The SRD gene was identified as an orthologue of α-glucan, water dikinase 1 (GWD1), whose expression is regulated under conditions of light/dark cycles in leaves and is associated with storage root development. The GWD1-RNAi cassava plants showed both retarded plant and storage root growth, as a result of starch excess phenotypes with reduced photosynthetic capacity and decreased levels of soluble saccharides in their leaves. These leaves contained starch granules having greatly increased amylose content and type C semi-crystalline structures with increased short chains that suggested storage starch. In storage roots of GWD1-RNAi lines, maltose content was dramatically decreased and starches with much lower phosphorylation levels showed a drastically reduced β-amylolytic rate. These results suggested that GWD1 regulates transient starch morphogenesis and storage root growth by decreasing photo-assimilation partitioning from the source to the sink and by starch mobilization in root crops.Regulation of storage root development by source strength remains largely unknown. The cassava  Manihot esculenta Crantz) is an important root crop that accumulates large amounts of starch in its storage roots, which is extensively cultivated in the tropics and subtropics2. Cassava is also recognized for its drought and infertility tolerance in marginal lands4. During cassava growth, photosynthetically-derived carbohydrates are appropriately partitioned from the leaves to storage roots, which are the major sink organs that reflects both cassava yield and starch productivity6. Unlike grain crops, photo-assimilates in cassava are translocated from the leaves to storage roots – an important process that contributes to sink productivity, although the regulatory mechanism is largely unknown8.Cassava  and phosphoglucan water dikinase (PWD), respectively. This biochemical reaction is completed by transferring the β-phosphate of ATP to the substrate and the γ-phosphate to water11. Constitutive suppression of GWD expression in maize and rice showed no impact on vegetative biomass24. Increased vegetative biomass and yield were reported in wheat following dampened activity of GWD in its endosperm25. In addition, no severe phenotypic changes and no effect on starch accumulation in the tubers were observed in GWD antisense potato lines18. Moreover, similar to the potato, cassava has an above-ground leaf source and an under-ground storage root sink, which results in long-distance downward carbon transport and partitioning between the leaves and storage roots26. Investigating its source strength, especially transient starch turnover, increases our understanding of the mechanisms that underlie the source-sink relationships in root crops. Further, the role of starch phosphorylation in starch metabolism needs to be determined in transient and storage starches of the cassava.In Arabidopsis, mutant plants that lacked GWD or PWD accumulated significant levels of excess starch and showed retarded growth. However, a recent study claimed that these enzymes exhibited a very low flux control coefficient with respect to starch synthesis and degradation in the leavesstorage root delay (srd) cassava mutant, which showed retarded storage root growth both in the greenhouse and the field. The mutation was traced to a T-DNA insertion in the eighth exon of α-glucan, water dikinase 1 gene (MeGWD1) in the cassava genome, which leads to reduced MeGWD1 production. Down-regulation of MeGWD1 expression in transgenic cassava by RNAi confirmed the starch excess phenotype in the leaves and retarded storage root growth. Consequently, physico-chemical properties of transient and storage starches and sugar content in GWD1-RNAi leaves and storage roots are also affected. We showed that the cassava GWD1 gene regulates transient starch morphogenesis and degradation in source and carbon partitioning by modulating starch phosphorylation.In the current study, we have identified the storage root delay (srd) mutant was identified after screening a cassava T-DNA population of more than 300 lines that were grown under the conditions of greenhouse. After three months of growth using in vitro shoot cultures as planting material, the srd plants showed normal foliage development but lacked storage roots  in one homologous chromosome, which was confirmed by PCR analysis using two sets of primer pairs  analysis  and the cDNA database of RIKEN27 (http://en.brc.riken.jp) using potato and Arabidopsis GWD1 protein sequences as queries. Their full open-reading frames (ORFs) were 4230 bp, 3687 bp and 3585 bp in length, respectively, and were cloned from cassava cDNAs. The MeGWD1, MeGWD2 and MeGWD3 proteins are made up of 1410, 1229, and 1195 amino acids, respectively . Further, MeGWD3 was predicted to have a single carbohydrate binding module 20 (CBM20), which was similar to the GWD3 of Arabidopsis and potato31 , cassava GWDs showed the lowest levels of expression as compared roots from other stages. A slight increase in MeGWD1 transcription was observed in M2 storage roots as compared that of M1 and M3 storage roots  were regenerated. The integration pattern of transgenes in these lines was assessed by Southern blot analysis using the digoxigenin-labelled hygromycin phosphor-transferase gene (HPT) probe after genomic DNA HindIII-digestion  in the leaves of G1i lines Fig. . A signiv/Fm) also confirmed the dramatic decrease in the photosynthetic rate of transgenic mature leaves , fewer median chains (DP 15–35) and slightly higher long chains (DP 37–48)  of transient starches of G1i transgenic lines was unchanged when compared to WT transient starch; however, the peak temperature (Tp) and conclusion temperature (Tc) increased slightly. Moreover, their enthalpy had increased to 11–13 J/g, which was much higher than that found in the WT  expression constitutively increased amylose content without altering the phenotype18. Silencing GWD1 expression by RNAi in monocots like maize or rice also led to an excess of leaf starch without negatively impacting biomass or growth rate24. Thus, our results provided direct evidence of the role of GWD1 in cassava storage root development by regulating transient starch turnover and carbohydrate partitioning.In leaves, starch phosphorylation is essential for normal starch metabolism, leading to increased sugar content in the light and decreased sugar content in the dark as confirmed in various plants like Arabidopsisves Fig. . Putativin vivo leaf starch degradation that is catalyzed by β-amylase45. In the cereal endosperm, ɑ-amylase initiates starch degradation10. In cassava, the rapid decrease in phosphorylation in amylolysis assay indicates that starch phosphorylation was necessary for both α- and β-amylases. However, starch degradation by β-amylase was greatly affected by the starch phosphorylation level, unlike α-amylase  was more than two-fold that of transient starch from cassava leaves. The significance of higher phosphorylation level of root starch still needs to be determined. HPAEC analysis also revealed a sharply reduced glucose-3-phosphate in the G1i transgenic lines, suggesting that reduced phosphorylation at C3 position was due to the absence of phosphoglucan substitute required for GWD3MeGWD1 function in the cassava, and especially linked source strength and storage root growth. Normally, transient starch is mobilized by synergistic interactions between GWD and β-amylase in dark phases of the day/light cycle, which provides source of carbon for leaf metabolism and sucrose synthesis that is then exported to storage roots to support storage root development. Carbohydrates that are imported from the source sustain expansion and starch accumulation in the cassava storage root. In its early stage of development, MeGWD1 might also mediate starch mobilization to support faster growth. When the storage roots reach maturity, cassava reduces the expression of MeGWD1 to facilitate storage starch granule formation. Overall, our study has revealed the role of GWD1 in root crops by affecting transient starch morphogenesis and a source/sink relationship that has not been previously reported.In summary, this study unraveled srd mutant was obtained from a batch of cassava T-DNA lines growing at SIPPE greenhouse, Shanghai, China. The T-DNA lines were transgenic cassava (cultivar TMS60444) that was integrated with T-DNA harboring a CaMV 35S::HPT expression cassette. After 3 months of growth, the potted plants were harvested to screen for the production of storage roots and subsequent molecular and physiological analysis. Their stem cuttings were planted in pots in the greenhouse for macropropagation and growth evaluation.The 55. The PCR products were purified, cloned into the pEASY- Blunt Simple vector  and sequenced.The T-DNA flanking region was isolated by inverse-nested PCR using the inverse primer designed from the 35S promoter and hygromycin sequences as described in Uchiyama and Watanabehttp://www.brc.riken.jp/inf/en/DB/) and cassava genome database (http://www.phytozome.net). The full length cDNAs of MeGWD1, MeGWD2 and MeGWD3 were cloned from a cassava cDNA library and the resulting sequences were submitted to the NCBI database . The GWD protein sequences from various plant species were collected from the NCBI database (cutoff e−4). Protein sequence alignment was performed with the ClustalX package and submitted to MEGA 5.0 software to generate a branched phylogenetic tree57. The neighbor-joining method with 1000 replicates of a bootstrap analysis was used to assess the statistical reliability of the tree topology.The sequence information of GWDs were derived from cassava full-length cDNA clones database  gene was used as probe for hybridization.The binary expression vector p35S::GWD1RNAi was constructed on the basis of the plasmid pRNAi-dsAC1in vitro and then transferred to pots in the greenhouse for macropropagation . For field evaluation, 10 stems per transgenic line and WT were planted in early May in the Wushe Plantation for Transgenic Crops, Shanghai, China , and harvested in early November. The performance of plants was phenotypically recorded.Transgenic lines (referred to as G1i for short) and wild type (WT) were propagated 2 for mRNA extraction. To quantify the expression of GWD genes, qRT-PCR was performed as described60. β-Actin was used as a reference for normalization. The primers were as follows:Leaves from at least three plants per line were harvested and then ground in liquid NMeGWD1 , MeGWD2 , MeGWD3 , and β-Actin .srd mutant was performed using the specific primers  located at 5′ end of insertion region. The GWD1 protein in cassava leaves was analyzed using Western blot with an antibody directed against MeGWD1. Total protein was extracted from leaves as described by Ritte et al.61. Protein was quantified according to the method of Bradford62. Approximately 60 μg of protein was loaded on an SDS-PAGE and immuno-blotting was performed with rabbit anti-MeGWD1 antiserum.The qRT-PCR analysis of GWD1 expression in Leaves and roots of six-month-old transgenic and WT cassava were harvested from three independent plants and then baked at 80 °C for two days to obtain a constant dry weight (DW). The same type of samples per plant line were mixed and ground together, respectively. The dried sample (30 mg) was dissolved in 0.7 ml of 80% ethanol, thoroughly vortexed and then incubated at 70 °C for 2 h. Aliquots