We studied the core bacterial communities of 19 sponge species from Nha Trang Bay , with particular emphasis on the contribution of planktonic seawater bacteria to the sponge core microbiomes. To ensure consistent sponge-microbe associations and accurate identification of planktonic bacteria transmitted from seawater, we were very restrictive with the definition of the sponge core microbiomes , and with the identification of valid biological 16S rRNA gene sequences (100% sequence identity) that belonged to potentially different bacterial taxa. We found a high overlap (>50% relative abundance) between the sponge species core microbiome and the seawater bacterial core in ca. a half of the studied species, including representatives of both, HMA and LMA sponges. From our restrictive analysis, we point to horizontal transmission as a relevant way of symbiont acquisition in sponges. Some species-specific recognition mechanisms may act in sponges to enrich specific seawater bacteria in their tissues. These mechanisms would allow the maintenance of bacterial communities in a species across geographical ranges. Moreover, besides contrasting preferences in bacteria selection from seawater, divergent physiological traits may also account for the different microbiomes in species of HMA and LMA sponges. In other words, to concentrate on those bacteria, which have established tight associations with the host through several evolutionary time scales2, independently of potential, mutual benefits or costs for the partners involved3. In this context, the core microbiota concept was adopted to ascertain the consistent associations of a metaorganism5, but also allows to study the core metabolic functions provided by the host–microbe interaction to the system6. The core concept was first applied to differentiate host–microbe interactions of the mammalian gut and in plant root systems6. Further, it was extended to marine animals with the aim of understanding the consistent contributions of the microbial symbionts to the host ecology, success, or decay1.The first step to study multi-microbial symbionts within animals is to focus on permanent symbionts by ruling out the background noise produced by transient microbes7 through spatial and temporal scales, concluding that the complexity of the reef habitat, and the life coral history traits likely influence the coral core microbiomes. Thus, more in deep research to explore accurately the core microbiome of many invertebrates is needed to overcome the constraints associated to their complex habitats. Although there is some controversial in the literature about how the core microbiota should be defined8, the most reasonable definition always depends on the question approached9. For instance, more or less restrictive criteria (i.e. present from 7% to 100% of the replicates) have been used for marine invertebrates11.In marine habitats, studies on coral and sponge microbiomes have proliferated in the last 10 years. For instance, the persistent microbial symbionts (core) of several coral species from a reef were identified13. They harbour the highest diversity of microsymbionts among marine invertebrates15. A wide range of studies using massive sequencing methods have retrieved thousands of microbial 16S rRNA gene sequences for each targeted sponge species11, which stressed the difficulty to unveil the mechanisms underlying these associations due to the high diversity of the partners involved. Indeed, some of the 16S rRNA gene sequences belonged to transient bacteria captured from the environment by the sponges while filtering seawater, and do not represent permanent symbionts11. Consequently, indirect analytical methods have been implemented trying to split stable symbionts from transient planktonic bacteria in sponge-microbial systems. The core microbiota concept was adopted to focus on permanent bacteria in a sponge species11 or phylum10, notwithstanding the host geographical and ecological origins, or temporal scales16. The rationale underlying the core concept is that stable symbionts should represent tight biological associations and thus, they are expected to be present in most, or all, host individuals.Sponges are a diverse group of sessile filtering invertebrates that play important ecological roles in benthic marine ecosystems9. Several studies have proposed that stable sponge microsymbionts should be present in the sponges but not (at least not in high abundance) in the surrounding seawater11. But, recording the presence of a sponge bacterium in seawater greatly depends on the abundance threshold used to include or exclude sequences from downstream analysis, the sequencing depth and the number of samples. Moreover, it has been proposed that stable symbionts are mainly inherited by the progeny from their parent sponges20. However, vertical and horizontal transmission of the same bacteria have been described21 and more recent investigations reported different microbiomes in adults and larvae of the same species, which highlights the role of seawater bacteria in the structural composition of sponge microbiomes22.Substantial differences in the resulting diversity metrics related to the core definition applied (from 12% to 100% occurrence), have been reported recently24. These differences have been related to contrasting structural and physiological traits of the two species groups25. HMA sponges have a denser mesohyle and a more complex aquiferous system, with smaller choanocyte chambers, than LMA sponges25. Also a partial trophic niche separation for HMA and LMA sponges has been proposed26. Comparisons of the core microbiomes of these two groups, which consisted in bacterial T-RFLPs that were present in all species replicates across seasons and study years, showed that HMA sponges had a larger number of core bacterial groups with a higher overlap with seawater than LMA hosts27.Differences in microbial diversity between High Microbial Abundance (HMA) and Low Microbial Abundance (LMA) sponges have been reported28, in particular for short reads obtained from Next Generation Sequencing (NGS). Instead, a 100% of sequence identity has been proposed to obtain true biologically informative sequences, which can underlie metabolic and ecological particularities28. To ensure that the 16S rRNA gene sequences recovered from the seawater samples were identical to those recovered from the sponges, we clustered OTUs  at 100% identity (Zero radius OTUS or ZOTUs28), which has only been recently applied in a couple of studies of sponge microbiomes30. Clustering sequences at 100% identity and restricting the core to microbes present in 100% of the analysed samples seem particularly relevant when trying to elucidate horizontal symbiont acquisition.In this study, we attempted to cast some light on the acquisition modes of microbial symbionts in sponges by i) unveiling the permanent microbiomes within a large number of sponge species, and ii) estimating the contribution of seawater bacteria to the sponge core microbiomes in representatives of both HMA and LMA sponges. To address these goals, we analysed the sponge microbiomes of the 19 most abundant sponge species inhabiting a small geographical area in Nha Trang Bay , as well as the bacterial assemblages of the surrounding seawater. We applied a restrictive approach to core community concept in terms of bacteria occurrence across species replicates and to OTU definition. Sequence identity thresholds <99% for the 16S rRNA V4 region have been proved to be inaccurate for bacterial species delimitation2 0.56, p-value < 0.01), which means that replicates from the same species were more closely related to each other than to any other species. However, dispersion within replicates greatly varied depending on the sponge species . A dichotomy  between the so-called HMA and LMA sponges regarding their bacterial composition was detected  was much lower than the dispersion within the 16 LMA species  varied from 54 in ra Table . The numN. hartmani was the species with the largest core microbiome, representing up to 94% of relative abundance  to 195 (in T. cf. raphidodophora). However, the abundance of species–specific ZOTUs did not surpass 25% in any case, and most often were below 5% of relative abundance in the respective core communities. Gellioides sp. and S. cf. laboutei were the species with the highest relative abundances of species-specific ZOTUs .The number of species-specific ZOTUs  varied from 0  and HMA or LMA species were detected with the A. paraviridis (99.5%), Gellioides sp. (93.2%) and C. reinwardti (90.4%), while Antho sp. (9.9%) and Pseudosuberites sp. (10.9%) showed the lowest overlap between the sponge and the SW core bacteria , rare SW ZOTUs (>0.01% of relative abundance) were abundant in the core of the sponge species. This is particularly visible in the example of C. reinwardti, which harboured ZOTUs that represented just a 5% of the SW core community and 90% of the sponge core. The opposite occurred in other sponge species such as Pseudosuberites sp. and T. cf. raphidophora, which had highly abundant (>1% of relative abundance) SW ZOTUs poorly represented in their core.The relative abundances of the shared core SW ZOTUs  with the sponge core microbiomes varied between 9% and 99% depending on the sponge species Fig. . The sporia Fig. , Table 1Gellioides sp. N. hartmani, Mycale sp. A. suberitioides, C. reinwardti, and S. cf. labutei , instead of the SW core ZOTUs, the relative abundances of bacteria shared with SW were drastically reduced in most of sponge core microbiomes, with most values below 40% Fig. . This reC. reinwardti, the proportion of SW OTUs changed from 10% (for ZOTUs) to 73% (for OTUs). Also remarkable were the differences for A. paraviridis, Gellioides sp., and Phorbas sp.Results of both comparisons for OTUs at 97% sequence identity are shown in Table Candidatus Branchiomonas (Betaproteobacteria) accounted for more than 30% and 50% of the Mycale sp. and A. paraviridis core microbiomes, respectively. Similarly, ZOTUs belonging to several Endozoicomonas (Gammaproteobacteria) were present at different relative abundances in the four species with the highest proportion of SW ZOTUs . Two main groups  were also differentiated according to the bacterial classes. The first group (C) corresponded to bacterial classes associated to LMA sponges, with high abundances of Alpha-, Beta-, and Gamma- Proteobacteria. The second group (D) showed bacterial classes associated to HMA sponges. ZOTUs belonging to PAUC34f, Chloroflexi, Acidobacteria, Actinobacteria, and Nitrospinae were almost exclusively found in the three HMA species .The abundance and distribution of the SW core ZOTUs in the sponge core microbiomes is shown as a heatmap representation Fig. . Two spo34. Selection of specific bacteria and competition among the selected bacteria31 may converge to shape the species-specific patterns of sponge microbiomes, which used to be similar in geographically distant individuals of the same genus35.The sponge-associated bacteria were species specific for the 19 study species, as previously reported for sponges from other geographical areas37. Indicator bacteria at class level for both groups are found, which match the indicator classes inferred from differential abundance analysis38. It has been reported that HMA sponges show higher microbiome similarity among species than LMA sponges27. Moreover, bacterial diversity is also claimed to be higher in HMA40. These aspects are confirmed in our study when considering the core members of both HMA and LMA sponges, although the number of representatives of each group is unbalanced.However, differentiated patterns of bacterial composition are observed according to the affiliation to the HMA or LMA groups, as