The domestic and foreign scholars have studied naked mole rats more focused on the respect such as its long life, resistant to low oxygen, little spontaneous tumor, but the study of the immune system is little. In this study, we compared the anatomy and tissue morphology of NMR and ICR mouse spleens and found that the gross appearance of the NNMR spleen differed from ICR. There were more macrophages in NNMR spleens than in ICR spleens. Furthermore, we focused on the differences of macrophages. We compared their phagocytic capabilities and the data showed that NNMR macrophages are more phagocytic than ICR mouse macrophages. We also used polyI:C and LPS to stimulate the NMR and ICR macrophages and then measured the immune response as expression of certain TLR signaling molecules. After stimulation, there was a lower increase in apoptosis of NMR macrophages than ICR macrophages and a non-significant increased expression of TLRs in NMR macrophages than in ICR macrophages. In contrast, NF-κB proteins increased more significantly in NMR’s than in ICR’s and the expression of downstream cytokines in NMR macrophages also increased more than in ICR macrophages. Based on these results, we hypothesize that in addition to being able to eat foreign matter, NMR macrophages can activate the TLRs, start the NF-κB and produce a large number of cytokines to enhance immune response, so as to protect the body from outside interference when the virus or bacteria invading. Heterocephalus glaber, the naked mole rat (NMR), is a prevalent underground rodent in Africa. In 1967, Jarvis first brought the NMRs into the laboratory for scientific research. Since then, more biologists have begun to cooperate with Jarvis for naked mole rat biological research [research –11.Immune system is an important index of biomedical research, which not only has a guiding significance for animal physiological state judgments and feeding management, also is the important basis of clinical medical treatment and scientific research. Immune system which is the most effective weapon defense pathogens invasion consists of immune organs, immune cells and immune molecules, and it can find and remove foreign bodies, exogenous pathogenic microorganisms, etc. There have been studies to uncover the link between the immune system and cancer. Along with the aging of the body, the immune system is gradually deteriorating, which is immune senescence. And immune senescence is related to systemic inflammation, chronic inflammatory diseases and many cancers.The spleen is the body’s largest lymphoid organ, which has important functions including immunity, hematopoiesis, and filtering blood . The splMacrophages are important immune cells in the body that mediate and maintain innate or acquired immune defense mechanisms , at the Herein, we started a pilot study to compare the immune system of NMRs to mice’s. First, we compared the spleens of NMRs and mice at the histological and cellular levels. We found that the proportion of NMR splenic macrophages was greater than that observed in mouse spleens. We also demonstrated that naked mole rat macrophages had greater phagocytic capability than mouse macrophages. We also profiled the immune response of NMR and mouse macrophages after stimulation with mimics of viral and bacterial pathogens. We found that NMR macrophages protected themselves from pessimal stimulation by secreting high levels of cytokines. In accordance with this data, we speculate that macrophages of NMRs play an important role in the innate immune response.We first compared the anatomical structure of the spleens of naked mole rats and mice. Through anatomical observation, we found that the mouse spleen was located in the left rib arch, the majority of it hidden behind the stomach, and its shape was smooth, flat, and oval Figure . The mouWe also compared the shape and structure of NMR and mouse spleens. By microscopy, the red pulp distribution area in the mouse spleen was small. The mouse splenic corpuscle area was large and had a concentrated distribution. Mice had fewer splenic trabeculae, which were short and thin Figure . In nakeAs macrophages usually phagocytose foreign bodies, we examined the phagocytic functions of NMR and mouse macrophages. At first, we microscopically observed ICR and NMR macrophages engulfing chicken red blood cells. Over the same time course, NMR macrophages swallowed more chicken red blood cells than ICR macrophages Figure . We simuIn addition to our study of phagocytosis by macrophages in naked mole rats and ICR mice, we tested the influence of apoptosis when macrophages were stimulated with analogs of viral and bacterial infections. Polyinosinic acid-polycytidylic acid (polyI:C) is a synthetic double-stranded RNA that is often used to simulate an infection because its structure is similar to the RNA of a variety of viruses . LipopolWe used polyI:C to simulate virus infection and studied the macrophage inflammatory or immune responses. Using real-time PCR, toll-like receptor 3 (TLR3) expressions significantly increased in ICR mouse macrophages and naked mole rat macrophages after treatment. There was a significant difference in TLR3 expression between ICR mouse macrophages and NMR macrophages. The expression levels of IFN-β in both ICR and NMR macrophages were significantly elevated, and the level was increased more in NMRs than in ICR mice Figure . In mousWe used LPS to simulate bacterial infection and studied the inflammatory immune responses of macrophages. Using real-time PCR, we found that TLR4 expression in ICR mouse macrophages increased after treatment, and the naked mole rat macrophages also showed significantly increased expression. There was no difference in TLR4 expression between ICR mouse macrophages and NMR macrophages. The expression levels of TNF-α in both ICR mouse and NMR macrophages were extremely significantly elevated, and the levels increased more in NMRs than in ICR mice Figure . In mousOur results indicated that Toll-like receptors in macrophages were activated when the cells were stimulated with polyI:C or LPS. PolyI:C acts through TLR3, and LPS mainly plays a role through TLR4 to activate NF-κBα, thus promoting the transcription and expression of cytokines. We measured cytokines by real-time PCR: we detected IFN-β in the polyI:C-treated group and TNF-α in the LPS-treated group. The expression of IFN-β increased in ICR mouse macrophages by 3.61±2.08, but it increased to 9.20±4.01 in NMR macrophages Figure . The expNaked mole rats have the longest life span of all rodents. As a group, the oldest individuals are more than 30 years old, and they are in generally good health in their 20s. Those who are 25 or older show some signs of weakness and most NMRs eventually die of old age. Some studies have suggested animal models of longevity are able to tolerate a broad spectrum of harmful stimuli. Compared with mice, naked mole rat cells have stronger resistance to many harmful insults  –11. ThesThe spleen, the largest lymphoid organ of the body, has many important functions in immunity, hematopoiesis, and blood filtration. The spleen is the main site of IgM antibody production, especially in the primary immune response. The spleen can also promote phagocytosis of peptides, complement, tumor necrosis factor, and many kinds of humoral factors. The white pulp is composed of dense lymphoid cells, and it is critical for maintaining the specificity of the immune response. Blood flow in the red pulp is slow, and it is the main site of phagocytosis by immune cells. There are more macrophages in the marginal zone, and these cells are important for identifying and capturing antigens and inducing immune responses , 23. In Macrophages are an important immune cell of the body, and they play a central role in the immune response. Macrophages play an important role in regulating innate and acquired immune defense mechanisms, and they are the main scavenger cells for responses to viral and bacterial infections, acting by engulfing and digesting pathogenic microorganisms to remove pathogenic materials. Macrophages are also antigen presenting cells that mediate immune responses , 25. In In the article, we studied the differences between ICR mouse macrophages and NMR macrophages by the detection of their phagocytic function and the response to polyI:C and LPS. We found that naked mole rat macrophages had increased phagocytic capability compared to mouse macrophages. After polyI:C and LPS stimulation, the TLRs in NMR macrophages had a non-significant increase in comparison to in ICR macrophages, but the expression of NF-κB and cytokines had remarkably elevated. These results suggest that a small amount of TLR activation in NMR macrophages can cause high expression of downstream NF-κB and increased expression of cytokines, resulting in a strong immune response to protect the body from damage by viruses and bacteria. Based on the results of our experiments, we hypothesize that the high phagocytic capability of naked mole rat macrophages is a powerful tool to resist invasion of exogenous substances. This phagocytic capability may be a factor that contributes to the long lifespan and low rate of spontaneous tumors in NMRs. There are many other cells, such as B cells, T cells, and dendritic cells, in immune system. What these cells function in the immune response of NMR and how these immune cells interact remain to be seen.ICR mice (6-8-week-old) were obtained from Joint Ventures Sipper BK Experimental Animals . Mice were kept and bred in pathogen-free conditions. Naked mole rats (over 10 months) were bred by the Second Military Medical University Laboratory Animal Center . All animal experiments were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals with approval of the Scientific Investigation Board of Second Military Medical University . PolyI:C and LPS were from Sigma-Aldrich . Antibodies specific to TLR3, TLR4, NF-κB and GAPDH used for immunoblotting were from Proteintech .After intraperitoneal injection of 0.3% sodium pentobarbital anesthetic, the abdominal cavity was opened for spleen removal and measurement. Spleens were placed in 10% neutral formalin; fixed for 24 hours; and subsequently dehydrated, paraffin embedded, sectioned, H&E stained, and imaged using optical microscopy to record morphological structure characteristics.2, 5% CO2, and 92% N2. After collecting and centrifuging the supernatant, cells were suspended using DMEM containing 60 ng/ml M-CSF and allowed to develop for 6 ∼ 7 d. The status of the cells was observed by an inverted microscope. Mouse cells were maintained at 35°C in Dulbecco’s Modified Eagle’s Medium with high glucose  supplemented with 10% fetal bovine serum  and 100 units/mL penicillin/streptomycin in a humidified incubator with 5% CO2 and 21% O2. NMR cells were maintained at 35°C in Dulbecco’s Modified Eagle’s Medium with low glucose  supplemented with 10% fetal bovine serum  and 100 units/mL penicillin/streptomycin in a humidified incubator under conditions of 92% N2, 5% CO2, and 3% O2.Naked mole rats were sacrificed using cervical dislocation and soaked for 2-3 min in 15% ethanol. Under sterile conditions, the femur and tibia were separated, cartilage tissue was removed, the bone marrow cavity was rinsed using a 1 ml syringe with complete medium, and cells were processed into a single cell suspension. After centrifugation, red blood cells were lysed using red blood lysis fluid, and complete medium was added to end lysis. After adding complete medium, the heavy suspension cells were cultured for 8 to 12 hours at 37°C in 3% OMacrophages were kept at room temperature away from light and incubated with fluorescent particles (FITC-Dextran) for 30 min. After PBS washing, we used flow cytometry to test the phagocytosis by each type of macrophage .An Annexin V-FITC assay  was usedGAPDH, TLR3, TLR4, IFN-β and TNF-α mRNA levels in the macrophages from naked mole rats and ICR mice were quantified by real-time PCR. Total RNA was extracted with RNAiso Plus   accordinAt the end of treatment, fibroblasts were lysed in lysis buffer . Lysates were centrifuged at 10,000× g for 10 min at 4°C, and protein concentration was determined. Samples (50μg/lane) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane. Blots were blocked for 1 h at 37°C in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.02% Tween-20 (TBST) containing 5% skimmed milk and probed using a 1:1000 dilution of the appropriate primary antibodies  for overnight incubation at 4°C. The blots were washed thrice with TBST and probed with goat-anti-mouse IgG or goat-anti-rabbit IgG conjugated to horseradish peroxidase . Quantitative analyses of protein band optical intensities were conducted using the Kodak Gel Logic 4000R Imaging System  and normalized to GAPDH for protein expression or to total protein for protein phosphorylation [Results are given as the means plus or minus standard deviation (SD). Comparisons between two groups were performed using Student’s t test. Statistical significance was determined as P values less than 0.01.