of 0.7 ml HPLC-grade water and 0.7 ml chloroform were added to the sample. After shaking several times, the mixtures were centrifuged at 12000 g for 10 min. The pellet was collected for starch content analysis while the supernatant was composed of soluble sugar. The starch pellet was washed three times with 80% ethanol and total starch content was analyzed using total starch kit . The aqueous supernatant of 0.7 ml was transferred into a 1.5-ml micro-centrifuge tube and mixed with 0.7 ml chloroform. After centrifugation at 12000 g for 10 min, 0.5 ml of the supernatant was transferred to a glass tube for HPLC analysis of each sugar component. The sugar-separation method that was used was slightly modified from the original protocol described by the manufacturer; the Agilent technologies HPLC column  with a differential refraction detector was then used. The mobile phase consisted of 75% acetonitrile with a flow rate of 0.8 ml/min and the temperature of the column was maintained at 35 °C. The sugar types were identified based on retention time of the standards, and the sample concentration was calculated from the external standard curve. Data was calculated from three biological replicates.v/Fm), which represented the maximum quantum yield of PSII, using an imaging chlorophyll fluorimeter . The measurements were conducted at room temperature (25 °C) using standard saturated light mode. The actinic light intensity was 10 µmol m−2 s−1. The Fv/Fm value was analyzed using ImagingWin Software. Leaves from three independent plants per line were used and data was calculated from three biological replicates.Leaves were collected from the same position of three-month-old plants in the greenhouse. Prior to detection, leaves were adapted to darkness for 30 min. The photosynthetic activity was recorded via chlorophyll fluorescence determinations of the photochemical yield  were harvested form at least three cassava plants per line in field and homogenized in liquid nitrogen. Transient starch in the homogenate was extracted and purified using 95% percollThe α-amylase and β-amylase were purchased from Sigma . A 1% native starch suspension was prepared using 1.0 M sodium citrate (pH 5.6). One unit of amylase was defined as the amount of enzyme that liberated 1 mg reducing sugar as maltose per 5 min with soluble starch substrate at 40 °C. Ten units of amylase were added into 10 ml of a 1% starch suspension and incubated at 40 °C. Each 1 ml of suspension was collected at 2, 4, 8, 14, 24, 34, and 50 h and then centrifuged. The reducing sugar of the aqueous fraction was analyzed using the DNS method. Maltose was used as a standard curve. The pellet was washed three times using 80% ethanol. The remaining phosphate of starch granules was analyzed using HPAEC-PAD after acid hydrolysis. Data was calculated from three biological replicates.et al.61 using high performance anion exchange chromatography (HPAEC) that was equipped with a CarboPac PA1 column. Starch was hydrolyzed in 0.7 N HCl before determining the phosphorylation level. The chain length distribution of amylopectin was started by boiling 5 mg starch and enzymatic digestion with isoamylase . Oligosaccharides with polymerization degree 4–7  were used as a standard.The starch phosphorylation and chain length distribution were carried out following the protocol previously described by Ritte θ range of 5–60° at a rate of 4° min−1. The thermal properties of the starch samples were analyzed using a differential scanning calorimeter  at a temperature scanning range of 30–95 °C. The gelatinization temperature and enthalpy were determined using the Universal Analysis 2000 . The amylose content was measured using the colorimetric amylose content determination as described in Knutson and Grove63. Amylose type III and amylopectin  from potato were used to establish a standard curve.X-ray diffraction analysis of the extracted starch powder was performed using a D8 Advance Bruker X-ray diffractometer  and scanned through the 23) were coated with 1,3-diformal propane and subjected to ultra-thin sectioning. The sections were photographed under a transmission electron microscope .Isolated starch granules in distilled water were dropped and sprinkled on double-sided sticky tape, air-dried, and coated with gold powder. Samples were observed and photographed by scanning electron microscopy . Pieces of fresh mature leaves . An alpha value of P < 0.05 was considered statistically significant.Basically, the leaf and root samples were collected from three independent plants per line and then mixed together for further analyses. Data from at least three biological replicates were presented as mean ± SD. Analysis of variance (ANOVA) by Duncan’s multiple comparison tests or independent samples Student’s Supplementary Information


While North China is one of the earliest independent centers for cereal domestication in the world, the earliest stages of the long process of agricultural origins remain unclear. While only millets were eventually domesticated in early sedentary societies there, recent archaeobotanical evidence reported here indicates that grasses from the Paniceae (including millets) and Triticeae tribes were exploited together by foraging groups from the Last Glacial Maximum to the mid-Holocene. Here we explore how and why millets were selected for domestication while Triticeae were abandoned. We document the different exploitation and cultivation trajectories of the two tribes employing ancient starch data derived from nine archaeological sites dating from 25,000 to 5500 cal BP (LGM through mid-Holocene) in North China. With this diachronic overview, we can place the trajectories into the context of paleoclimatic reconstructions for this period. Entering the Holocene, climatic changes increased the yield stability, abundance, and availability of the wild progenitors of millets, with growing conditions increasingly favoring millets while becoming more unfavorable for grasses of the Triticeae tribe. We thus hypothesize that climate change played a critical role in the selection of millet species for domestication in North China, with early domestication evidenced by 8700 cal BP. While pioneering research focused on the domesticated millets, recent research on archaeological plant remains indicates more complex patterns of foraging before domestication, with other grasses, in addition to wild millet, likely exploited as food sources9. Research must now be directed toward the human choices that led to the favoring of wild millet’s cultivation and domestication as a food source.Unraveling the mechanisms behind the selection of particular plants for cultivation and domestication is key to understanding the origins of dry-land farming in North China, one of the earliest independent domestication centers in the worldca. 25,000 to 5500 cal BP   Fig. . Because14. The cereal domesticates were barley (Hordeum vulgare) and wheat (Triticum spp.), members of the tribe Triticeae15. For North China, the more limited archaeological evidence can be analyzed in reference to southwestern Asia models1. In order to model the process leading to the farming of domesticated millet, we treat the starch evidence from the nine archaeological sites as representative of distinct stages that led to eventual domestication. Our evidence shows the differing consumption of grasses in both the Triticeae and Paniceae tribes and reflects changing patterns in human-plant relationships8. The stages in this process include collecting wild cereals by mobile foraging groups; early cultivation of wild cereals possibly involving management and selection; further cultivation by less mobile/sedentary bands; cultivation of wild millets with planting and harvesting near permanent Neolithic villages; and the agricultural production of domesticated millet. In particular, we document the decreasing consumption of members of the Triticeae grasses and increase in Paniceae members—likely the wild progenitors of foxtail millet  and broomcorn millet (Panicum miliaceum)—as they come to be cultivated, farmed, and domesticated. Other terminology used for such stages in this evolutionary continuum, after Harris16, include “pre-domestication cultivation”, “semi-domestication”, and a later “full domestication”17. Although starch granules cannot be identified to the species, we can infer the wild or domesticated millet species are represented at the various sites based on identifications of rare finds of charred macro-remains of these millets9. The starch evidence raises a critical question: why did the exploitation of Triticeae and Paniceae grasses in North China ultimately result in the selection of millets as the crop to be domesticated for a dry-land farming system while the grasses of the Triticeae tribe were abandoned?The origins of agriculture are best understood today in southwestern Asia, where broad spectrum exploitation of edible plants was followed by the selection of a limited number of species for cultivation, with resultant domestication emerging during the early Neolithic period18. Researchers in East Asia, following models from southwestern Asia, have argued that shifting climatic conditions played an important role in the selection of cereals for domestication15. Finer resolution, local environmental proxies from pollen, loess, and speleothem records19 can provide reconstructions of climate fluctuations and environmental changes from the Late Pleistocene to the Holocene Optimum in which to place our data, allowing us to hypothesize possible mechanisms responsible for the divergent paths of the grass tribes, with millets becoming more intensively cultivated and the Triticeae grasses being abandoned.Documenting the process of millet domestication requires evidence for cultivation of the wild species within a region where wild cultivation and dry-land farming could be successful in producing the surplus needed to feed growing semi-sedentary to sedentary populations. The region had to feature the required growing conditions for millets. Foxtail millet has a growing season of 60–120 days while broomcorn millet’s is 45–100 days; each has certain heat requirements, such as cardinal temperatures for germination for broomcorn millet of 30–40 °C and for foxtail millet of 20–30 °C, but also, these cereals are relatively unable to withstand frost (compared to wheat or barley)20. We collected seven residue samples from the surface of a grinding slab recovered from Layer 5  at Locality 1 of Longwangchan 21 and recovered 60 starch granules. Of them, 46 starch granules, or 76.7% of the total assemblage , are identified as derived from grasses of the tribe Paniceae, and 9 starch granules (15%) of the tribe Triticeae (recovered from five samples)  and Shizitan (Shanxi), located on the Loess Plateau along the middle Yellow River valley Fig. . The ecos) Table .6. These samples include 20 starch granules  identified as derived from the Paniceae, and 45 starch granules , as Triticeae  are obtained from six samples from three grinding slabs dating 23,000–19,500 cal BP, from Layer 4 GS2, GS3), and Layer 3 (GS1), GS3, an23 features flake tools (without microblades), polished axes, more querns and mullers, clearly defined activity areas, pottery, domesticated dogs, and wild pig and chicken. At Donghulin  23, microblades are still present, but appear along with pit burials, pit-houses, and hearth pits.Following the