previously reported41, but also questioned25, that LMA sponges contain mainly transient seawater bacteria. Representatives of both HMA and LMA study sponges contained high percentages of SW core bacteria (>50% of core relative abundance). However, species of each group acquire different SW bacteria that are indicators of either HMA or LMA sponges . Only recently, the sponge microbiomes have been analysed at the ZOTU level9. For example, 85% occurrence was used for a species with more than 47 replicates to conduct an interaction network analysis11. In our study, as we wanted to focus on the truly symbiotic bacterial community of each sponge species, we choose a restrictive approach to the species core by only considering bacteria that were present in all the replicates of each species (100% occurrence), disregarding whether they were present among the SW core or not. In this way we ensured that we were focusing on persistent symbionts rather than on transient microbes.Analysing the bacterial core at species level provides information about stable, purportedly fixed associations, which may be involved in sponge-bacteria interaction patterns. To define the species core, an appropriate percentage of bacteria occurrence across species replicates has to be selected depending on the study aims16. A high stability of the microbiome across sponge replicates can be indicative of the strength of the sponge-bacteria associations, whereas the opposite would indicate the presence of facultative /transient bacteria11. Most of the associations among bacteria and the studied species appeared to be highly constant, thus, suggesting a strongly fixed relationship, although we are aware that this might depend on the number of replicates analysed.The size of the bacterial core, which represents the permanent part of the sponge microbiome, seems to be intrinsic of each sponge species32. Schmitt et al.10 suggested that the species-specific bacteria would probably be vertically inherited. If this assumption is true, the percentage of vertically inherited symbionts in our sponges would be rather low, since we have found a few species-specific microbes and the majority of the sponge bacterial taxa are found in more than one species.The contribution of the species-specific ZOTUs to the core communities, in terms of relative abundance, was surprisingly low. Those values may be influenced by the number of sponge species taken into account in the study, the similarity between their microbiomes, and, more strongly, by the restrictions associated to the way species-specific OTUs are defined, whether being part of the species core community (our study) or not24. To avoid this potential contamination, we used the 100% occurrence core approach in both the sponges and seawater, because it is unlikely that a ZOTU contaminates all the replicates of the same source. Moreover, we took into account the differential abundances that a ZOTU can be present in both, the sponge and the seawater. In this way, to be conservative, we could only suspect of SW bacteria contamination in the cases in which abundant SW bacteria are in low abundance in a sponge for which only few replicates are available. These cases would merit further investigation.Both, sponge microbiomes and seawater communities are in close contact because of the filtering activity of sponges, which can result in occasional bacteria transfer from one source to another32. However, the relative abundance of SW bacteria in the sponge microbiomes was species dependent, ranging from almost 100% in A. paraviridis to less than 10% in Antho sp.Our results comparing the bacterial core of sponges and seawater showed that all the studied sponges contained SW bacteria, as previously reported for other sponge species24, but also allows inference about bacterial transmission modes. Thus, we estimated the relative abundance of the SW- sponge shared bacteria in each biome core trying to differentiate SW microbes that may represent stable symbionts from transient contaminant bacteria. We postulated that enrichment of seawater bacteria in the sponge occurred when low abundance SW core bacteria were found as the main components of the sponge microbiomes , reduced their relative abundance of SW ZOTUs from ~50%, ~70% and ~25%, respectively, to values below 5%  SW OTUs likely represent environmental contaminants and should be removed from the sponge samples, independently of their abundance in the sponge. Conversely, we propose, that only highly abundant SW bacteria that are rare in the sponge-species core are potential candidates to represent SW bacteria contamination, especially when only few replicates are available. Two sponge species from our dataset (e.g. Pseudosuberites sp. and T. raphidophora) are examples of possible contamination.We considered relevant the contribution of SW bacteria to the formation of the sponge microbiome when they were present across all species replicates. In contrast, some authorset al.17, Webster and Thomas43 and Moitinho-Silva et al.24. We detected widespread but rare44 SW bacteria forming part of sponge core microbiomes. Whether these taxa are metabolically active in the water column or represent dormant stages that reactivate after being incorporate to the sponge24 remains to be elucidated.Overall, we consider the comparisons using the SW core bacteria as a more accurate way to assess the true sponge-SW shared bacteria, as it considers bacteria that would be available across locations to be incorporated in the sponge microbiome. Our results support that “sponge-specific” bacteria are rather “sponge-enriched” bacterial clusters, and that seawater acts as a seed bank for sponge microbiomes, as suggested by Webster 22. Taking into account the high percentages of seawater bacteria detected in some of our sponge species and the species-specificity of many of them, we propose that environmental acquisition would play a major role in the establishment of species-specific sponge microbiomes.We find a high specificity of the associations between sponges and seawater bacteria. Each sponge species seems to incorporate different bacteria from the seawater in its microbiome. This suggests that some species-specific mechanisms have been fixed in the sponges to select some seawater bacteria and not others. Recognition mechanisms have been proposed to explain horizontal acquisition of microbes from the surrounding environmentTo summarize, sponge species is the main factor structuring microbiomes of the most common sponges from Nha Trang bay (Vietnam). By using a very restrictive approach of the “core species” concept and ZOTUs with 100% sequence identity for defining bacterial species, we proved that intra-species microbiome stability is the rule for most sponges. A high percentage of SW bacteria shaped the core microbiome in many study species. Our results point to horizontal transmission, as an ubiquitous mechanism of symbiont acquisition in sponges, while vertical transmission would represent a rather complementary acquisition way. Apparently, some highly specific recognition mechanisms may be acting in sponges to specifically enrich some SW bacteria in their tissues, and not others. Moreover, contrasting preferences in bacteria selection may account for differences in the microbiomes of HMA and LMA sponges and some physiological traits such as contrasting filtration rates might also contribute to enhance the differences. These mechanisms would allow the maintenance of stable bacterial communities disregarding environment conditions and geographical distance and merits to be confirmed by analysing in the same way as in the current study a larger number of sponge and water samples from different geographical regions.Sponge samples were collected in April 2015 by SCUBA diving along 13 transects, 25 m long each, randomly placed between 3 and 9 m deep in three neighbouring locations ~2 km apart  within Nha Trang Bay . This quantitative sampling method allowed us to detect the most representative sponges in the study area but not to collect the same number of replicates for all the species. For instance, only three HMA sponges were found in the whole sampling but two of them were present at high abundances. Overall, we collected 203 sponge samples, from which we only considered for this study the ones that were found at least twice. Thereby, 98 samples belonging to 19 sponge species with between 2 and 15 replicates each .Each sponge individual fitting within a transect was photographed and a piece of ca. 3 cmTriplicate plankton samples were taken from the three sampling locations where the ecological transects were performed . Two litres of water were collected and sequentially filtered throughout 5 μm, to remove undesired plankton components, and then throughout 0.22 μm polycarbonate membranes. The size fraction between 5 and 0.22 μm was processed for DNA extraction. Membranes were enzymatically digested with lysozyme, proteinase K and sodium dodecylsulfate and afterwards, DNA was extracted with phenol:chloroform-isoamyl alcohol  and chloroform:isoamyl alchohol . Purification and concentration of the DNA was carried out with Amicon® Ultra 4 Centrifugal Filter Units – 100000 NMWL (Millipore). The extraction procedures used for sponge samples and SW were the most appropriated according to their respective preservation.COI ~680 bp) were amplified and sequenced. Primers 1 F and 1795R45 were employed to amplify 18S rRNA, Por28S-830F and Por28S-1520R46 primers were used to amplify the D3-D5 partition of the 28S rRNA and LCO1490 and HCO219847 were used for COI. PCR amplifications were conducted in 50 μl reactions containing 1 ng of template genomic DNA, 5 μl of 10x PCR buffer (containing 1.5 mM MgCl2), 2 μl of dNTP mix (10 mM), 2 μl of bovine serum albumin, 1 μl pf each primer (10 mM) and 0.4 μl of Taq DNA polymerase (5 U μl−1). The temperature profile for the 18S rRNA was as follows: 94 °C/5 min;  × 35 cycles; 72 °C/5 min; for 28S rRNA: 94 °C/5 min;  × 30 cycles; 72 °C/5 min; and for COI: 94 °C/2 min;  × 35 cycles; 72 °C/7 min. Purification and sequencing were carried out by an external service . The obtained sequences were manually edited in Geneious v 9.0.2 and blasted against NCBI databaste (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm the morphological identification of the sponges at the lowest taxonomic level possible.We identified sponge species to the best possible taxonomic resolution by molecular markers and morphological features. Fragments of the nuclear genes encoding the 18S rRNA (~1700 bp) and 28S rRNA (~650 bp), as well as the cytochrome c oxidase subunit I  vs. large abundant choanocyte chambers (LMA), and mesohyle 25.Preparation of spicules and histological sections were made from specimens’ subsamples and observed under both, light and scanning electron microscopes. Morphological characters such as spicule types, shape, length, and width, as well as skeletal arrangementhttp://www.mrdnalab.com/). The variable V4 region of the 16S rRNA gene (c.a. 250 nt) was amplified using the primers 564F (5′AYTGGGYDTAAAGNG3′) and 785R (5′TACNVGGGTATCTAATCC3)49. Raw rRNA gene sequences were processed using the UPARSE pipeline50. A quality check and de-replication were applied to our dataset. Denoising (error-correction) of amplicons was performed to identify all correct biological sequences following the UNOISE pipeline51. This algorithm removed chimeras, reads with sequencing errors, PhiX, and low complexity sequences due to Illumina artifacts, and generates ZOTUs (“zero-radius” OTUs) consisting of sequences of 100% identity. For comparison purposes, sequences were also clustered at 97% threshold (Supplementary information). For this analysis, reads were dereplicated and clustered