Cryptorhynchus lapathi.The insect gut microbiota has been shown to contribute to the host’s digestion, detoxification, development, pathogen resistance, and physiology. However, there is poor information about the ranking of these roles. Most of these results were obtained with cultivable bacteria, whereas the bacterial physiology may be different between free-living and midgut-colonizing bacteria. In this study, we provided both proteomic and genomic evidence on the ranking of the roles of gut bacteria by investigating the anal droplets from a weevil, The gut lumen and the anal droplets showed qualitatively and quantitatively different subsets of bacterial communities. The results of 16S rRNA sequencing showed that the gut lumen is dominated by Proteobacteria and Bacteroidetes, whereas the anal droplets are dominated by Proteobacteria. From the anal droplets, enzymes involved in 31 basic roles that belong to 7 super roles were identified by Q-TOF MS. The cooperation between the weevil and its gut bacteria was determined by reconstructing community pathway maps, which are defined in this study. A score was used to rank the gut bacterial roles. The results from the proteomic data indicate that the most dominant role of gut bacteria is amino acid biosynthesis, followed by protein digestion, energy metabolism, vitamin biosynthesis, lipid digestion, plant secondary metabolite (PSM) degradation, and carbohydrate digestion, while the order from the genomic data is amino acid biosynthesis, vitamin biosynthesis, lipid digestion, energy metabolism, protein digestion, PSM degradation, and carbohydrate digestion. The PCA results showed that the gut bacteria form functional groups from the point of view of either the basic role or super role, and the MFA results showed that there are functional variations among gut bacteria. In addition, the variations between the proteomic and genomic data, analyzed with the HMFA method from the point of view of either the bacterial community or individual bacterial species, are presented.The most dominant role of gut bacteria is essential nutrient provisioning, followed by digestion and detoxification. The weevil plays a pioneering role in diet digestion and mainly digests macromolecules into smaller molecules which are then mainly digested by gut bacteria. Dendroctonus rhizophagus [Antheraea assamensis, Helicoverpa armigera, Plutella xylostella [Bombyx mori [Cassida rubiginosa) degrade pectin [The insect gut microbiota has been shown to contribute to the host’s digestion, detoxification, development, pathogen resistance, and physiology , as zophagus , Antheralostella , and Bombyx mori , showinge pectin . Howevere pectin .Anticarsia gemmatalis [Tenebrio molitor [Comparative studies with conventionally reared and aseptically reared insects showed that gut bacteria may contribute significantly to lipid digestion and protein digestion (mainly the latter), detoxification of secondary plant compounds, and modification of the volatile profiles of the insect host in mmatalis , Tenebri molitor , and als molitor . A very  molitor . However molitor . Thus, aP. xylostella has thousands of genes from six families that encode carbohydrate-active enzymes [For the past few years, metagenomic analysis has been extensively used for the study of the gut microbiome (reviewed in ). A very enzymes . However enzymes  or metat enzymes . Fortuna enzymes ). Compar enzymes .Cryptorhynchus lapathi (L.) (Coleoptera: Curculionidae), is a wood-boring pest of economic importance throughout Europe, China, Japan, the USA, and Canada [Semutophila saccharopa [Polyommatus coridon [Nicrophorus (burying beetles) [C. lapathi contain diverse proteins related to gut homeostasis [Metaproteome measurements of gut microbiota are typically conducted with fecal samples, and proteins in the fecal samples need to be extracted by either a direct or indirect enrichment protocol . In contd Canada . When did Canada –26. Lepiccharopa ) and som coridon ), also e coridon ). Proteibeetles)  and aphibeetles) . Furthereostasis . Insect Using anal droplets as the study material has great advantages. First, we do not need to extract proteins or DNAs from guts or frasses. Second, we do not need to sacrifice animals and we can design repeated experiments to obtain longitudinal data. In this paper, the proteome of the larval anal droplet was investigated to assess gut bacteria functions on digestion, detoxification, and nutrient provisioning. The genomic data of gut bacteria were also investigated, yielding a similar result to that of the proteomic data. The variation between the two data sets was also determined.Illumina paired-end sequencing yielded a total of 121,218 bacterial 16S ribosomal RNA (rRNA) gene sequences after trimming and quality control  are more than 15%, whereas the anal droplet community was dominated by Brenneria (~ 80% in RF). The Jaccard coefficient was 0.1746, indicating distinct communities on genus level. Pearson’s chi-squared test showed that there was a significant difference in genus abundance between the two groups . At the phylum level, the gut lumen is dominated by Proteobacteria (> 80% in RF) and Bacteroidetes (> 18% in RF), with minor populations of Actinobacteria, Firmicutes, Deferribacteres, and Epsilonbacteraeota, the last two of which were not detected in anal droplets . Although the anal droplet bacterial community is more diverse in phyla, the gut lumen bacterial community has higher species diversity (3.63 vs 1.47 in Shannon index)  into 3-methylcrotonyl-CoA, and androstanedione into HIP-CoA or pyruvate.l-cysteine, and GABA into succinate. We performed BLASTP against the sequence of the enzymes presented in ref. [Enzymes that degrade nonprotein amino acids were only identified as bacteria-derived enzymes. These enzymes can degrade beta-alanine into acetyl-CoA, taurine into  in ref. , and onl3 into l-glutamate, N2 or nitrate into NH3, taurine into l-cysteine, and sulfate into sulfide.In total, 94 proteins belonging to 23 enzymes involved in nitrogen and sulfur metabolism were identified from the anal droplets  from malonyl-ACP, tetrahydrofolate (VB9) from GTP, and cobalamin (VB12) from cobinamide.Bacterial enzymes involved in biosyntheses of EAAs and vitamins were identified from the anal droplets by module reconstruction  was used to investigate whether the gut bacteria form functional groups at the basic role level. The PCA results from the proteomic data showed that the gut bacteria form four functional groups: chh; rer and ala; dfe and bgj; and the others Fig. a. PC1 isMultiple factor analysis (MFA) was used to compare super roles among bacterial species. Based on the proteomic data of the anal droplets, a graph of partial individuals was plotted Fig. a. The reBased on the genomic data of the bacterial species identified from the gut of the weevil, a graph of partial individuals is also plotted Fig. b. The baThe results from MFA also indicate the functional variations among gut bacteria Fig. . The resTo compare the results from the proteomic and genomic data, a hierarchical multiple factorial analysis (HMFA) was carried out Fig. . As showC. lapathi, to reconstruct these biological pathways.In many insects, resident microbes often promote insect fitness by contributing to nutrition and detoxification . To undeNicrophorus defodiens [Nicrophorus vespilloides [Rhynchophorus ferrugineus [Dendroctonus spp. [Lissorhoptrus oryzophilus [Sphingomonas, Pseudomonas, and Brenneria, whereas D. rhizophagus is dominated by Stenotrophomonas and Rahnella [D. valens is dominated by Providencia (35%) and Enterobacter (31%) [We characterized the gut bacterial community of the larval weevil, although a diversity comparison of gut bacteria is not the focus of this study. Our results showed that the bacterial community of the gut is distinctive from that of the anal droplet  . The difer (31%) .Using metaproteomics, we identified both weevil- and bacteria-derived enzymes from the anal droplets. Venn plots show that both the weevil and the gut bacteria have distinctive enzymes for both nutrient digestion and PSM degradation, suggesting a cooperative interaction between the weevil and its gut bacteria.Firmicutes, degradative capacity is largely restricted to the cell surface and involves elaborate cellulosome complexes in specialized cellulolytic species. By contrast, in bacteria such as Bacteroidetes, utilization of soluble polysaccharides, encoded by PULs, entails outer membrane-binding proteins, and degradation is largely periplasmic or intracellular [Nasutitermes P3 microbiome [Amitermes wheeleri and N. corniger were also reconstructed based on gene/transcript abundance profiles [Bacteria often show incomplete metabolic pathways. For example, no complete pathways for the biosynthesis of biotin, pantothenate, pyridoxine, cobalamin, and menaquinone are present in any of the bifidobacterial genomes sequenced so far . The invcellular . It is ocellular . Howevercellular , which acellular . If bactcrobiome . Intraceprofiles . TherefoThis hypothesis also gives us an idea to rank the roles of the gut bacteria community. We calculated the percentage of reactions contributed by each species for each basic role or super role, and the percentages of all species were then summarized after weighted by the abundance of each species. We called this value the weighted sum functional score. The higher the functional score is, the more prominent the function is. The functional scores were calculated on either proteomic or genomic data. The results from the two data sets showed a similar functional order (0.68 Spearman coefficient). In light of these results, we conclude that the most dominant function of gut bacteria is essential nutrient provisioning, followed by digestion and detoxification.Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) is a very popular software for predicting the functional composition of a metagenome based on 16S rRNA . In thisC. lapathi is a poplar-boring weevil, celluloses (~ 50%), hemicellulose (~ 20%), and lignin (~ 23%) are the primary components of its diet [As its diet . Poplar its diet , and theits diet . By contlulose ~ 0%, and lInsect gut roles in digestion have been presented by two paradigms. One paradigm is that potential roles are extrapolated from the (meta) transcriptome , 51 or (D. ponderosae (https://www.genome.jp/kegg-bin/show_pathway?dpa00500), the pathway of cellulose digestion is missing. On the community pathway maps shown in Fig. Our results showed that for cellulose digestion, both cellulase and β-glucosidases were detected from the anal droplets. Additionally, there are more weevil-derived cellulases than those from bacteria , the reaction from maltose to glucose is missing. By reconstruction of community pathway maps, a complete digestive pathway of starch is established, as shown in Fig. The identified enzymes that digest amylose or hemicellulose were all bacteria-derived enzymes Fig. . These rthe host . For ther plates . TherefoProteases and lipases have been detected in insect feces  and gut Amitermes hastatus, have been shown to decolorize the dye RBBR and exhibit laccase and peroxidase activities [Nasutitermes P3 luminal community revealed no evidence for lignin degradation [In woody material, cellulolytic components of the plant cell wall are protected by lignin, representing a barrier for carbohydrate degradation in xylophagous insects . Unfortutivities , althougradation . In termradation . In our radation . Thereforadation . Howeverradation .Tenebrio