APO gene polymorphisms and ischemic stroke risk in a Chinese Han population. In this case-control study, we genotyped 14 single nucleotide polymorphisms (SNPs) in 3 APO genes in 488 cases and 503 controls using Sequenom Mass-ARRAY technology and evaluated their association with ischemic stroke using the χ2 and genetic model analysis. In the allelic model analysis, we determined three SNPs were significantly associated with ischemic stroke: rs693 with a p value of 0.042 , rs651821 with a p value of 0.007  and rs662799 with a p value of 0.006 . In the genetic model analysis, we found the minor allele “A” of rs693 was associated with an increased ischemic stroke risk in the additive model and dominant model. The minor allele “C” of rs651821 was associated with a decreased ischemic stroke risk in the additive model. The minor allele “G” of rs662799 was associated with a decreased ischemic stroke risk in the additive model. Additionally, strong linkage was found in 3 blocks constituted by rs1042034, rs676210, rs693, rs673548 in APOB; rs3791981, rs679899 in APOB; and rs651821, rs662799, rs17120035 in APOA5. Our data suggested that gene polymorphisms in the APO genes may exert influences ischemic stroke susceptibility in a Chinese Han population.This study aimed to assess the association of  HDAC9 [LTC4S, ALOX5 [APOA1, APOB [In recent years, ischemic stroke is becoming the most seriously disabling illness in industrialized countries . As the HDAC9 , LTC4S, S, ALOX5 , APOA1, A1, APOB .APOA1 is the major protein component of high density lipoprotein (HDL) in plasma. Defects in APOA1 are associated with HDL deficiencies, including Tangier disease and systemic non-neuropathic amyloidosis [APOA1 is closely linked with APOA5, which is another apolipoprotein gene on chromosome 11. The apolipoprotein encoded by APOA5 regulates the plasma triglyceride levels. Previous study have found that APOA5 mutations are associated with hypertriglyceridemia and hyperlipoproteinemia type 5 [The protein encoded by loidosis . APOA1 ia type 5 .APOB is the main apolipoprotein of chylomicrons and low density lipoproteins (LDL). Mutations in APOB or its regulatory region cause hypobetalipoproteinemia, normotriglyceridemic hypobetalipoproteinemia and hypercholesterolemia [The protein encoded by erolemia . Additioerolemia .APOA1, APOA5 and APOB genes polymorphisms to ischemic stroke risk, especially in the Chinese Han population. We therefore performed a case-control study to investigate the associations between single nucleotide polymorphisms (SNPs) in APOA1, APOA5 and APOB, and the risk of ischemic stroke in Chinese Han population.To date, several independent studies suggest that APOA1, APOA5 and APOB levels are risk factors for ischemic stroke risk in Western populations. However, little is known about the contribution of p < 0.001) and sex (p < 0.001) were significantly different between cases and healthy controls. Multivariate analyses were adjusted for age and sex.This study included 488 ischemic stroke cases  and 503 healthy controls . The clinical characteristics of cases and controls are shown in Table p > 0.05). We found three SNPs were significantly associated with ischemic stroke: rs693 with a p value of 0.042 , rs651821 with a p value of 0.007  and rs662799 with a p value of 0.006 .To design the multiplexed SNPs MassEXTEND assay, we used Sequenom MassARRAY Assay Design 3.0 Software  and dominant model  respectively. The minor allele “C” of rs651821 in APOA5 was associated with a decreased ischemic stroke risk in the additive model . The minor allele “G” of rs662799 in APOA5 was associated with a decreased ischemic stroke risk in the additive model .We further assessed the association between each SNP and ischemic stroke risk in an unconditional logistic regression analysis, which was performed using three models: additive, dominant and recessive model Table . The minAPOs using linkage disequilibrium (LD) and haplotype analyses. Figure APOB constructed by rs1042034, rs676210, rs693, rs673548 and rs3791981, rs679899 in chromosome 2 with D' = 1. Figure APOA5 constructed by rs651821, rs662799 and rs17120035 in chromosome 11 with D' = 1. The association analysis between the three blocks and ischemic stroke risk was shown in Table APOB constructed by rs1042034, rs676210, rs693 and rs673548 in chromosome 2 was associated with an increased ischemic stroke risk . The haplotype “CGC” in APOA5 constructed by rs651821, rs662799 and rs17120035 in chromosome 11 was associated with a decreased ischemic stroke risk .We further characterized the SNPs in APO genes and ischemic stroke risk in the Chinese Han population. We found the minor allele “A” of rs693 was associated with an increased ischemic stroke risk. The minor allele “C” of rs651821 and “G” of rs662799 have a protective role for ischemic stroke.In the current study, we evaluated the association between fourteen SNPs in three APOB gene. The minor allele “A” of rs693 in codon 3611 resulted in amino acid substitution of glutamine to arginine, which could further changed the LDL-receptor binding affinity [The SNP rs693 at chromosome 2p24.1 was located in the exon region of affinity –15. Our APOA5 gene respectively. As demonstrated by previous studies, the elevated triglyceride level may be an independent risk factor for ischemic stroke [APOA5 gene may play an important role in the development of ischemic stroke and triglyceride metabolism [APOA5, has been widely studied in ischemic stroke susceptibility. However, inconclusive results have been obtained. Some studies supported the conclusion that risk for ischemic stroke was associated with the polymorphism, whereas other studies drew converse conclusions [The SNP rs651821 and rs662799 at chromosome 11q23.3 was located in the 5′ UTR and promoter region of c stroke . Human atabolism , 18. APOclusions –22. Our The haplotype analysis suggested that the combination of certain SNPs could increase or decrease the risk of ischemic stroke since the change of a certain SNP may affect the change of another SNP thus generating a joint effect in the progressing of ischemic stroke. The detailed mechanism of this phenomenon deserved further investigation.APOs polymorphisms and environment factors in ischemic stroke must be evaluated in future studies.Our study had several intrinsic limitations. For example, ischemic stroke is a very complicated process, and environment factors such as eating and exercise habits are important risk factors for ischemic stroke. Because our study had a relatively small sample size, and it did not incorporate data regarding eating and exercise habits, we could not explore the interactions between genetic polymorphisms and environmental factors in ischemic stroke. Therefore, the relationship between In sum, our present study provided evidence that three SNPs rs693, rs651821 and rs662799 were associated with ischemic stroke risk in a Chinese Han population, which could be used in clinical diagnosis of ischemic stroke. Further investigations are deserved to discover more susceptible loci to other cardiovascular diseases, and to clarify the molecular mechanism of ischemic stroke.All the participants in this study were Han Chinese lived in Hainan Province. A total of 488 patients who diagnosed with ischemic stroke were recruited from the Haikou People's Hospital between January 2013 and February 2016. They were all newly diagnosed to be ischemic stroke patients according to the International Classification of Disease  on the basis of history, clinical symptoms, physical examination, and cranial computed tomography or magnetic resonance imaging. Patients with hemorrhagic stroke, subarachnoid hemorrhage, transient ischemic attack, traumatic brain injuries, infectious diseases, and tumors were excluded in this study.We also recruited 503 controls at the Haikou People's hospital. Controls were healthy, unrelated individuals selected randomly from the medical examination center of the hospital. All participants were informed of the procedures and purpose of the study, and each participant provided signed informed consent forms. The use of human samples in this study was approved by the Ethics Committees of the local participating hospitals and was in accordance with Department of Health and Human Services (DHHS) regulations for protection of human research subjects.APO genes were selected from previously published polymorphisms associated with stroke [APO genes including 2 SNPs in APOA1, 4 SNPs in APOA5 and 8 SNPs in APOB were selected for further genotyping. Genomic DNA was extracted from peripheral blood leukocytes using the GoldMag® nanoparticles method  according to the manufacturer's instructions and DNA concentration was measured using the NanoDrop 2000 . We used Sequenom MassARRAY Assay Design 3.0 Software to design Multiplexed SNP MassEXTEND assays [Candidate SNPs in the h stroke , 24. ValD assays . SNP genD assays . Data maD assays , 26.p values in this study were two-sided, and p ≤ 0.05 was considered as the statistical significance threshold [p values were calculated using unconditional logistic regression analysis with adjustment for age and gender [Statistical analyses were performed using Microsoft Excel  and SPSS 16.0 statistical package . All hreshold . An exacd gender , 29.http://www.cog-genomics.org/plink2/) to assess the association of single SNPs with the risk of ischemic stroke. The odds are a way of representing probability, especially familiar for betting. The confidence intervals are standard errors for the log odds ratio. The odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by unconditional logistic regression analyses adjusted for age and sex [The three genetic models  were applied by PLINK software ( and sex .We used the Haploview software package (version 4.2) to analyze the linkage disequilibrium (LD), haplotype construction, genetic association at polymorphism loci and a D' value greater than 0.8 indicated that the related SNPs formed one block . Marker