into operational taxonomic units (OTUs) at cut-off 0.03% identity after chimera removal (UCHIME) and excluding the singletons.PCR and high-speed multiplexed SSU rRNA gene Illumina MiSeq sequencing (NGS) were carried out following the genomic core facilities and methods of the MrDNA Lab  (52 using SILVA 128 database. SINA uses Lowest Common Ancestor method (LCA). We configurated a “Min identity” of 0.7 and a maximum number of search results of 1 per sequence results in “best match” type. Sequences with low alignment quality (<75%) and sequences identified as mitochondria or chloroplasts were removed from the analysis. In order to minimize biased effects for differences in sampling effort, the original ZOTU table was rarefied  contaminants11. On the other hand, we considered rare ZOTUs those with a relative abundance <0.01% and highly abundant ZOTUs those with relative abundance >1%32.We combined two approaches to estimate the real contribution of the seawater (SW) bacteria to the sponge core microbiomes. First, we looked for bacteria in each sponge species that were already present in the core SW community. We considered the core community of the SW as the community formed by the ZOTUs present in all water replicates. With this approach, we attempted to identify bacteria that commonly inhabited in the SW and that its presence was not merely circumstantial. Therefore, they could represent a potential source for the formation of the sponge microbiome over time. In the second approach, we made the comparison with the most abundant bacteria of the SW. We identified the ZOTUs with relative abundances higher than 0.01% in average across all water samples. This threshold was chosen since it has been used previously to remove from the sponge microbiome samples the OTUs that were likely to represent environmental contaminantsvegan package54 in R. A cluster dendrogram was built using the Bray-Curtis dissimilarity distance matrix of samples to visualize patterns of bacterial community structure in sponges and seawater. We tested the effect of host identity (species) as well as the effect of the HMA/LMA identity, on the structure of microbial communities with non-parametric Permutational Analysis of Variance (PERMANOVA). PERMUTEST was applied to detect differences in the dispersion between groups. A bias correction55 for the unequal sample size of HMA and LMA groups was applied in the betadisper function of the vegan package54. P-values of PERMANOVA and PERMUTEST were calculated using 999 permutations and significance cut-off for p-values was 0.05.We carried out a distance-based multivariate analysis at ZOTU level of the microbial communities of the sponges and seawater samples using the labdsv package56 in R to detect potential associations of certain bacterial phyla and classes to any of these two groups. We fixed an IndVal threshold of 0.6  to consider a bacterial taxon strongly associated to (or Indicator of) HMA or LMA sponges.The mean relative abundance of the bacterial phyla and classes was calculated for both, HMA and LMA groups. We applied an IndVal analysis using the Supplementary information


Carbon and nitrogen isotope composition of bone collagen indicate an omnivorous diet that included C3-based terrestrial plant and animal resources, in which plant food dominated. In both regions, there were no significant sex differences in dietary intakes. Higher δ15Ncoll values of younger infants presumably reflect the effect of weaning.We present the first comprehensive multi-isotopic data on human and animal remains from the Final Neolithic Corded Ware culture  in south-eastern Poland. The study focused on communities of two settlement areas located in the Małopolska Upland and in the Subcarpathian region. Carbon and nitrogen isotopes of bone collagen were investigated to obtain insights into human dietary preferences, whereas the strontium isotope composition of human tooth enamel was used to trace the mobility and provenance of individuals. Sr isotope data point to a non-local origin of at least one-quarter of the investigated individuals in the Subcarpathian region, consistent with associated allochthonous grave inventories of eastern or western origins. In contrast, all investigated individuals in the Małopolska Upland were of local origin. Furthermore, our study shows an example that the use of fauna for the assessment of the local  C4-based foods within terrestrial ecosystems [15Ncoll values correspond to trophic levels that organisms have in the food chain (e.g. [87Sr/86Sr) of tooth enamel enable identification of “local” vs. “non-local” members of a community and thus allow tracking of the mobility of individuals from the studied sites [87Sr/86Sr values reflect the Sr isotope composition of the environment where an individual lived when the enamel mineralization occurred. A local range of 87Sr/86Sr values is often defined as ±2 s.d. from the mean 87Sr/86Sr in tooth enamel from prehistoric animals that lived locally . Stronted sites , 11, 12 ly e.g. , 14, 15), 153- vsly  populations from the uplands of south-eastern Poland. Investigations have been concentrated in two settlement regions—one in the Małopolska Upland and the other in the Subcarpathian region .In this part of Central Europe, the Final Neolithic was a time of cultural and social changes connected with the origin and development of CWC communities , 19. PreThe archaeological record revealed that CWC societies in the Małopolska Upland and in the Subcarpathian region followed similar funeral rites. In the older phase of CWC development (28th-26th century BC), the deceased were buried mostly in central pits under mounds . In the The features included an entrance pit, passage and grave chamber. As a rule, the burials were richly furnished with pottery vessels, bone and lithic inventories, and sometimes copper objects .The male skeletons rested on their right side, accompanied by distinctive offerings, which were objects regarded as markers of gender and social status, such as stone battle-axes, flint axes and flint arrowheads. Apart from this type of burial, there were also graves with the body resting on its left side and furnished with pottery vessels and singular tools only. Most individuals resting on their left side were females, with only a small number of males found in this position . This spth century. In contrast to Małopolska, the Final Neolithic occupation of the Subcarpathian region remained poorly recognized until the last decade. Recent discoveries were made during excavations preceding motorway construction [Graves from the Małopolska Upland and Subcarpathian region have a similar construction type, and most of these graves surround mounds dated to the Middle or Final Neolithic age . The gratruction . The preMalżyce, site 30 : remnants of an extensive tumulus cemetery initiated by the Funnel Beaker Culture (FBC) community and later reused in the CWC period. Tumulus 1 of FBC was encircled by CWC niche graves 2, 4, 5, 7 [ 4, 5, 7  and tumu 4, 5, 7 . All the 4, 5, 7 . The matGabułtów, site 1  is a barrow cemetery of CWC comprising an older central grave (feature 1) covered by a mound and a younger secondary triple burial in the niche grave (feature 2) dug into its surroundings [oundings , 35. TheSzczytna, site 5  is a multicultural site occupied from the Neolithic to Early Bronze period and then in the Modern Period. The CWC graves were of a niche construction with single (no. 217) or double burials (no. 220) [no. 220) . The matSzczytna, site 6  is a multicultural site occupied from the Neolithic to the Early Bronze period and then in the Modern Period. The CWC single niche graves  surrounded the earlier barrow dated to the older phase of the culture [ culture . The matMirocin, site 24  is a multicultural site occupied from the Final Neolithic to Early Bronze period and then in the Modern Period. The CWC niche graves  surrounded the earlier barrow dated to the older phase of the culture [ culture . The matMirocin, site 27  is a multicultural site occupied from the Neolithic to Early Bronze period and then in the Modern Period. The CWC grave was a niche construction and contained remains of two individuals [ividuals . The matRozbórz site 42  is a multicultural site occupied from the early Neolithic to Modern Period. The CWC single burial of a male was discovered in a pit grave [it grave . The matChłopice, site 26  is a multicultural site occupied from the Neolithic to Early Bronze period. The CWC grave was of a niche construction and contained the remains of two individuals [ividuals . The mat13C, δ15N and 87Sr/86Sr were possible for each studied individual. The carbon (δ13Ccoll) and nitrogen (δ15Ncoll) stable isotope analyses were performed on 18 human bone samples recovered from the sites of both studied regions. In addition, to evaluate details of dietary preferences of humans in the CWC communities six animal samples (fragments of long bone diaphysis) were also selected for carbon and nitrogen isotope analyses. However, the investigated graves did not contain local faunal remains, and hence animal samples, broadly contemporary with the studied humans, were taken from other sites located in the Małopolska Upland and closely linked temporally with the CWC. These sites included: Wilczyce (Corded Ware culture), Szarbia , Smroków , and Koszyce . Unfortunately, no appropriate faunal material from the Subcarpathian region was available for the study.The Sr isotope analyses included human samples obtained from 21 individuals who were excavated from Malżyce and Gabułtów in the Małopolska Upland and from Chłopice, Mirocin, Rozbórz, and Szczytna in the Subcarpathian region. The individuals were selected for sampling based on preserved dental enamel and availability of contextual information. Enamel was taken from first molars (M1) whenever possible and occasionally from premolars . According to time of teeth mineralization they represent signals from childhood and adolescent period. In this study, faunal and environmental samples were also used as a means of establishing local signatures of the investigated areas. Because the investigated CWC features did not contain animal remains, the animal samples came from material excavated in the elaborated areas in younger archaeological sites, e.g. in Gabułtów . In other sites of the Małopolska Upland, faunal teeth were taken from sites at Michałowice  and Jakuszowice  and, in the Subcarpathian region, from a level associated with the Mierzanowice culture (Early Bronze Age) at Mirocin. In overall, tooth enamel samples from various domestic animals were analysed, including 2 samples of cattle, 2 samples of goat/sheep, 4 samples of horse, 2 samples of pig and one sample of dog. We have not collected skeletal remains of the modern fauna because a recent study [87Sr/86Sr.Individuals buried in these graves were subjected to multi-isotopic analyses. In most cases, analyses of δnt study  revealed87Sr/86Sr values around 0.7092 , a sing14C dating in the Poznań Radiocarbon Laboratory was transferred to the Isotope Dating and Environment Research Laboratory at the Institute of Geological Sciences of the Polish Academy of Sciences in Warsaw for carbon (δ13Ccoll) and nitrogen (δ15Ncoll) stable isotope analyses. Stable isotopic composition was determined using a Thermo Flash EA 1112HT elemental analyzer connected to a Thermo Delta V Advantage isotope ratio mass spectrometer in a Continuous Flow system. Isotope ratios were reported as delta (δ) values and expressed relative to VPDB for δ13Ccoll and to atmospheric nitrogen for δ15Ncoll. Delta values were normalized to a calibration curve based on international standards USGS 40, USGS 41, IAEA 600. Two bone samples were sent to the 14CHRONO Centre for Climate, Environment, and