Lactobacillus (>100-fold), Bifidobacterium (>100-fold), and Collinsella (>9-fold) than kittens fed canned diets. There were relatively few differences in the predicted microbiome functions associated with the pre-weaning diet. Post-weaning diet affected the abundance of functional gene groups. Genes involved in vitamin biosynthesis, metabolism, and transport, were significantly enriched in the metagenomes of kittens fed the canned diet. The impact of post-weaning diet on the metagenome in terms of vitamin biosynthesis functions suggests that modulation of the microbiome function through diet may be an important avenue for improving the nutrition of companion animals.Dietary format, and its role in pet nutrition, is of interest to pet food manufacturers and pet owners alike. The aim of the present study was to investigate the effects of pre- and post-weaning diets (kibbled or canned) on the composition and function of faecal microbiota in the domestic cat by shotgun metagenomic sequencing and gene taxonomic and functional assignment using MG-RAST. Post-weaning diet had a dramatic effect on community composition; 147 of the 195 bacterial species identified had significantly different mean relative abundances between kittens fed kibbled and canned diets. The kittens fed kibbled diets had relatively higher abundances of  There is increasing interest in the effects of diet on the composition of intestinal microbiota in domesticated cats and dogs due to the clear links between microbiota and pet health123While much research has investigated the effects of specific dietary components  on intestinal microbial composition in laboratory settings, pets in the home environment are typically fed specific formats of pet food, generally either kibbled or canned diets. There is relatively little research on the impact of dietary format, but its role in pet health is of great interest to pet food manufacturers and pet owners alike. Commonly six dietary components are discussed in terms of dietary format – carbohydrate, protein, fat, water and vitamin and mineral contents. Kibbled diets tend to have higher carbohydrate contents and lower protein levels, whereas canned diets have very low carbohydrate content, and medium to high levels of protein and fat. Canned diets also contain approximately 75–80% moisture. Our previous research has shown large differences in the composition of faecal microbiota in the domestic cat associated with either short-term changes in dietPrior research has shown that taurine status of cats is affected by dietary format, with taurine availability lower in canned diets compared to kibbled diets. Intestinal microbiota have a role in overall taurine status7de novo biosynthesis1113Although vitamins can be obtained from a variety of foods, some vitamins , vitamin B9 (folic acid) and vitamin B12 ) can also be provided through microbial Solobacterium, Peptococcaceae, Clostridium, and Megamonas.To our knowledge, four studies have examined the function of the intestinal microbiota in the domestic cat161718The aim of the present study was to investigate the effects of pre-weaning (gestation and lactation) diet and post-weaning diet on the composition and function of faecal microbiota in the domestic cat through metagenome shotgun sequencing, focussing on vitamin and taurine metabolism.P < 0.05) by 17 weeks of age than those fed the kibbled diets (1.3 vs 1.4 kg (SEM 0.1) kg). The apparent digestibility of energy and protein was altered (P < 0.05) in response to post-weaning diet (P = 0.063) was similar between diets. Faeces from cats fed the canned diet contained significantly more energy (P = 0.006) and fat (P = 0.012), while faecal protein content was similar between cats on the two diets (P = 0.503).The growth rate of the kittens has been published previouslying diet , while fPaired-end sequences were deposited in the MG-RAST database and are publically accessible with the following metagenome identifiers; 4629274.3, 4629275.3, 4629276.3, 4629277.3, 4629278.3, 4629279.3, 4629280.3, 4629281.3, 4629282.3, 4629283.3, 4629284.3, 4629285.3, 4629286.3, 4629287.3, 4629288.3, 4629289.3, 4629290.3, 4629291.3, 4629292.3, and 4629293.3.Alignment against the M5NR database resulted in an average of 3,663,452 paired end sequences per sample  that could be taxonomically classified.Lactobacillus, Bifidobacterium, and Collinsella genera compared to kittens fed the canned diet post-weaning  that could be functionally classified.P = 0.001). Although no effect from the maternal diet was observed (P = 0.687), there was a significant interaction between maternal diet and post-weaning diet (P = 0.031). This observation was supported by discriminant analyses which showed that the kitten’s faecal metagenomes could be differentiated based on the mother’s diet  “Metabolism of cofactors and vitamins” pathways, were significantly enriched (FDR < 0.05) in the metagenomes of kittens fed the canned diet  comparedthiH and ribB occurred in metagenomes with a prevalence between 0.26% and 0.70% in K-C and C-C kittens, but <0.12% in K-K and C-K kittens. Four other genes involved in riboflavin biosynthesis, predicted to encode UbiB (EC:1.16.1.3/EC:1.5.1.41), RibH (EC:2.5.1.78), SsuE (EC:1.5.1.38 ) and AphA (EC:3.1.3.2), were also more abundant in kittens fed the canned diet . UbiB and SsuE are also involved in the conversion of riboflavin to other metabolites, which may explain the reduced faecal and urinary riboflavin concentrations observed when feeding low carbohydrate diets in catsBacteroidetes, Fusobacteria and Proteobacteria are the dominant phyla responsible for riboflavin synthesisfolC, which encodes dihydrofolate/folylpolyglutamate synthase, and suID which encodes dihydroneopterin aldolase, are prevalent among Streptococcus and Lactobacillus genomes59606162The most relatively abundant vitamin B-related sequences included those with hits to ThiH (K03150), involved in thiamine biosynthesis, and RibB (EC:4.1.99.12), required for riboflavin biosynthesis . Both thmenA were relatively more abundant in kittens fed the canned diet. These differences raise the possibility that more vitamin K2 is being produced by the microbial community in kittens fed the canned diet.In addition to changes in vitamin B-related functions, the post-weaning diet also affected proportions of vitamin K-related genes and functions in the faecal metagenomes . VitaminTaurine is an essential nutrient for the cat and therefore must be supplied by all complete and balanced diets. This is in part due to the high reliance the cat has on taurine to conjugate bile acids. The type of diet fed to the cat – i.e., kibbled or canned, determines the required level of taurine required to be supplied by the diet. For example, 1 g taurine per kg DM is required in kibbled diets whereas canned diets require 2 g taurine per kg DM dietTaurine is readily degraded by the intestinal microbiota766677Cats preferentially use taurine to conjugate bile acids. The principal bile acid in cats is taurocholic acid, and the bacterial degradation of taurocholic acid leads to loss of taurine, thus increasing the dietary requirement. Previous work has shown that cats fed kibbled diets had a reduction in the levels and composition  of faecal bile acid secretion compared to cats fed canned dietsRecent reviews have shown that Maillard reaction products are high in commercially available pet foods, especially canned formulationsGiven the changes observed in both vitamin B and K metabolism associated with dietary format, the impact of dietary format on the long term nutrition via vitamin status of the cat is one that should be investigated in future studies.To our knowledge this is the first time the effects of pre- and post-weaning diets on the genetic functional potential of the faecal microbiota have been investigated in the domestic kitten. Although the mother’s diet did not significantly alter the composition of the microbiome, a significant interaction with post-weaning diet was observed. The impact of post-weaning diet on the metagenome in terms of energy and carbohydrate metabolism, and vitamin biosynthesis functions, suggests that modulation of microbiome function through diet may be an important avenue for improving the nutrition of companion animals.All procedures undertaken on the cats were performed in accordance with relevant guidelines and regulations of the Animal Welfare (Companion Cats) Code of Welfare 2007 and were approved by the Massey University Animal Ethics Committee (MUAEC 10/108). All cats used were owned by Massey University and were housed at the Centre for Feline Nutrition  according to the Animal Welfare (Companion Cats) Code of Welfare 2007.ad libitum daily to allow for normal growthAnimals and housing have been described in detail previouslyCommercially available kibbled  and canned diets  were utilised in this study  was determined during week 17. Individual total food intake and refusals were recorded daily and total faecal output was collected over a five day period and frozen (−20 °C), freeze dried, and ground for analysis. The diet and faeces were analysed for moisture using a convection oven at 105 °C , ash using a furnace at 550 °C (AOAC 942.05). Crude protein and crude fat were determined using the Leco total combustion method (AOAC 968.06) and acid hydrolysis/Mojonnier extraction (AOAC 954.02), respectively. Gross energy (kJ/g) was determined using bomb calorimetry.At 17 weeks of age, the kittens were housed individually for 24 h and a fresh faecal sample was collected from each animal within 15 min of excretion, snap-frozen in liquid nitrogen and stored at −85 °Cin silico, and taxonomically and functionally classified using the MG-RAST pipeline (version 3.2)−8 were removed from the dataset. Similarly, only taxa that occurred in at least 5 samples with a relative abundance greater than 1 × 10−4 were included.Metagenomic DNA extracted from faeces (week 17) was analysed by paired-end (PE100) shotgun sequencing using the Illumina Hi-Seq 2000 platform from Macrogen Inc. , a commercial sequencing provider. Paired-end sequences were joined P values for multiple testing was performed using the Benjamini & Hochberg false discovery rate (FDR) method, with FDR < 0.05 considered significant. Permutation MANOVA and Procrustes rotation analysis was performed using the adonis and procrustes functions, respectively, as implemented in the vegan packageStatistical tests were performed using R 3.1.3How to cite this article: Young, W. et al. Pre- and post-weaning diet alters the faecal metagenome in the cat with differences in vitamin and carbohydrate metabolism gene abundances. Sci. Rep.6, 34668; doi: 