molitor [Hylobius abietis, has the ability to degrade the diterpene acids of Norway spruce, and several genes of a diterpene degradation (dit) gene cluster were annotated via a metagenomic survey [Rhagoletis pomonella [Riptortus pedestris and allied stinkbugs [Plutella xylostella [Bactrocera dorsalis [Although the known PSMs are very large in number, the known PSM-degrading enzymes are few. The number of PSM-degrading enzymes detected from the anal droplet is also small. Nevertheless, our results showed that the weevil and gut bacteria contribute different enzymes Fig. b. In con molitor . The gutc survey . These rc survey ). Howeveomonella , bean butinkbugs , diamondlostella , orientadorsalis , and othdorsalis , as deduPSMs and xenobiotics may also stress gut microbes and change the host’s gut microbiota , and onlNonprotein amino acids (NPAAs) like γ-amino butyric acid (GABA), taurine, and β-alanine are abundant in the nervous systems of animals where they function in regulating neuronal excitability and thus behavior (reviewed in ). FeedinDendroctonus feed on phloem, which is a nitrogen-limited source. Nitrogen fixation and nitrogen recycling may compensate for or alleviate such a limitation. It has been demonstrated that the P3 segment microbiome of higher termites is capable of fixing nitrogen [15N2 incorporation in the gut of Odontotaenius disjunctus, a wood-feeding beetle native to the eastern USA [Dendroctonus rhizophagus and Dendroctonus valens are able to recycle uric acid and contribute to insect N balance [Many insects and other animals do not have the enzymatic capabilities required to produce ammonia from urate. In our study, community pathway maps of recycling nitrogenous wastes and fixing nitrogen were all identified as bacteria-derived pathways. Nitrogen recycling from stored uric acid in shield bugs and termites has been attributed to symbiotic microbes. Another hypothesis is that uric acid is transported to the gut, where it is catabolized by uricolytic microorganisms . The barnitrogen , which htern USA . The bac balance . Based o balance . Comparel-cysteine and sulfate into sulfide, which indicated that gut bacteria help the weevil utilize sulfur compounds.Most insect species are unable to use reduce oxidized sulfur compounds and incorporate them into biomolecules, depending on their diet or the activity of their endosymbionts. The endosymbionts can assimilate sulfate into sulfur-containing amino acids such as Cys or Met , 84. OurDrosophila melanogaster (axenic flies) revealed microbial sparing of dietary B vitamins and microbial promotion of protein nutrition [D. melanogaster does provide thiamine to its host, enough to allow the development of flies on a thiamine-free diet [13C stable isotope analyses revealed that gut microbes associated with Asian long-horned beetle Anoplophora glabripennis can serve as a source of EAAs when fed on nutrient-limited diets [l-histidine [l-isoleucine, l-leucine, and l-valine) were reconstructed and were similar to those of any other bacteria [Escherichia coli [The gut microbiota has been shown to provide nutrients such as vitamins and EAAs to their hosts by functional assay with axenic animals –88 or byutrition . Delayedutrition . The gutree diet . δ13C stistidine . The biobacteria . There abacteria , whereashia coli –96. Our hia coli . Bacterihia coli . In our hia coli . For thihia coli , suggestIt is proposed that every microbial taxon identified in an insect does not need to “have a function” . Our resPseudomonas or Acinetobacter from the gut of D. rhizophagus [B. mori [Saperda vestita [Pseudomonas, Sphingomonas, and Achromobacter have repeatedly isolated from agricultural field soils, and these bacteria can be very quickly captured by insects as gut symbionts after insecticide application [Pseudomonas capable of insecticidal degradation has been isolated from the apple maggot [Acinetobacter isolate degrading phenol has been isolated from the gut of the termite [Our results also showed the variation between bacterial species on either the basic role or super role level Fig. . The higzophagus , B. mori[B. mori , or Sape vestita  showed alication . Insect e maggot . Acineto termite . These rC. lapathi. A distinct bacterial community between the gut and the anal droplets was observed. The gut bacterial community structure is different from those of extensively studied wood-feeding higher termites. The proteome of the anal droplets of the weevil was investigated to rank the roles of the gut bacterial community. The most dominant role of the gut bacteria is essential nutrient provisioning, followed by digestion and detoxification. The results from genomic analyses of the gut bacteria showed a similar role order. The proteomic data were also used to reconstruct the community pathway maps showing the metabolically active portion of the gut bacterial community in the digestion of carbohydrates/proteins/lipids, detoxification of PSMs/xenobiotics, metabolisms of nitrogen/sulfur, and supply of essential amino acids/vitamins. The gut bacteria expand both the digestive and detoxifying spectrum of the weevil. The weevil probably plays a pioneering role in diet digestion and mainly digests macromolecules into smaller molecules, which are then mainly digested by gut bacteria. A possible cooperation mechanism of gut microbiota was also proposed in which members work in tandem to complete either extracellular or intracellular community pathways.To understand how gut bacteria function in vivo, we first investigated the bacterial microbiota of the gut lumen and anal droplets from a wood borer, Larval weevils were collected in the wild near Harbin  in June. Anal droplets were collected directly onto foils placed against the anal areas of each weevil, while gently squeezing their abdomens. Samples on the foil were then transferred into a mini glass bottle with a pipette and stored at − 20 °C when not immediately used. Five larval weevils were used to collect anal droplets and were subsequently dissected to obtain the gut lumen. Each of the five anal droplet samples was split into two parts: part I was used for 16S rDNA amplification, and part II was used for Q-TOF MS analysis after being pooled with the other four part II droplet samples.2O. The tube was shaken vigorously by a vortex to expose gut lumen. Then the five aqueous solutions containing gut lumen were used as templates for PCR using the 16S rRNA primers spanning the V3-V4 variable regions (PF: 5′-CCTACGGGAGGCAGCAG and PR: 5′-GGACTACHVGGGTWTCTAAT), and the DNA-free water was used as a template for the negative control. For each PCR, 25 μL of mixture was prepared, including 12.5 μL of Phusion® Hot Start Flex 2X Master Mix , 2.5 μL of each primer, 50 ng of template, and DNA-free water. The PCR involved a single denaturation step at 98 °C for 30 s, followed by 35 cycles of 98 °C for 10 s, 54 °C for 30 s, 72 °C for 45 s, and a final extension at 72 °C for 10 min. The five PCR products were pooled to construct a sequencing DNA library. The same process was performed on part I anal droplet samples. DNA libraries were validated by an Agilent 2100 Bioanalyzer  and quantified by a Qubit 2.0 Fluorometer. DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to the manufacturer’s instructions . Sequencing was performed using a 2 × 300/250 paired-end (PE) configuration; image analysis and base calling were conducted by the MiSeq Control Software (MCS) embedded in the MiSeq instrument HCO3, 50% acetonitrile), the gel pieces were dehydrated until they shrank and became white (approximately 2 min) with 25 mM (NH4)HCO3 with 50% acetonitrile and then once more for 30 s in 100% acetonitrile. The gel pieces were then rehydrated in freshly prepared 10 mM dithiothreitol for 1 h at 56 °C (water bath) and were alkylated with freshly prepared 55 mM iodoacetamide for 1 h at room temperature in the dark. Subsequently, the gel pieces were washed with 25 mM (NH4)HCO3 twice for 10 min and destained to become white as before. Trypsin digestion was performed overnight with trypsin working solution (1 μg/μL stock solution was diluted 15-fold with 25 mM (NH4)HCO3) at 37 °C. Digested proteins were extracted 4 times with 50 μL of 50 mM (NH4)HCO3, 50 μL of 0.1% (v/v) FA in water, 50 μL of 0.1% (v/v) FA in acetonitrile, and 50 μL of acetonitrile. All extracts were pooled, freeze-dried at − 20 °C, and resuspended in 0.1% FA for sequencing.Q-TOF MS sequencing was performed as described before . The ana2O with 0.1% FA and (b) 100% acetonitrile with 0.1% formic acid; gradient: 0–4 min, 5–5% B; 4–30 min, 5–40% B; 30–35 min, 40–80% B; 35–45 min, 80–80% B; 45–45.1 min, 80–5% B; 45.1–60 min, 5–5%; flow rate, 400 nL/min; drying gas temperature, 150 °C; capillary voltage, 1.5 kV; collision gas, argon. The results were exported as a .MGF file for X!tandem [The resuspended peptides were fractionated using reversed-phase high-pressure liquid chromatography , and the gradient-eluted peptides were analyzed using a MicrOTOF-QII system . The liquid chromatography columns were packed in-house with C18 . The LC-MS conditions were as follow: mobile phase: (a) 100% HX!tandem  analysisC. lapathi itself to identify weevil-derived proteins [C. lapathi was constructed by de novo assembly (Trinity software (v r20140717) [http://weizhongli-lab.org) to obtain unigenes. The unigenes were subsequently mapped to the proteome of Dendroctonus ponderosae with a cut-off E-value of 10−4 using BLASTX (v2.3.0) to obtain a proteomic database of C. lapathi.For protein identification, the peak list data from MS were searched against a protein database. In a classic metaproteomics analysis, it is difficult to ascribe the species origin of identified proteins based on a closely related metagenome . In thisproteins , and use0140717) ) of the D. ponderosae and bacteria were downloaded from UniProtKB (http://www.uniprot.org/). The database searches were performed by the R package, rTANDEM [For identification of bacterial proteins from the anal droplet, the peak list data from MS were also applied to search against proteomes of 13 pseudo-proteomes corresponding to the top 14 genera (the 10th genus was excluded because it only has family information) which accounted for 93.22% of the relative frequency of the gut bacteria community. The proteomes of  rTANDEM . The pro rTANDEM  score cohttp://www.uniprot.org) of D. ponderosae, and then, GO/KEGG IDs and EC numbers were obtained from UniProtKB using ID Mapping function. The modules and pathways were reconstructed by the KEGG Mapper function (https://www.genome.jp/kegg/mapper.html). Proteins were also annotated by local BLASTPs (v2.3.0) against sequences referring to the literature and/or NCBI conserved domain search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). For details, see Additional file The IDs of the weevil proteins identified by X!tandem were labeled with their homolog gene IDs (UniProtKB protein entry, BLASTPs v.3.0 agaihttps://github.com/Jingtz/R-codes-for-microbiome-2019/tree/master.The EC numbers were used to identify an enzyme, and the GO numbers, KEGG numbers, and enzyme names were also used if the EC numbers were not available or if an enzyme had several EC numbers. Venn plotting was carried out to show the origin specification of the detected enzymes. The roles of gut bacteria were assigned into seven super roles and 31 basic roles see the “” sectionAdditional file 1. Supplementary tables S1-S19.Additional file 2. Supplementary methods and Supplementary figures S1-S9.Additional file 3. barcode sequences.


