) for all the phytoplankton groups from both surface and DCM layers, reflecting the spatial variations of phytoplankton communities caused by ecological processes . Communin groups , suggestn groups .10.1128/mSystems.01203-21.4FIG S4FIG S4, TIF file, 0.3 MB.Testing of phylogenetic signal across short phylogeny distance. A Mantel correlogram was used to test the correlation between OTU niche difference and OTU phylogenetic difference. Optimal environmental factors  for OTUs in each phytoplankton community assembly were calculated as their niches. Solid and open symbols represent significant and nonsignificant correlations, respectively. Download Copyright © 2022 Xu et al.2022Xu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Synechococcus, and haptophyte communities, respectively, followed by ecological drift . Dispersal limitation had remarkable influences on the surface community assembly of Synechococcus (13.42%). Similar contributions of ecological processes were found in the DCM communities. We also showed that environmental factors were more important than spatial factors in accounting for community compositions of each type of phytoplankton at both surface and DCM layers (Synechococcus communities (P < 0.001 for both surface and DCM), indicating an important role of dispersal limitation.Our null model analysis showed that, at the basin scale, heterogeneous selection was the main ecological process regulating the community structure of each phytoplankton group . For theM layers . This wa10.1128/mSystems.01203-21.10TABLE S3R indicates a strong correlation, and significant correlations (P < 0.05) are in bold. Download Table S3, DOCX file, 0.02 MB.Correlations between environmental/spatial factors and community dissimilarities. Mantel and partial Mantel tests were performed to analyze the correlations between environmental and spatial factors and phytoplankton community compositions . Both surface (SUR) and DCM samples were analyzed. Env, environmental factors; Geo, spatial factors (transformed from geographic distance); Env|Geo, pure effects of environmental factors after spatial factors partitioned out; Geo|Env, pure effects of spatial factors after environmental factors partitioned out. A high Mantel Copyright © 2022 Xu et al.2022Xu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Synechococcus communities  but decreased in haptophyte communities . β-Nearest taxon index (βNTI) of the 3 phytoplankton groups displayed a similar pattern (H) of Synechococcus communities increased from subarctic  to tropic  with reduced community similarity , while haptophytes showed an opposite zonal pattern  and immigration rate (m) increased in haptophyte communities but decreased in Synechococcus communities (see R2 < −0.5). Moreover, diatom OTUs beyond the prediction threshold were mostly (>90%) below the predicted frequency, showing that they had low frequency of site occupation though their mean relative abundances were high  indicated that, from subarctic to tropic, both contribution of neutral processes  indicates the high species dispersal in the community assembly . Syn, Synechococcus. Download FIG S5, TIF file, 0.1 MB.Fitting of zonal phytoplankton community assemblies to the neutral community model (NCM). Rarefied OTU abundance table of a community assembly was used to fit the NCM. Samples of each phytoplankton group were divided by climatic zones (with surface and DCM layers combined). Copyright © 2022 Xu et al.2022Xu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01203-21.6FIG S6R2 indicates the fit to the NCM, and m indicates the immigration rate. Sur, surface. Download FIG S6, TIF file, 0.3 MB.Fit of the neutral community model (NCM) of diatom communities across zones and layers. The solid black dots are observed frequency (freq) of OTUs, and the solid red dots (freq.pred) represent the best fit to the NCM. Purple (pred.upr) and green (pred.lwr) dots are 95% confidence intervals around the model prediction. OTUs above the pred.upr value or below the pred.lwr value are considered to occur more or less frequently than predicted by the NCM, respectively. Copyright © 2022 Xu et al.2022Xu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the r = 0.30, P < 0.001), euphotic layer depth (ELD)  and temperature  contributed most to the phylogenetic turnover of diatom communities, followed by nitrate, silicate, and photosynthetically active radiation (PAR), with minor correlations (r < 0.2). (ii) Assembly processes of Synechococcus communities were mainly driven by physical factors, including temperature, ELD, salinity, PAR, and mixed-layer depth (MLD), with temperature as the most important factor . βNTI of Synechococcus was positively related to temperature difference between two samples, showing that a large difference in temperature between communities allowed heterogeneous selection to contribute more   were more important than physical factors (r < 0.4) in determining the assembly processes of haptophyte communities. In addition, nutrient factors were much more heterogeneous in the subarctic area   and the perature . (iii) Ntic area  and explhococcus . Diatom Synechococcus (2.49) communities remained low across all zones , such as OTUs from Prymnesium, Emiliania, and several other uncultured haptophyte species (Levins’ niche breadth of diatom (2.34) and ll zones . However species .10.1128/mSystems.01203-21.7FIG S7FIG S7, TIF file, 0.3 MB.Niche breadth of OTUs in haptophyte communities. Each triangle represents an OTU. Size of each OTU is proportional to its mean relative abundance (RA) in the metacommunity. OTUs are filled with different colors according to genus and are plotted in order of their taxonomies. Download Copyright © 2022 Xu et al.2022Xu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Synechococcus and haptophytes. This is in accordance with a common phenomenon, that relative contribution of ecological processes depends greatly on spatial scales, which has been widely reported but little mentioned with regard to phytoplankton . This could be explained by the varied zonal contribution of heterogeneous selection, which causes high community turnover due to the among-taxa fitness difference and leads to low community similarity  and heterogeneous selection overwhelmingly dominated the selection process  in our study, we focused on the changes in contribution of heterogeneous selection in communities across different zones.We mainly followed the trends of heterogenous selection to describe the ecological processes in phytoplankton communities in this study, although previous studies showed that the ratio of environmental selection to dispersal limitation can provide a better explanation for the microbial community than using the absolute value of contribution of each process , 40. In Synechococcus communities have rarely been reported. In this study, we show that the relative importance of environmental selection (represented by heterogeneous selection) in Synechococcus varied substantially across climatic zones and was mediated by several physical factors, including temperature, salinity, ELD, and MLD. Heterogeneous selection allows more species to coexist and leads to more dissimilar structures among communities due to the environmental heterogeneity of habitats  and high temperature divergence (between two sites) can cause more phylogenetic turnover in Synechococcus communities and lead the community assembly to be more deterministic with dominance of heterogeneous selection. Thus, our results may suggest an increasing species richness and greater community difference of Synechococcus in the ocean under warming effects, which would provide group-specific information for the global study of phytoplankton during climate changes  and appeared to dominate the assembly processes in  changes , 13. Tem changes .Synechococcus communities in our study to the following causes. First, from the perspective of evolution, marine Synechococcus has developed strains that are phylogenetically specialized to different thermal niches and respond  differentially due to their thermal preferences  than low latitudes  in the PO according to a previous global survey  with a high level of adaptation strategies  are favored  showed positive correlations with macronutrient concentrations in the northeast subarctic PO, and the ratio of nitrogen to phosphate is a key factor allowing its bloom  implies little contribution of neutral processes , which mainly rely on physical factors  with their controlling factors and the responses of phytoplankton communities across climatic zones in the PO . In geneSynechococcus, and haptophytes) simultaneously along the same gradients in the PO. We showed that heterogeneous selection, by promoting species richness and reducing similarity between communities, was the dominant process shaping the communities of each phytoplankton group at a basin scale. However, its relative importance varied greatly among different phytoplankton groups in different climate zones, explaining the uneven latitudinal distribution of α- and β-diversity of phytoplankton in the PO. By cross-group comparison of the relative magnitude of ecological processes influencing phytoplankton community assembly, our study leads to a new hypothesis that the importance of these processes may partially differ between taxa, a finding that needs to be verified. We also highlight the importance of identifying key factors affecting the relative importance of assembly processes  in phytoplankton communities across spaces, which will enhance our understanding of the biogeography of phytoplankton communities in the ocean and advance our ability to predict their patterns and variability in response to climate changes.In this study, for the first time, we disentangled the ecological