Purpose. We aimed to determine the value of lung function measurement for predicting cardiovascular (CV) disease by evaluating the association between FEV1 (%) and CV risk factors in general population. Materials and Methods. This was a cross-sectional, retrospective study of subjects above 18 years of age who underwent health examinations. The relationship between FEV1 (%) and presence of carotid plaque and thickened carotid IMT (≥0.8 mm) was analyzed by multiple logistic regression, and the relationship between FEV1 (%) and PWV (%), and serum uric acid was analyzed by multiple linear regression. Various factors were adjusted by using Model 1 and Model 2. Results. 1,003 subjects were enrolled in this study and 96.7% (n = 970) of the subjects were men. In both models, the odds ratio of the presence of carotid plaque and thickened carotid IMT had no consistent trend and statistical significance. In the analysis of the PWV (%) and uric acid, there was no significant relationship with FEV1 (%) in both models. Conclusion. FEV1 had no significant relationship with CV risk factors. The result suggests that FEV1 may have no association with CV risk factors or may be insensitive to detecting the association in general population without airflow limitation. Cardiovascular disease (CVD) is one of the main causes of mortality in the general population as well as in those with chronic obstructive pulmonary disease (COPD) . ReducedPrevious studies suggested that COPD and CVD might have this association because of their connection with regional or systemic inflammation –8. Low-gCurrently, evaluation of CVD risk is commonly and easily done by measurement of carotid intima-media thickness (IMT) and pulse wave velocity (PWV). Increased carotid IMT or abnormal PWV is used as a predictor of subclinical atherosclerotic burden –14 and bUntil now, most studies reporting the association of decreased lung function  with CVD risk factors have been conducted in patients with moderate to severe COPD. Their association in the general population without airflow limitation has not been clearly defined.This study aimed to determine the value of lung function measurement for predicting CVD by clarifying the relationship between decreased lung function represented by FEV1 and CV risk factors in general population without airflow limitation.n = 16), indefinite smoking status (n = 47), FEV1 < 80% (n = 70), FEV1/FVC < 70% (n = 89), and ankle-brachial index (ABI) < 0.9 (n = 35). We divided the final enrolled subjects  into four groups according to FEV1 (%). The study design was approved by the Institutional Review Board of the Dongguk University Ilsan Hospital.This was a cross-sectional, retrospective study of 1,260 subjects above 18 years of age who underwent health examinations at Dongguk University Ilsan Hospital  between 01 March 2006 and 29 February 2012. The exams included a questionnaire of smoking status, pulmonary function test (PFT), carotid ultrasonography (US), PWV test, and determination of serum uric acid level. Exclusion criteria were as follows: no questionnaire (2).All participants filled self-administered questionnaires which included questions about smoking status. The patients' height and weight were obtained from the database and body mass index (BMI) was calculated as weight in kilograms divided by height squared (kg/mVenous samples were collected after a 12 h overnight fast and serum white blood cell (WBC) count, serum lipids, high-sensitivity C-reactive protein (hsCRP), hemoglobin A1C (HbA1C), and serum uric acid were examined. The biochemical tests were done using a Roche/Hitachi cobas c702 automatic analyzer (Roche Diagnostics) at the Department of Laboratory Medicine at Dongguk University Ilsan Hospital, which is accredited by the Korean Society for Laboratory Medicine and the Korean Association of Quality Assurance.Spirometry was performed in the standing position by experienced technicians following the American Thoracic Society recommendations. A Vmax 2130 spirometer (Sensormedics) was used to measure FVC and FEV1 in absolute values (L), and predicted spirometry values (%) were calculated using the Morris equations . AirflowCarotid ultrasonography was conducted by a registered sonographer by high-resolution ultrasound system . The carotid arteries were examined both sides and the longitudinal images were obtained at the bulb of common carotid artery (CCA) and the carotid IMT was measured in the straight segment of the CCA 20 mm proximal to the carotid bifurcation.The thickened IMT was defined when both or one carotid IMT was ≥0.8 mm. The carotid IMT ≥ 0.8 mm was regarded as the cut-off of increased cardiovascular risk in previous studies , 21. Then = 35) were excluded because baPWV can be underestimated when the subject has a peripheral arterial disease, which can be defined as ABI <0.9 [Brachial-ankle PWV (baPWV) was obtained simply by wrapping blood pressure cuffs on the four extremities after at least a 5-minute rest and was calculated as the ratio of the virtual arterial path length derived from the subject's height and the time difference between the initiation point of systolic increases in brachial and ankle pressure waves. baPWV was measured using a Colin VP-2000 automated waveform analyzer . The percentage of PWV (PWV%) represented the difference from the value of calculated pulse wave velocity which was based upon age-matched Asian population . SubjectABI <0.9 , 25.p value was <0.05.We classified the subjects in quartiles by FEV1 (%): FEV1 80–93%, 94–100%, 101–109%, and 110–155%. To analyze subjects' baseline characteristics, we used one-way analysis of variance (ANOVA) for continuous variables and linear-by-linear association for categorical variables. The relationship between FEV1 and presence of carotid plaque or thickened IMT was analyzed by multiple logistic regression. The relationship between FEV1 and PWV (%) or serum uric acid was analyzed by simple and multiple linear regression. Model 1 was adjusted for age, BMI, and smoking status. Model 2 was adjusted for age, BMI, smoking status, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, systolic blood pressure (BP), HbA1C, and hsCRP. R version 3.2.4  was used as the statistical software, for windows and results were considered as statistically significant when the n = 970) of the subjects were men. Among them, 72.9% (n = 731) were current or ex-smokers; the mean BMI was 24.46 ± 3.22 kg/m2 and mean uric acid was 6.04 ± 1.31 mg/dL.Among the 1,260 participants who underwent health examinations that included spirometry, carotid US, PWV, and serum uric acid, 1,003 subjects were enrolled in this study . The basWe classified subjects in quartiles according to the FEV1 (%) . Age incWe analyzed the trend for the carotid plaque, thickened IMT, PWV (%), and uric acid by the FEV1 (%) quartiles . The perp = 0.034) in the 2nd quartile than in the 4th quartile, and there was no significant relationship among other quartiles (The odds ratio (OR) of the 4th quartile was considered as 1. In model 1, the adjusted OR of the presence of carotid plaque was lower , and uric acid after adjustment of confounding factors including age, BMI, smoking status, BP, cholesterol, HbA1C, and hsCRP. The age of the 4th quartile (higher FEV1) was the highest and the BMI was the lowest . This suThe methods of measurement of PWV are variable. Carotid-femoral pulse wave velocity (cfPWV) is the most validated and is regarded as the standard technique to measure arterial stiffness. But primary use of cfPWV in clinical fields is limited because of its difficulty to be performed. Therefore, our health examination program included baPWV, which is a popular modality of quantification of arterial stiffness and which has proven to be predictive of CV events as well as mortality –28.As the FEV1 increased, percentage of carotid plaque, thickened IMT, and PWV (%) had no consistent trend . Serum uMany studies reported the connection between airflow limitation and increased CV risk with systemic inflammation –8, 10. IBased upon the previous results that reduced lung function was related to increased CV risk in COPD patients, we thought that this concept could be applicable in subjects without airflow limitation. A few previous studies reported that decreased pulmonary function was related to CV risk factors in non-COPD patients but had some limitations , 32.One study reported the significant relationship between FEV1 and thickened carotid IMT and presence of carotid plaque in 1,625 subjects divided into 3 quartiles by FEV1 . But the3He magnetic resonance imaging (MRI) was related to carotid atherosclerosis measurements including IMT, vessel wall volume, and total plaque volume [In another small study of 27 asymptomatic ex-smokers, there was no association between FEV1 and thickened carotid IMT, but worse ventilation defect percent (VDP) measured by e volume .3He MRI can be a sensitive predictive method for detecting CV risk factors that cannot be detected by spirometry or computed tomography scan in ex-smokers without airflow limitation. This can be a novel suggestion but the interpretation has to be done with caution due to its limited sample size. However, the result that FEV1 was not related to carotid IMT but VDP on MRI related to it in the study is noteworthy.These results imply that The strength of our study is that we completely excluded subjects with airflow limitation and tried to clarify the relationship between FEV1 and CV risk factors in subjects without airflow limitation after adjustment of confounding factors. Furthermore, the sample size of this study was larger than the previous studies.However, our study showed no relationship between FEV1 and CV risk factors. We have to consider the limitations of the study that could have influenced the results. First, this was the cross-sectional study that included only subjects that had undergone the health examination. Therefore, we reviewed the data retrospectively so we could not obtain the time sequence of the decreased lung function and increased cardiovascular risk. In addition, self-reporting of smoking habit is prone to bias; hence, cotinine measurement should be included in future studies. Second, in our study, as the FEV1 increased, the mean age of the each quartile also increased and BMI decreased. We thought this could imply that more healthy old subjects compared to general population were enrolled in this study. Third, many important confounding factors were adjusted in our study, but some other factors that could affect the results might not have been considered, such as underlying comorbidities. Fourth, we did not conduct postbronchodilator spirometry tests because of the limitation of healthcare examination program. But in most previous related studies, prebronchodilator spirometry tests have been done and we initially excluded the subject with self-reported asthma or COPD. Fifth, this study focused on a Korean population. Therefore, it is unclear whether these results are applicable to a Western population in which mechanisms and risk factors for CVD may differ. Furthermore, 96.7% of the study subjects were men, which limits applicability to the general population. Future study including more female subjects will be needed to confirm these findings.In conclusion, the pulmonary function represented as FEV1 did not have significant association with CV risk factors, such as presence of carotid plaque, PWV abnormality, thickened carotid IMT, and uric acid after adjustment of confounding factors in a general population without airflow limitation. This implies that a relationship among them does not exist in subjects without airflow limitation or that FEV1 may be insensitive to the detection of the association with CV risk factors in general population without airflow limitation.Studies reporting the relationship between pulmonary function and cardiovascular risk of subjects without airflow limitation have been scarce. Therefore, this study provides meaningful data. Further large-scale prospective studies are needed to clarify the relationship and for finding the cost-effective diagnostic approaches in the subclinical state.


