Brassica rapa is an important Chinese vegetable crop that is beneficial to human health. The primary factor affecting B. rapa yield is low temperature, which promotes bolting and flowering, thereby lowering its commercial value. However, quickened bolting and flowering can be used for rapid breeding. Therefore, studying the underlying molecular mechanism of vernalization in B. rapa is crucial for solving production-related problems. Here, the transcriptome of two B. rapa accessions were comprehensively analyzed during different vernalization periods. During vernalization, a total of 974,584,022 clean reads and 291.28 Gb of clean data were obtained. Compared to the reference genome of B. rapa, 44,799 known genes and 2280 new genes were identified. A self-organizing feature map analysis of 21,035 differentially expressed genes was screened in two B. rapa accessions, ‘Jin Wawa’ and ‘Xiao Baojian’. The analysis indicated that transcripts related to the plant hormone signal transduction, starch and sucrose metabolism, photoperiod and circadian clock, and vernalization pathways changed notably at different vernalization periods. Moreover, different expression patterns of TPS, UGP, CDF, VIN1, and seven hormone pathway genes were observed during vernalization between the two accessions. The transcriptome results of this study provide a new perspective on the changes that occur during B. rapa vernalization, as well as serve as an excellent reference for B. rapa breeding. Arabidopsis have been identified, including the photoperiod, gibberellin (GA), vernalization, autonomous, and age pathways  were selected for RNA sequencing . In ‘JWW’, RNA sequencing was selected based on the following criteria: (1) underwent at least 25 DAT before flowering; (2) 35 DAT promoted ‘JWW’ flowering relatively quickly; (3) according to the difference in flowering times near 35 DAT, 30 and 45 DAT, these times were selected as representatives; (4) 50 DAT was the maximum treatment time; (5) 0 and 35 DAT at 25 °C were used as the controls. In ‘XBJ’, the criteria for RNA sequencing were as follows: (1) underwent at least 10 DAT before flowering; (2) 25 DAT promoted ‘XBJ’ flowering relatively quickly; (3) according to the difference in flowering times near 25 DAT, 15 and 40 DAT, these times were selected as representatives; (4) 50 DAT was the maximum treatment time; (5) 0 and 25 DAT at 25 °C were used as the controls.From the beginning of vernalization to the end of vernalization, the number of leaves of ‘JWW’ is intuitively greater than that of ‘XBJ’, which may be related to the degree of vernalization resistance A. At theB. rapa accessions were analyzed across other periods using 0 DAT as the control for each period. Illumina RNA-Seq analysis of the 42 samples produced 1,949,168,044 total reads and 291.28 Gb clean data. The Q30 was ≥ 93.51% and the average GC content was 47.36%  as the controls to identify vernalization-related DEGs . In ‘JWWB. rapa accessions exhibited a high degree of similarity in terms of unigene distribution. In the biological process category, unigenes were concentrated in the “metabolic process”, “cellular process”, and “single-organism process”. In the cell component category, most unigenes were related to “cell”, “organelle”, and “cell part”. In the molecular function category, unigenes were highly related to “catalytic activity” and “binding”. These highly enriched GO terms indicated the basic regulatory and metabolic functions of the two B. rapa accessions during vernalization. For the metabolic processes in the biological category, we selected the vernalization periods common to ‘JWW’ and ‘XBJ’ for comparison, which are 0 DAT (J1 and X1), 25 DAT (J2 and X4), and 50 DAT (J6 and X6). Compared to 0 DAT, we found that the change in 25 DAT and 50 DAT expression was larger in ‘XBJ’ than in ‘JWW’. Similarly, the catalytic activity-related genes in molecular functions are more pronounced in ‘XBJ’. J2 and J6 are enriched in this term by 2723 and 3303 DEGs, respectively, compared with J1.X4 and X6 are enriched in this term with 3461 and 3630 DEGs, respectively, compared with X1. Overall, in these three GO analysis categories, the expression of ‘XBJ’ in many terms has changed more than ‘JWW’.To determine the functional distribution of unigenes, GO annotation was used to classify the DEGs generated by ‘JWW’ and ‘XBJ’ during vernalization . The twoB. rapa accessions, SOM was used to identify the dynamic changes of the 21,035 DEGs in different vernalization periods, as well as a cluster analysis, which was performed on multiple groups of expression patterns. The expression of normalized DEGs in each period is represented by different colors of each hexagon in the square; the red to blue gradient indicated upregulation and downregulation, respectively , 9 auxin-responsive (IAA), 3 auxin response factor (ARF), 2 indole-3-acetic acid-amidosynthetase GH3 (GH3), and 6 auxin-responsive protein SAUR family genes (SAURs) were considerably upregulated (log2(J4FPKM/J1FPKM) > 1). Moreover, 2 AUX1, 1 protein transport inhibitor response 1 (TIR1) (BraA07g025310.3C), 3 IAA, 1 ARF, 3 GH3, and 9 SAUR family genes were downregulated (log2(J4FPKM/J1FPKM) < −1). Interestingly, the upregulated and downregulation of these genes had the same change in J4 versus JCK. In ‘XBJ’, 7 and 1 AUX1, 14 and 5 IAA, 4 and 1 ARF, 4 and 4 CH3, and 3 and 13 SAUR family genes were upregulated (log2(X4FPKM/X1FPKM) > 1) and downregulated (log2(X4FPKM/X1FPKM) < −1), respectively, in the transition from X1 (0 DAT) to X4 (25 DAT). These genes also had the same trend in X4 versus XCK. Seven DEGs were considerably expressed in the GA pathway. The two GA receptor GID1s were upregulated from J1 to J5 (0 to 45 DAT) in ‘JWW’, but clearly downregulated in J6 (50 DAT). In ‘XBJ’, the expression of GID1 genes varied throughout vernalization. Expression of the 2 GID2 genes in ‘JWW’ exhibited a fluctuating state of falling, rising, then falling and rising once more. Nevertheless, these levels rose after ‘XBJ’ showed a decline. The three DELLA protein RG (DELLA) genes exhibited the same trend in both accessions. Two of the four ethylene response sensor (ETR) genes were upregulated and two were downregulated in the transition from J1 to J4 in the ethylene (ET) pathway of ‘JWW’. In J4 versus JCK, all four ETRs were downregulated. In ‘XBJ’, among the four ETRs, X4 versus X1 and X4 versus XCK only have one ETR downregulated and three upregulated. Notably, a mitogen-activated protein kinase 6 (MPK6) gene (BraA04g002220.3C.gene) was expressed in trace amounts in ‘JWW’, or was almost absent, but decreased considerably in the vernalization of ‘XBJ’. ET-responsive transcription factor (ERF1/2) genes also behaved slightly differently in the B. rapa accessions. Compared to JCK, only one of the 17 abscisic acid (ABA) receptor PYL genes in the ABA pathway of ‘JWW’ was considerably upregulated in J4. In J4 versus J1, there were four PYL upregulated. Only one PYL (BraA10g000590.3C) remained the same upregulated level in both comparisons. However, compared to XCK, these PYL genes were upregulated and downregulated in ‘XBJ’, respectively, which also occurred in X4 versus X1. Protein phosphatase 2C (PP2C) genes and serine/threonine-protein kinase (SnRK2) genes exhibited more consistent trends during vernalization. In ‘JWW’ compared to JCK at J4, most PP2C genes were considerably upregulated. Compared to XCK and X4, most PP2C genes were considerably downregulated in ‘XBJ’. In the jasmonic acid (JA) pathway, 20 DEGs were identified. In the cytokinin (CK) pathway, 24 DEGs were identified, including 1 histidine kinase 4 X4 (AHK4), 3 histidine-containing phosphotransfer protein (AHP), and 2 two-component response regulator ARR-B family (B-ARR), and 13 two-component response regulator ARR-A family (A-ARR) genes. In the salicylic acid (SA) pathway, the expression patterns of the eight transcription factor TGA genes were slightly different between the two accessions, which were more conspicuous in ‘XBJ’.A total of 206 DEGs related to plant hormone signal transduction were identified in the two cessions . DEGs reSUS) genes were notably expressed during vernalization in ‘JWW’ and ‘XBJ’, and its expression levels increased at first then decreased. However, ‘JWW’ did not express the newly discovered SUS gene (newGene_3912). Of the five sucrose-phosphate synthase (SPS) genes from J1 to J4, three were considerably upregulated (log2(J4FPKM/J1FPKM) > 1), and one were considerably downregulated (log2(J4FPKM/J1FPKM) < −1) in JCK versus J4. In ‘XBJ’ from X1 to X4, 4 were considerably upregulated (log2(X4FPKM/X1FPKM) > 1) and 1 was slight downregulated. The alpha-glucan phosphorylase (PYG) and 1,4-alpha-glucan-branching enzyme (GBE) genes in the starch synthesis pathway were notably different between the two B. rapa accessions. Expression of the three PYG and three GBE genes in each vernalization period of ‘XBJ’ were expressed at higher levels than ‘JWW’. Vernalization promoted the upregulation of four maltose beta-amylase (BAM) genes and downregulation of two genes. However, in the same period, five BAMs were considerably higher than JCK in ‘JWW’ at J4. In X4, three BAM genes were expressed at higher levels in ‘XBJ’ than XCK. Alpha,alpha-trehalose-phosphate synthase (TPS) genes, which play important roles in sucrose and starch synthesis, were enriched in both accessions. Four TPSs in ‘JWW’ were considerably upregulated (log2(J4FPKM/J1FPKM) > 1) and three were considerably downregulated (log2(J4FPKM/J1FPKM) < −1) from J1 to J4. It is worth noting that in J4 versus JCK, only two TPSs were considerably upregulated (log2(J4FPKM/J1FPKM) > 1) and seven were considerably downregulated (log2(J4FPKM/J1FPKM) < −1). Unlike ‘JWW’, only seven TPSs were expressed, three of which were considerably upregulated (log2(X4FPKM/X1FPKM) > 1) in X1 to X4 of ‘XBJ’. Only one TPS was considerably upregulated (log2(X4FPKM/X1FPKM) > 1) in X4 versus XCK. Two of the three phosphoglucomutase (pgm) genes increased and then decreased during vernalization. Two UTP--glucose-1-phosphate uridylyltransferase (UGP) genes, which play roles in the intermediate regulation of sucrose and starch synthesis, were expressed. Specifically, UGP1 was continuously downregulated and UGP2 was steadily upregulated as vernalization progressed, which was more evident in ‘XBJ’. These results indicated that the starch and sucrose metabolism pathways play important roles in regulating the responses of B. rapa to vernalization.The enrichment pathways investigated by the KEGG pathway analysis included the starch and sucrose metabolism pathways, which were enriched in clusters 2, 7, and 10 of ‘JWW’, and in clusters 1, 2, and 10 of ‘XBJ’. Subsequently, 134 DEGs in these pathways were further analyzed . In the FPKM/J1FPKM) > 1). In ‘XBJ’ from X1 to X4, only 7genes were considerably upregulated (log2(X4FPKM/X1FPKM) > 1) and 6were considerably downregulated (log2(X4FPKM/X1FPKM) < −1). Phytochrome B(PHYB), two-component response regulator (APRR), and protein LHY (LHY) genes in the red-light pathway that positively regulate flowering locus T (FT) between the B. rapa accessions was slightly different during vernalization. It was worth noting that the two phytochrome B (PHYB) genes upregulated in J4 versus J1 were downregulated in J4 versus JCK. Ten APRRs were up- and downregulated during vernalization; the expression levels of APRRs in ‘JWW’ were generally higher than ‘XBJ’. The expression of LHYs, which play