Younger Dryas, new forms of sites appear in North China, represented by Nanzhuangtou (Hebei) and Donghulin (Beijing). Such sites, likely featuring more extensive wild cereal cultivation, could belong to less mobile or semi-sedentary groups. Nanzhuangtou 8, more than 1,000 starch granules were recovered from 18 samples collected at Nanzhuangtou and Donghulin. Paniceae dominate the starch assemblages , and are likely of the genera Setaria and Panicum, as each has been documented as undergoing cultivation at both sites5. Starches from Paniceae and Triticeae occur in all samples collected at Nanzhuangtou and those from the early occupational phase of Donghulin  contained large quantities of millet remains. All the finds demonstrate the ceremonial nature of this locality. The estimated 50,000 kg of stored millet shows that systematic farming was probably well established by this time25, with primarily Panicum miliaceum identified, while Setaria italica appears in late contexts in small quantities27. Panicum miliaceum is known to inhabit an arid ecological niche, while Setaria grasses tend to inhabit warm temperate, subtropical, and tropical areas, leading Bestel et al.28 to posit that Setaria grasses would not have been a weed of broomcorn millet. The 24 residue samples taken from three querns and seven mullers uncovered in the site yielded 10 starch grains (6.7%) from the Triticeae alongside 116 starch grains (77.9%) from (likely) millets.Sedentism marks a major turning point in the domestication process, as it gives rise to the earliest permanent villages and ceremonial centers and new requirements for local food production. The settlements feature planting and storage of wild millets and the earliest morphologically domesticated plants. This phase is represented by the North China Plain sites of Cishan  and Jiangjialiang . Cishan is an eight hectare site, culturally related to a number of village sites of the Cishan-Peiligang-Houli culture sphere on the lowland plains and dry grasslands of the middle and lower Yellow River catchment, perhaps dating ~8000–7500 cal BPThe Jiangjialiang excavations revealed nine square, semi-subterranean houses, marking it as a later settlement site than Cishan-Peiligang related sites with round houses. From 21 residue samples collected from three querns and two mullers found in a house, we recovered 13 starch grains from Triticeae alongside 104 starch grains from Paniceae (likely millets). The Paniceae starches make up 81.3% of the entire assemblage, and they are found in all samples at both sites, indicating widespread consumption. In contrast, the percentage of starches from the Triticeae is 10.2%. Triticeae are only found in 20–30% of the samples from both sites Tables  and S6. 25. We examined 12 residue samples collected from three slabs and three mullers. 290 starch granules of millets were identified in these samples making up 97% of the starch assemblage, but only four granules from Triticeae, 1.3% of the total, were recovered, from 3 samples , Luojiayingzi , and Sanjianfang —are generally located within a similar environment to the other sites, with millet cultivated here since the Xinglongwa Culture, ca. 8300 cal BP, with more domesticated millet macro-remains appearing by 7650 cal BPes Table .30.During the early Holocene, as Paniceae becomes the preferred species for cultivation, Triticeae cereals gradually fall out of favor. The Hongshan site starch data document the final decrease in the relative percentage of Triticeae by the mid-Holocene Fig. . PreservWhile previous starch studies in North China have focused on identifications at single sites, this study considers diachronic change in starch remains through the 20,000-year time period from the LGM to the Holocene Climatic Optimum. Our focus on cereals provides evidence of inter-related trajectories in the exploitation of Paniceae and Triticeae tribe members across five stages of human-plant interactions, from collecting wild cereals by mobile foragers to agricultural production.3. While preserved plant macro-remains are rare in earlier hunter-gatherer sites, remains from Neolithic settlements provide species-level identifications using wild vs. domesticated morphologies of millets; they also allow the recognition of broader spectra of exploited plant food resources at these sites than previously understood, including cereals, fruits, tubers, and especially nuts. Macro-remains are practically unknown for Late Pleistocene sites9, but based on Nanzhuangtou, Donghulin, and other sites, we would predict humans practicing both strategies of broad spectrum plant food consumption and of resource intensification.For Late Pleistocene sites, because macro-remains are poorly preserved, starch analysis often provides our only entryway to reconstructing plant exploitation patterns, in spite of small sample sizes and limited capability of only identifying to the tribe level. The small sample sizes, which are due to the ephemeral nature of site occupations and the complex issues of preservation and recovery, unfortunately, do not allow validation by statistical tests. The advantages of starch analysis, however, include the identification of plant use in the absence of macro-remains and the unique capability of enabling the identification of plants that were processed by specific tools, such as by grinding tools likely used in food processing, and thus can provide direct evidence of food consumptionWe posit that changes in the trajectories for the two exploited tribes, featuring the increasing preference for Paniceae (likely wild millets) leading to domestication for agricultural production with concomitant decreases in Triticeae consumption, are the result of a series of choices through time by Late Paleolithic and early Neolithic groups. These were in response to the increasing availability and productive yields of certain grasses that were resultant from changing global climate and local environments. Some changes in plant physiology and distribution were also the result of human agency, likely featuring selection and tending, management, and eventual cultivation. While detailed understanding of the dynamics of human-plant interactions must await further archaeological finds and studies, here we can begin by modeling the choices made in selecting millets over Triticeae, which we hypothesize were mostly influenced by changing environmental conditions in North China.31: these impact seasonal availability, abundance, and predictability of the plants, and observant foragers and early cultivators would have recognized such changes and perhaps promoted some through selection and management. To evaluate the role of climatic conditions in North China, we review the paleoclimatic sequence as known from recent proxies, including ice core, pollen, and speleothem profiles, and then place the archaeologically-known starch trajectories into their paleoenvironmental contexts.This environmental impact hypothesis for the selection of millets and their preference over Triticeae is supported by the known effects of climate on inherent physiological traits of the two tribes of grasses33, atmospheric CO2 concentrations were one-third lower (c. 180–235 ppmv) than early post-glacial levels  (c. 265–270 ppmv)34 )34 Fig. . With th41. Diversification of food resources and improved acquisition techniques specifically tailored to local environments, together with the onset of intentional cultivation of certain plants, become parts of viable food acquisition strategies, as is well-documented in southwestern Asia15. North China data, as well, indicate that similar measures for broadening the subsistence spectrum were adopted, as were new technologies such as microblades and grinding stones, and eventually pottery.During the Terminal Pleistocene, with amelioration, the global trend toward increasing population may apply in North China, as well13CTOC indicates that the area between latitudes 31°N to 40°N in the eastern North China Plain, where dry-land farming evolved, was originally covered by C3/C4 metabolic pathway mixed vegetation communities42. Although we cannot identify the species represented by the grass starches, it is known that Triticeae include early-maturing C3 grasses that poorly sustain frequent droughts43. As early maturing plants, humans could not rely on them for winter survival without using storage facilities, which are unknown for the Late Pleistocene. In contrast, grasses of the Paniceae tribe are warm-season and more drought-resistant C4 plants that mature in the fall and have a wider seasonal window for their growth, and thus could more successfully sustain climatic fluctuations during the Holocene45. Their ecological attributes make them available for human consumption during a longer period during the year, especially in the summer and fall seasons. Under any conditions, increasing reliance on cereals required the development of storage. The 8000–7500 cal BP Cishan deep storage pits demonstrate that this technique for conserving grain for winter season consumption and for security against bad seasons was already in place26. One can predict that older installations will be found in more Terminal Pleistocene sites such as Donghulin and Nanzhuangtou as archaeologists become more aware that such features can be present.A prerequisite for domestication is the availability of suitable plant resources in a given area. Analysis of surface soil δ 48 , archaeobotanical evidence indicates continuous intensive utilization of seed resources from both tribes. Although only the millets were selected for domesticationa56 Fig.  indicate58. This climatic pattern provided favorable conditions that likely increased the competitive advantages of Paniceae grasses and resulted in more productive and reliable yields, likely coupled with increasing areas of more highly tended fields as millet systematic agriculture is adopted, thus reducing available stands of Triticeae. At the same time, planting, harvesting, storage, and processing technologies improved, as new cooking and other cultural practices evolved that favored millet cereal consumption, all perhaps driving human selection of millets with higher yields that could then produce more than the “normal surplus” in order to have domestic storage to sustain farming communities during seasons of scarcity65, as well as for newly emerging ritual demands. Cishan and related sites support this hypothesis by the evidence mentioned, such as the 5-meter deep storage pits containing an estimated 50,000 kg of grain crops, as well as the great numbers of grinding stones , more elaboration in pottery vessel types and greatly increased amounts of pottery, and more polished stone axes and adzes (for land clearance), and polished stone sickles66. Millets proved to be a more competitive cereal, with their fast growth and early maturity, along with their drought resistance and tolerance for poor soils, thus requiring fewer human inputs for cultivation19. At the same time, rising winter temperatures during the Holocene Climatic Optimum could have been unfavorable for the growth of Triticeae grasses adapted to a relatively cold winter or spring for their development.As temperatures and precipitation increased with the Holocene Climatic Optimum after ~8000 cal BP, an intensified summer monsoon brought more rainfall to northern China during the warm seasonsca. 