processes of three key phytoplankton groups  and variation partitioning analysis (VPA). Three questions  were addressed in responding to the questions raised in the introduction. βNTI, β-nearest taxon index; RCCopyright © 2022 Xu et al.2022Xu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Synechococcus, and haptophytes) communities in the PO (Hakuho Maru (JAMSTEC/University of Tokyo)  and group-specific 18S rRNA genes (for diatoms and haptophytes) were performed, and products were sequenced with an Ion Torrent PGM system.We used the high-throughput sequencing data from two previous studies of phytoplankton  ; Fig. 1af Tokyo) . Briefly10.1128/mSystems.01203-21.8TABLE S1oC); Sal, salinity; NO3, nitrate (μM); PO4, phosphate (μM); SiO3, silicate (μM); NH4, ammonia (μM); PAR, photosynthetically active radiation; MLD, mixed-layer depth (m); ELD, euphotic layer depth (m); Fe, iron (μM). Download Table S1, DOCX file, 0.03 MB.Location of sampling station and distribution of environmental factors. DCM, deep chlorophyll maximum layer; Lon, longitude; Lat, latitude; Tem, temperature . The MLD was determined by CTD profiles following the method of Suga et al. (https://coastwatch.pfeg.noaa.gov/erddap/griddap/erdMH1par0mday.html).Environmental factors, including sampling sites, including temperature, salinity, nitrate, phosphate, silicate, iron, ammonia, photosynthetically active radiation (PAR), mixed-layer depth (MLD), and euphotic layer depth (ELD), were collected from previous studies where detailed methods of measurement were described , 65. Bril et al.  or detera et al. . InorganWe used the biogeographic distribution pattern, e.g., β-diversity, of phytoplankton communities to reflect the action of ecological processes. The following analyses were conducted using R software (v. 4.0.2) . First, R value indicates a larger difference of community composition between two clusters of samples. Geographic distance between two sites was calculated by the distm function in the geosphere package, according to their latitudes and longitudes , subtropic (23.5 to 40°N and S), and tropic (0 to 23.5°N and S). Before estimating ecological processes, we revealed the community biogeography of each phytoplankton group by comparing community similarity (Bray-Curtis). The significant difference in community composition between two clusters  of samples was tested by analysis of similarity (ANOSIM) by the anosim function in the vegan package. A higher ANOSIM ngitudes . Communingitudes . Here, dTo estimate the relative contribution of ecological processes to community assembly, we employed the null model based on the framework described by Stegen et al. . Before Bray) was measured and compared to the β-diversity obtained with the null model with 9,999 randomizations. RCBray values above 0.95 and below −0.95 indicate that community turnover is driven by dispersal limitation and homogenizing dispersal, respectively, while RCBray values between −0.95 and 0.95 indicate the dominance of drift  and zonal  levels.For the null model analysis, phylogenetic turnover using the abundance weighed β-mean nearest taxon distance (βMNTD) metric was measured, which quantifies the mean phylogenetic distances between the two evolutionarily closest OTUs in two communities. βMNTD values higher than expected by chance indicate heterogeneous (or variable) selection in community assembly, while βMNTD values lower than expected by chance indicate homogeneous selection. The null model expectation was performed using 999 randomizations, and the deviation between the observed βMNTD and the mean of the null model distribution is shown as the β-nearest taxon index (βNTI). A significant deviation  indicates the dominance of selection processes: a βNTI value below −2 indicates significantly less phylogenetic turnover than expected  while a βNTI value above 2 indicates significantly more phylogenetic turnover than expected  , 20. If of drift , 74. SinTo support the relative importance of ecological processes calculated from the null model, we supplemented the same data set with two kinds of statistical tests, each with a different focus.a level, nitrate level, phosphate level, silicate level, ammonia level, PAR, MLD, ELD, iron level, and depth) did not show normal distribution (by the shapiro.test function in R). Because some factors  exhibited large heterogeneity (10- to 100-fold differences) while other factors varied over small ranges , all environmental factors were log(x + 1) transformed to improve homoscedasticity and normality for multivariate statistical analysis. For spatial factors, principal coordinates of neighbor matrices (PCNM) analysis was used to generate a set of spatial variables based on the longitude and latitude coordinates of sampling stations, using the pcnm function in the vegan package  were retained for downstream analysis  and spatial  factors for both surface and DCM layers. All environmental factors  indicates the high species dispersal in the metacommunity . The overall fit of the community data to the NCM is indicated as R2, representing the proportion of neutral process in the metacommunity. The formulas for m and R2 are as follows: Freqi = 1 − I and R2 = 1 − SSerr/SStotal, where Freqi is the occurrence frequency of OTU i (a certain OTU in the metacommunity) across metacommunity; N is the number of individuals per community; m is the estimated immigration rate; ip is the mean relative abundance of OTU i across the metacommunity; I is the probability density function of beta distribution; 2R is the overall fit to the NCM; and SSerr is the sum of squares of residuals and SStotal is the total sum of squares, which come from the deviation between observed and predicted frequency. Both fitting statistics and predicted frequency of each OTU can be obtained following the R code in a previous study (m) and model fitness (R2) for phytoplankton metacommunities from both layer and zonal scales. In particular, we plotted the observed and predicted occurrence frequency (with a 95% threshold) of diatom taxa with their mean relative abundances. Notably, R2 can be a negative value when the deviations between observed frequency and predicted frequency are large, indicating the overwhelming dominance of the deterministic process .Second, NCM was used to compare the contribution of neutral process (as opposed to selection) in phytoplankton communities across climatic zones. The NCM is based on predicting the relationship between the frequency of OTUs  and their abundances across the metacommunity . It predus study . Here, wR value indicates the more important role of the environmental factor in controlling the phylogenic turnover in communities  were analyzed. The available environmental factors were divided into physical factors  and nutrient factors . Zonal differences  of both physical and nutrient factors were compared. The effects of physical and nutrient factors on phytoplankton community compositions were calculated by variation partitioning analysis (VPA), using the varpart function of vegan package. Before the VPA, selection of factors was conducted following the protocol described above for the Mantel test.To evaluate the environmental factors that mediate the relative contribution of ecological processes in community assemblies across different climatic zones, we analyzed the correlations between all the pairwise comparisons of βNTI values and environmental factors, by Mantel and partial Mantel tests. The significance of the correlation was tested with 999 permutations, and a larger Mantel munities . Due to B):jB is the habitat niche breadth of OTU j in a metacommunity, N is the total number of local communities in the metacommunity, and ijP is the proportion of OTU j in local community i. A high B value indicates that the OTU occurs widely and evenly across a large proportion of samples, representing a wide niche breadth. This calculation of niche breadth was conducted using the niche.width function in the spaa package  as an indicator of niche breadth at the community level  with accession number SRP148585; for haptophytes and diatoms, DNA Data Bank of Japan (DDBJ) with accession numbers DRA004899 to DRA004901. R scripts used for the statistical analysis can be found at https://github.com/xzhimenghkust/Pacific-PP.-R-scripts.Raw sequencing reads for analyses in this study were deposited in online open databases: for