intermediate roles in the red-light pathway, were not notably different between the two accessions. The cryptochrome-2 (CRY), gigantea (GI), and cyclic dof factor (CDF) genes in the blue light pathway were negatively regulated. CDFs were also regulated by red light-related genes. In the comparison between J4 and JCK, the GI and CDF genes in ‘JWW’ were upregulated during vernalization. Comparing X4 to XCK, vernalization slightly upregulated the GIs of ‘XBJ’, but downregulated CDFs. ‘XBJ’ also upregulated flowering genes, including zinc finger protein constants (CO) and FTs, during vernalization at higher levels than ‘JWW’. These results indicated that the photoperiod and circadian clock pathways may play important roles in regulating B. rapa flowering during vernalization.The photoperiod and circadian clock pathway-related genes are often referred to as flowering-related genes. The two pathways of the red and blue light absorbing plants were compared and analyzed to the 23 flowering-related genes identified in this study . In ‘JWWFLC and its related regulatory genes. In this study, FLCs and their related vernalization regulating genes were identified . SOC1 in ‘XBJ’ was upregulated more than ‘JWW’.Studies on the molecular mechanism of vernalization in dicotyledons have focused on the expression changes of entified . VIN3, Vates FLC , but wasDELLA, PYL, JAR1, AHP, and TGA), three starch and sucrose metabolism , two photoperiod and circadian clock , and two vernalization  genes  genes . The 12 B. rapa, the reference genome has been updated for several generations, and various regulatory pathways have been continuously studied [B. rapa at different periods. Thus, in order to clarify the associated regulatory mechanisms, two bolting accessions were selected for transcriptome analysis in this study. A total of 974,584,022 clean reads and 291.28 Gb clean data were obtained. After comparison with the B. rapa reference genome, 44,799 known genes and 2280 new genes were identified. A SOM analysis was performed and screened 21,036 DEGs in ‘JWW’ and ‘XBJ’  is an important part of the plant that helps complete the transition from vegetative to reproductive growth. During the vegetative growth stage, leaves formed around the SAM will generate lateral meristems. Then, a series of environmental influences and induced changes in endogenous signals will change the state of the SAM and induce flowering . Plant hbidopsis . Howevern detail .Aux/IAAs are primarily responsive to auxin genes. Aux/IAA stability and activity are regulated by auxin [Arabidopsis, the local accumulation of auxin releases ARF5 due to Aux/IAA inhibition, which subsequently promotes a rise in CH3 levels, as well as high expression levels of SAUR family genes, and is the leading cause of flowering on the lateral side of the SAM [ARFs, CH3s, and SAUR family genes with vernalization. These genes may be upregulated in the same way that auxin promotes flowering in Arabidopsis. This result indicates that, under the influence of vernalization, the auxin genes of the two B. rapa accessions may affect flower meristems, as well as their bolting and flowering abilities at different times.Auxin functions in the initiation of floral primordia and flower development . Aux/IAAby auxin . The intby auxin . Mutatioby auxin . Similarby auxin . In Arab the SAM . In thisArabidopsis and play a key role in controlling flowering time [Arabidopsis, GID1 is a positive regulator of GA signaling. The GID1−GA complex accelerates the degradation of the DELLA protein [GID1s were upregulated in the first four vernalization periods, but notably declined in the fifth period of ‘JWW’. However, in ‘XBJ’, GID1 levels varied depending on the vernalization stage. The three DELLAs exhibited the same trend in both accessions. As vernalization continued, two DELLAs were downregulated and one was upregulated. These results indicated that the GA signaling pathway may play a role in the transition from the growth period during vernalization of these two accessions.GAs are a plant hormone involved in the floral transformation process of ing time . In Arab protein . In thisArabidopsis, the ET receptor possesses fivemembers and has been classified based on certain characteristics into ET receptor 1 (ETR1) and ET receptor 2 (ETR2) [CTR1 gene plays an important role in ET signal transduction [MPK module located between CTR1 and ethylene-insensitive protein 2 (EIN2) [MPK6 loses its function, the ET signaling pathway will return to normal, indicating that MPK6 may not participate in this pathway [B. rapa accessions of this study, ‘XBJ’ exhibited a large change in two MPK6s during vernalization compared to ‘JWW’. Moreover, EIN2 is the positive regulator of the ET signaling pathway [EIN3 is located downstream of EIN2. EIN2 and EIN3 work together and act on the ET-responsive transcription factor (ERF1/2) [ERF1/2 genes were differentially expressed in the two B. rapa accessions of this study, which may explain the different conversion flowering periods.ET is a gas phytohormone that participates in plant stress responses and regulates a variety of developmental processes . In Arab2 (ETR2) . The sersduction . There i2 (EIN2) . A previ pathway . However pathway . EIN3 is(ERF1/2) . During PP2C in an ABA-dependent manner [PP2Cs interact with SnRKs and inactivate them due to phosphorylation [DELLA activities in the GA pathway [FLCs [PYL genes were differentially expressed in both accessions and exhibited different trends. In the fourth period of vernalization, PYLs in ‘JWW’ were considerably downregulated compared to the control, while the PYLs in ‘XBJ’ were upregulated and downregulated compared to the control. Moreover, vernalization continuously upregulated PP2C genes, thereby blocking the generation of some SnRK2 genes; the difference was more noticeable in ‘XBJ’. Therefore, the ABA signaling pathway may participate in regulating different growth period transformations in these two B. rapa accessions.ABA is a plant hormone that regulates plant development . PYL famt manner ,52. PP2Crylation . A previ pathway . A separay [FLCs . In thisJAR1) is one of 19 closely related Arabidopsis genes that issimilar to the auxin-induced soybean GH3 gene [TaVRN1 and TaFT1 genes, suggesting that MeJA may modulate vernalization and flowering time in wheat [Arabidopsis flowering [JA is a lipid plant hormone that plays multiple roles in plant growth and development . CKs areGH3 gene . Methyl in wheat . Previoulowering ,62. The lowering , which clowering .B. rapa accessions of this study. Clearly, this complex hormone signaling network accelerates the transition from vegetative to reproductive growth during vernalization in B. rapa.To summarize, different hormone biosynthesis and signaling pathways were differentially expressed during vernalization in the SUS is a key enzyme involved in the sucrose metabolism of plants [SPS is a key enzyme that catalyzes the first step of the sucrose synthesis pathway [SUS gene (newGene_3912), and the expression of SPSs in this accession were slightly higher than ‘JWW’ , GBEs, and PYGs [PYGs and GBEs in each vernalization period. These differences indicated that the starch and sucrose metabolism pathways may play important roles in regulating B. rapa with different bolting times, but different roles in activating the SAM. Maltose is produced during the conversion of starch to sucrose in leaves under dark conditions [BAM could potentially produce maltose as a key metabolite and osmolyte via starch hydrolysis [BAMs were upregulated during vernalization in the two B. rapa accessions. However, when comparing the same vernalization period to the control, five BAMs were upregulated in ‘JWW’ and three in ‘XBJ’. These results indicated that maltose may provide the two B. rapa accession with sufficient energy required for normal growth during vernalization at night.Starch is the main carbohydrate stored, which allows them to meet their long-term carbon needs . The maiand PYGs ,71. ‘XBJnditions . BAM coudrolysis . Maltosedrolysis . Four BAArabidopsis, TPS plays a critical role in controlling the transition from vegetative to reproductive growth [TPS catalyzes the formation of trehalose-6-phosphate (T6P) from glucose-6-phosphate and uridine diphosphate–glucose (UDP-glu) and is a signaling molecule that relays information regarding carbohydrate availability to other signaling pathways [TPS resulted in extremely delayed flowering in Arabidopsis. The TPS pathway also acts as a signal that coordinates the induction of flowering by regulating the expression of key floral integrators in the leaves and SAM [TPSs of ‘XBJ’ expressed during vernalization were less than ‘JWW’. It may be that the late-bolting ‘JWW’ accession requires higher TPS expression in order to promote faster flowering. The upregulation of pgms promotes the production of polysaccharides, which has been demonstrated in mushrooms [pgms increased at first and then decreased during vernalization. Therefore, it is speculated that pgms rose rapidly at the beginning of vernalization in order to provide sufficient energy for normal growth, and then it gradually decreased after the vernalization transformation period. In young and mature leaves, UGP is primarily involved in the sucrose biosynthesis pathway, the major form of transport of carbon in plants, which provides energy to plants [UGP genes exhibited opposing states during vernalization, in which, one was upregulated and the other was downregulated. The underlying mechanisms for these contrasting trends, however, require further investigation.In e growth ,76. TPS pathways ,78. The  and SAM . In thisushrooms . In thiso plants . In thisCO/FT mode is the core link between the photoperiod induction pathway [CO is a typical circadian clock control gene that acts on the FT promoter region and is the main positive regulator of FT [CO and FT, which were continuously regulated by CDFs through PHYBs, LHYs, and APRRs. Among them, APRRs were considerably different between the two B. rapa accessions and may affect bolting. The two PHYBs upregulated in J4 versus J1 were downregulated in J4 versus JCK, suggesting that PHYBs may not be affected by vernalization in ‘JWW’. CDFs are intermediate regulatory genes in this pathway that are regulated by both red and blue light. Specifically, vernalization downregulated the CDFs of ‘XBJ’, but upregulated the CDFs of ‘JWW’. Thus, CDF may be a gene that does not affect late-bolting B. rapa during vernalization.The photoperiod and circadian clock pathways function as a whole, as plants coordinate their metabolism according to changes in photoperiods, which thereby induces flowering. Expression changes in the  pathway . The puror of FT . The redFT undergo various changes, forming a complex regulatory network to achieve the transition from vegetative to reproductive growth [B. rapa, vernalization is the most important process that induces flowering. Currently, studies on the underlying molecular mechanism of the vernalization pathway in A. thaliana mainly focus on changes in the expression levels of FLCs. Among them, BrFLC1 and BrFLC2 of B. rapa respond to the regulation of vernalization and affect flowering time [VRN1, VRN2, and VIN3 are negative regulators of FLC, and previous studies demonstrated that VRN1 and VRN2 in Arabidopsis were not induced by vernalization and only play a role in the persistently low expression of FLC at the end of vernalization [B. rapa VRN1s exhibited opposing modes during