Chronology at Queen's University in Belfast for radiocarbon dating and stable isotope measurements. The Sr isotope analyses were carried out in the Isotope Laboratories at Adam Mickiewicz University  and the University of Copenhagen (Denmark). In Poznań, the sample material comprised human and animal tooth enamel, sediments and rainwater. The procedure included chemical separation of Sr and measurements of Sr isotope ratios. The mechanically isolated enamel was cleaned in an ultrasonic bath in ultrapure water to remove the sediment particles. Afterwards, about 10 mg of powdered enamel was treated sequentially with 0.1 ultrapure acetic acid (5 times) to eliminate the diagenetic Sr contamination, according to the procedure described by Dufour et al. [3. The powdered sediment samples (~60 mg) were dissolved on a hot plate  in closed PFA vials using a mixture of concentrated hydrofluoric and nitric acid (4:1). About 15 ml of the rainwater sample was evaporated on a hot plate in PFA vials. Subsequently, 340 ml of 2N HNO3 was added to the sample and left overnight to equilibrate. The miniaturized chromatographic technique developed by Pin et. al. [5 activator on a single Re filament and was analysed in dynamic multi-collection mode on a Finnigan MAT 261 mass spectrometer. Over the course of this study, the NBS 987 Sr standard yielded 87Sr/86Sr = 0.710229 ± 10 . The measured ratios were normalized to a nominal value of 0.710240 for the standard NBS 987. The total procedure blanks were less than 80 pg.Collagen extracted for AMS r et al. . Subsequ et. al.  and modi et. al.  was appl2O2. The samples dissolved within a few minutes, after which the solutions were dried on a hotplate at 80°C. Subsequently, the enamel samples were placed in a few drops of 3N HNO3 and then loaded onto disposable 100 μl pipette tip extraction columns, into which we fitted a frit that retained a 0.2 ml stem volume of intensively pre-cleaned mesh 50–100 SrSpec chromatographic resin. The elution recipe essentially followed that by Horwitz et al. [2O5-H3PO4-HF activator on a single Re filament and measured in a dynamic collection mode on a VG Sector 54 IT mass spectrometer. Over the course of this study, the NBS 987 Sr standard was typified by a 87Sr/86Sr ratio of 0.710241 ± 11 .In Copenhagen, the enamel’s surface was cleaned mechanically, after which the tooth enamel was sampled with a diamond bit. A few milligrams of enamel powder were dissolved in 7 ml Teflon beakers in a 1:1 solution of 0.5 ml 6 N HCl and 0.5 ml 30% Hz et al. . SamplesAlthough both areas under study in the Małopolska Upland and Subcarpathian region are situated approximately 130 km from each other, they share a nearly identical geological background. Both are located within a foredeep basin that developed due to thrust loading of the Central European lithospheric plate by the Carpathian orogen during the Miocene times , 50. TheThe topmost part of the Miocene sequence usually consists of claystones (Krakowiec Beds). The investigated CWC sites are in areas where these rocks are overlain by a Pleistocene sequence composed of loess and locally of glacial tills and fluvioglacial sands. The loess cover, which forms a typical plateau landscape, is generally only a few metres in thickness. However, locally, it has been removed by erosion, and the Miocene claystones are exposed , for exaRadiocarbon dating of bone material revealed that the analysed individuals in both regions lived almost simultaneously and dated graves to the younger stage of the CWC .15N coll values of the dated samples in the range of 9.5–13.5 do not indicate older 14C ages due to the freshwater effect. The calendar age was set using the OxCal v4.3.2 calibration programme of Ch. Bronk Ramsey from 2017 [However, single charcoal dating for grave 1 at Gabułtów (Poz-9451: 4115±30 BP) assigned its age to the older mound period which corresponds to 2855–2620 BC interval and it is compatible with the other dates from barrow graves from south-eastern Poland. The sample was taken from the burnt wooden construction of the grave. The measurements were carried out using the accelerator technique (AMS) at the Poznań Radiocarbon Laboratory (17 samples) and at the Belfast laboratory (2 samples). The δrom 2017 . The vasrom 2017 , 54, 55.In all 19 bone samples, the collagen was well preserved; its C/N ratios, below 3.6, show no significant alteration of in vivo isotopic signatures . The res13Ccoll values in the Subcarpathian region vary within a narrow range, between –20.4 and –19.3‰. In contrast, their nitrogen isotope compositions (δ15Ncoll) exhibit larger variations, from 9.5 to 13.5‰; this wide range, however, is due to the occurrence of two individuals at Mirocin that yield the highest and lowest signatures, respectively. Without these outliers, the remaining group is characterized by a narrow range of δ15Ncoll values, between 10.2 and 11.4‰. In the Małopolska Upland, the δ13Ccoll values of individuals are within an almost identical range (–20.2 to –19.3‰) as in the Subcarpathian region. Their nitrogen isotope values (δ15Ncoll) are also similar, ranging from 9.0 to 12.2‰. Here, the lowest and highest values, which expand the range, are from two infants, while the values of all adults range between 10.1 and 11.0‰. Overall, in both studied regions, there are no significant differences in collagen stable isotope values that are dependent on the age at death  and a single male from Rozbórz with an in-between Sr signature of 0.7097. Although our few animal enamel data from the Subcarpathian region cannot be considered statistically representative, most of them (except signature for one horse from Mirocin) correspond well with the distribution mode of obtained for humans  for loess deposited in western Ukraine. As expected, rainwater collected in the Małopolska Upland has a Sr isotope signature close to 0.7092, which is the composition of modern ocean water [Pleistocene loess deposits, with signatures from 0.7200 to 0.7234, are more radiogenic than the underlying Miocene claystones, which have uniform n Europe , 58, then Europe  who presan water . Hence, In the Subcarpathian region, the investigated sites are located not far away from valleys filled up with Holocene alluvial sediments, composed of detrital material eroded from the Carpathian Mountains. The Sr isotope composition of these sediments has not been studied so far. The Carpathians are predominantly built of monotonous sequences of various Cretaceous and Paleogene clastic rocks. Their siliciclastic composition  points tThe 3rd millennium BC was a time of significant cultural and social changes, which led to the emergence of Bronze Age societies. Understanding these change processes requires interdisciplinary investigations that combine standard archaeological and anthropological studies with modern analytical techniques. Among the latter are methods that utilize stable and radiogenic isotopes in soft or mineralized tissues of humans and animals to provide constraints on dietary preferences, provenance and mobility 13C values of collagen are at inland sites generally linked to those of the protein fraction of the diet [15Ncoll values of humans are higher than those of their average diet. However, they cannot be considered as absolute proxies for the diet since the δ15N coll values of plants can be influenced by environmental conditions [The carbon and nitrogen isotope composition of bone collagen enables the reconstruction of diet and nutritional customs of prehistoric humans; therefore, the method is routinely applied in archaeological research , 66, 67.the diet . The δ15nditions .13Ccoll values around –20‰ recognized in the Małopolska Upland and in the Subcarpathian region are typical for humans living in inland temperate environments that have a terrestrial diet based on C3-plant and animal resources , represent characteristic trophic enrichment values for one trophic level shift [e.g., 15Ncoll values in the studied population . It seems that after weaning, this child began to consume less animal protein than the adults. However, because this individual died at a young age, his low δ15Ncoll level could be connected to a physiological condition. Katzenberg and Lovell [15N ratio in collagen is affected by a variety of factors, including nutritional stress or ill health.These values compared to animal data  indicaten region . The difft e.g., , 75. Theft e.g., , 77. Thepulation  appears d Lovell  argued t15Ncoll signature of 9.5‰, discovered in the male individual buried in grave no. 360 (site 27) at Mirocin, is the lowest nitrogen value among all adults in the investigated areas. This person belongs to individuals identified in the Subcarpathian region as non-locally born , with signatures between rainwater and bedrock. The Sra Upland  are cons87Sr/86Sr values in the Subcarpathian region lie within a mixing space between rainwater and local bedrock, the local origin of all investigated individuals seems to be unlikely. It seems that the main cluster of 87Sr/86Sr values of humans, from 0.7104 to 0.7114, represents local Sr signatures and thus all individuals from Szczytna and Chłopice, and two Mirocin males can be recognized as “locals”. Their local origin is compatible with the indigenous ceramic identified in grave inventories [87Sr/86Sr values lower than that of rainwater. In the Subcarpathian region, around the studied CWC sites, natural sources of Sr  that are less radiogenic than rainwater do not occur, which is also why the horse found at Mirocin could not be from the local domestic fauna. Its unradiogenic Sr signature points to origin from an area where marine Neogene (Miocene) carbonate rocks constitute the local bedrock. The nearest areas that meet these conditions are located along the northern and eastern margins of the Carpathian Foredeep  included vessels, lithic artefacts and copper ornaments. One of the beakers was typical of the Middle Dnieper culture which developed in the basins of Dnieper and Pripyat rivers on the territory of the present-day Belarus and Ukraine . SimilarThe male buried at Rozbórz, with a Sr signature of 0.7097 , cannot In summary, the study underlines the importance of the Sr isotope method for understanding human mobility in the CWC communities, indicated only indirectly by the presence of non-local flint artefacts and vessels. The compliance of archaeological and isotopic data reveals additional important aspects of social life and funeral customs. The results allow us to claim that foreign individuals must have been successfully integrated into local CWC communities. Their burials were prepared in a planned way and were richly equipped. Their allochthonous origin was also marked by the addition of foreign inventories.87Sr/86Sr range in the Subcarpathian region, as suggested by Price et al. [87Sr/86Sr sourcing has shifted away from utilizing archaeological fauna to derive baselines”. For instance, Maurer et al. [87Sr/86Sr baseline for investigating past human migration. Very recently, Lengfelder et al. [Last, the study shows that the use of fauna for assessment of the local e et al.  and Bente et al. , would le et al.  that ther et al.  showed ir et al.  presente87Sr/86Sr values generated in plant, animal, and human tissues within a given area  were identified as people who died at a location other than place of their childhood. The Sr signatures of non-local individuals correspond well with associated allochthonous grave inventories of eastern or western provenience.We conclude also that a detailed Sr isotopic survey of the geological background and its hydrologic elements, rather than that of the fauna, seems to provide conclusive constraints for identification of local and non-local individuals in prehistoric communities.