10.1038/srep34668 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The use of endangered animal products in traditional Chinese medicine (TCM) and other ethno-medicines is culturally widespread across many regions of Asia. In the present study, traditional efficacies of seven types of animal horn including antipyretic, sedative and procoagulant activities were evaluated. Shotgun proteomic analysis was performed on material from horns following separation into soluble and insoluble fractions. Over 200 proteins were identified in each sample using nano LC-MS/MS, and these were classified according to their molecular function and cellular component using principal component analysis (PCA). The results indicated that seven horns showed antipyretic, sedative and procoagulant effect. Proteomic analysis showed that YH and WBH were similar to RH in terms of protein profile, and GH was similar to SAH. In addition, YH and GH were similar to RH in their cellular component classification profile. PCA based on the composition of keratin and keratin-associated proteins showed that constituents of WBH and GH were similar to RH and SAH, respectively. This is the first analysis of the protein content of animal horns used in TCM, and it is effective to substitute the horn of endangered animals with sustainable alternatives from domestic animals. Rhinoceri Asiatici Cornu, RH), Saiga antelope  and water buffalo  have been applied in traditional Chinese medicine (TCM) for over 2000 years143The horns of rhinoceros , in which RH was the most important medicine. Currently, RH in Xijiao Dihuang decoction was normally replaced by WBH and showed similar clinical curative effect. It was also reported that both SAH and GH showed apparent sedative activity and antihypertensive activityGazella subgutturosa horn and Mongolian gazelle horn as medicinal materials  due to their similar effects compared with SAH. In addition, in order to achieve considerable efficacy, the dosage of WBH and GH used as substitutes of RH and SAH should be 5–10 fold, or even more than 10 fold12There are scientists proposed to search for substitutes of threatened animal organs based on their pharmacological testing81011Animal horns are pointed projections of the skin on the heads of animals consisting of a covering of horn sheath surrounding a core of living bone. Unlike the horns of Antelope, water buffalo and other Artiodactyla animals, RH lacks the bony core and is composed entirely of horn material14Despite increasing research on WBH, GH, YH, etc. applied as alternatives of RH or SAH in TCM clinic, mechanisms of action remain unknown. The complex structure of animal horn makes it difficult to identify and separate the active components. Fortunately, the shotgun technique is known to be compatible with corneous materials such as hair, nail, and wool, and proved to be suitable for animal horn. Therefore, in the present study, shotgun proteomic analysis was applied to analyze constituents of animal horns. The large-scale identification of protein components is necessary for a molecular-level understanding of this complex material. In recent years, advances in mass spectrometry and database searching have assisted the identification of proteins in complex cross-linked structures18Bencao Gangmu  or some other traditional literatures, animal horn derived TCMs were usually applied for treating heat syndrome, relieving convulsion, and reducing blood bleeding. Therefore, bio-effect evaluation of animal horns including antipyretic, sedative, and procoagulant activities were evaluated in the present study, which may help to illustrate the bio-effect characteristics of various animal horns.Furthermore, according to the documentation of p < 0.05, p < 0.01). As shown in 240 min of each dose of animal horns was calculated. Among the seven horns, only the AUC240 min of Tibetan antelope horn (TAH) at three doses was higher than paracetamol. The antipyretic efficacy strength depicted by AUC240 min at high dose showed the order of seven horns was as follow: WBH > RH-1 > YH > RH-2 > GH > SAH > TAH. Based on these results, it is obvious that RH exhibit the strongest ability in lowering down rabbit body temperature. In addition, WBH continuously lower down the rabbit body temperature during the 240 min, whose AUC value was the lowest among seven horns at high dose level. In the present study, dosage of animal horns was identified according their clinical use, the high dosage of RH and SAH was 0.06 g/kg, while WBH and YH was 1.5 g/kg. Therefore, the dose of WBH or YH was 25 times higher than RH. At the present dose level, the AUC240 min of WBH or YH was similar with RH. It is indicated that WBH or YH exhibit similar efficacy strength compared with RH in the antipyretic activity test. It is consistent with the fact of WBH clinical dose over 10-fold higher than RH. RH, WBH and YH also can be observed stronger antipyretic efficacy than SAH, TAH and GH.The effect of animal horns on the fever rabbit was shown in p < 0.05). RH-1, RH-2, SAH, TAH, WBH and GH could significantly reduce the temperature of fever rats 2 h after oral administration . Furthermore, TNF-α, IL-6, and PGE2 levels in the plasma of rats were determined. One hour after oral administration with seven horns, RH-1 could significantly reduce the levels of TNF-α, IL-6, and PGE2 , RH-2 could reduce IL-6 level (p < 0.05), SAH and GH could reduce TNF-α and PGE2 level (p < 0.05), TAH could reduce TNF-α level (p < 0.05). Two hours after the oral administration, RH-1 also showed effects on reducing IL-6, and PGE2 level (p < 0.05), SAH, TAH, WBH, YH and GH could significantly reduce the levels of TNF-α and IL-6 , in addition, WBH significantly reduced the PGE2 level (p < 0.05). Fever model induced by yeast is considered as an infectious fever model and the inflammatory cytokines, such as TNF-α, IL-1β, IL-6, PGE2, etc. were stimulated by yeast, and then cytokines elevate the body temperature. These cytokines play key roles in regulating fever. Therefore, TNF-α, IL-6, and PGE2 in the plasma of fever rats were determined after treating with animal horns in the present study. As shown in the results, RH-1 could reduce the levels of TNF-α, IL-6, and PGE2 in two periods after oral administration. Compared with RH-1, WBH and YH do not show significant cytokines-reducing efficacy in the first period after oral treatment. Two hour after WBH or YH oral treatment, levels of TNF-α, IL-6, and PGE2 decreased significantly. It can be indicated that RH-1, WBH, and YH can relieve fever by reducing levels of cytokines, only but RH-1, WBH and YH exert their antipyretic effect at different time after the treatment. Furthermore, SAH and GH showed similar cytokines-reducing efficacy. In two different periods after oral treatment with both SAH and GH, the levels of cytokines significantly decreased. As available sustainable alternatives, WBH, YH and GH could reduce the levels of cytokines in the plasma of fever rats, which showed similar cytokines-reducing efficacy compared with RH and SAH.The effect of animal horns on the fever rat was shown in p < 0.05), TAH could decrease the mice spontaneous movements, but showed no significant difference with control (p > 0.05). 40 min after oral administration with horns, RH-1 and RH-2 significantly decreased mice voluntary movements (p < 0.05), 60 min after administration, RH, SAH, WBH, YH and GH could decreased mice voluntary movements significantly (p < 0.05). As shown in 120 min of each dose of animal horns was calculated. According to the AUC120 min value at high dosage, the sedative efficacy strength order was as follow: YH > RH-2 > RH-1 > GH > TAH > WBH > SAH. The AUC value could be used to evaluate the continuous effects, not only an efficacy data at certain time point. As a result, YH exhibit better sedative effect than RH and SAH based on the high dose AUC120 min value. AUC120 min value of GH and TAH was higher than SAH, which indicated that GH or TAH showed better sedative effect than SAH, despite TAH did not significantly decrease the mice movements at each time point. As a result, in general, sedative effect of RH, WBH and YH was better than GH, TAH and SAH.As shown in p < 0.05; p < 0.01). According to the results, the order of procoagulant activity at high dose was as follow: RH-2 > SAH > TAH > GH > RH-1 > WBH > YH. Except RH-2, the procoagulant activity of other six horns was similar. However, at mid dose the order of procoagulant activity was described as: TAH > SAH > RH-2 > GH > RH-1 > WBH > YH. RH-2, SAH and TAH exhibit significant procoagulant activity and the procoagulant efficacy of WBH and YH was slightly lower than other horns.As shown in Bencao Gangmu, Shennong Bencao Jing . Under their recommended dose level, horns all exhibit three traditional bio-effects above , but represent different efficacy priorities. RH-1 and RH-2 exhibit excellent antipyretic and sedative effect, followed by WBH and YH which exhibit satisfied antipyretic and sedative effect. At the high dosage level, RH, WBH, SAH, and GH could significant reduce the levels of cytokines, such as TNF-α, IL-6, and PGE2. The antipyretic and sedative effect WBH or YH was slightly lower than RH. Although SAH, TAH and GH also showed antipyretic and sedative effect, their effect was lower than RH, WBH or YH. In addition, RH-2, SAH and TAH exhibit apparent procoagulant activity, which slightly better than WBH and YH. Therefore, it was indicated that RH possesses wonderful antipyretic, sedative and procoagulant activity, WBH and YH tend to show significant antipyretic and sedative activity, while SAH, TAH and GH tend to exhibit significant procoagulant activity.Seven types of horn all possess antipyretic, sedative and procoagulant activity, which confirmed their documented traditional efficacies. All their dosage was identified according to the traditional literature, such as Animal horns such as RH, SAH, GH and WBH have been used in TCM for thousands of years, and TAH and YH have been used as ethnomedicines in Tibet for a comparably long time. In order to investigate the protein content and provide evidence for finding suitable sustainable substitutes to these endangered animal parts, we analyzed the proteomes of seven animal horns . In TCM, animal horns are usually powdered and swallowed with water directly, or extracted and decocted for oral administration6Among the proteins identified, keratins and KAPs, junctional proteins, ribosomal proteins, initiation/elongation factors, histones, heat shock proteins and 14-3-3 proteins were abundant. Between 12.6% and 23.7% of these proteins were present only in the solubilized fraction, while 54.9%–78.0% were