vernalization. However, these differences require further investigation. Changes in VRN2 were consistent with previous results [VIN3s were upregulated in the first five vernalization periods and downregulated in the last vernalization period. Therefore, it is speculated that B. rapa transitioned to reproductive growth just before the last vernalization period and, hence, the downregulation of VIN3s.Land plant flower development undergoes floral induction, floral primordia, and floral organ development phases. In the flowering induction stage, although there are no morphological changes in the SAM, a series of related genes centered around e growth . In B. ring time . In thislization ,13,14; tB. rapa have focused only on the comparison between the vernalized and unvernalized, and related research approaches have been relatively narrow. Vernalization promotes plant bolting and flowering as an important trait in productivity and reproduction; thus, it is necessary to clarify its effects on bolting and flowering in different vernalization periods. In this study, two B. rapa accessions with different bolting times were investigated over a series of vernalization periods. For the related flowering traits, the vernalization periods were selected for comparative transcriptome analysis to describe the effects of different vernalization periods on B. rapa. According to the results, the B. rapa accessions exhibited specific differences at each vernalization period, indicating that research on vernalization should be a process of vernalization, not a comparison between vernalized and unvernalized. In comparing the two accessions, key genes of seven hormone pathways of the plant hormone signal transduction pathway during vernalization were notably different, and the difference between late-bolting ‘XBJ’ was greater than early-bolting ‘JWW’. This difference was also confirmed in the vernalization pathway. In the starch and sucrose metabolism and photoperiod and circadian clock pathways, TPS, UGP, and CDF exhibited different expression patterns, but these differences require further analysis. Notably, VIN1, which does not play a role during vernalization in other dicotyledons, exhibited opposing expression patterns in this study, which may be a new avenue for research on vernalization in B. rapa in the future. The transcriptional regulation of B. rapa vernalization was summarized in this study (B. rapa with different abstractions during vernalization.To date, studies on the mechanisms related to vernalization of is study , which p


Vitis vinifera × Vitis labrusca ‘Shenhua’ is a tetraploid grape, a Franco-american species. This study first published the complete chloroplast genome of Vitis vinifera × Vitis labrusca ‘Shenhua’ was assembled. The chloroplast genome is 160928 bp in length, including a large single copy region , a small single-copy region  and a pair of inverted repeats of 26,354 bp. The chloroplast genome encodes 133 genes, comprising 88 CDSs, 37 tRNA genes and 8 rRNA genes. The phylogenetic tree demonstrated that Vitis vinifera × Vitis labrusca ‘Shenhua’ is different from the other 16 varieties. Vitis vinifera × Vitis labrusca ‘Shenhua’ shows strong heat-resistant and disease resistance. Thus, it is widely used for breeding of heat-resistant grape varieties. It is also an excellent wine and food grape variety because of its high anthocyanin, titratable acids and soluble solids contents  and can be perform to phylogenetic analysis. It supplies significant information for the basic research of grape resistance breeding.Vitis vinifera × Vitis labrusca ‘Shenhua’ originated from the Zhuang-hang Experimental Station of Shanghai Academy of Agricultural Sciences  and stored at the Center for Viticulture and Enology, Shanghai Jiao Tong University, the sample was called ‘Shenhua’. This DNA was used to prepare 400 bp tiny fragment DNA library and then sequenced by the NovaSeq-PE150 sequencing platform . 3.49 Gb clean reads were obtained in the aggregate. The complete chloroplast genome was composed by SPAdesv3.9.0  and A5-MiSeqv20150522 . The complete chloroplast genome of Vitis vinifera  serve as a reference , 4 rRNAs  and 7 tRNAs  appear as double-copies.The length of chloroplast genome is 160,928 bp, consistsing of a large single copy region , a small single-copy region 19,072 bp and a pair of inverted repeats of 26,354 bp. The chloroplast genome includes 133 single genes, including 88 protein-coding genes (CDS), 37 tRNA and 8 rRNA genes. 37.38% and 62.62% are the amounts of GC content and AT content of the grape genome. In these genes, most of them are single-copy, whereas 7 CDS  phylogenetic tree was builded by using 16  classes , which c classes . The phyClick here for additional data file.


Δ219–225 model (MGI: TtnMgotTM 2.1). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in TtnΔ219–225 muscles: a larger isoform that represents the TtnΔ219–225 N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in TtnΔ219–225 soleus muscle , but was reduced in EDL muscle . The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload  and hindlimb suspension  experiments were also conducted. TtnΔ219–225 mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy  in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin’s PEVK exons 219–225 have high passive tension, due in part to alterations elsewhere in splicing of titin’s spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin’s C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity.The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219–225 deleted, the Ttn Titin is a giant protein located inside the striated muscle sarcomere where it anchors inside the Z-disk and M-band and spans the half-sarcomere. This layout in the sarcomere uniquely positions titin to act as a molecular spring . The sprΔ30–38  = (muscle mass [g] × cos [θ])/(ρ [g × cm–3] × fiber length [cm])  . Stimuli were applied with a train duration of 400 ms and a 90 s interval. After completion of the contractility measurements, the weight of the muscle was determined. Cross-sectional area (in mm muscle) .0 by overnight immersion in 4% formaldehyde/1% glutaraldehyde. Intrinsic shrinkage was minimized by pinning the tissue during fixation. Thin full-length (tendon-tendon) fiber bundles were dissected from the fixed muscles and sarcomere length was measured by laser diffraction. The number of serial sarcomeres was calculated by dividing the length of the fiber bundles by the measured sarcomere length.A subset of these muscles was fixed at LSamples were run on 0.8% SDS-AGE gels for titin or 12% SDS-PAGE for signaling proteins, and transferred to PVDF membrane using a semi dry transfer unit . The blots were stained with Ponceau S to visualize total transferred protein. The blots were then probed with CSRP3/MLP, FHL1, MARP1, MARP2, Ttn C-term, Ttn N-term, Erk, Thr202/Tyr204 p-Erk, mTOR, S2481 p-mTOR, and GAPDH (see antibody table for details). Secondary antibodies conjugated with fluorescent dyes with near-infrared excitation spectra were used for detection. Two-color IR western blots were scanned  and the images analyzed with Image studio light (Li-Cor Biosciences). All proteins were normalized against GAPDH, and, subsequently, (for FO and HS samples) normalized to control samples (EDL or soleus) to determine relative changes in protein quantity.p < 0.05 as indicated in the figures. Statistical testing was performed using student t-test, one or two-way ANOVA as applicable. A linear model that accounts for experimental variability was used to analyze the microarray data. Curves were fitted for non-linear regression to determine the difference in fit between equations, an extra sum of squares F-test determined whether the data sets differed from each other.Data are presented as mean ± SEM. Significance was defined Δ219–225 skeletal muscle was assessed first. Calculations of the force per titin molecule using a worm-line chain model , showed a small predicted passive tension increase  in TtnΔ219–225 mice were not different in either soleus or EDL muscle  mice. Results for both the soleus and EDL muscles confirmed the expected deletion of exons 219–225 and revealed additional splicing in primarily the PEVK region of titin  mouse with the Rbm20RRMΔ (heterozygous) mouse model and studied titin isoform expression. The data reveal that reducing Rbm20 levels results in soleus and EDL muscles with single mutant N2A isoforms , caused by hypertrophy (discussed below). In contrast, the EDL muscle . Thus, in the TtnΔ219–225 mice, the maximal force generating capacity is differently affected in soleus than in EDL muscle.To determine if the increased passive tension of Ttn tension , intact s muscle , left shL muscle , right sΔ219–225 mice were shifted rightward  and EDL (Δ219–225). Tension-frequency relations were also obtained from female mice and similar trends were observed, except in female soleus muscle, where F50 was not different  and a minor reduction during maximal tetanic contractions . Similarly, in EDL . The time to maximal tension during tetanic contractions was not different . The relaxation kinetics (time for tension to reduce by 50%) followed similar temporal shifts. Soleus muscle showed reduced time to half relaxation during twitch contractions  and similarly during tetanic contractions . In EDL, the time to half relaxation was reduced during twitch contraction  and during tetanic contraction .The kinetics of force generation and force relaxation kinetics were studied during isometric twitch and maximal tetanic contractions. Soleus muscle  showed r, in EDL  the time50 and the faster tension kinetics reflected a change in fiber type composition, the MyHC isoform composition of muscles was determined. Unexpectedly, soleus shifted from 42.6 ± 1.9% type I MyHC in WT to 68.7 ± 3.3% type I MyHC in TtnΔ219–225 and EDL from 75.8 ± 1.1% type IIb MyHC in WT to 54.0 ± 4.5% type IIb MyHC in TtnΔ219–225 .To study whether the increase in FΔ219–225 . The chaΔ219–225 , as slowΔ219–225 . HoweverΔ219–225 soleus muscles of 3.5-month-old males , an increase that is absent in EDL muscle (10.4 ± 0.3 mg in WT to 10.6 ± 0.6 mg in TtnΔ219–225). This higher muscle mass was also present in soleus muscle of 6-months-old males , whereas EDL was not different between genotypes (10.8 ± 0.3 mg in WT to 11.2 ± 0.8 mg in TtnΔ219–225). To further study if this difference in muscle mass was dependent on muscle type, we also determined the mass of two other predominantly fast-twitch muscles: tibialis cranialis (TC) and diaphragm (DIA) muscles. At 3.5-months-old, both TC  and DIA  muscles were not different. However, at 6-months-old  and a trending increase in the DIA . These findings suggest that the hypertrophy is not limited to predominantly slow-twitch muscles, and the mass increase in soleus  and TC  is progressive, hinting at a continuously active hypertrophy program. TtnΔ219–225 male mice display early growth deficits, with a reduced body weight  and not different in EDL  muscles compared to WT. The sarcomere length data, taken together with the muscle length at L0, show that TtnΔ219–225 muscles have significantly more sarcomeres in series: TtnΔ219–225 soleus 4764 ± 95 versus WT soleus 4105 ± 154, EDL TtnΔ219–225 6031 ± 97 versus EDL WT 5572 ± 139. This represents a 16% increase in serial sarcomeres in the soleus and 8% in the EDL . In EDL no difference in mCSA was observed  of the soleus, by means of synergist ablation , or unloading by means of hindlimb suspension (HS). Following FO, soleus muscle mass was increased, with the increase being larger in TtnΔ219–225 mice . Interestingly, EDL muscles of TtnΔ219–225 mice also developed hypertrophy (21.6% increase) (Δ219–225 (mass -7.9% decrease). There was no effect of HS in EDL of either WT or TtnΔ219–225 mice  . Followi225 mice . This, t225 mice , suggestΔ219–225 muscles . HS only moderately affected MARP1 levels in soleus. EDL muscle showed the exact opposite response for MARP1, wherein FO showed no observable difference in MARP1 expression, but HS showed substantial upregulation of MARP in both WT and TtnΔ219–225 muscles (22-fold increase in WT to 33-fold increase in TtnΔ219–225). This opposing response of MARP1 in soleus compared to that in EDL muscle, following FO or HS, might imply a role during certain stages of hypertrophy development.To explore a potential signaling mechanism underlying the hypertrophy sensitization, we probed several target proteins with Western blot. We found marked upregulation of CSRP3, FHL1 and MARP2 in Ttn muscles  in respo muscles . FollowiΔ219–225 muscles coincided with faster kinetics of force generation and relaxation in slow-twitch soleus and fast-twitch EDL muscle, although the fraction of slow myosin heavy chain isoforms was increased. TtnΔ219–225 mice presented with longitudinal and cross-sectional hypertrophy in soleus muscle, and a stronger response to hypertrophic stimuli.In this study, we characterized skeletal muscles in a mouse model with a small C-terminal deletion in titin’s PEVK segment. This deletion resulted in alternative splicing of titin’s I-band region, leading to the appearance of a second smaller isoform (N2A2), and an accompanying increase in the passive tension of skeletal muscle. The increase in stiffness of the TtnΔ219–225 mouse model was originally generated to study the PEVK segment of the predominant titin isoform in the heart, N2B cardiac titin, and to resolve the function of the PEVK-segment in relation to biomechanics. In the heart, the TtnΔ219–225 model displayed increased passive tension, suggesting that the PEVK segment is a major contributor to titin-based passive tension  (Δ251–269 (IA-junction deletion)  mouse model (Δ219–225 model might be related to the deletion of the phosphorylation sites that regulate titin stiffness. The deleted section in the TtnΔ219–225 mouse contains 2 phosphorylation sites: S12742 (ex219) and S12884 (ex225) (Δ219–225 muscles (Δ30–38 muscles.A similar N2A2 isoform was previously described in the Ttneletion)  model. Seletion)  and TtnΔse model , present (ex225) . Both ar (ex225)  and CAMK (ex225) . These s (ex225) . However muscles , arguing muscles . Rbm20 sΔ112–158 muscle by Δ112–158 muscle). Recently, advances have been made in the understanding of the mechanisms by which titin stiffness modulates contraction kinetics. Stiffer titin molecules put more strain on the thick filaments  EDL muscle, suggesting that it is muscle specific. The muscles that do hypertrophy, show progressive growth  soleus muscle of the Ttnertrophy . The croe growth . Tibialie growth . This sme growth . Thus, tΔ112–158 model, which showed normalization of passive tension due to addition of sarcomeres in series  protocols (#09-095 & #13-488) of the University of Arizona.MG, CO, and HG designed the research studies. BH, RP, TG-N, FL, AK, JS, and CC conducted the experiments and acquired the data. RP, BH, FL, JS, and CC analyzed the data. RP, MG, HG, and CO wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The identification of food fish bearing anthropogenic contaminants is one of many priorities for Indigenous peoples living in the Arctic. Mercury (Hg), arsenic (As), and persistent organic pollutants including polychlorinated biphenyls (PCBs) are of concern, and these are reported, in some cases for the first time, for fish sampled in and around King William Island, located in Nunavut, Canada. More than 500 salmonids, comprising Arctic char, lake trout, lake whitefish, and ciscoes, were assayed for contaminants. The studied species are anadromous, migrating to the ocean to feed in the summers and returning to freshwater before sea ice formation in the autumn. Assessments of muscle Hg levels in salmonids from fishing sites on King William Island showed generally higher levels than from mainland sites, with mean concentrations generally below guidelines, except for lake trout. In contrast, mainland fish showed higher means for As, including non-toxic arsenobetaine, than island fish. Lake trout were highest in As and PCB levels, with salmonid PCB congener analysis showing signatures consistent with the legacy of cold-war distant early warning stations. After DNA-profiling, only 4–32 Arctic char single nucleotide polymorphisms were needed for successful population assignment. These results support our objective to demonstrate that genomic tools could facilitate efficient and cost-effective cluster assignment for contaminant analysis during ocean residency. We further suggest that routine pollutant testing during the current period of dramatic climate change would be helpful to safeguard the wellbeing of Inuit who depend on these fish as a staple input to their diet. Moreover, this strategy should be applicable elsewhere. The accumulation of anthropogenic contaminants in food fish has a long and tragic history as well as being a current global health concern. International conventions and risk management policies have been introduced to address individual contaminants, including those derived from burning fossil fuels or industrial infrastructure or processes that involve electrical transformers and plasticizers in the case of mercury (Hg) and persistent organic pollutants, respectively ,2,3. How2 form during the polar sunrise, after which it accumulates on ice and snow, and from there it moves to waterways where it is transformed by microbes to neurotoxic methylmercury )Salvelinus alpinus (Arctic char), S. namaycush (lake trout), Coregonus clupeaformis (lake whitefish), and the ciscoes, including C. autumnalis (Arctic cisco) and C. sardinella (least cisco), were chosen because they are targeted for local consumption both on the island and adjacent mainland sites, although the ciscoes are principally by-catch. It was our hypothesis that population genomic patterns combined with a geographical analysis would correlate with levels of anthropogenic contaminants and thus be a surrogate for impractical and expensive chemical analysis of hundreds of random fish samples. It is important to note that this exploratory evaluation represents an initial assessment, designed to determine the merit of such a novel strategy, rather than a definitive prescription to safeguard the health of consumers.Here, we investigated Hg, As, and PCB contamination in several important subsistence salmonids that were caught at fishing sites on or near King William Island, in the Kitikmeot region of Nunavut, Canada. According to ITK and as well as otolith-determined age-growth plots   from sampled salmonids were oven dried overnight at 70 °C or at 24 °C for two days for analysis of inorganic elements and Hg, respectively. Samples from a total of 531 fish were individually ground and submitted to the Queen’s University Analytical Services Unit . They were analyzed for 59 elements, including As but excluding Hg, by acid digestion followed by measurements using inductively coupled plasma mass spectrometry (ICP-MS), except for boron (B), phosphorus (P), and sulfur (S), which were measured using ICP-optical emission spectroscopy (OES). Analysis of total Hg  was accomplished by thermal decomposition of the solid sample, amalgamation, and atomic absorption spectroscopy in an Hg analyzer (DMA-80). Economic considerations dictated that PCBs were only analyzed in 20% (101/531) of the sampled fish analyzed for inorganic elements, but took into account age, species, and fishing site in an effort to target 10 samples for each site and species, although this was not always achieved. Samples were processed (dried and ground) as for Hg analysis (but as separate subsamples). Samples (1–2 g) were analyzed at QUASU for lipids and for PCBs; the latter analysis was by extraction and gas chromatography-electron capture detection (GC-ECD) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis for individual congeners using standard procedures . Quality assurance/quality control steps included analytical duplicates, certified reference materials (TORT-2) and appropriate controls and blanks for inorganic elements and Hg, and for PCBs, analytical duplicates, controls, blanks, and surrogates.Muscle tissue was used for all contaminant analysis. Frozen muscle samples  extraction procedure at 37 °C. The extracts were analyzed using anion exchange high performance liquid chromatography-ICP-MS. It should be noted that QUASU and ALS Global are accredited by the Canadian Association for Laboratory Accreditation Inc. to the standards of ISO/IEC 17025. Methods used were listed in the scopes of accreditation at the time of analysis.Corrections to wet weight (ww) values when required  were made using percent moisture obtained from a subset of samples according to fish species, either Arctic char, cisco, lake whitefish, or lake trout. All results were shared with community members in meetings with the Gjoa Haven Trappers and Hunters Association as well as the community, and additional fishing sites were recommended at each discussion, making ITK, site selection, and chemical analysis an interactive process.Hg (as ww concentrations) and As  concentrations, according to conventions in the contaminant literature, were analyzed for differences between means using non-parametric Kruskal–Wallis statistics, as analysis of variance (ANOVA) on log-transformed data that did not yield normally distributed residuals. Spearman rank correlations (a non-parametric measure of correlation) were calculated between age, Hg, As, selenium (Se), and nutritional elements including calcium (Ca), chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), P, potassium (K), sodium (Na), S, vanadium (V), and zinc (Zn). All statistical tests for Hg and As were performed in XLSTAT 2020.4.1, where As concentrations below the detection limits were imputed using log-normal regression on order statistics (ROS) methods in ProUCL 5.1.For the PCB analysis, data included the PCB congeners, inorganic elements, site, collection date, species, length, age, and % lipid. Where the reported congener datasets differed (because of different resolution of congeners on the GC column), separately reported congeners were summed to obtain the same congener dataset for each sample. Calculations were determined for total PCBs, total PCBs on a lipid weight basis (T-PCBlip), dioxin-like PCBs (DL-PCBs), non-dioxin-like PCBs, and the sum of six European Union (EU) PCBs (PCB6); details are provided . PCB andSbfI and MspI. In the last step of library preparation, samples with unique barcodes  were pooled and then sized with a BluePippin® DNA size selection system . SNP fragments of the appropriate size were then sequenced as single-end, 100 bp reads on a HiSeq2000 platform .DNA was extracted from fin clips and muscle samples from 429 Arctic char samples using either a Qiagen DNeasy Blood and Tissue kit  following the manufacturer’s protocol or a salt extraction method . DNA samRandomForestRC [gsi_sim [ST values computed from the training dataset, and the top 2, 4, 8, 16, 32, 64, 128, 200, 500, 1000, 2000, and 