ASNS) expression correlated with poorer survival for women. This is the first study to show a unique, nutrient-deplete metabolic subphenotype in women with RCC, with implications for tumor progression and outcomes in CRC patients.Women have a lower incidence of colorectal cancer (CRC) than men, however, they have a higher incidence of right-sided colon cancer (RCC). This is of concern as patients with RCC have the poorest clinical outcomes among all CRC patients. Aberrant metabolism is a known hallmark and therapeutic target for cancer. We propose that metabolic subphenotypes exist between CRCs due to intertumoral molecular and genomic variation, and differences in environmental milieu of the colon which vary between the sexes. Metabolomics analysis of patient colon tumors (n = 197) and normal tissues (n = 39) revealed sex-specific metabolic subphenotypes dependent on anatomic location. Tumors from women with RCC were nutrient-deplete, showing enhanced energy production to fuel asparagine synthesis and amino acid uptake. The clinical importance of our findings were further investigated in an independent data set from The Cancer Genomic Atlas, and demonstrated that high asparagine synthetase ( In 2019, there will be an estimated 145,600 new cases and 51,020 deaths attributed to the disease2. Cancers of the colorectum can be categorized by anatomic location. Right-sided colon cancers (RCCs) occur in the cecum, ascending colon and hepatic flexure, and left-sided colon cancers (LCCs) occur in the splenic flexure, descending, sigmoid and rectosigmoid colon. Mid and lower rectal cancers are sometimes grouped as LCCs3. Tumors in different anatomic locations have differing clinical outcomes, and recent epidemiologic studies have shown that patients with RCC have the poorest survival, even when adjusted for confounding factors, including clinical stage6. This suggests that differences exist in the underlying tumor biology based on anatomic location. Distinguishing molecular features of RCCs include high microsatellite instability (MSI-H), valine to glutamate BRAF mutations at codon 600, cytosine-guanosine (CpG) island methylation phenotype (CIMP)8, and diploid cells9. In contradistinction, patients with LCCs more frequently have chromosomal instability, mutations in the TP53 and APC genes, and aneuploidy10. Recently, expression arrays have revealed four molecular subtypes of CRCs, called “consensus molecular subtypes” which tend to occur more frequently in either the right- or left-sides of the colon. Consensus molecular subtype 1, for example, has a 77% frequency in RCCs and is enriched for high MSI, CIMP, and BRAF mutations. In addition, gene set enrichment analysis shows molecular pathways related to immune infiltration and PD-1 activation for this subtype11.Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States, and the second leading cause of cancer-related deaths for cancers that affect both men and women9. The reason for this sex difference is unclear and of concern for women, due to the association of RCCs with poorer clinical outcome.In addition to genetic variance in colorectal subsites, RCCs occur more frequently in women compared to men, whereas LCCs have an equal frequency between sexes. Data from the Clinical Outcomes Research Initiative (CORI) and Surveillance, Epidemiology, and End Results (SEER) databases show that the percentage of RCC cases is strikingly higher in women than in men (61.7% vs 38.3%), while only slightly more LCC cases are observed in women than men (52.1% vs 47.8%)10. This milieu is comprised of metabolites produced from the diet, microbiome, and environmental pollutants14. Sex-specific influences on metabolite production could additionally account for the differences observed in anatomical tumor location. Life-course exposure to sex-steroid hormones (estrogens and androgens), lifestyle and medication use  and microbiome diversity and metabolism, could contribute to specific metabolic phenotypes of colon cancer between men and women18. Compared to men, women have a much lower overall incidence of CRC, yet they are more frequently diagnosed with RCC, thus it is highly plausible that RCCs have a unique biology in women shaped by differences in life-course exposures which promotes their growth in this location and affects therapeutic response.Recent studies have indicated that in addition to genetic factors, the environmental milieu could differ along the length of the colorectum and influence tumor development and progression20. With advancements in high-throughput analytical techniques for metabolomics research, there are emerging studies that have revealed metabolic signatures in colon cancer22. However, there have been no studies investigating sex-associated differences in colon cancer metabolism. More importantly, it is not known if men and women have metabolic phenotypes specific to the anatomic location of the colon tumor.Aberrant metabolism, which is a salient feature of colon tumor cells, involves the alteration of metabolic pathways to increase macromolecules and energy for cell growth. Metabolic pathways known to be affected in tumor cells include glycolysis, glutaminolysis, one-carbon metabolism, and fatty acid synthesisIn this study, comprehensive untargeted metabolomics analysis was performed on normal colon and primary colon tumor tissues collected from a large cohort of CRC patients. Multiple comparative analyses were conducted identifying sex differences in colon tumor metabolites stratified by anatomic location and stage. The biological relevance of these sex-specific colon cancer metabolites was identified by understanding their involvement in metabolic pathways. We identified sex-specific differences in energy production, and widespread sex and anatomic differences in asparagine, methionine, and polyamine metabolism. We correlated our findings to clinical outcomes of patients with colon cancer using The Cancer Genome Atlas (TCGA) database, and identified a positive association of high asparagine synthesis and poorer survival in women with colon cancer. To our knowledge, this is the first high-throughput metabolomic study to identify sex differences in colon cancer metabolism. We have revealed novel insights into the biological differences in tumor metabolism within population groups, and identified asparagine metabolism as potential future therapeutic target for women with RCC.Comprehensive untargeted metabolomics was carried out on normal colon and primary colon tumor tissues from a prospectively collected cohort of patients with RCC or LCC (n = 236). The detailed information of the clinical cohort is listed in Supplementary Table Initially, metabolite levels were compared between all normal colon and primary tumor tissues from men and women independently to identify tumor-specific metabolites for both sexes. The tumor metabolites were therefore normalized to controls within each sex to determine whether they are tumor specific versus a normal feature of colon tissue differences between men and women. Supplementary Table p < 0.01) but not in men with RCC. However, lactate, typically produced at high rates by cancer cells under a condition termed aerobic glycolysis23, was upregulated in men with RCC  but not in women with RCC. Interestingly, the glycolysis intermediate glyceraldhyde-3-phosphate/dihydroxyacetone phosphate (G-3-P/DHAP) was only increased in women with RCC, and was upregulated across all stages (p < 0.05). Of note G-3-P and DHAP are isomers and could not be distinguished by mass spectrometry analysis, however, they are both important intermediates in the synthesis of lipids including lysophospholipids. The lysophospholipids lysophosphatidylcholine and lysophosphatidylethanolamine, were upregulated in women with RCC (stage I) but not in men , but its level was maintained in women with RCC, suggesting that the fatty acid supply was ensured by higher levels of lysophospholipids in women with RCC. In support of this hypothesis, increased fatty acyl carnitine levels  were observed in women with RCC, indicative of enhanced fatty acid transportation via the carnitine shuttle, and oxidation to supply energy to tumors from women with RCC; this difference between controls and tumor samples was not observed in men.The initial analysis revealed sex-specific differences to metabolites that are critical end-products or intermediates in the PPP, glycolysis pathway, and the carnitine shuttle of fatty acids. We further examined the influence of both sex and anatomic location on the tumor metabolites. With an initial focus on patients with RCC, sex-specific differences were identified Fig. . Ribulosp < 0.01), but not in women. The PPP and fatty acid metabolism was upregulated in women for all stages, p < 0.01 and p < 0.05 respectively. Subtle differences were seen for G-3-P/DHAP and R-5-P when comparing RCC and LCC for men. R-5-P was upregulated in LCCs stage I and G-3-P/DHAP upregulated LCCs stage I and II, and these metabolites were not increased in men with RCC, showing a possible utilization of both glycolysis and the PPP for men with LCC.For patients with LCC, similar differences in metabolites between normal and tumor tissues  were also observed . However, asparagine was only upregulated in men with RCC at tumor stage III, (p < 0.05). Adenosine monophosphate (AMP) was increased only in women with RCC across all stages, (p < 0.05)  which also supports increased asparagine synthesis. Glutamine was not increased in tumors from men with RCC.Differences in amino acid metabolism were observed between tumors from both women and men when compared to controls. A further stratification by anatomic location revealed that asparagine synthesis, which is adenosine triphosphate (ATP)- and glutamine-dependent, was upregulated in women with RCC across all stages, (05) Fig.  indicati25, we further investigated the differences in additional amino acid levels between men and women with RCC. As shown in Fig. p < 0.05, Pearson correlation). The increased uptake of amino acids, including serine in women with RCC, suggests increased nucleotide synthesis promoting cancer cell proliferation. Nucleotides such as guanosine monophosphate (GMP), deoxyinosine, uridine 5′-diphosphate (UDP) and the coenzyme flavin adenine dinucleotide (FAD) were upregulated in women with RCCs but no changes were present in tumors from men with RCC 26, however in LCCs, glutamine was not elevated in men or women with LCCs compared to control. Interestingly, the patterns of glutamine and asparagine between normal and LCCs across different stages in women are the same as in men with RCCs. However, serine, threonine and AMP are increased for women with stage III LCC, similar to the changes seen in these metabolites for women with RCC across all stages, but only serine is increased for men with stage I LCC  for women with colon cancer, and to validate our findings in an independent cohort, we carried out an analysis of TCGA gene expression data (RNA-Seq) deposited on cBioPortal28. This cohort contains ASNS gene expression data and clinical outcomes for 125 women with colon adenocarcinoma. Kaplan-Meier survival analysis . However, ASNS does not affect OS in men with colon cancer  . Colon cancer patient data was derived from the SEER database n = 267,472), and survival analysis showed that from 1990–2016, patients with RCC had poorer OS than those with LCC. This was seen for both women and men stages I-III, 72, and sIn addition to sex-related differences in energy metabolism and asparagine synthesis, methionine and polyamine metabolic differences were also observed. In tumors from women with RCC, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), serine (a cofactor for SAH production), and dimethylglycine were upregulated across all stages when compared to normal colon tissue. Methionine, a precursor for SAM production was not changed. For men with RCC, SAM was upregulated across all stages, but there was no upregulation of SAH when compared to control tissue SAH