Columba livia) have been reported worldwide. Currently, pigeon racing is becoming increasingly popular and considered to be a national sport in China, and even, the greatest competitions of racing pigeons are taking place in China. However, there are still no epidemiologic data regarding PiCV infections among racing pigeons in China. The purpose of our study was to provide information of prevalence, genetic variation and evolution of PiCV from racing pigeons in China.Pigeon circovirus (PiCV) infections in pigeons  and some of them were closely related to worldwide strains from different types of pigeons. A large number of recombination events (31 events) were also detected in the PiCV genomes from Chinese racing pigeons.To trace the prevalence, genetic variation and evolution of PiCV in sick and healthy racing pigeons, 622 samples were collected from 11 provinces or municipalities in China from 2016 to 2019. The results showed that the positive rate of PiCV was 19.3% (120/622) at the sample level and 59.0% (23/39) at the club level, thus suggesting that the virus was prevalent in Chinese racing pigeons. A sequence analysis revealed that the These findings indicate that PiCV strains circulating in China exhibit a high genetic diversity and also contribute to information of prevalence, genetic variation and evolution of PiCV from racing pigeons in China.The online version contains supplementary material available at 10.1186/s12864-022-08425-8. Circoviridae, within the order Circovirus, contain a small, circular, non-enveloped, single stranded DNA genome ranging from 1.7 to 2.5 kb in size [Circovirus of the family Circoviridae (http://www.ictvonline.org/). PiCV is a small, circular, non-enveloped virus that contains a single-stranded DNA genome with approximately 2 kb in size. The genome of PiCV has two main open reading frames (ORFs). The ORF-V1 located on the virion sense strand encodes a replication-associated protein (Rep), and the ORF–C1 located on the complementary sense strand encodes a capsid protein (Cap) [Members of the in (Cap) . The genin (Cap) . The othin (Cap)  and had Columba livia species.A variety of methods have been employed to detect PiCV infection in clinical specimens. The original diagnose methods electron microscopy, histology, dot blot hybridization, and in situ hybridization are time consuming –11. The In China, the first PiCV infection was detected from meat pigeons in Zhejiang province in 2009, and the full genome was sequenced . In receIn this study, PiCV was detected in samples from both sick and healthy racing pigeons. The results indicated that the positive rates of PiCV were 19.3% (120/622) at the sample level. Among the samples, 14 out of 51 sick pigeons  and 106 out of 571 healthy pigeons  were identified to be PiCV positive, respectively. In this study, 23 out of 39 racing pigeon clubs were positive for PiCV  (Table cap gene ranging from 813 to 828 nt in length and rep gene of 948 or 954 nt as shown in Table Sn = 21) and 2042 (n = 13) nucleotides, respectively  and the PiCV reference strains from other countries   among the Cap proteins including locations 7, 24, 29, 30, 35, 58, 130, 182, and 266  and other countries  as compared to the cap genes .The cap gene reported in GenBank, and cap genes of 90 PiCV strains obtained in our study was performed to investigate genetic relationship (Table Scap gene sequences reported in the present study were clustered into 7 clades: A (14.1%), B (60.9%), C (62.2%), E (25.0%), G (51.4%), H (28.6%), and I (40.0%). As shown in Fig. A phylogenetic analysis using 126 p Table S. The 90 ades: A 1.1%, B , 2044 nt (n = 2), and 2045 nt (n = 1) complete genome were present in the PiCV positive samples. Despite many genetic diversities were found in the genome of PiCV strains, no mutation was found in the conserved nonanucleotide motif (TAGTATTAC) located at the apex of a potential stem-loop which was putatively associated with the initiation of rolling circle replication (RCR) [cap gene [cap genes exhibited a higher diversity as compared with the reference strains. Some unique amino acid substitutions at 28 different positions were observed among the Cap proteins. In addition, the cap nucleotide sequence with 828 nt in length encoding a novel Cap protein of 275 amino acids was first identified in two Chinese PiCV strains, TY3/SN/2016/MW181931 (clade E) and WL4/SN/2018/MW181959 (clade E). Higher diversity of Cap protein versus the Rep protein is due to the fact that the Cap protein, as the protein shell of the virus, is exposed to the host’s immune cells, resulting in a stimulation of a cascade of immune responses. Positive selection occurs during this process. Some mutations can change the structure of Cap protein. This may lead to a better binding of virus with the receptors of target cells, which increases the infection ability of virus in cells. Moreover, these mutations may also protect virus from being neutralized by antibodies [cap gene, besides ATG and ATA [Previous studies showed that the length of PiCV genome was 2031–2043 nt , 28, 29.cap gene , 23, 34.tibodies , 36. In tibodies . Like BFtibodies , which mtibodies , 40. If tibodies . In additibodies , 42. Mortibodies . It mean and ATA , we foun and ATA  and BFDV and ATA . Since n and ATA , 50 and cap gene. Additionally, we found that PiCV strains isolated from the same club belonged to different clades and shared a low sequence identity. These data suggested that the infection and evolution of PiCV in Chinese racing pigeons might have different evolutionary origins. This may result from the fact that the import of racing pigeons from all around the world to China is very significant and a lack of oversight in the international pigeon trade (racing pigeons mainly) [cap gene were detected from the same pigeon, suggesting a horizontal transmission occurred among the racing pigeons in the same racing clubs [In the present study, the 90 PiCV strains were divided into seven clades  based on a phylogenetic analysis of  mainly) , 51. In ng clubs , 52. TheViral recombination had been proved to play a significant role in the evolution of many ssDNA viruses . The extIn conclusion, our study demonstrated that PiCV infection in racing pigeons was widespread in northern China and revealed the characteristics of the PiCV genome. Furthermore, the identified PiCV strains displayed a high genetic diversity. Our data also demonstrated that PiCV in Chinese racing pigeons had an extensive recombination for the first time. The high genetic diversity of PiCV may be derived from two possible facts: 1) the racing pigeons in China are imported from all around the world; and 2) a lack of oversight in the international pigeon trade (mainly refer to racing pigeons). In addition, another possible explanation is that the intensive trafficking of pigeons between clubs and racing events in China. Furthermore, the high intensity of recombination may suggest that frequent co-infections of different PiCV strains. Overall, clubs and racing events could provide a suitable chance for viral recombinants, which may pose a threat for the domestic pigeons, and has a possibility of spilling over onto wild species.In total, 622 samples were collected from 11 provinces or municipalities in China from November 2016 to August 2019. Of these samples, 571 serum samples were collected from 571 healthy racing pigeons from 39 racing pigeon clubs in 11 provinces or municipalities of China, including Beijing, Hebei, Liaoning, Shanxi, Gansu, Qinghai, Shandong, Shaanxi, Xinjiang Uygur Autonomous Region, Ningxia Hui Autonomous Region and Inner Mongolia Autonomous Region. Animals obtained from the racing pigeon clubs were returned to the club owners after collecting blood samples. Moreover, the corpses of 51 sick pigeons from 7 racing pigeon clubs in 5 provinces or municipalities were submitted to our laboratory at College of Veterinary Medicine, Northwest A&F University to determine the pathogen of the disease. The 0.2 g sections of internal organs  were obtained during the postmortem examination of all mentioned dead pigeons. All samples were stored at − 80 °C before DNA extraction.The tissue samples were ground to powder with liquid nitrogen and diluted with three volumes of phosphate‐buffered saline. The samples (1 ml) were centrifuged at 12,000 × g for 10 min at 4 °C and the supernatants were transferred into a 1.5 ml tube. Nucleic acids from tissues and serum samples were extracted by using an EasyPure® Viral DNA/RNA Kit  according to the manufacturer’s instructions. The extracted genomic DNA was stored at -20 °C before use.cap gene as described by Freick et al. (2008). The primer sequences were: PiCV-s, 5’-TTGAAAGGTTTTCAGCCTGGC-3’ and PiCV-as, 5’-AGGAGACGAAGGACACGCCTC-3’ [The PiCV was first detected using a PCR method targeting a 326-base fragment of GCCTC-3’ . The fulcap gene sequences, rep gene sequence and the full genome sequences of PiCV obtained in this study have been deposited in GenBank, respectively. The sequences obtained in the current study were compared with all available PiCV sequence data from different geographical locations within China (n = 60) and the rest of the world (n = 73) from the National Center for Biotechnology Information (NCBI) nucleotide database (Table STm (http://weblogo.threeplusone.com/), an online software for sequence logo generator [The e Table S. Multiplhina n =  and the enerator . A compacap gene sequences of PiCV obtained in this study were used for a phylogenetic analysis. All available PiCV sequence data from different geographical locations within China (n = 54) and the rest of the world (n = 72) were retrieved from the NCBI nucleotide database as reference sequences (Table Shttp://itol.embl.de/) [The entire s Table S. Maximummbl.de/) .p-value < 0.05 were considered plausible recombinant events. The sequences in the analyzed dataset that most closely resembled the parental sequences of recombinants were defined as either “minor parents” or “major parents” based on the size of the genome fragments which had contributed to the detected recombinants .In order to analyze the potential recombinant evidence, an integrated software package for the recombination detection program RDP4  was usedAdditional file 1: Table S1. Pigeon circovirus (PiCV) references sequences obtained from GenBank and used in this study. The information including strain name, year, country, host, genome length and accession number.Additional file 2: Table S2. Information of Pigeon circovirus (PiCV) stains obtained in this study. The information including strain name, collection date, collection region, gene length, initiation codons of cap gene, health status and accession number.Additional file 3: Table S3. Sequence analysis of genome sequences of Pigeon circovirus (PiCV) strains. The information including homology analysis of amino acid and nucleotide sequences.Additional file 4: Table S4. Details of recombination events (GenBank accession numbers) detected in the Pigeon circovirus (PiCV) strains obtained in this study. The information including location of recombination breakpoints, recombinant, potential minor parent(s), potential major parent(s), detection methods and p-value.