Low educational level (EL) and low physical fitness are both predictors of increased morbidity and mortality in patients with type 2 diabetes. It is unknown if EL is related to physical fitness. This would have important implication for the treatment approach of patients of low EL.In 2011/12, we invited participants of a new nationwide Swiss physical activity program for patients with type 2 diabetes to participate in this study. EL was defined by self-report and categorized as low (mandatory education), middle  or high (high school/university). Physical fitness was determined using 5 validated measures that assessed aerobic fitness, functional lower limb muscle strength, walking speed, balance and flexibility. Potential confounder variables such as other socio-cultural factors, physical activity level, body composition, diabetes-related parameters and complications/co-morbidities as well as well-being were assessed.2 and an HbA1c of 7.3±1.3%. The mean diabetes duration was 8.8±7.4 years. In the baseline assessment, higher EL was associated with increased aerobic fitness, increased functional lower limb muscle strength, and increased walking speed using linear regression analysis , 15 ± 4.7, 13.9 ± 2.7, 12.6 ± 2.9 seconds for strength (p = 0.001) and 8.8 ± 1.6, 8.3 ± 1.4, 7.8 ± 1.4 seconds for walking speed (p = 0.004)). These associations were independent of potential confounders. Overall, aerobic fitness was 46%, functional limb muscle strength 16%, and walking speed 11% higher in patients of high compared to those of low EL. EL was not related to balance or flexibility.All invited 185 participants  agreed to be included. Of all patients, 23.1% had a low, 32.7% a middle and 44.2% a high EL; 41.8% were professionally active. The study population had a mean BMI of 32.4±5.2 kg/mA main strength of the present study is that it addresses a population of importance and a factor (EL) whose understanding can influence future interventions. A second strength is its relatively large sample size of a high-risk population. Third, unlike studies that have shown an association between self-reported fitness and educational level we assessed physical fitness measures by a quantitative and validated test battery using assessors blinded to other data. Another novelty is the extensive evaluation of the role of many relevant confounder variables.In conclusion, we show that in patients with type 2 diabetes EL correlates favorably and independently with important health-related physical fitness measures such as aerobic fitness, walking speed, and lower limb strength. Our findings underline that diabetic patients with low EL should be specifically encouraged to participate in physical activity intervention programs to further reduce social disparities in healthcare. Such programs should be structured and integrate the norms, needs and capacities  of this population, and their effectiveness should be tested in future studies.NCT01289587University of Lausanne clinicaltrials.gov  Low socioeconomic status or educational level (EL) are risk factors for chronic diseases such as diabetes type 2, and related to worse metabolic control and diabetic complications in patients with diabetes . Low EL Physical activity  and physwww.diafit.ch), a program in Switzerland to promote regular exercise in patients with type 2 diabetes, should increase recruitment among low EL patients and adapt the program’s content to cover their needs with the aim to reduce morbidity and mortality.There is evidence for an impact of socioeconomic status on physical activity  and physThus, the goal of this study was to investigate if EL is related to five physical fitness measures in patients with type 2 diabetes: aerobic fitness, functional lower limb muscle strength, walking speed, balance and flexibility. Given that such a relationship might be affected by important potential confounders, such as physical activity or body composition, these were assessed and adjusted for.The clinical DIAfit program was introduced in 2011 in the French-speaking part of Switzerland. All 185 diabetic patients that were referred to this program between 2011 and 2012 from all 11 existing DIAfit treatment centers in the French-speaking part of Switzerland were invited to participate in this study.Inclusion criteria in the clinical DIAfit program patients were age ≥18 years and a diagnosis of type 2 diabetes for ≥3 months. Exclusion criteria were current diabetic foot ulceration; ischemia during an exercise test or state III peripheral vascular disease; untreated proliferative retinopathy and autonomic neuropathy with unstable blood pressure during the exercise stress test.This current analysis is based on the cross-sectional data of the study. All patients underwent the same structured medical visit by physicians collaborating in the DIAfit program and who had a one-day training. Referring physicians also filled out a questionnaire regarding detailed information about the different diabetes complications , presence, date of onset and extent of ischemic cardiac disease and peripheral arterial vascular disease), medical comorbidities and treatment. Patients had a fasting blood analysis, underwent physical fitness tests, wore a pedometer during three full days (Monday to Wednesday) and filled out questionnaires.The study was approved by the ethical committee of Lausanne in 2011 (protocol 252/10) and all participants signed a written informed consent. The trial was registered .Educational level has been shown to be the strongest predictor of socioeconomic status  and is rPhysical fitness was assessed by five parameters: aerobic fitness, lower limb muscle strength, walking speed, balance and flexibility.Aerobic fitness was evaluated with a maximal graded cycle ergometry test performed by a cardiologist blinded to the other data. Participants started at 20 Watts. Increments of 20 Watts per 2 min were made until exhaustion or until reaching one of the American College of Sports Medicine established criteria for maximal oxygen uptake . Heart rLower limb muscle strength was assessed with the Chair Stand Test. Participants sat with arms folded across the chest and with their back against the chair. The patient was instructed to stand up and sit down five times as quickly as possible and the required time (sec) was recorded . WalkingPhysical activity, foot deformity and falls: Physical activity, defined as number of steps per day, was recorded with the Yamax SW-200 digi Walker  during tThe presence of feet deformations such as hallux valgus, claw or hammer toe, and presence of flat foot were assessed by visual inspection.The number of falls within the last 12 months was reported during the medical interview or based on medical charts.Body composition: BMI was calculated based on measured height and weight. Waist circumference was measured midway between the iliac crest and the lowest border of the rib cage. Body composition was assessed by bioelectrical impedance using a 4-polar single frequency device .Diabetes-related parameters, presence or extent of diabetes complications: Information about diabetes duration, presence of retinopathy, nephropathy, ischemic cardiac disease, peripheral arterial vascular disease, and the current medical treatment were systematically retrieved from the referral letters, the existing medical charts, if the patient was referred from the same institution, or, if missing, collected during the medical DIAfit visit by asking the patient or the referring physician. Data were then reported in the medical charts. If data were missing or unclear regarding retinopathy, the patients’ ophthalmologists were also contacted and the presence of background, proliferative retinopathy and macular edema were noted. If the last exam was beyond 1 year, a repeat ophthalmology exam was requested. Regarding nephropathy, the presence of albuminuria or reduced glomerular filtration rate without any other etiology were used to define nephropathy. Albuminuria or plasma creatinine were tested during the visit, if no laboratory results performed within the last 3 months were provided by the referring physician or present in the chart. The presence of ischemic cardiac disease or peripheral vascular disease was diagnosed based on the report of the referring physician or the medical charts without performing another exam besides the standard cycle ergometry test done before and at the end of the program (see above). Vibration threshold was measured in every patient during the DIAfit medical visit using the mean value of the left and right foot measured by a graduated Rydel-Seiffer tuning fork  applied to the respective hallux of each foot . Of all Cardiovascular disease risk factors and well-being:Hypertension was defined as either treatment for hypertension or measured blood pressure values >140/90 mmHg . DyslipiWell-being was assessed using the WHO Well-Being Scale (WBT-5) as used previously in a large diabetic population . It is aStatistical analyses were conducted using Stata 12 . Descriptive statistics were calculated as mean±SD for continuous variables, or percentage for categorical variables. Missing data were not imputed.The associations between EL, coded as an ordinal variable from 1 (lowest education) to 3 (highest education), and the five physical fitness measures were assessed by least squares linear regression adjusting for age and sex.The relationships between the following potential confounders and the five physical fitness measures were also assessed separately after adjusting for age and sex ,37,38,39In order to identify the best modeling of fitness in this population, we also performed a backward-stepwise analysis on the five fitness measures including all above mentioned parameters as well as EL as predictors, keeping all terms with a significance level of p<0.1 and always including age and sex.All 185 recruited participants of the clinical DIAfit program agreed to participate in the study. One hundred fifty-six patients  had a valid dataset with information on EL and represent the population that was used for the present analyses . Baselin2 and a HbA1c of 7.3±1.3%. Their mean diabetes duration was 8.8±7.4 years. Microvascular  or macrovascular  diabetic complications were present in 2–14% of patients; 61% were treated with insulin, 24% with glucagon-like peptide-1 receptor agonists and 88% with oral anti-diabetic agents .Of all patients, 23.1% had a low, 32.7% a middle and 44.2% a high EL; 41.8% were professionally active. Forty-two percent of the study population were born outside of Switzerland. Patients had a mean BMI of 32.4±5.2 kg/mThere were no significant non-linear trends. A higher EL was related to (i) increased performance in aerobic fitness, (ii) increased functional lower limb muscle strength, and (iii) increased walking speed . For these three fitness tests, this observed increase was gradual across the three EL categories. Thus, an increase from each EL to the next was associated with a mean adjusted increase of 15.7 Watts in aerobic fitness, a mean decrease of 1.2 sec in the time needed to perform the Chair Stand Test, and a mean decrease of 0.4 sec to walk a 10 meter distance. Overall, aerobic fitness was 46%, functional limb muscle strength 16%, and walking speed 11% higher in patients with high compared to those with low EL. EL was not related to balance or flexibility.In bivariate analysis, higher lean body mass and a higher vibration threshold were related to increased aerobic fitness (p ≤ 0.05). None of the confounders were related to lower limb strength. Being professionally or physically active was related to a higher walking speed (p ≤ 0.02). Longer diabetes duration and neuropathy (lower vibration threshold) were related to decreased balance performance (p ≤ 0.02). BMI, waist circumference and lean body mass were inversely associated with flexibility (p ≤0.03). All other tested associations between potential confounders and physical fitness measures were not significant.Adjusting for the above mentioned significant confounder variables did not significantly alter the relationship between EL and aerobic fitness ; p = 0.003 after adjusting for lean body mass and neuropathy) or EL and walking speed ; p = 0.005 after adjusting for professional and physical activity).The results of the backward-stepwise analysis on the five physical fitness measures including all parameters are shown in Table 3.2 0.31 and 0.20, respectively, both p<0.0001). Lower limb strength was associated with EL and lower fat body mass. Better balance was associated with higher vibration threshold. Flexibility was associated with decreased lean body mass .While always including age and sex, aerobic fitness was best associated with EL. Walking speed was also associated with EL as well as with professional occupation  whether this same relationship exists for individuals with type 2 diabetes, 2) whether it holds true for different measured fitness parameters and 3) whether a potential relationship is independent of confounder variables.We have shown for the first time that in patients with type 2 diabetes higher EL is positively related to increases in aerobic fitness, functional lower limb muscle strength, and walking speed, three fitness measures that predict lower all-cause mortality in both the general and the diabetic population. In our population, a gradual increase in fitness was observed among the three EL categories. These relationships were independent of potential confounders such as other socio-cultural factors, physical activity, feet deformity, number of falls, body composition, diabetes duration and metabolic control, presence or extent of diabetes complications, presence of cardiovascular disease factors, and well-being.The finding of a strong and independent relationship between EL and different physical fitness measures in patients with type 2 diabetes is clinically relevant. For example, the difference between low and high EL in aerobic fitness was 46%. Importantly, both higher EL and higher physical fitness have been shown to be related to reduced morbidity and mortality. This relationship might be due to a higher consciousness of the importance of fitness and the relationship between physical activity and health in subjects with increased EL. Furthermore, family activity habits differ according to EL . HoweverThe observed relationship between EL and physical fitness measures are in line with two previous studies conducted in healthy non-diabetic populations: Coe et al.  found anIn our study, EL was not related to balance and flexibility. One explanation might be that balance in this specific population is predominantly influenced by the severity of neuropathy . In addiPhysical activity, body composition, diabetes duration, diabetic complications and professional occupation were also related to one or several fitness measures: Physical activity was related to aerobic fitness and walking speed in bivariate analyses. Increased lean body mass and less microvascular complications were related to aerobic fitness in bivariate analyses. This seems logical as a higher muscle mass is generally also related to a higher physical fitness , and becOverall, the results show that EL is independently related to physical fitness in patients with diabetes. Other factors such as number of steps per day, body composition and diabetic complications were also related to fitness. Based on these findings, the most important conclusion would be that a specific effort has to be undertaken to encourage physical activity promotion in this population. Structured physical-activity programs should be tailored in order to specifically target patients with low EL. The programs should increase knowledge about the important relationship between physical activity and health, as well as provide patients with tools to help them integrate physical activity in their daily life. Regarding other aspects of diabetes education, a recent study showed that a culturally sensitive, structured, group-based diabetes education based on theory of empowerment, food habits and health belief models can enhance biomedical and behavioral outcomes including patients with low EL . The preA main strength of the present study is that it addresses a population of importance in the health and a factor (EL) whose understanding can influence future interventions. A second strength is the fact that we recruited patients in non-specialized clinical centers. Importantly, all patients of the clinical program agreed to participate in the study which increases its representativeness considerably. The high prevalence of patients with diabetic complications and of patients that are treated with insulin further underlines its representativeness, as these patients are often excluded from trials related to physical activity or fitness. Only few data with a relatively large sample size of high-risk population do exist. Third, unlike studies that have shown an association between self-reported fitness and educational level we assessed physical fitness measures by a quantitative and validated test battery including five different fitness domains. A further strength is the extensive evaluation of many relevant known and potential confounder variables and predictors of fitness. One limitation is that we did not assess additional socio-cultural parameters such as income and wealth. Socioeconomic status is a rather complex construct usually held to comprise education, income, wealth and occupation . In pracIn conclusion, we show that in patients with type 2 diabetes EL correlates favorably and independently with important health-related physical fitness measures such as aerobic fitness, walking speed, and lower limb strength. Our findings suggest that patients with diabetes and low EL should be specifically encouraged to participate in physical activity intervention programs to reduce social disparities in healthcare. Such programs should be structured and integrate the norms, needs and capacities  of this population, and their effectiveness should be tested in future studies.S1 File(DTA)Click here for additional data file.


Results showed that the expression of CD31 and CD34 in serous and undifferentiated tumors was greater, whereas endothelial expression of connexin 43 was lower than in clear cell and endometrioid lesions. Serous cancers that formed in the presence of ascites displayed increased expression of connexin 43 in vascular smooth muscles as compared with tumors developed in the fluid's absence. Endothelial cells exposed to ascites from serous and undifferentiated tumors proliferated and migrated more vigorously than cells subjected to ascites from clear cell and endometrioid cancers. They also exhibited an increased level of HIF-1α and produced increased amounts of multiple proangiogenic agents. Our results indicate that high vascularization of aggressive ovarian tumors may be associated with profound angiogenic capabilities of ascites generated by these tumors.Here we examined whether malignant ascites may determine ovarian tumor angiogenesis, and if so whether ascites generated by highly aggressive serous and undifferentiated cancers are more proangiogenic than those from less aggressive clear cell and endometrioid tumors. Angiogenesis was analyzed according to expression of CD31, CD34, and connexin 43. Proliferation and migration of endothelial cells were tested using fluorescence-based methods. The quantification of angiogenic agents and hypoxia-inducible factor 1 Angiogenesis, considered to be the formation of new blood vessels and/or increased permeability of existing ones, is one of the most critical steps in successful progression of primary and metastatic tumors . The cenAlthough the role of angiogenesis in ovarian cancer progression, including the development of intraperitoneal metastases, is indisputable , there a in vivo and in vitro analyses in which we tried to conclusively answer the three following questions: (1) Does the presence of malignant ascites increase angiogenesis within ovarian tumors? (2) Is the proangiogenic potential of ascites from highly aggressive type II ovarian tumors (serous and undifferentiated histotypes) stronger as compared with those from less aggressive type I cancers (clear cell and endometrioid histotypes)? and (3) How does the proangiogenic activity of ascites involve altered proliferation and migration of vascular endothelial cells?Taking into account the above, we designed a project based on bothUnless otherwise stated, all chemicals and cell culture plastics were from Sigma .n = 12), endometrioid (n = 12), high-grade serous (n = 12), and undifferentiated (n = 12). The histopathology, grade, and stage of the tumors were assigned in accordance with the criteria of the International Federation of Gynecology and Obstetrics. The patients did not receive chemotherapy prior to surgery. The study included patients in whom tumors had developed in either the presence (n = 24) or absence (n = 24) of peritoneal ascites.The study was performed on tumors excised from the peritoneal cavity from 48 women with ovarian cancer (stages IIIC and IV) who were undergoing cytoreductive surgery. The analyses included four histological subtypes of ovarian cancer, that is, clear cell (n = 6) undergoing abdominal surgery due to the presence of noncancerous lesions, that is, cystadenoma mucinosum multiloculare. This study using tumors and ascites was approved by the bioethics committee at Poznań University of Medical Sciences  and all patients gave their informed written consent.Malignant ascites were collected in sterile conditions; the fluids were centrifuged at 2500 rpm for 5 min and then cell-free supernatants were stored in aliquots at −80°C until required. Control, benign fluids were obtained from age-matched patients  and cultured in DMEM medium supplemented with 10% fetal bovine serum, L-glutamine (2 mM), glucose (4500 mg/L), sodium pyruvate (110 mg/L), and antibiotics.