Subsphaeroidal SiC/polymer composite granules with good flowability for additive manufacturing/3D printing of SiC were prepared by ball milling with surface modification using polyvinyl butyral (PVB). PVB adheres to the particle surface of SiC to form a crosslinked network structure and keeps them combined with each other into light aggregates. The effects of PVB on the shape, size, phase composition, distribution and flowability of the polymer-ceramic composite powder were investigated in detail. Results show that the composite powder material has good laser absorptivity at wavelengths of lower than 500 nm. Silicon carbide, SiC, has excellent physical and chemical properties, such as high mechanical performance, high thermal conductivity, low thermal expansion, and good wear, oxidation and corrosion resistance ,2. TheseAs a new forming technique originated in the 20th century, additive manufacturing/3D printing is a versatile free-form technique characterized by its easy operation, high precision and near net shaping capability ,9. SelecTo address this issue, researchers have developed two approaches to fabricate ceramic parts using SLS technology, i.e., direct SLS and indirect SLS. In the case of direct SLS processing, the as-sintered ceramic parts normally have low density and poor mechanical properties. In particular, thermal cracks in the ceramic parts tend to emerge due to the high thermal stresses resulting from SLS processing ,17,18,19In this paper, the ball milling method was used to prepare SiC-polymer composite powders with good flowability and dispersity for indirect SLS processing. Polyvinyl butyral (PVB) was used as a binder to systematically investigate its effect on the surface modification of the SiC powders, and its effect on the shape, size, distribution and flowability of the polymer–ceramic composite powder agglomerates have been further discussed. Laser absorptivity of the composite powder was studied, as well to provide essential information for its SLS processing. Commercially purchased SiC  was used for the composite material development. In addition, polyvinyl butyral , polyvinylpyrrolidone (PVP), and anhydrous alcohol were used as a binder, dispersant and solvent, respectively; key information of the PVB material is listed in The SiC powder and PVP were mixed together with anhydrous alcohol and then ball milled at 120 rpm for 12 h. For the ball-milling process, two different sizes (Ф 5 mm and Ф 3 mm) of zirconia balls at the weight ratio of 3:2 were used and different weight contents of PVB were added and sequentially ball-milled for 3 h at a rotation speed of 120 rpm. The milled suspension was dried at 50 °C using a drying oven. After drying, the SiC/PVB mixtures were mechanically granulated. The SiC/PVB composite powders obtained were sieved through a 120 mesh screen . The preliminary spreading and forming tests of the SiC/PVB composite powders were carried out using a 3D printing machine . All tests were carried out at room temperature.λ = 1.5406 Å). The X-ray tube was operated at 40 kV and 40 mA. The scanning range for the XRD diagrams was from 20° to 80° in 2θ angle with a step of 0.02° and scanning time of 1 s per step. Hausner ratio (HR) and Carr index were also measured to characterize the flowability of granules. The apparent density and tap density were measured according to the national standards of China, GB5061-85 and GB5162-85, respectively. The size of the agglomerates was measured using laser diffraction . The UV-Vis analysis of the SiC/PVB composite powders was tested using a UV/Vis/NIR Spectrometer . Scanning electron microscopy (SEM) was used to examine the morphology and microstructure of the granulated particles. The phase composition of the composite powders was identified using X-ray diffraction (XRD) using a Rigaku diffractometer model UltimaIV . For SLS processing of ceramics, the first key aspect is that the powders should be able to spread out on the substrate of a 3D printing machine. To confirm this, the flowability of the SiC/PVB composite powders developed was tested, and the typical images of spreading and preliminary forming tests are shown in (1)The addition content of PVB has an optimal value (~3 wt. %). The modified SiC/PVB granules under the optimal addition of PVB exhibit good flowability and spreading performance.(2)The resultant composite powder shows good laser absorptivity when the wavelength is below 500 nm, suggesting that SLS additive manufacturing of the composite powder may be applicable using 3D printers of the corresponding wavelength .(3)Results show that the addition of the polymer binder improves the size distribution characteristic and flowability of the granulated particles within a certain range. However, when the PVB content increases to a higher value , greater addition of PVB will not have much influence on the apparent density, tap density, Carr index or Hausner ratio.Subsphaeroidal SiC/polymer composite granules with good flowability were prepared for SLS additive manufacturing/3D printing with surface modification by adding PVB binder. The effects of PVB on the shapes, sizes, distributions and flowability of polymer-ceramic composite powder agglomerates were investigated. There are several conclusions that can be drawn as shown below:


The optimization of bilayer coverage on particles is important for a variety of biomedical applications, such as drug, vaccine, and genetic material delivery. This work aims at optimizing the deposition of cationic bilayers on silica over a range of experimental conditions for the intervening medium and two different assemblies for the cationic lipid, namely, lipid films or pre-formed lipid bilayer fragments. The lipid adsorption on silica in situ over a range of added lipid concentrations was determined from elemental analysis of carbon, hydrogen, and nitrogen and related to the colloidal stability, sizing, zeta potential, and polydispersity of the silica/lipid nanoparticles. Superior bilayer deposition took place from lipid films, whereas adsorption from pre-formed bilayer fragments yielded limiting adsorption below the levels expected for bilayer adsorption. Biomimetic coatings on a variety of inorganic ,2,3 or pN-[1-propyl]-N,N,N-trimethylammonium chloride (DOTAP), from lipid films was determined in situ by means of an elemental analysis of the silica particles, and compared with the one obtained from pre-formed cationic bilayers in dispersion, such as the bilayer fragments (BF). Whereas, DODAB BF were extensively characterized and used before in drug and vaccine delivery [In this work, aiming at the optimization of cationic lipid bilayer coating on silica, the adsorbed amount onto silica of two different cationic lipids, dioctadecyldimethylammonium bromide (DODAB) and delivery ,23,24,25The results showed that the interaction between silica and cationic lipid films led to optimal bilayer adsorption in comparison to the reduced adsorption from previously dispersed DODAB bilayers. This was understood as being due to the occurrence of hydrophobic defects in the pre-formed bilayers that diminished their interaction with the hydrophilic silica NPs.2/g [DODAB, DOTAP and KCl were purchased from Sigma . Silica (AEROSIL OX-50) was purchased from Degussa . The mean particle diameter determined by the supplier using transmission electron microscopy (TEM) was 50 nm. Specific surface area was previously determined from Brunauer–Emmett–Teller (BET) method yielding 26.00 m2/g . The silThe DODAB or DOTAP chloroform solutions yielded the lipid films on the bottom of glass tubes from chloroform vaporization under a nitrogen flux . Any temg for 1 h at 4 °C). This yielded the DODAB BF [The DODAB BF were obtained from the DODAB powder added to water or 1 mM KCl water solution dispersed by sonication with tip at 85 W for 15 min above 47 °C, before centrifuging the dispersion for precipitation of titanium ejected from the tip (9300× DODAB BF . SonicatFilms or dispersions of the cationic lipids interacted with the silica particles (2 mg/mL) for 1 h at 56 °C before determination of colloidal stability at 1 and 24 h from photos see .Dz), zeta potential (ζ) and polydispersity (P) determinations [μ and the Smoluchowski equation: ζ = μη/ε, where η is the medium viscosity and ε is the dielectric constant of the medium. Measurements for silica or silica/lipid were obtained after diluting the dispersions by 1:20. The algorithm used by the Brookhaven apparatus to calculate the size distributions always tries to solve the size distributions with several peaks [Dz.Size, size distribution and zeta potential were measured after 24 h interaction between silica and films or BF, and only for the supernatants diluted in 1 mM KCl or in pure water. Precipitates did not allow the characterization measurements. The characterization of the silica/lipid particles was performed by means of dynamic light scattering (DLS) using a Brookhaven ZetaPlus-ZetaPotential Analyzer , equipped with a 677 nm laser and a correlator for DLS at 90° plus software for z-average diameter , withdrawing the supernatant and lyophilizing the precipitates for performing the elemental analysis using an Elemental Analyzer Perkin Elmer 2400 Series II . Thereby, carbon (C), hydrogen (H), and nitrogen (N) elements at the silica surface were analyzed to yield C, H, and N percentages in the samples [2/g [Silica (2 mg/mL) and lipid (0–2 mM) interacted for 24 h before centrifuging  and cationic lipid over a range of concentrations (0–2 mM) yielded extensive precipitation over a low range of lipid concentrations (0–0.3 mM); above 0.3 mM lipid, the colloidal stability improved and particles remained in dispersion though some precipitation still occurred over the long run . The col2/g, the silica concentration (2 mg/mL), and the mean molecular area for the lipid at the air–water interface taken as 0.6 nm2 for DODAB [2 for DOTAP [This behavior was previously obtained for the interaction between DODAB BF and silica under similar experimental conditions . It is ior DODAB  and 0.65or DOTAP . The comDz, ζ, and P determined from dynamic light scattering   . In partDAB ones , consistDAB ones .The anionic silica dispersions became cationic upon coverage with the cationic lipids DODAB or DOTAP . The zetThe elemental analysis of C, H, and N adsorbed on silica allowed a precise and reliable evaluation of the lipid concentration adsorbed onto the silica NPs as a function of the added lipid concentration. DOTAP adsorption on silica was determined in pure water a or in 1DODAB adsorption onto silica from films a,c or frThe mean DODAB adsorption calculated from all of the measurements derived from C, H, and N analysis yielded the adsorption isotherms on 2 surfaces was systematically evaluated by means of in situ ellipsometry [Over the last three decades, our group has been attempting to quantify the adsorption of a variety of lipids onto major classes of nanostructures and surfaces such as those of polymeric or mineral nanoparticles. This task was successfully fulfilled for phospholipids since analytical methods based on spectrophotometric detection of phosphorus and phosphate allowed the reliable and sensitive determination of nanomolar concentrations of phospholipids ,24,25,35psometry . High-afpsometry . In purepsometry . For thepsometry , or to cpsometry ,40 due tpsometry , once agIn 1 mM KCl, the results showed that DODAB adsorption from BF improved significantly, though not sufficiently to achieve a bilayer coating on all particles b. PossibThe experiments with DOTAP confirmed the importance of adding a small concentration of monovalent salt, such as 1 mM KCl, to achieve bilayer deposition onto all silica NPs c. This sThis work showed the optimization of cationic bilayer deposition onto silica from lipid films, a procedure that advantageously skipped the preliminary step of dispersing the lipids as bilayers before promoting their interaction with silica. In addition, the determination of lipid adsorption in situ from elemental analysis considerably improved the precision of analysis and the assessment of bilayer deposition in comparison with other methods available from the literature . Bilayer