Lixus subtilis Boheman is reported in this study. The circular genome is 15,223 bp long, including a standard set of 21 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), 13 protein-coding genes, and a non-coding control region. The trnI gene was not found in the L. subtilis mitogenome. All tRNAs had the typical cloverleaf structure, except for trnS1, which lacked the dihydrouridine arm. The phylogenetic tree of 13 Curculionidae species based on the concatenated nucleotide sequences of complete mitochondrial genomes strongly supported that L. subtilis is closely related to Curculioninae and Molytinae.The first complete mitochondrial genome of  Lixus subtilis Boheman, 1835 (Coleoptera: Curculionidae) is widely distributed  on 10 May 2018. One specimen was deposited in the College of Plant Protection, Shanxi Agricultural University, Taiyuan, China  under the accession no. YFMgnjP2018393.L. subtilis is a representative circular DNA molecule with a length of 15,223 bp (GenBank accession no. MW413392). Thirteen protein-coding genes (PCGs), 21 transfer RNA genes (tRNAs), the large and small ribosomal RNA unit genes (rrnL and rrnS), and a large non-coding region (putative control region) were contained by this mitogenome. In Coleoptera, the order and orientation of the mitochondrial genomes have been retained from the ancestral gene order, apart for the tRNA gene, which may be deleted or rearranged in some species  and Naupactus xanthographus   was present between trnF and nad5, in which intergenic spacers totaling 53 bp appeared in 11 positions and ranged in size from 1 to 18 bp. The control region had A + T content of 72.0% with 571 bp in length and was present between the rrnS and trnQ genes.Thirty specimens were used in the mitogenomic studies. The complete mitogenome of trnS1 lacked a stable DHU arm. This result was as the same as those reported in other insect mitogenomes , whereas one terminated with an incomplete stop codon TA (nad4).It was predicted that all 21 tRNAs had typical cloverleaf secondary structures, but the gene Acyrthosiphon pisum   were used. This phylogenetic analysis was performed using the maximum likelihood (ML) method with 1,000 bootstrap replicates using MEGA-X and via Bayesian inference (BI) using MrBayes  and outgroups from olytinae , indicat


Enterococcus faecalis infective endocarditis is commonly treated with intravenous ampicillin/ceftriaxone combination therapy. Ampicillin, however, is unsuitable for outpatient parenteral antibiotic therapy (OPAT) regimens due to its instability in 24 h continuous infusors, and has been successfully replaced by benzylpenicillin used together with ceftriaxone in a few small case series. Since in vitro synergy data of penicillin/ceftriaxone against E. faecalis are still lacking, checkerboard assays were performed for 28 clinical E. faecalis isolates and one laboratory standard strain. Synergistic effects (both lowest and median FICI) were observed for penicillin/ceftriaxone in 15/29 isolates, while ampicillin/ceftriaxone exhibited synergism in 22/29 isolates. For isolates with ceftriaxone MICs ≤ 256 mg/L, the addition of free ceftriaxone trough concentrations to penicillin or ampicillin resulted in comparable synergistic effects for both combinations. In contrast, for isolates with ceftriaxone MICs ≥ 512 mg/L free ceftriaxone trough concentrations were only sufficient to exhibit synergistic effects in combination with ampicillin, but not penicillin. This study suggests that benzylpenicillin/ceftriaxone would be expected to be suitable for the OPAT treatment of enterococcal endocarditis for E. faecalis isolates with ceftriaxone MICs ≤ 256 mg/L. However, combination therapy would be expected to provide no advantage over benzylpenicillin monotherapy for isolates with ceftriaxone MICs ≥ 512 mg/L. Further investigation is required to analyse the relationship between ceftriaxone susceptibility and penicillin/ceftriaxone synergy, especially for isolates with ceftriaxone MICs of 64 to 512 mg/L. Enterococcus faecalis is an increasingly common cause of infective endocarditis (IE) and should be treated with prolonged synergistic, bactericidal antibiotic combination therapy [ therapy ,2. Conte therapy . Ceftria therapy ,4.E. faecalis IE (EFIE) may be challenging to administer via an OPAT service due to the multiple doses required per day. The administration of the treatment by community nurses is often limited to once or twice daily injections or changing a 24 h elastomeric continuous infusor. While ampicillin/ceftriaxone has been proposed as an OPAT regimen for EFIE utilising either elastomeric continuous infusors or programmable pumps for the delivery of ampicillin [Although a previous guideline for the Outpatient Parenteral Antibiotic Therapy (OPAT) treatment of IE was restrictive , a recenpicillin , the litpicillin ,9,10,11.picillin  that enrE. faecalis endocarditis is common in New Zealand and also occurs in Australia and America. Benzylpenicillin has been successfully combined with ceftriaxone for the OPAT treatment of enterococcal endocarditis in a few small clinical case series [IV benzylpenicillin has been used as an alternative to IV ampicillin/amoxicillin for the treatment of EFIE, since benzylpenicillin is more stable in elastomeric continuous infusors and therefore better suited for OPAT regimens. The use of intravenous benzylpenicillin/ceftriaxone for the OPAT-guided treatment of e series ,14,15, bE. faecalis isolates and one laboratory standard strain, allowing for a comparison of both combinations at concentrations achieved by OPAT dosage regimens.Here, we report the checkerboard synergy analysis of penicillin/ceftriaxone and ampicillin/ceftriaxone in 28 clinical E. faecalis isolates were obtained from blood cultures from, or the heart valves of, patients with enterococcal infection at various foci, including eight patients with infective endocarditis . Test solutions of ampicillin (AMP) , ceftriaxone (CRO) , and benzylpenicillin (PEN)  were prepared immediately before use.Clinical carditis . The antCheckerboard assays for penicillin/ceftriaxone were performed as described previously , with 11Three different methods for interpreting the FICIs were used: (i) the lowest FICI value, with values ≤ 0.5 indicating synergism and 0.5 < FICI < 4 indicating no interaction; (ii) the median FICI value, taking 0.8 as the synergy threshold; (iii) single, one-point FICIs at free ceftriaxone trough concentrations.s) with a two-tailed CI of 95%.The correlation of the MICs and FICIs was tested using the nonparametric Spearman’s rank-correlation coefficient  ranged from 0.25 to 2 mg/L, while penicillin MICs (MICPEN) ranged from 0.5 to 4 mg/L, the highest MICPEN of 4 mg/L being below the CLSI susceptibility breakpoint of 8 mg/L. Ceftriaxone MICs (MICCRO) ranged from 1–2 to more than 1024 mg/L. The MICAMP showed no correlation with the MICCRO, but the MICPEN exhibited a weak positive correlation with the MICCRO . Both the MICPEN and MICCRO displayed a strong inverse correlation with the FICI values of penicillin/ceftriaxone (FICIPEN/CRO)—i.e., the higher the MICPEN or MICCRO, the lower the FICIPEN/CRO . The MICCRO inversely correlated with the FICIAMP/CRO , but the MICAMP did not.Synergistic effects were observed for penicillin/ceftriaxone in 16 (lowest FICI method) and 21 (median FICI method) of the 29 tested  strains . Ampicil strains . AmpicilPEN or MICAMP to one half, one quarter, and one eighth of the MICPEN or MICAMP are recorded in PEN but with FICIs above the synergy threshold of 0.5. In contrast, ampicillin/ceftriaxone exhibited synergism in 23 isolates at free ceftriaxone trough concentrations. For the isolates with MICCRO ≥ 512 mg/L, lower sub-MIC ceftriaxone concentrations were required in combination with ampicillin than in combination with penicillin to produce the same effect  centred around 8 mg/L ± 1–2× MIC and a smaller population of isolates (32%) centered around 512 mg/L ± 1× MIC  homologues, resulting in the inhibition of cell-wall synthesis. Little is known about the detailed functions of PBPs in mologues . The syntrations . Ceftriatrations . The disto PBP 5 . This fuE. faecalis isolates with an MICCRO ≤ 256 mg/L. No significant benefit from adding ceftriaxone to benzylpenicillin is expected for any isolate with an MICCRO ≥ 512 mg/L. These microbiological data support the use of OPAT treatment with the continuous infusion of benzylpenicillin and ceftriaxone for EFIE for isolates with an MICCRO ≤ 256 mg/L. As the penicillin/ceftriaxone synergy-testing data for isolates with MICCRO of 64 to 512 mg/L are limited in this study, further investigation is required to establish a reliable MICCRO cut-off above which the combination of benzylpenicillin and ceftriaxone is not superior to benzylpenicillin alone.The addition of ceftriaxone to benzylpenicillin provides synergy, or at least partial synergy, for


Background: Women ageing with HIV undergo sex-specific changes. There is limited evidence available with regards to how the menopause impacts HIV outcomes.Objective: To investigate whether menopausal age is associated with engagement-in-care (EIC), viral load (VL) suppression and rebound among women living with HIV.Methods: Women were grouped by age , corresponding to pre-, peri- and post-menopausal stages. EIC, HIV VL suppression (VL < 50 copies/mL) within 12 months of antiretroviral therapy initiation and VL rebound (two consecutive VL > 50 copies/mL) after VL suppression were compared across age groups using logistic/Cox proportional hazards regression. Associations were compared to those seen in heterosexual men.Results: Six thousand four hundred and fifty-five (6455) eligible women  contributed 44,226 person-years (PYRS) of follow-up; 29,846, 10,980 and 3,399 PYRS in those aged <40, 40–50 and >50, respectively. Women were engaged-in-care for 79.5% of follow-up time, 3,344 (78.0%) experienced VL suppression and 739 (22.1%) VL rebound. After adjustment, women aged >50 years had lower EIC than those aged <40. Women aged 40–50 were more likely to have VL suppression and were less likely to experience VL rebound than those aged <40 years. Trends in heterosexual men were similar for EIC but with no evidence of a higher VL suppression rate in those aged 40–50 years (pint. 0< .0001) and a stronger protective association between older age and VL rebound (pint. 0< .0001).Conclusion: Our findings warrant further research into the potential impact of the menopause to support women and clinicians through HIV care. In the UK, nearly a third of those living with HIV are women. With successes in treatment, the increasing life expectancy of people living with HIV now means this population is ageing; in 2018, 4.2% of women living with HIV were transitioning through the menopause and 33% were post-menopausal,Women undergo sex-specific changes as they age, specifically with hormone regulation. There is limited evidence available with regards to how these changes, more specifically the menopause, impact HIV outcomes. It is therefore imperative to understand the impact of the menopause on HIV care.,Only a few studies have specifically examined the association