levels Fig. . Serine N1, N12-diacetylspermine (DAS), was increased only in women with stage II-III RCCs, and it was not changed in men with RCCs. In tumors from women with RCC, spermidine was increased at stage I and III, and N1-acetylspermine increased across all stages, in men with RCC, tumors had increased spermidine at stage I, and N1-acetylspermine at stage II and III. Ornithine, a key metabolite in the urea cycle, is regulated by the tumor suppressor APC, and required for polyamine synthesis. Ornithine was upregulated in women with RCCs (stage I and stage III), similar to the changes seen for spermidine in women with RCC. Ornithine was not changed in men with RCC. For LCCs, DAS was increased across all stages for women, and not changed in men. Spermidine and N1-acetylspermine were both increased in stage III only in men, and increased in all stage tumors from women. Ornithine was not changed in any LCCs compared to normal tissues. Interestingly, formylmethionine, which is an initiator of protein synthesis, was upregulated in men at all stages for LCCs and RCCs whereas it was only increased in women at all stages for LCCs and advanced stage III for RCCs  were observed in tumors from women. This is indicative of enhanced fatty acid transportation via the carnitine shuttle, and β-oxidation to generate energy. Fatty acid metabolism and oxidation produces more ATP than oxidation of glucoseASNS, indicating that cells synthesize asparagine at the cost of cellular energy, and this reaction has been previously associated with tumor aggressiveness30. Previous studies have shown that ASNS is increased in high-grade anaplastic astrocytoma and glioblastoma, and correlates with poor prognosis for glioma and neuroblastoma patients31. We show for the first time, in an independent cohort, that high ASNS expression is associated with poorer OS in women with colon cancer but not in men. Further assessments of ASNS expression and its association with tumor location in the colon, show a trend of high expression and poorer OS in women with RCC. There was no such trend in ASNS expression and OS for LCC in both sexes or men with RCC. Therefore, asparagine metabolism could explain the link between tumor location difference and clinical outcomes in women with RCC compared to LCC32.Sex-associated differences in tumor metabolism by anatomic location were evident when examining asparagine production. Only women with RCC, had enhanced asparagine metabolism across all tumor stages. Asparagine is produced through a unidirectional ATP and glutamine-dependent reaction that converts aspartate to asparagine by 25, the increase of glutamine, serine, and threonine serve as important sensors to control mechanistic target of rapamycin (mTOR) signaling that governs cell growth and autophagy33. Here we show that asparagine levels positively correlate with threonine and serine levels which were increased in women with RCC. Of note, our findings of these sex differences in metabolism are consistent with gene expression results on nutrient deplete conditions and the potential regulation of mTOR in women with RCC34. The increased uptake of amino acids can serve as substrates for nucleotide synthesis promoting cancer cell proliferation. Our study supports this in showing increased levels of nucleotides and FAD seen in women with RCCs only. Our data collectively suggests a mechanism by which asparagine increases threonine and serine uptake for tumor cell growth and survival in women with RCC.Studies have shown that asparagine can be used as an amino acid exchange factor35, where asparaginase drugs are used to decrease circulating asparagine levels. Asparaginase deprives cells of asparagine, which leads to cell death, and could be a potential valuable for treatment of women with colon cancer. A recent study carried out in SW480 and SW620 colon cancer cells lines, showed that chloroquine and asparaginase decrease asparagine levels, which subsequently retards cell growth and autophagy, and activates apoptosis36. A role for KRAS in asparagine metabolism was also suggested, and ASNS expression was shown to be induced by the PI3K-AKT-mTOR pathway and mutated KRAS37. This therefore opens another potential therapeutic avenue for patients with mutated KRAS who cannot receive anti-epidermal growth factor receptor-based therapies such as cetuximab and panitumumab38.We propose a model for women with RCC, whereby enhanced energy production is driven by fatty acid metabolism, which facilitates asparagine synthesis and amino acid uptake under nutrient-deplete conditions in the colon Fig. . Asparagp16INK4α tumor suppressor gene, compared to men. Moreover, higher levels of methylated tumors are noted to occur within RCCs, however for women we do not observe differences in methionine cycling between RCCs and LCCs18. A previous study that targeted the analysis of SAM and SAH in the blood plasma of CRC patients, indicated that in men, both higher SAM and SAH were associated with decreased risk of colorectal adenoma. In contrast, both higher SAM and SAH were associated with increased risk of colorectal adenomas in women39. Therefore, understanding these metabolites in the context of methylation will provide more understanding as to their effects on CRC development.This study also revealed sex differences in methionine and polyamine metabolism in tumor tissues. Specifically, increased SAH and DAS were discovered only in women with RCCs but not in men with RCC. There are two plausible explanations for this. First, increased serine, dimethylglycine, SAH, and SAM indicates increased methionine cycling and potentially methylation in women with RCCs. This was also supported by increased polyamine methylation (DAS) seen in women with RCC. In men, SAM was increased in RCC, however SAH was not changed indicating that SAM is being shuttled for methylation purposes. For LCCs, SAM is upregulated across all stages for men, and SAH was upregulated at stages I-II. This indicates tumor location differences in methylation status for men. For women, these differences in SAM, SAH and serine, between LCCs and RCCs were not observed. These findings indicate perturbations to methionine cycling and potentially methylation in these patients. Unfortunately, it is not possible to determine the sex differences in methylation from this data. It has been previously observed that women were more likely to have methylation of the CpG island in the 5′ region of the 40. However, since the plasma metabolome encapsulates the systemic metabolome and multi-organ processing of metabolites, further studies in additional cohorts with clinical outcomes are necessary to validate this finding. Elevated DAS is also of interest in terms of CRC. This metabolite has been previously associated with multiple cancer types, including CRC, and is an end-product of polyamine metabolism. Our previous work showed that elevated DAS production in colon cancer was specific to tissues that had mucosal biofilms, which predominantly occurred on RCCs however, the sample size limitations did not allow for evaluation of sex-related differences41. However, it is possible that the microbiome plays a role in modulating metabolite production between men and women, as has been previously hypothesized42.The treatment implications of polyamine synthesis are evident in the Southwest Oncology Group-National Cancer Institute sponsored NCT01349881 clinical trial examining the effects of the polyamine inhibitor eflornithine on reducing development of premalignant adenomatous polypsIn addition to widespread differences between women and men, stage specific differences were seen. For example, lysophospholipids were only upregulated in stage I (node negative) tumors in women with RCC. Furthermore, in men with LCC, the metabolite G-3-P/DHAP was only upregulated in stage I and II (node negative) tumors, but not in advanced stage III (node positive) tumors. Our data suggests that sequential activation of metabolic pathways may exist for specific metabolites, and this may correlate with or drive colorectal cancer progression toward advanced stage disease. The progression of disease from stage II to stage III involves locoregional lymph node metastasis, which is a substantial change in cancer from being a process strictly localized in the colon to lymph (and then distant) metastasis. It is possible that lymph node metastasis is partly driven by lysophosphatidylethanolamine and G-3-P/DHAP, and these are necessary for development of metastasis. Additionally, lymph node metastasis may play a role in metabolites levels for advanced tumors. Whether or not these metabolites correlate with or drive metastasis needs further validation; it is the focus of experiments being tested in our lab to see if early stage biomarkers can be applied to clinical practice.In conclusion, this study highlights the widespread metabolic differences are present among tissue specimens collected from men and women with CRC and the effect of anatomic location of the tumor. Notably, tumor tissue samples from women with RCC display enhanced energy production driven by fatty acid metabolism, which facilitates asparagine synthesis and a subsequent increase in amino acid uptake and nucleotide production. These processes together indicate nutrient-depletion in tumors in women with RCC. The metabolic differences observed in this study could result from not only the distinct presence of environmental factors, but also the influence of molecular features including microsatellite instability, mutations to MLH1 and BRAF, and CIMP when accounting for anatomic location in CRC. Future stratification by these molecular features could be valuable to uncover the link between sex differences in metabolism and molecular biology. As with all discovery metabolomics studies, further validation in an independent cohort would further support our findings. Our study also has potential implications in clinical management using precision medicine approaches, wherein targeting of asparagine levels for women with CRC are employed. This study therefore enhances our understanding of the mechanisms governing sex-specific differences in colon cancer metabolism.2O), methanol (MeOH) and acetonitrile (ACN) were LC-MS grade and were purchased from Fisher Scientific .Ammonium acetate and formic acid were purchased from Fisher Scientific . Ammonium hydroxide was purchased from Honeywell . Water . Clinical data was recorded and updated retrospectively. Tumor tissue and normal colon tissue (away from the tumor at the resection margin) was acquired from surgical colectomy specimens. Each sample was snap frozen in liquid nitrogen and immediately stored in a −80 °C freezer. Pre-operative intravenous antibiotics  were administered within 60 min prior to resection. All patients received a standard mechanical bowel preparation (polyethylene glycol (PEG) solution) 24 h before scheduled surgery. For this study, samples were selected from patients that were ≥55 years old to reduce the confounding effects of estrogen signaling on metabolism before menopause. All normal colon tissues were selected from stage I-IV CRC patients (n = 39), and tumor tissue samples were selected from RCCs and LCCs stage I-III (n = 197). Stage IV tumor samples were not included as their metabolism may be affected by the presence of metastases in the liver or other site, therefore we cannot rule out this confounder. Normal samples were taken from stage IV patients, as their metabolic profiles were not significantly different from the normal colon tissues taken from stage I-III patients  was used with 2 mL lysing tube (Bertin Corporation) and 1.4 mm ceramic zirconium oxide beads (Bertin Corporation) to homogenize the tissues. Each sample was processed six times for 20 s, at 6,000 rpm with 5 s intervals. Dry ice was used to keep the temperature <10 °C during homogenization. From the homogenized solution, 100 μL was taken and added to 1.5 mL polypropylene microcentrifuge tubes for subsequent metabolite extraction. A