3055 markers were used as panels of loci to evaluate the impact of the number of markers used for assignment; (4) the holdout samples were then probabilistically assigned to either “Northern” or “Southern” populations; (5) steps 2–4 were repeated 30 times; (6) steps 1–5 were repeated three times ; and (7) the average performance was then assessed to represent assignment accuracy. Theoretical assignment to 1 of 17 fishing sites did not use steps 1 and 6 due to small sample sizes at some sites, but the rest of the steps were followed for both original and imputed datasets.Filtering and SNP calling have been described in detail  with seqForestRC  as impleForestRC  with 100[gsi_sim ,47 from [gsi_sim  was usedhttps://tsfn.gcrc.carleton.ca/index.html?module=module.tsfnatlas.quotas). Additional details of each fishing site including site number, location, equipment used, water type, and if fish from a particular site were sampled for contaminants and/or genetics are shown in ITK proved crucial for the mapping of subsistence fishing locations, including local context as to routes, equipment , as well as optimal times for western sampling protocols and resulted in robust knowledge exchange ,49. The −1. The lake trout samples representing the other Salvelinus species were generally older and slower growing, ranging from 8–62 years (mean = 25.4 ± 1.6), weighed 3.308 ± 186 g with a growth rate of 27.8 + 1.4 mm·year−1. Of the Coregonus salmonids sampled, the ciscoes were similar in age to Arctic char, ranging from 2–30 years (mean = 14.8 ± 1.4) but much smaller with an average weight of 504 ± 66 g and a growth rate comparable to lake trout at 28.5 ± 2.9 mm·year−1. The other Coregonus, lake whitefish ranged from 4–47 years (mean = 21.3 ± 2.2), with a weight of 935 ± 79 g and the slowest growth rate, perhaps reflecting their existence at the northern edge of their range at 23.8 ± 2.8 mm·year−1. All data have been deposited into the Polar Data Catalogue and shared as open access . Fish samples have been archived for access.There were differences in average age, weight, and growth rates depending on the salmonid species. Arctic char sampled ranged in age from 5–29 years with a mean of 14.2 ± 0.6 years (95% confidence limits), with an average weight of 3.086 ± 247 g and a linear growth rate of 47.1 ± 1.9 mm·yearp < 0.0001 unless stated otherwise) (p = 0.005), Cr (ρ = −0.280), Cu (ρ = −0.311), Mg (ρ = 0.282), Mn (ρ = 0.184), P (ρ = 0.266), K (ρ = 0.301), Na (ρ = 0.478), S (ρ = 0.518), V (ρ = −0.261), and Zn (ρ = 0.220). Fewer correlations (p < 0.0001 unless indicated) were observed for As, specifically with age (ρ = 0.403), Ca , Cr (ρ = −0.273), Cu , P , Na (ρ = 0.273), and V . As levels correlated with Se , but Hg did not . Similarly, as a group, older fish showed increased PCB levels (p = 0.008), Se , Hg , and Na  and negatively with Cr  and V , but no correlations were seen with % lipid . When PCBs were expressed on a % lipid basis (T-PCBlip), positive correlations were again seen with age , Hg , and Se , as well as with As . Correlations of T-PCBlip (p < 0.0001 unless indicated) were observed with the nutritional elements Zn , S (ρ = 0.535), Na (ρ = 0.545), Mg , P (ρ = 0.402), K (ρ = 0.397), and Mn  as a consequence of the strong negative correlation of these nutritional elements with % lipid . That is, fewer fatty fish, which result in higher T-PCBlip values, have higher concentrations of these nutritional elements. Within the PCB and associated inorganic elements dataset, Hg negatively correlated with % lipid , but As did not .Contaminant levels varied depending on the age of the sampled salmonids. As the fish aged, there was some accumulation of inorganic elements including Hg, independent of fish type . Correlaherwise)  were obsB levels . Total PB levels  positive−1 ww, with similar concentrations for lake whitefish (0.11 mg∙kg−1 ww) and ciscoes (0.09 mg∙kg−1 ww), although the average concentrations in the older salmonids were higher  and across all age classes had a higher mean level of 0.36 mg∙kg–1 ww, statistically higher than the mean Hg concentration for all other fish . Differences were seen between the overall means of other fish species as well (p < 0.0001), except for ciscoes and lake whitefish.Not all the salmonids accumulated contaminants to the same levels. The mean Hg in Arctic char in the sampled waters was 0.07 mg∙kge higher A. Lake t−1 dw), and a wide range was also seen when individual species were examined . As levels varied depending on the species, with the highest average values (25 mg·kg−1 for young fish and 59 mg·kg−1 for older fish) found in lake trout, with the overall mean significantly higher in these fish than in the other species . The lowest values were seen in lake whitefish (3.9 mg·kg−1 in young and 5.1 mg·kg−1 in older fish), with the overall mean significantly different from the other fish species (p< 0.0001 to p = 0.008); means for Arctic char and cisco were not significantly different from each other.Overall, As levels in the sampled fish varied over a broad range of concentrations  with Arctic char showing the broadest absolute range of all the salmonids . Lake trout had mean values of 7.1 in young and 26 µg·kg−1 in older fish, and, curiously, PCB levels in lake whitefish showed a reverse of the general trend for contaminant levels in all fish species in that they had higher mean values in young fish compared to older fish , with no significant differences between group 3 fish and any other groups or between group 1 and 4 fish. Species-specific comparisons showed that Arctic char obtained from nine fishing sites and grouped into four geographic regions showed low Hg levels independent of location, ranging from 0.05–0.13 µg·g−1 ww , but ciscoes from King William Island sites had significantly (p < 0.01) higher mean Hg levels than those obtained from mainland sites (groups 1 and 2 vs. 3 and 4) at 0.16 and 0.08 µg·g−1 ww, respectively. It should be noted that community members were interested in obtaining contaminant information from all individual sites, but this was not possible. For life history or logistical reasons, individual species were not obtained or were taken in insufficient numbers at all sites, particularly since this region represents the northern most distribution of lake whitefish [To understand site-specific contaminant levels and to keep sample numbers sufficiently high, all of the fishing sites were divided into four groups based on their geographic locations . When fig·g−1 ww A. Lake thitefish .n = 537, DF = 3, p = 0.861 for groups 1 and 2, p = 0.997 for groups 3 and 4, p < 0.0001 between the island sites and the mainland sites). This geographic trend was also seen for Arctic char, lake trout, and cisco , but only the group 1 lake whitefish mean, and not group 2, was significantly different from the means of lake whitefish groups 3 and 4 . Additionally, the mean As in Arctic char from group 3 was significantly lower than that of group 4 , but no differences were seen between group 3 and 4 means for other fish , and these were statistically significant  as well as mainland groups (3 and 4) in T-PCBlip . PCB content differences between species were not apparent at location group 4, the only location group for which all fish species were analyzed. On the other hand, Arctic char PCB concentrations in group 1 were significantly lower than group 1 lake whitefish  and ciscoes . Only Arctic char were analyzed from location groups 2 and 3  [−1 recommendation for subsistence consumption may be more appropriate, similar to those issued by several jurisdictions, including American states  , with pr mg·kg−1 , sport fkg−1 [−1 , the US  [−1 [−1 ; and Can [−1 [−1 ). AlthouBioaccumulation of high Hg levels in lake trout undoubtedly reflect the positive correlation between contaminant levels and fish weight , but, in−1 dw), with this much variation seen in older lake trout alone (−1) in Canada’s Food and Drug Regulations [−1 dw [−1 dw [−1 dw [−1 may be troubling and warrant further investigation. Overall, the As concentrations in these fish were higher than for other locations in the Arctic, with the highest concentrations similar to those seen in strictly marine fish [As levels in the sampled salmonids varied over a very large range, more than two orders of magnitude  had ~2-fold the As concentrations seen for the island group 2 and ~10-fold the concentration found for island group 1. Additionally, higher As concentrations in group 3 and 4 fish were seen in all individual fish species, and mean As concentrations of group 1 and 2 fish were consistently indistinguishable for all species . The hig−1 ww, similar to the range of PCB concentrations in fish from other Arctic locations, including former military sites at 0.5–364 µg·kg−1 ww [−1 PCBs, in the form of congener 126 [As indicated, economic considerations limited the amount of PCB testing, but when the PCB data from all species were amalgamated, total PCB values varied from 5–367 µg·kg·kg−1 ww ,77,78,79·kg−1 ww . In this·kg−1 ww , but als·kg−1 ww ). The ge·kg−1 ww . That thener 126 .−1 ww) used by the U.S. FDA [Of the total salmonids analyzed for PCBs, 12% (12/101) exceeded freshwater sportfish guidelines see . HoweverU.S. FDA , but theU.S. FDA . None ofU.S. FDA . There wU.S. FDA . AlthougThe numbers and the positions of chlorines on the biphenyl structure correspond to different PCB congeners, and these can be used as “fingerprints” for PCB mixtures. Nonetheless, PCB congeners 110, 153, 118, and 138 had the highest abundance in all the fish, and three or more were amongst the five predominant congeners in 84% of all samples . StrikinST values were used. In the latter case, low assignment success characterized 65% of the fishing sites, with four sites approaching a surprising 0% success (Geographic groupings of contaminants showed variation, but there was a generally higher level of Hg in salmonids as a whole and ciscoes in particular from island fishing sites than on the mainland, with the reverse for As contamination in all sampled fish, as well as a clustering of distinct PCB congeners from lake whitefish and Arctic char from island sites . Theoret success . This waIn contrast, population assignments were much more successful with random samples from 413 Arctic char correctly assigned as “Northern” residents, those fish caught on or near King William Island, as well as a mainland or “Southern” population. An individual Arctic char could be correctly assigned to one of these two populations more than 85–90% of the time with only 4–32 SNPs, respectively . TherefoOur original hypothesis was that levels of contaminants would vary geographically and that genomic tools would be useful to help target food fish at risk, even in mixed stocks in the open ocean, circumventing the necessity to analyze hundreds of fish at particular fishing sites. This exploratory evaluation shows that analysis with as few as 4–32 genetic markers can correctly assign random Arctic char to geographically distinct regional populations. With additional testing, SNPs for lake whitefish may also hold promise. It is our hope that this novel strategy can be pursued in the future to allow more widespread testing of contaminants from these remote Arctic regions so as to help safeguard the wellbeing of Inuit who depend on these anadromous salmonids. Further, we expect that this approach will be applicable for the monitoring of vulnerable food fish elsewhere particularly in extreme locations.