Canis lupis familiaris, we reveal the size of the hepcidin peptide in the canine, previous reports of which were contradictory to the evolutionary conservation predicted by genome annotation. Here, measurement of the peptide by mass spectrometry, following isolation from greyhound blood serum, revealed an amino acid sequence and peptide mass, differing from all accounts to date, yet demonstrating perfect sequence identity to that of the greater Canidae lineage of the Carnivora. Importantly, in the greyhound, the measured hepcidin peptide showed a similar temporal pattern to total serum iron, consistent with our understanding of hepcidin regulating iron homeostasis, in agreement with human diagnostics, and providing added translational evidence of the measured peptide being the iron regulatory hormone of the Canidae.Hepcidins are an evolutionarily conserved class of liver-expressed peptide, from which the twenty-five amino acid hormone, hepcidin-25 (herein hepcidin), has gained significant notoriety as the master regulator of iron homeostasis in mammals. Hepcidin maintains iron homeostasis by controlling the dietary absorption of iron and the mechanisms of recycling cellular iron stores. With the physiological significance of this hormone well established, it has emerged as an informative biomarker. In a comparison of the genome, transcriptome and peptidome of  Because there exists no mechanism of controlled iron removal from mammals, iron regulation by physiological and biochemical recycling mechanisms, finely balanced by controlled dietary intake, each mediated by hepcidin-25  are integral to maintaining the strict requirements of iron homeostasis2. By direct binding and resultant endocytosis of the iron exporter ferroportin, hepcidin regulates the release of iron into plasma from intestinal enterocytes, macrophages, hepatocytes, and placental cells5. Thus hepcidin maintains iron homeostasis by controlling the dietary absorption of iron and the mechanisms of recycling cellular iron stores. In mammals, low concentrations of systemic hepcidin promote iron absorption and are consistent with increased iron demand; such as is required for erythropoiesis6. Higher concentrations of hepcidin will deplete iron availability, by restricting absorption and promoting sequestration, including during infection7 or other inflammatory conditions8. Chronic deficiency in circulating hepcidin however is associated with the excessive absorption of dietary iron and clinical disorder of iron overload9. In excess, the negative regulation of ferroportin by hepcidin will reduce the transport of iron from the gut and sequester existing iron within cells3, reducing its physiological availability and which may lead to anaemias11.The only input to the total iron content of mammalian systems, other than by clinical intervention is through the diet13. It has been recognised that an availability of animal models with a more representative iron turnover to that of humans would also be advantageous for human translation14. More directly, hepcidin may yet prove useful in animal diagnostics15. It has been speculated that carnivores, such as dogs, can more readily absorb dietary heme than mice because they are evolutionarily carnivorous16, have a metabolic rate to body mass ratio more representative of humans17, and have a closer matched iron economy to humans; including of erythrocyte life-span20 and of total iron stores23. Further, whilst mice do not demonstrate equivalent toxicity of iron overload as humans24, iron is believed to contribute to the pathogenesis of diseases in dogs as it does in humans22.The use of laboratory mice as models of iron metabolism has greatly advanced our understanding of hepcidin and iron regulatory processes more broadlyHamp) gene, or permitted by cross-reactivity of orthologous hepcidin peptides by ELISA25. Relative quantitation of this peptide has therefore only been indirectly determined in the systemic circulation of animals other than of humans and mice28. Mass spectrometry (MS) provides the capability to measure the ionised hepcidin peptide itself, and with efforts toward standardisation29 may yet prove more clinically informative than other analytical techniques of hepcidin measurement30. Further, by leveraging the specificity of MS, evolutionary differences in the amino acid sequence of the native peptide between species can be determined. Whilst measurement by MS has not been achieved in many animals, the translational potential of hepcidin for describing the iron regulatory phenotype of animal models14 and relevance of the peptide as a diagnostic marker in veterinary medicine may prove substantial. To this end, we used a peptidogenomics approach to measure and validate the identity of the endogenous dog hepcidin; combining an intact mass measurement of the peptide by MS and validation against a series of genome and mRNA sequence alignments of the Canidae Family  ortholog of hepcidin was uncovered using a top-down peptidomics approach. Peptides were isolated from greyhound (C. lupis familiaris) serum and measured intact using liquid chromatography (LC) coupled to accurate-mass MS. Of the peptides measured, none were consistent with the expected mass of the canine hepcidin as determined in previous literature accounts26. Three ions matching the charge state distribution  and isotopic abundance pattern of hepcidin when previously measured, or predicted to result from, electrospray ionisation  peptide. Due to the discrepancy between this result and previous literature accounts of the mass, the analytical methods were checked for amenability to a commercially available recombinant “Canine Hepcidin” standard . Analysis of the recombinant peptide revealed a mass of 2,715 Da, consistent with the originally reported cDNA translation26, however inconsistent with the hepcidin isolated from greyhound serum. The measurements were repeated using serum sourced from the C. lupis familiaris breeds; kelpie X (Cross), giant schnauzer, cavalier King Charles spaniel, Labrador retriever X and Great Dane. The same ions were measured in each breed and served to confirm that our determined putative hepcidin mass was likely that of all C. lupis familiaris (and interbreeds thereof) and not an anomaly of the greyhound, with breeding practices potentially selecting for the heightened iron demand of an elite athlete. Hepcidin and the proteoforms thereof are highly conserved through evolution such that sequence orthology of the peptide amongst species should yield confirmatory product ion spectra arising from fragmentation in the highly conserved amino terminus of the peptide  and lysine (K) at these locations (Table de novo peptide sequence and the in silico prediction of dog hepcidin mRNA (Hamp) translated from the C. lupis familiaris genome assembly, each provided a consistent amino acid sequence to our measurements  and other database records34,  for other functional measurements was executed. The search revealed another mRNA measurement, submitted without annotation as a nucleic acid arising from dog liver, where ts Table .C. lupis familiaris hepcidin peptide, we sought to confirm its physiological role as an iron homeostatic regulator within the dog. An assessment of iron metabolic diagnostics was undertaken in a greyhound hemorrhagic shock model following transfusion with iron-rich stored red blood cells, and subsequent analysis by isolation of peptides from the blood serum, followed by analysis of the hepcidin by LC-MS. Analysis of an added recombinant peptide standard  determined good peak area reproducibility across the sample sequence of 117 (%RSD = 13.994), and all endogenous hepcidin measurements within the dynamic range of the standard curve. Physiologically, in agreement with expectations, an immediate and significant increase from baseline in total serum iron concentration was observed (p = 0.014) and followed more gradually by a significant increase in the identified C. lupis familiaris hepcidin concentration over time , C. lupis lupis (wolf) and C. lupis dingo (dingo) were aligned. The dog genome assembly (GCF_000002285.3) was complemented by the more recent wolf40 and dingo assemblies  and African wild dog (Lycaon pictus) were consistent with our described C. lupus familiaris hepcidin, and broadened the characterisation to the Canidae family. Previous reports of the Hamp sequence determined by cloning and sequencing a C. lupis familiaris Hamp cDNA categorised its evolutionary conservation as that of the “canine hepcidin”26. This level of ontology implied sequence conservation with the Canini (canine) genera, but not of the broader Canidae family from which the Canini originate and which also includes the Vulpes (Red Fox). Speciation between the Canidae and Felidae however, show diversion of the predicted amino acid sequence of hepcidin, revealing the measurements obtained here to represent the hepcidin (HAMP) of genera within the Canidae family, but not of the Felidae , the characterisation of hepcidin peptides for the given species under investigation should be undertaken. In this presented study, we used mass spectrometry to reveal the amino acid sequence identity of hepcidin in the dog, and exploited the high sequence conservation of hepcidin to challenge previous assumptions of the size of the molecule in the dog and in the greater Canidae. Complementary data resources of the genome and transcriptome of Canidae species and breeds thereof each provided a wealth of measurements for in silico prediction. Whilst typically, a peptidogenomics approach uses functional peptide measurement to curate genome annotations42, here rather, measurement of the functional peptide has been complemented by highly curated genome predictions, additionally serving to invalidate other functional measurements (in the form of incorrectly annotated mRNA transcripts and their translation). Indeed the only translations of the Canidae hepcidin which were consistent with the measured form within dog were identified in non-curated, or automated computed curations, of the nucleic acid sequence, rather than of (assumed) human-determined translations. Each of our independently determined genomic and previously non-annotated mRNA translations however were in agreement with the existence a Canidae hepcidin peptide of 2,777 Dalton, providing validation that our described identity and mass measurements of the Canidae hepcidin are indeed the Canidae ortholog of this hormone. With this determined discrepancy between the previously reported sequence and corresponding mass of the peptide, targeted MS methodology using a tandem MS instrument would not have resolved the Canidae hepcidin for measurement.Hepcidins are an evolutionarily conserved class of liver-expressed peptide. Here we have observed the 43. The induction of an acute haemorrhage resulting in iron depletion has been shown to reduce hepcidin to baseline, ensuring maximum physiological availability of iron toward erythropoiesis44. Here, greyhounds subjected to a significant reduction of blood volume (inducing hemorhagic shock) were quickly transfused with iron-rich stored red blood cells, resulting in a rapid rise from baseline in total serum iron, and as expected an increase in serum hepcidin  hepcidin to indeed be consistent with the evolutionary conservation of the greater Canidae, and in this determination, we provide an integral step toward the ready availability of this biomarker to translational studies employing dog models, and toward the establishment of hepcidin as a diagnostic marker in veterinary medicine.Together, these findings determine the dog  transfused with stored red blood cells. Ten healthy ex-racing greyhounds were anaesthetized and a large bore catheter was placed in the femoral or carotid artery. Nine hundred mL of blood was removed from the arterial catheter over a period of 15 minutes. Immediately after withdrawal of blood, 15 mL.kg−1 of 5–6 week old packed red blood cells was administered intravenously over a period of 20 minutes. Blood samples were collected at baseline (prior to transfusion), and then 2 hours, 4 hours, and 6 hours after baseline. Serum was collected and stored at −80 °C in polypropylene cryo-tubes until analysis. For qualitative analysis, additional blood sera were later collected, consisting of serum from Canis lupis familiaris breeds; kelpie X, giant schnauzer, cavalier King Charles spaniel, Labrador retriever X and Great Dane.Blood samples were obtained in a randomised controlled trial of dogs .C. lupis familiaris blood serum and measured by Ultra Performance Liquid Chromatography quadrupole time-of-flight Mass Spectrometry (UPLC-qTOF-MS), as a modification of previously described methodology48, using a 5600 