12,000 cal BP. These considerations very likely influenced the decisions of foragers, who then became cultivators, thus paving the road for millet domestication. The starch evidence presented here gives our first insights into cereal exploitation patterns from across the entire period of the lengthy domestication process, and these can serve to guide future research and the discovery of more macro-remains to support our hypothesis.In conclusion, we hypothesize that the interactions between the climatic fluctuations of the Terminal Pleistocene to early Holocene period and the particular physiological needs of grasses in both the Paniceae and Triticeae tribes resulted in their different relative availability, abundance, and yield stability after Ancient starches can be recovered from residues on the surfaces of tools, carbonized residues of potsherds, dental calculus, and cultural deposits. Here we selected just one type of lithic tool, the paired slab and muller Fig. , as thesTools from the sites of Longwangchan and Donghulin were sampled at the Key Laboratory of Land Surface Pattern and Simulation, Institute of Geographical Sciences and Natural Resources Research, Chinese Academy of Sciences. To allow for future studies of the same artifacts, one third of the length of each muller was sampled, as was one quarter of the slab.3. The recovered residue was mounted in 10% glycerine and 90% water on a slide and examined with both white and cross-polarized light at a magnification of 400×. Starch grains were counted, analyzed for morphological features, then recorded and compared with those from the modern reference collection.To dislodge adhering sediment and starch, tools were shaken in an ultrasonic water bath for 5 to 10 minutes, and starch was then isolated using heavy liquid flotation with a solution of CsCl at a density of 1.8 g/cmThe lithic tools from the sites of Nanzhuangtou, Cishan, and Jiangjialiang were sampled at the storage rooms of the Heibei Provincial Institute of Archaeology and Cultural Relics in Shijiazhuang, Hebei, and the tools from the sites of Jian’gou, Luojiayingzi and Sanjianfang were sampled at the storage rooms of the Ongniud Banner Museum, Inner Mongolia. Tools selected for sampling were initially cleaned by brush to remove adhering dust from storage and then washed clean with ultra-pure water. Cavities on the surface of the tools were then targeted for residue removal. 20–40 microlitres of ultra-pure water were applied to the areas of interest and left there to hydrate for 3–5 minutes. The wetted area was agitated with a metal pin to dislodge the sediment within the cavities. Finally, a sample of this material was removed with a micropipette and transferred to a clean, new, snap-cap vial for storage. These samples were processed in the laboratory where starches were floated by CsCl according to the methods described above.67.As a control for the presence of starch, during the previous studies, sediment samples were collected from the dust in the storeroom where the artifacts were curated, the surface soil at the sites, the sediments from the overlying layer and underlayer of the cultural deposits from which the artifacts were recovered, and the cultural deposits themselves. The samples were found to either lack starch granules completely or to have a density of starch that was much lower than the residues on the tools, indicating that the ancient starches were related to tool usehttp://cmsgd.igsnrr.ac.cn). All modern reference specimens were collected by the authors from botanical gardens and during field investigations. Starch grain identifications were based upon one-on-one comparisons between ancient starches and those derived from the modern reference collection.We have compiled a modern reference collection of over 200 starch-producing species from 20 families that are common in China . The morphological features of seed starches from the tribe Paniceae are typically, but not always, polyhedral , and some species occasionally contained spherical grains. The hila of starch grains from the Paniceae are centric with occasional fissures. The sizes range from a few microns to more than 20 microns in maximum length.Currently, the Tribe Triticeae includes 13 genera (one introduced) and 175 species  that have been identified in China; the Tribe Paniceae has 27 genera  and 145 species  that occur in China . The mean size of starch grains from these tribes often fall into the range of 20–30 microns.Starch grains from seeds of the Triticeae occur in bimodal populations of small, non-diagnostic forms and large, diagnostic types that are lenticular in morphology, with a side view that is oval with both ends attenuated. The equatorial groove, a remnant of the formation process, is frequently visible in the side view, and small indentations occur on the surface of a portion of the large grains in a population Fig. . The meaSupplementary information


Sudden cardiac arrest or death (SCA/SCD) in athletes has a low event rate. Pre-participation or eligibility screening is a widely accepted method of primary prevention of SCA/SCD in athletes. Most European countries and international sports governing bodies perform ECG-inclusive screening. However, implementation of a resting 12-lead ECG in pre-participation or eligibility cardiac screening is still a topic of debate. Recently, the ‘International recommendations for electrocardiographic interpretation in athletes’ was published in three leading international medical journals. These international ECG criteria are based on studies with detailed information on resting 12-lead ECG of Caucasian and Afro-Caribbean athletes or on consensus in case evidence was lacking. Normal, borderline and abnormal ECG findings in young athletes (age 12–35 years) are clearly described and illustrated to assist the screening physician in interpreting ECGs of athletes correctly.In this ‘point of view paper’ we will discuss whether these new ECG criteria actually help prevent SCA/SCD in athletes. Sudden cardiac arrest and/or death (SCA/SCD) in athletes is a very tragic event that attracts a lot of media attention. The key question conventionally raised after such an event resulting from lethal ventricular arrhythmia, i. e. ventricular tachycardia/fibrillation (VT/VF), is: could this have been prevented? SCD in competitive athletes aged 35 years and younger (young athletes) is rare . The incidence is considerably lower than in the overall population  and significantly lower than the incidence of VT/VF in Europe  . HoweverIn this manuscript we discuss the ‘International recommendations for electrocardiographic interpretation in athletes’, questioning if these new ECG criteria help preventing SCA/SCD in athletes –14.The purpose of pre-participation or eligibility cardiac screening in athletes is identifying CVD at risk of VT/VF and reducing SCA/SCD by disease management –10. Pre-In 2010, the European Society of Cardiology (ESC) for the first time classified athlete ECGs in training-related and training-unrelated or pathologic ECG findings . TraininThe Seattle 2013 criteria were based on ECG data in both Caucasian and Afro-Caribbean athletes , 24. ‘CoThe so-called ‘borderline ECG findings’ were introduced by Sheikh et al. because the issue of the resemblance of an athlete’s ECG and pathology had not been solved . The RefResearch, in particular that of Sharma et al. , conducted to describe in detail ECGs of athletes of different ethnicity, gender and intensity of sports participation has been of great value for the consensus of the international ECG criteria , 26, 27.The international consensus ECG criteria describe and illustrate each separate ECG finding at rest in young athletes –14. The Normal or training-related ECG findings are induced by long-term sports participation on a regular basis for at least four hours per week [per week –14. As iBorderline ECG findings are left atrial and right atrial enlargement, electrical axis deviation and complete right bundle branch block (RBBB). When found in isolation in asymptomatic athletes with a negative family history of inherited CVD or SCA/SCD these findings are classified as normal and need no further cardiac evaluation  recommending additional cardiac evaluation before clearing the athlete to participate in sports  and false negative rate (FNR) of pre-participation or eligibility cardiac screening remain a problem. On the one hand, false positive test results wrongly identify athletes at risk of VT/VF, lead to unnecessary additional cardiac evaluation at high costs and result in uncertainty about the athlete’s career , 28. On ECG-inclusive pre-participation cardiac screening to prevent SCA/SCD in athletes is implemented by most European countries and international sports governing bodies. The international ECG criteria, endorsed by international cardiac and sports medical societies and sports governing bodies, pose an updated and clear guide in interpreting ECGs of athletes to appropriately identify abnormal ECG findings at rest suspicious of CVD at risk of VT/VF. The screening physician must be trained in interpreting ECGs of athletes following the most recent ECG criteria to avoid wrong decision-making. Further studies are needed to determine if these updated international ECG criteria help to prevent SCA/SCD in athletes (12–35 years of age).


As an official nonprofit publication of the Bauru School of Dentistry (FOB), University of São Paulo (USP), this achievement deserves our respect and acknowledgements to all the authors that chose our journal to publish their contributions in the areas of Dentistry and Speech-Language Pathology and Audiology as well as related areas. The anonymous work of the reviewers and associate editors was also instrumental for the quality of the works published in the JAOS, which was translated into the indexation of the journal in internationally respected databases such as PubMed/MEDLINE, PubMed CentralGreat part of JAOS's success results from a dedicated work of all previous editors including those that started the former title “Revista da Faculdade de Odontologia de Bauru”. Therefore, this is the perfect time to acknowledge them and remember their names: Aguinaldo Campos Junior, Rumio Taga, José Mauro Granjeiro, Ricardo Marins de Carvalho, Carlos Ferreira Santos and Gustavo Pompermaier Garlet. We assure these colleagues that we are doing our best to keep the invaluable work they devoted to our journal.Once again we thank all the employees involved in the JAOS's publication: Valéria Cristina Trindade Ferraz, José Roberto Plácido Amadei, Deborah Schmidt Capella Junqueira, Sônia Cláudia Antonelli Pirola, Neimar Vitor Pavarini, Rubens Kazuo Kato, Allan Rodrigo Dias, Camila Medina and Zelma Batista Borges.Finally, we express our gratitude for the financial support provided over the last 25 years by the Rectory of USP, the Deans of FOB, the National Council for Scientific and Technological Development (CNPq), the Coordination for the Improvement of Higher Education Personnel (CAPES) and the State of São Paulo Research Foundation (FAPESP).Editor-in-ChiefVanessa Soares LaraCo-Editor-in-ChiefKarin Hermana Neppelenbroek