only in the particulate fraction, and 8.8%–21.3% were observed in both fractions . The higDesmosomal structural proteins such as desmoplakin, plakophilin, desmoglein, desmocollin, envoplakin and plakoglobin were identified, all of which play important roles in structural aspects of the cytoskeleton or in providing essential adhesion structures and linking transmembrane desmosomal cadherins to the cytoplasmic keratin filament networkGO analysis indicated that many of the identified proteins in the solubilized fraction (17.6%–24.8%) were related to the cytoskeleton, including keratins and KAPs that were the most abundant structural proteins in animal horns, and junction plakoglobin and tubulin. Keratins form a cytoplasmic network of intermediate filaments and KAPs interact with these filaments and stabilize disulfide linkages.The insoluble fractions contained between 23.8% and 28.5% cytoplasmic proteins including type I cytoskeletal keratin, plakoglobin, elongation factor, tubulin, and 40S and 60S ribosomal proteins. These results suggest that most keratins are solubilized, however the high degree of cross-linking results in a large number of soluble proteins interacting with or co-localizing with the insoluble material, and this may be functionally important in horn epidermal cellsIn the case of RH, 227 proteins were identified, of which 60 were found exclusive to the soluble fraction, and 208 proteins were detected only in the insoluble fraction. Among the identified proteins in the soluble fraction, over 95% were assigned to a predicted function and classified into seven categories according to molecular function. The majority of these  were classified as structural proteins, and 21 of these were keratins . Other aOf all proteins identified in the insoluble fraction, approximately 85% were assigned to various molecular functions. The majority of these were classified as having binding activity  including protein binding, nucleotide binding, metal ion binding, and DNA or RNA binding . The funCellular component classification analysis of the 227 identified RH proteins placed 60 soluble fraction proteins into 12 categories . The majThe proportion of soluble proteins with a structural function was higher than the insoluble fraction. The soluble fraction mainly consisted of keratins, while many of the insoluble proteins belonged to the binding activity category. The other animal horn samples were subjected to the same analysis, and the similarities and differences between the seven animal horn samples were investigated using PCA.PCA of molecular function reduced eight categories into three principal components  which accounted for >77% of the entire variance . Based oR2X of the PCA model was 0.771. The score plots of the PCA model , Rhinoceri Africani Cornu (RH-2), and Pantholopsis Hodgsoni Cornu  were purchased from Jiangsu Medicinal materials company . Saigae Tataricae Cornu (SAH), Bubali Cornu (WBH) and Caprae Hircus Cornu (GH) were purchased from Jiangsu Medicine Company . Bovis Grunniens Cornu (YH) was purchased from a slaughter house located on Linkuo North Road in Lhasa, Tibet. All horn samples were authenticated by Prof. Dr. Jin-ao Duan. All horn products were obtained as pieces form initially, and then were powdered. Detailed information is listed in ad libitum. Animal welfare and experimental procedures were strictly in accordance with the Guide for the Care and Use of Laboratory Animals. This study was approved by Nanjing University of Chinese Medicine.Adult New Zealand rabbits (2.0–2.5 kg) and mice (18–20 g) of both sexes, obtained from the Center of Experimental Animals, China Pharmaceutical University. Animals were housed under standard conditions of temperature (22 ± 2 °C); relative humidity (55 ± 5%) and light (12 h light/dark cycles) were used. The animals were fed with standard diet and water Escherichia coli endotoxin at a dose of 20 ng/kg. Rectal temperature was measured 6 h after the endotoxin injection, and rabbits, developing significant hyperthermia were used only. Then the rabbits were orally administered with horn powder (dosage of horns was shown in The antipyretic activity test was performed according to the previous method2 levels were measured by enzyme linked immunosorbent assay (ELISA) kit .The antipyretic activity test on rat was also performed according to the previous methodThe test was performed according to the method of Connor with slight modificationThe blood clotting time (CT) experiment was performed according to the method of Singh to evaluate the procoagulant activity of horns, with slight modificationTo quantify the antipyretic and sedative activity of horns, area under curve (AUC) were introduced to evaluate the effect and effective time in pharmacology experiments. AUC was calculated by Graphpad Prism 5 software.It was performed as described with slight modificationsAliquots of the soluble (first extract) and insoluble material were incubated for 0.5 h in 2% SDS, 20 mM DTT, and 50 mM phosphate buffer (pH 7.8) then incubated at room temperature for an additional 0.5 h following addition of 40 mM iodoacetamide. Proteins were precipitated from the soluble extract by addition of 3 volumes of ethanol. The soluble and insoluble protein samples were rinsed with 70% ethanol, then with freshly prepared 0.1 M ammonium bicarbonate, and were finally resuspended in fresh 0.1 M ammonium bicarbonate adjusted to 2 M urea. To each suspension bovine L-1-tosylamido-2-phenylethyl chloromethylketone-treated trypsin  was added to 1% (w/v). After 6–8 h at 37 °C, samples were stirred overnight at room temperature. Most of the detergent-insoluble material was solubilized, and virtually all of the detergent-soluble material was solubilized in this way.Mass spectrometric experiments were performed using a nano LC LTQ-Orbitrap mass spectrometer  equipped with a nano-electrospray ion source and an Easy II nano LC, as described previously with some modificationsth April, 2014). Spectra were also searched against an equal number of decoy sequences to estimate the false discovery rate (FDR) as described previouslyTandem mass spectra were extracted with Xcalibur version 2.0.7. All MS/MS samples were analyzed using X!Tandem  which was set up to search the bovidae_uniprot-taxonomy_9895 database  were performed using SPSS 16.0 and Simca-P.www.uniprot.org) to obtain Gene Ontology Annotation (GO) using the molecular functions and cellular component categories.GI numbers of identified proteins were matched to the UniProtKB database .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Increasing evidence supports the association between hyperuricemia and incident chronic kidney disease (CKD); however, there are conflicting data regarding the role of hyperuricemia in the progression of CKD. This study retrospectively assessed the longitudinal association between uric acid (UA) level and CKD progression in a Chinese population lived in Taiwan.2 or requiring renal replacement therapy.Patients with physician diagnosis of hyperuricemia or receiving urate-lowering therapy between 2003 and 2005 were identified in the electronic medical records (EMR) of a tertiary medical center and were followed up until December 31, 2011. Patients were divided into four UA categories at the cut-off 6, 8, and 10 mg/dL. CKD progression was estimated by the change of estimated glomerular filtration rate (eGFR) in the linear mixed models. Kidney failure was defined as an eGFR less than 15 mL/min/1.73 mβ = -9.6, 95% CI -16.1, -3.1), comparing to those with a UA level less than 6 mg/dL. When stratifying patients into four UA categories, all three hyperuricemia categories  associated with a greater decline in eGFR over the follow-up period with an increasing dose-response, comparing to the lowest UA category. The risk of progression to renal failure increased 7%  for each 1mg/dL increase in baseline UA level. The influences of hyperuricemia on eGFR decline and the risk of kidney failure were more prominent in patients without proteinuria than those with proteinuria.A total of 739 patients were analyzed. In the full-adjusted model, patients with a baseline UA level ≥6 mg/dL had greater decline in eGFR ((Our study showed a higher uric acid level is associated with a significant rapid decline in eGFR and a higher risk of kidney failure, particularly in patients without proteinuria. Our findings suggest hyperuricemia is a potential modifiable factor of CKD progression. Chronic kidney disease (CKD) is a global health care burden . IdentifAs uric acid is primarily excreted by the kidneys, it is difficult to evaluate the causal influence of uric acid on the progression of CKD in epidemiological research . AlthougThe current study aimed to contribute evidence from a longitudinal study to this ongoing debate about the role of serum UA level on CKD progression in a Chinese population-based sample from Taiwan. We also summarized published evidence about the effect of serum UA level on CKD progression.This study was approved by the Institutional Review Board of China Medical University Hospital (CMUH). Informed consent was not obtained from the study participants because the data was analyzed anonymously and was in accordance with Institutional Review Board guidelines. The Institutional Review Board has verified the anonymity of data analysis performed in this study.We conducted a retrospective cohort study at a tertiary medical center in Taiwan, which has adopted electronic medical records (EMR) since 2001. We consecutively selected patients who visited CMUH between 2003 and 2005 and have been diagnosed hyperuricemia   and/or r-1.154 × age-0.203 × 1.212 [if black] × 0.742 [if female]) [2 [2 and/or receiving chronic dialysis therapy) using cox proportional hazard model.The eGFR was estimated using the abbreviated Modification of Diet in Renal Disease (MDRD) equation  . The basale]) [2 . Daily pale]) [2 . DiabeteBaseline characteristics are presented for the total study population stratified by serum UA level  with UA<6 as the reference group, based on the saturation point for monosodium urate –26 and tThe primary outcome was individual eGFR trajectory during outpatient follow-up. As repeated eGFR measurements are correlated within each patient, linear mixed model (LMM) with random intercept and random slope was used to evaluate the longitudinal change in eGFR for each UA category at baseline. Based on Akaike Information Criterion (AIC), the unstructured covariance matrix was selected to handle the with-person correlation and improve the data fitting. For incident kidney failure, multiple Cox proportional-hazard regression model was performed using age as the