TripleTOF q-TOF-MS . For the preparation of serum, samples were thawed at room temperature for 90 min and subsequently mixed by vortex. A pooled sample was prepared by combining a 50 µl aliquot of each serum into a single polypropylene tube, which was mixed and dispensed into 400 µl replicate volumes. For quantitation, calibration samples were prepared from the pooled replicates by the addition of a recombinant hepcidin peptide  dissolved in 40%v/v acetonitrile (ACN), in a final concentration range of 0.6–75 nM; representing an 8-point calibration. To account for changes in sample preparative conditions and/or instrument performance the same peptide standard was added to each dog sera in 400 µL volumes, to a final concentration of 18.75 nM. All calibration and experimental samples were randomised and prepared in a single batch. Larger polypeptides were precipitated by the addition of 800 µL of ACN whilst gently mixing the sample by vortex, followed by incubation at room temperature for 20 min. Precipitate was collected by centrifugation at 10,000 g for 20 min and the supernatant was transferred to a fresh 2 mL polypropylene tube, frozen by submersion in liquid nitrogen and lyophylised to dryness in a Labconco Freezone 2.5 Freeze-dryer . Additional sample clean-up was by solid phase extraction using C18 silica , performed by dissolving, binding to the sorbent and washing the peptide lyophilisate in 20%v/v ACN, with subsequent elution in 60% ACN. For LC-MS analysis the samples were again randomised, transferred to a glass analytical vial by pipette, and the eluent diluted to 20% ACN by the addition of water. Samples were injected into the chromatograph in 10 µl volumes. Chromatography was by reversed phase separation performed using a Shimadzu Nexera FPLC comprised of a DGU-20A5 degasser, LC-30AD pump, SIL-30AC auto-sampler, and a CTO-20A column heater  coupled with an Aeris WIDEPORE 3.6 µ XB C18 column  operated at a column temperature of 50 °C at a constant flow rate of 500 µL.min−1. Mobile phase A was prepared using LCMS grade water (0.2% v/v formic acid), solvent B as LCMS grade acetonitrile (0.2% v/v formic acid). All solvents were degassed for 15 minutes after preparation using a Digital Benchtop Ultrasonic Cleaner water bath . The chromatographic system was equilibrated at 10% solvent B, and the binary gradient ramped to 40% B over 5 minutes, and again to 99% B at minute 6, before re-equilibration at 10% B for each subsequent sample. The ion source was operated in positive electrospray ionisation (ESI) mode with an ion spray voltage of 5,500, declustering potential of 80, and at a temperature of 550 °C. The ion source nebuliser, heater and curtain gases (N2) were operated at 50, 45 and 30 psi, respectively. Data were acquired between m/z 50 and 1,000 in high resolution, with an accumulation of 0.15 sec. The MS was acquired with a background collision energy of 5 eV. The (M + 5 H)+5 ion of the putative hepcidin peptide observed in the TOF-MS spectra of C. lupis familiaris peptide isolates were targeted for subsequent MS/MS using two collision energies, 20 and 40 eV. For MS/MS experiments, the quadrupole was operated at a resolution of 0.7 Da on the precursor ion m/z 556.6 (M + 5H)+5, with an accumulation of 0.30 sec. MS data were collected at an operating resolution of ~30,000 for MS and MS/MS data. LC/MS and MS/MS spectra were acquired and interrogated using Analyst TF 1.7.1 and PeakView version 1.2.0.3 softwares .Peptides were isolated from Raw spectral data were processed using MarkerView version 1.2.1.1 . From the TOFMS trace, peak integration was using a noise threshold of 3 and minimum spectral width of 0.05 Da; with peaks aligned using a retention time tolerance of 0.10 min and mass tolerance of 0.05 Da. The peak area of the recombinant peptide internal standard was assessed for analytical reproducibility by calculation of the percent relative standard deviation (%RSD). For quantitation, the peak area of the endogenous hepcidin peptide was compared against the calibration sample series.p < 0.05. Given the aim was to report on overall trends of concentration over time, post-hoc pairwise comparison tests were not performed.Concentrations of serum iron and hepcidin from the greyhound haemorrhagic shock model were assessed for normality at each time point by visualisation of histograms and Q-Q plots. Within-group change over time for both iron and hepcidin concentration was tested using the Friedman test (SAS Studio v3.91), with a significance level set at http://db.systemsbiology.net:8080/proteomicsToolkit/), the chemical composition of the putative hepcidin peptide was determined, following de novo elucidation of the amino acid sequence from the product ion (MS/MS) spectra. The mass was calculated with 8 hydrogen atoms removed to account for the post-translation modification of 4 disulfide bridges (consistent with the human hepcidin-2550), C117H172N32O31S8, and generating a monoisotopic mass prediction of 2777.06218 Da. Additionally, the monoisotopic mass of the serum-isolated hepcidin peptide, using the three observed charge states [M + 5H]+5 (m/z 556.420376), [M + 4H]+4 (m/z 695.273485) and [M + 3H]+3 (m/z 926.695333) was also determined, and found consistent with the ionisation of a 2777 Da peptide. The amino acid sequence was next entered into the Fragment Ion Calculator and following subtraction of a proton (−1.00794 Da) at each cysteine amino acid residue, a product ion mass spectrum was generated, calculated from an assumed [M + H+] charge state precursor, where M = 2777.06218 Da. The recombinant peptide standard (https://www.pepnet.com/Products/Detail/1863/Hepcidin-(canine)), although consistent with the mass of the published HAMP sequence, was significantly smaller than the mass of the putative hepcidin isolated from C. lupis familiaris serum.Using the Institute for Systems Biology Proteomics Toolkit, Composition Calculator and MS/MS Fragment Ion Calculator (Hamp). The search returned the originally described sequence NM_001007140.126, and which is the reference sequence cited as the translation for the commercially available recombinant “Canine Hepcidin-25” peptide standard . This sequence was Basic Local Alignment Search Tool (BLAST) searched against the Canis lupus familiaris genomic sequence51. The search returned a nucleotide sequence match of 117/119 (98%) in Range 1, 107/109 (98%) in Range 2 and 63/63 (100%) in Range 3 to the RefSeq CanFam3.1, Boxer breed sequence, chromosome 1 (NC_006583.3). Protein BLASTp (protein to protein) of the same amino acid translation26 also matched, and identified other sequences NP_001007141.134 and AY772532.133; each of which cite the same original authorship26. To investigate the imperfect alignment between these previously annotated mRNA translations and the Canis lupis familiaris genomic sequence, the predicted Hamp gene region was translated directly from the heavily curated genomic sequence of C. lupis familiaris51 using ExPASy nucleotide to protein sequence translation tool (https://web.expasy.org/translate/). As anticipated, translation from the genome predicts a discrepancy with both the previous literature and database accounts of the sequence26. Translation from the C. lupis familiaris genome identified; range 1 to contain two nucleotide differences, which translate to an amino acid sequence; DTHFPICIFCCGCCKTPKCGFCCRT, with previously unreported phenylalanine and arginine amino acids at the 21st and 24th positions (Table The NCBI Reference Sequence database was searched for nucleic acid (mRNA) entries of the canine hepcidin antimicrobial peptide  of the genomic-translation against the expressed sequence tag (est) database was executed and returned a C. lupis familiaris (beagle) liver mRNA sequence36 with a perfect alignment to the prediction. Finally, the predicted sequence were also searched using Ensembl Genebuild entry, generated using automated annotation from CanFam 2.052, which also returned an amino acid sequence with perfect alignment to the genomic Hamp prediction, though inconsistent with the earlier published sequence26.To establish if the originally reported amino acid sequence of canine hepcidin53. The predicted hepcidin nucleotide sequence from CanFam3.1 was BLAST Genome searched against the dingo genome. The gene regions accessions aligned with 100% sequence identity to the 3 matched ranges of Canis lupus dingo isolate Sandy unplaced genomic scaffold, ASM325472v1 SuperScaffold_32 (NW_020269188.1). The grey wolf genome is a recent release40 and not yet available for BLAST search. As such the FASTA sequence file for the entire genome was downloaded and viewed using Large Text File Viewer (Version 5.2 u). The 5′-3′ DNA nucleotide sequence (Hamp) which translates to the HAMP sequence DTHFPIC , and is highly conserved amongst mammals were searched and identified in Scaffold_31 at text line 2,657,851 . Within the surrounding sequence the same 3 gene ranges from dog and dingo were identified and found to match with 100% sequence identity.The BLAST workflow was repeated for the recently published dingo genome54.To observe the evolutionary relationship between the determined peptide sequences, the amino acid residues were aligned by ClustalW using Clustal Omega Multiple Sequence AlignmentAll genomics and transcriptomics data are freely available in the aforementioned databases and can be found using the referenced accession codes. All additional data supporting these described findings are provided within the paper and its supplementary information, and raw mass spectrometry data available through the Proteomics IDEntifications (PRIDE) database, Project PXD015661 (ebi.ac.uk/pride/archive).This study was approved by the Murdoch University Animal Ethics Committee (R2732/15 and NC2836/16 (510) and was conducted in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes.Supplementary information


This case–control study with a Fujian population investigated whether self-reported occupational and recreational physical activity may be associated with lung cancer.The population comprised 1622 patients with newly diagnosed primary lung cancer and 1622 age- and gender-matched healthy controls.P < .001), and also specifically nonsmall cell lung cancer (P < .001).High-intensity occupational physical activity was associated with significantly higher risk of lung cancer , especially nonsmall cell lung carcinoma . Moderate or low intensity recreational physical activity was associated with reduced risk of lung cancer. The protective effect of recreational physical activity was observed in current or former smokers, but not never-smokers, and in subjects with normal or high BMI, but not low BMI, as well as people without a history of chronic lung disease. The frequency of recreational physical activity was associated with a linear reduction in the risk of lung cancer (Occupational and recreational physical activity was associated with different effects on the risk of lung cancer in a Fujian population. While recreational physical activity was associated with decreased risk of lung cancer, occupational physical activity was associated with increased risk of lung cancer. Lung cancer accounts for the largest proportion (13.0%) of newly diagnosed cancer cases in the world, and highest proportion of cancer death (19.4%). In China, the age standardized incidence rate of lung cancer among men is 48.4/100,000, making it the most common cancer in men. Among women, lung cancer incidence ranks second only after breast cancer, with an age standardized incidence rate of 21.9/100,000. Regardless of gender, lung cancer is ranked the leading cause of death among malignant tumors.,3Lung cancer is one of the most common and serious types of cancer in the world. According to the GLOBOCAN 2012 report The risk of developing lung cancer is 14 times higher in smokers compared with nonsmokers. However, not all smokers suffer from lung cancer, suggesting that the etiology of lung cancer is multifactorial. Accumulating evidence suggests that lack of exercise may also be an important risk factor for cancer. Regular physical activity can improve lung function and reduce tobacco-induced lung and airway injury. Most studies on the subject have considered that smoking is a confounding factor. The results of a recent review suggested that the protective effect of physical activity is only found in current and former smokers, but not never-smokers. Other factors such as obesity