Current methods for detection and diagnosis of allergies do not provide epitope specific immunogenic information and hence lack critical information that could aid in the prediction of clinical responses. To address this issue, we developed a nanoparticle based platform, called nanoallergens that enable multivalent display of potential allergy epitopes for determining the immunogenicity of each IgE binding epitope. By synthesizing nanoallergens that present various epitopes from the major peanut allergen, Ara h2, we directly determined the immunogenicity of each epitope, alone and in combination with other epitopes, using patient sera. This information provided insights on which epitopes are most critical for physiological responses to Ara h2 and revealed the importance of both high and low affinity epitopes for allergic responses. We anticipate the nanoallergen platform to be used to provide information regarding allergic reactions and therefore potentially aid in more accurate diagnosis and design of personalized treatment options. Consequently, food allergies are a rapidly growing problem in developed nations, resulting in a dire need for more accurate and information-rich diagnostic testing and therapeutic options4. Effective allergy tests should provide a measure of how strongly an allergen stimulates a cellular immune response (immunogenicity) and how the intensity of this cellular response correlates to physiological symptoms. Allergy tests can be divided into two broad categories: (1) clinical allergy testing such as the skin prick test (SPT) which uses allergens to stimulate a controlled response on a patient and (2) in vitro assays that evaluate the level of allergen or epitope specific IgE (sIgE) antibodies in patients’ sera. The SPT test provides qualitative measurement for the intensity of the local degranulation, but has poor specificity when predicting the severity of physiological responses to allergen and cannot specifically test individual allergen epitopes. Recently, microarrays have been used to identify food allergen sIgEs in clinical samples not only to a specific allergen protein, but also to the allergen epitopes by generating linear peptides as epitope mimetics9. This information, while useful in evaluating the binding of major IgE epitopes, does not present any information regarding degranulation responses or how important these epitopes are for overall physiological reactions to allergens. This is because binding affinity can only provide a relative measure of immunogenicity and does not directly correlate, given the complexities of multivalent binding10. Immunogenicity also depends upon multivalent interactions between multiple IgEs and epitopes as well as intracellular regulatory responses and underestimates the contributions of moderate to low affinity epitopes12. Given these limitations, novel methods that can directly predict the clinical and physiological severity of an allergic reactionwould greatly improve both the current understanding of allergen immunogenicity.Food allergies are a type I hypersensitivity  to proteins within certain foods that can result in symptoms ranging from harmless skin irritation to a life-threatening anaphylactic reaction. Type I hypersensitivity is caused when an allergen binds multivalently to allergen specific IgEs bound to Fc epsilon receptors (FcεRIs) present on the surface of mast cells or basophils, causing receptor clustering and culminating in degranulation. While only 3% percent of adults suffer from food allergies, 8% percent of children under four years of age have a food allergy13. Liposomal nanoparticles have been used for many decades as drug delivery vehicles and for active targeting of diseased cells15. In our recent studies, we have developed a novel method for precise loading and efficient display of peptides on a liposomal surface. This method, combined with the nanoallergen platform enables a reliable means to incorporate one or more allergen epitopes on the same nanoparticle surface with a high degree of control over particle size and epitope stoichiometry18. As a result, nanoallergens can efficiently display potential IgE binding epitopes in a multivalent fashion and provide a novel and practical experimental model system to dissect epitope immunogenicity. In the past, liposomes have been employed as carriers for allergen proteins, either to aid sensitization or to prevent allergic reactions from pre-sensitized mice20. Nevertheless, liposomes have never been used to present individual epitopes from allergen proteins to trigger allergic reactions. In this manuscript, we introduce nanoallergens as a versatile platform to directly determine individual epitope immunogenicity for a particular patient by systematically evaluating the epitopes of the major peanut allergen, Ara h2, using clinical samples. This epitope specific information would provide a clearer picture of allergen potency, allowing more precise clinical diagnoses and potentially invaluable information about which epitopes future therapeutics should target.In order to address the weakness of current allergy testing methods, this manuscript describes a novel nanoparticle platform, called nanoallergens, that can individually determine the immunogenicity of each IgE binding epitope of an allergen21. A thorough evaluation of current literature on Ara h2 IgE binding epitopes revealed eight ubiquitously cited epitopes  response observed by the nanoallergens and the concentration of nanoallergens that induced half maximal of the degranulation response (EC50) .In order to evaluate degranulation using human patient sera, we modified the well-established rat basophilic leukemia (RBL) degranulation assay by using an engineered version of the RBL cell line, RBL-SX38, which was transfected to express the human FcεRI on their surface. We primed these RBL-SX38 cells using sera from a patient with peanut allergies (serum 1) and induced crosslinking using the synthesized nanoallergens. This serum was purchased commercially  and is taken from a patient with a history of peanut allergy reactions and with a high peanut specific IgE level Table . In our 25. We also observed that nanoallergens activated two intracellular pathways of RBL-SX38 cells in a similar manner as the natural Ara h2 allergen protein using western blot. Both the nanoallergen and Ara h2 protein induced phosphorylation of phosphatidylinositol-3, 4, 5-trisphosphate 5-phosphatase 1 (SHIP), an intracellular hydrolase commonly activated during antigen stimulation in degranulation responses, indicating that both by the nanoallergen and Ara h2 activate IgE mediated signaling pathways  of epitope 2 demonstrated significant degranulation response in the RBL-SX38 cells Fig. . We obseons Fig. . Furtherons Fig. . This is31. Importantly, the nanoallergens presenting epitope 2 were able to trigger significant degranulation at only 1 pM concentration, indicating it is more immunogenic than other epitopes . We synthesized nanoallergens that presented epitope 2 and 3 at various ratios  on the surface of the particle while keeping the total percentage of epitopes at 2%. When the lower immunogenic epitope 3 was introduced at a 0.1% ratio and epitope 2 decreased to 1.9% from 2%, we observed a significant decrease in the degranulation EC50 value, contrary to expectations given that there were fewer high immunogenic epitopes  in RBL-SX38 cells stimulated with nanoallergens and compared this data to EC50 values for degranulation responses. As demonstrated in Fig. max value, such as nanoallergens presenting epitope 2 and 3. This suggests an increase in pSHIP for certain epitope combinations, tempering the Dmax value, but not the EC50 value of the degranulation response. While further evaluation would be necessary to confirm this, this result suggests that an ideal composition of both high and low epitopes on antigens are necessary for triggering high degranulation responses, which avoids relegation by intracellular regulatory cascades. More importantly, this experiment demonstrates the proof of principle that nanoallergens can be used to elucidate the complex mechanistic aspects of degranulation.One of the most interesting results that emerged from the studies performed with combination nanoparticles was a significant drop in maximum degranulation response despite lower ECd 3 Fig. . We hypo50 < 2000 pM) and low responses to epitope 7, and no responses to epitope 8  demonstrated highly immunogenic responses to Ara h 6 epitope 3, similar to the EC50 values seen from Ara h2 epitope 2 with these patients , which would likely increase the size of potential FcεRI clusters, making nanoallergens intrinsically more immunogenic than natural allergens, therefore making direct comparisons difficult.Nanoallergens can also be used to study mechanistic aspects of degranulation. One of the key findings of this study is that nanoallergens that displayed a combination of high and low immunogenic epitopes can trigger more degranulation at a hundred-fold lower concentration than the nanoallergen displaying high immunogenic epitope alone for one of the patient samples, despite the reduction in the density of the high immunogenic epitope on particle surface. This finding is very significant as it demonstrates that the often-overlooked lower affinity epitopes can drastically alter overall immunogenicity and potential clinical responses of allergy proteins. We speculate this increase in immunogenicity as owing to the increased avidity of the nanoallergen presenting high and low immunogenic epitopes. Avidity of a multivalent entity depends on both the monovalent affinities of the moieties as well as the total number of binding interactions, hence the valency of the epitopes on nanoallergens as well as of the IgEs on cell surface carry significance. Since, immunogenicity depends upon both ECens Fig. 39. We coDespite these limitations, this platform provides quantitative measures to evaluate the immunogenicity of any individual epitope for a specific patient by isolating discrete epitopes and directly correlating them to a biological response. Our nanoallergen platform enabled the establishment of patient immunogenic profiles. This type of analysis was not possible with the currently existing methods and provides an invaluable tool that might aid diagnostics or future therapy options. As the study demonstrates, nanoallergens are also very adaptable and can be applied to any known allergen protein and could potentially be used to identify and evaluate new IgE binding epitopes. Hence, this study presents nanoallergens and established the proof of principle that this platform can elucidate factors determining allergen immunogenicity for individual epitopes.41. Therefore, a nanoallergen defined immunogenic profile could potentially be used to more accurately predict the severity of an individual’s prospective reaction to an allergen and provide an advantage over current diagnostic allergy tests. While any diagnostic value for nanoallergens will require further clinical evaluation with large patient populations, this initial study is important for characterizing the potential scientific and diagnostic features nanoallergens offer. Taken together, this study introduces nanoallergens as a versatile platform for studying allergic reactions and for providing clinically useful patient specific information regarding allergen epitopes that could improve diagnosis, enable targeted designs for future therapeutics and allow for personalized treatment options for patients.Overall, the results of the present study emphasize the significance of nanoallergen platform as an advanced and physiologically