volume of 400 μL ice cold MeOH:ACN  was added to each sample as the extraction solvent. The samples were vortexed for 30 s, and sonicated for 10 min. To precipitate proteins, the samples were incubated for 2 hours at −20 °C, followed by centrifugation at 13,000 rpm  and 4 °C for 15 min. The resulting supernatant was removed and evaporated to dryness for 12 hours using a vacuum concentrator . The dry extracts were then reconstituted in 100 µL of ACN:H2O , sonicated for 10 min, and centrifuged at 13,000 rpm  and 4 °C for 15 min to remove insoluble debris. The supernatant was transferred to UPLC autosampler vials . A pooled quality control sample was prepared by mixing 5 μL of extracted solution from each sample into a UPLC autosampler vial. All the vials were capped and stored at −80 °C prior to UPLC-MS analysis.50 ± 1 mg of each tissue was homogenized using 500 μL of UPLC-grade H4OH and 25 mM NH4OAc in water, while the mobile phase B was ACN for both electrospray ionization (ESI) positive and negative mode, respectively. The linear gradient was set as follows: 0~0.5 min: 95% B; 0.5~7 min: 95% B to 65% B; 7~8 min: 65% B to 40% B; 8~9 min: 40% B; 9~9.1 min: 40% B to 95% B; 9.1~12 min: 95% B. For RPLC-MS analysis, the mobile phases A was 0.1% formic acid in H2O, while the mobile phases B was 0.1% formic acid in ACN, respectively for both ESI+ and ESI−. The linear gradient was set as follows: 0~1 min: 1% B; 1~8 min: 1% B to 100% B; 8~10 min: 100% B; 10~10.1 min: 100% B to 1% B; 10.1~12 min: 1% B. Pooled samples were analyzed every eight injections during the UPLC-MS analysis to monitor the stability of the data acquisition and used for subsequent data normalization.Both HILIC-MS and RPLC-MS approaches were used for comprehensive analysis of the tissue metabolome. A UPLC system  coupled to a quadrupole time-of flight (QTOF) mass spectrometer  was used for MS data acquisition. A Waters ACQUITY UPLC BEH Amide column  × 2.1 mm (i.d.)) and Waters ACQUITY UPLC BEH C18 column  × 2.1 mm (i.d.)) were used for the UPLC-based separation of metabolites. The column temperature was kept at 25 °C for HILIC-MS analysis and 30 °C for RPLC-MS analysis. The solvent flow rate was 0.5 mL/min, and the sample injection volume was 1 μL. For HILIC-MS analysis, mobile phase A was 25 mM NHe data  were acquired for QC samples to enable metabolite identification. ESI source parameters on the Xevo GS-XS QTOF were set as following: capillary voltage 1.8 kV, sampling cone 40 V, source temperature 50 °C, desolvation temperature 550 °C, cone gas flow 40 L/Hr, desolvation gas flow 900 L/Hr.QTOF-MS scan data  was initially acquired for each biological sample for metabolite quantification. Then, both DDA (data-dependent acquisition) data ) and MS43 . The files were then processed in R (version 3.4.3) using the XCMS package44 for feature detection, retention time correction and alignment. The XCMS processing parameters were optimized and set as follows: mass accuracy for peak detection = 25 ppm; peak width c = ; snthresh = 6; bw = 10; mzwid = 0.015; minfrac = 0.5. The CAMERA package45 was used for subsequent peak annotation. The resulting data were normalized using the support vector regression algorithm in R to remove unwanted system error that occurred among intra- and inter- batches46. Initial metabolite identification was performed using the MetDNA algorithm (metabolomics standard initiative levels47 can be seen in Supplementary Table 248). Metabolites were further identified by matching retention time with an in-house metabolite standard library. In addition, metabolite identification was carried out by matching accurate mass and experimental MS/MS data against online databases (METLIN49 and HMDB50). The detailed identification information and identification confidence levels of dysregulated metabolites are listed in Supplementary Table The raw MS data (.raw) were converted to mzML files using ProteoWizard MSConvertp values were adjusted for multiple testing with Benjamini-Hochberg-based FDR. Pearson correlation analyses between metabolites were also performed using package “ggpubr” in R.Statistical analyses were performed in R (version 3.4.3). Nonparametric Kruskal–Wallis rank sum test with pairwise Wilcoxon Mann-Whitney U test were applied. https://seer.cancer.gov/seerstat/). Patients age 55 years and older who were diagnosed with primary adenocarcinoma of the colon from 1990 to 2016 were included in the analysis if they had a diagnosis of American Joint Committee on Cancer (AJCC) stage I to III colon adenocarcinoma. Tumor location within the colorectum was designated right-sided  or left-sided  according to the International Classification of Diseases for Oncology, 3rd edition (ICD-03). Patients with rectal, appendiceal, and transverse primary tumors were excluded. Kaplan-Meier survival analyses were conducted in R programming language using R packages “survminer” and “survival”. Log-rank test was utilized to analyze the statistical differences between different groups of colon cancer patients.Survival analyses on colon cancer patients  from SEER database were carried out to investigate outcomes between patients with RCC and patients with LCC. All data were downloaded using SEER*Stat software  recorded from 1992 to 2016. Data was extracted using SEER*Stat software (version 8.3.5). Patients with rectal, appendix, and transverse primary tumors were excluded. The incidence rates (using the 2000 U.S. standard population) express per 100,000 persons.28. Z-Scores (RNA Seq V2 RSEM) were used as a measure of mRNA expression. In addition to gene expression data, patient data including clinical parameters such as sex, age, survival status, and OS time were also downloaded. Patients with the colon cancer type colon adenocarcinoma (COAD) were selected. OS analyses were carried out only on patients with available survival and mRNA expression data. Two groups of samples: (a) High ASNS expression (with Z-Scores above upper quartile) and (b) Low ASNS expression  were categorized as previously described51. Kaplan-Meier survival analyses were conducted in R programming language using R packages “survminer” and “survival”. Log-rank test was utilized to analyze the statistical differences between different groups of colon cancer patients.In order to assess the correlation of our findings to patient outcomes we analyzed TCGA colon cancer data set . This was downloaded through cBioPortal v2.2.1Supplementary Information file.Supplementary Information Table S2.


Streptococcus pneumoniae strains before marketing and introduction of PCV-13.The 13-valent Pneumococcal Conjugate Vaccine (PCV-13) was introduced in the National Immunization Programme (NIP) schedule in Russia in March 2014. Previously, the 7-valent Pneumococcal Conjugate Vaccine (PCV-7) was marketed in Russia in 2009 but has never been offered for mass vaccination. A carriage study was performed among children in Arkhangelsk in 2006. The objective was to determine the prevalence of carriage, serotype distribution, antimicrobial susceptibility and the molecular structure of A cross-sectional study was conducted on a cluster-randomized sample of children and a self-administrated questionnaire for parents/guardians.  Nasopharyngeal samples were collected from 438 children younger than 7 years attending nurseries and kindergartens in the Arkhangelsk region, Russia. Detailed demographic data, as well as information about the child’s health, traveling, exposure to antimicrobials within the last 3 months and anthropometric measurements were collected for all study subjects. Variables extracted from the questionnaire were analysed using statistic regression models to estimate the risk of carriage. All pneumococcal  isolates were examined with susceptibility testing, serotyping and multilocus sequence typing.NT-42 and Poland6B-20, as well as penicillin- and macrolide-susceptible clone Netherlands3-31. A high level of antimicrobial consumption was noted by the study. According to the parent’s reports, 89.5% of the children used at least one antimicrobial regime since birth. None of the hypothesised predictors of S. pneumoniae carriage were statistically significant in univariable and multivariable logistic models. The overall prevalence of asymptomatic carriage was high and peaking at 36 months with a rate of 57%. PCV-13 covered 67.3% of the detected strains. High rates of non-susceptibility to penicillin, macrolides and multidrug resistance were associated with specific vaccine serotypes, pandemic clones, and local sequence types. Nine percent of isolates represented three globally disseminated disease-associated pandemic clones; penicillin- and macrolide-resistant clones NorwayThe study identified a high coverage of the PCV-13-vaccine, but serotype replacement and expansion of globally disseminated disease-associated clones with non-vaccine serotypes may be expected. Further surveillance of antimicrobial resistance and serotype distribution is therefore required. Streptococcus pneumoniae is a bacterial pathogen causing disease among all age groups. Despite the introduction of effective vaccines, invasive pneumococcal disease (IPD) is associated with high mortality and morbidity [, in 2015, pneumococci were estimated to have caused 318,000  deaths for both HIV-infected and HIV uninfected infants and young children in the age 1–59 months globally [orbidity –3. As waorbidity . Immunizorbidity . Still, globally .19F-14 [The epidemiology of pneumococcal disease prior to the introduction of pneumococcal vaccines was dominated by the spread of global disease-causing epidemic clones, both multidrug-resistant (MDR) and antimicrobial susceptible clones . The suc19F-14 .Haemophilus influenzae type b conjugate vaccine has not been available for mass vaccination [Russia is a large country with an estimated infant and child population aged up to 4 years of 9.0 million in 2019 . The immcination . The PCVcination , 15. Immcination . In 2017cination .Neither national nor regional surveillance of incidence for IPD cases exists in Russia , 18 . ThS. pneumoniae carriage in healthy pre-school children attending daycare centres (DCCs) 8 years before the introduction of PCV-13. All pneumococcal isolates were analysed with regard to serotypes, phenotypic antimicrobial resistance patterns and population structure based on multilocus sequence typing (MLST).The present study was conducted in the Arkhangelsk region in the northwest part of Russia where no data about the pre-PCV carriage are available. In order to determine pneumococcal carriage at baseline  and evalChildren and parents/guardians from ten DCCs were invited to participate in the study. All the DCCs were public childcare institutions that belonged to small towns and suburbs of the Arkhangelsk region and located within a range of 13 to 44 km from the city of Arkhangelsk. Each DCC consisted of a nursery and a kindergarten and was attended by 21 to 200 children. Besides, 32 randomly chosen healthy children living in the centrum of Arkhangelsk were sampled. Children and parents/guardians were invited to participate by the announcement in the local newspaper. All children were sampled during the last week of November 2006 by one otolaryngologist. None of the children experienced symptoms of a common cold like cough, runny or stuffy nose at the date of examination. The body temperature was normal for all children on the day of sampling.The questionnaires were filled out by parents or guardians for all participants in the study. Each questionnaire included questions concerning the child’s health, length of breastfeeding, travelling abroad or outside of the Arkhangelsk region within the last 6 months, smoking habits of family members, as well as the household size and the number of siblings and family members. Information on the use of antimicrobials agents during the last 3 months prior to sampling was also collected for all children. Anthropometric measurements were taken for all participants on the day of sampling.All parents/guardians