time scale. We treated age at each examination as a time varying co-variable. To handle left-truncation induced at the time of selection and appropriately align risk sets on the age scale, the late entry method was conducted using age at baseline as the individual entry time. Multivariable modeling was performed after adjusting for potential confounders: age at examination, sex, body mass index (BMI), presence of cardiovascular disease (CVD), systolic blood pressure, diabetes mellitus (yes vs. no), proteinuria (yes vs. no), baseline serum creatinine, ACEI/ARB use (yes vs. no) and allopurinol use (yes vs. no). A priori exploratory subgroup analysis stratified by proteinuria status was also performed using similar modeling process. The interactions between uric acid and proteinuria were tested in multivariable models for serial eGFR change and risk of ESRD. All analyses were conducted using Stata, version 12 . The 2-sided statistical significance level was set at α = 0.05.A total of 739 patients were analyzed with a mean follow-up of 4.25 years.p-trend <0.001 and <0.001, respectively, p -trend <0.001, Baseline characteristics across baseline UA categories are summarized in β = -9.6, 95% CI -16.1, -3.1), had a greater decline in eGFR during follow-up, comparing to those with UA level < 6 mg/dL. For every one mg/dl increase in baseline UA level, the decline in eGFR was significantly greater during follow-up   even after adjusted for baseline UA level  than in those with proteinuria    (d<0.001) , Model 4 p<0.001), so did UA level . In inteP value for interaction term between uric acid level and proteinuria status are <0.05 in model 3 and 4).A total of 144 patients developed kidney failure during the follow-up period. Using multivariable Cox proportional hazards models, the risk of progression to kidney failure increased by 7%  for every 1 mg/dL increase in baseline UA level . ACEI/AROur study supports that hyperuricemia is associated with a greater decline in renal function and a higher risk of progressing to kidney failure. The influences of hyperuricemia on renal function decline and the risk of kidney failure are greater in patients without proteinuria than those with proteinuria. We also found that patients treated with allopurinol had a greater eGFR decline, though the risk of progression to kidney failure did not significantly increase, comparing to those who did not use urate-lowering agents.2, the subjects with hyperuricemia had an average annual decline of eGFR of 2.5 ± 9.5 mL/min/1.73 m2, that was almost twice faster than those of patients with normal uric acid levels [et al. followed 824 healthy people for about 5 years and found that UA was an independent risk factor for a significant loss of renal function (defined as a decrease in eGFR ≥10 ml/min/1.73m2 over 5 years)[et al. also found the adjusted odds ratio for rapid progression of kidney function (defined as a decrease in eGFR ≥ 3 mL/min/1.73 m2 / year) was 1.14  for each 1 mg/dL increase in UA level in 5,808 elders in the Cardiovascular Health Study [Evidence for the role of hyperuricemia in the CKD progression is controversial. Althougd levels . Bellomo 5 years). Chonchoth Study . In our 2/year) [et al. found that UA levels at one month after transplantation were not associated with the 3-year graft function [The association between hyperuricemia and the rapid decline in renal function has also been evaluated in subpopulations, including patients with diabetes, IgA nephropathy, and kidney transplantation  20, 43––43–46. I2/year) . For IgA2/year) , 46. Reg2/year) –51. In tfunction –50. The Although our findings suggest UA might be a modifiable risk factor for the progression of CKD, we did not observe the beneficial effect of allopurinol on CKD progression. One important question is whether the effect of urate-lowering agents can directly abrogate the progression of CKD in humans. According to a recent systemic review including 8 randomized trials, meta-analysis of 5 trials (346 participants) using eGFR decline as the study outcome showed no difference between allopurinol and placebo group while the other 3 trials (156 participants) using change in serum creatinine as the end-point found patients received allopurinol therapy had significantly lower serum creatinine concentrations comparing to placebo group. The disSeveral potential mechanisms that UA may hasten the progression of renal function have been proposed. The precipitation of urate in renal tubules may cause “uric acid nephropathy” . BesidesStrengths of this study include the longitudinal methodology to avoid issues of cross-sectional design such as potential reverse causality and the laboratory’s consistency in the measurement of serum UA and creatinine throughout the duration of follow-up. From our study, uric acid is not just solely as a marker, but a true mediator for CKD. Moreover, compared to previous studies using relatively small sample size (<100) to evaluate the role of urate-lowering agents in the CKD progression in a shorter period of follow-up (less than 2 years), we estiOur study showed a higher UA level was significantly associated with a greater decline in renal function and a higher risk of progressing to kidney failure in a Chinese population. The influence of hyperuricemia on renal function decline and risk of renal failure was greater in subjects without proteinuria than in those with proteinuria. In addition, allopurinol did not have a benefit in mitigating CKD progression. Our findings support that hyperuricemia as a potential modifiable risk factor for CKD progression, particularly in patients without proteinuria.S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Society is increasingly demanding a more sustainable management of agro-ecosystems in a context of climate change and an ever growing global population. The fate of crop residues is one of the important management aspects under debate, since it represents an unneglectable quantity of organic matter which can be kept in or removed from the agro-ecosystem. The topic of residue management is not new, but the need for global conclusion on the impact of crop residue management on the agro-ecosystem linked to local pedo-climatic conditions has become apparent with an increasing amount of studies showing a diversity of conclusions. This study specifically focusses on temperate climate and loamy soil using a seven-year data set. Between 2008 and 2016, we compared four contrasting residue management strategies differing in the amount of crop residues returned to the soil (incorporation vs. exportation of residues) and in the type of tillage (reduced tillage (10 cm depth) vs. conventional tillage (ploughing at 25 cm depth)) in a field experiment. We assessed the impact of the crop residue management on crop production , soil organic carbon content, nitrate ( Once a crop is harvested, farmers have to decide what to do with the remaining crop residue (the above ground biomass that is cut but not harvested). Residues can be either exported and valorised as co-products , or restored to the soil as such or after being burnt. Returning straw directly to the field has been promoted as a source of organic matter and a way to increase soil water holding capacity and its overall quality. As such, it is thought to help maintain, or even to some extent restore, soil fertility . If the The precise impact of the restitution (or not) of residues and of the choice of tillage system to apply to the soil-plant system remains unclear and seems to be highly dependent on the pedo-climatic conditions  . For insAs The literature on the effect of residue management on nitrogen (N) or phosphorus (P) uptake by plant is equally dispersed with no for N: ; positivInteractions between crop residue management and the soil-water-plant system are complex and inherently depend on the pedo-climatic conditions. Local assessment and system approach are therefore necessary to come to relevant guidelines for residue management under specific pedo-climatic conditions. The objective of our study was therefore to determine the effects of contrasting crop residue management strategies on crop production and components of the soil fertility, over a period of several years. Regarding crop production, we studied how residues management strategy impacts on germination rate, biomass production and yield elaboration, along with N, P and K exportation. The soil fertility components that were dynamically followed were SOC, N, P and K contents and their repartition within the soil profile. The experiment was conducted in the loam belt under temperate climatic conditions, taking into account common crop rotations and local farming practices.The field experiment  was established on 1.7 ha of the experimental farm of Gembloux Agro-Bio Tech, University of Liège, Belgium in 2008 and yield measurements started in 2010. The soil is a Cutanic Luvisol . AccordiThe climate is temperate  the fate of the crop residue and (ii) the type of tillage. Firstly the residue fate can be restitution (IN) or exportation (OUT). It has to be noted that stubble and chaff are always left on the fields, even if the rest of the residue is exported. Secondly, we considered two tillage types see : conventBrassica napus) in 2008–09, three consecutive years of winter wheat (Triticum aestivum) in 2009–10, 2010–11 and 2011–12, mustard  cover crop in 2012–13, faba bean (Vicia Faba) in 2013, winter wheat in 2014, oats (Avena sativa) and peas (Pisum sativum) mixed as cover crop in 2014–15, and finally maize (Zea mays) in 2015. Sowing densities were 300 kernels/m2 for winter wheat, 50 kernels/m2 for faba bean, and 13 kernels/m2 for maize. Sowing process is detailed for each crop in The crop rotation during the experiment was: rapeseed  followed the regional standards depending on the type of crop. Rapeseed received two applications  are available in 3 and HClO4 (1:1) (15 ml per g of sample). K content was measured by a flame atomic absorption spectrometric method (Spectrometer Varian 220). P was measured by colourimetry with molybdate and ammonium vanadate at 430 nm . NPK content (kg/ha) were calculated by multiplying the nutrient content (%) by the biomass of the residue or grain (kg/ha).We monitored the germination rate and growth dynamics during the season with an adapted protocol for each crop type . The det2Cr2O7 and H2SO4; titration of the excess of K2Cr2O7 with Mohr Salt ((NH4)2Fe(SO4)2 ⋅ 6H2O). Available soil nutrients were measured by stirring a 10 g sample of soil (air-dried and sieved at 2 mm) for 30 min in 50 ml of solution  to form a composite sample per plot at 0–10 cm, 10–20 cm and 20–30 cm depth. The