relevant method for detailed characterization of allergen epitopes and degranulation responses in allergy research. Furthermore, we demonstrate how nanoallergens can be used to sketch out the immunogenic profile for a patient by presenting allergen epitopes separately as well as in combinations to elucidate their immunogenicities. The direct evaluation of epitope immunogenicity through tracking degranulation responses gives the nanoallergen platform a marked advantage over multi-epitope ELISA microarrays currently used to evaluate IgE binding epitopes. Nanoallergens can also take advantage of this microarray format, where nanoallergens could be used to display a library of potential epitopes, and degranulation assays or other immunological assays could provide the readout. Furthermore, it has been demonstrated in literature that immunoreactivity to certain epitopes is correlated with severe physiological reactions in a patientH-benzotriazol-1-yl)-1,1,3,3 tetramethyluroniumhexafluorophosphate], all Fmoc conjugated amino acids and BSA (Bovine Serum Albumin) from EMD Biosciences. DIEA , TFA (trifluoroacetic acid), Triisopropylsilane (TIS), hydrazine, Cholesterol, Dichloromethane, 2-proponol, ACN(acetonitrile), ethanol, all Kaiser test reagents, G418 salt and Bovine serum albumin  (>99.8%), chloroform, penicillin, L-glutamine and Eagle’s Minimum Essential Media from Thermo Fisher. DSPC , DSPE-mPEG2000 -2000] ), membranes and all mini extruder components were purchased from Avanti Polar Lipids . Fmoc-EG6-OH was purchased from Quanta Biodesign. DiD fluorescent dye -1,3-pentadienyl)-3,3-dimethyl-1-octadecyl-, perchlorate) and fluorescein 5(6) isothiocyanate was purchased from Invitrogen. Western Blot reagents, gels and equipment were obtained from Bio-Rad. Goat anti-rabbit IgG (ab6721), and anti-human IgE (ab99834) were purchased from Abcam. RIPA buffer and phosphatase inhibitor-I was purchased from Boston Bioproducts (Boston). Anti-rabbit-HRP IgG (cat# 111-035-003) was purchased from Jackson ImmunoResearch. All human serum samples were purchased from PlasmaLab International . Anti-SHIP conjugated beads (sc-8425 AC), Anti-SHIP antibodies (sc-8425) and anti phospho-tyrosine (p-tyr) antibodies (PY99) were purchased from Santa Cruz Biotechnologies. Tyrodes Buffer was made as previously described12.We purchased NovaPEG Rink Amide resin, HBTU [2- linker of exact repeating units of EG for a desired length, and two hydrocarbon (C16) tails to facilitate the molecule’s anchoring into lipid bilayer of the liposome (Table 6 spacers) and three lysines provide the optimal features for epitope presentation  of peptide-linker-lipid conjugate, 5% polyethylene glycol 2000-lipid conjugate to inhibit non-specific interactions, and the remaining mole percent of the molecules in liposomes was a bulk phospholipid (DSPC). Unless otherwise stated, the nanoallergens in this study typically included a 2% peptide-linker-lipid conjugate, and were 100 nm in diameter. Briefly, DSPC, mPEG-2000-DSPC, cholesterol, and peptide-linker-lipid conjugates were dissolved in chloroform, lyophilized for 30 minutes following rotate evaporation, rehydrated in PBS at 60 °C, and then extruded through a 100 nm polycarbonate filter (Avanti).Liposomal nanoallergens were prepared using a procedure as previously describedThe size of liposomes were confirmed using DLS analysis via the 90 Plus nanoparticle size analyzer , using 658 nm light observed at a fixed angle of 90° at 20 °C.42. RBL-2H3 cells were cultured in same media, however without the addition of G418.RBL-2H3 cells were purchased from ATCC. RBL-SX38 cells were a generous gift from Dr. Jean-Pierre Kinet from Harvard University. RBL-SX38 were cultured in Minimum Essential Media (Gibco) with 10% fetal bovine serum  and 1.2 mg/mL of G418 salt (Sigma) as previously described43. RBL-SX38 cells were plated into 96 well dishes for 24 hours and then incubated with 10% of human sera in cell culture media for an additional 24 hours prior to the degranulation assay.Degranulation assays were performed as previously described using nanoallergens as the allergen12. Degranulation maximum (Dmax) and EC50 values were calculated using Origin 7 software using Hill’s curve fit for sigmoidal curves using percent degranulation values taken from experiments.Percent degranulation was calculated as previously decribedA high binding 96 well plate was incubated with anti-Human IgE in Carbonate-Bicarbonate buffer (Sigma) at a concentration of 3 nM for 16 hours at 4 °C. Plate was washed with washing buffer (PBS with 0.5% tween 20) and then blocked with blocking buffer (5% BSA in PBS with 0.1% tween 20) for 1 hr at room temperature. Plate was washed with automatic plate washer (AquaMax 2000), then varying concentrations of either Ara h2-Biotin (Indoors Biotechnologies) or FITC-peptide 2 conjugate in blocking buffer for 1 hour. Plate was washed again and then either streptavidin-HRP (1:5 000 dilution) or anti-FITC HRP IgG (1:5000 dilution) was added in blocking buffer. After another washing step, Amplex Red substrate was added (Invitrogen) and plate fluorescence was read at 5 minute intervals according to manufacturer’s instructions.35. RBL-SX38 cells were plated at approximately 0.5 × 106 cells per mL into a 3 mL dish for 24 hours followed by incubation in 10% patient serum in cell culture media for another 24 hours. Cells were then washed with Tyrodes buffer and incubated with Tyrodes for 30 minutes at 37 °C. Ara h2 or nanoallergens were then added at varying concentrations and incubated at 37 °C for 3 minutes. Cells were washed with ice cold PBS, then incubated in RIPA with phosphatase inhibitor lysis buffer, scrapped and sonicated for one minute intervals over the course of 30 minutes on ice. Lysates were then spun down at 15,000 RPM for 10 minutes and their protein concentration determined by Bradford Assay. Laemmli buffer and PBS were added to all lysates so that their protein concentration was 0.5 mg/mL or would be 0.5 mg/mL prior to immunoprecipitation. Samples were boiled for 5 minutes; centrifuged and 20 uL of each was added to a 10% SDS-PAGE gel. Samples ran on the gel for 1 hr, were transferred to nitrocellulose paper for 1.5 hrs, and blocked with 5% BSA in tris buffered saline with 0.1% tween 20 (TBS-T)for 1 hr. Primary antibodies were added at the manufacturer’s suggested dilutions in blocking buffer (either 2000X or 500X), washed with TBS-T, then appropriate secondary antibodies with HRP conjugates were added according to manufacturer’s dilutions . The membranes were washed, and incubated in with Clarity™ Western ECL Blotting Substrate (Bio-Rad) for 5 minutes. Bands were exposed onto Kodak Chemiluminescence Film for times ranging from 1–30 minutes.Stimulated RBL cells were analyzed for intracellular activity with a western blotting technique previously described for expression of p-SHIPAll error bars represent the standard deviation of three independent replicates of cells. All p values were calculated using a one tailed student’s t test.All human samples used in this study were purchased from a third party vendor using anonymous donors.All data referenced in this study is included in the manuscript or supplemental file. All supplemental data is available online.Supplementary Information


G-protein-coupled receptor (GPCR)-related proteins are dysregulated and the GPCR CC-chemokine receptor 10 (CCR10) is significantly upregulated in inflammation-driven HCC. However, CCR10′s role in inflammation-driven hepatocarcinogenesis remains unknown. The aim of this study was to evaluate the role of CCR10 in inflammation-driven hepatocarcinogenesis. Via a targeted gene expression microarray screening alterations in GPCR family gene expression, we found CCR10 to be significantly upregulated in hepatocytes isolated from inflammation-driven human HCC tumors and matching paracancerous tissues. Tetrachloromethane (CCl4)-induced and diethylnitrosamine (DEN)-induced murine models of inflammatory hepatocarcinogenesis displayed significant hepatocellular TNF and CCR10 upregulation. Exogenous TNF applied to HepG2 and LO2 cell lines as well as wild-type (WT) mice significantly upregulated hepatocellular CCR10 expression, Akt phosphorylation, PCNA expression, and hepatocellular proliferation. Additionally, exogenous TNF significantly upregulated secretion of the natural CCR10 ligand-agonist CCL28 from both cell lines. Transgenic CCR10-knockout (CCR10 KO) in DEN-treated mice significantly increased hepatocellular apoptosis levels and significantly lowered compensatory hepatocellular proliferation but did not affect upstream TNF expression. In addition, DEN-treated CCR10 KO mice showed a significantly lower liver weight/body weight ratio, significantly lower liver tumor incidence, and significantly smaller tumors. Moreover, exogenous CCR10 expression significantly raised xenograft tumor growth in Balb/c nude mice. In vitro, CCR10 transfection or CCL28 treatment in HepG2 and LO2 cell lines significantly increased Akt phosphorylation, PCNA expression, and cell proliferation, while CCR10 silencing or Akt inhibition produced the opposite effects. In vivo, hepatocytes isolated from HCC tumor tissue and matching paracancerous tissue in DEN-treated CCR10 KO mice showed significantly lower Akt phosphorylation and PCNA expression relative to WT hepatocytes. In conclusion, inflammation-induced TNF promotes hepatocellular CCR10 expression and downstream PI3K/Akt-mediated hepatocarcinogenesis. CCR10 appears to function as a linkage between TNF stimulation and downstream PI3K/Akt pathway activation and shows promise as a potential therapeutic target for inflammation-driven HCC. Hepatocarcinogenesis displays strong linkages with underlying chronic inflammatory processes in liver parenchyma, such as chronic viral infection , hepatitis C virus), non-alcoholic fatty liver disease (NAFLD), alcoholism, and aflatoxin exposure2. These chronic inflammatory processes are initiated by Kupffer cell activation, resulting in secretion of pro-inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-1β) and interleukin-6 (IL-6)3. These pro-inflammatory cytokines create the microenvironment for hepatocarcinogenesis through promoting pre-malignant cell proliferation, malignant tumor growth, angiogenesis, and metastasis3.Hepatocellular carcinoma (HCC), the most common liver cancer, is a leading cause of cancer mortality globally4. GPCRs are the largest superfamily of cell-surface receptor complexes and uniformly consist of a seven transmembrane-helical receptor domain, a trimeric αβγ G-protein, and an effector molecule that transduces the receptor-initiated signal into the cytoplasm5. Various GPCRs are upregulated in HCC as well as other human cancers5, yet the exact mechanism(s) by which these GPCR-related changes promote inflammation-driven hepatocarcinogenesis remain unclear6.Although the precise mechanism(s) by which TNF promotes hepatocarcinogenesis still remains unclear, several G-protein-coupled receptors (GPCRs) have been linked to regulating TNF-driven inflammatory processes7. Yang et al.’s recent work revealed that one CC-chemokine receptor in particular, CC-chemokine receptor 10 , is significantly upregulated in inflammation-driven HCC tumors. However, no study to date has examined the role that CCR10 may play in inflammation-driven HCC. Therefore, the aim of this study will be to evaluate the role of CCR10 in