were informed about the study by informational letters and a majority of the parents/guardians participated in informational meetings. The written informed consents were filled out by parents or guardians for all participants of the study. Ethical approval was obtained from the Ethics Research Committee of North Norway, the reference number of the approval 5.2006.2086 from the 23rd of June 2006 and the Ethics Research Committee of the Northern State Medical University of Arkhangelsk, the reference number for the approval 06/06 from the 6th of June 2006. Permission to conduct the study was also obtained from the Health and Educational Services of the Arkhangelsk Region.2 at 35–37 °C for 18–24 h. Strains were identified as S. pneumoniae by colony morphology, negative catalase reaction, optochin susceptibility, agglutination in the Pneumo-Kit slidex test , and by the bile solubility test [The European Intervention Study (EURIS) manual was used for isolation of bacterial strains , 21. Nasity test . IsolateStrains were tested for antimicrobial susceptibility by disk diffusion on Iso-Sensitest Agar (ISA)  supplemented with nicotinamide adenine dinucleotide  and 5% defibrinated sheep blood. Antimicrobial paper disks  containing 1 μg oxacillin (OXA), 15 μg erythromycin (ERY), 30 μg tetracycline (TET), 25 μg trimethoprim-sulfamethoxazole (SXT) or 10 μg norfloxacin (NOR) were used. OXA-resistant isolates (inhibition zone < 18 mm) were further examined by penicillin G (PEN), cefuroxime (CXM), cefotaxime (CTX) and meropenem (MEM) Etests according to the manufacturer’s instructions (AB Biodisk). Unless otherwise stated, the breakpoints defined by the Norwegian Working Group for Antibiotics (NWGA) were used. NOR-resistant isolates were examined by Etest for their susceptibility to ciprofloxacin (CIP), NOR, moxifloxacin (MXF) and levofloxacin (LVX), using breakpoints from the Swedish Reference Group for Antibiotics (SRGA) –25. MultB-resistance, resistance to both ERY and CLI indicated constitutive resistance (cMLSB), whereas susceptibility to CLI and resistance to ERY indicated M-type resistance. Detection of the macrolide resistance determinants ermB and mefA was performed by PCRs as previously described [The double-disk diffusion (DDD) test with ERY and clindamycin (CLI)  was used for characterization of inducible macrolides, lincosamides, streptogramines (iMLS) resistance as described . Bluntinescribed .aroE, gdh, gki, recP, spi, xpt, and ddl. Alleles were identified and isolates were assigned into STs using the PubMLST database (https://pubmlst.org/spneumoniae/). The PHYLOViZ® programme was used for assigning the isolates into clonal complexes (CCs), defined as clusters sharing six out of seven common alleles.All strains were examined by MLST as described by Enright et al.  and assihttps://www.rstudio.com/} and R version 3.5.1 for Windows were used for calculation of odds ratios (OR), confidence intervals (95% CI), and p-values using two-tailed Fisher’s exact test. Carriage rates were calculated as incidence rate ratios (IRRs) with 95% CI. P-values < 0.05 were considered significant.RStudio© version 1.1.423 {We used Fisher’s exact test and univariable and multivariable logistic regression models to examine potential risk factors for pneumococcal carriage including sex, age, early life variables , family and socioeconomic status , medication and disease , and lifestyle factors , passive smoking).2 for 56% of the children and greater than 16 for 43% of the children on the day of examination. Only 1% of the children had a BMI of less than 13 on the day of sampling. The overall prevalence of antimicrobial consumption was high. According to the parent’s reports, 89.5% of the children used at least one antimicrobial regime since birth. Additionally, 38.7% (65/168) of the children with recognized carriage have been treated with antimicrobials within the last 3 months before sampling.Out of 766 children attending the ten selected DCCs, 438 (57.2%) agreed to participate in the study and nasopharyngeal swabs were collected from all these children. The demographic data for all 438 children are given in Table Pneumococci were isolated from 168 children (mean age 45.8 months), giving an overall carriage rate of 38.4% (CI 33.8–42.9%). The highest rate of carriage was found among the children aged from 19 to 36 months and it was lowest at the age less than 18 months  and did not display any statistically significant differences. Sex did not influence the carriage rate of S. pneumoniae significantly .The carriage rate of ly Table . None ofls Table . Receivin = 28; 16.7%), 23F , 6A  and 6B  were most prevalent  Table . SerotypPCV-13, including serotypes 1, 3, 4, 5, 6A/B, 7F, 9 V, 14, 18C, 19A/F, and 23F, would cover 67.3% (113/168) of the isolates in the study , TET , OXA , and ERY . Characterization of penicillin non-susceptible pneumococci (PNSP) and macrolide non-susceptible pneumococci (MNSP) is given below. Only a single strain was resistant to fluoroquinolones (< 1%). MDR was detected in 19 strains (11%). Only 26% (43/168) of nasopharyngeal carriage strains were susceptible to all examined antimicrobials  .The rates of non-susceptibility were as follows: SXT (n = 15). Ten percent of all STs were represented by single isolates. Thirty-five of the 60 STs were identified for the first time in Arkhangelsk.The isolates displayed 60 different STs Table . Forty-tThe majority of the pneumococcal isolates in each ST was related to a single serotype. Two STs, ST2859 and ST3244, were associated with two serotypes. To the authors’ knowledge, an association of ST2859 with both serotypes 15B and 15C, as well as ST3244 to both serotypes 6A and 6B has not been reported before. ST62 was represented by three strains with serotype 11A and a single NT strain.NT-42 clone  OXA-resistant strains were confirmed as non-susceptible to PEN (range 0.094–1 mg/L), and 19 of these strains (95.0%) were MDR. Ten of the 20 (50.0%) isolates were non-susceptible to both PEN and macrolides. The majority of PEN and MNSP isolates  were related to the Norwayne Table . Three aTen pneumococcal isolates displayed non-susceptibility to PEN, but were susceptible to macrolides. Seven of these strains expressed serotype 23F and belonged to ST1500 and were also resistant to STX. The three remaining isolates were non-serotypeable  (Table B (n = 7), cMLSB (n = 4), and M-type resistance (n = 5). The results were confirmed by ermB and mefA-PCRs. Macrolide non-susceptibility was associated with two globally disseminated clones. Six of the macrolide-resistant isolates belonged to CC315 (ST315 and ST3200), expressed the ermB-gene and were associated with the international Poland6B-20 clone. Six of the isolates belonged to ST344-NorwayNT-42 and possessed both mefA (n = 4) and ermB (n = 2) determinants. The other 4 macrolide-resistant isolates were represented by unrelated ST25, ST386, ST2991, and ST3201 and were connected to ermB (n = 3) and mefA (n = 1) genes.The analysis of all macrolide non-susceptible isolates by DDD method revealed the following phenotypes: iMLSS. pneumoniae carriage isolates in pre-school children of the Arkhangelsk region between separately located DCCs. The serotype distribution was diverse in the area, but globally reported epidemiological features, such as age-dependence, dominant serotype prevalence  and the presence of globally disseminated clones were confirmed in the study.This is one of the largest carriage surveys prior to the introduction of pneumococcal vaccines in Russia, where serotyping, antimicrobial susceptibility testing and MLST were performed on whole strain collection . MoreoveS. pneumoniae colonization [A 38.4% overall frequency of pneumococcal carriage rate was found in non-vaccinated pre-school children. The carriage rate among children aged 36 months was as high as 57.0%. Previous carriage studies in DCCs in Russia have described even higher overall frequencies of asymptomatic nization –34. Overnization . The preThe rates of asymptomatic carriage varied markedly between different age groups in our study, and also the diversity of serotypes displayed age variation. These findings have previously been observed by others –41. Youn19F-14 cluster previously associated with serotype 19F to 19A replacement [None of the isolates from our study collection expressed serotype 19A reported as the eighth most prevalent globally  and the lacement . Serotyplacement , 49, lealacement  demonstrIn our study, two isolates (1.8%) belonged to serotype 15A, which is not included in PCV-13. This serotype was previously associated with MDR and was isolated from most IPD cases in the post-PCV era in several post-industrial countries –53. Our A single carriage isolate from our collection displayed serotype 35B, which is a non-vaccine serotype associated with high capacity for biofilm production . The isoS. pneumoniae carriage strains may predict rates of resistance in invasive isolates [Since the pneumococcal disease is preceded by asymptomatic colonization, the distribution of antimicrobial resistance patterns in nasopharyngeal isolates , 57. Ratisolates –53, 58. isolates . The stuisolates , 34. Higisolates .Contrary to the reported low rates of SXT consumption in the area  the studRates of resistance are strongly associated with rates of antimicrobial consumption in local settings. According to the report from the European Surveillance of Antimicrobial Consumption (ESAC) network , the ratWe observed a high prevalence of various locally disseminated STs in the area. Although the Arkhangelsk region has rather low rates of migration and tourism and does not border any other countries, we found a close clonal relationship with the major globally disseminated pandemic clones, thus indicating possible import. Our study found ST1500 with serotype 23F to be associated with PEN non-susceptibility. A study carried out in Siberia found a high rate of ST1500 carriage isolates which were susceptible to PEN thus contrasting to the Arkhangelsk isolates . DiffereWe found that PCV-13 could be effective against 67% of the pneumococcal population and thus reduce the majority of penicillin, macrolide, and multidrug-resistant strains as previously shown in other countries –66. HoweNT-42, vaccination against which is so far unavailable.A high proportion of both penicillin and macrolide non-susceptible isolates in our study was found to be related to NT isolates. The recently published meta-analysis of estimated invasive disease potential for individual pneumococcal serotypes showed a low disease potential for non-serotypeable pneumococci . HoweverThe high effectiveness of PCVs against IPD in infants and young children has been proven in countries with well-established national surveillance. According to several reports –66 a shaS. pneumoniae population was found to be highly diverse and had common features such as age-dependency, dominant serotypes and the presence of major epidemic clones. High rates of resistance were linked to high rates of antimicrobial consumption in the area. Clonal expansion of several globally distributed pandemic clones was identified in a remote part of north European Russia with low rates of migration and tourism. The effectiveness of PCV-13 introduction cannot easily be predicted. According to international experience, clonal expansion due to replacement by the globally disseminated disease-associated pandemic clones with non-PCV-13-serotypes can be expected. The authors propose setting up a high-quality population-based national/regional surveillance system for asymptomatic colonization and serotype-specific IPD rates for monitoring effects of the vaccination programme.The present study has documented several important aspects of the local pneumococcal epidemiology specific for the north of European Russia. The Additional file 1: Table S1. Odds ratio of Streptococcus pneumoniae nasopharygeal carriage by hypothesised risk factors. A study among children in kindergartens in Arkhangelsk Russia 2006, N = 438*.