between menopausal symptoms and adherence to antiretroviral therapy (ART).per se, we also examine trends in men.We aim to investigate whether menopausal age is associated with engagement-in-care and virological outcomes  among women participating in the UK Collaborative HIV Cohort (UK CHIC) study. Given that age and menopausal status are highly correlated, and that associations between menopause and outcomes may reflect ageing rather than menopause The UK CHIC study is an ongoing cohort of individuals living with HIV (aged >16 years) who have accessed care at one or more of 25 HIV clinics in the UK at any time from 1996 onwards. The study methods are described elsewhere.The analyses described here are based on women who acquired HIV through heterosexual sex only, as factors associated with HIV outcomes may differ in those acquiring HIV through other routes, and as the number of women acquiring HIV through these routes in the cohort is too small to permit robust estimates. Data were collected up to 31 December 2017. Heterosexual women with >1 day of follow-up and a cohort entry date in January 2000 or later were followed from cohort entry until the earliest of the following: death, permanent loss to follow-up from HIV care (defined as failure to return for a follow-up visit within 12 months) or 31st December 2017. As UK CHIC does not record menopause specific data, women were grouped by age  to broadly corresponding to pre-, peri- and post-menopausal stages.Each woman’s follow-up was split into consecutive monthly intervals and the woman’s characteristics , combination ART (cART) use, previous AIDS diagnosis and calendar year) were determined at the start of each interval, allowing us to incorporate time-varying covariates into regression models. Ethnicity and Hepatitis B virus (HBV)/Hepatitis C virus (HCV) co-infection were treated as fixed covariates.Analyses considered three outcomes: (i) Engagement-in-care (EIC), which was defined using the Retention and Engagement Across Care services for HIV (REACH) algorithma priori. These included: ethnicity, CD4+ T-cell count, VL, ART use, previous AIDS, calendar year and hepatitis B/C infection. Analyses of EIC used generalized estimating equations (GEE) to model the association between age and the binary outcome of whether each month of follow-up was deemed to be in or out of care. For the analyses of viral suppression, individuals who initiated cART were followed from cART initiation to the earliest of the censoring date (described above) or 12 months after cART initiation. Time to VL suppression was compared across the three age groups using Cox proportional hazards regression. Among those with VL suppression, time to viral rebound was assessed from the date of VL suppression to the earliest of VL rebound, the censoring date or the first gap in treatment lasting >14 days, with comparisons between the age groups undertaken using Cox proportional hazards regression models.Univariable and multivariable regression were used to assess the association between age group and each outcome, adjusting for potential confounders selected We undertook further analyses within the whole UK CHIC cohort (including both men and women), to assess whether age associations (where present) differed significantly between men and women, through the inclusion of an interaction term between age group and sex.3. The majority of women were of black African ethnicity (70.6%). One-tenth (9.5%) of women had a prior AIDS event at study entry, and 0.4% and 0.2% were co-infected with HBV or HCV, respectively.A total of 6,455 heterosexual women in UK CHIC were included in analyses ; the womThe women were followed for a total of 44,226 person-years (PYRS); 29,846, 10,980 and 3,399 PYRS were contributed by women aged <40, 40–50 and >50 years, respectively. There were several key differences in the characteristics of the women in the three age groups. For example, women in the older age group tended to be followed up in more recent calendar years, generally had higher CD4+ T-cell counts, were more likely to have had a prior AIDS event, and were more likely to be on cART and to have a suppressed VL see .Women were determined to be engaged-in-care for 79.5% of their follow-up time, with the proportion of follow-up time spent engaged-in-care increasing from 73.6% among the younger age group to 82.0% in the middle age group to 85.9% in the older age group. Compared to those aged <40 years, in unadjusted analyses the odds of EIC increased by 27% (odds ratio (OR) 1.27 [95% confidence interval: 1.19–1.36]) and 41% (1.41 [1.27–1.57]) among those aged 40–50 and >50 years, respectively. After adjustment, however, both ORs fell below 1 (40–50 years: adjusted OR (aOR): 0.96 [0.89–1.03]; >50 years: 0.87 [0.78–0.96]) suggesting a gradual decrease in EIC as age increased.A total of 3,344 (78.0%) women who initiated cART became virally suppressed within the first 12 months (median time to VL suppression 4 months [IQR: 2–8 months]). Women initiating cART aged 40–50 years were more likely to become virally suppressed within a year (82.1%) than those aged <40 (75.8%) or >50 (78.9%) years, with crude hazard ratios (HRs) for those aged 40–50 and >50 years at cART initiation of 1.16 [1.08–1.25] and 1.06 [0.95–1.18], respectively, compared to those aged <40 years. After adjustment for potential confounders, the adjusted hazard ratio was increased for women aged 40-50 years (adjusted HR (aHR) 1.25 [1.14-1.37]) but remained similar for women aged >50 years (aHR: 1.08 [0.94–1.23]).Over one-fifth (22.1%) of women with viral suppression subsequently experienced virological rebound over 12,307 PYRS. Cumulatively, 13.9%, 20.1% and 23.9% had a viral rebound within the first two, four and six years after viral suppression, respectively. In unadjusted analyses, whilst VL rebound rates among women aged <40 and 40–50 years did not differ significantly (HR: 0.90 [0.77–1.06] for women aged 40–50 years compared to those aged <40 years), women aged >50 years were less likely to experience viral rebound (HR: 0.70 [0.56–0.88]) than those aged <40 years. After adjustment for confounders, however, the aHR for women aged 40–50 years was strengthened (aHR: 0.82 [0.68–0.98]) whereas that for women aged >50 years was attenuated (aHR: 0.89 [0.69–1.15]).3). A greater proportion were of white ethnicity (24.6%). As observed in women, the proportions with a prior AIDS event (11.1%), HBV and HCV co-infection  were low.A total of 5,843 heterosexual men from the UK CHIC study were included in comparative analyses, consisting of 3199 men aged <40 years, 1835 aged 40–50 years and 809 aged >50 years at study entry. This group contributed a total follow-up of 41,620 PYRS. Compared to women, heterosexual men in UK CHIC were older at study entry (median age: 38 [IQR: 32–46] years), with a lower median CD4+ T-cell count (264 [IQR: 115–430] cells/mmp = 0.36).Among the men, rates of EIC also showed an increasing trend with age  although after adjustment for potential confounders, there were no significant differences in rates of EIC across the age groups .In total, 3,353 (80.1%) of the men became virally suppressed within the first 12 months on cART, 78.8%, 80.2% and 83.6% in the three age groups, respectively. In contrast to women, there was no association between viral suppression and age amongst men after adjustment for confounding .Viral rebound occurred in 750 (22.4%) of those with viral suppression . Associations with age appeared to be stronger amongst heterosexual men  were more likely be virally suppressed and less likely to experience viral rebound than women aged <40. There have been only two studies to our knowledge that have explored the association of virological suppression with reported menopausal status.,We report no differences in the association between age and EIC in heterosexual men and women of similar age, suggesting that any reduction in EIC in the older age group may be related to ageing, rather than menopause specifically. In contrast, when exploring the association between age and viral outcomes, we found that the association differed significantly between men and women. Women of peri-menopausal age (40–50 years) had a higher likelihood of VL suppression compared to women aged <40 years, but this association was not present in men. Additionally, women in the peri-menopausal age group were less likely to experience virological failure compared to women aged <40, and this trend was not observed in the post-menopausal group. However, older men were consistently less likely to have VL rebound in both older age groups (40–50 and >50) compared to men aged <40 years. Previous analyses of the UK CHIC Study and American datasets exploring virological responses have reported no differences by sex.A strength of our study is the large number of women living with HIV from a representative sample of people attending HIV care in the UK included in our analyses. Longitudinal follow-up through routinely collected data in the UK CHIC Study cohort allows us to understand dynamic changes in clinical outcomes as people with HIV age.A key limitation is that we have no data on menopause status or menopausal symptoms, and instead use age as a surrogate marker. Although we selected our age groups to broadly correspond to menopausal stage, it is important to acknowledge that they may not capture this perfectly. Our age groups for this analysis were selected on the basis of the age distribution of women with HIV in the UK CHIC cohort as well as the expected age at onset of menopausal symptoms (with an average age of menopause in the UK of 51 years).,,Additionally, although our analyses focus on menopause, we recognize that many other factors may contribute to the differences seen in the age groups selected. Younger women, for example, may experience barriers which negatively impact their ability to engage in healthcare and to adhere to therapy, particularly when caring for a family. Ageing in general represents a whole host of new challenges not considered in these analyses, e.g. diagnoses of new comorbidities that may lead to polypharmacy. As such, the possibility of drug-drug interactions must always be considered to ensure there is no loss in virological efficacy or increased drug toxicities as people age.Further limitations include a lack of lifestyle data  which may have an impact on the timing and symptoms of menopause as well as ART adherence.In summary, using data from one of the largest and representative cohorts of people with HIV, we find an association between peri-menopausal age and viral outcomes among heterosexual women living with HIV. We suggest there is better adherence to ART amongst women aged 40–50 compared to that in younger women. Our findings highlight the importance of taking a life course approach when providing care to women living with HIV, and ensuring support is tailored to their needs at different life stages. We, therefore, welcome the national recommendations that HIV clinics provide information about the menopause to women attending their service, and establish care pathways with specialist menopause services and primary care.