fall sampling was usually either made after spring crop harvest and before winter wheat sowing or after winter wheat harvest and cover crop sowing. The spring sampling was made when climatic conditions were again favourable for winter wheat growth or after spring crop sowing. TOC was determined on a 1g of dry soil (ground at 200 µm) by the Walkley-Black method : oxidati pH 4.65 ). After In addition to the two overall soil sampling campaigns per growing season, soil nitrate content was measure more frequently to catch the dynamic of uptake during the growing phase of the main crops. Composite humid soil samples based on eight subsamples (sampled with gouge auger of 2 cm diameter) were used per plot at three depths: 0–30 cm, 30–60 cm and 60–90 cm. We used KCl extraction and a colourimetry method of reduction of nitrate to nitrite (using Cadmium or Hydrazine) with a determination of nitrite ions by the modified Griess-Ilosvay reaction .versus OUT treatments, and, on the other hand, RT versus CT treatments. These comparisons were then immediately made on the basis of the results of the 2-way ANOVA test. Contrarily, when an interaction between the fixed factors was significant, the four treatments  were intercompared and ranked using a post-hoc test (Student-Newman-Keuls—SNK). Analyses of variance (2-way ANOVA) and SNK tests were performed with the agricolae package  for IN plots (55.7 t/ha) compared to OUT plots (26.8 t/ha)  and S4 tWhile the stock , although significant differences were observed during some specific years , was 11.7 g/kg. After 8 years, the different treatments (tillage or residue management) did not have a significant effect on the overall TOC content evolution  in the reduced tillage treatments  and RT-IN from spring 2014 onwards ). In the top layer (0–10 cm), these treatments showed a higher decrease in P content than deeper in the profile. Our data do not suggest any significant impact of crop residue on P stocks in the soil .Over the course of the experiment, P content in soil significantly decreased in the 0–30 cm soil layer for all crop residue treatments, due to the absence of fertilisation during the course of the experiment . In addiF = 0.06, p = 0.81), except for the last 2 years of the experiment (2015–2016), where IN plots showed a higher K content. Also for K a stratification was visible  with decreasing concentrations from top to bottom.As for P, K content in soil decreased from the beginning of the trial, as no K fertilisation was applied . No partThe presence or absence of extra residues did not affect germination rate, except for winter wheat in 2013–14 where we observed a lower germination rate in the RT-IN treatment. Tillage type, however, did result in differences in germination rate. In When looking at the dynamics of crop growth, except for two sampling dates on ears’ growth and two sampling dates on shoot’s growth, no interactions were found between tillage and residue management treatment. Observations and results of the ANOVA and SNK analysis are reported in the supplementary material separately for ears and shoot growth –S11. Thevs. OUT) were observed for crop development. Incorporation of residues (IN plots) negatively impacted the dynamic of ears and shoots during the first two crop seasons (winter wheat seasons 2009–10 and 2010–11). These differences appeared in both cases after the ear emergence stage. These were more pronounced for the shoot than the ear biomasses. In 2010, with weather conditions close to historical means, lower biomass under IN treatments decreased over the crop growth period, but no negative impact on yields were observed when residues were returned to the soil. In 2011, characterised by a spring drought, the exportation of residue (OUT) was favourable to the shoot development and to the early growth of ears. However, for this season, the final yield was not impacted by the residue management treatment (IN vs. OUT), as detailed in the next section. Finally, one could also notice that at the end of the 2014 crop season, while no differences were observed all along the season, a statistical difference was reported on the last sampling date in favour of IN plots. For all other sampling dates, no statistical differences were reported.Differences between residues management treatments (IN Reduced tillage mostly negatively impacted shoot crop development of winter wheat (2010–11), faba bean (2013) and maize (2015)  and S9. Yearly grain yields were in general not influenced by residue fate or soil tillage  and S3.The cumulative grain yield since 2010 was significantly lower under reduced compared to conventional tillage −3.4%, . No effeP-value: 0.03) and a slightly lower K content under conventional tillage in 2 years out of 5 .There was no significant effect of the treatments on NPK content of the harvested grain or seeds , except No correlation was seen between germination rate and shoot dry weight except for faba bean (2013) and a slight effect for winter wheat (2010–11) . Grain yA principal component analysis was performed to study the relationship between soil and plant parameters . Plant agrain, Pgrain and Ngrain) were favoured by conventional tillage, as illustrated by the discrimination of treatments along the second component (Y-Axis) of the PCA. Similarly, it seemed that the soil parameters  were more positively influenced by the crop residue retention (X-Axis) of the PCA.The two principal components allowed to explain respectively 35% and 25% of the variance. Treatments were easily differentiated by the two principal components . It appeetention , as illuOverall, tillage influenced crop production more strongly than import or export of residues. The strongest effect was seen in terms of germination rate and was even stronger for residue incorporation treatments. RT resulted consistently in lower germination rates as also shown by Several studies report higher crop production levels under CT under temperate climate, when compared to RT . HoweverThe differences observed for germination rates or during crop growth seem to have little influence on crop yield. A similar lack of correlation between shoot biomass and grain yield, as observed most strongly for faba bean and wheat (2014) in our study, has previously been observed for legume crops . For winExcept for the winter wheat 2010–11, our results did not show an increase in N grain stock. This observation is in agreement with vs. OUT).Over the seven years of this experiment, the effects of crop residue management on soil fertility parameters showed few statistical differences in the early time of the trial. However, the results were slowly magnifying, up to the point where differences became more systematically significant, with clear stratification occurring over the different soil layer of the ploughing depth. Furthermore, our results  confirmed a clear link between soil TOC, K content, and to a lower extent P content, with residues management treatment  but this decrease is slight. Compared to P content, K content of residues was much higher. Furthermore, as the mineralisation process is not involved, K is released in soil solution as soon as the plant cells were dead . These cWhen looking within the available choices among the soil and crop management techniques, crop production remains one of the most important drivers of farmer’s decisions. However, the impact of residue management and tillage treatment on crop production, and also on soil fertility, are known to be highly dependent on the local pedo-climatic conditions, and the literature usually focus on one of the aspects (soil or crop) and barely on multiple aspects at the same time.vs. OUT), the cumulative grain yield was found to be significantly lower (−3.4%) under reduced tillage (RT) compared to conventional tillage (CT).This study aimed at analysing the impacts of crop residue management techniques on different soil and crop parameters together, as interacting components of the agro-ecosystem, and as a response to the local pedo-climatic conditions. We found out that, at the annual scale, crop production was generally not significantly impacted by the different residue management strategies. However, over the duration of the trial, while no effect of residue fate was reported , other factors than the sole crop productivity should be included in the farmer’s decision process, such as fuel consumption, required working hours, greenhouse gas emissions ; analysi10.7717/peerj.4836/supp-1Figure S1Data were collected in 2011. Maps were obtained by interpolating using the kriging methods in ArcGIS.Click here for additional data file.10.7717/peerj.4836/supp-2Figure S2Orange line is the 30 year mean, grey line is the observations for each year.Click here for additional data file.10.7717/peerj.4836/supp-3Figure S3Orange line is the 30 year mean, grey bars show the observation for the year.Click here for additional data file.10.7717/peerj.4836/supp-4Figure S4p-value <0.05) between crop residue management strategies per sampling date.Bottom ribbons represent the period covered by crops. Error bars depict the standard error between plots. Stars represent significant differences .Significance code: ‘***’ Click here for additional data file.10.7717/peerj.4836/supp-8Table S3p-value < 0.05). .For each crop, treatments means with different letters are significantly different .Significance code: ‘***’ Click here for additional data file.10.7717/peerj.4836/supp-10Table S5p-value < 0.05). .For each crop, treatments means with different letters are significantly different .Significance code: ‘***’ Click here for additional data file.10.7717/peerj.4836/supp-12Table S7p-value < 0.001; ‘**’ p-value < 0.01; ‘*’ p-value < 0.05. .Significance code: ‘***’ Click here for additional data file.10.7717/peerj.4836/supp-13Table S8p-value < 0.05). .For each crop, treatments means with different letters are significantly different .Significance code: ‘***’ Click here for additional data file.10.7717/peerj.4836/supp-15Table S10p-value < 0.05). .For each crop, treatments means with different letters are significantly different .Significance code: ‘***’ Click here for additional data file.10.7717/peerj.4836/supp-17Table S12p-value < 0.001; ‘**’ p-value < 0.01; ‘*’ p-value < 0.05; ‘.’ p-value < 0.1. .Significance code: ‘***’ Click here for additional data file.10.7717/peerj.4836/supp-18Table S13p-value < 0.001; ‘**’ p-value < 0.01; ‘*’ p-value < 0.05. .Significance code: ‘***’ Click here for additional data file.10.7717/peerj.4836/supp-19Table S14p < 0.05). .For each crop, treatments means with different letters are significantly different (ANOVA, Click here for additional data file.