Inflammation plays an important role in the initiation and progression of atrial fibrillation (AF), but data about the relationship between subclinical inflammation and recurrence of AF after catheter ablation remains poorly studied. We aimed to assess whether plasma levels of C-reactive protein (CRP) are associated with long-term AF recurrence following catheter ablation.Prior to the intervention, plasma CRP concentrations were measured in patients who underwent first catheter ablation for AF. AF recurrence was evaluated after 12 months and defined as any AF episode longer than 30 s recorded on either 12-lead electrocardiogram, 24-h Holter or 7-day Holter monitoring. Multivariable adjusted Cox models were constructed to examine the association of CRP levels and AF recurrence.P = 0.004). The adjusted hazard ratios (aHR) of recurrent AF across increasing quartiles of CRP were 1.0 (reference), 1.26 , 1.15  and 1.60  (P trend = 0.015). A similar effect was observed when CRP was analyzed as continuous variable . When a predefined CRP cut-off of 3 mg/l was applied, patients with CRP levels of 3 mg/l or above had a higher risk of AF recurrence than those with levels below .Of the 711 patients  included in this study, 247 patients (35%) experienced AF recurrence after ablation. Patients who were in the highest CRP quartile had a higher rate of recurrent AF compared to those who were in the lowest quartile (53.4 vs. 33.1% at 1 year of follow-up; Increasing pre-interventional CRP levels are associated with a higher risk of AF recurrence in patients undergoing catheter ablation for AF.ClinicalTrials.gov identifier, NCT03718364. Atrial fibrillation (AF) currently affects more than 18 million Europeans and its prevalence is increasing rapidly . PatientEpidemiologic and histological evidence suggests that inflammation may play an important role in the initiation and maintenance of AF , 12. In The study population was derived from the Swiss Atrial Fibrillation Pulmonary Vein Isolation (SWISS-AF PVI) study, an ongoing prospective cohort study in Switzerland enrolling patients > 18 years of age with diagnosed AF who are scheduled to undergo first elective catheter ablation. Exclusion criteria were presence of longstanding persistent or permanent AF, and inability or unwillingness to participate in the study. Also, patients were excluded if they were experiencing acute inflammatory conditions (such as pneumonia or urosepsis) or showed symptoms of acute illness at baseline. However, this group of patients was eligible for enrolment 4 weeks after stabilization of their acute episode. None of the patients had diagnosed rheumatoid arthritis at baseline. The study protocol was approved by the local ethics committee, and written informed consent was obtained from all participants.Data on personal and medical characteristics were collected at baseline by study personnel using standardized case report forms. Information on demographics, lifestyle habits, medical history, and current medications was obtained. Prior to the ablation procedure, we performed transthoracic echocardiography to quantify left atrial dimension and left ventricular ejection fraction (LVEF). Type of AF was classified according to the current guidelines either as paroxysmal  or persistent AF  .Venous blood samples were obtained and processed at the day prior to ablation. Plasma levels of CRP were measured using an immunoturbidimetry on latex assay . This assay allows quantification of CRP plasma levels below 2 mg/L (detection range of 0.3 to 350 mg/L), which is sufficient for cardiovascular risk assessment –26.Catheter ablation procedures aimed to restore sinus rhythm through isolation of all pulmonary veins. Ablation strategy and mapping system selection was left to the discretion of the electrophysiologist. Only experienced electrophysiologists who have had performed several AF ablation procedures, including pulmonary vein isolation, using the same approach were allowed to perform the ablation procedures. At the discretion of the operators, additional lesions were placed. The operators were unaware of the patients measured CRP levels.Regular follow-up visits in outpatient clinics were conducted by physicians at 3, 6, and 12 months. At each visit, a detailed medical and physical examination, 12-lead electrocardiogram, 24-h Holter, and 7-day Holter monitoring at 12 months were performed. In some patients the 7-day Holter was performed beyond the scheduled 12-month visit. All recordings were centrally collected at the University Hospital Basel and outcomes were adjudicated by trained study personnel and cardiologists. The outcome was AF recurrence defined as any AF or atrial tachycardia episode lasting longer than 30 s, which is in accordance with the 2017 Heart Rhythm Society expert consensus statement .A blanking period was applied to exclude events that occurred during the first 90 days after ablation, as recommended by the current guidelines . BaselinWe used a multistage process to address the effect of CRP levels on the rates of AF recurrence that occurred after the blanking period. First, we divided patients into quartiles of CRP levels; the lowest quartile was defined as the reference. Incidence rates stratified by CRP quartiles were calculated at 1 year of follow-up and compared using the trend test for survival functions. Kaplan-Meier survival curves were used to estimate the cumulative incidence of recurrent AF across CRP quartiles and compared using log-rank test. We constructed Cox proportional hazard models to assess the risk of AF recurrence across CRP quartiles. The first model was unadjusted, the second model was adjusted for age and sex, and the third model was adjusted for age, sex, body mass index, AF type , history of hypertension (yes vs. no), history of heart failure (yes vs. no), history of obstructive sleep apnea (yes vs. no), AF duration in years, and left atrial diameter (LAD). Second, we log-transformed CRP levels and repeated the analysis process using CRP as a continuous variable. Multivariable models were adjusted for the same set of variables as described above.In additional analyses, we stratified the study population at a CRP cut-off value of 3 mg/l, a level known to be associated with high cardiovascular risk –30. We eP-value < 0.05 was considered to indicate statistical significance.All analyses were performed using Stata, version 13  and a two-sided n = 33) and because blood samples were drawn after catheter ablation (n = 4). The median follow-up duration was 1.0 years . Of the 711 AF patients included in our analyses, 247 (35%) experienced AF recurrence after ablation. Table From April 29, 2010, through May 11, 2017, we enrolled 847 AF patients in the study. Of those, 748 patients completed at least one follow-up visit . In the fully adjusted model, the hazard ratios (aHR) for those in the second, third and highest quartiles of CRP, as compared to those in the in lowest quartile (reference), were 1.26 , 1.15 , and 1.60 , respectively (Table P = 0.009).Table P = 0.009) (Table P = 0.019)  and interleukin-6 (IL-6) were found to be significantly associated with recurrent AF after ablation , 38. AnoThe findings of the current analysis may have several implications. First, given that failure rates of AF ablation remain significant, our results provide potential targets for therapeutic strategies to improve ablation success. Prospective epidemiologic studies demonstrated that obesity, smoking, high blood pressure and diabetes are independently associated with elevated levels of CRP and other inflammatory biomarkers –44. ThisClinicalTrials.gov number, NCT04160117). From a mechanistic point of view, the association between pre-interventional subclinical inflammation and AF recurrence requires further investigations into the underlying biologic mechanism.Second, previous trials have evaluated whether administration of anti-inflammatory therapy shortly after catheter ablation would reduce AF recurrence , 49. AltThe strength of this study is the prospective design including a large sample of well-characterized AF patients. Furthermore, the large number of confirmed events, the availability of many covariates and the low rate of missing values allowed us to perform meaningful analyses. Nonetheless, this study has some potential limitations that require discussion. First, patient recruitment started in 2011 and ablation techniques may have changed over time, which could have impacted procedural success rates. Second, we measured CRP only at baseline and thus, could not evaluate the effects of changes in levels over time, or the effect of postinterventional CRP levels. Such measures may have provided a better overview about subclinical inflammatory state of the patients included in this study. Third, the study sample included patients who were recruited at a Swiss study center and the generalizability of the results to other populations is unclear. Forth, more than 10% of patients who had undergone initial catheter ablation were lost to follow-up (Figure Our data demonstrate that increasing CRP levels measured prior to catheter ablation were associated with a higher risk of AF recurrence. These findings support the hypothesis that inflammation may be involved in the recurrence of AF after catheter ablation.Additional file 1: Figure S1. Flow diagram of patient selection and follow-up.