Dietary composition was more diverse in winter than in summer, while Gramineae and Rosaceae were consumed frequently all year. Turnover of seasonal diet and gut microbiota composition occurred consistently. Yaks shifted enterotypes in response to dietary change between warm and cold seasons to best utilize nitrogen and energy, in particular in the harsh cold season. Our findings provide insights into understanding seasonal changes of diet–microbiota linkages in the adaptation of mammals to high altitudes.Dietary selection and intake affect the survival and health of mammals under extreme environmental conditions. It has been suggested that dietary composition is a key driver of gut microbiota variation; however, how gut microbiota respond to seasonal dietary changes under extreme natural conditions remains poorly understood. Sequencing plant  The yak (Bos grunniens), an iconic symbol of high altitude and a mainstay for Tibetan people2, has anatomical and physiological adaptations and a genetic basis for mammalian adaptations3, as well as a co-evolved microbiome4, that equip the animal for the high altitude and extreme environment. Yet gut microbiota of the yak and their relationship to seasonal dietary shifts in their natural habitat remain largely unknown, although this information could contribute to the understanding of adaptation to the high-altitude Tibetan plateau.The Tibetan plateau, called ‘the third pole’, forms the largest and highest year-round grazing area in the world. The region is characterized by a harsh climate of extreme cold and aridity, and high ultraviolet radiation and hypoxia, which challenge the survival of humans and other mammals5, being sensitive to perturbations, such as dietary changes, environmental factors6 and enteric pathogens. They play an integral role in nutrient intake, behavior, metabolism, immune function, and development of the host8. Substantial changes in mammalian microbiota composition have been observed in response to seasonal diet availability among and within individuals, as evidenced by longitudinal analyses of gut microbiota in Hadza hunter-gatherers9, wild wood mice (Apodemus sylvaticus)10, red squirrels (Tamiasciurus hudsonicus)6, giant pandas (Ailuropoda melanoleuca)11, wild great apes13 and North American bison (Bison bison)14. However, most studies on mammalian gut microbiota dynamics were done in non-stressful environments and without quantitative dietary information related to habitats. Recent reports on gut microbiota composition from large herbivores in the semi-arid East African savanna revealed a greater seasonal turnover and diet–microbiota association in domesticated than in wild species15. These studies provide a better understanding of intra-specific and inter-specific diet–microbiota associations in wild and domesticated species. Yet studies on seasonal diet and microbiota relations are lacking in high-altitude mammals.Gut microbiota are complex and dynamicTo examine the fine-scale relationship between quantitative dietary consumption and gut microbiota, we conducted a spatio-temporal study of the impact of seasonal diet on gut microbiota from 302 individual yaks. Using DNA metabarcoding and 16S rRNA gene analyses, measurements were made in free-grazing yaks across four seasons on the eastern part of the Tibetan plateau, in which either a transhumance (TH) or open-continuous grazing (OCG) regime was followed. These in-depth and longitudinal analyses could contribute to the understanding of the adaptations of mammals to the harsh, high altitude environment.trnL (UAA) intron marker genes16 (see the “Methods” section). This revised library was used for DNA metabarcoding analysis. In total, our library comprised 411 plant species  throughout the alpine grassland area of the study in the TH and OCG regimes.According to the National Center for Biotechnology Information (NCBI) and Bold system databases, there were 199 plant species in the study areas. Combined with plant taxonomic identification, this study generated an additional 212 local plant species DNA barcode reference library using P6 loop of the chloroplast The diet data included 30,534,414 high-quality sequences, 81,530 unique sequences after removal of singletons and 2010 operational taxonomic units (OTUs)  was highest in the warm season (from June to September), with 2891 ± 148 kg DM/ha in September, and was lowest in the cold season (from October to May) with 99 ± 10.8 kg DM/ha in May Fig. . NeutralRuminococcaceae_UCG-010, Ruminococcaceae_UCG-005, Rikenellaceae_RC9_gut_group, unclassified Ruminococcaceae and unclassified Bacteroidales the most abundant across seasons  dissimilarities, seasonal diet composition was associated with microbiota composition consistently in both TH  revealed that the samples formed three distinct enterotype clusters based on BC dissimilarities. Each cluster was driven by the variation of its representative genera level: Akkermansia and uncultured Eubacterium WCHB1-41 in Enterotype 1, Ruminococcaceae_UCG-005 in Enterotype 2 and Ruminococcaceae_UCG-010 in Enterotype 3  and Enterotype 3 was prevalent throughout the year  database. Both Akkermansia and Kiritimatiellaeota showed convergent enrichment of enzymes that are involved in arginine and fatty acid biosynthesis pathways (map00220 and map00061)  cycle and involves arginine biosynthesis, and also regulates fatty acid synthesis. Notably, we observed 12 enzymes that play key roles in arginine biosynthesis, but no enzyme is involved in urea synthesis to reduce nitrogen loss in urine under conditions of low-nitrogen stress. Six of a total of seven enzymes participated directly in fatty acids biosynthesis and contributed to energy deposition. These results indicated that both arginine and fatty acid biosynthesis pathways evolved in high altitude mammals for efficient nitrogen utilization and energy deposition in the cold season.These seasonal distributions of gut enterotypes led us to hypothesize that the fixed gut enterotype, represented by 61) Fig. . Pyruvat19 and differ in composition from microbiota of lowland ruminants4.We determined seasonal shifts in diet and gut microbiota in a high-altitude large herbivore. Yaks graze the grasslands of the Tibetan plateau all year, and face severe challenges, in particular extreme cold and limited food availability during the cold season. To survive under such conditions, yaks have evolved anatomical and physiological adaptations. Furthermore, convergent evolution was reported for yaks and their rumen microbiota. The rumen microbiota follow unique maturation strategies10. In response to cyclic seasonal dietary fluctuations of the host, gut microbiota could alter their metabolic rate and energy extraction from complex carbohydrates, and, ultimately, promote the co-evolution of hosts and microbes6. Furthermore, our findings support the present grassland polices of TH of moving yaks to different landscapes and searching for favorable diet. This practice mitigates grazing pressure, thereby enhancing animal performance. As demonstrated by our diet and microbiota analyses, a better understanding of relations between yaks and plants can yield key insights in supporting TH with a proper stocking rate and improving grassland management strategies. The key microbiota associated with feed efficiency could provide an alternative solution for strengthening microbiota-led breeding programs to increase yak performance, particularly in the cold season.In this study, seasons constrained both diet and gut microbiota, but less so for microbiota than diet. Such effect of season on microbiota composition in response to food availability and chemical composition might be due to a re-assembly of the community structure of the gut microbiota resulting from substrate availability. Surprisingly, among seasons, the changes in gut microbiota composition were relatively stable compared with changes in dietary composition, which suggests that high altitude mammals evolved stable patterns of gut microbiota composition across seasons. By contrast, gut microbiota in humans and other mammals differed markedly with seasonal dietary shifts21. However, these methods are not suitable for diet analyses of herbivorous mammals feeding on pasture with high plant diversity. Field observations require high visibility and are prone to omission of plant species22, while histology is very tedious and often inaccurate23. The n-alkane method is limited when the animal consumes a large number of plant species24. When the n-alkane method was used to determine diet composition of grazing yaks on the Tibetan plateau, only the two or three main dietary components could be detected20. Studies of DNA metabarcoding identified the level of forb dominance in megafaunal diets from permafrost sediment samples25 and allowed fine-grained niche separation from seven large mammalian herbivores26. These studies indicated that ingested plants can be effectively identified and quantified using the DNA metabarcoding method27.The lack of precise methods to assess and identify the complex diet consumed by grazing herbivorous mammals has been a long-standing obstacle in field studies. Conventional methods include field observations, microhistology and natural n-alkanes from plant cuticular waxesRuminococcaceae_UCG-005 enterotype was associated with high protein and low fiber diets in the warm season and the Akkermansia and uncultured Eubacterium WCHB1-41 enterotype with low protein and high fiber diets in the cold season. It was suggested that the proportion of protein and carbohydrate contents in the diet mediates the host’s enterotype shift, at least in baboons29. Seasonal variations in dietary protein and carbohydrate contents could provide an attractive explanation in terms of enterotype dynamics, and also contribute to determine the enterotype for high altitude herbivores. Gut enterotype in humans remained stable when on a 10-day low-fat/high-fiber diet intervention30. However, the most compelling evidence for seasonal diet shifts in enterotypes in the present study was that enterotypes 1 and 2 shifted consistently between seasons and Ruminococcaceae_UCG-01 enterotype 3 remained stable throughout the year. This might be the result