R0 resection in primary surgery, it’s unclear if it’s oncologically safe after neoadjuvant chemotherapy (NAC). Only limited data demonstrate that surgery within new margins in cases of a pathological complete response (pCR) is safe. We therefore investigated the influence of different margins and pCR on local recurrence and survival rates after NAC.While “no tumour on ink” is an accepted margin width for R ≤ 1 mm, R > 1 mm and RX (pCR) for local recurrence-free survival (LRFS), disease-free survival (DFS) and overall survival (OS).We retrospectively analysed data of 406 women with invasive breast cancer, treated with NAC and breast-conserving therapy between 1994 and 2014 in two certified Austrian breast health centres. We compared R ≤ 1 mm: 94.2%, R > 1 mm: 90.6%, RX: 95.0%; p = 0.940), the 5-year DFS  and the 5-year OS  did not differ significantly between narrow, wide, nor RX resections. Regarding DFS and OS, a negative nodal status reduced the hazard ratio significantly.After a median follow-up of 84.3 months, the 5-year LRFS (There is no significant difference in LRFS, DFS and OS comparing close, wide or unknown margins after pCR. We suggest that resection in new margins after NAC is safe according to “no tumour on ink”. Resection of the clipped area in cases of pCR is emphasized. It downsizes large tumours1,6,R0 resection after neoadjuvant therapy, there are insufficient data and only expert recommendations, such as that from the St. Gallen panellists, who suggest that the approach of no tumour cells in the resection margin is sufficient.8Originally, a wide tumour-free resection margin was necessary to guarantee a favourable oncological outcome. In the meantime, the definition of a tumour-free margin in primarily operated patients changed to the “no tumour on ink” approach.4Breast cancer shrinks with a scattered profile in up to 39% of patients during neoadjuvant chemotherapy.10This study aims to investigate whether narrow or wide tumour margins impact differently on the oncological outcome of patients with residual disease after NAC. Moreover, we compare oncologic outcomes of patients having residual disease and negative margins with patients having no residual disease (pCR) and thus unclear margins.R1 resection was defined as a surgical procedure resulting in the excision of a specimen with invasive tumour cells adjacent to one or more margin, whereas a R0 described the absence of invasive tumour cells on the inked margin of the specimen. A pathologist performed the evaluation of the margin involvement. The presence of ductal carcinoma in situ (DCIS) or lymphatic vascular invasion, even in the absence of stromal tumour invasion, was not compatible with the diagnosis of pCR. Nevertheless, the pCR referred to the lumpectomy specimen only, not to the axillary lymph node status. The majority of included patients received post-surgery radiotherapy according to local standards; in the main, conventional fractionated irradiation up to a total dose of 50 Gy with or without local boost therapy. Women who suffered from a lobular carcinoma were excluded with the intention to generate a homogenous study population. Cases where definitive histology showed a R1 resection (n = 17) and a mastectomy, or no further operation was performed, were excluded in order to avoid a bias regarding oncological outcome. Of the 17 patients with a R1 resection, 14 patients underwent a subsequently performed mastectomy, therefore contradicting our inclusion criteria of breast conserving therapy. Three women didn’t undergo a further operation, either for personal or for unspecified reasons. Thus, they were expected to have higher local recurrence rates and decreased survival rates. Data was extracted from a prospectively maintained patient database, histopathological reports and patients’ charts. Relevant data for this study included age, menopausal status, TNM-stage before and after NAC, preoperative biopsies including the histopathological grading, immunohistochemical hormone receptor and human epidermal growth factor receptor 2 (HER2) status—after its establishment—as well as the Ki67 labelling index, clinical stage of the tumour and the axillae, date of the operation and the final histological results. The study was approved by the local ethics committees both at the Medical University Vienna and the Hospital of the Sisters of Charity (now Ordensklinikum) Linz, Austria.We performed a retrospective analysis of 416 patients with BC and breast-conserving therapy (BCT) after neoadjuvant chemotherapy. Treatment was performed between 1994 and 2014 either at the Department of General Surgery of the Vienna General Hospital or at the Breast Health Centre of the Hospital of the Sisters of Charity in Linz. Before NAC, the tumour was clipped in order to facilitate identification of the primary tumour bed in case of pCR. Regimens of NAC were administered depending on the local oncologist’s choice . The operation included lumpectomy and sentinel lymph node (SLN) biopsy. Breast cancer tissue was clip-marked at the time of biopsy. The day before excision, a wire was used to mark non-palpable lesions. SLNs as well as the breast tissue specimen were assessed routinely by intraoperative frozen section. The sentinel lymph node was intraoperatively identified by using blue dye. If available, dual tracer mapping by using a radiocolloid and blue dye was applied. In case of SLN metastasis, SLN biopsy was followed by level I and II axillary lymph node dissection. If resection margins were found to be involved, immediate further resection was performed. If involved margins weren’t identified in the frozen section but in the definitive histological report, a secondary operation for re-excision was mandatory. A R ≤ 1 mm” and wide margins “R > 1 mm”. Tumour margins in patients without invasive or non-invasive tumour cells (pCR) in the breast specimen were grouped as “RX”. Only thirteen women had a positive margin at final histology without re-excision and were excluded from the analysis.Margin widths were collected from histology reports. Negative tumour margins in patients with residual disease were grouped into close margins “Endpoints of this study were local recurrence-free survival (LRFS), which we defined as the time from surgery until recurrence in the ipsilateral breast as well as disease-free survival (DFS) and overall survival (OS).Statistical analysis was performed by using SAS . Categorical variables were described by frequencies and percentages, normally distributed continuous variables were described by mean ± standard deviation, non-normally distributed data by median with minimum and maximum. Survival curves of LRFS, DFS and OS were computed by the Kaplan–Meier method. For 5-year survival estimates, 95% confidence intervals (CI) were calculated using the asymptotic normality assumption of the log–log transformation. Group differences were compared by log-rank test and quantified by hazard ratios (HR) with corresponding 95% confidence intervals from the Cox regression model. A multiple Cox regression model was used to adjust group differences by other prognostic factors as follows: age, nodal stage after NAC (ypN stage), estrogen receptor (ER), progesterone receptor (PR), HER2 and Ki67 status, a cell proliferation index, as well as grading. ER and PR status were combined as hormone receptor (HR) status. All statistical tests were based on a two-sided significance level of 0.05.In total, 406 patients were included in the analysis. Of those, 44% of the patients underwent surgery at the Department of Surgery at the General Hospital of Vienna, and 56% at the Breast Health Centre at the Sisters of Charity Hospital in Linz. Median patient age was 51.5 years (range 20.5–82.6 years). Median follow-up was 84.3 months (95% CI 71.6–97.1). One hundred and eighty-two (44.8%) women were pre- whereas 224 patients (55.2%) were post-menopausal. A palpable tumour at diagnosis was reported in 377 patients (92.8%). At the time of presentation, in 144 patients (35.5%) clinically lymph-node positive disease was reported, whereas 155 women (38.1%) were clinically lymph-node negative. In 107 patients (26.4%) clinical lymph node status was not documented. In 51 patients HER2 status was neither determined before nor after chemotherapy. These patients were counted as ‘nd—not done’. In the preoperative biopsy, breast cancer subtypes were represented in descending order as follows: 190 (46.8%) HR+/HER2−/nd, 118 (29.1%) HR−/HER2−/nd, 58 (14.3%) HR+/HER2+ and 40 (9.9%) HR−/HER2+ disease. All patients underwent BCT; in 48 patients a re-excision due to positive margins of the initial specimen was necessary. This represents a re-excision rate of 11.8%. Patients’ characteristics are shown in Table R ≤ 1 mm was reported in 74 patients (18.2%), R > 1 mm in 284 patients (70.0%) and RX due to pCR in 48 patients (11.8%). During follow-up 64 patients died (15.8%). Local recurrence was diagnosed in 39 patients (9.6%) whereas 13 patients (3.2%) suffered from contralateral recurrence. Therefore, in 354 cases (87.2%) no disease recurrence was observed. Axillary recurrence was found in 9 cases (2.2%) whereas distant metastasis was reported in 86 patients (21.2%).A margin width of R0”, because the existence of residual disease was not considered as pCR.The definitive histological report showed ypT0/X in 50 patients (12.3%), ypT1 in 210 (51.7%), ypT2 in 102 (25.1%), ypT3/4 in 14 (3.5%) and ypTis in 30 (7.4%). Positive nodal status (ypN+) was observed in 175 patients (43.2%). ypN- was found in 230 patients (56.8%). In one patient the lymph node status was not available. In one case ypT0/X was classified by pathologists although lymphangiosis was found in the tumour bed. This patient was classified as “Postoperative whole breast irradiation with or without additional local boost therapy was administered in 389 of 406 patients (95.8%). Seventeen patients (4.2%) did not receive radiotherapy due to individual decisions: either patients’ or interdisciplinary tumour board decisions.R0” or “RX” . There was a non significant trend of a higher 5-year overall survival in patients with Rx with an absolute amount of 9.1%. A trend towards a better DFS, yet not significant, was seen in women after RX margins when compared to R0 margins . Survival curves are shown in Fig. Regarding LRFS as well as OS, no significant difference was found for either margins described as “R0 resections into “R ≤ 1 mm” and “R > 1 mm”, no significant difference was found for either LRFS  or for DFS  or for OS . Survival curves for those three groups are shown in Fig. Even after further dividing of p = 0.004, 95% CI 0.27–0.78) and DFS  but not for LRFS  .p = 0.0001).10Intratumoural heterogeneity causes heterogeneous response to NAC that further leads to unpredictable tumour shrinkage.11R1 resections, the influence of positive margins on the oncological outcome should be considered. Gentilini et al. focused on the difference in local recurrence rates between R0 and R1 resections. They found that positive margins have a marginally significant higher incidence of local recurrence after 3 years than negative margins .R1 resections were excluded; consequently, a comparison with our findings cannot be done. Nevertheless, the optimal margin width remains unclear in the neoadjuvant setting.Given that NAC can lead to higher rates of Therefore, the primary objective of our trial was to answer the question as to whether the “no tumour on ink” approach is safely applicable in the neoadjuvant setting. We primarily focused on the difference of oncological outcome parameters in patients with wide and narrow specimen margins.R ≤ 1 mm) do not lead to a statistically significant difference in local recurrence-free, overall, or disease-free survival in comparison to wider resections (R > 1 mm), we can confirm that “no tumour on ink” is reliable and safe after NAC. Nevertheless, a trend towards improved survival of patients with RX resections was observed in Kaplan–Meier curves.As we could show that narrow resections margins  or wider (≥ 2 mm) margins.15Likewise, Tyler et al. investigated whether close margins (< 2 mm) have an impact on patient outcomes. In a study cohort of over 10,000 patients and a median follow-up of 8 years, where 87% of patients were administered systemic chemotherapy, close margins were not associated with increased local recurrence or decreased breast cancer specific survival. In conclusion, they suggest that omitting re-excisions of positive or close margins might be an acceptable approach in selected cases scheduled for adjuvant radiotherapy.A further aim of our study was to clarify whether pCR influences the oncological outcome.,19–21Pathologic complete response in general is associated with improved survival.17R0 resection. Nevertheless, we also observed a noticeable trend towards improved DFS . Taken together, in cases of pCR, a resection of the clipped tumour bed within the new boundaries is safe as well.In our study cohort, we determined that residual DCIS in the operation specimen didn’t match the criteria of pCR. Applying this, neoadjuvant chemotherapy led to pCR in 12.3% of our patients. Our study might have been underpowered to correlate pCR with improved oncological outcome, as LRFS, OS and DFS didn’t differ significantly when compared to patients with In the presented study population, we observed a re-operation rate due to positive margins after the first breast conservation attempt of 11.8%. An earlier study of our patients without neoadjuvant therapy demonstrated a 9% re-resection rate, suggesting no significant difference in re-resection rate with or without neoadjuvant treatment.22In an adjusted Cox model we found that a negative nodal status after NAC was independently correlated with the HR for events regarding OS and DFS. This finding is supported by a study conducted by Van Nijnatten et al., who observed that ypN1-3 was significantly associated with worse prognosis.Our study has some limitations, which must be taken into consideration when interpreting our findings. In particular, the retrospective as well as the non-randomized design must be mentioned. Furthermore, the small number of patients with “RX” in their final pathologic report might weaken the conclusion in this subgroup. However, it must not be assumed that these limitations influence the validity of the results presented here regarding close or wide margins an their impact on local recurrence and survival rates. In some cases, data regarding HER2 (in 51 patients) and Ki67 status (in 177 patients) could not be obtained, as several women were treated at times when HER2 and Ki67 immunohistochemistry was not yet routinely performed. Therefore, a detailed subgroup analysis of the impact of tumour subtypes on outcome is restricted. Detailed documentation on adjuvant radiotherapy regimes including local boost therapy is not available.Taken together, large, randomized controlled trials investigating the oncological outcome of patients undergoing NAC followed by BCT would be desirable and necessary to confirm our findings.R0 resections as “no tumour on ink” is applied.In conclusion, the data presented here suggests that there is no significant difference in oncological outcome, measured by LRFS, DFS and OS, between narrow or wider margins or RX resections due to pCR. This implies that breast conserving surgery after neoadjuvant chemotherapy is oncologically safe even if the definition of