inflammation-driven hepatocarcinogenesis.One type of GPCR, CC-chemokine receptors, are upregulated in various kinds of cancer cells in reaction to pro-inflammatory cytokinesThe clinical protocols of this study were approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University  and the Ethics Committee of Renji Hospital . Written informed consent was obtained from each participant prior to inclusion in this study. All animal procedures were approved by the Animal Care and Use Committee at Renji Hospital (Shanghai Jiao Tong University School of Medicine).HBV-infected HCC patients and hepatic hemangioma patients were recruited for this study. The inclusion criteria for HCC patients were: (i) aged 18–75 years, (ii) hepatitis B surface antigen-positive, or HBsAg-negative and HBV DNA-positive, (iii) Child-Pugh score of 5–6, (iv) presence of at least one resectable hepatic tumor, and (v) definitive HCC diagnosis based on postoperative histopathological analysis. The exclusion criteria for HCC patients were: (i) history of antiviral, radiotherapy, or transcatheter arterial chemoembolization (TACE) therapy; (ii) infection with hepatitis C, hepatitis D, or human immunodeficiency viruses; (iii) presence of other malignant tumors, (iv) presence of extra-hepatic metastases; (v) history of drug abuse, (vi) pregnancy; or (vii) presence of immunocompromising disease.The inclusion criteria for hepatic hemangioma patients were: (i) aged 18–75 years, (ii) Child-Pugh score of 5–6, (iii) presence of at least one resectable hepatic tumor, and (iv) hepatic hemangioma diagnosis based on postoperative histopathological analysis. The exclusion criteria for hepatic hemangioma patients were: (i) history of antiviral, radiotherapy, or TACE therapy; (ii) infection with hepatitis B, hepatitis C, hepatitis D, or human immunodeficiency viruses; (iii) presence of cirrhosis or hepatitis, (iv) presence of other tumors, (v) history of drug abuse, (vi) pregnancy; or (vii) presence of immunocompromising disease.n = 81). Healthy liver tissue specimens were surgically extracted from hepatic hemangioma patients for use as normal controls (n = 28). Demographic and clinical characteristics are detailed in Supplementary Table Paired liver tissue specimens from HCC tumors and paracancerous tissues were surgically extracted from HBV-infected HCC participants n = 8. Healthy8. Human CCL28 is a natural ligand-agonist for human CCR109. A6730 produces a conformational change in Akt that prevents it from associating with upstream kinases, thereby abrogating Akt phosphorylation10.HepG2, Hep3B, and LO2 cell lines were purchased from the Shanghai Institute of Cell Biology . Cell lines were tested for mycoplasma contamination prior to culture. HepG2 and Hep3B cells were cultured in Dulbecco’s Modified Eagle’s Medium, while LO2 cells were cultured in RPMI-1640 medium plus with 10% fetal bovine serum . For some in vitro experiments, HepG2 and LO2 cell lines were pre-treated with the pro-inflammatory cytokine TNF  for 4 h, recombinant human CC chemokine ligand 28 (CCL28)  for 2 h, or the allosteric Akt inhibitor A6730  for 2 h11. Briefly, a targeting construct consisting of a 3.6-kb fragment exactly 5′ of the CCR10 start codon (the 5′ arm), followed by the EGFP coding sequence, followed by the loxP-flanked neo cassette, and followed by a 0.5-kb fragment containing a 0.2-kb 3′ portion of CCR10 as well as a 0.3-kb 3′ noncoding region (the 3′ arm). The 3.6-kb 5′ arm and the 0.5-kb 3′ arm were amplified by PCR from murine genomic DNA and sequence confirmed. Then, the linearized targeting construct was transfected into the J1 murine embryonic stem cell line, of which G418/gancyclovir-resistant clones were PCR-screened for KO recombinants with a primer set amplifying a 0.9-kb fragment from the 3′ end of CCR10 . CCR10+/+ wild-type (WT) and CCR10−/− knockout (CCR10 KO) litter mates on a C57BL/6 background were purchased from Shanghai Genechem Company Ltd. . Construction of CCR10 KO mice was performed according to Jin et al.’s well-established protocol . All mice were held in pathogen-free, micro-isolator cages on a 12 h/12 h light/dark cycle. All mice were fed standard chow and water ad libitum.Total RNA from isolated hepatocytes was extracted with a TRIzol reagent  and then purified using a mirVana miRNA Isolation Kit . RNA purities and concentrations were spectrophotometrically assessed using OD260/280 absorbance readings from a NanoDrop ND-1000 . Fluorescently-labeled cDNA (Cy3-dCTP) was synthesized with Eberwine’s linear RNA amplification method using a cRNA Amplification and Labelling Kit  followed by enzymatic reaction. Double-stranded cDNA (dsDNA) was synthesized and then purified with a NucleoSpin Extract II Kit . The resulting dsDNA was eluted in elution buffer , vacuum-evaporated to a total volume of 16 μl, and in vitro transcribed with a T7 Enzyme Mix  in a total reaction volume of 40 μl at 37 °C for 14 h. CbcScript II reverse transcriptase was used for reverse transcription followed by Klenow enzyme labeling.A gene microarray was performed with a SurePrint G3 Human Gene Expression 8 × 60 K Array . There were eight identical arrays per slide. Each array consisted of 27958 Entrez Gene RNA probes as well as 1280 Agilent control probes (www.chem.agilent.com/ library/brochures/5989–3805EN.pdf). An Agilent hybridization oven was used to perform overnight hybridization . The array was consecutively rinsed with two solutions: the first solution was 0.2% SDS, 2 × SSC, , and the second solution was 0.2 × SSC .p-value of less than 0.05 (post-Benjamini-Hochberg correction). The “Adjust Data” function of CLUSTER 3.0 was used to log2 transform and median center the array data. We then performed hierarchical clustering using average linkage. Java Treeview  was used to perform tree visualization. The microarray data has been deposited to the Gene Expression Omnibus database under the accession number GSE95698.GeneSpring version 12 (Agilent) was used to analyze array data. Differentially-expressed genes were defined as those with a greater than 1.5 × absolute fold change and a 4)-induced model of cirrhosis was constructed by i.p. injection of 20% CCl4 in olive oil  into mice at 8 weeks of age for an 8-week period twice weekly. Vehicle control mice were i.p. injected with matching volumes of olive oil following the same regimen.A tetrachloromethane  into mice at eight to 12 weeks of age. All mice were sacrificed after 10 days on a standard chow diet. TNF treatment was performed through two i.p. injections of TNF (40 μg/kg) 6 h prior to sacrifice. For in vivo experiments, recombinant murine CCL28 (R&D Systems) or the Akt inhibitor A6730 was i.p. injected (both 50 mg/kg) 6 h prior to sacrifice. Murine CCL28 is a natural ligand-agonist for murine CCR10 The long-term diethylnitrosamine (DEN)-induced model of inflammation-driven hepatocarcinogenesis was constructed by a single intraperitoneal (i.p.) injection of DEN in physiological saline (15 mg/kg) into mice at 15 days of age. All mice were sacrificed after 9 months on a standard chow diet. After sacrifice, all surface liver tumor nodules were counted and measured via a caliper.15 followed by fluorescence-activated cell sorting (FACS). Two-step collagenase-isolated hepatocytes were centrifuged down at 75 g for 5 min. The pellet was rinsed with Dulbecco’s PBS and frozen at −80 °C until the FACS procedure.Isolation of hepatocytes from liver tissue samples was performed by two-step collagenase isolation as previously described by LeCluyse et al.16. As CD95 is solely expressed by hepatocytes among all liver cell types, the morphological hepatocyte gate was defined by size, which was confirmed by a high frequency of CD95-expressing cells. To eliminate any immune cell contamination within the hepatocyte gate, we also applied sorting to eliminate cells expressing the immune cell marker CD45. After selecting for CD95+CD45- hepatocytes, we then comparatively assessed hepatocyte CCR10 expression with a phycoerythrin-conjugated anti-human CCR10 antibody .FACS was then applied to sort hepatocytes with a FITC-conjugated anti-human CD95 antibody  and a biotin-conjugated anti-human CD45 antibody  as previously described with minor modificationsTotal RNA was extracted with a TRIzol reagent (Invitrogen). First-strand cDNA was synthesized from RNA with Superscript Reverse Transcriptase (Invitrogen). Real-time PCR was conducted using iQ SYBR Green Supermix  and gene-specific primers on an ABI 7500 instrument . Real-time reverse transcriptase PCR (RT-PCR) was conducted using SYBR Green Master Mix (Invitrogen) on an ABI 7500 instrument (Applied Biosystems). Primer sequences are detailed in Supplementary Table 4, DEN, etc.) were used as positive controls for CD45 PCR assays.Total liver samples from the experimental groups  , anti-cleaved caspase-3 , anti-cleaved PARP , anti-PCNA , and β-actin . Bands were detected with ECL reagents . Band signals were quantified through densitometry. Results were reported as the ratio of the target protein’s densitometry units to β-actin’s densitometry units.Western blotting was conducted with the following primary antibodies : anti-CCR10 , anti-GFP , anti-TNF , anti-PI3K ,anti-Akt , anti-phospho-Akt2O2. Sections were then boiled in a Tris/EDTA-based antigen retrieval solution (pH 9.0) for 2 min. Then, goat serum (15%) was applied for 30 min to block non-specific antibody binding. For tissue specimen staining, slides were incubated with the following primary antibodies : anti-CD68 , anti-MPO , and anti-Ki-67 . Apoptosis levels were assayed via a terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labelling (TUNEL) assay kit . Cells with nuclei staining brown-to-black were deemed TUNEL + apoptotic cells. One thousand cells were counted in each specimen in order to calculate a TUNEL + percentage (%).Liver sections were deparaffinized, rehydrated, and placed in 3% H17. The CCR10 siRNA targeting CCR10 mRNA’s coding region was constructed through BLOCK-iT RNAi Designer (Invitrogen) with a sense strand of GCUGGAUACUGCCGAUCUA and an anti-sense strand of UAGAUCGGCAGUAUCCAGC. Cells were seeded and cultured in six-well plates. Cells were then transfected with the CCR10 siRNA or a scrambled oligonucleotide negative control siRNA  using Lipofectamine 2000 (Invitrogen).All experiments were performed in triplicate wells and repeated in triplicate.In vitro silencing of CCR10 via siRNA was conducted as previously described by Chen et al.Cell proliferation was assayed with a Cell Count Kit-8 . Briefly, using a 96-well plate, the parent cell lines (1000 cells/well) were transfected with pcDNA3.0-EGFP control vector or pcDNA3.0-CCR10-EGFP vector or transfected with a CCR10 siRNA or a control siRNA (pcDNA3.0-EGFP vector was a gift from Doug Golenbock [Addgene plasmid # 13031]). Untreated parent cell lines as well as transfected cell lines were incubated for 24 h to 96 h. Then, a WST-8 solution was added to the culture (20 μl in 180 μl medium), left for 3 h at 37 °C, and 450-nm absorbance readings were collected.2O2. MPO activity was spectrophotometrically assessed at 460-nm absorbance.Liver tissue specimens were homogenized in 0.5% hexadecyltrimethylammonium bromide in phosphate buffer . Homogenates were then sonicated for 10 s, freeze-thawed thrice, and centrifuged for 15 min at 15,000 RPM. The resulting supernatant (100 μl) was mixed in O-dianisidine hydrochloride (0.167 mg/ml) and 0.0005% H6 cells) were subcutaneously injected into flanks of athymic nude mice bilaterally at 6 weeks of age. Post-injection, calipers were used to monitor tumor growth at 3-day intervals over the course of 24 days. Tumor volumes were calculated as follows: tumor volume = 0.5 × tumor length × tumor width2.Cultured Hep3B cells were stably transfected with a pcDNA3.0-CCR10-HA overexpression vector or a pcDNA3.0-HA null vector. To construct the xenograft tumor model, Hep3B cells .