μm sections. Deparaffinization, rehydration, and epitope retrieval were conducted using Envision Flex Target Retrieval Solution . The cancerous tissue was identified using H+E staining. Tumor angiogenesis was evaluated according to immunohistochemical detection of endothelial cell markers, that is, CD31 and CD34. Briefly, the specimens were incubated with an antibody against CD31  and against CD34 , both diluted 1 : 25. Mouse lung preparations were used as the positive and negative controls. The staining was performed using Autostainer Link 48 .The tumors were fixed in 4% formalin, embedded in paraffin, and cut into 3 Expression of connexin 43 was performed using Connexin-43 Polyclonal Antibody , diluted 1 : 200. The reaction was visualized using the Novolink Polymer Detection System .Planimetric analysis of the brown-stained area reflecting the presence of CD31/CD34/connexin 43-positive cells was conducted using ImageJ 1.47v . Pictures of all immunoreactions were taken using an Axio Vert.A1 microscope .Proliferation of EA.hy926 cells exposed to 10% malignant or benign ascites (for 72 h) was examined using Cell Proliferation Kit I . Migration of EA.hy926 towards 100% malignant ascites used as a chemoattractant (for 4 h) was quantified using ChemoTx migration chambers . Both assays were performed strictly according to the manufacturer's instructions.5 of cells were seeded into 25 cm5 flasks, allowed to attach for 4 hours, and incubated with 10% malignant and benign ascites for 72 h. Afterwards the ascites were removed and the cells were carefully washed and incubated for 72 h to generate an autologous conditioned medium. Samples of conditioned media were centrifuged, filtered through a 0.2 μm pore size filter, and stored at −80°C until required. Measurement of angiogenic agents was performed using appropriate DuoSet® Immunoassay Development kits . The concentration of transcription factor HIF-1α in EA.hy926 cells exposed to ascites  was determined in cell extracts using Human/Mouse Total HIF-1α ELISA (R&D Systems). All assays were performed strictly according to the manufacturer's instructions. Fluorescence was recorded using a Synergy™ 2 spectrofluorometer .Concentrations of EGF, FGF2, GRO-1, HGF, IL-8, MCP-1, and VEGF were quantified in conditioned medium generated by EA.hy926 cells. Briefly, 3 × 10P value < 0.05 were considered to be statistically significant.Statistical analysis was conducted using GraphPad Prism™ 5.00 software . The means were compared with repeated measures analysis of variance (ANOVA) with the Newman-Keuls test as post hoc. When appropriate, the Mann–Whitney test was used. Results were expressed as means ± SEM. Differences with a What is definitely known about the relationship between the presence of malignant ascites, angiogenesis, and the progression of ovarian tumors is that the suppression of blood vessel development, for example, by neutralization of VEGF, results in a reduction of ascite formation and tumor growth , and thaIn the study presented here, we tried to address the issue of the role that malignant ascites may play in ovarian cancer neovascularization by using an optimal model, that is, samples of tumors excised during cytoreductive surgery from patients with various types of ovarian cancer and tumor histotype-specific malignant fluids. Initially, however, we used high-grade serous ovarian carcinoma, which is the most frequent type of ovarian cancer , as a reFollowing this observation, we conducted analogical immunoreactions using tumors representing type I (clear cell and endometrioid) and type II (serous and undifferentiated) cancers that had developed in the presence of comparable volumes of malignant ascites and we found that tumors belonging to highly aggressive histotypes, that is, serous and undifferentiated ones , were chThis effect coincided with markedly decreased expression of gap junction protein connexin 43 in the vascular endothelium within serous and undifferentiated cancers, which may suggest that the ascites also stimulated the second  critical aspect of angiogenesis, that is, increased permeability of existing blood vessels  Figure . It is wUnexpectedly, measurements of connexin 43 in the same cancer histotype but within vascular smooth muscle cells revealed that expression of the protein was very high in the presence of malignant ascites, whereas connexin 43 was almost undetectable in the absence of the ascites . This ef in vitro model. Experiments with EA.hy926 endothelial cells, which are commonly used in studies on angiogenesis [Having phenomenologically established that malignant ascites seem to promote angiogenesis within ovarian tumors and that ascites from serous and undifferentiated histotypes act more proangiogenically than ascites from clear cell and endometrioid tumors, we then decided to analyze the mechanistic aspects of these phenomena by using anogenesis , showed ogenesis , may impβ1, HGF, SDF-1, IL-8, CXCL5, GRO-1, and MCP-1 [It is possible that the predominance of fluids derived from aggressive cancers is associated with their biochemical composition, in particular with an increased concentration of soluble proangiogenic stimuli, including VEGF, TGF-nd MCP-1 . This wond MCP-1 . In addind MCP-1 .Furthermore, we found that malignant ascites from serous and undifferentiated tumors alter the secretory profile of endothelial cells; that is, they trigger the overproduction of such angiogenic agents as EGF, FGF2, GRO-1, HGF, IL-8, MCP-1, and VEGF . This maIt is worth noting that the secretion of five out of seven of the tested proteins was the highest in response to ascites generated by undifferentiated tumors, which may be another factor explaining the very high aggressiveness and the worst prognosis in patients suffering from this type of disease . It shouα (HIF-1α). This molecule's level was the highest in endothelial cells subjected to ascites from undifferentiated tumors, whereas it was considerably lower in cells exposed to ascites from clear cell and endometrioid tumors (α is upregulated under hypoxic conditions characterizing the tumor environment and that the production of above listed proangiogenic agents is known to depend on this factor [α [Another type of activity of the malignant ascites discovered in this study was their effect on the concentration of hypoxia-inducible factor 1d tumors . Taking s factor , it seemactor [α , which iactor [α .Taken together, our findings indicate that augmented angiogenesis may be an important determinant of the high aggression of serous and undifferentiated ovarian tumors, and that the diversified neovascularization level of both more and less aggressive cancer histotypes may be associated with the presence and activity of malignant ascites. From a clinical point of view, these findings may additionally show the necessity of therapeutic strategies aimed at targeting ascites, particularly in patients with highly aggressive peritoneal tumors.


Escherichia coli and Buchnera aphidicola, which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location.Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of  Escherichia coli and Buchnera aphidicola and show that they occur at rates approaching those of base substitutions. Knowledge of the full extent of sequences subject to transcription indels supports a new model of bacterial transcription slippage, one that relies on the number of complementary bases between the transcript and the DNA template to which it slipped.The high level of mistakes generated during transcription can result in the accumulation of malfunctioning and misfolded proteins which can alter global gene regulation and in the expenditure of energy to degrade these nonfunctional proteins. The transcriptome-wide occurrence of base substitutions has been elucidated in bacteria, but information on transcription insertions and deletions—errors that potentially have more dire effects on protein function—is limited to reporter gene constructs. Here, we capture the transcriptome-wide spectrum of insertions and deletions in  In addition to the errors that occur during DNA synthesis, which form the basis for adaptation and heritable genetic variation, nonheritable errors are generated during the process of transcription. These transient errors are produced at rates that are orders of magnitude higher than replication error rates –4, such –5–9–Escherichia coli of ~8 × 10−5 per nucleotide that was relatively constant across different growth states and growth phases  along the template DNA, causing a portion of the template to be either skipped (resulting in a deletion) or retranscribed (leading to an insertion) –20. Prev–17–E. coli, which has an equitable occurrence of each nucleotide, and a low-G+C bacterial endosymbiont, Buchnera aphidicola, whose genome is greatly enriched in long tracts of adenosine and thymine. We found that while insertions predominated in homopolymeric runs in both species, deletions occurred in more complex sequences. These results led us to develop a general model of transcription slippage that is driven by RNA:DNA hybrid complementarity at the site of the new DNA template.The focus on error-prone repeats led to the notion that transcription indels occur primarily at homopolymeric runs –20, whic–Escherichia coli and Buchnera aphidicola by applying a circularization method that prevents the inclusion of sequencing artifacts  Despititutions . Conside10.1128/mBio.01230-17.2DATA SET S1 E. coli transcription deletions used in our analyses. Column 1 contains the SRA accession number of the source transcriptome, column 2 indicates the biological replicate, column 3 provides the genomic location of the deletion, column 4 provides the nine bases directly upstream of the transcription deletion, column 5 provides the deleted sequence, and column 6 provides the sequence immediately downstream of the transcription deletion. Download DATA SET S1, XLSX file, 0.1 MB.Copyright © 2017 Traverse and Ochman.2017Traverse and OchmanCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01230-17.3DATA SET S2 E. coli transcription insertion used in our analyses. Column 1 contains the SRA accession number of the source transcriptome, column 2 indicates the biological replicate, column 3 provides the genomic location of the insertion, column 4 provides the nine bases directly upstream of the transcription insertion, column 5 provides the inserted sequence, and column 6 provides the sequence immediately downstream of the transcription insertion. Download DATA SET S2, XLSX file, 0.01 MB.Copyright © 2017 Traverse and Ochman.2017Traverse and OchmanCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01230-17.4DATA SET S3 Buchnera transcription deletions used in our analyses. Column 1 contains the SRA accession number of the source transcriptome, column 2 indicates the biological replicate, column 3 provides the genomic location of the deletion, column 4 provides the nine bases directly upstream of the transcription deletion, column 5 provides the deleted sequence, and column 6 provides the sequence immediately downstream of the transcription deletion. Download DATA SET S3, XLSX file, 0.01 MB.Copyright © 2017 Traverse and Ochman.2017Traverse and OchmanCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01230-17.5DATA SET S4 Buchnera transcription insertions used in our analyses. Column 1 contains the SRA accession number of the source transcriptome, column 2 indicates the biological replicate, column 3 provides the genomic location of the insertion, column 4 provides the nine bases directly upstream of the transcription insertion, column 5 provides the inserted sequence, and column 6 provides the sequence immediately downstream of the transcription insertion. Download DATA SET S4, XLSX file, 0.02 MB.Copyright © 2017 Traverse and Ochman.2017Traverse and OchmanCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli and 11 nt in Buchnera [Among the insertions that occur during transcription, 80% involve the addition of an individual nucleotide . These sBuchnera ). In eveE. coli and Buchnera can be ascribed almost entirely to the incidence of homopolymeric runs in these genomes. The low (26%) G+C content of the Buchnera genome increases the likelihood and lengths of homopolymeric runs of adenine or thymine, which comprise 80% of the sequenced homopolymeric runs in the E. coli transcriptome and 97% of the sequenced homopolymeric runs in the Buchnera transcriptome. In both organisms, 100% of insertions within homopolymeric runs occurred in runs of adenine or thymine, indicating that homopolymeric runs of guanine and cytosine are not prone to slippage. A minority of transcription insertions (19 in E. coli and 6 in Buchnera) did not occur in these repeat tracts, but in 15 of 19 such cases in E. coli, the inserted nucleotide(s) matched the preceding nucleotide, suggesting that they originated by backward slippage followed by retranscription of the slipped region . In Buchnera, the most common transcription deletion involves single nucleotides . Moreover, guanine and cytosine are significantly underrepresented within transcription