The Scythians are frequently presented, in popular and academic thought alike, as highly mobile warrior nomads who posed a great economic risk to growing Mediterranean empires from the Iron Age into the Classical period. Archaeological studies provide evidence of first millennium BCE urbanism in the steppe while historical texts reference steppe agriculture, challenging traditional characterizations of Scythians as nomads. However, there have been few direct studies of the diet and mobility of populations living in the Pontic steppe and forest-steppe during the Scythian era. Here, we analyse strontium, oxygen, and carbon isotope data from human tooth enamel samples, as well as nitrogen and carbon isotope data of bone collagen, at several Iron Age sites across Ukraine commonly associated with ‘Scythian’ era communities. Our multi-isotopic approach demonstrates generally low levels of human mobility in the vicinity of urban locales, where populations engaged in agro-pastoralism focused primarily on millet agriculture. Some individuals show evidence for long-distance mobility, likely associated with significant inter-regional connections. We argue that this pattern supports economic diversity of urban locales and complex trading networks, rather than a homogeneous nomadic population. Histories), who describes Scythian populations living in wagons while also engaging in raiding and warfare , and NO3− . All international standards were obtained from the National Bureau of Standards in Gaithersburg, Maryland. Internal standards were measured repeatedly during the analysis and provided the following results: peptone ; glycine . The %C and %N values were calibrated against known quantities of the internal peptone and glycine standards. Isotopic values are reported in permil (‰) relative to the Vienna Pee Dee Belemnite (VPDB) standard for δ13C and atmospheric nitrogen (AIR) for δ15N. Collagen quality was assessed using %C, %N, and C:N ratios [Sample isotope ratios were compared to a secondary gas standard with an isotope ratio that was calibrated to international standards. For N ratios ,100.Climate and precipitation across Ukraine are discussed relative to the sites under study and the broader region. We have also included a discussion of terrestrial vegetation and the ecological zones that were inhabited. See Supplemental Text  values of human enamel from different sites and periods were compared using an analysis of variance (ANOVA) paired with Tukey’s test in R [87Sr/86Sr and δ13C values from the sites of Medvin and Mamai-Gora  and Mamai-Gora (n = 25), are shown in 87Sr/86Sr ratios of human teeth from Medvin range from 0.7106 to 0.7111, with all teeth (15 of 15) sitting within the local range (0.7104 to 0.7111). Medvin is located on a Cenozoic substrate but surrounded by Palaeozoic substrates (0.7110 to 0.7130) and individuals had 87Sr/86Sr ratios  falling within the local range estimated as 0.7096 to 0.7103 (Median Sr ratio ± 3MADnorm).We measured strontium isotope ratios for individuals from two cemeteries that have material culture associated with the Scythians. Both cemeteries are considered key sites with regard to understanding the mobility of Pontic steppe populations as they are well excavated and in different environmental zones. The results of our study, which measured dvin n = 5 and MamGora n = 5, are shGora n = 5, are shios Figs  that sugBased on modern geologic substrates, the site of Mamai-Gora is located on Cenozoic substrates, while Palaeozoic substrates with estimated ‘high’ strontium isotope values (0.7110–0.7130) are located along the northern bank of the Dnieper as well as ~130 km to the southeast. The closest geological locales with ‘low’ strontium values (<0.709) are located far to the northeast (~220 km away), in Crimea (~260 km), and west of Cherkasy (~280 km). At Mamai-Gora, one individual  had an early forming molar (M1) exhibiting a low strontium value and a late forming molar (M3) with a high strontium value suggesting that they were born far outside of the region, then lived north or southeast of the site until adolescence, perhaps coming to live at Mamai-Gora after 16 years of age. A second individual  had molars  with high strontium values suggesting that they lived in another region, either to the north or southeast of the site, and came to Mamai-Gora after adolescence. Three other individuals  exhibited smaller variation in strontium isotope values between teeth of ≥0.0002, suggesting they engaged in less restrictive forms of landscape use than individuals that had little variation between teeth.18O (C-VPDB) from the sites of Medvin (n = 12) and Mamai-Gora (n = 26)  with an M1 value of −3.9‰. The total range of oxygen isotope values for these two sites fall within the normal amount of variation (~3‰) for a population  . δ18O vaulation ,103–105)–10518O , with an estimated baseline of 0.7098±0.0003 and three outliers. At Vinogradnoe, one outlier from the Eneolithic was an individual with a high value (0.7101) that fell just outside of our baseline, while in the Early Catacomb period, two individuals fell outside our estimated baseline, one with a high value (0.7102) and one with a low value (0.7095). The site of Peshtchanka  has an estimated baseline of 0.7104±0.0008, with one outlier with a low Sr ratio of 0.7096. Spanning both the Yamnaya and Catacomb periods, the site of Kirovograd has an estimated baseline of 0.7107±0.0016 with no outliers. A single outlier (0.7098) was identified at the site of Shakhta Stepnaya, falling just outside the estimated baseline of 0.7100±0.0001. The site of Nevskoe has an estimated baseline of 0.7091±0.0004, with no outlying individuals. Thus, there were five outlying individuals in total for Eneolithic, Early Catacomb, and Catacomb period sites, and no outlying individuals dating to Yamnaya period.Archaeological sites across Ukraine have consistently been identified in areas with Cenozoic substrates (0.7090–0.7110), except for the site of Nevskoe that is located within Mesozoic substrates (0.7070–0.7090) but in close proximity to Cenozoic substrates. Independent Sr reference datasets were not available for some of the sites under study  with a low Sr ratio of 0.7092, well outside the baseline estimated at 0.7099±0.0005. At the site of Bel’sk, the local baseline was estimated as 0.7099±0.0010, with six outliers. Five individuals  had low Sr ratios which are similar to areas located north or east of the site, at distances of 90 km and 160km respectively.18O values have a range from −4.3 to −7.8‰, similar to individuals from Yamnaya sites with a range from −4.2 to −7.2‰ [18O values of human teeth from Scythian era sites overlap with these values, ranging from −3.9 to −7.2‰, except for the site of Bel’sk that had lower δ18O values ranging from −4.6 to −8.5‰ [18O values  were identified between the site of Bel’sk and several other sites including Ordzhonikidze, Mamai-Gora, Vinogradnoe, and Peshtchanka  at Medvin (n = 12) and Mamai-Gora (n = 26) . There is a significant difference between the mean values for these two cemeteries . At Medvin and Mamai-Gora, intra-individual variation ranged from 0.6 to 4.3‰, with an average difference between paired teeth of 2.4‰ and 1.6‰, respectively. Large differences in carbon isotope values between paired teeth were evident for some individuals, suggesting a shift in diet over time. For example, at Mamai-Gora one individual (#33) had an M1 measured at −12.0‰ and an M3 measured at −7.4‰ (difference = 4.6‰), while at Medvin there was an individual (#29) with an M1 measured at −7.1‰ and an M2 measured at −3.4‰ (difference = 3.8‰). Neither of these individuals exhibited differences in their strontium isotope ratios between teeth. The wide range of carbon isotope values of individuals at these sites suggests diversity in the amount of C3 and C4 plant consumption, or the consumption of livestock ingesting these plants. Enamel bioapatite δ13C values approaching −4.0‰ (dietary δ13C of approximately −13.0‰) align with those of individuals from regions of the globe with a documented reliance on C4 plants  . Stable ts (e.g. ).13C for humans at Medvin (−3.4 to −9.8‰) and Bel’sk (−3.9 to −12.6‰) indicate a reliance on C4 food sources for at least some individuals. At Mamai-Gora, δ13C values for humans (−6.6 to −12.3‰), suggest more moderate consumption of C4 food sources. At the sites of Alexandropol, Ordzhonikidze, and Zolotaya Balka, δ13C values range from −9.3 to −14.0‰ [4 food sources. Finally, at the sites of Babina Mogila, Drana Kokhta, and Zolotaya Balka average values ranged from −10.4 to −14.3‰ [3 plants, or livestock ingesting C3 vegetation. Significant differences were identified between Medvin  and all other sites, except Bel’sk  values of human enamel indicates that there was no consistent correlation (strongly positive or negative) for the sites under study  At Bel’sk there was a significant positive relationship (r = 0.41) between carbon and strontium values (p = 0.015).A comparison of 13C values at Mamai-Gora were slightly higher, ranging from −17.7 to −14.9‰ (average = −16.6‰). At the site of Bel’sk, δ13C values of humans ranged from −17.6 to −12.8‰, with an average value of −15.5‰, while fauna (n = 3) had an average value of −18.1‰ . At Bel’sk, δ15N values of humans ranged from 10.3 to 12.9‰, with an average value of 11.4‰, while fauna (n = 3) had an average value of 8.6‰.We analysed a total of 58 human bone collagen samples from the sites of Bel’sk (n = 29), Mamai-Gora (n = 15), and Medvin (n = 10) . Very feThe carbon isotopic values for animals at Bel’sk are contextualized within a discussion of available data on ancient faunal values, modern vegetation, and controlled studies. See Supplemental Text . While a subset of populations apparently engaged in inter-regional mobility, there is also evidence for intensive localized mobility at some sites such as Bel’sk. One caveat is that at some sites there is a lack of variation in geological substrates across a wide area, thus masking smaller-scale local and regional movement. Scythian era populations across Eurasia were engaging in pastoralism and horseback riding, drawing on this component of their lifestyle to develop shared motifs, while simultaneously engaging in agricultural production ,107. Hig3 vegetation [4 sources in human or animal diets are likely from the consumption of domesticated crops, namely millet. Observed δ13C values for faunal bone collagen at Bel’sk range from −19.8 to −16.3‰, indicating a mixed diet of C3 vegetation as well as small amounts of C4 plants such as millet. Among societies practicing pastoralism (lacking domesticated grains) isotopic data demonstrate a shared carbon isospace of humans and livestock, from the same region and time period [In addition to this direct assessment of individual mobility, evidence for variability in diets among Iron Age populations has highlighted the complexity of Scythian lifeways. Floral biomes in Ukraine support mainly Cgetation ,109, thue period . Howevere period . The pop3 plants. In contrast, individuals with low levels of mobility had a wider spectrum of dietary intake varying from high levels of millet consumption, to diets based almost exclusively on pastoral products or C3 plants. Linear regression analysis demonstrates that for all sites (except Bel’sk) there was no significant relationship between strontium and carbon isotope values, nor was there a consistent positive or negative correlation , as well as between individuals with different grave goods and apparent social status. More detailed primary mapping work will enable a greater understanding of isotopic variation across space in this understudied region (for discussion see ). AlthouS1 File(PDF)Click here for additional data file.