of long-term co-evolution between the host and environment and suggests that the fixed seasonal enterotype dynamics play a key role in species formation and adaptation of mammals to the extreme environment at high altitude.This present study provides biological insights into the clusters and functionality of gut enterotypes. In particular, we describe gut enterotypes as well as the functional genomic information following seasonal dietary changes in free grazing yaks. The Akkermansia resides in the mucus layer of the gut and is known for its degradation of mucin31. It was reported that A. muciniphila plays a pivotal role in the prevention of obesity and type 1 and type 2 diabetes and, hence, promotes health in humans33. In addition, there is evidence that A. muciniphila functions as an energy sensor and regulates energy homeostasis for host microbial mutualism35. The present study demonstrated a substantial increase in Akkermansia and uncultured Eubacterium WCHB1-41 enterotype 1 with low nitrogen and low energy intake in the cold season, suggesting that this enterotype responds to a high fiber and low protein diet. Previous studies reported that a high fat diet decreased mucus thickness32, whereas, a fiber-rich diet increased mucus thickness, which improved the gut positive barrier function36. Akkermansia and uncultured Eubacterium WCHB1-41 degraded mucins and converted them into short-chain fatty acids (SCFAs), which provided nutrients for other resident bacteria and cells37.Whsc1, Glul), fatty acid biosynthesis and metabolism gene (Hsd17b12)3, and up-regulation in 36 genes that were related to volatile fatty acids transport and absorption in the ruminal epithelium of high-altitude ruminants4. These studies provide additional adaptive responses to insufficient energy intake that yaks experience. In addition, it was reported that yaks have a relatively low N requirement and utilize dietary N more efficiently than cattle in the cold season41. The current study also provides a mechanistic explanation as Akkermansia and Kiritimatiellaeota are involved in arginine and fatty acid biosynthesis pathways to utilize dietary N and generate energy in the cold season. In the context of the increased metabolic disease prevalence, this study provides an insight on the influence of Akkermansia and uncultured Eubacterium WCHB1-41 enterotype in nitrogen and energy utilization in the host, and implies that this enterotype plays a key role in mediating the nutritional homeostasis in high altitude animals with large therapeutic potential of metabolic diseases in humans. Further analyses of genomic functions of gut microbiota of yaks would be beneficial for a better understanding of mammals living in an extreme environment. Findings in the present study demonstrate that gut microbiota respond to seasonal dietary shifts, which allows yaks to better utilize poor forage of low protein content. The understanding of diet–gut microbiota interaction improves our understanding of how yaks adapt to extreme environments.Genetic studies in yaks demonstrated enrichments in the amino-acid metabolism gene  in winter-spring; at 3130–3300 m above sea level  in summer; and at 3015–3100 m above sea level  in autumn. The dominant and associated species in winter-spring pasture included E. nutans, K. humilis, K. capillifolia, S. capillata, Leymus secalinus, P. viviparum, and A. smithii; in summer included P. fruticose, P. viviparum, Juncus himalensis, Deschampsia cespitosa, Festuca ovina, Saussurea amara, Carex atrofusca, and C. moorcroftii; in autumn included P. fruticose, Salix oritrepha, P. viviparum, E. nutans, D. cespitosa, K. tibetica, K. humilis, C. kansuensis, J. castaneus, P. anserine, T. alpinum, and S. katochaete. The annual mean temperature was −0.1 °C in the TH and 0 °C in the OCG with a peak in summer (June–August) and a trough in winter (December–February). The grassland was alpine meadow and alpine shrub meadow and the plant growing season was 90–120 days.The Tibetan plateau forms the high-altitude core of Asia and presents a challenge for mammals to survive43, and the average monthly temperature and precipitation recoded by a nearby meteorological station in the study area, four seasons can be identified as spring (April–May), summer (June–August), autumn (September–October), and winter (November–March the following year) . Fresh yak feces were collected from the TH and OCG grasslands, mixed thoroughly in an unused freezing tube, placed immediately into liquid nitrogen containers in the field, and transported to Lanzhou University until further processing. An amount of 0.2 g of fresh feces was used for DNA extraction with QIAamp® Fast DNA Stool Mini Kit  and an extraction blank was processed to monitor for cross-contamination. DNA was quantified using the NanoDrop-2000 UV–Vis Spectrophotometer . The DNA samples were used for both diet (n = 302) and microbiota (n = 300) analyses. The studies and all procedures involving the animals were approved by experimental field management protocols (EAF2021012) of Lanzhou University.According to previous studies44. NDF and ADF were measured according to Van Soest et al.45, with heat stable alpha amylase and sodium sulfite used in the NDF procedure.Three plots (100 m × 100 m) were selected randomly in each of the TH and OCG grasslands in each of the four seasons. Within each plot, five quadrats (50 cm × 50 cm) were selected randomly. AGB was harvested in each quadrat, oven-dried at 65 °C to a constant weight, ground to pass through a 1-mm screen and stored at room temperature for chemical composition analysis. Dry matter (DM) was determined by oven-drying at 105 °C for 24 h, EE by extraction with petroleum ether and N content by the Kjeldahl methodtrnL (UAA) region was used for DNA metabarcoding with primers trnL (UAA) g and trnL (UAA) h46 (Supplementary Table The P6 loop of the chloroplast 47 (Supplementary Table The V3–V4 region of the 16S rRNA gene was sequenced on Illumina MiSeq 2500 platform with primers (341F/806R)To identify diet plant sequences from fecal samples, we established an extensive DNA reference database from plant species throughout the alpine grassland area of the study, Yongfengtan and Wushaoling. The collection included 212 species that were most abundant in the study area. All plants were identified to species-level by expert botanists.trnL-P6 (UAA) using established primers and protocols . trnL (UAA) was sequenced in 25 μL PCR reaction that included 2.5 μL MgCl2, 4 μL dNTP, 0.5 μL of each primer (trnL(UAA)c/trnL(UAA)d), 12.5 μL 2 × GC Buffer I, 0.25 μL TaKaRa LA Taq®, and 0.5 μL DNA template. Thermocycling for trnL (UAA) proceeded at 94 °C for 1 min, 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, with a 5 min extension at 72 °C. Programs Geneious and MEGA7.0.14 were used for sequence alignment and analysis.Reference plant DNA was extracted with DNeasy Plant Mini Kit  using 0.2 g of leaves, and sequenced chloroplast 49. Sequences shorter than 10 bp and mean Illumina fastq quality scores <20 were not considered. Paired reads were merged using USEARCH1150 and then all merged sequences of each sample were pooled. Quality filtering was performed on the pooled sequence with more than 0.5 expected error using fastq_filter command in USEARCH11 and only sequence lengths ≥ 10 were retained. Fastx_uniques command in USEARCH11 was used to find a set of unique sequences from filtered sequences, and singletons  were removed. The remaining sequences were denoised (cluster at 100% similarity) using UNOISE algorithm51, during which OTU representative sequences were generated and potential chimeras were excluded. All the initially pooled sequences were mapped into the denoised sequence (zOTU) to generate an OTU table using otutab command implemented in USEARCH11. Strictly identical sequences were merged and assigned plant species based on their unique sequences to DNA metabarcode sequences with exact matches (100% identity) to reference sequences. Only unique sequences with an identity of 100% to reference sequences were kept for further analysis. When 100% identities were acquired from the local reference and NCBI libraries; preference was given to the local reference library. When a diet sequence matched multiple reference sequences exactly, assignments were revised to the finest taxonomic level by blasting with NCBI. We used the summarize_taxa command to group identical sequences, tally them within samples and then quantify the relative read abundance of each sequence, which is widely used to quantify the proportional foods consumed by animals54, and has been confirmed in several studies56. The resulting OUT counts per sample were rarefied to 40,000.Sequence demultiplexing, quality, and preliminary identifications were conducted by QIIME 1.9.1. Demultiplexing used split_libraries_fastq.py51 with a threshold of 100% identity and seeded with SILVA rRNA gene databases57. Sequences identified as archaea, mitochondria, and chloroplast were removed. After filtering and identification, the bacterial 16S rRNA gene data included sequences across 300 samples. The resulting OTU counts per sample were rarefied to 10,000.Quality control, merging of pair ends, OTU clustering, and taxonomic assignation were performed using the QIIME 1.9.1. Illumina fastq quality scores < 20, ambiguous nucleotides and chimeras were discarded. The reads were assigned to OTUs using UNOISE358 to test for the presence of enterotypes in high-altitude yak. The genus-level relative abundance profiles of samples were clustered using Jensen–Shannon divergence (JSD) and BC dissimilarity and partitioning around medoid (PAM) clustering in R. The robustness of clusters was assessed by the Calinski–Harabasz (CH) index and silhouette score59. We applied the PAM, CH index, and silhouette score to clustering using BC and JSD methods, for which results did not differ  in vegan61. Pairwise differences within and across seasonal diets and microbiota variation were permutested with 9999 permutations and false discovery rate (FDR) correction. To assess which plant and microbiota taxa were most responsible for seasonal differences in diet and microbiota variations, we performed indicator species analysis62 with indicspecies in R. We used the multipatt function with 9999 permutations to the list of species that were related with a group of samples and r.g. function determined the correlation between two binary vectors. Within seasons, the relationship between diet and microbiota richness in each sample was assessed by linear regression using the data across seasons in TH and OCG regimes. The correlations between diet and microbiota composition were computed based on Procrustes analysis, a program that compares the relative positions of points in two multivariate datasets63, and conducted in R using the vegan package. Monte Carlo p-values for rotational agreement significance testing were determined from 9999 permutations. For enterotype comparisons, samples were pooled into bins , and the significance among seasons were identified using Fisher’s exact test with FDR correction of p-values. FDR was applied at a level of 0.05 per tested correlation and significance for multiple comparison.Standard R commands were performed to generate variations in relative abundance across seasons, and the Wilcoxon test (two-sample comparisons) or the Kruskal–Wallis test (multiple groups) was used to measure significance in non-parametric relative abundance profiles. The Further information on research design is available in the Supplementary InformationReporting Summary