Background. While cancer/testis antigens (CTAs) are restricted in postnatal tissues to testes and germ line-derived cells, their role in cancer development and the clinical significance of their expression still remain to be better defined. Objective. The aim of this study was to investigate the level of CTA expression in colon samples from patients with colorectal cancer (CRC) in relation to patient clinical status. Methods. Forty-five patients with newly diagnosed colorectal cancer were included in the study. We selected a panel of 18 CTAs that were previously detected in CRC as well as some new gene candidates, and their expression was detected at the mRNA level by employing RQ-PCR. Additionally, we evaluated CTA expression in three colon cancer cell lines  after exposure to the DNA methylation-modifying drug 5-azacytidine. Results. We report that 6 out of 18 (33%) CTAs tested  were significantly (p < 0.05) overexpressed in tumor tissue compared with healthy colon samples isolated from the same patients. Conclusions. Moreover, we found that MAGEA3, PLU-1, and DKKL expression positively correlated with disease progression, evaluated according to the Dukes staging system. Finally, 5-azacytidine exposure significantly upregulated expression of CTAs on CRC cells, which indicates that this demethylation agent could be employed therapeutically to enhance the immune response against tumor cells. In postnatal life, the cancer/testis antigens (CTAs) are typically restricted to the testes; however they may be aberrantly expressed in several types of cancers. There are more than 100 CTA genes reported to date, and their specific expression pattern makes them useful as markers of malignancies . AlthougThe abnormal expression of CTA on tumor cells is intriguing. On the one hand, it seems to support the so-called “embryonic rest hypothesis of cancer development.” Specifically, during the 19th and early 20th centuries, it was proposed that cancer originates in populations of cells that are left in a dormant state in developing organs during embryogenesis. This hypothesis was initially postulated by Recamier 1829), Remak (1854), and Virchow (1858) and later elaborated by Durante (1874) and Cohnheim (1875), who suggested that adult tissues contain embryonic remnants that normally lie dormant but that can be “activated” to become cancerous. In agreement with these theories, Wright (1910) proposed a germinal cell origin of Willms tumor (nephroblastoma), and Beard (1911) proposed that some tumors may arise from displaced trophoblasts or germ cells  is the third most common cancer and the fourth most common cause of mortality worldwide, accounting for 9% of all malignancies , 13. CRCIn the present study we evaluated the expression of a panel of CTAs, comprising 18 genes in three CRC cell lines and 45 patient samples. The selected panel of CTAs evaluated was previously detected in CRC; however, we also included new gene candidates. Here, we report that some CTAs are potential markers of CRC and that their expression correlates with disease progression. What is most important, CTAs are potential targets for immune cells, and their expression increased upon treatment with 5-azacytidine. In support of this notion, 5-azacytidine has been reported to upregulate CTA expression in certain cancer cell lines, which may render the cancer cells susceptible to the immune response .® . Samples were stored at −80°C until the time of mRNA extraction. The stage and histological type of cancer were assessed from routine examination of paraffin sections obtained from surgical specimens. Evaluation of the stage was performed according to the criteria of Dukes [n = 36, 84%). Patient data are summarized in Forty-five patients with newly diagnosed colorectal cancer at the Department of Gastroenterology, Pomeranian Medical University in Szczecin, were included in the study. The median age of the patients was 75 years , and 58% of the patients were male. All patients had a malignancy confirmed by histology and gave written informed consent to participate in the study. The study conforms to the Code of Ethics of the World Medical Association (Declaration of Helsinki), printed in the British Medical Journal (18 July 1964). Twelve tumors (27%) were located in the rectum, 18 (44%) in the sigmoid colon, and 15 (33%) in the right colon. For statistical analysis, carcinomas were divided into rectal and nonrectal lesions. None of the patients received radiotherapy or immunotherapy before biopsy. Patients were also categorized according to smoking and body mass index (BMI). Clinical data covered localization of the tumor, lymph node involvement, the presence of distant metastases, and stage. Tissue samples from colon cancer and normal colonic mucosa, located 5 cm away from the tumor edge, were obtained at the time of colonoscopy. Tissue fragments were cut into small fragments and immediately frozen in RNAlaterof Dukes . There wμg/mL streptomycin, and 50 μg/mL neomycin (Life Technologies) in the presence of 10% heat-inactivated fetal bovine serum . The cells were cultured in a humidified atmosphere at 37°C in 5% CO2 at an initial cell density of 2.5 × 104 cells/flask, and the medium was changed every 48 hours.We used the human colon cancer cell lines, CL-188 (LS 147T), HTB-39 (SK-CO-1), and HTB-37 (Caco-2), which were obtained from ATCC. Cells were cultured in Eagle's Minimum Essential Medium (EMEM) 1640 , supplemented with 100 IU/mL penicillin, 10 μM for 48 h. After the indicated time, the cells were lysed, and the RNA was isolated.Cells were treated with 5-azacytidine (Sigma-Aldrich) at a concentration of 5 μg of RNA from each sample was reverse transcribed into cDNA in a total volume of 20 μL with the RevertAid First Strand cDNA Synthesis Kit  according to the manufacturer's instructions.Tissues were stored in RNAlater Solution (Life Technologies) at −80°C. After defrosting, the tissues were rinsed with PBS buffer  for 1 min to remove any residual RNAlater Solution. Samples were homogenized with the Ultra-Turrax T-10 basic dispersing tool in 600 mL RLT buffer  for 5 min at 30,000 rpm/min. Total RNA was extracted from homogenates using the RNeasy Fibrous Tissue Mini Kit (Qiagen) in accordance with the manufacturer's protocol. The concentration and purity of RNA samples were determined by measuring the absorbance using a spectrophotometer (PerkinElmer Lambda Bio). In the next step, 0.3 β-2 microglobulin and β-actin as internal calibrators, was calculated as the fold difference and further processed using statistical analysis.Quantitative assessment of mRNA levels was performed by real-time RT-PCR on an ABI 7500 Fast instrument with Power SYBR Green PCR Master Mix reagent (Life Technologies). Real-time conditions were as follows: 95°C (15 sec), 40 cycles at 95°C (15 sec), and 60°C (1 min). According to melting point analysis, only one PCR product was amplified under these conditions. The relative quantity of a target, normalized to the endogenous control Rs). p < 0.05 was considered statistically significant.CTA expression data were compared between samples from colon cancer and normal colonic mucosa with the Wilcoxon signed-rank test. Data were analyzed as cancer tissue absolute expression (AE) and cancer tissue relative expression (RE) to normal tissue in the same patient, calculated as the ratio of expression levels: cancer tissue/normal tissue. The Mann-Whitney test was used to compare expression between groups of patients. Correlations between expression and other quantitative or rank variables (including Dukes stages) were analyzed using the Spearman rank correlation coefficient  –20 and iμM) of this compound , and observed that several CTAs were expressed in response to a subtoxic dose  CTAs tested, MAGEA3, OIP5, TTK, PLU1, DKKL1, and FBXO39, were significantly (p < 0.05) overexpressed in the tumor compared with normal tissue located ~5 cm away from the primary lesion. At the same time, PLAC1 was overexpressed in tumors, but with borderline significance (p = 0.07)  .p < 0.05). Additionally, we found a borderline positive correlation between the RE for OIP5 (p = 0.086) and NY-ESO-1 (p = 0.051). However, the RE of OIP5 was significantly higher (p = 0.03) in advanced stages of the disease (stages C or D) compared with stages A or B  of most of the significantly overexpressed CTAs  correlated positively with disease progression . At the s A or B . By contRs = 0.43, p = 0.003, Additional analysis showed a correlation between KU-CT-1 expression in cancer tissue and patient age. Specifically, its expression in cancer tissue (AE) progressively increased with age . Our results are in accordance with previous studies, where colon cancer cell lines also did not express CTAs frequently and uniformly . Antigenp < 0.05) in CRC patient samples  of colon cancer cell lines and in 12.8% of patient samples . In our OIP5 was also significantly overexpressed in CRC cell lines and patient samples . ReporteFurthermore, an interesting CTA overexpressed in our study is PLU1 (CT31), which belongs to the JARID1 histone demethylase family and plays an important role in histone methylation . Ohta etWe also report here the high expression of TTK, both in CRC cell lines and in patient samples. TTK was previously reported to be highly expressed in prostate cancer  and HodgAnother CTA, DKKL1 ), is a protein related to the Dickkopf protein family that negatively regulates Wnt-mediated effects . While DThere is a growing body of evidence that cancer, including CRC, may be considered as a stem cell disease that results from the existence of self-renewing and pluripotent cancer-initiating cells. Among the cell populations identified, very small embryonic-like stem cells residing in adult tissues are potential candidates for such malignant transformation . SeveralAlthough colon cancer is regarded as a low-CTA-expressing tumor, there are several strategies by which the number of expressed CTAs may be increased. One method is the use of epigenetic drugs, specifically demethylating agents such as 5-azacytidine . AmplifiIn conclusion, several CTAs, such as MAGEA3, PLU-1, or DKKL, may serve as biomarkers of CRC, and since their expression correlates with advanced stages of the disease, they are of prognostic relevance. Finally, since 5-azacytidine treatment upregulates several CTAs on CRC cells, application of this DNA demethylating agent should be explored to enhance the immune response against CRC.


This study aimed to determine the association between the clinical reference range of serum glycated hemoglobin A1c (HbA1c) and thyrotropin (TSH) and the risk of coronary heart disease (CHD) in non-diabetic and euthyroid patients. We examined baseline HbA1c and TSH in 538 healthy participants, and then analyzed the associations and potential value of these indicators for predicting CHD using Cox proportional hazard and support vector machine analyses. During the median follow-up of 120 months, 39 participants later developed CHD. The baseline HbA1c and TSH within the reference range were positively associated with CHD risk. No correlation and interaction were found between the baseline HbA1c and TSH for the development of CHD. Disease event-free survival varied among participants with different baseline HbA1c quintiles, whereas disease event-free survival was similar for different TSH tertiles. The combination of these baselines showed sensitivity of 87.2%, specificity of 92.7%, and accuracy of 92.3% for identifying the participants who will later develop CHD. Relatively high but clinically normal HbA1c and TSH levels may increase the risk of CHD. Therefore, the combination of these indicators can serve as a biomarker for identifying healthy individuals from those who would later develop CHD. Coronary heart disease (CHD) has one of the highest mortality rates among adults worldwide, especially in developed areas, such as Europe, North America, and China123568Glycated hemoglobin A1c (HbA1c), a form of hemoglobin, is formed via a non-enzymatic glycation pathway through the exposure of hemoglobin to plasma glucose13141612in vitro is capable of stimulating glucose oxidation26Thyroid hormones are important regulators of glucose homeostasis and have a definite association with diabetes and CHD. Thyrotropin (TSH), a hormone secreted by the pituitary gland, acts on and stimulates the thyroid gland to produce thyroid hormones. TSH significantly and precisely responds to minor changes in the concentrations of circulating thyroid hormones. However, increasing evidence suggests that TSH exerts extra-thyroidal effectsIn summary, HbA1c and TSH play important roles in glucose metabolism and development of CHD. These two parameters are considered biomarkers for this disorder. Hence, we speculated that there may have a link between HbA1c and TSH for developing CHD. However, this link and the ideal value of relatively high