To investigate and compare the eating behavior and food neophobia ofchildren and adolescents from different age groups, body mass index per age,and sex. This was a cross-sectional study, with a convenience sample, involving 150children and adolescents aged 3 to 13 years, of both sexes, treated at apediatric outpatient clinic of a teaching hospital in the municipality ofUberaba-MG, Brazil. Subscales of the Child Eating Behavior Questionnaire(CEBQ) were used to evaluate eating behavior, and the Child Food NeophobiaScale (CFNS) was used to evaluate food neophobia. Higher scores were found in the subscales “food responsiveness” (p=0.015),“enjoyment of food” (p=0.002), and “emotional overeating” (p=0.009) amongolder children and adolescents. Younger children had higher scores in thesubscales “satiety responsiveness” (p=0.004) and “slowness in eating”(p=0.001). There was a tendency toward higher scores for “foodresponsiveness” (p=0.005) and “emotional overeating” (p=0.013) inparticipants with severe obesity. There were no differences in the scale offood neophobia. Overall, food neophobia positively correlated with lack ofinterest in food and negatively correlated with interest in food. The study showed significant differences in some domains of eating behavioramong children and adolescents of the sample; however, no differences werefound regarding food neophobia. These results may contribute to theimprovement of future interventions related to infant eating behavior andfood neophobia. However,there is a gap in studies that assess the relationship between eating behavior andfood neophobia in this population. The objective of the present study was toinvestigate and compare eating behavior and food neophobia between children andadolescents from different age groups, body mass index according to age(BMI-for-age), and sex.This is a cross-sectional study conducted from June to November 2018 in a pediatricoutpatient clinic of a teaching hospital in the city of Uberaba, State of MinasGerais, Brazil. A convenience sampling was carried out, involving 150 children andadolescents aged from 3 to 13 years, of both sexes, who only consumed foods orally,and were accompanied by parents or caregivers who knew their daily eating habits andbehaviors well enough. This population was chosen due to the increasing prevalenceof overweight and obesity in this age group.Parents and caregivers completed three self-administered questionnaires in thepresence of the researcher to minimize data loss, considering that the outpatientclinic serves patients from several municipalities: the Child Eating BehaviorQuestionnaire (CEBQ), the Child Food Neophobia Scale (CFNS), and a structuredsocioeconomic questionnaire in order to characterize the sample. Moreover, theweight and height of children and adolescents were measured after completing thequestionnaires.,,sumos” (European Portuguese word for“juices”) with “sucos” (Brazilian Portuguese word for“juices”). CEBQ is a specific questionnaire applied to investigate eating behavior in childrenand young people according to the answers provided by their parents or guardians,focusing on behavioral determinants of obesity. Conversely, the CFNS is a version consisting of ten items similar to those of theFood Neophobia Scale applied to adults, but used for parents or guardians to reportchildren’s traits of food neophobia and appetite to try new foods. The socioeconomic questionnaire consisted of questions about the number of familymembers living in the same residence , area of residence , residence status ,education of parents or caregivers and family income , using the minimum wage value as a reference . Children and adolescents were classifiedin groups, such as age groups , sex, andBMI-for-age, for later comparison between CEBQ and CFNS scores.®, Santa Bárbara d’Oeste, Brazil) with a capacity of up to 200kg and precision of 0.1 kg; and height (cm) was measured in a stadiometer with a0.1-cm graduation; BMI was subsequently calculated (BMI=weight(kg)/height(m)2). To assess the nutritional status, the BMI-for-age z-score wasused, which was classified according to the growth curves proposed by the WorldHealth Organization (WHO)® and AnthroPlus® software wereused .It is worth noting that the previously trained research team collected anthropometricmeasurements, in which weight (kg) was measured on a digital scale software, version 22.0 . The relative frequencies of the following variables werepresented: sex, age, socioeconomic characterization, and BMI-for-age classification.Normality of data was assessed by the Kolmogorov-Smirnov test. The scores of theCEBQ subscales and the final CFNS score were indicated as mean and standarddeviation. The Kruskal-Wallis test was used to compare the scores of thequestionnaires between the age groups and the BMI-for-age, and the Mann-Whitney testwas used to compare the scores between sex. For correlations between the CEBQsubscales and the final CFNS score, the Pearson’s coefficient was employed. Theadopted significance level was 5% (p<0.05). The project was approved by the Research Ethics Committee of Universidade Federal doTriângulo Mineiro (UFTM) under protocol No. 2,625,851, and all those responsible forchildren and adolescents signed an informed consent form.A total of 150 children and adolescents participated in the study, and 64.0% (n=96)were male. Age varied between 3 and 13 years old, with a mean of 7.7±2.9 years,distributed into four groups with approximately the same sample size: <6 years(n=41), 6-7 years (n=32), 8-9 years (n=36), and ≥10 years (n=41). The average age ofparents and caregivers was 35.8±9.9 years, and the education level that prevailedwas high school , followed by some elementary school .Regarding the socioeconomic characterization of the sample, most  hadmore than three family members living in the same residence, and 96.7% (n=145) livedin an urban area. In addition, 70.0% (n=105) of the sample had their own residence,32.7% (n=49) had a family income of 1 to 1.9 minimum wages, followed by 31.3% (n=47)with 2 to 3 minimum wages.When analyzing the BMI-for-age of children and adolescents, based on the z-score, aprevalence of normal weight was found , followed by 14.7% (n=22)overweight, 12.0% (n=18) obesity, 7.3% (n=11) severe obesity, and 2.7% (n=4) risk ofoverweight, indicating that 36.7% of the sample was overweight. No participantsgrouped in the thinness category were found in the sample.The means and standard deviation of the scores of the CEBQ subscales and the CFNS,according to age group, were demonstrated in As for scores related to BMI-for-age classifications, significant differences wereobserved only in the CEBQ subscales “emotional undereating” (p=0.041), “foodresponsiveness” (p=0.005), and “emotional overeating” (p=0.013) . In thesThere were no significant differences in the scores between sexes, but a tendency forhigher scores in the subscale “emotional overeating” was observed in males .With regard to “slowness in eating,” there was a positive correlation with “emotionalundereating” (p=0.042), and a negative correlation with “food responsiveness”(p=0.002) and “enjoyment of food” (p=0.000). “Food fussiness” negatively correlatedwith “enjoyment of food” (p=0.012) and CFNS (p=0.000). As for “emotionalundereating,” a positive correlation with “emotional overeating” (p=0.004), and anegative correlation with CFNS scores (p=0.023) were observed. “Food responsiveness”positively correlated with “enjoyment of food” (p=0.000) and “emotional overeating”(p=0.000). Yet, “enjoyment of food” positively correlated with “emotionalovereating” (p=0.000) and CFNS (p=0.002) as well.The study indicated that only a few domains of eating behavior significantly differedbetween age groups, with a tendency for higher score in “interest in food” on thepart of older children, and in “lack of interest in food” on the part of youngerchildren. Regarding the BMI-for-age classification, there was a tendency for higherscores in “food responsiveness” and “emotional overeating” by participants withsevere obesity compared with those with normal weight, and no differences inrelation to sex were observed. CFNS did not present significant differences inrelation to age group, BMI-for-age, and sex. Overall, subscales related to interestin food positively correlated between each other, and negatively correlated withthose related to lack of interest in food.,The subscales “slowness in eating” and “satiety responsiveness” tended to higherscores on the part of younger children. This datum is in agreement with the findingsof Viana et al.It is worth defining that “slowness in eating” is associated with lack of interest infood, whereas “satiety responsiveness” assesses the counterregulatory eatingcapacity, i.e., the regulation of appetite,In a study on school-age children, the authors inferred that the CEBQ subscale“satiety responsiveness” can be an effective indicator of characteristics ofresponse to internal signs of satiety, and “food responsiveness” and “enjoyment offood” are effective indicators of the capacity to respond to external foodstimuli.,The category “interest in food” tended to be higher in children with severe obesity. Studies indicate a positiverelation between BMI and the subscales “food responsiveness,” “emotionalovereating,”,,,,,None of the scales used in this research showed significant differences in relationto sex, corroborating data found in similar studies.,,,CFNS scores negatively correlated with “satiety responsiveness,” “food fussiness,”and “emotional undereating,” indicating that the higher the scores of thesesubscales, the lower the CFNS score, i.e., the more severe the food neophobia. TheseCEBQ subscales correspond to the lack of interest in food. In this sense, it can beconsidered that this characteristic is also present in food neophobia, consideringthat it includes resistance to eating and trying new foods.This study is one of the first to relate food neophobia to infant eating behavior inthe Brazilian population. Nevertheless, it has some limitations that must beconsidered when interpreting the results, such as the cross-sectional study design,which does not allow confirming a causal relationship between the variables, theconvenience sample, and the small sample size. Despite the limitations, the studyindicated differences in some domains of the eating behavior between children ofdifferent ages and BMI-for-age, but not in relation to sex. Although no differenceswere found regarding food neophobia, overall, it positively correlated with lack ofinterest in food, and negatively correlated with interest in food.Understanding how eating behavior is displayed at different ages, sexes, andnutritional statuses contributes to the implementation of nutritional interventions,seeking to assist children and adolescents in learning healthy behaviors accordingto each demand. Furthermore, understanding the relationship between eating behaviorand food neophobia enables studying strategies that can be used by parents andcaregivers without including persuasion, coercion, bribery, or the use of rewards togetting children to try new foods.