χ2) test was used to calculate positive rate differences among normal liver tissue specimens, paracancerous liver tissue specimens, and HCC tumor specimens. A one-way analysis of variance (ANOVA) test was used to compare CCR10 transcript levels among normal liver tissue specimens, paracancerous liver tissue specimens, and HCC tumor specimens. We applied a P-value threshold of 0.05 for statistical significance to all statistical tests.Statistical analysis was conducted with GraphPad Prism and GraphPad InStat software version 6 . Results are reported as means ± standard deviations. An independent Student’s t-test was used to test for statistically significant differences in the following experiments: gene expression, protein expression, cell proliferation, colony formation, and tumorigenicity. A Chi-squared  and myeloperoxidase (MPO) (a marker of neutrophil infiltration) in HCC tumor specimens, matching paracancerous specimens, and normal liver specimens. Both paracancerous specimens and HCC specimens displayed significantly higher levels of CD68 and MPO relative to normal liver specimens  and exclusively focus on the hepatocellular effects, we stringently isolated hepatocytes using a two-step collagenase isolation procedure followed by FACS (CD45−/CD95 + cell selection) for the remainder of this study16.CCR10 has been shown to be expressed on several cell types in the liver, most prominently infiltrating immune cellsThe isolated hepatocytes from human HCC and matching paracancerous specimens displayed significant CCR10 mRNA and protein upregulation relative to those isolated from normal liver specimens Figs. . Moreove4), and the other model was constructed through applying the pro-inflammatory hepatocarcinogen diethylnitrosamine (DEN)6. The pro-inflammatory markers IL-1α, IL-1β, IL-6, TNF, ICAM-1, and VCAM-1 were all significantly upregulated under both hepatitis models  mice and wild-type (WT) mice. Specifically, mice subjects were injected with DEN at 15 days of age; 9 months post-DEN injection, tumor sizes and tumor weights were measured. HCC tumors developed in all DEN-treated subjects Fig. . CCR10 K21. Thus, we investigated CCR10′s effects upon hepatocellular apoptosis as well as compensatory hepatocellular proliferation in the DEN-treated murine model of hepatitis. TUNEL staining revealed that DEN-treated mice displayed significantly higher apoptosis levels, with DEN-treated CCR10 KO mice showing significantly greater apoptosis levels , hepatocytes isolated from CCR10 KO mice showed significantly lower DEN-induced Akt phosphorylation and PCNA expression relative to hepatocytes isolated WT mice Fig. . CCR10 KTo validate the above findings, we next investigated hepatocellular PI3K/Akt pathway activity in reaction to short-term TNF exposure. Injection of TNF into WT mice significantly elevated CCR10 expression as well as Akt phosphorylation in isolated hepatocytes Fig. . NotablyTo understand CCR10′s role in hepatocellular proliferation and PI3K/Akt pathway activation in reaction to long-term DEN-induced inflammatory hepatocarcinogenesis, we next investigated hepatocellular PI3K/Akt pathway activity in long-term DEN-treated CCR10 KO and WT mice. Following long-term DEN-induced inflammatory hepatocarcinogenesis (9 months), hepatocytes isolated from HCC tumor tissue or surrounding paracancerous tissue displayed significant upregulation in CCR10 expression, PI3K expression, Akt expression, Akt phosphorylation, and PCNA expression relative to hepatocytes isolated from matching normal liver tissue Fig. . Hepatoc24. In this study, we demonstrated that hepatic inflammation drives production of the pro-inflammatory cytokine TNF, which promotes hepatocellular CCR10 expression. In turn, we also showed that hepatocellular CCR10 activates PI3K/Akt pathway signaling via phosphorylation of Akt, which drives hepatocarcinogenesis through inhibiting apoptosis and promoting compensatory proliferation. Our findings reveal that inflammation-driven hepatocarcinogenesis operates via activation of the CCR10/PI3K/Akt axis and that CCR10 is a key promoter of inflammation-driven hepatocarcinogenesis.Improving our understanding of key pathways involved in inflammation-driven hepatocarcinogenesis remains an important goal of current HCC research24. TNF has been established as a key promoter of inflammation-driven hepatocarcinogenesis in NAFLD, alcoholic cirrhosis, and chronic viral hepatitis27. Although liver inflammation has been shown to drive hepatocarcinogenesis via TNF in DEN-induced and CCl4-induced murine models of hepatitis, TNF’s downstream target(s) that promote tumorigenic change in hepatocytes remain largely unknown. However, it is known that TNF-driven liver inflammation is associated with dysregulated expression of GPCR-associated proteins. Here, we initially observed elevated levels of pro-inflammatory cytokines (most notably TNF), infiltration of inflammatory cells, and upregulation of four GPCR-associated genes  in human HCC tumor and paracancerous specimens. Notably, all four GPCR-associated genes have been previously associated with human carcinogenesis,30. Through RT-PCR and Western blotting validation, we confirmed that CCR10 expression was significantly elevated in hepatocytes isolated from matching human HCC tumor and paracancerous specimens. Moreover, FACS revealed strong CCR10 expression in paracancerous and HCC hepatocytes with negligible CCR10 expression in normal hepatocytes.Inflammatory processes play a critical role in HCC development4)6, we next demonstrated that DEN-induced and CCl4-induced hepatitis significantly elevated hepatocellular TNF and CCR10 expression in WT mice. In addition, TNF significantly promoted the expression of hepatocellular CCR10 and its natural ligand-agonist CCL28 in a dose-dependent manner. Accordingly, TNF has been shown to induce pro-inflammatory chemokine and cytokine expression by HCC progenitor cells and surrounding cells that directly contributes to HCC oncogenesis31. These combined findings suggest that hepatic inflammation drives hepatocellular TNF and CCR10 upregulation and that CCR10 upregulation lies downstream of TNF.Although these initial findings suggested an association between CCR10 upregulation and inflammation-driven hepatocarcinogenesis, it remained unclear as to whether CCR10 upregulation was induced via hepatic inflammation. Employing two well-established models of murine hepatitis (DEN and CCl32. These findings suggest that CCR10 is a key contributor to inflammation-driven hepatocarcinogenesis.Having established that CCR10 is significantly upregulated in short-term murine models of hepatitis, we next investigated CCR10′s role in long-term DEN-induced murine hepatocarcinogenesis using CCR10 KO and WT mice. Nine months after DEN treatment, tumor counts and sizes were both significantly reduced in CCR10 KO mice relative to WT mice. Moreover, implantation of CCR10-transfected Hep3B xenograft tumors in nude mice significantly promoted in vivo tumor growth. Similarly, previous work has shown that activation of CCR10 contributes to carcinogenesis and invasive progression in melanoma and glioma cells in vivo33. Thus, we next investigated CCR10′s effects upon hepatocellular apoptosis as well as compensatory hepatocellular proliferation in DEN-treated CCR10 KO and WT mice. Knocking-out CCR10 produces significantly greater hepatocellular apoptosis levels along with significantly lower compensatory proliferation in vivo. Consistent with our present findings, CCR10 activation has been associated with promoting the development of malignant melanoma and glioma through stimulating cell proliferation32.Previous research suggests that hepatocarcinogenesis is buttressed by compensatory proliferation of hepatocytes following apoptosis32. With respect to HCC, Akt phosphorylation has also been shown to be a key factor in hepatocarcinogenesis, with p-Akt levels positively correlating with inferior outcomes in HCC patients34. Accordingly, here we found that artificial CCR10 expression significantly enhanced hepatocellular Akt phosphorylation and expression of the cell proliferation marker PCNA in vitro. Moreover, employing CCR10′s natural ligand-agonist CCL2835 that we demonstrated to be expressed and secreted by hepatocytes in vitro, we found that application of CCL28 significantly promoted CCR10-driven hepatocellular Akt phosphorylation and PCNA expression in vitro, while application of the Akt inhibitor A6730 produced the opposite effects. Validating these in vitro findings, we confirmed that short-term DEN-treated CCR10 KO mice displayed significantly lower levels of hepatocellular Akt phosphorylation and PCNA expression. Notably, however, DEN-treated CCR10 KO and WT mice showed similar levels of hepatocellular TNF expression. Moreover, short-term stimulation with TNF significantly enhanced hepatocellular CCR10 expression, Akt phosphorylation, and PCNA expression, supporting our claim that CCR10/PI3K/Akt activation lies downstream of TNF. In order to investigate the longer-term effects of CCR10 expression, we finally investigated hepatocellular PI3K/Akt pathway activity in long-term DEN-treated CCR10 KO and WT mice. Consistent with our short-term findings, HCC tumor tissue and matching paracancerous tissue in long-term DEN-treated CCR10 KO mice displayed significantly lowered hepatocellular Akt phosphorylation and PCNA expression. These combined findings indicate that CCR10/PI3K/Akt pathway activation occurs at the pre-malignant/malignant hepatocyte level. That being said, this evidence does not completely rule out ancillary upstream effects from leukocytes or other cell types that may also contribute to HCC development.Moreover, previous studies in malignant melanoma and glioma suggest that CCR10 promotes cellular proliferation through PI3K/Akt pathway activationIn sum, these findings demonstrate that hepatic inflammation drives production of the pro-inflammatory cytokine TNF, which promotes hepatocellular CCR10 expression and downstream PI3K/Akt-mediated hepatocarcinogenesis. These findings reveal that inflammation-driven hepatocarcinogenesis operates via activation of the CCR10/PI3K/Akt axis. As CCR10 appears to function as a linkage between TNF stimulation and downstream PI3K/Akt pathway activation, CCR10 may show promise as a potential therapeutic target for inflammation-driven HCC.All data generated or analyzed during this study are included in this published article and its supplementary information files.Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Information


Dear fellow researchers,Welcome to the first issue of the Journal of Kidney Cancer and VHL (JKCVHL).JKCVHL is a peer-reviewed, open access journal created to fill the vacuum for a specialized journal to disseminate information on the advances in kidney cancer and VHL research. I am confident JKCVHL will fulfil its intended purpose, and will be a leading journal in this field in the days to come.