baseline HbA1c and TSH in reference range for discriminating individuals who will later develop CHD are poorly understood. In the present study, we aim to detect the association of baseline HbA1c and TSH with the risk of CHD through a 10-year follow-up study involving healthy individuals in a Chinese cohort. In addition, the relationship between baseline HbA1c and TSH was also investigated. Support vector machine (SVM) analysis was conducted to determine whether the combination of baseline HbA1c and TSH in healthy individuals can serve as a predictor for CHD with ideal accuracy and stability.r = 0.103, p = 0.017), TCH , TG , and LDL  in the baselines. However, the correlation between baseline HbA1c and age of participants was no longer significant after the Bonferroni corrections (p < 0.05/8 = 0.00625). The details are shown in r = −0.009, p = 0.847; F = 0.940, p = 0.593).The correlations between baseline HbA1c and TSH and serum lipid profiles in type 2 diabetic patients are revealed by previous studies. However, in the healthy examination cohort, we found that HbA1c but not TSH was positively correlated with age .During the median follow-up of 120 months, 33 participants were withdrawn due to the changes of home address or telephone or other indefinite reasons, 43 participants later developed to diabetes, 9 participants later developed thyroid dysfunction, and 39 participants  later developed CHD. Twenty-six of the CHD cases were acute coronary syndrome and the remaining cases were chronic coronary artery disease. Baseline HbA1c and TSH were 5.40% ± 1.19% and 2.64 ± 1.00 mIU/L for participants who developed CHD during the follow-up. These values were significantly higher than the 4.32% ± 1.32% and 2.26 ± 1.08 mIU/L for the participants who did not develop CHD . Therefore, the combination of baseline HbA1c and TSH had a sensitivity of 87.2% (34/39), a specificity of 92.7% (432/466), and an accuracy of 92.3% (466/505) for discriminating the participants who later developed CHD  men and women aged 18~80 years; (2) without history of and not suffering from diabetes, hyperlipidemia, thyroid dysfunction, and cardiovascular disease; and (3) with available and complete medical reports of healthy examination and contact information. However, individuals were excluded if they have: (1) baseline HbA1c ≥6.5% or significant abnormal baseline TSH; (2) medications that might affect HbA1c and TSH levels and serum lipid profiles; and (3) serious neurologic diseases, psychiatry, renal disorders, or hepatic disorders. A total of 974 individual data during this period were screened from the medical records. Finally, 538 individuals who satisfied the inclusion and exclusion criteria were included in the study. The study was conducted in compliance with the Declaration of Helsinki. All participants wrote and signed their informed consents. The study was approved by the ethics committee of the Xinxiang Central Hospital.The baseline data including HbA1c, TSH, and serum lipid profiles, as well as the clinical characteristics  were obtained from the medical reports of healthy individuals. HbA1c was estimated using the Tina-Quant turbidimetric inhibition immunoassay , TSH was measured using electrochemiluminescence immunoassay , and the lipid profiles were measured using an automatic biochemistry analyzer  and commercial kits. Laboratory reference ranges for HbA1c and TSH were <6.5% and 0.3~5.50 mIU/L, respectively. Clinical baselines of these participants are shown in Long-term follow-up was conducted twice per year through an outpatient interview, telephone call, home visit, or self-reporting of participants. Follow-up time was calculated as the time between the date of the baseline examination and the date of the endpoint event or June 30, 2015. The endpoint of this study was the prevalence of CHD. When the endpoint event occurred, the date of incident and the type (acute coronary syndrome or chronic coronary artery disease) and disease extent (single or multi-vessel) of CHD were recorded.et al.et al.Referenced to the study of Pfister χ2) test. Relative risk and 95% confidence interval of baseline HbA1c and TSH for incident CHD event were estimated from the Cox proportional hazard analysis. Tests of linear trends were computed using median values within each of the HbA1c quintiles or TSH tertiles. The first models were adjusted only for age. In the subsequent models, we added the possible confounding factors for the adjustment, including age, smoking and drinking history, BMI, blood pressures, history of CHD in first-degree relatives, and serum lipid profiles. Kaplan-Meier survival curves were plotted and the differences in event-free survival were assessed using the log-rank test for multiple group comparisons. Interaction between HbA1c and TSH for developing CHD was detected through covariance analysis. Two-sided p values < 0.05 indicate statistical significance.SPSS 18.0 software was used for statistical analysis. The relationships between baseline HbA1c and TSH or between the two parameters and other clinical variables were performed using Pearson’s correlation analysis. Bonferroni correction was applied to minimize type I error in the multiple tests. The participants were categorized according to baseline HbA1c quintiles or TSH tertiles. Differences between the tertiles or quintiles were tested with Chi-square (http://www.csie.ntu.edu.tw/~cjlin/libsvm/)C is the most important step for the classifier analysis in SVM. Grid search method and Gaussian radial basis function kernels were used for parameter optimization. The “leave-one-out” cross-validation approach, which takes one instance from the original sample as the validation data and leaves the remaining observations as the training dataSVM using the LIBSVM software package .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


During infection, there is an activation of the L-arginine-nitric-oxide pathway, with a shift from nitric oxide synthesis to a degradation of L-arginine to its metabolites, asymmetric and symmetric dimethylarginine (ADMA and SDMA). However, the prognostic implications for short-term or long-term survival remains unclear. We investigated the association of L-arginine, ADMA, and SDMA with adverse clinical outcomes in a well-defined cohort of patients with community-acquired pneumonia (CAP).We measured L-arginine, ADMA, and SDMA in 268 CAP patients from a Swiss multicenter trial by mass spectrometry and used Cox regression models to investigate associations between blood marker levels and disease severity as well as mortality over a period of 6 years.r = 0.32, p < 0.001 and r = 0.56, p < 0.001 for ADMA and SDMA, respectively) and higher in 6-year non-survivors versus survivors . Both ADMA and SDMA were significantly associated with long-term mortality  1.84 to 10.74] and 2.81 [95% CI 1.45 to 5.48], respectively). The effects were no longer significant after multivariate adjustment for age and comorbidities. No association of L-arginine with severity and outcome was found.Six-year mortality was 44.8%. Admission levels of ADMA and SDMA (μmol/L) were correlated with CAP severity as assessed by the pneumonia severity index (Both ADMA and SDMA show a severity-dependent increase in patients with CAP and are strongly associated with mortality. This association is mainly explained by age and comorbidities.ISRCTN95122877. Registered 31 July 2006. Nitric oxide (NO), which is synthesized from L-arginine by nitric oxide synthases (NOS), is a vaso- and bronchodilator . Its defCirculating levels of ADMA are elevated in multiple diseases with endothelial dysfunction  , 11–14, Community-acquired pneumonia (CAP) is to date one of the leading causes of death due to an infection . CAP is A total of 268 patients with a definite diagnosis of CAP and available blood samples from a previous Swiss multicenter trial  , 28 were9 cells/L), or one clinical finding upon auscultation . An infiltrate was radiologically confirmed in all patients with CAP. Exclusion criteria included language restriction or dementia that precluded patients from giving written informed consent, intravenous drug use, a terminal condition, or hospital-acquired pneumonia (HAP). Participants were accepted into the study if they had received short-term antibiotic pretreatment or corticosteroids, but were excluded if they had obtained long-term antibiotic pretreatment or suffered from severe immunosuppression at ED presentation.The initial trial was a noninferiority randomized controlled trial evaluating the economical use of antibiotics at six Swiss secondary and tertiary care centers between October 2006 and March 2008 . The priBlood samples collected from each patient and frozen in aliquots at ED admission were used for measuring the different biomarkers. Baseline arginine, ADMA, and SDMA levels were measured in blood samples of all 268 CAP patients included in the study, with 109 of them also having blood specimens available at day 7 of hospitalization for these measurements. If any of above biomarkers had a value of 0, it was considered to be a measuring error and excluded from the analysis. Due to technical errors or insufficient volume of blood specimens during biomarker measurement, not all patients had a complete set of markers. The final analysis included 268 patients of which 268 (100%) had complete markers of arginine, while 233 (87%) and 242 (90%) had ADMA and SDMA markers available, respectively.AbsoluteIDQ p180 Kit   and quick Sepsis Related Organ Failure Assessment [qSOFA] score) . Vital sp value <0.05. Categorical variables are expressed as percentages (numbers) and continuous variables as medians (interquartile range [IQR]) unless stated otherwise. The distribution of ADMA, SDMA, and L-arginine (just referred to as biomarkers henceforth) were skewed. The distribution of the biomarkers approximated a normal distribution after logarithmic transformation at a base of 10. Chi-square  tests were used for frequency comparisons, and nonparametric tests  were executed for comparisons of two or more groups. The significance levels were subjected to Bonferroni adjustment. To study the association of comorbidities with initial biomarker levels, we used univariate and multivariate linear regression models. To investigate associations between biomarker levels at baseline and all-cause mortality, we utilized univariate and multivariate Cox regression models. These associations are reported as hazard ratios (HRs) with 95% confidence intervals (CIs) and significance levels for the chi-square  test. Odds ratios were calculated and reported with 95% confidence intervals (CIs). Area under the receiver operating characteristics curves (AUCs) with 95% CIs are presented to illustrate discrimination and predictive power. Mortality based on biomarker quartiles (highest versus lower three) has been illustrated through the use of Kaplan-Meier curves. All analyses were performed with the ratio of arginine to ADMA as well.All statistical analyses were performed using STATA 12.1 . Statistical significance was considered at a N = 268) as well as for patients stratified by 6-year survival status are presented in Table Baseline characteristics of the entire cohort (n = 14), increasing to 17.9% (n = 48) after 1 year and 44.8% (n = 120) after 6 years of follow up. Figure Mortality at day 30 was 5.2% . There were no significant differences in arginine levels between non-survivors and survivors   for short- and mid-term mortality and 0.64 (95% CI 0.57 to 0.71) for long-term mortality at 6 years. The AUCs for ADMA did not reach these levels Table .Table 2Aρ = 0.31, p <0.001 and ρ = 0.5307, p <0.001). In contrast, there was no association between all the investigated biomarkers and the qSOFA score.In Spearman’s rank correlation analyses with Bonferroni adjustment, we found significant associations of ADMA and SDMA with the Pneumonia Severity Index . We also did not measure NO levels and so cannot be sure that our conclusions are complete. Finally, we did not have a complete marker set available for all patients due to low blood specimen volume. Nevertheless, our hypothesis-generating study provides valuable information that will facilitate the design and conduct of additional studies in this area.Deducing from our findings, we may assume that neither SDMA nor ADMA nor L-arginine are independently associated with all-cause mortality in CAP patients, although strong unadjusted associations of SDMA—more than ADMA—with mortality and comorbidities are seen in these patients. It remains unclear if higher ADMA and SDMA levels cause higher mortality or if higher mortality causes high levels of ADMA and SDMA, and the precise role NO plays in this scheme of events.Given our findings, future studies should focus on the causal connection between ADMA and SDMA levels and mortality and on investigating the clinical benefit of antagonising ADMA and SDMA .