and chronic lung disease are also possible confounding factors that may obscure an association between physical activity and lung cancer.Smoking tobacco is the single most important risk factor for lung cancer. Most previous studies have focused on the role of recreational  physical activity. A systemic review and meta-analysis of 28 relevant studies concluded that regular recreational physical activity may reduce lung cancer risk.The association between physical activity and the risk of lung cancer is complex, and a recent study suggested that not all physical activity has the same effect. and that men with standing occupations had higher risk of lung cancer than did men with sitting occupations.,11 However, other studies showed that occupational physical activity does not affect lung cancer risk.,13Studies on the effect of occupational physical activity have been less common. Some studies have reported that occupational physical activity may increase the risk of lung cancer in men,The present case–control study explored an association between self-reported occupational and recreational physical activity and lung cancer in a Fujian population.22.1The Ethics Committee of Fujian Medical University approved the study and all the subjects signed the consent form. The present study applied a hospital-based on-going case–control design.From January 2006 to June 2015, we enrolled cases of newly diagnosed primary lung cancer who were admitted to First Affiliated Hospital of Fujian Medical University, Fujian Medical University Affiliated Union Hospital, or Fuzhou General Hospital of Nanjing Military Region. The diagnoses were made by clinical examination and bronchoscopy. Control subjects were selected by frequency matching according to age (±2 years) and gender, among the healthy visitors to nononcology departments during the same period and healthy people in the community. All the subjects had lived in Fujian province in China for more than 10 years and were willing to cooperate with the survey and able to answer the questions clearly.2.2,15 The interview included questions concerning the following broad areas: baseline characteristics; smoking status; exposure to environmental tobacco smoke (ETS); physical activity; history of chronic lung disease; family history of lung cancer; and alcohol and tea consumption.The survey participants were interviewed by uniformly trained investigators according to a structured questionnaire.2 (m2), and categorized as low (<18.5), normal (18.5 ≤ BMI < 25.0), and overweight or obese (≥25.00). A smoker was defined as someone who had cumulatively smoked >100 cigarettes. A former smoker was a person who previously smoked, but had not smoked for more than 3 consecutive months. Exposure to ETS was defined as never directly smoked, but inhales the smoke produced by cigarettes or exhaled by smokers >15 minutes per day. Drinking alcohol was considered as ≥1 time/week for more than 6 months, and drinking tea as ≥1 cup/week for more than 6 months.Body mass index (BMI) was calculated as body weight (kg)/height Specifically, low intensity refers to activities performed by office workers; repairers of electrical appliances, watches, and clocks; sales staff; hotel attendants; chemical experiment operators; and lecturers. Moderate intensity occupations include those in which participants sit or stand for 25% of the work time, while 75% is spent on moderate intensity special vocational activities such as student daily activities; motor vehicle driving; electrical installation; lathe operation; and metalwork cutting. High intensity occupations are those in which 40% of the time is spent sitting or standing, while 60% is spent on high intensity activities such as nonmechanized agricultural labor, steel, dance, sports, loading and unloading, and mining.Physical activity during the past 2 years was quantified. Occupational physical activity was rated as low, moderate, or high intensity, in accordance with the Reference Standard of Labor Intensity recommended by the Chinese Nutrition Society in the year 2000.The intensity of recreational physical activity or exercise was classified as moderate (sweat inducing) or low . Recreational physical activity was also rated as occasional (2–3×/week) or frequent (>3×/week).2.3t test were used to compare the baseline demographic characteristics of the case and control groups. An unconditional logistic regression model was used to analyze the correlation between physical activity and lung cancer. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Associations between physical activity and lung cancer were analyzed with logistic regression analysis based on smoking status, ETS exposure, history of chronic lung diseases, and BMI. Multiple logistic regression analysis was performed using the Backward Stepwise Wald method to identify the risk factors for lung cancer. The P-value of the results was 2-tailed, with a test level alpha (α) = 0.05.All statistical analyzes in this study were performed using SPSS 23.0 statistical software. The Chi-squared test and 33.1The study population comprised 3244 individuals, including 1622 patients with lung cancer and 1622 age- and gender-matched healthy controls. The baseline characteristics are shown in Table 3.2P < .001), that is, the more frequent the physical activity, the higher the risk reduction. A similar linear correlation (P < .001) was also observed when only nonsmall cell lung cancers were included. The risk of nonsmall cell lung cancer in those exposed to high intensity occupational activity was 1.384-fold (95% CI: 1.087–1.762) that of those only exposed to low intensity  in subjects who engaged in high intensity occupational physical activity compared to low intensity. Recreational physical activity at both moderate  and low intensity  was associated with a lower risk of any lung cancer  and frequently . In overweight and obese subjects, low intensity recreational physical activity was associated with lower risk of lung cancer  lower risk when performed frequently and 60.1% (95% CI: 0.268–0.596) when performed occasionally. Low intensity recreational physical activity was associated with 48.1% (95% CI: 0.380–0.708) lower risk when performed frequently and 47.9% (95% CI: 0.381–0.712) when performed occasionally.In former smokers, frequent moderate-intensity recreational physical activity was associated with lower 39.6% (95% CI: 0.366–0.998) lower risk of lung cancer. Frequent low-intensity physical activity was associated with 52.6% (95% CI: 0.296–0.761) lower risk, while occasional low-intensity physical activity was associated with 49.3% (95% CI: 0.282–0.911) lower risk. Physical activity was not associated with altered risk for lung cancer in never-smokers  for frequent moderate intensity recreational physical activity and 0.693 (95% CI: 0.494–0.974) for occasional moderate intensity recreational physical activity. Low intensity recreational physical activity was also associated with lower lung cancer risk when performed frequently . However, in ETS-exposed subjects, low-intensity recreational physical activity was only associated with 30.3% (95% CI: 0.547–0.890) lower risk when conducted frequently, and 29.4% (95% CI: 0.530–0.941) when conducted occasionally . Subjects who had never suffered from a chronic lung disease had a reduced risk of lung cancer when involved in moderate intensity recreational physical activity. Frequently performing moderate intensity recreational physical activity  and occasionally performing such  was associated with lower lung cancer risks (Table 3.6P = .021), low intensity (P = .012) and moderate intensity recreational physical activity (P = .005) have all been shown to affect lung cancer risk  represents the intensity of a particular physical activity, and according to the activity type and intensity, different physical activity has a specific MET value. In the present study, information about the activity intensity and activity frequency were acquired through questionnaires or interviewing the subjects, and asking the subject to define their own intensity level, generally defined as moderate or low. Moderate intensity refers to physical activity or physical exercise that reaches the degree of sweating, and low intensity to nonsweat-inducing exercise such as walking. The subjects were also asked to state their physical activity frequency in terms of the number of times physical activity was performed (times/week) during the last 2 years. Occasional was defined at recreational physical activity 2 to 3 times per week, and frequent as more than 3 times per week. The intensity of occupational physical activity was defined according to the Reference Standard of Labor Intensity recommended by the Chinese Nutrition Society in 2000.The intensity of activity can also be measured according to the energy metabolic equivalent (MET) of the participant. The ratio of metabolic rate during exercise to the standard resting metabolic rate  and current smokers , but had no association with lung cancer risk of nonsmokers . Adjusting for the level of smoking did not change these results (Pinteraction = .73). This is highly consistent with the results of the present study. In addition, this study further found that exposure to ETS did not affect the association between physical activity and lung cancer (Pinteraction = .16). However, in the present study, high intensity occupational physical activity increased the risk of lung cancer in subjects exposed to ETS, but does not affect subjects that are not ETS-exposed. Recreational physical activity reduces lung cancer risk in both ETS-exposed and not ETS-exposed . To the authors’ best knowledge, no previous study has included history of chronic lung disease as an adjustment factor or to stratify their analysis. The present study found that moderate intensity recreational physical activity negatively correlated and high intensity occupational physical activity positively correlated with lung cancer risk only in people without a history of chronic lung disease.The present study found that low-intensity physical activity had a protective effect on subjects with BMI in the normal or overweight/obese range, but no effect in subjects with low BMI Table . These rSeveral hypotheses may potentially explain a biological mechanism underlying a reduction in lung cancer risk through recreational physical activity. For example, exercise can affect growth factors such as insulin-like growth factor (IGF) and IGF binding protein (IGFBP), thereby altering tumor progression. High levels of circulating IGF-1 may increase lung cancer risk, while high levels of IGFBP-3 may reduce the risk of lung cancer. Another possibility is that physical activity may enhance immune system function,38 by increasing the number and activity of macrophages, natural killer cells, lymphokine-activated killer cells, and lymphokine regulated cytokines, as well as increasing mitogen-induced lymphocyte proliferation rates. Smoking and ETS exposure can lead to respiratory inflammation and increase oxidative stress. Physical activity can increase the secretion of endogenous free radical scavengers which decrease oxidative stress and inflammation, thereby increasing lung ventilation and perfusion to reduce the risk of lung cancer.–56In addition, physical activity can significantly reduce insulin, glucose, and triglyceride levels, and increase the level of high-density lipoprotein cholesterol, which may be associated with reduction in lung cancer risk.5In conclusion, we found a significant association between the type and intensity of physical activity and lung cancer in this Fujian population. Occupational and recreational physical activity was associated with different effects on lung cancer risk. High intensity occupational physical activity was associated with increased risk of lung cancer, especially nonsmall cell lung cancer, while recreational physical activity was associated with reduced risk of lung cancer, especially for nonsmall cell lung cancer, in current and former smokers, people with no history of chronic lung disease and people with normal or high BMI. Further research with a prospective design would be valuable to confirm the effect of physical activity on lung cancer.We thank all the staff at the Department of Thoracic Surgery, First Affiliated Hospital of Fujian Medical University. We also express our appreciation to all the patients and healthy volunteers who participated in our study.