Patients with renal cell carcinoma (RCC) usually develop drug resistance and have poor prognosis owing to its insensitive property. However, the underlying mechanisms of RCC are still unclear. We implemented an integrative analysis of The Cancer Genome Atlas and Gene Expression Omnibus datasets. Three genes  were found to be potential biomarkers in ccRCC and validated by four independent cohorts. Then, ccRCC patients with a decreased expression of CRHBP in tumor tissues had significantly poor survival by TCGA ccRCC datasets and verified by clinical samples as well as RCC cell lines. Overexpression of CRHBP suppressed cell proliferation, migration, invasion as well as apoptosis in vitro and in vivo. Moreover, the results of western blot analysis showed the effects of CRHBP via upregulating NF-κB and p53-mediated mitochondria apoptotic pathway. Our results suggested that CRHBP may be an effective target to treat ccRCC patients. Renal cell carcinoma (RCC), the third most common malignancy of the urinary system, is the sixth most frequently diagnosed malignant neoplasms in men and tenth in women accounting for 4% of all cancers , 2. CleaIn the last decade, great advancement of high-throughput sequencing technology enabled us to bring a comprehensive molecular insight on ccRCC , 6. AccoCRHBP, PSAT1 and RAB25 were significantly linked with the progression of ccRCC by support vector machine recursive feature elimination (SVM-RFE) and least absolute shrinkage and selector operation (LASSO) regression model as well as cox regression model. Four independent ccRCC microarrays were utilized to validate the crucial genes. At the same time, these genes were validated by 24 pairs of clinical ccRCC samples as well as four ccRCC cell lines. CRHBP, the gene with the most significant variation in expression was selected to elucidate the underlying molecular mechanism in vitro and in vivo. We explored the influences of CRHBP on tumor cell migration and invasion. In addition, we demonstrated that CRHBP could affect inflammation and apoptosis of ccRCC by targeting NF-κB and p53-mediated mitochondria apoptotic pathway.In our study, two popular microarray databases from The Cancer Genome Atlas (TCGA) and gene expression omnibus (GEO) are selected to integratively analyze and explore DEGs between ccRCC tumors and adjacent normal samples. We then performed survival analysis and regress analysis to define the relationship between ccRCC patients and important genes, and constructed a predictive model to assess the contribution of important genes that acted as suitable and valuable biomarkers for ccRCC. Three genes named http://www.ncbi.nlm.nih.gov/geo/) database and TCGA database, respectively. Besides, a total of 539 ccRCC clinical informations were also obtained from TCGA database (https://tcga-data.nci.nih.gov/tcga/). After excluding patients without entire follow-up information, 491 patients with clinicopathological parameters including age, gender, pathological stage (according to the seventh edition AJCC) and neoplasm grade remained available for analysis.The mRNA data expressions of GSE16449, GSE36895, GSE53757, GSE66270, GSE68417, GSE66272 and GSE76351, as well as other mRNA expression files (level 3), were downloaded from GEO (http://asia.ensembl.org/index.html). After preprocessing, Linear Models for Microarray (Limma) package and empirical analysis of digital gene expression data in R (EdgeR) package [P-value were individually calculated by the two packages as well as Benjamini–Hochberg method [P < 0.05 and |log2FC| ≥2 were considered statistically significant.Briefly, all raw gene datasets in GEO were background corrected, normalized separately by Robust Multi-Array Average method , and ann package  were useg method  in each http://david.abcc.ncifcrf.gov/) was used for elucidating the biological properties such as biological process (BP), cellular component (CC) and molecular function (MF) of the intersecting DEGs. Moreover, pathway enrichment analysis of the DEGs was performed using gene set enrichment analysis (GSEA). LASSO cox regression model [Database for annotation, visualization and integrated discovery (DAVID)  and pLVX-CRHBP-Puro obtained from Genechem  was used to infect 769P and ACHN according to the manufacturer’s protocol.HK-2 derived from normal human kidney, and Caki-1,769-P, ACHN as well as 786-O human RCC cell lines were purchased from the Cell Bank of Wuhan University and the Cell Bank of Type Culture Collection . Minimum essential medium and RPMI 1640 medium  supplemented with 10% fetal bovine serum (FBS)  and 1% streptomycin–penicillin  were, respectively, used for HK-2 and ACHN as well as Caki-1, 769-P and 786-O cell culture. All cell lines were maintained in 37 °C with 5% COCRHBP was calculated by 2−ΔΔCt method and GAPDH expressed level served as an internal control to normalize the expression. The primers involved in our study are listed as follows:Total RNA from RCC specimens and RCC cell lines were extracted using a Trizol reagent  and reverse transcribed to produce complementary DNA performing with RevertAid First Strand cDNA Synthesis Kit . qRT-PCR was performed using with SYBR Green Mix  in the CFX Connect Real-Time PCR Detection System  according to the manufacturer’s instructions. The relative expression of CRHBP forward: 5′-TTCATTACCATCCACTACGA-3′;CRHBP reverse: 5′-CTCCTGCTAAGACCACTC-3′;GAPDH forward: 5′-CCTTCATTGACCTCAACTACA-3′;GAPDH reverse: 5′-GCTCCTGGAAGATGGTGAT-3′.Briefly, total protein from tumor samples and cell lines was isolated using RIPA lysis buffer , and protein concentration was measured by BCA Protein assay kit . Western blot analysis of CRHBP , P-AKT , AKT , p-p65 , p65 , P-IκBα , IκBα , p-p53 , p53 , Bax , Bcl2 , Bcl-2 , Bcl-xl , Cytochrome c , β-actin  and GAPDH  was according to standard protocols. The blots were scanned with two-color infrared imaging system  after incubating with goat-anti-rabbit secondary body , and analyzed by image J software.4 cells per well in 100 µl of culture medium. CCK8 solution was added into each well, and the plate was then incubated for 1 h at 37 °C. Finally, treated cells were measured at the absorbance of 450 nm at the indicated time points.Cell Counting Kit-8  was employed to test the proliferation rate based on the manufacturer’s protocols. Briefly, 769P and ACHN cells with CRHBP overexpression and vector ccRCC cells were plated in 96-well plates in triplicate at 104 cells with serum-free medium were added into each upper chamber and complete medium with 10% FBS was added into the corresponding lower chambers to provoke the cell invasion. After incubation for 24 h, the cells that have passed through the membrane and attached into the bottom surface were fixed and stained with 4% paraformaldehyde and 0.1% crystal violet solution. Then, the stained cells were observed and counted in five random fields under inverted microscope, and the results were analyzed by ImagePro Plus 5.0 .769-P and ACHN cell lines in each group were respectively plated into 6-well plates for 24–48 h, and vertical wound was created by 200 µl sterile pipette tip when the density of cell reached at a density of 90%. Then, the cells were cultured in serum-free media and the image of cell migration was obtained 0 and 24 h later by inverted microscope . Matrigel chamber  was used for validating the migration ability of cells. Briefly, 3 × 10Cells were fixed with 4% formaldehyde for 20 min and washed with PBS, then permeabilized with 0.1% Triton X-100 and blocked with 0.5% goat serum for 1 h at room temperature. Subsequently, cell lines were incubated with CRHBP  and cleave caspase 3  antibodies for overnight, followed by Alexa Fluor 488 and 555  goat anti-rabbit secondary antibodies. The nuclei of the cells were stained with DAPI in dark, and images were captured by fluorescence microscope , and the results were analyzed by ImagePro Plus 5.0 .769-P and ACHN cells were infected with lentivirus, and stably overexpressed CRHBP; then the cells were stained with Annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry based on the manufacturer’s instructions .n = 5 per group). A total of 200 µl of cell suspension of 769-P cells (2 × 106) stably transfected with CRHBP lentivirus was subcutaneously injected into the left armpits of the mice. All mice were sacrificed after 28 days injection, and the xenografts were harvested for further assessment. The animal experiments involved in this study were approved by the Animal Care and Use Committee of Wuhan University Renmin Hospital.Five-week-old male BALB/c nude mice (20 ± 2 g) were obtained from the Animal Center of Wuhan University  and divided randomly into two group . All statistical analysis was performed with GraphPad Prism version 7.0 software , and the results were represented as mean ± standard deviation (SD) at three independent experiments. Differences with P value < 0.05 were considered statistically significant.Two groups were compared using Student's ariables , 15, andGene expression dataset of TCGA, GSE66272, GSE16449 and GSE76351 were download from TCGA and GEO database. After quality detection and normalization of raw data, 2334, 981, 574 and 692 DEGs were respectively screened, and the results of Veen plot showed that 100 DEGs are intersected differentially expressed identified from four datasets Fig. . As expeCRHBP, PSAT1, RAB25 were the most significant (P < 0.05) linked with patients clinicopathological characteristics  of CRHBP also showed the same result  Fig. . MoreoveAccumulating evidence indicated that ccRCC is not sensitive to chemotherapy, radiotherapy as well as targeted therapy, resulting in recurrence rate and 5-year survival rate of the patient is still up to 40% and 20% after radical nephrectomy, respectively . TherefoIn the present study, we revealed that the expression of CRHBP is aberrantly decreased in ccRCC samples and cell lines, and positively associated with overall survival rate of patients. Besides, the levels of CRHBP are closely related to patients’ clinical stage and histologic grade, implying that it has important role in the pathogenesis of ccRCC. Then, lentivirus with overexpressed CRHBP was transfected into ccRCC cell lines to prove the results of bioinformatic analysis. Functionally, CRHBP could suppress the cellular capacity of proliferation, migration and invasion. In addition, it promoted inflammation and apoptosis in 769P and ACHN via regulating NF-κB and p53-related apoptotic pathways. Consistently, the data of tumor xenograft model indicated that overexpression of CRHBP inhibited tumor growth and metastasis in mice. All these results support a notion that CRHBP may be acted as a potential biomarker for ccRCC diagnosis and therapy.CRHBP, a kind of circulating peripheral secreted protein expressed in hypothalamus cortical regions and pituitary gland, was reported to have effect on pregnancy, binge drinking as well as stress through binding corticotropin releasing factor –21. ReceUp to now, accumulating evidence reported that CRHBP could attenuate the detriment of binge drinking, stress and pregnancy mainly through binding to its receptor such as corticotropin releasing factor type 2 and 3 –22. CRHBAs we all know, inflammation is a vital trigger to the progression of cellular apoptosis . The NF-Together, present results demonstrate that CRHBP is a novel suppressor of ccRCC and further extend the notion that the underlying mechanisms of CRHBP anti-tumor capacity are associated with NF-κB pathway and p53-mediated mitochondrial apoptosis pathway. Nevertheless, there remain some limitations that are required to be solved. Whether there are two proteins or receptors that could interact with CRHBP to effect ccRCC progress because it is a secreted protein. Moreover, it is unclear how CRHBP regulates NF-κB signaling pathway and p53-mediated mitochondrial apoptosis pathway and whether it accepts the stimuli from environment and then still activates the pathways. In addition, CRHBP may act as a prognostic biomarker or potential target for ccRCC patient.