The elderly colon cancer (CC) patients are increasing and represent a heterogeneous patient group. The objectives of this study were to identify the features of lymph node examination and to explore the optimal minimum lymph node count after CC resection for patients aged ≥80. Using the Surveillance, Epidemiology, and End Results (SEER) database, we identified 65719 CC patients in stage I-III between 2004 and 2012, 26.0% of patients were aged ≥80. The median node count decreased with increasing age, which were 25.5, 20.2, 17.8 and 16.9 for patients aged 20–39, 40–59, 60–79, and ≥80. The rate of ≥12 nodes and the rate of node positivity for patients aged ≥80 were obviously lower than younger patients. Using X-tile analysis, we determined 9 nodes as the optimal node count for patients aged ≥80. Then, we compared the 5-year cancer specific survival (CSS) between patients with ≥9 nodes and <9 nodes. The results showed the 5-year CSSs were improved for patients with ≥9 nodes. Furthermore, the rate of node positivity and survival under the 9-node measure were equal to 12-node measure. Therefore, the lymph node examination should be discriminately evaluated for elder patients, and 9-node measure was available for patients aged ≥80. Elderly patients currently account for a significant proportion of patients who are diagnosed with colon cancer (CC), which bring a challenge of dealing with an aging population to medical oncologistsAccurate cancer staging is highly dependent on sufficient number of lymph nodes examination to detect the positive nodes45781011Elder CC patients have distinct characteristics that need to be taken into account regarding lymph node evaluation, especially for patients aged ≥8013We totally identified 65719 patients diagnosed with stage I-III CC from 2004 to 2012 in the SEER database, 17058 patients (26.0%) were aged 80 years and older. Baseline characteristics significantly differed among four subgroups . PatientIn this study, patients with adequate lymph node count (≥12) accounted for 77.9% in all patients, and hence it was confidently staged according to their nodal status. The median number of lymph node count was decreased with increasing age, which were 25.5, 20.2, 17.8 and 16.9 for patients aged 20–39, 40–59, 60–79, and ≥80, respectively (P < 0.001) . For lymWe applied X-tile analysis to determine the optimal cut-off node count for prediction of CSS according to different lymph node count. The result showed that 9 was the optimal cut-off node count for patients aged ≥80 (P < 0.001) . TherefoThe median CSSs were 55.0 months for patients with ≥9 nodes, and 39.0 months for patients with <9 nodes. The 5-year CSS was 46.7% for patients with ≥9 nodes and 38.6% for those with <9 nodes (P < 0.001) . Then, wUsing univariate and multivariate regression analyses, we identified the risk factors that associated with long-term survival outcomes for patients aged ≥80. The results showed that retrieval of <9 nodes was identified as independent adverse prognostic factors in patients aged ≥80 . In addiAdequate lymph node retrieval was higher related to accurate tumor staging and improved long-term survival. To confirm the value of 9-node measure for patient aged ≥80, we separately evaluate the effect of lymph node count on the rate of node positivity and long-term survival. Firstly, we separately calculated the rate of node positivity based on different node count from ≥6 to ≥15. The result showed that the rate of node positivity changed between 33.0% and 33.5% according to the node count from ≥6 to ≥15 . The ratIn addition, we assessed the effect of node count on the long-term survival outcomes. The 3-, 5- and 8-year CCSs were separately calculated according to node count from ≥6 to ≥15. For patients with ≥9 nodes, the 3-, 5- and 8-year CSSs were 61.6%, 46.7% and 29.4%. For patients with ≥12 nodes, the 3-, 5- and 8-year CSSs were 62.6%, 47.8% and 29.9% . TherefoFurthermore, we performed the survival comparison in patients with different nodes examined (<9 nodes vs. 9–12 nodes vs. ≥12 nodes). The CC patients aged ≥80 represented a large proportion in all patients. Here, we totally identified 17058 CC patients aged ≥80, which accounted for 26.0% in all patients from 2004 to 2012. Elder patients, especially for patients aged ≥80, not only has been thought as an independent prognostic factor regarding with survival, but also has been associated with different clinical and histological features91314Lymph node examination after CC resection is considered to be one crucial factor in assessing the accuracy of tumor staging, which determine further therapeutic planning and prognosis. Patients with positive nodes have to accept adjuvant chemotherapy regarding with the higher risk of tumor recurrence and metastasis18In this study, we tentatively explore the optimal cut-off node count, instead of 12 nodes, for patients aged ≥80 based on the prediction of CSS. Our results identified that 9 nodes was the optimal cut-off point for patients aged ≥80. All CC patients in stage I, II and III all could obtain much survival benefit from examining ≥9 nodes, and examining <9 nodes was also identified as independent adverse prognostic factors in patients aged ≥80. In addition, 9-node measure did not decrease the chance of obtaining positive lymph node compared with the 12-node measure. Hence, the use of the 9-node measure might be more reasonable for patients aged ≥80.In identifying the difference of lymph node examination for CC patients aged ≥80, the SEER databases provide sufficient CC cases with uniformly collected data, which highly represented the CC patients across the United States. However, there had some potential limitations in SEER database including lack of information regarding the adjuvant chemotherapy for CC patients, lack of central histological review, and lack of information on operative morbidity and comorbidities. The study may also be confounded by other contributing factors, such as general ASA status, which may indirectly affect the lymph node examination in the elderly patients. These noted limitations in SEER are common to most of other large epidemiological databases, which have been well addressed in literatures. Furthermore, the selection biases could not be avoided in this retrospective cohort study, because the elderly patients who underwent CC resection should be fitter, and the radical surgery of CC might be not regularly performed for this group of patients as young patients. Despite these limitations, SEER remains a valuable resource to analyze trends and patterns in patient characteristics, tumor features, cancer treatments, and survival outcomes.In conclusion, this population-based study demonstrated that patients aged ≥80 accounted for a large proportion of CC patients, they had obviously lower rate of ≥12 nodes examined compared with the younger patients. Instead of 12-node measure, we identified that the 9-node measure may be more feasible and reasonable in patients aged ≥80. However, whether this finding could impact the adjuvant treatment decision-making for the elderly patient who cannot tolerate chemotherapy, more investigations of outcomes for this group of CC patients are still need to be performed.We extracted cancer data from the Surveillance, Epidemiology, and End Results (SEER) database between 2004 and 2012. The SEER database includes the information regarding cancer incidence, treatment and survival outcomes from 17 population-based cancer registries, which represented 28% of the US populationThis study was approved by the Ethics Committee of The Second Affiliated Hospital of Harbin Medical University institutional review board. The SEER database is openly accessed, and we have got permission to access the cancer data from the SEER database by National Cancer Institute, and the reference number was 11228-Nov2014. All methods were performed in accordance with the relevant guidelines and regulations of SEER database.We collected cases based on the International Classification of Diseases Oncology, Third Edition (ICD-O-3) codes for anatomic site (colon excluding rectum). We used the term “age” which was referred to “age at diagnosis” in SEER database. Patients were divided into four age subgroups by 20-year intervals, including 20–39, 40–59, 60–79, ≥80. Patients included in this study should undergo radical resection of the CC as the first course of therapy, which were more available and accurate for the lymph node evaluation. Race/ethnicity was categorized as white, black and Asian/Pacific Islander (API), including American Indian/Alaska Natives. Right CC included tumors being located at cecum, ascending colon, hepatic flexure and transverse colon. Left CC included tumors being located at splenic flexure, descending colon and sigmoid colon. The exclusion criteria should include patients: dead due to other causes, with an unknown number of nodes examined, aged <20 years, who received preoperative radiotherapy in the consideration of the decreased number of node examined, and who underwent a local procedure, partial colon resection or total colectomies.2 test. The cancer specific survival (CSS) was defined as the time from the CC diagnosis until cancer recurrence or metastasis, cancer-associated death and the end of follow up. The 5-year CSS was estimated with Kaplan-Meier method and log-rank test was used to compare the difference of CSS curves. Univariate and multivariate Cox’s regression model were performed to estimate hazard rate (HR) and exact 95% confidence intervals (CIs). P < 0.05 (two sides) was considered to be statistical significance. The statistical analyses were conducted with SPSS statistical software, version 20 .The comparisons of lymph node evaluation among different age subgroups were performed in three ways including the median number of lymph node, the rate of ≥12 lymph nodes and the rate of node positivity. All categorical variables were compared between groups using χ2 value is calculated for every possible division of the population. The optimal cutoff node count for survival was calculated by selecting minimum P value with the maximum χ2 value.X-tile plots is a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization, which illustrates the presence of substantial patients subpopulations and shows the robustness of the relationship between a marker and survival outcome by construction of a two dimensional projection of every possible subpopulation. X-tile plots divided population into different divisions based on every possible cutoff point. X-tile data are presented in a right triangular grid where each point represents a different cutoff point. The X-tile plots statistically test each divisions based on each cutoff point. All possible divisions of the cutoff point are assessed. Then, a χHow to cite this article: Guan, X. et al. Exploration of the Optimal Minimum Lymph Node Count after Colon Cancer Resection for Patients Aged 80 Years and Older. Sci. Rep.6, 38901; doi: 10.1038/srep38901 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.