deletions , whereas many of the G-rich dinucleotides  were less likely to promote a deletion (P < 0.001)  . The dincleotide . All sigIn the current model for transcription deletions, the elongation complex and transcript lose register with the template DNA (such that the transcript and DNA template are no longer paired), slip forward, and then resume transcription at a downstream point on the template DNA . After aE. coli and Buchnera, the reconstructed RNA:DNA hybrids from observed deletions had more complementary base pairing than expected (P < 0.0001), indicating that after a slippage event, transcription is more likely to resume in regions that impart high complementarity within the new RNA:DNA hybrid.To calculate the extent of complementarity between the slipped transcript and the new DNA template location, we reconstructed the RNA:DNA hybrids after slippage by comparing the nine nucleotides immediately preceding the start of each deletion with the nine nucleotides preceding the end of each deletion, each from the annotated start and endpoints of the deletion in the reference sequence. In both expected  and B (cP < 0.01). Overall, about half of all two-nucleotide deletions occurred in a dinucleotide repeat, and 37 of the 143 three-nucleotide deletions occurred in a trinucleotide repeat. Unlike insertions, these short deletions did not increase in frequency with repeat number: 34 of the 37 repeating runs that promoted deletions consisted of only two repeats, with the second instance of the repeat experiencing the deletion.We next examined the extent to which nonhomopolymeric repeat sequences were associated with transcriptional deletions. Deletions of two or three nucleotides were significantly more likely to occur in regions containing di- or trinucleotide repeats, respectively  (Wilcoxon = 265) of all deletions had complementary base pairing in the last two positions in the RNA:DNA hybrid, a percentage significantly higher than that expected by chance (P < 0.01)  . In sum,Previous studies of the insertions and deletions that arise during transcription examined only those errors that occurred within long synthetic homopolymeric repeats, and the reported findings provided neither the absolute rate of transcription indels nor the full spectrum of sequence motifs prone to such errors –20. Thes–E. coli and 227 indels (70 deletions and 157 insertions) in Buchnera, yielding rates of 1.7 × 10−5 and 3.1 × 10−5 indels per transcribed nucleotide, respectively. The transcription error rates for indels were several-fold lower, but within the same order of magnitude as the transcription error rate for base substitutions reported for E. coli and Buchnera  in Buchnera , yieldinproteins , 23, 24.udogenes , 26. It E. coli and Buchnera, a majority of transcription insertions involve the addition of a single base into homopolymeric runs of either A or T  that occurred in nonhomopolymeric sequences—errors that were previously never assayed—mostly involved duplications of the preceding nucleotide, suggesting that virtually all transcription insertions, whether in homopolymers or not, are caused by retranscription after an event of backward slippage.Based on early models of transcription slippage, previous assays of transcription indels were designed to detect errors occurring in homopolymeric runs. With respect to transcription insertions, our results largely corroborate prior findings, because our genome-wide approach showed that in both r A or T –20. ThosBuchnera were initiated within uninterrupted homopolymeric runs, and only 3 (less than 1%) transcription deletions in E. coli were initiated within uninterrupted homopolymeric runs, a difference likely attributable to the very high incidence of homopolymeric runs in Buchnera.In contrast to transcription insertions, most transcription deletions occurred in sequences that are more complex and were therefore missed by previous assays, which focused solely on transcription errors in homopolymeric runs. Only 15 (21%) transcription deletions in E. coli, suggesting that selection serves to avoid or eliminate frameshifting deletions. Transcription deletions are common in E. coli, and these ≤6-nt deletions comprise 58% of all deletions in E. coli, so it possible that they occur at frequencies high enough to impact fitness. Despite similarities in the rates of transcription deletions in E. coli and Buchnera, the periodicity in transcription deletions was not apparent in Buchnera, most likely because selection is less effective due to the small effective population sizes.Although the relatively high frequencies of transcription errors compared to replication errors imply that transcription errors are generally of little consequence to cellular fitness, we detected a 3-nt periodicity in deletions of 6 or fewer nucleotides in Knowledge of the full scope of transcription errors provides several insights into the mechanisms by which transcription indels arise. In brief, most deletions were greater than 1 nucleotide in length, whereas most insertions were 1 nucleotide in length, arising from the backward slippage of the elongation complex by only 1 base before transcription resumes . BecauseE. coli. Once an elongation complex and transcript lose register with the template and slip backward, the forward translocation from an array of actively transcribing elongation complexes will most likely result in a deletion rather than an insertion. This process likely operates in Buchnera as well, but the high incidence of homopolymeric runs makes for many more backward slippage events, thereby elevating the number of insertions.A process by which upstream arrays of elongation complexes (i) prevent large insertions by blocking further backward slippage, (ii) help restore the original position of a slipped elongation complex, and (iii) facilitate translocation to distant positions helps to explain, based on all three of these aspects of the process, the low insertion-to-deletion rate in E. coli and Buchnera: (i) for insertions at homopolymeric runs, a backward slip of the elongation complex at these sites usually retains complementary base pairing between the 3′ end of the transcript and the template DNA, allowing transcription to resume because the template sequence before and after the slippage event remains identical. (ii) For those insertions occurring at tri- or tetranucleotide repeats, the elongation complex slipped backwards by one repeat and then transcribed an extra repeat , which Because the stability of the elongation complex is affected by the RNA:DNA hybrid, low complementarity after slippage may cause the slipped elongation complex to dissociate , 30, 32.The mechanisms that generate indels during replication are similar to those that result in transcription slippage, but there are fundamental differences between the processes. First, the majority of indels in DNA occur in short repetitive regions , whereasOverall, our model of transcription slippage ; Fig. S110.1128/mBio.01230-17.1FIG S1 FIG S1, PDF file, 1.8 MB.Model of transcription slippage and the resulting insertions. Based on locations and sequence contents of insertions genome-wide, the degree of complementarity of the RNA:DNA hybrid after a transcription slippage event (I and II) determines whether transcription is aborted, produces a truncated transcript (III and IV), or resumes, producing a transcript containing an insertion (V and VI). Steps in the model use the following notations: black, template DNA; blue, transcript RNA and incoming ribonucleotides; orange, the original RNA:DNA hybrid location; green, the retranscribed  region; mismatched bases are shown as angled contacts between noncomplementary nucleotides, and the RNA polymerase transcription elongation complex is represented by a yellow bubble. In this model, normal transcription (I) becomes interrupted when the elongation complex and transcript lose register with the DNA template (II). Possible outcomes include the elongation complex slipping backward to a region of low complementarity (III), and in the example depicted, the elongation complex slips backward 1 base, landing on a template location where 6 of the 9 bases in the RNA:DNA hybrid are not complementary. If transcription cannot resume due to the extent of mispairing in the RNA:DNA hybrid and/or fraying at the end of the transcript, the transcript is aborted (IV). Alternatively, if the elongation complex slips to template location with fewer mismatches, in this case a homopolymeric run (V), the 3′ end of the RNA bonds sufficiently to the DNA template, and transcription resumes (VI) after the retranscribed region, generating an insertion. Download Copyright © 2017 Traverse and Ochman.2017Traverse and OchmanCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the Escherichia coli MG1655 and two biological replicates of Buchnera aphidicola LSR1 by using the CirSeq library preparation protocol (http://andino.ucsf.edu/CirSeq) to generate a consensus sequence for each read (NC_000913.3 for E. coli and NZ_ACFK01000001 for Buchnera). Additionally, we edited the run.sh script to retain the intermediate output  generated in the CirSeq_v3 pipeline, since these outputs contain candidate insertions and deletions. Additional strain information and library preparation protocols have been described elsewhere  as the observed deletions.In many cases, it is possible to identify the precise location of a deletion by aligning reads to the reference sequence; however, many deletions occur in regions of low sequence complexity or involved the deletion of a repeat in a repetitive sequence. Such cases can result in ambiguities in ascertaining which of the multiple, identical repeat units has been deleted, and so these were resolved by positioning the ambiguous portion to the 3′ end of the deletion. Because this procedure may artificially increase base complementarity at the 3′ end of reconstructed RNA:DNA hybrids (see below), we controlled for any introduced biases by treating simulated deletions in the same manner.E. coli and Buchnera were calculated for each replicate by dividing the total number of insertions or deletions in protein-coding transcripts by the sequencing coverage of the corresponding regions and averaging across replicates. To calculate the indel error rates at homopolymeric runs, we first identified all homopolymeric runs of ≥4 nucleotides in length within protein-coding genes of E. coli MG1655 and B. aphidicola LSR1, and then we determined the numbers of insertions and deletions originating in runs of each length category. When evaluating error rates in homopolymeric runs, or across any gene category, indel frequencies were normalized to the sequence coverage for each category. To determine the effect of transcript abundance on error rate, all genes were binned by their average coverage, and the errors and total coverage were tabulated for each bin. Coverage bins increased in 1× increments from 0- to 10-fold coverage, 10× increments from 10- to 100-fold coverage; 100× increments from 100- to 500-fold coverage, and subsequently in 500- to 1,000-fold, 1,000- to 2,000-fold, and >2,000-fold coverage bins. Transcriptomes were analyzed using custom python scripts, and all statistics were performed using Prism GraphPad and R.Transcriptome-wide rates of insertions and deletions of The nucleotide compositions of deleted nucleotides and of the 15-bp regions preceding and succeeding each deletion were calculated by direct count for each observed or simulated replicate and then pooled across replicates. We inferred the complementarity of bases within the RNA:DNA hybrids after a slippage event by comparing the nine nucleotides directly preceding the start of each deletion to the nine nucleotides directly preceding the end of each deletion. The nine nucleotides preceding the start of each deletion represent the nucleotides transcribed before the slippage event and constitute the RNA portion of the RNA:DNA hybrid, and the nine nucleotides preceding the end of each deletion represent the region in which slippage stopped and thus constitute the new portion of DNA in the RNA:DNA hybrid.SRX1694197, SRX1694194, SRX1694017, SRX1694016, SRX1694007, SRX1694003, SRX1693946, SRX1693944, SRX1686622, and SRX1686515.The sequencing data are publicly available from the NCBI SRA (SRP072992). The following individual accession numbers for sequences were assigned:


The intestinal microbiome is critically important in shaping a variety of host physiological responses. However, it remains elusive on how gut microbiota impacts overall growth and more specifically, adipogenesis. Using the pig as an animal model, we compared the differences in bacterial community structure throughout the intestinal tract in two breeds (Landrace and Jinhua) of pigs with distinct phenotypes. The Landrace is a commercial purebred and the Jinhua is a Chinese indigenous, slow-growing breed with high propensity for fat deposition. Using 16S rRNA gene sequencing, we revealed that the bacterial communities are more diverse in the duodenum, jejunum, and cecum of Jinhua pigs than in those of Landrace pigs, whereas the ileal and colonic microbiota show a similar complexity between the two breeds. Furthermore, a number of bacterial taxa differentially exist in Jinhua and Landrace pigs throughout the entire intestinal tract, with the jejunal and ileal microbiome showing the greatest contrast. Functional prediction of the bacterial community suggested increased fatty acid biosynthesis in Jinghua pigs, which could partially explain their adiposity phenotype. Further studies are warranted to experimentally verify the relative contribution of each enriched bacterial species and their effect on adipogenesis and animal growth. Several recent studies characterized the gut microbiome in pigs regarding its relationship with diet4, developmental stage5, lipid metabolism6, antibiotic resistance7, meat quality8, feed efficiency9, and growth performance10. However, relatively little is known about the biogeography of gut microbiota in pigs and their correlation with adipogenesis.Pigs are an important livestock species that serve as both a major food source and an important animal model to study human diseases due to their large size and similar anatomy to humans12. In contrast, the Landrace pig is a lean, fast-growing breed of Danish origin selected for high carcass yield13. To understand the correlation between intestinal microbiota, swine growth and adipogenesity, we compared the biogeography of gut microbiotas collected from different segments of the intestinal tract of Jinhua and Danish Landrace pigs.The objective of this study was to characterize and compare the biogeography of gut microbiota in two swine breeds with distinct phenotypes, namely Jinhua and Landrace pigs. The Jinhua pig is a well-known indigenous breed of Zhejiang Province in eastern China with a slow growth rate but a high propensity for adipogenesis and intramuscular fat deposition14. Intramuscular fat in the longissimus muscle was also measured according to the AOAC (1990) procedures.All animal procedures were approved by the Institutional Animal Care and Use Committee of the Zhejiang Academy of Agricultural Sciences and all methods were performed in accordance with the relevant guidelines and regulations. Three Jinhua and three Landrace sows of a similar age were selected, fed the same diet, and housed in an environmentally controlled room. After parturition, a total of 36 weanling pigs, with three males and three females of similar weight from each sow, were selected and weaned on day 35. All piglets were then housed in six pens in an environmentally controlled room and fed ad libitum commercial diet under standard management, with six pigs from each sow housed in a single pen. Five healthy male and five female piglets of similar weight were selected from each breed and slaughtered on day 240. The digesta from the middle of the duodenum, jejunum, cecum, ileum and colon were collected, snap frozen in liquid nitrogen, and stored at −80 °C. The average thickness of the backfat was measured on the first rib, last rib, and last lumbar vertebrae in the midline using a sliding caliper as described15. The PCR reaction was carried out using Phusion High-Fidelity PCR Master Mix (New England Biolabs) under the following condition: initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and elongation at 72 °C for 30 s, and a final extension at 72 °C for 5 min. PCR products were purified using GeneJET Gel Extraction Kit (Thermo Scientific). Amplicons from different samples were mixed in equal amounts. Libraries were generated by using the Illumina TruSeq DNA PCR-Free Library preparation kit and sequenced by Novogene  on Illumina HiSeq 2500.Bacterial DNA was isolated from each intestinal digesta using the QIAamp DNA Stool Mini Kit  according to the manufacturer’s instructions. The concentration and purity of the extracted DNA was measured using a NanoDrop ND-1000 Spectrophotometer , and the integrity was evaluated on a 1% (w/v) agarose gel. The V4 hypervariable region of the 16S rRNA gene was then amplified with degenerate primers, 515 F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806 R (5′-GGACTACHVGGGTWTCTAAT-3′)http://metagenomics.anl.gov/mgmain.html?mgpage=project&project=mgp82198).The datasets generated during the current study are available in the MG-RAST repository (16 following MiSeq SOP (http://www.mothur.org/wiki/MiSeq_SOP)17. The 2 × 250 bp paired-end reads with ambiguous bases or longer than 275 bp were removed. The UCHIME18 and preclustering methods19 were used to remove chimeric sequences and to reduce sequencing noise, respectively. Sequences related to Archaea, Chloroplast, Mitochondria, and Eukaryota were removed according to the MiSeq SOP. Singletons were removed to reduce the size of the distance matrix. Sequences were aligned with the SILVA reference database 20 and assigned to operational taxonomic units (OTUs) based on the sequence identity threshold of 97% and were classified using the RDP classifier21.Sequences were processed and analyzed using mothur v.1.38.022. The Bray-Curtis distance23 was calculated to estimate the dissimilarity in the community structure, which was visualized using principal coordinates analysis (PCoA). Analysis of similarities (ANOSIM) was performed in mothur v1.380. Enriched OTUs of gut microbiota in both Jinhua and Landrace pigs were identified using LEfSe  24, which first compares the relative abundance of OTUs using the Kruskal-Wallis test at an alpha level of 0.05. The resulting significantly different OTUs were then used as input for linear discriminant analysis (LDA) to calculate their effect sizes. OTUs with the logarithmic LDA score threshold of >2.0 were selected as significantly different OTUs between groups. PICRUSt was further utilized to infer the functional pathways from 16 S rRNA gene sequencing data and the Greengenes database 13.525. The statistical differences between pairs of sample or multiple groups of KEGG pathways were visualized using the STAMP  Software package26.To reduce the biases caused by the sequencing depth, the reads of each sample were randomly subsampled to 51,612 for downstream α- and β-diversity analyses. The α-diversity was examined using Shannon Indexp < 0.05.ANOVA test was used to test the differences in alpha diversity. Significance was set at P < 0.001; Fig. P < 0.001; Fig. Longissimus muscle than Landrace pigs  , which accounted for 23–28% of all bacteria  was also dominant in both the cecum and colon of Landrace pigs. On the other hand, a Bifidobacterium species (OTU #14) was more prevalent in the duodenum, ileum, and cecum of Jinhua pigs, and a Lactobacillus species (OTU #13) was dominant throughout the small intestinal tract of Jinhua pigs. Two members in the genus of Clostridium sensu stricto  were more abundantly associated with Jinhua pigs in the ileum and jejunum, while OTU #18 was more dominant in the cecum and colon of Jinhua pigs that those of Landrace pigs. Similarly, an Allobaculum species (OTU #16) showed over-representation in the duodenum, ileum, and cecum of Jinhua pigs.To identify enrichment of specific bacterial taxa between the two breeds, we performed LEfSeigs Fig. . While mria Fig. , was overia Fig. . A membeLactobacillus (OTU #2) was more abundant in both the duodenum and colon of Landrace pigs, it became more prevalent in the ileum of Jinhua pigs. Another Lactobacillus (OTU #7) was clearly abundant in the duodenum of Landrace pigs, but also had a prevalence in the ileum of Jinhua pigs. A Megasphaera species (OTU#16) was over-represented in the cecum of Landrace pigs, but was more dominant in the duodenum of Jinhua pigs.Interestingly, a few OTUs showed a different pattern of dominance in the two breeds Fig. . For exa25. Many bacterial genes that could potentially trigger inflammatory responses  were predicted to be enriched in both the ileum and jejunum of Landrace pigs. On the other hand, the bacterial genes involved in the metabolism of fatty acids, proteins, amino acids, nucleotides, and carbohydrates were predicted to be enriched in Jinghua pigs  harbors more diverse microbiome than the small intestine  in both Jinhua and Landrace pigs, which is consistent with previous studiesEscherichia were abundant in the small intestinal sections  in the Landrace pigs which is consistent with previous reports30. However, these OTUs were significantly less abundant in the small intestine of Jinhua pigs, which partially explains the higher abundance of genes in Landrace pigs that potentially trigger inflammatory responses such as LPS biosynthesis, motility, secretion, chemotaxis, and flagellar assembly predicted by PICRUSt. Chronic inflammation has been associated with obesity. In a recent study, Yang et al. found that Escherichia spp. was significantly enriched in Laiwu pigs, another Chinese indigenous breed with extremely high intramuscular fat content28. The discrepancy in the abundance of Escherichia between the two obese breeds, Jinhua (this study) and Laiwu , might be due to the differences in genetics, diet, environment, and developmental stages. Of note, there are several limitations of this study. First, despite PICRUSt being used in a wide range of host-microbiota studies, it was mainly developed by using reference genomes tailored to humans. Second, there might be several factors affecting the differences in the biogeography of the gut microbiota between the Jinhua and Landrace piglets such as the sow’s microbiota, which might contribute to the differences in the piglets’ gut microbiota by serving as an initial inoculator of the piglets’ gut microbiota. Furthermore, significantly differences in body weight observed between the Jinhua and Landrace piglets, very likely due to the differences in breeds, might also affect the gut microbiota. Nevertheless, the purpose of this study is not to pinpoint the origins and confounders of the swine gut microbiota, rather, we want to describe the longitudinal variation along the GI-tract microbiota and the cross-sectional variation between the two breeds. Further experiments are desired to determine if the intra- and inter-breed variation in the biogeography of the intestinal microbiome explains the differences in growth performance and fat deposition between different swine breeds.OTUs associated with Supplemental information