The prognostic value of intratumor T regulatory cells (Tregs) in colorectal cancer (CRC) was previously reported, but the role of these cells in tumor draining lymph nodes (TDLNs) was less addressed.A total of 150 CRC stages I-IV were retrospectively enrolled. Intratumor and TDLN Tregs were examined by immunohistochemical assay. The association of these cells was estimated by Pearson correlation. Survival analyses of subgroups were conducted by Kaplan–Meier curves, and the log-rank test and risk factors for survival were tested by the Cox proportional hazard model.P < 0.01) and OS  in the patients.High accumulation of Tregs in tumors was significant in patients with younger age and good histological grade, where enrichment of these cells in TDLNs was more apparent in those with node-negative disease and early TNM stage disease, both of which were more common in early T stage cases. A significant correlation of intratumoral and TDLN Tregs was detected. Patients with higher intratumoral Tregs displayed significantly better PFS and OS than those with lower Tregs. However, no such differences were found, but a similar prognostic prediction trend was found for these cells in TDLNs. Finally, intratumoral Tregs were an independent prognostic factor for both PFS (HR = 0.97, 95% CI 0.95–0.99, Higher intratumor Tregs were associated with better survival in CRC. Although no such role was found for these cells in TDLNs, the positive correlation and similar prognostic prediction trend with their intratumoral counterparts may indicate a parallelized function of these cells in CRC.The online version contains supplementary material available at 10.1186/s12876-022-02205-0. Colorectal cancer (CRC) remains one of the main cancers worldwide, accounting for 9.8% of all new cases and 9.2% of all deaths for all cancers combined in 2021 [T regulatory cells (Tregs), which are specifically labeled by forkhead box transcription Factor 3 (Foxp3) . The notNotably, Tregs were also found to be increased in other locations, such as peripheral blood in CRC . TDLNs, In this study, we aimed to detect Tregs in tumors as well as TDLNs and to determine the underlying correlation and their individual prognostic value. Furthermore, we compared their prognostic efficacy with other robust prognostic indicators, including the neutrophil to lymphocyte ratio (NLR), lymphocyte to monocyte ratio (LMR), and prognostic nutritional index (PNI), in CRC.From December 2012 to January 2018, CRC patients with radical recession of primary lesions in Hainan Hospital of Chinese PLA General Hospital were enrolled, and patients were excluded if they met any one of the following criteria: 1. neoadjuvant therapies; 2. missing laboratory tests within a week before surgery or key information in postoperative pathological reports; 3. multiple or recurrent malignancies or in situ lesions; or 4. loss to follow-up or less than 36 months. Other clinicopathological parameters, including body mass index (BMI) and tumor size (TS), were collected as previously reported –31. The Resected fresh samples were immediately processed by standard histological methods, and the staining was carried out as follows. In brief, 5-µm slides were cut for both tumors (samples with necrosis and broken areas were excluded) and TDLNs, and one node was randomly selected either in cases with mTDLNs or mfTDLNs (including those with tumor deposits). After deparaffinization, sodium citrate solution was used for antigen retrieval for 20 min and then cooled to room temperature. Subsequently, 3% hydrogen peroxide was used for blocking for 10 min. After washing, nonspecific antigen blocking was then performed with 5% goat serum for 30 min at room temperature . After washing, the primary rabbit anti-human Foxp3 antibody  was incubated at 4 °C overnight, and the negative control was conducted by replacement of the primary antibody with identically diluted 5% nonimmunized rabbit serum . After washing, goat anti-rabbit IgG H&L (HRP)-preadsorbed secondary antibody  were added for 30 min at room temperature. Staining was then achieved by adding 3,3′diaminobenzidine  according to the manufacturer’s protocol, the slides were then washed and mounted. The results were read under a light microscope at a magnification of × 200  by two independent pathologists who were blinded to the clinical information. The absolute number of Tregs in the tumor and in TDLNs was counted as follows: for each slides, five hot spots in 10 high power field (HPF) with the highest number of positive cells were selected, and JPEG images were taken by using a digital camera, then the number of positive cells in the images were counted by using ImageJ software as previous report ; patientRoutine laboratory data were collected as described in our previous report , and theThe follow-up was conducted as scheduled in a previous report . PFS wasP < 0.05 was considered statistically significant.Statistical analyses were conducted by SPSS 20.0 . Differences in clinicopathological parameters in Tregs-low or high in tumors and TDLNs were determined by χ2-test, Student’s t, or nonparametric rank sum test when appropriate. The association of Tregs in tumors and TDLNs, as well as with systemic inflammation markers, was determined by Pearson correlation. Survival differences for Treg-low or Treg-high groups were determined by Kaplan–Meier (K-M) analysis followed by log-rank tests. Risk factors for survival were estimated by a Cox proportional hazards model. Double-sided P < 0.01), where TDLNs with high frequencies were more apparent in node-negative (P < 0.01) and early TNM stage (P = 0.02) patients, and both of which were more common in early T stages .In total, 290 patients were enrolled, and 150 patients  were included in the final analysis  and OS  rates could be detected, but no such differences could be found for TDLN Tregs in PFS  and OS . By K-M analyses, patients with intratumor high Tregs had a significantly better PFS  and OS  than those with Tregs-low  and OS , a similar survival difference trend could be found  and OS .Univariate tests indicated that CEA status, invasive depth, node involvement, distant metastasis, TNM stages, preoperative LMR and PNI, and intratumor Tregs were significant prognostic factors for PFS Table  and OS , and wheIn the present study, we found that Tregs accumulated differently in tumors and TDLNs. Although only intratumoral Tregs showed a significant prognostic value for patient survival, a positive correlation of these cells was established for those located in tumors and TDLNs; in addition, a similar prognostic trend of these cells in TDLNs as in tumors was observed. To the best of our knowledge, our study includes the largest sample to explore Tregs in TDLNs and examine their prognostic role as well as correlation with their intratumoral counterparts in CRC.hi and Foxp3lo subsets, and the latter could release inflammatory cytokines and correlate with better prognosis in CRC [hiCD45RA−), nonsuppressive Tregs (Foxp3loCD45RA−), and resting Tregs (Foxp3loCD45RA+); only activated Tregs correlated with tumor metastases [Taking into consideration its negative role in manipulating antitumor immune responses, the accumulation of Tregs is regarded as an indicator of poor survival in many cancers –9. Commos in CRC . In additastases . NonetheP < 0.01) [P = 0.01 and P = 0.07, respectively, data not shown). More importantly, we detected that these cells presented a parallel prognostic trend to the intratumoral ones, which supports our aforementioned speculation that these cells may functionally correlate in CRC irrespective of their different localization.TDLNs play an important role in inhibiting the spread of cancer , 21 and  < 0.01) . Additio < 0.01) , we alsoIt was reported that Tregs could not only release inflammatory cytokines such as interleukin (IL)-10, IL-35, tumor necrosis factor-α (TNF-α), and interferon-γ (INF-γ) , 52 but Notably, we found that 17.33% (26/150), 12% (18/150) cases were blank for Tregs in tumor and TDLNs, respectively. In fact, absence of Tregs in tumor has been registered in previous reports –63. As aOur study has some limitations: first, its retrospective nature cannot completely exclude confounding factors; second, patients with T3 or higher disease with other risk factors or those with mTDLNs would receive subsequent therapies after surgery, which would affect not only survival but also Tregs ; third, Overall, our study indicated that higher intratumoral Tregs were associated with better survival in CRC, and a positive correlation of these cells in TDLNs could be found with intratumor Tregs. The similar prognostic prediction trend for these cells in TDLNs as intratumor Tregs suggested that these cells may share a parallelized function in patients; however, more studies are still needed in the future.Additional file 1. Dataset to generate the results of the study.